EBV Book

Current Topics in Microbiology and Immunology
Christian Münz Editor
Epstein
Barr Virus
Volume 1
One Herpes Virus: Many Diseases
Current Topics in Microbiology
and Immunology
Volume 390
Series editors
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School of Medicine, Rollins Research Center, Emory University, Room G211, 1510 Clifton Road, Atlanta, GA 30322,
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Klaus Aktories
Medizinische Fakultät, Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Abt. I,
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Influenza Research Institute, University of Wisconsin-Madison, 575 Science Drive, Madison, WI 53711, USA
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Centre d’Immunologie de Marseille-Luminy, Parc Scientifique de Luminy, Case 906, 13288, Marseille Cedex 9,
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More information about this series at http://www.springer.com/series/82
Christian Münz
Editor
Epstein Barr Virus Volume 1

One Herpes Virus: Many Diseases
Responsible Series Editor: Peter K. Vogt
13
Editor
Christian Münz
Institute of Experimental Immunology
University of Zürich
Zürich
Switzerland
ISSN 0070-217X ISSN 2196-9965 (electronic)

Current Topics in Microbiology and Immunology
ISBN 978-3-319-22821-1 ISBN 978-3-319-22822-8 (eBook)
DOI 10.1007/978-3-319-22822-8
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Preface
We celebrated the 50th anniversary of the discovery of Epstein Barr virus (EBV)
in Burkitt’s lymphoma last year. During these 50 years of research on EBV, this
first human candidate tumor virus has been found associated with many more
malignant diseases in addition to Burkitt’s lymphoma, including Hodgkin’s lym-
phoma, nasopharyngeal carcinoma, a subset of gastric carcinomas, rare T/NK cell
lymphomas, and many more. However, not only malignant diseases, but also some
autoimmune diseases and the lymphocytosis of infectious mononucleosis have
been found to be linked to EBV. In addition, we have learned to appreciate that
continuous cell-mediated immune control prevents these EBV associated diseases,
but cannot inhibit persistent infection, which the virus establishes in more than
90 % of the human adult population. Thus, EBV serves both as a paradigm for
viral oncogenesis in humans and life-long immune control of chronic infection at
the same time. The changes in the viral host cell and the host’s immune control
that determine the switch between these two states, continue to fascinate us and
new experimental developments allow us to address this question in much more
detail. Our ability to sequence EBV genomes faster and at lower cost allows us to
explore the genetic diversity of EBV and its possible disease association for the
first time. The recombinant EBV system allows us to generate mutant viruses to
address the functional relevance of this diversity and new in vivo models of EBV
infection, tumorigenesis, and immune control provide valuable insights into the
pathologic relevance of the EBV characteristics that we have mapped during the
last 50 years. With these tools in hand we should be able to unravel many more
secrets that this human tumor virus keeps and develop vaccines against some of
the EBV associated diseases in the next 50 years.
This exciting journey is summarized in the two book volumes in front of you.
It starts with personal accounts of the discovery, tumor association, and immune
control by pioneers of EBV research (Anthony Epstein, George Klein, Vivianna
Lutzky, and Dennis Moss). It then continues with the knowledge on EBV genet-
ics and epigenetics that has been gained (Paul Farrell, Paul Lieberman, Wolfgang
Hammerschmidt, Regina Feederle, Olaf Klinke, Anton Kuthikin, Remy Poirey,
Ming-Han Tsai, and Henri-Jacques Delecluse). An overview of EBV associated
v
vi Preface
diseases ranging from infectious mononucleosis and primary immune deficiencies

to EBV associated tumors and autoimmune diseases completes the first volume
(David Thorley-Lawson, Kristin Hogquist, Samantha Dunmire, Henri Balfour,
Jeffrey Cohen, Ann Moormann, Rosemary Rochford, Paul Murray, Andrew Bell,
Jane Healy, Sandeep Dave, Nancy Raab-Traub, Kassandra Munger, and Alberto
Ascherio). In the second volume individual latent EBV gene products are then dis-
cussed (Lori Frappier, Bettina Kempkes, Paul Ling, Martin Allday, Quentin Bazot,
Robert White, Arnd Kieser, Kai Sterz, Osman Cen, Richard Longnecker, Rebecca
Skalsky, and Bryan Cullen). Viral entry and exit complete the virology chap-
ters (Lindsey Hutt-Fletcher, Luidmila Chesnokova, Ru Jiang, Jessica McKenzie,
and Ayman El-Guindy). The remainder of volume two is dedicated to the EBV
specific immune response (Martin Rowe, Anna Lünemann, David Nadal, Jaap
Middeldorp, Andrew Hislop, Graham Taylor, Maaike Ressing, Michiel van Gent,
Anna M. Gram, Marjolein Hooykaas, Sytse Piersma, and Emmanuel Wiertz), in
vivo models of EBV infection (Fred Wang, Janine Mühe, and Christian Münz),
and EBV specific therapies (Stephen Gottschalk, Cliona Rooney, Corey Smith,
Rajiv Khanna, Jennifer Kanakry, and Richard Ambinder). The resulting picture of
32 chapters on EBV biology will hopefully inspire many more young scientists to
join research on this paradigmatic human tumor virus.
Indeed we might just have now the toolbox in hand not only to transfer discove
ries in preclinical infection models to EBV, but also use EBV itself as a human
model pathogen to learn more about the human immune system, viral dynamics in
the human population, and the intricacies of EBV infection.
Zürich, Switzerland Christian Münz

Contents
Part I History
Why and How Epstein-Barr Virus Was Discovered 50 Years Ago. . . . . . . 3

Anthony Epstein
Tumor Associations of EBV—Historical Perspectives. . . . . . . . . . . . . . . . . 17

George Klein
EBV-Specific Immune Response: Early Research

and Personal Reminiscences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
D.J. Moss and V.P. Lutzky
Part II Virus Genetics and Epigenetics
Epstein–Barr Virus Strain Variation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

Paul J. Farrell
Chromatin Structure of Epstein–Barr Virus Latent Episomes . . . . . . . . . 71

Paul M. Lieberman
The Epigenetic Life Cycle of Epstein–Barr Virus. . . . . . . . . . . . . . . . . . . . 103

Wolfgang Hammerschmidt
Epstein–Barr Virus: From the Detection of Sequence Polymorphisms

to the Recognition of Viral Types. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Regina Feederle, Olaf Klinke, Anton Kutikhin, Remy Poirey,
Ming-Han Tsai and Henri-Jacques Delecluse
vii
viii Contents
Part III Viral Infection and Associated Diseases
EBV Persistence—Introducing the Virus. . . . . . . . . . . . . . . . . . . . . . . . . . . 151

David A. Thorley-Lawson
Infectious Mononucleosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211

Samantha K. Dunmire, Kristin A. Hogquist and Henry H. Balfour
Primary Immunodeficiencies Associated with EBV Disease. . . . . . . . . . . . 241

Jeffrey I. Cohen
Burkitt’s Lymphoma. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267

Rosemary Rochford and Ann M. Moormann
Contribution of the Epstein-Barr Virus to the Pathogenesis

of Hodgkin Lymphoma. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
Paul Murray and Andrew Bell
The Role of EBV in the Pathogenesis of Diffuse Large

B Cell Lymphoma. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
Jane A. Healy and Sandeep S. Dave
Nasopharyngeal Carcinoma: An Evolving Role

for the Epstein–Barr Virus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339
Nancy Raab-Traub
EBV and Autoimmunity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365

Alberto Ascherio and Kassandra L. Munger
Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387
Part I
History
Why and How Epstein-Barr Virus
Was Discovered 50 Years Ago
Anthony Epstein
Abstract An account is given of the experiences and events which led to a search
being undertaken for a causative virus in the recently described Burkitt’s lym-
phoma and of the steps which ultimately culminated in the discovery of the new
human herpesvirus which came to be known as Epstein-Barr virus (EBV).
Contents
1 Introduction........................................................................................................................... 4
1.1 Early Chance Events Essential for Both “Why” and “How”....................................... 4
1.2 A Subsequent Key Chance Leading to “Why”............................................................. 4
2 The Search for a Virus........................................................................................................... 6
2.1 Reflections on Research Funding in the 1960s............................................................ 7
2.2 The Beginning of “How”—Persistent Early Failures.................................................. 7
2.3 An Idea Giving a Glimmer of Hope for “How”........................................................... 8
2.4 Chance Provides the Key to “How”............................................................................. 8
2.5 The End of the Beginning to “How”............................................................................ 10
3 The Final Breakthrough to “How”........................................................................................ 11
3.1 “How” the Virus Was Found........................................................................................ 11
3.2 Naming the Virus......................................................................................................... 13
3.3 Characterization of the Uniqueness of the Virus.......................................................... 13
4 Concluding Remarks............................................................................................................. 14
References................................................................................................................................... 14
Based on a lecture presented at EBV@50, the International Meeting in Oxford held in the week
of 28 March 2014 to celebrate the 50th Anniversary of the first publication on the virus by
Epstein, Achong and Barr on 28 March 1964.
A. Epstein (*)
Wolfson College, University of Oxford, Linton Road, Oxford OX2 6UD, UK
e-mail: anthony.epstein@wolfson.ox.ac.uk
© Springer International Publishing Switzerland 2015 3

C. Münz (ed.), Epstein Barr Virus Volume 1, Current Topics in Microbiology
and Immunology 390, DOI 10.1007/978-3-319-22822-8_1
4 A. Epstein
1 Introduction
The story I am recalling here arose from a sequence of linked chances which fol-
lowed from one to the next in an extraordinary chain, with each coming at exactly
the right moment for its significance to be recognized. As was memorably pointed
out by Louis Pasteur 160 years ago, “Dans les champs de l’observation le hasard
ne favorise que les esprits préparés” (In the field of observation chance favours
only the prepared mind).
1.1 Early Chance Events Essential for Both “Why”

and “How”
The chain started some 65 years ago when I began research at the Middlesex
Hospital Medical School in London (founded 1836; since 1987, incorporated into
University College London) and a quite unexpected death gave me access to one
of the very earliest electron microscopes at a time when such things were excep-
tionally rare. Made in UK by Metropolitan-Vickers in Manchester in 1946 it was
the first commercially available instrument of this kind, but is now an exhibit in
the Manchester Museum of Science and Industry; sadly it was not persisted with
and the subsequent market went to Holland, Germany, Japan and the USA.
It was also a lucky chance that the Middlesex Hospital Medical School had an
interest in the then deeply unfashionable chicken cancer viruses. So it was that
I came to work on the Rous fowl sarcoma virus, the first virus known to cause
malignant tumours. It was studied then by only a handful of people worldwide;
indeed, so unfashionable at that time was the idea of viruses causing cancer in
general that Peyton Rous (1879–1970) only got the Nobel Prize (1966) 55 years
after he made his discovery (Rous 1911) because he lived to 86 by which time
views on this subject had changed radically.
With the Rous virus I was able, using the electron microscope, to demonstrate
its morphology and show for the first time that it was an RNA not a DNA virus
(Epstein 1958; Epstein and Holt 1958). All this made me keenly aware of viruses
which cause cancer and of the possibilities of electron microscopy.
1.2 A Subsequent Key Chance Leading to “Why”
This further chance was critically significant. In the 1950s a British Colonial
Service medical officer based in Uganda came, when on leave in UK, to the
Middlesex Hospital, London (Fig. 1), where he had a connection with the
Academic Department of Surgery and his enthralling seminars were usually about
Why and How Epstein-Barr Virus … 5
Fig. 1 The Middlesex

Hospital, London, UK
(founded 1745; 2005,
replaced by the new
University College London
Hospital). Image courtesy of
University College London
Hospital NHS Trust archive
Fig. 2 Photograph of the

notice of a talk by Denis
Burkitt in 1961 at which he
gave the first account outside
Africa of the lymphoma
which came to carry his
name. The original notice is
still extant
the exotic and extreme cases encountered in a developing country. Early in 1961
he came again, but this time he gave a very different kind of talk—the speaker
was in fact Denis Burkitt (1911–1993), an unknown bush surgeon as he described
himself, and his lecture (Fig. 2) was the first account he had ever given outside
Africa of the lymphoma which brought him worldwide fame. Quite by chance I
saw the notice of Burkitt’s talk, and probably out of curiosity, I went.
After the first 20 min I was greatly excited by this strange malignant tumour
of children in Africa affecting bizarre sites and fatal in a few months (reviewed in
Burkitt 1963). But even stranger, Burkitt went on to present unprecedented pre-
liminary data which showed that geographical distribution depended on tempera-
ture and rainfall. This suggested to me that a biological agent must play a part in
causation and with my knowledge of tumour viruses I immediately postulated a
climate-dependant arthropod vector spreading a cancer-causing virus. It turned out
later that it was a cofactor which was arthropod borne (Burkitt 1969), but my idea
focused correctly on the need to search for a viral cause.
Even as Burkitt was talking, I decided to stop my current work in order to seek
for viruses in what became known as Burkitt’s lymphoma—so excited was I that
6 A. Epstein
after the talk I took the notice off a board (Fig. 2) and I have had it ever since.
When Burkitt finished speaking, I was introduced to him, I invited him to my lab-
oratory, and we agreed to collaborate. It was these quite unrelated chances which
were responsible for “Why” the virus was discovered.
2 The Search for a Virus
So what about “How” the virus was discovered? That started with generous sup-
port from the then British Empire Cancer Campaign (founded 1923, became the
Cancer Research Campaign 1970, became Cancer Research UK 2002) which
funded me to visit Uganda a few weeks later. A first visit to Africa was quite
daunting in the 1960s for unlike now, when even teenagers backpack widely,
exotic travel was rare then. However, here too chance leant a hand because after
World War II ended in Europe I had been posted to the Far East (Fig. 3) where
the conflict continued with Japan, and having learned how things were done in the
Indian Empire under the British Raj, it was easy to find my way around the British
Ugandan Colonial Administration modelled on it.
Fig. 3 Inspection at Bareilly

Cantonment in 1946 by
Field Marshall Sir Claude
Auchinleck, C-in-C India
Command. Capt. M.A.
Epstein (right)
The purpose of my visit to Uganda was, of course, to work out how a regular
supply of lymphoma samples from Burkitt’s patients in the capital Kampala could
be flown overnight to my laboratory in London.
2.1 Reflections on Research Funding in the 1960s
Commenting on these events a much later Editorial aptly remarked “It is hard to
imagine any current funding agency supporting a project based on the gut feeling
of a young worker without any supporting data. Thank goodness that was not the
case 40 years ago!”
2.2 The Beginning of “How”—Persistent Early Failures
For 2 years I applied the virus isolation techniques then in use to lymphoma sam-
ples with depressing negative results. Tumour material was inoculated into test cell
cultures, embryonated hen eggs and newborn mice but without effects and direct
examination in the electron microscope also proved fruitless. Failure to gain any-
thing with this tool in relation to the lymphoma was especially disappointing in
view of my early access to it. But additionally so since in 1956 I had gone, thanks
to the Anna Fuller Fund of New Haven, to the Rockefeller Institute in New York
(now the Rockefeller University) specifically to learn from George Palade (1912–
2008; Nobel Prize 1974) at the time of his outstanding contributions to the earliest
phases of biological electron microscopy and, indeed, to the very foundations of
the whole of modern cell biology.
This long period without results was extremely alarming at a very insecure
stage in my career. There was no employment law at that time—I had no letter of
appointment, no terms and conditions, and no idea from year to year whether the
Head of Department would feel inclined to reappoint me.
At this very low point I managed, unusually for a UK scientist then, to get a
very small grant from the US National Cancer Institute. This $45,000 gave me
some very modest independence and enabled me at the end of 1963 to recruit
Dr Bert Achong (1928–1996) to help, once I had taught him, with the electron
microscopy and Miss Yvonne Barr (as she then was) to assist with tissue culture
of which she already had some experience. Before this I had worked for 15 years
only with George Ball (Fig. 4), an absolutely reliable and completely unflappable
young laboratory technician, who had provided indispensable support and contin-
ued to do so in the decades to come.
8 A. Epstein
Fig. 4 George Ball in 1961,

the absolutely reliable young
laboratory technician who
provided indispensable
support before, throughout
and long after the search for
EBV. Image courtesy of
Mr. G.R. Ball
2.3 An Idea Giving a Glimmer of Hope for “How”
In the event, an idea at this time proved more important than the grant. It occurred
to me that if the tumour cells could be grown in culture away from host defences,
a latent cancer virus might be activated and become apparent as I knew happened
with certain chicken tumours (Bonar et al. 1960). However, doing this with a human
lymphoma seemed unlikely since no type of human lymphocytic cell had ever been
maintained in vitro for more than an hour or two (Woodliffe 1964). Nevertheless
I tried repeatedly with the lymphoma using fragments in plasma clots, fragments
floating on rafts and so on, but depressingly and quite predictably all failed.
2.4 Chance Provides the Key to “How”
Yet once again chance intervened in a big way. On Friday 5 December 1963 the
overnight flight from Kampala was diverted to Manchester by fog and we were
only able to retrieve our biopsy in the afternoon after the plane finally reached
London. As usual the tissue was floating in transit fluid, but unusually this was
cloudy. As it was getting late and the cloudiness was likely to be due to bacterial
contamination, the feeling was that we could leave the laboratory for the weekend.
But instead of discarding the specimen and going home I put a drop of the cloudy
fluid on a slide and examined it with the light microscope as a wet preparation.
Rather than seeing the expected contaminating bacteria I was astonished to
find that the cloudiness was due to large numbers of viable-looking free-floating
tumour cells (Fig. 5) which had been shaken from the cut edges of the lymphoma
sample during the flight.
This chance was in turn assisted by another, for I was immediately reminded
that earlier that year on a visit to Yale Medical School I had learned that their
Mouse Lymphoma Research Group had only succeeded in culturing mouse lym-
phoma cells by starting with suspensions of free-floating single cells (Fischer
1957, 1958) obtained in their case in ascitic fluid after growing the tumours in the
abdominal cavities of mice.
Fig. 5 Wet preparation of

free-floating viable-looking
lymphoma cells shaken
from the cut edges of the
lymphoma sample sent
overnight from Uganda
4/5 December 1963. The
appearance was reminiscent
of a mouse ascites tumour.
Phase-contrast light
micrograph

author’s laboratory notebook
for 5 December 1963. Note
the delayed lymphoma
sample described as “like
ascites tumour” (arrow) and
set up for the first time in
suspension culture—“free
cells” (arrow)
Because of this, the free-floating cells in our delayed sample were described in
my laboratory book (Fig. 6) as “like ascites tumour” and were set up for the first
time in suspension culture (Fig. 6) as “free cells”.
10 A. Epstein
2.5 The End of the Beginning to “How”
Shortly after setting up the suspension culture a continuous lymphoma-derived

cell line grew out (Fig. 7) which we labelled EB (Epstein and Barr) to distinguish
its containers from the HELA, OMK, BHK and other banal cell lines we had in
the laboratory, and suspension culture rapidly gave us more such lines from further
Burkitt’s lymphomas. It should be noted that 50 years ago there were no hoods
and we worked on the open bench with rigorous aseptic technique in the updraft of
a lighted Bunsen burner which carried atmospheric contaminants away. Very early
on we used extremely small conical flasks which allowed more culture fluid with-
out increasing the depth than with straight-sided containers, since depth critically
affected the diffused oxygen tension around the cells resting on the bottom, and
this system had the advantage that it could readily be scaled up as the cells became
plentiful.
This was the first time that human lymphocytic cells had ever been grown long
term in vitro, and when the account of the successful procedure was sent for pub-
lication, a leading journal’s expert referees were unwilling to believe that human
lymphocytic cells could be cultured at all. Yet suspension is now the standard
technique to grow such cells used worldwide today for a huge number of different
types of research.
Fig. 7 Light micrographs

of the first ever culture of
Burkitt’s lymphoma-derived
lymphoblasts designated
EB1. Phase contrast of live
cells (left) and Giemsa-
stained fixed cells (right)
3 The Final Breakthrough to “How”
All efforts to show a virus in EB cells using standard contemporary biological

tests failed, so as soon as material could be spared, some cells were fixed, pel-
leted and embedded for electron microscopy. But it should be emphasized that
this was not accepted then as a method for demonstrating viruses; dogma required
that they should be shown by their biological activity or by finding the antibodies
they induced. It was not credited that they could be recognized morphologically.
Indeed, at this time when electron microscopy was rare and little understood, the
images obtained of biological material were considered by many as artefacts of
fixation and processing.
It is worth mentioning here that a notable exception to such views was provided
by Oxford’s Professor Sir Howard Florey (1898–1968; penicillin Nobel laure-
ate 1945, later Lord Florey of Adelaide and Marston); not one to miss a new and
important advance he had come himself to my very small laboratory in London on
21 January 1959 to see what electron microscopy was about in preparation for set-
ting up a unit in his department.
3.1 “How” the Virus Was Found
As regards images of viruses, my time with George Palade had convinced me that
they could be recognized, and classified at least into families, by their appearance
as had been done for bacteria with the light microscope for 100 years.
I examined the first EB cell preparation with the electron microscope on 24
February 1964 and was exhilarated to see unequivocal virus particles in a cultured
lymphoma cell in the very first grid square I searched. I was extremely agitated in
case the specimen might burn up in the electron beam—I switched off, I walked
round the block in the snow without a coat, and when somewhat calmer I returned
to record what I had seen.
I recognized at once that I was looking at a typical member of the herpesvirus
group (Fig. 8) with which I was already very familiar and noted it as such in my
electron microscope laboratory book (Fig. 9) “virus, like herpes”, but there was
no means of knowing which herpesvirus it might be. However, it did seem quite
extraordinary that a herpesvirus was producing virus particles in a cell line yet was
so biologically inert that it did not destroy the whole culture as the known herpes-
viruses would have. Accordingly I rapidly set about reporting the discovery with
my new assistants Bert Achong and Yvonne Barr (Fig. 10). The resulting paper
(Epstein et al. 1964) appeared on 28 March 1964 and became a Citation Classic in
1979, and the 50th anniversary of its publication was celebrated at an International
Meeting in the week of 28 March 2014.
The unusual inertness was reinforced when biological tests for herpesviruses
were applied to the EB cells and all proved negative. At this point I became
12 A. Epstein
Fig. 8 The first electron

micrographs of EBV.
Immature virions assembling
in the cytoplasm of a
cultured EB1 lymphoma cell;
inset, a mature enveloped
particle. These images were
recognized at once as a
herpesvirus

author’s electron microscopy
notebook dealing with the
EB1 cells harvested on 18
February 1964 and examined,
after the usual delays for
processing, on 24 February
1964. Note entry “virus, like
herpes” (arrow)
Fig. 10 M.A. Epstein, B.G.

Achong (1928–1996) and
Y.M. Barr in 1964 at the time
of the first publication on
EBV (Epstein et al. 1964)
concerned that something unnoticed in our procedures was inactivating the virus
and it was clearly urgent to have the tests repeated in some other laboratory.
I approached two leading British herpes virologists, but neither was interested
in our unorthodox findings, and so it came about that I contacted my friends the
husband and wife virologists Werner and Gertrude Henle (1910–1987; 1912–
2006) at the Children’s Hospital in Philadelphia.
EB cells were flown from my laboratory to Philadelphia, the Henles rapidly
confirmed the biological inertness of the virus, and we then reported jointly that it
was a new member of the herpes family (Epstein et al. 1965).
3.2 Naming the Virus
Following my sending the virus to the Henles, they soon subsequently referred to
it as “EBV” (Henle et al. 1968) after the EB cells in which it had come to them,
and this name caught on and was rapidly universally adopted.
3.3 Characterization of the Uniqueness of the Virus
In addition to the biological inertness of the virus, its immunological singularity

was soon demonstrated in Philadelphia (Henle and Henle 1966) and in my labora-
tory using quite different techniques (Epstein and Achong 1967). Shortly after this
its novel biochemical nature was also established (zur Hausen et al. 1970), and
14 years later the complete viral genome was sequenced (Baer et al. 1984).
In the light of subsequent knowledge of the very limited range of cells with
receptors for the virus, the failure to show biological activity is readily understand-
able, but it was very puzzling at the time. It was indeed fortunate that work on the
lymphoma cells and the search for a virus was undertaken in a laboratory where
a rare electron microscope was in daily use (yet another chance) as otherwise the
extreme inertness could have left it undiscovered.
14 A. Epstein
Table 1 Epstein-Barr 28 March 1964–28 March 2014 30,995

virus—research publications
1984 525
(from PubMed)
2004 1079
2013 25/week
4 Concluding Remarks
EBV was in fact the first virus to be found solely by electron microscopy, and
the story of its discovery thus acted out a little joke published over 100 years ago
before viruses were known or electron microscopes dreamt of:
The microbe is so very small
You cannot make him out at all
But many sanguine people hope
To see him through the microscope
(Belloc 1897)
But the huge extent of work on EBV following its finding by electron micros-
copy is not generally realized even by experts in the field and is therefore worth
a comment. In the first 50 years since the discovery there were more than 30,000
peer-reviewed publications on EBV (Table 1). Of course in the early years the
numbers were very small, but as the decades went by they increased dramatically
(Table 1, cf 1984 and 2004) and finally in 2013 they were running at 25 per week.
It is an arresting thought that each author of a chapter in the present book is mak-
ing a contribution, however small, to the vast worldwide undertaking of accumu-
lated EBV research.
The reason for the wide interest in EBV has been, of course, because it was
the first putative and then the first definitive human cancer virus. Interestingly, in
the 50 years that the virus has been known to science human tumour virology has
moved from the distant margins of the biomedical agenda to the very centre and in
recent years to the very top with the introduction of anti-tumour virus vaccine pro-
grammes to prevent significant human cancers.
References
Baer R, Bankier AT, Biggin MD et al (1984) DNA sequence and expression of the B95-8
Epstein-Barr virus genome. Nature 310:207–211
Belloc H (1897) The microbe in more beasts for worse children. Duckworth & Co., London
Bonar RA, Weinstein D, Sommer JR et al (1960) Virus of avian myeloblastosis. XVII.
Morphology of progressive virus-myeloblast interactions in vitro. Natl Cancer Inst Monogr
4:251–290
Burkitt DP (1963) A lymphoma syndrome in tropical Africa. In: Richter GW, Epstein MA (eds)
International review of experimental pathology, vol 2. Academic Press, New York, pp 69–138
Burkitt DP (1969) Etiology of Burkitt’s lymphoma—an alternative hypothesis to a vectored
virus. J Natl Cancer Inst 42:19–28
Epstein MA (1958) Composition of the Rous virus nucleoid. Nature 181:1808

Epstein MA, Achong BG (1967) Immunologic relationships of the herpes-like EB virus of cul-
tured Burkitt lymphoblasts. Cancer Res 27:2489–2493
Epstein MA, Achong BG, Barr YM (1964) Virus particles in cultured lymphoblasts from
Burkitt’s lymphoma. Lancet 1:702–703
Epstein MA, Henle G, Achong BG, Barr YM (1965) Morphological and biological studies on a
virus in cultured lymphoblasts from Burkitt’s lymphoma. J Exp Med 121:761–770
Epstein MA, Holt SJ (1958) Observations on the Rous virus; integrated electron microscopical
and cytochemical studies of fluorocarbon purified preparations. Br J Cancer 12:363–369
Fischer GA (1957) Tissue culture of mouse leukemic cells. Proc Am Assn Cancer Res 2:201
Fischer GA (1958) Studies of the culture of leukemic cells in vitro. Ann NY Acad Sci
76:673–680
Henle G, Henle W (1966) Studies on cell lines derived from Burkitt’s lymphoma. Trans NY Acad
Sci 29:71–79
Henle G, Henle W, Diehl V (1968) Relation of Burkitt’s tumor-associated herpes-type virus to
infectious mononucleosis. Proc Natl Acad Sci USA 59:94–101
Rous P (1911) A sarcoma of the fowl transmissible by an agent separable from the tumor cells.
J Exp Med 13:397–411
Woodliffe HJ (1964) Blood and bone marrow cell culture. Eyre and Spottiswoode, London
zur Hausen H, Schulte-Holthausen H, Klein G et al (1970) EBV DNA in biopsies of Burkitt
tumours and anaplastic carcinomas of the nasopharynx. Nature 228:1056–1058
Tumor Associations of EBV—Historical
Perspectives
George Klein
Abstract This is a brief history of our collaborative work with Werner and
Gertrude Henle, Francis Wiener, George and Yanke Manolov, and others on the
association of Epstein-Barr virus (EBV) with Burkitt lymphoma and other human
tumors. Special emphasis is put on the question where EBV is a true cancer virus.
Contents
1 Introduction........................................................................................................................... 17
2 Is EBV a Cancer Virus?........................................................................................................ 21
3 What Is the Role of EBV in the Genesis of BL?.................................................................. 21
4 The Lessons of HART Therapy............................................................................................ 21
References................................................................................................................................... 22
1 Introduction
The inspiring articles of Dennis Burkitt and Dennis Wright in the early 60s made
the scientific community aware of the African childhood lymphoma prevalent in
hot and humid regions of Africa and the “starry sky” like histology. The sugges-
tion that an insect transmitted virus may cause the disease triggered researchers
in numerous laboratories to look for the hypothetical agent. The search was facili-
tated by the fact that the tumor readily fell apart into single cell suspensions with-
out any trypsinization and grew readily into cell lines.
The Virus Cancer Program of the NIH was in full swing. One day—probably
in 1963 or 1964—I visited John Moloney, who headed the program, to tell him
the latest news about our project on virus-induced mouse tumors. Tony Epstein
G. Klein (*)
Department of Microbiology, Tumor and Cell Biology (MTC),
Karolinska Institutet, 17177 Stockholm, Sweden
e-mail: gk-secretary@mtc.ki.se; georg.klein@ki.se

18 G. Klein
was the other visitor. I knew Tony from his earlier visits to Torbjörn Caspersson’s
department in Stockholm where I worked. He showed EM images of cell lines
from what was now called Burkitt lymphoma (BL) to John. In some lines, herpes-
type virus particles could be seen in a small minority of the cells that were clearly
degenerating and dying. John and I thought that the tumor cells may have picked
a common herpesvirus as a passenger. But Tony said: It may be a wild goose but it
is a goose that has to be chased. Right he was.
Soon thereafter, Werner and Gertrud Henle in Philadelphia performed immu-
nofluorescence tests on the same lines and showed that the virus containing cells
failed to react with antibodies against any known herpesvirus. This was, therefore,
a new human herpesvirus. The Henles and we decided to call it EBV.
We were ready to join the adventure of looking for the footsteps of a virus
in proliferating BL cells, using the experience we had from work with virus-
induced mouse lymphomas. We were fortunate to establish an “air bridge” with
Peter Clifford, Head of the ENT Department at the Kenyatta National Hospital in
Nairobi. Getting in touch with him, we followed the percept that if you look for a
collaborator to do a really hard job with you, find the busiest person and he will do
it.
Peter was the only ENT surgeon between Johannesburg and Cairo with an
immense working load, but passionately interested in BL. He has developed its
chemotherapy in parallel with Dennis Burkitt. Unlike Burkitt, he gave only mod-
erate doses to spare the immune system. The frequency of long-term survivors—
or, as it turned out later, cures—was higher in his material than in Burkitt’s more
drastically treated patients.
On my request for biopsies and sera, the material started coming with clock-
work regularity every Tuesday, with the only weekly SAS plane from Nairobi, fro-
zen sera in dry ice, live tumor tissue in wet ice, in great abundance. Every Tuesday
night was Burkitt night at our laboratory for about ten years. In addition to what
we did with the material, we also fanned it out to other laboratories in Europe,
Japan, and the USA.
Our most significant finding was the discovery of EBNA, the EBV-encoded
nuclear antigen which later turned out to be a conglomerate of six different pro-
teins. When we first detected EBNA, it was still not clear whether the virus was
present in some hidden form in the proliferating cells of the tumor that have not
entered the lytic viral cycle which inevitably led to cell death.
To detect EBNA, Beverly Reedman and I departed from the observation of
John Pope in Australia, showing that an EBV-specific complement fixing antigen
was present in a BL line that did not make any virus. We decided to look for it
by anticomplement fluorescence. EBNA soon appeared in all its magnificence
(Reedman and Klein 1973).
The detection of EBNA by anticomplement fluorescence was tricky, and some-
times, it did not work. Years later, under the rule of Idi Amin, a note appeared in
Newsweek saying that the African radiotherapist, Charles Olweny, Head of the
Uganda Cancer Center at that time, was found in the forest with his head cut-
off together with two other colleagues, because they opposed the renaming of
Tumor Associations of EBV—Historical Perspectives 19
Makerere Medical College to Idi Amin University. I tried to call Charles. He was
not there. I called a week later and he was still not there. But he was going to
come the week after. When I called the third time, he came on the line. Charles! I
shouted. Are you all right? No George, the EBNA test is not working, he said.
Lloyd Old and Herbert Oettgen at Sloan Kettering were among the recipient
laboratories in the USA. They detected an EBV-specific soluble antigen by immu-
noprecipitation in BL and also nasopharyngeal carcinoma (NPC) specimens. The
EBV/NPC association was confirmed by the serology of the Henles. But it was
not clear whether the virus was carried by the carcinoma cells or by the abundant
lymphoid infiltrate. We were inclined to blame the latter until we found that the
lymphoid infiltrate of NPC consisted mostly of T cells, not known as EBV harbor-
ing cells at the time. Importantly, the EBNA test clearly showed that latent virus
was carried by the carcinoma cells themselves. With Harald zur Hausen, we could
also confirm the presence of multiple EBV genomes in both BL and NPC cells, by
DNA–DNA hybridization (zur Hausen et al. 1970).
The fanning out of the Nairobi material had many other interesting and
some amusing byproducts. Blood and biopsies were sent to Philip Fialkow,
human geneticist at the University of Seattle who was looking for unusual
isozyme markers. One of his letters came with a big red label saying “top secret”!
Not to be opened by anyone, but Professor Klein. Inside there was another enve-
lope with a wax seal on it and the text “under no circumstances must this letter
be opened by anyone but Professor Klein”. The letter said: Dear George, destroy
this letter immediately when you have read it and make sure that its content is not
communicated to Nairobi. You have sent us the blood of a Masai chief, living on
the southern slope of the Kilimanjaro. On our request you have also supplied us
with blood of his three wives and nine children. So far, we have tested eight of
the children. None of them can be the descendants of the Masai chief. PS We just
completed the ninth test. Paternity excluded.
Knowing a little about the Masai, I made a copy of the letter and sent it to
the Svensson sisters, our Swedish secretaries in Nairobi, girls with a good sense
of humor. They immediately wrote to Fialkow as follows: Dear Dr. Fialkow,
the Masai are a highly ethical and moral people but there customs differ from ours.
After the Masai chief has entertained a close friend of his age group for dinner, he
walks around with the friend among the huts of his wives. He puts down his spear
in front of one of the huts. That is where he invites his friend to spend the night.
However, if anyone would try to get into a wife without being invited, he would be
immediately killed. PS there is no need to keep this information confidential.
Following the confirmation that BL and NPC are the two most regularly EBV-
carrying malignant tumors, their dominating position remained. The low differen-
tiated or anaplastic form of NPC was found to carry latent EBV in nearly 100 %.
This was independent of geography. It was equally true for the high-incidence
Southern Chinese and the rare Western cases. Such a regular association must have
a profound significance.
Today, several decades after the discovery of the association, the role of the
virus for the etiology of the tumor is still unknown.
20 G. Klein
In BL, the situation is somewhat different. The virus is associated with about
90 % of the high endemic African tumors, but the worldwide sporadic BLs carry
EBV in only 20–30 %. The rest contains no virus and has no traces of its genome.
An important missing link in the genesis of BL was provided by cytogenetic
studies. A curious, unexpected convergence occurred at our laboratory. On the sec-
ond floor, Francis Wiener, just about the best mouse tumor cytogeneticist of this
time, worked on mouse plasmacytomas, together with Shinsuke Ohno from Japan.
On the third floor, George Manolov and his wife Yanka Manolova, human cytoge-
neticists from Bulgaria, looked at the chromosomes of the incoming BL biopsies
from Nairobi. The two cytogenetic teams did not have much communication.
Frankly, they did not have much respect for one another. The mouse cytogeneticist
did not think much about the work on human material that did not permit experi-
mentation. For the human cytogeneticist, the mouse work appeared irrelevant.
And then suddenly, the two floors converged. Almost precisely, the same chro-
mosomal translocations were discovered in mouse plasmacytoma and human BL.
Subsequently, many other laboratories became involved in this work. The outcome
was that the translocations act by juxtaposing the powerful c-myc oncogene to one
of the three immunoglobulin loci. To my great surprise, the work of Phil Leder
and Carlo Croce on BL and of Susan Cory and Ken Marcu on MPC confirmed
my speculation that an eminent molecular biologist friend, Lennart Philipsson,
called the most hair-raising extrapolation from the centimorgans to the kilo-
bases, postulating that these translocations act by juxtaposing an oncogene to a
highly active normal gene in that particular cell type (Klein 1981). It was the only
hypothesis I ever made that turned out to be correct. The work was crowned by
Michel Cole’s demonstration that the oncogene was c-myc in both BL and MPC.
I do not know of any other example in cancer biology where tumors of two differ-
ent species, with entirely different pathogenetic histories, arise due to basically the
same oncogene activation event in the same broad cell lineage as the only common
denominator.
Thus, the Ig/myc translocation, rather than EBV, is the common feature of all BLs.
But what is the role of EBV?
Prior to and in parallel with the cytogenetic developments, some monumental dis-
coveries were made in the EBV field. They include the transforming and immor-
talizing activity of the virus for B cells, the role of the virally encoded growth
transformation-associated proteins, and particularly EBNA2 and LMP1 for sus-
tained B-cell proliferation and the causative role of the virus for mononucleosis.
Moreover, the virus turned out to be the driving force for the immunoblastomas
that arise from it genetically, iatrogenically (e.g., in transplant recipients) or by
coinfection (HIV immunosuppressed patients). Importantly, EBV-carrying African
BL cells do not express the full growth program. They do not need it because they
are driven by myc.
In addition to BL and anaplastic NPC, other consistent tumor-EBV associations
were discovered later. The relationship with Hodgkin’s lymphoma (HL) is particu-
larly noteworthy since there is both epidemiological and molecular evidence that
the virus may be essential for the proliferative potential of the HLs that carry it.
Tumor Associations of EBV—Historical Perspectives 21
Similar evidence is emerging for the EBV-carrying form of diffuse large B-cell
lymphoma. The virus can also be carried by T-cell lymphomas. Its association
with the T-cell-derived lethal midline granuloma is particularly consistent.
2 Is EBV a Cancer Virus?
Having completed the sequencing of the powerfully tumorigenic mouse polyoma

virus, a considerable feat at the time, Fred Sanger felt that a human tumor virus
should come next. On the advice of Beverly Griffin, he chose EBV, considered as
the first human tumor virus. Paul Farrell carried out the job which had important
consequences.
But is EBV really a tumor virus?
The immunoblastomas of the immunosuppressed are clearly driven by EBV as
already mentioned. But are the immunoblastomas “truly malignant”? They tend
to remain diploid and do not carry characteristic tumor-associated mutations. The
frequently mutated p53 pathway remains wild type, as a rule.
3 What Is the Role of EBV in the Genesis of BL?
This question has no straightforward answer. But the presence of the virus in 90 %
of the high endemic tumors cannot be a coincidence. I also attach considerable
significance to the argument of Bill Sugden. He pointed out that the lack of syn-
chrony between cell division and EBV episome duplication must lead to the loss
of the viral genomes unless they are needed for the sustainability of the tumor.
In fact, EBV-carrying BL lines can loose the virus in vitro, but a similar loss has
never been observed in vivo. This indicates that some viral function may provide
the in vivo tumor cell with a selective growth advantage.
4 The Lessons of HART Therapy
Malignant lymphomas have been a frequent complication of untreated AIDS.

Part of them were EBV-driven immunoblastomas, but a substantial portion were
Ig/myc-carrying BL or BL-like lymphomas. This picture changed radically after
the introduction of HART therapy. In parallel with increase of the CD4+ T-cell
count, the EBV-driven immunoblastomas practically disappeared. This makes
sense in view of the highly efficient T-cell-mediated immune response against
EBV-driven immunoblasts. The Ig–myc-carrying BL and BL-like lymphomas
showed no similar tendency to decrease and are by now the most frequent lym-
phoma complication in HART-treated HIV carriers.
22 G. Klein
Ig–myc translocation-carrying cells with constitutively activated myc genes are

prone to apoptosis. They need to be rescued by the cognate antigen or by B-cell
stimulatory lymphokines and cytokines. HIV proteins linger around in the lymph
nodes for a long time even under HART therapy. They may contribute to the sur-
vival of the Ig/myc translocation-carrying cells that are known to arise continu-
ously as accidents of normal B-cell differentiation.
This argument is also pertinent to the question why chronic hyperendemic
malaria and HIV are the only two conditions where translocation-carrying BLs
arise in a relatively high frequency. A B-cell stimulatory environment is the
characteristic for both conditions. Rescue from apoptosis by B-cell stimulatory
cytokines may be the common responsible mechanism.
References
Klein G (1981) Nature 294:313–318

Reedman BM, Klein G (1973) Int J Cancer 11:499–520
zur Hausen H et al (1970) Nature 228:1056–1058
EBV-Specific Immune Response: Early
Research and Personal Reminiscences
D.J. Moss and V.P. Lutzky
Abstract Early research on Epstein–Barr virus (EBV) developed from serological

observations that were made soon after the discovery of the virus. Indeed, the defi-
nition of the humoral response to a variety of EBV proteins dominated the early
literature and was instrumental in providing the key evidence for the association
of the virus with infectious mononucleosis (IM), Burkitt’s lymphoma (BL), and
nasopharyngeal carcinoma (NPC). Each of these disease associations involved
a distinct pattern of serological reactivity to the EBV membrane antigens (MA),
early antigens (EA), and the EBV nuclear antigen (EBNA). When it became gen-
erally accepted that the marked lymphocytosis, which is a hallmark of acute IM,
was dominated by T cells, considerable effort was directed toward untangling the
specificities that might be associated with restricting the proliferation of newly
infected B cells. Early evidence was divided between support for both EBV non-
specific and/or HLA non-restricted components. However, all results needed to be
reassessed in light of the observation that T cells died by apoptosis within hours
of separation from fresh blood from acute IM patients. The observation that EBV-
infected cultures from immune (but not non-immune) individuals began to die
(termed regression) about 10 days post-seeding, provided the first evidence of a
specific memory response which was apparently capable of controlling the small
pool of latently infected B cells which all immune individuals possess. In this
early era, CD8+ T cells were thought to be the effector population responsible for
this phenomenon, but later studies suggested a role for CD4+ cells. This historical
review includes reference to key early observations in regard to both the specific
humoral and cellular responses to EBV infection from the time of the discovery
of the virus until 1990. As well, we have included personal recollections in regard
to the events surrounding the discovery of the memory T cell response since we
believe they add a human dimension to a chapter focussed on early history.
D.J. Moss (*) · V.P. Lutzky

QIMR Berghofer Medical Research Institute, Brisbane, Australia
e-mail: denis.moss@qimrberghofer.edu.au
V.P. Lutzky
e-mail: Viviana.lutzky@qimrberghofer.edu.au

24 D.J. Moss and V.P. Lutzky
Contents
1 Humoral Responses to EBV Infection.................................................................................. 24
1.1 Antibody to the Virus Capsid Antigen......................................................................... 24
1.2 Antibody to the EBV Membrane Antigen ................................................................... 25
1.3 Antibody to the EBV Early Antigen............................................................................ 26
1.4 Antibody to EBV Nuclear Antigen.............................................................................. 26
1.5 EBV-Specific IgA Response as a Serological NPC Marker........................................ 27
2 EBV Seroepidemiology........................................................................................................ 27
3 Cellular Response to EBV Infection..................................................................................... 29
3.1 Early Studies to Define an EBV-Specific T Cell Response
in Acute EBV Infection................................................................................................ 29
3.2 Memory T Cell Response in Healthy Immune Individuals:
Personal Reminiscences of Denis Moss from 1977 to 1980....................................... 30
3.3 Defining the First EBV CTL Epitope: Personal Reminiscences
of Denis Moss from 1988 to 1990............................................................................... 36
3.4 Early Contributions of Other Investigators.................................................................. 36
4 Final Remarks....................................................................................................................... 37
References................................................................................................................................... 37
1 Humoral Responses to EBV Infection
1.1 Antibody to the Virus Capsid Antigen
In 1966, the Henle laboratory observed that a small proportion of cells in a series
of Burkitt’s lymphoma (BL) cell lines (EB-1 and EB-2) showed strong immuno-
fluorescence when stained with sera from a variety of patient groups as well as
most (but not all) healthy Americans (Henle and Henle 1966). The authors noted
in this publication that while it was not possible to absolutely ascribe Epstein–Barr
virus (EBV) specificity to this strong immunofluorescent staining, several obser-
vations supported this conclusion. Firstly, both virus particles and strong immu-
nofluorescence were associated with cells undergoing degeneration. Secondly,
exposure of cell lines to the anti-herpesvirus drug 5-methylamino-2′-deoxy-uridine
also reduced the proportion of stainable cells. Irrefutable proof of the EBV speci-
ficity of the immunofluorescence was provided by Harald zur Hausen, who dem-
onstrated by electron microscopy that individual fluorescent cells all contained
virus particles, while no virus particles were demonstrated in non-immunofluo-
rescent cells (zur Hausen et al. 1967). This and related studies clearly established
that this staining was directed to the EBV capsid antigen (VCA) and was sub-
sequently extensively used to establish a prior EBV infection (clinical or silent).
However, the anti-VCA IgG immunofluorescence assay had diagnostic limitations
since (a) the low titers seen in acute infectious mononucleosis (IM) overlapped
with the high titers seen in some healthy individuals (b) only 10–20 % of sera
showed a significant difference between acute and convalescent sera. Of particular
EBV-Specific Immune Response … 25
Table 1 Typical antibody responses against the EBV proteins VCA, EA and EBNA in IM, BL
and NPC patients along with healthy controls
Disease Antibody to
VCA EA EBNA
IgM IgA IgG D R
IM ++ 0 ++ + 0 0
BL 0 0 ++ 0 ++ +
NPC 0 ++ ++ ++ 0 ++
Controls 0 0 + 0 0 +
0, Not detectable as a rule; +, Antibody titers ≤1:160; ++, Antibody titers ≥1:320
Table reproduced (with permission) from Henle and Henle (1979)
diagnostic importance, however, was the observation that an IgM response to

EBV VCA was detectable only during the acute phase of primary infection and
this response faded within two months of clinical presentation (Edwards and
McSwiggan 1974; Henle and Henle 1979; Nikoskelainen and Hanninen 1975;
Nikoskelainen et al. 1974; Schmitz and Scherer 1972). A summary of the anti-
body patterns against VCA and other EBV antigens (see below) for different EBV-
associated diseases is presented in Table 1.
1.2 Antibody to the EBV Membrane Antigen
Early studies indicated that some EBV antibody-positive human sera detected a
specific membrane antigen (MA) on the surface of BL cells (Klein et al. 1966,
1967). Further investigation indicated two components associated with this reac-
tivity—an early EBV antigen (early MA) on live BL tumor cells in vivo and a
late MA antigen (late MA) in later stages of virus replication. This latter reactiv-
ity was seen in association with VCA. Indeed, Henle in delivering a special lec-
ture at the First International Symposium on EBV in Greece (Henle and Henle
1984) recalled that in 1967 George Klein reported to a conference of the American
Cancer Society that there was a strong correlation between those cell lines that
exhibited MA reactivity and those, in which Henle demonstrated VCA immuno-
fluorescence, thus inferring that MA was virus-induced. These MA reactivities
were originally demonstrated using three different serological assays (1) immuno-
fluorescence using sera from BL as well as other sources (Klein et al. 1966, 1967,
1968), (2) antibody-dependent cellular cytotoxicity (Pearson and Orr 1976), and
(3) 125I-labeled protein A binding (Pearson and Qualtiere 1978). The immunofluo-
rescence test for the detection of anti-MA was later replaced by a blocking proce-
dure to avoid interactions with membrane antigens unrelated to EBV (Klein et al.
1969). Because MA is a complex of several components (early and late MA), the
utility of this assay was limited. Sera used to detect this reactivity included a wide
spectrum of EBV specificities, but subsequent studies indicated that MA reactiv-
ity was closely associated with neutralization of the virus (Pearson et al. 1970;
Thorley-Lawson and Geilinger 1980). The early literature refers to three different
methods of measuring virus neutralization: (1) inhibition of EA antigens (Henle
et al. 1970b), (2) inhibition of colony formation (Rocchi and Hewetson 1973), and
(3) inhibition of transformation (Henle et al. 1967; Miller et al. 1972; Pope et al.
1968). When neutralization levels were assessed in a group of human sera by each
of these assays, there was a reasonable, but not a perfect, correlation between each
of these technologies (de Schryver et al. 1974).
Since it was well established that anti-MA reactivity closely correlated with
virus neutralization, it is not surprising that there was an early attempt to develop
a vaccine to EBV based on the high molecular weight glycoprotein component of
MA (gp340) (Epstein and Morgan 1986; North et al. 1980, 1982; Thorley-Lawson
and Geilinger 1980). This effort was based largely on the ability of immunized
cotton-top tamarins to inhibit the formation of malignant lymphomas following
challenge with EBV. These efforts to develop a vaccine were based on the hypoth-
esis that a specific humoral response would be protective from the appearance of
clinical symptoms.
1.3 Antibody to the EBV Early Antigen
A further antigen and antibody specificity was discovered following superinfec-

tion of lymphoblasts from non-producer lymphoblastoid cell lines (LCLs) with
EBV derived from the P3HR-1 cell line. This superinfection was shown to reduce
cell growth but failed to stain for VCA in immunofluorescence assays using sera
from healthy individuals (Henle et al. 1970b), yet stained strongly in up to 90 % of
cells when sera from IM, BL, or nasopharyngeal carcinoma (NPC) patients were
used. This serum reactivity (referred to as early antigen, EA) disappeared when
clinical symptoms abated (Henle et al. 1971b). Later studies (Henle et al. 1971a)
divided this reactivity on the basis of methanol sensitivity (R and D). Since the
anti-D reactivity was strongly associated with acute IM and rarely seen in healthy
individuals, this assay was historically valuable in the diagnosis of IM (Table 1)
(Henle et al. 1974).
1.4 Antibody to EBV Nuclear Antigen
The EBV nuclear antigen (EBNA) was first detected using EBV immune serum
in an anti-complement immunofluorescence (ACIF) assay. The high sensitivity
and specificity of this three-step assay relies on the ability of complement from an
EBV-negative donor serum to bind to the EBNA antibody/EBNA protein complex
followed by conjugated anti-human β1C/β1A (Reedman and Klein 1973). Antibody

to this protein is widely found in EBV immune sera except in acute IM where it
appears weeks or months after onset of illness (Henle et al. 1974). Interestingly,
high levels of EBNA antibody are detected in NPC patients (Table 1).
1.5 EBV-Specific IgA Response as a Serological NPC

Marker
The discovery of a strong IgA response to EBV in NPC patients heralded the begin-
ning of its serological utility as an outstanding feature of this malignancy (Wara
et al. 1975). Stimulated by this early report, the Henle and Klein laboratories (Henle
and Henle 1976) tested a total of 372 sera from patients with NPC, other carcinomas
of the head and neck, BL, IM, and healthy controls. The results demonstrated that
prior to therapy, 93 % of NPC patients had an IgA VCA response and that these
titers increased from stages I or II to stages III or IV, i.e., a strong correlation with
the total tumor burden. Conversely, many of the NPC patients examined 2–6 years
after initial therapy had only low levels of EBV-specific IgA or none at all, and the
majority of those with high titers were known to have residual or recurrent disease.
In contrast to untreated NPC patients, less than 5 % of 73 patients with other carci-
nomas and none of 76 healthy individuals revealed a specific IgA response. These
observations provided a stepping stone for the widespread use (particularly in main-
land China) of the detection of IgA to VCA and EA as a means of mass population
screening (Zeng et al. 1979, 1982, 1983).
2 EBV Seroepidemiology
Within a decade of the discovery of the virus, most of the important serological
trends linked with different EBV-associated diseases had been defined. Initial
interest in this field was intense in regard to BL since this was the first human
malignancy shown to have an apparent viral etiology. Anti-VCA assays were used
to determine whether the serological titers in BL endemic areas were significantly
higher than those in non-endemic areas. These epidemiological surveys confirmed
that infection with EBV among healthy individuals was extremely widespread,
with higher titers being recorded among east African (typically by three years of
age) as well as in low socioeconomic individuals in other regions of the world,
compared to those in higher socioeconomic regions (Gerber and Monroe 1968;
Henle and Henle 1966; Moore et al. 1966). When these studies were extended to
defining anti-VCA titers in a range of other EBV-associated diseases along with
controls including non-EBV-associated malignancies (Fig. 1), it became appar-
ent that anti-VCA levels in IM, BL, and NPC patients were generally higher than
Fig. 1 Distribution (expressed as a percentage of cases) of antibody titers to VCA in patients

(PTS) with various diseases and appropriate control (CO) groups. IM infectious mononucleosis;
BL Burkitt’s lymphoma; NPC nasopharyngeal carcinoma; HD Hodgkin’s disease; CLL chronic
lymphocytic leukemia; GM geometric mean. Figure adapted (with permission) from Henle and
Henle (1979)
those recorded in healthy individuals living in the same area (de-The et al. 1978;
de Schryver et al. 1969; Henle and Henle 1979; Niederman et al. 1968). Evidence
for the association of EBV with these diseases was further strengthened when
these assays were extended to include the EA complex (Henle and Henle 1979;
Henle et al. 1970a, 1971b).
3 Cellular Response to EBV Infection
3.1 Early Studies to Define an EBV-Specific T Cell Response

in Acute EBV Infection
One of the pathological hallmarks of acute IM is the presence of atypical mono-

nuclear cells which permeate not only into the peripheral blood but also into many
tissues (Carter 1975). It should be pointed out that this heterogenous population
is not specific for acute IM but was referred to much earlier in relation to bacte-
rial infection (Turk 1898) and graft rejection (Parker and Mowbray 1971). It was
generally understood that these atypical mononuclear cells would be dominated
by recently transformed B cells, and it was thus somewhat unexpected when it
was established that this population possessed sheep erythrocytes receptors rather
than B cell markers (Sheldon et al. 1973). This result was confirmed in a study of
acute IM in which five independent B and T cell markers were assessed (surface
immunoglobulin, complement receptors, B and T lymphocyte-specific markers,
and sheep erythrocyte receptors). It was concluded that these atypical mononu-
clear cells were indeed largely of T cell origin (Pattengale et al. 1974) resulting in
intense interest in the possibility that they might either include a specific compo-
nent capable of limiting the proliferation of virus-infected B cells or were homeo-
static, non-specific suppressor cells. Attempts were made to identify EBV-specific
T cell activity by exposing cultured lymphocytes from acute IM donors to spe-
cific EBV antigens and monitoring the response in a leukocyte migration inhibi-
tion (LMI) assay (Lai et al. 1977) and leukocyte adherence inhibition (LAI) assay
(Chan et al. 1977). Both of these approaches showed some indication of a specific
response but were somewhat awkward to use and did not find wide-spread appli-
cation. Subsequently, two distinct T cell activities were identifiable in vitro (1) a
suppressor cell activity that was demonstrated using pokeweed mitogen-induced
B cell activation (Henderson et al. 1977; Johnsen et al. 1979; Tosato et al. 1979),
which was also later revealed to suppress the proliferative responses of T cells to
mitogenic and antigenic stimuli (Reinherz et al. 1980) and (2) an apparently EBV-
selective cytotoxicity following removal of conventional NK cell activity (Bakacs
et al. 1978; Royston et al. 1975; Svedmyr and Jondal 1975). Thus, for example,
the publication from Royston and colleagues was based on a collection of mono-
nuclear cells from 21 acute IM patients and demonstrated a higher level of lysis
toward autologous LCLs than non-EBV-infected target cells (non-EBV tumor
cells). It should be pointed out, however, that the effector to target ratio used was
high (100:1) and the level of lysis of the autologous cell line fairly modest (10 %)
compared to the non-EBV-infected control (3 %). These conditions of assay would
currently be regarded as extreme, particularly when compared with the high levels
of NK cell activity included in the blood of acute IM patients. One of the criti-
cisms that could reasonably be leveled at these and other earlier studies (Bakacs
et al. 1978; Lipinski et al. 1979; Seeley et al. 1981; Tursz et al. 1977) is that they
purport to detect EBV specificity but lack obvious HLA restriction (Zinkernagel
and Doherty 1979). Killing of this type led to the concept of a lymphocyte-deter-
mined membrane antigen (LYDMA), which was said to be expressed on the sur-
face of all LCLs (Klein et al. 1976; Svedmyr and Jondal 1975). The concept that
cytotoxic T cells (CTLs) recognized a surface protein was quite understandable
and predated the historic discovery that CTL recognized small peptides in associa-
tion with class I MHC and that these small peptides were frequently derived from
proteins, which by serological criteria appeared to have an intracellular location
(Townsend et al. 1985).
It should be pointed out that these early studies needed to be assessed carefully
in light of the observation that T cells from acute IM patients die by apoptosis
(Fig. 2) within hours of isolation (Bishop et al. 1985; Moss et al. 1985) and that
the early assays to detect bioactivity were almost certainly significantly affected
by this phenomenon. Indeed, when T cells from IM patients were isolated after
15 h, less than 5 % of T cells were recovered which is in stark contrast to that seen
from blood of healthy donors (Moss et al. 1985). Interestingly, apoptosis can be
largely avoided by inclusion of IL-2 in the culture medium, both at the time of iso-
lation of the mononuclear cells as well as in all of the steps involved in the analy-
sis of these cells (Bishop et al. 1985).
The contemporary view regarding the T cell response has clarified much of the
early uncertainty regarding its presence or otherwise during acute infection. It is
now generally accepted that the CD8+ lymphocytosis seen during acute infection
includes a large specific T cell component and that following resolution of the dis-
ease there is a disproportionate culling of the CD8+ population and restoration of
the CD4/CD8 ratio back into the normal range (Annels et al. 2000; Callan et al.
1996, 2000; Roos et al. 2000).
3.2 Memory T Cell Response in Healthy Immune

Individuals: Personal Reminiscences of Denis Moss
from 1977 to 1980
In 1973, Beverley Reedman from the EBV laboratory at QIMR, spent a year
in Stockholm at the Karolinska Institute with George and Eva Klein. This col-
laboration resulted in the first description of the presence of the EBNA pro-
tein (Reedman and Klein 1973), which was very important in that it allowed a
ready visualization of the EBV protein present in all endemic BL cell lines and
LCLs (Pope et al. 1969). Based on this momentous discovery of an EBV protein
Fig. 2 a Electron micrograph of T cells 15 h after fractionation from a representative acute IM
patient. A lymphocyte of normal appearance (L) is shown with three dying lymphocytes display-
ing characteristic features of apoptosis (loss of microvilli, cytoplasmic and nuclear condensation,
crescent shaped margination of chromatin, nuclear fragmentation, and cellular budding). Mag-
nification ×10,000. b Light micrograph of T cells 15 h after fractionation from the same patient
as above. A lymphocyte of normal appearance (L) is surrounded by dying cells showing features
associated with cell death by apoptosis (condensation and basophilic nuclear fragments). Magni-
fication ×1300. Figure reproduced with permission from Moss et al. (1985)
invariably associated with EBV infection, our laboratory decided to embark on

a project to map the kinetics with which EBNA was induced following infection
of human lymphocytes. I had anticipated that this should not present significant
technical problems as the EBNA staining assay was both sensitive and specific.
We demonstrated that by three days, about 10 % of lymphocytes showed strong

EBNA staining, while the majority of cells were similarly stained by five days
(Moss and Pope 1975). This study was exclusively carried out using lymphocytes
from EBV immune adults. It came as somewhat a surprise that these strongly pro-
liferating cultures began to show cell concentration-dependent microscopic evi-
dence of cell death by day 10, followed by a total collapse of the lymphoblastoid
clumps by day 21, together with the complete disappearance of EBNA staining
(Fig. 3). In the discussion section of this publication, reference was made to an
earlier observation (Moss and Pope 1972) reporting the difficulty in establishing
cell lines in some instances and we hypothesized that this phenomenon of cell
death might be due to “an immune response to developing EBV antigens” (Moss
and Pope 1975). Indeed, we went on to show in preliminary experiments (unpub-
lished) that lymphocyte cultures from an EBV seronegative individual contin-
ued to expand without any evidence of cell death. This concept of the effect of
the immune status of the lymphocyte donor on the course of EBV transforma-
tion was further reinforced by our later observation that transformation of lym-
phocytes from EBV seropositive (but not seronegative) individuals was reversibly
blocked when cultured on adult fibroblasts (Moss et al. 1977). Unfortunately,
the mechanism involved in this inhibition was never determined since the phe-
nomenon proved to be very sensitive to minor changes in culture conditions. By
the time Alan Rickinson (Bristol) arrived in John Pope’s laboratory in 1977, I
was able to discuss with him a working hypothesis compatible with these pub-
lished and unpublished observations. In particular, we suggested that cell death
in these cultures from EBV immune individuals may well have an immunologi-
cal basis. Our laboratory had not regularly seen this cell death phenomenon ear-
lier because at that time we had only recently switched from using cord blood
Fig. 3 The relationship between EBNA production (shown as the percentage of EBNA-positive
cells/ml) and initial cell concentration following infection of human leukocytes with EBV at a
multiplicity of 0.003 TD50/cell. Initial cell concentrations were 106/ml ( ); 5 × 105/ml
( 5
); 2.5 × 10 /ml ( 5
); 10 /ml ( ). Figure reproduced with permission from
Moss and Pope (1975)
(always EBV seronegative) to adult blood (usually EBV seropositive) in our

experiments due to new hospital regulations regarding the use of cord blood for
experimental purposes. Alan and I decided to work together to either prove or
disprove the immunological basis of regression. We were fortunate in that I had
only recently returned from a visit to the laboratory of Tony Basten (University
of Sydney) who had demonstrated to me the new technique of separating human
T cells rosettes using sheep red blood cells coated with the highly purified sulfhy-
dryl reagent, 2 aminoethylisothiouronium bromide hydrobromide (AET) (Kaplan
and Clark 1974). It was unfortunate that the first experiment we jointly set up
included my regular EBV seronegative donor as a control and the results indi-
cated that these cells began to die after 10 days of apparent proliferation. We were
greatly relieved to learn that this individual had recently seroconverted. Indeed,
this single experiment convinced us of the validity of our hypothesis and during
that year we unequivocally proved that cell death (which Alan termed regression)
was mediated by a population of EBV-specific T cells present in the blood of all
healthy immune individuals (Fig. 4) (Moss et al. 1978; Rickinson et al. 1979). In
a subsequent study, we proposed that previously infected individuals possessed
a pool of virus-specific memory T cells capable of mounting a specific response
when challenged in vitro (Moss et al. 1979). Given the marked T cell expansion
associated with primary infection, we were surprised to subsequently demonstrate
that the lymphocyte population from acute IM patients showed weak regression
(Rickinson et al. 1980). Indeed, more than five years were to elapse before an
explanation for this result was forthcoming (Fig. 2) (Bishop et al. 1985; Moss
et al. 1985).
One of the useful features of the regression assay is that it can be used to pro-
vide a semiquantitative estimate of the overall level of EBV-specific CTL immu-
nity in the blood of any immune individual based on a 50 % endpoint of the
incidence of transformation in EBV-infected cultures (Moss et al. 1978). In our
experience, this regression endpoint was surprisingly reproducible and was applied
to compare the level of EBV CTL immunity in malaria endemic and non-endemic
individuals in Papua New Guinea (Moss et al. 1983a), as well as in NPC patients
(Moss et al. 1983b).
A series of publications sought to investigate the mechanism of regression.
The results of double chamber experiments argued against the involvement of any
soluble factors including antibody, whereas there was an absolute requirement for
T cells in the initial cultures along with a requirement for proliferation of effec-
tor T cells within the first 14 days (Rickinson et al. 1979). Furthermore, T cells
prepared by ammonium chloride lysis of T cell rosettes from regressing cultures
showed growth inhibition of autologous and HLA-related target cells (Moss et al.
1979). This lysis was neither dependent on the target cells expressing serologically
defined envelope antigens on their cell surface nor did it involve an artifact in the
culture medium (e.g., fetal calf serum). Moreover, lysis was not intrinsically asso-
ciated with B cell proliferation per se.
Fig. 4 Photomicrographs of EBV-infected cultures of blood mononuclear cells seeded initially

at 106 cells/ml (×125). a Seronegative adult donor cells 14 days post-infection. b Seronegative
adult donor cells 28 days post-infection. c Seropositive adult donor cells 14 days post-infection.
d Seropositive adult donor cells 28 days post-infection. Figure reproduced with permission from
Moss et al. (1978)
Overall, this year’s collaboration with Alan was very important since it pro-
vided for the first time experimental proof for the widely held notion that an
immune memory response must exist in all previously infected individuals to pre-
vent uncontrolled proliferation of the latently infected B cells which are a found in
all immune donors (Nilsson et al. 1971).
After Alan returned to Bristol, I realized that regression, although most likely
mediated by CTLs, may have been due to some non-cytotoxic, suppressor mecha-
nism. At this stage, Ihor Misko (Australian National University, Canberra) joined
the Pope’s laboratory and brought with him additional immunological skills
including experience in HLA restriction and the use of the 51chromium release
assay. We thus set about formally proving that regressing cultures included a CTL
cell component capable of killing autologous and HLA-related LCLs (Misko
et al. 1980). Interestingly, this finding in healthy immune donors contrasted with
the earlier result in acute IM patients in which no HLA-associated killing was
reported (Svedmyr and Jondal 1975; Svedmyr et al. 1979).
In 1979, the Queensland Cancer Council provided me with the opportunity of
working in Tony Epstein’s laboratory in Bristol for 12 months, once again with
Alan but also with Lesley Wallace and Martin Rowe. Notable findings during this
era included detailed investigations into EBV-specific HLA restriction (Fig. 5)
(Moss et al. 1981b; Rickinson et al. 1981b; Wallace et al. 1981) as well as demon-
strating the use of irradiated autologous LCLs to activate a specific CTL response
in vitro (Rickinson et al. 1981a). This method went on to become a cornerstone in
the activation of specific CTLs in patients with various EBV-associated lymphoid
malignancies (Gottschalk et al. 2005; Rooney et al. 1998).
In order to derive some notion of the possible nature of the target for T cell rec-
ognition, we went on to demonstrate that LYDMA was expressed on the cell sur-
face soon after the appearance of the EBNA protein(s) and coincident with, but not
dependent on, the initiation of cellular DNA synthesis (Moss et al. 1981a). This
was a significant finding in relation to the development of an EBV vaccine since it
Fig. 5 Allogeneic target cell recognition, measured either in a 51chromium release assay or in
a growth inhibition assay and expressed each time as a percentage of that activity shown by the
same effector cells against their autologous cell line, plotted against the number of HLA-A and
HLA-B antigens shared between effector and target cells. Composite data from 14 donors cover-
ing 153 allogeneic combinations; mean values (±1 standard deviation) are shown for groups
of effector: target cell combination sharing 0, 1, 2, 3, or 4 HLA-A and HLA-B antigens. Figure
reproduced with permission from Moss et al. (1981b)
provided evidence that immunological control over EBV-infected B cells appeared

to be exercised soon after infection, raising the possibility that a vaccine might
provide protection before clinical symptoms appeared. I found the intellectual
environment in Bristol very stimulating and scientifically productive. As well, the
close proximity to America and Europe gave me the chance of meeting a host of
EBV luminaries including George and Eva Klein and Maria Masucci (Karolinska
Institute, Stockholm) along with Guy de Thé (Pasteur Institute, Paris) and Werner
Henle (Children’s Hospital of Philadelphia).
3.3 Defining the First EBV CTL Epitope: Personal

Reminiscences of Denis Moss from 1988 to 1990
Following the discovery by the Oxford group (Townsend et al. 1985) that T cells
recognized small peptides (typically nine amino acids long), it became clear that
our laboratory would need to develop a similar molecular approach if our long-
term goal of developing a vaccine to EBV was to be realized. Up to this point,
peptide epitopes had only been defined in relation to comparatively small viruses
such as influenza and it was not clear how a similar technology could be directly
applied to EBV which included more than 10 times as many proteins. In hind-
sight, the critical observation involved the isolation of a series of CD4+ and CD8+
CTL clones that recognized autologous type 1 (formerly known as type A) but not
type 2 (formerly known as type B) LCLs (Moss et al. 1988). These two strains
were believed at the time to differ on the basis sequence divergence within the
BamH1 WYH region of the genome encoding EBNA2 (Dambaugh et al. 1984).
Subsequently, these clones were used to screen a series of peptides showing
sequence divergence between both strains and which also corresponded to pre-
dicted epitope algorithms (Delisi and Berzofsky 1985; Rothbard and Taylor 1988).
This resulted in the identification of the first EBV CTL epitope (Burrows et al.
1990).
3.4 Early Contributions of Other Investigators
The literature includes a significant body of work in this early era from other
investigators. Notable among these was the observation that T cells from adult
individuals could delay the appearance of foci of EBV-transformed cells in com-
parison with corresponding cultures from cord blood donors (Thorley-Lawson
et al. 1977). Despite the problems inherent in the interpretation of a morphological
study of this kind, the data indicated that the effect was observable over a range
of initial cell concentrations as well as different B:T cell ratios. Furthermore, a
subsequent study using EBNA and 3thymidine induction confirmed the initial
observation (Thorley-Lawson 1980). Curiously, it was subsequently shown that

mitomycin C-treated T cells could delay transformation suggesting that this delay
was independent of T cell proliferation. It should be pointed out that the serologi-
cal status of the individuals involved in these studies was not recorded. However,
subsequent studies demonstrated that this effect occurred with both seropositive
and seronegative donors (Shope and Kaplan 1979), inferring that this delay did
not have an immunological basis and was quite distinct from the observation of
regression of transformation. Indeed, a role for α interferon has been suggested
raising the possibility of a contribution of NK cells to this delay in transformation
(Shope and Kaplan 1979).
The sensitivity of BL cells to different mechanisms of killing was another impor-
tant aspect of research in this early era. These studies involved a comparison of the
killing sensitivity of so-called paired cell lines, i.e., BL and LCLs derived from
the same patient. It appeared that BL cells, although sensitive to allo-specific lysis,
were generally insensitive to EBV-specific T cell killing (Rooney et al. 1985a, b).
Subsequent studies revealed that this phenomenon was related to the different pat-
tern of latency present in BL cell lines compared to LCLs (Rowe et al. 1985), thus
excluding the possibility that BL arose from an EBV-specific T cell defect.
Although there was widespread agreement in the early literature that regression
was mediated by virus-specific CTLs that had been reactivated in vitro from mem-
ory T cells, there existed a degree of confusion as to whether all the components of
the phenomenon were included within the CD8+ subset or whether CD4+ may also
play a role (Crawford et al. 1983; Rickinson et al. 1984; Tsoukas et al. 1982). More
contemporary investigations indicate that CD4+ cells do indeed play a role in con-
trolling the expansion of newly infected CD23+ B cells (Nikiforow et al. 2001).
4 Final Remarks
The link between EBV with various human diseases provided the impetus for the
use of EBV serology as a biomarker. This was particularly the case in IM, NPC,
and BL. Subsequently, the discovery of a memory T cell response to the virus pro-
vided an additional perspective of the dynamics of the control of the latent EBV
infection in all immune individuals. In conclusion, this early era provided a basis
for the subsequent developments in EBV immunobiology as well as a platform for
immunological intervention in EBV-associated diseases.
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Part II
Virus Genetics and Epigenetics
Epstein–Barr Virus Strain Variation
Paul J. Farrell
Abstract What is wild-type Epstein–Barr virus and are there genetic differences
in EBV strains that contribute to some of the EBV-associated diseases? Recent
progress in DNA sequencing has resulted in many new Epstein–Barr virus (EBV)
genome sequences becoming available. EBV isolates worldwide can be grouped
into type 1 and type 2, a classification based on the EBNA2 gene sequence. Type
1 transforms human B cells into lymphoblastoid cell lines much more efficiently
than type 2 EBV and molecular mechanisms that may account for this difference
in cell transformation are now becoming understood. Study of geographic varia-
tion of EBV strains independent of the type 1/type 2 classification and system-
atic investigation of the relationship between viral strains, infection and disease
are now becoming possible. So we should consider more directly whether viral
sequence variation might play a role in the incidence of some EBV-associated
diseases.
Contents
1 Introduction........................................................................................................................... 46
2 History of EBV Sequencing.................................................................................................. 47
3 Broad Aspects of Genome Variation—EBV Types, Selection Forces and Recombination.... 49
4 Variation in EBV Genes........................................................................................................ 51
4.1 LMP1........................................................................................................................... 51
4.2 LMP2A........................................................................................................................ 53
4.3 EBNAL........................................................................................................................ 53
4.4 EBNA3 Family............................................................................................................ 54
4.5 Other Latent Cycle Elements....................................................................................... 54
4.6 Lytic Cycle................................................................................................................... 55
P.J. Farrell (*)

Section of Virology, Department of Medicine, Imperial College, St Mary’s Campus,
Norfolk Place, London W2 1PG, UK
e-mail: p.farrell@imperial.ac.uk

46 P.J. Farrell
5 Functional Difference Between Type 1 and Type 2 EBNA2................................................ 56

6 How to Identify Variation Potentially Relevant to EBV-Associated Diseases...................... 58
References................................................................................................................................... 59
1 Introduction
EBV infection is prevalent all over the world, but some EBV-associated diseases
have unusual geographic distributions, for example nasopharyngeal carcinoma
(NPC) or endemic Burkitt’s lymphoma (BL) (Rickinson 2014). Genetic varia-
tion of the host, local environmental co-factors and co-infections all play a role
in this, but natural variation in the EBV may also be important. There have been
many studies which show substantial geographic variation in the virus sequence
in normal infected populations, so the endemic strain of EBV in some parts of the
world might be inherently more able to contribute to cancers. Heterogeneity of the
endemic virus circulating in the general population might also result in multiple
strains of EBV being present in one person and selection of a specific variant in a
cancer, as proposed for an LMP1 variant in a case of NPC (Edwards et al. 2004).
It is additionally possible that specific mutations in EBV might arise in an
infected person. The normal life cycle of EBV is thought to involve infected
cells passing through a germinal centre in a lymph node, which is a highly muta-
genic environment, and the virus replication itself might also accumulate errors
(although herpesviruses are generally thought to have very stable genomes).
Examples of infrequent mutations that appear to be relevant to disease could be
the deletions in some BL EBV that allow BHRF1 expression from Wp to prevent
apoptosis (Kelly et al. 2009) and the mutation of EBNA-3B causing diffuse large
B cell lymphoma (DLBCL) in mice, with some EBNA-3B mutants also found in
human DLBCL (White et al. 2012).
All these speculative ideas highlight the importance of knowing more about
natural variation of EBV, particularly identifying what constitutes wild-type EBV
so that mutation or variation from this can be recognised. Most EBV strains that
have been characterised come from cancer cell lines or have been selected by B
cell transformation to make a lymphoblastoid cell line (LCL). The recent descrip-
tion of M81 EBV from a Hong Kong nasopharyngeal carcinoma (NPC) patient
emphasises this point; M81 BAC (bacterial artificial chromosome) EBV is more
spontaneously lytic and infects epithelial cells better than the B95-8 reference
strain (Tsai et al. 2013); its properties were interpreted as being relevant to devel-
opment of NPC.
The renewed interest in an EBV vaccine (Cohen et al. 2011) also empha-
sises the importance of ensuring that this will be directed to the wild-type EBV
sequence. In general, we need to know what is wild-type EBV. This chapter inte-
grates and updates some previously published reviews on EBV sequence variation
(Jenkins and Farrell 1996; Tzellos and Farrell 2012).
Epstein–Barr Virus Strain Variation 47
2 History of EBV Sequencing
Because of the relatively large genome for a virus (175 kb) and the presence of
several tandem repeat arrays (which are difficult to sequence), sequencing of EBV
genomes was initially quite limited. The sequences of some small fragments of
B95-8 EBV were published in 1982 (Cheung and Kieff 1982; Dambaugh and
Kieff 1982), but the first complete EBV sequence (accession number V01555) of
the B95-8 strain was published in 1984 (Baer et al. 1984). At that time, it was the
largest DNA sequence that had been determined.
The B95-8 strain was sequenced because B95-8 was the only EBV cell line
available that produced virus at a sufficient level to make it practical to clone the
viral restriction fragments. B95-8 cells were originally derived (Miller et al. 1972)
using EBV from 883L (a spontaneous human LCL from an infectious mononu-
cleosis patient) to transform lymphocytes from the cotton top marmoset (Saguinus
oedipus). EBV is secreted into the medium spontaneously, giving useful amounts
of infectious, transforming virus but B95-8 cells can also be induced into the lytic
cycle with phorbol myristate acetate (PMA, also called TPA). To obtain the DNA
for sequencing, B95-8 EBV was produced from PMA-treated B95-8 cells and the
EBV DNA was cloned as restriction fragments, Eco RI fragments in a cosmid vec-
tor pHC79 or Bam HI fragments in pBR322 (Arrand et al. 1981). Sequencing was
by the Sanger method (Baer et al. 1984).
It was already known from restriction site mapping that there is a 13.6-kb dele-
tion in B95-8 relative to other known EBV strains (Raab-Traub et al. 1980) and
this sequence was subsequently determined from cloned restriction fragments of
EBV from the Raji BL cell line (Parker et al. 1990). The error rate in the original
sequencing proved to be very low (about 1/50,000), so predictions made of the
open reading frames (Baer et al. 1984) turned out to be an accurate guide to the
genetic content and these have been the basis of much of the subsequent investiga-
tion of EBV. Three single nucleotide errors discovered in the genome sequence
were corrected (de Jesus et al. 2003), but the sequence has not been adjusted for
loss of a small part of the repeat array in oriP (Fruscalzo et al. 2001; Kanda et al.
2011) selected by cloning in plasmids. To facilitate studies on the whole viral
genome, a “wild-type” EBV sequence (EBVwt, AJ507799) was assembled from
the corrected B95-8 and Raji sequence, the number of major internal repeat units
in the sequence was reduced to 7.5 to be more typical and annotation was updated
(de Jesus et al. 2003).
A more standard annotation of wild-type EBV including three additional small
open reading frames that could now be recognised from sequence comparison
was released by AJ Davison and PJ Farrell in 2010 as the RefSeq HHV4 (EBV)
sequence NC_007605. This is the current standard reference sequence.
Key insights into EBV sequence variation have come from publication of fur-
ther complete EBV sequences from Africa, China, Japan and the USA. The EBV
sequence with accession number DQ279927 from the AG876 African BL cell
line (Dolan et al. 2006) is a type 2 EBV strain (see below). EBV from the African
48 P.J. Farrell
Mutu BL cell line (KC207814) and the Japanese Akata BL cell line (KC207813)
were reported in 2013 (Lin et al. 2013), the same year as two isolates K4413-Mi
(KC440851) and K4123-Mi (KC440852) from spontaneous LCLs are produced
in the USA (Lei et al. 2013). The African BL Raji EBV has also been fully
sequenced (KF717093).
The Chinese GD1 isolate (AY961628) was from a LCL (LCL) made by immor-
talising cord B cells with EBV from saliva of an NPC patient in Guangzhou
(Zeng et al. 2005). The GD2 sequence (HQ020558) is a direct determination of
EBV sequence from a Guangzhou NPC biopsy (Liu et al. 2011). Further EBV
sequences from Hong Kong NPCs, HKNPC1 from a biopsy (JQ009376) (Kwok
et al. 2012) and an additional series of 8 Hong Kong NPCs from the same group
(Kwok et al. 2014) have been reported. EBV from two cell lines derived from
Hong Kong has also been sequenced; M81 (KF373730.1) is from a marmoset
LCL prepared using EBV from an NPC patient (Tsai et al. 2013; Desgranges et al.
1976) and C666.1 (KC617875.1) is the only epithelial cell line derived from an
NPC that is known to retain its EBV in cell culture (Tso et al. 2013).
The methods used to determine these EBV sequences have advanced with
technological development. The AG876 sequence was determined using Sanger
sequencing of cosmid-cloned fragments, supplemented by PCR amplification of
selected regions. GD1 and HKNPC1 were amplified as a set of PCR fragments,
which were then sequenced. Many of the more recent sequences were obtained by
selecting the EBV reads from “next-generation” Illumina sequencing of the whole
cell and viral DNA.
The small amount of starting material required for determination of a complete
EBV sequence by the Illumina methods makes it realistic to analyse large num-
bers of primary human EBV strains, but the cost of this without first enriching
the EBV DNA is still quite high. Although next-generation sequencing has greatly
increased the production of sequences, the number of tandem repeat arrays in the
EBV genome and relatively high GC content still make sequencing of EBV rela-
tively difficult.
An interesting approach to concentrating sequencing effort on the EBV con-
tent of human samples involves first enriching the viral sequences on custom
SureSelect beads (Depledge et al. 2011). This can greatly reduce the cost of the
sequencing if many samples are to be tested. This approach has been used by a
group at the Sanger Institute to sequence 71 novel EBV strains (Palser et al.
2015) and was also used on some of the Hong Kong samples (Kwok et al. 2014).
Combining that data with the sequences already published give sufficient infor-
mation to draw some general conclusions about natural EBV variation, even
though many parts of the word are still not represented in the data. It is already
clear that the genetic standard map of the EBV genome annotated in the RefSeq
genome NC_007605 is generally consistent with all the known strains, although a
few strains have deletions in various parts of the viral genome. The open reading
frames are mostly conserved, providing further evidence for their validity.
3 Broad Aspects of Genome Variation—EBV Types,

