viruses

Opinion
A Perspective on Organoids for Virology Research
Adithya Sridhar 1,2 , Salvatore Simmini 3 , Carla M. S. Ribeiro 4 , Caroline Tapparel 5,6 ,
Melvin M. Evers 7 , Dasja Pajkrt 1,2,† and Katja Wolthers 1, *,†
1 OrganoVIR Labs, Department of Medical Microbiology, Amsterdam UMC, Location Academic
Medical Center, University of Amsterdam, 1100 AZ Amsterdam, The Netherlands;
a.sridhar@amsterdamumc.nl (A.S.); d.pajkrt@amsterdamumc.nl (D.P.)
2 Department of Pediatric Infectious Diseases, Emma Children’s Hospital, Amsterdam UMC,
Location Academic Medical Center, University of Amsterdam, 1100 AZ Amsterdam, The Netherlands
3 Gastrointestinal Biology Group, STEMCELL Technologies UK Ltd., Cambridge CB28 9TL, UK;
salvatore.simmini@stemcell.com
4 Department of Experimental Immunology, Amsterdam Institute for Infection and Immunity,
Amsterdam UMC, University of Amsterdam, 1100 AZ Amsterdam, The Netherlands;
c.m.ribeiro@amsterdamumc.nl
5 Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva,
1211 Geneva, Switzerland; Caroline.Tapparel@unige.ch
6 Division of Infectious Diseases, Geneva University Hospital, 1205 Geneva, Switzerland
7 Department of Research and Development, uniQure Biopharma B.V., 1105 BE Amsterdam, The Netherlands;
m.evers@uniqure.com
* Correspondence: k.c.wolthers@amsterdamumc.nl
† These authors contributed equally to this work.




Received: 15 October 2020; Accepted: 22 November 2020; Published: 23 November 2020


Abstract: Animal models and cell lines are invaluable for virology research and host–pathogen
interaction studies. However, it is increasingly evident that these models are not sufficient to fully
understand human viral diseases. With the advent of three-dimensional organotypic cultures, it is
now possible to study viral infections in the human context. This perspective explores the potential
of these organotypic cultures, also known as organoids, for virology research, antiviral testing, and
shaping the virology landscape.

Keywords: human organoids; virology; standardization

Because viruses are obligate intracellular parasites, model systems comprising various cellular
components are necessary for elucidating their biology. Historically, animal models and immortalized
(cancerous) cell lines enabled virology research and host–pathogen interaction studies. Although these
model systems immensely contributed to expand our knowledge of virology, the limitations of these
models are clear [1]. Animal models do not adequately reproduce human disease pathology, and
in some instances, viral pathogens have a unique human tropism that cannot be replicated in a
non-natural host. Similarly, cell lines display intrinsic alterations in major signaling pathways, and thus
do not recapitulate the homeostatic functions of a normal cell. Cell lines also lack the structural and
functional complexity of an organ. These deficiencies often lead to a virus adapting to a cell line after a
single passage. This caveat calls for novel validated models that can recapitulate human physiology
and are predictive of human disease. Human organoids and organotypic cultures could address this
unmet need for human model systems in virology research and antiviral testing. Therefore, in this
perspective, we (1) provide an introduction to organoid technology, (2) explore how it could shape the
virology landscape, (3) comment on challenges for widespread implementation, and (4) speculate on
the future of this technology in the context of viral research.

Viruses 2020, 12, 1341; doi:10.3390/v12111341 www.mdpi.com/journal/viruses

Viruses 2020, 12, 1341 2 of 10

1. Organoid Technology
Organoids are self-organized 3D cultures derived from stem cells. They are a miniature and
simplified version of an organ that recapitulates the genetic phenotype, organization, and (limited)
functionality of the organ. They consist of organ-specific cell types with spatially restricted
lineage commitment. Organoids can be derived from induced pluripotent or multipotent tissue
stem cells that can be embryonic, fetal, or adult in origin. Organoids have been established for several
different organ systems: intestine, stomach, lung, liver, thyroid, kidney, blood vessel, brain, prostate,
pancreas, and ovaries. For the sake of simplicity, we include human airway epithelial (HAE) cultures
under this umbrella, as they are widely used in virology. HAE cultures are derived by culturing primary
airway epithelial cells on permeable inserts and differentiating them in an air–liquid interface [2].
HAE cultures, by definition, are not organoids, but these organotypic cultures recapitulate the cellular
heterogeneity and function of the airway, offering the same advantages as those of organoids. The main
benefit of using these stem-cell-derived organoid and HAE models is their ability to mimic pathologies
at the organ level. Furthermore, the use of human stem cells allows for recapitulating human-specific
features that make them relevant for translational studies. For a complete overview of organoid
systems, and their potential and applications, several excellent reviews cover this topic in depth [3–5].

2. Organoids Broaden the Scope for Virology

Specific viruses cause diverse diseases in different animal models, and infection follows distinct
routes within the host. For this reason, it is important to employ the most natural system to study the
effects of viral infections. In order to increase the translatability of results from an in vitro or ex vivo
model to an in vivo situation, it is imperative to rely on results from human-based model technology.
Virus-infected human organoids can provide a more accurate image of what host factors are essential
for the establishment of viral infection in humans, and improve the study of effects between donors with
varying age, sex, or genetic make-up. The identification of such factors is essential in understanding
why some individuals experience only mild disease after a specific viral infection, while others fall
severely ill or even die. If the response to a viral infection can be predicted for individuals or groups,
this leverages more personalized medicine, such as who to hospitalize or treat.

