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Volume 10
Issue 12
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Viruses, Volume 10, Issue 12 (December 2018) – 71 articles

Cover Story (view full-size image): We employed two different approaches to mutate Sulfolobus islandicus rod-shaped virus 2 (SIRV2). The anti-CRISPR (Acr) gene acrID1 was used as a selection marker to knock out genes from an acrID1-null mutant of SIRV2. Moreover, the endogenous CRISPR-Cas systems of its host were repurposed to knock out all the accessory genes. The results are relevant for future functional studies, and such approaches are applicable to other virus–host systems. View Paper here.
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9 pages, 1435 KiB  
Article
Engineering RNA Virus Interference via the CRISPR/Cas13 Machinery in Arabidopsis
by Rashid Aman, Ahmed Mahas, Haroon Butt, Zahir Ali, Fatimah Aljedaani and Magdy Mahfouz
Viruses 2018, 10(12), 732; https://doi.org/10.3390/v10120732 - 19 Dec 2018
Cited by 86 | Viewed by 9673
Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems are key immune mechanisms helping prokaryotic species fend off RNA and DNA viruses. CRISPR/Cas9 has broad applications in basic research and biotechnology and has been widely used across eukaryotic species for genome [...] Read more.
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems are key immune mechanisms helping prokaryotic species fend off RNA and DNA viruses. CRISPR/Cas9 has broad applications in basic research and biotechnology and has been widely used across eukaryotic species for genome engineering and functional analysis of genes. The recently developed CRISPR/Cas13 systems target RNA rather than DNA and thus offer new potential for transcriptome engineering and combatting RNA viruses. Here, we used CRISPR/LshCas13a to stably engineer Arabidopsis thaliana for interference against the RNA genome of Turnip mosaic virus (TuMV). Our data demonstrate that CRISPR RNAs (crRNAs) guiding Cas13a to the sequences encoding helper component proteinase silencing suppressor (HC-Pro) or GFP target 2 (GFP-T2) provide better interference compared to crRNAs targeting other regions of the TuMV RNA genome. This work demonstrates the exciting potential of CRISPR/Cas13 to be used as an antiviral strategy to obstruct RNA viruses, and encourages the search for more robust and effective Cas13 variants or CRISPR systems that can target RNA. Full article
(This article belongs to the Special Issue Applications of CRISPR Technology in Virology 2018)
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<p>Cas13a interference with TuMV-GFP in <span class="html-italic">A. thaliana</span> plants. (<b>A</b>) Schematic representation of the engineered system for in planta expression. The T-DNA consists of pCas13a and crRNA driven by the 35s and AtU6-26 promoter, respectively. In addition, it also contains Kan<sup>r</sup> (kanamycin resistance) driven by the Nos promoter for selection of transgenic plants. (<b>B</b>) Confirmation of pCas13a protein expression in T<sub>1</sub> <span class="html-italic">A. thaliana</span> plants. Western blot analysis with anti-HA antibody was used to detect the expression of Cas13a in T<sub>1</sub> <span class="html-italic">A. thaliana</span> lines. NB-pCas13a represents protein extracted from transiently expressed pCas13a in <span class="html-italic">Nicotiana benthamiana</span>. (<b>C</b>) Interference of TuMV-GFP in pCas13a expressing transgenic <span class="html-italic">Arabidopsis</span> lines. Stably transformed <span class="html-italic">Arabidopsis</span> lines expressing pCas13 and crRNA were inoculated with TuMV-GFP sap from <span class="html-italic">N. benthamiana</span>. Plants were imaged for GFP fluorescence to examine TuMV-GFP systemic spread under UV light in the dark. (<b>D</b>) Western blot analysis to confirm TuMV-GFP accumulation in <span class="html-italic">Arabidopsis</span> systemic leaves. Protein resolved on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was blotted with GFP antibody to detect virus accumulation. Coomassie brilliant blue (CBB) stained membrane (lower panel) was used as loading control. (<b>E</b>) Northern blot to analyze TuMV-GFP virus titer in plants. Northern blot confirms that Hc-crRNA and GFP-T2 crRNAs give better interference with TuMV-GFP followed by GFP-T1 and CP crRNAs. RNA blots from (C) were probed with a DIG-labeled TuMV complementary (250-nt) RNA fragment and detected with anti-DIG antibody. The arrow indicates the accumulation of the TuMV-GFP RNA genome. (<b>F</b>) Quantification of TuMV-GFP RNA genome. The graph represents the relative expression of the TuMV-GFP RNA virus as calculated on the bases of three independent biological replicates of northern blot. Error bars represents STDEV.</p> Full article ">Figure 2
<p>A schematic representation of CRISPR/Cas13-mediated RNA virus interference in plants. The diagram illustrates the mechanism of molecular immunity against RNA viruses in <span class="html-italic">A. thaliana</span> plants expressing CRISPR/Cas13a. When plants are infected with virus, Cas13 guided by virus-targeting crRNA recognizes and degrades the RNA virus genome, providing immunity against the virus.</p> Full article ">
28 pages, 5702 KiB  
Article
Global Interactomics Connect Nuclear Mitotic Apparatus Protein NUMA1 to Influenza Virus Maturation
by Md Niaz Rahim, Ludger Klewes, Ali Zahedi-Amiri, Sabine Mai and Kevin M. Coombs
Viruses 2018, 10(12), 731; https://doi.org/10.3390/v10120731 - 19 Dec 2018
Cited by 5 | Viewed by 6528
Abstract
Influenza A virus (IAV) infections remain a major human health threat. IAV has enormous genetic plasticity and can rapidly escape virus-targeted anti-viral strategies. Thus, there is increasing interest to identify host proteins and processes the virus requires for replication and maturation. The IAV [...] Read more.
Influenza A virus (IAV) infections remain a major human health threat. IAV has enormous genetic plasticity and can rapidly escape virus-targeted anti-viral strategies. Thus, there is increasing interest to identify host proteins and processes the virus requires for replication and maturation. The IAV non-structural protein 1 (NS1) is a critical multifunctional protein that is expressed to high levels in infected cells. Host proteins that interact with NS1 may serve as ideal targets for attenuating IAV replication. We previously developed and characterized broadly cross-reactive anti-NS1 monoclonal antibodies. For the current study, we used these mAbs to co-immunoprecipitate native IAV NS1 and interacting host proteins; 183 proteins were consistently identified in this NS1 interactome study, 124 of which have not been previously reported. RNAi screens identified 11 NS1-interacting host factors as vital for IAV replication. Knocking down one of these, nuclear mitotic apparatus protein 1 (NUMA1), dramatically reduced IAV replication. IAV genomic transcription and translation were not inhibited but transport of viral structural proteins to the cell membrane was hindered during maturation steps in NUMA1 knockdown (KD) cells. Full article
(This article belongs to the Special Issue CSV2018: The 2nd symposium of the Canadian Society for Virology (CSV))
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<p>Identification of non-structural protein 1 (NS1)-interacting host proteins. (<b>A</b>) Western blot analyses of influenza A virus (IAV) NS1 immunoprecipitations. Samples were collected from the cytosols or nuclei of Mock- or PR8-infected cells in P150 dishes at indicated times and resolved in 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) before (Input; 30 µg) and after (Immppt; 10% of total sample) treatment with Dynabeads to which the indicated antibodies (α-NS1 or Isotype-matched controls) had been pre-bound. Resolved proteins were transferred to Immobilon-P polyvinylidene difluoride (PVDF) membranes, probed with α-NS1 primary antibody, and re-probed with VeriBlot secondary α-mouse antibody. (<b>B</b>) Venn diagram indicating degree of overlap in protein identifications from 3 different biological replicates (Bio 1–3). Biological replicate #3 was repeated as 2 technical replicates (Bio 3 Tech1 and Bio 3 Tech 2).</p> Full article ">Figure 2
<p>Pathway analyses of NS1 interacting host factors. DAVID-Panther analyses of (<b>A</b>) biological processes, (<b>B</b>) molecular functions and (<b>C</b>) reactome and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Gene Ontologies of (<b>D</b>) Biological processes and (<b>E</b>) Molecular functions. STRING analyses of NS1-interacting proteins in (<b>F</b>) Gene expression pathway, (<b>G</b>) Processing of capped intron-containing pre-mRNA pathway, and (<b>H</b>) Spliceosome pathway. Additional interacting protein networks are shown in <a href="#app1-viruses-10-00731" class="html-app">Supplementary Figure S1A–D</a> and a STRING interaction network of all 183 identified proteins is shown in <a href="#app1-viruses-10-00731" class="html-app">Supplementary Figure S1E</a>.</p> Full article ">Figure 3
<p>Genetic knockdown of candidate genes by siRNA array screen. Reverse transfections of indicated genes in A549 cells grown in 96-well plates were checked for cell viability with WST-1 at (<b>A</b>) 48 h after knockdown, and (<b>B</b>) after knockdown and PR8 infection at multiplicity of infection (MOI) = 0.05 for an additional 43 h. (<b>C</b>) Virus yields from PR8 infection after MOI = 0.05 infection at 43hpi were determined by plaque assay on canine kidney (MDCK) cells. All values were normalized to the corresponding non-silencing (N-Si) controls, which were set as 100%. Error bars represent standard error of the mean (SEM) from three independent replicates. *: <span class="html-italic">p</span> &lt; 0.05; **: <span class="html-italic">p</span> &lt; 0.005. The 11 genes, knockdown (KD) of which significantly reduced the infectious virus production to between 2.6–30% of the N-Si controls, are depicted as black bars, and cell viabilities after 91 h of knockdown, and the ratios of virus production to cell viability for these 11 genes are shown in <a href="#app1-viruses-10-00731" class="html-app">Supplementary Figure S2</a>.</p> Full article ">Figure 4
<p>Characterizations of protein, RNA, and infectious virus production in NUMA1 KD cells. (<b>A</b>), Upper panel: confirmation that NUMA1 is immunoprecipitated and recognized by α-NUMA1 antibody in Western blot. NUMA1 could not be detected in non-concentrated cell lysates. Cell lysates were prepared from P100 dishes of A549 cells, reacted with Dynabeads to which α-NUMA1 Abs had been coupled, and ½ of the reaction dissolved in SDS-PAGE sample buffer, resolved in 10% SDS-PAGE, proteins transferred to Immobilon-P PVDF membranes, and probed with α-NUMA1 antibody. Lower panel: Cell extracts prepared from P100 dishes of A549 cells infected with PR8 at MOI = 5 PFU/cell were probed for NS1 prior to immunoprecipitation (Input; 30 µg), or were immunoprecipitated with beads to which NUMA1 or an irrelevant isotype control antibody had been bound. Co-precipitated products were resolved by SDS-PAGE and blots were immunoprobed with α-NS1 antibody. (<b>B</b>) Confirmation of NUMA1 KD efficiency in A549 cells. Sets of P100 dishes were treated with 25 nM of non-silencing (N-Si) control or with <span class="html-italic">NUMA1</span>-specific siRNA twice, 24 h apart for a total of 48 h treatment. Cell extracts were prepared and a 1/40th dilution probed for β-actin to confirm equivalent starting amounts (middle panel). Extracts were then immunoprecipitated with α-NUMA1-Dynabeads. After washing, beads were dissolved in SDS-PAGE sample buffer, proteins resolved by SDS-PAGE, and immunoprobed for NUMA1 (upper panel) or IgG heavy chain (lower panel). (<b>C</b>) Densitometry confirms NUMA1 was knocked down to ~16% of N-Si levels. (<b>D</b>) Percentages of infectious virus production from NUMA1 A549 KD cells compared to N-Si cells. Cells were infected at MOI of 0.05 and harvested at 43 hpi for plaque assay. (<b>E</b>) Percentages of indicated infectious IAV produced from <span class="html-italic">NUMA1</span> KD A549 and HBEC cells compared to N-Si cells at 43 hpi after MOI = 0.05 infection. The horizontal dashed line at 100% corresponds to each virus’ yield from matching N-Si cells. (<b>F</b>) mRNA levels of NS1 and of NP in A549 cells infected with PR8 at MOI = 5. <span class="html-italic">NUMA1</span> KD and N-Si cell lysates were quantified by real-time RT-PCR and normalized to both 18S RNA and to NUMA1 quantities produced in the N-Si cells. (<b>G</b>) Cell extracts prepared from N-Si- and <span class="html-italic">NUMA1</span> KD-infected cells were immunoprobed with the indicated viral proteins (right) or with actin. (<b>H</b>–<b>J</b>) Analyses of infected supernatants from N-Si or <span class="html-italic">NUMA1</span> KD cells, before ultracentrifugal concentration (<b>H</b>; left-most pair of bars) or after concentration (<b>H</b>; rightmost pair of bars, and <b>I</b> and <b>J</b>). Concentrated viruses were tested for infectivity (<b>H</b>) and immunoprobed for indicated structural proteins (<b>I</b>). (<b>J</b>) Densitometry confirms <span class="html-italic">NUMA1</span> KD cells produce particles with ~20–40% protein content compared to N-Si cells. Error bars represent SEM from two independent replicates. *: <span class="html-italic">p</span> &lt; 0.05; **: <span class="html-italic">p</span> &lt; 0.01; ***: <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 5
<p>Ultrastructural examination of Mock-infected, and of PR8-infected N-Si and <span class="html-italic">NUMA1</span> KD cells at 20 hpi after MOI = 3 infection. Mock-infected, and N-Si and <span class="html-italic">NUMA1</span> KD PR8-infected, A549 cells were harvested, processed with EM Grade Karnovsky fixative and stained with uranyl acetate. All processed samples were analyzed with a Philips CM-10 electron microscope by the histology lab, Department of Human Anatomy. Numerous ~100 nm particles appear to be budding from infected N-Si KD cells (black arrows, center panel) whereas virus production was significantly reduced in PR8-infected <span class="html-italic">NUMA</span> KD A549 cells (right panel). Boxed regions are enlarged in the lower left insets and scale bars for the micrographs, and for the insets, are indicated.</p> Full article ">Figure 6
<p>Immunofluorescent localization of cellular NUMA1 and of viral NS1, M1 and M2 proteins in A549 cells infected with PR8 at MOI = 3. (<b>A</b>), Mock (top row), non-KD infected wild-type (2nd row), non-silencing KD infected (N-Si; 3rd row), and infected <span class="html-italic">NUMA1</span> KD cells (bottom row) were stained with 4’, 6-diamidino-2-phenylindole (DAPI) to detect nuclei (left-most column; blue), with anti-IAV NS1 (2nd column; red), and with anti-NUMA1 (3rd column; green). Merged images are shown in the 4th column and boxed regions are enlarged at far right. The NUMA1 merged box was rotated clockwise 90°. Scale bars are 25 µm for the low-magnification images and 5 µm for the enlarged images. (<b>B</b>), Mock infected N-Si (left), PR8-infected N-Si (middle) and PR8-infected <span class="html-italic">NUMA1</span> KD (right) A549 cells were stained with Alexa Fluor 546 anti-M1 (red; top) or with Alexa Fluor 488 anti-M2 (green; bottom) and analyzed by super resolution structured illumination microscopy (SIM) at 20 hpi. Nuclei were stained with DAPI (blue). White arrows indicate the cluster of M1 in <span class="html-italic">NUMA1</span> KD cells (top, right) or accumulation of M2 near cytoplasmic membranes of <span class="html-italic">NUMA1</span> KD cells (lower, right). Scale bars represent 10 µm for the <span class="html-italic">X</span> and <span class="html-italic">Y</span> axes, and 2µm for the <span class="html-italic">Z</span> axis, respectively.</p> Full article ">Figure 7
<p>Proposed model for the role of NUMA1 during IAV replication. Major trafficking pathway, involving NUMA1, tubulin and exocytosis via the Golgi are indicated in the right of each panel. Lack of NUMA1 on the right results in significant attenuation of this pathway. An alternate, presumably minor pathway involving microfilaments (left side of each panel in dashed box), accounts for minor amounts of progeny virus production in normal, wild-type infection and in <span class="html-italic">NUMA1</span> KD cells.</p> Full article ">
19 pages, 3778 KiB  
Article
Simultaneous Detection of Beta and Gamma Human Herpesviruses by Multiplex qPCR Reveals Simple Infection and Coinfection Episodes Increasing Risk for Graft Rejection in Solid Organ Transplantation
by Yessica Sánchez-Ponce, Gustavo Varela-Fascinetto, José Carlos Romo-Vázquez, Briceida López-Martínez, José Luis Sánchez-Huerta, Israel Parra-Ortega, Ezequiel M. Fuentes-Pananá and Abigail Morales-Sánchez
Viruses 2018, 10(12), 730; https://doi.org/10.3390/v10120730 - 19 Dec 2018
Cited by 25 | Viewed by 5573
Abstract
Herpesviruses are common components of the human microbiome that become clinically relevant when a competent immunosurveillance is compromised, such as in transplantation. Members of the beta and gamma subfamilies are associated with a wide diversity of pathologies, including end-organ disease and cancer. In [...] Read more.
Herpesviruses are common components of the human microbiome that become clinically relevant when a competent immunosurveillance is compromised, such as in transplantation. Members of the beta and gamma subfamilies are associated with a wide diversity of pathologies, including end-organ disease and cancer. In this study, we developed a multiplex qPCR technique with high specificity, sensitivity, efficiency and predictability that allowed the simultaneous detection and quantification of beta and gamma human herpesviruses. The technique was tested in a cohort of 34 kidney- or liver-transplanted pediatric patients followed up for up to 12 months post-transplant. Viral load was determined in 495 leukocyte-plasma paired samples collected bi-weekly or monthly. Human herpesvirus (HHV) 7 was the herpesvirus most frequently found in positive samples (39%), followed by Epstein-Barr virus (EBV) (20%). Also, EBV and HHV7 were present in the majority of coinfection episodes (62%). The share of positive samples exclusively detected either in leukocytes or plasma was 85%, suggesting that these herpesviruses tended to take a latent or lytic path in an exclusive manner. Infection by human cytomegalovirus (HCMV) and HHV6, as well as coinfection by EBV/HHV7 and EBV/HHV6/HHV7, were associated with graft rejection (RR = 40.33 (p = 0.0013), 5.60 (p = 0.03), 5.60 (p = 0.03) and 17.64 (p = 0.0003), respectively). The routine monitoring of beta and gamma herpesviruses should be mandatory in transplant centers to implement preventive strategies. Full article
(This article belongs to the Section Animal Viruses)
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<p>Assessment of specificity of primers in a human DNA background. MELT curves from each Sybr Green-based PCR detected the indicated virus. A single peak of dissociation (red arrow) was observed from each individual reaction ran in the presence of human genomic DNA from the MOLT3 cell line. Agarose gel electrophoresis (bottom right) showed that a unique PCR product of the expected size was amplified in each individual Sybr green-based PCR. MM: 100 bp (base pairs) molecular weight marker. Black arrowheads point at bands corresponding to 100 bp and 500 bp.</p> Full article ">Figure 2
<p>Standardization of the two-tube multiplex qPCR. (<b>a</b>) Serial dilutions of standard plasmids (10<sup>6</sup>–10<sup>1</sup> copies) were used to construct standard curves. The number of copies detected for each standard plasmid dilution was plotted against the average of the cycle threshold. Efficiencies and coefficient of determination R<sup>2</sup> values (predictability) are shown in <a href="#viruses-10-00730-t002" class="html-table">Table 2</a>. (<b>b</b>) Correlation of viral copy numbers detected in multiplex vs. single assays. Correlation coefficients (R) are shown in <a href="#viruses-10-00730-t002" class="html-table">Table 2</a>.</p> Full article ">Figure 3
<p>Cross-reactivity assay for HCMV. The primers and fluorescent probe used for detecting HCMV were tested for cross-reactivity with the other viruses (as is indicated in the top of each panel). Positive fluorescent signal was only detected when HCMV DNA or serial dilutions (10<sup>6</sup>–10<sup>1</sup>) of HCMV standard plasmid were used as template. For all panels, amplification cycle (Cycle) is shown in the X axis, while the normalized fluorescence (Norm. Fluoro.) is shown in the Y axis. Horizontal red lines indicate the Cycle Threshold. Dotted vertical red lines indicate the LOD. Similar results were obtained for EBV, HHV6, HHV7 and KSHV (<a href="#app1-viruses-10-00730" class="html-app">Supplementary Figure S1</a>).</p> Full article ">Figure 4
<p>Detection of beta and gamma herpesviruses in pediatric transplanted patients. (<b>a</b>) Distribution of viral positivity considering leukocytes and plasma samples together (<span class="html-italic">N</span> = 171). (<b>b</b>) Example of a patient’s dynamics of infection. The follow-up period is shown in the X axis. Samples of day 0 were collected on the same day of transplant just before the surgery. Viral loads determined as the copy number/μg of DNA (leukocytes) or copy number/mL (plasma) are shown in the Y axis. This patient was chosen because presented intermittent, persistent and coinfection episodes as indicated with arrows and bracket. Viruses are color-coded. Infections detected in leukocytes (leuk) are drawn as continuous lines while those detected in plasma (pl) are drawn as dotted lines. Horizontal lines below one viral copy indicate that virus was undetected.</p> Full article ">Figure 5
<p>Single and multiple infections by beta and gamma herpesviruses detected in transplanted patients. (<b>a</b>) Venn diagram showing number of positive samples for each herpesvirus. Number of single infections can be seen at the ends of the diagram, while coinfections can be seen at overlapping areas (<span class="html-italic">N</span> = 125). (<b>b</b>) Pie diagram showing proportions of multiple infections (<span class="html-italic">N</span> = 24).</p> Full article ">Figure 6
<p>Comparison between viral loads found in leukocytes vs. plasma samples. Panel (<b>a</b>) depicts percentages of positive samples. Virus positive in leukocytes or plasma is represented in closed or open bars, respectively. Panel (<b>b</b>) depicts the number of viral copies. Leuk: leukocytes, pl: plasma.</p> Full article ">Figure 7
<p>Comparison between viral loads found in leukocytes vs. plasma blood fractions. (<b>a</b>) Venn diagrams showing the distribution of the different viruses. For each virus the filled circle indicates the number of positive samples detected in leukocytes, while the empty circle indicates the number of positive samples detected in plasma. Circles are scaled according to the number of positive samples. (<b>b</b>) Correlation of viral loads in leukocytes and plasma for samples with viruses detected in both fractions. Coefficient of correlation (R) were the following: HCMV (<span class="html-italic">N</span> = 7): R = 0.8648, <span class="html-italic">p</span> = 0.012; HHV6 (<span class="html-italic">N</span> = 3): R = 0.994, <span class="html-italic">p</span> = 0002; HHV7 (<span class="html-italic">N</span> = 7): R = 0.8192, <span class="html-italic">p</span> = 0.0495. For HHV7 one sample did not show correlation and was not considered for the analysis. EBV was not estimated.</p> Full article ">Figure 8
<p>Plots showing frequency of intermittent and persistent infections. Large pie charts (<b>left</b>) show percentages of intermittent and persistent infections for each virus as indicated. Small pie charts (<b>right</b>) show proportions of 2, 3, and ≥4 sustained episodes of consecutive positive detection.</p> Full article ">
35 pages, 3672 KiB  
Review
Cervical Cancer Screening Programs in Europe: The Transition Towards HPV Vaccination and Population-Based HPV Testing
by Andreas C. Chrysostomou, Dora C. Stylianou, Anastasia Constantinidou and Leondios G. Kostrikis
Viruses 2018, 10(12), 729; https://doi.org/10.3390/v10120729 - 19 Dec 2018
Cited by 185 | Viewed by 15453
Abstract
Cervical cancer is the fourth most frequently occurring cancer in women around the world and can affect them during their reproductive years. Since the development of the Papanicolaou (Pap) test, screening has been essential in identifying cervical cancer at a treatable stage. With [...] Read more.