Selection Forces and Recombination
The first major variation to be identified in EBV was the type 1 or type 2 clas-
sification based on differences in EBNA2 (Dambaugh et al. 1984; Adldinger et al.
1985; Rickinson and Kieff 2007). The types were also known as type A and B,
respectively. Most EBV genes differ by less than 5 % in sequence in different iso-
lates, but EBNA2 clearly sorts into type 1 or type 2, with only 70 % identity at the
nucleotide level and 54 % identity in the protein sequence. There is linked varia-
tion in EBNA3 genes (Rowe et al. 1989), but the degree of sequence difference is
less. Type 1 is the main EBV prevalent worldwide, but in sub-Saharan Africa, type
2 EBV is equally abundant and there are many mixed infections. In Argentina,
type 1 was present in 76 % of healthy carriers, type 2 in 15 %, and co-infections
with both types in 7 % (Correa et al. 2004); in Mexico, 33 % had type 1, 57 % had
type 2 and 10 % had mixed infection (Palma et al. 2013), but in Australia, 98 % of
infections were type 1 (Lay et al. 2012).
Sequencing of type 2 EBV from the AG876 cell line enabled a comparison
between type 1 and type 2 EBV genomes (Dolan et al. 2006). This revealed that
the two types are co-linear and very similar, with the exception of the known
divergent alleles. The type variation has a clear phenotypic consequence in cell
culture; type 2 EBV is much less effective at establishing LCLs than type 1 EBV
(Rickinson et al. 1987). There has been considerable progress in understanding the
mechanism of this, described in detail separately below. There is strong but not
complete genetic linkage of EBNA2 and EBNA3 genes in relation to the types.
The significance of these types has recently been put on a much firmer basis by
analysis of the 71 novel strains and the previously published 12 strains (Palser
et al. 2015).
Recent comparison of the genome sequences of 20 HSV isolates demonstrated
the extensive historical recombination that has occurred (Szpara et al. 2014).
Preliminary results from the Sanger Institute study of 71 sequences also demon-
strate this in EBV (Palser et al. 2015). At this point, it is not possible to identify
preferred sites of recombination or haplotype blocks of sequence in the whole
group of EBV sequences that are known. Considering smaller sets of sequences
from single geographic areas, however, makes it possible to recognise regions of
similarity to other strains. This extensive recombination means that it is not appro-
priate to use phylogenetic trees to compare the whole genomes directly. Principal
component analysis can be used on the whole genomes and this has shown that the
type 1/type 2 classification is quantitatively the greatest variation in EBV strains
worldwide (Palser et al. 2015).
The extent to which EBV can be meaningfully classified into types based on
individual gene markers depends partly on the extent of inter-typic recombina-
tion that may occur. There is clear evidence for inter-typic recombination based on
the polymorphisms distributed along the genome (Midgley et al. 2000; Yao et al.
1996; Kim et al. 2006). A more direct study of 83 EBV genome sequences (Palser
50 P.J. Farrell
et al. 2015) showed that 69 had EBNA2 and EBNA3s both type 1, 12 EBVs had
both EBNA2 and EBNA3s type 2, and 2 EBVs had type 2 EBNA3s but type 1
EBNA2. This describes the genetic linkage between EBNA2 and EBNA3s. A
comparison of the B95-8, GD1 and AG876 sequences led to a proposal for a mini-
mum theoretical number of recombination events that would be required to result
in the current genome arrangements of those viruses (McGeoch and Gatherer
2007), but the extent of historical recombination accumulated in the panel of 83
EBV genomes studied (Palser et al. 2015) is very complex. It will be simpler to
study recombination in a less diverse panel of strains, perhaps from a single geo-
graphic area.
Points of variation have been studied within type 1 EBNA2 (Schuster et al.
1996), but type 1 and type 2 EBNA2 sequences are sufficiently different to pre-
clude their recombination within EBNA2. So the type 1 and type 2 EBNA2
characteristics will tend to survive irrespective of recombination that may occur
elsewhere in the genome. Interpreting the significance of the persistence of these
types will therefore depend on understanding the mechanism and phenotypic con-
sequences of the type differences in vivo. The recent discovery that APOBEC3
cytidine deaminase RNA editing can modify EBV genomes in cell culture demon-
strates another possible mechanism for generating virus heterogeneity in vivo, in
addition to infection with multiple strains and virus recombination (Suspene et al.
2011).
Factors that affect in vivo selection of viral recombinants and variants might
be expected to include immune surveillance and the ability to infect and persist
through the complex life cycle of EBV. Immune surveillance would be expected
to correlate with MHC type since functional epitopes will vary according to the
presentation on MHC. Since predominant MHC types differ between racial groups
and geographically, this could be a major factor in world wide variation of EBV.
Many epitopes for CTL surveillance have been mapped in EBV antigens and cor-
related with MHC type. There is some clear evidence for epitope selection based
on immune surveillance (Burrows et al. 2004; Midgley et al. 2003a, b; Nagamine
et al. 2007; Tang et al. 2008; Lin et al. 2004, 2005; Duraiswamy et al. 2003).
Evidence for positive selection also comes from ratios of non-synonymous to syn-
onymous changes in the SNPs present in reading frames (Palser et al. 2015). This
showed an excess of non-synonymous changes in EBNA3 genes and LMP1, as
predicted from the immunological studies, but also in the immune evasion pro-
tein BNLF2a (Horst et al. 2012), which binds TAP and in the glycoproteins gp350
(BLLF1) and gL (BKRF2). So far little is known about the extent to which EBV
selection in vivo may be affected by other polymorphisms that could affect, for
example, ability to promote cell proliferation or viral replication. Detailed study
of the non-synonymous variation in EBNA3 genes (Palser et al. 2015) surpris-
ingly revealed that most of the amino acids contributing to the positive selec-
tion were not in known T cell epitopes. This result and the apparent clustering of
genes showing positive selection in the genome suggest that there may be addi-
tional mechanisms determining the positive selection, which are not understood at
present.
There are several examples of deletions of part of the EBV genome in endemic
BL cell lines. The best characterised of these are the approximately similar dele-
tions in P3HR1, Daudi, Sav, Oku and Ava BL cells (Kelly et al. 2002) that remove
EBNA2, most of BHLF1 and the C-terminal part of EBNA-LP. The significance of
these was interpreted as a mechanism for avoiding EBNA2 antagonism of c-MYC
function (Kelly et al. 2002) in the tumour cells. Further investigation indicated that
enhanced expression of BHRF1 (the anti-apoptotic, viral BCL2 homologue) as a
result of the deletion could also play a role in some BL cells harbouring EBV with
this type of deletion (Kelly et al. 2009).
Some other well-characterised EBV deletions are in the Raji BL cell line. Two
separate deletions result in a loss of EBNA3C and some genes essential for lytic
DNA replication (Hatfull et al. 1988). Complementation of the defective lytic
cycle genes by expression of BALF2 was sufficient to restore lytic DNA replica-
tion in Raji cells (Decaussin et al. 1995). As mentioned above, the B95-8 strain of
EBV also has a deletion relative to most EBV isolates (Raab-Traub et al. 1980;
Parker et al. 1990); the B95-8 deletion removes many of the BART miRNA
sequences and one of the lytic origins of replication, but this does not seem to
adversely affect lytic replication or immortalisation of B cells.
Rearranged, defective EBV genomes (known as het DNA) have been character-
ised in detail in the P3HR1 BL cell line (Jenson et al. 1986). The rearrangements
cause constitutive expression of BZLF1 (Rooney et al. 1988) and the result-
ing lytic cycle activation allows persistence of the defective genomes in the cell
population. There have been reports of similar rearranged EBV genomes in vivo
(Patton et al. 1990; Gan et al. 2002), but a recent study concluded that they are
only present quite rarely in vivo (Ryan et al. 2009).
4 Variation in EBV Genes
4.1 LMP1
The locus of variation that has attracted most investigation in relation to disease
is LMP1, which contains a higher degree of polymorphism than most EBV genes
(Palser et al. 2015). LMP1 is a membrane protein which makes many interactions
through its C-terminal region that mediate important signal transduction. These
interactions regulate NF-kB and cell survival in several ways, so it is easy to envis-
age how sequence variation could alter those processes. A key step forward in
understanding the many points of polymorphism in LMP1 came with classification
of LMP1 variants into 7 main groups (Mainou and Raab-Traub 2006) and devel-
opment of a rapid heteroduplex assay that allows classification of LMP1 type in a
large number of samples. The sequence variants of LMP1 relative to B95-8 were
named Alaskan, China 1, China 2, Med+, Med−, and NC. This more general
insight into LMP1 variation taking into account the whole LMP1 sequence (Miller
et al. 1994; Walling et al. 2004; Mainou and Raab-Traub 2006) has also been used
52 P.J. Farrell
by many other groups (Sung et al. 1998; Kanai et al. 2007; Nagamine et al. 2007;
Lin et al. 2005; Zhao et al. 2005; Shibata et al. 2006; Saechan et al. 2006, 2010;
Dardari et al. 2006; Li et al. 2009; Nguyen-Van et al. 2008; Diduk et al. 2008;
Pavlish et al. 2008; Wang et al. 2007), but there was generally little evidence for a
specific disease association of variants. Investigation of variant LMP1 sequences
in HIV patients in Switzerland also identified polymorphisms (I124 V/I152L and
F144I/D150A/L151I), which were markers of increased NF-κB activation in vitro
but were not associated with EBV-associated Hodgkin’s lymphoma (Zuercher
et al. 2012).
It is still not clear how many EBV variants are present within one individual
or even whether being infected offers immune protection against acquiring addi-
tional EBV strains. Studies using a heteroduplex assay confirmed that individuals
can be infected with multiple variants (Edwards et al. 2004; Tierney et al. 2006).
Multiple LMP1 variants can be found in people with infectious mononucleosis
(Fafi-Kremer et al. 2005), Hodgkin’s lymphoma or NPC (Rey et al. 2008), and
there is also evidence from people who are immunosuppressed, for example AIDS
patients, for infection with multiple EBV strains (Palefsky et al. 2002; Walling
et al. 2003). Based on LMP1 analysis, variants differ in abundance between throat
wash samples and peripheral blood samples in a variety of conditions (Sitki-Green
et al. 2002, 2003, 2004; Tierney et al. 2006), although a recent study reported the
polymorphisms in EBV from throat washings and tumour in Chinese NPC patients
to be the same (Nie et al. 2013). Evidence for a specific variant of LMP1 being
involved in a cancer could be provided by finding selective presence of that allele
in cancer cells relative to the virus in the saliva or peripheral circulation. This is
what was found in an analysis of an NPC patient (Edwards et al. 2004), but the
interpretation made was in the context of evasion of immune surveillance of the
LMP1 in the MHC background of the patient rather than specifically enhanced
transforming activity of the LMP1.
Interest in LMP1 variation and function was stimulated by reports that a vari-
ant with a 30-bp deletion (Cao LMP1) isolated from an NPC tumour had a greater
transforming activity than the reference LMP1 (Hu et al. 1991, 1993). There are
many points of sequence difference between Cao LMP1 and the reference B95-8
protein, but attention was focussed on the 30-bp deletion in Cao LMP1. The 30-bp
deletion (amino acids 346–355) includes part of C-terminal activating region 2
(CTAR2, amino acids 351–386) of LMP1. CTAR2 (Huen et al. 1995) mediates
signalling to NF-kB and AP-1, but the 30-bp deletion does not alter the parts of
LMP1 that activate NF-kB (Farrell 1998). An analysis of 249 patients in Taiwan
showed that patients with the Cao CTAR2 variant had an increased risk of dis-
tant metastasis compared with the non-Cao variant (Pai et al. 2007). The Cao
CTAR2 was also a negative predictor for overall survival and post-metastasis dis-
ease-specific survival in that series. Another study indicated enhanced ability in
tumour-derived variants to activate ERK kinase and induce c-Fos (Vaysberg et al.
2008), although that was accounted for by G212S or S366T rather than the 30-bp
deletion.
Since references given in a previous review (Jenkins and Farrell 1996), many
groups have investigated the presence of the 30-bp deletion LMP1 in normal car-
riers (Correa et al. 2004) or a variety of EBV-associated diseases (Cheung et al.
1998; Hayashi et al. 1998; Zhang et al. 2002; Tai et al. 2004; Jen et al. 2005;
Chang et al. 2006; Zhao et al. 2005; Correa et al. 2007; See et al. 2008; Lorenzetti
et al. 2012; Banko et al. 2012; Giron et al. 2013). A rarer 69-bp deletion in the
C-terminus has also been studied (Hadhri-Guiga et al. 2006); it was reported to
have a reduced ability to activate the cell AP1 transcription factor (Larcher et al.
2003). In general, it is clear that these LMP1 variants are widely distributed with
somewhat different frequencies in different parts of the world, but in most studies,
there was no evidence for a specific association with disease (Senyuta et al. 2014;
Gantuz et al. 2013). Other reports of LMP1 variation associated with specific pop-
ulations and disease contexts, for example gastric and oral carcinoma (Chen et al.
2010, 2011; BenAyed-Guerfali et al. 2011; Higa et al. 2002), have mostly lacked
sufficient numbers or control samples to interpret in the context of the relation-
ship between variation and disease, but some studies have suggested an associa-
tion with disease (Corvalan et al. 2006).
4.2 LMP2A
Low levels of LMP2A enhance cell survival, but high levels of LMP2A can also
interfere with signalling from the B cell receptor by binding of lyn and fyn tyros-
ine kinases to the hydrophilic N-terminal part of LMP2A. Sequence polymor-
phism has been described in the N-terminal region but it does not affect the key
phosphorylated residues (Busson et al. 1995). Although sequence polymorphism
has been detected in isolates from various parts of the world and various diseases
(Berger et al. 1999; Tanaka et al. 1999; Wang et al. 2010a; Han et al. 2012), there
is no evidence for a disease association at present. Sequence variation is present,
for example in South-east Asia and New Guineau (Lee et al. 1993), in mapped
epitopes for class I restricted cytotoxic T cells, but this is not sufficient to prevent
the possibility of LMP2A being a target for immunotherapy of EBV-associated
cancers (Rickinson and Moss 1997; Khanna et al. 1999; Taylor et al. 2014).
4.3 EBNAL
EBNA1 proteins frequently differ in size due to variation in the length of the Gly–
Ala repeat, but further differences in the unique parts of EBNA1 have been used
to define P (prototype, B95-8) and V (variant) EBNA1, which differ at about 15
amino acids (Bhatia et al. 1996). These each have two subtypes defined by the
amino acid at position 487 (P-ala, P-thr, V-pro and V-leu). In the initial report
(Bhatia et al. 1996), P-thr was most frequent in peripheral blood lymphocytes of
54 P.J. Farrell
African and American samples and in African tumours, but most American EBV-
associated lymphomas had V-leu EBNA1. A subsequent report confirmed the
subtypes but found no association with lymphoma (Habeshaw et al. 1999). The
variation might affect immune recognition of EBNA1 (Wrightham et al. 1995;
Chen et al. 1999; Bell et al. 2008) and has been noted in Chinese NPC samples (Ai
et al. 2012; Snudden et al. 1995; Zhang et al. 2004; Wang et al. 2010a) and gastric
cancer (Chen et al. 2012), but there is no substantial evidence of disease associa-
tion. It has been reported that the V-val subtype of EBNA1 has increased ability to
activate the enhancer functions of oriP in transfection assays (Do et al. 2008; Mai
et al. 2010).
4.4 EBNA3 Family
Some of the polymorphism in the EBNA3 genes is linked to EBNA2-type varia-

tion (Rowe et al. 1989; Palser et al. 2015), but additional subvariation in EBNA3A
and 3C has been noted with no relationship to disease (Gorzer et al. 2006;
Wu et al. 2012).
Since EBNA-3B is not required for immortalisation by EBV, variation in
EBNA-3B was originally considered mainly in the context of immune surveil-
lance. For example (Chu et al. 1999), polymorphisms of EBNA-3B (called
EBNA4 in that publication) were found to be frequent in EBV-associated
Hodgkin’s lymphoma, gastric carcinoma and AIDS-lymphoma but not related
to patients’ HLA-A11 status. Sequence variation in EBNA-3B is now being re-
examined since it has been realised that EBNA-3B acts as a tumour suppressor
gene (White et al. 2012). Loss of function mutants of EBNA-3B have much higher
B cell transforming activity than wild type and a propensity to cause DLBCL in
a mouse model (White et al. 2012). It will therefore be important to determine
which of the many polymorphisms in EBNA-3B affect its function so as to
increase the viral transforming activity.
4.5 Other Latent Cycle Elements
The EBER RNAs are strongly expressed in all types of EBV-associated cancer.
The extent of sequence variation in EBERs is quite small and has been linked to
the type 1/type 2 EBNA2 status (Arrand et al. 1989; Schuster et al. 1996). A recent
report identified some new variants in Chinese samples (Wang et al. 2010b). At
present, there is little information on sequence variation in the BART or BHRF1
miRNAs, although attention has been drawn to a G155849A polymorphism near
the RPMS1 open reading frame in the BART region of EBV from EBV associated
with NPC in China (Li et al. 2005). Variation in promoter activity affecting expres-
sion in reporter assays has also been reported for the Cp and Qp promoters in EBV
isolates from Chinese NPC samples (Wang et al. 2012a), but there is no evidence
for a role in disease. In contrast, it is clear that the number of copies of the major
internal repeat (IR1), which contains the Wp promoter, does affect the ability of
EBV to express EBNA2 and EBNA-LP efficiently and has a major effect on trans-
formation efficiency (Tierney et al. 2011).
4.6 Lytic Cycle
BZLF1 is the transcription factor that initiates the lytic cycle reactivation in
B cells and its promoter is tightly regulated since it mediates the switch from
latency to the lytic cycle. There has been considerable interest in BZLF1 vari-
ants that might affect its function or expression (Lorenzetti et al. 2012; Jin et al.
2010; Imajoh et al. 2012; Yang et al. 2014). Although there are suggestions of vari-
ant sequence in Zp correlating with disease (Martini et al. 2007; Lorenzetti et al.
2014), further functional analysis would be required to substantiate this.
Natural variation in the BZLF1 protein sequence has been described in Chinese
isolates but showed no relationship to disease (Ji et al. 2008; Luo et al. 2011).
Variation in the BZLF1 dimerisation sequence was studied using synthetic pep-
tides but found to have only a small effect on its activity (Hicks et al. 2001).
Polymorphism has also been noted in BRLF1 (Jia et al. 2010; Yang et al. 2014),
the EBV transcription factor which cooperates with BZLF1 to activate the early
lytic cycle genes. Although a possible disease association was noted, this would
require substantiation with more samples and controls.
Variation has also been reported in the early lytic cycle genes BHRF1 (Jing
et al. 2010) and BNLF2a (Horst et al. 2012). BHRF1 is similar to BCL2 with anti-
apoptosis activity and BNLF2a has a role in immune evasion, reducing cell surface
HLA class I levels. Protein function was not affected by these sequence polymor-
phisms. In an earlier study, sequence variants of the BHRF1 protein were identi-
fied (Khanim et al. 1997), but no effect of the variation was found in the ability to
protect against apoptosis induced by cisplatin.
BARF1 is expressed in the early lytic cycle in B cell reactivation but is also
expressed in NPC cells. It is secreted and binds to colony stimulating factor 1
(CSF-1), inhibiting the binding of CSF-1 to its receptor (Strockbine et al. 1998).
Sequence polymorphism has been identified in BARF1 in Indonesian and Chinese
samples (Hutajulu et al. 2010; Wang et al. 2012c) but most likely reflects natural
selection of EBV strains unconnected to carcinogenesis.
The late lytic cycle gene gp350 encodes a surface glycoprotein on the EBV par-
ticle which is the target of neutralising antibodies for EBV infection. Sequence
variation of this protein has been identified but again appears to show geographic
restriction rather than tumour specific polymorphism (Kawaguchi et al. 2009; Luo
et al. 2012).
56 P.J. Farrell
5 Functional Difference Between Type 1 and Type 2

EBNA2
At present, there is little evidence for a disease relationship to the EBV types. One
study found that type 1 EBV was more likely to cause infectious mononucleosis
than type 2 EBV (Crawford et al. 2006), but a second investigation found no sig-
nificant difference (Tierney et al. 2006). Although type 2 EBV is prevalent in the
same sub-Saharan region of Africa as endemic BL, the frequency of type 1 or type
2 EBV in BL from that region seems to reflect the incidence in the population
rather than a specific-type association (Rickinson and Kieff 2007). Some subtype
variation of EBNA2 has been noted in Chinese EBV-associated carcinomas (Wang
et al. 2012b) and in European lymphomas (Schuster et al. 1996), but again there is
little evidence that it is related to disease.
The greatest biological and functional difference between the two viral types
is that type 1 EBV immortalises B cells in vitro much more efficiently than type 2
EBV (Rickinson et al. 1987). When LCLs are transformed with type 1 EBV, they
grow more quickly and to a higher saturation cell density in comparison with type
2 transformants (Rickinson et al. 1987). This difference in in vitro transforming
efficiency between type 1 and type 2 EBV has been mapped to the EBNA2 locus
(Cohen et al. 1989). When a type 2 P3HR1 EBV strain was engineered to carry a
type 1 EBNA2 sequence, this virus gained the type 1 immortalisation phenotype
(Cohen et al. 1989). In contrast, EBNA3 type does not affect the immortalisation
ability of the virus, as replacing the type 2 EBNA3 gene locus with corresponding
type 1 sequences in the P3HR1 EBV genome showed no difference in primary
B lymphocyte growth transformation (Sample et al. 1990; Tomkinson and Kieff
1992).
This in vitro transformation phenotype of type 1 and type 2 EBV correlates
with tumour formation frequency in SCID mice that were inoculated intraperito-
neally with type 1 or type 2 in vitro-transformed LCLs (Rowe et al. 1991; Cohen
et al. 1992). Also, similar rates of tumour induction were observed for EBV-LCLs
generated in vitro with a wild-type type 1 strain or with a type 2 P3HR1 strain car-
rying a type 1 EBNA2 in the SCID mice model (Cohen et al. 1992).
More recently, a transfection assay with an LCL (EREB2.5) infected with
EBV containing conditional EBNA2 function was used to compare the abilities
of type 1 and type 2 EBNA2 to maintain cell proliferation (Lucchesi et al. 2008).
Type 1 EBNA2 maintained the normal growth of the cells, but the type 2 EBNA2
did not, providing a simple cell growth assay for this aspect of EBNA2 activity.
The reduced proliferation in cells expressing type 2 EBNA2 correlated with loss
of expression of some cell genes that are known to be targets of type 1 EBNA2.
Microarray analysis of EBNA2 target genes identified a small number of genes
that are more strongly induced by type 1 than by type 2 EBNA2, and one of these
genes (CXCR7) was shown to be required for proliferation of LCLs. The EBV
LMP1 gene was also more strongly induced by type 1 EBNA2 than by type 2,
but this effect was transient. The results indicated that differential gene regulation
by EBV type 1 and type 2 EBNA2 might be the basis for the much weaker B cell
transformation activity of type 2 EBV strains compared to type 1 strains (Lucchesi
et al. 2008).
To map the part of the EBNA2 protein responsible for the enhanced cell growth
activity of type 1 EBNA2, the effect on EREB2.5 cell growth of chimaeras of
type 1 and type 2 EBNA2 was tested in the EREB2.5 cell growth assay (Cancian
et al. 2011). Although the major sequence differences between type 1 and type
2 EBNA-2 lie in N-terminal parts of the protein, the superior ability of type 1
EBNA-2 to induce proliferation of EBV-infected lymphoblasts was found to be
mostly determined by the C-terminus of EBNA-2. Substitution of the C-terminus
of type 1 EBNA-2 into the type 2 protein was sufficient to confer a type 1 growth
phenotype in the EREB2.5 cell growth assay and type 1 expression levels of
LMP-1 and CXCR7. Within this region, the RG, CR7 and TAD domains were the
minimum type 1 sequences required. The results indicated that the C-terminus of
EBNA-2 accounts for the greater ability of type 1 EBV to promote B cell pro-
liferation, through mechanisms that include higher induction of genes (LMP-1
and CXCR7) required for proliferation and survival of EBV-LCLs (Cancian et al.
2011).
More detailed analysis showed that converting a single amino acid of type 2
EBNA2 from serine to the aspartate found in type 1 EBNA2 (S442D) was suf-
ficient to give the type 1 growth maintenance of LCLs (Tzellos et al. 2014). That
amino acid is in the transactivation domain (TAD) of EBNA2, and the TAD of
type 1 EBNA2 was about 2-fold more active in a standard TAD reporter assay.
However, this did not explain the specific superior induction of a small group of
cell genes and the LMP1 gene by type 1 EBNA2. About 9 genes were directly
induced more strongly by type 1 EBNA2 (Lucchesi et al. 2008), including
CXCR7, which was shown to be required for LCL proliferation. Recent ChIP-
seq analysis of binding sites for EBNA2 in LCL and BL cell lines has shown that
many EBNA2 binding sites are quite distant from the transcription start sites of
genes and regulated genes frequently have several EBNA2 binding loci within
about 50 kb of the transcription start sites. By comparing the DNA sequences
of EBNA2 binding sites in differentially regulated genes, a conserved motif was
identified that is present in the differentially regulated promoters, including the
LMP1 promoter (Tzellos et al. 2014). The motif contains the consensus binding
sites for PU.1 and IRF transcription factors, and it is thought that the function of
EBNA2 at these sites may be mediated by its association with these factors rather
than the usual RBP-Jk DNA binding protein.
It seems surprising that such a complex functional difference between the
EBNA2 types would be mediated by a single amino acid when the proteins are
only 56 % identical. Perhaps there are other phenotypes mediated by other
sequence differences, but these have not yet been measured in these B cell assays.
The physiological significance of the type 1/type 2 variation is not known at
present. One interesting speculation is that type 2 EBV might be favoured in con-
ditions of chronic immune activation. This might be present in the parts of Africa
where type 2 EBV is most abundant due to other co-infections including malaria.
58 P.J. Farrell
Other situations where detection of type 2 seems relatively common also involve
disturbance of the immune system, for example in AIDS patients. It has also
recently been reported that type 2 EBV can infect T lymphocytes and cause a tran-
sient cell proliferation with concomitant cytokine secretion (Coleman et al. 2015).
6 How to Identify Variation Potentially Relevant

to EBV-Associated Diseases
This can be considered using NPC in southern China as an example. Chinese

strains of EBV have characteristic polymorphisms in several regions of the
genome, including some of the regions relevant to NPC, such as the BART
miRNA region and LMP1. At present, there is little direct evidence that these
polymorphisms contribute to the high incidence of NPC in southern China, but it
has recently been shown that the BART miRNAs can play an important role in
growth of NPC cells (Qiu et al. 2015). To investigate the role of the sequence pol-
ymorphisms, two types of experiment will be needed. First, it will be necessary to
sequence large numbers of EBV samples from people with or without NPC from
the endemic area, sampling directly from the tumour in NPC patients and from
their saliva. A correlation could indicate whether there are tumour specific vari-
ants within individuals—studies of this type are currently in progress. Secondly,
it would be necessary to test whether some of these specific polymorphisms alter
the tumorigenic properties of the virus in an experimental system. There is a great
need for a meaningful animal model for EBV-associated NPC so that mutations
could be introduced into the virus and the role of polymorphisms could be ana-
lysed. At present, no system of that type exists, but it represents an important chal-
lenge for future research on NPC. The detailed analysis of differences between
type 1 and type 2 EBNA2 described above, with emphasis on a single amino acid
(S442D) determining transcriptional function on a specific set of genes important
for cell growth, shows how challenging it may be to identify the crucial sequence
differences among a large number of inconsequential polymorphisms. If the mech-
anism became clear from model systems and were obviously relevant, this might
be sufficient to convince. A more rigorous proof through prevention of infection
by immunisation seems a distant possibility at present, but it is possible that some
form of immunisation would modify disease without actually blocking infection.
In general to understand natural variation of EBV, there is currently a great need
for more viral genome sequences, much better worldwide coverage and more
whole EBV genome analysis relative to disease to give the statistical power that
will be required to identify variation relevant to disease.
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PLoS ONE 7(2):e32168. doi:10.1371/journal.pone.0032168
Chromatin Structure of Epstein–Barr Virus
Latent Episomes
Paul M. Lieberman
Abstract EBV latent infection is characterized by a highly restricted pattern of

viral gene expression. EBV can establish latent infections in multiple different tis-
sue types with remarkable variation and plasticity in viral transcription and rep-
lication. During latency, the viral genome persists as a multi-copy episome, a
non-integrated-closed circular DNA with nucleosome structure similar to cellular
chromosomes. Chromatin assembly and histone modifications contribute to the
regulation of viral gene expression, DNA replication, and episome persistence dur-
ing latency. This review focuses on how EBV latency is regulated by chromatin
and its associated processes.
Contents
1 Introduction......................................................................................................................... 72
2 History and Discovery of the EBV Latent Episomes......................................................... 73
3 Nuclear Entry, DNA Recognition, and Chromatinization.................................................. 73
4 Early Viral Gene Expression............................................................................................... 76
5 Chromatin Organization of the Latent Episome................................................................. 78
6 OriP as a Chromosome Organizing Element...................................................................... 80
7 EBNA1 as a Critical Regulator of EBV Chromatin........................................................... 82
8 Barriers to Lytic Reactivation............................................................................................. 83
9 Late Gene Transcription and Replication........................................................................... 85
10 Network and Homeostatic Control of Viral Chromatin...................................................... 86
11 Chromatin Modifiers that Disrupt Latency—Environment, Pathogenesis,
and Therapeutics................................................................................................................. 88
12 Variation and Heterogeneity of Viral Chromatin Control................................................... 89
13 Conclusions......................................................................................................................... 89
References................................................................................................................................... 90
P.M. Lieberman (*)

The Wistar Institute, Philadelphia, PA 19104, USA
e-mail: Lieberman@wistar.org

72 P.M. Lieberman
Abbreviations
3C Chromatin conformation capture

BAH Bromo-adjacent homology
BL Burkitt’s lymphoma
CBD Chromosome binding domain
ChIP Chromatin immunoprecipitation
CMV Cytomegalovirus
CTD Carboxy-terminal domain
DDR DNA damage response
DS Dyad symmetry
EBV Epstein–Barr virus
FGARAT Phosphoribosylformylglycinamidine synthase
FR Family of repeats
HDAC Histone deacetylase
INR Initiator element
KSHV Kaposi’s sarcoma-associated herpesvirus
LCL Lymphoblastoid cell line
MCFA Medium chain fatty acids
MNase I Micrococcal nuclease I
NPC Nasopharyngeal carcinoma
NK/T Natural killer/T cell
OriP Origin of plasmid replication
PML-NB Promyelocytic leukemia-nuclear body
SCFA Small chain fatty acids
SUMO Small ubiquitin-like modifier
TPA Phorbol ester 12-O-Tetradecanoylphorbol-13-acetate
VPA Valproic acid
1 Introduction
Epstein–Barr Virus (EBV) can act as a cofactor in numerous and diverse human
cancers, ranging from B- and NK/T-cell lymphomas to epithelial carcinomas of
the stomach and nasopharynx. In all instances, EBV-associated pathogenesis cor-
relates with the persistence of the viral genome in the majority of tumor cells. This
persistence is referred to as a “latent” infection, because infectious viral particles
are rarely produced. Importantly, EBV latent infection is a highly active, dynamic,
and “programmed” process, where the viral DNA co-opts host-cell fate decisions,
including proliferation, differentiation, and survival. During latency, the viral
genome expresses a restricted set of latency-associated genes, replicates once and
only once, and segregates faithfully to newly divided daughter cells. The latency
gene expression programs are highly dynamic and respond to host-cell-specific
factors and environmental signals.
Chromatin Structure of Epstein–Barr Virus Latent Episomes 73
This review considers how viral latency is controlled by chromatin. In it broad-

est definition, chromatin refers to the nucleoprotein complexes that assemble on
DNA to form a functional chromosome. At the most basic level, chromatin is com-
posed of the histone core proteins that form nucleosomes by wrapping two turns
of ~145 bp of DNA around an octamer of H2A-H2B/H3-H4 (review in Zentner
and Henikoff 2013). The histones can be extensively post-translationally modified
to produce epigenetic marks that have profound effects on DNA metabolism and
gene expression. These modifications are commonly referred to as the “marks”
of a histone code that is interpreted by protein “readers,” “writers,” and “erasers”
(Ruthenburg et al. 2007; Jenuwein and Allis 2001). Viruses, such as EBV, utilize
and manipulate the histone code and chromatin structure to execute their own life-
cycle programs. For EBV, chromatin formation is critical for the persistence of the
viral genome and the control of viral gene expression during latent infection.
2 History and Discovery of the EBV Latent Episomes
EBV was identified in 1964 as a latent infection associated with Burkitt lym-
phoma (BL) tissue and BL-derived cell lines (Epstein et al. 1964). EBV DNA
from latently infected cells could be separated from chromosomal DNA by gradi-
ent centrifugation (Nonoyama and Pagano 1972) and sedimented as closed circu-
lar episomes (Adams and Lindahl 1975). The chromatin structure of EBV was first
examined MNase I nuclease digestion assays that revealed a nucleosome organiza-
tion that was indistinguishable from the host bulk chromosome (Shaw et al. 1979).
Later studies identified several regions of the genome that were DNase I sensitive
and likely to be nucleosome-free regions (Dyson and Farrell 1985). This was con-
firmed by Sexton and Pagano who showed that the regions within the origin of
plasmid replication (OriP) were nucleosome free, suggesting that chromatin struc-
ture was not uniform throughout the latent genome (Sexton and Pagano 1989) and
therefore subject to regulation.
3 Nuclear Entry, DNA Recognition, and Chromatinization
The early events that lead to a stable latent infection have been referred to as the
pre-latency phase, since latency is the predominant outcome of primary infection
in resting B-lymphocytes (Kalla and Hammerschmidt 2012). Several major events
occur on the path to latency, all of which are highly regulated, both by the cell and
virus. The major events that lead to assembling a functional latent episome include
membrane receptor engagement and signal transduction, capsid transport to the
nucleus, nuclear entry of viral DNA, genome circularization, genome chromati-
nization, transcription of latency-associated genes, repression of lytic cycle genes,
and genome copy number maintenance (Fig. 1).
74 P.M. Lieberman
Fig. 1 Early events in the establishment of EBV episomal chromatin. EBV virions enter through
CD21 receptor and activate signal transduction pathways that can influence nuclear transcription
factors, including NF-kB and AP1. Linear, viral DNA lacking nucleosomes enter the nuclear pore
and confront host-cell factors, including IFI-16, PML-NBs, and components of the DDR. Viral
genomes circularize and assemble into chromatin (chromatinization). At least one viral tegument
protein, BNRF1, and early latency gene product, EBNA-LP, can alter the PML-NBs and influ-
ence the chromatinization of the viral genome to promote latency-associated gene expression
Like other herpesviruses, EBV is thought to enter the nucleus as naked linear
DNA without any associated chromatin proteins. Mass-spectrometry and pro-
teomic studies of EBV virions fail to detect histone proteins (Johannsen et al.
2004). This is consistent with previous studies showing that newly replicated DNA
and virion-associated DNA lack typical nucleosome structure (Dyson and Farrell
1985). It is also consistent with a more recent study finding that EBV produc-
tive replication occurs in histone-deficient replication compartments and in cells
depleted of histone chaperones (Chiu et al. 2013). This strongly suggests that
histones are not incorporated into the newly synthesized and encapsidated viral
genomes. How the viral DNA gets inserted into the nucleus is also not known for
EBV. For HSV-1, the DNA is thought to enter the nucleus through nuclear pores
(Meyring-Wosten et al. 2014; Liashkovich et al. 2011). Nuclear pore interactions
may be highly significant, since nuclear pore proteins can function in chromatin
assembly and transcriptional regulation (Ptak et al. 2014).
Among the first nuclear events known to occur when viral genomes enter
the nucleus is the confrontation with the host intrinsic resistances and anti-viral
responses (Weitzman et al. 2010; Boutell and Everett 2013). These include the
DNA damage response (DDR), the inflammatory response, and the formation of
PML-nuclear bodies. The promyelocytic leukemia (PML) protein assembles into
a PML-nuclear body (PML-NB) at sites where viral DNA localizes in the nucleus
(reviewed in Van Damme and Van Ostade 2011; Everett and Chelbi-Alix 2007).
PML-NB formation depends on SUMO modification of PML (Cuchet-Lourenco
et al. 2011) and the recruitment of SUMO interacting proteins, such as Daxx, and
other interacting proteins, like ATRX and SP100, that collectively form a nuclear
body where viral DNA-structures may be captured (Reichelt et al. 2011). PML-
NBs are typically destroyed or disabled by viral-encoded proteins, such as HSV-1
ICP0 and CMV pp75 (Saffert and Kalejta 2008). Destruction of PML-NBs is nec-
essary for productive infection of HSV and CMV, and likely to be necessary for
EBV lytic replication, as well.
At least one EBV tegument protein, BNRF1, has been implicated in modulat-
ing PML-NBs through its interaction with Daxx (Tsai et al. 2011). BNRF1 is a
member of the FGARAT family of proteins, conserved among gammaherpesvirus,
and includes KSHV ORF75, MHV68 ORF75a, b, c, and HVS ORF75 and ORF3.
While these genes have been duplicated or triplicated in other gammaherpesvirus,
EBV retains a single member of this family. The FGARAT domain has homology
to de novo purine biosynthesis enzymes conserved from prokaryotes to humans.
However, no enzymatic activity has yet been attributed to the viral FGARAT pro-
teins and amino acid mutations in catalytic amino acids suggest that gammaher-
pesvirus FGARAT proteins have lost conventional purine biosynthesis capability,
but may have acquired other, as yet unknown, activities.
Recent studies have revealed that all gammaherpesvirus FGARAT proteins
interact with components of the PML-NBs. The EBV-encoded BNRF1 protein
has been shown to interact directly with Daxx (Tsai et al. 2011). Daxx has been
shown to collaborate with ATRX to assemble histone variant H3.3 into repressive
chromatin at GC-rich repetitive DNA (Lewis et al. 2010; Goldberg et al. 2010).
BNRF1 binding to Daxx displaces ATRX from binding Daxx (Tsai et al. 2011).
The implication of these studies is that BNRF1 prevents ATRX association with
Daxx, and thereby inhibits ATRX-dependent formation of repressive H3.3 on viral
genomes. Interesting, other gammaherpesvirus FGARAT targets different compo-
nents of the PML-NBs and in most cases leads to the degradation of one or more
components (Full et al. 2014; Ling et al. 2008). The fact the BNRF1 does not
cause the degradation of PML, Daxx, or ATRX suggests that EBV has a different
strategy for interacting with PML-NBs than other herpesviruses. The modulation
of Daxx–ATRX without its degradation may be related to the propensity of EBV
to establish a latent, rather than a lytic infection.
A potentially related response to viral DNA is the interaction with interferon
inducible protein 16 (IFI-16). IFI-16 was shown to recognize the newly infect-
ing DNA genomes of KSHV as they enter the nucleus (Kerur et al. 2011). IFI-
16 interaction with KSHV DNA results in the nuclear export of IFI-16 and the
activation of the ASC-linked inflammasome (Singh et al. 2013). IFI-16 also inter-
acts with the latent EBV episome in the nucleus to cause the chronic activation of
the inflammasome (Ansari et al. 2013). IFI-16 interacts with structured DNA with
a preference for four-way junction cruciform DNA and superhelical DNA (Brazda
et al. 2012). It is proposed that IFI-16 preferentially recognizes EBV latent
genome relative to cellular DNA (Ansari et al. 2013). IFI-16 has been linked to
76 P.M. Lieberman
DNA damage recognition and response (Aglipay et al. 2003). IFI-16 also interacts
with the KSHV (Singh et al. 2013; Kerur et al. 2011), CMV (Horan et al. 2013;
Gariano et al. 2012), and HSV-1 (Johnson et al. 2013; Orzalli et al. 2013; Conrady
et al. 2012) genome upon entry in the nucleus, and leading to inflammasome sign-
aling. In HSV-1, IFI-16 has been implicated in transcriptional repression of viral
genes (Orzalli et al. 2013). IFI-16 signaling and cytoplasmic export is regulated by
lysine acetylation (Li et al. 2012). Whether IFI-16 regulates chromatin formation
on the EBV episome is not known.
Foreign DNA entering the nucleus is expected to elicit a DNA damage response
(Weitzman et al. 2010). EBV infection has been shown to activate the DNA dam-
age response (DDR) through the ATM/Chk2 kinasepathway during the pre-latency
phase of infection (Nikitin et al. 2010). However, this DDR response occurs at
~7 days of post-infection and correlates best with EBV-induced host-cell hyper-
proliferative response. An earlier DDR to linear, non-chromatinized DNA may
be masked by EBV-encoded factors, but these have not yet been characterized.
Pharmacological inhibitors of ATM and Chk2 increase EBV transformation effi-
ciency upon primary infection (Nikitin et al. 2010), indicating that DDR pathways
are an inherent block to EBV latency. EBV encodes a number of other tegument
proteins that are likely to influence these early events in chromatin assembly and
gene regulation. Among these are BPLF1 which has deubiquitinating and dened-
dylating activity (Gastaldello et al. 2010, 2012). BPLF1 has been shown to inter-
act with cellular protein complex Rad6/18 and deubiquinate cellular PCNA and
to functionally inhibit ribonulceotide reductase activity and attenuate DNA repli-
cation lesion repair synthesis (Kumar et al. 2014; Whitehurst et al. 2009, 2012).
Whether BPLF1 regulates the DDR response during early stages of infection, and
how this may affect viral chromatinization is not yet known.
4 Early Viral Gene Expression
The earliest viral gene expression detected after de novo infection of resting human
primary B-lymphocytes is the EBNA2 transcript derived from the Wp promoter
(Tierney et al. 2000a, b, 2007; Alfieri et al. 1991; Shannon-Lowe et al. 2005).
The B-cell transcription factor, BSAP/PAX5, was found to bind and regulate Wp
transcription during primary infection (Tierney et al. 2000a, 2007). Wp-initiated
transcriptscan make a short form of EBNA-LP and EBNA2 only. EBNA-LP and
EBNA2 interact with chromatin regulatory factors to further promote the formation
of the viral chromosome and latency gene expression program.
EBNA-LP can bind and inactivate SP100, a member of the PML-NBs that has
been implicated in transcription repression (Echendu and Ling 2008; Ling et al.
2005). EBNA-LPtargets a different component of the PML-NBs than does BNRF1
and at a time when BNRF1 may be degraded. This suggests that temporal remod-
eling of PML-NBs by EBV is necessary for coordinated chromatin assembly and
latency transcription during the pre-latency phase.
EBNA2 recruits several transcriptional co-activators and chromatin regu-

latory factors to the viral episome. Biochemical studies reveal that EBNA2 can
interact with SNF2 family of ATP-dependent chromatin remodeling factors (Wu
et al. 1996), p300/CBP family of histone acetylases (Wang et al. 2000), and RNA
pol II initiation factor TFIIH (Chabot et al. 2014). EBNA2 does not bind to DNA
directly, but associates with several sequence-specific transcription factors, most
notably RBP-jK(also referred to as CBF1and CSL) (Ling et al. 1993). RBP-jKis
the scaffold for intracellular Notch binding, which leads to activation of specific
class of genes important for growth and differentiation in response to extracellu-
lar signaling through Delta (reviewed in Hayward 2004). EBNA2 can also interact
with other transcription factors, including Pu.1, which is implicated in the regula-
tion of LMP1 transcription (Sjoblom et al. 1995a, b). More recent genome-wide
studies indicate that EBNA2 colocalizes with many B-cell-specific transcription
factors throughout the host chromosome, including the BATF/IRF4or SPI1/IRF4
(Jiang et al. 2014; Zhao et al. 2011). Whetherthese sites are also found on the viral
chromosome are not yet known.
Once EBNA2 is expressed, it triggers a promoter switch to the upstream start
site referred to as Cp (Woisetschlaeger et al. 1991). EBNA2-driven Cp is compe-
tent for generating a much longer (~100 kb) multicistronic transcript that encodes
EBNA3A, 3B, and 3C, as well as EBNA1. Enhancing RNA pol II elongation may
be an important component of the transition to type III latency where all EBNA3
genes are generated from this long Cp-initiated transcript. EBNA2 binding at Cp
enhances recruitment of the pTEF-b elongation factor for RNA pol II (Palermo
et al. 2008, 2011). EBNA2 stimulates the CDK9-dependent phosphorylation of
RNA pol II CTD and enhances its ability to overcome negative elongation factor
blocks to transcription elongation. Recent studies in transcription and chromatin
regulation have implicated the control of RNA pol II elongation as a major step
in transcription control throughout the host genome, as well as in the formation of
chromatin organization and higher-order structures (Gilchrist and Adelman 2012).
EBNA1 can be generated from an alternative promoter, referred to as Qp
(Schaefer et al. 1995). Qp can be expressed prior to Cp utilization (Schlager et al.
1996), and Qp is thought to be auto-repressed by high levels of EBNA1 that are
generated through Cp (Yoshioka et al. 2008). Transcription initiation at Qp may
be driven by several different transcription factors, including Initiator element
(INR) (Nonkwelo et al. 1997), interferon regulatory factors IRF1, IRF2 (Schaefer
et al. 1997), and IRF7 (Zhang and Pagano 2000), STATs (Chen et al. 1999), Rb
(Ruf and Sample 1999), histone demethylase LSD1 (Chau et al. 2008), chromatin-
organizing factor CTCF (Salamon et al. 2009; Day et al. 2007), and heat shock
factor 1 (HSF1) (Wang et al. 2011). EBNA1 also binds to the transcription ini-
tiation site of Qp where it can auto-inhibit transcription through a mechanism that
involves binding to its own pre-mRNA (Yoshioka et al. 2008). Thus, Qp regulation
depends on both EBNA1 levels and host-cell factors.
Some latency-associated viral genes can be activated by cellular, as well
as viral transcriptional regulators. LMP1 transcription can be activated by
EBNA2, but EBNA2-independent promoter activity has also been observed in
78 P.M. Lieberman
different latency and tumor types. Factors that regulate LMP1 expression include
Pu.1(Sjoblom et al. 1995a, b), ATF/CRE (Sjoblom et al. 1998), E-box proteins
(Sjoblom-Hallen et al. 1999), NF-kB (Johansson et al. 2009), STAT3/5 (Chen
et al. 2001, 2003), C/EBP (Noda et al. 2011), and CTCF (Chen et al. 2014a).
Similar regulation can be observed for LMP2. LMP1 and LMP2 expression
independent of EBNA2 is important since they are frequently expressed in EBV-
positive epithelial tumors lacking EBNA2 (Tsao et al. 2002). In addition, many
epithelial tumors have high levels of EBV miRNAs and BART transcripts (Pratt
et al. 2009), which initiate from a regions that may not depend upon EBNA2 co-
activation (Kim do and Lee 2012). The temporal regulation of BART and miRNA
transcription is not known with respect to establishment of latency, but it is likely
that these are activated as early transcripts in epithelial infections, which have
been difficult to model efficiently in vitro. Among the BART and miRNA pro-
moter regulatory elements are C/EBPbeta and cMyc (Kim do and Lee 2012; Chen
et al. 2005). It is not known what factors control the different expression levels of
BART and miRNAs in different cell and tumor types.
5 Chromatin Organization of the Latent Episome
The coordination or competition between chromatin assembly and transcription

factor binding on the EBV genome is likely to play an important role in establish-
ing the latent episomal chromosome. The conserved cellular factor CTCF has been
strongly implicated in chromatin organization in all metazoan organisms (Ong and
Corces 2014; Phillips and Corces 2009). CTCF is an 11-zinc-finger DNA binding
protein that can organize both nucleosome position and higher-order DNA loop
interactions including mediating promoter–enhancer interactions. CTCF sites are
often co-occupied by cohesin subunits (SMC1, SMC3, and RAD21), which can
mediate sister chromatid cohesion as well as facilitate DNA–loop interactions
important for gene regulation (Merkenschlager and Odom 2013). In the estab-
lished EBV episome in latently infected B-lymphocytes, CTCF and cohesin bind
at ~19 binding sites throughout the viral genome, with some sites showing higher
co-occupancy (Arvey et al. 2012, 2013) (Fig. 2).
CTCF binding sites can influence nucleosome position and histone modifica-
tion patterns. For EBV, CTCF binding sites upstream of Cp and Qp have been
implicated as chromatin boundaries that prevent the spreading of repressive het-
erochromatin into the promoter control regions (Tempera et al. 2010; Chau et al.
2006). Mutation of CTCF binding sites in EBV bacmids reintroduced into 293
cells showed an increase in heterochromatin formation at Cp and Qp after sev-
eral weeks in culture. CTCF binding sites are CpG-rich, and DNA methylation can
inhibit CTCF binding. At Qp, CTCF has been shown to prevent DNA methylation
(Tempera et al. 2010), and most latently infected cells lack detectable DNA meth-
ylation at Qp (Takacs et al. 2010). Thus, one major function of CTCF is to prevent
Fig. 2 EBV latent transcript promoters aligned with CTCF-cohesin chromatin organization.
Schematic of the EBV genome depicting the position of transcriptionstart sites for the major
latency transcripts, including the RNA pol III-dependent EBERS, and the RPMS1/BARTp pro-
moter that expresses BART miRNAs. Lytic immediate early genesBZLF1(Z) and BRLF1 (R)are
indicatedby green arrows. Below shows the ChIP-Seq tracks for EBNA1, CTCF, and Cohesin in
EBV-infected lymphoblastoid cell lines
DNA methylation at critical promoters, such as Qp. In this capacity, CTCF func-
tions as an insulator.
Both CTCF and cohesins were found to be highly enriched at the LMP1/LMP2
locus, binding within the first intron of LMP2A and the 3’ UTR of LMP1. LMP1
and LMP2 are convergently transcribed, and CTCF may function to coordinate
some of RNA pol II activity to avoid head-to-head collisions. Mutations of the
CTCF binding site at the LMP1/LMP2 locus led to a loss of LMP1 and LMP2A
expression, and a surprising increase in LMP2B expression initiating from down-
stream promoters near the terminal repeats (Chen et al. 2014a). Cohesin deple-
tion did not have the same effect as CTCF binding site mutations, suggesting that
these factors have different fundamental activities in gene regulation. Interestingly,
shRNA depletion of cohesins leads to the reactivation of KSHV, but not EBV in
latently infected B-lymphoma cell lines (Li et al. 2014; Chen et al. 2012). This
suggests that cohesins have a different regulatory function for EBV and KSHV,
or that their function is cell-type and latency-type dependent. The function of
CTCF and cohesin at the LMP1/2 promoter appears to be more complex than
chromatin boundary function, and DNA looping may mediate more complex 3D
regulatory interactions. DNA looping has been assayed using chromosome con-
formation capture (3C) for the LMP1/LMP2 region, and an interaction with OriP
was identified (Chen et al. 2014a; Arvey et al. 2012). DNA loop interactions were
80 P.M. Lieberman
also found between OriP and Cp or Qp, depending on latency type and correlating
with promoter activity (Tempera et al. 2011). These findings suggest that higher-
order chromosome conformation is important for regulating viral gene expression
during latency. In this capacity, CTCF functions to facilitate DNA loops and hubs
between regulatory elements.
6 OriP as a Chromosome Organizing Element
The unusual chromosome structure of OriP was recognized in early studies exam-
ining EBV nucleosome patterns and DNAse sensitivity (Sexton and Pagano 1989;
Dyson and Farrell 1985). More recent 3C chromatin conformation capture stud-
ies suggest that Orip may serve as a central hub in mediating multiple interactions
with the viral genome (Tempera and Lieberman 2010, 2014). OriP has been shown
to function as a transcriptional enhancer of Cp and LMP1 promoters (Gahn and
Sugden 1995; Nilsson et al. 2001; Puglielli et al. 1996), and 3C data provide evi-
dence for a physical interaction between OriP and these promoters when transcrip-
tionally active. The chromatin structure around OriP has been examined by MNase I
digestion and by genome-wide ChIP-Seq studies (Arvey et al. 2012, 2013) (Fig. 3).
First, EBNA1 binding to FR can prevent nucleosome binding in vitro, and the FR
region is either nucleosome-free or has irregular unphased nucleosomes. Purified
Fig. 3 Organization of the latency control region around the OriP enhancer. The EBNA1 bind-
ing sites (purple boxes) at the family of repeats (FR) and Dyad Symmetry (DS) that constitute
OriP are depicted as the central regulator of EBV transcription and chromatin during latency.
OriP functions as a transcriptional enhancer of the promoters at LMP1 and Cp (controlling
EBNA multicistronic transcript). CTCF-cohesinbinding sites are also indicated as they occupy
sites upstream or close to promoter elements at Cp, EBERs, and LMP1/2. The DS is shown to
bind telomere repeat factors (TRFs) and ORC and have positioned nucleosomes enriched in
H3K4me3. PAX5 is shown to be enriched at the terminal repeats. Binding sites for B-cell tran-
scription factors RBP-jKand Pu.1that dock EBNA2 at Cp or LMP1 are indicated
EBNA1 can efficiently assemble onto chromatinized templates and destabilize his-
tones bound to OriP (Avolio-Hunter and Frappier 2003; Avolio-Hunter et al. 2001).
Genome-wide ChIP-Seq studies indicate that the region surrounding OriP is
enriched in euchromatic marks for H3K4me3 (Arvey et al. 2013). Some of the
H3K4me3 may be due to the very high levels of RNA pol III transcription at the
EBERs which are immediately adjacent to OriP. However, early genetic studies
have suggested that EBER deletion has no effect on the ability of EBV to immor-
talize B-lymphocytes or establish latent infection (Swaminathan et al. 1991).
Other factors are known to bind within, or near OriP, and these may also influence
chromatin-organizing functions and related OriP activities. These include cMyc
binding to the EBER promoter (Niller et al. 2003), and Oct2 and E2F factors inter-
acting with FR elements (Borestrom et al. 2012; Almqvist et al. 2005).
The DS element of OriP can function as an efficient origin of DNA replication,
but does not appear to be essential for virus genome replication during latency
(Ott et al. 2011; Norio and Schildkraut 2004; Norio et al. 2000). DNA replication
can initiate at several other locations in the genome, including Rep* and a region
within the BamHI A locus (Kirchmaier and Sugden 1998). This is very similar to
how host chromosome DNA replication initiates in loosely defined zones associ-
ated with euchromatic histone modifications. Nevertheless, the DS has complex
chromatin regulation that suggests it contributes to the overall success of EBV
latency in some cell types. In addition to recruiting ORC and MCMs (Ritzi et al.
2003; Chaudhuri et al. 2001; Dhar et al. 2001), essential for replication origin
function, the DS binds to telomere repeat factors TRF1 and TRF2, which enhance
DNA replication and episome maintenance (Deng et al. 2002; Lindner et al. 2008).
The telomere repeat factor binding sites flank EBNA1 sites within OriP and con-
tribute to nucleosome position and histone modification (Zhou et al. 2005). ORC
may also contribute to chromatin regulation, as it has recently been shown to
interact with specific histone modifications (e.g., H4K20me2) through the BAH
domain of ORC1 (Kuo et al. 2012).
Like most cellular enhancers, OriP associates with many protein factors, and
it is not clear which of these are essential for transcription activation function.
However, unlike cellular enhancers, OriP is unique in its repetitive DNA structure,
aberrant nucleosome structure, and ability to tether to metaphase chromosomes.
Although no specific tethering sites have been identified, OriP generally asso-
ciates with active regions of the nucleus (Deutsch et al. 2010). IF FISH studies
reveals that viral genomes associate with chromatin regions enriched in H3K4me3
and H3K9ac. Loss of DS showed a slight increase in association of EBV genomes
with H3K27me3 domains. The FR region was more critical for this association,
than DS. These studies suggest that OriP can determine what type of chromatin
domain EBV episomes associate with during interphase, and possibly tether to
during metaphase chromosome formation and mitotic division (Fig. 3).
82 P.M. Lieberman
7 EBNA1 as a Critical Regulator of EBV Chromatin
EBNA1 has essential functions in viral transcription, DNA replication, episome

maintenance, and host-cell survival (reviewed in Frappier 2012; Smith and Sugden
2013). EBNA1 binds with high affinity and selectivity to three major positions in
the EBV genome: FR, DS, and Qp (Rawlins et al. 1985). EBNA1 can also bind to
sequence-specific sites in the host genome (Lu et al. 2010; Dresang et al. 2009).
Sequence-specific DNA binding is mediated by the C-terminal DNA binding
domain, which shares structural homology to KSHV LANA and HPV E2 DNA
binding domains (Bochkarev et al. 1995). EBNA1 can also tether to metaphase
chromosomes through two amino-terminal chromosome binding domains termed
CBD1 (aa40-54) and CBD2 (328-377) (Kanda et al. 2013). The arginine-rich
(RGG-like) CBDs can interact with numerous chromatin-associated substrates
including AT-rich DNA, G-quadruplex RNA, histone H1, and EBP2 (Norseen
et al. 2008; Sears et al. 2004; Hung et al. 2001; Shire et al. 1999). EBNA1 CBDs
can also interact with nucleosome core particles through electrostatic interactions
(Kanda et al. 2013), similar to that observed for KSHV LANA (Barbera et al.
2006).
Among the many functions of EBNA1 is its ability to induce higher-order chro-
matin structures. Recent studies suggest that EBNA1 can condense host chromo-
some by mimicking the architectural protein HMG1A (Coppotelli et al. 2013).
EBNA1 can also form homo-typic oligomeric interactions through a Zn-hook
formed by conserved cysteine and histidine residues in the amino-terminal domain
(Singh et al. 2009; Sears et al. 2004). This interaction is likely to be critical for
OriP transcriptional enhancer function as mutations in these amino acid resi-
dues caused the loss of EBNA1-dependent transcription activation of Cp-driven
EBNA2 during primary B-cell infection (Altmann et al. 2006). How these differ-
ent chromatin interacting and chromosome organizing activities contribute to each
of EBNA1’s functions in viral latency remain to be delineated.
Proteomic studies reveal that EBNA1 interacts with several chromatin modu-
latory proteins, including NAP1, TAF1b, USP7, PRMT5, nucleophosmin (Malik-
Soni and Frappier 2012, 2014), and nucleolin (Chen et al. 2014b). Nucleolin
affects EBNA1 DNA binding and transcription activation function, and this is
dependent on RNA-binding domain of both nucleolin and EBNA1. USP7 and
GMP synthase (GMPS) can be recruited by EBNA1 to OriP as a complex that
regulates histone H2B deubiquitination (Sarkari et al. 2009). USP7-dependent
deubiquitination of H2B correlates with a loss of EBNA1-dependent transcription
activation (Sarkari et al. 2009). Thus, USP7 and GMPS may downregulate OriP
enhancer function at some stages of EBV latency.
EBNA1 is an essential chromatin regulatory protein that has multiple func-
tions on and off the viral genome. During latency, EBNA1 binds to several cellular
genes (Lu et al. 2014; Smith and Sugden 2013) and colocalizes with regions of the
interphase chromatin that is newly replicated (Ito et al. 2002). During lytic replica-
tion, EBNA1 localizes to and degrades PML nuclear bodies, and this is facilitated
by EBNA1 interaction partners CK2 and USP7 (Sarkari et al. 2011). Thus, there
remain many unanswered questions as to how EBNA1 and associated cellular fac-
tors mediate the essential functions of OriP, including enhancer–promoter interac-
tions and chromosome tethering during latency. Notably, most of these EBNA1
functions are chromatin-associated processes.
8 Barriers to Lytic Reactivation
To maintain latency, lytic gene expression must be repressed. Leaky lytic gene
expression, especially of the immediate early gene BZLF1 (also known as EB-1,
Zta, Z, ZEBRA), would enable progression into the lytic cycle. BZLF1 expres-
sion and function is regulated at multiple levels and reviewed elsewhere (reviewed
in Murata and Tsurumi 2013; Miller et al. 2007; Kenney and Mertz 2014). The
chromatin regulation of the BZLF1 promoter is closely linked to transcrip-
tion factor binding and signal transduction pathways involved in reactivation.
Transcription of the BZLF1 gene can be activated by various signaling pathways
depending on the latently infected cell type. Among the more commonly studied
lytic-inducing agents are phorbol esters (TPA), halogenated nucleotides, calcium
ionophores, sodium butyrate, and anti-IgG activation of the B-cell receptor in
latently infected B-lymphocytes (reviewed in Speck et al. 1997; Amon and Farrell
2005; Tsurumi et al. 2005). Numerous transcription factors can bind BZLF1 pro-
moter and respond to these signaling pathways. Transcription activators that bind
BZLF1 promoter include MEF2 (Liu et al. 1997b; Murata et al. 2013) (Gruffat
et al. 2002), Sp1/3 (Liu et al. 1997a), CREB, ATF, AP1, C/EBP (Wu et al. 2004),
XBP-1 (Bhende et al. 2007), JDP2 (Murata et al. 2011), YY1 (Montalvo et al.
1995), SMAD (Iempridee et al. 2011), and ZEB1/2 (Ellis et al. 2010; Kraus et al.
2003). Other transcription factors, such as STAT3 (Hill et al. 2013) and Ikaros
(Iempridee et al. 2014), have been shown to regulate EBV reactivation indirectly
(Fig. 4).
In the absence of positive activation signals, several of these factors can repress
BZLF1 transcription. MEF2 recruitment of class II HDAC to EBV BZLF1 links
latency and histone modification (Gruffat et al. 2002). Depletion of ZEB1/2 or
mutation of ZEB binding sites in BZLF1 promoter induces BZLF1 transcrip-
tion and EBV reactivation (Ellis et al. 2010; Kraus et al. 2003). ZEB1/2 represses
transcription through interactions with corepressor CtBP and HDACs (Postigo
and Dean 1999). CTCF, which can function as a transcriptional repressor, was
also found to bind the BZLF1 promoter (Holdorf et al. 2011; Arvey et al. 2012).
However, a mutation in this CTCF site did not effect BZLF1 expression levels, at
least in 293 HEK cells (Murata and Tsurumi 2013).
A search for epigenetic marks that control EBV latency has been initiated on
several fronts. DNA methylation plays an important role in the epigenetic regula-
tion of EBV latency, and this is reviewed extensively elsewhere (Woellmer and
Hammerschmidt 2013; Takacs et al. 2010; Miller et al. 2007; Ambinder et al. 1999).
84 P.M. Lieberman
Fig. 4 Lytic control region chromatin regulatory factors. Schematic of the BZLF1 promoter reg-
ulatory region and some of the factors that have been implicated in its regulation. These include
the direct DNA binding factors SP1/3, MEF2D, AP1, ATF, CREB, XBP1, and Zif268 that have
been implicated in transcription activation (green boxes) and the factors implicated in repressing
Zp (YY1, ZEB, JDP2). BZLF1-encoded protein (also referred to as Z, Zta, ZEBRA, EB1) is an
important auto-activator of Zp and Rp. Rp-encoded transcription activator Rta (or R) is a potent
activator of Zp, and other viral lytic genes, especially in epithelial cell latency. ChIP-Seq tracks
for histone H3K4me3 show an absence of euchromatic at Zp in LCLs, with enrichment of CTCF
and Rad21 immediately upstream of Zp regulatory factors
DNA methylation levels at the BZLF1 promoter were shown to be relatively low
compared to most regions of the latent genome (Hernando et al. 2013). However,
treatment of some latently infected cell types with demethylating agents such as 5’
azacytidine can induce BZLF1 transcription and reactivation (Murata et al. 2012).
It is possible that demethylating agents may also induce DNA damage and DDR
which may function as more effective triggers of EBV reactivation.
Histone modifications have been implicated in the control of BZLF1 gene
expression and EBV lytic reactivation. HDAC inhibitors are known to stimulate
reactivation in many, but not all, latently infected cells (Luka et al. 1979; Jenkins
et al. 2000). EBV chromatin histone acetylation levels are generally low during
latency and increased uniformly during reactivation, including the region encom-
passing the BZLF1 promoter (Murata et al. 2012). ChIP assays have revealed that
H3K27me3, H3K9me2/3, and H4K20me3 are enriched at the BZLF1 promoter in
latently infected Raji cells (Murata et al. 2012), and H3K27me3 and H3K9me3 in
Akata cells (Ramasubramanyan et al. 2012). In Raji cells, HP1 binding and H2A
ubiquitination were associated with BZLF1 in latency, while histone acetylation
and H3K4me3 dominate during reactivation (Murata et al. 2012). Interestingly,
while treatment of Raji cells with DZNep (inhibitor of the EZH2 H3K27me3
methylase) or TSA alone had only a modest effect on EBV reactivation, the com-
bination of these two drugs led to a synergistic activation of BZLF1 transcription
and viral reactivation. This suggests that both derepression of H3K27me3 and
activation through histone acetylation (HDAC inhibition) are required for effi-
cient activation of BZLF1 promoter in Raji cells. H3K27me3-mediated repres-
sion has also been implicated in KSHV latency, as inhibitors of EZH2, such as
DZNep, are sufficient to induce reactivation as single agents (Toth et al. 2010,
2013a). However, these effects may be cell-type dependent. DZNep can also
inhibit the methylation of H3K20me3 through SUV420h1, and knockdown of this
gene could also lead to induction of BZLF1 transcription (Murata et al. 2012).
H3K9me3 was also found to be elevated at the BZLF1 promoter, but inhibitors of
G9a (BIX01294) and chaetocin (inhibitor of SUV39a) did not induce BZLF1 tran-
scription. However, it was not clearly demonstrated that BIX01294 or chaetocin
efficiently removed H3K9me3 from BZLF1 promoter. Histones at the BZLF1
promoter were also found to be enriched in histone H2A monoubiquitination on
lysine 119 as well as enriched for HP1 (Murata et al. 2012). These findings sug-
gest that multiple different forms of chromatin repression contribute to Zp repres-
sion during latency.
The DNA methylation state of the virus has also been investigated in great
detail, and while this plays an integral role in the epigenetic control of gene
expression and is deeply coordinated with chromatin structure and function, much
of this subject is the focus of other chapters in this volume and reviewed elsewhere
(Woellmer and Hammerschmidt 2013; Takacs et al. 2010; Ambinder et al. 1999).
9 Late Gene Transcription and Replication
The EBV lyticgenome evades histone assembly during lytic cycle replication
(Chiu et al. 2013). During lytic replication, the nuclear morphology is reorgan-
ized, and replication compartments containing viral replication proteins, as well as
cellular proteins, such as PCNA, form higher-order structures. Cellular DNA and
histones are excluded from these compartments, and cellular PCNA does not colo-
calize with replicating viral DNA. Since PCNA is implicated in histone chaperone
recruitment and histone assembly during cellular DNA replication, the segregation
of PCNA away from viral replicating DNA may partly account for the escape from
histone assembly. This study also found that EBV lytic cycle leads to the selective
loss of cellular histone chaperones CAF1 and ASF1a at the protein and mRNA
levels, providing an additional mechanism for the reduction of histone assembly
on viral DNA during lytic amplification (Chiu et al. 2013).
86 P.M. Lieberman
EBV, like all herpesviruses, requires lytic cycle DNA replication to enable tran-
scription of viral late genes. In gammaherpesviruses, late genes have an unusual
TATT sequence present in the promoter regions where the TATA box is typically
found (Serio et al. 1998; Wong-Ho et al. 2014). Recent studies have identified a
herpes-encoded TATA-box protein (TBP)-related factor that can bind directly
to the TATT element and is required for late gene transcription (Wyrwicz and
Rychlewski 2007; Gruffat et al. 2012). The viral TBP is found among gamma and
beta-herpesviruses, but is not found in the alpha-herpesviruses. This has several
implications, including potential differences among herpesvirus family mem-
bers in the regulation of late gene transcription. For EBV, late gene transcription
appears to require a specialized viral-encoded transcription initiation complex that
may function in the absence of normal cellular histone assembly, as suggested by
the live cell imaging of histone-free replication compartments.
10 Network and Homeostatic Control of Viral Chromatin
Several signaling pathways are known to control EBV gene expression, and these
are almost always linked to changes in chromatin structure. Inflammatory and
DNA damage response pathways have particularly important roles in regulating
EBV latency, and recent studies have revealed how these pathways intersect at the
level of viral chromatin (Fig. 5).
STAT-associatedinflammatory pathways have been implicated in several differ-
ent control nodes of EBV latency. STAT1/3/5 binding sites have been identified in
the viral genome, and best characterized at the Qp and at sites upstream of LMP1,
regulating standard ED-L1 promoter, and the alternative promoter, L1-TR, in the
terminal repeats (Chen et al. 1999, 2001). LMP1 can stimulate STAT3 activa-
tion, suggesting that it provides a positive feedback loop (Chen et al. 2003; Kung
et al. 2011; Buettner et al. 2006). STAT3 was identified as a cellular factor that
prevents lytic reactivation when upregulated in BL and LCL cells (Daigle et al.
2010; Hill et al. 2013). Several cellular genes activated by STAT3 were shown
to repress EBV lytic gene transcription, including several Zn-finger repressors
(ZNF253, ZNF257, SNF589) and SETDB1. STAT3 is also shown to be impor-
tant for EBV-induced B-cell immortalization, as hypomorphic mutations lead to
a reduction of in vitro transformation (Koganti et al. 2014a). STAT3 was found to
disrupt the DDR signaling by interfering with ATR activation of Chk1 (Koganti
et al. 2014b). In these studies, activation of STAT3 is found to be an early event
prior to viral gene expression, and STAT3 modifies DDR response through activa-
tion of Caspase 7 and cleavage of DDR component Claspin.
CpGactivation of TLR9 induces a G1/S cell cycle arrest that restricts EBV-
induced B-cell proliferation. TLR9 signaling in BL cells was found to sup-
press BZLF1 transcription through histone modification (Zauner et al. 2010;
Ladell et al. 2007). This response was mediated partly by IL12 and INFgamma.
EBV latent genomes can also induce chronic stimulation of the IFI-16-mediated
Fig. 5 Network interactions that impact the chromatin control of EBV latent/lytic switch. The
interaction network regulating the decision to establish latent chromosome formation rather than
lytic productive infection is depicted schematically and highlighting only a few of the many
known regulatory interactions. Regulation of latency through DDR and ATM signaling, as well
as inflammatory pathways mediated by STAT3 are likely to play central roles in the regulation of
EBV latent to lytic switch at the level of chromatin control
inflammasome (Ansari et al. 2013), but it is not yet known whether this inflam-
matory signal restricts lytic reactivation or otherwise influences latent gene
expression.
The DNA damage response (DDR)appears to play a major role in regulat-
ing EBV latency and reactivation. As DNA damage signaling often results in the
modification of chromatin, it is likely that this pathway modifies the EBV chro-
mosome and chromatin. Consistent with this, DNA damaging agents, especially
ATM and ATR agonists, were found to be potent stimulators of EBV lytic reacti-
vation (Feng et al. 2004; Hagemeier et al. 2012). Latency-associated EBNA3C can
alter ATM/ATR signaling by interacting with downstream target Chk2 and prevent
a G2/M cell cycle block (Choudhuri et al. 2007). By inhibiting ATM signaling,
EBNA3C may function to block lytic cycle reactivation.
P53 plays a central role in DDR-dependent transcription. P53 has been shown
to contribute to EBV reactivation induced by HDAC inhibitors in NPC cells
(Chang et al. 2008). Post-translational modification of p53 at serine 46 and 392
was found to be important for this function. In contrast, p53 family member
ΔNp63 is reported to inhibit EBV reactivation in epithelial cells (Kenney and
Mertz 2014). Perhaps, HDAC inhibitors lead to a DNA damage response that is
p53 dependent, and this has an important regulatory role in controlling EBV reac-
tivation. P53 may support EBV reactivation through direct interactions with Sp1
protein bound to Zp, as well as increase in EGR1 (Kenney and Mertz 2014).
88 P.M. Lieberman
ATM kinase activity also plays a central role in regulating EBV reactivation. In
addition to p53, ATM targets KAP1, TIP60, or H2AX may also effect EBV chro-
matin to toggle the latent to lytic switch (Kenney and Mertz 2014; Hagemeier et al.
2012; Li and Hayward 2011). The EBV protein kinase BGLF4 can mimic aspects
of ATM kinase, as it can phosphorylate substrates such as H2AX and p27 that can
prolong the pseudo S-phase necessary for viral DNA replication (Iwahori et al.
2009). BGLF4 can also phosphorylate topoisomerase II and condensins, which
effect chromatin structure and chromosome organization (Chang et al. 2012; Lee
et al. 2007, 2008). Like BGLF4, MHV68 ORF27 kinase activates the DDR through
H2AX phosphorylation and promotes viral replication (Tarakanova et al. 2007).
Interestingly, H2AX knockdown reduces the number of latently infected cells in
mouse studies (Tarakanova et al. 2010), suggesting that lytic replication is required
for MHV68 to achieve latent infection in vivo. BGLF4 kinase is thought to func-
tion at early stages of the lytic replication cycle and is important for remodeling
chromatin and nuclear morphology, as well as arresting the cell cycle in a pseudo
S-phase (Chang et al. 2012). The many targets of BGLF4 reflect host-cell factors
that play important roles in maintaining viral chromatin during latency.
Homeostatic mechanisms also control viral latency by acting on chroma-
tin (Fig. 5). LMP2a suppresses lytic reactivation by blocking signaling through
surface IgG cross-linking and histone acetylation of BZLF1 (Miller et al. 1994,
1995). LMP1 induces cellular miR155, which in turn suppresses lytic reactiva-
tion by targeting RBL1 to derepress DNA methyltransferase 1 (DNMT1) (Lu et al.
2008). The increase in expression DNMT1 leads to de novo DNA methylation and
stable epigenetic repression of the latent viral episome.
11 Chromatin Modifiers that Disrupt Latency—

Environment, Pathogenesis, and Therapeutics
Many environmental factors alter EBV chromatin structure. Periodontal pathogen

Porphyromonas gingivalis induces EBV lytic switch by histone modification of Zp
(Imai et al. 2012). A major metabolite of P. gingivalis (and other bacteria) is small
chain fatty acids (SCFAs), such as butyrates, that are potent inhibitors of class
II HDACs. However, not all SCFAs stimulate EBV reactivation, and particular
SCFAs have cell-type and viral-specific effects (Gorres et al. 2014). Interestingly,
several SCFAs that are inhibitors of HDACs failed to reactivate EBV, while these
were capable of activating KSHV. Similarly, some medium chain fatty acids
(MCFAs), such as valproic acid (VPA) and phenylbutyrate, are capable of block-
ing EBV reactivation in B-cell lymphoma (BL) cell lines. Like VPA, the HDAC
inhibitor sulforaphan can also inhibit EBV reactivation in NPC cells (Wu et al.
2013). This is consistent with related studies showing that lipophilic small mol-
ecules, including resveratrol (De Leo et al. 2012; Yiu et al. 2010) and moronic acid
(Chang et al. 2010), could block EBV reactivation. Together, these studies suggest
that a specific receptor or membrane perturbation may be playing a role in regulat-
ing EBV chromatin and reactivation.
Potential pathogen-associated lytic activators have been isolated from malaria

membrane proteins (Chene et al. 2007) and the lignins in salted fish (Bouvier
et al. 1995). These reactivators may contribute to the carcinogenesis of Burkitt’s
lymphoma and NPC, respectively. On the other hand, identification of safe and
effective reactivators of EBV may be of clinical value for lytic therapy to treat
EBV-positive tumors (Kenney 2006).
12 Variation and Heterogeneity of Viral Chromatin

Control
EBV and KSHV share many common properties, including the formation of chro-
matinized episomes during latent infection. Nevertheless, there are many differ-
ences in the mechanisms of regulation for these two gammaherpesviruses during
latency. Reactivation of EBV in response to HDACsand protein kinase C ago-
nists requires new protein synthesis, but this is not the case for KSHV (Ye et al.
2007). KSHV latency is regulated by polycomb-mediated H3K27me3, while EBV
latency is less dependent on this type of chromatin repression (Toth et al. 2010,
2013b). This was shown in co-infected cells treated with DZNep, inhibitor of
EZH2, demonstrating that only KSHV, but not EBV, lytic cycle was reactivated.
Similarly, KSHV latency can be disrupted by cohesin depletion, while cohesin
depletion leads to a change in EBV latency gene expression, but not lytic reactiva-
tion (Li et al. 2014; Chen et al. 2014a).
Many factors may influence the chromatin structure and epigenetic regulation
of EBV latency. Heterogeneity may exist among viral genomes in a single cell,
among cells in a population, and between different cell and tissue types. Different
BL-derived cell lines have different chromatin regulation, as Akata have reduced
histone acetylation, while Raji have elevated H3K27me3 and H4K20me3. Also,
lytic activators such as TPA, A23187, and sodium butyrate stimulate BZLF1
transcription in Raji, but do not alter the repressive marks for H3K27me3 and
H4K20me3. Genome copy number and heterogeneity may play a factor in the
complex behavior and response to reactivation (only a subset of genomes is
responsive). Additional histone remodeling factors or acetylation of non-histone
proteins may help to explain the refractory population of latent EBV (Murata et al.
2012; Ramasubramanyan et al. 2012).
13 Conclusions
The predominant structure of EBV genomes during latency is the chromatinized

episome. However, it is also true that some EBV-positive tumors have integrated
EBV (e.g., Namalwa), and many cells have both episomes, and integrated sub-
genomic fragments of EBV (Wuu et al. 1996; Lestou et al. 1996; Kripalani-Joshi
and Law 1994; Srinivas et al. 1998). The more recent discoveries of EBV in
90 P.M. Lieberman
epithelial cancers, such as nasopharyngeal carcinoma and gastric carcinoma, have

been more problematic to conclude definitively that EBV is exclusively episomal
since these tumors have been very difficult to expand in tissue culture without the
loss of EBV genomic DNA. However, deep sequencing of EBV-positive tumors
fails to find evidence of chromosomally integrated viral genomes (Khoury et al.
2013). Furthermore, the frequent loss of EBV genomes from cultured NPC cells
suggests that these genomes are episomal. EBV episome maintenance in epithe-
lial tumors may involve factors associated with the tumor microenvironment that
is not recapitulated in tissue culture. Chang and Moore have proposed that tumor
viruses transform cells only when their normal life-cycle pathways are disrupted
(Moore and Chang 2010). It is interesting to consider this with EBV, which may
establish aberrant latent infections in epithelial cells or rearrange in lymphoid cells
to drive host-cell carcinogenesis.
Much remains to be discovered with respect to the chromatin structure (s) of
latent EBV genomes in normal and pathogenic infection. While EBV may have
chromatin structure “indistinguishable” from host-cell chromosome, it is becom-
ing apparent that host chromosomes have very complex, dynamic, and heteroge-
neous chromatin structures that are only beginning to be understood at the level
of histone modification code and higher-order chromatin structures. Detailed char-
acterizations of viral-specific events will be necessary to determine whether EBV
has chromatin structures distinct from the host chromosome. As EBV has unique
chromosome dynamics, especially the establishment and maintenance of a chro-
matinized episome, it seems likely that it may shed new light and insight into the
broader field of chromatin biology. It is also likely that EBV’s unique chromatin
structure and regulation will be an opportunity for therapeutic intervention.
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The Epigenetic Life Cycle of Epstein–Barr
Virus
Wolfgang Hammerschmidt
Abstract Ever since the discovery of Epstein–Barr virus (EBV) more than
50 years ago, this virus has been studied for its capacity to readily establish a
latent infection, which is the prominent hallmark of this member of the herpes-
virus family. EBV has become an important model for many aspects of herpes-
viral latency, but the molecular steps and mechanisms that lead to and promote
viral latency have only emerged recently. It now appears that the virus exploits
diverse facets of epigenetic gene regulation in the cellular host to establish a latent
infection. Most viral genes are transcriptionally repressed, and viral chromatin is
densely compacted during EBV’s latent phase, but latent infection is not a dead
end. In order to escape from this phase, epigenetic silencing must be reverted effi-
ciently and quickly. It appears that EBV has perfected a clever strategy to over-
come transcriptional repression of its many lytic genes to initiate virus de novo
synthesis within a few hours after induction of its lytic cycle. This review tries
to summarize the known molecular mechanisms, the current models, concepts,
and ideas underlying this viral strategy. This review also attempts to identify and
address gaps in our current understanding of EBV’s epigenetic mechanisms within
the infected cellular host.
Contents
1 Introduction........................................................................................................................... 104
2 The Pre-latent Phase............................................................................................................. 105
3 The Latent Phase................................................................................................................... 108
4 Lytic Induction and Virus Synthesis..................................................................................... 109
5 Open Questions..................................................................................................................... 112
References................................................................................................................................... 114
W. Hammerschmidt (*)
Research Unit Gene Vectors, Helmholtz Zentrum München, German Research Center
for Environmental Health, and German Centre for Infection Research (DZIF),
Partner site Munich, Marchioninistr. 25, 81377 Munich, Germany
e-mail: hammerschmidt@helmholtz-muenchen.de