2.1. Organoids Enable Culturing the Unculturable

Most virology research groups use immortalized cell lines to grow viruses. For example, HeLa cells
are widely used to study rhinoviruses that cause respiratory disease, even though this cell line is derived
from human cervical tissue. Therefore, to efficiently grow in these artificial culture models, viruses often
need to adapt. For some viruses, e.g., the norovirus, this adaption is not possible, and despite intensive
trials in multiple cell lines, they remain unculturable. Stem-cell-derived human intestinal organoids
have allowed for the culture of these unculturable or hardly culturable enteric viruses, such as
norovirus, although it is still limited to one round of infection [6]. Similarly, respiratory viruses that
are unculturable in cell lines were successfully isolated from clinical specimens using HAE cultures.
These models were used to grow and characterize human coronavirus HKU1, human bocavirus,
and human rhinovirus C [7–9]. The permissiveness of these models for the rapid propagation of viruses
directly from clinical isolates makes them an interesting and relevant platform for the characterization
of unknown viruses [10].

2.2. Organoids Recapitulate the Natural Virus Host Environment

Viruses can be directly amplified in organoid models from clinical isolates without the need to
mutate or adapt. In contrast to laboratory-adapted viral strains or American Type Culture Collection
(ATCC) strains grown in immortalized cell lines, viruses from infected human materials (such as
feces, blood, or nasopharyngeal swabs), grown only in organoids or HAE, more closely retain their
original characteristics and infectivity profiles [11–13]. Consequently, findings about their tropism,
Viruses 2020, 12, 1341 3 of 10

receptor usage, and innate immunity induction are highly relevant and exclude any bias linked to
laboratory cell adaptation.

2.2.1. Organoids Allow for Identification of Entry Host Receptors and Pathways
The expression of surface molecules may be completely different in immortalized cells or animal
models compared to human host tissue, and results regarding receptor usage can be misleading
and result from lab adaptation [14]. An example of this is the acquired dependency on heparan
sulfate binding through cell-culture adaptation [15]. Only a few immortalized cell lines are polarized,
and do not always recapitulate the receptor distribution observed on polarized organoid cultures or
in vivo situations. Here, organoids allow for dissecting the polarized entry of viruses and their ability
to cross epithelial barriers. The pathogenesis of the measles virus (MV) is an example where this has
long been misunderstood. For years, it was thought on the basis of in vitro findings that the infection
started with entry at the apical side of respiratory epithelia. The first discovered MV cellular receptor
was CD46, a cell-surface molecule ubiquitously expressed by most nucleated cells, but subsequent
studies showed that the CD46-mediated entry is limited to vaccine- and laboratory-adapted MV
strains [16]. Recently, the use of complementary in vitro, ex vivo, and in vivo approaches highlighted
that respiratory epithelial cells cannot be the initial targets of MV because nectin-4, the true receptor
of wild-type MV strains, is only expressed at the basolateral side of epithelial cells, and is thus not
accessible upon entry from the lumen side of respiratory tissue [17]. Epithelial cells are, therefore,
infected later from the basolateral side and, combined with epithelial damage, lead to the release of
particles in the respiratory tract and MV transmission [18].

2.2.2. Infected Organoids Resemble Clinical Observation In Vivo

Brain organoids were extensively used to confirm the neurotropism of the Zika virus and its
preferential infection of neural progenitor cells [19]. Zika virus infections gained global attention
because children born with a Zika virus infection showed severe neurological complications such as
microcephaly [20]. This phenomenon was illustrated with the use of human-brain organoids derived
from induced pluripotent stem cells (iPSC). The Zika virus infection reduced the size of human brain
organoids, mimicking the Zika-virus-induced microcephaly in affected children. Besides increased
insight into the mechanism of Zika virus pathogenesis in humans, efforts are now underway to
investigate routes of infection, neurotropism, neurovirulence, and related effects in iPSC-derived
human-brain organoids for herpes-simplex-virus and human-cytomegalovirus infections [21,22].
Similar to Zika virus infection, neonatal herpes-simplex-virus and congenital-cytomegalovirus
infections can elicit major neurological deficits such as microcephaly. With the use of human-organoid
technology, these viral infections can be more effectively studied, potentially leading to new effective
therapeutic interventions [23].