Cervical cancer is the fourth most frequently occurring cancer in women around the world and can affect them during their reproductive years. Since the development of the Papanicolaou (Pap) test, screening has been essential in identifying cervical cancer at a treatable stage. With the identification of the human papillomavirus (HPV) as the causative agent of essentially all cervical cancer cases, HPV molecular screening tests and HPV vaccines for primary prevention against the virus have been developed. Accordingly, comparative studies were designed to assess the performance of cervical cancer screening methods in order to devise the best screening strategy possible. This review critically assesses the current cervical cancer screening methods as well as the implementation of HPV vaccination in Europe. The most recent European Guidelines and recommendations for organized population-based programs with HPV testing as the primary screening method are also presented. Lastly, the current landscape of cervical cancer screening programs is assessed for both European Union member states and some associated countries, in regard to the transition towards population-based screening programs with primary HPV testing. Full article
(This article belongs to the Section Animal Viruses)
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<p>Management algorithm in primary HPV screening. Abnormal cytology refers to a borderline or more severe cytological result. This algorithm was developed based on “The supplements of the second edition of the European Guidelines for Quality Assurance in Cervical Cancer Screening of 2015” [<a href="#B55-viruses-10-00729" class="html-bibr">55</a>].</p> Full article ">Figure 2
<p>The implementation status of HPV vaccination in E.U. member states and some E.U. associated countries as of 15 May 2018, based on the World Health Organization (WHO) “Vaccine in National Immunization Program Update”. Source: <a href="http://www.who.int/immunization/monitoring_surveillance/data/en/" target="_blank">http://www.who.int/immunization/monitoring_surveillance/data/en/</a>; assessed for the last time on 16 July 2018 [<a href="#B131-viruses-10-00729" class="html-bibr">131</a>]. The magnifying glass serves to enlarge the island of Malta.</p> Full article ">Figure 3
<p>The implementation status of primary HPV testing in E.U. member states and some E.U. associated countries. The magnifying glass serves to enlarge the island of Malta. It is important to state that this is a rapidly changing field and that the status of implementation could not be confirmed for all countries from two independent sources.</p> Full article ">Figure 4
<p>Health care providers that act as sample takers in cervical cancer screening programs in E.U. member states and some E.U. associated countries. The magnifying glass serves to enlarge the island of Malta. It is important to state that this is a rapidly changing field and that the status of implementation could not be confirmed from two independent sources and that this is a rapidly changing field. This figure was designed based on information available in <a href="#viruses-10-00729-t001" class="html-table">Table 1</a> and Basu et al., 2017 [<a href="#B115-viruses-10-00729" class="html-bibr">115</a>].</p> Full article ">
17 pages, 2031 KiB  
Article
Contemporary Zika Virus Isolates Induce More dsRNA and Produce More Negative-Strand Intermediate in Human Astrocytoma Cells
by Trisha R. Barnard, Maaran M. Rajah and Selena M. Sagan
Viruses 2018, 10(12), 728; https://doi.org/10.3390/v10120728 - 19 Dec 2018
Cited by 17 | Viewed by 5221
Abstract
The recent emergence and rapid geographic expansion of Zika virus (ZIKV) poses a significant challenge for public health. Although historically causing only mild febrile illness, recent ZIKV outbreaks have been associated with more severe neurological complications, such as Guillain-Barré syndrome and fetal microcephaly. [...] Read more.
The recent emergence and rapid geographic expansion of Zika virus (ZIKV) poses a significant challenge for public health. Although historically causing only mild febrile illness, recent ZIKV outbreaks have been associated with more severe neurological complications, such as Guillain-Barré syndrome and fetal microcephaly. Here we demonstrate that two contemporary (2015) ZIKV isolates from Puerto Rico and Brazil may have increased replicative fitness in human astrocytoma cells. Over a single infectious cycle, the Brazilian isolate replicates to higher titers and induces more severe cytopathic effects in human astrocytoma cells than the historical African reference strain or an early Asian lineage isolate. In addition, both contemporary isolates induce significantly more double-stranded RNA in infected astrocytoma cells, despite similar numbers of infected cells across isolates. Moreover, when we quantified positive- and negative-strand viral RNA, we found that the Asian lineage isolates displayed substantially more negative-strand replicative intermediates than the African lineage isolate in human astrocytoma cells. However, over multiple rounds of infection, the contemporary ZIKV isolates appear to be impaired in cell spread, infecting a lower proportion of cells at a low MOI despite replicating to similar or higher titers. Taken together, our data suggests that contemporary ZIKV isolates may have evolved mechanisms that allow them to replicate with increased efficiency in certain cell types, thereby highlighting the importance of cell-intrinsic factors in studies of viral replicative fitness. Full article
(This article belongs to the Special Issue New Advances on Zika Virus Research)
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Figure 1
<p>ZIKV isolates demonstrate unique plaque morphology and different growth kinetics in A549 and U-251 MG cell lines. (<b>A</b>) Representative images of Vero cell plaque assays of the indicated ZIKV isolates. (<b>B</b>–<b>E</b>) Cell culture supernatants were collected at the indicated time points and viral titer was determined by plaque assay. (<b>B</b>) A549 and (<b>C</b>) U-251 MG cells were infected with ZIKV at MOI = 10. (<b>D</b>) A549, and (<b>E</b>) U-251 MG cells were infected with ZIKV at MOI = 0.01. Values represent mean ± SD of at least three independent experiments. Asterisks indicate significant differences in viral titer relative to ZIKV<sup>AF</sup>: * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001.</p> Full article ">Figure 2
<p>ZIKV isolates elicit different cytopathic effects in A549 and U-251 MG cell lines. (<b>A</b>) A549 and (<b>B</b>) U-251 MG cells were infected with ZIKV at MOI = 10 and cell viability was determined by MTT assay at 24 h post-infection. (<b>C</b>) A549 and (<b>D</b>) U-251 MG cells were infected with ZIKV at MOI = 0.01 and cell viability was determined by MTT assay 72 h post-infection. % Cytopathicity = 100% − ((Uninfected Absorbance − Infected Absorbance)/(Uninfected Absorbance) × 100%). Values represent the mean ± SEM of three independent experiments. Asterisks indicate significant differences in % cytopathicity: * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001.</p> Full article ">Figure 3
<p>Contemporary ZIKV isolates induce more dsRNA than pre-epidemic isolates, despite similar numbers of infected cells. (<b>A</b>) A549 and (<b>B</b>) U-251 MG cells were infected with ZIKV at MOI = 10 and at 24 h post-infection cells were stained with the pan-flavivirus (4G2) antibody and the percentage of infected cells was determined by flow cytometry. (<b>C</b>) A549 and (<b>D</b>) U-251 MG cells were infected with ZIKV at MOI = 10 and 24 h post-infection the percentage of dsRNA-positive cells was determined by flow cytometry. The percentage of positive cells was determined by comparison to mock-infected cells. Values represent mean ± SEM of at least three independent experiments. Asterisks indicate significant differences in % infected cells: * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 4
<p>Isolate-specific differences are observed in number and fluorescence intensity of dsRNA foci in infected cell. (<b>A</b>) A549 and (<b>B</b>) U-251 MG cells were infected with ZIKV at MOI = 10 and 24 h post-infection dsRNA expression was analyzed by immunofluorescence microscopy. Scale bar, 20 μm. The number of dsRNA foci per cell in (<b>C</b>) A549 and (<b>D</b>) U-251 MG cells was quantified using Imaris software (&gt;100 cells/condition). (<b>E</b>) A549 and (<b>F</b>) U-251 MG cells were infected with ZIKV at MOI = 10 and 24 h post-infection the mean fluorescence intensity (MFI) of dsRNA-positive cells was determined by flow cytometry. Values represent mean ± SEM of at least three independent experiments. Asterisks indicate significant differences: * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 5
<p>Asian lineage ZIKV isolates induce a higher ratio of negative:positive strand RNA. (<b>A</b>) A549 and (<b>B</b>) U-251 MG cells were infected with ZIKV at MOI = 10 and 24 h post-infection intracellular positive strand viral RNA was quantified by qRT-PCR. Data are normalized to GAPDH and expressed relative to a standard curve of PFU equivalents per ng input RNA. (<b>C</b>) A549 cells and (<b>D</b>) U-251 MG cells were infected with ZIKV at MOI = 10 and 24 h post-infection the relative amounts of positive and negative strand ZIKV genomes was quantified by qRT-PCR. Data are expressed as a ratio of negative:positive strand RNA. Values represent mean ± SEM of two or three independent experiments. Asterisks indicate significant differences: * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">
11 pages, 2570 KiB  
Article
SARS-Like Coronavirus WIV1-CoV Does Not Replicate in Egyptian Fruit Bats (Rousettus aegyptiacus)
by Neeltje Van Doremalen, Alexandra Schäfer, Vineet D. Menachery, Michael Letko, Trenton Bushmaker, Robert J. Fischer, Dania M. Figueroa, Patrick W. Hanley, Greg Saturday, Ralph S. Baric and Vincent J. Munster
Viruses 2018, 10(12), 727; https://doi.org/10.3390/v10120727 - 19 Dec 2018
Cited by 18 | Viewed by 12390
Abstract
Severe acute respiratory syndrome (SARS)-like WIV1-coronavirus (CoV) was first isolated from Rhinolophus sinicus bats and can use the human angiotensin converting enzyme 2 (ACE2) receptor. In the current study, we investigate the ability of WIV1-CoV to infect Rousettus aegyptiacus bats. No clinical signs [...] Read more.
Severe acute respiratory syndrome (SARS)-like WIV1-coronavirus (CoV) was first isolated from Rhinolophus sinicus bats and can use the human angiotensin converting enzyme 2 (ACE2) receptor. In the current study, we investigate the ability of WIV1-CoV to infect Rousettus aegyptiacus bats. No clinical signs were observed throughout the experiment. Furthermore, only four oropharyngeal swabs and two respiratory tissues, isolated on day 3 post inoculation, were found positive for viral RNA. Two out of twelve bats showed a modest increase in coronavirus specific antibodies post challenge. In conclusion, WIV1-CoV was unable to cause a robust infection in Rousettus aegyptiacus bats. Full article
(This article belongs to the Section Animal Viruses)
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Figure 1

Figure 1
<p>Egyptian fruit bat ACE2 is a suitable receptor for WIV1-CoV. (<b>A</b>) Schematic overview of coronavirus spike proteins. (<b>B</b>) VSV particles pseudotyped with coronavirus spike proteins were concentrated and analyzed for spike incorporation by Western blot. (<b>C</b>) BHK cells were transfected with coronavirus receptors and infected with pseudotyped particles in triplicate. (<b>D</b>) BHK cells were transfected with ACE2 plasmids and inoculated with WIV1-CoV at a MOI of 0.01, 24 h after transfection. Supernatants were harvested at 0, and 48 hpi and viral titers were determined by endpoint titration in quadruplicate in VeroE6 cells.</p> Full article ">Figure 2
<p>ACE2 immunohistochemistry of (<b>A</b>) nasal turbinate—400×, multifocal apical immunoreactivity in ciliated epithelial cells (arrows); (<b>B</b>) trachea—400×, negative immunostaining; (<b>C</b>) lung—400×, multifocal cytoplasmic endothelial cell immunoreactivity (arrowheads): (<b>D</b>) kidney—200×, multifocal cytoplasmic endothelial cell immunoreactivity (arrowheads); (<b>E</b>) stomach—400×, multifocal cytoplasmic endothelial cell immunoreactivity (arrowheads): (<b>F</b>) intestine—100×, diffuse immunoreactivity of brush border.</p> Full article ">Figure 3
<p>Disease symptoms, shedding and tissue tropism after inoculation with WIV1-CoV. (<b>A</b>) Relative weight change and (<b>B</b>) body temperature change in Egyptian fruit bats following inoculation with WIV1-CoV. (<b>C</b>) Viral RNA in oropharyngeal, rectal, and urogenital swabs obtained daily. (<b>D</b>) Lung:body weight ratio of Egyptian fruit bats at 3, 7, and 28 dpi. (<b>E</b>) Viral RNA load in tissues obtained 3 dpi (<span class="html-italic">N</span> = 4 bats).</p> Full article ">Figure 4
<p>Hematology of Egyptian fruit bats inoculated with WIV1-CoV. Pre-challenge values (black) were obtained at D-95 (<span class="html-italic">N</span> = 12) and D-2 (<span class="html-italic">N</span> = 11). Post-challenge values were obtained at 3 dpi (red), 7 dpi (blue), and 28 dpi (green). All values were measured using the IDEXX ProCyte DX Analyzer. Dotted line = average of pre-challenge values.</p> Full article ">Figure 5
<p>Serology titers in sera obtained pre- and post-challenge. ELISA assays were performed using SARS-CoV proteins N and S. Bats with an increase in serology titer are shown in bold.</p> Full article ">
21 pages, 3480 KiB  
Article
The Amino-Terminal Region of Hepatitis E Virus ORF1 Containing a Methyltransferase (Met) and a Papain-Like Cysteine Protease (PCP) Domain Counteracts Type I Interferon Response
by Eugénie Bagdassarian, Virginie Doceul, Marie Pellerin, Antonin Demange, Léa Meyer, Nolwenn Jouvenet and Nicole Pavio
Viruses 2018, 10(12), 726; https://doi.org/10.3390/v10120726 - 18 Dec 2018
Cited by 15 | Viewed by 4978
Abstract
Hepatitis E virus (HEV) is responsible for large waterborne epidemics of hepatitis in endemic countries and is an emerging zoonotic pathogen worldwide. In endemic regions, HEV-1 or HEV-2 genotypes are frequently associated with fulminant hepatitis in pregnant women, while with zoonotic HEV (HEV-3 [...] Read more.
Hepatitis E virus (HEV) is responsible for large waterborne epidemics of hepatitis in endemic countries and is an emerging zoonotic pathogen worldwide. In endemic regions, HEV-1 or HEV-2 genotypes are frequently associated with fulminant hepatitis in pregnant women, while with zoonotic HEV (HEV-3 and HEV-4), chronic cases of hepatitis and severe neurological disorders are reported. Hence, it is important to characterize the interactions between HEV and its host. Here, we investigated the ability of the nonstructural polyprotein encoded by the first open reading frame (ORF1) of HEV to modulate the host early antiviral response and, in particular, the type I interferon (IFN-I) system. We found that the amino-terminal region of HEV-3 ORF1 (MetYPCP), containing a putative methyltransferase (Met) and a papain-like cysteine protease (PCP) functional domain, inhibited IFN-stimulated response element (ISRE) promoter activation and the expression of several IFN-stimulated genes (ISGs) in response to IFN-I. We showed that the MetYPCP domain interfered with the Janus kinase (JAK)/signal transducer and activator of the transcription protein (STAT) signalling pathway by inhibiting STAT1 nuclear translocation and phosphorylation after IFN-I treatment. In contrast, MetYPCP had no effect on STAT2 phosphorylation and a limited impact on the activation of the JAK/STAT pathway after IFN-II stimulation. This inhibitory function seemed to be genotype-dependent, as MetYPCP from HEV-1 had no significant effect on the JAK/STAT pathway. Overall, this study provides evidence that the predicted MetYPCP domain of HEV ORF1 antagonises STAT1 activation to modulate the IFN response. Full article
(This article belongs to the Special Issue Emerging Viruses)
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Figure 1

Figure 1
<p>Effect of the expression of full-length HEV ORF1 and several of its domains on IFN-stimulated response element (ISRE) promoter activation. (<b>a</b>) Schematic representation of the different domains of HEV ORF1. Met: Methyltransferase domain; Y: Y domain; PCP: Papain-like cysteine protease; HVR: Hypervariable region; X: Macrodomain; Hel: Helicase domain; RdRp: RNA-dependent RNA polymerase. The position of the different putative functional domains present in the ORF1 amino acid sequence of the HEV-3 strain used in this study is indicated. The different fragments of ORF1 that were cloned and expressed in 293T cells are represented by arrows. (<b>b</b>) Expression of FLAG-tagged full-length and domains of ORF1 in 293T cells detected by immunoblotting using an anti-FLAG antibody. Bands corresponding to PCP (arrow) and PCP products of higher molecular weight (asterisks) are indicated. Actin served as a loading control. Cells were lysed 18 h post-transfection. (<b>c</b>) Effect of full-length ORF1, MetYPCP, Y, PCP, macrodomain (X), Met, MetY and YPCP on ISRE promoter activation: 293T cells were transfected with pISRE-Luc, pCMV-Luc and a pCINeo-3xFLAG empty vector (EV) or a plasmid coding for ORF1, MetYPCP, Y, PCP, X, Met, MetY, YPCP or MV-V. Forty hours later, cells were treated or not (NT) with IFN-β for 7 h and lysed to determine firefly and <span class="html-italic">Renilla</span> luciferase activities. Mean ratios between firefly and <span class="html-italic">Renilla</span> luciferase activities were calculated and are presented as percentages of the treated EV control (± standard deviations). Results shown represent the mean of four independent experiments performed in triplicate. Here, * <span class="html-italic">p</span> &lt; 0.05; *** <span class="html-italic">p</span> &lt; 0.0005 compared to EV control for treated samples (unequal variance <span class="html-italic">t</span>-tests). Raw data are shown in <a href="#app1-viruses-10-00726" class="html-app">Supplementary Materials Table S1</a>. (<b>d</b>) Cell viability assays at 40 h post-transfection: 293T cells were transfected or not with a pCINeo-3xFLAG empty vector or a plasmid coding for ORF1, MetYPCP, Y, PCP, X, Met, MetY, YPCP or MV-V fused to a 3xFLAG tag. Forty hours after transfection, cells were lysed and cell viability determined using a luminescent-based assay. Luciferase activities (± standard deviations) are expressed as percentage relative to nontransfected cells. No significant difference was found between the cells transfected with the pCINeo-3xFLAG empty vector and the one transfected with the plasmid coding for the different FLAG-tagged proteins (unpaired <span class="html-italic">t</span>-tests). Results represent the mean of three independent experiments performed in triplicate.</p> Full article ">Figure 2
<p>Expression of MetYPCP of HEV ORF1 downregulated mRNA levels of several ISGs following IFN-β treatment. (<b>a</b>–<b>c</b>) Here, 293T cells were transfected with a pCINeo-3xFLAG empty vector or a plasmid coding for MetYPCP, PCP or MV-V fused to a 3xFLAG tag. Forty hours post-transfection, cells were stimulated or not (NT) with 500 IU/mL of IFN-β for 6 h. Total RNA was extracted, and expression of the mRNA coding for (<b>a</b>) ISG56, (<b>b</b>) melanoma differentiation-associated protein (MDA)5 and (<b>c</b>) 2′,5′-oligoadenylate synthetase (OAS)1 were measured by RT-qPCR. <span class="html-italic">Glyceraldehyde 3-phosphate dehydrogenase</span> (<span class="html-italic">GAPDH</span>) was used as a reference gene. Relative mRNA expression was calculated for each condition and is presented as percentages of the treated EV control (± standard deviations). Results shown represent the mean of three independent experiments performed in triplicate: *, <span class="html-italic">p</span> &lt; 0.05 compared to EV control for treated samples (unequal variance <span class="html-italic">t</span>-tests). Results from the three independent experiments are presented in <a href="#app1-viruses-10-00726" class="html-app">Supplementary Materials Figure S2</a>.</p> Full article ">Figure 3
<p>Expression of MetYPCP of HEV ORF1 decreased signal transducer and activator of transcription protein (STAT)1 nuclear translocation upon IFN-β treatment. (<b>a</b>) Here, 293T cells were transfected with a pCINeo-3xFLAG empty vector or a plasmid coding for MetYPCP, PCP or MV-V fused to a 3xFLAG tag. Twenty-four hours post-transfection, cells were stimulated or not for 30 min with 1000 IU/mL of IFN-β. Cells were then washed, fixed and stained with primary antibodies raised against STAT1 and FLAG, followed by fluorescent dye-conjugated secondary antibodies. Intracellular localization of 4,6-diamidine-2-phenylindole dihydrochloride (DAPI)-stained nuclei (blue), FLAG (green) and STAT1 (red) was visualized by microscopy (magnification, 630×). Scale bars: 10 μm. Cells showing diffuse cytoplasmic/nuclear localisation of STAT1 upon IFN-β treatment are indicated by arrows. (<b>b</b>) STAT1 localization was visualized after immunostaining as described in (<b>a</b>) in 293T cells transfected with a pCINeo-3xFLAG empty vector or a plasmid coding for MetYPCP, PCP or MV-V fused to a FLAG tag. For each condition, STAT1 localisation (predominant nuclear localisation or diffuse localisation within the cytoplasm and nucleus) was determined in 70 to 172 cells expressing the corresponding FLAG-tagged protein (except for the EV control, for which 356 to 384 cells were randomly assessed). The mean percentage (± standard deviation) of cells showing a predominant nuclear localization of STAT1 from three independent experiments is shown: ** <span class="html-italic">p</span> &lt; 0.005; *** <span class="html-italic">p</span> &lt; 0.0005 compared to EV control for treated samples (unpaired <span class="html-italic">t</span>-tests).</p> Full article ">Figure 4
<p>Expression of MetYPCP of HEV ORF1 inhibited STAT1 but not STAT2 phosphorylation upon IFN-β treatment. (<b>a</b>) Here, 293T cells were transfected with a pCINeo-3xFLAG empty vector or a plasmid coding for MetYPCP, PCP or MV-V fused to a 3xFLAG tag. Twenty-four hours post-transfection, cells were stimulated for 30 min with 500 IU/mL of IFN-β. Cell lysates were extracted and used for the detection of FLAG-tagged proteins, total STAT1, phosphorylated STAT1 (p-STAT1), total STAT2 and phosphorylated STAT2 (p-STAT2) by immunoblotting. Actin served as an internal control. (<b>b</b>) Here, 293T cells were transfected with an empty vector or a plasmid coding for MetYPCP, PCP or MV-V and treated as described in (<b>a</b>). Cell lysates were extracted and used for the detection of total STAT1, p-STAT1 and actin by immunoblotting. Band intensities were quantified using ImageJ software, and relative levels of STAT1, p-STAT1 and actin were determined for each treated sample. Ratios between p-STAT1 and actin, STAT1 and actin, and p-STAT1 and STAT1 were calculated and expressed as a relative percentage in comparison to the EV control. (<b>c</b>) Here, 293T cells were transfected with an empty vector or a plasmid coding for MetYPCP or MV-V and treated as described in (<b>a</b>). Cell lysates were extracted and used for the detection of total STAT2, p-STAT2 and p-STAT1 by immunoblotting. Band intensities were quantified using ImageJ software, and relative levels of STAT2, p-STAT2, p-STAT1 and actin were determined for each treated sample. Ratios between p-STAT2 and actin and STAT2 and actin were calculated and expressed as a relative percentage in comparison to the EV control. The ratio between p-STAT1 and actin was also determined to ensure significant inhibition of the p-STAT1 level by MetYPCP in this set of experiments. In (<b>b</b>–<b>c</b>), the mean percentage (± standard deviation) of four independent experiments is presented for each panel: * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.005; *** <span class="html-italic">p</span> &lt; 0.0005 compared to the EV control for IFN-treated samples (unequal variance <span class="html-italic">t</span>-tests).</p> Full article ">Figure 5
<p>Expression of MetYPCP of HEV ORF1 inhibited weakly STAT1 translocation but not STAT1 phosphorylation in response to IFN-II. (<b>a</b>) Here, 293T cells were transfected with a pCINeo-3xFLAG empty vector or a plasmid coding for MetYPCP or MV-V fused to a 3xFLAG tag. Twenty-four hours post-transfection, cells were stimulated for 30 min with 1000 IU/mL of IFN-β or 250 ng/mL of IFN-γ. Cells were then washed, fixed and stained with primary antibodies raised against STAT1 and FLAG, followed by fluorescent dye-conjugated secondary antibodies. STAT1 localization was determined in 64 to 102 cells expressing the corresponding FLAG-tagged protein (except for the EV control, for which 299 to 328 cells were randomly assessed). The mean percentage (± standard deviation) of cells showing a predominant nuclear localization of STAT1 from three independent experiments is shown: * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.005; *** <span class="html-italic">p</span> &lt; 0.0005 compared to the EV control for treated samples (unpaired <span class="html-italic">t</span>-tests). (<b>b</b>) Here, 293T cells were transfected with a pCINeo-3xFLAG empty vector or a plasmid coding for MetYPCP, PCP or MV-V fused to a 3xFLAG tag. Twenty-four hours post-transfection, cells were stimulated for 30 min with 500 IU/mL of IFN-β or 250 ng/mL of IFN-γ. Cell lysates were extracted and used for the detection of FLAG-tagged proteins, total STAT1, phosphorylated STAT1 (p-STAT1) and actin as an internal control by immunoblotting. (<b>c</b>) Band intensities were quantified using ImageJ software, and relative levels of STAT1, p-STAT1 and actin were determined for each sample treated with 125 or 250 ng/mL of IFN-γ or 500 IU/mL of IFN-β. Ratios between p-STAT1 and actin and STAT1 and actin were then calculated and expressed as a relative percentage in comparison to the EV control. The mean percentage (± standard deviation) of three independent experiments is presented: * <span class="html-italic">p</span> &lt; 0.05 compared to the EV control (unequal variance <span class="html-italic">t</span>-tests).</p> Full article ">Figure 6
<p>Comparison of the effect of MetYPCP from HEV-1 and HEV-3 on the Janus kinase (JAK)/STAT pathway. (<b>a</b>) Expression of FLAG-tagged MetYPCP and PCP from a strain of HEV-1 (MetYPCP-G1 and PCP-G1) and HEV-3 (MetYPCP-G3 and PCP-G3) in 293T cells detected by immunoblotting using an anti-FLAG antibody. Actin served as a loading control. Cells were lysed 24 h post-transfection. (<b>b</b>) Effect of MetYPCP and PCP from HEV-1 and HEV-3 on ISRE promoter activation: 293T cells were transfected with pISRE-Luc, pCMV-Luc and a pCINeo-3xFLAG empty vector or a plasmid coding for MV-V, MetYPCP-G1, MetYPCP-G3, PCP-G1 or PCP-G3. Forty h later, cells were treated or not (NT) with IFN-β for 7 h and lysed to determine firefly and <span class="html-italic">Renilla</span> luciferase activities. Mean ratios between firefly and <span class="html-italic">Renilla</span> luciferase activities were calculated and are presented as percentages of the treated EV control (± standard deviations). Results shown represent the mean of five independent experiments performed in triplicate: * <span class="html-italic">p</span> &lt; 0.05; *** <span class="html-italic">p</span> &lt; 0.0005 compared to the EV control for treated samples (unequal variance <span class="html-italic">t</span>-tests). Raw data are presented in <a href="#app1-viruses-10-00726" class="html-app">Supplementary Materials Table S2</a>. (<b>c</b>) Here, 293T cells were transfected with a pCINeo-3xFLAG empty vector or a plasmid coding for MetYPCP-G3, MetYPCP-G1 or MV-V fused to a 3xFLAG tag. Twenty-four hours post-transfection, cells were stimulated for 30 min with 1000 IU/mL of IFN-β. Cells were then washed, fixed and stained with primary antibodies raised against STAT1 and FLAG, followed by fluorescent dye-conjugated secondary antibodies. STAT1 localization was determined in 70 to 117 cells expressing the corresponding FLAG-tagged protein (except for the EV control, for which 311 to 328 cells were randomly assessed). The mean percentage (± standard deviation) of cells showing a predominant nuclear localization of STAT1 from three independent experiments is shown: * <span class="html-italic">p</span> &lt; 0.05; *** <span class="html-italic">p</span> &lt; 0.0005 compared to the EV control for treated samples (unpaired <span class="html-italic">t</span>-tests).</p> Full article ">
4 pages, 179 KiB  
Editorial
One Health (r)Evolution: Learning from the Past to Build a New Future
by Ilaria Capua and Giovanni Cattoli
Viruses 2018, 10(12), 725; https://doi.org/10.3390/v10120725 - 18 Dec 2018
Cited by 19 | Viewed by 5965
Abstract
The One Health concept recognizes that the health of human beings, animals, plants and the environment is interconnected and interdependent. This idea has been shaped over the centuries and has gained momentum and traction as anatomy, physiology, microbiology and other disciplines have substantiated [...] Read more.