104 W. Hammerschmidt
1 Introduction
The infection of resting primary human B lymphocytes is a versatile and tracta-

ble model to study EBV. As a consequence of infection with EBV, lymphoblas-
toid cells lines (LCLs) emerge that can be easily obtained from any donor and
cultivated in bulk or as single cell clones in vitro. It is said that EBV immortal-
izes or growth transforms B cells, which become latently infected and prolifer-
ate for months. Infection of human B lymphocytes has been most informative to
study the early steps of viral infection that lead to latent infection. EBV not only
establishes a latent infection in LCLs but also in all EBV-infected tumors. From
biopsies, certain tumor cell lines can be obtained and cultivated as permanent cell
lines in vitro. For example, a large number of Burkitt’s lymphoma cell lines are
available that maintain their EBV-positive state in cell culture and remain latently
infected. Together with LCLs, they are a rich source to study EBV latency and its
epigenetic regulation.
Established Burkitt’s lymphoma cell lines have been the preferred model to
study the induction of EBV’s lytic phase. Incubation with TPA and butyrate or
cross-linking of the surface immunoglobulin of certain Burkitt’s lymphoma cell
lines readily induces the lytic phase of EBV and leads to virus de novo synthe-
sis and egress of infectious virus. These measures induce the expression of a viral
transcription factor termed BZLF1 (also called Z, Zta, ZEBRA, or EB1), which
has been identified as the key molecular switch gene and inducer of EBV’s lytic
phase (Takada et al. 1986; Countryman and Miller 1985). Expression of BZLF1
can revert the latent phase and induce the expression of all viral lytic genes, which
are epigenetically silenced during EBV’s latent phase.
EBV not only infects human B cells, but also other cell types. Two human
carcinomas, endemic nasopharyngeal carcinomas and a small fraction of gastric
carcinomas, are characterized by latently EBV-infected tumor cells (see also the
chapter by Nancy Raab-Traub in this book). EBV has also been found occasion-
ally in normal, non-transformed epithelial cells in the oropharynx (Walling et al.
2001) indicating EBV does infect epithelial cells in vivo. Unfortunately, ex vivo
infection of primary human epithelial cells with EBV is very inefficient. The cells
do not synthesize virus progeny upon infection nor is the virus maintained long
term in cell culture.
Latently, EBV-infected cells are characterized by the expression of certain viral
genes. Different sets of viral genes, termed EBNAs (Epstein–Barr nuclear anti-
gens) and LMPs (latent membrane proteins) together with noncoding transcripts
such as viral microRNAs and long noncoding RNAs, the EBERs, are expressed
in latently infected cells. Depending on the type and origin of the cell, different
latency programs classify the extent of latent gene expression. For example, six
EBNA and three LMP genes are expressed along with noncoding RNAs in LCLs.
In Burkitt’s lymphoma cells, only a small subset of the latent genes is commonly
The Epigenetic Life Cycle of Epstein–Barr Virus 105
expressed. In all instances, the viral genes support the extrachromosomal mainte-
nance of several copies of the EBV genome, which is about 160 kbps in size. In
addition, the latent gene products induce and support proliferation of the latently
infected cells and contribute to their transformed phenotype.
All herpesviruses deliver their genomic DNA in an epigenetically naïve form.
Besides viral DNA, the herpesviral capsids do not appear to contain histones or
other DNA-associated proteins of cellular or viral origin (Johannsen et al. 2004).
The viral DNA is also free of 5′-methyl cytosine residues (Gibson and Roizman
1971; Kintner and Sugden 1981), which typically occur in a CpG dinucleotide
context in mammalian DNA and are associated with epigenetic repression of tran-
scription. EBV DNA has a high overall content of CpG dinucleotides of 5.4 %. In
fact, DNA methylation plays an important role in the latent phase of EBV infec-
tion during which chromatin architecture and the epigenetic landscape also deter-
mine the transcriptional state of genomic EBV DNA.
Recent molecular data indicate that EBV is unusual in that it cannot replicate
itself upon infection. This statement appears surprising given the fact that all other
human herpesviruses readily support their own de novo synthesis in permissive
cells, which release progeny within a short period after infection. Rather, EBV
establishes a latent infection during which the viral genomic DNA acquires all fea-
tures of cellular chromatin. They include extensive CpG DNA methylation, global
epigenetic modifications, and histone marks, which are mostly repressive leading
to transcriptional silencing of all but the virus’ latent genes.
EBV’s lifestyle seems to consist of three phases, which are illustrated in Fig. 1.
Upon infection, the virus attaches to its receptors at the cell surface, internalizes
and delivers its epigenetically naïve linear DNA genome to the nucleus of the cell.
The circularization of the viral DNA is one of the very first events in order to pro-
tect the DNA ends from degradation and minimize induction of the DNA dam-
age response of the infected cell (Hurley and Thorley-Lawson 1988). Within a few
hours post-infection, a number of viral lytic genes are found expressed together
with latent genes. About ten days later, EBV’s latent gene expression prevails and
the expression of lytic genes becomes nearly undetectable. The latent phase is
established. Upon appropriate stimuli, i.e., the activation of the surface immuno-
globulins on memory B cells, a signaling cascade induces the expression of the
viral transcription factor BZLF1, which is the prologue to lytic reactivation and
leads to virus synthesis, eventually.
2 The Pre-latent Phase
Upon infection of primary human B lymphocytes, the virus does not induce its de
novo synthesis (Kalla et al. 2010) but initiates a “pre-latent phase” during which
a subset of lytic genes together with viral latent genes are expressed (reviewed in
establishment lytic reactivation
pre- latent phase latent phase lytic phase
Time course 0-2 weeks weeks to months within 48 hours
Functions of cellular activation EBV genome viral DNA replication and

viral genes and proliferation maintenance structural components
(and cellular
proliferation)
Fig. 1 EBV’s lifestyle encompasses three different phases. The scheme shows a single EBV
virion, which infects a B lymphocyte. The virus delivers its linear, double-stranded DNA to the
nucleus of the cell where the EBV DNA is present in several copies (not shown) of an EBV
minichromosome (red circle). During latency, this state is maintained even in proliferating cells.
Upon antigen contact via the surface immunoglobulin, EBV’s molecular switch gene BZLF1
is activated and induces the lytic, productive phase. The cell releases virus progeny now. The
timing and length of infection during the three different phases of EBV infection are shown
together with the functions of viral genes expressed in the different phases. In latently infected
B cells in vitro, the virus induces their continued proliferation, which depends on the expres-
sion of certain viral latent genes. (As discussed in the review, this model is primarily based on
experiments with primary human B lymphocytes and HEK293 cells, an established cell line with
epithelia-like characteristics. The proposed model is debatable. In the past, the reported expres-
sion of individual lytic viral genes in epithelial cells has been interpreted to mean that they can
support EBV’s full-fledged viral phase immediately upon infection. However, recently published
data and the current state of knowledge as discussed in this review support the three-phase model
shown here.)
Woellmer and Hammerschmidt 2013; Kalla and Hammerschmidt 2012). EBV also
infects primary human epithelial cells and expresses lytic as well latent viral genes
upon infection (Shannon-Lowe et al. 2006 and references therein). It is likely that
epithelial cells also fail to support the ready synthesis of viral progeny (Walling
et al. 2001). The reason for it can be found in EBV’s intricate and peculiar epige-
netic lifestyle (see below).
In newly infected cells, viral transcripts are detectable prior to transcription of
the incoming EBV DNA. Cells that support the synthesis of herpesviruses release
exosomes, virus-like particles, and virions, which contain viral transcripts as cargo
(Kalamvoki et al. 2014; Jochum et al. 2012b; Bresnahan and Shenk 2000; Bechtel
et al. 2005; Cliffe et al. 2009; Greijer et al. 2000; Sciortino et al. 2001). The deliv-
ery of viral transcripts to the infected cell allows the immediate expression of viral
factors that can elicit viral immune escape mechanisms and modulate cellular gene
expression (Kalamvoki et al. 2014). As a consequence, EBV transcripts delivered
upon infection activate the resting B lymphocytes and support their latent infection
(Jochum et al. 2012a, b).
De novo transcription of the incoming EBV DNA probably starts shortly after
infection of B lymphocytes. To my knowledge, no systematic study provides a
complete picture of newly transcribed viral genes in these cells. It is known that
as early as six hours post-infection, transcripts that originate from the so-called
W promoter (Wp) encode the latent EBNA-LP and EBNA2 genes, which are
also the first “latent” EBV proteins detectable in newly infected B lymphocytes
(Schlager et al. 1996; Woisetschlaeger et al. 1990; Alfieri et al. 1991). It was sur-
prising to learn that lytic viral genes are also transcribed initially and fulfill impor-
tant functions in the newly infected B lymphocytes. Among these, lytic viral genes
are EBV-encoded members of the anti-apoptotic BCL-2 family (Altmann and
Hammerschmidt 2005), the molecular switch gene BZLF1 (Wen et al. 2007; Kalla
et al. 2010), viral immunoevasins, and lytic genes with transactivating and regula-
tory functions such as BMRF1 and BRLF1 (Jochum et al. 2012a, b; Kalla et al.
2010). It is important to note certain viral genes essential for lytic amplification
of viral DNA and genes encoding structural viral proteins are not expressed in the
pre-latent phase consistent with the notion that viral progeny is not produced ini-
tially (Kalla et al. 2010).
Viral transcription early after infection appears to be chaotic. It seems that the
epigenetically naïve EBV genomic DNA, which lacks histones and nucleosomes
and is free of methylated CpG dinucleotides, serves as a permissive template for
the cellular transcription machinery. Transcription of viral genes is not universal
but seems to be restricted to viral promoters, which recruit cellular transcription
factors and are transcribed by RNA polymerase II. Among them are viral pro-
moters, which drive the expression of latent genes, e.g., EBNA1, EBNA2, and
EBNA-LP (Shannon-Lowe et al. 2005; Tierney et al. 2007; Schaefer et al. 1995;
Schlager et al. 1996 and references therein), but also lytic, so-called immedi-
ate early genes such as BZLF1 and BRLF1 (Wen et al. 2007; Kalla et al. 2010),
and lytic early genes such as BHRF1, BALF1, BMRF1, BCRF1, and BNLF2a
(Jochum et al. 2012a, b). Currently, the complete catalogue of viral genes tran-
scribed within the first days after infection of primary B lymphocytes is not
known. This promiscuous state of transcription seems to come to an end as
EBV DNA acquires cellular nucleosomes and becomes methylated later during
infection.
During the pre-latent phase, the resting small B lymphocytes enlarge into B cell
blasts and eventually begin to proliferate. About four days post-infection, nucleo-
some occupancy of viral DNA becomes detectable at a select viral locus, the dyad
symmetry element of the plasmid origin of DNA replication, where nucleosome
positioning is accurate and highly ordered (Zhou et al. 2005). Nucleosomal occu-
pancy increases gradually at this locus within the next days and reaches high lev-
els genome-wide about two to three weeks post-infection (Schmeinck 2011).
In contrast, methylation of viral DNA is a slow process in infected primary B
lymphocytes. After two weeks post-infection, CpG methylation becomes first detect-
able and gradually increases for the next two to three months to considerable levels
(Kalla et al. 2010). Few regions in EBV DNA are spared from CpG methylation,
which is extensive in established LCLs (Fernandez et al. 2009; Minarovits 2006;
Paulson and Speck 1999; Tierney et al. 2000; Woellmer et al. 2012) as well as in
latently infected peripheral memory B lymphocytes in vivo (Woellmer et al. 2012).
EBV DNA acquires an epigenetic signature during the pre-latent phase. The
early expression of a plethora of viral genes might result from the epigenetically
“naked” EBV genome. During the pre-latent phase, EBV DNA nucleosomes are
positioned and repressive chromatin marks are presumably introduced, which are
the characteristic for EBV’s chromatin during the latent phase (see below). The
acquisition of cellular chromatin and a repressive epigenetic pattern driven by the
host cell leads to the eventual silencing of all lytic genes but also certain promot-
ers of latent genes. This process of epigenetic shutoff is completed about ten to
14 days post-infection (Fig. 1).
3 The Latent Phase
The programs of latent viral gene expression differ according to the type and state
of differentiation of the latently EBV-infected cell. The expression of lytic genes
is strictly controlled and repressed in this phase presumably to prevent immune
recognition in vivo and block virus synthesis, both of which would jeopardize the
survival of the latently infected cell. Recent data indicate that epigenetic modifica-
tions of viral DNA define viral latency. In other words, latency is encoded in the
state of EBV’s chromatin.
Four types of global epigenetic modification are known that repress EBV’s lytic
viral genes during latency: histone modifications, nucleosomal density and com-
paction of DNA, DNA methylation, and chromatin architecture.
Transcriptionally silenced lytic viral genes and their promoters are associated
with H3K27me3 histone marks, which are repressive modifications introduced
by the histone methyltransferase EZH2, a component of the Polycomb repres-
sive complex 2, PRC2 (Woellmer et al. 2012). This histone modification seems
to be important because trimethylation of H3K27 is erased upon induction of
EBV’s lytic phase (see below). Less frequent is the repressive mark H3K9me3
(Ramasubramanyan et al. 2012), which is not removed upon viral reactivation sug-
gesting that this modification is less important for the maintenance of repressed
viral chromatin during latency. In contrast to cells latently infected with Kaposi’s
sarcoma-associated herpesvirus (KSHV), H3K4me3 histone marks and RNA poly-
merase II are absent in epigenetically repressed EBV’s chromatin during latency.
In this respect, chromatin in latently KSHV-infected cells is bivalent at most early
lytic promoters (Gunther and Grundhoff 2010; Toth et al. 2010; Lieberman 2013).
It thus appears that EBV uses cellular functions of epigenetic repression and pre-
fers static, repressive chromatin marks at transcriptionally repressed lytic genes
during latency.
Repressed promoters of lytic genes are enriched in nucleosomes, and their
high local concentrations argue for compact and condensed viral chromatin dur-
ing latency (Woellmer et al. 2012). In fact, high nucleosome occupancy correlates
with promoter silencing in many different cells latently infected with EBV (Arvey
et al. 2012). Nucleosomal density is particularly high at early lytic promoters of
EBV, which are controlled and transactivated by BZLF1 upon induction of EBV’s
lytic phase (see below) suggesting that nucleosomes provide an obstacle to trans-
activating factors in general and BZLF1 in particular. It is currently elusive but
certain positioned nucleosomes and compacted chromatin might directly contrib-
ute to the epigenetic stability of repressed viral genes ensuring EBV’s latent phase.
DNA methylation is an important epigenetic parameter and is thought to con-
tribute to overall gene silencing. The state of CpG methylation in EBV DNA in
latently infected cells is generally extremely high at repressed early lytic promot-
ers and genes consistent with this notion (Woellmer et al. 2012; Fernandez et al.
2009). In vivo, EBV DNA in memory B cells seems to adopt a similar pattern
(Woellmer et al. 2012; Paulson and Speck 1999). CpG methylation of EBV DNA
is not global but bimodal because promoters of latent genes, which are active, are
spared. Among others, the Fp/Qp and Cp promoters that drive EBNA1 in certain
Burkitt’s lymphoma cell lines, the promoters of the LMP1/LMP2A genes, and
the two EBER genes, are free of methylated CpG dinucleotides similar to cer-
tain DNA sequences that co-localize with nucleosome-free regions and are sus-
pect to mediate higher order chromatin architecture of EBV DNA (Kalla et al.
2010; Woellmer et al. 2012 and unpublished data). It thus appears that CpGs,
which escape methylation, are located at functionally active sites in latently EBV-
infected DNA, while the overwhelming majority of CpGs are almost entirely
methylated and functionally silent. Lack of CpG methylation commonly coincides
with histones bearing active or permissive histone marks indicating that key regu-
latory features of EBV’s chromatin follow textbook rules. Promoter usage seems
to be controlled by DNA methylation during latency.
The state of EBV’s genomic architecture and organization in latently infected
cells is just beginning to emerge. The cellular CCCTC-binding factor (CTCF)
and members of the cohesion complex often co-localize on DNA and promote the
formation of chromatin loops to demarkate and organize chromosomal domains.
These factors seem to be involved in the architecture of genomic EBV DNA in
latently infected cells (Arvey et al. 2012; Holdorf et al. 2011; Tempera et al. 2010,
2011) (see also the chapter by Paul Lieberman in this book). It is currently unclear
whether binding and looping of EBV DNA lead to gene activation or repression
by connecting or separating functionally distinct regulatory elements in latently
infected cells. How EBV’s genome architecture changes upon induction of the
lytic phase of EBV’s lifestyle has not been addressed, yet.
4 Lytic Induction and Virus Synthesis
During de novo virus synthesis, about 70 lytic genes of EBV are expressed that
support viral asynchronous DNA amplification independent of the cellular DNA
replication and encode viral structural components to allow virus morphogenesis
and release of virus progeny. Two genes, BZLF1 and BRLF1, which encode viral
transcription factors, orchestrate the transition from viral latency to productive,
lytic infection. The former acts as a master switch regulator, which can induce the
lytic phase of EBV’s life cycle in latently infected cells (Sinclair 2003), and the
latter is equally indispensable for the expression of all viral lytic genes (Feederle
et al. 2000). Cellular differentiation of latently EBV-infected B cell or epithelial
cells likely drives the expression of both BZLF1 and BRLF1 genes (Reusch et al.
2015).
EBV genuinely repressed chromatin poses the problem of efficient reactivation
to support de novo virus synthesis in latently infected cells. Besides compacted
chromatin with dense nucleosomes and repressive histone modifications, CpG
methylation is the key features of repressed lytic genes of EBV, but, counterin-
tuitively, DNA methylation is even a prerequisite for the escape from EBV’s latent
phase.
The induction of EBV’s lytic phase in latently infected cells is initiated by the
expression of the viral BZLF1 gene encoding the transcription factor BZLF1,
which is a basic leucine zipper (bZIP) transcription factor. It is modular in struc-
ture and contains a basic domain that mediates sequence-specific binding to DNA,
a coiled-coil domain that confers to homodimerization, and a transcriptional acti-
vation domain (Sinclair 2003).
BZLF1 binds sequence specifically to one class of DNA motifs, termed ZREs,
but prefers a second class that contains methylated 5′-cytosine residues (5mC),
termed meZREs (Bhende et al. 2004; Bergbauer et al. 2010; Kalla et al. 2010;
Karlsson et al. 2008; Petosa et al. 2006; Dickerson et al. 2009). Binding to both
motifs can activate viral promoters that drive a number of lytic genes. meZREs
are the exclusive BZLF1-binding motifs in certain early viral promoters, which,
for example, encode factors essential for the lytic amplification of EBV DNA
(Bergbauer et al. 2010) such as the viral helicase, DNA polymerase, and DNA
polymerase accessory protein among others (Fixman et al. 1992). The BZLF1-
mediated expression of these viral genes depends on meZREs. Paradoxically, their
CpG methylation is instrumental for the expression of certain essential lytic genes
and indispensable for virus synthesis (Kalla et al. 2012), changing the conven-
tional view of DNA methylation solely as a repressive epigenetic feature.
BZLF1 binds with high affinity to meZREs in a methylation-dependent fash-
ion (Bergbauer et al. 2010). This peculiar feature also explains why the virus fails
to induce synthesis of progeny virus during the initial, pre-latent phase of infec-
tion during which BZLF1 is expressed (Fig. 1). Key lytic viral promoters that
depend on BZLF1 and contain meZRE sites cannot be expressed during this phase
because they lack DNA methylation, which abrogates binding of BZLF1 to its
meZRE target sequences and prevents progeny virus synthesis in newly infected
cells. Thus, EBV invented a marvelous system to regulate its different phases with
the help of DNA methylation.
In latently infected cells, binding of BZLF1 protein to methylated meZRE sites
induces an epigenetic reprogramming at the repressed and silenced target promot-
ers. Nucleosomes are removed locally, and chromatin adopts an open, accessible
configuration. Epigenetic repression by Polycomb proteins is erased, activation

marks like H3K4me3 are introduced, and, eventually, RNA polymerase II is bound
and transcription of the target genes is increased massively. Interestingly, tran-
scription of lytic genes occurs on a fully methylated template without the need of
active DNA demethylation at promoters or gene bodies. Thus, DNA methylation
per se does not prevent the binding or block the initiation and elongation activity
of RNA polymerase II providing a new paradigm for gene regulation (Woellmer
et al. 2012).
One of the interesting aspects of BZLF1 binding to its target promoters is the
local changes that occur in nucleosome occupancy. BZLF1 binding to chromatinized
DNA can evict nucleosomes in vivo indicating that the BZLF1 transcription fac-
tor directly or indirectly can induce a change in the chromatin structure (Woellmer
et al. 2012). Therefore, BZLF1 could act as a pioneer factor (Woellmer and
Hammerschmidt 2013). Pioneer factors can bind to compacted chromatin and trig-
ger its local opening in order to prepare promoters or other cis-acting elements for
transcriptional activation with or without the help of chromatin remodeling factors.
During their existence, herpesviruses have regularly acquired cellular genes
by random recombination with host cell DNA, and certain adopted genes have
evolved to serve specific viral functions. BZLF1 shares amino acid sequence
homology with the AP-1 family of transcription factors. Its binding domain is
very similar to that of c-FOS protein (Taylor et al. 1991; Farrell et al. 1989). It
could be that BZLF1 has adopted its genuine binding properties to methylated
DNA from its cellular counterpart c-FOS or other members of the AP-1 family. In
fact, the prototypical c-JUN/c-FOS heterodimer is able to bind to methylated DNA
sequence motifs in cellular chromatin and activate gene transcription from methyl-
ated templates as BZLF1 does (Gustems et al. 2014). DNA binding to methylated
AP-1 sequence motifs is infrequent in cellular chromatin as compared to the con-
sensus normal AP-1 target sites that lack CpG dinucleotide pairs, but it appears
that EBV has optimized an existing cellular principle of gene regulation, which it
can use to establish latent infections in all cells it infects.
In the lytic phase, the expression of early lytic genes precedes the induction of
late lytic promoters. As with other herpesviruses, the expression of a number of
late lytic genes of EBV requires newly replicated viral DNA (Amon et al. 2004),
which is massively amplified via the virus-encoded replication machinery in the
lytic phase. The concept of late gene regulation of herpesviruses has been elusive
so far, but the viral components that drive the expression of late EBV genes have
been identified, recently (Aubry et al. 2014). The virally encoded, TBP-like pro-
tein, BcRF1 is part of a set of six viral gene products, which is essential and suf-
ficient to assemble the transcriptional pre-initiation complex including Pol II at
late promoters (Gruffat et al. 2012). It appears plausible to argue that transcription
of late viral genes occurs on newly replicated viral DNA, which is known to be
free of methylated CpGs (Kalla et al. 2010 and references therein). Recognition of
late lytic EBV promoters by this dedicated multicomponent transcription complex
depends on a TATA-like viral sequence motif and replicated viral DNA, which is
also free of nucleosomes (Chiu et al. 2013). Again, viral epigenetic mechanisms
are crucial and timely regulated even during late viral transcription supporting
escape from latency and virus morphogenesis.
5 Open Questions
How does viral DNA acquire cellular histones and/or nucleosomes?

When EBV infects a cell, it delivers the epigenetically naïve and histone-free
viral DNA into the nucleus of the cell. Do cellular histone loading factors such
as HIRA, DAXX, or NAP1L1 help to chromatinize the epigenetically naïve viral
DNA during EBV’s pre-latent phase? When are classical cellular histones syn-
thesized, which are normally deposited in a cell cycle-dependent fashion? Virion-
delivered BNRF1, a tegument protein, suppresses DAXX–ATRX-mediated H3.3
loading on viral DNA supporting latent gene expression and preventing repressive
chromatin formation onto the EBV genome during primary infection of B lympho-
cytes (Tsai et al. 2014). Which cellular or viral factors drive de novo assembly of
chromatin on epigenetically naïve EBV DNA is currently elusive.
How does latent EBV chromatin acquire repressive histone marks?
The majority of lytic viral genes and their promoters are governed by PRC2-
induced trimethylation of H3K27, which is a prevalent repressive histone mark
together with H3K9me3. It is unclear what directs PRC2 to these regions and
whether mechanisms exist that promote the silencing of lytic genes sparing the
promoters of those genes, which are active during EBV’s latent phase. It is equally
unclear whether additional histone methyltransferases are active during EBV’s dis-
tinct phases of infection.
What causes the slow but extensive CpG methylation of viral genomic DNA?
Enzymes catalyzing the addition of a methyl group to cytosine residues are termed
DNA methyltransferases (DNMTs). In newly infected B lymphocytes, the mainte-
nance methyltransferase DNMT1 is expressed already at day one post-infection.
One week post-infection expression of DNMT1 increased further together with
DNMT3a and DNMT3b, which encode the two cellular de novo methyltrans-
ferases (Schmeinck 2011). EBV’s latent membrane protein 1 (LMP1) induces the
expression of all methyltransferases in nasopharyngeal carcinoma cell lines via
the JNK pathway (Tsai et al. 2006) suggesting a role for LMP1 in upregulating
DNMT expression also in newly infected B lymphocytes. The nuclear protein of
95 kDa (NP95), a cellular protein associated with maintenance and de novo DNA
methylation, recruits DNMT1 to hemi-methylated CpGs, but also interacts with
de novo DNMTs, histone methyltransferases, and trimethylated H3K9 connecting
the DNA methylation pathway to the establishment of repressive histone marks
(Meilinger et al. 2009; Rottach et al. 2010). NP95 is also upregulated in EBV-
infected B lymphocytes seven days post-infection (Schmeinck 2011) indicating
that NP95 could be involved in introducing methylated CpG dinucleotides in EBV
DNA. The slow increase in DNA methylation of EBV’s DNA could probably be
the reason for the lack of virion synthesis until twelve days post-infection (Kalla
et al. 2010).
Is the promoter of BZLF1 epigenetically regulated?
BZLF1 can induce the lytic phase in latently EBV-infected cells, but its expres-
sion is tightly repressed in these cells. Several cellular repressors of transcription
have been identified (Kenney and Mertz 2014 and references therein) that block
the promoter of BZLF1 and prevent its reactivation. In contrast to the major-
ity of EBV’s lytic genes and their promoters, CpG dinucleotides are exception-
ally rare and exempt from DNA methylation in the promoter of BZLF1 (Woellmer
et al. 2012). It appears that epigenetic mechanisms, which increase or alleviate
the repressed state of the BZLF1 promoter, have not been completely analyzed
(Murata and Tsurumi 2014).
What causes decompaction of EBV DNA and transcriptional reactivation of higher
order viral chromatin?
Reactivation of epigenetically repressed viral chromatin requires chromatin open-
ing and removal of inactivating, repressive histone marks. How this profound
change in the status of viral chromatin is achieved is not known. It is also not
known how the BZLF1 factor gains access to compacted chromatin and whether
it mediates, directly or indirectly, chromatin remodeling. Whether BZLF1 or other
viral factors play a role in changing the architecture of viral chromatin upon lytic
induction is equally uncertain.
How are CpG methylation and histones lost prior to encapsidation of virion DNA?
EBV encodes its own replication machinery of viral enzymes and cofactors that
mediate unlicensed amplification of viral DNA during the virus’ lytic phase.
DNA amplification occurs in the nucleus at distinct sites and is accompanied by
nuclear reorganization. Histones are not part of the sites of lytic DNA amplifica-
tion, and PCNA is equally excluded. Moreover, cellular histone chaperone levels
decrease during EBV’s lytic phase indicating that EBV follows a strategy that is
very different from cellular DNA replication (Chiu et al. 2013). PCNA mediates
the recruitment of cytosine methyltransferases to replication forks during cellu-
lar DNA replication, but in the DNA replication machinery, PCNA is replaced
by EBV’s own DNA clamp BMRF1. It is speculative but BMRF1 might fail to
recruit the cellular hemi-methyltransferase DNMT1 that would maintain the sta-
tus of CpG methylation in newly replicated viral DNA (Chiu et al. 2013; Kalla
et al. 2012).
Acknowledgments This review is based on the works of many colleagues. I sincerely
apologize to all scientists whose important contributions could not be cited here due to
space limitations. I would like to thank Bill Sugden for reading the manuscript and his
valuable suggestions. This review and work in my laboratory is supported by Institutional
Intramural Grants, grants from the Deutsche Forschungsgemeinschaft SFBTRR36/TPA04,
SFB1064/TPA13, SFB1054/TPB05, grants from German Centre for Infection Research (DZIF),
and National Institutes of Health Grant CA70723.
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Epstein–Barr Virus: From the Detection
of Sequence Polymorphisms to the
Recognition of Viral Types
Regina Feederle, Olaf Klinke, Anton Kutikhin, Remy Poirey,

Ming-Han Tsai and Henri-Jacques Delecluse
Abstract The Epstein–Barr virus is etiologically linked with the development of

benign and malignant diseases, characterized by their diversity and a heterogene-
ous geographic distribution across the world. The virus possesses a 170-kb-large
genome that encodes for multiple proteins and non-coding RNAs. Early on there
have been numerous attempts to link particular diseases with particular EBV
strains, or at least with viral genetic polymorphisms. This has given rise to a
wealth of information whose value has been difficult to evaluate for at least four
reasons. First, most studies have looked only at one particular gene and missed
the global picture. Second, they usually have not studied sufficient numbers of
diseased and control cases to reach robust statistical significance. Third, the func-
tional significance of most polymorphisms has remained unclear, although there
are exceptions such as the 30-bp deletion in LMP1. Fourth, different biological
properties of the virus do not necessarily equate with a different pathogenicity.
This was best illustrated by the type 1 and type 2 viruses that markedly differ in
terms of their transformation abilities, yet do not seem to cause different diseases.
Reciprocally, environmental and genetic factors in the host are likely to influence
the outcome of infections with the same virus type. However, with recent develop-
ments in recombinant virus technology and in the availability of high throughput
sequencing, the tide is now turning. The availability of 23 complete or nearly com-
plete genomes has led to the recognition of viral subtypes, some of which pos-
sess nearly identical genotypes. Furthermore, there is growing evidence that some
genetic polymorphisms among EBV strains markedly influence the biological
and clinical behavior of the virus. Some virus strains are endowed with biological
R. Feederle · O. Klinke · A. Kutikhin · R. Poirey · M.-H. Tsai · H.-J. Delecluse (*)

Unit F100, Inserm unit U1074, DKFZ, German Cancer Research Centre (DKFZ),
69120 Heidelberg, Germany
e-mail: h.delecluse@dkfz.de
R. Feederle
Helmholtz Zentrum München, German Research Center for Environmental Health,
Institute of Molecular Immunology, Marchioninistrasse 25, 81377 Munich, Germany

120 R. Feederle et al.
properties that explain crucial clinical features of patients with EBV-associated

diseases. Although we now have a better overview of the genetic diversity within
EBV genomes, it has also become clear that defining phenotypic traits evinced by
cells infected by different viruses usually result from the combination of multi-
ple polymorphisms that will be difficult to identify in their entirety. However, the
steadily increasing number of sequenced EBV genomes and cloned EBV BACS
from diseased and healthy patients will facilitate the identification of the key poly-
morphisms that condition the biological and clinical behavior of the viruses. This
will allow the development of preventative and therapeutic approaches against
highly pathogenic viral strains.
Contents
1 Introduction........................................................................................................................... 121
2 Next-Generation Sequencing Gives a Detailed Picture of Sequence Heterogeneity
at the Genome Level............................................................................................................. 122
3 The Degree of Protein Heterogeneity Varies Among Viral Proteins..................................... 126
4 The EBV Genome Evinces a Large Number of Genetic Polymorphisms
of Unknown Biological and Medical Significance............................................................... 131
5 EBV Type 1 and Type 2, Two Strains with Different Biological Properties
but Apparently not Associated with Particular Diseases....................................................... 133
6 Polymorphisms in EBNA3B Modulate the Transformation Abilities
of the Virus In Vitro and In Vivo........................................................................................... 135
7 Polymorphisms Within Latent Genes are Submitted to Immune Pressure
and Influence the Geographic Distribution of Virus Isolates................................................ 141
8 The NPC-Associated Virus M81 Is Endowed with Unique Properties
that Distinguishes It from Other Strains In Vitro and In Vivo............................................... 142
9 Conclusions........................................................................................................................... 143
References................................................................................................................................... 144
Abbreviations
BAC Bacterial artificial chromosomes

EBV Epstein–Barr Virus
EBNA Epstein–Barr virus nuclear antigen
LMP Latent membrane protein
miRNAs MicroRNAs
NGS Next-generation sequencing
RFLP Restriction fragment length polymorphism
PTLD Posttransplant lymphoproliferative disorder
Epstein–Barr Virus: From the Detection … 121
1 Introduction
The Epstein–Barr virus (EBV) possesses a double-stranded 170-kb-large DNA

genome that encodes for more than 70 genes and non-coding RNAs including 25
pre-microRNAs (Rickinson 2007; Cullen 2011). EBV is etiologically linked with
a large number of benign and malignant diseases, characterized by the histologi-
cal diversity (Delecluse et al. 2007). Infectious mononucleosis is typically encoun-
tered in industrialized countries and results from a primary infection delayed to
late teenage (Rickinson and Kieff 2007). EBV-associated tumors include lympho-
mas of several types, as well as carcinomas of the nasopharynx (NPC), stomach
(GC), parotid, and thymus (Delecluse et al. 2007). Rare cases of sarcomas have
also been reported in children with AIDS (McClain et al. 1995). These broad
categories of tumors are very heterogeneous in terms of geographic distribution.
While NPC occurs at high and intermediate incidence in Southeast Asia, Northern
Africa, and Alaska, EBV-positive GC is encountered all around the world,
although the incidence varies somehow between countries (Rickinson and Kieff
2007). EBV-positive Burkitt’s lymphoma (BL) is endemic in equatorial Africa and
America, and EBV-positive nodal T-cell lymphomas are frequent in Japan, and T
or NK nasal-type lymphomas are mainly encountered in Asia and South America
(Rickinson and Kieff 2007; Young and Rickinson 2004). Independently of their
country of origin, individuals with congenital or acquired immunodeficiency are
at high risk of EBV-associated lymphoproliferations (Rickinson and Kieff 2007;
Young and Rickinson 2004). These posttransplant lymphoproliferative disorders
(PTLD) or HIV-associated EBV-positive lymphomas or lymphoproliferations are
usually B-cell lymphomas, but may also be T-cell lymphomas and Hodgkin’s lym-
phomas (Rickinson and Kieff 2007; Young and Rickinson 2004).
The diversity in histological subtypes of tumors and their geographic hetero-
geneity has early been suspected to reflect infection with different virus subtypes
but the large size of the virus has precluded sequencing of multiple virus isolates
(Rickinson and Kieff 2007). However, it was recognized early on that sequence
polymorphisms are very common all across the genome (reviewed in Chang et al.
2009). This led to a very large number of studies that investigated multiple genes
from viruses present in tumors or in healthy population controls (Chang et al.
2009). However, these studies usually investigated a small number of virus iso-
lates, lacked a sufficient number of control cases from healthy donors, and were
often limited to a single gene (Chang et al. 2009). The inhomogeneity of the inves-
tigated population can also be a source of flawed results. This is particularly true
for studies that were conducted in countries with a large population in which dis-
eases might particularly affect one region such as Hong Kong and the Cantonese
Province in China in which the incidence of NPC is highest worldwide. In such a
case, it is very important to make sure that both the diseased and the control popu-
lations were born in the region studied since it was proposed that particular EBV
polymorphisms may be restricted to some populations rather than to geographical
areas (Lung et al. 1994). Crucially, the functional consequences of the viral poly-
morphisms for the infected hosts remained relatively unexplored until recently. In
their 2009 review of the topic (Chang et al. 2009), Chang and colleagues noted
that ‘definite conclusions regarding the link between EBV genotypes, disease and
geography are not possible.’ We also refer to the comprehensive review by Jan
Gratama and Ingemar Ernberg that covers the earlier developments in this com-
plex field (Gratama and Ernberg 1995). The aim of the present review was to
record the progress that new-generation sequencing and the increasing availabil-
ity of tractable genetic systems made possible in this field. We also only briefly
address the functional consequences of protein polymorphisms as these issues are
more extensively addressed by Paul Farrell in another chapter of this book.
The necessary background on the biology of the virus can be found in other
chapters from this book or for example in Kieff et al. (2007), Rickinson and Kieff
(2007), or in Longnecker et al. (2014), and we limit ourselves to a brief summary
of the virus’ defining features. EBV infects B lymphocytes with a uniquely high
efficiency. Shortly after infection, B lymphocytes resume cell growth and generate
so-called lymphoblastoid cell lines in vitro. This process requires expression of the
set of latent proteins that belong to the Epstein–Barr nuclear antigen (EBNA 1, 2,
3A, 3B, 3C, EBNA-LP) and latent membrane protein (LMP1 and LMP2) families.
EBV gene expression can also lead to new viral progeny. Viral replication requires
the sequential expression of immediate-early, early, and late viral lytic genes. The
protein encoded by the BZLF1 gene is endowed with transactivating properties
and can initiate virus replication in permissive cells.
2 Next-Generation Sequencing Gives a Detailed Picture

of Sequence Heterogeneity at the Genome Level
The increasing availability of next-generation sequencing technology has allowed

sequencing of 23 EBV genomes so far, although the extent of the sequence avail-
able varies (see Table 1 and references therein). Some sequences are complete,
others do not include the repeats and yet others cover only partly the viral genome.
16 sequences were obtained from viruses present in malignant tumors or from
patients with these diseases (13 with NPC, 3 with BL). From these, 14 arose in
Asian patients (13 Chinese and 1 Japanese) and the remaining two in African
patients. The other 7 viruses were isolated from individuals with IM or from
endogenous LCLs that were generated with the blood of healthy patients. Three
of these viruses were isolated from individuals who reside in the USA; the oth-
ers were sequenced from cell lines used in the 1000 Genomes Project and were
established with blood from healthy African or European individuals. Thus, the
currently available information is very much skewed toward EBV isolates associ-
ated with tumors, in particular NPC.
However, this information has already allowed a first global assessment of
genetic diversity between EBV isolates isolated in Asia, Africa, in the USA, and
in Europe. We have evaluated genetic distance between these strains based on an
alignment of the comparable portions of the genomes (roughly 159 kb). No matter
Table 1 Currently available EBV genome sequences and common investigated polymorphisms
Strain, EBV subtype Type Type C/D wt/mut-W1/l1 Type XhoI+ BZLF1 protein BZLF1 pro- Sample Sample geographical
reference 1/2 (A/B) F/f (I/i) /Xho− variant moter variant origin localization
AG876 (Dolan et al. 2 (B) F D (i) wt-W1/I1 + BZLF1-A2 Zp-V3 BL Africa (Ghana)
2006)
Mutu (Lin et al. 2013) 1 (A) F D (i) wt-W1/I1 + BZLF1-C Zp-P BL Africa (Kenya)
NA19114 (Santpere 1 (A) F D (i) wt-W1/I1 + BZLF1-C Zp-P PBL Africa
et al. 2014) (Nigeria)
NA19315 (Santpere 1 (A) F D (i) wt-W1/I1 + BZLF1-C Zp-P PBL Africa (Kenya)
et al. 2014)
NA19384 (Santpere 1 (A) F D (i) wt-W1/I1 + BZLF1-C Zp-P PBL Africa (Kenya)
et al. 2014)
1 (A) F C (I) wt-W1/I1 BZLF1-A1 Zp-V3 Saliva NPC Asia (China)
Epstein–Barr Virus: From the Detection …
GD1 (Zeng et al. −

2005)
GD2 (Liu et al. 2011) 1 (A) f C (I) mut-W1/I1 − BZLF1-A1 Zp-V3 NPC Asia (China)
C666-1 (Kwok et al. 1 (A) f C (I) mut-W1/I1 − BZLF1-A1 Zp-V3 NPC Asia (China)
2014)
HKNPC1 (Kwok et al. 1 (A) f C (I) mut-W1/I1 − BZLF1-A1 Zp-V3 NPC Asia (China)
2012)
HKNPC2 (Kwok et al. 1 (A) F C (I) mut-W1/I1 − BZLF1-A1 Zp-V3 NPC Asia (China)
2014)
2014)
2014)
2014)
(continued)
123
Table 1 (continued)
124
Strain, EBV subtype Type Type C/D wt/mut-W1/l1 Type XhoI+ BZLF1 protein BZLF1 pro- Sample Sample geographical
reference 1/2 (A/B) F/f (I/i) /Xho− variant moter variant origin localization
HKNPC6 (Kwok et al. 1 (A) F C (I) mut-W1/I1 − BZLF1-C Zp-P NPC Asia (China)
2014)
HKNPC7 (Kwok et al. 1 (A) F C (I) mut-W1/I1 − BZLF1-C Zp-P NPC Asia (China)
2014)
2014)
2014)
M81 (Tsai et al. 1 (A) f C (I) mut-W1/I1 − BZLF1-A1 Zp-V3 NPC Asia (China)
2013)
Akata (Lin et al. 1 (A) F C (I) wt-W1/I1 − BZLF1-A1 Zp-V3 BL Asia (Japan)
2013)
B95.8 (Baer et al. 1 (A) F Δ Δ + BZLF1-C Zp-P IM North America
1984, de Jesus et al. (USA)
2003)
K4123-Mi (Lei et al. 1 (A) F D (i) wt-W1/I1 + BZLF1-C* Zp-P PBL North America
2013) (USA)
K4413-Mi (Lei et al. 1 (A) F D (i) wt-W1/I1 + BZLF1-C* Zp-P PBL North America
2013) (USA)
BL Burkitt’s lymphoma, PBL peripheral blood B lymphocytes, NPC nasopharyngeal carcinoma, IM infectious mononucleosis
Subtypes 1 and 2 (A/B) are based on the sequence of the EBNA2 protein. Type F viruses lack a BamHI site in the BamHI-F region present in the type f
viruses. Type C viruses (also known as I) lacks the BamHI site at BamHI W1*/I1* boundary region (in the non-coding region between BART14 and BILF1)
whereas type D viruses (also know as i) possess it. The Wt-W1/I1 polymorphism has a T at position 148,972 in the reference strain NC_007605.1 (in the
non-coding region between BART14 and BILF1) whereas mut-W1/I1 has a C at the same position. Type XhoI+ viruses have an XhoI restriction site in the
exon1 of LMP1 in contrast to type XhoI-viruses. The BZLF1 types are defined by clusters of amino acid variations in this protein and its promoters
R. Feederle et al.
Fig. 1 Sequence heterogeneity among EBV isolates. A divergence tree of the 22 strains whose
genome sequence has been published. Distances are based on a multiple alignment of 159 kb
from these genomes
what model of DNA evolution was used (e.g., Felsenstein or Jukes-Cantor) and no
matter how a tree was built from these distances (we used the maximum likelihood
method), the overall picture remained the same. Figure 1 depicts the genetic dis-
tances as a tree but this depiction should not be mistaken for a phylogenetic tree,
as many of the assumptions commonly made for phylogenies may not be valid for
the Epstein–Barr virus evolution. Furthermore, this approach does not distinguish
between divergence that results from polymorphisms concentrated in a small num-
ber of genes such as EBNA2 and the EBNA3 family in type 1 and type 2 viruses
(see below) and divergence caused by polymorphisms that are more homogenously
distributed along the genome. Nevertheless, the clustering that is apparent in Fig. 1
is globally concordant with previous analyses that looked at only a subset of these
sequenced genomes (Tsai et al. 2013; Kwok et al. 2014). Indeed, we previously
reported that viruses isolated from individuals who lived in different geographic area
show a maximum divergence rate of approximately 1 % at the DNA level, and 1.3 %
at the protein level (Tsai et al. 2013). These data are concordant with the 0.65–
0.73 % variation in EBV genomes from NPC tissues relative to B95-8 observed by
other authors, in particular because this analysis did not take into account repeated
sequences that we found to be also polymorphic (Kwok et al. 2014).
Inspection of this tree immediately suggests the existence of three large
groups of viruses, one containing viruses isolated from tumors that arose in Asian
patients, one from viruses isolated in healthy or diseased individuals in Africa
and in the USA and one that contains the type 2 virus AG876. Among the virus
isolated in Asia, one very homogenous subgroup that comprises viruses that infect
NPCs and mainly arose in the Hong Kong area is easily recognized and will be
referred to as NPC I. Another 2 viruses, although clearly related to NPC I and also
isolated in infected patients from Hong Kong with NPC, displayed some important
differences from IR2 to IR3 genomic region and will be referred to as NPC group
II. Interestingly, HKNPC2 is likely to have arisen from a recombination between a
virus from NPC group I and a virus from NPC group II. Intertypic recombinants
between type 1 and type 2 viruses have previously been reported in the Chinese
population (Midgley et al. 2000).
Crucially, type 2 viruses and one virus from the NPC I group display particular
biological properties with likely important clinical consequences as described in
paragraphs 5 and 8.
However, the currently available series of viral genomes will soon be massively
expanded to include 71 novel EBV genomes isolated in malignant tumors of more
diverse lineages and that developed in patients from other geographic regions.
Furthermore, this series will also comprise viruses isolated from EBV-positive
healthy carriers from multiple regions of the world including Australia, Kenya,
and the United Kingdom (P. Kellam, P. Farrell, R. White, personal communica-
tion). Therefore, our picture of viral diversity will soon be drastically enriched and
will probably render this review rapidly obsolete. Nevertheless, it is very likely
that an even much larger number of complete virus sequences from all continents
will be needed to capture the full extent of virus diversity.
3 The Degree of Protein Heterogeneity Varies

Among Viral Proteins
The EBV genome contains more than 25 types of repeated sequences, and many
of which are located within coding EBV genes and are present in varying numbers
across different isolates. This, combined to amino acid polymorphisms that affect
the molecular weight and the presence of posttranslational modifications, gives
rise to proteins of different sizes. This phenomenon strongly affects the EBNA
proteins whose size can vary within a variable range and has been used to define
an ‘EBNotype’ that defines different viral isolates (Gratama and Ernberg 1995).
EBNA1 also displays many polymorphisms across EBV isolates but the functional
significance of these mutations remains unknown (Bhatia et al. 1996; Gutierrez
et al. 1997; Habeshaw et al. 1999).
In contrast, the role of polymorphisms within the EBNA2 gene has been char-
acterized in detail and led to the definition of the type 1 and type 2 viruses that
will be discussed in the sequel. Similarly, important polymorphisms have been
identified within the EBNA3B gene (see paragraph 6).
The LMP1 gene carries multiple repeats that are present in different numbers
in the different strains. This includes the 33-bp repeats, as well as a 15-bp inser-
tion in some cases (Fig. 2) (Sandvej et al. 1997; Cheung et al. 1996). Finally,
Fig. 2 Amino acid sequence of the LMP1 gene from different virus types showing polymorphisms, repeats, insertions, and deletions. ins15: insertion of
15bp/5aa, Rep33: 33bp/11aa repeat. Dots indicate base identity
127
many strains carry a 30-bp deletion at the C-terminal end of the protein. There
are also multiple single amino acid polymorphisms within the carboxy terminus
of the LMP1 protein. These polymorphisms have given rise to multiple variants
that carry some mutations and/or the 30-bp deletion and that have been named
according to the country where the virus was identified (China 1, 2, 3, Alaskan,
North Carolina, Mediterranean, etc.) (Chang et al. 2009; Sung et al. 1998). A more
recent paper found that these different LMP1 alleles do not vary in their ability
to transform Rat-1 fibroblasts, increase mobility of Madin-Darby canine kid-
ney cells, or induce homotypic adhesion of BJAB cells (Mainou and Raab-Traub
2006). At the molecular level, while these LMP1 variants activated the PI3K/AKT
signaling pathways at similar levels, some LMP1 variants such as Alaskan induced
NF-kB signaling with an approximately 2 times higher efficiency than B95-8. This
explains reports from the early nineties that already found that LMP1 variants
differed in their ability to activate the NF-kB pathway (reviewed in Jenkins and
Farrell 1996). Altogether, although it is clear that multiple LMP1 alleles exist,
there is currently no overwhelming evidence that particular LMP1 alleles are
associated with particular diseases or geographical locations (Sandvej et al. 1997;
Edwards et al. 1999; Mainou and Raab-Traub 2006).
BZLF1 is also a highly polymorphic EBV protein (Fig. 3a, b). The BZLF1 pro-
moter has also been investigated in detail in an attempt to understand why some
EBV isolates supported lytic replication more efficiently than others and gave rise to
the current recognition of 5 variants (Zp-P, Zp-PV, Zp-V1, Zp-V3, and Zp-V4) that
are themselves subdivided into multiple subvariants (Chang et al. 2009; Gutierrez
et al. 2002; Tong et al. 2003; Martini et al. 2007; Lorenzetti et al. 2009; Jin et al.
2010; Yu et al. 2012; Lorenzetti et al. 2012). BZLF1 proteins have been subdivided
into groups A1, A2, B1, B2, B3, B4, B5, B6, C, D, E, F, some of which such as A1,
A2, and C also contain variants (Chang et al. 2009; Luo et al. 2011; Lorenzetti et al.
2014). There is growing evidence that some BZLF1 variants are endowed with dif-
ferent properties in recombinant viruses (Tsai et al. 2013, see paragraph 8).
The data obtained by NGS confirm and extend these data (Tsai et al. 2013;
Kwok et al. 2014). Importantly, the sequence heterogeneity at the protein level
is unequally distributed across the genome (Tsai et al. 2013; Kwok et al. 2014).
Comparison between 22 EBV isolates reveals that most proteins diverge by less
than 1 % (Fig. 4). However, other proteins, including most latent proteins, show
higher divergence (Fig. 4). In case of LMP1, it reaches 8 %, making it one of
the most polymorphic EBV proteins (Lin et al. 2013). Polymorphic proteins also
include tegument proteins such as the large tegument proteins BOLF1 and BPLF1,
or surface glycoproteins such as gp350 or gp110 that are encoded by BLLF1 and
BALF4, respectively (Kwok et al. 2014). Some of these polymorphisms were
found to influence the viral tropism (Tsai et al. 2013). In contrast, the early lytic
proteins that perform DNA replication and include BMRF1, BMRF2, and BRLF1
are much more conserved (Tsai et al. 2013; Kwok et al. 2014). This implies that
mutations within these genes are deleterious for viral propagation and are elimi-
nated. The EBV miRNAs and BCRF1, and the viral homolog of IL10 are also well
conserved (Kwok et al. 2014).
(a)
Fig. 3 a The promoter region of the BZLF1 gene, from position −221 to +12 relative to the transcription start and its different types. The sequence of the
prototype B95.8 is compared to the published variants. Dots indicate base identity. b An alignment of the BZLF1 amino acid sequence. Representatives from
A1 to F types are shown. The B95-8 sequence that corresponds to the C type is taken as a reference. Dots indicate amino acid identity
129
(b)
130
Fig. 3 (continued)
R. Feederle et al.
10
8
percent divergence from consensus
0
LMP1
LMP2A
BZLF1
BRLF1
BNLF2a
BcRF1
BLLF1
BDLF3
BALF4
BRRF2
BOLF1
BPLF1
BDLF1
BcLF1
BMRF1
BMRF2
BALF5
LF2
B95.8 HKNPC2 HKNPC7 MUTU NA19384
M81 HKNPC3 HKNPC8 K4123 AG876
GD1 HKNPC4 HKNPC9 K4413
GD2 HKNPC5 C666-1 NA19114
HKNPC1 HKNPC6 AKATA NA19315
Fig. 4 The percentage of divergence at the protein level across the completely sequenced EBV
strains. Proteins involved in virus replication and transformation are shown. LF2 is a putative
protein
4 The EBV Genome Evinces a Large Number

of Genetic Polymorphisms of Unknown
Biological and Medical Significance
The genetic diversity among EBV isolates has initially been recognized by restric-
tion analysis of viral DNA from EBV-infected cells and by sequencing of DNA
obtained from any EBV-positive tissue or cell line (Chang et al. 2009). The
sequence heterogeneity within isolates gives rise to various restriction sites and
thus to RFLPs, some of which such as the BamHI site within the F fragment (Type
F/f), the BamHI site I fragment (type C/D), or the XhoI site within the LMP1
gene (type XhoI+ versus XhoI−) have been extensively studied (Fig. 2) (Chang
et al. 2009). A very high number of epidemiological studies have tried to cluster
the existence of polymorphisms with EBV-associated diseases, among which the
XhoI restriction site plays a prominent role (Chang et al. 2009). In all the studies
that found a difference in the frequency of some LMP1 alleles in tumors relative
to normal controls, this difference was mild to moderate. One recurrent prob-
lem with these studies is the paucity of information on the distribution of LMP1
alleles within the normal population (Chang et al. 2009). This is particularly true
for countries with a very large population in which a cohort of patients from a
large university hospital might include individuals from different regions in which
the molecular characteristics of the local EBV strains might vary. A middle-sized
study from Europe did not find any differences in the distribution of LMP1 vari-
ants in the control versus diseased population (Sandvej et al. 1997). Altogether,
the extent to which some LMP1 variants might be more transforming than others
remains to be appreciated (see paragraph 3).
Reciprocally, there are many more polymorphisms within the EBV genome that
are not necessarily identifiable by RFLPs and whose importance might have not
yet been recognized.
Despite the limitations inherent to sampling that have already been mentioned,
we assessed the value of these markers to identify the viral strains whose sequence
is known. Figure 1 and Table 1 show their distribution within the diversity tree.
Interestingly, these markers clustered well with groups of viruses identified by the
global genome analysis. The f variant was expressed only by the very homogenous
NPC group I. The C and XhoI− markers were exclusively present in viruses iso-
lated from Chinese or Japanese individuals; all other isolates carried the D and
the XhoI+ markers. The mut-W1/I1 marker was found in all group I and group II
NPC viruses but not in GD1 (Chen et al. 2012). All other isolates carried wt-W1/
I1 (Chen et al. 2012). Other markers such as those present in the BZLF1 ORF
or its promoter did not match perfectly the groups defined by the divergence tree.
Nevertheless, it is clear that within this group of viruses, BZLF1-A1, Zp-V3, and
BR1-A are always present together in a given virus, as are BZLF1-C and Zp-P.
Furthermore, the first group of markers is present in all viruses found in Asian
countries with the exception of the group II NPCs. What can be deducted from this
analysis? First it appears that particular combinations of markers correlate very
well with the groups defined by the divergence tree. This means that they might
be used in the future as a set of markers to identify particular viral strains when
they are used in combination. However, in agreement with previous interpreta-
tions, it remains unclear whether they identify clustering with particular groups
of individuals or with particular diseases (Chang et al. 2009). As more complete
virus sequences become available, their validity as predictive markers will be
reevaluated.
Is there any evidence that these markers can identify EBV strains associated
with particular diseases? It is certainly not the case for the gastric carcinoma in
which viruses from cases that arose in different parts of the world carried different
markers (reviewed in Chen et al. 2012). The viruses that infect NPC can be of type
f or F. Altogether, there is currently not sufficient evidence to support the concept
that these polymorphism can identify tumor types.
5 EBV Type 1 and Type 2, Two Strains with Different

Biological Properties but Apparently not Associated
with Particular Diseases
It has long been recognized that two viral types, type 1 and type 2 or type A and
type B exist (Dambaugh et al. 1984; Adldinger et al. 1985). Both types were ini-
tially identified on the basis of extensive sequence polymorphisms within the
EBNA2 gene, but it was also soon recognized that this divergence extends to the
EBNA3 gene family (Rowe et al. 1989; Sculley et al. 1989; Sample et al. 1990).
The divergence is strongest in EBNA2 (only 54 % homology between the 2 types),
justifying the distinction of EBNA2 into type A and type B (Dambaugh et al.
1984). EBNA2A and B display many single nucleotide polymorphisms but also
larger deletions/insertions (Aitken et al. 1994) (Fig. 5). Type 1 viruses transform
primary B lymphocytes more readily than type 2 viruses in vitro and a recombi-
nant type 2 virus that carries a type 1 EBNA2A acquires the transforming abilities
of type 1 viruses (Rickinson et al. 1987; Cohen et al. 1989). Although the epidemi-
ological data remain sparse, the geographic distribution of both virus types seems
to vary extensively across the world. Type 1 viruses seem to largely dominate in
Asia and in Europe, but both type 1 and type 2 viruses are found at more compara-
ble rates in Africa, New Guinea, Australia, the USA, and more specifically Alaska,
as well as in transplant recipients and in HIV-positive individuals (Aitken et al.
1994; Young et al. 1987; Sculley et al. 1988; Sixbey et al. 1989; Kunimoto et al.
1992; Shu et al. 1992; Kyaw et al. 1992; Apolloni and Sculley 1994). In the lat-
ter group, EBV type 2 is transmitted by sexual route in individuals with multi-
ple partners, some of which of non-European origin. Both virus types have been
found in BL NPC and HL (Young et al. 1987; Zimber et al. 1986; Sculley et al.
1988; Abdel-Hamid et al. 1992; Boyle et al. 1993; van Baarle et al. 2000). In con-
trast, type 1 viruses predominate in PTLD observed in US transplant recipients
that express EBNA2 and EBNA3 (Frank et al. 1995). As precise information about
the distribution of type A and type B viruses in the normal population of different
countries is lacking, it remains unclear whether type 1 and type 2 viruses differ
in the spectrum of diseases they cause or in the frequency with which they cause
them. The current view is that both viruses can cause disease with the same effi-
ciency but we would like to argue that the studied population is much too small to
be able to identify subtle differences. Future large-scale studies that address viral
diversity should be able to readdress these issues.
The division into type 1 and type 2 viruses does not sum up the diversity within
the EBNA2 gene. Sequencing of the EBNA2 locus in a large panel of viruses iso-
lated in diseased or healthy patients from multiple countries have revealed the
existence of different strains described as 1.1a, 1.1b, 1.1c, 1.1d, 1.1e, 1.2, 1.3a,
1.3b, 1.3c, 1.3d, 1.3e (Schuster et al. 1996; Midgley et al. 2003). Strains not
only 1.1a and 1.3b but also 1.3a and 1.2 are more common in Europe, whereas
the strain 1.1b, also known as Wu, and 1.3e, also named Li, are frequent in Asia
(Midgley et al. 2003). Interestingly, only the 1.1b allele was found in Japan
134
Fig. 5 Some of the differences between type 1 and type 2 viruses. It shows the first 227 amino acids of the EBNA2 protein from different virus isolates
that belong to type 1 and type 2 EBV families. The consensus sequence is also shown. The names of type 2 strains are underlined. Dots indicate amino acid
identity. Light gray slashes signify amino acid deletion
R. Feederle et al.
(Sawada et al. 2011). Such a heterogeneity is hardly visible in type 2 viruses. The
type 1 EBNA3 gene family was also submitted to diversification as shown by the
recognition of alleles that co-segregate with EBNA2 in the infected Chinese popu-
lation. Thus, Wu and Li EBNA3A, -B, and -C alleles exist and are tightly linked
to the EBNA2 alleles that bear the same name. In contrast to the EBNA2 1.1b
that is common between isolates from China and Japan, the EBNA3 alleles found
in Japanese healthy and diseased individuals slightly differ. These sub-alleles are
called Wu′ and Li′, Li′′ and differ from their Chinese relatives by one or two point
mutations (Sawada et al. 2011) (Fig. 6a). These polymorphisms, however, did not
cluster with viruses identified from patients with T/NK lymphoproliferations.
6 Polymorphisms in EBNA3B Modulate

the Transformation Abilities of the Virus
In Vitro and In Vivo
Another layer of complexity has been added by the recognition that EBNA3B can
display additional polymorphisms that further influence the transforming poten-
tial of the virus (White et al. 2012). Indeed, B cells transformed with EBNA3B-
negative viruses have a growth advantage in vitro and in vivo in humanized mice
relative to their wild-type counterparts, and this property renders them more tumo-
rigenic in these animals (White et al. 2012). This is concordant with the previous
recognition that EBNA3B is a negative regulator of EBV-induced proliferation.
These data are particularly important because a virus that carries a 245-bp dele-
tion of EBNA3B that presumably inactivates the protein was previously found in
a posttransplant lymphoproliferative disorder (PTLD) that was resistant to T-cell
therapy (Gottschalk et al. 2001). Another PTLD cell line also carried viruses with
a truncation of EBNA3B that resulted from a missense mutation, as did an ABC-
type large-cell lymphoma that arose in an HIV-infected individual (White et al.
2012) (Fig. 6b). Thus, viruses with EBNA3B deletions seem to possess a stronger
transforming potential in immunosuppressed individuals (White et al. 2012). This
fits with the observation that these tumors frequently express the EBNA3 genes
(Rickinson 2007). Additional EBNA3B mutations have been identified in cell lines
or primary EBV-positive tumors such as Hodgkin’s lymphomas or Burkitt’s lym-
phomas whose distribution differs from those observed in non-diseased controls
(White et al. 2012) (Fig. 6b). However, it remains unclear at this stage whether
they have biological and medical consequences, in particular because recombi-
nant viruses that carry these mutations have not been tested yet and because these
tumors are usually EBNA3-negative. Particular EBNA3 alleles could nevertheless
play a role at an early stage of these diseases.
(a)
136
Fig. 6 a An alignment of the sequence of the 315-466 codons from EBNA3B with B95-8 taken as the reference. Multiple polymorphisms that define the
Li, Li′, Li′′, Sp, Sp′, Wu and Wu′ alleles are shown, as are the position of two HLA A11-restricted epitopes. Dots indicate amino acid identity. We have also
indicated the position of multiple epitopes encoded in this region. b EBNA3B mutations and deletions identified in the genomes of viruses isolated from
B-cell tumors or from spontaneous LCLs isolated from healthy individuals (sLCL series). The EBNA3B B95-8 sequence is taken as a reference. The posi-
tion of some virus epitopes is given; dots indicate amino acid identity; light gray dashes indicate amino acid deletion. c Some EBNA3B epitopes identified in
B95-8 as well as their HLA restriction type. The figure also shows polymorphisms within these epitopes encoded by virus strains that have been completely
sequenced. We then focus on the HLA-A11 restricted epitopes AVF and IVT that were found in different geographic areas: C/HK (Canton/Hong Kong), Eur/
R. Feederle et al.
USA (European/USA), NGC (New Guinean coast), PNG (Papua New Guinean), SEA (Southeast Asia). Dots indicate amino acid identity and light gray
dashes indicate amino acid deletion
Fig. 6 (continued)
(b)
Fig. 6 (continued)
Fig. 6 (continued)
(c)
Fig. 6 (continued)
7 Polymorphisms Within Latent Genes are Submitted

to Immune Pressure and Influence the Geographic
Distribution of Virus Isolates
Although it has now been well established that EBV strains around the world are
polymorphic, it remains unclear why these strains are unequally distributed. One
possibility is that the virus is submitted to immune selection, i.e., mutations in
some viral epitopes would alter the immune response in individuals with a given
HLA type. This assertion stems from the observation that the amino acids 416-424
IVTDFSVIK nonamer epitope, and to a lesser extent 399-408 AVFDRKSDAK,
both from B95-8 EBNA3B, are efficiently presented by HLA-A11 (de Campos-
Lima et al. 1993) but that mutations within IVTDFSVIK, particularly at position
2 or 9, nearly completely abrogate the immune response (de Campos-Lima et al.
1993, 1994; Midgley et al. 2003) (Fig. 6c). As a consequence, infected HLA
A11-positive B cells cannot present the EBNA3B allele present in type 2 viruses
or in viruses isolated from Papua New Guinea or China that all have mutations at
this position and/or mutations in position 1 or 2 of the AVFDRKSDAK epitope
(de Campos-Lima et al. 1993). Crucially, in individuals that express HLA-A11,
the cytotoxic response against the virus is mainly directed against this EBNA3B
epitope (Gavioli et al. 1993). This implies that individuals with HLA-A11 can effi-
ciently control viruses such as B95-8 that express IVTDFSVIK or AVFDRKSDAK,
and presumably limit their propagation within the population. This fits with the
observation that viruses whose genomes encode the IVTDFSVIK epitope are rarely
found in populations that express HLA-A11 at high frequency, such as those who
live in South China or in the coastal areas of Papua New Guinea (de Campos-Lima
et al. 1993, 1994; Midgley et al. 2003) (Fig. 6b). Thus, a high frequency of HLA-
A11 is not compatible with a high frequency of B95-8-type viruses. However, this
model does not necessarily imply that Caucasian or African viruses must all be of
B95-8 type. Indeed, Caucasian populations in Australia or from the Papua New
Guinea highlands, only a minority of which is A11-positive, have been reported to
be infected by viruses with mutations in AVFDRKSDAK and or IVTDFSVIK. This
observation in our opinion does not argue against immune pressure. Indeed, the
immune selection model only predicts that a high frequency of infection with B95-
8 type EBNA3B in a population with a high frequency of HLA-A11 is incompat-
ible. However, other authors interpret the equal distribution of the same EBNA3B
alleles, more precisely the ones that are not presented by HLA A11, within PNG
individuals who live in coastal areas and are frequently A11-positive within those
who live in the highlands and are rarely A11 as an indication that immune pres-
sure does not exist (Burrows et al. 1996). This group also extended their analysis
to other EBV epitopes in EBNA3A, EBNA3B and EBNA3C as well as in LMP2A
(Khanna et al. 1997). Furthermore, there was no direct correlation between the
frequency of a particular HLA allele and the distribution of alleles of these viral
epitopes. These data were interpreted by these authors as evidence that amino acid
changes within CTL epitope regions are not influenced by the host immune system.
They did not find any evidence for a higher frequency of non-synonymous versus
synonymous mutations and therefore of selection for particular viral epitopes. We
feel that this argument would be more convincing if it was proven that some of
the epitopes they interrogated were as immunodominant as IVTDFSVIK. If, on the
contrary, the immune response is directed against multiple epitopes presented by
different HLA haplotypes, it would be very difficult to argue for or against anti-
genic drift caused by immune pressure simply by looking at singular epitope HLA
allele combinations. Moreover, Alan Rickinson and his group performed similar
experiments with a panel of 31 virus isolates from China. They found that only the
two A11-restricted epitopes in EBNA3B were under positive selection pressure
(Midgley et al. 2003). It would be interesting to study large communities of HLA
A11-positive individuals who have been living for multiple generations in areas
where B95-8-type viruses are common. If these individuals remain only rarely
infected by B95-8, it would definitely settle the case.
8 The NPC-Associated Virus M81 Is Endowed

with Unique Properties that Distinguishes
It from Other Strains In Vitro and In Vivo
We mentioned earlier that viruses in Southeast Asia, and in particular in the area
around Hong Kong, have been extensively studied for the search of single nucleo-
tide polymorphisms that might explain the high incidence of NPC in these regions
and how these attempts proved altogether rather inconclusive. Nevertheless,
the case for particular EBV strains involved in NPC development is compelling
because of the presence of the virus in epithelial cells, which suggest a tropism of
the virus for this cell type. However, EBV is generally only weakly epitheliotropic
in vitro. Furthermore, individuals who display strong serological titers against
viral proteins involved in replication are at a much higher risk of disease, suggest-
ing that they are infected with strongly replicating strains.
We tested this hypothesis by studying the properties of a virus isolated from a
Chinese patient (M81) in vitro and in vivo in humanized mice (Tsai et al. 2013).
M81 is genetically very close to NPC group I viruses and therefore likely to be
representative of this group of viruses. We found that M81 induces lytic repli-
cation in infected B cells at much higher levels than other EBV isolates in both
experimental settings (Fig. 7). Indeed, infected B cells express all replication
proteins, including BZLF1, gp110, and gp350 at much higher levels than B cells
infected with Akata or B95-8 (Tsai et al. 2013). Furthermore, these cells produce
infectious viruses, although most of these are found bound to neighbor B cells
that express CD21 and CD35 at high level. M81 viruses also infect epithelial cells
more efficiently than other EBV types (Tsai et al. 2013). Figure 1 shows the long
genetic distance between M81 and B95-8, with mutations present in the large
majority of viral genes. Construction of recombinant viruses from highly repli-
cating viruses that carry genes from weakly replicating ones allows identification
Fig. 7 B cells infected

with M81 show high degree
of lytic replication. Upper
picture gp350 staining of an
LCL infected with the M81
strain. The arrow shows a
gp350-positive replicating
B cell, whereas the
arrowhead points to viruses
bound to neighbor B cells.
Lower picture DAPI staining
of the same cells
of the viral proteins that are responsible for the different behavior of strains. This
approach showed that BZLF1 and its promoter play a role in the acquisition of a
highly replicating ability but other genes, probably many, also contribute to the
phenotype (Tsai et al. 2013). The challenge of the coming years will be to identify
the genes that collaborate to influence the viral behavior. Although M81’s proper-
ties explain many clinical features observed in patients with NPC, it remains to be
formally demonstrated that NPC development is due to a particular EBV strain.
This will presumably require an animal model that allows infection of epithelial
tissues in a humanized animal model.
9 Conclusions
The combination of genetic analysis and NGS strategies has allowed identifica-
tion of polymorphisms in EBNA2 and EBNA3B that modulate the immortalizing
potential of the virus, but also of genetic variants that radically change defining
properties of the virus such as cell tropism or ability to replicate in B cells. Some
of these properties are very likely to be involved in the development of EBV-
associated diseases. This would represent a shift in paradigm in which the virus
type is put at the center of the attention, although this does not in anyway exclude
that environmental factors potentiate the effects of infection by particular viral
types. It will be vital in the next years to further increase the number of viral
sequences obtained from both healthy and diseased patients and to clone repre-
sentative examples of these isolates on BACs to allow a functional approach of
these polymorphisms. This should facilitate the definition of genotypic markers
that allow unequivocal identification of the main viral strains and would even-
tually allow the design of a world map that gives the distribution of the differ-
ent isolates. We now know that the distribution of EBV strains is heterogeneous
across the different countries and or populations. It remains a tremendous chal-
lenge to understand in detail the forces that drive this heterogeneous distribution.
The immunological pressure hypothesis remains in our opinion the most attrac-
tive one at this point. Although contested, we feel the EBNA3B/A11 case remains
valid and could explain the relative paucity of B95-8-type isolates in China. There
might be other, so far unknown, dominant epitopes that would explain the distribu-
tion of other viral strains.
This body of knowledge would have important consequences in terms of pre-
vention strategies as vaccination with particular viral type or proteins thereof
might only protect against a particular disease in particular populations. Similarly,
the recognition of viral strains with enhanced transforming potential could have an
important role in the monitoring of immunosuppressed patients who are at risk of
PTLD.
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Part III
Viral Infection and Associated Diseases
EBV Persistence—Introducing the Virus
David A. Thorley-Lawson
Abstract Persistent infection by EBV is explained by the germinal center model

(GCM) which provides a satisfying and currently the only explanation for EBVs
disparate biology. Since the GCM touches on every aspect of the virus, this chap-
ter will serve as an introduction to the subsequent chapters. EBV is B lympho-
tropic, and its biology closely follows that of normal mature B lymphocytes. The
virus persists quiescently in resting memory B cells for the lifetime of the host in
a non-pathogenic state that is also invisible to the immune response. To access this
compartment, the virus infects naïve B cells in the lymphoepithelium of the ton-
sils and activates these cells using the growth transcription program. These cells
migrate to the GC where they switch to a more limited transcription program, the
default program, which helps rescue them into the memory compartment where
the virus persists. For egress, the infected memory cells return to the lymphoe-
pithelium where they occasionally differentiate into plasma cells activating viral
replication. The released virus can either infect more naïve B cells or be amplified
in the epithelium for shedding. This cycle of infection and the quiescent state in
memory B cells allow for lifetime persistence at a very low level that is remark-
ably stable over time. Mathematically, this is a stable fixed point where the mecha-
nisms regulating persistence drive the state back to equilibrium when perturbed.
This is the GCM of EBV persistence. Other possible sites and mechanisms of per-
sistence will also be discussed.
D.A. Thorley-Lawson (*)

School of Medicine, Tufts University, Boston, MA 02111, USA
e-mail: david.thorley-lawson@tufts.edu

152 D.A. Thorley-Lawson
Contents
1 Introduction......................................................................................................................... 153
2 The Germinal Center Model (GCM) of EBV Persistence—A Historical Perspective....... 155
3 EBV Infection in the Healthy Host—A Summary of the GCM......................................... 156
3.1 Crossing the Epithelial Barrier and the Activation/Infection of Naïve B Cells......... 159
3.2 Migration to the Follicle and the Germinal Center (GC) Reaction........................... 164
3.3 EBV Persistence in the Peripheral Memory B Cell Compartment............................ 171
3.4 Viral Replication—Plasma Cell Differentiation,
Stress, and the Role of Epithelial Cells...................................................................... 174
4 The Cyclic Pathogen Refinement of GCM......................................................................... 177
5 The Model of Persistence—A Summary............................................................................ 180
6 Disease Pathogenesis—Insights from the GCM................................................................. 180
6.1 Infectious Mononucleosis—Acute Infection (AIM).................................................. 181
6.2 Autoimmune Disease................................................................................................. 183
6.3 Cancer........................................................................................................................ 184
7 Other Sites of EBV Persistence.......................................................................................... 189
7.1 The Epithelium........................................................................................................... 189
7.2 The Tonsil Intraepithelial (Marginal Zone)
B Cell—A Second Route to Persistence?.................................................................. 189
7.3 GC-Independent Maturation of Infected Naïve Blasts.............................................. 191
7.4 Two Pathways to Persistence?................................................................................... 192
7.5 Direct Infection of Memory Cells.............................................................................. 193
8 Conclusions......................................................................................................................... 194
9 To Be Continued................................................................................................................. 195
10 Final Thought—EBV Is Not As Safe As You Might Think!............................................... 195
References................................................................................................................................... 196
Abbreviations
AID Activation-induced cytidine deaminase

AIM Acute infectious mononucleosis
APOBEC Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like
BAFF B cell activating factor
BCR B cell receptor
BLC B lymphocyte chemoattractant CXCL13
CD40L CD40 ligand
cIg Cytoplasmically expressed immunoglobulin
CPM Cyclic pathogen model
CtBP C-terminal-binding protein
CTL Cytotoxic T cell
DZ Dark zone
eBL Endemic Burkitt’s lymphoma
EBV Epstein-Barr virus
EBNA Epstein-Barr virus nuclear antigen
GC Germinal center
GCM Germinal center model
EBV Persistence—Introducing the Virus 153
HD Hodgkin’s disease
HEV High endothelial venules
HIV Human immunodeficiency virus
IE Immediate early
Ig Immunoglobulin
IL Immunoblastic lymphoma
LZ Light zone
RBPJk Recombining binding protein
RTPCR Real-time polymerase chain reaction
SDF1 Stromal cell-derived factor 1 CXCL12
sIg Surface-expressed immunoglobulin
sBL Sporadic Burkitt’s lymphoma
Th CD4+ T helper cell
1 Introduction
Persistent latent infection for the lifetime of the host is a defining feature of her-
pesviruses. Each herpesvirus has a target tissue(s) in which it persists and each has
evolved a strategy for getting there and back out again. Once at the site of persistent
latent infection, the strategies coalesce in the sense that the goal is to persist latently
within a very small number of cells and to minimize or eliminate viral gene expres-
sion, at least at the protein level. This in turn allows the virus to evade immune reg-
ulation and persist with minimal impact on the host where it will stay for the rest of
its life. Acute infection and viral reactivation to allow spread to new hosts similarly
seem to have evolved for minimal impact on the host. Acute infection should occur
in childhood and is often silent. It is not a coincidence that some of the human her-
pesviruses are so benign and non-pathogenic that they went unnoticed until the age
of AIDS where chronic immunosuppression revealed their presence.
Usually, in the struggle between virus and host, one or the other wins—if it
is the host, the virus is eliminated, for example influenza. Flu goes through an
acute viremic stage and then is cleared within a week or two (Fig. 1a). If the virus
wins, then the host dies, for example HIV. HIV also has an acute viremic stage but
resolves into a low-level infection. However, this is unstable and the virus eventu-
ally returns to kill the host. EBV also has an acute viremic stage that resolves into
a low-level infection, but unlike HIV the virus then simply persists stably at this
very low level (something like 1 infected cell per 5 ml of blood) for the lifetime
of the host (Hadinoto et al. 2009; Khan et al. 1996; Thorley-Lawson and Allday
2008). Mathematically, this is referred to as a stable fixed point. Dynamically, it is
a situation that requires the mechanisms regulating the state (persistent infection)
to drive it back to the fixed point whenever it is perturbed (Fig. 1b). Biologically,
i.e., in the presence of perturbations, a stable fixed point is the only way to achieve
stable long-term behaviors.
Fig. 1 EBV establishes a stable, benign, low-level, lifetime persistent infection. a EBV is a safe
virus. EBV establishes a persistent, benign infection in virtually every human being for their
entire life. This is in comparison with a virus like flu whose infection resolves in a few days or
HIV which undergoes an acute infection that resolves into a long-term low-level persistent infec-
tion that eventually returns to kill the host. EBV also undergoes acute infection but then enters
into a low-level persistent infection which remains stable for the life of the host. b The stable
fixed point. The type of equilibrium EBV achieves is referred to mathematically as a stable fixed
point. This means that the forces regulating the system act to return it to the same place after per-
turbation, e.g., a marble in the bottom of a bowl, whereas in an unstable fixed point, small pertur-
bations irrevocably destroy the fixed point, e.g., a marble on top of the bowl. In real-life biology,
where there are always perturbations, the only way to achieve long-term stability is through a
stable fixed point
EBV is a paradigm for studying the mechanism by which persistent infec-

tion is maintained in vivo. It is an unlikely candidate for this status. We lack an
in vitro lytic system that would allow viral genetics to be studied—the production
of a single viral mutant is a laborious and technically challenging task (Delecluse
and Hammerschmidt 2000). Certainly, no system exists for screening large num-
bers of viral variants and selecting mutants of choice. For a detailed discussion on
the production of EBV recombinants, see the chapter authored by Henri-Jacques
Delecluse. Similarly, we lack a malleable animal model to perform these studies.
The animal models available are limited to primates which are expensive, diffi-
cult to work with, and lacking in sophisticated reagents (Wang 2013) and mouse
models. For a detailed discussion of primate models, see the chapter authored by
Fred Wang, and for mouse models, see the chapter authored by Christian Munz.
Mouse models fall into two classes: reconstitution of genetically immunocompro-
mised mice with human cells (Chatterjee et al. 2014) and studies on the murine
gammaherpesvirus MHV68 (Barton et al. 2011). Of the two systems, the latter has
proved highly tractable for studying and analyzing, at the molecular, genetic, and
immunological level, the basis and details of persistent infection by a gammaher-
pesvirus. Of the human herpesviruses, EBV is the most amenable to study in vivo
because it infects readily accessible tissue, namely B lymphocytes of the lymphoid
tissue (tonsils) and peripheral blood. With the advent of sophisticated and sensi-
tive flow cytometric techniques to characterize lymphoid populations and PCR to
detect very rare infected cells and their gene expression, EBV became accessible
for in vivo study.
2 The Germinal Center Model (GCM) of EBV

Persistence—A Historical Perspective
Epstein-Barr virus was discovered in Burkitt’s lymphoma in 1964. It is a reflec-

tion of the complex and subtle biology of the virus that 50 years later, we are only
just beginning to understand the role of the virus in the development of this tumor
(Speck 2002; Thorley-Lawson and Allday 2008; Vereide and Sugden 2009). By
1999, a large body of work had been accumulated pertaining to EBV’s molecular
and cellular biology, immunology, virology, epidemiology, clinical manifestations,
and disease associations. However, this work existed as a series of independent
pieces of information that did not hang together in a consistent way to explain
viral biology and persistence [for a discussion of the specific issues, see Thorley-
Lawson (2005)].
For example, it has long been known that, unlike most other human herpesvi-
ruses, EBV is able to establish latent persistent infection in tissue culture (Henle
et al. 1967; Pope et al. 1968). The sine qua non of EBV infection in vitro is that
the virus always persists latently in proliferating B lymphoblasts whose growth is
driven by viral latent proteins. This process is often referred to as “immortaliza-
tion.” However, an apparent contradiction arose when it was discovered that the
virus did not persist in this state in vivo but in a diametrically opposite type of cell,
namely quiescent, resting memory B cells where viral protein expression has been
extinguished (Babcock et al. 1998; Hochberg et al. 2004; Miyashita et al. 1997).
The GCM arose to resolve this contradiction (Thorley-Lawson and Babcock
1999) and in doing so provided a way to understand the complex biology of EBV.
It has stood for 15 years and many tests of its reliability and predictive power
(Thorley-Lawson et al. 2013). To date, it remains the only model that consistently
provides a conceptual framework for understanding the complex and subtle behav-
iors of the virus (Thorley-Lawson and Allday 2008; Thorley-Lawson and Gross
2004). It is built on the simple idea that the virus uses the normal pathways of
B cell biology in the lymphoid tissue of Waldeyer’s ring (tonsils and adenoids)
(Fig. 2) to establish infection, persist, and replicate. Today, the questions that arise
are not as to the validity of the general model but the extent to which the virus
goes along for the ride or actively manipulates the process and whether there are
additional mechanisms/sites of viral persistence.
Fig. 2 The lymphoepithelium of the tonsil where EBV performs its biology. a Waldeyer’s
ring consists of the adenoids and tonsils which form a ring of lymphoid tissue at the back of
the throat. b The structure of the lymphoepithelium underlying the saliva. Inset is an expanded
view of the marginal zone/epithelium. B cells exit the circulation and enter the lymphoid tissue
through the HEV and migrate to the mantle zone of the follicle. Here, they reside for a period
of time and then either leave or, if they see antigen, enter the follicle to undergo a GC reaction
which produces memory cells that can then enter the peripheral circulation. This is the B cell
system that EBV exploits. For more details, see Figs. 3 and 4 and text (Figure provided by Marta
Perry)
3 EBV Infection in the Healthy Host—A Summary

of the GCM
A sea change in thinking about EBV was the recognition that under normal condi-
tions, it should not be thought of as an oncogenic virus. This despite its discovery
in and association with tumors and its ability to latently infect B cells in culture
and continuously drive their proliferation. The essence of its biological behavior is
that it initiates, establishes, and maintains persistent infection by subtly using vari-
ous aspects of normal B cell biology and has evolved to minimally perturb the nor-
mal behavior of the infected B cells. A summary of normal mature B cell biology
Fig. 3 EBV biology mirrors B cell biology. To the left is diagrammed a typical mucosal humoral
immune response. Antigen in saliva crosses the epithelial barrier of the tonsil to be sampled by
naïve B cells in the underlying lymphoid tissue. When naïve B cells recognize cognate antigen,
they become activated blasts and migrate to the follicle to undergo a GC reaction. If they receive
signals from antigen and antigen-specific Th cells, they can leave to become resting memory B
cells that occasionally undergo division as part of memory B cell homeostasis. To the right is dia-
grammed how EBV uses the same pathways. EBV is spread through saliva, crosses the epithelial
barrier, and infects naïve B cells. These become B cell blasts that enter the GC. Here, the viral
latent proteins LMP1 and LMP2 have the capacity to provide surrogate antigen and Th survival
signals that allow the latently infected B cells to leave the GC as resting memory cells that also
divide through homeostasis. To the right are listed in orange the transcription programs used at
each stage. The blue circles represent the viral DNA which is a circular episome
and the parallels with EBV is given in Fig. 3, and a full description of the GCM is
presented in Fig. 4. A summary of the steps from Fig. 4 is as follows:
1. Oral antigens enter in saliva, are sampled by the epithelium of Waldeyer’s ring ,
and then presented to naïve B cells in the underlying lymphoid tissue (Fig. 3).
When the naïve B cells see cognate antigen, they become activated into a pro-
liferating blast. Similarly, EBV is spread through saliva contact and crosses the
epithelial barrier of Waldeyer’s ring to interact with naive B cells. Upon infec-
tion of the naïve B cell, it drives the infected cell to become a proliferating
blast using the growth transcription program (a summary of the viral transcrip-
tion programs is provided in Table 1).
Fig. 4 The germinal center model (GCM) of EBV persistence. The stages 1–4 follow those in
the text from Sect. 3. “EBV Infection in the healthy host—a summary of the GCM.” For details,
see the text
Table 1 The latency transcription programs of EBV in vivo

Program Alternate Site in vivo Lymphoma Expressed Proteinsa
Growth Latency 3 Tonsil Naïve B mmunoblastic EBNA1(Cp) EBNA2 EBNA 3A-C LMP1 LMP2
Default Latency 2 Tonsil GC B Hodgkin’s EBNA1(Qp)b LMP1 LMP2
cell
EBNA1 Latency 1 Periphery Burkitt’s EBNA1(Qp)b
only dividing
memory B
Latency Latency 0 Periphery
resting
memory B
aThe noncoding RNAs which includes EBERs and the BART miRNAs are expressed in all nor-
mal and tumor cells irrespective of transcription program. The exception is a subset of BART
miRNAs that are absent from the GC and memory compartments.
bEBNA1 is expressed from the Cp promoter in the growth program but a different promoter (Qp)
in the default and EBNA1 only programs.
2. Antigen-activated naïve blasts migrate into the follicle to initiate a GC reaction

where survival of the B cell requires signals from cognate antigen and antigen-
specific helper T cells. Similarly, EBV-infected naïve blasts migrate into the
follicle where they switch their transcription program to the default program
which provides surrogate antigen and T cell help signals.
3. A successful, antigen-specific, GC B cell leaves the GC to enter the mem-

ory compartment as a resting, long-lived, memory B cell which is sustained
through occasional homeostatic driven division. Similarly, the latently infected
GC B cells leave the follicle as resting memory B cells which are quiescent
with respect to viral latent protein expression (the latency transcription pro-
gram). These cells occasionally divide in the periphery. Proliferation is not
driven by the virus but by the normal memory homeostatic mechanisms. At this
time, the virus expresses the genome tethering protein EBNA1 which allows
the viral genome to replicate with the cells (EBNA1 only program).
4. Antigen-specific, memory B cells in lymphoid tissue can be signaled by cog-
nate antigen to terminally differentiate into plasma cells and produce antibody.
Similarly, if an infected, resting, memory B cell latently infected with EBV returns
to Waldeyer’s ring and receives signals that initiate terminal differentiation, it will
trigger the release of infectious virus. The released virus can initiate a new round
of naïve B cell infection or infect the epithelium. This results in transient plaques
of lytic epithelial infection that greatly amplifies the amount of infectious virus
that ultimately is shed into saliva for infectious spread to new hosts.
In this model, EBV gene expression is tightly regulated in a tissue-specific fash-
ion. Dysregulation can lead to lymphomas which arise from each of the three pro-
liferative stages of EBV infection predicted by the model. It is the context and
location combined with the stage-specific viral transcription program that defines
the lymphoma (Fig. 5). These are immunoblastic lymphoma (IL) from cells
expressing the growth program (new infection), Hodgkin’s disease (HD) from
cells expressing the default program (GC cells), and Burkitt’s lymphoma (BL)
from cells expressing EBNA1 only (late GC cell).
The following sections will discuss evidence and relevant information for each
of these 4 steps in more detail.
3.1 Crossing the Epithelial Barrier and the Activation/Infection

of Naïve B Cells
3.1.1 Crossing the Epithelial Barrier
It is generally believed that EBV is spread through salivary contact (Hoagland