2.3. Organoids Provide New Insights

The use of intestinal organoids highlighted the critical role of an enterovirus upstream open
reading frame (uORF) present in the 50 UTR genomic region. Many enteroviruses present a small open
reading frame (ORF) upstream of the main polyprotein ORF. Early studies performed in cell lines
concluded that this uORF is not used for translation initiation [24]. Recently, Lulla and colleagues
could show that, although dispensable in cell lines, the small protein encoded by this uORF plays an
essential role for virus release in human intestinal organoids and that viruses lacking this uORF are
attenuated in this model [25]. Further, the specific cellular tropism and response to infection can also be
dissected in tissue culture models. Recent infection studies in intestinal organoids showed that different
enteroviruses infect different cell types and induce a cell-type-specific antiviral response [26,27].
Another interesting advantage of using organoid models is the ability to study the interactions
between codetected pathogens, which is not always possible in cell lines because viruses might be
culturable on only two different cell lines. For example, respiratory syncytial virus grows better on
Viruses 2020, 12, 1341 4 of 10

A549, influenza virus on MDCK, and rhinovirus on HeLa cells; thus, it is difficult to find a cell line
that is equally suited for the growth of these three pathogens [28]. These organoid models can also be
used to assess the effect of host conditions such as age and comorbidities on viral infections [29,30].
Respiratory viruses exacerbate asthma or other respiratory comorbidities such as cystic fibrosis or
chronic obstructive pulmonary disease. HAE infection derived from healthy and asthmatic donors
with rhinovirus highlighted a different airway epithelial structure and inflammatory signaling in
asthmatic patients [29].

2.4. Organoids Are Valuable Tools in the Severe Acute Respiratory Syndrome Coronavirus 2 Pandemic
Organoids were widely applied to study severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2) recapitulating clinical disease and they provide novel insights highlighting their
utility for virology research [31]. HAE cultures were used as a model of the primary replication
site, demonstrating efficient replication in this airway through the infection of ciliated cells [32].
A plaquelike cytopathic effect (CPE) was observed with loss of epithelial integrity, shrinking cilium,
cell fusion, and apoptosis [33]. Therapeutic evaluation on organoid models showed remdesivir and
remdesivir–diltiazem to be effective against SARS-CoV-2 infection [34].
In addition to enabling indepth studies of clinical observations of lung epithelial infection, the
potential to infect secondary tissue was also observed using organoid models. Lamers et al., showed that
SARS-CoV-2 productively infects the human gut epithelium, targeting differentiated enterocytes [35].
Similarly, Stanifer et al., showed productive infection of human intestinal epithelial cells with a robust
intrinsic immune response, and demonstrated the critical role of Type III interferon in controlling
infection [36]. These organoid studies provided novel insights, as the SARS-CoV-2 genome is detectable
in feces even after the virus is not detectable in oropharyngeal swabs, raising concerns of intestinal
infection and potential fecal transmission [37].
Another area of investigation using organoids has been determining the neuroinvasive potential
of SARS-CoV-2 [38]. Epidemiological studies showed that SARS-CoV-2 infection can present with
neurological complications such as headaches, ischemic stroke, and encephalitis along with cranial-
nerve-related complications such as anosmia, hyposmia, and ageusia. This has led researchers
to employ brain organoids for assessing if SARS-CoV-2 has neuroinvasive potential. Zhang and
colleagues showed that SARS-CoV-2, but not SARS-CoV, can infect human neural progenitors [39].
Furthermore, they demonstrated that SARS-CoV-2 can productively infect human brain organoids,
targeting cortical neurons. Similarly, Ramani et al. also showed infection of brain organoids through
neurons, resulting in neuronal death [40]. Moreover, recent data from Pellegrini et al. using choroid
plexus organoids demonstrated potential viral tropism for choroid plexus epithelial cells, resulting in
damage to the epithelium [41]. Damage to this barrier could present a potential route of entry for the
virus into the cerebrospinal fluid and the brain. Taken together, these studies illustrated the promise of
organoids for studying emerging viral infections, and elucidated their full disease potential against
clinical observations.

3. Technical Challenges
While the previous section highlights the potential that organoids hold for virology, they are
still a nascent technology with broader and virology-specific issues that need to be addressed as
summarized below.

3.1. Technical Challenges Related to Virology

Organoids, as they were originally established, are closed round structures embedded in Matrigel® .
These round structures are difficult to infect with viruses: receptors needed for infection are mostly
located inside the organoid. As with HAE cultures, round gut organoids can be transformed to an
open organoid model where cells grow on a Transwell® , so that they can be reached from the upper
Viruses 2020, 12, 1341 5 of 10

and lower sides to establish infections [6,26,42,43]. This model system is ideal for infectious-disease
studies and to test antimicrobial drugs.
Another important consideration is readouts used for analysis after infection. For instance, virus
cultures in primary models often do not result in a CPE. This is likely due to the release of viral particles
in a nonlytic manner, as seen in the case of enterovirus A71. Huang and colleagues demonstrated that
enterovirus A71 infection of human intestinal organoids resulted in viral release through exosomes
rather than lytic processes observed when using a classical RD cell line [44]. Moreover, the production
of infectious viral particles after infection is assessed by performing back titration or plaque assays
using cell lines. However, as described earlier, distinct behavior in primary culture versus cell lines
raises concerns about using titer data from cell lines as a measure for evaluating the production of
infectious viral particles in primary culture. More suitable methods need to be developed for directly
assessing this in primary cultures.