The One Health concept recognizes that the health of human beings, animals, plants and the environment is interconnected and interdependent. This idea has been shaped over the centuries and has gained momentum and traction as anatomy, physiology, microbiology and other disciplines have substantiated earlier theories. Here we recall major historical milestones which have contributed to shaping the One Health concept as it is today, and discuss the past and future drivers in view of future challenges in an evolving scenario. Full article
(This article belongs to the Special Issue Zoonotic and Vectorborne Viral Infections: Applying the One Health Vision—from Challenges to Solutions)
6 pages, 180 KiB  
Opinion
Advances in Influenza Virus Research: A Personal Perspective
by Kanta Subbarao
Viruses 2018, 10(12), 724; https://doi.org/10.3390/v10120724 - 18 Dec 2018
Cited by 2 | Viewed by 4560
Abstract
Technical advances in the last decade have made it possible to investigate influenza virus infection from the cellular and subcellular level to intact animals and humans. As a result, we have gained a new understanding of the virus and disease. Full article
(This article belongs to the Special Issue What’s New with Flu?)
13 pages, 5689 KiB  
Review
Prion Strain-Specific Structure and Pathology: A View from the Perspective of Glycobiology
by Ilia V. Baskakov, Elizaveta Katorcha and Natallia Makarava
Viruses 2018, 10(12), 723; https://doi.org/10.3390/v10120723 - 18 Dec 2018
Cited by 29 | Viewed by 5192
Abstract
Prion diseases display multiple disease phenotypes characterized by diverse clinical symptoms, different brain regions affected by the disease, distinct cell tropism and diverse PrPSc deposition patterns. The diversity of disease phenotypes within the same host is attributed to the ability of PrP [...] Read more.
Prion diseases display multiple disease phenotypes characterized by diverse clinical symptoms, different brain regions affected by the disease, distinct cell tropism and diverse PrPSc deposition patterns. The diversity of disease phenotypes within the same host is attributed to the ability of PrPC to acquire multiple, alternative, conformationally distinct, self-replicating PrPSc states referred to as prion strains or subtypes. Structural diversity of PrPSc strains has been well documented, yet the question of how different PrPSc structures elicit multiple disease phenotypes remains poorly understood. The current article reviews emerging evidence suggesting that carbohydrates in the form of sialylated N-linked glycans, which are a constitutive part of PrPSc, are important players in defining strain-specific structures and disease phenotypes. This article introduces a new hypothesis, according to which individual strain-specific PrPSc structures govern selection of PrPC sialoglycoforms that form strain-specific patterns of carbohydrate epitopes on PrPSc surface and contribute to defining the disease phenotype and outcomes. Full article
(This article belongs to the Special Issue Deciphering the Molecular Targets of Prion and Prion-Like Strains)
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Figure 1

Figure 1
<p>Staining of PrP<sup>Sc</sup> plaques with SNA lectin. Images of SO5 PrP<sup>Sc</sup> plaques (<b>A</b>–<b>C</b>) or atypical PrP<sup>Sc</sup> plaques (<b>E</b>,<b>F</b>) in hamster brains stained with anti-PrP 3F4 antibody (<b>A</b>,<b>E</b>), SNA lectin (<b>B</b>,<b>F</b>), or secondary antibody used for SNA staining as negative control (<b>C</b>). Plaques are shown by arrows. Staining of normal age-matched control with SNA lectin is shown in (<b>D</b>). Brains fixed in 10% neutral buffered formalin were treated with 95% formic acid for 1 h before embedding in paraffin wax and sectioning into 4 µm sections. After a standard rehydration procedure, slides were submerged in 10 mM tri-sodium citrate buffer, pH 6.0, boiled for 5 min by microwaving at 20% power, and cooled for 1 h before proceeding with lectin staining. Incubation in 3% hydrogen peroxide in methanol for 20 min was used to remove endogenous peroxidase activity. After 5 min wash in running water, slides were incubated for 1 h with 5 µg/mL biotin-labeled elderberry bark lectin (SNA, Vector laboratories, Burlingame, CA) diluted in lectin buffer, pH 7.6 (50 mM Tris, 150 mM NaCl, 1 mM MgCl2, 0.75 mM CaCl2). Following triple 5 min wash in lectin buffer, the slides were incubated for 30 min in 5 µg/mL horse radish peroxidase-labeled streptavidin (Thermo Fisher scientific, Waltham, MA), then again washed three times with lectin buffer, and developed with 3,3’ Diaminobenzidine (DAB) Quanto chromogen and substrate (VWR, Radnor, PA).</p> Full article ">Figure 2
<p>Schematic diagram illustrating selective recruitment of PrP<sup>C</sup> sialoglycoforms in a strain-specific manner according to the PrP<sup>C</sup> sialylation status. The left panel shows distribution of PrP<sup>C</sup> molecules according to their glycosylation status (in horizontal dimension) and sialylation status (in vertical dimension) ranging from non-sialylated to highly sialylated molecules. PrP<sup>C</sup> molecules are shown as blue circles and sialic acid residues as red diamonds. The panels on the right show 2D Western blots of three prion strains with different recruitment selectivity. While 263K (strain #1) recruits PrP<sup>C</sup> sialoglycoforms without strong preferences, hypersialylated PrP<sup>C</sup> molecules are preferentially excluded from RML (strain #2) and excluded even stronger from atypical PrP<sup>Sc</sup> (strain #3). As a result of strain-specific exclusion of highly sialylated PrP<sup>C</sup>, ratios of glycoforms within PrP<sup>Sc</sup> shift toward mono- and unglycosylated glycoform, as illustrated by corresponding 1D Western blots. Adapted from Baskakov and Katorcha 2016 [<a href="#B35-viruses-10-00723" class="html-bibr">35</a>].</p> Full article ">Figure 3
<p>Correlation between strain-specific sialylation status and glycoform ratio. Strain-specific percentages of diglycosylated glycoforms plotted as a function of strain-specific percentage of hypersialylated glycoforms within PrP<sup>Sc</sup>. Mean and standard deviations are shown (n = 3 animals). Black solid line shows the result of linear fitting of the percent of diglycosylated as a function of the percent of hypersialylated for brain-derived PrP<sup>Sc</sup>. Adapted from Katorcha et al. 2015 [<a href="#B37-viruses-10-00723" class="html-bibr">37</a>].</p> Full article ">Figure 4
<p>Schematic diagram illustrating differences in quaternary assembly between non-selective (left panels) and selective (right panels) strains. It is proposed that non-selective strains can accommodate diglycosylated sialoglycoforms because of rotation between neighboring PrP molecules that allows spatial separation of glycans and reduces electrostatic repulsion. In selective strains, the rotation between neighboring PrP molecules is very small (<span class="html-italic">i</span>) or absent (<span class="html-italic">ii</span>). Recruitment of diglycosylated molecules by selective strains would lead to spatial interference and electrostatic repulsion between glycans (<span class="html-italic">iii</span>). Negative selection of diglycosylated molecules helps to minimize spatial and electrostatic interference between glycans (<span class="html-italic">iv</span>). While the same principles are applicable to both three- and four-rung solenoid structure, for simplicity of presentation only three-rung solenoid structures are shown here.</p> Full article ">Figure 5
<p>Modeling of N-linked glycans in PrP<sup>Sc</sup> consisting of three-rung solenoid. Two views of three-rung solenoid structures carrying tri-antennary N-glycans. Polypeptide chains are represented in the tube form, whereas N-glycans are represented in the ball-and-stick form. Each PrP molecule with corresponding N-glycan is of a different color. Sialic acid residues are colored in red. The structure of a tri-antennary N-linked glycan (shown in inset) was taken from PDB entry 3QUM, a crystal structure of human prostate specific antigen (PSA) [<a href="#B45-viruses-10-00723" class="html-bibr">45</a>]. Both calculations of electrostatic surfaces and generation of images were performed with CCP4MG software. The model based on three-rung solenoid structure is shown here for simplicity of presentation and should not be considered as preferable over the four-rung solenoid model.</p> Full article ">Figure 6
<p>Schematic representation of the hypothesis proposing that carbohydrate epitopes on PrP<sup>Sc</sup> surface determine response of glia. A. High density of glycans with terminal sialylation leads to chronic neuroinflammation. B. Desialylation of PrP<sup>Sc</sup> that results in a high density of exposed galactose triggers “eat me” signal in glia. C. Atypical PrP<sup>Sc</sup> has low density of glycosylation and sialylation, similar to those of sialoglycocalyx. Atypical PrP<sup>Sc</sup> does not trigger “eat me” signal or chronic neuroinflammation.</p> Full article ">
9 pages, 1296 KiB  
Meeting Report
“FAGOMA: Spanish Network of Bacteriophages and Transducer Elements”—V Meeting Report
by Modesto Redrejo-Rodríguez and Pilar García
Viruses 2018, 10(12), 722; https://doi.org/10.3390/v10120722 - 18 Dec 2018
Viewed by 4189
Abstract
The Spanish Network of Bacteriophages and Transducer Elements (FAGOMA) was created to answer the need of Spanish scientists working on phages to exchange knowledge and find synergies. Seven years and five meetings later, the network has become a fruitful forum where groups working [...] Read more.
The Spanish Network of Bacteriophages and Transducer Elements (FAGOMA) was created to answer the need of Spanish scientists working on phages to exchange knowledge and find synergies. Seven years and five meetings later, the network has become a fruitful forum where groups working on distinct aspects of phage research (structural and molecular biology, diversity, gene transfer and evolution, virus–host interactions, clinical, biotechnological and industrial applications) present their work and find new avenues for collaboration. The network has recently increased its visibility and activity by getting in touch with the French Phage Network (Phages.fr) and with different national and international scientific institutions. Here, we present a summary of the fifth meeting of the FAGOMA network, held in October 2018 in Alcalá de Henares (Madrid), in which the participants shared some of their latest results and discussed future challenges of phage research. Full article
(This article belongs to the Section Bacterial Viruses)
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<p>Geographic location of research group members of the Spanish Network of Bacteriophages and Transducer Elements (FAGOMA II) and their main research topic.</p> Full article ">Figure 2
<p>Participants of the V FAGOMA II meeting.</p> Full article ">
25 pages, 6053 KiB  
Review
Development of Small-Molecule MERS-CoV Inhibitors
by Ruiying Liang, Lili Wang, Naru Zhang, Xiaoqian Deng, Meng Su, Yudan Su, Lanfang Hu, Chen He, Tianlei Ying, Shibo Jiang and Fei Yu
Viruses 2018, 10(12), 721; https://doi.org/10.3390/v10120721 - 17 Dec 2018
Cited by 51 | Viewed by 12266
Abstract
Middle East respiratory syndrome coronavirus (MERS-CoV) with potential to cause global pandemics remains a threat to the public health, security, and economy. In this review, we focus on advances in the research and development of small-molecule MERS-CoV inhibitors targeting different stages of the [...] Read more.
Middle East respiratory syndrome coronavirus (MERS-CoV) with potential to cause global pandemics remains a threat to the public health, security, and economy. In this review, we focus on advances in the research and development of small-molecule MERS-CoV inhibitors targeting different stages of the MERS-CoV life cycle, aiming to prevent or treat MERS-CoV infection. Full article
(This article belongs to the Special Issue MERS-CoV)
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<p>Summary of morbidity statistics with country- and quarter-level panel data.</p> Full article ">Figure 2
<p>Schematic diagram of Middle East respiratory syndrome coronavirus (MERS-CoV) infection. MERS-CoV enters host cells by plasma membrane fusion (membrane fusion) or endosomal membrane fusion (endocytosis), and then releases the viral RNA into the cytoplasm. The RNA genome is replicated and viral proteins are produced. The progeny virus is generated and released from the infected cells.</p> Full article ">Figure 3
<p>Schematic representation of MERS-CoV S (spike) protein S1 subunit and S2 subunit. RBD, receptor binding domain; FP, fusion peptide; HR1, heptad repeat 1 domain; HR2, heptad repeat 2 domain; TM, transmembrane domain; CP, cytoplasmic domain. The residue numbers of each region correspond to their positions in the S protein of MERS-CoV. HR2P, the peptide derived from the HR2 domain of MERS-CoV S protein S2 subunit; HR2P-M2, HR2P analogous peptide with mutations.</p> Full article ">Figure 4
<p>Schematic representation of the inhibition mechanism of HR2P and HR2P-M2. ① Target cell membrane; ② MERS-CoV; ③ dipeptidyl peptidase-4 (DPP4). (<b>A</b>) Mechanism of normal binding between a host cell and MERS-CoV. MERS-CoV enters the host cell by binding the viral particle via the RBD in spike protein to the cellular receptorDPP4 on the surface of the host cell. The HR2 binds to the HR1 to form a six-helix bundle (6-HB) fusion core, which brings viral and cell membranes into close apposition for fusion. (<b>B</b>) HR2P and HR2P-M2 block six-bundle fusion core formation between HR1 and HR2 peptides by binding to the viral S protein HR1 domain.</p> Full article ">Figure 5
<p>Chemical structure formulae of small-molecule inhibitors of MERS-CoV described in this review.</p> Full article ">Figure 5 Cont.
<p>Chemical structure formulae of small-molecule inhibitors of MERS-CoV described in this review.</p> Full article ">Figure 5 Cont.
<p>Chemical structure formulae of small-molecule inhibitors of MERS-CoV described in this review.</p> Full article ">Figure 5 Cont.
<p>Chemical structure formulae of small-molecule inhibitors of MERS-CoV described in this review.</p> Full article ">Figure 5 Cont.
<p>Chemical structure formulae of small-molecule inhibitors of MERS-CoV described in this review.</p> Full article ">Figure 5 Cont.
<p>Chemical structure formulae of small-molecule inhibitors of MERS-CoV described in this review.</p> Full article ">Figure 5 Cont.
<p>Chemical structure formulae of small-molecule inhibitors of MERS-CoV described in this review.</p> Full article ">Figure 5 Cont.
<p>Chemical structure formulae of small-molecule inhibitors of MERS-CoV described in this review.</p> Full article ">
9 pages, 5118 KiB  
Article
A Divergent Hepatitis D-Like Agent in Birds
by Michelle Wille, Hans J. Netter, Margaret Littlejohn, Lilly Yuen, Mang Shi, John-Sebastian Eden, Marcel Klaassen, Edward C. Holmes and Aeron C. Hurt
Viruses 2018, 10(12), 720; https://doi.org/10.3390/v10120720 - 17 Dec 2018
Cited by 63 | Viewed by 7373
Abstract
Hepatitis delta virus (HDV) is currently only found in humans and is a satellite virus that depends on hepatitis B virus (HBV) envelope proteins for assembly, release, and entry. Using meta-transcriptomics, we identified the genome of a novel HDV-like agent in ducks. Sequence [...] Read more.
