[go: up one dir, main page]
Next Issue
Volume 11, March
Previous Issue
Volume 11, January
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
7.3
3.8
Journals
Viruses
Volume 11
Issue 2
share announcement

Viruses, Volume 11, Issue 2 (February 2019) – 107 articles

Cover Story (view full-size image): Many enveloped viruses employ late domains such as PPXY to recruit the host ESCRT machinery needed for budding and particle release. Paramyxoviruses typically lack late domain sequences, yet budding of these viruses is often ESCRT-dependent. Here, we provide evidence for a model in which paramyxoviruses use AMOTL1 as a linker to indirectly recruit the same WW domain-containing NEDD4 ubiquitin ligases for budding that other enveloped viruses recruit directly through PPXY late domains. View this paper.
  • Issues are regarded as officially published after their release is announced to the table of contents alert mailing list.
  • You may sign up for e-mail alerts to receive table of contents of newly released issues.
  • PDF is the official format for papers published in both, html and pdf forms. To view the papers in pdf format, click on the "PDF Full-text" link, and use the free Adobe Reader to open them.
Order results
Result details
Section
Show export options expand_more Show export options expand_less
Select all
Export citation of selected articles as:
14 pages, 3302 KiB  
Review
Antiviral Drug Discovery: Norovirus Proteases and Development of Inhibitors
by Kyeong-Ok Chang, Yunjeong Kim, Scott Lovell, Athri D. Rathnayake and William C. Groutas
Viruses 2019, 11(2), 197; https://doi.org/10.3390/v11020197 - 25 Feb 2019
Cited by 47 | Viewed by 9954
Abstract
Proteases are a major enzyme group playing important roles in a wide variety of biological processes in life forms ranging from viruses to mammalians. The aberrant activity of proteases can lead to various diseases; consequently, host proteases have been the focus of intense [...] Read more.
Proteases are a major enzyme group playing important roles in a wide variety of biological processes in life forms ranging from viruses to mammalians. The aberrant activity of proteases can lead to various diseases; consequently, host proteases have been the focus of intense investigation as potential therapeutic targets. A wide range of viruses encode proteases which play an essential role in viral replication and, therefore, constitute attractive targets for the development of antiviral therapeutics. There are numerous examples of successful drug development targeting cellular and viral proteases, including antivirals against human immunodeficiency virus and hepatitis C virus. Most FDA-approved antiviral agents are peptidomimetics and macrocyclic compounds that interact with the active site of a targeted protease. Norovirus proteases are cysteine proteases that contain a chymotrypsin-like fold in their 3D structures. This review focuses on our group’s efforts related to the development of norovirus protease inhibitors as potential anti-norovirus therapeutics. These protease inhibitors are rationally designed transition-state inhibitors encompassing dipeptidyl, tripeptidyl and macrocyclic compounds. Highly effective inhibitors validated in X-ray co-crystallization, enzyme and cell-based assays, as well as an animal model, were generated by launching an optimization campaign utilizing the initial hit compounds. A prodrug approach was also explored to improve the pharmacokinetics (PK) of the identified inhibitors. Full article
(This article belongs to the Special Issue Noroviruses)
►▼ Show Figures

Figure 1

Figure 1
<p>Proteolysis of the scissile bond between P<sub>1</sub> and P<sub>1</sub>’. Standard nomenclature P<sub>1</sub>… P<sub>n</sub> and P<sub>1</sub>’… P<sub>n</sub>’ for amino acid residues of the substrates. S<sub>1</sub>... S<sub>n</sub> and S<sub>1</sub>’… S<sub>n</sub>’ are the corresponding binding sites on the enzyme (per Berger and Schechter nomenclature (3)).</p> Full article ">Figure 2