3.2. Standardization
Organoids are generated from primary cells, and culture conditions to maintain appropriate
long-term cell composition and function are based on the knowledge of their in vivo requirements.
For example, intestinal organoid culture systems typically require the activation of Wnt and EGF
pathways, and the inhibition of BMP pathways. However there are variations of culture conditions for
modulating these pathways, resulting in shifts in the cell composition of these organoid systems [3].
A striking example is illustrated by differences in cellular composition observed in human intestinal
organoids derived in different medium formulations. The first described organoids exhibited a high
proportion of stem cells shown by high expression levels of Wnt-related genes such as LGR5, ASCL2,
AXIN2, and OLFM4, and the inhibition of goblet and enterocyte differentiation [45]. The subsequent
withdrawal of Wnt from this medium triggered the generation of differentiated intestinal cell types.
Fujii et al. developed culture media that gave rise to organoids exhibiting increased multilineage
differentiation while sustaining high levels of self-renewal, as demonstrated by the expression of LYZ,
CHGA, and MUC2 with LGR5 in a single culture condition [46].
Another factor that could introduce variability in organoid cultures is related to the starting
material. Varying performance is often seen in different batches of conditioned media because of the
difficulty to precisely control which factors are secreted into the medium. Further, Matrigel® is not
completely defined and may lead to some variability. In addition, the costs of 3D culture systems tend
to be higher than those of standard 2D culture systems due to their complexity. Many laboratories
find alternative approaches that influence the selection of media-component suppliers or the type of
extracellular matrix, and develop internal production of serum-based conditioned media.
Reducing variability is hindered by a lack of widely accepted criteria and guidelines for assessing
the quality and characteristics of organoid cultures that are generated using different reagents.
During the past decade, protocols using different reagents and culture conditions were used for
deriving and culturing organoids from a range of organs [47]. As discussed above, these limitations
of current culture conditions in some of these systems can create bias toward specific cell types
within organs. Thus, it is important to continue to improve and standardize culture media to make
organoids more closely resemble their respective tissues in vivo. In fact, donor-source variability
and organoid cultures derived from these tissue types are intrinsically heterogeneous; the use of
different media can increase the variability and limit the reproducibility of outcomes measured in
downstream applications. This lack of standardization is a major bottleneck and limits the comparison
of results based on organoids generated through different protocols and research groups. A joint
effort by the academic and industrial sectors is needed to optimize media formulations and enable
the production of highly reproducible organoids with the goal of reducing experimental variability,
increasing result accuracy, and reducing the cost of current commercial solutions.
Viruses 2020, 12, 1341 6 of 10

3.3. High Throughput

Apart from the heterogeneous nature of currently developed organoids, the next challenge is
the development of high-throughput platforms that support organoid technology. For the industrial
and commercial use of organoid technology for translational applications such as antiviral screening,
organoid technology needs to be scaled up with standardized quality control while maintaining
the complex nature of the organoids. This quandary is one of the major limitations for widespread
implementation of organoid technology in a commercial setting. For the adequate implementation
of organoid models, validated cell-culture protocols are critically needed that demonstrate organoid
cultures in high-density microplates (96-well, 384-well, or higher) with high quality-controlled standard
performance to facilitate high-throughput screening (HTS). Validated HTS standards would facilitate
reproducibility and reduce associated costs. The HTS methodology would be essential to commercially
increasing the current use of organoids and facilitating personalized medicine.

4. Future Perspectives

4.1. Increasing Complexity

At present, organoids are mainly single-organ systems representing the epithelium. For instance,
gut organoids comprise the gut epithelial layer and lack the mesenchymal or immune-cell elements
present in the gut mucosa [48]. The organoid community is undertaking efforts to address this
issue through the establishment of cocultures of epithelial organoid systems with other organ-
specific components. For instance, the cocultures of immune cells (such as macrophages and T
cells) are possible with organotypic cultures such as intestinal organoids. This expedites studies
on the role of the mucosal compartment and epithelial-immune cell communication in antiviral
immunity [49,50]. Furthermore, the complexity of these in vitro model systems could be further
improved by interconnecting multiple organ systems. Stem-cell-derived organoid models implemented
in an organ-on-chip system enable the modeling of multiorgan complexity such as the gut–brain axis,
allowing for the study of disease progression from primary to secondary infection sites.

4.2. Functionality Measurement

One of the advantages of animal models (in viral research) over human organoids is the availability
of functionality readouts in animal systems. For example, in a monkey model, intravenous EV-A71
resulted in direct neurological signs such as tremor, ataxia, and brain edema [51]. Similarly, in mice
models, poliovirus causes poliomyelitis with paralysis resembling paralytic disease in humans [52].
These functional observations are not yet possible in human organoid models. However, progress was
made over the last couple of years. For example, in human brain organoid technology, Quadrato and
Giandomenico demonstrated that the functional sensory input and motor output of organoids can be
measured [53,54]. This indicates that human cerebral organoids could be useful in studying neural
connectivity and other functional modalities of the brain.

4.3. Replacing the Golden Standard

Currently, 500–1000 animals are needed for optimizing a new antiviral compound for clinical trials.
However, 95% of drugs that proceed to the clinical-trial stage do not make it to the market despite
promising results in animal models. On the basis of the resemblance to the in vivo situation, organoid
models show potential superiority to animal models in predicting efficacy and toxicity of an antiviral
compound and therefore reducing costs in the development of novel antiviral therapies. In the
pharmaceutical industry, selecting and testing lead compounds to assess their potency and safety
typically involves the use of animal models to show in vivo proof of concept and provide evidence for
clinical benefit, which is time-consuming and resource-intensive. For some indications, animal models
might be unavailable or not translatable due to a lack of human components that are required for their
mechanism of action. For systemic indications, disease-relevant, patient-derived cellular models very
Viruses 2020, 12, 1341 7 of 10

often exist. However, for central or peripheral nervous system (CNS/PNS) disorders, relevant in vitro
models derived from patients hardly exist due to the inaccessibility of nervous tissue. To overcome
these limitations, patient stem-cell-derived brain organoids provide an important translational bridge:
a reliable model for CNS/PNS indications in the human context, thereby reducing the number of animal
studies required for both efficacy and mechanism-of-action safety studies, decreasing development
time into the clinic, and increasing the success rate due to the better-translatable in vitro platform.