Hepatitis delta virus (HDV) is currently only found in humans and is a satellite virus that depends on hepatitis B virus (HBV) envelope proteins for assembly, release, and entry. Using meta-transcriptomics, we identified the genome of a novel HDV-like agent in ducks. Sequence analysis revealed secondary structures that were shared with HDV, including self-complementarity and ribozyme features. The predicted viral protein shares 32% amino acid similarity to the small delta antigen of HDV and comprises a divergent phylogenetic lineage. The discovery of an avian HDV-like agent has important implications for the understanding of the origins of HDV and sub-viral agents. Full article
(This article belongs to the Section Animal Viruses)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Characteristics of the genome of an avian hepatitis delta virus (HDV)-like agent. (<b>A</b>) Avian HDV-like agent genome, annotated with ORFs, genomic, and antigenomic ribozyme sites. Metadata rings include the read coverage, proportion of polymorphisms in reads, followed by GC content. (<b>B</b>) Abundance of transcripts in the metatranscriptomic library. Total avian viral abundance was dominated by that of the influenza A virus. However, the abundance of HDV is higher than that of Ribosomal protein S13 (RPS13), a stably expressed reference gene in Mallards (<span class="html-italic">Anas platyrhynchos</span>). (<b>C</b>) Maximum likelihood phylogeny of the HDAg protein. Representative human HDAg sequences fall into the currently described clades HDV1-8 (5). The scale bar represents the number of amino acid substitutions per site. The phylogeny is rooted between the human and avian/snake viruses. (<b>D</b>) Location of genomic and antigenomic ribozyme sequences, and the predicted ORF of the delta antigen in the avian HDV-like genome compared to their location in the HDV genome sequence (GenBank accession X04451.1).</p> Full article ">Figure 2
<p>A circle graph showing the base pairing of the circular RNA genome structure of the avian HDV-like agent into an unbranched rod-like structure. The circle circumference represents the genome sequence, and the arcs represent the base pairing. Colouring of arcs: Red for G-C pairing, blue for A-U pairing, green for G-U pairing, and yellow for other types of pairings.</p> Full article ">Figure 3
<p>HDV ribozymes. (<b>A</b>) Secondary structures of the genomic and antigenomic ribozymes inferred using the TT2NE algorithm. The HDV ribozyme models were used as reference to screen for the ribozyme sequences in the avian HDV-like genome sequence. (<b>B</b>) Re-drawn secondary structures of the genomic and antigenomic ribozymes based on the secondary structures shown in the review by Webb and Luptak (21).</p> Full article ">Figure 4
<p>Features of the predicted HDAg protein. Alignment of the amino acid sequences (small delta antigen) translated from the genomes of HDV and the avian HDV delta-like agent. The potential coiled-coil region is highlighted, including the presence of leucine residues in the correct spacing for a leucine zipper (filled red circle). The delta antigen does not have a strict requirement for leucine in the d-position of the heptad repeat. Additional leucine residues are shown by circles in light red. Serine residues that are conserved between different HDV genotypes and post-translationally modified (phosphorylated) are highlighted with an asterisk. The conserved arginine and lysine residues modified by methylation (Arg-Me) and acetylation (Lys-Ac) are indicated. NLS: Nuclear localisation signal.</p> Full article ">
10 pages, 240 KiB  
Review
Chrysanthemum Stunt Viroid Resistance in Chrysanthemum
by Tomoyuki Nabeshima, Yosuke Matsushita and Munetaka Hosokawa
Viruses 2018, 10(12), 719; https://doi.org/10.3390/v10120719 - 17 Dec 2018
Cited by 5 | Viewed by 5644
Abstract
Chrysanthemum stunt viroid (CSVd) is one of the most severe threats in Chrysanthemum morifolium production. Over the last decade, several studies have reported the natural occurrence of CSVd resistance in chrysanthemum germplasms. Such CSVd-resistant germplasms are desirable for the stable production of chrysanthemum [...] Read more.
Chrysanthemum stunt viroid (CSVd) is one of the most severe threats in Chrysanthemum morifolium production. Over the last decade, several studies have reported the natural occurrence of CSVd resistance in chrysanthemum germplasms. Such CSVd-resistant germplasms are desirable for the stable production of chrysanthemum plants. Current surveys include finding new resistant chrysanthemum cultivars, breeding, and revealing resistant mechanisms. We review the progress, from discovery to current status, of CSVd-resistance studies, while introducing information on the improvement of associated inoculation and diagnostic techniques. Full article
(This article belongs to the Special Issue Viroid-2018: International Conference on Viroids and Viroid-Like RNAs)
18 pages, 2028 KiB  
Article
CD8+ T Cells Responding to the Middle East Respiratory Syndrome Coronavirus Nucleocapsid Protein Delivered by Vaccinia Virus MVA in Mice
by Svenja Veit, Sylvia Jany, Robert Fux, Gerd Sutter and Asisa Volz
Viruses 2018, 10(12), 718; https://doi.org/10.3390/v10120718 - 16 Dec 2018
Cited by 36 | Viewed by 6032
Abstract
Middle East respiratory syndrome coronavirus (MERS-CoV), a novel infectious agent causing severe respiratory disease and death in humans, was first described in 2012. Antibodies directed against the MERS-CoV spike (S) protein are thought to play a major role in controlling MERS-CoV infection and [...] Read more.
Middle East respiratory syndrome coronavirus (MERS-CoV), a novel infectious agent causing severe respiratory disease and death in humans, was first described in 2012. Antibodies directed against the MERS-CoV spike (S) protein are thought to play a major role in controlling MERS-CoV infection and in mediating vaccine-induced protective immunity. In contrast, relatively little is known about the role of T cell responses and the antigenic targets of MERS-CoV that are recognized by CD8+ T cells. In this study, the highly conserved MERS-CoV nucleocapsid (N) protein served as a target immunogen to elicit MERS-CoV-specific cellular immune responses. Modified Vaccinia virus Ankara (MVA), a safety-tested strain of vaccinia virus for preclinical and clinical vaccine research, was used for generating MVA-MERS-N expressing recombinant N protein. Overlapping peptides spanning the whole MERS-CoV N polypeptide were used to identify major histocompatibility complex class I/II-restricted T cell responses in BALB/c mice immunized with MVA-MERS-N. We have identified a H2-d restricted decamer peptide epitope in the MERS-N protein with CD8+ T cell antigenicity. The identification of this epitope, and the availability of the MVA-MERS-N candidate vaccine, will help to evaluate MERS-N-specific immune responses and the potential immune correlates of vaccine-mediated protection in the appropriate murine models of MERS-CoV infection. Full article
(This article belongs to the Special Issue MERS-CoV)
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<p>Generation and characterization of recombinant Modified Vaccinia virus Ankara expressing the Middle East respiratory syndrome coronavirus N protein (MVA-MERS-N); (<b>a</b>) Schematic diagram of the MVA genome indicating the major deletion sites I-VI on the top. Flank-1 and flank-2 refer to MVA DNA sequences adjacent to corresponding insertion site. Deletion III was used to insert MERS-N encoding gene sequences under the transcriptional control of the vaccinia virus promoter PmH5. Repetitive sequences (FR) were designed to remove the mCherry marker by intragenomic homologous recombination (marker gene deletion); (<b>b</b>,<b>c</b>) PCR analyses of genomic viral DNA using oligonucleotide primers to confirm the correct insertion of recombinant MERS-N gene into deletion III (<b>b</b>), and the genetic integrity of the MVA genome for the C7L gene locus (<b>c</b>); (<b>d</b>) Multi-step growth analysis of recombinant MVA-MERS-N and non-recombinant MVA (MVA); Chicken embryo fibroblasts (CEF) and human HaCat or HeLa cells were infected at a multiplicity of infection (MOI) of 0.05 with MVA-MERS-N or MVA. Infected cells were collected at different time points after infection and titrated on CEF cells.</p> Full article ">Figure 2
<p>Analysis of recombinant MVA-MERS proteins; (<b>a</b>) Western Blot analysis of MERS-CoV N protein produced in CEF or HaCat cells. Lysates from cells infected with recombinant MVA (MVA-MERS-N, MVA-MERS-S) or non-recombinant MVA (MVA) at a MOI of five, or from non-infected cells (mock) were prepared at eight, 12, or 24 hpi. Proteins were analyzed by immunoblotting with a monoclonal anti-MERS-N antibody; (<b>b</b>–<b>d</b>) Western Blot analysis of MERS-CoV N and S proteins produced in CEF. Total cell extracts from CEF infected with recombinant MVA (MVA-MERS-N, MVA-MERS-S) or non-recombinant MVA (MVA) at a MOI of five, or from non-infected cells (mock) were prepared at 24 hpi. Cell lysates and proteins were tested by immunoblotting using monoclonal anti MERS-N and anti MERS-S antibody (<b>b</b>) or polyclonal sera from MERS-CoV infected rabbits (<b>c</b>) or cynomolgus macaques (<b>d</b>). Arrows indicate the N- or S-specific protein bands.</p> Full article ">Figure 3
<p>Screening for H2-d restricted T cell epitopes in MERS-CoV N protein using matrix peptide pools; (<b>a</b>–<b>b</b>) groups of BALB/c mice (<span class="html-italic">n</span> = 2 to 6) were vaccinated twice (21-day interval) by i.p. (<b>a</b>) or i.m. (<b>b</b>) application with 10<sup>8</sup> plaque-forming-units (PFU) of recombinant MVA-MERS-N (MVA-N). Mice inoculated with non-recombinant MVA (MVA) or phosphate-buffered saline (PBS) were used as controls. Splenocytes were restimulated in vitro with pools of overlapping peptides corresponding to MERS-CoV N protein. IFN-γ spot-forming CD8+ T cells (IFN-γ SFC) were measured by ELISPOT. The lines represent means.</p> Full article ">Figure 4
<p>Mapping of H2-d restricted T cell epitopes in MERS-CoV N protein; (<b>a–b</b>) BALB/c mice (n = 2 to 4) were immunized twice (21-day interval) i.p. or i.m. with 10<sup>8</sup> PFU of recombinant MVA-MERS-N (MVA-N), non-recombinant MVA (MVA) or PBS. Splenocytes from vaccinated mice were incubated in the presence of subpools (V8.1, V8.2, H8.1, H8.2) from positive matrix pools (<b>a</b>) or individual 15-mers peptides #89 or #90 (<b>b</b>). IFN-γ spot-forming CD8+ T cells (IFN-γ SFC) were quantified by ELISPOT. The lines represent means.</p> Full article ">Figure 5
<p>Identification of an H2-d restricted T cell epitope in MERS-CoV N protein; (<b>a–d</b>) Groups of BALB/c mice (<span class="html-italic">n</span> = 3 to 8) were vaccinated in a prime-boost regime with 10<sup>8</sup> PFU of MVA-MERS-N via i.p. (<b>a</b>) or i.m. (<b>b–d</b>) application. Mice immunized with non-recombinant MVA (MVA) and PBS served as negative controls. (<b>a-b</b>) Splenocytes were stimulated with individual 8-11-mer peptides and IFN-γ spot-forming CD8+ T cells (IFN-γ SFC) were measured by ELISPOT. (<b>c–d</b>) Splenocytes were stimulated with positive MERS-CoV N 10.2 peptide (<b>c</b>) or F2L<sub>26-34</sub> peptide (<b>d</b>) and IFN-γ producing CD8+ or CD4+ T cells were measured using intracellular cytokine staining assay and FACS analysis. The lines represent means. *&lt; 0.05, **&lt; 0.005.</p> Full article ">
18 pages, 6012 KiB  
Article
Strawberry Vein Banding Virus P6 Protein Is a Translation Trans-Activator and Its Activity Can be Suppressed by FveIF3g
by Shuai Li, Yahui Hu, Lei Jiang, Penghuan Rui, Qingqing Zhao, Jiying Feng, Dengpan Zuo, Xueping Zhou and Tong Jiang
Viruses 2018, 10(12), 717; https://doi.org/10.3390/v10120717 - 15 Dec 2018
Cited by 8 | Viewed by 4098
Abstract
The strawberry vein banding virus (SVBV) open reading frame (ORF) VI encodes a P6 protein known as the RNA silencing suppressor. This protein is known to form inclusion like granules of various sizes and accumulate in both the nuclei and the cytoplasm of [...] Read more.
The strawberry vein banding virus (SVBV) open reading frame (ORF) VI encodes a P6 protein known as the RNA silencing suppressor. This protein is known to form inclusion like granules of various sizes and accumulate in both the nuclei and the cytoplasm of SVBV-infected plant cells. In this study, we have determined that the P6 protein is the only trans-activator (TAV) encoded by SVBV, and can efficiently trans-activate the translation of downstream gfp mRNA in a bicistron derived from the SVBV. Furthermore, the P6 protein can trans-activate the expression of different bicistrons expressed by different caulimovirus promoters. The P6 protein encoded by SVBV from an infectious clone can also trans-activate the expression of bicistron. Through protein-protein interaction assays, we determined that the P6 protein could interact with the cell translation initiation factor FveIF3g of Fragaria vesca and co-localize with it in the nuclei of Nicotiana benthamiana cells. This interaction reduced the formation of P6 granules in cells and its trans-activation activity on translation. Full article
(This article belongs to the Special Issue Fruit Tree Viruses and Viroids)
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<p>Strawberry vein banding virus (SVBV) P6 is the only translation trans-activator; (<b>A</b>) map of the SVBV genome and bicistron construct. The ring symbolizes the 7863-bp double-stranded SVBV DNA and the arrangement of seven open reading frames (ORFs). US-US71GFP contains the SVBV-US isolate promoter (pUS) and a fused p7-p1-gfp fragment; (<b>B</b>) leaves were co-infiltrated with pUS-US71GFP and the empty vector (Vec, pCAM2300), pCAM-USP1 (P1), pCAM-USP2 (P2), pCAM-USP3 (P3), pCAM-USP4 (P4), pCAM-USP5 (P5), pCAM-USP6 (P6), or pCAM-GFP (GFP). The infiltrated leaves were photographed under the UV illumination at 3 days post agro-infiltration (dpai); (<b>C</b>) Western blot and Northern blot assays of the GFP protein and GFP mRNA accumulation in various infiltrated leaves. P1 through P6 indicate the six vectors expression SVBV P1, P2, P3, P4, P5 and P6 proteins. The same samples were used for these two assays. Western blot was probed with a GFP specific monoclonal antibody and the Northern blot was probed with a DIG-labeled GFP-specific probe. Coomassie blue staining was used to estimate protein loadings and ethidium bromide staining was used to estimate RNA loadings.</p> Full article ">Figure 1 Cont.
<p>Strawberry vein banding virus (SVBV) P6 is the only translation trans-activator; (<b>A</b>) map of the SVBV genome and bicistron construct. The ring symbolizes the 7863-bp double-stranded SVBV DNA and the arrangement of seven open reading frames (ORFs). US-US71GFP contains the SVBV-US isolate promoter (pUS) and a fused p7-p1-gfp fragment; (<b>B</b>) leaves were co-infiltrated with pUS-US71GFP and the empty vector (Vec, pCAM2300), pCAM-USP1 (P1), pCAM-USP2 (P2), pCAM-USP3 (P3), pCAM-USP4 (P4), pCAM-USP5 (P5), pCAM-USP6 (P6), or pCAM-GFP (GFP). The infiltrated leaves were photographed under the UV illumination at 3 days post agro-infiltration (dpai); (<b>C</b>) Western blot and Northern blot assays of the GFP protein and GFP mRNA accumulation in various infiltrated leaves. P1 through P6 indicate the six vectors expression SVBV P1, P2, P3, P4, P5 and P6 proteins. The same samples were used for these two assays. Western blot was probed with a GFP specific monoclonal antibody and the Northern blot was probed with a DIG-labeled GFP-specific probe. Coomassie blue staining was used to estimate protein loadings and ethidium bromide staining was used to estimate RNA loadings.</p> Full article ">Figure 2
<p>P6 can trans-activate the translation of different bicistrons; (<b>A</b>) schematic representations of bicistron constructs. p35S-Ca71GFP contains the CaMV 35S promoter and a fused p7-p1-gfp fragment. pSY-SY71GFP contains the SVBV-SY isolate promoter (pSY) and a fused p7-p1-gfp fragment; (<b>B</b>) and (<b>C</b>) P6 trans-activated the translations of different bicistrons. <span class="html-italic">N. benthamiana</span> plants co-infiltrated with p35S-Ca71GFP, pUS-US71GFP, or pSY-SY71GFP with the empty vector pCAM2300 or with pCAM-USP6 were photographed under the UV illumination at 3 dpai; (<b>D</b>) Western blot and Northern blot assays using <span class="html-italic">N. benthamiana leaves</span> co-infiltrated with pCAM2300 and p35S-Ca71GFP (lane 1), pCAM2300 and pUS-US71GFP (lane 2), pCAM2300 and pSY-SY71GFP (lane 3), pCAM-USP6 and p35S-Ca71GFP (lane 4), pCAM-USP6 and pUS-US71GFP (lane 5), or pCAM-USP6 and pSY-SY71GFP (lane 6) at 3 dpai. The Western blots were probed with a GFP or SVBV P6 specific antibody. The Northern blot was analyzed as described in <a href="#viruses-10-00717-f001" class="html-fig">Figure 1</a>D).</p> Full article ">Figure 2 Cont.
<p>P6 can trans-activate the translation of different bicistrons; (<b>A</b>) schematic representations of bicistron constructs. p35S-Ca71GFP contains the CaMV 35S promoter and a fused p7-p1-gfp fragment. pSY-SY71GFP contains the SVBV-SY isolate promoter (pSY) and a fused p7-p1-gfp fragment; (<b>B</b>) and (<b>C</b>) P6 trans-activated the translations of different bicistrons. <span class="html-italic">N. benthamiana</span> plants co-infiltrated with p35S-Ca71GFP, pUS-US71GFP, or pSY-SY71GFP with the empty vector pCAM2300 or with pCAM-USP6 were photographed under the UV illumination at 3 dpai; (<b>D</b>) Western blot and Northern blot assays using <span class="html-italic">N. benthamiana leaves</span> co-infiltrated with pCAM2300 and p35S-Ca71GFP (lane 1), pCAM2300 and pUS-US71GFP (lane 2), pCAM2300 and pSY-SY71GFP (lane 3), pCAM-USP6 and p35S-Ca71GFP (lane 4), pCAM-USP6 and pUS-US71GFP (lane 5), or pCAM-USP6 and pSY-SY71GFP (lane 6) at 3 dpai. The Western blots were probed with a GFP or SVBV P6 specific antibody. The Northern blot was analyzed as described in <a href="#viruses-10-00717-f001" class="html-fig">Figure 1</a>D).</p> Full article ">Figure 3
<p>P6 can trans-activate the translation of bicistrons expressed by different 35S promoters; (<b>A</b>) schematic representations of four bicistron constructs with different 35S promoters and P7-P1:GFP fragments. The 35S promoters were from SVBV US or CaMV isolate, and the P7-P1:GFP fragments were from SVBV US, SVBV SY or CaMV isolate; (<b>B</b>) <span class="html-italic">N. benthamiana</span> leaves were co-infiltrated with pCAM-P6 (P6) and pUS-US71GFP, pUS-SY71GFP, p35S-SY71GFP, or pUS-Ca71GFP. Leaves infiltrated with pUS-US71GFP alone were used as negative controls. The infiltrated leaves were examined for GFP expression under a fluorescence microscopy at 70 h post agro-infiltration (hpai). Bars = 100 μm.</p> Full article ">Figure 4
<p>The P6 protein expressed during SVBV infection can trans-activate the translation of a US71GFP bicistron; (<b>A</b>) <span class="html-italic">N. benthamiana</span> leaves were co-infiltrated with pUS-US71GFP, pBinPLUS, and pCAM-2b (2b), pUS-US71GFP, pSVBV-US, and pCAM-2b (2b), or pCAM-GFP alone (2300-GFP). The infiltrated plants were photographed under the UV illumination at 3 dpai; (<b>B</b>) Western blot assay of GFP and P6 protein accumulation, and Northern blot assay of GFP RNA accumulation in the infiltrated leaves. Lane 1, a sample from the pUS-US71GFP, pBinPLUS, and 2b co-infiltrated leaves. Lane 2, a sample from the pUS-US71GFP, pSVBV-US, and 2b co-infiltrated leaves. Lane 3, a sample from the pUS-US71GFP and pCAM2300 co-infiltrated leaves. Procedures of the Western blot and Northern blot assays were the same as described in <a href="#viruses-10-00717-f001" class="html-fig">Figure 1</a>.</p> Full article ">Figure 5
<p>Yeast two-hybrid (Y2H) and BiFC assays for the interaction between the FveIF3g and the P6 protein; (<b>A</b>) Y2H gold cells were co-transfected with the indicated plasmids and cultured on the synthetic drop out medium SD-Leu-Trp followed by the SD-Ade-His-Leu-Trp medium. The cells co-transfected with pGAD-RecT and pGBK-53 were used as a positive control and the cells co-transfected with pGAD-RecT and pGBK-Lam were used as a negative control. Dilutions of the transfected cells are indicated at the top of the images; (<b>B</b>) leaves of <span class="html-italic">N. benthamiana</span> plants were co-infiltrated with various combinations of plasmids: YFP<sup>N</sup>-P6 and YFP<sup>C</sup>-FveIF3g, YFP<sup>N</sup>-P6 and YFP<sup>C</sup>-FvL18, YFP<sup>N</sup>-P6 and YFP<sup>C</sup>-FvL24, YFP<sup>N</sup>-4A and YFP<sup>C</sup>-P2 (positive control), or YFP<sup>N</sup> and YFP<sup>C</sup> (negative control). YFP fluorescence was observed by Confocal Microscopy at 70 hpai; (<b>C</b>) subcellular localization of FveIF3g-mCherry and P6:CFP in the infiltrated <span class="html-italic">N. benthamiana</span> leaf cells was examined and imaged by Confocal Microscopy at 70 hpai. DAPI staining was used to visualize the nuclei in cells. The top left and right images show the FveIF3g-mCherry and P6:CFP fusion fluorescence, respectively. The lower left and right images show the DAPI stained nuclei and the merged image of the three, respectively. Arrows are pointed at the co-localized FveIF3g-mCherry and P6-GFP granules. Bars = 100 µm.</p> Full article ">Figure 5 Cont.
<p>Yeast two-hybrid (Y2H) and BiFC assays for the interaction between the FveIF3g and the P6 protein; (<b>A</b>) Y2H gold cells were co-transfected with the indicated plasmids and cultured on the synthetic drop out medium SD-Leu-Trp followed by the SD-Ade-His-Leu-Trp medium. The cells co-transfected with pGAD-RecT and pGBK-53 were used as a positive control and the cells co-transfected with pGAD-RecT and pGBK-Lam were used as a negative control. Dilutions of the transfected cells are indicated at the top of the images; (<b>B</b>) leaves of <span class="html-italic">N. benthamiana</span> plants were co-infiltrated with various combinations of plasmids: YFP<sup>N</sup>-P6 and YFP<sup>C</sup>-FveIF3g, YFP<sup>N</sup>-P6 and YFP<sup>C</sup>-FvL18, YFP<sup>N</sup>-P6 and YFP<sup>C</sup>-FvL24, YFP<sup>N</sup>-4A and YFP<sup>C</sup>-P2 (positive control), or YFP<sup>N</sup> and YFP<sup>C</sup> (negative control). YFP fluorescence was observed by Confocal Microscopy at 70 hpai; (<b>C</b>) subcellular localization of FveIF3g-mCherry and P6:CFP in the infiltrated <span class="html-italic">N. benthamiana</span> leaf cells was examined and imaged by Confocal Microscopy at 70 hpai. DAPI staining was used to visualize the nuclei in cells. The top left and right images show the FveIF3g-mCherry and P6:CFP fusion fluorescence, respectively. The lower left and right images show the DAPI stained nuclei and the merged image of the three, respectively. Arrows are pointed at the co-localized FveIF3g-mCherry and P6-GFP granules. Bars = 100 µm.</p> Full article ">Figure 6
<p>FveIF3g can suppress the accumulation of the P6 protein and its trans-activation activity; (<b>A</b>) pCAM-P6:GFP and p2300-FveIF3g (P6-GFP + FveIF3g) or pCAM-P6-GFP and pCAM-2300 (P6-GFP + Vec) were co-infiltrated into <span class="html-italic">N. benthamiana</span> leaves. The infiltrated leaves were examined and photographed under the UV light or under a fluorescence microscope at 70 hpai. Bars = 100 μm; (<b>B</b>) Northern blot assay of GFP RNA accumulation in the infiltrated leaves; (<b>C</b>) Western blot assay of P6 protein accumulation in the infiltrated leaves; (<b>D</b>) <span class="html-italic">N. benthamiana</span> leaves were co-infiltrated with pUS-US71GFP, pCAM-USP6 (P6), and p2300-FveIF3g (FveIF3g), or pUS-US71GFP, P6, and pCAM-2300 (Vec). The infiltrated plants were photographed under the UV light at 3 dpai. GFP fluorescence in the pUS-US71GFP, P6, and FveIF3g co-infiltrated leaves was weaker than that in the pUS-US71GFP, P6, and Vec co-infiltrated leaves; (<b>E</b>) Western blot assays of GFP and P6 protein accumulation in the co-infiltrated leaves at 3 dpai. Coomassie brilliant blue staining was used to estimate the sample loadings.</p> Full article ">Figure 6 Cont.