1955) and that the virus enters through the epithelium that lines the nasophar-
ynx. The lymphoid system that surrounds the nasopharyngeal region includes the
adenoids and tonsils and is called Waldeyer’s ring (Fig. 2a). Together with the
overlying epithelium, it forms a continuous structure referred to as the lymphoe-
pithelium (Fig. 2b) (Perry and Whyte 1998). The epithelium is invaginated to form
crypts below which resides the lymphoid tissue (Perry 1994; Tang et al. 1995).
Deep in the crypts, the epithelium can be only a single epithelial cell in thickness.
Environmental antigens are sampled directly through the epithelium (Perry and
Whyte 1998; Brandtzaeg et al. 1999a, b). The involuted nature of the crypts allows
Fig. 5 The origin of EBV-positive lymphomas. EBV lymphomas arise from different stages of
the infection process. The figure shows diagrammatically the flow of virus from infectious virions
to latently infected resting memory B cell as detailed in Figs. 3 and 4 and the text. To the right are
shown the 3 EBV-associated lymphomas and their proposed origin and to the left are listed the
viral transcription programs expressed in the tumors and at the equivalent stage of infection. IL is
proposed to arise from a latently infected blast that is unable to differentiate and so continues to
proliferate. HD is derived from a GC B cell, and BL is a GC cell that has left the follicle. Note that
a tumor is proposed to arise from each of the three stages of EBV biology that involve proliferation
for a massive surface area for detecting antigens as they come in with food and,
when exposed to EBV-bearing saliva, provides a large target for EBV infection.
It is likely that the virus, in saliva, enters the crypts and crosses the thin layer of
epithelial cells to infect naïve B cells that reside below. The sponge-like nature and
deep invaginations of the crypts ensure that all of the lymphocytes in the under-
lying lymphoid tissue are effectively close to the surface where EBV crosses the
epithelium. How the virus crosses the epithelial barrier is unclear although there is
evidence that the virus may cross passively via transcytosis (Tugizov et al. 2013).
It has been speculated that the virus actually infects the epithelial cells, replicates,
and then is released to infect B cells in the underlying areas, but there is no direct
evidence for this and epithelial cells appear to be resistant to infection from the
apical (i.e., mucosal) side (Tugizov et al. 2003).
3.1.2 The Activation/Infection of Naïve B Cells
As far as we know whenever EBV encounters and infects a resting B cells, it

always latently infects it and uses the growth program to drive that cell to become
a proliferating lymphoblast (Thorley-Lawson and Mann 1985). Phenotypically,
the newly infected B cells look remarkably like antigen-activated B cell blasts
(Thorley-Lawson et al. 1982, 1985; Nilsson 1979); however, in this case, the B
cell is activated not through interaction with antigen and T cell help but through
the activity of the latent proteins encoded by the growth program (Kempkes et al.
1995). The population that expresses the growth program in the tonsils of healthy
carriers of the virus is activated, naive B cells (Joseph et al. 2000a; Babcock et al.
2000). These cells express CD19 (B cell lineage marker) CD23 and CD80 (B cell
activation markers) and IgD (a marker of naïve B cells) and all of the latent pro-
teins associated with the growth program. They lack CD10 (GC cell marker) and
CD27 (memory B cell marker). Therefore, the target of the incoming virus is the
resting naive B cell. This is the first example we will encounter of a latent gene
transcription program used in lymphoma, being found in a normal infected B cell
counterpart in vivo. In this case, the growth program, which is used in immunoblas-
tic lymphoma (IL), is found in newly infected naïve B cell blasts (Table 1, Fig. 5).
Naive B cells continuously recirculate throughout the body. They extravasate
from the peripheral circulation into secondary lymphoid tissue such as the ton-
sils through specialized structures called high endothelial vesicles (HEVs) which
reside in the lymphoepithelium (inset in Figs. 2b and 6). The naïve B cells migrate
through the epithelium to the mantle zone (Fig. 2b) of the follicles which resides
just below the epithelium. They remain there for a few days and then reenter the
circulation (Brandtzaeg et al. 1999a) unless they encounter cognate antigen in
which case they migrate into the follicle.
The migration of naïve B cells from HEV in the epithelium to the mantle zone
is critical for them to become exposed to the virus. This is because microdissec-
tion studies reveal that virus production and infection of new naive B cells occur
in the intraepithelial layer not the mantle zone (Roughan et al. 2010). Thus, naive
B cells are becoming infected, as they traverse the epithelium, by EBV that has
either crossed the epithelial barrier during primary infection or been produced by
the lymphoepithelium during persistent infection (Fig. 6). It follows that by the
time the infected B cell arrives at the follicle, it will already be a blast so will not
migrate to the mantle zone.
3.1.3 The Growth Program
Because the target for EBV infection is a resting cell, the virus must initiate latent
gene transcription in a quiescent environment. It infects cells through the inter-
action of the viral glycoproteins gp350/220 with CD21 (Nemerow et al. 1985;
Fingeroth et al. 1984) and gp42/gH/gL with MHC class II on the B cell (Li et al.
1997). For a detailed discussion of viral entry, see the chapter authored by Lindsey
Hutt-Fletcher. CD21 is a receptor for C3d (a component of complement) and forms
part of a multimeric signal transduction complex with CD19, CD81 (TAPA-1),
and Leu-13 (Matsumoto et al. 1993). The high density of gp350/220 on the virion
(Thorley-Lawson and Poodry 1982) ensures that the binding of viral particles will
cause extensive cross-linking of the CD21 signaling complex which provides the
signal to begin moving the resting B cells from G0 into the G1 phase of the cell
Fig. 6 The first steps of EBV infection. Naïve B cells emerge from the HEV and migrate toward
the mantle zone of the follicle. On the way, they encounter EBV that either has crossed the epi-
thelial barrier or is derived from lytically infected plasma cells. The newly infected lymphoblast
upregulates the chemokine receptor EBI2 and follows a gradient of oxysterol chemokine into the
follicle
cycle (Sinclair and Farrell 1995). During this time, the earliest expressed latent
protein (EBNA2) is detected (Allday et al. 1989; Rooney et al. 1989). This pro-
tein is expressed from a promoter (Wp) that is present in multiple copies in the
viral genome and may be designed to function in the transcriptionally sparse envi-
ronment of a resting B cell (Woisetschlaeger et al. 1990). EBNA2 drives the cells
through the first G1 (Sinclair et al. 1994). EBNA2 is a transcription factor that acti-
vates the promoters necessary to produce all nine of the latent proteins expressed
in the growth program [reviewed in Kieff and Rickinson (2007)]. For a detailed
discussion of EBNA2, see the chapter authored by Bettina Kempkes. At this point,
transcription of the EBNA2 gene switches from Wp to Cp (Woisetschlaeger et al.
1990), a promoter that works optimally in B lymphoblasts and allows expression of
all the EBNA proteins. The result is that infected normal B cells become activated
lymphoblasts and begin to proliferate in response to the actions of viral latent pro-
teins. Although they should not be thought of as classically transformed cells, such
as are obtained with other DNA tumor viruses (e.g., SV40, papillomavirus, and
adenovirus) (Allday et al. 1995), EBV-driven cells are not completely normal either
as evidenced by deregulation of their cell cycle control that can result in immortal
growth in culture (O’Nions and Allday 2003; Wade and Allday 2000) [reviewed in
Allday (2013), O’Nions and Allday (2004)]. Thus, they rather should be thought
of as undergoing a hyperplastic or preneoplastic proliferation that can develop into
full-blown neoplasia if allowed to proceed unchecked and accumulate additional
oncogenic mutations. However, at this point, it is necessary to mention an impor-
tant caveat to these studies. Almost all have been conducted with the B95-8 strain
of EBV that is often referred to as the “wild-type” “strain.” In fact, this is not a
wild-type strain, but a highly defective laboratory strain that is carried in marmoset
cells and was selected for its ability to transform those cells in culture and make
them oncogenic in marmosets, not a natural host for the virus. This virus has multi-
ple genomic deletions (Raab-Traub et al. 1980) among which are those that deregu-
late expression of the major glycoproteins (Edson and Thorley-Lawson 1981) and
delete virtually all of the miRNAs (Skalsky et al. 2012). The latter, in particular, are
of concern for interpreting studies on how EBV makes B cells grow and how and
to what extent the virus deregulates cell cycle controls.
The nine latent proteins of the growth program include six nuclear proteins
(EBNAs—Epstein-Barr virus nuclear antigens—1, 2, 3a, 3b, 3c, and LP) and three
membrane proteins (LMPs—latent membrane proteins) [reviewed in Kieff and
Rickinson (2007) but see this textbook for the most recent information]. Several
of the latent proteins have potent growth-promoting activity and can act as onco-
genes. These include EBNA2 (Kempkes et al. 1995), EBNA3a (Hickabottom et al.
2002), EBNA3c (Parker et al. 1996), and LMP1 (Wang et al. 1985).
In addition to the nine latent proteins, EBV-infected lymphoblasts express two
small non-polyadenylated RNAs, termed EBER1 and EBER2 (Arrand and Rymo
1982), and ~40 microRNAs. Neither EBERs nor the miRNAs are essential for
EBV infection in vitro, suggesting that their functions are most important in vivo
(Kuzembayeva et al. 2014). For a detailed discussion of EBV-encoded non-trans-
lated RNAs, see the chapter authored by Bryan Cullen.
The latent genes are transcribed from the viral genome which exists as a cova-
lently closed episomal circle (Adams and Lindahl 1975). For a detailed description
of genomic structure, see the chapter authored by Paul Farrell. The linear genome
from the virion forms this circle when the newly infected cell begins proliferating
(Hurley and Thorley-Lawson 1988). Interestingly, only a single episome forms
upon initial infection, but this then begins to amplify over time as the infected cells
proliferate till a steady-state distribution of episomes is found in cells that have pro-
liferated extensively (Hurley and Thorley-Lawson 1988; Roughan et al. 2010). The
forces that produce this distribution are not well understood (Nanbo et al. 2007),
but it serves as a useful marker to distinguish cells that have been recently infected
from ones that have proliferated extensively. Thus, the status of the viral genome
in a tissue provides a considerable amount of useful information. Linear genomes
indicate viral replication, whereas episomal genomes, in the absence of linear
genomes, are indicative of latently infected cells (Decker et al. 2001) and the episo-
mal copy number is a measure of proliferation history (Roughan et al. 2010).
The viral growth program has evolved to drive the activation and proliferation of
new latently infected human B cells. It achieves this, not through some rare random
event, such as the integration of the viral genome and disruption of cellular genes
employed by retroviruses, but by a highly intricate transcriptional program that is
uniquely designed to control the growth of human B cells. This ensures that EBV
will efficiently and predictably establish latency and initiate cell growth whenever
it encounters a resting naive B cell in the lymphoepithelium of the nasopharynx.
This program puts the host, in which the virus intends to persist, at risk for devel-
oping neoplastic disease (see Sect. 6.3.1), but it is essential, so the virus can drive
the newly infected cell into a state, the proliferating blast, from where it can differ-
entiate into a resting memory B cell. Once there, the virus can shut down, become
non-pathogenic, and persist for the life of the host. How does an antigen-activated
B blast and, by analogy, the EBV-infected B blast become a resting memory B cell?
3.2 Migration to the Follicle and the Germinal

Center (GC) Reaction
To understand how latently infected, naive B lymphoblasts expressing the growth

program can become resting memory B cells, with no viral gene expression, it is
first necessary to describe how a normal naive B cell blast becomes a memory cell.
3.2.1 Entering the Follicle
Naïve B cells, activated by antigen, migrate toward the GC following a gradient

of the oxysterol lipid 7a,25-dihydroxycholesterol. This lipid is produced by fol-
licular lymphoid stromal cells and is recognized by the chemokine receptor EBI2,
also known as G protein-coupled receptor 183, on the activated B cell (Gatto and
Brink 2013). When EBV activates the newly infected naïve B cell with the growth
program, one of the phenotypic changes it causes involves induction of EBI2
(Birkenbach et al. 1993), thus insuring that the virus-infected blasts will migrate
toward the follicle (Fig. 6).
3.2.2 The Germinal Center Reaction
Once an antigen-specific B cell enters the follicle as an activated blast, it undergoes

a period of rapid expansion for about 3 days, with a cell division time ~8–12 h to
form the GC which consists of antigen-specific B cells (Figs. 2 and 3) [reviewed
in Liu and Arpin (1997), MacLennan (1994), Victora and Nussenzweig (2012)].
These cells loose surface IgD and acquire GC-specific markers including CD10,
CD77, and CD38 and they express AID and bcl-6. AID is an enzyme of the
APOBEC family that is highly expressed in GCs. It is the enzyme necessary to
initiate somatic hypermutation (SHM) and class switch recombination (CSR)
(Muramatsu et al. 2007), functions of the GC. bcl-6 on the other hand is the mas-
ter transcription factor of the GC (Basso and Dalla-Favera 2010). Its expression
is restricted to GC cells (Cattoretti et al. 1995), it is required for GC production
(Ye et al. 1997), and its downregulation is essential for B cells to leave the GC
(Calame et al. 2003). When proliferating, the cells reside in the dark zone (DZ)
of the germinal center and are referred to as centroblasts. Here, the cells undergo
CSR to express a single isotype, which can be IgM, IgG, IgA, or IgE and they
also undergo SHM. After several divisions, the cells rest and migrate to the light
zone (LZ) of the GC. These cells are referred to as centrocytes, and they com-
pete for help delivered by antigen-specific T helper (Th) cells (Schwickert et al.
2011). The Th cell delivers its rescue signal to the B cell through the interaction of
CD40 ligand on Th cells with CD40 on B cells (Banchereau et al. 1994). Signaling
through CD40 also turns off expression of bcl-6 and turns on bcl-2 which allows
the cell to leave the GC and differentiate (Calame et al. 2003).
Cells in the GC go through multiple rounds of proliferation, migration, and
selection so that ultimately those expressing the highest affinity B cell recep-
tor (BCR) are selected—a process referred to as affinity maturation. Migration
between the light and dark zones is controlled through the expression of specific
chemokine receptors CXCR4 and CXCR5 and their cognate ligands (SDF1 and
BLC, respectively) (Allen et al. 2004). The cells that survive ultimately have two
fates depending on the length and type of exposure to Th cells and specific lym-
phokines (Banchereau et al. 1994). They can either terminally differentiate into
antibody-secreting plasma cells or enter the long-lived memory compartment as
resting isotype-switched memory B cells. As the name implies, these cells carry
immunological memory and are responsible for a heightened secondary response
upon reexposure to the specific antigen.
Unswitched, IgM+/IgD+, memory cells also exist, but they do not arise
through the GC (Weill et al. 2009; Weller et al. 2004). These are generally referred
to as marginal zone memory B cells because they were originally described in

the marginal zone of the spleen (Spencer et al. 1985, 1998) and in the circulation
(Weller et al. 2004). A phenotypically related population has also been described
in the epithelium of the tonsil (Dono et al. 2003; Spencer et al. 1998); however,
they appear to be functionally distinct (Weill et al. 2009).
What is clear then is that a series of events must occur if an EBV-infected naive
B lymphoblast, expressing the growth program, is to become a memory cell. First,
the cells should enter the GC where the latent genes that drive proliferation are
turned off, and then the cells must receive the requisite survival signals and finally
leave as resting memory B cells.
3.2.3 EBV-Infected Cells Reside and Participate in the GC
Newly infected B cells are driven by the growth program to undergo an ini-
tial phase of rapid expansion with a division time of ~8 h for ~3 days—closely
mimicking the dynamics of the early phase of GC development (Nikitin et al.
2010; Thorley-Lawson and Strominger 1978). In vitro, such cells then switch to
long-term indefinite proliferation as lymphoblasts with a division time of ~24 h.
However, in vivo, the cells do not continue to proliferate driven by the growth pro-
gram; instead, they become GC cells and switch to a more limited form of viral
gene expression—the default program.
Cells in the GC latently infected with EBV are by all measures true GC B cells.
They express the classic GC surface phenotype CD10+, CD77+, CD38+, the
functional markers AID and bcl-6 (Roughan and Thorley-Lawson 2009), and the
correct set of chemokine receptors being CXCR4+ CXCR5+ and CCR7−. The lat-
ter ensure that the cells will be retained in and migrate throughout the germinal
center. They are positive for the proliferation marker Ki67 and undergo multiple
rounds of cell division (≥20) (Roughan et al. 2010). Despite this, microdissection
studies revealed that there are only on average 3–4 latently infected cells per GC
(for reference, there are about 105 total B cells in a typical GC). Consequently, the
vast majority of latently infected cells produced from the GC must die; otherwise,
the memory compartment would be overwhelmed. This death could represent
some version of affinity maturation/selection (if the emerging memory cells are
truly antigen-selected) or simply destruction by CTL. However, functional CTLs
do not appear to enter GCs (Quigley et al. 2007), so the cells would have to be
continuously leaving and then killed.
Taken together, these data imply that latently infected B cells in the GC are
truly undergoing a GC reaction, that the virus is having a minimal impact on the
process and the cells may even be undergoing some form of affinity maturation
and selection. Confirmation of this mechanism has come from studies with another
B lymphotropic gammaherpesvirus: MHV68 in the mouse. The ability to geneti-
cally manipulate both host and virus in this system has allowed for a direct and
convincing demonstration that latently infected B cells traverse the GC in order to
enter memory (Barton et al. 2011; Collins and Speck 2014).
3.2.4 EBV-Infected Cells in the GC Express the Default

Not the Growth Program
Microdissection and flow cytometric analysis have provided compelling and une-
quivocal evidence that the EBV-infected cells in the GC express the default pro-
gram not the growth program (Babcock et al. 2000; Roughan and Thorley-Lawson
2009). The demonstration that infected GC cells express the default program
means that this latency transcription program is consistent with the retention of
GC phenotype and functionality in vivo. This is crucial because it identifies the
critical intermediate between the lymphoblastoid growth program and the resting
memory B cells. It is known that direct infection and the growth program ablate
GC functionality and phenotype, i.e., they are not consistent with GC function
(Babcock et al. 2000; Siemer et al. 2008). Thus, for a newly infected naïve blast
to differentiate into memory, it must switch to the default program in the GC. This
is the second example we will encounter of a latent gene transcription program
used in lymphoma, being found in a normal infected B cell counterpart in vivo. In
this case, the default program, which is used in Hodgkin’s disease (HD), is found
in latently infected GC cells (Table 1, Fig. 5). The default program involves only
three of the nine latent proteins, EBNA1, LMP1, and LMP2a (Kieff and Rickinson
2007; Thorley-Lawson 2001). Here, the Q promoter (Qp) is employed so that
EBNA1 may be expressed without the other EBNAs (Tsai et al. 1995; Schaefer
et al. 1995; Nonkwelo et al. 1996). EBNA1 is essential because it is required for
retaining the viral genome by tethering it to cellular DNA and allowing it to be
replicated (Yates et al. 1985). For a detailed discussion of EBNA1, see the chapter
authored by Lori Frapier.
3.2.5 Turning Off the Growth Program
When EBNA2 is turned off in the presence of an activated c-myc, which is

expressed in GC cells (Dominguez-Sola et al. 2012; Martinez-Valdez et al. 1996),
the cells downregulate surface markers’ characteristic of B blasts, such as CD23,
and acquire GC-specific markers, such as CD10 (Polack et al. 1996). Therefore,
the infected lymphoblast appears free to acquire a GC phenotype once the dif-
ferentiation block, imposed by EBNA2, is removed. One of the direct targets of
EBNA2 is c-myc, a known regulator of cell growth and apoptosis (Kaiser et al.
1999). We can assume therefore that upon arrival in the follicle, the EBV lympho-
blast receives a signal that turns EBNA2 and the growth program off while allow-
ing c-myc expression to continue. How this is achieved remains unknown, but
there is in vitro evidence to suggest that it may depend in part upon signals origi-
nating in the GC from cytokines such as IL-10, IL-21, and Type 1 IFN in combi-
nation with CD40 ligand (CD40L) (Kis et al. 2006, 2010; Salamon et al. 2012).
The actual mechanism by which cells switch from the growth to the default
program probably depends on a negative feedback loop involving EBNA2 and the
EBNA3s. These are believed to act as functional homologues of the intracellular
components in the Notch signaling pathway (Kempkes et al. 1995; Speck 2002).
For a detailed discussion of this hypothesis, see Thorley-Lawson and Allday
(2008), and for a review of the Notch system, see Artavanis-Tsakonas et al.
(1995). Upon infection of B cells, the first viral protein expressed is EBNA2
which interacts with the enhancer elements of cellular and viral latent genes to
block differentiation and drive cellular proliferation. At the same time, EBNA2
activates the major EBV latent promoter Cp which leads directly to expression of
all the EBNAs including EBNA3a and 3c. For a detailed discussion of the EBNA3
proteins, see the chapter authored by Martin Allday. Based on their known func-
tions, EBNA3a and 3c could displace EBNA2 from Cp (Zimber-Strobl and Strobl
2001) and recruit repressor proteins that would lead to the stable epigenetic silenc-
ing of Cp and the suppression of EBNA2 production (Hickabottom et al. 2002;
Knight et al. 2003; Radkov et al. 1999; Touitou et al. 2001). For a detailed dis-
cussion of EBV-associated chromatin and epigenetics, see the chapters authored
by Paul Lieberman and Wolfgang Hammerschmidt. Cessation of EBNA2 pro-
duction would cause growth arrest and allow the cells to assume a GC phenotype
and express the default program. In this model, growth driven by EBV is a self-
regulating feedback loop involving EBNA2 and the EBNA3s where the balance is
tilted in favor of growth arrest by signaling from T cell-associated cytokines and
CD40L (Kis et al. 2006, 2010; Salamon et al. 2012). It follows that the in vitro
phenomenon of immortalization may be a biological artifact where the balance has
been shifted in favor of EBNA2 by the absence of T cell-derived signals and the
powerful selection pressure of in vitro growth.
3.2.6 EBV Can Provide the Rescue Signals—LMP1 and LMP2
Once the growth program is turned off, we have good evidence that the expression
of LMP1 and LMP2 in the default program is capable of providing the two sig-
nals, T cell help and BCR, necessary to rescue the GC cell into memory.
LMP1 is a membrane protein that acts as a ligand-independent, constitutively
activated receptor (Gires et al. 1997). For a detailed discussion of LMP1, see the
chapter authored by Arnd Kieser. It does this by engaging signaling molecules
(Izumi and Kieff 1997; Mosialos et al. 1995) which normally transmit signals
from CD40 when it engages its ligand on Th cells [reviewed in Lam and Sugden
(2003)]. Thus, in principle, LMP1 is able to deliver a Th signal to the infected
B cell in the absence of Th cells. The parallel between CD40-mediated Th and
LMP1 signaling extends to the ability of LMP1 to drive immunoglobulin class
switching (He et al. 2003; Uchida et al. 1999). LMP1, like CD40, also turns off
expression of bcl-6 (Panagopoulos et al. 2004) and turns on bcl-2 (Henderson
et al. 1991). Through its ability to regulate bcl-2 and bcl-6, LMP1 (Carbone et al.
1998) almost certainly plays a role in driving the latently infected B cell to leave
the GC and differentiate into a memory cell (Fig. 7).
LMP2 is also a membrane protein, but it delivers a constitutive, ligand-inde-
pendent BCR signal (Caldwell et al. 1998). For a detailed discussion of LMP2, see
the chapter authored by Richard Longnecker. LMP2a contains the same signaling
Fig. 7 A summary of the functions of LMP1 and LMP2a demonstrated in vitro or in vivo with
transgenic mice that could contribute to the GC processing of a latently infected B cell
motifs (ITAMs) (Beaufils et al. 1993), as the α- and β-chains of the BCR. These
motifs allow it to engage signaling molecules employed by the BCR (Miller et al.
1995; Kurosaki 1999). The BCR produces two types of signals (MacLennan
1998): One (tonic) is required to ensure the survival of resting B cells (Lam et al.
1997; Maruyama et al. 2000), while the other (activating) leads to cellular acti-
vation, proliferation, and ultimately differentiation into immunoglobulin-secreting
plasma cells (MacLennan 1994; Liu and Arpin 1997). LMP2a is able to provide
the tonic but not the activating signal (Caldwell et al. 1998) and in the absence of
a BCR is able to drive GC formation in mucosal tissue where the cells show evi-
dence of having undergone mutation of their immunoglobulin genes (Casola et al.
2004b). Thus, LMP2a almost certainly plays a role in driving the latently infected
B cell into and through the GC (Fig. 7).
In sum, LMP1 and LMP2a have the capacity to provide the latently infected B
cell with a whole range of signals associated with GC development (Fig. 7).
3.2.7 Does EBV Do It All—The Conundrum of LMP1 and LMP2
One critical remaining question is: does EBV do it all? The signaling properties
of LMP1 and LMP2 imply that together they could potentially provide all the
signals necessary to rescue a latently infected B cell from the GC into memory,
bypassing the normal mechanisms of antigen selection. If so, the immunoglobu-

lin genes of latently infected memory B cells should either be unmutated or show
an unselected pattern of mutations. However, the expressed immunoglobulins in
latently infected memory B cells from the blood have undergone CSR, have no
stop codons, and display the SHM pattern expected for antigen-selected memory
cells (Souza et al. 2005, 2007). Thus, it seems that the expression of LMP1 and
LMP2 has little discernible impact on the selection process as EBV-infected cells
transit the GC into memory.
Experiments involving the expression of either LMP1 or LMP2 in the B cell com-
partment of transgenic mice indicate that alone these molecules can have devastat-
ing physiologic effects. In such studies, LMP1 could exclude B cells from the GC
(Uchida et al. 1999) and even drive the development of B cell lymphomas (Kulwichit
et al. 1998). LMP2 on the other hand was shown to replace the BCR allowing BCR-
negative B cells to survive and enter the periphery (Caldwell et al. 1998) (a particu-
larly relevant observation for Hodgkin’s lymphoma see below) and in some models
break tolerance allowing autoreactive cells to survive in the periphery (Chang et al.
2012; Swanson-Mungerson and Longnecker 2007; Swanson-Mungerson et al. 2005).
These observations suggest that deregulated expression of LMP1 or LMP2 may play
an important role in the pathogenesis of lymphoma and autoimmune disease devel-
opment but seemed strangely at odds with the striking lack of B cell lymphoma and
autoimmunity in the vast population of EBV-infected people. However, in humans,
LMP1 and LMP2 are usually expressed together and a follow-up study on double
transgenic mice revealed that now the mice did not develop lymphoma or autoim-
mune disease and their B cells were able to comfortably transit the GC, undergo
affinity maturation, and enter the memory compartment (Vrazo et al. 2012).
Thus, it seems that LMP1 and LMP2, when coexpressed in vivo, can modu-
late each other’s signaling. For example, in vitro, LMP1 when expressed alone
can downregulate bcl-6 (Panagopoulos et al. 2004) and upregulate bcl-2, yet in the
GC, LMP1 expression, in the presence of LMP2, is fully compatible with bcl-6
expression and is not associated with the upregulation of bcl-2 (Roughan and
Thorley-Lawson 2009). What then is the role of these proteins in the GC? Because
their functions are so tuned to the requirements of the GC and they are specifically
expressed there, it seems certain that they must play some important role. What
could this be? A clue comes from the analysis of the small subset of bcl-2-positive
cells in the GC, those about to leave, which revealed that they only express LMP1,
not LMP2, i.e., LMP1 expression in vivo, just as in vitro, is associated with upreg-
ulation of bcl-2 but only in the absence of LMP2. It seems likely therefore that
the expression of LMP1 or LMP2 alone in the GC is strictly regulated to occur
only at specific moments to achieve specific ends. Based on what we know so far,
LMP2 expression alone in latently infected cells would ensure that the cells form
GCs in mucosal epithelium; LMP1 and LMP2a together drive CSR and SHM and
provide the requisite survival signals, and LMP1 alone ensures exit from the GC
and terminal differentiation by switching off bcl-6 and switching on bcl-2 (Fig. 7).
To test this hypothesis will require careful dissection of infected GC populations.
Previous attempts at this showed no differences (Babcock et al. 2000; Roughan
and Thorley-Lawson 2009), but were based on the now discredited marker CD77
(Victora et al. 2010) and were therefore artifacts. Recently, an accurate phenotype
for GC subsets has been described (Victora and Nussenzweig 2012; Victora et al.
2010), making these studies now feasible.
3.3 EBV Persistence in the Peripheral Memory

B Cell Compartment
How the transition from GC to resting long-lived memory B cell is achieved for
any cell is not fully understood, but we may assume that once the mechanism is
uncovered, we will find that the virus exploits it to gain access to the memory
compartment.
3.3.1 The Resting Memory B cell
EBV, in the peripheral blood, is found only in B cells (Miyashita et al. 1995) that
have the phenotype expected of a latently infected, long-lived, GC-derived, rest-
ing, memory B cell, i.e., classical memory B cells (Table 2) (Babcock et al. 1998;
Decker et al. 1996; Joseph et al. 2000b; Miyashita et al. 1997). Persistence in
memory B cells, first demonstrated for EBV, may be a common strategy for all B
lymphotropic gammaherpesviruses (Barton et al. 2011). Restriction of EBV in the
periphery to the GC-derived memory compartment is so tight that less than 1 in
10,000 latently infected cells in the blood are in the naïve compartment (Hochberg
et al. 2004). They have the phenotypic hallmarks of classical GC-derived memory
B cells being CD27+ (Joseph et al. 2000b; Klein et al. 1998) and having under-
gone CSR and SHM (Babcock et al. 1998; Joseph et al. 2000b; Souza et al. 2007).
They are also CD23− and CD80− (B cell activation markers) (Miyashita et al.
1995), and >90 % are in the G0 stage of the cell cycle (Miyashita et al. 1997;
Hochberg et al. 2004) all characteristics of resting B cells.
The latently infected cells occupy a skewed niche within the memory compart-
ment, being excluded from the IgD+ memory subset, but otherwise are evenly
distributed among B cells carrying the different immunoglobulin isotypes. This
Table 2 Phenotype of EBV Infected Cells in the Blood

Phenotype Implication
CD19+, CD20+, CD3- B Cell
CD23-, CD80-, Ki67-, G0 stage of cell cycle Resting Cell
CD27+, Ig genes hypermutated and class switched GC Derived Memory Cell
IgD- Not marginal zone B cells
CD5- Not B1 cells
Episomal viral genomes, no linear form Latently Infected
suggests that once they enter memory, the EBV-infected cells cannot be distin-
guished from uninfected cells by host homeostasis mechanisms. The pattern of
SHM (Souza et al. 2005) they display is that expected for antigen-selected mem-
ory cells (Souza et al. 2005). They tend to accumulate more mutations than their
uninfected counterparts and actually showed a reduced proclivity to be self-reac-
tive (Tracy et al. 2012). However, these differences were modest and may sim-
ply reflect differences between mucosal (EBV+) and splenic (peripheral) derived
memory B cells. What is apparent though is that EBV does not significantly dis-
rupt the normal processing of latently infected cells into memory. Deviations from
normal B cell biology are not tolerated in these cells despite the potentially potent
signaling capacities of LMP1 and LMP2.
EBV is not found in the CD5+ B1 subset (Joseph et al. 2000b), nor in circulat-
ing IgD+/IgM+/CD27+ marginal zone memory cells (Joseph et al. 2000b; Souza
et al. 2007). These are both long-lived compartments of B cells (Youinou et al.
1999; Kantor 1991) that frequently have specificity for polyantigens such as bacte-
rial cell wall components (Hardy 2008) but neither of which develop through GCs.
The absence of EBV from these subsets provides further support for the conclu-
sion that transit of the GC is required for the production of memory B cells latently
infected with EBV. Studies claiming to find EBV preferentially in IgA-bearing B
cells (Ehlin-Henriksson et al. 1999) or in IgD+ memory cells (Chaganti et al. 2009)
were technically flawed and have not been substantiated [for a detailed discussion of
the issues, see Joseph et al. (2000b) and Thorley-Lawson et al. (2013), respectively].
Memory cells latently infected with EBV in the peripheral blood do not express any
of the known latent proteins (Hochberg et al. 2003a; Hochberg and Thorley-Lawson
2005). This is an important point to stress. Several studies have identified EBV latent
gene expression in the peripheral blood based on RT-PCR analysis. However, these
were not quantitative studies and were performed on bulk preparations of B cells
(Babcock et al. 1999; Chen et al. 1995; Qu and Rowe 1992; Tierney et al. 1994).
Because the assays used are so sensitive and variable in their sensitivity, it is impossi-
ble to know whether the signals are from rare infected cells expressing the transcript or
are representative of the whole infected population of cells. It turns out that the former
is true. By performing a limiting dilution RT-PCR analysis (Hochberg et al. 2003a;
Hochberg and Thorley-Lawson 2005), it was possible to show that >99 % of the
infected cells do not express transcripts for any of the known latent proteins. Indeed,
the single-cell analysis afforded by this approach revealed that when latent gene tran-
scripts were found, they were not part of any known transcription programs, indicating
that they almost certainly are residual transcripts of no biological significance.
We may conclude therefore that the memory B cell is the site of long-term viral
persistence. Here, it can remain for the lifetime of the host because immunologi-
cal memory is for life, but the virus is no longer pathogenic to the host because the
genes that drive cellular proliferation and threaten neoplastic disease are turned off.
Similarly, the virus is safe from immunosurveillance because no viral proteins are
expressed to act as targets of the immune system. The transcription program used
in these cells, where no viral proteins are expressed, is called the latency program
(Hochberg et al. 2003a and Table 1) reflecting its role at the site of latent persistence.
The frequency of infected memory B cells for an individual healthy carrier is

very stable over time (Hadinoto et al. 2009; Khan et al. 1996). However, the level
of infected cells in a population ranges widely from 5 to 3000 for every 107 memory
B cells both in the peripheral blood (mean 110/107) and in Waldeyer’s ring (mean
175/107—the virus is evenly distributed throughout the ring) (Laichalk et al. 2002).
The level of infected cells is similar between peripheral blood and Waldeyer’s ring
but at least 20-fold lower in the other lymphoid tissue tested (spleen and mesenteric
lymph node) (Laichalk et al. 2002), suggesting preferential homing to the lymphoe-
pithelium of Waldeyer’s ring. Based on these measurements, the total body load
calculates to 104–107 (mean 0.5 × 106) infected memory B cells per person repre-
senting a small, stable, and, most critically, “safe” pool of infected cells that guaran-
tees long-term persistence. Only ~1 % of these cells reside in the peripheral blood.
3.3.2 Memory B Cell Homeostasis—The Maintenance of Long-Term

Memory and Persistent Infection
The survival of memory B cells requires a tonic signal from the BCR (Maruyama
et al. 2000), and the number of cells is controlled by homeostasis mediated by
cytokines such as BAFF (Mackay and Schneider 2009; Stadanlick and Cancro
2008). The tonic BCR signal can be completely replaced by LMP2 (Caldwell et al.
1998), raising the possibility that persistently infected cells could be BCR inde-
pendent. However, this is not the case, infected cells in the periphery do not express
LMP2 (Hochberg et al. 2003a), and as already noted, they express a functional, pos-
sibly, antigen-selected BCR. A number of independent lines of evidence suggest
that memory B cells latently infected with EBV are also maintained by homeostasis:
1. EBV-infected memory B cells in the periphery of adult humans are >90 % in
a resting state, but at any given time, around 2–3 % of the cells are undergoing
cell division (Miyashita et al. 1997; Hochberg et al. 2004). This is exactly the
same rate as has been reported (2.7 %) for normal memory B cells (Hochberg
et al. 2004; Macallan et al. 2005; Miyashita et al. 1997).
2. The half-life of both EBV-infected and EBV-uninfected memory B cells is
virtually identical –7.5 ± 3.7 days (Hadinoto et al. 2008) and 11 ± 4 days
(Macallan et al. 2005), respectively.
3. Latently infected memory cells in the periphery express no viral latent proteins.
Therefore, when they divide, it must be driven by normal homeostasis signals.
We may conclude therefore that the pool of latently infected memory B cells is
indistinguishable to the host from normal memory B cells.
When EBV-infected cells divide, they must express EBNA1 because the viral
genome cannot replicate in its absence (Yates et al. 1985). Predictably, there-
fore, latently infected memory cells in the periphery express EBNA1 when they
undergo cell division (Hochberg et al. 2003a). This is the third example we
will encounter of a latent gene transcription program used in lymphoma, being
found in a normal infected B cell counterpart in vivo. In this case, the EBNA1
only program, which is used in Burkitt’s lymphoma (BL), is found in dividing,

latently infected memory B cells in the blood (Table 1, Fig. 5). EBNA1 expres-
sion during cell division is the only potential point of attack for the immune sys-
tem against the pool of latently infected memory cells. It is perhaps not surprising
therefore that EBNA1 has evolved so as not to be processed and presented effi-
ciently to the immune system [Levitskaya et al. (1995, 1997) reviewed recently in
Daskalogianni et al. (2014)], thus minimizing the risk of attack.
3.4 Viral Replication—Plasma Cell Differentiation,

Stress, and the Role of Epithelial Cells
3.4.1 Terminal Differentiation—Maintenance of Stable Antibody

Production and Viral Shedding
The last component of persistent infection to be discussed is that infectious virus is

continuously shed into the saliva (Golden et al. 1973; Hadinoto et al. 2009). Unlike
the level of latently infected memory cells, which is strikingly stable over long time
periods, virus shedding fluctuates dramatically. The level of shedding is relatively
stable over short periods (hours–days) but varies through 3.5–5.5 orders of magni-
tude over longer periods (Hadinoto et al. 2009). This variation means, contrary to
what is generally believed, that the definition of high and low shedder is not so much
a function of variation between individuals but within individuals over time. Also
an important but simple insight, that had gone unrealized in the field, was that EBV
shedding into saliva must be continuous and rapid. This is because the virus must
be replaced ~2 min which is how frequently, on average, a normal individual swal-
lows. Thus, the mouth is not, as often cited, a reservoir of virus but a conduit through
which a continuous flow stream of virus passes in saliva (Fig. 8). Consequently, virus
is being shed at a much higher rate than is generally appreciated.
Memory B cells, transiting the nasopharyngeal lymphoid tissue, presumably
must occasionally initiate virus replication and release the virus. From cell sur-
face phenotyping of fractionated tonsil cells, it is clear that the B cells replicat-
ing the virus in the lymphoepithelium of the tonsils are plasma cells (CD38hi,
CD10−, CD19−, CD20lo, sIg−, and cIg+) (Laichalk and Thorley-Lawson 2005),
a conclusion consistent with histological observations (Niedobitek et al. 2000;
Anagnostopoulos et al. 1995). Quantitative estimates suggest that somewhere
in the region of ~250 cells are undergoing replication in Waldeyer’s ring at any
one time (Hawkins et al. 2013; Laichalk and Thorley-Lawson 2005). However,
sequentially fewer cells express the immediate early, early, and then late antigens
of the lytic cycle such that only ~10 % of the cells complete the replicative cycle.
Thus, only a handful of B cells are actually releasing virus in Waldeyer’s ring at
any given time. This sequential diminution in the numbers of cells replicating
the virus as they proceed through the cycle may indicate that replication is fre-
quently abortive or may be the result of aggressive immunosurveillance by CTL
Fig. 8 A model of EBV reactivation and shedding. The known data fit a model where a single
latently infected memory B cell in the tonsil occasionally differentiates into a plasma cell and
releases virus that infects epithelial cells. The infection spreads exponentially through the epi-
thelium, resulting in the shedding of virus. The plaque is eventually eliminated by the immune
response. Meanwhile, another plaque initiates elsewhere in the Waldeyer’s ring. The data are
consistent with their being no more than three such plaques in Waldeyer’s ring at any one time.
Virus is continuously shed into the mouth where it mingles with saliva for about 2 min before
being swallowed. Thus, the mouth is a flow stream of EBV not a static reservoir
(Callan et al. 1998b). This despite mechanisms that the virus employs during its
lytic cycle to reduce CTL surveillance (Ressing et al. 2008). For a detailed discus-
sion of immune evasion by EBV, see the chapter authored by Emmanuel Wietz.
It has been shown that differentiation into plasma cells, and not the signals that
induce differentiation, initiates viral replication (Laichalk and Thorley-Lawson
2005). Again, the biology of the virus is intimately tailored and responsive to nor-
mal B cell biology. This was confirmed by in vitro studies in cells showing that the
promoter for BZLF1, the gene that begins viral replication, becomes active only
after memory cells differentiate into plasma cells, that it is active in plasma cell
lines and is activated by the plasma cell-associated transcription factors XBP-1
and Blimp1. The molecular mechanism behind this activation process has been
comprehensively reviewed recently (Kenney and Mertz 2014). For a detailed dis-
cussion, see the chapter authored by Ayman El-Guindy.
The signal that causes latently infected memory B cells to undergo terminal
differentiation is unclear. It has been suggested that immunological B cell mem-
ory may be sustained through bystander T cell help (Bernasconi et al. 2002) such
that a memory B cell transiting through a lymph node will, when it encounters
bystander T cell help, undergo a cell division that will generate one memory cell
and one plasma cell. This ensures the stability of the memory pool, while a con-
tinuous supply of plasma cells is produced that will guarantee stable production
of antibody. Applied to EBV, this could explain how the population of latently
infected memory cells could be maintained for years, while, through the genera-
tion of plasma cells, virus can also be continuously produced.
An alternate hypothesis is that the generation of plasma cells replicating EBV

is stimulated by cognate antigen and T cell help. This hypothesis has the attractive
feature that latency would need to be established in antigen-specific memory cells
in the tonsil. These cells would then enter the peripheral circulation where they
would maintain persistent infection. As these cells reenter secondary lymphoid tis-
sue, the site where they would most likely reencounter cognate antigen would be
the tonsil. This would provide a mechanism for preferential homing and reactiva-
tion of the latently infected memory cells in the tonsil compared to other lymph
nodes. Although it is difficult to conceive of a mechanism by which the virus
could access antigen-specific naive B cells with a high enough probability and fre-
quency to be feasible, this model is very consistent with the observation that the
latently infected memory cells appear to bear antigen-selected BCRs.
3.4.2 Stress—An Alternate Pathway to Viral Replication
Indications of a second pathway to viral replication come from in vitro studies

that a number of stress-inducing agents including TGFb and chemotherapy agents,
BCR cross-linking and hypoxia can also initiate viral replication in cell lines
(Kenney and Mertz 2014). In these systems, however, there is acute activation of
the BZLF1 promoter within minutes of receiving the stimulus, and not surprisingly,
the cells do not undergo plasma cell differentiation prior to viral replication. In a
similar fashion, explanted infected peripheral memory cells will acutely undergo
spontaneous reactivation (Rickinson et al. 1977), presumably in response to the
stress induced upon being placed in culture. What these systems have in common
is the induction of apoptosis in the B cells in response to the stress signal (Inman
et al. 2001). However, EBV encodes a homologue of the antiapoptotic gene bcl-2
that is expressed during viral replication in vitro (Henderson et al. 1993) and this
protects these cells from stress-induced death and apoptosis, while the virus repli-
cates (Inman et al. 2001). It is known that B cells are particularly prone to apopto-
sis. It seems therefore that in addition to replication in plasma cells located in the
epithelium of the tonsil for infectious spread, the virus has developed an escape
hatch that allows it to exit any infected B cell that may begin to die by apoptosis.
3.4.3 Replication in Epithelial Cells
Although we lack a direct demonstration that EBV replicates in epithelial cells in

vivo, the indirect evidence is compelling:
1. The strongest evidence comes from numerical arguments. Put simply, there are
not enough B cells replicating the virus in Waldeyer’s ring to account for either
the amount or extreme variability of EBV shedding in saliva. For a detailed
discussion of the numbers, see Hadinoto et al. (2009). The dynamics of virus
shedding is most simply explained by single B cells sporadically releas-
ing virus that infects neighboring epithelial cells (Fig. 8). (This mechanism is
analogous to the neurotropic herpesviruses (HSV and VZV) that persist silently
in ganglia but when reactivated travel down the neurons to replicate in fibro-
blasts.) Epithelial infection by EBV spreads at an exponential rate and is ter-
minated randomly, resulting in infected plaques of epithelial cells ranging in
size from 1 to 105 cells, more than sufficient to account for the observed rate
of shedding. At any one time, there would be a very small number (≤3) of such
infected epithelial plaques in the entire Waldeyer’s ring that would be transient
and usually small, explaining why they have previously gone undetected.
2. Cell cultures of primary epithelial cells from tonsils carry already infected cells
that are both latently infected and replicating the virus (Pegtel et al. 2004).
3. EBV is found in oral hairy leukoplakia which represents a lesion where EBV
is actively replicating in the epithelium of the tongue (Greenspan et al. 1985).
This indicates that EBV can replicate in epithelial cells in vivo.
4. The glycoprotein patterns on the virus differ depending on whether the virus
emerges from a B cell or an epithelial cell (Borza and Hutt-Fletcher 2002).
This happens in such a way that the virus bears an epithelial tropic pattern of
viral glycoproteins when it emerges from B cells and a B lymphotropic pattern
when it emerges from epithelial cells. These results imply that the virus has
evolved to efficiently shuttle back and forth between epithelial and B cells.
5. A unique receptor, α5β1 integrin, for EBV is expressed on epithelial cells that
allows infection only on the basolateral surface (Tugizov et al. 2003). As with
the glycoprotein patterns, this implies that epithelial cell infection by EBV
is only used in one direction, in this case specifically restricted to exit by the
virus.
Taken together, this evidence presents a strong circumstantial argument that ton-
sillar epithelium is actively infected with replicating EBV as an ongoing part of
normal viral persistence and provides an explanation for the presence of the virus
in the associated diseases of epithelial cells.
4 The Cyclic Pathogen Refinement of GCM
If a biological model is correct, then it should be logically rigorous and able to

be expressed mathematically. Mathematical modeling is not really that different
from how biology has always been done, it is just a more rigorous way to organ-
ize data and a more logical way to make testable predictions based on hypotheses.
However, most biological systems are not well enough characterized quantita-
tively to be amenable to this type of analysis. This is under appreciated by biolo-
gists who tend to see the failing of modeling (despite its obvious utility in other
more quantitative sciences such as physics and engineering) as a consequence
of the limitations of modeling itself rather than the lack of rigor in understand-
ing the biological system being studied. Persistent EBV infection is an exception.
Fig. 9 The cyclic pathogen model (CPM). a CPM is a mathematical description of the GCM.
It consists of a cycle of 6 infected stages (blue circles based on the biological GCM illustrated
in Fig. 4). These are blast, GC, memory and immediate early, early and late lytically infected B
cells, each of which is potentially controlled by the immune response (red circles). The single
lytic stage in the GCM is broken down into three discrete stages which are known to be rec-
ognized independently by the immune response. Biologically, there is never a CTL response
against the memory stage; however, the model allows analysis of theoretical conditions such as
the memory compartment being regulated by CTL. This model can be described by a system of
differential equations employing rate constants for the stimulation of CTL (blue arrows), kill-
ing of CTL targets (red arrows), and the proliferation and death of each stage (green arrows).
For this system, there is one and only one mathematical solution that is stable and biologi-
cally credible. This solution accurately describes biologically persistent infection. b Shows the
infected populations as circles whose area is proportional to their frequency within all tonsils
(1:5:1.5.102:104:104:0.5.104, Late:Early:ImmEarly:Memory:GC:Blast). This highlights the very
large range in the sizes of these populations
The GCM, as generalized in Fig. 9, can be described by a system of differential

equations—the cyclic pathogen model (CPM) (Delgado-Eckert and Shapiro 2011)
for which there is one and only one solution that is stable and biologically cred-
ible. We have sufficient quantitative information to be able to know, derive pre-
cisley, or estimate approximately values for all the parameters (rate constants)
governing these equations. When solved with this parameter set, the model very
precisely replicates the actual dynamics of the infection (Hawkins et al. 2013).
This includes predicting which and to what extent each infected stage is recog-
nized by CTL and even precisely predicting the expected sizes of the infected
memory and GC populations and the extent to which they vary between infected
individuals. Furthermore, when marginally non-biological values are assigned to
parameters, the model fails to replicate infection. This is an important result that
seems to have gone unappreciated in the biological community. The chances that
one could randomly pluck a complex model such as the one shown in Fig. 9 and
have it predict correctly when and only when biological values are applied are
vanishingly small. The fact that this model works so well is a convincing argu-
ment for the biological accuracy of the GCM in explaining EBV persistence.
The mathematical description of the cyclic pathogen model and its subsequent
analysis also provided important new insights including:
1. There are two possible mechanisms for EBV persistence in B cell memory. In
one, the virus persists through homeostasis independently of new infection and
addition to that compartment. In the second (predicted by the CPM), it is the
cycle of infected states that accounts for persistence. Aggressive intervention
with antivirals should distinguish these since they should have no impact on
the memory compartment if the former is true but will reduce the overall level
of viral infection if the latter is true. Indeed, long-term treatment with antivi-
rals, which dramatically reduce viral shedding, produced a parallel decline in
the level of infected memory B cells (Hoshino et al. 2009). This confirms the
prediction from the CPM. For a detailed discussion of antiviral interventions,
see the chapter authored by Richard Ambinder.
2. Based on the same arguments, CPM predicts that an effective vaccine against
primary EBV infection will also be effective over time in reducing and eventu-
ally eliminating persistent infection because it will interdict the cycle of infec-
tion required for long-term persistence. For a detailed discussion of vaccine
strategies, see the chapter authored by Rajiv Khanna.
3. To a biological eye, it is apparent that EBV persists because it can attain latent
infection of resting memory B cells that are invisible to the immune system.
However, the CPM provides a different interpretation, fully compatible with
all the biological data, namely that it is the cycle of infection that allows per-
sistence. Persistence is possible even if the memory compartment were highly
immunogenic; however, the overall structure and dynamics of the persistent
infection would look nothing like what is actually observed (in passing, it is
worth noting the utility of modeling in allowing such biologically impossi-
ble experiments to be performed mathematically). This is a further validation
of the accuracy of the mathematical model. Thus, access to the immunologi-
cally protected memory compartment defines the overall pattern, features, and
dynamics of persistent infection but alone does not account for it.
4. It explains how infection can be stable at a very low level. This is crucial for
both the host and the virus because it imposes the minimum burden on the host
within which EBV wants to persist for life. For an average person, there is ~1
infected cell per 5 ml of blood. Such a low level of infection leaves the virus
vulnerable to extinction through stochastic variation, yet the value only varies
by a factor of perhaps ±25 % over many years (Hadinoto et al. 2009). This is
because the cycle of infection ensures that an obliterated population can be rap-
idly repopulated returning the system to the same equilibrium as before.
5. The absolute levels of infection are defined by the level of the immune
response against viral proteins. This includes cytotoxic responses to infected
cells expressing latent and lytic proteins and neutralizing antibody against
infectious virus. The prediction that the immune system only moderates
the overall viral load, not the form of persistence, is confirmed in studies of
immunosuppressed individuals. Here, in the presence of a minimally effective
immune response, the levels of virus-infected memory B cells increase on aver-

age 50-fold (Babcock et al. 1999). However, the regulation of viral persistence
is intact and the virus in the blood remains restricted to resting memory B
cells. This means that the immune response per se plays no role in regulating
the mechanisms of viral persistence, but it only regulates absolute levels of the
infection.
6. That the system is a simple circle is amply demonstrated by studies on acutely
infected individuals. Here, the system is allowed to run unchecked until the
immune response is activated. In this case, as many as a staggering 50 % of all
memory cells may become latently infected with EBV until the immune system
begins to reduce the overall load of infection (Hadinoto et al. 2008; Hochberg
et al. 2004). Impressively, the regulation still holds and the virus remains
restricted to resting memory cells in the blood again highlighting that the
immune system only functions to regulate the level not the form of the infection.
5 The Model of Persistence—A Summary
In summary, persistent infection by EBV can be seen as a self-perpetuating cir-

cle of infection, differentiation, persistent infection, reactivation, and reinfec-
tion (Figs. 4 and 9) that exploits virtually every aspect of mature B cell biology.
The expansion of the virus is counterbalanced by the immune response. It is this
cycle of infection together with the quiescent infection of peripheral memory B
cells that allows the virus to be maintained at the extremely low and stable levels
characteristic of persistent infection. In doing so, EBV does not disrupt the normal
processing of latently infected cells into memory, and in so far as the presence of
the virus may cause deviations from normal B cell biology, they are not detectable
by the time the cells enter the memory compartment.
6 Disease Pathogenesis—Insights from the GCM
The GCM explains that EBV needs to transit the GC to access the resting mem-
ory compartment. EBV-infected and GC B cells are tightly regulated because they
both proliferate rapidly—a risk factor for cancer. GC cells also actively undergo
DNA breakage and mutagenesis during CSR and SHM, additional risk factors for
tumor development and the production of autoreactive B cells. Furthermore, in the
GC, EBV expresses LMP1, a growth-promoting potential oncogene, and LMP2,
a pro-survival molecule able to rescue autoreactive B cells. Thus, the presence
of EBV in GC B cells presents a nexus for disease risk, especially cancer (EBV-
positive Hodgkin’s disease and Burkitt’s lymphoma both arise from EBV-infected
GC cells) and autoimmunity. It is not surprising, therefore, that EBV has been
linked with a number of such diseases.
6.1 Infectious Mononucleosis—Acute Infection (AIM)
Delayed infection by EBV can cause infectious mononucleosis (AIM). Why ado-
lescence and adults get AIM is not clear. It is likely immunopathologic in nature,
meaning the disease symptoms are caused by the inflammatory response of the
immune system rather than the virus itself. For a detailed discussion of AIM, see
the chapter authored by Kristin Hogquist. The intensity of the disease varies but
can last for weeks or months before finally resolving (Hoagland 1967). IM is char-
acterized by a lymphocytosis (Wood and Frenkel 1967) due to the appearance of
large numbers of “atypical” lymphocytes which are predominantly CD8+ T cells,
representing a vigorous CTL response to the virus (Strang and Rickinson 1987;
Callan et al. 1998b).
6.1.1 AIM and the GCM
Virologically and immunologically, we know nothing about what is happening in

the newly infected host until they arrive at the clinic with symptoms some 5 weeks
into the infection (Hoagland 1964). We may assume though that when the virus
initially infects, there is nothing to control the cycle of infection, latency, reac-
tivation, and reinfection, shown in Fig. 4. Consequently, the memory compart-
ment begins to fill up with latently infected B cells. A staggering level of infection
is achieved that can reach ≥50 % of all memory B cells (Hochberg et al. 2004).
Despite this overwhelming invasion of the B cell compartment by EBV no cells
expressing the lymphoblastoid form of latency/infection are detected in the periph-
ery, the virus remains restricted to resting memory B cells. This is fully consistent
with the GCM which predicts that the lymphoblastoid form of latency is restricted
to the lymphoid tissue and tightly regulated such that the cells rapidly transit into
the GC to become memory cells before entering the circulation.
By the time patients experience symptoms and arrive at the clinic, the infection
is always resolving (Hadinoto et al. 2008). Viral shedding and the levels of newly
infected B cells are all falling. All that is left is the massive level of infection in
the memory compartment. Since these cells are not seen by the immune response,
their levels decrease simply by attrition as they initiate viral replication and are
immediately killed by CTLs that recognize the immediate early lytic antigens.
Consequently, at this time, as many as half of all the CTLs in the body are directed
against EBV-infected cells expressing these targets (Callan et al. 1998b). It is most
likely that this destruction of large numbers of infected B cells is responsible for
the inflammatory response leading to the fever and malaise characteristic of IM.
There then ensues a parallel decrease in the number of latently infected mem-
ory B cells (dying as they enter viral replication and are destroyed by CTL) and
the number of CTLs that they stimulate (Catalina et al. 2001; Hadinoto et al.
2008). For the next few weeks, there is an exponential decrease in the levels of
latently infected memory B cells (half-life ~7.5 days) and CTL against the IE
proteins (half-life ~73 days). Eventually, the level of infected memory B cells
drops to a point where the rate of attrition is matched by the steady-state low-level
production of newly infected memory B cells. At this time, the level of infected
memory cells and CTL against IE proteins begins to stabilize. Thus, the acute
infection is eventually limited by the immune response but at an excruciatingly
slow rate. The ensuing events are strikingly different from infection with most
other viruses. Shedding of EBV does not stop, but continues for life (Hadinoto
et al. 2009; Yao et al. 1985). B cells expressing the growth and default programs
persist in Waldeyer’s ring (Babcock et al. 2000), and latently infected memory
cells, expressing the latency program, remain in the blood for life (Babcock et al.
1998; Khan et al. 1996). At the same time, levels of neutralizing antibodies (Henle
and Henle 1979) and CTL (Callan et al. 1998a; Steven et al. 1996) also continue
at significant and stable levels for the lifetime of the host. For a detailed discussion
of the immune responses that regulate EBV, see the chapters authored by David
Nadal, Martin Rowe, Jaap Middeldorp, and Andrew Hislop et al. It is apparent
therefore that the immune response ameliorates and counterbalances the infection
but never clears it. An equilibrium is established between the immune response
and the various states of viral latency allowing the virus to persist at stable lev-
els without causing significant impairment to the host. The CPM states that these
countervailing forces are responsible for maintaining the stable fixed point that
mathematically describes EBV persistence.
6.1.2 Why Do Adolescents Get AIM?
The age dependence of symptoms has led to the suggestion that IM is a disease of
a mature immune response. What this means mechanistically is less clear. It has
also been suggested that the tonsil is an immune-privileged site during AIM, since
the majority of circulating CTLs at this time lack the requisite mucosal homing
receptors to enter the tonsil (Hislop et al. 2005). However, a much simpler and
more likely explanation for this observation is that the majority of EBV-infected
cells reside in the spleen during acute infection as evidenced by the well-docu-
mented symptom of splenomegaly. Thus, most of the CTL should be homing to
this non-mucosal site until the infection resolves.
A more compelling explanation for AIM is the theory of heterologous immu-
nity (Clute et al. 2010; Selin et al. 1998). In this theory, the CTL response to new
infections early in life is by naive T cells which produce high-affinity CTLs that
efficiently and rapidly clear the virus infection and then become memory CTL. As
the organism ages, memory CTLs accumulate to a variety of pathogens. Exposure
to a novel infection later in life is more likely to trigger cross-reacting memory
CTL than naive CTL. These memory CTLs are of lower affinity and may produce
an ineffectual response allowing extensive viral replication and spread that triggers
a massive inflammatory response that lasts until the virus is finally brought under
control.
6.2 Autoimmune Disease
EBV has variously been linked with a number of autoimmune diseases including
SLE (James et al. 1997), rheumatoid arthritis (Lotz and Roudier 1989), Sjorgen’s
syndrome (Fox et al. 1987), and most recently and aggressively with multiple scle-
rosis [reviewed in Ascherio and Munger (2010)]. For a detailed discussion of EBV
and autoimmune disease, see the chapter authored by Alberto Ascherio. The driv-
ing concept behind these associations is the knowledge that EBV can cause the
activation and proliferation of infected B cells in an antigen-independent fashion
and that such cells are immortal in tissue culture. This raises the possibility that,
by infecting them, EBV infection could allow the survival of autoreactive clones
of B cells. However, EBV does not persist by immortalizing B cells in vivo, but
by differentiating the infected cells into a resting memory state. Consequently,
the fundamental rationale for the association must be modified to suggest that
the EBV latent genes may rescue a forbidden clone from a GC into the memory
compartment. Such a latently infected cell itself could not produce antibodies
because upon plasma cell differentiation it would produce infectious virus and die.
However, theoretically, such a cell could present autoimmune antigens and break
tolerance. There is evidence to support this idea in that LMP2, which is expressed
in the GC, is capable of breaking tolerance in a mouse model of autoreactivity
(Swanson-Mungerson and Longnecker 2007; Swanson-Mungerson et al. 2005),
and has even been shown to exacerbate disease in a mouse model of MS (Chang
et al. 2012). Such behavior must be anomalous, however, because the expressed
immunoglobulins of latently infected memory cells in healthy humans are, if any-
thing, skewed away from self-reactivity (Tracy et al. 2012).
Demonstrating experimentally a causative role for EBV in autoimmune disease
is difficult because infection usually occurs early in life and by adulthood >90 %
of the population is infected. Analysis is further complicated by the fact that EBV
is carried in the peripheral circulation by infected memory B cells, so sensitive
tests will detect EBV in any inflamed tissue, regardless of the virus’s role in caus-
ing the inflammation. The GCM/CPM adds another layer of complication which
is that EBV uses virtually every aspect of mature B cell biology to establish and
maintain persistent infection and virus shedding which is counterbalanced by the
immune response to produce a defined stable level of infection. The corollary is
that EBV is exquisitely sensitive to changes in the immune system. Any disease
that affects the immune system will have an impact on the regulation of EBV
persistence. This could result in an increase in the numbers of infected cells in
the blood (peripheral blood burden) and/or an increase in virus shedding. Thus,
changes in virological or immunological parameters of EBV infection associated
with an autoimmune disease are most likely an indirect effect of a compromised
immune system caused by the disease as is the case with SLE (Gross et al. 2005)
rather than a cause of the disease.
6.3 Cancer
The motivating force behind associating EBV with cancer is obvious. It is well
established that EBV has latent proteins that can drive cellular proliferation, at
least in B lymphocytes, and it is highly likely that inappropriate or deregulated
expression of these genes could play a causative role in tumor development. EBV-
associated cancers fall into three discrete groups.
1. Tumors for which there are claims that remain to be substantiated. These
would include, but not be limited to, breast (Bonnet et al. 1999) and hepato-
cellular carcinoma (Sugawara et al. 1999). In these cases, the doubt usually
exists through the inability of investigators to reproducibly detect the virus
in the tumor cells. Since the assays used are usually based on either PCR or
immunohistochemistry, they are subject to the vagaries of those techniques
which include a high level of false positives and dependence on technical
skill to perform well-controlled studies. Thus, it becomes difficult to resolve
whether conflicting results are caused by false-positive artifacts or technical
inconsistencies.
2. Tumors for which there is strong supportive evidence, but the tumors arise
in cell types for which no latently infected biological equivalent has been
established. Such tumors may arise through accidental infection leading
to inappropriate viral latent gene expression. This includes such tumors as
nasopharyngeal (Raab-Traub 2002), gastric (Shibata et al. 1991; Shibata and
Weiss 1992), and salivary gland (Raab-Traub et al. 1991) carcinomas. For
a detailed discussion of EBV-positive carcinoma, see the chapter authored
by Nancy Raab-Traub. Also included are leiomyosarcoma (van Gelder et al.
1995; Timmons et al. 1995) and T and NK lymphomas (Chiang et al. 1996;
Tao et al. 1995). In these cases, there is a high degree of correlation between
the disease and EBV [e.g., 100 % of undifferentiated NPC contains EBV
(Andersson-Anvret et al. 1977)] and reproducible detection of the virus in the
tumor cells. Since the viral episome is lost from cells absent some selective
pressure for its retention (Kirchmaier and Sugden 1995), the consistent pres-
ence of viral DNA in any tumor is prime facie evidence that the virus is play-
ing a role in the growth/survival of the tumor cells (Vereide and Sugden 2009).
These tumors also frequently express the latent gene LMP1 which is known
to be highly oncogenic when constitutively expressed (Baichwal and Sugden
1988; Moorthy and Thorley-Lawson 1992; Nicholson et al. 1997; Uchida et al.
1999; Wang et al. 1985). This type of tumor is relevant to the GCM which pos-
its that EBV latent gene expression patterns have evolved to be regulated in
concert with normal B cell activation and differentiation, with the ultimate goal
of establishing persistent infection in a memory B cell where the latent genes
are no longer expressed. It follows that if EBV fortuitously gains access to a
cell type which is not a natural target of infection, i.e., a non-B cell, this could
lead to aberrant latent gene expression that would not be regulated appropri-
ately. This could result in constitutive expression of LMP1 for example.
3. Tumors for which there is good evidence linking EBV. These are the lympho-
mas IL, HD, and BL. There is convincing epidemiological, serological, and
molecular biological evidence associating EBV with these tumors. The GCM
provided the first and, to date, only explanation for the origin of these lympho-
mas and the reason they express restricted patterns of latent proteins (Thorley-
Lawson and Gross 2004). Indeed, it is supportive of the GCM and cannot be a
coincidence that tumors arise from each of the three proliferative stages of EBV
infection predicted by the model (Fig. 5). These are IL from cells expressing the
growth program (new infection), HD from cells expressing the default program
(GC cells), and BL from cells expressing EBNA1 only (late GC cell).
6.3.1 Lymphoma in the Immunosuppressed—IL
Patients who are immunosuppressed are at risk for diseases such as post-transplant
lymphoproliferative disease (PTLD) in organ transplant patients and immunoblas-
tic lymphoma in AIDS patients. These are a heterogeneous collection of B cell dis-
orders [reviewed in Hopwood and Crawford (2000)] that usually carry the virus
and express the growth program (Thomas et al. 1990). The obvious explanation
for PTLD is that suppression of the immune response allows uninhibited growth
of EBV-infected cells; however, it is not that simple. If EBV was able to freely
drive cell growth in the absence of an immune response, there would be several
consequences. First, all immunosuppressed patients who are EBV-infected should
develop the disease. Second, it should be a polyclonal and disseminated disease
since EBV would drive the growth of many infected cells throughout the body.
Finally, the lymphomas would be expected to arise most frequently in the places
where infected cells are known to express the growth program—Waldeyer’s ring
(Joseph et al. 2000a). In reality, the disease has none of these features. Only a
small fraction of patients (0.1–10 % depending on the setting) develop the dis-
ease, it is usually oligoclonal arising from one or a few infected cells, and it often
occurs in extranodal sites such as the brain and gut (Penn 1998; Hopwood and
Crawford 2000). This indicates that the disease is not simply EBV-driven growth
but involves a rare event where EBV infection has gone wrong.
The GCM states that the growth program of EBV is used specifically to activate
newly infected naïve B cells in Waldeyer’s ring, so they can then differentiate into
resting memory B cells. It follows that for a lymphoblastoid cell, proliferating due
to the growth program, to survive and evolve into a lymphoma, the cell must be
unable to exit the cell cycle. This could occur if infection of the wrong B cell type
or in the wrong location occurs (inappropriate infection).
IL in the immunosuppressed is therefore a consequence of two events. The
rare specific event is the expression of the growth program in a B cell that can-
not exit the cell cycle. The global event is immunosuppression that prevents the
elimination of these rare cells. At this stage of the disease, the tumor cells are still
susceptible to immunosurveillance and regression can be achieved by reducing
immunosuppression (Starzl et al. 1984) or by treatment with autologous CTL

(Rooney et al. 1995). For a detailed discussion of the application of adoptive trans-
fer for treatment of EBV tumors, see the chapter authored by Stephen Gottschalk
and Cliona Rooney. However, in the absence of T cell immunity, the proliferating
cells acquire additional genetic damage and more malignant clones arise (Knowles
et al. 1995). These cells ultimately become unresponsive to reduced immunosup-
pression or immunotherapy and are usually fatal.
6.3.2 Hodgkin’s Disease (HD)
For a detailed discussion of HD, see the chapter authored by Paul Murray and
Andy Bell. HD is a tumor of germinal center cells (Kuppers 2012; Kuppers and
Rajewsky 1998) and is characterized by the unusual Hodgkin’s Reed–Sternberg
(HRS) tumor cells. AIM and elevated antibody titers to EBV are both risk factors
for HD (Ambinder 2007; Henle and Henle 1979; Hjalgrim et al. 2007), and up
to 40 % of the tumors contain EBV (Glaser et al. 1997). The virus in the tumors
is clonal and expresses the default transcription program (Oudejans et al. 1996;
Deacon et al. 1993; Herbst et al. 1991; Niedobitek et al. 1997), the same transcrip-
tion program used by latently infected GC B cells (Babcock et al. 2000). Thus, the
cell origin and the viral gene expression data agree that HD arises from an EBV-
infected GC B cell expressing the default program (Fig. 5). In effect, the viral gene
expression pattern in HD is not created within and selected by the tumor, but is
a natural consequence of the cellular origin of the tumor. The presence of EBV
in ~40 % of the tumors would seem to rule out a chance association of the virus
with the tumor. But this does not take into account that the levels of EBV-infected
B cells reach extremely high levels during AIM, frequently 10–50 % (Hochberg
et al. 2003b), so there is a very high probability (as high as 50 %) that the prema-
lignant GC cell will have EBV in it by chance. Therefore, it remains a possibility
that it is the immunological disruption of AIM which is the risk factor and EBV is
simply a passenger that plays no role in tumor development.
However, retention of the virus in HD strongly argues that it must be contrib-
uting something to tumor cell survival/growth (Vereide and Sugden 2009). One
specific contribution has been identified for the subset of HD tumors that express
immunoglobulin genes crippled by mutation. These cases are almost universally
EBV positive (Bechtel et al. 2005) and express LMP2 which has been shown inde-
pendently to replace the missing BCR-derived tonic signal necessary for the sur-
vival of B cells with crippled BCRs (Mancao and Hammerschmidt 2007).
6.3.3 Burkitt’s Lymphoma (BL)
For a detailed discussion of BL and diffuse large B cell lymphoma, see the chap-
ters authored by Ann Moorman and Rosemary Rochford and Sandeep Dave. BL
has the pedigree of being the tumor in which EBV was originally discovered, but
its contribution to BL still remains enigmatic. The defining genetic lesion in BL

is deregulated activation of the c-myc oncogene due to reciprocal translocation
with one of the immunoglobulin genes (Klein 1983; Leder 1985; Manolov and
Manolova 1972). BL can occur without EBV, and expression in transgenic mice
of c-myc, in the context of the immunoglobulin translocation, has been shown to
be sufficient to produce Burkitt’s lymphoma-like tumors (Kovalchuk et al. 2000),
suggesting that deregulated c-myc is sufficient to produce the tumors. This raises
the question as to what role EBV may play. The most compelling evidence of
EBV’s involvement in BL is the retention of the genome by the tumors (Vereide
and Sugden 2009) and the high frequency of tumors carrying the virus (de-Thé
1985) in the endemic (eBL) regions of Africa (>95 % contain EBV DNA). The
frequency is lower (15–85 %) in the sporadic form of the tumor (sBL). The pres-
ence of clonal EBV in the tumors (Gulley et al. 1992) has also been interpreted
as evidence of EBV’s role, but in actuality, this only means that EBV was pre-
sent prior to the last event that produced the tumor. Curiously, none of the viral
growth-promoting latent genes are expressed in the tumor cells, the only latent
protein present being EBNA1 (Gregory et al. 1990), along with the non-coding
small RNAs EBERs and the microRNAs. Transcriptionally, this looks like a situ-
ation where the virus is just along for the ride since EBNA1 has to be expressed
to allow duplication of the viral genome. However, there is evidence for all the
EBV genes expressed in BL that they may contribute to pathogenesis, usually by
limiting sensitivity to apoptosis (Iwakiri 2014; Kennedy et al. 2003; Vereide et al.
2014; Wilson et al. 1996).
What is required for understanding how EBV may predispose to BL is an
explanation for why only EBNA1 is expressed. In the case of HD, we have shown
that the default program is expressed because the tumor derives from an EBV-
infected GC cell and the default program is what the virus naturally expresses in
a GC cell. Applying this thinking to BL, there is currently only one way known to
produce the “EBNA1-only” phenotype of BL in a non-tumor cell. This is when
a latently infected GC cell that becomes a memory cell expressing the latency
program, i.e., no latent proteins, divides, as part of normal B cell homeostasis
(Figs. 3 and 4). At this time, the virus turns on expression of “EBNA1 only” to
ensure replication of the viral DNA with the cell. BL has the phenotypic (Gregory
et al. 1987) and gene expression profile of a light zone (LZ) GC cell (Victora et al.
2012). The LZ is where GC cells express c-myc (Dominguez-Sola et al. 2012)
before they begin to proliferate again and also where GC cells reside prior to exit.
Thus, this would be the location where viral gene expression would be expected to
shut down prior to exiting the GC. Lastly, although BL has the characteristics of a
GC cell, the tumor actually grows in extrafollicular locations (Klein et al. 1995).
Therefore, a consistent scenario is that BL is derived from a LZ GC cell that has
left the follicle to become a resting memory cell but cannot achieve this because
it continues to proliferate due to an activated c-myc and therefore constitutively
expresses EBNA1 only. This scenario also accounts for the presence of clonal
EBV in the tumors because the virus would already be present when the major
transformation event, c-myc translocation, occurs.
There are two major infectious players in the predisposition to eBL: malaria
and EBV. Recently, experimental evidence has been presented to account for this
based on the GCM of EBV and the notion that BL arises from a latently infected
GC B cell (Thorley-Lawson and Allday 2008; Torgbor et al. 2014). It is known
that expression of the growth program that occurs prior to entry into the GC
includes the epigenetic silencing of proapoptotic functions, including bim, an
important regulator of myc-induced apoptosis (Allday 2009, 2013). This, together
with the antiapoptotic activities associated with the EBNA1, EBERs and micro-
RNAs expressed in latently infected GC cells leave these cells more resistant to
apoptosis induced by a translocated/deregulated myc gene. The myc translocation
itself is believed to be mediated by the enzyme AID which is uniquely expressed
in the GC (Ramiro et al. 2004; Robbiani et al. 2008). Infection by P. falciparum
malaria has two consequences. First, it increases the viral burden of EBV, result-
ing in higher numbers of latently infected cells transiting the GC and able to resist
apoptosis. Second, it drives deregulated expression of AID in B cells, potentially
increasing the frequency of translocation events (Torgbor et al. 2014). Taken
together, the increased levels of virus-infected cells and rate of myc translocations
in the GC induced by malaria can account for the close association of eBL with
malaria and EBV.
6.3.4 X-linked Lymphoproliferative Disease—XLP
For a detailed discussion of EBV infection in primary immunodeficiencies, see

the chapter authored by Jeffrey Cohen. XLP is a rare X-linked immunodefi-
ciency (Purtilo et al. 1975; Seemayer et al. 1995) which frequently results in
lymphoma or fulminating AIM [reviewed in Bassiri et al. (2008), Purtilo et al.
(1975), Seemayer et al. (1995)]. Responses to other virus infections are typi-
cally normal, but ~75 % of the boys typically succumb within one month of
primary EBV infection. Death is due to the accumulation of EBV-infected
B cells expressing the growth program in tissues such as the liver or subse-
quently by the widespread tissue damage associated with a pronounced virus-
associated hemophagocytic syndrome. Surviving boys typically have severely
disrupted immune systems, resulting in varying degrees of hypogammaglobu-
linemia. The XLP gene itself, SH2D1A or SAP (Sayos et al. 1998; Coffey et al.
1998), encodes for a small signaling molecule of 128 amino acids that consists
essentially of a single SH2 domain with a small C-terminal extension that is an
important regulator of T and NK cell interactions and activation. It has been
suggested that the inefficient recognition of SAP-deficient B cells, the target cell
for EBV-driven growth, accounts for the disease (Dupre et al. 2005). However,
studies in SAP-deficient mice and humans have demonstrated defects in long-
term B cell memory (Crotty et al. 2003; Ma et al. 2006) due to their inabil-
ity to develop functional GCs. This suggests an alternative scenario based on
the GCM, namely that these patients may be unable to process latently infected
blasts into memory because of their defective GCs. This would result in the
infected cells being permanently stuck in the proliferative phase driven by the
growth program which, together with the defective T cell response, could lead
to uncontrolled proliferation and death.
7 Other Sites of EBV Persistence
7.1 The Epithelium
The role of the epithelium in persistence as a site of viral replication is now

broadly accepted. Whether it itself is an independent site of persistent infection
is less clear. Two early studies suggested that it is not. Patients undergoing com-
plete bone marrow ablation as part of a bone marrow transplant lost their EBV
(Gratama et al. 1988), and patients with XLA, an X-linked genetic disorder where
the patients lack B cells, showed no signs of being infected (Faulkner et al. 1999).
However, the technical aspects of these papers leave much to be desired and there
is a need to reproduce them using modern sensitive, quantitative techniques.
This is especially true in light of recent work from the laboratory of Katherine
Luzuriaga (Renzette et al. 2014). They have presented intriguing evidence from
deep sequencing of EBV within infected individuals that variants are primarily
generated over time in saliva not in the blood. This is completely consistent with
the GCM concept that the site of latency is a resting memory B cell in the blood
which divides only rarely and therefore would accumulate variants extremely
slowly, whereas the virus replicates in the epithelium, providing a site for the more
rapid accumulation of mutations. However, since (1) the variants would arise as
single virions and need to be amplified by reinfection to be detected and (2) the
variants are retained over time, this is striking evidence suggesting that the epithe-
lium may be a site where the virus can persist through continuous reinfection and
replication in new epithelial plaques.
7.2 The Tonsil Intraepithelial (Marginal Zone)