5. Conclusions
Over the past decade, organoids showed promise as a human model for studying human
viral diseases. However, as a nascent technology, organoids still have room for advancement
and standardization. As the complexity of these model systems increases with cocultures and
organ-on-chip systems, new opportunities and challenges for the field arise, and the virology
landscape benefits.

Author Contributions: Original-draft preparation, A.S., S.S., C.T., C.M.S.R., M.M.E., D.P., and K.W.;
writing—review and editing, A.S., S.S., C.T., C.M.S.R., M.M.E., D.P., and K.W. All authors have read and
agreed to the published version of the manuscript.
Funding: This work was cofunded by OrganoVIR (grant 812673) and the PPP Allowance made available by
Health~Holland, Top Sector Life Sciences and Health, to Amsterdam UMC, location Amsterdam Medical Center
to stimulate public–private partnerships.
Conflicts of Interest: M.E. is an employee and shareholder of uniQure B.V.; S.S. is an employee of STEMCELL™
Technologies UK Ltd. The other authors declare no conflict of interest. The funders had no role in the design of
the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision
to publish the results.

References
1. Louz, D.; Bergmans, H.E.; Loos, B.P.; Hoeben, R.C. Animal models in virus research: Their utility
and limitations. Crit. Rev. Microbiol. 2013, 39, 325–361. [CrossRef]
2. Hasan, S.; Sebo, P.; Osicka, R. A guide to polarized airway epithelial models for studies of host–pathogen
interactions. FEBS J. 2018, 285, 4343–4358. [CrossRef] [PubMed]
3. Kim, J.; Koo, B.K.; Knoblich, J.A. Human organoids: Model systems for human biology and medicine.
Nat. Rev. Mol. Cell Bio. 2020, 21, 571–584. [CrossRef] [PubMed]
4. Schutgens, F.; Clevers, H. Human Organoids: Tools for Understanding Biology and Treating Diseases.
Annu. Rev. Pathol. Mech. 2020, 15, 211–234. [CrossRef] [PubMed]
5. Lehmann, R.; Lee, C.M.; Shugart, E.C.; Benedetti, M.; Charo, R.A.; Gartner, Z.; Hogan, B.; Knoblich, J.;
Nelson, C.M.; Wilson, K.M. Human organoids: A new dimension in cell biology. Mol. Biol. Cell 2019, 30,
1129–1137. [CrossRef]
6. Ettayebi, K.; Crawford, S.E.; Murakami, K.; Broughman, J.R.; Karandikar, U.; Tenge, V.R.; Neill, F.H.;
Blutt, S.E.; Zeng, X.L.; Qu, L.; et al. Replication of human noroviruses in stem cell-derived human enteroids.
Science 2016, 353, 1387–1393. [CrossRef]
7. Pyrc, K.; Sims, A.C.; Dijkman, R.; Jebbink, M.; Long, C.; Deming, D.; Donaldson, E.; Vabret, A.; Baric, R.;
van der Hoek, L.; et al. Culturing the Unculturable: Human Coronavirus HKU1 Infects, Replicates, and
Produces Progeny Virions in Human Ciliated Airway Epithelial Cell Cultures. J. Virol. 2010, 84, 11255–11263.
[CrossRef]
8. Dijkman, R.; Koekkoek, S.M.; Molenkamp, R.; Schildgen, O.; van der Hoek, L. Human Bocavirus Can Be
Cultured in Differentiated Human Airway Epithelial Cells. J. Virol. 2009, 83, 7739–7748. [CrossRef]
9. Hao, W.D.; Bernard, K.; Patel, N.; Ulbrandt, N.; Feng, H.; Svabek, C.; Wilson, S.; Stracener, C.; Wang, K.;
Suzich, J.; et al. Infection and Propagation of Human Rhinovirus C in Human Airway Epithelial Cells.
J. Virol. 2012, 86, 13524–13532. [CrossRef]
10. Farsani, S.M.J.; Deijs, M.; Dijkman, R.; Molenkamp, R.; Jeeninga, R.E.; Ieven, M.; Goossens, H.; van der Hoek, L.
Culturing of respiratory viruses in well-differentiated pseudostratified human airway epithelium as a tool to
detect unknown viruses. Influenza Other Resp. 2015, 9, 51–57. [CrossRef]
Viruses 2020, 12, 1341 8 of 10