<p>FveIF3g can suppress the accumulation of the P6 protein and its trans-activation activity; (<b>A</b>) pCAM-P6:GFP and p2300-FveIF3g (P6-GFP + FveIF3g) or pCAM-P6-GFP and pCAM-2300 (P6-GFP + Vec) were co-infiltrated into <span class="html-italic">N. benthamiana</span> leaves. The infiltrated leaves were examined and photographed under the UV light or under a fluorescence microscope at 70 hpai. Bars = 100 μm; (<b>B</b>) Northern blot assay of GFP RNA accumulation in the infiltrated leaves; (<b>C</b>) Western blot assay of P6 protein accumulation in the infiltrated leaves; (<b>D</b>) <span class="html-italic">N. benthamiana</span> leaves were co-infiltrated with pUS-US71GFP, pCAM-USP6 (P6), and p2300-FveIF3g (FveIF3g), or pUS-US71GFP, P6, and pCAM-2300 (Vec). The infiltrated plants were photographed under the UV light at 3 dpai. GFP fluorescence in the pUS-US71GFP, P6, and FveIF3g co-infiltrated leaves was weaker than that in the pUS-US71GFP, P6, and Vec co-infiltrated leaves; (<b>E</b>) Western blot assays of GFP and P6 protein accumulation in the co-infiltrated leaves at 3 dpai. Coomassie brilliant blue staining was used to estimate the sample loadings.</p> Full article ">
12 pages, 3951 KiB  
Article
Human Respiratory Syncytial Virus NS 1 Targets TRIM25 to Suppress RIG-I Ubiquitination and Subsequent RIG-I-Mediated Antiviral Signaling
by Junsu Ban, Na-Rae Lee, Noh-Jin Lee, Jong Kil Lee, Fu-Shi Quan and Kyung-Soo Inn
Viruses 2018, 10(12), 716; https://doi.org/10.3390/v10120716 - 14 Dec 2018
Cited by 58 | Viewed by 5674
Abstract
Respiratory syncytial virus (RSV) causes severe acute lower respiratory tract disease. Retinoic acid-inducible gene-I (RIG-I) serves as an innate immune sensor and triggers antiviral responses upon recognizing viral infections including RSV. Since tripartite motif-containing protein 25 (TRIM25)-mediated K63-polyubiquitination is crucial for RIG-I activation, [...] Read more.
Respiratory syncytial virus (RSV) causes severe acute lower respiratory tract disease. Retinoic acid-inducible gene-I (RIG-I) serves as an innate immune sensor and triggers antiviral responses upon recognizing viral infections including RSV. Since tripartite motif-containing protein 25 (TRIM25)-mediated K63-polyubiquitination is crucial for RIG-I activation, several viruses target initial RIG-I activation through ubiquitination. RSV NS1 and NS2 have been shown to interfere with RIG-I-mediated antiviral signaling. In this study, we explored the possibility that NS1 suppresses RIG-I-mediated antiviral signaling by targeting TRIM25. Ubiquitination of ectopically expressed RIG-I-2Cards domain was decreased by RSV infection, indicating that RSV possesses ability to inhibit TRIM25-mediated RIG-I ubiquitination. Similarly, ectopic expression of NS1 sufficiently suppressed TRIM25-mediated RIG-I ubiquitination. Furthermore, interaction between NS1 and TRIM25 was detected by a co-immunoprecipitation assay. Further biochemical assays showed that the SPRY domain of TRIM25, which is responsible for interaction with RIG-I, interacted sufficiently with NS1. Suppression of RIG-I ubiquitination by NS1 resulted in decreased interaction between RIG-I and its downstream molecule, MAVS. The suppressive effect of NS1 on RIG-I signaling could be abrogated by overexpression of TRIM25. Collectively, this study suggests that RSV NS1 interacts with TRIM25 and interferes with RIG-I ubiquitination to suppress type-I interferon signaling. Full article
(This article belongs to the Section Animal Viruses)
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Figure 1

Figure 1
<p>Suppression of RIG-I dependent interferon signaling by RSV NS1. (<b>A</b>) HEK293T cells were transfected with RIG-I-2Cards (RIG-IN; 10 ng/well) together with increasing amount of RSV NS1 (63, 125, 250 ng/well) as indicated together with Interferon (IFN)-β promoter firefly luciferase and Thymidine kinase (TK) renilla luciferase reporter plasmids. Promoter activities were determined by dual-luciferase assays. Data were presented as the mean ± SEM. * <span class="html-italic">p</span> &lt; 0.05. (<b>B</b>–<b>D</b>): HEK293T (<b>B</b>) A549 (<b>C</b>) and HEp-2 (<b>D</b>) cells were transfected with RIG-IN together with increasing amounts of NS1 (63 and 250 ng/well). Total RNAs were prepared 24 h after transfection and subjected to RT-qPCR to determine mRNA levels of IFN-β and ISG15. Data were presented as the mean ± SEM. * <span class="html-italic">p</span> &lt; 0.05. All experiments were repeated at least three times. The results show the most representative data from a single experiment conducted in triplicate.</p> Full article ">Figure 2
<p>Inhibition of RIG-I ubiquitination by RSV NS1. (<b>A</b>) HEK293T cells were transfected with vector (pEBG-GST; 7 μg/dish) or GST-RIG-I-2Cards (GST-RIG-IN; 10 μg/dish) and incubated for 24 h. Cells were infected with RSV (m.o.i. = 4) as indicated and further incubated for 24 h. Cell lysates were subjected to GST-pulldown and immunoblotting using indicated antibodies to analyze the ubiquitination of GST-RIG-IN. Ubiquitinated forms were detected using anti-ubiquitin antibody (P4D1). (<b>B</b>) RSV NS1 and NS2 expression plasmids were transfected to HEK293T cells together with RIG-IN and TRIM25 as indicated. Cell lysates were subjected to GST-pulldown assays. Ubiquitination of RIG-IN was analyzed by immunoblotting of GST pulldown samples and whole cell lysates (WCL). (<b>C</b>) RIG-IN and TRIM25 were transfected with increasing amounts of RSV NS1. RIG-IN ubiquitination was analyzed as in (<b>B</b>). Ubiquitination of RIG-I was quantitatively analyzed by densitometry and normalized to pulled-down GST-RIG-IN (unubiquitinated form) of each sample. All experiments were conducted at least three times with similar results.</p> Full article ">Figure 3
<p>Interaction between RSV NS1 and TRIM25. (<b>A</b>) To test whether RSV NS proteins interact with TRIM25, V5-TRIM25 was expressed with RSV NS1-FLAG or NS2-HA in HEK293T. Whole cell lysates (WCL) were subjected to co-immunoprecipitation (co-IP) and immunoblotting using indicated antibodies. (<b>B</b>) FLAG-tagged RSV NS1 was overexpressed in HEK293T. Cell lysates were immunoprecipitated with anti-FLAG antibody and analyzed by immunoblotting. (<b>C</b>) HEK293T cells were transfected with FLAG-tagged NS1 and V5-tagged TRIM25 expression plasmids. Localization of NS1 and TRIM25 were visualized by primary antibodies and FITC- and PE-labeled secondary antibodies as described in Material and Methods. The lower panel shows the intensity of NS1 (green) and TRIM25 (red) along the white line of the upper panel images. (<b>D</b>) TRIM25 truncated mutants including V5-tagged RING, B-box/CCD and SPRY domain constructs were expressed in HEK293T cells together with RSV NS1. Co-IP was performed followed by immunoblotting. The arrowheads indicate the bands with correct size of each construct. (<b>E</b>) Wild type TRIM25 or SPRY deletion (ΔSPRY) mutant plasmid was transfected with RSV NS1-FLAG followed into HEK293T cells. Interaction was analyzed by co-IP and immunoblotting as indicated. All experiments were conducted at least three times with similar results.</p> Full article ">Figure 4
<p>Inhibition of RIG-IN interaction with MAVS-CARD-PRD by RSV NS1. (<b>A</b>) GST-RIG-IN or GST vector was transfected to HEK293T cells together with MAVS-CARD-PRD-FLAG plasmid as indicated. After 24h, cells were infected with RSV (moi=4) as indicated and further incubated for 24 h. Cell lysates were subjected to GST-pulldown and immunoblotting using indicated antibodies to analyze the ubiquitination of GST-RIG-IN. (<b>B</b>) MAVS-CARD-PRD-FLAG, RSV NS1-V5 and GST-RIG-IN or GST vector plasmids were transfected into HEK293T cells as indicated. The lysates (WCL) were analyzed by co-IP and immunoblotting. Ubiquitinated forms were detected using anti-ubiquitin antibody (P4D1). (<b>C</b>) Densitometric analysis of the precipitated MAVS-CARD-PRD-FLAG. The levels of co-precipitated MAVS-CARD-PRD-FLAG were analyzed by densitometry using Vision-Capt software and normalized to the levels of pulled-down GST-RIG-IN. (<b>D</b>) HA- and V5-tagged TRIM25 (10 μg/Φ10 cm dish) with or without FLAG-tagged NS1 (0, 4 or 8 μg/Φ10 cm dish) were transfected into HEK293T cells as indicated. Interaction between HA-TRIM25 and V5-TRIM25 was analyzed by co-IP and immunoblotting using indicated antibodies. All experiments were conducted at least three times with similar results.</p> Full article ">Figure 5
<p>Ablation of interferon-β promoter suppressive effect of NS1 by ectopic expression of TRIM25. (<b>A</b>,<b>B</b>) RSV NS1 was expressed with increasing amount of TRIM25 in HEK293T cells. To induce interferon-β promotor activity, low molecular weight polyI:C (<b>A</b>) or RIG-IN (<b>B</b>) were transfected at the same time as indicated. Interferon (IFN)-β promoter firefly luciferase and Thymidine kinase (TK) renilla luciferase reporter plasmids were co-transfected. Interferon-promoter activities were analyzed by luciferase assays. Data were presented as the mean ± SEM. * <span class="html-italic">p</span> &lt; 0.05. The experiments were repeated at least three times. The results show the most representative data from a single experiment conducted in triplicate.</p> Full article ">
22 pages, 5272 KiB  
Article
Seasonality Drives Microbial Community Structure, Shaping both Eukaryotic and Prokaryotic Host–Viral Relationships in an Arctic Marine Ecosystem
by Ruth-Anne Sandaa, Julia E. Storesund, Emily Olesin, Maria Lund Paulsen, Aud Larsen, Gunnar Bratbak and Jessica Louise Ray
Viruses 2018, 10(12), 715; https://doi.org/10.3390/v10120715 - 14 Dec 2018
Cited by 19 | Viewed by 6010
Abstract
The Arctic marine environment experiences dramatic seasonal changes in light and nutrient availability. To investigate the influence of seasonality on Arctic marine virus communities, five research cruises to the west and north of Svalbard were conducted across one calendar year, collecting water from [...] Read more.
The Arctic marine environment experiences dramatic seasonal changes in light and nutrient availability. To investigate the influence of seasonality on Arctic marine virus communities, five research cruises to the west and north of Svalbard were conducted across one calendar year, collecting water from the surface to 1000 m in depth. We employed metabarcoding analysis of major capsid protein g23 and mcp genes in order to investigate T4-like myoviruses and large dsDNA viruses infecting prokaryotic and eukaryotic picophytoplankton, respectively. Microbial abundances were assessed using flow cytometry. Metabarcoding results demonstrated that seasonality was the key mediator shaping virus communities, whereas depth exerted a diversifying effect within seasonal virus assemblages. Viral diversity and virus-to-prokaryote ratios (VPRs) dropped sharply at the commencement of the spring bloom but increased across the season, ultimately achieving the highest levels during the winter season. These findings suggest that viral lysis may be an important process during the polar winter, when productivity is low. Furthermore, winter viral communities consisted of Operational Taxonomic Units (OTUs) distinct from those present during the spring-summer season. Our data provided a first insight into the diversity of viruses in a hitherto undescribed marine habitat characterized by extremes in light and productivity. Full article
(This article belongs to the Special Issue Viruses of Microbes V: Biodiversity and Future Applications)
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Figure 1

Figure 1
<p>Sampling stations during the five cruises performed in 2014. Symbol colors are uniform for sampling month, whereas station labels indicate the names of sampling stations. The same color scheme is utilized throughout this study.</p> Full article ">Figure 2
<p>Scatterplot showing abundances of key eukaryotic microbial populations as determined by flow cytometric enumeration. (<b>A</b>) Pico, (<b>B</b>) nano, (<b>C</b>), HNF, and (<b>D</b>) cyanobacteria. Depth (m) is shown on the <span class="html-italic">y</span> axis. Sampling months are represented with different colors. Note logarithmic <span class="html-italic">x</span> axis.</p> Full article ">Figure 3
<p>Scatterplot showing abundances of heterotrophic prokaryotes (HPs) as determined by flow cytometric enumeration. Depth (m) is shown on the <span class="html-italic">y</span> axis. Sampling months are represented with different colors. Note logarithmic <span class="html-italic">x</span> axis.</p> Full article ">Figure 4
<p>Scatterplot showing abundances of virus-like particles (VLPs) as determined by flow cytometric enumeration. (<b>A</b>) Small viruses, (<b>B</b>) medium viruses, (<b>C</b>) large viruses, (<b>D</b>) total viral abundance. Depth (m) is shown on the <span class="html-italic">y</span> axis. Sampling months are represented with different colors. Note logarithmic <span class="html-italic">x</span> axis.</p> Full article ">Figure 5
<p>Virus-to-prokaryote ratios (VPRs). VPRs were calculated by dividing flow cytometry counts of small viruses (V1) by flow cytometry counts of heterotrophic prokaryotes (HPs). Depth (m) is shown on the <span class="html-italic">y</span> axis. Sampling months are represented with different colors.</p> Full article ">Figure 6
<p>Rarefaction analysis of (<b>A</b>) g23 and (<b>B</b>) MCP OTU diversity. OTUs with a relative abundance &gt;0.1% were included.</p> Full article ">Figure 7
<p>Nonmetric multidimensional scaling (NMDS) analysis of OTU diversity for (<b>A</b>) <span class="html-italic">g23</span> and (<b>B</b>) <span class="html-italic">mcp</span> genes. Numbers indicate sampling depth (m), and colors indicate sampling month. Samples clustered together based on similarity in OTU composition (Bray–Curtis dissimilarity), and the axes indicate separation between samples.</p> Full article ">Figure 7 Cont.
<p>Nonmetric multidimensional scaling (NMDS) analysis of OTU diversity for (<b>A</b>) <span class="html-italic">g23</span> and (<b>B</b>) <span class="html-italic">mcp</span> genes. Numbers indicate sampling depth (m), and colors indicate sampling month. Samples clustered together based on similarity in OTU composition (Bray–Curtis dissimilarity), and the axes indicate separation between samples.</p> Full article ">Figure 8
<p>Two-way hierarchical clustering heat maps showing clustering of samples according to similarity in OTU composition. The analyses were based on the relative abundance of dominant OTUs (overall relative abundance &gt;0.1%) in the individual samples. (<b>A</b>) <span class="html-italic">g23</span> and (<b>B</b>) <span class="html-italic">mcp</span> genes. The blue shading represents a continuous scale of OTU relative abundance from high (dark blue) to low (light blue).</p> Full article ">Figure 9
<p>Maximum likelihood tree constructed from the 29 most abundant MCP OTUs (<span class="html-fig-inline" id="viruses-10-00715-i001"> <img alt="Viruses 10 00715 i001" src="/viruses/viruses-10-00715/article_deploy/html/images/viruses-10-00715-i001.png"/></span>). The evolutionary history was inferred using the maximum likelihood method based on the JTT matrix-based model [<a href="#B54-viruses-10-00715" class="html-bibr">54</a>] with 100 bootstraps. Branch lengths indicate the number of amino acid substitutions per site. Abbreviations: CroV = Cafeteria roenbergensis virus; Moumou = Moumouvirus goulette; Mimi = Mimivirus; Mega = Megavirus chiliensis; AaV = Aureococcus anophagefferens virus; PoV = Pyramimonas orientalis virus; PkV = Prymnesium kappa virus; HeV = Haptolina ericina virus; HhV = Haptolina hirta virus; CeV = Chrysochromulina ericina virus; PgV = Phaeocystis globosa virus; PpV = Phaeocystis pouchetii virus; PBCV = Paramecium bursaria chlorella virus; MpV = Micromonas pusilla virus; OsV = Ostreococcus sp. virus; OlV = Ostreococcus lucimarinus virus; BpV = Bathycoccus prasinos virus. Scale bar represents 0.2 substitutions per site.</p> Full article ">
22 pages, 4515 KiB  
Article
Simultaneous Detection of Different Zika Virus Lineages via Molecular Computation in a Point-of-Care Assay
by Sanchita Bhadra, Miguel A. Saldaña, Hannah Grace Han, Grant L. Hughes and Andrew D. Ellington
Viruses 2018, 10(12), 714; https://doi.org/10.3390/v10120714 - 14 Dec 2018
Cited by 9 | Viewed by 5975
Abstract
We have developed a generalizable “smart molecular diagnostic” capable of accurate point-of-care (POC) detection of variable nucleic acid targets. Our isothermal assay relies on multiplex execution of four loop-mediated isothermal amplification reactions, with primers that are degenerate and redundant, thereby increasing the breadth [...] Read more.