B Cell—A Second Route to Persistence?
The study of persistent infection by EBV has been driven from the start by the
property that EBV is able to establish latent persistent infection in vitro by driv-
ing newly infected B cells to become latently infected proliferating lymphoblasts
expressing the growth program. As a consequence, it was initially assumed that
proliferating latently infected lymphoblasts represented the mechanism by which
the virus persisted in vivo. We now know from the GCM that this is incorrect.
Rather EBV uses lymphoblastoid activation of newly infected naïve B cells tran-
siently in vivo to gain access to the resting memory compartment—the actual site
of viral persistence. However, lymphoblastoid activation by EBV infection in vitro
is not transient; it results in indefinite proliferation. The question remains there-

fore: Does extended lymphoblastoid proliferation driven by EBV have a biologi-
cally relevant role in vivo? Is this an in vitro counterpart of an in vivo infected,
proliferating cell where the virus might persist or is it an artifact of selection for
growth in culture?
For a proliferating blast to persist for a long period of time in vivo, it would
need to evade CTL, but it turns out that is not so hard to do. One can envision a
scenario where infection occurs in vivo, driving the expansion of latently infected
lymphoblasts that subsequently stimulate a robust CTL response. The CTLs begin
to kill the blasts reducing their numbers, and therefore the antigenic load, lead-
ing in turn to attrition of the CTLs. Thus, both populations will be reduced till
they reach a point where the time it takes for a CTL to find its target, the lympho-
blastoid cell expressing the growth program, exactly equals the time it takes for
that cell to die and an equilibrium will have been established (Hawkins et al.
2013). Besides avoiding CTL, long-term proliferation of lymphoblastoid cells
also requires that latent proteins have functions specifically evolved to override
the cellular mechanisms that normally limit proliferation, i.e., the virus would
have to fundamentally change the nature of the B cell (Allday 2013; Price and
Luftig 2014). It is now clear that in vitro at least this is indeed the case since
EBNA3A and 3C specifically act to override cell cycle checkpoints (Allday 2013).
Moreover, this activity is not required for the initial phase of rapid proliferation
only coming into play as late as 7 days post-infection. Clearly, this activity must
exist to sustain long-term proliferation. Confirmation that these arguments are cor-
rect requires that such cells must be demonstrated to exist in vivo. What type of
cell might this be?
When naïve B cells are infected in culture, they become activated proliferat-
ing blasts that express AID and the memory cell marker CD27 and undergo SHM
(Siemer et al. 2008). However, they are unable to undergo CSR (Heath et al.
2012), remain IgD+, and do not express bcl-6 (Siemer et al. 2008), and hence,
they would be unable to enter a GC (Kitano et al. 2011). Thus, the phenotype
of a lymphoblastoid cell derived from an infected naïve B cell in vitro is IgD+,
CD27+, AID+, and bcl-6−, with Ig genes that are somatically mutated but not
class-switched [for a complete gene expression profile, see White et al. (2010) and
http://www.epstein-barrvirus.org/]. This is reminiscent of resident, tonsil intraepi-
thelial (marginal zone) B cells (Dono et al. 2003; Spencer et al. 1985; Weill et al.
2009; Xu et al. 2007) and completely distinct from GC cells, which are bcl-
6+ and undergoing Ig class switching, and GC-derived memory cells, which are
IgD− and AID−, and have class-switched Ig genes (Table 3). Can we find such
cells in tonsils? The answer is tentatively yes. We have found a population of
IgD+ CD27+ B cells in the tonsil that express the growth program and have pro-
liferated extensively (Torgbor and Thorley-Lawson unpublished observations). If
it can be confirmed that they are also AID+ and bcl-6− this will be compelling
evidence that EBV is able to enter into and persist for some period of time in the
resident tonsil intraepithelial (marginal zone) memory B cell compartment.
Table 3 Lymphoblasts transformed in vitro by EBV most closely resemble activated marginal
zone memory B cells
Marker Lymphoblasts Marginal Zone B Cells Memory B Cells GC B Cells
AID + + - +
bcl-6 - - - +
CD27 + + + +/-
SHM + + + +
CSR - - + +
It is important to reiterate that what we are discussing here is the resident, mar-
ginal zone-like, intraepithelial, memory compartment of the tonsils. These are
thought to be distinct from circulating marginal zone memory cells (Weill et al.
2009; Weller et al. 2004) which have been shown in repeated studies to lack EBV
(Joseph et al. 2000b; Souza et al. 2007). The potential presence of EBV in the
tonsil subset and absence from the circulating subset support a separate origin for
these two types of cells.
There remains much work to be done to investigate this hypothesis not the least
of which is whether lymphoblastoid proliferation is a form of long- or short-term
persistence, do the cells somehow transition to a resting state, why do they not
usually enter the periphery, and how does the virus get back out again from these
cells. But the central question remains: What is the biological significance, if any,
of long-term proliferation driven by EBV in vivo?
7.3 GC-Independent Maturation of Infected Naïve Blasts
As noted above, direct infection of naïve B cells leads them to become blasts that
have many characteristics of memory cells including somatically mutated Ig genes
and expression of CD27. This has led to the suggestion that EBV could drive the
differentiation of infected naïve B cells all the way to a memory phenotype with-
out the need to access the GC (Heath et al. 2012). This model does not provide a
mechanism for the cells to leave the cell cycle and does not account for the dif-
ferent viral latency programs nor the origin of the different lymphomas. More
critically, the cells produced in vitro did not undergo CSR and presumably do not
express antigen-selected patterns of SHM, two well-known characteristics of the
latently infected memory B cells seen in vivo. Rather than contradicting the GCM,
these studies actually provide an elegant refinement because the authors reported
that the cells would undergo CSR if provided exogenous T cell help which can
only be found in the GC (Victora and Nussenzweig 2012). Thus, these studies sug-
gest that infection of naïve B cells in vivo can initiate the GC process, but the cells
need to migrate into and through a GC to emerge as class-switched memory B
cells with antigen-selected patterns of SHM.
7.4 Two Pathways to Persistence?
The results discussed in the two previous sections raise the intriguing possibility
that a newly infected naïve B cell in vivo in Waldeyer’s ring may have two routes
to persistence (Fig. 10). The key may lie in the observation that upon initial infec-
tion in vitro, cells undergo a brief period (~3 days) of rapid proliferation before
transitioning to a stable slower proliferative state that goes on indefinitely—the
lymphoblastoid cell line [Nikitin et al. (2010), Thorley-Lawson and Strominger
(1978) and see Sect. 3.2.3]. If this occurs in vivo, then the initial phase of rapid
proliferation may indicate the initiation of the GC reaction; hence, the cells
express AID and begin SHM in the absence of bcl-6 (see preceding sections).
Because these cells lack bcl-6, they likely will be unable to physically enter the
GC (Kitano et al. 2011). Instead, they will arrest at the T cell/B cell boundary of
Fig. 10 Are there 2 pathways to persistence? The current data suggest the following possi-
ble hypothetical model. Infected naive blasts will migrate to the follicle because they express
the chemokine receptor EBI2. They express AID and undergo SHM but will not enter the GC
because they are bcl-6 negative. If they receive the necessary signals (cytokines/T cell help), they
will enter the follicle switch on bcl-6, undergo CSR, and eventually leave as resting memory
B cells as described by the GCM (Route 1). If, however, the cells do not receive the necessary
signal to turn on bcl-6, they will continue to proliferate as marginal zone memory B cells (Route
2). The ultimate fate of such cells is unclear. For example, to be biologically relevant, they would
need to release infectious virus at some point. What is clear is that they appear capable of exten-
sive proliferation despite the presence of CTL
the GC. The speculation is that if they access T cell help and/or other signals at
this time, they would become bcl-6+, proceed into the GC, switch to the default
program, and undergo CSR and some version of affinity maturation. It is inter-
esting to note here that one of the latent genes expressed in the growth program,
EBNA3B, specifically activates the expression of cytokines that would attract
Th cells (White et al. 2012). Eventually, they exit into the periphery as a latently
infected GC-derived memory cell as described by the GCM. If, however, after
2–3 days of hyperproliferation, the cells cannot access the necessary signals at the
T/B cell boundary, they would transition to the phase of slower, long-term prolif-
eration, remain bcl-6 negative, fail to enter the GC, and instead remain in the inter-
follicular lymphoepithelium as latently infected marginal zone B cells.
7.5 Direct Infection of Memory Cells
Direct infection of memory B cells was first raised as a possibility over 15 years
ago (Babcock et al. 1998) and was subsequently proposed by Rajewsky and cow-
orkers (Kurth et al. 2000, 2003). However, no further evidence for or explanation
of a mechanism behind this idea has been produced. Problems with the model
include the following:
1. In repeated experiments, we have never detected evidence for the presence of
directly infected memory B cells in the tonsil.
2. It fails to provide an explanation for the different latency transcription pro-
grams and especially why EBV would have a program (the default program)
specifically designed to allow the survival of GC B cells.
3. It has failed to provide evidence or a mechanism for how the directly infected
memory B cells transit to a resting state.
4. It does not explain why EBV in the periphery is restricted only to GC-derived
memory B cells.
5. Predictions made by the model were incorrect when tested experimentally,
instead supporting the GCM. Thus, infected GC B cells express the viral
default transcription program in vivo (Babcock et al. 2000; Roughan and
Thorley-Lawson 2009) (as predicted by the GCM), not the growth program
[as predicted by the direct infection model (Siemer et al. 2008)], and in a
transgenic mouse model, one of the EBV latent proteins expressed in the GC
(LMP2a) was shown to drive B cells to form GCs in the absence of antigen as
required by the GCM and contrary to the idea that EBV directly infects mem-
ory cells (Casola et al. 2004a).
8 Conclusions
The GC model of EBV infection demonstrates that persistent infection by EBV

is a self-renewing circle of infection, differentiation, persistent infection, reac-
tivation, and reinfection (Fig. 4) that elegantly exploits virtually every aspect of
mature B cell biology to:
1. Establish persistent infection (B cell activation with the growth program and
GC differentiation with the default program);
2. Maintain persistent infection (latency in the long-lived memory pool, main-
tained and regulated through the processes of homeostasis);
3. Replicate for reinfection and infectious spread (reactivation of viral replication
in response to terminal differentiation into plasma cells).
It is this cycle of infection together with the quiescent infection in memory that
allows the virus to be maintained at the extremely low and stable level of infection
observed. In doing so, EBV does not detectably disrupt the normal processing of
latently infected cells into memory.
This remains the only model, consistent with experimental observation that
provides a framework for uniting and understanding the disparate behaviors of
EBV, for example:
Why does EBV drive the activation and proliferation of B cells which put the host
at risk for neoplastic disease? Because the latently infected resting naïve B cell
has to become activated so that it can subsequently differentiate through the GC to
become a resting memory B cell where it can persist in a state that is no longer a
pathogenic risk to the host.
EBV uses a different transcription program in different forms of lymphoma (IL,
HD, and BL). Why? The pattern of genes expressed by the different lymphomas is
indicative of the infected cell of origin. The existence of lymphomas expressing all
three of the transcription programs associated with the proliferation of infected B
cells—growth program (IL), default program (HD), and EBNA1 only (BL)—sug-
gests that each of these stages in the EBV life cycle is vulnerable to deregulation
leading to lymphoma.
EBV infects and persists in >90 % of the adult human population almost always
benignly despite its ability to make cells grow. The proliferating cells are short-
lived and not normally a pathogenic threat because the virus is programmed to
ensure that they rapidly differentiate into resting memory cells.
LMP1 and LMP2 have signaling properties analogous to T cell help and the BCR,
respectively. Why would two EBV latent proteins mimic B cell survival and differ-
entiation signals? LMP1 and LMP2 have these properties because they are repli-
cating the signals that are normally used to rescue and differentiate normal GC B
cells into memory. Hence, LMP1 and LMP2 are the only viral regulatory proteins
expressed in infected GC cells.
The epitopes on the latent proteins recognized by cytotoxic T cells are conserved
(Khanna et al. 1997). Why would the virus do this? Once the virus has colonized
the memory compartment, any infected cell that continues to express the growth
program is a threat to the host. The virus ensures that any cell population that con-
tinues to expand due to the growth program will be eliminated by conserving the
targets of EBV-specific CTL.
The GCM has also provided insights into the behavior of memory B cells.
Notably, it has overturned the belief that memory cells do not recirculate (Gray
et al. 1982) because latently infected memory B cells clearly do recirculate
(Laichalk et al. 2002). In addition, the restriction of EBV to the isotype-switched
GC-derived memory pool and absence from the marginal zone memory pool in the
periphery support the view that these marginal zone memory B cells arise inde-
pendently of the GC (Weill et al. 2009; Weller et al. 2004). Lastly, the presence
of EBV in tonsil intraepithelial (marginal) zone B cells supports the idea that this
subset is functionally distinct from the circulating/splenic marginal zone B cell.
9 To Be Continued
Although the details of the GCM are likely to change and much is still to be
learned, it seems certain that an ultimate understanding of EBV infection will
involve a model, whereby EBV uses the normal biology of mature B lymphocytes
to establish and maintain persistent infection. The most interesting unanswered
questions that remain about EBV persistence are as follows:
1. Is/are there GC-independent mechanisms/sites of persistent infection?
2. What, if any, is the biological significance in vivo of the in vitro phenomenon
of long-term lymphoblastoid proliferation?
3. Why does the virus encode for a BCR surrogate if it is persisting in B cells
with an apparently normal BCR?
4. What is the relative contribution of viral latent proteins (especially LMP1 and
LMP2a) and physiologic signals (Th and BCR) to the production of latently
infected memory cells? Could the requirement for both be providing us new
insights into the complexities involved in producing and maintaining immuno-
logical memory?
10 Final Thought—EBV Is Not As Safe As

You Might Think!
EBV seems like a pretty safe virus. It infects virtually every human being for
life, and the infection is almost always benign. However, XLP arises during acute
EBV infection and almost always results in death. It is caused by mutations in the
SH2D1A gene (Sayos et al. 1998; Coffey et al. 1998). So all that stands between
EBV switching from a benign lifetime persistent infection to a life-threatening
acute disease is a single point mutation in the XLP gene. Put another way, the
reader of this chapter would likely have expired and not be around to read this if it
was not for that single mutation.
Acknowledgments The work described here is in large part the consequence of research
carried out by a number of graduate students in my own laboratory too numerous to mention
individually but hopefully appropriately referenced in the text. I would also like to express my
thanks to Michael Lawson for a very careful and thorough editing of the text. To the extent that
this chapter is comprehendible, it is due to him. Finally, I would like to acknowledge NIH, who
have supported my laboratory continuously through Public Health Service grants R01 CA65883
and R01 AI18757.
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Infectious Mononucleosis
Samantha K. Dunmire, Kristin A. Hogquist and Henry H. Balfour
Abstract Infectious mononucleosis is a clinical entity characterized by sore

throat, cervical lymph node enlargement, fatigue, and fever most often seen
in adolescents and young adults and lasting several weeks. It can be caused by
a number of pathogens, but this chapter only discusses infectious mononucleosis
due to primary Epstein–Barr virus (EBV) infection. EBV is a γ-herpesvirus that
infects at least 90 % of the population worldwide. The virus is spread by intimate
oral contact among teenagers and young adults. How preadolescents acquire the
virus is not known. A typical clinical picture with a positive heterophile test is usu-
ally sufficient to make the diagnosis, but heterophile antibodies are not specific
and do not develop in some patients. EBV-specific antibody profiles are the best
choice for staging EBV infection. In addition to causing acute illness, there can
also be long-term consequences as the result of acquisition of the virus. Several
EBV-related illnesses occur including certain cancers and autoimmune diseases,
as well as complications of primary immunodeficiency in persons with the certain
genetic mutations. A major obstacle to understanding these sequelae has been the
lack of an efficient animal model for EBV infection, although progress in primate
and mouse models has recently been made. Key future challenges are to develop
protective vaccines and effective treatment regimens.
S.K. Dunmire (*) · K.A. Hogquist

Center for Immunology, University of Minnesota, Minneapolis, MN 55455, USA
e-mail: dunmi002@umn.edu
K.A. Hogquist
e-mail: hogqu001@umn.edu
H.H. Balfour
Department of Laboratory Medicine and Pathology, Department of Pediatrics,
University of Minnesota, University of Minnesota Medical School,
Minneapolis, MN 55455, USA
e-mail: balfo001@umn.edu

212 S.K. Dunmire et al.
Contents
1 Introduction......................................................................................................................... 213
2 Epidemiology of Primary EBV Infection........................................................................... 213
2.1 Age-Specific Prevalence of EBV Antibodies............................................................. 213
2.2 Routes of Transmission of Primary EBV Infection................................................... 214
3 Clinical Manifestations of Primary EBV Infection............................................................ 215
3.1 Acute Illness............................................................................................................... 215
3.2 Complications of the Acute Illness............................................................................ 216
3.3 EBV-Associated Diseases.......................................................................................... 218
4 Virus–Host Interactions During Primary EBV Infection.................................................... 219
4.1 Incubation Period....................................................................................................... 219
4.2 Acute Infection........................................................................................................... 220
4.3 Convalescence............................................................................................................ 224
5 Diagnosis of Infectious Mononucleosis Due to EBV......................................................... 224
6 Genetic Susceptibility......................................................................................................... 226
7 Prevention of Primary EBV Infection................................................................................. 229
8 Treatment............................................................................................................................ 230
9 Animal Models of Infectious Mononucleosis..................................................................... 230
9.1 Humanized Mice........................................................................................................ 230
9.2 Rabbits....................................................................................................................... 231
9.3 Non-human Primates................................................................................................. 232
10 Summary and Outlook........................................................................................................ 232
References................................................................................................................................... 233
Abbreviations
CAEBV Chronic active Epstein–Barr virus

DC Dendritic cells
EBNA Epstein–Barr nuclear antigen
eBL Endemic Burkitt’s lymphoma
EIA Enzyme immunoassay
HL Hodgkin’s lymphoma
HLA Human leukocyte antigen
HLH Hemophagocytic lymphohistiocytosis
IFN Interferon
LCV Lymphocryptovirus
MHC Major histocompatibility complex
MS Multiple sclerosis
NK Natural killer
NHANES National Health and Nutrition Examination Survey
NIH National Institutes of Health
SAP Signaling lymphocytic activation molecule-associated protein
VCA Viral capsid antigen
XLP X-linked lymphoproliferative disease
Infectious Mononucleosis 213
1 Introduction
Infectious mononucleosis is a clinical entity characterized by sore throat, cervical

lymph node enlargement, fatigue, and fever. It can be caused by a number of path-
ogens, but this chapter considers it as disease resulting from primary Epstein–Barr
virus (EBV) infection and is focused on the immunocompetent host. Infectious
mononucleosis was the name coined by Sprunt and Evans (1920) to describe
a syndrome that resembled an acute infectious disease accompanied by atypical
large peripheral blood lymphocytes. These atypical lymphocytes, also known as
Downey cells (Downey and McKinlay 1923), are actually activated CD8 T lym-
phocytes, most of which are responding to EBV-infected cells. Infectious mono-
nucleosis is medically important because of the severity and duration of the acute
illness and also because of its long-term consequences especially the development
of certain cancers and autoimmune disorders.
2 Epidemiology of Primary EBV Infection
2.1 Age-Specific Prevalence of EBV Antibodies
EBV infection is extremely common worldwide and approximately 90 % of adults

become antibody-positive before the age of 30 (de-The et al. 1975; Venkitaraman
et al. 1985; Levin et al. 2010). A recent example is that 1037 (90 %) of 1148 sub-
jects 18 and 19 years old participating in the US National Health and Nutrition
Examination Surveys (NHANES) between 2003 and 2010 had IgG antibodies
against EBV viral capsid (VCA) antigen, indicative of prior infection (Balfour
et al. 2013a, b).
The prevalence of EBV antibodies in preadolescent children is lower, varying
from 20 to 80 % depending on age and geographic location. Factors clearly related
to early acquisition of primary EBV infection include geographic region (reviewed
in Hjalgrim et al. 2007), and race/ethnicity (Balfour et al. 2013a, b; Condon
et al. 2014). Other factors implicated are socioeconomic status (Henle et al.
1969; Hesse et al. 1983; Crowcroft et al. 1998), crowding or sharing a bedroom
(Sumaya et al. 1975; Crowcroft et al. 1998), maternal education (Figueira-Silva
and Pereira 2004), day care attendance (Hesse et al. 1983), and school catchment
area (Crowcroft et al. 1998).
Regarding race/ethnicity, it was recently shown that antibody prevalence across
all age groups of USA children 6–19 years old enrolled in NHANES between
2003 and 2010 was substantially higher in non-Hispanic blacks and Mexican
Americans than non-Hispanic whites (Balfour et al. 2013a, b). The greatest dispar-
ity in antibody prevalence was among the younger children, especially the 6- to
8-year-olds. Interestingly, the difference in antibody prevalence between whites
and non-whites diminished during the teenage years. Thus, family environment
and/or social practices may differ among white and non-white families, which
could account for this disparity in antibody prevalence in younger children. Within
each race/ethnicity group, older age, lack of health insurance, and lower household
education and income were statistically significantly associated with higher anti-
body prevalence.
These NHANES findings were confirmed (Condon et al. 2014) and extended to
include younger children (18 months to 6 years of age) living in the Minneapolis-
St. Paul metropolitan area. The Twin Cities study showed that the divergence in age-
specific antibody prevalence between blacks and whites was clearly apparent by the
age of 5 years.
The age at which primary EBV infection is acquired may be increasing in
developed countries (Morris and Edmunds 2002; Takeuchi et al. 2006; Balfour
et al. 2013a, b). This is important to monitor because there is a complex inter-
play between age of acquisition, symptomatic versus asymptomatic infection,
and the subsequent risk of EBV-associated cancers or autoimmune diseases. For
example, younger age at the time of primary EBV infection among Kenyan infants
was associated with elevated levels of EBV viremia throughout infancy, leading
the investigators to postulate that these infants were at higher risk for endemic
Burkitt’s lymphoma (Piriou et al. 2012; Slyker et al. 2013). Another study found
that Greenland Eskimo children acquired primary EBV infection at an earlier
age and had higher titers of IgG antibody against VCA than age-matched Danish
children (Melbye et al. 1984). The authors speculated that early infection with
“a large inoculum of EBV” explained why Eskimos were at high risk for naso-
pharyngeal carcinoma versus Danes who were not. Nevertheless, late acquisition
of primary EBV infection is also detrimental in several contexts. Adolescents and
young adults are more likely to experience infectious mononucleosis during pri-
mary infection than children (Krabbe et al. 1981). Furthermore, multiple sclerosis
(MS) is an inflammatory autoimmune disease that involves EBV infection and risk
of MS is higher among individuals who have experienced infectious mononucleo-
sis (Ascherio and Munger 2010). Infectious mononucleosis also increases the risk
of Hodgkin’s lymphoma (Hjalgrim et al. 2000). Thus, since age of primary EBV
infection is an important factor in infectious mononucleosis, it is an important
consideration for EBV-related diseases.
2.2 Routes of Transmission of Primary EBV Infection
Kissing is the major route of transmission of primary EBV infection among ado-
lescents and young adults. This was elegantly documented by Hoagland’s careful
clinical observations (Hoagland 1955) and confirmed many decades later by a pro-
spective study at the University of Minnesota (Balfour et al. 2013a, b). Penetrative
sexual intercourse has been postulated to enhance transmission (Crawford et al.
2006), but we have found that subjects reporting deep kissing with or without coi-
tus had the same risk of primary EBV infection throughout their undergraduate
years (Balfour et al. 2013a, b).
The incubation period of infectious mononucleosis is approximately 6 weeks.

Hoagland’s clinical records suggested an incubation period of 32–49 days
based on the dates of kissing episodes until the onset of infectious mononucleo-
sis (Hoagland 1955). A well-documented case was reported by Svedmyr et al.
(1984) in which the kissing event occurred 38 days prior to onset of symptoms.
Behavioral data from our medical history questionnaires collected during pro-
spective studies are consistent with an incubation period of 42 days (Balfour et al.
Unpublished observations).
Besides deep kissing, primary EBV infection can also be transmitted by blood
transfusion (Gerber et al. 1969), solid organ transplantation (Hanto et al. 1981), or
hematopoietic cell transplantation (Shapiro et al. 1988), but these routes account
for relatively few cases overall. Alfieri et al. (1996) used polymorphisms in the
EBV BAMHI-K fragment length and size polymorphisms in EBV nuclear anti-
gen EBNA-1, EBNA-2 and EBNA-3 proteins to identify the specific blood donor
responsible for transmitting EBV to a 16-year-old liver transplant recipient who
subsequently developed infectious mononucleosis.
The way young children contract EBV is unknown. A reasonable supposition
is that they are infected by their parents or siblings who are “carriers” of the virus
and who intermittently shed it in their oral secretions (Sumaya and Ench 1986).
An extreme example of this is the very early acquisition of EBV among three dis-
tinct Melanesian populations whose infants have multiple caregivers that premasti-
cate the baby’s food (Lang et al. 1977).
3 Clinical Manifestations of Primary EBV Infection
3.1 Acute Illness
Our prospective studies have determined that 75 % of young adults between the
ages of 18 and 22 develop typical infectious mononucleosis after primary EBV
infection. Approximately 15 % have atypical symptoms and 10 % are completely
asymptomatic (Balfour et al. 2013a, b); Balfour et al. Unpublished observations).
There are two common presentations among symptomatic patients. The first is the
abrupt onset of sore throat, which many patients say is the worst sore throat they
have ever had. Patients may also notice a swollen neck that results from cervical
lymph node enlargement. Parenthetically, anterior and posterior cervical nodes are
usually equally enlarged. The second common presentation is the gradual onset of
malaise, myalgia (“body aches”) and fatigue. Table 1 shows the frequency of signs
and their median duration in 72 undergraduate students studied prospectively.
Most findings have a median duration of 10 days or less, but fatigue and cervical
lymphadenopathy persist for a median of 3 weeks. Other findings, seen in fewer
than 20 % of cases in our experience, include abdominal pain, hepatomegaly,
splenomegaly, nausea, vomiting, palatal petechiae, periorbital and eyelid edema,
and rash. Rash is seen more often in patients given penicillin derivatives, which is
most likely due to transient penicillin hypersensitivity (Balfour et al. 1972).
Table 1 Clinical features of primary EBV infections in 72 undergraduate students studied

prospectively (48 women, 24 men; age range, 18–22 years)
Feature No. of subjects (%) Median duration (days)
Sore throat 68 (94 %) 10
Cervical lymphadenopathy 58 (81 %) 21
Fatigue 52 (72 %) 20
Upper respiratory symptoms 46 (64 %) 4.5
Headache 38 (53 %) 9.5
Decreased appetite 38 (53 %) 9.5
Feels feverish 34 (47 %) 4
Myalgia (body aches) 33 (46 %) 3
Subclinical hepatitis documented by elevated levels of alanine aminotransferase

occurs in approximately 75 % of prospectively followed patients and, in some
cases (5–10 %), overt hepatitis develops with tender hepatomegaly and jaundice
(Balfour et al. Unpublished observations).
Primary EBV infection in preadolescents has not been thoroughly investigated
most likely because prospective studies in young children are logistically difficult
to conduct. The assumption has been that the majority of primary EBV infections
in children before puberty are asymptomatic but that is not necessarily so. Young
children, especially those under the age of 4 years, may not develop a positive het-
erophile antibody response during primary EBV infection (Horwitz et al. 1981),
and unless specific EBV assays are performed, the diagnosis will be missed.
3.2 Complications of the Acute Illness
Fortunately, serious complications during the acute phase of primary EBV infec-
tion are rare. Table 2 shows reported complications divided into those estimated to
occur in at least 1 % of patients and those that are seen in fewer than 1 % of cases
(Hoagland and Henson 1957; White and Karofsky 1985; Robinson 1988; Connelly
and DeWitt 1994; Jenson 2000). Splenic rupture is the most feared complication,
which has kept many athletes out of competition for weeks. Current consensus is
that athletes may return to contact sports 3 weeks after onset of infectious mono-
nucleosis provided they are afebrile, their energy has returned to normal, and they
have no other abnormalities associated with primary EBV infection (Putukian
et al. 2008).
Although most symptoms associated with infectious mononucleosis resolve in
a matter of months, there can be severe and lasting disease that develops following
primary EBV infection. One of these complications may manifest in the form of
chronic active EBV (CAEBV). Patients presenting with CAEBV generally exhibit
signs that can occur during infectious mononucleosis such as fever, lymphadenop-
athy, splenomegaly, and hepatitis and show markedly elevated levels of EBV DNA
Table 2 Complications during acute primary EBV infection

Frequency of complications Complication
≥1 % Airway obstruction due to oropharyngeal inflammation
Meningoencephalitis
Hemolytic anemia
Streptococcal pharyngitis
Thrombocytopenia
<1 % Conjunctivitis
Hemophagocytic syndrome
Myocarditis
Neurologic disorders (other than meningoencephalitis)
Neutropenia
Pancreatitis
Parotitis
Pericarditis
Pneumonitis
Psychological disorders
Splenic rupture
in the blood (Kimura et al. 2001). Less frequently, patients may also present with
lymphoma or hemophagocytic disease, a complication of EBV that is discussed in
greater detail below (Kimura et al. 2003). Interestingly, in many cases of CAEBV
outside of the USA, particularly in Japan, EBV is reported to infect T or NK cells
rather than its usual reservoir of B cells (Quintanilla-Martinez et al. 2000; Kimura
et al. 2001). Several instances of B cell tropic CAEBV have also been reported,
but these fall into the minority of documented incidences (Schooley et al. 1986;
Kimura et al. 2003). While some patients have T or NK cell dysfunction, none of
the subjects in a National Institutes of Health (NIH) study had mutations typically
associated with EBV-related immunodeficiencies and thus the disease observed in
that study was considered to be largely insidious. The most successful treatment
for CAEBV has been hematopoietic stem cell transplant. In the same NIH study,
all but one of the patients who presented with CAEBV died within an average of
six years unless a transplant was received. Those that survived all subsequently
became negative for EBV DNA in the blood (Cohen et al. 2011a, b).
EBV may also cause hemophagocytic disease, which is alternately referred to
in the literature as EBV-associated hemophagocytic syndrome or EBV-associated
hemophagocytic lymphohistiocytosis (HLH). The relative rarity of any form
of HLH stands as a barrier to diagnosis, and thus, cases of EBV-HLH are even
more uncommon. The disease is characterized by fever, splenomegaly, and cyto-
penias, though the key laboratory signs are high levels of ferritin and soluble
CD25 (Jordan et al. 2011; Janka 2012). The distribution of EBV-HLH seems to
be similar to CAEBV, focused mainly in Asian populations and infecting T or NK
cells in those groups (Kawaguchi et al. 1993) though B cells are also infected in
other populations (Beutel et al. 2009). EBV-HLH may be related to one of several
primary immunodeficiencies discussed below, but EBV infection may be a trig-

gering event even in the absence of an identified genetic condition. Interestingly,
transcriptome profiling studies showed that the peripheral blood gene expression
signature observable during infectious mononucleosis highly resembles that of
HLH (Dunmire et al. 2014). This reinforces links between primary EBV infections
as a trigger in the initiation of HLH.
X-linked lymphoproliferative syndrome (XLP) is a disease characterized by
anemia, hypergammaglobulinemia, and lymphohistiocytosis. Generally, the boys
who present with this disease exhibit massive cellular responses to primary EBV
infection that result in hemophagocytic pathology, even though they are simulta-
neously unable to control EBV-transformed B cells (Cannons et al. 2011). It was
discovered that the main deficiency involved with XLP is in the signaling lym-
phocytic activation molecule-associated protein (SAP), which is encoded by the
human gene SH2D1A. Mutations in this gene disrupt the ability of T cells and NK
cells to interact with B cells, resulting in a lack of immunoglobulin class switching
and meaning that T cells and NK cells cannot efficiently recognize B cell targets
to induce death (Hislop et al. 2010; Zhao et al. 2012).
3.3 EBV-Associated Diseases
EBV has been shown to be the causative agent of about 1 % of the worldwide
human cancer burden. In particular, EBV infection is associated with neoplasia of
lymphoid and epithelial origins including endemic Burkitt’s lymphoma (eBL) and
Hodgkin’s lymphoma (HL) in the case of the former, as well as nasopharyngeal
carcinoma and gastric carcinoma in the case of the latter. EBV is considered the
etiologic agent in 95 % of cases of eBL, which occur in regions where malaria is
common (Brady et al. 2007). Likewise, EBV can be detected in a high proportion
of HL cases in underdeveloped nations, but accounts for less than half of cases in
Western countries (Flavell and Murray 2000). It is important to note that incidence
of infectious mononucleosis is exceptionally low in Southeast Asia and equatorial
Africa where EBV infection during childhood is nearly ubiquitous; thus, it might
be extrapolated that infectious mononucleosis does not have a strong correlation
with either eBL or HL in these areas (de-The et al. 1978). Emerging evidence sug-
gests that previous presentation with infectious mononucleosis can increase the
risk of HL (Hjalgrim et al. 2000). While associations between infectious mononu-
cleosis and epithelial carcinomas have not been explored, the presence of EBV in
tumors from nasopharyngeal and gastric carcinoma patients is well documented.
About 10 % of human gastric carcinomas are EBV-positive (Iizasa et al. 2012).
Like eBL and HL seen in underdeveloped nations, nasopharyngeal carcinomas
from endemic regions are virtually all positive for EBV DNA (Raab-Traub 2002),
with these tumors thought to be derived from a single EBV-infected epithelial cell
(Raab-Traub and Flynn 1986; Pathmanathan et al. 1995).
It is possible that achieving a very high viral titer in the blood at any point in life
predisposes individuals to subsequent EBV-related cancers. For example, patients
who present with eBL and endemic nasopharyngeal carcinomas live in malaria
endemic areas. Evidence shows that patients being treated for malarial disease can
have extremely high titers of EBV in the blood (Nijie 2009). Titers of this magnitude
are seen exclusively in patients presenting with infectious mononucleosis in devel-
oped countries. Thus, it may be possible to reduce occurrences of cancer with pro-
phylactic or therapeutic vaccines aimed at preventing primary EBV infection or at the
very least reducing the set point at which the virus is maintained in these individuals.
In recent years, infectious diseases have been emerging as possible triggers for
autoimmune disorders. EBV infection in particular has come to be highly associ-
ated with occurrence of MS. EBV as a causation factor in MS was first proposed
over thirty years ago (Warner and Carp 1981). Many correlative observations for
this trend exist, including a low incidence of infectious mononucleosis and MS
in developing countries, and MS usually first manifests after the adolescent years
during which EBV would be acquired, increasing at a rate of 11 % per year fol-
lowing primary EBV (Ascherio and Munger 2010). There is also a high associa-
tion between patients who recall having infectious mononucleosis and subsequent
development of MS (Alotaibi et al. 2004; Pohl et al. 2006; Banwell et al. 2007).
Furthermore, MS in EBV-negative individuals occurs very infrequently (Levin
et al. 2010; Pakpoor et al. 2013).
A causative role for EBV was supported by examination of the antibody pro-
files of patients with MS, scrutinizing the viral loads, epitope specificity, and
quantity of antibodies, especially those against EBNA. The risk of MS increases
positively with levels of circulating anti-EBNA antibodies (Ascherio et al. 2001;
DeLorenze et al. 2006; Levin et al. 2005; Sundstrom et al. 2004). The ability to
discriminate MS cases and controls was substantially enhanced by the inclusion
of quantitative measures of the anti-EBNA-1 response to EBV infection (Strautins
et al. 2014). Interestingly, the strong genetic association of MS with particular
human leukocyte antigen (HLA) alleles primarily reflects the association with
anti-EBV responses (Rubicz et al. 2013). Researchers recently treated an MS
patient with autologous T cells expanded by exposure to EBV antigens. Transfer
of the EBV-specific CD8 T cells resulted in a decrease in anti-EBV antibody as
well as the size and number of MS related lesions in the brain (Pender et al. 2014).
4 Virus–Host Interactions During Primary EBV Infection
4.1 Incubation Period
The long incubation period of EBV continues to be poorly understood due to

a lack of samples obtained between the time of infection and presentation with
EBV-related symptoms. During primary infection, viral replication is first detected
in the oral cavity (Balfour et al. 2013a, b). The virus infects tonsillar epithelial
cells as well as B cells in the parenchyma of the tonsil (Wang et al. 1998). There
may be a cyclic nature to the pattern of infection in the oral cavity as it has been
shown in vitro that virus derived from epithelial cells has a much higher entry
efficiency for infecting B cells and vice versa, resulting a switched viral tropism
depending on the cell type in which the virus replicates (Borza and Hutt-Fletcher
2002). At some point during the incubation period, the virus moves from the oral
cavity to the blood. Little is known about the kinetics or means of this transition.
A type I interferon response was detected by gene expression profiling approx-
imately 2 weeks prior to symptom onset in some subjects experiencing primary
EBV infection (Dunmire et al. 2014). Viral genomes can sometimes be detected
in the peripheral blood as early as three weeks prior to symptom onset and con-
sistently at least one week prior to illness (Dunmire et al. unpublished observa-
tions), where it is probably maintained latently in resting memory phenotype B
cells (Hadinoto et al. 2008).
4.2 Acute Infection
The kinetics of the EBV viral loads and EBV-specific antibody responses during
primary EBV infection are illustrated in Figs. 1 and 2. High viral loads in both the
Oral cell virus VCA IgM

Saliva virus VCA IgG
Blood virus EBNA IgG
Antibody titer or log10 viral genomes/mL
0 50 100 150 200 250 300 350 400

Days post symptom onset
Fig. 1 Kinetics of EBV viral load and antibody responses in subjects with primary EBV infec-
tion. Depicted are viral loads in whole blood, saliva, and oral cell pellets (black lines) as well as
IgM and IgG antibodies to VCA and IgG to EBNA1 (colored lines). Note the limit of detection
of the EBV viral genomes in blood was 200 copies per mL of whole blood
Oral cell virus BZLF1 IgG (IE)

Saliva virus p54 IgG (BMRF1, E)
Relative antibody index or log10 viral genomes/mL
Blood virus p138 IgG (BALF2, E)

p18 IgG (BFRF3, L)
p23 IgG (BLRF2, L)
0 50 100 150 200 250 300 350 400

Days post symptom onset
Fig. 2 Kinetics of antibody responses to additional EBV antigens as determined by immunoblot.

Depicted are viral loads in whole blood, saliva, and oral cell pellets (black lines) as well as the
relative kinetics of antibodies generated against the EBV antigens p18, p23, BZLF1, p138, and
p54 (colored lines). The gene name of each antigen and the stage of expression (IE immediate
early, E early, or L late) are indicated in parentheses
oral cavity and blood are detected around the time of symptom onset in infectious
mononucleosis and accompanied by production of IgM antibodies to EBV VCA
and an extraordinary expansion of CD8 T lymphocytes (Balfour et al. 2013a, b).
While CD8 T cell responses during primary EBV infection have been thoroughly
discussed elsewhere (Hislop et al. 2007; Odumade et al. 2011), a brief discussion
of CD8 T cell and B cell responses follows. Of particular interest is the response
of cytotoxic CD8 T cells, which have been shown to be important in the control
of EBV-infected B cells as evidenced by the disease that occurs in patients who
lack elements of CD8 T cell function such as the ability to interact with and kill
infected B cells (Rigaud et al. 2006; Palendira et al. 2011).
During infectious mononucleosis when there are very high numbers of circu-
lating CD8 T cells, many of these cells are EBV-specific and directed toward lyt-
ically expressed proteins from the immediate early and early stages of the lytic
cycle with particular predilection for the immediate early. Cells specific for some
late antigens tend to emerge only after the patient has been infected for some time
as discovered by generating T cell clones from infectious mononucleosis patients
(Abbott et al. 2013). The case of latency, however, is rather different and is often
dependent on the relevant HLA type of the EBV-infected individual in question.
Immunodominant epitopes for the most prevalent HLA types generally include
those derived from latently expressed proteins, especially EBNA-2 and EBNA-3,
although some patients develop a strong population of CD8 T cells specific for
less readily expressed antigens such as EBNA-1 (Blake et al. 2000; Hislop et al.
2007).
Both CD8 and CD4 T cells require cell-to-cell contact in order to become acti-
vated and perform related functions (Adhikary et al. 2007; Merlo et al. 2010).
Although total CD4 numbers do not increase appreciably during infectious mono-
nucleosis, evidence exists to support the concept that CD4 T cells are activated
and help control infected B cells. Using major histocompatibility complex (MHC)
II tetramers, it was shown that several lytic antigens are recognized by CD4 T
cells during acute infection and that these cells are maintained at low levels in the
blood of EBV-infected hosts (Long et al. 2013). That study also revealed that the
response to different antigens varies: CD4(+) T cell responses to EBNA1 did not
develop until much later, which likely explains the delay in EBNA1 IgG antibody
responses.
Natural killer (NK) cells are also emerging as important players during infec-
tious mononucleosis. Several immunodeficiencies involving T and NK cells and/
or their cytolysis pathways result in severe EBV-related outcomes (Menasche
et al. 2005; Parvaneh et al. 2013). These include familial hemophagocytic lym-
phohistiocytosis 2 (FHL2), x-linked lymphoproliferative syndrome (XLP), XIAP
deficiency, and x-linked immunodeficiency with Mg+2 defect (XMEN) disorders
and are discussed in detail in another chapter of this book. The value of NK cells
specifically was suggested by the observation that NK cells preferentially killed
EBV-infected cells as the virus entered the lytic cycle (Pappworth et al. 2007).
The role of NK cells in vivo was investigated using a humanized mouse model,
where NOD-scid γc−/−(NSG) mice were reconstituted with CD34+ lin-hemat-
opoietic stem cells (Strowig et al. 2010; Ramer et al. 2011) and infected with the
B95.8 strain of EBV and monitored for signs of infectious mononucleosis such as
CD8 lymphocytosis and viremia. Animals depleted of NK cells experienced more
severe EBV-related disease (Chijioke et al. 2013). It is interesting to note that NK
depletion after an established EBV infection did not have significant effects, in
contrast to the effect of depletion before infection. Given the gap in the robustness
of responses observed between tonsillar and peripheral blood NK cells, it is pos-
sible that peripheral NK cells during the systemic infectious mononucleosis phase
are less important than those during early infection in the oropharynx (Strowig
et al. 2008). Indeed, studies in humans have disagreed about the state of NK cells
in peripheral blood during infectious mononucleosis. Work from Williams et al.
showed an inverse correlation between NK cell numbers in the periphery and virus
in the blood (Williams et al. 2005). In contrast, a larger prospective study found a
positive correlation (Balfour et al. 2013a, b). Thus, the interplay between NK cells
and blood virus in human subjects needs further study. Of course, the specific type
or subset of NK cells may be more important than total numbers. NKG2C+ NK
cells were shown to specifically respond to and play a crucial role in immunity to
cytomegalovirus (Lopez-Verges et al. 2011). However, NKG2C+ NK cells do not
expand upon EBV infection (Hendricks et al. 2014). Rather NK cells expressing
NKG2A and CD54 could be found in higher numbers in the tonsils of EBV car-
riers (Lunemann et al. 2013) and in the peripheral blood during acute infection
(Hendricks et al. 2014). In a study of pediatric infectious mononucleosis patients,
it was recently shown that CD56dim NKG2A+ KIR-NK cells preferentially pro-
liferate in response to EBV-infected cells during acute infection (Azzi et al. 2014).
EBV also seems to have evolved mechanisms to interfere with the activation of
NK cells during viral replication. The protein encoded by the EBV open reading
frame BILF1 downregulates expression of HLA-A and HLA-B on the surface of
infected cells but not HLA-C, which is inhibitory to NK cells (Griffin et al. 2013).
Several specific populations of NK cells have been implicated in limiting the
transformation of B cells by EBV in vitro. When exposed to dendritic cells (DCs)
prepared with EBV, CD56bright CD16-NK cells were preferentially primed and
were able to limit B cell transformation in vitro in an interferon (IFN) γ-dependent
manner. Interestingly, tonsillar NK cells were much more efficient than NK cells
derived from peripheral blood (Ferlazzo et al. 2004; Strowig et al. 2008). Further
understanding of NK cell recognition of EBV-infected cells and their responses
during human infection are needed at this point.
Despite our growing understanding of the innate and adaptive immune
response to EBV, it remains unclear why primary EBV infection leads to infec-
tious mononucleosis in adolescents yet is most often asymptomatic in young
children. It is possible that adolescents receive a larger initial virus inoculum
when transmission occurs through deep kissing. Our data did not find any cor-
relation between virus copy number in the oral cavity and severity of illness
(Balfour et al. 2013a, b); however, peak virus copy number in the oral cavity
may not directly reflect the initial virus inoculum, nor is it possible to measure
the initial virus inoculum with natural infection. Another idea put forward is that
infectious mononucleosis in adolescents may reflect the response of cross-reac-
tive memory CD8 T cells. For example, influenza-specific CD8+ T cells might
cross-react with EBV (Clute et al. 2005), and as adolescents are presumably
more likely to have high numbers of influenza-specific CD8+ T cells, they could
react more strongly with EBV. However, we have not seen evidence of influ-
enza–EBV dual-specific CD8+ T cells in our cohort (Odumade et al. 2012) and
it remains debatable whether preexisting (cross-reactive) CD8+ T cell immunity
to EBV would increase or decrease infectious mononucleosis. A CD8+ T cell
peptide epitope vaccine was effective in generating EBV-specific CD8+ T cell
responses, and there was no incidence of infectious mononucleosis in the vac-
cinated group, although the study was small (Burrows et al. 1990). An exciting
proposition has arisen from recent data that implicate an NKG2A+ NK cell as
important in EBV control (Azzi et al. 2014; Hendricks et al. 2014). Azzi and
colleagues showed that CD56dim NKG1A+ KIR-NK cells were found at signifi-
cantly lower levels in the peripheral blood of adolescents and adults compared to
children, suggesting that decreased NK-mediated immune control of EBV could
explain why adolescents and adults experience infectious mononucleosis more
frequently than children.
4.3 Convalescence
During the convalescent period of infectious mononucleosis (3–6 months postin-

fection), the number of CD8+ T cells declines to normal levels (Balfour et al.
2013a, b). Previous work in mouse suggested that infection with herpesviruses
may cause long-term changes to the “readiness” of host immune cells, thus prim-
ing them for subsequent bacterial infections (Barton et al. 2007). These effects
were later shown to be transient in that model (Yager et al. 2009), but work in
human subjects showed that there were no long-term gene expression changes
observable in peripheral blood mononuclear cells following acquisition of EBV
(Dunmire et al. 2014). The virus is maintained in resting memory-like B cells.
Probably, the most interesting phenomenon that occurs during this phase of
infection is related to the levels of antibody that are produced and maintained by
latently infected hosts.
Some peculiar trends exist in the antibody response to EBV. For example,
antibodies against EBNA-1 display an unusually long delay between virus acqui-
sition and the presence of anti-EBNA-1 IgG, as it generally appears only after a
patient with infectious mononucleosis has convalesced (Henle et al. 1987; Hille
et al. 1993). This is especially odd given the high levels of class-switched anti-
body toward EBV antigens that can be measured in many patients presenting with
infectious mononucleosis, including responses to latently expressed gene prod-
ucts of EBNA-2 and EBNA-3 (Long et al. 2013) (Figs. 1 and 2). The delayed
kinetic might be associated with alanine–glycine-rich regions within the structure
of EBNA-1’s protein product, which has been shown to inhibit proteosomal pro-
cessing of relevant epitopes (Levitskaya et al. 1997). That this is the case, how-
ever, is not clear and may have to do more with accumulation of protein released
from cells for cross-presentation. In contrast, high levels of antibody specific for
the immediate early antigen BZLF1 are maintained for the life of the host. This is
likely because BZLF1 is expressed by cells undergoing viral reactivation and thus
is more frequently presented to B cells in latently infected hosts in comparison with
proteins that are coded for later in the viral replication process (Massa et al. 2007).
5 Diagnosis of Infectious Mononucleosis Due to EBV
Infectious mononucleosis due to EBV should be suspected in patients, especially

teenagers and young adults, who present with an acute illness characterized by
sore throat, cervical lymphadenopathy, fever, and fatigue. Clinical signs that make
the diagnosis more likely are exudative pharyngitis with swelling of the uvula and
tonsils; periorbital and eyelid edema; and symmetrical cervical and postauricular
lymphadenopathy.
A heterophile test using one of the numbers of commercially available antibody
kits is most often done to support the clinical diagnosis (Table 3). Heterophile tests
are relatively inexpensive and easy to perform. However, heterophile antibodies
Table 3 Staging EBV infection by enzyme immunoassay patterns

Stage of infection VCA IgM VCA IgG EBNA-1 IgG
Naïve Negative Negative Negative
Acute primarya 1—2+ Negative—1+ Negative
Subacuteb 3—4+ 2—4+ Negative—1+
Convalescentc Negative—3+ 3—4+ Negative—2+
Pastd (90–95 % of cases) Negative 3+ 3—4+
Pastd (5–10 % of cases) Negative 3+ Negative
a0–3 weeks after onset of illness
b3 weeks to 3 months after onset of illness
c3–6 months after onset of illness
d>6 months after onset of illness
by definition are not specific. They are IgM class antibodies directed against
mammalian erythrocytes. False-positive heterophile tests have been reported in a
myriad of conditions including other acute infections, autoimmune disease, and
cancer (Sadoff and Goldsmith 1971; Phillips 1972; Horwitz et al. 1979; Hendry
and Longmore 1982; Fisher and Bhalara 2004). Although heterophile tests are
most commonly used to diagnose infectious mononucleosis, the US Centers for
Disease Control and Prevention has recently advised against them “for general
use” because of their non-specificity and the possibility of false-negative results
especially in young children (Centers for Disease Control and Prevention 2014).
In our experience, however, if the clinical picture is typical of infectious mononu-
cleosis and the heterophile antibody test is positive, no additional diagnostic pro-
cedures are necessary.
No single antibody test is perfect for confirming the diagnosis of primary EBV
infection. Most patients (75 %) will have VCA IgM antibodies at the onset of
clinical illness and 95 % eventually make them (Table 3). In prospective studies
of EBV-naïve college students, we detected VCA IgM by EIA as early as 8 days
before onset of symptoms. The median first day of detection was 2 days after onset
of illness (Fig. 1). However, a problem with IgM class antibody tests in general is
cross-reactivity with related pathogens. In the case of VCA IgM antibodies, false-
positive results have been reported especially with cytomegalovirus infections
(Guerrero-Ramos et al. 2014).
Depending on the assay platform and antigen used in the assay, VCA IgG
antibodies are first detected during the first month of illness. IgG class antibody
against the p18 component of VCA develops later than against the p23 component
(see Fig. 2). Using an EIA with p18 as the antigen, we found in prospective stud-
ies that the median first day of detection after onset of illness was 31 with a very
wide range of 1–118 days. Everyone who experiences a primary EBV infection
develops IgG antibodies to VCA (Balfour et al. 2013a, b), so this is the best single
test to verify a previous EBV infection. It is superior to EBNA-1 antibody tests
because 5–10 % of patients never make EBNA-1 antibodies. Trends in viral load
and antibody titer are shown in Figs. 1 and 2.
Antibodies against EBNA-1 are slow to appear with a median first day of detec-
tion of 91 days (Fig. 1). Because of this, the presence of EBNA-1 antibodies dur-
ing an acute illness rules out primary EBV infection. In general, the vast majority
of EBV infections in immunocompetent patients can be staged by assaying a
blood sample for VCA IgM, VCA IgG, and EBNA-1 antibodies and interpreting
the results as shown in Table 4. Western blots or immunoblots can be used to con-
firm results of screening tests and also stage EBV infections (Schubert et al. 1998;
Bauer 2001).
Measurement of IgG antibodies against EBV early antigen (EA) is not use-
ful for the diagnosis of primary infection because only 60–80 % of acutely ill
patients are positive and EA antibodies can be found for years in 20 % of healthy
individuals (Hess 2004; Centers for Disease Control and Prevention 2014).
When the available antibody data do not distinguish the stage of an EBV infec-
tion, IgG avidity assays may be useful (Schubert et al. 1998; Nystad and Myrmel
2007). The principle is that during the course of infection, antibodies with high
binding strength to their target are selected. In other words, IgG antibodies dur-
ing the acute phase of infection do not bind to their target as tightly as antibod-
ies produced during convalescence. Low-avidity antibodies can be dissociated
from their target by exposure to urea or another chaotropic reagent. Avidity is
determined by comparing the amount of antibody detected with and without urea
treatment.
The best test for diagnosing and monitoring EBV infections in the immuno-
compromised host is the blood viral load (or quantitative EBV DNAemia assay)
usually performed on a PCR platform (Holman et al. 2012). Most of these infec-
tions are not primary, but there are a few that are and result in classic infectious
mononucleosis. The monitoring concept is to anticipate the risk of impending
EBV disease based on sequential changes in blood viral load. A threshold amount,
which varies from center to center, is established that triggers intervention with
changes in immunosuppressive and/or antiviral drug regimens.
6 Genetic Susceptibility
Given the disparities in antibody prevalence among populations of different racial

backgrounds observed in surveys of children in the USA, there may exist vari-
ances in the genetic susceptibility of certain race/ethnicities to EBV. This theory is
supported by the incidence of infectious mononucleosis in family groups. A study
examining infectious mononucleosis concordance in twins from the California
Twin Program found that monozygotic twins were twice as likely to both develop
infectious mononucleosis than dizygotic twin pairs. When the analysis was lim-
ited to same-sex dizygotic twins, the risk in those groups was higher (Hwang et al.
2012). These findings were then further expanded to include first-degree relatives
in a large study of Danish families surveyed by governmental registries. The rate
Table 4 Diagnostic tests for EBV infections
Test Advantages Disadvantages Proportion of patients Median day detected after onset
positivea of illness (range of days until
detection)a
Heterophile antibody Inexpensive, easy to Non-specific (false positives 85 % (72 % the first week) 0 (6–31)
perform, becomes negative due to acute infections and
3–12 months postinfection autoimmune diseases); may
be negative during first week
Infectious Mononucleosis
of illness and persistently

negative in young childrenb
VCA IgM antibody Specific, becomes negative Not usually performed at 95 % (85 % the first week) 2 (21–20)
3–12 months postinfection point of care sites
VCA IgG antibody Best test for diagnosis of past Not usually performed at 100 % 31 (1–118)
EBV infection point of care sites
EBNA-1 IgG antibody Best test to distinguish acute Not usually performed at 90–95 % 91 (6–479)
from convalescent EBV point of care sites; 5–10 %
infection of patients never become
positive
EA antibody A marker of acute infection Absent in 20–40 % of acute Not tested Not tested
illnesses; persists for years
in ~20 % of cases
Immunoblot antibodies Can be used to stage infection Relatively expensive 100 %c 2 (25–60)
(acute, convalescent, past)
Blood viral load Correlates with severity Viremia is short-lived 80 % 4 (8–38)
of illness; best test to monitor and may be missed in
infection in the immunocompetent
immuncompromised host patients
(continued)
227
Table 4 (continued)
228
Test Advantages Disadvantages Proportion of patients Median day detected after onset
positivea of illness (range of days until
detection)a
Oral viral load Noninvasive, confirms past Cannot be used to diagnose 100 % −4 (21–31)
infection acute infection because
virtually all antibody-
positive adults shed oral virus
intermittently
aBased on prospective studies of EBV-naive college students who developed primary EBV infections (Balfour et al. 2005, 2013a; Balfour et al. Unpublished
observations)
bAlthough heterophile tests are most commonly used to diagnose infectious mononucleosis, the dendritic cell (DC):conventional (cDC) has recently advised
against them “for general use” (Centers for Disease Control and Prevention 2014)
cTo one or more antigens
S.K. Dunmire et al.
ratios for same-sex twins were highest, followed by groups of siblings (Rostgaard
et al. 2014). It is important to note that siblings tend to have similar environments
and behaviors, which may explain why the degree of concordance is so high.
Further evidence lends credence to a genetic basis for susceptibility to infec-
tious mononucleosis. Recently, the HLA locus was identified as a major factor
influencing antibodies to EBNA-1 in large Mexican American families (Rubicz
et al. 2013). Although the authors interpreted this genetic influence on suscep-
tibility to EBV infection, not all individuals who are infected with EBV make a
strong antibody response to EBNA-1. Therefore, it is possible that the HLA locus
influences the response to infection rather than the infection rate per se. In regard
to infectious mononucleosis, the effect on adaptive response to primary infec-
tion may be sufficient to achieve a change in whether or not primary infection is
symptomatic. Class II MHC is also required for viral entry into the cell, although
whether or not there are alterations in viral entry efficiency between HLAs preva-
lent in different racial groups has not been explored.
7 Prevention of Primary EBV Infection
Given the disease burden associated with acute and chronic EBV diseases, devel-
opment of an EBV vaccine has long been a priority for researchers in the field.
The National Cancer Institute recommended that more clinical trials be conducted
to test the safety and efficacy of a vaccine to prevent infectious mononucleosis and
cancers caused by EBV (Cohen et al. 2011a, b). Although the first phase 1 trial for
a prophylactic EBV vaccine occurred almost twenty years ago (Gu et al. 1995),
there has been relatively little progress since. In total, three prophylactic vaccines
have been tested in humans, and although all proved at least moderately immu-
nogenic, none provided sterilizing immunity (Balfour 2014). However, sterilizing
immunity is probably not necessary to impact symptomatic disease caused by pri-
mary EBV infection. For example, a phase 2 trial in Belgium showed that vaccina-
tion with a gp350 subunit adjuvant vaccine could reduce the number of cases of
infectious mononucleosis (Sokal et al. 2007).
Whether or not a vaccine exclusively targeting gp350 is sufficient to prevent
EBV-related disease, however, is unknown. In the case of epithelial neoplasia, it
seems less convincing on the grounds that gp350 is not strictly required for viral
entry into epithelial cells and can be achieved via the viral proteins gH and gL,
albeit less efficiently (Hutt-Fletcher 2007). Increasing the range of the vaccine
to include other proteins necessary for this entry such as the aforementioned gH
or gL, especially given what is known concerning the switch tropism of EBV
between B cells and epithelial cells, might greatly improve the efficacy of a vac-
cine with the goal of preventing EBV-positive lymphomas and carcinomas.
8 Treatment
There is no currently accepted specific treatment for infectious mononucleosis.

While it is clear that acyclovir and valacyclovir have an antiviral effect in vivo,
a clinical benefit has not been convincingly demonstrated to date (Tynell et al.
1996). Ganciclovir and valganciclovir have been used to treat EBV infections in
immunocompromised hosts, but there are no controlled trials demonstrating clini-
cal efficacy. Corticosteroids are often prescribed to treat inflammatory complica-
tions such as airway obstruction or autoimmune phenomena such as anemia and
thrombocytopenia, but the value of these drugs is controversial, and they may
impair clearance of the viral load (Luzuriaga and Sullivan 2010).
9 Animal Models of Infectious Mononucleosis
9.1 Humanized Mice
One of the major barriers to studying many human viruses is the lack of a small
animal model. EBV belongs in this group because it only infects primates.
Although studies have been performed with murine gammaherpesvirus-68 (MHV-
68), there are important genetic differences between that virus and EBV. In order
to directly evaluate the effects of EBV on various lymphoid compartments in vivo,
efforts toward developing feasible methods for modeling human infections have
resulted in the creation of humanized mice. Humanized mice have only become
a viable option for studying human diseases within the last ten years or so due to
low engraftment of human cells even in animals with severe combined immuno-
deficiency (SCID) or knockout of one of the recombinase activating genes (RAG).
With the advent of common gamma chain (a receptor component for IL-2, 4, 7, 9,
15, and 21) knockouts, engraftment improved dramatically.
The two types of mice most commonly employed are the previously mentioned
NSG and the BALB/c RAG−/− γc−/−. Cells derived from either human fetal
liver, human fetal thymus, or CD34+ hematopoietic stem cells are then transferred
into neonatal mice to reconstitute their immune systems (Leung et al. 2013). Of
these two mouse strains, the NSG mouse has more complete reconstitution of
the T and NK compartments and could be maintained for 22 weeks with latent
virus detectable and without developing tumors or other EBV-related pathology
(Strowig et al. 2009). This model has also been shown to give an approximation
of human immune components (Strowig et al. 2010; Ramer et al. 2011). Mice are
bred onto a transgenic HLA background. This then allows for thymic selection of
the human-derived cells and later specific responses during infection of these ani-
mals with EBV (Yajima et al. 2008; Shultz et al. 2010).
It is important to note, however, that study of EBV within this context may
neglect important aspects of interplay between EBV and epithelial cells, which
have been shown to be important during the replication of EBV, chiefly within the
oropharynx (Borza and Hutt-Fletcher 2002). Nevertheless, humanized mice repre-
sent a significant step forward to investigating cellular responses in vivo.
In particular, the roles of certain subsets of cells have been interrogated through
the depletion of these populations prior to infection with EBV. A significant gap
in our understanding of the early innate response to EBV existed and had only
been addressed in vitro prior to the advent of this model. Using the NSG mouse
and virus obtained from the B95-8 cell line, investigators can now effectively emu-
late events that occur early during infection in the peripheral blood. Humanized
mice exhibit classical features of infectious mononucleosis such as elevated CD8
counts and high levels of IFNγ (Chijioke et al. 2013). Specific deletion of sub-
sets of NK cells could then be performed to examine which were most important
during the response to primary EBV infection. Furthermore, the roles of adaptive
immune cells may be examined as well. Investigators looking at CD4 and CD8
T cells found EBV-specific T cells were HLA restricted and responded to autolo-
gous lymphoblastoid cell lines. When either CD4 or CD8 T cells were depleted
from these mice, they developed EBV-related pathologies (Strowig et al. 2009).
These results are not particularly surprising given the same might be observed in
primary human immunodeficiencies as previously described, but provided impor-
tant evidence that the humanized mouse can be an appropriate tool for asking
questions about the response to EBV. Mouse models, however, neglect nearly all
aspects of initial infection as events in the oral cavity cannot occur as EBV does
not have tropism for murine epithelial cells. In order to examine infection of the
tonsil and oral epithelium, primate models might be preferred as there is estab-
lished infection in the oral cavity similar to EBV in humans.
9.2 Rabbits
Studies from Japan have suggested that EBV infection may be modeled in rab-
bits (Takashima et al. 2008; Okuno et al. 2010). Animals in these studies were
alternately inoculated intravenously, intranasally, and perorally with EBV derived
from the B95-8 cell line. Most animals had only transient levels of virus detect-
able in the blood, but two had consistent viral titers. Both T and B cells appeared
to be infected in these incidences (Takashima et al. 2008; Okuno et al. 2010). It
is important to note that while early antigen IgG titers were maintained, VCA
IgG antibodies were transient even when very high quantities of virus were used
(Okuno et al. 2010). Though not a model of infection with a great deal of similar-
ity to infectious mononucleosis, rabbits may still provide interesting insight with
regard to the kinetics and magnitude of antibody responses to EBV, which has
implications for the development and testing of humoral component vaccines such
as the gp350 subunit vaccine.
9.3 Non-human Primates
Non-human primates are the other major option for investigating EBV infection
in vivo. The gamma herpesvirus lymphocryptovirus (LCV) exists in two types:
that which infects old world and new world primates. The LCV that infects old
world primates has higher genetic similarity to EBV than new world LCV. The
open reading frames of rhesus LCV have 28–98 % amino acid identity with EBV
(Wang 2013). Thus, rhesus LCV is used to model EBV in rhesus macaques. The
symptoms of EBV and LCV are very similar, and LCV can be given orally to
emulate natural infection routes in humans. One difference is that the incubation
period of LCV is generally shorter, lasting about three weeks rather than six.
LCV can be manipulated using a bacterial artificial chromosome system to
allow for mutation of the LCV genome. This enables researchers to examine the
possible effects of EBV homologues in vivo. For example, BARF-1 has been
shown to bind and inhibit the signaling of colony stimulating factor 1 (Elegheert
et al. 2012; Shim et al. 2012), which can promote the maturation and maintenance
of type I interferon producing plasmacytoid DCs (Fancke et al. 2008). Knocking
out BARF-1 in LCV resulted in more favorable outcomes and lower viral loads in
infected rhesus macaques (Ohashi et al. 2012).
The LCV model can also be used to test potential prophylactic or therapeutic
vaccines. An LCV gp350 subunit vaccine protected against infection and reduced
the viral set point in rhesus macaques (Sashihara et al. 2011). More recently,
Leskowitz and colleagues showed that an adenovirus-based vaccine encoding LCV
EBNA-1 induced expansion of CD4+ and CD8+ T cells specific for EBNA-1 in
rhesus macaques with natural persistent LCV infection (Leskowitz et al. 2014).
10 Summary and Outlook
EBV is one of the most important human pathogens. Although this virus was dis-
covered more than 50 years ago and infects more than 90 % of the worldwide
population, there are large gaps in our knowledge of its epidemiology and patho-
genesis. Our future challenge is to focus research on the following five gaps.
1. We don’t know how EBV is transmitted to young children.
2. We don’t know why some adolescents and young adults become very ill from a
primary EBV infection, while others remain completely asymptomatic.
3. We don’t have an approved specific treatment for EBV infections.
4. We don’t have an approved EBV vaccine.
5. Finally, we don’t know the mechanism by which EBV induces malignancies or
autoimmune diseases. In terms of EBV-associated cancer, we do know a rea-
sonable amount about how this virus infects and transforms lymphocytes and
epithelial cells. What we don’t understand is how these cells escape immune
recognition.
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Primary Immunodeficiencies Associated
with EBV Disease
Jeffrey I. Cohen
Abstract Epstein-Barr virus (EBV) infects nearly all humans and usually is
asymptomatic, or in the case of adolescents and young adults, it can result in
infectious mononucleosis. EBV-infected B cells are controlled primarily by NK
cells, iNKT cells, CD4 T cells, and CD8 T cells. While mutations in proteins
important for B cell function can affect EBV infection of these cells, these muta-
tions do not result in severe EBV infection. Some genetic disorders affecting T
and NK cell function result in failure to control EBV infection, but do not result
in increased susceptibility to other virus infections. These include mutations in
SH2D1A, BIRC4, ITK, CD27, MAGT1, CORO1A, and LRBA. Since EBV is the
only virus that induces proliferation of B cells, the study of these diseases has
helped to identify proteins critical for interactions of T and/or NK cells with B
cells. Mutations in three genes associated with hemophagocytic lymphohistocyto-
sis, PRF1, STXBP2, and UNC13D, can also predispose to severe chronic active
EBV disease. Severe EBV infection can be associated with immunodeficiencies
that also predispose to other viral infections and in some cases other bacterial and
fungal infections. These include diseases due to mutations in PIK3CD, PIK3R1,
CTPS1, STK4, GATA2, MCM4, FCGR3A, CARD11, ATM, and WAS. In addition,
patients with severe combined immunodeficiency, which can be due to mutations
in a number of different genes, are at high risk for various infections as well as
EBV B cell lymphomas. Identification of proteins important for control of EBV
may help to identify new targets for immunosuppressive therapies.
J.I. Cohen (*)

Medical Virology Section, Laboratory of Infectious Diseases, National Institutes of Health,
50 South Drive, MSC 8007, Bethesda, MD 20892, USA
e-mail: jcohen@niaid.nih.gov

242 J.I. Cohen
Contents
1 Introduction........................................................................................................................... 243
2 Immunodeficiencies Specific for EBV Disease.................................................................... 244
2.1 X-Linked Lymphoproliferative Disease 1 (XLP1)...................................................... 244
2.2 X-Linked Lymphoproliferative Disease 2 (XLP2)...................................................... 251
2.3 IL-2-Inducible T Cell Kinase (ITK)............................................................................ 252
2.4 CD27............................................................................................................................ 252
2.5 Magnesium Transporter 1 (MagT1) Protein................................................................ 253
2.6 Coronin Actin Binding Protein 1A.............................................................................. 254
2.7 LPS-Responsive Beige-Like Anchor (LRBA) Protein................................................ 254
3 Proteins Associated with EBV Disease and Familial Hemophagocytic
Lymphohistiocytosis (FHL).................................................................................................. 254
3.1 Perforin........................................................................................................................ 254
3.2 Munc13-4..................................................................................................................... 255
3.3 Munc18-2..................................................................................................................... 255
4 Genes Associated with EBV and Other Infections............................................................... 256
4.1 Phosphatidylinositol-3-OH Kinase (PI3K) Catalytic Subunit P110δ.......................... 256
4.2 Cytidine 5′-Triphosphate Synthase 1 (CTPS1)............................................................ 258
4.3 Serine/Threonine Kinase 4 (STK4)............................................................................. 258
4.4 GATA Binding Protein 2 (GATA2).............................................................................. 259
4.5 Minichromosome Maintenance Complex Component 4 (MCM4).............................. 259
4.6 Fcγ Receptor 3A (CD16a)........................................................................................... 259
4.7 Caspase Recruitment Domain-Containing Protein 11 (CARD11):
Gain of Function Mutations......................................................................................... 260
4.8 Other Immunodeficiencies Associated with Multiple Infections................................. 260
References................................................................................................................................... 260
Abbreviations
APDS Activated PI3Kδ syndrome

CAEBV Chronic active EBV disease
CARD11 Caspase recruitment domain family, member 11
CTL Cytotoxic T lymphocyte
CTPS1 Cytidine 5′-triphosphate synthase
FHL Familial hemophagocytic lymphohistiocytosis
GATA2 GATA binding protein 2
HLH Hemophagocytic lymphohistiocytosis
HPV Human papillomavirus
HSCT Hematopoietic stem cell transplantation
iNKT Invariant NKT
ITK IL-2-inducible T cell kinase
LRBA LPS-responsive beige-like anchor
MagT1 Magnesium transporter 1
MCM4 Minichromosome maintenance complex component 4
MST1 Mammalian sterile 20-like protein
Primary Immunodeficiencies Associated with EBV Disease 243
MTOR Mammalian target of rapamycin

NK Natural killer
PASLI P110δ-activating mutation causing senescent T cells, lymphadenopa-
thy, and immune deficiency
PI3K Phosphatidylinositol-3-OH kinase
PIP2 Phosphatidylinositol-(4,5)-biphosphate
PIP3 Phosphatidylinositol-(3,4,5)-triphosphate
PKC Protein kinase C
PLC Phospholipase C
PML Progressive multifocal leukoencephalopathy
SAP SLAM-associated protein
SCID Severe combined immunodeficiency
SH2 Src homology 2
SM Sec1/munc18
SNARE Soluble NSF attachment protein receptor
STK4 Serine/threonine kinase 4
XIAP X-linked inhibitor of apoptosis
XLP X-linked lymphoproliferative disease
XMEN X-linked immunodeficiency with magnesium defect, EBV infection,
and neoplasia
1 Introduction
Epstein-Barr virus (EBV) infects 95 % of the human population, and most indi-
viduals are infected asymptomatically and are unaware that they shed virus from
the oropharynx throughout their life. Infection of adolescents and young adults
often results in infectious mononucleosis with fever, sore throat, lymphadenop-
athy, and splenomegaly. EBV infection of B cells can result in a lytic infection
with death of the cells. More often, virus infection of B cells results in a latent
infection with expression of a very limited number of viral proteins. Certain
B cell abnormalities affect the interaction of EBV with the host. Patients with
Bruton’s agammaglobulinemia lack mature B cells, and their B cells cannot be
infected with EBV (Faulkner et al. 1999). Patients with the hyperIgM syndrome
have mutations in CD40L, and their cells are impaired for transformation by EBV
in vitro (Imadome et al. 2003). B cells from patients with mutations in STAT3
have impaired proliferation in response to virus infection (Koganti et al. 2014).
However, no B cell defects have been described to date that result in more severe
EBV infection.
Control of EBV-infected cells is mediated by natural killer (NK cells), invari-
ant NKT (iNKT) cells, CD4 T cells, and CD8 T cells (Chung et al. 2013; Hislop
et al. 2007). Impairment in the function of these cell types can result in failure to
adequately control EBV infection. This can lead to persistent EBV viremia, lym-
phoproliferation, and ultimately EBV lymphomas.
244 J.I. Cohen
Surprisingly, many of the genes important for controlling EBV infection are not
critical for controlling other pathogens including other members of the herpesvi-
rus family. This indicates a unique characteristic of EBV relative to other patho-
gens. EBV is the only human virus that induces growth proliferation of B cells.
Therefore, host cell proteins important for interactions of T or NK cells with B
cells are often critical for control of EBV. Identification of cellular gene mutations
associated with severe EBV infection helps us to identify proteins important for
the B cell–T cell or B cell–NK cell immunologic synapse. These cellular proteins
may also serve as targets for new immunosuppressive medications.
2 Immunodeficiencies Specific for EBV Disease
See Tables 1 and 2.
2.1 X-Linked Lymphoproliferative Disease 1 (XLP1)
The first genetic disorder linked with severe and often fatal EBV disease was
XLP1, also known as Duncan’s disease. Purtillo and colleagues described an
X-linked lymphoproliferative disease (XLP) in boys that often presented with
fulminant infectious mononucleosis with lymphocytic and histiocytic infiltra-
tion of the bone marrow, central nervous system, and other organs (Purtilo et al.
1975). Female carriers are not affected. If untreated, patients often die from bone
marrow failure with bleeding and infection, or liver failure. Subsequent stud-
ies showed that the disease has a variety of phenotypes including fatal infec-
tious mononucleosis, aplastic anemia, or hypogammaglobulinemia after primary
EBV infection, immunoblastic B cell lymphoma, Burkitt lymphoma, or plasma-
cytoma and that the underlying cause is an abnormal immune response to infec-
tion with EBV (Purtilo 1976; Purtilo et al. 1977). More recent studies show that
the presenting symptoms of XLP1 include hemophagocytic lymphohistiocytosis
(HLH) in 31.9 % of persons, dysgammaglobulinemia in 22 %, a family history
of XLP1 alone in 16.5 %, lymphoma in 14.3 %, fulminant infectious mononu-
cleosis in 7.7 %, and other symptoms in 7.7 % (Booth et al. 2011). These other
symptoms include aplastic anemia, lymphomatoid granulomatosis, and vasculitis.
Patients have impaired antibody responses to vaccination. Most lymphomas are
EBV-positive non-Hodgkin’s B lymphomas, although T cell lymphomas and EBV-
negative B cell lymphomas have also been reported. In most cases, there is not a
clear correlation between specific mutations and clinical phenotype (Booth et al.
2011; Filipovich et al. 2010). Within families, different members can have differ-
ent severity of presentation despite the same mutation.
The gene responsible for XLP1 was identified in 1988 and is referred to as
SH2D1A (Coffey et al. 1998), DSHP (Nichols et al. 1998), and SAP (Sayos et al.
Table 1 Clinical features of immunodeficiencies predisposing to EBV disease
Protein Gene Name of Transmission EBV-associated symptoms Non-infectious diseases in
syndrome the absence of EBV infection
SLAM-associated protein SH2D1A XLP1 X-linked Fulminant infectious mononu- EBV-negative B cell lym-
cleosis, B cell lymphoma, HLH, phoma, aplastic anemia,
lymphomatoid granulomatosis vasculitis
X-linked inhibitor of BIRC4 XLP2 X-linked Fulminant infectious mononucleosis, Colitis, HLH, inflammatory
apoptosis HLH, splenomegaly, cytopenias bowel disease
IL-2-inducible T cell kinase ITK EBV-associated Autosomal recessive Lymphoproliferation, Hodgkin’s Autoimmune kidney disease
autosomal lym- lymphoma, HLH, hepatospleno-
phoproliferative megaly, lung disease, lymphoma-
syndrome toid granulomatosis
CD27 CD27 CD27 deficiency Autosomal recessive Lymphoproliferative disease, None
HLH, lymphoma, aplastic anemia
Magnesium transporter 1 MAGT1 XMEN X-linked B cell lymphoma Autoimmune cytopenias
protein
Coronin actin binding CORO1A Coronin 1A Autosomal recessive B cell lymphoma, lymphoprolif Neurocognitive impairment
protein 1A deficiency erative disease
Primary Immunodeficiencies Associated with EBV Disease
LPS-responsive beige-like LRBA Autosomal recessive B cell lymphoproliferative disease, Inflammatory bowel disease,
anchor protein EBV viremia chronic diarrhea, autoim-
mune cytopenias
Perforin PRF1 FHL2 Autosomal recessive HLH, CAEBV, splenomegaly HLH
Munc13-4 UNC13D FHL3 Autosomal recessive CAEBV, vasculitis, hepatitis, HLH
splenomegaly
Munc18-2 STXBP2 FHL5 Autosomal recessive CABEV, lymphoma, HLH, colitis, bleeding
splenomegaly
PI3K catalytic subunit 110δ: PIK3CD PASLI, APDS Autosomal dominant Lymphoma Lymphoid nodules in upper
gain of function mutations airway and gastrointestinal
tract, autoimmune cytopenias
(continued)
245
Table 1 (continued)
246
Protein Gene Name of Transmission EBV-associated symptoms Non-infectious diseases in

syndrome the absence of EBV infection
PI3K catalytic subunit 110δ: PIK3CD Autosomal recessive EBV viremia Lymphadenopathy, hepatos-
loss of function mutation plenomegaly, autoantibodies
CTP synthase 1 CTPS1 CTP synthase 1 Autosomal recessive Severe infectious mononucleo- None
deficiency sis; lymphoproliferative disease,
lymphoma
Serine threonine kinase 4 STK4 STK4 deficiency Autosomal recessive B cell lymphoma, lymphopro Dermatitis, autoimmune
liferative disease, autoimmune cytopenias
hemolytic anemia
GATA binding protein 2 GATA2 MonoMac Autosomal dominant Severe infectious mononucleosis, Myeloid malignancies,
EBV-positive smooth muscle myelodysplastic syndrome,
tumors, CAEBV, HLH autoimmune disease, pul-
monary alveolar proteinosis,
primary lymphedema.
Minichromosome mainte- MCM4 Classical NK cell Autosomal recessive EBV lymphoproliferative disease, Adrenal insufficiency, growth
nance complex component 4 deficiency type 2 lymphoma retardation
Fcγ receptor 3A (CD16a) FCGR3A Autosomal recessive EBV-positive Castleman’s disease None
Caspase recruitment domain- CARD11 Autosomal dominant EBV viremia Autoimmune neutropenia
containing protein 11: gain of
function mutations
Abbreviations: XLP X-linked lymphoproliferative disease; HLH hemophagocytotic lymphohistiocytosis; XMEN X-linked immunodeficiency with magne-
sium defect; EBV infection, and neoplasia; CAEBV chronic active EBV disease; FHL familial hemophagocytic lymphohistiocytosis; PI3K phosphoinositide
3-kinase; PASLI p110δ-activating mutation causing senescent T cells, lymphadenopathy, and immune deficiency; APDS activated PI3Kδ syndrome; CTP
cytidine 5′-triphosphate
J.I. Cohen
Table 2 Immunologic and infectious features of immunodeficiencies predisposing to EBV disease
Protein Humoral immune Cellular immune findings Chronic EBV Other infections References
findings viremia
SLAM-associated Low IgG, increased Absent iNKT cells, normal No None Coffey et al. (1998);
protein IgA, increased IgM, numbers of B, T cells; reduced Nichols et al.
reduced antibody memory B cells; impaired T (1998); Sayos et al.
response to vaccinations and NK cell killing (1998)
and infections
X-linked inhibitor of Low IgG Normal or low numbers of No None Rigaud et al. (2006);
apoptosis iNKT cells; normal numbers of Speckmann et al.
B, T, NK cells (2013)
IL-2-inducible T cell Low or normal IgG Absent iNKT cells; low num- Yes None Huck et al. (2009);
kinase bers of WBC, CD4 T cells; Linka et al. (2012)
normal numbers of B cells
CD27 Low or normal IgG Normal or reduced iNKT and Yes None van Montfrans et al.
memory B cells (2012); Salzer et al.
(2013)
Magnesium transporter Low or normal IgG Low numbers of CD4 cells; Yes Bacterial sinusitis, chronic Li et al. (2011,
1 protein normal numbers of iNKT cells; diarrhea 2014); Chaigne-
reduced levels of NKG2D on Delalande et al.