11. Tapparel, C.; Sobo, K.; Constant, S.; Huang, S.; Van Belle, S.; Kaiser, L. Growth and characterization of
different human rhinovirus C types in three-dimensional human airway epithelia reconstituted in vitro.
Virology 2013, 446, 1–8. [CrossRef] [PubMed]
12. Bochkov, Y.A.; Watters, K.; Basnet, S.; Sijapati, S.; Hill, M.; Palmenberg, A.C.; Gern, J.E. Mutations in VP1 and
3A proteins improve binding and replication of rhinovirus C15 in HeLa–E8 cells. Virology 2016, 499, 350–360.
[CrossRef] [PubMed]
13. Palmer, S.G.; Porotto, M.; Palermo, L.M.; Cunha, L.F.; Greengard, O.; Moscona, A. Adaptation of Human
Parainfluenza Virus to Airway Epithelium Reveals Fusion Properties Required for Growth in Host Tissue.
MBio 2012, 3. [CrossRef] [PubMed]
14. Karelehto, E.; Cristella, C.; Yu, X.; Sridhar, A.; Hulsdouw, R.; de Haan, K.; van Eijk, H.; Koekkoek, S.; Pajkrt, D.;
de Jong, M.D.; et al. Polarized Entry of Human Parechoviruses in the Airway Epithelium. Front. Cell
Infect. Microbiol. 2018, 8. [CrossRef]
15. Cagno, V.; Tseligka, E.D.; Jones, S.T.; Tapparel, C. Heparan Sulfate Proteoglycans and Viral Attachment: True
Receptors or Adaptation Bias? Viruses 2019, 11, 596. [CrossRef]
16. Dorig, R.E.; Marcil, A.; Chopra, A.; Richardson, C.D. The Human Cd46 Molecule Is a Receptor for
Measles-Virus (Edmonston Strain). Cell 1993, 75, 295–305. [CrossRef]
17. Ludlow, M.; Rennick, L.J.; Sarlang, S.; Skibinski, G.; McQuaid, S.; Moore, T.; de Swart, R.L.; Duprex, W.P.
Wild-type measles virus infection of primary epithelial cells occurs via the basolateral surface without
syncytium formation or release of infectious virus. J. Gen. Virol. 2010, 91, 971–979. [CrossRef]
18. Laksono, B.M.; de Vries, R.D.; Duprex, W.P.; de Swart, R.L. Measles pathogenesis, immune suppression and
animal models. Curr. Opin. Virol. 2020, 41, 31–37. [CrossRef]
19. Qian, X.Y.; Nguyen, H.N.; Jacob, F.; Song, H.J.; Ming, G.L. Using brain organoids to understand Zika
virus-induced microcephaly. Development 2017, 144, 952–957. [CrossRef]
20. Wen, Z.X.; Song, H.J.; Ming, G.L. How does Zika virus cause microcephaly? Gene Dev. 2017, 31, 849–861.
[CrossRef]
21. D’Aiuto, L.; Bloom, D.C.; Naciri, J.N.; Smith, A.; Edwards, T.G.; McClain, L.; Callio, J.A.; Jessup, M.; Wood, J.;
Chowdari, K.; et al. Modeling Herpes Simplex Virus 1 Infections in Human Central Nervous System
Neuronal Cells Using Two- and Three-Dimensional Cultures Derived from Induced Pluripotent Stem Cells.
J. Virol. 2019, 93. [CrossRef] [PubMed]
22. Brown, R.M.; Rana, P.S.J.B.; Jaeger, H.K.; O’Dowd, J.M.; Balemba, O.B.; Fortunato, E.A. Human Cytomegalovirus
Compromises Development of Cerebral Organoids. J. Virol. 2019, 93. [CrossRef] [PubMed]
23. Depla, J.A.; Sogorb-Gonzalez, M.; Mulder, L.A.; Heine, V.M.; Konstantinova, P.; van Deventer, S.J.;
Wolthers, K.C.; Pajkrt, D.; Sridhar, A.; Evers, M.M. Cerebral Organoids: A Human Model for AAV
Capsid Selection and Therapeutic Transgene Efficacy in the Brain. Mol. Ther. Meth. Clin. D 2020, 18, 167–175.
[CrossRef] [PubMed]
24. Meerovitch, K.; Nicholson, R.; Sonenberg, N. Invitro Mutational Analysis of Cis-Acting Rna Translational
Elements within the Poliovirus Type-2 50 Untranslated Region. J. Virol. 1991, 65, 5895–5901. [CrossRef]
25. Lulla, V.; Dinan, A.M.; Hosmillo, M.; Chaudhry, Y.; Sherry, L.; Irigoyen, N.; Nayak, K.M.; Stonehouse, N.J.;
Zilbauer, M.; Goodfellow, I.; et al. An upstream protein-coding region in enteroviruses modulates virus
infection in gut epithelial cells. Nat. Microbiol. 2019, 4, 280–292. [CrossRef]
26. Drummond, C.G.; Bolock, A.M.; Ma, C.R.; Luke, C.J.; Good, M.; Coyne, C.B. Enteroviruses infect human
enteroids and induce antiviral signaling in a cell lineage-specific manner. Proc. Natl. Acad. Sci. USA 2017,
114, 1672–1677. [CrossRef]
27. Essaidi-Laziosi, M.; Brito, F.; Benaoudia, S.; Royston, L.; Cagno, V.; Fernandes-Rocha, M.; Piuz, I.; Zdobnov, E.;
Huang, S.; Constant, S.; et al. Propagation of respiratory viruses in human airway epithelia reveals persistent
virus-specific signatures. J. Allergy Clin. Immun. 2018, 141, 2074–2084. [CrossRef]
28. Essaidi-Laziosi, M.; Geiser, J.; Huang, S.; Constant, S.; Kaiser, L.; Tapparel, C. Interferon-Dependent and
Respiratory Virus-Specific Interference in Dual Infections of Airway Epithelia. Sci. Rep. 2020, 10, 10246.
[CrossRef]
29. Bai, J.W.; Smock, S.L.; Jackson, G.R.; MacIsaac, K.D.; Huang, Y.S.; Mankus, C.; Oldach, J.; Roberts, B.; Ma, Y.L.;
Klappenbach, J.A.; et al. Phenotypic Responses of Differentiated Asthmatic Human Airway Epithelial
Cultures to Rhinovirus. PLoS ONE 2015, 10, e0118286. [CrossRef]
Viruses 2020, 12, 1341 9 of 10