We have developed a generalizable “smart molecular diagnostic” capable of accurate point-of-care (POC) detection of variable nucleic acid targets. Our isothermal assay relies on multiplex execution of four loop-mediated isothermal amplification reactions, with primers that are degenerate and redundant, thereby increasing the breadth of targets while reducing the probability of amplification failure. An easy-to-read visual answer is computed directly by a multi-input Boolean OR logic gate (gate output is true if either one or more gate inputs is true) signal transducer that uses degenerate strand exchange probes to assess any combination of amplicons. We demonstrate our methodology by using the same assay to detect divergent Asian and African lineages of the evolving Zika virus (ZIKV), while maintaining selectivity against non-target viruses. Direct analysis of biological specimens proved possible, with crudely macerated ZIKV-infected Aedes aegypti mosquitoes being identified with 100% specificity and sensitivity. The ease-of-use with minimal instrumentation, broad programmability, and built-in fail-safe reliability make our smart molecular diagnostic attractive for POC use. Full article
(This article belongs to the Special Issue New Advances on Zika Virus Research)
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Figure 1
<p>Schematic depicting (<b>A</b>) loop-mediated isothermal amplification (LAMP) integrated with (<b>B</b>) one-, (<b>C</b>) two-, or (<b>D</b>) four-input oligonucleotide strand exchange signal transducers. LAMP uses 2 inner (FIP and BIP) and 2 outer (F3 and B3) primers along with the optional stem (SP) and loop (LP) primers to prime strand displacement DNA amplification by Bst DNA polymerase. The resulting continuous amplification (initiated by both new primer-binding and by self-priming) generates double-stranded concatameric amplicons containing single-stranded loops to which non-priming oligonucleotide strand exchange signal transducers can hybridize. The one-input OSD signal transducer composed of one long and one short DNA strand can hybridize to a single LAMP amplicon loop sequence leading to separation of the fluorophore (F) and quencher (Q). The OR Boolean logic processing two-input strand exchange transducer, 2GO, is composed of two labeled strands, S<sub>I</sub> and S<sub>II</sub>, and a third bridging strand S<sub>III</sub>. Either S<sub>I</sub> and/or S<sub>II</sub> can hybridize to their specific LAMP loop sequences resulting in separation of F and Q. The four-input 4GO probe composed of 5 DNA strands (S1–S5) can hybridize to any combination of up to four different LAMP amplicon loops and perform an OR Boolean operation to produce fluorescence signal. The 4GO probe is denoted in terms of lettered domains (<span class="html-italic">a</span>–<span class="html-italic">g</span>), each of which represents a short fragment of DNA sequence in an otherwise continuous oligonucleotide strand. Complementarity is denoted by a single prime symbol.</p> Full article ">Figure 2
<p>Detection of Zika virus <span class="html-italic">capsid</span>, <span class="html-italic">NS1</span>, <span class="html-italic">NS3</span>, and <span class="html-italic">NS5</span> genes using real-time and visually-read reverse transcription LAMP-OSD assays. Indicated copies of <span class="html-italic">capsid</span> (<b>A</b>), <span class="html-italic">NS3</span> (<b>B</b>), <span class="html-italic">NS1</span> (<b>C</b>), and <span class="html-italic">NS5</span> (<b>D</b>) synthetic RNA templates were amplified by degenerate LAMP-OSD assays specific to each template. OSD fluorescence signals measured in real-time during LAMP amplification are depicted as red (10<sup>5</sup> template copies), blue (10<sup>4</sup> template copies), orange (10<sup>3</sup> template copies), gray (100 template copies), and black (0 template copies; 10<sup>6</sup> non-template RNA) traces. The <span class="html-italic">x</span>-axis depicts the duration of LAMP amplification. OSD fluorescence was also imaged at amplification endpoint using a cellphone (images depicted at the bottom of each panel). Numbers on each assay tube in these images indicate the RNA template copies used. Representative results from three replicate experiments are depicted.</p> Full article ">Figure 3
<p>Detection of Asian and African lineage ZIKV using degenerate reverse transcription LAMP-OSD assays. Genomic RNA from DENV, CHIKV, or Asian or African lineage ZIKV (indicated by their GenBank accession numbers) were used as templates for amplification in degenerate RT-LAMP-OSD assays for Zika virus <span class="html-italic">capsid</span>, <span class="html-italic">NS1</span>, <span class="html-italic">NS3</span>, and <span class="html-italic">NS5</span> genes. OSD fluorescence signals measured at amplification endpoint using LightCycler 96 real-time PCR machine are depicted as blue (<span class="html-italic">capsid</span>), orange (<span class="html-italic">NS1</span>), gray (<span class="html-italic">NS3</span>), and yellow (<span class="html-italic">NS5</span>) bars. Representative results from three replicate experiments are depicted.</p> Full article ">Figure 4
<p>Simultaneous detection of four Zika virus genes using multiplex reverse transcription degenerate LAMP-OSD (multiplex LAMP-OSD) assay. (<b>A</b>) Real-time multiplex LAMP-OSD—synthetic RNA mixtures containing indicated copies of each of the four ZIKV synthetic RNA templates (<span class="html-italic">CA</span>, <span class="html-italic">NS1</span>, <span class="html-italic">NS3</span>, and <span class="html-italic">NS5</span>) were amplified using multiplex LAMP-OSD assays containing 21 degenerate primers and 4 degenerate OSD probes for simultaneous LAMP amplification and sequence-specific detection of all four ZIKV targets. OSD fluorescence signals measured in real-time during LAMP amplification are depicted as blue (10,000 copies each of <span class="html-italic">CA</span>, <span class="html-italic">NS1</span>, <span class="html-italic">NS3</span>, and <span class="html-italic">NS5</span> RNA) and orange (0 ZIKV RNA; 10<sup>6</sup> copies of DENV RNA) traces. The <span class="html-italic">x</span>-axis depicts the duration of LAMP amplification. (<b>B</b>) Endpoint multiplex LAMP-OSD assay with visual detection—synthetic RNA mixtures containing indicated copies of each of the four ZIKV synthetic RNA templates (<span class="html-italic">CA</span>, <span class="html-italic">NS1</span>, <span class="html-italic">NS3</span>, and <span class="html-italic">NS5</span>) were amplified using degenerate multiplex LAMP-OSD assays. OSD fluorescence was imaged after 90 min of amplification using a cellphone. Numbers above each assay tube indicate the RNA template copies used. The reaction with ‘0’ ZIKV RNA received 10<sup>6</sup> copies of DENV RNA. (C-F) Performance of individual assays in the multiplex LAMP-OSD system—synthetic RNA mixtures containing indicated copies of each of the four ZIKV synthetic RNA templates (<span class="html-italic">CA</span>, <span class="html-italic">NS1</span>, <span class="html-italic">NS3</span>, and <span class="html-italic">NS5</span>) were amplified using multiplex LAMP-OSD assays containing LAMP primers for all four targets but only one type of OSD for either <span class="html-italic">capsid</span> (<b>C</b>), <span class="html-italic">NS1</span> (<b>D</b>), <span class="html-italic">NS3</span> (<b>E)</b>, or <span class="html-italic">NS5</span> (<b>F</b>) amplicons. OSD fluorescence signals measured in real-time during LAMP amplification are depicted as blue (10,000 copies each of <span class="html-italic">CA</span>, <span class="html-italic">NS1</span>, <span class="html-italic">NS3</span>, and <span class="html-italic">NS5</span> RNA) and orange (0 ZIKV RNA; 10<sup>6</sup> copies of DENV RNA) traces. The <span class="html-italic">x</span>-axis depicts the duration of LAMP amplification. (<b>G</b>) Detection of Asian and African lineage ZIKV genomic RNA using degenerate multiplex LAMP-OSD assays. Genomic RNA from DENV, CHIKV, or Asian or African lineage ZIKV (indicated by their GenBank accession numbers) were used as templates for amplification. Real-time OSD fluorescence signals are depicted as blue (Asian), red (African), black (CHIKV), and green (DENV) traces. The <span class="html-italic">x</span>-axis depicts the duration of LAMP amplification. For all experiments, representative results from three replicate tests are depicted.</p> Full article ">Figure 5
<p>Simultaneous detection of four Zika virus genes using degenerate 4GO probes and multiplex degenerate reverse transcription LAMP (multiplex LAMP-4GO) assays. Indicated copies of <span class="html-italic">capsid</span>, <span class="html-italic">NS1</span>, <span class="html-italic">NS3</span>, and <span class="html-italic">NS5</span> synthetic target RNA were amplified either individually (panels <b>A</b>–<b>D</b>, respectively) or as a mixture (panel <b>E</b>) using multiplex LAMP-4GO assays containing LAMP primers for all four ZIKV targets and the four-input 4GO probe. 4GO probe fluorescence, measured in real-time at 37 °C after 90 min of LAMP amplification, is depicted as red (10,000 template copies), blue (1,000 template copies), yellow (100 template copies), and black (non-specific LAMP primers with 10<sup>5</sup> copies of its target RNA) traces. The <span class="html-italic">x</span>-axis depicts the duration of endpoint signal measurement. (<b>F</b>) Detection of Asian and African lineage ZIKV genomic RNA using degenerate multiplex LAMP-4GO assays. Genomic RNA from DENV, or Asian or African lineage ZIKV (indicated by their GenBank accession numbers) were used as templates for amplification. 4GO probe fluorescence, measured in real-time at 37 °C after 90 min of LAMP amplification, is depicted as blue (Asian), red (African), and green (DENV) traces. The x-axis depicts the duration of endpoint signal measurement. (<b>G</b>) Detection of Asian and African lineage ZIKV genomic RNA using TaqMan qRT-PCR assay specific for Asian lineage ZIKV NS2b gene. Same amount of viral genomic RNA as was used in panel <b>F</b> were amplified and real-time measurements of assay fluorescence are depicted as blue (Asian), red (African), and green (DENV) traces. (<b>H</b>) Detection limit of degenerate multiplex LAMP-4GO assay for ZIKV genomic RNA. Indicated copies of an Asian lineage ZIKV genome or non-specific DENV genomes were amplified using multiplex LAMP-4GO assays. 4GO probe fluorescence, measured in real-time at 37 °C after 90 min of LAMP amplification, is depicted as blue (Asian) and black (DENV) traces with template copies indicated by open squares (2000 genomes), open circles (189 genomes), and open diamonds (2 genomes). The <span class="html-italic">x</span>-axis depicts the duration of endpoint signal measurement. For all experiments, representative results from three replicate tests are depicted.</p> Full article ">Figure 6
<p>Detection of ZIKV RNA using two-input 2GO probes and degenerate reverse transcription LAMP. (<b>A</b>) Sequence-dependent activation of 2GO probes—synthetic RNA mixtures of 10<sup>6</sup> copies of <span class="html-italic">CA</span>, <span class="html-italic">NS1</span>, <span class="html-italic">NS3</span>, and <span class="html-italic">NS5</span> RNA were amplified using individual or multiplex (Mx) degenerate LAMP assays containing either one or both CAN3.2GO and N1N5.2GO probes. 2GO probe fluorescence signals measured at amplification endpoint using LightCycler 96 real-time PCR machine are depicted as blue (LAMP with only CAN3.2GO), orange (LAMP with only N1N5.2GO), and gray (LAMP with both CAN3.2GO and N1N5.2GO) dots. LAMP primer specificities are indicated on the <span class="html-italic">x</span>-axis. (<b>B</b>) Visual readout of degenerate LAMP-2GO assays. Cellphone image depicts 2GO probe fluorescence at amplification endpoint in individual or multiplex ZIKV LAMP assays containing both CAN3.2GO and N1N5.2GO probes and 10<sup>6</sup> copies of all four synthetic ZIKV RNA and a non-specific LAMP assay (“Non”) containing its cognate RNA. (<b>C</b>) Detection limit of visually-read degenerate multiplex LAMP-2GO assays. Cellphone image depicts endpoint 2GO probe fluorescence of multiplex degenerate RT-LAMP assays containing primers and indicated template RNA copies of all four ZIKV targets. The reaction without any ZIKV RNA contained a non-specific RNA and its cognate LAMP primers. For all experiments, representative results from three replicate tests are depicted.</p> Full article ">Figure 7
<p>Detection of Asian and African lineage ZIKV genomes using degenerate multiplex LAMP-2GO assays. (<b>A</b>) Genomic RNA from DENV, or Asian or African lineage ZIKV (indicated by their GenBank accession numbers) were used as templates for amplification. 2GO probe fluorescence signals measured at amplification endpoint using LightCycler 96 real-time PCR machine are depicted as blue (Asian ZIKV), red (African ZIKV), and black (DENV) markers. (<b>B</b>) Detection limit of degenerate multiplex LAMP-2GO assay for ZIKV genomic RNA. Indicated copies of an Asian lineage ZIKV genome (left panel), indicated dilutions of an African ZIKV genome (right panel), and non-specific DENV genomes (“Non”) were amplified using multiplex LAMP-2GO assays. 2GO probe fluorescence was imaged at amplification endpoint using a cellphone. For all experiments, representative results from three replicate tests are depicted.</p> Full article ">Figure 8
<p>Detection of Zika virus-infected mosquitoes using individual- and multiplex degenerate reverse transcription LAMP assays. Zika virus-infected (panels <b>A</b>–<b>D</b>) and uninfected (panels <b>E</b>–<b>H</b>) <span class="html-italic">Aedes aegypti</span> mosquitoes were directly analyzed using <span class="html-italic">NS1</span> and <span class="html-italic">capsid</span> LAMP-OSD assays or with multiplex LAMP-2GO assays. As a positive control, mosquitoes were tested using the <span class="html-italic">A. aegypti coi</span> LAMP-OSD assay (panels <b>D</b> and <b>H</b>). Smartphone images acquired after 2 h of amplification are depicted. P: positive control; M+: mosquito analyte with LAMP primers; M-: mosquito analyte without LAMP primers; N: no template control. Results of NS2b TaqMan qRT-PCR analysis of all mosquitoes are tabulated.</p> Full article ">
15 pages, 1855 KiB  
Article
Transcriptional and Small RNA Responses of the White Mold Fungus Sclerotinia sclerotiorum to Infection by a Virulence-Attenuating Hypovirus
by Shin-Yi Lee Marzano, Achal Neupane and Leslie Domier
Viruses 2018, 10(12), 713; https://doi.org/10.3390/v10120713 - 14 Dec 2018
Cited by 26 | Viewed by 5317
Abstract
Mycoviruses belonging to the family Hypoviridae cause persistent infection of many different host fungi. We previously determined that the white mold fungus, Sclerotinia sclerotiorum, infected with Sclerotinia sclerotiorum hypovirus 2-L (SsHV2-L) exhibits reduced virulence, delayed/reduced sclerotial formation, and enhanced production of aerial [...] Read more.
Mycoviruses belonging to the family Hypoviridae cause persistent infection of many different host fungi. We previously determined that the white mold fungus, Sclerotinia sclerotiorum, infected with Sclerotinia sclerotiorum hypovirus 2-L (SsHV2-L) exhibits reduced virulence, delayed/reduced sclerotial formation, and enhanced production of aerial mycelia. To gain better insight into the cellular basis for these changes, we characterized changes in mRNA and small RNA (sRNA) accumulation in S. sclerotiorum to infection by SsHV2-L. A total of 958 mRNAs and 835 sRNA-producing loci were altered after infection by SsHV2-L, among which >100 mRNAs were predicted to encode proteins involved in the metabolism and trafficking of carbohydrates and lipids. Both S. sclerotiorum endogenous and virus-derived sRNAs were predominantly 22 nt in length suggesting one dicer-like enzyme cleaves both. Novel classes of endogenous small RNAs were predicted, including phasiRNAs and tRNA-derived small RNAs. Moreover, S. sclerotiorum phasiRNAs, which were derived from noncoding RNAs and have the potential to regulate mRNA abundance in trans, showed differential accumulation due to virus infection. tRNA fragments did not accumulate differentially after hypovirus infection. Hence, in-depth analysis showed that infection of S. sclerotiorum by a hypovirulence-inducing hypovirus produced selective, large-scale reprogramming of mRNA and sRNA production. Full article
(This article belongs to the Special Issue Mycoviruses)
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Figure 1

Figure 1
<p>Size distributions of small RNA sequences that aligned to the <span class="html-italic">S</span>. <span class="html-italic">sclerotiorum</span> genome. (<b>A</b>) Size distribution of small RNA libraries from combined virus-free and hypovirus-infected <span class="html-italic">S</span>. <span class="html-italic">sclerotiorum</span> cultures. White and black columns represent unique and total reads of the sRNAs, respectively. (<b>B</b>) Frequency of 5′ terminal nucleotides from pooled small RNA samples. Size distribution of small RNA reads aligning to (<b>C</b>) coding regions, intergenic regions, retrotransposon sequences, and (<b>D</b>) ribosomal RNA and tRNA sequences. (<b>E</b>) Mature tRNA structures predicted by tRNAscan-SE with sequences of the two most abundant small RNA sequences that resembled stress-induced tRNA halves. I: 4.5 × 106 reads (5.0% of total reads); tRNA Glu-derived tRF5-Glu(GAA) on <span class="html-italic">S</span>. <span class="html-italic">sclerotiorum</span> chromosomes 4, 7, and 16 (two copies); and <span class="html-italic">B</span>. <span class="html-italic">cinerea</span> chromosomes 2, 8, 14, and 16 (two copies). II: 1.6 × 106 reads (1.9%); tRNA Asp on <span class="html-italic">S</span>. <span class="html-italic">sclerotiorum</span> chromosomes 1, 5, 11, 12, and 14; and <span class="html-italic">B</span>. <span class="html-italic">cinerea</span> chromosomes 9, 10, and 13 (two copies).</p> Full article ">Figure 2
<p>Size distribution of small RNA sequences that aligned to the Sclerotinia sclerotiorum hypovirus 2 L (SsHV2L) genome sequence. (<b>A</b>) Size distribution and (<b>B</b>) frequency of 5′ terminal nucleotides of small RNAs that aligned to the SsHV2L genome. (<b>C</b>) Distribution of small RNA reads that aligned to the SsHV2L genome. Bars above zero indicate alignment to the positive strand, and bars below zero indicate alignment to the negative strand.</p> Full article ">Figure 3
<p>Examples of sRNA producing loci in the <span class="html-italic">S</span>. <span class="html-italic">sclerotiorum</span> genome (<b>A</b>) capable of folding into structures similar to pre-microRNA and (<b>B</b>) conserved in the genomes of other members of the family Sclerotiniaceae. Arrows indicate the positions of mature microRNA-like sequences. Connected shaded boxes indicate regions of conserved base pairing in predicted stem-and-loop structures.</p> Full article ">Figure 4
<p>Genome-wide identification of loci producing phased small RNAs in <span class="html-italic">S</span>. <span class="html-italic">sclerotiorum</span>. (<b>A</b>) Distribution of loci producing phased small RNAs (red horizontal bars) on <span class="html-italic">S</span>. <span class="html-italic">sclerotiorum</span> chromosomes. Loci marked with asterisks showed significantly different accumulation of small RNAs between mock-inoculated and SsHV2L-infected samples. Asterisks on the left of the bars indicate reduced small RNA accumulation; bars to the right indicate increased small RNA accumulation. (<b>B</b>) Small RNA abundances and phasing score distributions across two loci producing phased small RNAs in the <span class="html-italic">S</span>. <span class="html-italic">sclerotiorum</span> genome.</p> Full article ">
16 pages, 263 KiB  
Review
Suppression of Type I Interferon Signaling by Flavivirus NS5
by Stephanie Thurmond, Boxiao Wang, Jikui Song and Rong Hai
Viruses 2018, 10(12), 712; https://doi.org/10.3390/v10120712 - 14 Dec 2018
Cited by 39 | Viewed by 5173
Abstract
Type I interferon (IFN-I) is the first line of mammalian host defense against viral infection. To counteract this, the flaviviruses, like other viruses, have encoded a variety of antagonists, and use a multi-layered molecular defense strategy to establish their infections. Among the most [...] Read more.
Type I interferon (IFN-I) is the first line of mammalian host defense against viral infection. To counteract this, the flaviviruses, like other viruses, have encoded a variety of antagonists, and use a multi-layered molecular defense strategy to establish their infections. Among the most potent antagonists is non-structural protein 5 (NS5), which has been shown for all disease-causing flaviviruses to target different steps and players of the type I IFN signaling pathway. Here, we summarize the type I IFN antagonist mechanisms used by flaviviruses with a focus on the role of NS5 in regulating one key regulator of type I IFN, signal transducer and activator of transcription 2 (STAT2). Full article
(This article belongs to the Special Issue New Advances on Zika Virus Research)
12 pages, 4012 KiB  
Article
Parechovirus A Detection by a Comprehensive Approach in a Clinical Laboratory
by Bao-Chen Chen, Jenn-Tzong Chang, Tsi-Shu Huang, Jih-Jung Chen, Yao-Shen Chen, Ming-Wei Jan and Tsung-Hsien Chang
Viruses 2018, 10(12), 711; https://doi.org/10.3390/v10120711 - 12 Dec 2018
Cited by 4 | Viewed by 4399
Abstract
Parechovirus A (Human parechovirus, HPeV) causes symptoms ranging from severe neonatal infection to mild gastrointestinal and respiratory disease. Use of molecular approaches with RT-PCR and genotyping has improved the detection rate of HPeV. Conventional methods, such as viral culture and immunofluorescence assay, together [...] Read more.
Parechovirus A (Human parechovirus, HPeV) causes symptoms ranging from severe neonatal infection to mild gastrointestinal and respiratory disease. Use of molecular approaches with RT-PCR and genotyping has improved the detection rate of HPeV. Conventional methods, such as viral culture and immunofluorescence assay, together with molecular methods facilitate comprehensive viral diagnosis. To establish the HPeV immunofluorescence assay, an antibody against HPeV capsid protein VP0 was generated by using antigenic epitope prediction data. The specificity of the anti-HPeV VP0 antibody was demonstrated on immunofluorescence assay, showing that this antibody was specific for HPeV but not enteroviruses. A total of 74 HPeV isolates, 7 non–polio-enteroviruses and 12 HPeV negative cell culture supernatant were used for evaluating the efficiency of the anti-HPeV VP0 antibody. The sensitivity of HPeV detection by the anti-HPeV VP0 antibody was consistent with 5′untranslated region (UTR) RT-PCR analysis. This study established comprehensive methods for HPeV detection that include viral culture and observation of cytopathic effect, immunofluorescence assay, RT-PCR and genotyping. The methods were incorporated into our routine clinical practice for viral diagnosis. In conclusion, this study established a protocol for enterovirus and HPeV virus identification that combines conventional and molecular methods and would be beneficial for HPeV diagnosis. Full article
(This article belongs to the Special Issue Emerging Viruses)
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<p>Epitope analysis and antibody production. (<b>A</b>) The 289 amino acid-polypeptide sequence of parechovirus A (Human parechovirus type 1, HPeV1) VP0 (Genbank: AGO28196.1) was used to predict an epitope by using the Bepipredlinear epitope prediction method (IEDB analysis resource, <a href="http://tools.immuneepitope.org/bcell/" target="_blank">http://tools.immuneepitope.org/bcell/</a> or <a href="http://www.cbs.dtu.dk/services/BepiPred/" target="_blank">http://www.cbs.dtu.dk/services/BepiPred/</a>). The eight-peptide sequence with antigenicity was shown. (<b>B</b>) Multiple sequence alignment of HPeV type 1-8, 14 and 17-19 VP0 peptides involved use of Clustal Omega, <a href="https://www.ebi.ac.uk/Tools/msa/clustalo/" target="_blank">https://www.ebi.ac.uk/Tools/msa/clustalo/</a>. The VP0 sequence of HPeV type 9-11 and 15-16 are not available in NCBI Genbank for analysis. (<b>C</b>) Schematicfor anti-HPeV VP0 antibody generation. (<b>D</b>) Antibody titration (dilution ranging 1:1000~1:27,000) of the anti-serum was measured by dot blotting with 1 to 1000 ng VP0 peptide spotted.</p> Full article ">Figure 2
<p>Specificity of anti-HPeV VP0 antibody. Immunofluorescence assay (IFA) was conducted in A549 cells (1 × 10<sup>5</sup>) infected with HPeV1, HPeV3, echovirus 9 (ECHO 9), ECHO 11, enterovirus 71 (EV71), coxsackievirus A2 (CVA2), CVA10 and CVA16, coxsackievirus B3 (CVB3) and CVB5, Aichi virus (AiV) and mock control for 24 h. Green fluorescence indicates viral-infected cells identified by anti-HPeV VP0 antibody. The DAPI staining indicated the nuclear location, scale bar: 100 μm.</p> Full article ">Figure 3
<p>Immunoblotting assay with anti-HPeV VP0 antibody. (<b>A</b>) A549 cells (1 × 10<sup>5</sup>, upper panels) and DBTRG-05MG cells (1 × 10<sup>5</sup>, lower panels) were infected with HPeV1 and HPeV3 (MOI = 5), respectively. Cell lysates were harvested at various times post-infection (2 to 48 h) for VP0 detection by anti-HPeV VP0antibody. β-actin was a loading control. (<b>B</b>) pCMV-3X-FLAG-HPeV1 VP0 or -HPeV3 VP0plasmid (2 µg) was transfected into Vero cells (3 × 10<sup>5</sup>) for 30 h. The ectopic expression of FLAG-HPeV1 VP0 or -HPeV3 VP0 was detected by immunoblotting with anti-HPeV VP0 and anti-FLAG antibodies. GAPDH was a loading control.</p> Full article ">Figure 4
<p>Schematic of HPeV diagnosis protocol. The schematic shows the process of HPeV diagnosis in a clinical virology laboratory. The procedures include specimen inoculation, cytopathic effect (CPE) evaluation, IFA staining and RT-PCR analysis of HPeV.</p> Full article ">Figure 5
<p>HPeV detection in clinical specimens from patients underwent HPeV diagnosis by IFA with anti-HPeV VP0 antibody. Green fluorescence indicates HPeV-infected cells (upper panel). Evans blue staining shows the cell location (red fluorescence). Cells infected with HPeV1, HPeV3 HPeV4 and HPeV6 were used as staining controls, scale bar: 20 µm.</p> Full article ">
23 pages, 2745 KiB  
Article
The N-Terminus of the HIV-1 p6 Gag Protein Regulates Susceptibility to Degradation by IDE
by Adrian Schmalen, Julia Karius-Fischer, Pia Rauch, Christian Setz, Klaus Korn, Petra Henklein, Torgils Fossen and Ulrich Schubert
Viruses 2018, 10(12), 710; https://doi.org/10.3390/v10120710 - 12 Dec 2018
Cited by 4 | Viewed by 4457
Abstract
As part of the Pr55Gag polyprotein, p6 fulfills an essential role in the late steps of the replication cycle. However, almost nothing is known about the functions of the mature HIV-1 p6 protein. Recently, we showed that p6 is a bona fide [...] Read more.