NK and T cells; impaired T cell (2013)
killing of EBV-transformed B
cells; impaired NK cell function
Coronin actin binding Normal or low normal Reduced numbers of CD4 T, Yes Recurrent ear, nose, throat, Moshous et al.
protein 1A IgG, normal or low IgM CD8 T, naïve T cells, and B and upper respiratory tract (2013)
cells; low or absent iNKT cells infections
LPS-responsive beige-like Normal or low IgG Normal or low B cell numbers; Yes Usually none, may have Alangari et al.
anchor protein normal T, NK cell numbers recurrent otitis media and (2012)
pneumonia
(continued)
247
Table 2 (continued)
248
findings viremia
Perforin Normal IgG Low numbers of B cells, CD4 No None Katano et al. (2004)
cells, and neutrophils; impaired
NK cell and CTL killing
Munc13-4 Low IgG Low numbers of neutrophils, No Recurrent upper airway Rohr et al. (2010)
impaired NK cell and CTL infections
killing
Munc18-2 Low IgG Low numbers of neutrophils, Yes Recurrent upper airway Rohr et al. (2010);
impaired NK cell and CTL infections Cohen et al. (2015)
killing
PI3K catalytic subunit Low, normal, or high IgG Low numbers of CD4 cells, Yes Sinus and pulmonary infec- Lucas et al. (2014);
110δ: gain of function memory T cells, and naïve tions, CMV viremia Angulo et al. (2013)
mutations CD4 T cells; increased CD8
cells and senescent effector
CD8 T cells
PI3K catalytic subunit High IgG and IgM Increased numbers of B cells, Yes Recurrent otitis media and Kuehn et al. (2013)
110δ: loss of function reduced memory B cell, and sinusitis
mutation diminished NK cell killing
CTP synthase 1 Normal or increased IgG Lymphopenia, low CD4/CD8 Yes Herpesviruses, encapsulated Martin et al. (2014)
ratios, low naïve CD4 T cells, bacteria
low CD27 memory B cells;
absent iNKT cells; increased
effector memory T cells
Serine threonine kinase 4 High IgG Low numbers of CD4 cells, Yes Severe HPV and molluscum Nehme et al. (2012);
naïve T cells, B cells, and contagiosum infection, can- Abdollahpour et al.
neutrophils dida, recurrent bacterial and (2012)
virus infections
(continued)
J.I. Cohen
Table 2 (continued)
findings viremia
GATA binding protein 2 Normal IgG Low numbers of B cells, CD4 T Yes Severe HSV, VZV, CMV, Spinner et al. (2014)
cells, NK cells, dendritic cells, HPV, non-tuberculous myco-
and monocytes bacteria, fungal infections
Minichromosome mainte- Normal IgG Low numbers of NK cells, Unknown Respiratory infections, recur- Gineau et al. (2012);
nance complex compo- absent CD56dim NK cells rent herpesvirus infections Eidenschenk et al.
nent 4 (2006)
Fcγ receptor 3A Normal IgG Variable numbers of NK cells, Unknown Respiratory infections, severe deVries et al.
but impaired function herpesvirus infections (1996); Grier et al.
(2012)
Caspase recruitment Normal or slightly B cell lymphocytosis, normal T Yes Respiratory infections, Snow et al. (2012)
domain-containing pro- increased IgG, low cell numbers sinusitis, otitis media, mol-
tein 11: gain of function IgM, reduced antibody luscum contagiosum
mutations response to polysaccha-
ride-based vaccines
Abbreviations: iNKT invariant NKT; HPV human papillomavirus; PML progressive multifocal leukoencephalopathy; CTL cytotoxic T lymphocyte; CTP cyti-
dine 5′-triphosphate
249
250 J.I. Cohen
1998) and is expressed on T, NK, and iNKT cells. Mutations in SAP account
for 60–70 % of cases of XLP. The protein encoded by the gene, SAP (SLAM-
associated protein), is an adapter molecule that is expressed on T, NK, and iNKT
cells. SAP consists of a single Src homology 2 (SH2) domain and interacts with
several proteins including SLAM, 2B4, NTB-A, CD84, Ly108, and Ly9 (Cannons
et al. 2010). The interaction of SAP with SLAM reduces production of IFN-γ
(Latour et al. 2001) and T cell killing of virus-infected cells (Dupré et al. 2005).
The interaction of SAP with 2B4 (Parolini et al. 2000) and NTB-A (Bottino et al.
2001) increases NK cell cytotoxicity. SAP-deficient cells are impaired for forma-
tion of immunologic synapses and killing of B cells, but not dendritic cells (Qi
et al. 2008; Zhao et al. 2012). This results in inefficient recruitment and reten-
tion of T cells to germinal centers. CD84 and Ly108 are critical for T and B cell
contacts, and CD84 is required for germinal center formation; in the absence of
SAP, germinal centers are defective (Cannons et al. 2011). SAP is also important
for control of T cell proliferation and apoptosis during antigen stimulation; in the
absence of SAP, T cells are resistant to apoptosis mediated by T cell receptor res-
timulation (Snow et al. 2009). Taken together, these effects result in impaired T
and NK cell cytotoxicity, with massive proliferation of T and NK cells, excessive
cytokine production, and HLH.
XLP1 patients have impaired controlled of EBV, but not other virus infec-
tions or bacteria. These patients lack class-switched memory B cells (Chaganti
et al. 2008). SAP knockout mice lack virus-specific memory B cells and long-
lived plasma cells, due to a defect in CD4 T cells (Crotty et al. 2003). Patients
who survived EBV infection were found to have impaired recognition of SLAM-
ligand EBV-transformed B cells expressing EBV protein, but were able to recog-
nize SLAM-ligand-negative EBV-infected B cells (Hislop et al. 2010). Somatic
reversion of SAP-mutated cells in patients who survived XLP1 occurred solely in
memory CD8 T cells, and these T cells proliferated and were cytotoxic for EBV-
infected B cells (Palendira et al. 2012). A study of female XLP carriers (who have
SAP+/SAP− alleles) showed that memory CD8 T cells specific for influenza and
CMV were present in both SAP+ and SAP− cells; however, EBV-specific T cells
were only present in SAP+ cells (Palendira et al. 2011). These differences are due
to the failure of SAP− cells to respond to antigens presented by B cells, and since
EBV is the only human virus that latently infects B cells, these results help to
explain why XLP1 is a disease confined to EBV and does not predispose to infec-
tions by other pathogens. Blocking NTB-A and 2B4, both of which bind to SAP,
restores the ability of SAP− T cells to respond to antigen presentation by B cells.
While patients with XLP1 have normal numbers of T and B cells, they lack iNKT
cells (Nichols et al. 2005) which are critical for T cell receptor-induced cellular
cytotoxicity (Das et al. 2013). These patients also have impaired T cell production
of IL-10 (Ma et al. 2005). Thus, patients with XLP1 have impaired recognition of
antigens presented by B cells, absent iNKT cells, impaired T cell cytotoxicity, and
reduced expression of IL-10 by T cells.
While intravenous immunoglobulin, which contains neutralizing antibody
to EBV, has been used to try to prevent EBV infection in patients with XLP1,
breakthrough infections have occurred resulting in death. Rituximab, an anti-

CD20 monoclonal antibody, was reported to reverse fulminant infectious mono-
nucleosis in two patients with XLP1 (Milone et al. 2005). Reduced intensity
conditioning hematopoietic stem cell transplantation (HSCT) resulted in an 80 %
one-year survival rate in patients presenting with XLP1, regardless of whether
they had a history of HLH (Marsh et al. 2014). In the future, genetic therapy cor-
recting the SAP gene directly may be possible, and this has been demonstrated
using retrovirus-mediated gene transfer in a mouse model of XLP1 (Rivat et al.
2013).
2.2 X-Linked Lymphoproliferative Disease 2 (XLP2)
XLP2, due to a mutation in BIRC4 which encodes the X-linked inhibitor of apop-
tosis (XIAP), was initially described in patients from three families who presented
with HLH, often but not always, associated with EBV, splenomegaly, hypogam-
maglobulinemia, and colitis (Rigaud et al. 2006). XIAP is expressed in B, T, NK,
and dendritic cells as well as non-hematopoietic cells. Mutations in XIAP account
for about 20–30 % of cases of XLP. While the initial paper reported that patients
had low numbers of iNKT cells, a later report indicated that patients had normal
numbers of these cells (Marsh et al. 2009). These patients have normal numbers of
B, T, and NK cells. A review of 25 cases of XLP2 found that only 8 presented with
HLH; other patients presented with colitis, severe infectious mononucleosis, or
splenomegaly; iNKT cells were not reduced (Speckmann et al. 2013). There was
no clear correlation of specific mutations with the severity of disease (Filipovich
et al. 2010; Speckmann et al. 2013). Patients who underwent reduced condition-
ing HSCT had a better long-term survival rate (55 %) than those who had ablative
conditioning (14 %) (Marsh et al. 2013).
Mutations in XIAP result in enhanced T cell apoptosis in response to stimula-
tion with anti-Fas antibody, anti-CD3 antibody, and trimeric TRAIL (Rigaud et al.
2006); however, a subsequent report did not find enhanced apoptosis with anti-Fas
antibody (Marsh et al. 2010). T cells from patients with XIAP have enhanced T
cell reactivation-induced cell death. XIAP is also important for NOD2-mediated
signaling (Krieg et al. 2009) as well as activation of NF-κB. Thus, patients with
XLP2 have enhanced T cell apoptosis to various stimuli including T cell receptor
restimulation.
A comparison of patients with XLP1 and XLP2 found that HLH was more
common in XLP2 (76 %) versus XLP1 (55 %), but was more likely to be fatal in
patients with XLP1 (61 %) versus XLP2 (23 %) (Pachlopnik Schmid et al. 2011).
Infection with EBV triggered the onset of HLH in 92 % of persons with XLP1 and
83 % with XLP2. Significantly, more patients with XLP1 had hypogammaglobu-
linemia (67 %) and lymphoma (30 %) than those with XLP2 (33 and 0 %, respec-
tively). In contrast, significantly more patients with XLP2 had cytopenias (52 %)
and splenomegaly (87 %) in the absence of HLH, and hemorrhagic colitis (17 %)
252 J.I. Cohen
than XLP1 (12, 7, 0 %, respectively). The large number of differences between

XLP1 and XLP2 has led some authors to propose that XLP2 should be reclassified
instead as an X-linked familial HLH disorder (Filipovich et al. 2010).
2.3 IL-2-Inducible T Cell Kinase (ITK)
IL-2-inducible T cell kinase (ITK) deficiency was first reported in two girls with
homozygous mutations who died with B cell proliferation due to EBV (Huck et al.
2009). The disease has been called EBV-associated autosomal lymphoproliferative
syndrome. ITK is a member of the TEC family of kinases and has a critical role in
T cell receptor signaling. It is important for T cell proliferation and differentiation.
Both girls had high levels of eomesodermin in CD8 T cells, and iNKT cells were
absent from the one girl who was tested. A review of seven patients (four girls
and three boys) with ITK deficiency from 4 families found that all patients pre-
sented with fever, lymphadenopathy, and all of whom were tested had markedly
elevated EBV DNA in the peripheral blood (Linka et al. 2012). Most had hepat-
osplenomegaly, pulmonary disease, hypogammaglobulinemia, leukopenia, and
CD4 lymphopenia. Pathology showed Hodgkin’s lymphoma in four patients, B
cell lymphoproliferative disease in two patients, and both large B cell lymphoma
and lymphomatoid granulomatosis in one patient. Two patients underwent HSCT,
one survived, and the other died of complications associated with graft-versus-
host disease. All patients that were tested had low numbers of iNKT cells and an
impaired calcium flux in T cells after stimulation of the T cell receptor with anti-
CD3 antibody.
2.4 CD27
Two brothers were reported with homozygous mutations in CD27 and per-
sistent EBV viremia: One had aplastic anemia and the other had hypogamma-
globulinemia (van Montfrans et al. 2012). Both patients had undetectable CD27
on all lymphocytes, but normal numbers of lymphocyte subsets. T cell prolifera-
tive responses to CD27-dependent mitogens (CD2 and pokeweed mitogen) were
reduced, and T cell-dependent B cell responses to vaccines were impaired. CD27
is a member of the tumor necrosis receptor family and binds to its ligand, CD70,
and provides costimulatory signaling to activate B, T, and NK cells. CD27 is a
marker for memory B cells and enhances B cell differentiation, and T and NK cell
function.
Eight patients in three additional families with CD27 deficiency were subse-
quently reported: Three patients had asymptomatic deficiencies in memory B
cells, three had EBV HLH and lymphoproliferative disease, and two had lym-
phoma (Salzer et al. 2013). Three patients developed hypogammaglobulinemia
after primary EBV infection. Two with severe disease had reduced NK cell func-
tion and diminished numbers of iNKT cells. One patient received repeated courses
of rituximab and two underwent HSCT.
2.5 Magnesium Transporter 1 (MagT1) Protein
MAGT1 encodes a magnesium transporter protein located in the plasma cell mem-
brane. MagT1 protein allows an influx of magnesium into cells after stimulation of
the T cell receptor which results in activation of T cells (Li et al. 2011). The influx
of magnesium results in increased calcium signaling and activation of PLC (phos-
pholipase C)-γ1, PKC (protein kinase C)-θ, and NF-κB. Thus, magnesium, like
calcium, can act as an intracellular second messenger to couple events on the cell
surface with changes in the cytoplasm and nucleus.
Seven patients have been reported with mutations in MAGT1 who had markedly
elevated levels of EBV DNA in the blood; these patients ranged in age from 3 to
45 years old with a mean age of 16 years (Chaigne-Delalande et al. 2013). Four
patients had B lymphomas, three of whom were tested for EBV and were positive.
Stimulation of the T cell receptor in PBMCs from the patients resulted in impaired
calcium signaling and reduced activation of PLC γ1, PKC-θ, and NF-κB.
Patients with mutations in MAGT1 often have low CD4 cell counts with an
inverted CD4:CD8 ratio, reduced NKG2D (an NK cell activating receptor) on
NK cells and cytotoxic T lymphocytes (CTLs), and impaired T cell activation.
Their CTLs showed reduced killing of autologous EBV-transformed B cells, and
their NK cells were impaired for killing other target cells. In addition to elevated
levels of EBV in the blood, some patients had hypogammaglobulinemia, sinusi-
tis, and chronic diarrhea. Two patients had autoimmune cytopenias. All patients
had splenomegaly; one had hemophagocytosis. Two patients underwent HSCT and
both died of complications related to the transplant. The disease has been termed
XMEN (X-linked immunodeficiency with magnesium defect, EBV infection, and
neoplasia).
Patients with mutations in MAGT1 had lower levels of intracellular magne-
sium, which suggested that supplemental magnesium might improve their immune
responses. In vitro supplemental magnesium of PBMCs from patients resulted in
increased levels of intracellular magnesium, NKG2D, and improved NK cell and
CTL cytotoxicity. Treatment of two patients with magnesium supplementation
resulted in increased levels of intracellular magnesium, increased expression of
NKG2D on CTLs, improved CTL activity against autologous EBV-transformed B
cells, and a reduction in the percentage of EBV-infected cells in the blood.
254 J.I. Cohen
2.6 Coronin Actin Binding Protein 1A
Three siblings in one family that presented with EBV B cell lymphoproliferative
disease in early childhood were found to have homozygous mutations in CORO1A
which encodes coronin actin binding protein 1A (Moshous et al. 2013). One
patient had an EBV-positive lymphoproliferative process and two had EBV lym-
phomas. Two of the patients in whom EBV DNA levels in the blood were meas-
ured had elevated levels. All three patients had recurrent ear, nose, and throat as
well as upper respiratory tract infections. Two of the patients died: one in prepara-
tion for HSCT and one from graft-versus-host disease after HSCT. Coronin actin
binding protein 1A binds to actin-related protein 2/3 and is important for T cell
synapse formation and T cell receptor signaling. The patients had reduced num-
bers of CD4, CD8, CD19, and naïve T cells, a reduced T cell repertoire, low or no
iNKT cells, and few mucosal-associated invariant T cells.
2.7 LPS-Responsive Beige-Like Anchor (LRBA) Protein
Patients with mutations in LRBA present with inflammatory bowel disease, chronic
diarrhea, and autoimmune cytopenias (Alangari et al. 2012). One patient presented
with EBV lymphoproliferative disease, elevated EBV DNA in the blood, and auto-
immune pancytopenia. The LRBA protein has domains that are conserved with
the Chediak–Higashi syndrome protein and is important for endocytosis of ligand-
activated receptors; however, its role in immunity is not well understood.
3 Proteins Associated with EBV Disease and Familial

Hemophagocytic Lymphohistiocytosis (FHL)
See Tables 1 and 2.
3.1 Perforin
Familial hemophagocytic lymphohistiocytosis (FHL) is a group of diseases due

to mutations in proteins important for maturation or release of cytotoxic granules
from CTLs and NK cells, or for entry of cytotoxic proteins from these granules
into target cells. Four genes have been identified in which mutations cause FLH-
PRF1, UNC13D, STX11, and STXBP2, which are responsible for FHL2, FHL3,
FHL4, and FHL5, respectively.
Perforin is encoded by PRF1 and is expressed in cytotoxic granules of CTLs

and NK cells. When foreign antigens are expressed on antigen-presenting cells,
CTLs become activated and granules containing perforin and granzymes dock on
the plasma membrane and are released. Perforin oligomerizes to form pores in tar-
get cells which allows entry of granzymes into these cells resulting in activation
of caspases and death of the cells. Mutations in perforin result in an autosomal
recessive disorder known as FHL2. Perforin mutations result in impaired killing of
target cells by CTLs and NK cells.
We described a boy who presented with EBV-positive infectious mononucleo-
sis followed by persistent splenomegaly and lymphadenopathy and was diagnosed
with chronic active EBV disease (CAEBV) and HLH (Katano et al. 2004; Cohen
et al. 2011). The patient had different mutations in the two alleles of perforin
which resulted in reduced expression of the native form of the protein. The patient
only expressed the immature form of perforin, since his perforin was not cleaved
at the carboxyl terminus to yield the active form of the protein. Accordingly, his T
cells were impaired for killing target cells.
3.2 Munc13-4
Munc13-4 is encoded by UNC13D. Munc13-4 interacts with syntaxin 11 to

change the conformation of syntaxin from a closed to an open conformation; a
soluble NSF attachment protein receptor (SNARE) complex is formed between
v-SNARE on cytotoxic granules and the target membrane t-SNARE syntaxin 11.
This allows priming of cytotoxic granules and ultimately results in fusion of the
granules with the membrane of the cell with exocytosis of granules. Mutations
in munc13-4 result in an autosomal recessive disease referred to as FHL3 with
impaired NK and T cell cytotoxicity.
Mutations in munc13-4 were reported in one patient with CAEBV who had
cerebral vasculitis, hypogammaglobulinemia, chronic hepatitis, splenomegaly, and
recurrent respiratory infections (Rohr et al. 2010). The patient was a compound
heterozygote for munc13-4 mutations. The patient was initially EBV seroposi-
tive and then developed CAEBV with a high viral load and HLH and died of the
disease.
3.3 Munc18-2
Munc18-2 is encoded by STXBP2, a member of the sec1/munc18 (SM) family

of proteins that are important for SNARE-mediated membrane fusion. Munc18-2
binds to syntaxin 11, on the plasma membrane of NK cells, and to v-SNARE, on
cytotoxic granules. Thus, munc18-2 forms a bridge assisting in the docking of
256 J.I. Cohen
cytotoxic granules to the plasma membrane of CTLs or NK cells. Mutations in

munc18-2 result in an autosomal recessive disease referred to as FHL5. Deficiency
in munc18-2 results in impaired binding of munc18-2 to syntaxin 11, reduced sta-
bility of both proteins, and impaired exocytosis of cytotoxic granules from CTLs
or NK cells (Côte et al. 2009; zur Stadt et al. 2009). Mutations in munc18-2 affect
folding of the protein which impair its binding activity (Hackmann et al. 2013).
These patients have impaired NK and T cell killing of target cells.
Mutations in munc18-2 were reported in four patients with CABEV (Rohr
et al. 2010). Three patients had homozygous mutations and one was a com-
pound heterozygote. Two patients developed HLH after primary EBV infection:
one presented with HLH-like symptoms and then severe HLH after primary EBV
infection, and one was initially EBV seropositive and then developed CAEBV
with a high viral load and HLH. All four patients had hypogammaglobuline-
mia, three had persistent splenomegaly, two had recurrent infections, and one
had Hodgkin’s lymphoma. Three underwent HSCT at ages 6, 16, and 16 and one
survived; the fourth patient with lymphoma had a HSCT and remains alive and
well. Another patient with compound heterozygous mutations in munc18-2 pre-
sented with late onset CAEBV and did well after HSCT (Cohen et al. 2015).
4 Genes Associated with EBV and Other Infections
See Tables 1 and 2.
4.1 Phosphatidylinositol-3-OH Kinase (PI3K) Catalytic

Subunit P110δ
4.1.1 PI3K P110δ Gain of Function Mutations
Thirty-one patients with gain of function mutations in the p110δ catalytic subunit
of phosphatidylinositol-3-OH kinase (PI3K) have been reported who had impaired
control of EBV (Lucas et al. 2014; Angulo et al. 2013). PI3K is activated in T cells
after ligand binding to the T cell receptor (Okkenhaug and Vanhaesebroeck 2003).
This results in binding of the p85 regulatory domain of PI3K to phosphorylated
tyrosine residues on proteins and its dissociation from the p110 catalytic subunit
of PI3K. p110δ is found exclusively in lymphocytes. Free p110δ is then recruited
to the plasma membrane, and it phosphorylates PIP2 (phosphatidylinositol-(4,5)-
biphosphate) to PIP3 (phosphatidylinositol-(3,4,5)-triphosphate). This results in
phosphorylation of Akt (also known as protein kinase B) which phosphorylates
mammalian target of rapamycin (mTOR). The mTOR complex (composed of
mTOR, raptor, and GβL) phosphorylates 4E-BP1 (a protein translation initiation
inhibitor) and p70S6 kinase (which promotes protein translation). Phosphorylation
of the former inhibits its ability to block eukaryotic translation initiation fac-
tor eiF4E, while phosphorylation of the latter activates the S6 ribosomal protein
to increase protein translation. This results in increased protein synthesis, cell
growth, proliferation, differentiation, and survival.
Heterozygous mutations (N334K, E525K, and E1021K) in p110δ result in gain
of function mutations (Lucas et al. 2014; Angulo et al. 2013). These likely block the
interaction of p110δ with p85 (to allow unbridled activity of p110δ) or promote the
association of p110δ with the cell membrane. This results in increased activation
(phosphorylation) of PI3K either in the presence or in the absence of T cell receptor
stimulation. Stimulation of peripheral blood mononuclear cells from these patients
with antibody to CD3 and CD28 results in reduced IL-2 secretion and decreased pro-
liferation compared with controls. Surprisingly, these patients have increased num-
bers of EBV-specific T cells based on tetramer staining and increased EBV-specific
effector memory cells. Patients with mutations in p110δ have reduced memory CD8
T cells, reduced naive CD4 T cells, increased senescent effector CD8 T cells, reduced
class-switched IgG and IgA cells, and increased activation-induced cell death.
Patients present early in childhood with sinus and pulmonary infections, per-
sistent EBV and/or CMV viremia, lymphoproliferative disease with lymphoid
nodules in mucosa that can obstruct the lungs and gastrointestinal tract, and auto-
immune cytopenias. Patients have normal or elevated IgM, variable levels of IgG,
reduced IgA, and impaired antibody production after vaccination. Patients have
decreased CD4 cells and increased CD8 cells with an inverted CD4/CD8 ratio,
and a progressive B and T cell immunodeficiency. Two patients developed EBV-
positive B cell lymphomas and one patient had a marginal zone lymphoma. The
impaired ability to control EBV may be due to the low numbers of CD4 cells and/
or the reduction in memory CD8 T cells.
This disease has been termed PASLI (p110δ-activating mutation causing senes-
cent T cells, lymphadenopathy, and immune deficiency) (Lucas et al. 2014) or
APDS (activated PI3Kδ syndrome) (Angulo et al. 2013). Treatment of one patient
with rapamycin, an mTOR inhibitor, resulted in reduced CD8 T cell numbers,
increased IL-2 secretion, and increased T cell proliferation after stimulation with
anti-CD3 and anti-CD28 antibody in vitro (Lucas et al. 2014). The patient had
a reduction in the size of his lymph nodes, liver, and spleen. Treatment of cells
in vitro from patients with mutations in p110δ with specific inhibitors of p110δ
reduced the activity of the protein (Angulo et al. 2013).
A similar disease due to gain of function mutations in the p85 subunit of PI3K
(PIK3R1) has been associated with EBV viremia (Deau et al. 2014).
4.1.2 PI3K P110δ Loss of Function Mutation
One patient has been reported who initially presented with recurrent otitis media and
sinusitis, generalized lymphadenopathy, hepatosplenomegaly, B cell lymphocytosis,
and persistent EBV viremia (Kuehn et al. 2013). The patient’s serum had autoanti-
bodies to several cellular proteins, and his NK cells had diminished cytotoxicity.
258 J.I. Cohen
4.2 Cytidine 5′-Triphosphate Synthase 1 (CTPS1)
Eight patients from 5 families were reported with mutations in cytidine 5′-triphos-
phate synthase (CTPS1): Four patients presented with severe infectious mononu-
cleosis (three of which had chronic EBV viremia), three with lymphoproliferative
disease involving the central nervous system (two of whom had EBV-positive
non-Hodgkin’s lymphoma), and one with asymptomatic chronic EBV viremia
(Martin et al. 2014). All of the patients had other severe herpesvirus infections dur-
ing childhood and infections with encapsulated bacteria. One had Streptococcus
pneumoniae sepsis and meningitis, and one had Neisseria meningitidis meningitis.
Six of the eight underwent HSCT and four survived; one died of graft-versus-host
disease and one from disseminated varicella-zoster virus.
Most patients with CTPS1 had lymphopenia with low CD4:CD8 ratios dur-
ing infections. One patient who was studied more intensively had low numbers
of naïve CD4 T cells, increased effector memory T cells, low numbers of CD27
memory B cells, and absent iNKT cells. Proliferation and DNA synthesis of T
cells in response to anti-CD3 antibody and proliferation of B cells in response to
anti-B cell receptor and CpG were impaired. Reduced levels of CTP were pre-
sent in stimulated T and B cells from the patients. CTPS1 expression is normally
increased with T cell activation; therefore, deficiency of the protein presumably
limits the ability of T cells to proliferate and control virus and bacterial infections.
4.3 Serine/Threonine Kinase 4 (STK4)
Three patients from two families with mutations in serine/threonine kinase 4

(STK4) were reported with high levels of EBV DNA in the blood. One patient
developed an EBV-positive Hodgkin’s lymphoma and survived, a second patient
developed disseminated EBV B-cell lymphoproliferative lesions and died after
HSCT from graft-versus host disease and infectious complications, and a third
patient with autoimmune hemolytic anemia underwent HSCT and also died from
graft-versus-host disease and infection (Nehme et al. 2012). The patients had a
history of recurrent bacterial and viral infections, dermatitis, CD4 lymphopenia,
reductions in the numbers of naïve T cells, impaired T cell proliferative responses
to phytohemagglutinin and to anti-CD3 antibody, reduced T cell receptor reper-
toires, and increased levels of IgG. Increased Fas expression was present on the
surface of the cells, which showed increased sensitivity to Fas-induced apoptosis.
Another study reported three patients from one family with STK4 mutations;
one patient with generalized lymphadenopathy had a biopsy which showed a
monoclonal EBV lymphoproliferative process that was reported to resemble a
lymphoplasmacytic lymphoma (Abdollahpour et al. 2012). These patients had bac-
terial and viral infections (including extensive warts and molluscum contagiosum),
mucocutaneous candidiasis, neutropenia, CD4 lymphopenia, and B cell lymphope-
nia, and most had elevated levels of IgG. STK4 is also referred to as mammalian
sterile 20-like protein (MST1) and is involved in signaling pathways important for
cell proliferation and apoptosis; STK4 is cleaved by caspases and is thought to be
a pro-apoptotic protein.
4.4 GATA Binding Protein 2 (GATA2)
Patients with mutations in GATA binding protein 2 (GATA2) can have vari-
ous signs and symptoms including acute myeloid leukemia, myelodysplastic
syndrome, autoimmune disease, pulmonary alveolar proteinosis, and primary
lymphedema. Patients with GATA2 mutations have presented with chronic active
EBV disease, EBV-positive smooth muscle tumors, and persistent EBV viremia
(Hsu et al. 2011; Spinner et al. 2014). In addition, these patients also are suscepti-
ble to other severe herpesvirus infections as well as severe human papillomavirus,
fungal, and non-tuberculous mycobacterial infections. GATA2 encodes a tran-
scription factor important for hematopoiesis; accordingly, patients with mutations
in GATA2 often have low numbers of B cells, CD4 T cells, NK cells, dendritic
cells, red blood cells, neutrophils, monocytes, and platelets.
4.5 Minichromosome Maintenance Complex

Component 4 (MCM4)
Patients with mutations in minichromosome maintenance complex component 4

(MCM4) present with adrenal insufficiency, growth retardation, low numbers of
NK cells, and absent CD56dim NK cells (Gineau et al. 2012). These latter cells
are cytotoxic and produce cytokines after recognition of target cells. One patient
developed an EBV lymphoma (Eidenschenk et al. 2006). MCM4 is a DNA heli-
case that is important for DNA replication.
4.6 Fcγ Receptor 3A (CD16a)
Two patients with mutations in Fcγ receptor 3A (CD16) were described with
EBV diseases; one patient had a prolonged illness with fever and malaise associ-
ated with EBV infection (deVries et al. 1996) and the second was reported to have
recurrent lymphadenopathy due to EBV-positive Castleman’s disease (Grier et al.
2012). The former patient also had severe infections with Bacille Calmette–Guerin
and varicella-zoster virus, while the latter patient also had severe HPV infections
and deficient NK cell cytotoxicity. Fcγ receptor 3A is expressed on NK cells
and neutrophils; mutations in this receptor are responsible for classical NK cell
deficiency.
260 J.I. Cohen
4.7 Caspase Recruitment Domain-Containing Protein 11

(CARD11): Gain of Function Mutations
Patients with germ line gain of function mutations in CARD11 present with B cell
lymphocytosis, splenomegaly, lymphadenopathy with florid follicular hyperplasia,
recurrent sinusitis, and otitis media (Snow et al. 2012). One patient presented with
persistently elevated EBV DNA in the blood as well as splenomegaly, lymphad-
enopathy, bronchiectasis, recurrent otitis media, and molluscum contagiosum. The
CARD11 protein is required for activation of NF-κB by antigen receptor in B and
T cells. Somatic gain of function mutations of CARD11 are present in many dif-
fuse large B cell lymphomas.
4.8 Other Immunodeficiencies Associated

with Multiple Infections
Other primary immunodeficiency diseases associated with multiple infections can

present with EBV lymphoproliferative disease or lymphomas. Ataxia telangiecta-
sia is an autosomal recessive disease due to a mutation in ATM which encodes a
serine/threonine kinase that is important for DNA repair. In addition to neurologic
and skin disease, these patients often develop sinopulmonary infections, interstitial
lung disease, and are at increased risk for malignancies. These patients often have
increased levels of EBV DNA in the blood and can develop EBV lymphomas.
Wiskott–Aldrich syndrome is an X-linked disorder due to mutations in WAS. The
Wiskott–Aldrich syndrome protein is important for the formation of the immuno-
logic synapse which is the site of interaction between antigen-presenting cells and T
cells. In addition to increased propensity of infections, these patients have thrombo-
cytopenia, eczema, and autoimmune disease. Patients may develop EBV lymphomas.
Patients with severe combined immunodeficiency (SCID) can have mutations
in a number of different genes; these result in impaired B cell and T cell immunity.
In addition to increased infections, these patients often have chronic diarrhea and
failure to thrive. These patients are susceptible to EBV-positive B cell lymphomas.
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Burkitt’s Lymphoma
Rosemary Rochford and Ann M. Moormann
Abstract Endemic Burkitt’s lymphoma (BL) remains the most prevalent pediatric
cancer in sub-Saharan Africa even though it was the first human cancer with a
viral etiology described over 50 years ago. Epstein–Barr virus (EBV) was discov-
ered in a BL tumor in 1964 and has since been implicated in other malignancies.
The etiology of endemic BL has been linked to EBV and Plasmodium falciparum
malaria co-infection. While epidemiologic studies have yielded insight into EBV
infection and the etiology of endemic BL, the modulation of viral persistence in
children by malaria and deficits in EBV immunosurveillance has more recently
been reified. Renewed efforts to design prophylactic and therapeutic EBV vaccines
provide hope of preventing EBV-associated BL as well as increasing the ability to
cure this cancer.
Contents
1 The Early Work..................................................................................................................... 268
2 The Role of EBV in Endemic BL......................................................................................... 271
3 The Role of Malaria on EBV................................................................................................ 273
4 EBV-Specific T-Cell Immunosurveillance............................................................................ 276
5 Other Potential Co-factors in Endemic BL........................................................................... 278
6 Future Directions.................................................................................................................. 279
References................................................................................................................................... 280
R. Rochford
Department of Microbiology and Immunology, SUNY Upstate Medical University,
Syracuse, NY, USA
e-mail: rochforr@upstate.edu
A.M. Moormann (*)
Program in Molecular Medicine, University of Massachusetts Medical School,
373 Plantation Street, Worcester, MA 01605, USA
e-mail: ann.moormann@umassmed.edu

268 R. Rochford and A.M. Moormann
1 The Early Work
Endemic Burkitt’s lymphoma was first described by Denis Burkitt, a British sur-
geon living in Uganda in the late 1950s. In his initial report (Burkitt 1958), he
described a malignant tumor which was occurring in young children between the
ages of 2 and 14 years. The striking feature of this cancer was the involvement of
the jaw, which led Burkitt to classify this as a sarcoma. Additionally, Burkitt noted
the involvement of other anatomical sites in many of these children, including the
abdomen. Following histological review, Burkitt and O’Conor (1961) reported that
these jaw and abdominal tumors were identical and that this was a distinct patho-
logical entity. Upon further examination, it was concluded that the tumor was in
fact a malignant lymphoma, not a sarcoma. Strangely, this lymphoma seemed to
only involve extranodal sites. Referring to this disease generally as malignant lym-
phoma, it was reported to be very common in Uganda and accounted for approxi-
mately half of the cases of cancer that occurred in children. The authors examined
106 cases and reported an age distribution similar to what was previous described
by Burkitt. Interestingly, it was noted that there was approximately a 2:1 male-
to-female sex ratio in the distribution of these cases. By collecting information
through personal contact and questionnaire, it was reported that malignant lym-
phomas were seen in a relatively limited geographic area across most of central
Africa, which was referred to as the “lymphoma belt.”
In order to elucidate the cause of this tumor, Burkitt began examining its geo-
graphic occurrence in Uganda (Burkitt 1962a) and traveled on a series of “tumor
safaris” across much of central and east Africa, attempting to visit as many hos-
pitals as possible along the “edge” of this belt (Burkitt 1962a, b). He further
traveled to areas of West Africa in order to examine the occurrence of the cancer
there. Burkitt found the only factor that seemed to be related to the incidence of
this lymphoma was altitude, whereby he noted that this cancer only appeared in
populations residing in areas with an altitude of less than 5000 feet and near the
equator. When moving geographically away from the equator, this altitude cut-
off decreased along with the incidence of BL. By mapping the occurrence of BL
along the edge of the lymphoma belt, it was determined that altitude was a proxy
measure of the minimum temperature. That is, this lymphoma only occurred in
areas where the minimum temperature during the coldest season of the year was
higher than 60 °F. This hypothesis was supported by the fact that the only area of
Africa that met these temperature requirements was a belt across central Africa.
This belt was strikingly similar geographically to Burkitt’s so-called lymphoma
belt. At a time when no infectious agents were known to cause malignancies in
humans, Burkitt suggested that the temperature dependency indicated that a vector
may be responsible for transmission of the disease and hypothesized the transmit-
ted agent to be a virus. After examining rainfall levels in West Africa, Burkitt fur-
ther hypothesized that the occurrence of this cancer was also humidity dependent,
suggesting that this was perhaps an indication of vegetation dependence (Burkitt
1962a).
Burkitt’s Lymphoma 269
The geographic restriction of this cancer, which was now referred to as

Burkitt’s lymphoma (BL), was examined in more detail by Haddow (1963). He
noted that the temperature and rainfall requirements described by Burkitt corre-
sponded closely with the distribution of certain species of mosquitoes and that at
temperatures between 60 and 65 °F, viruses, such as the yellow fever virus and
dengue virus, fail to develop in a mosquito vector. Because many mosquito species
rely on an annual rainfall of at least 20 inches, it was hypothesized that BL would
only be seen in areas where the annual rainfall was greater than 20 inches a year.
To test this hypothesis, a map of the areas in Africa which met these geographic
requirements was obtained and the location of all known cases of BL was overlaid
on this map. Upon examination, 95 % of geographic locations which had reported
cases of BL occurred in these areas. It was concluded that BL was geographically
associated with a mean average temperature of greater than 60 °F (15.5 °C) for the
coolest month and an annual rainfall of at least 20 inches (500 mm).
Therefore, Haddow made the following deductions (Haddow 1964):
1. If an arbovirus was involved in the generation of BL, the number of cases
occurring was far too small to permit maintenance of the disease by direct
case-to-case transmission.
2. If this was a zoonotic agent that occasionally escapes into humans, one would
expect definite outbreaks to occur in limited areas. There was yet no evidence
of this. In addition, the curious age distribution would remain unexplained.
3. Thus, the infection, if it did occur essentially in humans, must be an exceed-
ingly common and widespread one in order to permit continuous transmission,
with the tumor being a rare and extreme manifestation.
In order to explore the arbovirus hypothesis, Haddow analyzed the age distribu-
tion of 363 cases collected by Burkitt from Uganda, Tanganyika (now Tanzania),
Mozambique, and Nigeria. The age distribution showed a peak between the ages
of 6 and 7 years and only 2 % of cases occurring in individuals above the age
of 20 years. From this, it was concluded that the risk of responding to the postu-
lated infection by development of the tumor was eliminated by age 20, suggesting
a very common viral infection. Furthermore, the fact that the tumor did not occur
in extremely young children suggested that they were protected by maternal pas-
sive immunity following birth, consistent with the hypothesis that this agent was
common.
In a report publish in 1966 (Burkitt and Wright 1966), Burkitt et al. exam-
ined the occurrence of 450 histologically determined cases of BL over an eight
year time period. He found the occurrence of the cancer to be closely corre-
lated geographically with temperature. High rates, ranging from 8.7 to 13.4 per
100,000 children per year, were seen in lowland areas along the Nile, while rates
were almost 20 times lower in the mountainous area of southwestern Uganda.
Consistent with the previously reported sex bias, the overall incidence was 2.3
males per female, even though the admission rates to pediatric wards were equal
for sexes in the hospitals from which records were obtained. In areas with high
rates of BL, the average age of tumor occurrence was 8.1 years old, similar to
previous reports. Interestingly, in areas with a low rate of BL, the average age of
incidence was 16.2 years, markedly older. Furthermore, among immigrants who
moved to areas of high BL incidence, similar rates of disease to native residents
were seen. However, BL occurred among older age groups for those migrating to
high incident areas with immigrants accounting for nearly 50 % of patients over
the age of 15 year in these areas. Using this evidence, the authors suggested that in
highly endemic areas, exposure to the “mysterious” arbovirus occurred more com-
monly early in life, causing the tumor to develop soon thereafter, whereas in areas
where BL was less common exposure tended to occur later in life. The authors
reported that all tribes living in lowland areas had similar rates of BL and that the
ratio of African to Asian cases of BL was consistent with the population ratio of
these two groups [also see (Burkitt 1962c)], suggesting that geographic differences
in the occurrence of this cancer could not be explained by human genetic factors.
While visiting the territories of Papua and New Guinea in 1960, Ten Seldam
observed 2 cases of lymphosarcoma in young boys and was struck by the simi-
larity to the BL patients which had been described in Africa. Ten Seldam et al.
(1966) examined 35 cases of childhood lymphoma that occurred in these territo-
ries between 1958 and 1963. Of these, approximately one-third had the typical
jaw involvement described by Burkitt which was about half the ratio of tumors
with jaw involvement found in Africa. However, this group reported an extraor-
dinarily uniform histological picture in the appearance of these tumors with those
described in Africa. Geographically, these cases occurred in areas which met the
temperature and rainfall requirements previously described. Interestingly, it was
noted that Plasmodium falciparum malaria was also holoendemic in both areas of
Africa and New Guinea, where repeated malarial infections were nearly universal
during the first years of life (Dalldorf et al. 1964).
Following this lead, Burkitt collected and examined evidence suggesting that
malaria could play an etiologic role in BL and in 1969 published an extensive
review that used a number of arguments to suggest that malaria was involved in
the generation of BL (Burkitt 1969). In this review, Burkitt discussed in detail
epidemiologic evidence for the association between malaria and BL, most impor-
tantly the fact that holoendemic malaria was only known to occur in Africa and
New Guinea, as well as pathological evidence, such as the possibility that the
occurrence of BL in extranodal sites could be explained by hyperreactivity of
the reticuloendothelial system caused by chronic malaria infections, also known
as tropical splenomegaly syndrome (Fakunle and Greenwood 1976). This review
further discussed the evidence for the involvement of a virus, such as the virus
isolated by Epstein and colleagues from BL tissue culture cells. With increasing
evidence pointing toward the role of an infectious agent in the etiology of BL,
yet no conclusive results to date, Burkitt stated, “Some of the strongest evidence
implicating an infective agent as partly responsible for the occurrence of Burkitt’s
lymphoma is the demonstration of case clustering in space and time. This would
be consistent with the postulation of a viral infection, either as a vectored agent
primarily responsible for the tumor or as a virus not necessarily vectored but pro-
moting actual tumor formation.”
In 1964, EBV was discovered in a BL tumor from a Ugandan patient when

Burkitt sent a biopsy specimen to Anthony Epstein, Yvonne Barr, and Bert
Achong who were able to visualize the virus within this B-cell tumor using elec-
tron microscopy (Epstein et al. 1964). Details of this discovery of EBV can be
found in the chapter by Dr. Epstein. Since then, EBV has been associated with
nearly all endemic (African) BL tumors, with clear evidence that EBV infection
precedes B-cell clonal expansion (Neri et al. 1991). Yet BL can present without
detectable EBV within the tumor even though all BL tumors have the c-myc trans-
location (Dalla-Favera et al. 1982). Other forms of BL have been classified as spo-
radic BL in which only 20 % of the patients have detectable EBV within their
tumors, immune deficiency (AIDS) associated BL with EBV in situ in 30–40 %
of the patients’ tumors, and an “intermediate” form of BL found in less developed
countries such as Brazil, Middle East, and other areas that do not have malaria. It
is tempting to speculate that EBV triggered these forms of BL but was lost dur-
ing rapid cell replication hijacked by the myc-oncogene. For the purposes of this
review, we will limit our discussion to the EBV-associated form of BL.
2 The Role of EBV in Endemic BL
Despite inconsistencies between studies investigating space-time clustering, sea-

sonality, sex ratios, etc. (Biggar and Nkrumah 1979; Morrow et al. 1977; Rainey
et al. 2007; Siemiatycki et al. 1980), these early descriptive studies provided a
great deal of evidence implicating EBV and holoendemic malaria as etiologic
agents in the generation of BL. The advent of a serologic test to diagnose primary
EBV infection and follow the stages of infection heralded a means to investigate
the temporal role of EBV in BL etiology, and EBV gene expression patterns are
discussed in more detail in the chapter on EBV serology. In brief, Gertrude and
Werner Henle developed a diagnostic assay using immunoglobulin G (IgG) and
IgM antibodies to EBV following the hierarchy of antigen expression (Henle et al.
1969). Antibody titers to viral capsid antigen (VCA) , early antigen-diffuse (EA-D)
and Epstein–Barr virus nuclear antigen (EBNA-1) are used to determine the stage
of infection. There is some debate as to the interpretation of EBV serology pat-
terns, however, IgM to VCA indicates early-acute primary infection, whereas ele-
vated IgG to VCA and EA-D indicate viral reactivation. The development of IgG to
EBNA-1 marks the recovery phase and is an indicator of remote infection.
Serological surveys conducted by Guy de Thé in the 1970s demonstrated that
nearly all African children were EBV seropositive by three years of age (de-The
et al. 1978), in contrast to children in developed countries who seroconvert later
in life with ~30 % of the population remaining seronegative until adolescence
(Balfour et al. 2013). This suggested that EBV may be necessary but not suffi-
cient to cause BL tumorigenesis and raised the question as to whether changes in
serological profiles to EBV antigens could be predictive of BL. The first serosur-
vey study, reported in 1978, was conducted by de Thé et al. (1978) in the West
Nile district of Uganda from 1971 to 1976. Serum samples were taken from
42,000 children below the age of 8 years. Over the course of the study, 14 children
enrolled were clinically diagnosed with BL. Serum antibodies from these cases
were then compared to the following 3 controls: (1) serum from a neighbor of the
same age and sex, (2) 4 control sera selected from the sera bank of the same age,
sex, and approximate locality, and (3) sera from a random sample of the surveyed
population. Whereas no difference was seen in pre-BL sera antibodies to EBV EA
and EBNA antigens for patients in comparison with matched controls, higher titers
of antibodies to VCA were present in pre-BL sera of cases, with an average geo-
metric mean titer (GMT) approximately 3.4 times higher. When GMTs to VCA
were compared to a random sample of the population, all but 2 cases were higher
than the mean for children in that corresponding age group in the normal popula-
tion, indicating that the age, sex, and geographically adjusted risk of developing
BL was approximately 30 times higher for children with a VCA titer 2 dilutions
above the mean than for children with average sera titers. Antibody levels to her-
pes simplex virus and cytomegalovirus showed no difference between cases and
controls, indicating that EBV plays a specific role in the development of BL, not
just that elevated VCA titers were a marker for overall immune activation. Further
support for the role of EBV in the etiology of endemic BL came from studies
showing that the virus was clonal in the tumors suggesting that infection preceded
the development of malignancy (Neri et al. 1991).
In 1977, Guy de Thé proposed that perinatal infection with EBV was a risk fac-
tor for BL (de-The 1977) but not until 2012, there was evidence to support this
hypothesis (Piriou et al. 2012). In a prospective study by Piriou et al. based in
Kenya, two groups of infants were followed from 1 month of age through 2 years
of age. The first group of 68 children was from a malaria endemic region and the
second group of 82 children was from an area with low malaria transmission. EBV
infection was detected either by evidence of EBV DNA in the blood or by levels
of IgG and IgM to VCA and of IgG to EBNA-1. Several observations were made.
First, infants born in a malaria holoendemic region were infected with EBV ear-
lier in life than infants born in a region with low malaria transmission (mean age
7.28 months compared to 8.39 months, respectively). Second, ~35 % of infants
from the malaria holoendemic region were infected before 6 months of age. And
finally, infection with EBV early in life predicted higher viral load over time sug-
gesting that early age of infection resulted in poor control of the virus.
At a molecular level, several lines of evidence also point to a role for the virus
in driving tumorigenesis. These include as follows: the maintenance of the viral
genome in BL tumors (zur Hausen et al. 1970), the viral genome is present in
all cells within the tumors (zur Hausen et al. 1970), the virus is clonal within the
tumors (Neri et al. 1991), and the viral protein EBNA-1 (Lindahl et al. 1974) along
with viral non-coding BART microRNAs (Tao et al. 1998; Xue et al. 2002) are
consistently expressed within the tumors. In addition, EBV infection within the
context of type I latency program promoted BL cell growth by inhibiting c-myc-
induced apoptosis through the upregulation of Bcl-2 and a commensurate decrease
in c-myc expression (Ruf et al. 2001). The consequences of EBV gene expression
patterns that diverge from restricted Qp promotor latency I may have implications
for responsiveness to chemotherapy. Studies conducted by Griffin et al. found that
BL tumors from Malawian patients expressed an array of lytic genes and expression
of EBV BZLF1 replication activator intermediate early promotor (ZEBRA) corre-
lated with responsiveness to cyclophosphamide (Labrecque et al. 1999). Studies of
BL cell lines concluded that Wp-restricted tumors were more aggressive and resist-
ant to apoptosis (Kelly et al. 2013). In addition, the transformation capacity of EBV
also highlights the virus’ oncogenic potential. EBNA-1 induces lymphomas in a
transgenic mouse model (Wilson et al. 1996) supporting a potential direct role for
this EBV latent protein in oncogenesis. An alternative model for the role of EBV
in oncogenesis is that EBV infection of a B cell increases the risk for other cellu-
lar changes. These could be either through epigenetic modifications (Kaneda et al.
2012) or by promoting genetic instability (Gruhne et al. 2009).
3 The Role of Malaria on EBV
In an attempt to retrospectively determine malaria infection history of BL patients,

a case–control study was conducted in Uganda from 1994 to 1999 (Carpenter et al.
2008). Carpenter et al. found that children diagnosed with BL received more fre-
quent treatments for malaria in the preceding year and were less likely to live in
a household using insecticide-treated bednets compared to controls. IgG VCA
titers were measured on 126 BL patients and 70 controls. Consistent with the ear-
lier studies, IgG VCA titers were significantly higher in the BL patients surveyed
compared to children without BL. Antibodies to malaria were measured by indi-
rect immunofluorescence assay (Sulzer et al. 1969). The odds ratio (OR) estimates
for high antimalarial antibody titers verged on significance, however, when the
cases and controls were matched according to residential district, antimalarial anti-
body titers were no longer a risk factor, whereas elevated antibody levels to VCA
remained highly significant. A replication case–control study was conducted by
Mutalima et al. in Malawi in 2005–2006 (Mutalima et al. 2008). IgG titers to VCA
and malaria parasite schizont extract (Verra et al. 2007) were higher in 137 BL
patients compared to 91 controls, OR = 14.8 (95 % CI 5.8–38.5) and OR = 2.4
(1.2–4.4), respectively, supporting an interaction between malaria and EBV in
increasing the risk for BL.
Studies conducted by Piriou et al. (2009) in western Kenya compared EBV
serological profiles in 67 children residing in a malaria holoendemic area to 102
children residing in an area with sporadic malaria transmission. The cumulative
effect of chronic and/or repeated P. falciparum malaria infections resulted in ele-
vated IgG antibody titers to VCA, EBNA-1, EA-D, and another immediate early
protein, Z trans-activation antigen, Zta (also known as ZEBRA), which is typi-
cally not induced in healthy individuals (Fachiroh et al. 2006). These data suggest
that over time, children co-infected with malaria experienced more EBV reactiva-
tion than children not infected with malaria. A complementary study went on to
compare EBV and malaria serological profiles in 32 children with BL and 25 con-
trols who were frequency matched for age and sex (Asito et al. 2010). This study
used a panel of liver- and blood-stage malaria antigens in a multiplex serological
assay to generated IgG antibody titers and found no difference in median antibody
levels to specific antimalarial proteins between BL cases and age-, sex- and geo-
graphically matched controls. In contrast, BL cases had elevated antibody titers to
Zta and VCA compared to controls, consistent with the Ugandan study (Carpenter
et al. 2008) but contradicting the Malawian study (Mutalima et al. 2008). In a
more recent study in Uganda by Orem et al. (2014), 46 BL patients and 50 chil-
dren with other forms of non-Hodgkin’s lymphoma had significantly elevated IgG
levels to EA-D, yet IgG titers to VCA did not differ between cases and controls.
Activation of EBV could be due to poor general health that may affect control over
EBV latency and may be an effect modifier in children residing in malaria holoen-
demic areas.
The first intervention study attempting to examine the causative role of malaria
in BL was conducted by Geser et al. whereby they attempted to prevent malaria in
the entire childhood population of the North Mara lowlands of Tanzania from 1977
to 1982 and compare the rates of BL during this study period to previous and sub-
sequent years (Geser and Brubaker 1985; Geser et al. 1989). The group carried out
baseline malaria surveys in 1974, 1975, and 1976 as well as continuous surveys
from 1978 to 1982, while chloroquine tablets were given twice monthly to all chil-
dren in the region from 1977 to 1982. Prior to the study period, from 1964 to 1976,
the annual incidence of BL averaged 4.3 per 100,000 children, ranging from 2.6
to 6.9 per 100,000 children. Immediately following the start of the intervention,
the incidence of BL began dropping rapidly, hitting a minimum of 0.5 per 100,000
children in 1980 and 1981. Subsequently, from 1982 onward the rate rose sharply,
hitting a peak of 7.1 per 100,000 children in 1984. Using simple linear regression,
the overall drop in incidence from 1964 to 1982 was highly significant, whereas
the slight decline in the rate prior to intervention (1964–1976) was only margin-
ally significant. Upon preliminary analysis, this drop seemed to correspond with
the effects of the intervention. However, following a large drop in the prevalence
of malaria parasitemia in the population (48 % in 1976 to 11 % in 1977), the levels
of parasitemia subsequently rose during the study period and returned to pre-inter-
vention levels by 1981, primarily due to inefficiency of drug distribution. The fact
that this intervention was only marginally successful in preventing malaria makes
it difficult to determine whether the drop in BL was actually due to the antima-
larial intervention. Additionally, the researchers found upon closer scrutiny that
BL incidence in the North Mara district may have started dropping in 1972, five
years prior to the initiation of this intervention. Serological work from this study
did show that neither the prevalence of EBV nor the geometric mean titer to EBV
antibodies in children varied throughout the study, indicating declining EBV prev-
alence was not associated with the decline in BL. Furthermore, it was found from
the malaria surveys that P. falciparum was responsible for about 90 % of malaria
infections; consistent with the observation from other areas that P. falciparum is
the dominant parasite species in areas with a high incidence of BL.
Transient lymphopenia and selective immune suppression has long been

recognized as a complication of acute malaria infections (Greenwood et al.
1972; Williamson and Greenwood 1978). Acute malaria has also been shown to
induce pathophysiological changes in B-cell homeostasis (Asito et al. 2008) as
well as long-term changes in memory B-cell subsets (Portugal et al. 2012; Weiss
et al. 2009) leading to a possibility that malaria could alter EBV persistence. A
direct effect of P. falciparum on EBV-infected B cells was shown by Chêne et al.
(2007). BL cell lines were incubated with the cysteine-rich inter-domain region 1
α (CIDRα) of the P. falciparum erythrocyte membrane protein which resulted in
lytic cycle activation. In support of this as a potential mechanism for altering EBV
persistence, children from Uganda with acute malaria had EBV viremia (Donati
et al. 2006) and healthy children from a malaria endemic region had evidence of
viral reactivation as indicated by viremia (Rasti et al. 2005). Serologic evidence
for increased viral reactivation in children living in malaria holoendemic regions
was also shown in a study by Piriou et al. (2009) in Kenya. That there might also
be an increase in latently infected cells was first shown by Lam et al. (1991). A
separate study by Nije et al. (2009) of Gambian children with acute malaria con-
firmed this observation by demonstrating an elevated viral load in peripheral blood
mononuclear cells from children with acute malaria.
More recent studies to elucidate a mechanism by which malaria could play a
role in BL pathogenesis have focused on the enzyme activation-induced cyti-
dine deaminase (AID) which is highly expressed in germinal center B cells
(Muramatsu et al. 2000; Ramiro et al. 2004, 2006) and is responsible for somatic
hypermutation and class switch recombination. In a mouse model, AID was also
found to be required for the c-myc translocation (Robbiani et al. 2008). Incubation
of human B cells with P. falciparum extracts induces AID and class switch recom-
bination (Potup et al. 2009). Subsequently, a study by Torgbor et al. (2014) using
B-cell lines and palatine tonsil tissue obtained from 12 Ghanaian children com-
pared to one North American, malaria-naïve control demonstrated that P. falcipa-
rum malaria, more specifically the malaria-derived hemozoin/DNA complex that
is a TLR9 agonist (Parroche et al. 2007), deregulates AID expression. Another
study by Wilmore et al. of healthy Kenyan children with divergent malaria expo-
sure examined AID expression in peripheral blood mononuclear cells and dem-
onstrated that malaria-exposed children with detectable EBV circulating in their
peripheral blood had higher AID expression compared to EBV-seropositive chil-
dren with no detectable virus by PCR (Wilmore et al. 2014). This study is impor-
tant for solidifying the link between malaria and EBV co-infection in endemic
BL etiology in that malaria-non-exposed children with measurable EBV load had
similar AID levels compared to EBV PCR negative children. Repeated or chronic
malaria-induced upregulation of AID could in turn increase EBV load due to the
preference of EBV to infect B cells with mutated immunoglobulin (Heath et al.
2012). This mechanism may explain in part the synergy between malaria and
EBV leading to BL and is compatible with malaria-induced alternations in T-cell
immunosurveillance.
4 EBV-Specific T-Cell Immunosurveillance
T-cell responses that control persistent EBV infections in immune competent indi-
viduals and T-cell responses in individuals suffering from acute infectious mono-
nucleosis and X-linked lympho-proliferative disorder are covered in more detail
in other chapters of this book and in a recent review by Rickinson et al. (2014).
Suffice it to mention, studies of EBV-specific T-cell immunity developed by chil-
dren infected within the first few years of life and in children diagnosed with BL
are few due to challenges in gaining access to pediatric patients and appropriately
matched controls as well as limitations in blood volumes ethically obtainable from
children.
Early studies to assess the effects of malaria infection on EBV-specific T-cell
immunity were conducted by Moss et al. (1983) using blood samples from adults
residing in two areas of Papua New Guinea: a lowland coastal region with hol-
oendemic malaria and a high incidence of BL, and a highland area where malaria
was not endemic and the incidence of BL was low. Antibody titers to both P. fal-
ciparum and EBV antigens were determined for both study groups as well as with
a group of Caucasian controls. Levels of EBV-specific T-cell-mediated immunity
were determined by regression assay. Antibody titers to malaria were higher in the
lowland group in comparison with the other two groups and there was no signifi-
cant difference in anti-VCA and anti-EBNA between the three groups. Regression
assays indicated a highly significant difference in the levels of EBV-specific
T-cell-mediated immunity associated with holoendemic malaria. Additionally,
spontaneous B-cell transformation was seen in 14 of 55 cultures from periph-
eral blood samples from individuals residing the area with holoendemic malaria,
whereas no transformation was seen in any of the cultures from the other two
study groups. This study concluded that adults in areas with holoendemic malaria
had decreased T-cell-mediated immunity to EBV. It should be noted that no other
viruses were examined in this study; therefore, it was not possible to determine
whether this was a generalized T-cell immunosuppression or if it was specific to
only EBV. After stratifying by study group, it was found that there was no sig-
nificant correlation between regression end-points and antibody levels to EBNA or
VCA, consistent with the results from the study by Biggar et al. (1981) in Ghana
that found no significant differences in EBV antibody titers between African com-
munities in malarious and non-malarious regions.
A similar study examining T-cell-mediated immunity to EBV was conducted
by Whittle et al. (1984) in the Gambia. In this study, blood samples were taken
from nine children ranging in age from 5 to 18 years during and 3 weeks following
an acute infection with P. falciparum malaria. Regression assays for T-cell con-
trol of EBV showed significantly higher regression indices during acute infection,
which fell to normal levels following recovery, implying that a transient loss of
T-cell-mediated immunity to EBV occurs only during acute malarial infection.
Analysis of the cell types in the blood indicated that during acute malaria infec-
tion, children had a reduced total number of T cells, as well as reduced ratio of
T-helper to T-suppressor cells. Furthermore, there was an increased ratio of B

cells to T-suppressor cells. Using this information, the group hypothesized the
following model: In children destined to develop BL, a large number of their B
cells are infected by EBV during initial infection [based of the work of de Thé
et al. (1978)]. Repeated attacks with malaria result in the loss of T-cell control of
EBV, allowing EBV-infected B cells to proliferate and increase in number, which
increases the likelihood that chromosomal translocation and malignancy will
occur.
It was not until years later that this model could be put to the test. Studies con-
ducted by Moormann et al. (2005) in Kenya measured EBV loads by real-time
quantitative PCR in 104 children residing in a malaria holoendemic area and com-
pared them to 127 children from a highland area that experienced sporadic malaria
transmission. EBV load was highest in the malaria-exposed children (1–4 years
of age) prior to the peak age of BL onset. This study went on to investigate the
specificity of the T-cell immune deficiency described by earlier studies by com-
paring IFN-γ ELISPOT responses to pools of EBV lytic and latent antigen pep-
tides specific to CD8+ T cell (Moormann et al. 2007). Immune responses to EBV
were contrasted to those against malaria peptides. Children were stratified by age
group: 1–4 years of age, when malaria morbidity is the highest; 5–9 years of age,
when premunition to malaria is being developed characterized by semi-protective
immunity permissive of asymptomatic parasitemias and coinciding with the peak
age-incidence for BL; and 10–14 years of age, when children have developed anti-
disease immunity to malaria (Riley and Stewart 2013) and have not succumbed to
BL. This study demonstrated deficient IFN-γ responses to EBV lytic and latent
antigen peptides restricted to 5- to 9-year-old children who had been residing in a
malaria holoendemic area compared to age-matched control children from an area
with sporadic malaria transmission (Moormann et al. 2007). Longitudinal stud-
ies conducted by Snider et al. (2012) suggest that T-cell immunity to EBV lytic
antigens may diminish prior to those against EBV latent antigens. This would be
consistent with a model whereby malaria induces lytic reactivation, and lytic-spe-
cific T cells become exhausted due to chronic or repeated antigen stimulation (Wei
et al. 2013), thereby allowing infectious virions to incrementally establish a higher
frequency of latently infected B cells.
In support of an incremental loss of EBV-specific T-cell immunosurveillance
hypothesis in BL etiology is a study conducted by Moormann et al. (2009) in
2005–2006, which was the first to directly examine T-cell immunity to EBV and
malaria in BL patients. This study compared IFN-γ responses to HLA Class I
restricted EBV lytic and latent antigen peptides (Rickinson et al. 1992; Khanna and
Burrows 2000) in addition to an overlapping pool of longer HLA Class II peptides
to EBNA-1 (Heller et al. 2007) and a recombinant protein to malaria merozoite sur-
face antigen 1 (MSP-1) (Singh et al. 2003). EBNA-1 is of particular interest since
it is the only EBV antigen expressed in all BL tumor cells (Crawford 2001) and
is only rarely detected by cytotoxic CD8+ T lymphocytes (Munz 2004). Children
diagnosed with BL had a significant deficiency in IFN-γ responses to EBNA-1
compared to healthy age-matched controls and yet had robust responses to MSP-1
comparable to healthy malaria-exposed children (Moormann et al. 2009). Of note,

the BL children had IgG1 subclass to EBNA-1 similar to healthy EBV-seropositive
children suggesting T-cell depletion in the BL patients rather than Th2 polarization.
In addition, non-BL, malaria-exposed children lacking IFN-γ responses to EBNA-1
had higher median EBV loads compared to healthy children with EBNA1-specific
T-cell immunity. Another study by Chattopadhyay et al. used HLA-A2 tetram-
ers to phenotype CD8+ T cells specific to EBV lytic (BMFL1 and BRLF1) and
latent (LMP1, LMP2, and EBNA3C) peptides and multidimensional analysis of
CD45RO, CD27, CCR7, CD127, CD57, and PD-1 expression (Chattopadhyay
et al. 2013). They found that CD8+ T cells against lytic antigens tended to dis-
play an exhausted phenotype lacking homeostatic potential and individuals resid-
ing in malaria holoendemic areas had more differentiated CD8+ T cells to EBV
latent antigens with fewer central memory subsets compared to those living in
regions with little to no malaria transmission. Malaria did not skew CMV-specific
T-cell subsets nor affect the global CD8+ memory T-cell pool. These studies fur-
ther solidified a malaria-associated detrimental impact on the generation and main-
tenance of EBV-specific T cells that may contribute to the etiology of BL.
5 Other Potential Co-factors in Endemic BL
The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), a phor-

bol ester isolated from croton oil was found to efficiently induce the production
of EBV from BL cell lines (zur Hausen et al. 1978) and enhance the ability of
EBV to transform B cells (Mizuno et al. 1983; Yamamoto and zur Hausen 1979).
As croton oil is extracted from a plant in the Euphorbiaceae family, extracts of
other plants from the same plant family (Croton tiglium, Euphorbia lathyris, and
Euphorbia tirucalli) were also found to induce EBV reactivation (Ito et al. 1981).
Dose-dependent treatment of the Jijoye BL cell line with crude E. tirucalli latex
(10-fold serial dilutions ranging from 10−3 to 10−7) resulted in induction of lytic
cycle gene expression (sixfold for BZLF1 to 19-fold for gp350) over background
levels (MacNeil et al. 2003). Additionally, dual treatment of EBV+ cell lines with
n-butyrate and extracts from E. tirucalli induced EBV antigen expression and
treatment of cord blood lymphocytes with the extract following EBV infection
enhanced the transforming ability of EBV over 10-fold. The active ingredient in
these extracts was isolated by silica gel column chromatography and found to be
the chemical 4-deoxyphorbol ester (Osato et al. 1987, 1990). Following infection
of cord blood lymphocytes with EBV and treatment with purified 4-deoxyphor-
bol ester, this group noted a high frequency of chromosomal rearrangements, often
involving chromosome 8, the chromosome commonly implicated in translocations
in BL (Aya et al. 1991). A more recent study (Mannucci et al. 2012) treated EBV
transformed lymphoblastoid cell lines with E. tirucalli extracts and found that
within 5-day evidence of chromosomal abnormalities were present.
Armed with data indicating an interaction between extracts from plants of

the Euphorbiaceae family and EBV, Osato et al. (1987) performed surveys from
1984 to 1986 in Kenya and Tanzania, looking at the geographic distribution of
Euphorbiaceae with relation to the occurrence of BL. In a brief but compelling
letter, this group reported that profusion of the plant E. tirucalli coincided with
the endemicity of BL. According to their research, E. tirucalli was used daily in
the Lake Victoria Basin of Kenya as a traditional medicine. In a separate study,
45 residents and 6 traditional healers in the Lake Victoria region of Kenya were
interviewed about their uses for and exposure to E. tirucalli (MacNeil et al. 2003).
E. tirucalli was an extremely common, domesticated plant with many traditional
uses. Exposure to the plant among children could occur through a variety of ways
including through the use of the latex as a topical medicine, an ingested medicine,
a play item, and as glue. Epidemiologically, only one study to date has examined
the association between BL and E. tirucalli. In this case–control study, conducted
by van den Bosch et al. in Malawi (van den Bosch et al. 1993), the presence of
E. tirucalli around households was investigated for 67 cases of BL and 228
matched controls. This group found there to be a significant association between
the prevalence of E. tirucalli at homesteads and the occurrence of BL (OR = 7.96,
p-value = 0.012). However, it should be noted that the plant was only identified
at the homes of 5 cases and 2 controls, making it difficult to assess the validity of
these results.
Other co-factors that could contribute to the increased risk of endemic BL
have included micronutrient deficiency (Sumba et al. 2010) and arboviruses, spe-
cifically Chikungunya virus (van den Bosch and Lloyd 2000). Further studies are
needed to determine whether these agents are causal in the etiology of endemic
BL.
6 Future Directions
After 50 years since the discovery of EBV, its role in BL etiology and continued
role in preventing apoptosis once B-cell oncogenesis has been initiated remain
active areas of investigation. There appears to be a sequence of events whereby
EBV infection occurs early in life, followed by repeated and often chronic, asymp-
tomatic P. falciparum malaria infections that in turn modulates EBV persistence
and erodes EBV-specific T-cell immunity. The peak incident age for the devel-
opment of BL (5–9 years) indicates a cumulative effect of these two infectious
agents; the chronology of which is depicted in Fig. 1. The possible involvement
of other co-factors such as an arboviral infection or environmental exposure that
would trigger malignant transformation after EBV and malaria has set the stage
warrant further exploration. Future studies to prevent BL include the development
of a prophylactic EBV vaccine to prevent infection (Balfour 2014; Cohen et al.
2013). Decreases in BL incidence may be coincidental with the implementation of
a malaria vaccine to reduce the burden of malaria in young children and with other
Early-age primary EBV infection and

malaria-associated EBV reactivation
Peak age-incidence of eBL
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Age in years
Erosion of EBV-specific
T cell immunosurveillance
Highest prevalence of
symptomatic Pf-malaria
Development of premunition or immune tolerance to malaria

permissive of asymptomatic parasitemia
Fig. 1 Cumulative impact of Epstein–Barr virus and Plasmodium falciparum malaria co-infec-
tions in the etiology of endemic Burkitt’s lymphoma. Proposed model indicating early-age EBV
infection with repeated EBV reactivation illustrated by blue triangle mountain peaks of EBV
viremia which precedes the development of eBL. Blue line represents distribution curve for
eBL incidence by age, rising at 5 years of age and decreasing by 9 years of age with a range of
2–14 years of age. Average age of eBL clinical presentation is 7.5 years old. Malaria co-infec-
tions frequently occur before 5 years of age and contribute to repeated EBV reactivation. After
cumulative exposure to malaria, children develop immune tolerance allowing malaria to become
a chronic rather than acute infection which contributes to the detrimental impact of malaria on
EBV-infected B cells and the erosion of EBV-specific T-cell immunosurveillance. Abbreviations:
EBV Epstein–Barr virus; Pf Plasmodium falciparum; eBL endemic Burkitt’s lymphoma
malaria control programs. There is also interest in a therapeutic EBV vaccine that
could be used in combination with conventional chemotherapy to improve survival
for children diagnosed with BL (Neparidze and Lacy 2014). These and other chal-
lenges await the next generation of scientists.
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Contribution of the Epstein-Barr Virus
to the Pathogenesis of Hodgkin
Lymphoma
Paul Murray and Andrew Bell
Abstract The morphology of the pathognomonic Hodgkin and Reed-Sternberg

cells (HRS) of Hodgkin lymphoma was described over a century ago, yet it was only
relatively recently that the B-cell origin of these cells was identified. In a propor-
tion of cases, HRS cells harbour monoclonal forms of the B lymphotropic Epstein-
Barr virus (EBV). This review summarises current knowledge of the pathogenesis of
Hodgkin lymphoma with a particular emphasis on the contribution of EBV.
Contents
1 Introduction........................................................................................................................... 288
2 Origin of HRS Cells.............................................................................................................. 289
3 Suppression of the B-Cell Phenotype in HRS Cells............................................................. 290
4 Deregulated Cellular Signalling in Classical HL.................................................................. 291
5 EBV and Classical HL.......................................................................................................... 294
6 Contribution of EBV to the Pathogenesis of Classical HL................................................... 296
7 Loss of BCR Functions as a Potential Pathogenic Event in EBV-Positive HL.................... 298
8 EBV and the HL Microenvironment..................................................................................... 299
9 Conclusions........................................................................................................................... 301
References................................................................................................................................... 302
Abbreviations
BCR B-cell receptor

B2m Beta-2 microglobulin
CTL Cytotoxic T lymphocyte
P. Murray (*) · A. Bell

School of Cancer Sciences and Centre for Human Virology, College of Medical and Dental
Sciences, University of Birmingham, Birmingham, Edgbaston B15 2TT, UK
e-mail: P.G.Murray@bham.ac.uk
A. Bell
e-mail: A.I.BELL@bham.ac.uk

288 P. Murray and A. Bell
DDR1 Discoidin domain receptor 1

DLBCL Diffuse large B-cell lymphoma
EBERS EBV-encoded RNAs
EBNA EBV nuclear antigen
EBF1 Early B-cell factor 1
HAART Highly active anti-retroviral therapy
HRS Hodgkin and Reed-Sternberg
HL Hodgkin lymphoma
HLA Human leucocyte antigen
IM Infectious mononucleosis
ITAM Immunoreceptor tyrosine activation motif
JAK Janus kinase
L&H Lymphocytic and histiocytic
NF-κB Nuclear factor kappa B
NLP Nodular lymphocyte predominant
PD-L1 Programmed cell death-ligand 1
PI3K Phosphatidylinositol-3-kinase
REAL Revised European American Lymphoma
RTK Receptor tyrosine kinase
STAT Signal transducer and activator of transcription
TGFβ Transforming growth factor β
TNF Tumour necrosis factor
WHO World Health Organization
1 Introduction
Hodgkin lymphoma (HL) is one of the most common lymphomas in the Western
world, with an annual incidence of approximately three new cases per 100,000. On
the basis of morphological, immunophenotypic and clinical differences, the Revised
European American Lymphoma (REAL)/World Health Organization (WHO) clas-
sification divides HL into two major types: classical HL and nodular lymphocyte
predominant (NLP) HL (Swerdlow et al. 2008). In both cases, involved lymph nodes
show a disrupted architecture in which the malignant cells represent a minority of
the tumour mass, with the remaining cells comprising a cellular infiltrate of non-
neoplastic cells including T lymphocytes and B lymphocytes. Cross talk between
this tumour microenvironment and the malignant cells plays a crucial role in the
growth, survival and immune escape of the tumour (Aldinucci et al. 2010). In
NLPHL, the tumour cells are referred to as lymphocytic and histiocytic (L&H) cells,
while classical HL is characterised by the presence of malignant Hodgkin/Reed-
Sternberg (HRS) cells (Kuppers 2009). Classical HL is further subdivided into four
morphological subtypes known as mixed cellularity, nodular sclerosis, lymphocyte
Contribution of the Epstein-Barr Virus … 289
Table 1 Comparison of classical and nodular lymphocyte predominant Hodgkin lymphoma

REAL/WHO Morphology and EBV status Immunoglobulin gene status
classification immunophenotype
of HRS cells
Classical HL Typical HRS cells 20–40 % Lack of BCR expression
Mixed cellularity CD15+ CD20− positive Destructive or non-functional
Nodular sclerosis CD30+ CD45− IgH rearrangements
Lymphocyte rich Loss of Ig-specific
Lymphocyte depletion transcription factors
Nodular lymphocyte Atypical ‘popcorn’ Usually nega- Express BCR
predominant HL cells tive Functional Ig rearrangements
CD15− CD20+ Evidence of intra-clonal
CD30− CD45+ diversity indicating ongoing
somatic hypermutation
rich and lymphocyte depletion (Table 1). While L&H cells retain the expression of
typical B-cell antigens such as CD20 and CD19, HRS cells of classical HL have an
unusual phenotype in which the B-cell gene expression programme is largely extin-
guished. Although recent studies suggest that a small proportion of NLPHL cases
harbour the Epstein-Barr virus (EBV) (Wang et al. 2014; Huppmann et al. 2014),
here we focus on classical HL in which the link with EBV is well established.
2 Origin of HRS Cells
The cellular origin of HL tumour cells remained elusive for many years with
early studies suggesting that HRS cells might be derived from macrophages,
dendritic cells or granulocytes. However, based on the detection of clonally rear-
ranged immunoglobulin heavy and light chain genes, it is now clear that HRS
cells of classical HL originate from mature B lymphocytes (Kuppers et al. 1994).
Moreover, the immunoglobulin variable region sequences of HRS cells show evi-
dence of somatic hypermutation, indicating that HRS cells are derived from post-
germinal centre B cells (Kuppers et al. 1994; Kanzler et al. 1996; Vockerodt et al.
1998; Marafioti et al. 2000). Notably, in approximately one-quarter of cases, the
rearranged immunoglobulin sequences carry ‘crippling’ mutations which render
the surface immunoglobulin molecule or B-cell receptor (BCR) non-functional
(Kanzler et al. 1996). Since a functional BCR is required for B-cell survival within
the germinal centre, it is now widely believed that classical HL originates from
pre-apoptotic germinal centre B cells that have been rescued from apoptosis by
cellular transformation events (Fig. 1). However, in rare instances, classical HL
can be of T-cell origin, as evidenced by the presence of T-cell receptor gene rear-
rangements (Muschen et al. 2000; Seitz et al. 2000).
The lack of expression of a functional BCR is one of the hallmarks of classi-
cal HL. While in some cases, this is a consequence of destructive immunoglob-
ulin mutations, other mechanisms can also result in the loss of BCR expression.
Affinity selection
Somatic hypermutation
Ig class switching Memory B cell
Favourable IgV
mutations
Plasma B cell
Unfavourable
Antigen-activated IgV mutations Rescue by EBV and/or
naive B cell other genetic events
unmutated IgV
HRS cell
Fas-mediated
apoptosis
Fig. 1 HRS cells may originate from pre-apoptotic germinal centre B cells. When naive B cells are
activated as a result of binding to their cognate antigen via the B-cell receptor (BCR), they migrate
into B-cell follicles, proliferate rapidly and establish germinal centres. This B-cell proliferation is
accompanied by somatic hypermutation of the immunoglobulin variable (IgV) region genes, thus
generating BCR variants. Only B cells carrying a BCR with an increased affinity for antigen will
survive and leave the germinal centre as either class-switched memory B cells or plasma cells. In
contrast, B cells with unfavourable BCR mutations, such as reduced antigen affinity, premature stop
codons or frameshifts, should be eliminated by Fas-mediated apoptosis. However, these pre-apop-
totic germinal centre B cells may be rescued by a combination of EBV and cellular genetic altera-
tions, generating a pool of cells which are thought to be the progenitors of HRS cells
For example, immunoglobulin gene expression can be downregulated by the

epigenetic silencing of critical transcription factors such as BOB-1/POU2AF1,
OCT2/POU2F2 and the Spi-1/PU.1 proto-oncogene (Re et al. 2001; Jundt et al.
2002a; Torlakovic et al. 2001; Stein et al. 2001; Hertel et al. 2002).
Immunoglobulin transcription can also be suppressed by epigenetic modifications
of the immunoglobulin promoter region (Doerr et al. 2005; Ushmorov et al. 2004),
while in a minority of cases, mutations in the octamer region of the immunoglob-
ulin promoter disrupt transcription factor binding (Theil et al. 2001). In addition,
many downstream components of the BCR signalling pathway are either absent or
expressed at low levels in HRS cells (Schwering et al. 2003).
3 Suppression of the B-Cell Phenotype in HRS Cells
HRS cells have a highly unusual phenotype characterised by a striking downreg-

ulation of global B-cell lineage gene expression and the aberrant co-expression
of markers of other haematopoietic cell types including T cells, NK-cells and
myeloid cells (Hertel et al. 2002; Ushmorov et al. 2004; Schwering et al. 2003;
Kuppers et al. 2003; Tiacci et al. 2012; Steidl et al. 2012). This loss of B-cell
identity is linked to the disruption of a number of transcription factor networks
involving PAX5, early B-cell factor 1 (EBF1) and TCF3/E2A that regulate B-cell
development (Nutt and Kee 2007; Bohle et al. 2013). The downregulation of the
B-cell commitment factor EBF1 is a critical event, since EBF1 cooperates with
TCF3 and PAX5 to orchestrate the activation of numerous B-cell genes. Loss of
EBF1 also contributes to the aberrant expression of the NK-cell-associated tran-
scription factor, ID2, which in turn further represses the B-cell gene expression
programme by binding and inhibiting TCF3 (Kuppers et al. 2003; Renne et al.
2006). ID2 overexpression may also result from amplification of the ID2 locus,
which has been reported in approximately one-half of HL patients (Renne et al.
2006). In addition, HRS cells express the helix-loop-helix protein ABF1 which
further inhibits TCF3 activity (Mathas et al. 2006). The importance of silencing
TCF3 activity is underlined by a recent meta-analysis of genome-wide associa-
tion studies that identified a TCF3 polymorphism which is protective against HL
(Cozen et al. 2014). This polymorphism might increase the expression of TCF3,
thereby preventing loss of the B-cell phenotype in HRS cell progenitors and
reducing the risk of developing HL. In this respect, it is notable that the HL cell
line, SUPHD1, contains the N551K substitution previously described to alter the
DNA binding specificity of TCF3 in Burkitt lymphoma cells (Schmitz et al. 2012).
This raises the possibility that certain mutations may redirect TCF3 to a different
set of target genes in HRS cells.
Activation of the Notch pathway also leads to remodelling of the B-cell-specific
transcription factor network and to the aberrant expression of T-cell genes in HRS
cells (Jundt et al. 2002, 2008). Notch suppresses the B-cell programme by induc-
ing the degradation of TCF3, while increasing expression of the TCF3 inhibitor
ABF1 (Mathas et al. 2006; Jundt et al. 2008; Nie et al. 2003; Smith et al. 2005).
Notch may also bind and negatively regulate the master B-cell regulator, PAX5,
expression of which is usually retained in HL (Jundt et al. 2008). Several mecha-
nisms contribute to Notch signalling in HRS cells. Notch activity is triggered by
the Notch ligand, Jagged1, which is expressed both by HRS cells and by cells
present in the tumour infiltrate (Jundt et al. 2008). HRS cells are also character-
ised by lack of expression of deltex1, the major Notch1 inhibitor, as well as high-
level expression of the Notch co-activator, mastermind-like-2 (Jundt et al. 2008;
Kochert et al. 2011). In addition, EBV-encoded LMP2A can induce Notch expres-
sion in human B-cell lines, suggesting that EBV infection may also contribute to
increased Notch activity in HL (Portis et al. 2003; Portis and Longnecker 2004).
4 Deregulated Cellular Signalling in Classical HL
A critical step in the pathogenesis of HL is the ability of HRS progenitor cells

to evade apoptosis that would be the normal fate of germinal centre B cells lack-
ing BCR expression. In this regard, HRS cells show deregulation of multiple cell
signalling pathways that contribute to HRS survival and proliferation (Fig. 2).
CD30 PDGFRA
CD40 BCMA Cytokine TRKA/B
RANKL LMP1 TACI receptor CCR5
TNFAIP3
NEMO NIK JAK MEK

IKKα IKKβ
Non-canonical SOCS1
Canonical NF B
PTPN1
NF B pathway pathway
I Bα I Bε IKK α STAT MAPK