<p>Examples of commercially available protease inhibitors and their targets. Dabigatran and Rivaroxaban are anti-coagulants; Lisinopril is for hypertension; Sitagliptin is for diabetes; and Bortezomib is for multiple myeloma.</p> Full article ">Figure 3
<p>Crystal structure of HIV-1 protease (<b>A</b>), and its cleavage sites (<b>B</b>). The catalytic site resides between two monomers (blue and green), and Asp25 and Asp25’ (red) from each dimer orchestrate the cleavages (PDB: 2NMZ). The flap region and dimerization domain are also shown. Cleavage sites (amino acids) of mature proteins of HIV-1 are listed. The red color indicates P<sub>1</sub> specificity.</p> Full article ">Figure 4
<p>FDA-approved HIV protease inhibitors with approved year and EC<sub>50</sub> values against HIV-1 in cell culture.</p> Full article ">Figure 5
<p>Crystal structure of hepatitis C virus (HCV) NS3/4A (genotype 1), and its cleavage sites. (<b>A</b>) The two β-barrel sub-domains of the NS3/4A protease domain are indicated in a ribbon diagram (adapted from PDB:1A1R). His57, Asp99, and Ser139, which form the catalytic triad, are shown in ball-and-stick representation. The protease structural zinc ion (blue sphere) is indicated. The NS4A peptide is rendered as a light orange ribbon. (<b>B</b>) Cleavage sites (amino acids) between mature proteins of HCV genotype 1 are listed. Red color indicates P<sub>1</sub> specificity.</p> Full article ">Figure 6
<p>FDA-approved HCV protease inhibitors with approved year and EC<sub>50</sub> values against genotype 1 in cell culture (replicon harboring cells).</p> Full article ">Figure 7
<p>Crystal structure of NV 3CL<sup>Pro</sup> (PDB: 2LNC) (<b>A</b>) and its cleavage sites (<b>B</b>). The active site resides between the two domains and the catalytic triad with Cys139, His30 and Glu54 are shown. Cleavage sites (amino acids) between mature proteins of Norwalk virus are listed. The red color indicates P<sub>1</sub> specificity.</p> Full article ">Figure 8
<p>Examples of optimization strategy employed for the dipeptidyl compounds against norovirus from one of the initial hits (<span class="html-italic">GC373</span>). Potency increased by making changes in R<sub>1</sub> and R<sub>2</sub>. Red color indicates the most optimized <span class="html-italic">GC583</span>.</p> Full article ">Figure 9
<p>X-ray crystal structure of NV 3CL<sup>Pro</sup> and <span class="html-italic">GC543</span> (<b>A</b>,<b>C</b>, PDB: 4XBC) and <span class="html-italic">GC583</span> (<b>B</b>,<b>D</b>, PDB: 4XBB). The structures revealed that increased potency is correlated to interactions between the S<sub>4</sub> subsite and the cap residue. The <span class="html-italic">m</span>-Cl benzyl group in the cap allows for interactions with hydrophobic residues in S<sub>4</sub>.</p> Full article ">Figure 10
<p>Examples of macrocyclic NV 3CL<sup>Pro</sup> inhibitors. (<b>A</b>–<b>C</b>). Three different classes (<b>A</b>–<b>C</b>) of macrocyclic inhibitors were synthesized. The P<sub>2</sub> position, and the length and nature of the linker were examined for optimal efficacy against the enzyme and viral replication. (<b>D</b>–<b>E</b>) X-ray co-crystallization of NV 3CL<sup>Pro</sup> and an oxadiazole-based macrocycle (<b>B</b>, PDB: 5DG6).</p> Full article ">
16 pages, 6722 KiB  
Article
Duck Plague Virus Promotes DEF Cell Apoptosis by Activating Caspases, Increasing Intracellular ROS Levels and Inducing Cell Cycle S-Phase Arrest
by Chuankuo Zhao, Mingshu Wang, Anchun Cheng, Qiao Yang, Ying Wu, Renyong Jia, Dekang Zhu, Shun Chen, Mafeng Liu, Xinxin Zhao, Shaqiu Zhang, Yunya Liu, Yanling Yu, Ling Zhang, Bin Tian, Mujeeb Ur Rehman, Leichang Pan and Xiaoyue Chen
Viruses 2019, 11(2), 196; https://doi.org/10.3390/v11020196 - 24 Feb 2019
Cited by 14 | Viewed by 4911
Abstract
Background: Duck plague virus (DPV) can induce apoptosis in duck embryo fibroblasts (DEFs) and in infected ducks, but the molecular mechanism of DPV-induced apoptosis remains unknown. Methods: We first used qRT-PCR and a Caspase-Glo assay to determine whether the caspase protein family plays [...] Read more.