30. Kessler, M.; Hoffmann, K.; Fritsche, K.; Brinkmann, V.; Mollenkopf, H.J.; Thieck, O.; da Costa, A.R.T.;
Braicu, E.I.; Sehouli, J.; Mangler, M.; et al. Chronic Chlamydia infection in human organoids increases
stemness and promotes age-dependent CpG methylation. Nat. Commun. 2019, 10, 1194. [CrossRef]
31. Clevers, H. COVID-19: Organoids go viral. Nat. Rev. Mol. Cell Bio. 2020, 21, 355–356. [CrossRef] [PubMed]
32. Milewska, A.; Kula-Pacurar, A.; Wadas, J.; Suder, A.; Szczepanski, A.; Dabrowska, A.; Owczarek, K.;
Marcello, A.; Ochman, M.; Stacel, T.; et al. Replication of Severe Acute Respiratory Syndrome Coronavirus 2
in Human Respiratory Epithelium. J. Virol. 2020, 94. [CrossRef] [PubMed]
33. Zhu, N.; Wang, W.L.; Liu, Z.D.; Liang, C.Y.; Wang, W.; Ye, F.; Huang, B.Y.; Zhao, L.; Wang, H.J.; Zhou, W.M.; et al.
Morphogenesis and cytopathic effect of SARS-CoV-2 infection in human airway epithelial cells. Nat. Commun.
2020, 11, 3910. [CrossRef] [PubMed]
34. Pizzorno, A.; Padey, B.; Julien, T.; Trouillet-Assant, S.; Traversier, A.; Errazuriz-Cerda, E.; Fouret, J.; Dubois, J.;
Gaymard, A.; Lescure, F.X.; et al. Characterization and Treatment of SARS-CoV-2 in Nasal and Bronchial
Human Airway Epithelia. Cell Rep. Med. 2020, 1, 100059. [CrossRef]
35. Lamers, M.M.; Beumer, J.; van der Vaart, J.; Knoops, K.; Puschhof, J.; Breugem, T.I.;
Ravelli, R.B.G.; Paul van Schayck, J.; Mykytyn, A.Z.; Duimel, H.Q.; et al. SARS-CoV-2 productively infects
human gut enterocytes. Science 2020, 369, 50–54. [CrossRef]
36. Stanifer, M.L.; Kee, C.; Cortese, M.; Zumaran, C.M.; Triana, S.; Mukenhirn, M.; Kraeusslich, H.G.;
Alexandrov, T.; Bartenschlager, R.; Boulant, S. Critical Role of Type III Interferon in Controlling SARS-CoV-2
Infection in Human Intestinal Epithelial Cells. Cell Rep. 2020, 32, 107863. [CrossRef]
37. Wu, Y.; Guo, C.; Tang, L.; Hong, Z.; Zhou, J.; Dong, X.; Yin, H.; Xiao, Q.; Tang, Y.; Qu, X.; et al. Prolonged
presence of SARS-CoV-2 viral RNA in faecal samples. Lancet Gastroenterol. Hepatol. 2020, 5, 434–435.
[CrossRef]
38. DosSantos, M.F.; Devalle, S.; Aran, V.; Capra, D.; Roque, N.R.; Coelho-Aguiar, J.M.; Spohr, T.; Subilhaga, J.G.;
Pereira, C.M.; D’Andrea Meira, I.; et al. Neuromechanisms of SARS-CoV-2: A Review. Front. Neuroanat.
2020, 14, 37. [CrossRef]
39. Zhang, B.Z.; Chu, H.; Han, S.; Shuai, H.; Deng, J.; Hu, Y.F.; Gong, H.R.; Lee, A.C.; Zou, Z.; Yau, T.; et al.
SARS-CoV-2 infects human neural progenitor cells and brain organoids. Cell Res. 2020, 30, 928–931.
[CrossRef]
40. Ramani, A.; Muller, L.; Ostermann, P.N.; Gabriel, E.; Abida-Islam, P.; Muller-Schiffmann, A.; Mariappan, A.;
Goureau, O.; Gruell, H.; Walker, A.; et al. SARS-CoV-2 targets neurons of 3D human brain organoids.
EMBO J. 2020, 39, e106230. [CrossRef]
41. Pellegrini, L.; Bonfio, C.; Chadwick, J.; Begum, F.; Skehel, M.; Lancaster, M.A. Human CNS barrier-forming
organoids with cerebrospinal fluid production. Science 2020, 369. [CrossRef]
42. Roodsant, T.; Navis, M.; Aknouch, I.; Renes, I.B.; van Elburg, R.M.; Pajkrt, D.; Wolthers, K.C.; Schultsz, C.;
van der Ark, K.C.H.; Sridhar, A.; et al. A Human 2D Primary Organoid-Derived Epithelial Monolayer Model
to Study Host–Pathogen Interaction in the Small Intestine. Front. Cell Infect. Microbiol. 2020, 10. [CrossRef]
[PubMed]
43. Stanifer, M.L.; Mukenhirn, M.; Muenchau, S.; Pervolaraki, K.; Kanaya, T.; Albrecht, D.; Odendall, C.;
Hielscher, T.; Haucke, V.; Kagan, J.C.; et al. Asymmetric distribution of TLR3 leads to a polarized immune
response in human intestinal epithelial cells. Nat. Microbiol. 2020, 5, 181–191. [CrossRef] [PubMed]
44. Huang, H.I.; Lin, J.Y.; Chiang, H.C.; Huang, P.N.; Lin, Q.D.; Shih, S.R. Exosomes Facilitate Transmission
of Enterovirus A71 From Human Intestinal Epithelial Cells. J. Infect. Dis. 2020, 222, 456–469. [CrossRef]
[PubMed]
45. Sato, T.; Stange, D.E.; Ferrante, M.; Vries, R.G.J.; van Es, J.H.; van den Brink, S.; van Houdt, W.J.; Pronk, A.;
van Gorp, J.; Siersema, P.D.; et al. Long-term Expansion of Epithelial Organoids From Human Colon,
Adenoma, Adenocarcinoma, and Barrett’s Epithelium. Gastroenterology 2011, 141, 1762–1772. [CrossRef]
46. Fujii, M.; Matano, M.; Toshimitsu, K.; Takano, A.; Mikami, Y.; Nishikori, S.; Sugimoto, S.; Sato, T.
Human Intestinal Organoids Maintain Self-Renewal Capacity and Cellular Diversity in Niche-Inspired
Culture Condition. Cell Stem. Cell 2018, 23, 787–793. [CrossRef]
47. Lancaster, M.A.; Huch, M. Disease modelling in human organoids. Dis. Models Mech. 2019, 12, dmm039347.
[CrossRef]
48. Garcia-Rodriguez, I.; Sridhar, A.; Pajkrt, D.; Wolthers, K.C. Put Some Guts into It: Intestinal Orgnaoid Models
to Study Viral Infection. Viruses 2020, 12, 1288. [CrossRef]
Viruses 2020, 12, 1341 10 of 10