NF- B
NF- B
Cytoplasm
Nucleus
NF- B STAT TFs
BCL3 IRF5
Fig. 2 Deregulated cell signalling pathways in HRS cells. Simplified schematic illustrating the
major signalling pathways deregulated in HRS cells. HRS cells constitutively activate both the
canonical and non-canonical NF-κB pathways. Multiple receptors including CD30, CD40 and
RANKL constitutively activate the canonical pathway through the IKK complex (composed of
IKKα, IKKβ and NEMO), leading to phosphorylation and degradation of the NF-κB inhibitors
IκBα and IκBε. Consequently, active NF-κB complexes translocate to the nucleus where they
activate target genes in cooperation with BCL3 and IRF5. In some cases, NF-κB activity can
be induced by overexpression of the c-Rel subunit. Note this pathway is negatively regulated by
TNFAIP3, which prevents activation of the IKK complex. In the non-canonical pathway, recep-
tor stimulation leads to activation of NIK (MAPK3K14) and IKKα, enhancing the processing
of NF-κB precursors which subsequently translocate to the nucleus. In EBV-positive cases of
HL, LMP1 can activate both the canonical and non-canonical NF-κB pathways. Stimulation of
cytokine receptors by various cytokines secreted by HRS cells and by other cells in the micro-
environment results in activation of the JAK/STAT signalling pathway, which can be further
augmented by mutation of the suppressor of cytokine signalling 1 (SOCS1) and the protein tyrosine
phosphatase PTPN1/PTP1B. Various receptor tyrosine kinases, including PDGFRA, TRKA,
TRKB and CCR5, are also aberrantly activated in HRS cells leading to transcription factor (TF)
activation. Activating mutations which lead to increased cell signalling in HRS cells are indicated
by green stars, while loss-of-function mutations are indicated by red circles
A key pathogenic feature of HRS cells is the constitutive activation of a fam-

ily of transcription factors referred to as nuclear factor kappa B (NF-κB) (Bargou
et al. 1996). NF-κB signalling contributes essential functions in HL, since
inhibition of this pathway in HL cell lines leads to increased sensitivity to apopto-

sis after growth factor withdrawal and impaired tumorigenicity in severe combined
immunodeficiency mice (Izban et al. 2001; Bargou et al. 1997). Several mecha-
nisms contribute to the activation of both the canonical and non-canonical NF-κB
pathways in HL. HRS cells abundantly express multiple TNF receptors, including
CD30, CD40, TACI, BCMA and RANK which trigger NF-κB activation (Carbone
et al. 1995a; Fiumara et al. 2001; Horie et al. 2002; Chiu et al. 2007). Importantly,
the cellular infiltrate surrounding the HRS cells includes eosinophils, neutrophils
and CD4 T cells which activate these receptors in a paracrine manner (Carbone
et al. 1995; Pinto et al. 1997). A recent genomic analysis of transcription factor
binding sites in HL cell lines also revealed that IRF5, aberrantly activated in HRS
cells, cooperates with NF-κB to fully activate the HRS cell phenotype (Kreher
et al. 2014). In addition, there may be cross talk between the Notch and NF-κB
pathways which further induces NF-κB activity in HL (Schwarzer et al. 2012).
NF-κB activation also results from a wide variety of genetic lesions in HRS
cells. The c-REL subunit of NF-κB is amplified in HL cell lines and in around
one-half of all cases of classical HL (Barth et al. 2003; Joos et al. 2003; Martin-
Subero et al. 2002). In around 20 % of HL cases, NF-κB activity is upregulated
by mutation of the IκB inhibitor proteins, IκB alpha and IκB epsilon, which nor-
mally sequester NF-κB as an inactive complex in the cytoplasm (Cabannes et al.
1999; Emmerich et al. 1999, 2003; Jungnickel et al. 2000; Lake et al. 2009),
while rare HL cases overexpress the NF-κB transcriptional co-activator BCL3
(Martin-Subero et al. 2006; Mathas et al. 2005). In addition, there are recurrent
mutations that affect TNFAIP3/A20, a ubiquitin-modifying enzyme that negatively
regulates NF-κB signalling (Schmitz et al. 2009; Reichel et al. 2015). The non-
canonical NF-κB pathway also contributes to the survival of HL cell lines through
NIK/MAP3K14-mediated activation of RelB (Ranuncolo et al. 2012), and chro-
mosomal gains of NIK have been reported in around 30 % of primary HL cases
(Otto et al. 2012).
JAK/STAT signalling, a central pathway involved in mediating the effects of
cytokines, is also strongly implicated in the proliferation and survival of HRS
cells. HRS cells abundantly produce multiple cytokines, including IL-7, IL-9 and
IL-13 (Cattaruzza et al. 2009; Skinnider et al. 2001; Kapp et al. 1999), which
induce JAK/STAT activation leading to elevated levels of phosphorylated STAT3,
STAT4 and STAT6 (Hinz et al. 2002; Skinnider et al. 2002; Kube et al. 2001). In
addition, HRS cells also respond in a paracrine manner to cytokines secreted by a
number of different cell types in the tumour microenvironment, including IL3 pro-
duced by infiltrating T cells (Aldinucci et al. 2002). JAK/STAT signalling can be
further dysregulated by genetic events including amplification of JAK2 and loss-
of-function mutations of the negative regulators SOCS1 and PTPN1/PTPB1 (Joos
et al. 2000; Weniger et al. 2006; Gunawardana et al. 2014). Numerous receptor
tyrosine kinases (RTKs), including PDGFRA, TRKA, TRKB and TIE1, are also
aberrantly activated in HRS cells, although co-expression of several RTKs appears
to be largely restricted to EBV-negative cases of HL (Renne et al. 2005, 2007).
While the above findings provide important clues to the genetic events under-
pinning the pathogenesis of HL, genomic studies will undoubtedly increase our
knowledge of the broader mutational landscape in HRS cells. In this regard,
a recent exome analysis of seven HL cell lines identified 463 genes that were
mutated in at least two of the cell lines (Liu et al. 2014a). While a number of these
mutated genes had previously been described in the context of other B-cell lym-
phomas, the majority of mutations were unique, suggesting that the genetic events
during the malignant transformation in HL are distinct from those in non-Hodgkin
lymphomas. A second study, describing the exome analysis of flow-sorted primary
HRS cells, recently identified novel alterations affecting genes involved in antigen
presentation, transcription regulation and genome integrity, in addition to well-
known HL-associated genes such as TNFAIP3 and SOCS1 (Reichel et al. 2015).
Interestingly, this study also revealed frequent mutations in beta-2 microglobulin
(B2M) and showed that the absence of B2M was associated with a lower stage of
disease, younger age at diagnosis and better clinical outcome (Reichel et al. 2015).
Beta-2 microglobulin forms part of the MHC class I complex, and loss of MHC
class I expression is a common feature of HL (Oudejans et al. 1996a). Since previ-
ous reports have suggested a link between clinical outcome and the presence of
certain cell types, including regulatory T cells and CD68+ macrophages, in the
tumour infiltrate (Alvaro-Naranjo et al. 2005; Tzankov et al. 2008, 2010; Kamper
et al. 2011), it is possible that the loss of MHC class I may confer a better clini-
cal response due to differences in the interactions between HRS cells and immune
cells in the tumour microenvironment.
5 EBV and Classical HL
The detection of raised antibody titers to EBV antigens present in HL patients

both at the time of, and preceding, diagnosis provided the first evidence that EBV
might be involved in the pathogenesis of HL (Levine et al. 1971; Mueller et al.
1989). The detection by in situ hybridisation of EBV DNA and expression of the
abundant EBV-encoded RNAs (EBERs) within HRS cells provided strong evi-
dence in support of an aetiological role for EBV in this disease (Weiss et al. 1989;
Wu et al. 1990). The detection of clonal EBV DNA in 20–25 % of HL tissues sug-
gests that EBV infection of the HRS tumour progenitor occurred prior to its clonal
expansion (Anagnostopoulos et al. 1989).
The fraction of classical HL which harbours EBV genomes varies dramatically
with age, gender, histological subtype, ethnicity and country of residence (Glaser
et al. 1997). Thus, EBV rates in HL from North America and Europe vary between
20 and 50 %, but much higher rates are observed in underdeveloped countries
(Chang et al. 1993; Weinreb et al. 1996). EBV-positive rates are also higher: in
males compared with females; in Asians and Hispanics compared with whites or
blacks (Glaser et al. 1997); and in the UK in South Asian children compared with
non-South Asian children (Flavell et al. 2001). In developed countries, the propor-
tion of EBV-positive cases is higher in older people and in children, especially
in those under 10 years of age, whereas the lowest rates of EBV-positive disease
are found in young adults (Glaser et al. 1997; Jarrett et al. 1991; Armstrong et al.
1998). These differences have led to the hypothesis that there are three forms of
classical HL: paediatric HL (EBV-positive, mixed cellularity type), HL of young
adults (EBV-negative, nodular sclerosis type) and HL of older adults (EBV-
positive, mixed cellularity type) (Armstrong et al. 1998). EBV-positive classical
HL in older adults has been attributed to an age-related decline in EBV-specific
immunity (Jarrett et al. 1991; Armstrong et al. 1998). Senescence of EBV immu-
nity is also suspected in a related tumour, known as EBV-positive diffuse large
B-cell lymphoma (DLBCL) of the elderly (Oyama et al. 2003, 2007).
The incidence of classical HL is only modestly increased during human immu-
nodeficiency virus (HIV) infection, and most cases are EBV-positive and of
mixed cellularity type (Glaser et al. 2003; Uccini et al. 1990; Biggar et al. 2006).
Classical HL occurs more commonly in HIV patients with intermediate lev-
els of immune impairment (Biggar et al. 2006). This is in contrast to the peak in
Burkitt lymphoma incidence which occurs early in HIV infection when circulat-
ing CD4+ T cell numbers are normal or only slightly decreased, and the peak in
the incidence of the ‘immunoblastic’ form of DLBCL which occurs when circulat-
ing CD4+ T cells are very low and the patient is severely immunocompromised.
Furthermore, the incidence of classical HL in HIV-positive patients has not fallen
in the post-highly active anti-retroviral therapy (HAART) era; indeed, some stud-
ies suggest HL risk may be increased in the first few months following immune
reconstitution on HAART (Kowalkowski et al. 2013; Gotti et al. 2013; Bohlius
et al. 2011). These findings not only provide further evidence that defects in EBV-
specific immunity are important for the development of EBV-positive HL, but also
suggest tumour-promoting functions for CD4+ T cells in the microenvironment.
First-degree relatives of patients with classical HL have a threefold to ninefold
increased risk (Crump et al. 2012a, b), and monozygotic twins a 100-fold increase
compared with dizygotic twins (Mack et al. 1995). Genetic association studies
based on segregation and linkage analysis in families have identified susceptibility
loci in the human leucocyte antigen (HLA) region (Kushekhar et al. 2014). Thus,
an increased and decreased risk of EBV-positive HL is associated with HLA-
A*01 and HLA-A*02 alleles, respectively (Diepstra et al. 2005; Niens et al. 2007;
Hjalgrim et al. 2010). Both self-reported and laboratory-confirmed prior infec-
tious mononucleosis (IM) is also associated with an increased risk of developing
EBV-positive; an association not observed in EBV-negative HL (Hjalgrim et al.
2000, 2007). These data have led to an immunological model for the development
of EBV-positive HL in which the levels of circulating EBV-infected lymphocytes
regulated by cytotoxic T-cell responses are a critical determinant of disease risk
(Farrell and Jarrett 2011).
6 Contribution of EBV to the Pathogenesis of Classical HL
EBV is a potentially oncogenic virus which can induce resting B cells into immor-
talised lymphoblastoid cell lines. This growth transformation process is driven by
the cooperative action of a small number of viral gene products: six EBV nuclear
antigens (EBNA1, -2, -3A, -3B, -3C and –LP), three latent membrane proteins
(LMP1, LMP2A and LMP2B), two highly expressed non-polyadenylated RNAs
(EBER1, EBER2) and around 40 miRNAs (Longnecker et al. 2013). In contrast,
virus gene expression in EBV-infected HRS cells is limited to EBNA1, LMP1
and LMP2A/B, together with the non-coding EBERs and miRNAs (Deacon et al.
1993; Young et al. 1991; Niedobitek et al. 1997; Murray et al. 1992; Grasser et al.
1994). In the absence of pro-survival signals mediated by a functional BCR, these
EBV latent gene products contribute to the survival of HRS progenitors through
multiple routes, most of which converge on a limited number of cell signalling
pathways. Importantly, LMP1 and LMP2 mimic the physiological functions of
CD40 and BCR, respectively, which are normally required for the survival and
proliferation of germinal centre B cells.
LMP1 is a member of the TNF receptor superfamily and functions as a consti-
tutively active homologue of the cellular CD40 receptor (Lam and Sugden 2003).
LMP1 expression is especially critical in the pathogenesis of HL since it activates
multiple cell signalling pathway which could potentially rescue HRS progenitors
from apoptosis (Gires et al. 1999; Huen et al. 1995; Kieser et al. 1997). These
include the NF-κB, JAK/STAT and phospatidylinositol-3-kinase (PI3K)/AKT
pathways which are known to be constitutively expressed in HRS cells (Bargou
et al. 1997; Kube et al. 2001; Dutton et al. 2005). LMP1 induces the overexpres-
sion of FLICE-inhibitory protein (c-FLIP), a negative regulator of Fas signal-
ling, thus providing further protection from apoptosis (Dutton et al. 2004). The
importance of LMP1 in HL pathogenesis is further emphasised by the finding
that LMP1 expression in primary germinal centre cells, the presumed progenitor
of HL, induces many gene expression changes characteristic of HRS cells; these
include downregulation of B-cell transcription factors and BCR signalling compo-
nents required to maintain B-cell identity and upregulation of survival genes such
as BCL2, BFL-1 and c-FLIP that protect B cells from apoptosis (Vockerodt et al.
2008; Henderson et al. 1991; D’Souza et al. 2004). Some of these transcriptional
changes may be mediated through the induction of ID2 (Vockerodt et al. 2008),
while others may be result from epigenetic mechanisms involving polycomb pro-
teins, DNA methyltransferases and protein arginine methyltransferases (Anderton
et al. 2011; Dutton et al. 2007; Leonard et al. 2011, 2012).
Unlike LMP1, LMP2A is not a classical transforming gene but engages a num-
ber of signalling pathways important for B-cell survival, including activation of
RAS/PI3K/AKT (Dutton et al. 2005; Swart et al. 2000; Fukuda and Longnecker
2004). LMP2 exists as two isoforms, LMP2A and LMP2B, which share 8 com-
mon coding exons but have different 5′ exons. While the 5′ exon of LMP2B is
non-coding, the unique N-terminus of LMP2A includes an ITAM motif, which
resembles the signalling domain of the BCR (Longnecker and Kieff 1990). In
transgenic mice experiments, LMP2A functions as a BCR surrogate, allowing B
cells to develop in the absence of normal BCR signalling (Caldwell et al. 1998;
Merchant et al. 2000). Gene expression profiling experiments have demonstrated
that LMP2A interferes with the expression of numerous B-cell transcription fac-
tors, including EBF1 and E2A, and mimics many of the gene expression changes
seen in HRS cells (Portis et al. 2003; Portis and Longnecker 2004; Vockerodt et al.
2013). LMP2A also activates the Notch pathway, and recent evidence suggests
that these two factors may cooperate to further reinforce the loss of B-cell identity
in HRS cells (Anderson and Longnecker 2008). Paradoxically, however, many of
the adaptor molecules necessary for both BCR and LMP2A signalling are absent
in HRS cells, and therefore, it remains unclear whether LMP2A acts as a BCR sur-
rogate in HL. One possible explanation is that LMP2A provides essential survival
signals to the HRS progenitor cells, which retain the downstream BCR signalling
molecules, but subsequent transformation events followed by downregulation of
B-cell-specific genes replace some of these critical LMP2A functions. However,
given that EBV-positive cases of HL are consistently LMP2A positive, it is likely
that LMP2A also has BCR-independent functions that are important for mainte-
nance of the HRS phenotype.
Other viral genes expressed in HL include EBNA1, EBERs and the BART
miRNAs. Little is known about the role of EBERs and viral miRNAs in the con-
text of HL, although it is interesting to speculate that export of these non-coding
RNAs from HRS cells via exosomes may affect gene expression in cells of the
surrounding microenvironment (Pegtel et al. 2010). By contrast, EBNA1 poten-
tially contributes several functions in HRS cells. EBNA1 is absolutely required for
EBV infection as it acts as a viral replication factor and tethers the viral genome
to the chromosomes of daughter cells during cell division (Smith and Sugden
2013). In addition to its genome maintenance function, EBNA1 is also a transcrip-
tion factor that regulates both viral and cellular gene expression (Altmann et al.
2006; Frappier 2012). EBNA1 inhibits TGFβ signalling, in part through increas-
ing the turnover of SMAD2, and promotes the growth and survival of HL cells
by downregulating the TGFβ target gene PTPRK (Flavell et al. 2008). EBNA1
also enhances expression of the T-cell chemokine CCL20 which may dampen
the immune response to HRS cells by recruiting regulatory T cells to the tumour
microenvironment (Baumforth et al. 2008). In the context of another B-cell
tumour, Burkitt lymphoma, EBNA1 has also been shown to contribute anti-apop-
totic functions through destabilisation of p53 and induction of the anti-apoptotic
gene, survivin (Holowaty et al. 2003; Lu et al. 2011).
While EBV only appears to have subtle effects on the overall transcriptional
programme in the final HRS clone (Tiacci et al. 2012), it is clear that EBV can
potentially make a major contribution to the pathogenesis of HL. Furthermore,
there is increasing evidence that EBV-positive and EBV-negative cases of HL arise
through different pathogenetic routes and that the role of EBV is to functionally
substitute for cellular genetic changes. First, almost all cases of classical HL bear-
ing destructive immunoglobulin gene mutations are EBV-positive (Brauninger
et al. 2006), suggesting that progenitor cells carrying such mutations can only
survive and undergo malignant transformation if infected with EBV. This is sup-
ported by the fact that EBV immortalises BCR-negative B cells in vitro, demon-
strating that EBV has the potential to rescue pre-apoptotic germinal centre cells
(Chaganti et al. 2005; Mancao et al. 2005; Bechtel et al. 2005). Second, TNFAIP3
mutations are more common in EBV-negative cases of the disease, suggesting that
inactivation of TNFAIP3 and EBV infection may represent alternative pathways to
deregulate NF-κB signalling during HL development (Schmitz et al. 2009). Third,
the aberrant co-expression of several RTKs is largely restricted to EBV-negative
HL cases, arguing that the functions of these RTKs can be replaced by LMP1 and
LMP2 signalling (Renne et al. 2007).
7 Loss of BCR Functions as a Potential Pathogenic

Event in EBV-Positive HL
As we have seen, EBV can provide the necessary anti-apoptotic functions required
for the survival of BCR-negative HRS cell progenitors. However, it is not clear
why the loss of a functional BCR should be important for the pathogenesis of
EBV-associated classical HL. To attempt to answer this question, we need to
consider another important aspect of EBV biology, namely regulation of the
virus replication cycle. Productive EBV infection is associated with the temporal
expression of immediate early, early and late viral genes which ultimately leads
to the production of new virus particles. This switch from latency to virus replica-
tion can be triggered by two different mechanisms: plasma cell differentiation or
activation of BCR signalling. Since productive infection leads eventually to cell
death, it is assumed that suppression of EBV lytic replication is an important event
in HL pathogenesis. To this end, both mechanisms appear to be disrupted in EBV-
infected HRS cells.
Plasma cell differentiation is dependent on a series of transcription fac-
tors which coordinately silence genes associated with the germinal centre B-cell
programme while activating genes required for terminal B-cell differentia-
tion (Lin et al. 2003). One of these factors is BLIMP1, which has been reported
to activate the EBV immediate early genes BZLF1 and BRLF1, thus provid-
ing a mechanistic link between plasma cell differentiation and EBV reactivation
(Reusch et al. 2015). LMP1 suppresses plasma cell differentiation by abrogating
BLIMP1α expression (Vrzalikova et al. 2011, 2012a) and inducing the expres-
sion of BLIMP1β, a BLIMP1 isoform which antagonises the activity of BLIMP1α
(Vrzalikova et al. 2012b). Thus, it is likely that disruption of BLIMP1α function is
an essential step in the pathogenesis of EBV-positive germinal centre-derived lym-
phomas, preventing not only the terminal differentiation of the tumour cells but
also inhibiting virus replication.
EBV replication can also be induced following BCR activation, and there-
fore, loss of a functional BCR is likely to be an important event in HL develop-
ment because it would prevent entry into the replicative cycle and the ensuing cell
death. As described above, EBV-positive HRS cells frequently have non-functional
immunoglobulin genes and lack expression of critical downstream molecules of
the BCR signalling pathway. Importantly, while LMP2A can still induce lytic
cycle entry in the absence of a functional BCR, it cannot do so when the essential
downstream BCR components are missing (Vockerodt et al. 2013). Thus, the loss
of BCR, as well as of BCR signalling components, could combine to prevent both
BCR- and LMP2A-induced virus replications.
8 EBV and the HL Microenvironment
The HRS cells of classical HL are surrounded by a non-neoplastic cell infiltrate

composed of T cells, B cells, macrophages, mast cells, eosinophils and fibroblasts
which support the proliferation and survival of HRS cells (Aldinucci et al. 2010;
Liu et al. 2014b). There is now increasing evidence that HRS cells actively shape
this reactive microenvironment by secreting multiple chemokines and cytokines
which aid in the recruitment of certain cell types. For example, Th2 and FoxP3+
regulatory T cells are attracted by CCL5 (RANTES), CCL17 (TARC), CCL20
and CCL22 (Skinnider and Mak 2002; Aldinucci et al. 2008; Fischer et al. 2003;
van den Berg et al. 1999), eosinophils are attracted by IL5, CCL5 and CCL28
(Aldinucci et al. 2008; Hanamoto et al. 2004), and neutrophils are attracted by IL8
(Foss et al. 1996). In addition, activated fibroblasts in the tumour infiltrate pro-
duce CCL11 (eotaxin) and CCL5, which further contribute to the attraction of
eosinophils, mast cells and regulatory T cells (Buri et al. 2001; Jundt et al. 1999).
Multiple cytokines, including IL3, IL5, IL6, IL8, IL9 and IL13, are also abun-
dantly expressed in HL tissues, either by the HRS cells themselves or by the cel-
lular infiltrate (Aldinucci et al. 2010). In EBV-positive cases, EBV-encoded LMP1
may further alter this microenvironment (Fig. 3). Thus, LMP1 signalling through
the p38/MAPK and PI3K pathways increases the expression of IL6, IL8 and IL10
(Vockerodt et al. 2008; Lambert and Martinez 2007; Eliopoulos et al. 1999) which
can exert both autocrine and paracrine effects, while LMP1 also stimulates the
production of immune modulators such as CCL5, CCL17 and CCL22 (Vockerodt
et al. 2008; Nakayama et al. 2004).
In addition to supporting the growth and survival of the HRS cells (Liu et al.
2014b), the tumour infiltrate also plays an important role in suppressing the host
immune response. In this regard, CCR4+ regulatory T cells, attracted by high
levels of CCL17, CCL20 and CCL22, inhibit the activity of infiltrating effector
CD4+ T cells (Ishida et al. 2006). T-cell effector functions are further blocked by
engagement between the PD-1 receptor and the programmed cell death-ligand 1
(PD-L1) expressed on HRS cells, resulting in functional exhaustion of the T cells
(Muenst et al. 2009; Yamamoto et al. 2008). Notably, amplification of the PD-L1
Inhibition
CD8+ T cells
Treg CTL Paracrine signals
IL13
Collagen IL 21 Jagged1
Cytokine
DDR1 PD-1L receptors Notch
CCL5 TGFβ IL6

CCL17 Galectin-1 IL8
DDR1 CCL22 PD-1L IL10
ZEB2
LMP1 STAT BZLF1
EBNA2 independent Inhibition

expression lytic cycle
Fig. 3 Interactions between EBV and cellular environment in HL. LMP1 contributes to the
highly suppressive immune environment of HL through several different mechanisms. LMP1-
mediated NF-κB signalling increases the expression of numerous chemokines, including CCL5,
CCL17 and CCL22, which dampen CD8+ T cell activity by recruiting regulatory T cells (Treg).
LMP1 also upregulates surface expression of PD-1L, which induces T cell anergy through inter-
action with the PD-1 receptor, and increases the secretion of immunosuppressive regulators
such as IL10, galectin-1 and TGFβ. LMP1 signalling through the MAPK and PI3K pathways
enhances the production of several cytokines including IL6, IL8 and IL10, leading to auto-
crine activation of JAK/STAT signalling in HRS cells. Importantly, IL10, IL13 and IL21 acti-
vate STAT6 which stimulates LMP1 transcription from an alternative promoter independent of
EBNA2 expression. LMP1 also upregulates expression of the receptor tyrosine kinase and dis-
coidin domain receptor 1 (DDR1) which in the presence of its ligand, collagen, may promote
the survival of HRS cells through activation of NF-κB and the PI3K pathways. Notch signalling
extinguishes the expression of many B-cell-specific genes and also prevents entry into lytic cycle
by ZEB2-mediated repression of the viral immediate early gene BZLF1
and PD-L2 genes at 9p24 is common in nodular sclerosis HL (Green et al. 2010),
while the PD-L1 gene can also be deregulated as a result of a reciprocal translo-
cation involving CIITA in a proportion of classical HL cases (Steidl et al. 2011).
In the context of EBV-positive HL, EBV upregulates the expression of numer-
ous immunosuppressive factors, including IL10, galectin-1 and TGFβ, all of
which can inhibit cytotoxic T-cell responses (Marshall et al. 2007; Juszczynski
et al. 2007; Gandhi et al. 2007), while LMP1 has also been reported to induce
PD-L1 expression (Green et al. 2012). These immune evasion mechanisms may
be particularly important in EBV-positive HL, which express virus antigens. The
microenvironment of HL is an emerging therapeutic target as exemplified by a
recent study in which the vast majority of patients with relapsed or refractory HL
responded to PD-1 blockade therapy (Ansell et al. 2015).
Loss of HLA expression is another important mechanism that enables the
tumour cells to avoid the host immune response. Immune evasion is particu-
larly important in the context of EBV-positive HL, since virus-encoded LMP1
and LMP2A proteins are targets for CD8+ cytotoxic T lymphocytes (CTLs)
(Rickinson and Moss 1997). In vitro, HL cells can process and present epitopes
from LMP1 and LMP2A in the context of multiple HLA class I alleles and are
sensitive to lysis by EBV-specific CTLs (Khanna et al. 1998; Lee et al. 1993).
Loss of HLA class I expression is frequently observed in classical HL, and a
recent study showed that most cases of nodular sclerosis HL carry inactivating
mutations in the B2M (Reichel et al. 2015). Notably, however, HLA loss is more
common in EBV-negative cases of classical HL, while EBV-positive cases express
normal or even elevated levels of HLA and contain more activated CTLs than
EBV-negative cases (Oudejans et al. 1996b, 1997; Lee et al. 1998). This suggests
that EBV-positive cases of HL must exploit additional mechanisms to avoid recog-
nition by CTLs.
Recent evidence also suggests that the microenvironment of classical HL plays
a role in modulating virus gene expression in HRS cells (Fig. 3). For example,
LMP1 expression in HRS cells is driven from an alternative STAT-responsive
promoter which is stimulated by IL-4, IL-10, IL-13 and IL-21 (Kis et al. 2006,
2010, 2011). Importantly, this provides a mechanism for LMP1 expression in the
absence of EBNA2, which is a hallmark of the typical type II latency pattern seen
in HRS cells. Recent data also suggest that Notch can inhibit EBV entry into the
lytic cycle in a non-Hodgkin B-cell lymphoma cell line by upregulating the cel-
lular transcription factor Zeb2, which subsequently represses the transcription of
the lytic switch regulator BZLF1 (Rowe et al. 2014). There is also emerging evi-
dence that the microenvironment can modulate EBV gene functions. Thus, LMP1
induces expression of a receptor tyrosine kinase known as discoidin domain recep-
tor 1 (DDR1) (Cader et al. 2013), but this receptor is only active in the presence
of its ligand, collagen, which is a major constituent of the HL microenvironment.
Notably, ligation of DDR1 by collagen promotes the survival of lymphoma cells in
vitro (Cader et al. 2013). These observations suggest that some of the oncogenic
effects LMP1 may be dependent on cues from the tumour microenvironment.
9 Conclusions
The study of Hodgkin lymphoma has been a challenge, not least because of the
low abundance of the malignant cells in the tumour; this is one of the reasons why
the genetic investigation of this tumour has lagged behind that of many other solid
cancers. Furthermore, although the association with EBV has been known for many
years, we still do not fully understand how the virus contributes to the development
of this tumour. Although there is already some evidence that the EBV-positive and
EBV-negative forms are genetically distinct, future studies attempting to unravel these
differences will have to take into account the complex epidemiology of Hodgkin lym-
phoma; EBV may have different aetiological roles in different patient groups, for
example, at different ages. Other important questions remain. For example, we still
do not fully understand why the loss of B-cell identity, including the loss of B-cell
receptor signalling, is seemingly so important for Hodgkin lymphoma development,
especially for those cases which are EBV-negative. Further studies are warranted to
identify which are the critical driver mutations in HRS cells and investigate whether
alterations in multiple signalling pathways can cooperate during lymphomagenesis.
As we move into an era in which personalised medicine is becoming increasingly
common, large-scale studies will be required to determine the clinical relevance of
these different genetic alterations and how they impact upon survival and response to
treatment. In addition, the successful application of approaches such as immunother-
apy will require an improved understanding of how the microenvironment contributes
to the immune evasion of HRS cells, especially in the context of EBV.
Acknowledgments We are grateful to Leukaemia Lymphoma Research and to Cancer
Research UK for support.
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The Role of EBV in the Pathogenesis
of Diffuse Large B Cell Lymphoma
Jane A. Healy and Sandeep S. Dave
Abstract Epstein-Barr virus (EBV) infection is a common feature of B cell

lymphoproliferative disorders (LPDs), including diffuse large B cell lymphoma.
Approximately 10 % of DLBCLs are EBV-positive, with the highest incidence in
immunocompromised and elderly patients. Here, we review the clinical, genetic,
and pathologic characteristics of DLBCL and discuss the molecular role of EBV
in lymphoma tumorigenesis. Using EBV-positive DLBCL of the elderly as a
model, we describe the key features of EBV-positive DLBCL. Studies of EBV-
positive DLBCL of the elderly demonstrate that EBV-positive DLBCL has a dis-
tinct biology, related to both viral and host factors. The pathogenic mechanisms
noted in EBV-positive DLBCL of the elderly, including enhanced NFκB activity,
are likely to be a generalizable feature of EBV-positive DLBCL. Therefore, we
review how this information might be used to target the EBV or its host response
for the development of novel treatment strategies.
Contents
1 Introduction........................................................................................................................... 316
2 Classification Challenges of DLBCL.................................................................................... 316
2.1 Disease Etiology.......................................................................................................... 321
2.2 EBV and Lymphoma................................................................................................... 322
3 Clinical and Pathologic Aspects of EBV-Positive DLBCL.................................................. 325
3.1 EBV-Positive DLBCL as a Disease Model.................................................................. 325
3.2 Historical Perspective.................................................................................................. 326
J.A. Healy
Department of Hematology/Oncology, Duke University, DUMC Box 3841,
Durham, NC 27710, USA
e-mail: jane.healy@duke.edu
S.S. Dave (*)
Center for Genomic and Computational Biology, Duke University,
CIEMAS, 2177C, 101 Science Drive, Box 3382, Durham, NC 27708, USA
e-mail: sandeep.dave@duke.edu

316 J.A. Healy and S.S. Dave
3.3 Epidemiology................................................................................................................ 326
3.4 Clinical Characteristics................................................................................................. 327
3.5 Histopathology.............................................................................................................. 328
3.6 Genetics........................................................................................................................ 328
3.7 Biology.......................................................................................................................... 329
4 Novel Treatment Approaches for EBV-Positive DLBCL....................................................... 330
4.1 Antiviral Therapy.......................................................................................................... 330
4.2 EBV-Targeted Adoptive Immunotherapy...................................................................... 330
4.3 Biology of Targeted Therapies...................................................................................... 331
5 Conclusions............................................................................................................................. 331
References................................................................................................................................... 332
1 Introduction
Epstein-Barr virus (EBV) has been linked to a wide number of human cancers.
Among these neoplasms, B cell lymphoproliferative disorders (LPDs) are the
most frequent and strongly associated. Diffuse large B cell lymphoma is the most
common lymphoid malignancy in adults, accounting for nearly a third of non-
Hodgkin’s lymphoma cases (NHL) globally (Fisher and Fisher 2004; Menon et al.
2012; Jemal et al. 2011).
DLBCL is characterized by rapidly proliferating cells expressing B cell-asso-
ciated antigens CD19, CD20, CD22, and CD79a (Martelli et al. 2013; Swerdlow
et al. 2008). Despite these common features, DLBCL is a heterogeneous disease
from the standpoints of biology and clinical outcome (Dave 2010). Prognosis
reflects this heterogeneity, with long-term survival rates ranging between 30 and
90 %, dependent on clinical stage and disease subtype (Ziepert et al. 2010; Corti
et al. 2010).
Roughly 10 % of DLBCLs are EBV-positive, with a significantly higher inci-
dence in setting of immunocompromised and elderly patients (Heslop 2005).
There are additional biological differences that distinguish EBV-positive DLBCLs
from other DLBCLs, particularly the activation of the NF-kB and JAK/STAT sign-
aling pathways (Montes-Moreno et al. 2012; Kato et al. 2014).
This chapter will review the challenges of classifying DLBCL, particularly
with regard to EBV-positive lymphoid tumors. We will further review the clinical,
genetic, and biological studies that have led to our current understanding of EBV-
positive DLBCL. Finally, implications for these findings on treatment strategies
and patient care will be considered.
2 Classification Challenges of DLBCL
The general organizing principles of DLBCL as it understood today were initially

presented in the REAL classification of 1994, and were subsequently incorporated
into the WHO classification (Menon et al. 2012) which is now the widely accepted
The Role of EBV in the Pathogenesis … 317
standard. While these evolving classifications have provided a clearer categoriza-

tion of these tumors, the diagnosis does not capture all the observed clinicopatho-
logic heterogeneity that is commonly observed in the disease.
Significant effort has been dedicated to the subclassification of DLBCL into
more clearly delineated disease entities (Swerdlow et al. 2008; Balague Ponz et al.
2008). Table 1 depicts the subtypes of DLBCL defined in the 2008 WHO clas-
sification. Details of each category, including cellular immunophenotype, cytoge-
netics, gene expression patterns, and EBV association, are provided. The complex
subdivisions in this scheme highlight diverse characteristics such as anatomic
location (DLBCL leg type), patient age (EBV-positive DLBCL of the elderly), and
presence of viral coinfections (HHV8+ DLBCL arising in the setting of multicen-
tric Castleman’s disease) (Swerdlow et al. 2008).
In addition to immunophenotyping and cytogenetics, microarray technology
has provided new insight into molecular patterns of DLBCL through the examina-
tion of global gene expression alterations occurring in a given tumor. Gene expres-
sion profiling (GEP) of DLBCL tumors demonstrates that these tumors can be
divided into two major subtypes based on their gene signatures (Alizadeh et al.
2000; Rosenwald et al. 2002; Lenz et al. 2008a). These two entities, germinal
center B cell (GCB) and activated B cell (ABC), show distinct clinical behavior
and treatment outcomes. The five-year overall survival of ABC and GCB DLBCL
is 30 and 59 %, respectively (Lenz et al. 2008a). The complexity of processing
and interpreting microarray data has restricted its global applicability and has thus
far prevented GEP classification from being incorporating into the WHO classi-
fication (Balague Ponz et al. 2008). However, immunohistochemistry provides a
useful surrogate to GEP (Hans et al. 2004; Choi et al. 2009). GEP surrogates are
a reproducible, less expensive alternative to microarrays and have been widely
adopted by pathologists. A combination of five immunohistochemical mark-
ers, GCTE1, CD10, BCL-6, IRF4, and FOXP1, predicted clinical outcome and
achieved 90 % concordance with GEP (Choi et al. 2009).
GEP data suggest that most DLBCLs originate either during a B cell’s tran-
sit through the germinal center reaction (GCB type) or in post-germinal center
B cells (ABC type). Next-generation sequencing studies of DLBCL, including
whole exome sequencing, genome copy number analysis, and RNA sequencing
show that, in addition to having unique gene expression signatures, GCB and ABC
tumors have different patterns of genetic mutation that likely reflect their distinct
biology (Zhang et al. 2013; Morin et al. 2013; Pasqualucci et al. 2011). Distinct
features of GCB and ABC DLBCL will be reviewed next.
Germinal centers (GCs) are transient structures that form in secondary lym-
phoid tissue in response to antigenic stimulation. During their passage through
the GC, B cells undergo rapid proliferation, somatic hypermutation (SHM) of the
variable chains of their immunoglobulin genes, class-switch recombination (CSR),
and affinity selection (Victora and Nussenzweig 2012). The GC reaction is the
process by which mature B cells are generated, and is thus absolutely critical to
adaptive immunity. It is also a common site of lymphomagenesis. B cell transfor-
mation can occur during the GC reaction from the acquisition of mutations that
Table 1 DLBCL subtypes from the WHO 2008 classification of lymphomas
318
Subtype Defining features Immunophenotype Cytogenetics GEP subtype % EBV tumor EBV latency References
positivity pattern
DLBCL, NOS – Most common – (+)CD19/20, – 3q27 rear- ABC or GCB 10 % I/II (Martelli et al.
NHL subtype CD22, CD79a, rangement 2013; Swerdlow
– Diffuse immunob- PAX5 (pan B cell (30 %)– t(14:18) et al. 2008; Stein
lasts effacing normal markers) (20 %)– MYC et al. 1984)
tissue architecture rearrangement
– No defining age/ (10 %)
disease location/
virus
T cell/histiocyte- – Scattered immuno- – Neoplastic cells – High burden Intermed. 20 % I/II (Lim et al. 2002;
rich large B cell blasts (<10 % cells) express pan B cell of chromosomal between GCB Franke et al. 2002;
lymphoma with predominant markers alterations and Reed– Pittaluga and Jaffe
T cell/histiocyte – (−) CD30/15 – 3q27 rearrange- Sternberg cells 2010)
background – T cells (+) ment (50 %)
– Splenomegaly BM CD3/4, TIA-1, (−) – X gain
involvement Granzyme B – 17p−
– Can progress to
DLBCL NOS
DLBCL – Arising at sites of – Pan B cell – See DLBCL ABC 100 % III (Loong et al. 2010;
associated chronic inflamma- markers NOS Narimatsu et al.
with chronic tion (e.g., metal- 2007; Nakatsuka
inflammation lic prostheses, et al. 2002)
pyothorax)– “Local
immune deficiency”
due to chronic
inflammation
EBV-positive – Age >50y, immu- – Pan B cell mark- – See DLBCL ABC 100 % II/III (Oyama et al.
DLBCL of the nocompetent, no ers NOS 2003, 2007; Ok
elderly previous lymphoid – CD30+ (50 %) et al. 2013)
malignancy
J.A. Healy and S.S. Dave
(continued)
Table 1 (continued)
Subtype Defining features Immunophenotype Cytogenetics GEP subtype % EBV tumor EBV latency References
positivity pattern
Primary – Commonly young – (+) Pan B cell – 9p24 gain Thymic pattern, 0% N/A (Addis and
mediastinal adults markers common not ABC or Isaacson 1986;
large B cell – Derived from – (+) CD30 GCB Cazals-Hatem
lymphoma medullary thymic – (−) Surface Ig et al. 1996)
B cells
Primary – Aggressive cutane- – (+) Pan B cell – See DLBCL ABC Unknown Unknown (Martelli et al.
cutaneous ous lymphoma markers NOS 2013; Swerdlow
DLBCL, leg – Poor prognosis – (+) BCL2, IRF4 et al. 2008;
type – (−) CD10 Nakatsuka et al.
2002)
The Role of EBV in the Pathogenesis …
Plasmablastic – HIV associated – (−) CD20, PAX5 – MYC rear- ABC 50–70 % II/III (Castillo et al.
lymphoma – Typically arises in – (+) CD79a, rangement com- 2008; Valera et al.
oral cavity CD138 mon (50 %) 2010; Capello
et al. 2010)
Intravascular – Large B cells in – Pan B cell mark- – DLBCL NOS ABC 0% N/A (Murase et al.
large B cell blood vessel lumens ers 2007; Ponzoni
lymphoma – Symptoms due – (−) CD29/54 et al. 2007)
to microvascular
infarcts
− Rapidly fatal
Large B cell – Coincident HHV8 – Pan B cell mark- – DLBCL NOS ABC Rare III (Corti et al. 2010;
lymphoma aris- and (±) and HIV ers Carbone and
ing in HHV8- infections – IRF4/MUM1 Gloghini 2005)
associated – Arises in severely
multicentric immunocompro-
Castleman’s mised
disease
319
either promote sustained proliferation or impair apoptosis. These mutations may

be acquired during repeated cycle cellular replication or may result from off target
effects of activation-induced cytidine deaminase (AID), the enzyme that initiates
the processes of SHM and CSR in GC B cells (Orthwein and Di Noia 2012).
Gene mutations that occur more frequently in GCB compared to ABC-type
DLBCL include C-MYC, EZH2, and GNA13 (Zhang et al. 2013). BCL2 translo-
cations have also been identified in roughly one-quarter of GCB tumors (Schuetz
et al. 2012). BCL2 activation protects cells from programmed cell death (Kroemer
1997). Chromosomal rearrangements involving C-MYC, or gain of function
mutations, promote unregulated cellular proliferation in affected cells (Ott et al.
2013). Gain of function mutations in EZH2, a histone methyltransferase, promote
lymphoma by silencing cell cycle regulation genes and tumor suppressor genes
(Béguelin et al. 2013). Loss of function mutations in GNA13, a G protein involved
in cell–cell adhesion, enhance AKT signaling and cellular motility and are
strongly associated with GCB DLBCL (Morin et al. 2013; Muppidi et al. 2014).
In contrast to GCB DLBCL, ABC tumors have gene expression profiles simi-
lar to those seen in activated B cells (Lenz et al. 2008a). The hallmark of ABC
biology is believed to be chronically active B cell receptor signaling, leading to
upregulated NFκB activity (Davis et al. 2001; Lenz et al. 2008b). Gene expres-
sion profiling in primary tumor samples, as well as ABC lymphoma-derived cell
lines, demonstrates enhanced expression of NFκB target genes (Bea et al. 2005;
Rosenwald and Staudt 2003). Augmented NFκB activity is driven by activating
mutations in signaling proteins downstream of the B cell receptor and/or Toll-like
receptors. Common genetic defects in the ABC subtype of DLBCL include gain of
function mutations in CD79B (B cell receptor), MyD88, MALT1, and Card11, all
of which promote canonical NFκB activity (Zhang et al. 2013; Morin et al. 2013).
A20, a negative NFκB regulator, is subject to inactivating mutations (Kato et al.
2009). These events promote oncogenesis by enhancing cell proliferation and sup-
pressing apoptotic signals. Constitutive NFκB activity may also explain the post-
germinal center phenotype of ABC DLBCL. NFκB promotes enhanced expression
of IRF4 (interferon regulatory factor-4), a transcription factor that drives the plas-
mablastic differentiation (Staudt 2010). Another important aspect of ABC biology
is IL-6 and IL-10 generation. These cytokines exert autocrine effects on the tumor
cells, resulting in the activation of STAT3 (Ding et al. 2008). A subset of DLBCL
tumors have high STAT3 target gene expression and nuclear localization of phos-
phorylated STAT3. These tumors also demonstrate higher expression of NFκB tar-
get genes (Lam et al. 2008).
At a molecular level, constitutive NFκB activity is related to activating muta-
tions in proteins upstream of NFκB. In B cells, CARD11, MALT1, and BCL10
form a signaling complex regulated by activation of the B cell receptor (Schulze-
Luehrmann and Ghosh 2006). Upon antigen stimulation, cytoplasmic CARD11
is phosphorylated by PKCβ (Tan and Parker 2003). CARD11 is then recruited to
the plasma membrane, where it serves as a molecular scaffold for the assembly
of MALT1 and BCL10. This recruits the NFκB IKK complex, eventually leading
to the activation of IKKβ. IKKβ is a kinase that phosphorylates the tonic NFκB
inhibitor IκBα. The inhibitor then dissociates with NFκB subunits p50/p65, leav-
ing them free to dimerize, translocate to the nucleus, and activate target genes
(Staudt 2010). This process is critical for clonally expanding populations of anti-
body-producing B cells in response to an antigenic challenge. ABC tumors co-opt
BCR signaling with activating mutations in CARD11 or CD79A/B (Lenz et al.
2008c; Davis et al. 2010). Unbound to antigenic stimulation, affected B cells are
now capable of sustained NFκB activity. This leads to the enhanced proliferation
and evasion of cell death, both hallmarks of cancer.
2.1 Disease Etiology
DLBCL usually occurs as a de novo malignancy, or less frequently through the

“transformation” of an indolent B cell neoplasm, such as follicular lymphoma or
chronic lymphocytic leukemia (CLL). In latter instance, acquisition of additional
mutations results in transformation to a more aggressive neoplasm, often referred
to as Richter’s transformation (Giles et al. 1998).
A causative agent is not identifiable for most cases of DLBCL, though there
are a number of known risk factors associated with its development, includ-
ing mutagens, toxins, immune dysfunction, and infections (Martelli et al. 2013).
Chemical exposures including alkylating chemotherapeutics, industrial chemicals,
pesticides, and fertilizers have been shown to increase a person’s risk of develop-
ing DLBCL (Fisher and Fisher 2004). This is due to the ability of these agents to
mutagenize DNA. The high proliferation rate of hematopoietic cells renders them
particularly vulnerable to these toxins.
Problems of immune dysregulation are common in patients with DLBCL
(Smedby et al. 2006; Miranda et al. 2013). These include genetic and acquired
causes of immunodeficiency, chronic inflammation, and autoimmune disease. The
cause of this association is likely multifactorial, but T cell suppression resulting in
impaired antitumor immunity is felt to play an important role (Yang et al. 2006).
Immune suppression also permits the reactivation of lymphoma-associated viral
pathogens, particularly EBV (Rickinson 2014).
Viral infections that increase the risk of DLBCL development include EBV,
human immunodeficiency virus (HIV), hepatitis C (HCV), and human herpes-
virus-8(HHV8) (Rickinson 2014). Viral infections can increase the risk of lym-
phoma through diverse mechanisms. They can alter T cell suppressor function,
such as in the case of HIV, which diminishes the body’s immune-mediated anti-
tumor surveillance (Carbone and Gloghini 2005). Viruses can also promote lym-
phoma by driving B cell hyperstimulation, as is the case with EBV infection
(discussed in detail below), HCV, and HHV8 (Rickinson 2014).
Environmental exposures, immune dysfunction, and infections can operate syn-
ergistically to promote mutagenesis, suppress T cell function, and activate B cell
stimulation. The cumulative effects of these processes increase the likelihood of
lymphoma development.
2.2 EBV and Lymphoma
Over 90 % of the world’s population is infected with EBV, though this infec-
tion is asymptomatic in the vast majority of individuals (Niederman et al. 1970).
However, the oncogenic potential of EBV is undeniable, as it has been linked to a
broad range of tumor subtypes, most of which are of B cell origin (De Martel et al.
2012). The addition of EBV to primary B cells grown in culture leads to growth
transformation and the generation of immortalized lymphoblastoid B cell lines
(Nilsson et al. 1971). B cell neoplasms linked to EBV infection include DLBCL,
post-transplant lymphoproliferative disease (PTLD), Burkitt lymphoma (BL),
AIDS-related lymphoma (Primary CNS Lymphoma), plasmablastic lymphoma,
and primary effusion lymphoma. These tumors are characterized by malignant
B cells that frequently express EBV transcripts and proteins indicating EBV
infection.
In PTLD, rates of EBV infection range from 70 to 100 % and are related to the
length and degree of immunosuppression (Juvonen et al. 2003; Taylor et al. 2005).
In the HIV-positive population, 80 % of DLBCL is EBV-positive (Park et al. 2007)
and 100 % of primary CNS lymphomas are EBV-positive (Swerdlow et al. 2008;
Gloghini et al. 2013). Primary effusion and plasmablastic lymphomas, which
mostly occur in HIV-positive patients, are also positive for EBV in most cases
(Hsi et al. 2011; Verma et al. 2005). Immunosuppression is a common pathogenic
cofactor in these B cell neoplasms, and the relevance of this association requires a
discussion of EBV virology.
EBV is a gamma-1-type herpesvirus that first discovered fifty years ago in
Burkitt lymphoma tumors from pediatric patients in equatorial Africa (Epstein
et al. 1964). EBV is similar to other herpesviridae in its capacity to persist in a
latent state in infected cells. It is distinctive from other herpesvirus genera in its
restriction to primate hosts, its tropism for establishing latency in B lymphocytes,
and its ability to promote oncogenic transformation of B lymphocytes through its
latent gene expression repertoire (Rickinson 2014; Nilsson et al. 1971).
EBV infection in humans occurs in three distinct stages: lytic phase, latent
phase, and reactivation. First, EBV enters a host by infecting the polarized res-
piratory epithelial cells of the nasopharynx. This initial infection is followed by
the entry of EBV into the surrounding mucosal lymphoid cells through transcyto-
sis, leading to infection of B cells. This initial lytic phase results in the cell death,
sloughing of the respiratory epithelial cells and release of high titers of virus, a
process that can also occur during viral reactivation (Lemon et al. 1977). During
this phase, EBV binds to CD21 receptors on naive B lymphocytes (Carel et al.
1990). Once EBV infects a B lymphocyte, the expression of viral genes initially
promotes growth transformation in the infected cell. Transformed B cells are then
believed to transit though the germinal center reaction, differentiating into long-
lived quiescent memory B cells (Thorley-Lawson and Gross 2004; Roughan and
Thorley-Lawson 2009). At this stage, the viral genome persists in an episomal
state, expressing only a limited number of viral antigens (Young and Rickinson
2004). This is the latent phase of infection.
The latent infection is often punctuated by brief periods of viral reactivation

caused by perturbations in the EBV-infected memory B cell pool. EBV-positive
memory B cells are thought to function similarly to other memory B cells. Thus,
if a given cell encounters cognate antigen, it will awake from its resting state and
undergo plasma cell differentiation. Plasma cell differentiation is a trigger of
viral reactivation (Laichalk and Thorley-Lawson 2005). Reactivation is believed
to occur as a result of the actions of XBP-1, a B cell transcription factor critical
for plasma cell differentiation. XBP-1 is capable of activating the viral BZLF pro-
moter, which controls the expression of viral lytic genes (Sun and Thorley-Lawson
2007). During this process, infected cells reinitiate the expression of viral antigens
on their cell surface. In immunocompetent individuals, viral reactivation results
in brisk humoral and cell-mediated immune responses (Jones and Straus 1987;
Rickinson and Moss 1997). While antibodies to viral membrane proteins decrease
viral shedding and infectivity, CD4 and CD8+ T cells are primarily responsible for
suppressing lytic and latent EBV infections (Jones and Straus 1987). The immune
system ensures that EBV-positive B cells that reactivate are promptly eliminated,
thus re-establishing the steady state of latent infection. This is the usual cycle of
EBV infection present in a normal, healthy individual.
Immunosuppressive states upset the virus–host balance by weakening the
body’s principle defense against EBV reactivation: cell-mediated immunity. In the
absence of the CD8+ T cell response, EBV-positive B cells are able to proliferate
and express viral antigens. Hence, patients suffering from immune-deficient states
associated with T cell dysfunction are particularly vulnerable to EBV reactivation.
These include HIV infection and post-transplant immunodeficiency, where med-
ication-induced T cell suppression is necessary to prevent graft rejection. Aging
can also result in T cell dysfunction due to reduced numbers of CD4/8+ T cells
and reduction in naïve T cell receptor diversity (Miller 1996). Immune senescence
is defined as age-related immune alterations that result in increased infections,
autoimmunity, cancer, and reduced response to prophylactic vaccines. Consistent
with these observations, the incidence of polyclonal EBV-positive lymphoprolif-
erative disease increases with age, as does the risk of DLBCL133. Figure 1 summa-
rizes the effect of EBV on the pathogenesis of lymphoma formation.
The oncogenic effect of EBV on B cells occurs through the action of a number
of viral microRNAs and the protein LMP-1 (Rickinson 2014). EBV produces 44
viral microRNAs, which are believed to regulate viral and cellular mRNA during
the latent phase of infection (Lopes et al. 2013). These noncoding RNAs promote
cell growth and immune evasion and prevent the transcription of proapoptotic
signaling molecules. LMP-1 is a well-studied viral oncogene that is believed to be
the prime actor in EBV-mediated B cell transformation (Kaye et al. 1993).
LMP-1 is expressed during the latent and lytic phases of infection and is pre-
sent in tumors corresponding to higher degrees of immune compromise (latency
pattern II and III). LMP-1 is an integral membrane protein that behaves as a func-
tional mimic of CD40, a costimulatory receptor required for B cell activation
(Uchida et al. 1999; Eliopoulos et al. 1997; Huen et al. 1995). Under physiologic
conditions, helper T cells recognize antigens presented by B cells. During this
Cell Mediated
Immunity
Immune
Competent
Host
EBV EBV
infection latency EBV
Reactivation
GC reaction Antigen stimulation
naive memory plasma
B cell B cell B cell
Cell Proliferation
Apoptosis
Immune
Mutations Compromised
Lymphoma Lymphoproliferative Host
Disease
Fig. 1 The relationship of EBV to the pathogenesis of lymphoma. Naive B lymphocytes are
infected with EBV and subsequently undergo transformation to memory cells via transit through
the GC reaction. EBV establishes a latent infection in memory B cells. Upon memory B cell
reactivation, EBV enters the lytic stage of infection, where it expresses viral antigens on the sur-
face of the infected cell. In immune competent hosts (green), this activates cell-mediated immu-
nity and the lytic phase cells are targeted for destruction. By contrast, immune compromise is
associated with deficiency in cell-mediated immunity. In these individuals, EBV sets up a pro-
gram of proliferation and increased cell survival which promotes polyclonal lymphocyte expan-
sions or lymphoproliferative disease (red). Ongoing rounds of cellular division lead to the acqui-
sition of additional mutations. If a sufficient number of oncogenic driver and tumor suppressor
mutations are achieved, then lymphoma occurs
interaction, the extracellular portion of CD40 binds its ligand on the T cell mem-
brane. This activates CD40, inducing conformational shape changes that promote
receptor oligomerization and nucleation of TRAF signaling proteins to its cyto-
plasmic domain (Tsubata et al. 1993). This leads to activation of canonical NFκB
pathway signaling, cell cycle entry, and protection against apoptosis. Under nor-
mal conditions, these processes lead to adaptive immunity, resulting in the rapid
generation of high affinity antibodies against foreign antigens. The viral LMP-1
protein co-opts this process by behaving as a constitutively active CD40 (Uchida
et al. 1999). In doing so, LMP1 uncouples B cell activation from antigen selection
and activates AICDA (activation-induced cytidine deaminase), and leads to poly-
clonal lymphocytosis and the acquisition of additional mutations that increase the
likelihood of transformation into overt lymphoma.
Dependent on the degree of host immune compromise, transformed B cells
may express all, or just a portion of the EBV latency genes (Tierney et al. 1994).
Latency I pattern corresponds to the expression profile present in a typical active
Table 2 EBV latency patterns and associated malignancies

Latency pattern Associated EBV Tumors
proteins
I LMP2Aa Burkitt lymphoma
EBNA1a DBCL NOS
T cell-rich DLBCL
II #I proteins + Classic Hodgkin’s lymphoma
LMP1 Angioimmunoblastic T cell lymphoma
LMP2B NK/T cell lymphoma, N
Nasopharyngeal carcinoma
Gastric carcinoma
III #I/II proteins + Primary EBV infection
LPa Post-transplant lymphoproliferative disease
EBNA2 AIDS-related lymphomas (plasmablastic DLBCL,
EBNA3A primary CNS lymphoma, primary effusion lym-
EBNA3B phoma)
EBNA3C EBV + DLBCL of the elderly
aEBNA, EBV virus nuclear antigen. EBNAs promote EBV genome maintenance and regulate
gene expression. LMP, latent membrane protein. LMPs interfere with signaling pathways from
various receptors in the B cell membrane to induce cellular proliferation and inhibit programmed
cell death. LP, leader protein. LPs co-opt hormone receptors in the B cell nucleus to promote
growth transformation (Igarashi et al. 2003)
infection, latency II pattern is marked by the presence of a subset of viral anti-

gens, and latency III pattern results in the expression of the entire EBV repertoire
(Young and Murray 2003). There may also be a role for viral genetic variation in
the efficiency of B cell transformation; however, this remains to be fully defined
through a careful study of cases and controls. Latency patterns may be significant
as they may reflect distinct aspects of tumor biology. Table 2 depicts the proteins
associated with each latency pattern and shows the malignancies possessing each
pattern. The EBV latency patterns associated with different DLBCL subsets are
indicated in Table 1.
3 Clinical and Pathologic Aspects of EBV-Positive DLBCL
3.1 EBV-Positive DLBCL as a Disease Model
Much of our current understanding of the clinical impact of EBV in DLBCL

comes from studies of EBV-positive DLBCL of the elderly. This DLBCL classifi-
cation appeared first in the WHO classification of tumors in 2008 as a provisional
entity (Swerdlow et al. 2008). EBV-positive DLBCL is defined as an EBV-positive
monoclonal large B cell lymphoproliferation occurring in an immunocompetent
patient greater than 50 years of age with no history of prior lymphoma. The age
cutoff emphasizes the tendency of these tumors to arise in individuals of advanced
age (Balague Ponz et al. 2008; Cho et al. 2008; Cohen et al. 2014), though
EBV-positive DLBCL has been reported in younger immunocompetent patients,

albeit rarely (Cohen et al. 2014; Ao et al. 2014; Beltran et al. 2011a). It is pres-
ently unclear whether elderly patients can be truly considered immune compe-
tent, and thus distinct from other EBV-positive DLBCL subtypes, or whether the
EBV reactivation in these patients is due to age-related T cell dysfunction (Miller
1996). Regardless, compared to other EBV-positive DLBCL subtypes, EBV-
positive DLBCL of the elderly is not associated with concomitant immunosup-
pression, or use of transplant rejection medications, HIV or HHV8 coinfection,
secondary malignancies, or chronic inflammatory disease. For this reason, EBV-
positive DLBCL of the elderly appears to be a good model for the study of how
EBV affects DLBCL disease course and treatment response and it is instructive to
review this disease entity in some detail.
3.2 Historical Perspective
EBV-positive DLBCL was initially described by Oyama et al. in 2003 as “senile

EBV-positive lymphoproliferative disorder” (Oyama et al. 2003). In that case
series of 22 Japanese patients, the authors described a spectrum of EBV-positive
lymphoproliferative disease (LPD) ranging from polyclonal B cell lymphocytosis
to DLBCL. Compared to EBV-negative LPD, patients with EBV-positive tumors
had a higher rate of extranodal involvement, a more aggressive clinical course, fre-
quent refractory disease or early relapse, and worse overall survival. Since this ini-
tial observation, groups in other countries have confirmed the existence of DLBCL
patients with EBV-positive tumors and without known immunodeficiency (Gibson
and Hsi 2009; Beltran et al. 2011; Hoeller et al. 2010; Hofscheier et al. 2011; Uner
et al. 2011; Al-Humood et al. 2014). Consistent with the initial report, these EBV-
positive immune competent individuals are almost exclusively elderly.
3.3 Epidemiology
Though there have been a number of studies assessing the prevalence rate of EBV
positivity in DLBCL tumors from elderly, immune competent patients, the geo-
graphic prevalence of the disease appears variable. Groups from the USA and
European countries have reported incidences <5 % (Gibson and Hsi 2009; Hoeller
et al. 2010; Hofscheier et al. 2011) and some Asian and Latin American countries
report rates as high as 10–15 % of DLBCLs (Beltran et al. 2011b; Hofscheier et al.
2011), implicating potential genetic polymorphisms, coinfections, or environ-
mental factors in these geographic differences. However, some studies have dem-
onstrated significant variability within the same geographic region (Wada et al.
2011), suggesting that there might be an additional confounding variable in the
lack of standardized criteria for determining EBV positivity in clinical cases.
Laboratories use differing cutoffs for the percentage of antigen expressing

cells necessary for a tumor to be deemed EBV-positive (20 % vs. 50 %) (Wada
et al. 2011). Furthermore, sample processing is not uniform. For example, there
are various methods for separating EBV-positive tumor cells from contaminating
background cells (Ok et al. 2013) and these methods are not always applied con-
sistently. Finally, there are different means of detecting EBV infection (EBERin
situ hybridization or LMP-1 immunohistochemistry) that may affect the assay sen-
sitivity. Universal standardization of EBV testing is needed before definitive con-
clusions about geographic variation of this disease can be made.
3.4 Clinical Characteristics
The median age for EBV-positive DLBCL is 71, with the greatest proportion (20–
25 %) of cases occurring in patients greater than age 90 (Ok et al. 2013; Castillo
et al. 2011). Initial descriptions of EBV-positive DLBCL of the elderly stressed
that patients presented in later stage of the disease, measured by IPI and Ann Arbor
stage, and possessed a high degree of extranodal involvement (Oyama et al. 2003,
2007). Extranodal extension to GI tract, lung, liver, skin, soft tissue and bone were
described. However, later studies in North American patients showed that, simi-
lar to EBV-negative DLBCL, both nodal and extranodal disease sites are common
(Gibson and Hsi 2009; Hoeller et al. 2010; Hofscheier et al. 2011). No distinguish-
ing clinical features have been reliably associated with EBV positivity, except a
trend toward higher Ann Arbor stage at presentation (Gibson and Hsi 2009).
Studies in Asia and Europe demonstrate that patients with EBV-positive
DLBCL of the elderly respond poorly to treatment and have worse overall survival
compared to those with EBV-negative tumors (Park et al. 2007; Hofscheier et al.
2011; Oyama et al. 2007; Chang et al. 2014). Age, by itself, is a risk for poorer
outcomes in DLBCL. However, EBV-positive DLBCL is also associated with an
ABC GEP, which is known to have a worse outcome than GCB tumors (Kato et al.
2014; Ok et al. 2014). Montes-Moreno et al. (2012) explored whether the differ-
ence in survival was merely due to a higher prevalence of ABC phenotype. They
compared elderly patients with either EBV-positive DLBCL or EBV-negative
DLBCL stratified by ABC versus GCB GEP and found that EBV positivity con-
ferred a worse outcome than ABC subtype alone, suggesting that EBV is an inde-
pendent risk factor for poor outcome.
One caveat to the survival studies is that most patients were treated prior to
the time when rituximab, a monoclonal antibody directed against CD20, became
a standard addition to chemotherapy regimens targeting B cell lymphoma. A
study performed on DLBCL patients treated with R-CHOP demonstrated no sur-
vival difference between EBV-positive and EBV-negative patients, suggesting that
rituximab alone may overcome the survival difference noted in previous reports
(Montes-Moreno et al. 2012). The possibility of rituximab having activity in
EBV-positive DLBCL is not unexpected, since rituximab monotherapy effectively
eliminates the majority of mature B cells and is a highly effective treatment for
EBV-positive PTLD (Taylor et al. 2005). More studies are needed to clarify the
impact of rituximab on the clinical outcomes of this lymphoma.
3.5 Histopathology
EBV-positive DLBCLs typically demonstrate an effacement of nodal and extran-

odal tissue architecture by large, rapidly proliferating immunoblasts with inter-
dispersed areas of geographic necrosis (Al-Humood et al. 2014; Oyama et al.
2007; Dojcinov et al. 2011). The cellular makeup of the tumor is variable, with
both polymorphic and monomorphic subtypes described. Polymorphic tumors
display numerous reactive cells, including histiocytes, plasma cells, and normal
lymphocytes intermingled with malignant large cells. The monomorphic subtype
is characterized by sheets of uniform-appearing large cells with minimal reactive
component. Both entities may contain Reed–Sternberg cells (Oyama et al. 2007),
which are commonly present in Hodgkin’s lymphoma, another B cell tumor asso-
ciated with EBV. Despite differing appearances, histological subtypes do not have
prognostic significance (Oyama et al. 2007).
The immunophenotype of EBV-positive DLBCL is that of an aggressive B cell
tumor of post-germinal center origin. Malignant cells are typically positive for B
cell markers CD19, CD20, CD79a, and PAX-5. Ki67, a marker of proliferation, is
usually present in greater than 50 % of tumor cells (Swerdlow et al. 2008; Montes-
Moreno et al. 2012; Al-Humood et al. 2014). Using immunohistochemical mark-
ers, post-germinal center (ABC-associated) proteins IRF4, MUM1, are typically
positive. GC markers CD10 and BCL6 are usually negative (Montes-Moreno et al.
2012). In comparison with Reed–Sternberg cells found in Hodgkin’s lymphoma,
CD15 immunostaining of the neoplastic cells in EBV-positive DLBCL is negative,
though most specimens (50–89 %) are CD30 positive (Montes-Moreno et al. 2012).
EBV positivity is measured either by fluorescent in situ hybridization of the
EBERRNA (Chang et al. 1992) or by immunohistochemical detection of the
LMP-1 protein (Gulley 2001). The expression of EBV latency genes in EBV-
positive DLBCL reveals EBNA-1 and LMP-1 expression in >90 % of tumors, and
28 % positive for EBNA2, which is consistent with a viral latency II or III pattern,
similar to that seen in PTLD (Oyama et al. 2003; Hofscheier et al. 2011; Oyama
et al. 2007; Nguyen-Van et al. 2011; Shimoyama et al. 2008).
3.6 Genetics
Cytogenetic and FISH studies in tumors from EBV-positive DLBCL of the

elderly have revealed no characteristic abnormalities (Al-Humood et al.
2014). Chromosomal translocations involving the heavy chain locus occur in
approximately 15 % of samples (Montes-Moreno et al. 2012). Cytogenetic alter-

ations have been reported, including copy number gains of the C-MYC, BCL2,
and BCL6 loci (Dojcinov et al. 2011). Al-Humood et al. (2014) reported that the
mean total number of chromosomal alternations per case was less than that seen
for EBV-negative disease. This suggests that the EBV itself, rather than acquired
mutations, may be driving the pathogenesis of infected tumor cells.
B cell clonality in EBV-positive DLBCL is common, as measured by PCR of
VH-JH rearrangements in the Ig locus. Most patients demonstrate light chain restric-
tion by Kappa and Lambda immunostaining (Al-Humood et al. 2014). The EBV
genomes of these specimens also demonstrate clonality, as evidenced by FISH
probes design to detect the EBV terminal repeat regions (Oyama et al. 2007).
There is also a high incidence of T cell clonality in EBV-positive DLBCL patients,
with 24 % of cases demonstrating monoclonality in at least one TCR-gamma gene
(Oyama et al. 2007). The significance of this finding is unclear, however, since
clonal T cells have also been demonstrated in many healthy elderly individuals.
Some reports show a prevalence of T cell clones greater than 80 % in asymptomatic
patients over the age of 75 years (Hadrup et al. 2006). These clones are believed to
reflect the reduction of T cell diversity inherent to age-related immune senescence.
3.7 Biology
EBV-positive DLBCL is associated with ABC phenotype, which is characterized

by upregulated NFκB signaling (Staudt 2010). In 2012, Montes Moreno et al.
assessed the state of NFκB activation in EBV-positive DLBCL tumors by Western
blot analysis and subcellular localization of classical NFκB subunits p105/p50 and
alternative pathway subunits p100/p52 (Montes-Moreno et al. 2012). They found
nuclear localization of these factors in 79 and 74 % of tumors, respectively. Over
half of tumors demonstrated nuclear expression of both canonical and alternative
NFκB pathways, significantly greater NFκB activity than that seen in ABC lym-
phoma alone. Furthermore, Kato et al. (2014) found that infecting human ABC
DLBCL-derived cell lines with EBV enhanced NFκB activity measured by elec-
trophoretic mobility shift assay.
There is only one report of gene expression profiling for EBV-positive DLBCL
thus far. Kato et al. (2014) studied a total of 61 patients meeting criteria for the
diagnosis of EBV-positive DLBCL of the elderly and compared these tumors to 36
EBV-negative DLBCL specimens. The authors found that immune and inflamma-
tory gene pathways are highly expressed in EBV-positive DLBCL of the elderly,
including NFκB, JAK/STAT, NOD receptor, and Toll-like receptor signaling
pathways. Expression analysis of the transcriptional targets of NFκB and STAT3
signaling revealed that these pathways are overactive in EBV-positive tumors.
The authors went on to evaluate the effect of EBV status on the subset of ABC
tumors. They found that NFκB and STAT3 expression gene sets were enriched in
EBV-positive tumors. Finally, the authors evaluated other EBV-positive lymphoma
subtypes to determine whether EBV positivity promoted NFκB and JAK/STAT

signaling in other tumor types. NFκB target gene enrichment was the character-
istic of HIV-associated DLBCL, EBV-positive Hodgkin’s lymphoma, and NK cell
lymphoma, whereas the STAT3 signature was only associated with B cell lym-
phoma subtypes. These data suggest that NFκB and STAT3 activity may be defin-
ing features of EBV pathogenesis as it relates to lymphoma.
4 Novel Treatment Approaches for EBV-Positive DLBCL
Recent insights into the biology of EBV-positive DLBCL of the elderly have
revealed the distinct biology of B cell lymphomas that arise in the setting of EBV
infection. Further, there are many reports suggesting that patients with EBV-
positive lymphoma have worse prognosis than their EBV-negative counterparts.
For these reasons, new treatments are needed to address the unique pathogenesis
of this disorder. Possible therapeutic approaches include the following: antiviral
strategies, EBV-targeted adoptive immunotherapy, and/or agents that target the
NFκB or STAT3 signaling pathways.
4.1 Antiviral Therapy
Antiviral drugs offer clear potential for the treatment of EBV-positive lymphoma.
There are other lymphoma subtypes that have previously demonstrated response
to iradication of an associated microorganism. Gastric MALT (mucosa-associated
lymphoma) is an extranodal marginal zone lymphoma that is highly associated
with Helicobacter pylori infection and can be effectively treated with antibiotics in
70 % of patients (Bayerdörffer et al. 1995). Owing to its potential for latent infec-
tion in resting B cells, EBV is a less straightforward treatment target than H. pylori.
Treatment of EBV with antiviral medications would first require activation of the
virus into the lytic phase of infection. Known EBV lytic phase inducers include
DNA methylase transferase inhibitors, HDAC inhibitors, and chemotherapeutics
(Feng et al. 2004). Recently, HDAC inhibitors panobinostat and vorinostat have
demonstrated potent induction of EBV lytic genes in cell lines (Ghosh et al. 2012),
as well as activity in EBV-associated lymphoma (Piekarz et al. 2011; Younes 2009).
Induction therapy with an EBV lytic phase inducing agent, followed by EBV antivi-
ral therapy, would provide a potential solution to the latency issue.
4.2 EBV-Targeted Adoptive Immunotherapy
EBV-targeted adoptive immunotherapy is a strategy in which T cells isolated

from a patient’s peripheral blood are expanded in vitro and activated by exposure
to EBV-specific antigens. These cells are then re-introduced into the patient,
where they colonize tissues and attack lymphoma cells expressing EBV anti-
gens (Gattinoni et al. 2006). Adoptive transfer of EBV-specific CD8+ T cells in
solid organ transplant recipients has been undertaken successfully (Sherritt et al.
2003). Adoptive immunotherapy used in combination with DLBCL chemotherapy
regimens may result in improved response compared to chemotherapy alone for
patients with EBV-postive DLBCL.
4.3 Biology of Targeted Therapies
The NFκB and JAK/STAT pathways are attractive therapeutic targets in EBV-
positive DLBCL. If given in combination with traditional DLBCL regimens, tar-
geted agents may mitigate the survival differences seen between EBV-positive and
EBV-negative tumors. Therapeutic strategies that directly inhibit NFκB signaling
have been fraught with difficulties. NFκB is critical to physiologic processes in many
cells. Deficiency in genes IKKβ and p65 provokes massive hepatocyte apoptosis
during development (Strnad and Burke 2007). Adult hepatocytes are less perturbed
by reductions in these proteins, but still show high sensitivity to toxin and cytokine-
related injury. It is still possible that these inhibitors could be useful in tumors that
are highly reliant on NFκB activity and provided that the appropriate concentra-
tion of inhibitor can be achieved. Bortezomib is a proteasome inhibitor that is capa-
ble of inhibiting both canonical and noncanonical NFκB signaling (Staudt 2010).
Bortezomib is widely used in the treatment of multiple myeloma as is well tolerated
both alone and in combination with other agents. Bortezomib suppresses NFκB acti-
vation by degrading IαBα, an inhibitor of NFκB nuclear translocation. Bortezomib
is cytotoxic to human EBV-infected lymphoblastoid cell lines (Zou et al. 2007).
Drugs that target JAK/STAT signaling would also be of potential therapeutic benefit
to EBV-positive DLBCL. At present, there is much interest in the development of
potent, selective STAT3 inhibitors for the treatment of lymphoid malignancies.
5 Conclusions
In this chapter, the clinical, genetic, and pathologic characteristics of DLBCL

were presented, followed by an explanation of the role of EBV in DLBCL tum-
origenesis. Using EBV-positive DLBCL of the elderly as a model, we describe
the key clinical and pathologic characteristics of EBV-positive DLBCL. We also
discussed the recent insights into EBV-positive lymphoma biology and potential
treatment strategies.
Studies of EBV-positive DLBCL of the elderly have provided key insight into
the pathogenic role that EBV plays in DLBCL. These data demonstrate that EBV-
positive DLBCL has a distinct tumor biology, which is related to the tenuous
relationship that the EBV virus establishes with its B cell host. The pathogenic
mechanisms noted in EBV-positive DLBCL of the elderly, including enhanced
NFκB activity, are likely to play a role in all forms of EBV-positive DLBCL.
More work is needed to determine whether EBV-positive DLBCLs occurring
in distinct contexts of immune dysfunction are biologically different tumors. The
current WHO classification scheme includes subgroups for plasmablastic DLBCL,
DLBCL associated with chronic inflammation, and EBV-positive DLBCL of the
elderly, all of which are EBV-positive DLBCL tumors. It would be interesting
and informative to compare gene expression profiles from these subtypes to see
whether they are similar. If EBV is contributing to the tumor pathogenesis in each
case, the other subtypes may also demonstrate marked NFκB and JAK/STAT acti-
vation. This work would confirm that EBV-positive DLBCL has a unique biology
and provide new clues to treating this disease by methods that disrupt the life cycle
of EBV viral infection or the signaling pathways induced in these tumors.
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Nasopharyngeal Carcinoma: An Evolving
Role for the Epstein–Barr Virus
Nancy Raab-Traub
Abstract The Epstein–Barr herpesvirus (EBV) is an important human pathogen

that is closely linked to several major malignancies including the major epithelial
tumor, undifferentiated nasopharyngeal carcinoma (NPC). This important tumor
occurs with elevated incidence in specific areas, particularly in southern China
but also in Mediterranean Africa and some regions of the Middle East. Regardless
of tumor prevalence, undifferentiated NPC is consistently associated with EBV.
The consistent detection of EBV in all cases of NPC, the maintenance of the viral
genome in every cell, and the continued expression of viral gene products sug-
gest that EBV is a necessary factor for the malignant growth in vivo. However,
the molecular characterization of the infection and identification of critical events
have been hampered by the difficulty in developing in vitro models of NPC.
Epithelial cell infection is difficult in vitro and in contrast to B-cell infection does
not result in immortalization and transformation. Cell lines established from NPC
usually do not retain the genome, and the successful establishment of tumor xen-
ografts is difficult. However, critical genetic changes that contribute to the onset
and progression of NPC and key molecular properties of the viral genes expressed
in NPC have been identified. In some cases, viral expression becomes increas-
ingly restricted during tumor progression and tumor cells may express only the
viral nuclear antigen EBNA1 and viral noncoding RNAs. As NPC develops in the
immunocompetent, the continued progression of deregulated growth likely reflects
the combination of expression of viral oncogenes in some cells and viral noncod-
ing RNAs that likely function synergistically with changes in cellular RNA and
miRNA expression.
N. Raab-Traub (*)
Department of Microbiology, Lineberger Comprehensive Cancer Center, CB#7295,
University of North Carolina, Chapel Hill, NC 27599-7295, USA
e-mail: nrt@med.unc.edu

340 N. Raab-Traub
Contents
1 Introduction........................................................................................................................... 341
2 Aspects of EBV and Cancer Biology Discovered in NPC.................................................... 342
3 Classes of EBV-Associated Malignancies............................................................................ 345
4 Latent Membrane Protein 1.................................................................................................. 347
5 Latent Membrane Protein 2.................................................................................................. 350
6 EBNA1.................................................................................................................................. 352
7 EBV BART Noncoding RNAS............................................................................................. 353
8 Conclusions........................................................................................................................... 354
8.1 EBV and Carcinoma: A Contest of Hide and Seek...................................................... 354
References................................................................................................................................... 355
Abbreviations
EBV Epstein–Barr Virus

mRNA Messenger ribonucleic acid
VCA Viral capsid antigen
EAd Early antigen diffuse
IgA Immunoglobulin subtype A
AIDS Acquired immunodeficiency syndrome
EBNA EBV nuclear antigen
LP Leader protein
CTL Cytotoxic lymphocyte
TR Terminal repeat
CIS Carcinoma in situ
LOH Loss of heterozygosity
FHIT Fragile histidine triad protein
RASSF1A Ras association domain family 1 isoform A
Rb Retinoblastoma gene
Cdk4 Cyclin-dependent kinase 4
BART BamHI A rightward transcript
EBER Epstein–Barr encoded RNA
TNFR Tumor necrosis factor receptor
TRAF TNFR-associated factors
NFkB Nuclear factor kappa B locus
CTAR Carboxy terminal activation region
IRF7 Interferon response factor 7
NIK NFkB inducing kinase
EGFR Epidermal growth factor receptor
PI3kinase Phosphoinositol 3 kinase
Nasopharyngeal Carcinoma … 341
Akt Protein kinase B—v-akt murine thymoma viral oncogene homolog 1

ERK Extracellular signal-regulated kinases
ICAM Intercellular adhesion molecule 1
ID Inhibitor of DNA binding
1 Introduction
The Epstein–Barr herpesvirus (EBV) is an important human pathogen that is

closely linked to several major malignancies (Raab-Traub 2007). After the dis-
covery of EBV in Burkitt’s lymphoma (BL) and the determination that primary
infection could cause infectious mononucleosis (IM), an association of EBV with
nasopharyngeal carcinoma (NPC) was revealed in early studies of EBV serol-
ogy (Henle et al. 1968, 1970). These studies showed that patients with NPC had
exceptionally high titers to the EBV antigens, viral capsid antigen (VCA) , and
early antigen diffuse (EAd) (Henle et al. 1970). The viral genome was subse-
quently detected in the malignant epithelial cells by in situ hybridization and rena-
turation kinetics (Wolf et al. 1975; Pagano et al. 1975). The finding of EBV in
NPC revealed that EBV, in addition to B lymphocytes, also could infect epithe-
lial cells. This finding was supported by later studies identifying EBV and viral
RNAs in sloughed epithelial cells in throat washings (Sixbey et al. 1984; Lemon
et al. 1977). An additional important aspect of these findings was that IgA titers
were specifically indicative of NPC, suggesting that these antigens were produced
within the tumor and inducing a mucosal antibody response (Henle and Henle
1976). Detailed subsequent studies using EBV serology have shown that IgA anti-
bodies to multiple antigens associated with EBV replication are specific markers
for the development, prognosis, and reoccurrence of NPC (Dardari et al. 2000).
IgA titers to these antigens begin to rise 1–2 years prior to the development of
NPC (Zeng et al. 1985; Levine et al. 1981). More recent studies also show that
detection of viral DNA in the sera of NPC patients is also informative (Shao et al.
2004).
Lifelong infection with EBV begins with infection and replication in oral epi-
thelial and lymphoid cells followed by persistence in memory B lymphocytes
(Babcock et al. 1998). EBV-associated cancers develop in both epithelial and
lymphoid cells and include NPC, gastric cancer, Hodgkin’s lymphoma, BL, and
lymphomas that develop in the immunocompromised, including both AIDS and
post-transplant lymphomas (Raab-Traub 2007). EBV readily infects B lympho-
cytes in vivo and in vitro and usually persists in the infected cell as an extrachro-
mosomal episome (Gussander and Adams 1984; Nonoyama and Pagano 1972).
The infected cells that have acquired the ability to continue to proliferate in vitro
are growth immortalized (Pope et al. 1973). The majority of cells do not produce
virus and are considered latently infected. However, the cells express multiple
viral proteins including the EBV nuclear antigens (EBNA) 1, 2, 3A, 3B, and 3C,
and leader protein (LP) and the latent membrane proteins (LMP) 1, 2A, and 2B
342 N. Raab-Traub
(Kieff and Rickinson 2001). The EBNA3 proteins are major targets of cytotoxic
lymphocytes (Murray et al. 1992). The ability to expand and proliferate EBV-
specific cytotoxic T cells (CTL) has led to immunotherapy that is highly effec-
tive in the prevention and treatment of immunodeficiency-associated lymphomas
(Rooney et al. 1998).
In the absence of immunodeficiency, EBV-associated malignancies often have
distinct geographical distributions. BL was quickly shown to have elevated inci-
dence in areas of high malarial infection, while NPC is highly prevalent in south-
ern China, but also occurs with elevated incidence among Alaskan Inuits, and in
Mediterranean Africa (Burkitt 1971; Chang and Adami 2006). In southeast Asia,
NPC develops with a peak incidence of 20–50 cases per 100,000 and constitutes
approximately 18 % of all cancers in that area. The World Health Organization
distinguishes 3 subtypes of NPC based on their degree of differentiation
(Shanmugaratnam 1978). EBV is found in all three subtypes but is consistently
associated worldwide with the most common subtype (subtype 3), characterized as
an undifferentiated carcinoma (Raab-Traub et al. 1987).
The consistent detection of EBV in all cases of NPC, the maintenance of
the viral genome in every cell, and the continued expression of viral gene prod-
ucts suggest that EBV is a necessary factor for the malignant growth in vivo.
Surprisingly, the viral genome is frequently lost in epithelial cell lines established
from EBV (Dittmer et al. 2008; Lin et al. 1990). In BL cell lines, the loss of EBV
results in cell death (Vereide and Sugden 2009; Kennedy et al. 2003). This chap-
ter will summarize our understanding of the potential contribution of EBV to the
development and growth of malignant epithelial cells.
2 Aspects of EBV and Cancer Biology Discovered in NPC
Studies of NPC have been highly informative in the characterization of the biologic
and molecular properties of EBV and the contribution of genetic changes during
cancer progression. Multiple studies have shown that despite major differences in
NPC occurrence, all cases of undifferentiated NPC, including those that develop
with low incidence in nonendemic areas, contain EBV (Pathmanathan et al.
1995a). Interestingly, multiple studies have identified EBV in more differentiated
forms of NPC, especially in areas of high incidence (Pathmanathan et al. 1995a;
Teng et al. 1996). Early studies using renaturation kinetics determined that the viral
genome was essentially the same as that found in BL and during IM and that the
viral genome was maintained as an extrachromosomal episome within the nucleus
(Pagano et al. 1975; Nonoyama and Pagano 1972). However, unlike EBV infection
of B cells, epithelial cells are extremely difficult to infect and the viral genome is
frequently not maintained in stable cell lines established from the tumors (Dittmer
et al. 2008; Cheung et al. 1999; Lin et al. 1990). Thus, much of the early molecular
biology of NPC utilized biopsy material and rare examples of NPC that could grow
as xenografts in nude mice (Raab-Traub et al. 1983; Busson et al. 1988).
Early restriction enzyme mapping and cloning of the viral genome facilitated
these studies (Raab-Traub et al. 1987; Dambaugh et al. 1980). Identification of the
terminal fragments of the viral genome revealed that virion DNA had heterogene-
ous terminal fragments with varying number of terminal repeats (TRs) (Dambaugh
et al. 1980). Additionally, the fused terminal fragments of the episomes could be
distinguished by hybridization to Southern blots with probes from either end of
the linear genome (Raab-Traub and Flynn 1986). This led to the surprising find-
ing that in NPC and BL, the episomal DNA was homogeneous with a single
restriction fragment representing the heterogeneous terminal restriction fragments
that are fused in the episome. The identification of clonal viral DNA suggested
by extension that the cells were also clonal and that the tumor had arisen from
a single EBV-infected cell (Raab-Traub and Flynn 1986). Previously, only the
clonality of lymphomas could be determined based on immunoglobulin gene (Ig)
rearrangements (Arnold et al. 1983). However, the clonality of the viral episome
enabled the identification of clonality in carcinomas. Analyses of Ig rearrangement
and EBV termini confirmed the relationship between viral and cellular clonality
(Katz et al. 1989; Walling et al. 2004). Multiple subsequent studies have identified
clonal EBV in most of malignancies associated with EBV including Hodgkin’s
lymphoma, salivary gland carcinoma, and gastric cancer, and clonal termini have
become one of the criteria indicating a etiologic role for EBV in these cancers
(Raab-Traub et al. 1991; Weiss et al. 1988). This first identification of the terminal
fragments also identified faint ladder arrays of fragments, indicating that virus rep-
lication did occur in a subset of cells and was likely the source of the link between
elevated antibody titers to replicative antigens and development or reoccurrence
of NPC (Raab-Traub and Flynn 1986). This was confirmed in later studies that
identified the Z immediate early protein and EA (Martel-Renoir et al. 1995;
Luka et al. 1988).
In contrast to many malignancies, particularly cervical cancer, identification of
early dysplastic or premalignant lesions was rare in NPC. In an initial survey, only
11 examples of atypical hyperplasia or carcinoma in situ (CIS) were identified in
screening over 5000 biopsies. Clonal EBV was detected in the few examples of
CIS with detection of the EBERs and latent membrane protein 1 (LMP1) in all
cells (Pathmanathan et al. 1995b). In subsequent studies, examples of dysplasia
and CIS were identified that contained EBV in a subset of the cells and intriguing
findings revealed genetic changes, such as loss of p16 expression, that preceded
cancer development (Chan et al. 2000, 2002). These findings have led to a model
of carcinogen-induced genetic changes that initiate dysplastic growth. These envi-
ronmental exposures also potentially activate EBV replication resulting in produc-
tion of IgA antibodies to replicative antigens. A recent study using epithelial raft
cultures has shown that EBV replication is activated as cells differentiate; how-
ever, evidence of latent infection was not detected within the basal cells in this
in vitro model (Temple et al. 2014). In the genesis of NPC, the potential increase
in viral replication induced by environmental factors likely results in increased
epithelial infection. The dysplastic growth induced by potential genetic changes
may enable the establishment of a latent epithelial cell infection. However, after
344 N. Raab-Traub
infection with EBV and expression of EBV transforming genes, the cells rapidly
progress to aggressive malignancy. Thus, it is a matter of discussion whether trans-
forming EBV infection should be considered a late event in the development of
NPC or perhaps the precipitating event leading to rapid progression to invasive
malignancy.
The endemic pattern of incidence of NPC led to many thorough epidemiologic
studies that identified potential contributing environmental and genetic contribu-
tions. Indeed, very early insightful studies by Dr. John Ho revealed a strong cor-
relation between consumption of preserved food at an early age and subsequent
development of NPC, 30–50 years later (Ho et al. 1978). One major factor thought
to contribute to the high incidence of NPC among Hong Kong fisherman was the
consumption and weaning of children to foods containing preserved, salted fish.
Perhaps, this is the first environmental insult that begins the malignant process. Dr.
Ho’s lifelong campaign to stop this custom is now likely coming to fruition with
dropping incidence of NPC in the affected populations (Li et al. 2014). Indeed,
preserved foods containing carcinogens and compounds that lead to EBV reacti-
vation and replication are also consumed in other areas with elevated incidence,
including Alaskan and Greenland Inuits, and Mediterranean countries such as
Tunisia and Algeria (Jeannel et al. 1990).
Dr. Dolly Huang continued these studies and initiated the first genetic studies
of NPC to identify common genetic changes. Although very difficult to perform
on solid tumors, cytogenetic studies identified consistent changes on multiple
chromosomes including 1, 3p, 5p, 9p, 11q, and 12q (Huang et al. 1989). The
development of comparative genomic hybridization and identification of loss of
heterozygosity (LOH) further pinpointed important changes. LOH was identi-
fied with very high frequency at many of the same sites identified by cytogenet-
ics, including 3p, 9p, and 14q in almost all samples (Wong et al. 2003). Many of
these regions were subsequently shown to contain important tumor suppressor
genes. One major factor is either homozygous loss of the p16 gene at 9p or p16
promoter methylation (Lo et al. 1996). These genetic and epigenetic changes result
in the loss of p16 protein in the majority of primary NPC samples and in early
EBV negative dysplasia. A recent study has shown that a region adjacent to p16
that encodes a cellular miRNA, miR31, was also deleted in NPC and early lesions
(Cheung et al. 2014). These genetic changes may work in combination with viral
proteins and miRNAs to alter cell growth regulation. Potential tumor suppressor
genes that contribute to NPC development include FHIT and RASSF1A at the
3p locus and additional genes at multiple other loci (Chow et al. 2004; Ko et al.
2002). Interestingly, mutation of the canonical tumor suppressor genes, p53 and
Rb, is rarely detected in Chinese NPC (Effert et al. 1992; Sun et al. 1993). This
finding suggested that viral proteins may reduce the selection for mutation of these
tumor suppressors. It was shown that LMP1 could specifically block p53-mediated
apoptosis (Fries et al. 1996; Okan et al. 1995). Additionally, cyclin D1 overexpres-
sion has been detected in premalignant lesions, and its expression or expression
of p16 resistant cdk4 facilitates EBV infection (Tsang et al. 2012). Identification
of chromosomal amplification at specific regions also suggested that PI3kinase
and p63 may contribute to NPC development (Chiang et al. 2009). It is intriguing
that EBV genes, LMP1 and LMP2, expressed in NPC also affect these pathways
(Dawson et al. 2003; Scholle et al. 2000; Fotheringham et al. 2010).
The use of cDNA cloning enabled the identification of viral transcription in
tumor samples. Surprisingly, the pattern of EBV expression in NPC was quite dis-
tinct from that determined in transformed lymphocytes (Raab-Traub et al. 1983).
The DNA sequences subsequently shown to encode the viral transforming proteins
EBNA2, 3A, 3B, and 3C were not transcribed, and transcription was restricted to
the sequences that encode EBNA1, LMP1, and LMP2, and abundant transcription
from the BamHI A restriction fragment (BART RNA) was not transcribed in trans-
formed lymphocytes (Kieff and Rickinson 2001).
This novel pattern of expression led to the identification of distinct states of
viral latency in EBV-associated tumors which could also be recapitulated in
some cell lines (Rowe et al. 1992). The most restricted form of latency, Type
I, was characteristic of BL with expression limited to EBNA1 and the EBV-
encoded RNAs (EBERs). Type II latency was considered characteristic of NPC
and included expression of the latent membrane proteins, LMP1 and LMP2, and
BART transcription. In Type III latency, which is characteristic of transformed
B lymphocytes, all of the EBNAs, LMP1, LMP2, and the EBERs are expressed
(Kieff and Rickinson 2001). This pattern of expression is also found in post-trans-
plant lymphoma (Young et al. 1989). However, continued study has revealed that
additional patterns of expression can occur and that a malignancy may have some
cells in different patterns of latency (Niedobitek et al. 1995).
Further dissection of the mechanisms underlying these differences in expres-
sion led to the discovery of different promoters used for latent genes in different
forms of latency. An important difference was the identification of a previously
unidentified promoter, Qp, that initiated transcription of an RNA that only encoded
EBNA1 (Sample et al. 1991). This is in contrast to the RNAs that are initiated
from B-cell specific promoters, Cp and Wp, that encode EBNA2, 3A, 3B, 3C, and
leader protein (Zetterberg et al. 1999; Kieff and Rickinson 2001). Additionally,
Northern blotting and RNA mapping revealed that the LMP1 RNA initiated from
a promoter within the TR (Sadler and Raab-Traub 1995a). It has subsequently
been suggested that the number of TRs may affect transcription of both LMP1 and
LMP2 (Repic et al. 2010; Moody et al. 2003). These differences in viral regulation
reflect the clearly distinct interactions of EBV with epithelial or lymphoid cells.
3 Classes of EBV-Associated Malignancies
These first studies in NPC led to the understanding that the EBV-associated malig-
nancies are distinguished by distinct patterns of expression. Interestingly, it was
quickly determined that the lymphomas that develop after immune suppression
post-organ or bone marrow transplantation express the same genes expressed in
growth transformed lymphocytes including the EBNA2 and EBNA3 genes (Young
346 N. Raab-Traub
et al. 1989). Characterization of the EBV immune response had revealed that the
EBNA2 and 3 proteins were the major targets for recognition by cytotoxic lym-
phocytes (Murray et al. 1992). Thus, in the absence of recognition by cytotoxic
lymphocytes, EBV-infected lymphocytes are able to switch to the transforming
growth mode of expression resulting in lymphoma. In some cases, multiple clonal
tumors are identified in different organs (Walling et al. 2004). This detailed under-
standing of the EBV immune response has led to the first clearly effective viral-
specific immunotherapy or prophylaxis where EBV-specific CTLs are expanded
ex vivo by exposure to EBV transformed lymphocytes (Heslop et al. 1999). These
CTLs can be infused back into the patient and cure or prevent tumor development.
This confirms the etiologic role for EBV in these lymphomas that develop during
immunosuppression with the lymphomas dependent on EBV transforming genes.
In contrast to NPC, gastric cancer, Hodgkin’s lymphoma, and many EBV-
associated cancers that develop in the immunocompetent, the major CTL targets,
the EBNAs 2 and 3 genes, are not expressed. Expression is restricted to EBNA1
which is essential for maintenance of the viral genome and also contributes to
the regulation of expression of both viral and cellular genes (Yates et al. 1984).
Additionally, the viral membrane proteins, LMP1 and LMP2, are expressed
although the consistency and strength of their expression has clear variation, and
the proteins are not necessarily detected in all cells or all samples. This may indi-
cate that during cancer progression, genetic changes may occur that can substitute
for the functions of the viral proteins. These proteins are also much less immuno-
genic than the EBNA proteins which might contribute to their successful expres-
sion in immunocompetent individuals (Hislop et al. 2007). Multiple studies have
distinguished consistent markers of distinct EBV genomes with many studies that
have analyzed sequence variation within LMP1 (Edwards et al. 1999; Miller et al.
1994; Lung et al. 1991). One study identified at least 6 consistent variants with a
predominance of sequence changes within the epitopes predicted to be presented
by their HLA types (Edwards et al. 2004). The successful enrichment of CTLs
directed against these proteins has been achieved, and some efficacy has been
shown in clinical trials (Haigh et al. 2008). Thus, the potentially reduced recogni-
tion of cells expressing a LMP1 variant within a person of a specific HLA type
could be one contributing factor to the ability to evade immune recognition.
Additionally, both NPC and gastric cancer have very high levels of expression
of the EBV BART RNAs (Gilligan et al. 1990b; Hitt et al. 1989). These RNAs
have since been shown to be the primary RNA template for the production of viral
miRNAs (Cai et al. 2006; Pfeffer et al. 2004). Interestingly, the BART RNAs are
not expressed or expressed at very low levels in transformed lymphocytes (Cai
et al. 2006; Gilligan et al. 1990b). Thus, the elevated expression of these RNAs
in the epithelial tumors that lack many of the EBV transforming proteins suggests
that the effects of BART miRNAs contribute to transformation in these restricted
forms of infection. This leads to the intriguing possibility that in the presence of
the highly tuned immune response to EBV in the immunocompetent individual,
deregulated growth continues due to the evolving transforming powers of nonim-
munogenic, noncoding RNAs.
4 Latent Membrane Protein 1
Latent membrane protein 1 has long been considered the EBV oncogene as it was
the first viral protein shown to have transforming properties in vitro (Wang et al.
1985). Genetic studies have shown that LMP1 is also essential for EBV transfor-
mation of B lymphocytes (Kaye et al. 1999). Importantly, transgenic mice that
express LMP1 in B lymphocytes develop lymphoma, while mice that express
LMP1 using epithelial specific promoters develop hyperplasia and carcinoma
(Kulwichit et al. 1998; Curran et al. 2001). The LMP1 mRNA was shown early
on to be an abundant transcript expressed in NPC and is frequently transcribed
from a distinct promoter from that utilized in lymphocytes (Sadler and Raab-
Traub 1995a). The promoter is within the TR which results in a larger sized 3.5 kb
mRNA. This larger mRNA is also detected in the permissive EBV infection, hairy
leukoplakia, suggesting that its utilization is more frequent in epithelial cells
(Gilligan et al. 1990a). Although the mRNA is abundant, the detection of the viral
protein is less consistent. However, the aggressive growth and invasive properties
of NPC, which is a highly metastatic malignancy, have been linked to both EBV
LMP1 and 2.
LMP1 has the ability to alter many cellular growth properties and cellular reg-
ulatory networks. The first molecular clues to its mechanisms of action were the
identification of interaction of LMP1, in yeast two hybrid assays, with the tumor
necrosis factor receptor (TNFR) associated factors (TRAFs) (Mosialos et al.
1995). This finding led to the understanding that LMP1 functioned as a consti-
tutively active member of the TNFR family. Two domains of LMP1 were identi-
fied that could activate NFκB and interacted with different TRAF proteins (Huen
et al. 1995). The carboxy terminal activation region 1 (CTAR1) is thought to
interact with TRAF1, 2, 3, and 5 with different avidities, while CTAR2 may inter-
act with RIP, TRADD, and BS69 and also affects the binding of IRF7 to LMP1
(Fig. 1) (Mosialos et al. 1995; Izumi and Kieff 1997; Song et al. 2008; Miller et al.
1998). Different biological and molecular properties are linked to either CTAR1
or CTAR2. CTAR2 has the strongest activation of the canonical NFκB pathways,
and inhibition of NFκB leads to irrevocable cell death (Cahir-McFarland et al.
2004). Additionally, CTAR2 is required for activation of the jun kinase pathway
(JNK) and the p38 MAPK (Fig. 1) (Eliopoulos et al. 1999). Activation of CTAR2
is also required for the binding of IRF7 to an adjacent region of LMP1 (Song et al.
2008). Interestingly, EBV containing LMP1 deleted for CTAR2 can still transform
B lymphocytes (Kaye et al. 1999). Additionally, LMP1-mediated rodent and human
fibroblast transformation requires LMP1-CTAR1 but not CTAR2 (Mainou et al.
2005). Initial studies showed that LMP1-CTAR1 activated multiple forms of NFκB
including canonical p50/p65 heterodimers, abundant homodimers of p50, and
increased processing of p100 to p52 (Paine et al. 1995; Eliopoulos et al. 2003). The
formation of p52/relB heteroimers is now termed “noncanonical NFκB activation”
and is dependent on LMP1-CTAR1 and activation of the NFκB inducing kinase
(NIK). LMP1-CTAR1 also uniquely activates the PI3kinase/Akt pathway and ERK
348 N. Raab-Traub
lipid raft
LMP1 exosome immune