Background: Duck plague virus (DPV) can induce apoptosis in duck embryo fibroblasts (DEFs) and in infected ducks, but the molecular mechanism of DPV-induced apoptosis remains unknown. Methods: We first used qRT-PCR and a Caspase-Glo assay to determine whether the caspase protein family plays an important role in DPV-induced apoptosis. Then, we used an intracellular ROS detection kit and the mitochondrial probe JC-1 to respectively detect ROS levels and mitochondrial membrane potential (MMP). Finally, flow cytometry was used to detect apoptosis and cell cycle progression. Results: In this study, the mRNA levels and enzymatic activities of caspase-3, caspase-7, caspase-8, and caspase-9 were significantly increased during DPV-induced apoptosis. The caspase inhibitors Z-DEVD-FMK, Z-LEHD-FMK, and Q-VD-Oph could inhibit DPV-induced apoptosis and promote viral replication. Subsequently, a significant decrease in MMP and an increase in the intracellular ROS levels were observed. Further study showed that pretreating infected cells with NAC (a ROS scavenger) decreased the intracellular ROS levels, increased the MMP, inhibited apoptosis, and promoted viral replication. Finally, we showed that DPV infection can cause cell cycle S-phase arrest. Conclusions: This study shows that DPV causes cell cycle S-phase arrest and leads to apoptosis through caspase activation and increased intracellular ROS levels. These findings may be useful for gaining an understanding of the pathogenesis of DPV and the apoptotic pathways induced by α-herpesviruses. Full article
(This article belongs to the Section Animal Viruses)
►▼ Show Figures

Figure 1

Figure 1
<p>Cytopathic effects (CPEs) induced by duck plague virus (DPV) in duck embryo fibroblasts (DEFs). (<b>A</b>) Cellular morphological changes in cells infected with DPV for the indicated number of hours. At 24, 36, 48, and 60 hpi (hours postinfection), the arrows indicate that infected cells appeared to have cellular fragmentation and plaques. (<b>B</b>) Nuclear morphological changes in cells infected with DPV for the indicated number of hours. At 24, 36, 48, and 60 hpi, the arrows indicate that nuclei of infected cells appear appeared as fragmented and marginated typical apoptotic bodies. (<b>C</b>) Viral titers were determined at the indicated time points by measuring the TCID<sub>50</sub> for the DEFs. All titrations were carried out in three independent experiments. The titers obtained were averaged, and the standard error of the mean was calculated for each time point. (<b>D</b>) Quantitative analysis of viral DNA by quantitative real-time PCR assay. Viral DNA detection was carried out in three independent experiments. The titers obtained were averaged, and the standard error of the mean was calculated for each time point.</p> Full article ">Figure 2
<p>Effects of DPV infection on the caspase family. (<b>A</b>) mRNA expression levels of caspase-3, caspase-7, caspase-8, and caspase-9. (<b>B</b>) Activities of caspase-3, caspase-7, caspase-8, and caspase-9. The data are presented as the means ± SD of three independent experiments. * <span class="html-italic">p</span> &lt; 0.05 and ** <span class="html-italic">p</span> &lt; 0.01, compared with the control group.</p> Full article ">Figure 3
<p>Z-IEHD-FMK, Z-DEVD-FMK, Z-IETD-FMK, and QVD-P-Oh inhibit apoptosis induced by DPV. (<b>A</b>) Cellular morphological changes following treatment with Z-IEHD-FMK, Z-DEVD-FMK, Z-IETD-FMK, and QVD-P-Oh for 2 h and detected at 48 h. (<b>B</b>) Changes in DEF viability following treatment with Z-IEHD-FMK, Z-DEVD-FMK, Z-IETD-FMK, and QVD-P-Oh for 2 h were measured using an MTT assay kit at 48 h. (<b>C</b>) DEF cells were pretreated with inhibitors for 2 h and then infected with DPV for 48 h. After incubation, the viruses were collected, and the viral titers were determined and presented as log10 TCID<sub>50</sub>/mL. (<b>D</b>) DEFs