49. Noel, G.; Baetz, N.W.; Staab, J.F.; Donowitz, M.; Kovbasnjuk, O.; Pasetti, M.F.; Zachos, N.C. A primary human
macrophage-enteroid co-culture model to investigate mucosal gut physiology and host–pathogen interactions.
Sci. Rep. 2017, 7, 46790. [CrossRef]
50. Schreurs, R.R.C.E.; Baumdick, M.E.; Sagebiel, A.F.; Kaufmann, M.; Mokry, M.; Klarenbeek, P.L.; Schaltenberg, N.;
Steinert, F.L.; van Rijn, J.M.; Drewniak, A.; et al. Human Fetal TNF-alpha-Cytokine-Producing CD4(+) Effector
Memory T Cells Promote Intestinal Development and Mediate Inflammation Early in Life. Immunity 2019, 50,
462–476. [CrossRef]
51. Nagata, N.; Iwasaki, T.; Ami, Y.; Tano, Y.; Harashima, A.; Suzaki, Y.; Sato, Y.; Hasegawa, H.; Sata, T.;
Miyamura, T.; et al. Differential localization of neurons susceptible to enterovirus 71 and pollovirus type 1 in
the central nervous system of cynomolgus monkeys after intravenous inoculation. J. Gen. Virol. 2004, 85,
2981–2989. [CrossRef] [PubMed]
52. Ida-Hosonuma, M.; Iwasaki, T.; Taya, C.; Sato, Y.; Li, J.F.; Nagata, N.; Yonekawa, H.; Koike, S. Comparison of
neuropathogenicity of poliovirus in two transgenic mouse strains expressing human poliovirus receptor
with different distribution patterns. J. Gen. Virol. 2002, 83, 1095–1105. [CrossRef] [PubMed]
53. Quadrato, G.; Nguyen, T.; Macosko, E.Z.; Sherwood, J.L.; Yang, S.M.; Berger, D.R.; Maria, N.; Scholvin, J.;
Goldman, M.; Kinney, J.P.; et al. Cell diversity and network dynamics in photosensitive human brain
organoids. Nature 2017, 545, 48–53. [CrossRef] [PubMed]
54. Giandomenico, S.L.; Mierau, S.B.; Gibbons, G.M.; Wenger, L.M.D.; Masullo, L.; Sit, T.; Sutcliffe, M.;
Boulanger, J.; Tripodi, M.; Derivery, E.; et al. Cerebral organoids at the air–liquid interface generate diverse
nerve tracts with functional output. Nat. Neurosci. 2019, 22, 669–679. [CrossRef] [PubMed]

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional
affiliations.

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).