As part of the Pr55Gag polyprotein, p6 fulfills an essential role in the late steps of the replication cycle. However, almost nothing is known about the functions of the mature HIV-1 p6 protein. Recently, we showed that p6 is a bona fide substrate of the insulin-degrading enzyme (IDE), a ubiquitously expressed zinc metalloprotease. This phenomenon appears to be specific for HIV-1, since p6 homologs of HIV-2, SIV and EIAV were IDE-insensitive. Furthermore, abrogation of the IDE-mediated degradation of p6 reduces the replication capacity of HIV-1 in an Env-dependent manner. However, it remained unclear to which extent the IDE mediated degradation is phylogenetically conserved among HIV-1. Here, we describe two HIV-1 isolates with IDE resistant p6 proteins. Sequence comparison allowed deducing one single amino acid regulating IDE sensitivity of p6. Exchanging the N-terminal leucine residue of p6 derived from the IDE sensitive isolate HIV-1NL4-3 with proline enhances its stability, while replacing Pro-1 of p6 from the IDE insensitive isolate SG3 with leucine restores susceptibility towards IDE. Phylogenetic analyses of this natural polymorphism revealed that the N-terminal leucine is characteristic for p6 derived from HIV-1 group M except for subtype A, which predominantly expresses p6 with an N-terminal proline. Consequently, p6 peptides derived from subtype A are not degraded by IDE. Thus, IDE mediated degradation of p6 is specific for HIV-1 group M isolates and not occasionally distributed among HIV-1. Full article
(This article belongs to the Section Animal Viruses)
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<p>p6 derived from the HIV-1 isolates SG3 and 4lig7 is not degraded by IDE. (<b>A</b>) <span class="html-italic">v</span>p6 derived from NL4-3, SG3 or 4lig7 was incubated with S10 extract. p6 and p24 probings were detected by Western blotting. <span class="html-italic">s</span>p6 served as staining control. Representative Western blots of three independent experiments are shown. (<b>B</b>) Alignment of the p6 aa sequences of HIV-1 NL4-3, SG3, and 4lig7. Aa that differ in the sequence of SG3 and 4lig7 compared to NL4-3 are written in bold. Common polymorphisms of SG3 and 4lig7 are highlighted by arrows. Conserved aa positions in the sequence alignment are indicated by asterisks. Furthermore, aa exchanges between strongly (colon) and weakly (dot) similar aa residues are specified [<a href="#B45-viruses-10-00710" class="html-bibr">45</a>,<a href="#B46-viruses-10-00710" class="html-bibr">46</a>,<a href="#B47-viruses-10-00710" class="html-bibr">47</a>]. (<b>C</b>) Sequence alignment of IDE insensitive p6 peptides from HIV-2, SIV and EIAV p9 peptide [<a href="#B16-viruses-10-00710" class="html-bibr">16</a>]. The sequence of HIV-2 p6 originates from the isolate ROD10, SIV p6 from SIVmac239, and EIAV p9 from the isolate EIAV<sub>Wyoming</sub> [<a href="#B16-viruses-10-00710" class="html-bibr">16</a>,<a href="#B58-viruses-10-00710" class="html-bibr">58</a>]. Colors of the sequence alignments according to the physicochemical properties of the aa, as proposed by the Clustal Omega multiple sequence alignment software [<a href="#B45-viruses-10-00710" class="html-bibr">45</a>,<a href="#B46-viruses-10-00710" class="html-bibr">46</a>,<a href="#B47-viruses-10-00710" class="html-bibr">47</a>].</p> Full article ">Figure 2
<p>Pro-1 in p6 impairs IDE-mediated degradation. Lysates of VLPs derived from pNLenv1 <span class="html-italic">wt</span> and L1P (<b>A</b>) or viral particles of SG3 <span class="html-italic">wt</span> and P1L (<b>B</b>) were incubated with S10. Reactions were stopped by heat inactivation either immediately or after incubation for 60 min at 37 °C. Representative Western blots of three independent experiments are shown. <span class="html-italic">v</span>p6 derived from NL4-3 <span class="html-italic">wt</span> and L1P (<b>C</b>) or SG3 <span class="html-italic">wt</span> and P1L (<b>D</b>) were incubated with S10 for the times indicated. Samples were analyzed by Western blotting. (<b>E</b>) Time kinetics of three independently performed experiments are shown. Values represent the arithmetic mean ± SD.</p> Full article ">Figure 3
<p>Longtime degradation kinetics of p6 derived from NL4-3 <span class="html-italic">wt</span> and L1P. NL4-3 <span class="html-italic">v</span>p6 derived from the mutants NL4-3 <span class="html-italic">wt</span> (<b>A</b>) and L1P (<b>B</b>) were incubated with S10 derived from TZMbl <span class="html-italic">wt</span> or IDE KO cells, respectively. After incubation for the times indicated, reactions were stopped by heat inactivation. Samples were analyzed by Western blotting using a p6-reactive antiserum. Representative Western blots of three independent experiments are shown. (<b>C</b>) Time kinetics of three independently performed experiments is shown. Values represent the arithmetic mean ± SD.</p> Full article ">Figure 4
<p>Influence of the mutation L1P on virus release and Gag-processing. HeLa SS6 cells were transfected with pNL<span class="html-italic">env</span>1 <span class="html-italic">wt</span> or pNL<span class="html-italic">env</span>1 L1P, respectively. Cell lysates and VLP fractions were analyzed by Western blotting using a p24-reactive (<b>A</b>) or a p6-reactive (<b>B</b>) antiserum. (<b>C</b>) The efficiency of virus release was calculated as the ratio of Gag (Pr55 and p24) present in the VLP fraction relative to the total amount of Gag detected in cells and released VLPs. (<b>D</b>) The rate of p24 processing was determined by calculating the ratio of p24 vs. Gag (Pr55 and p24) detected in released VLPs. (<b>E</b>) The rate of p6 processing was determined by calculating the ratio of p6 vs. Gag (Pr55, NC-p6, and p6) detected in released VLPs. (C–E) Band intensities were densitometrically quantified with AIDA. Values of pNL<span class="html-italic">env</span>1 <span class="html-italic">wt</span> were set to 100%. Scattered blots with columns representing mean values of four (C,D) or three (E) independent experiments ± SD. One sample <span class="html-italic">t</span>-test was conducted to determine statistically significant differences in virus release (C), p24 processing (D) and p6 processing (E) between the mutant pNL<span class="html-italic">env</span>1 L1P and pNL<span class="html-italic">env</span>1 <span class="html-italic">wt</span> (*<span class="html-italic">p</span> &lt; 0.05; not significant (n.s.) <span class="html-italic">p</span> ≥ 0.05).</p> Full article ">Figure 5
<p>Influence of the IDE inhibitor 6bk on the replication capacities of X4-tropic HIV-1 NL4-3 L1P and SG3 P1L. Representative replication profiles are shown for infected PHA-IL2-stimulated PBMCs. Cells were infected with X4-tropic HIV-1<sub>NL4-3</sub> <span class="html-italic">wt</span> (<b>A</b>), HIV-1<sub>NL4-3</sub> L1P (<b>B</b>) (each 30 pg p24, MOI 10<sup>−4</sup>), SG3 <span class="html-italic">wt</span> (<b>C</b>) or SG3 P1L (<b>D</b>) (each 60 pg p24, MOI 10<sup>−2</sup>) with or without permanent treatment with 10 μM 6bk (left). Uninfected and untreated PBMCs served as mock control. Replication was assessed by quantification of the virus-associated RT activity contained in cell culture supernatant collected on the indicated dpi. The replication capacity of X4-tropic HIV-1<sub>NL4-3</sub> wt, HIV-1<sub>NL4-3</sub> L1P, SG3 <span class="html-italic">wt</span> or SG3 P1L following infection of PHA-IL2-stimulated PBMCs with and without permanent treatment with 10 µM 6bk was assessed by calculating the area under the curve (AUC) from each replication profile (right). The replication capacity of untreated cells in each experiment was set to 100%. Scattered blots with columns representing mean values of three (A,B) or five (C,D) independently performed experiments ± SD. One sample <span class="html-italic">t</span>-test was conducted to determine statistically significant differences between the replication capacity of treated and untreated cells (*<span class="html-italic">p</span> &lt; 0.05; n.s. <span class="html-italic">p</span> ≥ 0.05).</p> Full article ">Figure 6
<p>Phylogenetic background of the aa at position one of p6. (<b>A</b>) SATé HIV-1/SIV/HIV-2 phylogeny in circular form. In the phylogenetic tree, the isolates have been colored corresponding to the first aa of p6. Isolates with a proline at position one of p6 are depicted in red, isolates in which p6 begins with leucine are colored green, and all other isolates are written in blue. The labeled rings indicate the virus species HIV-1 or HIV-2, respectively (outer), groups (middle) and subtypes (inner). (<b>B</b>) A total of 2088 representative sequences from the Los Alamos Sequence Database have been analyzed in silico regarding the first aa of p6. Depicted are the aa occurrences at position one of the p6 peptides of all HIV-1 groups and subtypes, SIVsm (including SIVmac sequences) and HIV-2. Sequences of p6, which begin neither with proline (red) nor with leucine (green), have been summarized as others (blue). The number of analyzed sequences is indicated in brackets and was set to 100% for each column.</p> Full article ">Figure 7
<p>Degradation of p6 derived from various subtypes by IDE. (<b>A</b>) rIDE was incubated with <span class="html-italic">v</span>p6 derived from the indicated field isolates. Reactions were stopped by heat inactivation either immediately or after incubation for 60 min at 37 °C. p6 incubated with IDE buffer for 60 min at 37 °C served as negative control. Representative Western blots of four independent experiments are shown. (<b>B</b>) Sequence alignment of the isolates tested for sensitivity towards IDE-mediated degradation. Colors of the sequence alignments according to the physicochemical properties of the aa, as proposed by the Clustal Omega multiple sequence alignment software. Conserved aa positions in the sequence alignment are indicated by asterisks. Furthermore, aa exchanges between strongly (colon) and weakly (dot) similar aa residues are specified [<a href="#B45-viruses-10-00710" class="html-bibr">45</a>,<a href="#B46-viruses-10-00710" class="html-bibr">46</a>,<a href="#B47-viruses-10-00710" class="html-bibr">47</a>].</p> Full article ">
18 pages, 2569 KiB  
Article
RVFV Infection in Goats by Different Routes of Inoculation
by Andrea L. Kroeker, Valerie Smid, Carissa Embury-Hyatt, Estella Moffat, Brad Collignon, Oliver Lung, Robbin Lindsay and Hana Weingartl
Viruses 2018, 10(12), 709; https://doi.org/10.3390/v10120709 - 12 Dec 2018
Cited by 7 | Viewed by 4520
Abstract
Rift Valley fever virus (RVFV) is a zoonotic arbovirus of the Phenuiviridae family. Infection causes abortions in pregnant animals, high mortality in neonate animals, and mild to severe symptoms in both people and animals. There is currently an ongoing effort to produce safe [...] Read more.
Rift Valley fever virus (RVFV) is a zoonotic arbovirus of the Phenuiviridae family. Infection causes abortions in pregnant animals, high mortality in neonate animals, and mild to severe symptoms in both people and animals. There is currently an ongoing effort to produce safe and efficacious veterinary vaccines against RVFV in livestock to protect against both primary infection in animals and zoonotic infections in people. To test the efficacy of these vaccines, it is essential to have a reliable challenge model in relevant target species, including ruminants. We evaluated two goat breeds (Nubian and LaMancha), three routes of inoculation (intranasal, mosquito-primed subcutaneous, and subcutaneous) using an infectious dose of 107 pfu/mL, a virus strain from the 2006–2007 Kenyan/Sudan outbreak and compared the effect of using virus stocks produced in either mammalian or mosquito cells. Our results demonstrated that the highest and longest viremia titers were achieved in Nubian goats. The Nubian breed was also efficient at producing clinical signs, consistent viremia (peak viremia: 1.2 × 103–1.0 × 105 pfu/mL serum), nasal and oral shedding of viral RNA (1.5 × 101–8 × 106 genome copies/swab), a systemic infection of tissues, and robust antibody responses regardless of the inoculation route. The Nubian goat breed and a needle-free intranasal inoculation technique could both be utilized in future vaccine and challenge studies. These studies are important for preventing the spread and outbreak of zoonotic viruses like RVFV and are supported by the Canadian-led BSL4ZNet network. Full article
(This article belongs to the Special Issue Animal Models for Viral Diseases)
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Figure 1

Figure 1
<p>Sequence of RVFV strain UAP. Next generation sequencing was used to sequence the three genome segments of RVFV: L (<b>A</b>), M (<b>B</b>), and S (<b>C</b>). In each graph, the <span class="html-italic">y</span>-axis indicates the number of reads and <span class="html-italic">x</span>-axis represents the coverage over the length of the gene in base pairs. The nucleotide (<b>D</b>) and corresponding amino acid (<b>E</b>) sequences of the three RVFV genome segments of UAP were aligned with ZH-501 and Kenya-128-b RVFV strains; results of % homology between the strains are presented for each genome segment.</p> Full article ">Figure 2
<p>Phylogenetic analysis of RVFV strain UAP A phylogenetic analysis of the RVFV strain UAP using the Maximum-Likelihood Model was performed using MEGA7 software for each of the three RVFV genome segments: L (<b>A</b>), M (<b>B</b>), and S (<b>C</b>).</p> Full article ">Figure 2 Cont.
<p>Phylogenetic analysis of RVFV strain UAP A phylogenetic analysis of the RVFV strain UAP using the Maximum-Likelihood Model was performed using MEGA7 software for each of the three RVFV genome segments: L (<b>A</b>), M (<b>B</b>), and S (<b>C</b>).</p> Full article ">Figure 2 Cont.
<p>Phylogenetic analysis of RVFV strain UAP A phylogenetic analysis of the RVFV strain UAP using the Maximum-Likelihood Model was performed using MEGA7 software for each of the three RVFV genome segments: L (<b>A</b>), M (<b>B</b>), and S (<b>C</b>).</p> Full article ">Figure 3
<p>Experimental design and clinical signs. The differences between and number of animals in the experimental groups are summarized for the Nubian (<b>A</b>) and LaMancha (<b>B</b>) goats. Nubian (<b>C</b>) and LaMancha (<b>D</b>) goats were examined daily for signs of illness and each experimental group was collectively given a clinical score between 0 and 11. Rectal temperatures were taken and recorded daily for the Nubian (<b>E</b>) and LaMancha (<b>F</b>) goats. The threshold for fever was considered to be a temperature of 41 °C or higher. Temperature values are shown for individual goats (<span class="html-italic">n</span> = 4 at 0–1 dpi; <span class="html-italic">n</span> = 3 at 2–7 dpi; and <span class="html-italic">n</span> = 2 at 8 dpi); the horizontal line represents the average for the group; the vertical line represents the standard deviation for the group. C6 refers to virus that was grown in C6/36 mosquito cells; VE6 refers to virus that was grown in VE6 mammalian cells. The Nubian and LaMancha uninfected groups received an injection of PBS but no virus.</p> Full article ">Figure 4
<p>Quantification of viremia. (<b>A</b>–<b>C</b>) Quantification of RVFV viremia in Nubian goats after subcutaneous infection (<b>A</b>), mosquito-primed subcutaneous infection (<b>B</b>), and intranasal infection (<b>C</b>). (<b>D</b>–<b>F</b>) Quantification of RVFV viremia in LaMancha goats after subcutaneous infection (<b>D</b>), intranasal infection with mosquito cell-derived virus (<b>E</b>), and intranasal infection with mammalian cell-derived virus (<b>F</b>). Triangles indicate the presence of viral RNA as measured by RT-PCR; circles indicate the presence of infectious virus as measured by plaque assay. Values are shown for individual goats (<span class="html-italic">n</span> = 4 on day 1; <span class="html-italic">n</span> = 3 on days 2–7); the horizontal line represents the average for the group; the vertical line represents the standard deviation for the group. C6 refers to virus that was grown in C6/36 mosquito cells; VE6 refers to virus that was grown in VE6 mammalian cells. The PCR and plaque detection thresholds are indicated. Dpi: days post infection. * threshold for RNA detection; ** threshold for plaque detection.</p> Full article ">Figure 5
<p>Oral and nasal shedding. (<b>A</b>–<b>C</b>) Quantification of RVFV in nasal swabs in Nubian goats after subcutaneous infection (<b>A</b>), mosquito-primed subcutaneous infection (<b>B</b>), and intranasal infection (<b>C</b>). (<b>D</b>–<b>F</b>) Quantification of RVFV in oral swabs in Nubian goats after subcutaneous infection (<b>D</b>), mosquito-primed subcutaneous infection (<b>E</b>), and intranasal infection (<b>F</b>). All LaMancha swabs were negative for virus and data is not shown. Triangles indicate the presence of viral RNA as measured by RT-PCR; circles indicate the presence of infectious virus as measured by plaque assay. Values are shown for individual goats (<span class="html-italic">n</span> = 4 on day 1; <span class="html-italic">n</span> = 3 on days 2–7; and <span class="html-italic">n</span> = 2 on days 14–28); the horizontal line represents the average for the group; the vertical line represents the standard deviation for the group. C6 refers to virus that was grown in C6/36 mosquito cells; VE6 refers to virus that was grown in VE6 mammalian cells. The PCR and plaque detection thresholds are indicated. Dpi: Days post infection. * threshold for RNA detection; ** threshold for plaque detection.</p> Full article ">Figure 6
<p>Viral load in tissues. Quantification of RVFV in tissues in Nubian goats at 1 dpi (<span class="html-italic">n</span> = 1), 7 dpi (<span class="html-italic">n</span> = 1), and 28 dpi (<span class="html-italic">n</span> = 2). Only data for the detection of viral RNA by RT-PCR is shown; virus isolations for all tissues were negative. All LaMancha tissues were negative for virus and data is not shown. Dpi: Days post infection. C6 refers to virus that was grown in C6/36 mosquito cells. LN: Lymph node.</p> Full article ">Figure 7
<p>In situ hybridization for RVFV in tissues. In-situ hybridization staining for RVFV in spleen tissues from Nubian goats. (<b>A</b>) subcutaneous infection at 7 dpi (<span class="html-italic">n</span> = 1), (<b>B</b>) subcutaneous infection at 28 dpi (<span class="html-italic">n</span> = 2), (<b>C</b>) mosquito-primed subcutaneous infection at 28 dpi (<span class="html-italic">n</span> = 2), and (<b>D</b>) intranasal infection at 28 dpi (<span class="html-italic">n</span> = 2). The large images are taken at 40× magnification and arrows indicate individual dots in the slides where weak staining is present. The inserted panels in (<b>B</b>–<b>D</b>) are taken at 100× magnification to help visualize an area of positive staining, as shown within the dotted lines.</p> Full article ">Figure 8
<p>Quantification of neutralizing antibodies against RVFV in serum. The titers are given as a log<sub>2</sub> reciprocal dilution for the Nubian (<b>A</b>) and LaMancha (<b>B</b>) goats. Data for individual animals is shown with the horizontal bars representing the standard deviations (<span class="html-italic">n</span> = 3 on days 2–7; <span class="html-italic">n</span> = 2 on days 14–28). Dpi: Days post infection.</p> Full article ">
27 pages, 1385 KiB  
Review
Modulation of Innate Immune Responses by the Influenza A NS1 and PA-X Proteins
by Aitor Nogales, Luis Martinez-Sobrido, David J. Topham and Marta L. DeDiego
Viruses 2018, 10(12), 708; https://doi.org/10.3390/v10120708 - 12 Dec 2018
Cited by 70 | Viewed by 9226
Abstract
Influenza A viruses (IAV) can infect a broad range of animal hosts, including humans. In humans, IAV causes seasonal annual epidemics and occasional pandemics, representing a serious public health and economic problem, which is most effectively prevented through vaccination. The defense mechanisms that [...] Read more.