were pretreated with Z-IEHD-FMK or Z-DEVD-FMK for 2 h and then infected with DPV for 36 h. DEFs were pretreated with Z-IETD-FMK or QVD-P-Oh for 2 h and then infected with DPV for 48 h. Apoptosis was detected by FCM. (<b>E</b>) Histogram of the percentage of apoptotic cells; DEFs were pretreated with Z-IEHD-FMK or Z-DEVD-FMK for 2 h and then infected with DPV for 36 h. DEFs were pretreated with Z-IETD-FMK or QVD-P-Oh for 2 h and then infected with DPV for 48 h. The data are presented as the means ± SD of three independent experiments. * <span class="html-italic">p</span> &lt; 0.05 and ** <span class="html-italic">p</span> &lt; 0.01, compared with the control group.</p> Full article ">Figure 4
<p>Determination of ROS levels in DEFs. (<b>A</b>) Assessment of ROS levels in DEFs using an intracellular ROS detection kit and fluorescence microscopy; the red color indicates intracellular ROS. (<b>B</b>) Assessment of ROS levels in DEFs using an intracellular ROS detection kit and a multifunctional microplate reader. The data are presented as the means ± SD of three independent experiments. * <span class="html-italic">p</span> &lt; 0.05 and ** <span class="html-italic">p</span> &lt; 0.01, compared with the control group.</p> Full article ">Figure 5
<p>Mitochondrial membrane potential (MMP) determination in DEFs. (<b>A</b>) Assessment of DEF MMP through JC-1 staining and fluorescence microscopy. Mitochondria with normal membrane potential are indicated in red, and mitochondria with reduced membrane potential are indicated in green. (<b>B</b>) Assessment of DEF MMP though JC-1 staining and a multifunctional microplate reader. The data are presented as the means ± SD of three independent experiments. * <span class="html-italic">p</span> &lt; 0.05 and ** <span class="html-italic">p</span> &lt; 0.01, compared with the control group.</p> Full article ">Figure 6
<p>The ROS scavenger NAC reduces ROS levels, increases the MMP, and inhibits apoptosis. (<b>A</b>) Changes in cell viability following DEF pretreatment with 5 or 10 mM NAC for 2 h and detected by MTT assay at 36 h. (<b>B</b>) Pretreatment with 5 or 10 mM NAC decreased ROS in DPV-infected cells; uninfected cells were used as a control. (<b>C</b>) NAC (5 or 10 mM) increased the MMP in DPV-infected cells; uninfected cells were used as a control. (<b>D</b>) NAC (5 or 10 mM) promoted viral replication. (<b>E</b>) NAC (10 mM) inhibited apoptosis induced by DPV. The data are presented as the means ± SD of three independent experiments. * <span class="html-italic">p</span> &lt; 0.05 and ** <span class="html-italic">p</span> &lt; 0.01, compared with the control group.</p> Full article ">Figure 7
<p>DPV induces cell cycle arrest at the S-phase. (<b>A</b>) DEFs were mock-infected (control) or infected with DPV. Cells were collected postinfection (p.i.) at the times indicated for FACS analysis of the cell cycle. (<b>B</b>) Histogram of the percentage of DEFs in the G0/G1-, S-, and G2/M-phases of the cell cycle, with the percentage of cells in each phase of the cell cycle shown. The data are presented as the means ± SD of three independent experiments. * <span class="html-italic">p</span> &lt; 0.05 and ** <span class="html-italic">p</span> &lt; 0.01, compared with the control group.</p> Full article ">
15 pages, 1893 KiB  
Article
Classifying the Unclassified: A Phage Classification Method
by Cynthia Maria Chibani, Anton Farr, Sandra Klama, Sascha Dietrich and Heiko Liesegang
Viruses 2019, 11(2), 195; https://doi.org/10.3390/v11020195 - 24 Feb 2019
Cited by 43 | Viewed by 9739
Abstract
This work reports the method ClassiPhage to classify phage genomes using sequence derived taxonomic features. ClassiPhage uses a set of phage specific Hidden Markov Models (HMMs) generated from clusters of related proteins. The method was validated on all publicly available genomes of phages [...] Read more.