regulation
pathway
EGFR
CD63
galectin-9
p p
p110 p85
CTAR2 CTAR1
NIK
TRAF1
TRAF2
TRAF5
TRAF3
noncanonical
A20
canonical RIP IKKα
TRAF6
AKT
TRADD
TAK1 BS69 IKKα
IKKβ IKKγ vimentin
filaments
IKKα
RelB
PKCδ NFκB2/p100
canonical MEK
JNK
atypical p38
transformation IκBα IκBα
ERK
p65/RelA p50
transformation cytoplasm
p50 p50 p50 p

p50 p50 p52 STAT3
p65/RelA p50 p52 RelB
BCL3 nucleus
transformation BCL3
EGFR
Fig. 1 Latent membrane protein 1: structure and molecular interactions. The proteins that have
been identified to interact with LMP1 and the resultant effects of cellular signaling pathways
including distinct forms of NFκB and activation of kinases are indicated
activation, both of which are required for rodent fibroblast transformation (Mainou
et al. 2007; Dawson et al. 2003). Surprisingly, although activation of NFκB is criti-
cally required for continued B-lymphocyte proliferation, it is not required for the
altered growth properties of rodent or human fibroblasts (Mainou et al. 2005).
One of the first unique properties identified for CTAR1 was the ability to induce
expression of the epidermal growth factor receptor (EGFR) (Miller et al. 1997).
This induction is thought to reflect the ability of LMP1-CTAR1 to induce a unique
form of NFκB consisting of p50 homodimers in a complex with bcl3 (Thornburg
et al. 2003). Additionally, LMP1 increases bcl3 expression through activation of
STAT3 (Fig. 1) (Kung and Raab-Traub 2008). Activation of STAT3 and ERK are
linked to PKCδ activation (Fig. 1) (Kung et al. 2011). Additional genes that are
specifically induced by LMP1-CTAR1 include TRAF1, ICAM, and the ID proteins
(Devergne et al. 1998; Everly et al. 2004). A recent study has shown that in NPC,
the primary forms of NFκB consist of p50/p50/bcl3 and p50/relB heterodimers,
both of which are NFκB forms uniquely induced by LMP1-CTAR1 (Chung et al.
2013). Intriguingly, this study also showed that most NPC had missense mutations
in the TRAF3, TRAF2, NFκB p105, or A20, suggesting that NFκB is constitu-
tively activated in NPC through effects of LMP1 or perhaps genetic mutation. It is
possible that in the early stages of cancer such as carcinoma in situ, LMP1 expres-
sion is required for growth while as the tumor evolves, genetic changes occur that
reduce the requirement for the viral transforming proteins.
The constitutive activity of LMP1, which is independent of any ligand binding,

reflects the aggregation of LMP1 within membranes due to its six hydrophobic
transmembrane domains. LMP1 has been shown to localize to lipid-rich mem-
brane domains, termed lipid rafts (Ardila-Osorio et al. 2005). LMP1 is associated
with TRAF3 in lipid rafts and also modifies raft contents. Subsequent studies have
shown that LMP1 induces raft localization of the p85 subunit of PI3kinase and the
cellular intermediate filament, vimentin, which was one of the first proteins shown
to interact with LMP1 (Meckes et al. 2013b). The integrity of LMP1 raft structures
and polymerization of vimentin filaments are required for rodent fibroblast trans-
formation as disruption of rafts with cholesterol or inhibition of vimentin polymer-
ization blocked activation of Akt, ERK, and growth transformation (Meckes et al.
2013b). It is likely that the association of cellular proteins with LMP1 is dynamic
and perhaps distinct in different subcellular components. Most of the TRAF pro-
teins that associate with LMP1 are ubiquitin ligases, and the A20 protein which
also binds LMP1 is both a ubiquitin ligase and deubiquitinase (Fries et al. 1999).
The interaction of LMP1 with the ubiquitin pathway could likely modulate the lev-
els and location of various critical signaling molecules.
The LMP1 effects on lipid rafts are also reflected in exosomes produced
by LMP1-expressing cells (Meckes et al. 2010). Exosomes are microvesicles
thought to be specifically produced through budding into multivesicular bod-
ies. Initial studies showed that LMP1 was excreted from infected B cells in
exosomes and that exosomes produced by EBV-infected cells impaired T-cell
function (Flanagan et al. 2003; Keryer-Bibens et al. 2006). Using mass spectrom-
etry of purified lipid rafts, an interaction of LMP1 with galectin 9 was identified
(Fig. 1) (Keryer-Bibens et al. 2006). It was shown that LMP1 induced galectin
9 expression resulting in the secretion of exosomes containing LMP1 and galec-
tin 9. Subsequent studies showed that exosomes produced by LMP1 expressing
cells could transfer LMP1 and activated signaling molecules into recipient cells
leading to Akt and ERK activation (Meckes et al. 2010). Recent studies have
shown that LMP1 induces exosomal transfer of HIF1α and activation of HIF1α-
regulated transcription (Aga et al. 2014). Further characterization of the effects
of LMP1 on exosome content analyzed purified exosomes from EBV and KSHV,
and EBV/KSHV dually infected B cell lines using 2D difference gel electropho-
resis and spectral counting (Meckes et al. 2013a). Multiple significant changes
were identified with 360 of 870 identified proteins specific to the viral exosomes.
Bioinformatic analysis suggested that the viral exosomes likely modulate cell
death and survival, ribosomal function, and mTOR signaling. Viral distinct effects
on exosomes suggested that the KSHV exosomes would alter cellular metabo-
lism, while the EBV exosomes would activate cell signaling through effects on
integrins, actin, and NFκB. LMP1 was identified as a significant principal com-
ponent that affected exosome content (Meckes et al. 2013a). These findings sug-
gest that virally induced changes in exosome content likely modulate the tumor
environment. This would be critical for inhibiting immune function but also could
enable a rare subset of cells expressing LMP1 to affect cell growth regulation in
neighboring cells. In many cases of NPC, LMP1 expression is found in a small
350 N. Raab-Traub
subset of cells; however exosomal transfer of LMP1 itself or its activated signaling
molecules could contribute to the growth of neighboring cells. Additionally, it is
likely that the subset of cells within NPC that become permissively infected also
modulate the tumor environment and possibly contribute to infected cell growth.
Several studies have revealed that replicative infection enhances tumor induction
in humanized mice which may reflect effects on cytokine production or potential
exosome transfer of important growth inducing factors (Ma et al. 2011; Jones et al.
2007).
5 Latent Membrane Protein 2
LMP2 is also considered an EBV transforming protein although it is not strictly

required for B-lymphocyte transformation (Longnecker and Miller 1996;
Longnecker et al. 1993). LMP2 is transcribed across the terminal repeats of the
episome with two forms that contain the first coding exon which contains the criti-
cal signaling motifs (LMP2A) or do not contain this exon (LMP2B) (Laux et al.
1989). LMP2A is consistently detected in NPC at the RNA and protein level and
is also expressed in many EBV-associated lymphomas with high levels of expres-
sion in HD (Busson et al. 1992; Morrison et al. 2004). Recent studies show syn-
ergy between LMP2A and activated c-myc and suggest that LMP2 may also
contribute to BL (Bieging et al. 2010).
LMP2A contains 12 hydrophobic transmembrane domains with multiple tyros-
ines that likely form signaling motifs within the intracellular amino terminus of
LMP2A (Longnecker and Kieff 1990). The protein contains a putative src kinase
binding motif, YEEA, and an immunoreceptor tyrosine-based activation motif
(ITAM) that binds the syk kinase (Longnecker et al. 1991). Additionally, LMP2A
contains two PY domains that interact with WW-containing ubiquitin ligases such
as Itch (Ikeda et al. 2003). In B cells, LMP2A is thought to induce survival by mim-
icking B-cell receptor signaling. In both B lymphocytes and epithelial cells, LMP2A
Akt activation leads to translocation of Β-catenin to the nucleus (Morrison et al.

activates the Akt pathway (Swart et al. 2000; Scholle et al. 2000). In epithelial cells,
2003). Expression of LMP2A can induce growth transformation in some epithelial

cell lines, perhaps in association with ras activation, resulting in anchorage inde-
pendence and tumorigenicity (Scholle et al. 2000; Fukuda and Longnecker 2007).
LMP2A also inhibits epithelial cell differentiation possibly reflecting the induction
of ΔNp63 by LMP2A (Fotheringham et al. 2010; Scholle et al. 2000). Additionally,
LMP2 induces cell migration, and this induction of migration is dependent on
effects on integrin expression and localization and activation of src, syk, and focal
adhesion kinase (Fotheringham et al. 2012; Lu et al. 2006).
In transgenic mice where LMP2 is expressed in B cells under the control of the
Ig promoter, LMP2 can provide prosurvival signals enabling survival of B cells
lacking a functional B-cell receptor (Swanson-Mungerson et al. 2006). However,
transgenic mice expressing LMP2 in epithelial cells lacked a discernable phenotype
(Longan and Longnecker 2000). In classical skin painting experiments in mice

that distinguish tumor initiation, promotion, and progression, LMP1 functioned as
a weak tumor promoter and increased the formation of papillomas (Curran et al.
2001; Shair et al. 2012). In contrast, expression of LMP2 alone did not affect pap-
illoma or carcinoma formation. Interestingly, the formation of squamous cell car-
cinoma was significantly increased in the LMP1 and LMP2 doubly transgenic
animals with elevated levels of activated ERK and STAT3 in all tumors (Shair et al.
2012). This was the first identification of synergy between LMP1 and LMP2. These
studies all indicate that the effects of LMP2 are manifest in specific situations.
An interesting system that has been used to characterize the properties of mul-
tiple viral and cellular oncogenes is based on the nontumorigenic mammary epi-
thelial cell line, MCF10A (Debnath and Brugge 2005). When grown in Matrigel,
the normal cells differentiate and form hollow, spherical acinar structures that
maintain normal glandular features. The effects of multiple cellular oncogenes and
requirements for specific signaling pathways involved in the sequential cell pro-
liferation, apoptosis, and potential malignant progression have been identified in
this system. Deregulation of the cell cycle through overexpression of cyclin D1 or
inactivation of the retinoblastoma tumor suppressor gene by the human papilloma
virus (HPV) E7 protein resulted in continued proliferation of apical cells result-
ing in enlarged acini; however, formation of the hollow lumen, a process depend-
ent upon apoptosis, was not impaired. In contrast, activation of Akt repressed the
formation of acini lumen, while expression of the ErbB2 oncogene inhibited cell
death within the acinar lumen and also induced continued proliferation leading to
the formation of irregularly shaped spheroids (Debnath et al. 2003). Expression
of LMP2A was also evaluated in this system to determine the relationship of the
effects of LMP2A to previously identified properties of other oncogenes (Fig. 2).
Surprisingly, LMP2A had profound effects on MCF10A growth and differentia-
tion and induced the formation of filled lobular structures, similar to those induced
by the potent oncogene, ErbB2 (Fotheringham and Raab-Traub 2013). The block
in differentiation and lumen formation and enhanced proliferation all required
LMP2-mediated activation of src and Akt. The ITAM, src binding, and PY
motifs were also required to block the anoikis-mediated lumen formation and the
increased proliferation (Fig. 2). Surprisingly, expression of LMP2A with mutation
of the PY motif alone resulted in the formation of large, hollow acini with contin-
ued proliferation of the apical cells, a phenotype reminiscent of the E7 oncogene
that inhibits the retinoblastoma tumor suppressor gene. These findings reveal that
the activation of multiple pathways by LMP2 can induce a highly transformed,
proliferative phenotype with loss of growth control and inhibition of the induction
of cell death during differentiation. Similarly to LMP1, the ability to bind ubiqui-
tin ligases is an essential component in the LMP2A-induced uncontrolled prolif-
eration and protection from cell death. As both LMP1 and LMP2 have profound
effects on cellular signaling networks and can affect differentiation, migration,
anchorage independence, and tumorigenicity, it is likely that the combined effects
of LMP1 and LMP2 contribute to the pathogenesis of undifferentiated and highly
metastatic NPC.
352 N. Raab-Traub
pBabe LMP2A PY ITAM YEEA
DAPI
6-integrin
-catenin
Fig. 2 Latent membrane protein 2: structure and activation of signaling pathways. Top sec-
tion The effects of LMP2 and LMP2 signaling mutants on acini formation in MCF10a cells are
shown. The vector control (pBabe), wild type (LMP2A), mutation in the ubiquitin ligase bind-
ing domains (PY), mutation of the ITAM domain, and mutation of the src family kinase bind-
ing domain (YEEA) are compared. Nuclei are identified by DAPI staining (blue), and cell mem-
branes and junctions are shown by staining for α6-integrin (red) and β-catenin (green). Bottom
section Schematic representation of the requirement for the YEEA, ITAM, and PY mutants and
specific signaling pathways on distinct processes in acini formation. Effects of known oncogenes
in this system are indicated. Adapted from Fotheringham and Raab-Traub (2013)
6 EBNA1
EBNA1 is clearly a critical viral protein which is essential for maintenance of the
EBV genome and for the controlled segregation with the dividing cells (Yates et al.
1984; Sivachandran et al. 2011). EBNA was the first viral protein detected in NPC
and is expressed within all cells (Huang et al. 1974). In addition to its essential
role in maintaining the viral genome, EBNA1 also has signaling activity and trans-
forming effects. EBNA interacts with USP7, a protein that prevents degradation of
p53 (Saridakis et al. 2005). EBNA1 has also been shown to induce disruption of
promyelocytic nuclear bodies (PML-NB) in NPC cells (Sivachandran et al. 2010).
PML bodies contribute to DNA repair, and the loss induced by EBNA1 may con-
tribute to increase genetic instability in NPC cells (Sivachandran et al. 2008). These
same properties have been identified in EBV-infected AGS gastric carcinoma cells
(Sivachandran et al. 2012). EBNA1 may also regulate expression of other EBV
proteins through its enhancer function (Altmann et al. 2006). It is likely that the
molecular properties of EBNA1 contribute to altered growth regulation perhaps
synergistically with viral oncogenes, miRNAs, and cellular genetic changes.
7 EBV BART Noncoding RNAS
The BamHI A rightward transcripts (BARTs) were originally identified as the

most abundant transcripts in studies of EBV transcription in NPC biopsy samples
(Hitt et al. 1989; Gilligan et al. 1990b). These RNAs were shown to be a set of
alternatively spliced transcripts (Sadler and Raab-Traub 1995b). The BART RNAs
can be detected by PCR in all forms of EBV latency, although expression is con-
siderably more abundant in Type II latency, and particularly in the infected epithe-
lial cells in NPC and gastric carcinomas (Brooks et al. 1993; Gilligan et al. 1990b;
Cai et al. 2006). The variable splicing of the RNA forms multiple open read-
ing frames (Sadler and Raab-Traub 1995b). The potential protein products have
intriguing properties; however, the proteins have not been identified in infected
cells and the transcripts have been shown to remain in the nucleus (Kusano and
Raab-Traub 2001; Thornburg et al. 2004; Smith et al. 2000). Importantly, the
BART transcripts have been shown to be the template for the precursors for as
many as 44 microRNAs (miRNAs) (Cai et al. 2006). MicroRNAs are ~22 nucleo-
tide RNAs that are processed and function much like siRNAs in inhibiting pro-
tein translation through base pair interactions with target mRNAs. Interestingly,
the EBV B95-8 laboratory strain that readily transforms primary B lymphocytes is
deleted for most of the BART miRNAs which indicates that they are not required
for B-cell transformation (Raab-Traub et al. 1980; Pfeffer et al. 2004). This is in
agreement with the minimal BART expression in B-cell lines (Cai et al. 2006). In
contrast, a cluster of three miRNAs that are produced from the primary EBNA2
transcript and are not expressed in NPC have been shown to contribute to B-cell
transformation (Feederle et al. 2011). As the function of miRNAs is dependent
upon their relative abundance to a given target, multiple studies have attempted
to determine patterns of EBV miRNA expression that distinguish the diseases
associated with EBV and even the type of B-cell infection (Qiu et al. 2011; Chen
et al. 2010; Amoroso et al. 2011). The relative abundance of the BART miRNAs
determined by different methods varies greatly across studies (Marquitz and Raab-
Traub 2012). This variation appears to be due to more than just differences in the
samples used, as the relative abundance of miRNAs present in the C666-1 cell line
is very different depending on the method of PCR detection or direct sequencing.
Considering the limited viral protein expression in NPC and gastric carcinoma
and the abundance of the BART miRNAs, it is likely that these miRNAs contrib-
ute to the development of EBV-associated carcinomas. Several genes involved in
apoptosis are potential targets of various BART miRNAs, including PUMA, Bim,
and TOMM22 (Marquitz et al. 2011; Choy et al. 2008; Dolken et al. 2010). These
targets have been identified in different cells in different studies but point to a
common targeting of mitochondrial proteins. It is possible that the targets differ
between cell lines and varies from one clonal growth to another.
The AGS gastric cell line is a rare epithelial cell line that can be consistently
infected with EBV. It has been shown that EBV infection of this cell line results in
altered growth properties (Marquitz et al. 2012; Kassis et al. 2002). Although the
EBV negative AGS cell line normally grows very poorly in soft agar, cells latently
354 N. Raab-Traub
by EBV become anchorage independent, a hallmark of transformation. The expres-

sion pattern of these cells is basically Type I latency, with very limited protein
expression but high levels of the BART miRNAs. Analysis of changes in cellular
expression using microarray analysis revealed dramatic changes after infection
with EBV. The majority of changes reflected decreased expression of genes after
EBV infection. Additionally, the downregulated genes were significantly enriched
for potential BART miRNA targets, suggesting that the BART miRNAs are major
contributors to the dramatic expression changes (Marquitz et al. 2012). Ingenuity
Pathway Analysis identified genes involved with migration, cellular movement, inva-
sion, growth, and proliferation as highly and significantly enriched in the affected
gene set. The genes affected by the BARTs may be distinct in different infections
and also be different in the presence or absence of viral protein expression. The
effect of EBV infection on cellular miRNA expression was also determined using
RNA-Seq. This study revealed that EBV had significant effects on cellular miRNA
expression. In EBV-infected AGS cells, 15 % of all miRNAs were BART miRNAs.
In the C15 NPC tumor, 57 % were BART miRNAs, and in the C666 cell line estab-
lished from an NPC xenograft, the BART miRNAs represented 40 % of all miRNAs.
These high levels of BART miRNAs detected in the NPC tumors may indicate that
tumorigenicity selects for high levels of BART miRNA expression (Marquitz et al.
2014). Interestingly, multiple tumor suppressor miRNAs were consistently down-
regulated. Additionally, other studies have identified tumor suppressor genes such
as WIF1 and APC as miRNA targets (Wong et al. 2012). A recent study provided
evidence for selection for high levels of BART miRNA expression in EBV-positive
cells that form tumors in immunodeficient mice (Qiu et al. 2015).
However, many likely targets of the BART miRNAs and their biologic effects
have yet to be determined. As our analyses of tumors become more accurately
detailed, unique features of tumors are increasingly identified. It is known that
miRNAs have many potential targets; thus, the actual effects on growth during
infection are likely a combination of the abundance of viral proteins, the cellular
target mRNAs, the viral miRNA(s), and cellular miRNAs. Indeed, a recent study
indicated that the cellular miR31 which is close to the p16 locus is frequently
deleted or not expressed in NPC and in premalignant epithelial tissue (Cheung
et al. 2014). It is possible that in different cell lines and individual tumors, differ-
ent targets may be responsible for malignant growth. However, it is likely that the
same cellular pathways are affected either directly by viral proteins or indirectly
through viral noncoding RNAs.
8 Conclusions
8.1 EBV and Carcinoma: A Contest of Hide and Seek
The many years of study of EBV and NPC have revealed many unique properties
of EBV and its proteins. The epidemiology and the studies of viral infection reveal
a required contribution for genetic changes likely induced by carcinogenic insult.

These may modulate the ability of EBV to infect these cells and also influence the
type of infection from replication to latent and transforming. In the early stages
of tumor development, the viral proteins in combination with the genetic changes
likely are essential for deregulated cell growth. The viral proteins expressed in this
restricted form of latency are poorly immunogenic. Additionally, variants of these
proteins are found within NPC that have sequence changes that would affect rec-
ognition by cytotoxic lymphocytes.
As the tumor continues to evolve, the requirement for viral expression may be
reduced such that viral oncogenes may be expressed in a subset of cells and trans-
ferred through exosomes to induce growth. Alternatively, the occurrence of addi-
tional genetic changes may supplant the need for viral protein expression through
effects on the same critical cellular signaling pathways.
Additionally, it appears that the viral noncoding RNAs in combination with the
EBNA1 may be sufficient to induce deregulated growth by targeting distinct cell
functions through a combination of changes in cellular expression and cellular
and viral miRNAs. However, the continued retention of the viral genome in vivo
despite this ongoing evolution points to a continued requirement for EBV despite
its evolving role in contributing to oncogenic growth. The ability to transform
cells without expression of immunogenic viral proteins would be a major and sig-
nificant mechanism through which oncogenic viruses induce cancer. Importantly,
the identification of specific cellular functions and pathways that may be affected
through multiple mechanisms could lead to specific therapies that target the acti-
vated pathways.
Acknowledgments These studies represent the work of many graduate students and
postdoctoral fellows from my laboratory who are indicated in the references. I would also like
to thank Rachel Edwards and Anuja Mathur for assistance with figure preparation and Dr. Aron
Marquitz for helpful comments. These studies have been supported by the NCI through grants
RO1 CA32979, RO1138811, and PO1 19014.
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EBV and Autoimmunity
Alberto Ascherio and Kassandra L. Munger
Abstract Although a role of EBV in autoimmunity is biologically plausible and

evidence of altered immune responses to EBV is abundant in several autoim-
mune diseases, inference on causality requires the determination that disease risk
is higher in individuals infected with EBV than in those uninfected and that in
the latter it increases following EBV infection. This determination has so far been
possible only for multiple sclerosis (MS) and, to some extent, for systemic lupus
erythematosus (SLE), whereas evidence is either lacking or not supportive for
other autoimmune conditions. In this chapter, we present the main epidemiological
findings that justify the conclusion that EBV is a component cause of MS and SLE
and possible mechanisms underlying these effects.
Contents
1 Introduction........................................................................................................................... 366
2 Multiple Sclerosis................................................................................................................. 368
2.1 Definition and Epidemiology....................................................................................... 368
2.2 Epidemiological Evidence that EBV Is a Cause of MS............................................... 369
2.3 Potential Mechanisms Relating EBV to MS............................................................... 372
2.4 Implications for MS Prevention and Treatment........................................................... 374
2.5 Summary on EBV and MS.......................................................................................... 375
A. Ascherio (*)
Channing Division of Network Medicine, Department of Medicine,
Brigham and Women’s Hospital, Boston, MA, USA
e-mail: aascheri@hsph.harvard.edu
A. Ascherio
Harvard Medical School, Boston, MA, USA
A. Ascherio · K.L. Munger
Department of Epidemiology and Nutrition, Harvard T.H. Chan School of Public Health,
Boston, MA, USA

366 A. Ascherio and K.L. Munger
3 Systemic Lupus Erythematosus............................................................................................ 376

3.1 Definition and Epidemiology....................................................................................... 376
3.2 Epidemiological Evidence that EBV Is a Cause of SLE............................................. 376
3.3 Potential Mechanisms Relating EBV to SLE.............................................................. 378
3.4 Implications for SLE Prevention and Treatment......................................................... 379
References................................................................................................................................... 379
Abbreviations
CMV Cytomegalovirus
CNS Central nervous system
CSF Cerebrospinal fluid
DoDSR Department of defense serum repository
EBERs Epstein–Barr virus-encoded small RNAs
EBNA Epstein–Barr virus nuclear antigen
HLA Human leukocyte antigen
MS Multiple sclerosis
OR Odds ratio
SLE Systemic lupus erythematosus
VCA Epstein–Barr virus viral capsid antigen
1 Introduction
Autoimmune diseases encompass a broad range of conditions that collectively affect

about 5 % of the population (Davidson and Diamond 2001). Recognition of self-
antigens is normally required to maintain the broad repertoire of adaptive immunity
(Goldrath and Bevan 1999), but disease occurs when the strength and persistence of
autoimmune responses disrupt normal physiological functions or cause tissue dam-
age. Autoimmune disease can be induced experimentally by injecting self-antigens
with strong adjuvants, but human autoimmune diseases occur spontaneously, as a
result of interactions between genetic susceptibility, related mostly to variations in
human leukocyte antigen (HLA) class I and II genes, and poorly understood envi-
ronmental factors. Associations between infectious agents and autoimmune disease
suggest that infections play an important role in autoimmunity, but there are only a
few well-documented conditions in which a specific infection can be identified as
the cause. For example, rheumatic heart disease following infection with group A
streptococci is caused by molecular mimicry between streptococcal and cardiac anti-
gens such that the antibodies the immune system develops against the streptococcal
infection also recognize and attack cardiac self-antigens (Marijon et al. 2012).
Epstein–Barr virus (EBV), by causing a persistent latent infection with periodic
reactivations, immortalizing B lymphocytes, and eliciting a strong T-cell response,
seems a uniquely plausible cause of autoimmunity (Pender 2003), and numerous
EBV and Autoimmunity 367
claims of causality have been made based on altered humoral and cellular immune
responses to EBV in several autoimmune diseases. These alterations, however, could
as well be a consequence rather than a cause of the disease. A demonstration of cau-
sality, broadly defined as a state of nature in which a reduction in the frequency or
severity of infection would be followed by a reduction in the frequency of the auto-
immune disease (MacMahon and Trichopolous 1996), would require proof that pre-
vention of EBV infection is followed by a reduction in disease incidence. Lacking a
suitable vaccine for such an experiment, the strongest evidence of causality rests on
the results of an experiment of nature: Are those individuals who are not infected with
EBV protected from the disease, and do they lose protection after EBV infection?
This protection does not need to be complete, because EBV could be the underlying
cause in only a subset of cases. The relation between the hepatitis B virus and liver
cancer provides a useful analogy: Liver cancer occurs in non-infected individuals, but
infection causes a 50- to 100-fold increase in risk (Beasley et al. 1981). On the other
hand, if the frequency of liver cancer was the same among infected and non-infected
individuals in the same population, we would conclude that causality is unlikely.
More generally, we would say that EBV is a cause of a disease X if the future
frequency of X in a healthy and virus naïve population would be higher if the
members of this population became infected with EBV than if they remained unin-
fected. This counterfactual occurrence is unobservable, but given two groups of
EBV-negative individuals with similar characteristics, we would expect a higher
disease incidence in those who become infected with EBV than in those who do
not. If so, and lacking any credible alternative explanation for the difference in
frequency of X, we could infer that EBV is a cause of X, even if we do not fully
understand the underlying mechanisms.
Most individuals worldwide are EBV infected, making determination of the
autoimmune disease frequency in those who are EBV-negative challenging. To
overcome this difficulty, two complementary strategies have been pursued:
(i) meta-analyses combining the data from several case–control studies and
(ii) case-control studies of pediatric populations in which prevalence of EBV
positivity is still low at ~75 % or less.
Although EBV has been suspected as an etiological factor in multiple autoim-
mune diseases, evidence of a higher disease frequency in EBV infected as com-
pared to non-infected individuals has been found only for multiple sclerosis (MS)
and systemic lupus erythematosus (SLE). There is little to no epidemiological evi-
dence in favor or against a role of EBV in most autoimmune diseases, whereas for
a few others, including juvenile rheumatoid arthritis and myasthenia gravis, evi-
dence does not support causality. The dysregulation of immune responses to EBV
observed in these conditions is thus likely to be secondary to autoimmune and
inflammatory reactions (Costenbader and Karlson 2006; Ferraccioli and Tolusso
2007). A summary of selected data is shown in Table 1.
The results in Table 1 support, but do not demonstrate, a causal link between
EBV infection and MS or SLE. One of the main limitations of these data is their
cross-sectional nature, because of which the possibility of reverse causation
(i.e., MS or SLE increasing susceptibility to EBV infection) cannot be ruled out.
Table 1 Association between EBV infection and risk of autoimmune diseases in selected
case–control studies
Age group Disease Cases N Controls N Odds ratio for EBV
(% negative) (% negative) negative (p value)
Adults Multiple sclerosisa 1779 (0.5) 2499 (6.4) 0.06 (p < 10−8)
Pediatric SLE (James et al. 117 (0.9) 153 (30.0) 0.02 (p < 10−10)
onset 1997) 26 (0) 26 (30.0) 0.04 (p < 10−3)
SLE (Harley and
James 1999)
Multiple sclerosisb 281 (8.9) 350 (35.7) 0.18 (p < 10−6)
Myasthenia gravisc na (41) na na
(Klavinskis et al.
1985)
Myositis (James 36 (28.0) 153 (30.0) 0.91 (ns)
et al. 1997)
Juvenile rheumatoid 38 (28.0) 153 (30.0) 0.91 (ns)
arthritis (James
et al. 1997)
aMeta-analysis (Ascherio and Munger 2007a, b)
bCombined data from three investigations (Alotaibi et al. 2004; Pohl et al. 2006; Banwell et al.
2007a), approximate p value estimated by authors of this review
cCases with onset <20 years; number of cases not provided, and no matched controls available,
but the 41 % negativity is similar to expected among UK adolescents
Longitudinal studies demonstrating that EBV infection indeed precedes the first
clinical manifestation of disease have been conducted only for MS and SLE. Thus,
in the remainder of this chapter, we will focus on these conditions, specifically
discussing the epidemiological evidence supporting EBV causality and some of
the possible mechanisms that seem to converge with the epidemiological findings.
2 Multiple Sclerosis
2.1 Definition and Epidemiology
MS is a chronic and disabling inflammatory and neurodegenerative disease of the

central nervous system that affects 350,000 people in the USA and approximately
2 million worldwide (Pugliatti et al. 2002). In over 80 % of cases, the disease
starts with a relapsing–remitting course. Relapses are caused by discrete demy-
elinating lesions pathologically consistent with an immune-mediated process.
Macrophages, B cells, T cells, and immune complexes with evidence of comple-
ment activation have been recognized in biopsy or autopsy material, with evidence
of some heterogeneity across individuals (Lucchinetti et al. 2000). There is also
evidence in peripheral blood cells of altered cellular immunity against myelin
antigens (Lovett-Racke et al. 1998; Markovic-Plese et al. 2001) and of impaired
function of regulatory T cells (Viglietta et al. 2004), which support the autoim-
mune etiology. Symptoms of MS are variable depending on the localization of the
lesions within the brain or spinal cord. Although most lesions resolve completely
or nearly completely within weeks, and relapses decrease over time and eventually
cease completely, an underlying slow loss of brain tissue ultimately leads to pro-
gressive disability.
MS reaches a peak incidence between 20 and 40 years of age and affects women
more often than men, and it is common in Europe, USA, Canada, Australia, and
New Zealand. There is evidence that incidence is increasing among women in both
Europe and North America and in both sexes in some countries, but MS remains
rare in Africa and most of Asia. Although genetic factors, particularly the prevalence
of the MS-risk allele HLA-DR15, contribute to this geographical distribution, the
results of studies among migrants provide compelling evidence for a role of envi-
ronmental factors (Gale and Martyn 1995). The most consistent risk factors are vita-
min D insufficiency (Ascherio et al. 2010), cigarette smoking (Ascherio and Munger
2007b), and, as described below, infectious mononucleosis (IM) and EBV infection.
2.2 Epidemiological Evidence that EBV Is a Cause of MS
MS and IM share many epidemiological similarities—both diseases are uncommon

in the tropics, as well as in Japan, China, and among Eskimos, display a latitude gra-
dient in incidence within temperate zones in North America, Europe, and Oceania,
and occur at somewhat younger age in women than in men (Warner and Carp 1988;
Ascherio and Munger 2007a, b). Further, MS risk is about 2.3-fold higher among
individuals with clinical history of IM, as compared with those without such history,
as demonstrated in a meta-analysis of case-control studies (Thacker et al. 2006) and
confirmed in a longitudinal investigation based on the Danish national data (Nielsen
et al. 2007). One interpretation of this finding is that IM and MS share a common
cause: a high level of hygiene in childhood that predisposes to IM, by delaying the
age of primary EBV infection, and MS, by reducing exposure to helminthes and
other immune-modulating infections. This hypothesis, known as the hygiene hypoth-
esis, could explain why MS is rare in the tropics and subtropics where sanitation
tends to be low. If hygiene was the common cause of IM and MS, however, indi-
viduals who escape EBV infection (lack of EBV infection being a marker of high
hygiene) should have a high MS risk (Fig. 1). In striking contrast with this predic-
tion, there is compelling evidence that individuals who are not infected with EBV
rarely, if ever, develop MS. This evidence includes the following:
(i) Consistent results of numerous case–control studies demonstrating
extremely low odds of MS in individuals with negative EBV serology. In a
meta-analysis including 13 studies, the odds ratio for MS in EBV-negative
individuals was 0.06 (95 % confidence interval: 0.03–0.13; p value <10−8)
(Ascherio and Munger 2007a). This low MS risk in EBV-negative indi-
viduals and the increased risk in individuals with a history of IM imply a
dramatic increase in MS risk following IM, which cannot be attributed to
hygiene alone (Fig. 2).
Fig. 1 The hygiene hypothesis of MS causation. a The formulation of the hygiene hypothesis
that states MS and IM arise from the common cause of “high hygiene” in childhood and are asso-
ciated due to this common cause. b The “EBV variant” of the hygiene hypothesis, which states
that high hygiene in childhood increases the likelihood of a late age at infection with EBV (IM),
which then leads to an increased risk of MS. Current epidemiological data support this latter for-
mulation of the hypothesis. Source Ascherio and Munger, J Neuroimmune Pharmacol 2010
RR of MS according to EBV infection and history of mononucleosis.
2.3*
2
RR of MS
1.0 (Ref)
1
0.06
0
EBV positive, no history EBV negative EBV positive, history of
of mononucleosis mononucleosis
Mostly infected with High hygiene/sanitation, escaped EBV

EBV in early infection in early childhood
childhood
Fig. 2 Relative risk (RR) of developing MS according to EBV infection and history of mononu-
cleosis. Bars represent the 95 % confidence intervals of the RR estimates. †,*p < 10−8. Data from
Thacker et al. (2006), Ascherio and Munger (2007a). Reprinted from Thieme Medical Publish-
ers, Inc., Ascherio and Munger, Semin Neurol 2008:28(1);17–28
Fig. 3 a No incident cases

of MS were observed among
individuals who were EBV
seronegative at recruitment
and did not seroconvert
during follow-up. In contrast,
ten incident MS cases
were confirmed after EBV
seroconversion. b Time
between first EBV positive
serum and MS onset. The
shortest interval between
seroconversion and onset
of first MS symptoms was
>21 months. Data from Levin
et al. (2010)
(ii) Investigations in pediatric onset MS. The odds ratio comparing children with
negative EBV serology to children with evidence of past EBV infection was
0.11 (p < 0.001) in a study in Canada (Alotaibi et al. 2004), 0.04 (p < 0.001)
in a study in Germany (Pohl et al. 2006), and 0.36 (p = 0.02) in a multisite
international study (Banwell et al. 2007b).
(iii) The demonstration in a longitudinal study based on the Department of Defense
Serum Repository with samples from over seven million young adults that
individuals who are EBV negative do not appear to develop MS until after
they seroconvert for EBV and that the onset of MS symptoms occurs only sev-
eral months after EBV seroconversion (Fig. 3) (Levin et al. 2010).
Because the extremely low incidence of MS in EBV-negative individuals is critical to
determine causality, it is important to explore alternative explanations, which include:
(i) MS causes an activation of the immune system and increased antibody titers
against multiple antigens. The higher titer of anti-EBV antibodies among
cases reduces the number of false negative serology, creating a spuri-
ous difference in prevalence of EBV infection between MS cases and con-
trols. This argument has some foundation, because it has been documented
that some individuals infected with EBV are indeed serologically negative
(Savoldo et al. 2002). However, longitudinal studies of adults with nega-
tive EBV serology have demonstrated that these individuals seroconvert at
high rates (10–11 % per year) and have high IM incidence when exposed
to EBV (Balfour et al. 2013), which is consistent with their experiencing a
primary infection. Similarly, EBV-negative adolescents are at high risk of
IM and thus unlikely to have false negative serology (Balfour et al. 2013).
Finally, this difference in prevalence of infection is unique to EBV and is not

seen for other viruses, including cytomegalovirus (CMV) and other herpes
viruses (Ascherio and Munger 2007a).
(ii) Higher rate of EBV positivity among MS cases is due to false positives. This
explanation is unlikely because serological results are based on a combina-
tion of highly specific tests. Further, there is no evidence of IM occurring in
individuals with MS.
(iii) Individuals who are EBV negative are genetically resistant to both EBV
infection and MS. This possibility is ruled out by the results of a longitu-
dinal study that demonstrated that EBV-negative young adults are suscep-
tible to both EBV infection and MS (Levin et al. 2010). Pediatric MS data
also provide compelling evidence against this explanation, because almost
all EBV-negative children will become EBV-positive adults, which proves
their genetic susceptibility. Some HLA-DR alleles have been linked to risk
of IM (Ramagopalan et al. 2011) or anti-Epstein–Barr virus nuclear anti-
gen (EBNA)1 IgG titers (Waubant et al. 2013; Rubicz et al. 2013), but none
of these explain the associations of these factors with MS. Rather, MS-risk
alleles appear to have additive or multiplicative effects with history of IM
(Disanto et al. 2013; Simon et al. 2014) and anti-EBNA1 titers (De Jager
et al. 2008; Sundqvist et al. 2012; Sundstrom et al. 2009).
(iv) Increased susceptibility to EBV infection is a feature of early, preclinical
MS—i.e., MS causes EBV infection. This theoretical possibility is difficult
to exclude empirically, but it is rather implausible. There is no evidence
that MS increases susceptibility to infection in general or to EBV specifi-
cally. Also, there is no evidence of EBV infection after the onset of MS.
Most importantly, it has been demonstrated in a longitudinal study of EBV-
negative young adults that MS occurs only after EBV infection, with the
onset of the first MS symptoms occurring at least 21 months after serocon-
version (Fig. 3) (Levin et al. 2010).
Artifacts and alternative explanations excluded, the exceedingly low MS inci-
dence in EBV-negative individuals provides compelling evidence that EBV plays a
causal role in most cases of MS.
2.3 Potential Mechanisms Relating EBV to MS
The molecular mechanisms linking EBV infection to MS have not been elucidated
though numerous hypotheses have been proposed. Some of these hypotheses,
however, do not explain several observations emerging from epidemiological and
clinical studies, which include:
(i) MS has not been reported as a complication of immunosuppression in post-
transplant lymphoproliferative disease or after prolonged treatment with immu-
nosuppressive drugs. In fact, immunosuppression is used to treat MS that is
resistant to first-line treatment.
(ii) There is a lag of at least several months between EBV infection and onset of
the first MS symptoms, as demonstrated in a longitudinal study (Levin et al.
2010), and, indirectly, by lack of reported MS cases in children or adults
with evidence of recent EBV infection (Yea et al. 2013).
(iii) EBV viral load in serum or peripheral blood cells is only modestly
increased in MS (Wagner et al. 2004; Buljevac et al. 2005; Lindsey et al.
2009) (although children with MS shed EBV in saliva more frequently than
healthy controls) (Yea et al. 2013).
(iv) Monoclonal antibodies (natalizumab) that block α4 integrin and thus prevent
T-cell migration into the central nervous system (CNS) provide a very effec-
tive treatment against MS relapses (Polman et al. 2006). Notably, interruption
of treatment is associated with a rebound of disease activity (Fox et al. 2014).
(v) Monoclonal anti-CD20 antibodies that deplete B cells (rituximab) are
extremely effective in reducing MS relapses (Hauser et al. 2008). Rituximab
does not directly deplete plasma cells, and therefore, its effectiveness within
a few weeks and before any reduction in immunoglobulin titers suggests that
the role of B cells in MS goes beyond antibody production (von Budingen
et al. 2011). This role may include antigen-presenting activity and release of
cytokines or cytotoxic factors (Lisak et al. 2012).
(vi) Serum antibody titers against the EBV nuclear antigens (EBNA com-
plex, EBNA-1, and EBNA-2) in healthy young adults are strongly related
to risk of developing MS (Ascherio et al. 2001). This association has been
confirmed in numerous independent longitudinal studies (Sundstrom et al.
2004; Levin et al. 2005; DeLorenze et al. 2006), and it is not explained by
the MS-risk haplotype HLA-DR1501 (De Jager et al. 2008). In the largest
investigation, MS risk was up to 30-fold higher in individuals with the high-
est titers of anti-EBNA complex antibodies as compared to those with the
lowest (Munger et al. 2011).
The above observations suggest that uncontrolled lytic viral replication or over-
proliferation of EBV-infected B cells is not the primary cause of MS, but rather
the pathological process is driven by the immune response to EBV, including T
cells, B cells, and antibodies. The rebound of inflammatory activity follow-
ing interruption of natalizumab, which keeps T cells, including EBV-specific
T cells, out of the CNS, may result from increased EBV replication in the CNS
during treatment. The several months interval between EBV infection and MS
onset is consistent with an important specific role of anti-EBNA1 antibodies
and anti-EBNA1 CD4+ T cells, which are usually absent during acute infection
and increase over a period of months after recovery (Long et al. 2013). Notably,
CD4+ T cells specific for EBNA1 peptides, which are an important part of
immune control of EBV in healthy individuals, have been found to be increased
in frequency and to have enhanced proliferation capacity, interferon-γ produc-
tion, and broadened specificity in individuals with MS than in HLA-DR and
demographically matched healthy controls (Lunemann et al. 2006). Further, these
cells have been found to have a T-helper 1 central memory or effector memory
phenotype and to recognize myelin antigens more frequently than other antigens
not associated with MS (Lunemann et al. 2008). There is thus a convergence of
epidemiological, clinical, and immunological evidence that anti-EBNA1 antibod-
ies and CD4+ positive EBNA1-specific T cells cross-reacting with myelin anti-
gens contribute to the pathological process in MS. CD4+ T cells recognizing other
EBV antigens and myelin epitopes have also been reported in blood (Lang et al.
2002) and the cerebrospinal fluid (Holmoy and Vartdal 2004).
These findings do not exclude an important role for EBV-specific CD8+ T
cells, although comparisons of their frequency and function in blood and cerebro-
spinal fluid (CSF) from individuals with MS and matched healthy controls have
given somewhat mixed results, which could be in part attributable to methodologi-
cal differences (Hollsberg et al. 2003; Gronen et al. 2006; Jilek et al. 2008, 2012;
Jaquiery et al. 2010; Angelini et al. 2013; Pender et al. 2014a; Lossius et al. 2014).
B cells must also play an important role, either through their antigen-presentation
activity or other mechanisms. In particular, EBV-infected B cells have a survival
advantage and even when encoding cross-reacting antibodies they could pass the
checkpoints that eliminate most autoreactive cells (Pender 2003). Other mecha-
nisms that could relate EBV infection to MS include activation of superantigens
such as HERV-K18 (Tai et al. 2008) or induction of autoimmune responses against
alpha–beta crystallin, an important antigen in CNS myelin (van Sechel et al. 1999).
According to a report in 2007, large numbers of EBV-infected B cells were
found postmortem in meningeal follicles and MS lesions in the brain of the major-
ity of patients with relapsing–remitting or secondary progressive MS (Serafini et al.
2007). The more active lesions also showed evidence of lytic infection and cytotoxic
responses to EBV-infected B cells, suggesting that these cells drive MS pathology.
Several discordant reports (Willis et al. 2009; Peferoen et al. 2010; Sargsyan et al.
2010) raised questions on these findings (Ascherio and Bar-Or 2010), but the pres-
ence of latently infected EBV-positive cells in perivascular infiltrate of all active MS
lesions has been recently confirmed in a new rigorous investigation (Tzartos et al.
2012). By itself, the presence of EBV-positive cells in MS lesions does not prove
that these cells have a causal role, because these cells could be attracted to areas
of inflammation by locally produced cytokines, but when considered in the context
of the epidemiological evidence, it provides a plausible scenario of MS causation.
Infected B cells, in addition to activating EBV-specific T cells, can also promote
inflammation by releasing Epstein–Barr virus-encoded small RNAs (EBERs)
that bind to the Toll-like receptor 3 resulting in activation of innate immunity and
interferon-α production (Iwakiri et al. 2009; Tzartos et al. 2012).
2.4 Implications for MS Prevention and Treatment
If, as we propose, EBV infection is a component cause of most cases of MS, pre-
vention of EBV infection would be expected to substantially reduce MS incidence.
Although complete prevention of EBV infection is not in the foreseeable future, a
vaccine under development was found to be effective in preventing IM in young

adults exposed to EBV (Moutschen et al. 2007; Sokal et al. 2007). Conceivably,
by reducing the intensity of the immune response to EBV such a vaccine could
reduce MS risk, but this may depend on the specific effects of the vaccine on the
relevant immune responses. Theoretically, the incidence of both IM and MS could
also be reduced by facilitating EBV infection in early childhood, but in the case of
MS, this intervention would only be partially effective. Whether EBV continues
to play a pathological role after MS onset or whether it contributes to initiate a
self-perpetuating autoimmune response remains uncertain. The presence of EBV-
infected cells in MS lesions suggests that EBV is an important factor in determin-
ing relapses and MS progression, but evidence relating serological signs of EBV
reactivation with MS relapses or disease activity (Wandinger et al. 2000; Buljevac
et al. 2005; Lindsey et al. 2009) remains sparse and unconvincing. This, however,
does not exclude the possibility that EBV reactivation within the CNS drives MS
pathology. Conflicting results have also been reported on the relation between
EBNA-1 IgG antibodies and MS outcomes (Farrell et al. 2009; Lunemann and
Ascherio 2009). The results of three placebo-controlled trials of the antiviral ala-
cyclovir or its precursor valacyclovir suggested overall a nonsignificant benefit in
the treated patients, but these studies were too small to be conclusive, and most
importantly, alacyclovir does not decrease the number of latently infected B cells
which are likely to drive the immune response and thus MS pathology (Bech et al.
2002; Friedman et al. 2005; Lycke et al. 1996). Treatment of progressive MS
with in vitro expanded autologous EBV-specific CD8+ T cells has been proposed
and attempted in a single patient with progressive MS (Pender et al. 2014b). The
patient did well, but clearly more data are needed to determine whether this is a
valuable therapeutic approach.
2.5 Summary on EBV and MS
The overall epidemiological evidence and the demonstration in a longitudinal

study that EBV-negative individuals do not get MS unless they first acquire EBV
provides unquestionable evidence that EBV is an important component cause of
MS, but it is neither necessary, as proven by rare cases of pediatric MS in EBV-
negative children, nor sufficient. Rather, in most cases, MS is a rare complication
of EBV infection in genetically predisposed individuals. Other factors are likely
to influence MS risk. Some are known, such as vitamin D insufficiency or cig-
arette smoking, whereas others remain to be discovered. However, neither EBV
infection nor other known risk factors provide a convincing explanation for several
features of MS epidemiology. Among the observations that remained unexplained
are the change in risk among migrants (Gale and Martyn 1995), a probable MS
outbreak in the Faroe islands (Kurtzke and Heltberg 2001), and the increasing MS
frequency among African-Americans in the USA (Wallin et al. 2012) and among
women in several countries in North America (Orton et al. 2006) and Europe
(Koch-Henriksen and Sorensen 2010). One possibility is that there are genetic var-
iations in EBV itself that modulate its propensity to cause MS. A few reports have
addressed this hypothesis (Munch et al. 1998; Lindsey et al. 2008; Mechelli et al.
2011) as well as interactions with genetic or environmental factors, which could
include other infectious agents.
3 Systemic Lupus Erythematosus
3.1 Definition and Epidemiology
SLE is a multisystem autoimmune disease with varied presentations, progression,

and symptoms experienced by patients. Some common manifestations include
skin rash (malar and/or discoid), photosensitivity, and arthritis, and many patients
suffer with general pain and fatigue (Tsokos 2011). Despite the heterogeneity of
the disease, the majority of SLE patients have detectable autoantibodies, the most
common being antinuclear antibodies in nearly 100 %, but others include anti-
Sm, anti-Ro (~40 % combined), and anti-dsDNA (Arbuckle et al. 2003). Immune
complexes between autoantibodies and their respective antigens have a key role
in tissue injury (Tsokos 2011), but multiple mechanisms contribute to pathogen-
esis, including abnormalities in B-cell and T-cell signaling and transcription, IL-2
production, and a deficiency in cytotoxic T-cell activity, which predispose to an
increased risk of infection (Tsokos 2011). The incidence of SLE is about ninefold
higher in women than in men and higher among African-Americans and Asians
than Caucasians. The etiology of SLE is unknown, but many potential lifestyle
and infectious agents, including EBV, have been studied (Simard and Costenbader
2007). As for MS, genetic susceptibility plays an important role, as demonstrated
by higher concordance rates in monozygotic twins (24–60 %) than in dizygotic
twins (2–5 %) (Simard and Costenbader 2007); numerous genes have been found
to be associated with risk, but, as for MS, the strongest associations are with poly-
morphisms within the HLA-II region (Armstrong et al. 2014).
3.2 Epidemiological Evidence that EBV Is a Cause of SLE
While, as discussed above, history of IM is a strong, consistent risk factor for MS,
there has been no similar association found with SLE (Strom et al. 1994; Cooper
et al. 2002; Ulff-Moller et al. 2010). Thus, neither hygiene nor age at infection
with EBV seems to be important factors in SLE etiology. There is evidence, how-
ever, that individuals who are not infected with EBV have a low risk of SLE. This
evidence includes the following:
(i) In a meta-analysis of 16 case–control studies, there was a modest but signifi-

cant association between EBV infection as assessed by positive serology for
anti-EBV viral capsid antigen (VCA) IgG and SLE (odds ratio [OR] for EBV
negative = 0.48; p = 0.007) (Hanlon et al. 2014). This result, however, should
be interpreted cautiously, because most of the studies were small and of ques-
tionable quality and there was significant heterogeneity between studies.
(ii) The results of two case–control studies of EBV and pediatric SLE: The
first study included children (age 4–19 years) with SLE in Oklahoma City
(n = 59 SLE, n = 95 controls) and San Diego (n = 58, n = 58 controls)
(James et al. 1997). Few details on control selection were given other
than that the Oklahoma City controls were similar to cases on a variety of
demographic factors and the San Diego controls were siblings of the cases.
Seropositivity to anti-VCA IgG was determined; 99 % of SLE cases versus
70 % of controls were positive for VCA IgG (OR for EBV negative vs. EBV
positive = 0.02; p < 10−10). The second study in a smaller group of chil-
dren (n = 26 cases, n = 26 age-, race-, and sex-matched controls) confirmed
these findings (OR = 0.04, p = 0.00024) (Harley and James 1999). Odds
ratios of this magnitude, which suggest a 25- to 50-fold increase in risk of
SLE following EBV infection, rarely occur as a result of confounding or
other sources of bias. Further, as with the pediatric MS studies, the lower
prevalence of EBV infection in children as compared to adults makes these
investigations particularly robust.
(iii) Evidence from a longitudinal study based on the Department of Defense
Serum Repository (DoDSR) that EBV infection tends to precede not only
the clinical onset of SLE, but also the appearance of autoantibodies. In this
study, which included 130 incident cases of SLE with multiple blood sam-
ples collected before onset of clinical symptoms, positivity for anti-VCA
antibodies preceded or was simultaneously detected with the circulating
autoantibodies that support the diagnosis of SLE (McClain et al. 2005).
In four cases, it was possible to observe a sequence of positivity with the
cases becoming positive for anti-VCA, then anti-EBNA1, and then anti-Ro
(McClain et al. 2005). While this is a small series of cases, these observa-
tions, coupled with the experimental data (see below), support a causal role
for EBV in SLE development.
Because SLE is a systemic disease that affects virtually all the components of the
immune system and increases susceptibility to infection, it is difficult to exclude
the possibility that the higher prevalence of EBV infection among cases is a con-
sequence of SLE rather than its cause and that the increased viral titers and EBV-
specific immune responses in SLE merely reflect the sensitivity of the virus to
perturbation of the immune system (Gross et al. 2005). The conclusion that EBV
infection indeed precedes the first serological evidence of SLE is based mainly
on the four cases from the longitudinal study described above, and it is thus less
compelling than the comparable evidence for MS. On the other hand, as discussed
below, there is clear mechanistic evidence supporting a link between EBV and SLE.
3.3 Potential Mechanisms Relating EBV to SLE
EBV was first proposed as a possible causal agent in SLE in the early 1970s when
a small study among 100 SLE patients (34 of whom were age-, sex-, and race-
matched to 34 tuberculosis patients) found that 62 % had high titers to EBV and
among the matched subset, six times as many SLE as TB patients had elevated
titers to EBV (Evans et al. 1971). However, results of similar studies over the
following years were mixed, and the hypothesis largely fell out of favor. About
20 years later, a series of studies began to elucidate the peptide sequences of some
of the targeted SLE antigens that the autoantibodies recognized, and whether there
was cross-reaction with infectious agents including EBV.
One of the first studies to utilize this approach focused on the anti-Sm B/B′
antibodies that develop in some SLE patients (James et al. 1994). Investigators
systematically deconstructed the Sm B/B′ protein into overlapping octapeptides
and discovered that the peptide sequence PPPGMRPP was the most strongly
antigenic, as shown by reactivity with SLE patients’ anti-Sm B/B′ antibodies.
The EBNA-1 protein contains a similar peptide sequence, PPPGRRP, also recog-
nized by the anti-Sm B/B′ antibodies from SLE patients, suggesting that molecu-
lar mimicry could be a mechanism of SLE development in some patients (James
et al. 1994). James et al. went on to show that this antigenic Sm B/B′ octapep-
tide can induce autoimmunity and epitope spreading in rabbits, leading to an SLE-
like disease, and that rabbits immunized with the EBNA-1 peptide PPPGRRP
develop an early auto-antigen profile followed by epitope spreading similar to
that seen in SLE patients positive for anti-Sm B/B′ antibodies, further support-
ing a link between EBV and SLE (James et al. 1997; Poole et al. 2008). A high
sequence similarity has also been identified between a glycine–arginine repeat in
the C-terminal region of SmD protein and an EBNA-1 peptide. Similarly to results
with Sm B/B′, it has been demonstrated that anti-Sm D antibodies bind this gly-
cine–arginine-rich sequence of EBNA-1 and mice immunized with the EBNA-1
peptide develop autoimmunity (Sabbatini et al. 1993).
In the longitudinal study utilizing the DoDSR mentioned above, the develop-
ment of autoimmune antibodies in 130 SLE cases with serial pre-diagnostic sam-
ples was examined (Arbuckle et al. 2003). One of the earliest appearing antibodies
was to 60 kDa Ro. In a subsequent study building on this, and also including cases
from the Oklahoma Clinical Immunology Serum Repository, 29 SLE cases who
were anti-Ro negative and then became anti-Ro positive were identified. In nine
of these cases, the earliest sample positive for anti-Ro recognized only one epitope
(amino acids 169–180) where in the majority of the remaining cases, the first
positive anti-Ro sample already recognized multiple epitopes including Ro169–180
(McClain et al. 2005). The anti-Ro169-180 antibodies, but not antibodies to the
other Ro epitopes, cross reacted with EBNA-1. Interestingly, there is no over-
lap between the Ro169–180 and the EBNA-1 peptide sequence recognized by the
anti-Ro169–180 antibody; however, immunization of rabbits with the EBNA-1 pep-

tide also developed antibodies to Ro and symptoms consistent with SLE (McClain
et al. 2005).
With experimental evidence suggesting molecular mimicry between antibod-
ies against EBNA-1 and at least three targeted antigens in SLE (Sm B/B′, Sm
D, and Ro), there is a strong case for biological plausibility for EBV as a causal
agent in at least some cases of SLE. Interestingly, however, anti-VCA and anti-
early antigen antibody titers tend to be more prominently elevated in SLE patients
than anti-EBNA1 titers (Hanlon et al. 2014). This observation is consistent with
the hypothesis that there is some degree of defective immune control of EBV in
SLE, as further suggested by a ~40-fold increase in EBV viral load in peripheral
blood cells not explained by immunosuppressive treatment (Kang et al. 2004), an
increase in the frequency of EBV-specific CD4+ T cells producing gamma inter-
feron (Kang et al. 2004), and evidence of defective cytotoxic T-cell activity against
EBV (Kang et al. 2004; Larsen et al. 2011).
3.4 Implications for SLE Prevention and Treatment
Unlike in MS, late age at EBV infection and IM do not appear to be risk factors
for SLE; thus, early exposure to EBV is unlikely to be beneficial. The potential
effects of vaccines that do not provide sterilizing immunity are also uncertain, as
the outcome is likely to depend on the specific effect of the vaccine on the genera-
tion of cross-reacting antibodies. On the other hand, elimination of the infection or
reduction of the viral load by reducing the antigenic stimulation could reduce titers
of autoantibodies and possibly have a clinical benefit.
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Index
A BART, 51, 54, 58, 78, 79, 272, 297, 345, 346,
α5β1, 177 353, 354
A20, 293, 320, 348, 349 BATF, 77
Achong, Bert, 7, 11, 13, 271 B cell
Activation-induced deaminase (AID), 275, B cell factor 1 (EBF1), 291, 297
320 B cell receptor (BCR), 165, 168–170, 173,
Acyclovir, 230, 375 176, 186, 194, 195, 289–291, 296–298,
Adenovirus (Ad), 232 321
AIDS, 21, 52, 54, 58, 121, 153, 185, 271, 325, Bcl2, 51, 55, 296, 320, 329
341 Bcl6, 328, 329
Antibody BcRF1, 107, 111, 128
anti-capsid antibody (VCA), 24–27, 213, BGLF4, 88
214, 220, 225, 226, 231, 271–273, 341, BHRF1, 46, 51, 54, 55, 107
377 BILF1, 223
anti-CD20 antibody (Rituxan, rituximab), Bim, 188, 353
251, 373 BLIMP1, 175, 298
anti-early antigen (EA) antibody, 226 BMRF1, 107, 113, 128
anti-EBNA1 antibody, 372–374, 377, 379 BMRF2, 128
anti-glycoprotein (gp340) antibody, 26 BNLF2a, 50, 55, 107
anti-membrane antigen (MA) antibody, 25 BNRF1, 74–76, 112
anti-Ro antibody, 376–378 BPLF1, 76, 128
anti-Sm antibody, 255 BRLF1, 55, 79, 107, 110, 128, 278, 298
heterophile antibody, 216, 225 Burkitt, Denis, 5, 268
IgA, 27, 165, 257, 341, 343 Burkitt’s lymphoma (BL)
IgM, 25, 165, 220, 221, 225, 226, 243, endemic, 27
257, 271, 272 sporadic, 20, 271
ATM, 76, 87, 88, 260 BZLF1, 51, 55, 83, 84, 86, 88, 104–107,
ATR, 86, 87 109–111, 113, 122, 128, 129, 132, 142,
175, 176, 221, 224, 273, 278, 298, 301
B
Bacterial artificial chromosome (BAC), 232 C
BARF1 Caspase recruitment domain-containing
rhBARF1, 55, 232 protein 11 (CARD11), 246, 260
Barr, Yvonne, 7, 11, 271 CBF1, 77
CCR5, 292

and Immunology 390, DOI 10.1007/978-3-319-22822-8
388 Index
CCR7, 166, 278 EBNA1

Chronic active EBV (CAEBV), 216, 217, 255, gly–ala repeat domain, 53
256 gly–arg-rich region, 224
Clifford, Peter, 18 EBNA2
Cluster of differentiation (CD) associated cellular proteins, 20
CD19, 161, 174, 254, 289, 316, 328 cellular target gene, 56
CD27, 161, 171, 172, 190, 191, 252, 258, C-TAD, 57
278 N-TAD, 57
CD30, 292, 293, 328 protein, 18, 20
CD40, 165, 167, 168, 243, 292, 293, 296, responsive promoter elements, 301
323, 324 EBNA3
CD79a, 316, 321, 328 EBNA3A, 77, 135, 141
CD81, 161 EBNA3B, 126, 135, 136, 141, 143
C-myc, 20, 51, 167, 187, 271, 272, 275, 350 EBNA3C, 141
Complement receptor 1 (CD35), 142 EBNA-LP
Complement receptor 2 (CD21), 74, 142, 161, expression, 76, 107
322 IR1 repeats, 55
Coronin, 254 protein, 76
CtBP, 83 Electron microscopy, 4, 7, 11, 12, 14, 24, 271
CTCF, 77–80, 83, 84, 109 Extracellular signal-regulated kinases (ERK),
CXCR4, 165, 166 52, 347–349, 351
Cytidine 5’-triphosphate synthase 1 Exosome, 106, 297, 349, 350, 355
(CTPS1), 258 EZH2, 85, 89, 108, 320
D F
Dendritic cell (DC) Fcγ receptor 3A (CD16a), 259
conventional (cDC), 228
plasmacytoid (pDC), 232
De The, Guy, 36, 271, 272 G
Diffuse large B cell lymphoma (DLBCL) Galectin-1, 300
ABC type, 317, 320 Galectin-9, 349
germinal center B cell (GCB) type, 317, Ganciclovir, 230
320 GATA Binding Protein 2 (GATA2), 259
Discoidin domain receptor 1 (DDR1), 300, Genome
301 Cp, 54, 77, 78, 80, 86, 105, 107, 110, 345
DNA damage response (DDR), 74, 76, 86, EBV type 1, 57, 133
87, 105 EBV type 2, 133
DNA methylation, 78, 83, 85, 88, 105, Fp, 109
108–110, 112, 113 dyad symmetry element (DS), 80
DNA polymerase (BALF5), 110 family of repeats (FR), 80
DNAse (BGLF5), 80 Qp, 54, 77, 78, 80, 86, 109, 167, 273, 345
Wp, 46, 55, 76, 107, 163, 273, 345
Germinal center model (GCM)
E default program, 159, 166–168, 185, 193
EBER, 54, 79–81, 104, 109, 163, 187, 188, growth program, 159, 160, 167, 185, 188,
294, 296, 297, 327, 328, 343, 345, 374 189
EBP2, 82 latency program, 104, 172, 182, 187, 191,
Episome, 21, 73, 75, 77, 78, 81, 88–90, 157, 272
164, 184, 341–343, 350 Glycoprotein
Epstein, Anthony, 271 gp42, 161
Epstein Barr nuclear antigen (EBNA) gp110, 128, 142
Index 389
gp340/gp350, 26, 50, 55, 128, 142, 143, IRF

161, 229, 231, 232, 278 IRF4, 77, 317, 320, 328
gL, 50 IRF5, 292, 293
IRF7, 77, 347
H
HDAC, 83–85, 87–89, 330 J
Hemophagocytic lymphohistiocytosis (HLH), Janus kinase (JAK), 292, 293, 296, 316, 329,
217, 222, 244, 254 331, 332
Henle, Gertrude, 13, 271 JNK, 112, 347
Henle, Werner, 13, 18, 36, 271
Herpes virus, 11, 12, 105, 106, 108, 111,
153–155, 166, 171, 177, 232, 322, 372 K
Heterologous immunity, 182 Kaposi Sarcoma associated herpes virus
Heterophile antibody (HA), 216, 225 (KSHV, HHV8), 75, 79, 82, 85, 88, 89,
Hodgkin’s lymphoma (HD or HL) 108, 349
classical Klein, Eva, 30, 36
lymphocyte depleted, 288 Klein, George, 25
lymphocyte rich, 288
mixed cellularity, 295
nodular sclerosis, 288, 295, 300, 301, L
288, 289, 293–295, 297, 299–301 Latency
Hodgkin and Reed Sternberg cell (HRS), latency 0, 158
186, 288–291, 293, 294, 296–299 latency I, 158
nodular lymphocyte predominant (NLP), latency II, 158
288 latency III, 158
pediatric, 223, 269, 276, 322 Latent membrane protein (LMP)
Human leucocyte antigen (HLA) LMP1
HLA-A*01, 295 AP1, 53, 74, 82–84
HLA-A*02 (HLA-A2), 295 carboxy terminal activation region 1
HLA-A11, 54, 136, 141 (CTAR1), 347, 348
HLA-DRB1*1501, 372 CTAR2, 52, 347
Human immunodeficiency virus (HIV), 20, LMP2
22, 52, 121, 135, 153, 154, 295, 321, ITAM, 168, 296, 350–352
322, 326, 330 proline-rich motif (PY motif), 351
Humanized mice (huMice) LPS-responsive beige-like anchor (LRBA)
NSG, 222, 230, 231 protein, 254
Lymphoblastoid cell line (LCL), 46–48
Lymphocryptovirus (LCV)
I rhesus LCV (rhLCV), 232
IFI16, 75, 76 Lymphoma in the immunosuppressed (IL), 185
IKK, 292, 320 Lyn, 53
IL-2-inducible T cell kinase (ITK), 252 Lytic EBV infection
IL-6, 320 early (E) gene, 79, 107
IL-10, 167, 250, 301, 320 immediate early (IE) gene, 79, 83, 174,
Immunosurveillance, 172, 174, 185, 267, 276, 178, 181, 298, 300
277 late (L) gene, 85, 86, 111
Infectious mononucleosis (IM, AIM)
Lymphocytosis, 23, 30, 181, 222, 257, 260,
324, 326 M
INKT, 243, 250–254, 258 MagT1, 253
Interferon Major capsid protein (p160, BcLF1), 131
type I (alpha and beta), 220, 232, 374 Malaria
type II (gamma), 223, 373, 379 Plasmodium falciparum, 270
390 Index
MAPK, 299, 300, 347 Primary immunodeficiency

MHC class II, 161 X-linked agammaglobulinemia (XLA), 189
Micro RNA (miRNA), 51, 54, 58, 78, 79, 128, X-linked lymphoproliferative disease
163, 296, 297, 344, 346, 352–355 (XLP), 188, 195, 218, 222, 244, 250,
Middlesex Hospital, 4, 5 251
Minichromosome maintenance complex X-linked immunodeficiency with
component 4 (MCM4), 259 magnesium defect, EBV infection,
Moss, Denis, 30, 36 and neoplasia (XMEN), 222, 253
Multiple sclerosis (MS), 183, 214, 367 Proteasome, 331
Munc PU.1, 57, 78, 80, 290
Munc13-4, 255 PUMA, 353
Munc18-2, 255, 256
R
N Ras, 296, 344
Nasopharyngel carcinoma (NPC) RBP-Jk (CBF1), 57, 77, 80
EBV latency pattern, 325 Regression assay, 33, 276
geography, 19, 122 Rheumatoid arthritis (RA), 183, 367
Natural killer (NK) cells, 222 Rickinson, Alan, 32, 35, 142
NF-Kb
canonical pathway, 292
non-canonical pathway, 292 S
NF-kB inducing kinase (NIK), 347 Saliva, 48, 52, 58, 156, 157, 159, 160,
NK cell lymphoma, 330 174–176, 189, 220, 373
NKG2D, 253 SAP, 188, 218, 244, 250, 251
NKT cells, 250–252 Serine/threonine kinase 4 (STK4), 57, 87,
Non-Hodgkin’s lymphoma (NHL), 316 258, 260
Notch, 77, 168, 291, 293, 297, 300, 301 Severe combined immunodeficiency (SCID),
230, 260, 293
Sjögren’s syndrome, 183
O Small non-coding RNA (scRNA), 163
Oral hairy leukoplakia, 177 SP1, 83, 84, 87
Src, 250, 350–352
STAT, 77, 86, 292, 293, 296, 300, 301, 316,
P 329, 331, 332
P16INK4a, 344 Strains
P53, 21, 87, 88, 297, 344, 352 Akata, 48, 85, 89, 142
PAX5, 76, 80, 291 B95-8, 46, 47, 50, 51, 53, 125, 128, 136,
PCNA, 76, 85, 113 141, 142, 144, 163, 231, 353
PD-L1, 299–301 CAO, 52
Perforin, 254, 255 M81, 46, 48, 142, 143
PI3K, 128, 256, 257, 296, 299, 300 Raji, 47, 48, 51, 84, 85, 89
Plasma cell, 159, 162, 165, 169, 174–176, 183, SUMO, 75
194, 250, 253, 290, 298, 323, 328, 373 Survivin, 297
PML nuclear bodies (PML-NB), 82 Syk, 350
Pope, John, 18, 32 Systemic lupus erythematosus (SLE), 367
Posttransplant lymphoproliferative disease
(PTLD), 121, 133, 135, 144, 185, 322,
328 T
Primary CNS lymphoma, 322 T cells
Primary effusion lymphoma, 322 CD4+ T cell, 295, 299
Index 391
CD8+ T cell, 23, 181, 223, 224, 232, 277, V

278, 300, 323, 331, 375 Valacyclovir, 230, 375
cytotoxic T lymphocytes (CTL), 30, 33, Virus like particle (VLP), 106
34, 36, 37, 50, 141, 166, 174, 178, 181,
182, 186, 190, 192, 195, 253, 255, 301,
342, 346 W
memory T Cell, 23, 30, 33, 37, 258, 278 Waldeyer’s ring, 155, 157, 159, 173–176, 182,
T cell lymphoma, 121, 244 185, 192
Template activating factor Iβ (TAF-Iβ), 82 Wiskott–Aldrich syndrome (WAS), 260
TNF-receptor-associated death domain protein
(TRADD), 347
Toll-like receptor (TLR) X
TLR9, 86, 275 XBP1, 84
TOMM22, 353 X-linked inhibitor of apoptosis (XIAP), 222,
Tonsil, 155–157, 159, 161, 166, 174–177, 182, 251
189–191, 193, 195, 220, 223, 224, 231,
275
Transforming growth factor β (TGFβ), 176, Z
297, 300 ZEB1/2, 83
Transporter associated with antigen processing Zur Hausen, Harald, 13, 19, 24
(TAP), 50
Tumor necrosis factor receptor (TNFR)
associated factors (TRAFs), 347
U
Ubiquitin specific protease 7 (USP7), 82, 83,
352

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Current Topics in Microbiology and Immunology

Christian Münz Editor

Michael B.A. Oldstone

Honorary Editor: Hilary Koprowski (deceased)

Epstein Barr Virus Volume 1

ISSN 0070-217X ISSN 2196-9965 (electronic)

Library of Congress Control Number: 2015948721

Springer Cham Heidelberg New York Dordrecht London

Printed on acid-free paper

Springer International Publishing AG Switzerland is part of Springer Science+Business Media

diseases ranging from infectious mononucleosis and primary immune deficiencies

Zürich, Switzerland Christian Münz

Why and How Epstein-Barr Virus Was Discovered 50 Years Ago. . . . . . . 3

Tumor Associations of EBV—Historical Perspectives. . . . . . . . . . . . . . . . . 17

EBV-Specific Immune Response: Early Research

Part II Virus Genetics and Epigenetics

Epstein–Barr Virus Strain Variation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

Chromatin Structure of Epstein–Barr Virus Latent Episomes . . . . . . . . . 71

The Epigenetic Life Cycle of Epstein–Barr Virus. . . . . . . . . . . . . . . . . . . . 103

Epstein–Barr Virus: From the Detection of Sequence Polymorphisms

Part III Viral Infection and Associated Diseases

EBV Persistence—Introducing the Virus. . . . . . . . . . . . . . . . . . . . . . . . . . . 151

Infectious Mononucleosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211

Primary Immunodeficiencies Associated with EBV Disease. . . . . . . . . . . . 241

Burkitt’s Lymphoma. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267

Contribution of the Epstein-Barr Virus to the Pathogenesis

The Role of EBV in the Pathogenesis of Diffuse Large

Nasopharyngeal Carcinoma: An Evolving Role

EBV and Autoimmunity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365

© Springer International Publishing Switzerland 2015 3

1.1 Early Chance Events Essential for Both “Why”

1.2 A Subsequent Key Chance Leading to “Why”

Fig. 1 The Middlesex

Fig. 2 Photograph of the

2 The Search for a Virus

Fig. 3 Inspection at Bareilly

2.1 Reflections on Research Funding in the 1960s

2.2 The Beginning of “How”—Persistent Early Failures

Fig. 4 George Ball in 1961,

2.3 An Idea Giving a Glimmer of Hope for “How”

2.4 Chance Provides the Key to “How”

Fig. 5 Wet preparation of

Fig. 6 Photograph of the

2.5 The End of the Beginning to “How”

Shortly after setting up the suspension culture a continuous lymphoma-derived

Fig. 7 Light micrographs

3 The Final Breakthrough to “How”

All efforts to show a virus in EB cells using standard contemporary biological

3.1 “How” the Virus Was Found

Fig. 8 The first electron

Fig. 9 Photograph of the

Fig. 10 M.A. Epstein, B.G.

3.2 Naming the Virus

3.3 Characterization of the Uniqueness of the Virus

In addition to the biological inertness of the virus, its immunological singularity

Table 1 Epstein-Barr 28 March 1964–28 March 2014 30,995

Epstein MA (1958) Composition of the Rous virus nucleoid. Nature 181:1808

© Springer International Publishing Switzerland 2015 17

2 Is EBV a Cancer Virus?

1.1 Early Chance Events Essential for Both “Why”

1.2 A Subsequent Key Chance Leading to “Why”

2 The Search for a Virus

2.1 Reflections on Research Funding in the 1960s

2.2 The Beginning of “How”—Persistent Early Failures

2.3 An Idea Giving a Glimmer of Hope for “How”

2.4 Chance Provides the Key to “How”

2.5 The End of the Beginning to “How”

3 The Final Breakthrough to “How”

3.1 “How” the Virus Was Found

3.2 Naming the Virus

3.3 Characterization of the Uniqueness of the Virus

2 Is EBV a Cancer Virus?

3 What Is the Role of EBV in the Genesis of BL?

4 The Lessons of HART Therapy

1 Humoral Responses to EBV Infection

1.1 Antibody to the Virus Capsid Antigen

1.2 Antibody to the EBV Membrane Antigen

1.3 Antibody to the EBV Early Antigen

1.4 Antibody to EBV Nuclear Antigen

1.5 EBV-Specific IgA Response as a Serological NPC

3 Cellular Response to EBV Infection

3.1 Early Studies to Define an EBV-Specific T Cell Response

3.2 Memory T Cell Response in Healthy Immune

3.3 Defining the First EBV CTL Epitope: Personal

3.4 Early Contributions of Other Investigators

5 Functional Difference Between Type 1 and Type 2 EBNA2................................................ 56

2 History of EBV Sequencing

3 Broad Aspects of Genome Variation—EBV Types,

4 Variation in EBV Genes

4.5 Other Latent Cycle Elements

5 Functional Difference Between Type 1 and Type 2

6 How to Identify Variation Potentially Relevant

3C Chromatin conformation capture

2 History and Discovery of the EBV Latent Episomes

3 Nuclear Entry, DNA Recognition, and Chromatinization

4 Early Viral Gene Expression

5 Chromatin Organization of the Latent Episome

6 OriP as a Chromosome Organizing Element

7 EBNA1 as a Critical Regulator of EBV Chromatin

8 Barriers to Lytic Reactivation

9 Late Gene Transcription and Replication

10 Network and Homeostatic Control of Viral Chromatin

11 Chromatin Modifiers that Disrupt Latency—

12 Variation and Heterogeneity of Viral Chromatin