Influenza A viruses (IAV) can infect a broad range of animal hosts, including humans. In humans, IAV causes seasonal annual epidemics and occasional pandemics, representing a serious public health and economic problem, which is most effectively prevented through vaccination. The defense mechanisms that the host innate immune system provides restrict IAV replication and infection. Consequently, to successfully replicate in interferon (IFN)-competent systems, IAV has to counteract host antiviral activities, mainly the production of IFN and the activities of IFN-induced host proteins that inhibit virus replication. The IAV multifunctional proteins PA-X and NS1 are virulence factors that modulate the innate immune response and virus pathogenicity. Notably, these two viral proteins have synergistic effects in the inhibition of host protein synthesis in infected cells, although using different mechanisms of action. Moreover, the control of innate immune responses by the IAV NS1 and PA-X proteins is subject to a balance that can determine virus pathogenesis and fitness, and recent evidence shows co-evolution of these proteins in seasonal viruses, indicating that they should be monitored for enhanced virulence. Importantly, inhibition of host gene expression by the influenza NS1 and/or PA-X proteins could be explored to develop improved live-attenuated influenza vaccines (LAIV) by modulating the ability of the virus to counteract antiviral host responses. Likewise, both viral proteins represent a reasonable target for the development of new antivirals for the control of IAV infections. In this review, we summarize the role of IAV NS1 and PA-X in controlling the antiviral response during viral infection. Full article
(This article belongs to the Special Issue Cytokine Responses in Viral Infections)
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Figure 1

Figure 1
<p>A schematic representation of an influenza A virus (IAV) non-structural (NS) segment, viral transcripts, and NS1 domains. (<b>A</b>) An IAV NS RNA segment is indicated by a blue box and non-coding regions (NCR) are indicated with white boxes. IAV NS1 and nuclear export protein (NEP) transcripts are indicated with yellow and red boxes, respectively. IAV NS1 and NEP open reading frames (ORFs) shared the first 30 nucleotides in the N-terminus. The numbers on the top of the bars represent the ORF length and nucleotide splice positions. (<b>B</b>) The NS1 protein is divided into four distinct regions: The N-terminal RNA-binding domain (RBD; amino acids 1–73, yellow), the linker sequence (L; amino acids 74–88, red), the effector domain (ED; amino acids 89–202, blue), and the C-terminal tail (CTT; amino acids 203 to the end, gray). Note that both the L and the CTT can vary in length among different IAV strains, and, although a 237 amino-acids-length NS1 has been represented, the NS1 can be 219, 230, and 237 amino acids in length. Nuclear localization and export signals (NLS and NES, respectively) are indicated with black boxes at the bottom.</p> Full article ">Figure 2
<p>Direct and indirect effects of the IAV NS1 protein on innate immune responses. (<b>A</b>) IAV NS1 decreases RIG-I activation, and IFN responses, through the sequestration of dsRNA [<a href="#B88-viruses-10-00708" class="html-bibr">88</a>,<a href="#B89-viruses-10-00708" class="html-bibr">89</a>,<a href="#B90-viruses-10-00708" class="html-bibr">90</a>], or by interaction with TRIM25 or Riplet, which results in the suppressed ubiquitination and activation of RIG-I, which signals through the mitochondrial antiviral signaling protein (MAVS) to induce IFN responses [<a href="#B91-viruses-10-00708" class="html-bibr">91</a>,<a href="#B92-viruses-10-00708" class="html-bibr">92</a>]. (<b>B</b>) NS1 inhibits the IRF3 [<a href="#B99-viruses-10-00708" class="html-bibr">99</a>], NF-κβ [<a href="#B117-viruses-10-00708" class="html-bibr">117</a>], and AP-1 [<a href="#B100-viruses-10-00708" class="html-bibr">100</a>] transcription factors, impairing IFN production, and, therefore, the induction of IFN-stimulated gene (ISG) products. In addition, NS1 directly inhibits the antiviral activities of the ISGs PKR and OAS-RNaseL. (<b>C</b>) The IAV NS1 protein binds dsRNA and PKR, leading to decreased PKR activity to phosphorylate eIF2α, and host translation inhibition [<a href="#B101-viruses-10-00708" class="html-bibr">101</a>,<a href="#B102-viruses-10-00708" class="html-bibr">102</a>,<a href="#B103-viruses-10-00708" class="html-bibr">103</a>]. (<b>D</b>) The IAV NS1 protein, via the dsRNA-binding activity of its RBD, inhibits OAS activation, blocking RNA degradation [<a href="#B87-viruses-10-00708" class="html-bibr">87</a>]. (<b>E</b>) The IAV NS1 protein also inhibits NLRP3 inflammasome activation [<a href="#B105-viruses-10-00708" class="html-bibr">105</a>,<a href="#B106-viruses-10-00708" class="html-bibr">106</a>,<a href="#B107-viruses-10-00708" class="html-bibr">107</a>], impairing the cleavage of pro-interleukin (IL)-1β and pro-IL-18 into their mature forms IL-1β and IL-18, respectively, which are released from the cell to stimulate inflammatory processes. (<b>F</b>) NS1 proteins from some human and avian IAV strains bind to CPSF30, blocking the cleavage of immature mRNAs (pre-mRNAs) and the recruitment of the poly(A) polymerase to add the poly(A) tail [<a href="#B62-viruses-10-00708" class="html-bibr">62</a>,<a href="#B72-viruses-10-00708" class="html-bibr">72</a>,<a href="#B74-viruses-10-00708" class="html-bibr">74</a>,<a href="#B108-viruses-10-00708" class="html-bibr">108</a>,<a href="#B109-viruses-10-00708" class="html-bibr">109</a>,<a href="#B110-viruses-10-00708" class="html-bibr">110</a>,<a href="#B123-viruses-10-00708" class="html-bibr">123</a>]. The IAV NS1 protein also binds to the poly(A)-binding protein II (PABPII), inhibiting its ability to stimulate the synthesis of long poly(A) tails [<a href="#B111-viruses-10-00708" class="html-bibr">111</a>]. These last two processes lead to host shutoff of protein synthesis [<a href="#B111-viruses-10-00708" class="html-bibr">111</a>]. (<b>G</b>) Additionally, the NS1 of influenza A/WSN/33 H1N1 (WSN) binds NXF1, p15, RAE1, and E1B-AP5, which interact with both mRNAs and nucleoporins to direct mRNAs through the nuclear pore complex, blocking their function, and likely facilitating host cellular shutoff [<a href="#B116-viruses-10-00708" class="html-bibr">116</a>].</p> Full article ">Figure 3
<p>The tridimensional structure of the IAV NS1 ED coupled to the F2/F3 domain of CPSF30. A/Udorn/72 H3N2 strain NS1 ED bound to the F2/F3 fragment of the human CPSF30 was previously crystalized [<a href="#B108-viruses-10-00708" class="html-bibr">108</a>] (protein data bank (PDB) entry 2RHK). Colors were included using the MacPyMOL Molecular Graphics system (pymol.org). Each monomer of the NS1 ED is represented in green colors. Monomers of the F2/F3 fragment of the human CPSF30 are represented in blue colors. The artificially introduced NS1 amino acid residues 108, 125, and 189 restoring NS1–CPSF0 binding (<b>A</b>) [<a href="#B125-viruses-10-00708" class="html-bibr">125</a>], and the residues 90, 123, 125, and 131 found in naturally circulating pH1N1 viruses (<b>B</b>) [<a href="#B126-viruses-10-00708" class="html-bibr">126</a>], are indicated in reddish colors (orange, purple, and pink). Figure adapted from [<a href="#B126-viruses-10-00708" class="html-bibr">126</a>].</p> Full article ">Figure 4
<p>A schematic representation of the IAV PA viral segment and the PA and PA-X open reading frames (ORFs). Blue and red boxes indicate the ORF for PA and PA-X, respectively. The +1 frameshift motif (UCC UUU <b><span class="html-italic">C</span></b>GU C) at position 191 is indicated. Bold and italics in the frameshift motif (C nucleotide) indicate that the nucleotide C is not read during PA-X translation. PA-X proteins containing 232 or 252 amino acids if the C-terminal region has a 41 or 61 amino acid extension, respectively, are indicated.</p> Full article ">
12 pages, 2761 KiB  
Article
Metatranscriptomic Analysis and In Silico Approach Identified Mycoviruses in the Arbuscular Mycorrhizal Fungus Rhizophagus spp.
by Achal Neupane, Chenchen Feng, Jiuhuan Feng, Arjun Kafle, Heike Bücking and Shin-Yi Lee Marzano
Viruses 2018, 10(12), 707; https://doi.org/10.3390/v10120707 - 12 Dec 2018
Cited by 21 | Viewed by 4858
Abstract
Arbuscular mycorrhizal fungi (AMF), including Rhizophagus spp., can play important roles in nutrient cycling of the rhizosphere. However, the effect of virus infection on AMF’s role in nutrient cycling cannot be determined without first knowing the diversity of the mycoviruses in AMF. Therefore, [...] Read more.
Arbuscular mycorrhizal fungi (AMF), including Rhizophagus spp., can play important roles in nutrient cycling of the rhizosphere. However, the effect of virus infection on AMF’s role in nutrient cycling cannot be determined without first knowing the diversity of the mycoviruses in AMF. Therefore, in this study, we sequenced the R. irregularis isolate-09 due to its previously demonstrated high efficiency in increasing the N/P uptake of the plant. We identified one novel mitovirus contig of 3685 bp, further confirmed by reverse transcription-PCR. Also, publicly available Rhizophagus spp. RNA-Seq data were analyzed to recover five partial virus sequences from family Narnaviridae, among which four were from R. diaphanum MUCL-43196 and one was from R. irregularis strain-C2 that was similar to members of the Mitovirus genus. These contigs coded genomes larger than the regular mitoviruses infecting pathogenic fungi and can be translated by either a mitochondrial translation code or a cytoplasmic translation code, which was also reported in previously found mitoviruses infecting mycorrhizae. The five newly identified virus sequences are comprised of functionally conserved RdRp motifs and formed two separate subclades with mitoviruses infecting Gigaspora margarita and Rhizophagus clarus, further supporting virus-host co-evolution theory. This study expands our understanding of virus diversity. Even though AMF is notably hard to investigate due to its biotrophic nature, this study demonstrates the utility of whole root metatranscriptome. Full article
(This article belongs to the Special Issue Mycoviruses)
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Figure 1

Figure 1
<p>(<b>A</b>) Agarose gel electrophoresis of the RT-PCR product showing a ~3 kb nested PCR amplicon that was confirmed by Sanger sequencing as cDNA amplicon of RirMV1. Left lane: 1 kb ladder. Right lane: RirMV1 amplicon of the predicted size of 3 kb and (<b>B</b>) Agarose gel electrophoresis of the RT-PCR product showing no amplification, suggesting the viral contig of RNA-Seq was not originated from <span class="html-italic">Medicago</span> root without <span class="html-italic">R</span>. <span class="html-italic">irregularis</span> strain 09 infection. Left to right lanes: 1 kb ladder, viral primers, plant primers.</p> Full article ">Figure 2
<p>Maximum likelihood tree (with bootstrap consensus) depicting the relationships of the predicted amino acid sequences of RNA-dependent RNA polymerase (RdRp) of the <span class="html-italic">Rhizophagus</span> mitoviruses, and other confirmed and proposed members of the <span class="html-italic">Narnaviridae</span>. Predicted RdRp amino acid sequences were aligned with ClustalW [<a href="#B31-viruses-10-00707" class="html-bibr">31</a>], and the phylogenetic tree was inferred using Mega 7.0 software [<a href="#B32-viruses-10-00707" class="html-bibr">32</a>]. Branch lengths are scaled to the expected underlying number of amino acid substitutions per site. The Saccharomyces 20S RNA narnavirus RdRp amino acid sequence was used as an outgroup to root the tree. Five newly identified mitoviruses (in bold) formed two separate monophyletic clusters between the Rhizophagus-associated mitoviruses. The following abbreviations were used for the Mitovirus (MV) sequences: Sc, <span class="html-italic">Saccharomyces cerevisiae</span>; Gm, <span class="html-italic">Gigaspora margarita</span>; Rd, <span class="html-italic">Rhizophagus diaphanum</span>; Rc, <span class="html-italic">Rhizophagus clarus</span>; Sc, <span class="html-italic">Sclerotinia sclerotiorum</span>; Rir, <span class="html-italic">Rhizophagus irregularis;</span> Ta, <span class="html-italic">Tuber aestivum</span>; Te, <span class="html-italic">Tuber excavatum</span>.</p> Full article ">Figure 3
<p>The genome organization of <span class="html-italic">Rhizophagus</span> spp. mitoviruses. The comparisons are of the organizations of RdMV1, RdMV2, RdMV3, RdMV4 and RirMV1. RdRp coding regions are labeled in blue (see also <a href="#viruses-10-00707-t001" class="html-table">Table 1</a>).</p> Full article ">Figure 4
<p>Conserved motifs identified in the RdRp domain of the genus <span class="html-italic">Mitovirus</span> based on the multiple sequence alignment of the amino acid sequences. Similar to the other mitoviruses, six conserved motifs were found. These conserved regions were labeled A-F as RdRp associated motifs described previously [<a href="#B36-viruses-10-00707" class="html-bibr">36</a>].</p> Full article ">
11 pages, 3410 KiB  
Review
Vertical and Horizontal Transmission of Pospiviroids
by Yosuke Matsushita, Hironobu Yanagisawa and Teruo Sano
Viruses 2018, 10(12), 706; https://doi.org/10.3390/v10120706 - 12 Dec 2018
Cited by 31 | Viewed by 12014
Abstract
Viroids are highly structured, single-stranded, non-protein-coding circular RNA pathogens. Some viroids are vertically transmitted through both viroid-infected ovule and pollen. For example, potato spindle tuber viroid, a species that belongs to Pospiviroidae family, is delivered to the embryo through the ovule or pollen [...] Read more.
Viroids are highly structured, single-stranded, non-protein-coding circular RNA pathogens. Some viroids are vertically transmitted through both viroid-infected ovule and pollen. For example, potato spindle tuber viroid, a species that belongs to Pospiviroidae family, is delivered to the embryo through the ovule or pollen during the development of reproductive tissues before embryogenesis. In addition, some of Pospiviroidae are also horizontally transmitted by pollen. Tomato planta macho viroid in pollen infects to the ovary from pollen tube during pollen tube elongation and eventually causes systemic infection, resulting in the establishment of horizontal transmission. Furthermore, fertilization is not required to accomplish the horizontal transmission. In this review, we will overview the recent research progress in vertical and horizontal transmission of viroids, mainly by focusing on histopathological studies, and also discuss the impact of seed transmission on viroid dissemination and seed health. Full article
(This article belongs to the Special Issue Viroid-2018: International Conference on Viroids and Viroid-Like RNAs)
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<p>Pathways of tomato planta macho viroid during vertical and horizontal transmission through pollen in infected petunia plants.</p> Full article ">Figure 2
<p>In situ hybridization shows the presence of potato spindle tuber viroid (PSTVd) in the generative nucleus and vegetative nucleus of, respectively, infected mature pollen grains (<b>A</b>) and infected germinating pollen grains on healthy stigma (<b>B</b>) in PSTVd-infected petunia. cp, cytoplasm; gn, generative nucleus; pg, pollen grain; pt, pollen tube; st, stigma; vn, vegetative nucleus. Scale bars = 50 μm.</p> Full article ">Figure 3
<p>In situ hybridization shows the presence of potato spindle tuber viroid in the floral apical meristem of an infected tomato plant (<b>A</b>) and a healthy (<b>B</b>) tomato plant. Scale bars = 50 μm.</p> Full article ">Figure 4
<p>In situ hybridization shows the presence of potato spindle tuber viroid in the placenta, ovule, and ovary wall in flowers of an infected tomato plant (<b>A</b>) and a healthy tomato plant (<b>B</b>) at the flower opening stage. es, embryo sac; in, integuments; ov, ovule; ow, ovary wall; pl, placenta. Scale bars = 50 μm.</p> Full article ">Figure 5
<p>In situ hybridization shows the presence of potato spindle tuber viroid in the ovary wall (ov) and placenta (pl), but not the ovule (ov), which comprise the embryo sac (es) and integument (in), respectively, in a flower of an infected eggplant (<span class="html-italic">Solanum melongena</span>) at the flower opening stage. Scale bars = 100 μm.</p> Full article ">
28 pages, 945 KiB  
Article
The Revisited Genome of Bacillus subtilis Bacteriophage SPP1
by Lia M. Godinho, Mehdi El Sadek Fadel, Céline Monniot, Lina Jakutyte, Isabelle Auzat, Audrey Labarde, Karima Djacem, Leonor Oliveira, Rut Carballido-Lopez, Silvia Ayora and Paulo Tavares
Viruses 2018, 10(12), 705; https://doi.org/10.3390/v10120705 - 11 Dec 2018
Cited by 12 | Viewed by 6038
Abstract
Bacillus subtilis bacteriophage SPP1 is a lytic siphovirus first described 50 years ago. Its complete DNA sequence was reported in 1997. Here we present an updated annotation of the 44,016 bp SPP1 genome and its correlation to different steps of the viral multiplication [...] Read more.
Bacillus subtilis bacteriophage SPP1 is a lytic siphovirus first described 50 years ago. Its complete DNA sequence was reported in 1997. Here we present an updated annotation of the 44,016 bp SPP1 genome and its correlation to different steps of the viral multiplication process. Five early polycistronic transcriptional units encode phage DNA replication proteins and lysis functions together with less characterized, mostly non-essential, functions. Late transcription drives synthesis of proteins necessary for SPP1 viral particles assembly and for cell lysis, together with a short set of proteins of unknown function. The extensive genetic, biochemical and structural biology studies on the molecular mechanisms of SPP1 DNA replication and phage particle assembly rendered it a model system for tailed phages research. We propose SPP1 as the reference species for a new SPP1-like viruses genus of the Siphoviridae family. Full article
(This article belongs to the Special Issue Bacteriophage Genomes and Genomics: News from the Wild)
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<p>Organization of the SPP1 genome. The continuous bar represents the 44,016 bp-long genome where coordinate 1 is the main <span class="html-italic">pac</span> cleavage position [<a href="#B35-viruses-10-00705" class="html-bibr">35</a>]. The two origins of replication <span class="html-italic">ori</span>R and <span class="html-italic">ori</span>L (magenta), the DNA packaging signal <span class="html-italic">pac</span> (black) and non-essential regions of the SPP1 genome defined by deletions (dashed) are highlighted in the bar. The sequence inverted in SPP1<span class="html-italic">invmir</span> is displayed by a pink line underneath the genome bar. The position of promoters (<a href="#viruses-10-00705-f002" class="html-fig">Figure 2</a>) and potential Rho-independent transcriptional terminators that form stem loops (red) in mRNA (<a href="#viruses-10-00705-t002" class="html-table">Table 2</a>) is displayed on top of the bar. Transcription is from left to right. DNA packaging initiated at <span class="html-italic">pac</span> occurs in the same direction (arrow on the top left). The set of SPP1 genes and ORFs, identified as described in Materials and Methods (see <a href="#sec2-viruses-10-00705" class="html-sec">Section 2</a>), are presented above the genome bar and colored according to their function assignment shown on the bottom legend.</p> Full article ">Figure 2
<p>SPP1 promoters. The sequence of SPP1 promoters and the initiation codon of their downstream gene (double underline) are displayed. The −35 and −10 promoter regions are shaded in grey for promoters whose transcription start position (+1) was determined experimentally. A dashed box denotes the atypical −35 sequence of <span class="html-italic">P</span>L1. SPP1 putative early promoters identified by sequence similarity to <span class="html-italic">B. subtilis</span> vegetative promoters are highlighted in black with sequence characters in white. Note that the approximate position of transcription initiation and promoter strength was determined for all early promoters by electron microscopy of DNA-RNA polymerase complexes [<a href="#B41-viruses-10-00705" class="html-bibr">41</a>,<a href="#B42-viruses-10-00705" class="html-bibr">42</a>].</p> Full article ">
17 pages, 953 KiB  
Review
Pathogen at the Gates: Human Cytomegalovirus Entry and Cell Tropism
by Christopher C. Nguyen and Jeremy P. Kamil
Viruses 2018, 10(12), 704; https://doi.org/10.3390/v10120704 - 11 Dec 2018
Cited by 109 | Viewed by 9615
Abstract
The past few years have brought substantial progress toward understanding how human cytomegalovirus (HCMV) enters the remarkably wide spectrum of cell types and tissues that it infects. Neuropilin-2 and platelet-derived growth factor receptor alpha (PDGFRα) were identified as receptors, respectively, for the trimeric [...] Read more.
The past few years have brought substantial progress toward understanding how human cytomegalovirus (HCMV) enters the remarkably wide spectrum of cell types and tissues that it infects. Neuropilin-2 and platelet-derived growth factor receptor alpha (PDGFRα) were identified as receptors, respectively, for the trimeric and pentameric glycoprotein H/glycoprotein L (gH/gL) complexes that in large part govern HCMV cell tropism, while CD90 and CD147 were also found to play roles during entry. X-ray crystal structures for the proximal viral fusogen, glycoprotein B (gB), and for the pentameric gH/gL complex (pentamer) have been solved. A novel virion gH complex consisting of gH bound to UL116 instead of gL was described, and findings supporting the existence of a stable complex between gH/gL and gB were reported. Additional work indicates that the pentamer promotes a mode of cell-associated spread that resists antibody neutralization, as opposed to the trimeric gH/gL complex (trimer), which appears to be broadly required for the infectivity of cell-free virions. Finally, viral factors such as UL148 and US16 were identified that can influence the incorporation of the alternative gH/gL complexes into virions. We will review these advances and their implications for understanding HCMV entry and cell tropism. Full article
(This article belongs to the Special Issue Recent Advances in Cytomegalovirus Research)
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<p>Receptors for HCMV gH/gL complexes. The trimeric gH/gL/gO complex (trimer) interacts with PDGFRα to drive a pH-independent mode of entry that involves macropinocytosis. The pentameric gH/gL/UL128–131 complex (pentamer) interacts with Nrp2 in a mode of entry that involves endocytosis and a decrease in pH. CD147 also appears to be required in the latter mode of entry. See text for additional details.</p> Full article ">Figure 2
<p>Regulation of alternative gH/gL complexes by UL148 and US16. UL148, a viral endoplasmic reticulum (ER)-resident glycoprotein, promotes high-level expression of the trimer during infection (wild-type) by stabilizing gO within the endoplasmic reticulum, resulting in the production of trimer-rich progeny virions. In <span class="html-italic">UL148</span>-null infections, decreased levels of trimer are synthesized, leading to the production of virions that more efficiently infect and spread between epithelial cells. US16, in contrast, localizes to the viral assembly compartment to promote incorporation of the pentamer into progeny virions, perhaps via physical interaction with UL130. <span class="html-italic">US16</span>-null mutants produce progeny virions lacking pentamer that are unable to infect endothelial and epithelial cells. See text for additional details.</p> Full article ">
6 pages, 593 KiB  
Communication
Complete Nucleotide Sequence of a Partitivirus from Rhizoctonia solani AG-1 IA Strain C24
by Chen Liu, Miaolin Zeng, Meiling Zhang, Canwei Shu and Erxun Zhou
Viruses 2018, 10(12), 703; https://doi.org/10.3390/v10120703 - 11 Dec 2018
Cited by 16 | Viewed by 3580
Abstract
The complete genome of a novel double-stranded (ds) RNA mycovirus, named as Rhizoctonia solani partitivirus 5 (RsPV5), isolated from rice sheath blight fungus R. solani AG-1 IA strain C24, was sequenced and analysed. RsPV5 consists of two segments, dsRNA-1 (1899 nucleotides) and dsRNA-2 [...] Read more.
The complete genome of a novel double-stranded (ds) RNA mycovirus, named as Rhizoctonia solani partitivirus 5 (RsPV5), isolated from rice sheath blight fungus R. solani AG-1 IA strain C24, was sequenced and analysed. RsPV5 consists of two segments, dsRNA-1 (1899 nucleotides) and dsRNA-2 (1787 nucleotides). DsRNA-1 has an open reading frame (ORF) 1 that potentially codes for a protein of 584 amino acid (aa) containing the conserved motifs of a RNA-dependent RNA polymerase (RdRp), and dsRNA-2 also contains a ORF 2, encoding a putative capsid protein (CP) of 513 aa. Phylogenetic analysis revealed that RsPV5 clustered together with six other viruses in an independent clade of the genus Alphapartitivirus, indicating that RsPV5 was a new member of the genus Alphapartitivirus, within the family Partitiviridae. Full article
(This article belongs to the Special Issue Mycoviruses)
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<p>Schematic representation of the genomic organization of RsPV5 isolated from R. solani AG-1 IA strain C24, the causal agent of rice sheath blight. (<b>a</b>) Schematic representation of the genomic organization of dsRNA-1. The rectangle represents open reading frame (ORF 1) and the nucleotide positions of the start and end codons are listed above the box. The gray bar represents the conserved RNA-dependent RNA polymerase (RdRp), the predicted molecular masse and the nucleotide positions of the start and termination codons are listed above the bar. The arrows under the single lines represent the length of the non-coding sequence. (<b>b</b>) Schematic representation of the genomic organization of dsRNA-2. The rectangle represents the open reading frame (ORF 2) and its encoded protein, capsid protein (CP), the nucleotide positions of the start and end codons are listed above the box. The arrows under the single lines represent the length of the non-coding sequences. (<b>c</b>) Alignments of 5′- and 3′-untranslated regions (UTRs) of RsPV5 dsRNA-1 and dsRNA-2. The letters with black shading represent conserved sequences at both ends.</p> Full article ">Figure 2
<p>Phylogenetic analysis of RsPV5. A phylogenetic tree was generated for the putative amino acid sequences of the deduced RdRp proteins using the neighbor-joining method with the program MEGA 6.0 and Bootstrap 1000 replicates. The RdRp sequences were obtained from GenBank and the accession numbers of viruses are given in the brackets behind the virus names. The scale means a genetic distance of 0.1 amino acid substitutions per site. Viral lineages are marked based on their taxonomic status.</p> Full article ">
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