This work reports the method ClassiPhage to classify phage genomes using sequence derived taxonomic features. ClassiPhage uses a set of phage specific Hidden Markov Models (HMMs) generated from clusters of related proteins. The method was validated on all publicly available genomes of phages that are known to infect Vibrionaceae. The phages belong to the well-described phage families of Myoviridae, Podoviridae, Siphoviridae, and Inoviridae. The achieved classification is consistent with the assignments of the International Committee on Taxonomy of Viruses (ICTV), all tested phages were assigned to the corresponding group of the ICTV-database. In addition, 44 out of 58 genomes of Vibrio phages not yet classified could be assigned to a phage family. The remaining 14 genomes may represent phages of new families or subfamilies. Comparative genomics indicates that the ability of the approach to identify and classify phages is correlated to the conserved genomic organization. ClassiPhage classifies phages exclusively based on genome sequence data and can be applied on distinct phage genomes as well as on prophage regions within host genomes. Possible applications include (a) classifying phages from assembled metagenomes; and (b) the identification and classification of integrated prophages and the splitting of phage families into subfamilies. Full article
(This article belongs to the Special Issue Diversity and Evolution of Phage Genomes)
►▼ Show Figures

Figure 1

Figure 1
<p>Markov Models (HMM) scan of phage family derived models own input “CDS” and coding sequences of other families. The scan of the protein sequences derived from <span class="html-italic">Ino</span>-, <span class="html-italic">Myo</span>-, <span class="html-italic">Podo</span>-, and <span class="html-italic">Siphoviridae</span>, was conducted by the profile HMMs. The names of all phages grouped into phage-families are marked at the bottom of heatmap. The bit-score of the HMM matches was normalized by the size (in bp) of the HMM’s consensus sequence (data see <a href="#app1-viruses-11-00195" class="html-app">Table S9</a>). The results are color-coded from blue (low-score) to red (high-score).</p> Full article ">Figure 2
<p>Alignment of <span class="html-italic">Caudovirales</span> genomes. (<b>A</b>) <span class="html-italic">Myoviridae</span>, (<b>B</b>) <span class="html-italic">Podoviridae</span>, and (<b>C</b>) <span class="html-italic">Siphoviridae</span>. Genomes of phages that have not yet been assigned by ICTV are marked in pink. Four phages JSF9, JSF10, JSF12, and JSF15 are boxed in red. JSF12 has been assigned to <span class="html-italic">Podoviridae</span> based on transmission electron micrographs (TEM) the complete genome alignment indicates a close relation to the <span class="html-italic">Siphoviridae</span> phage JSF10. The data has been visualized with Easyfig.</p> Full article ">Figure 3
<p>Taxonomic classification of vibriophages. This heatmap shows a profile HMM scan on the proteins of 58 unclassified bacteriophages genomes. Forty-one unclassified genomes generated sufficient with enough hits to be assigned to a taxonomic group. The HMMs have been integrated in the heatmap (<span class="html-italic">x</span>-axis). The HMMs are grouped (on the <span class="html-italic">y</span>-axis) into the respective phage families. The indicator for the quality of a hit is color coded to the normalized bit-score assigned for the respective match by hmmscan.</p> Full article ">Figure 4
<p>HMM scan results of all <span class="html-italic">Inoviridae</span> phages.This heatmap shows an <span class="html-italic">Inoviridae</span> derived profile HMM (<span class="html-italic">y</span>-axis) scan on the proteins of 119 <span class="html-italic">Inoviridae</span> genomes grouped by host genome (<span class="html-italic">x</span>-axis). HMMs ranged from hits specific to <span class="html-italic">Inoviridae</span> infecting <span class="html-italic">Vibrionaecea</span> to general hits for <span class="html-italic">Inoviridae</span> infecting other hosts. The indicator for the quality of a hit is color coded to the normalized bit-score assigned for the respective match by hmmscan.</p> Full article ">Figure 5
<p>Profile HMM scan of <span class="html-italic">Podoviridae</span> HMMs from <span class="html-italic">Vibrionaceae</span> versus genomes from <span class="html-italic">Podoviridae</span> phages infecting non-vibrio hosts. This heatmap shows a profile HMM scan on the proteome of 1066 <span class="html-italic">Podoviridae</span> genomes. Sufficient hits were generated to discriminate four groupings of <span class="html-italic">Podoviridae.</span> The HMMs have been integrated in the heatmap (<span class="html-italic">y</span>-axis). The HMMs are grouped (on the <span class="html-italic">x</span>-axis) into general <span class="html-italic">Podoviridae</span> subclassifications. The indicator for the quality of a hit is color coded to thenormalized bit-score assigned for the respective match by hmmscan. The generated hmmscan output was visualized using matplotlib library in Python 3.5.</p> Full article ">Figure 6
<p>HMM search for prophages in <span class="html-italic">Vibrio</span> genomes with proven phage activities.Family specific HMMs constructed for <span class="html-italic">Ino</span>-, <span class="html-italic">Myo</span>-, <span class="html-italic">Podo</span>-, <span class="html-italic">andSiphoviridae</span> (grouped on <span class="html-italic">x</span>-axis) were used to scan all proteins derived from the genome of nine <span class="html-italic">V. alginolyticus</span> and one <span class="html-italic">V. typhli</span> genomes (<span class="html-italic">x</span>-axis per phage family grouping). In all of the <span class="html-italic">V. alginolyticus</span> genomes, regions encoding proteins matching to the profile HMMs were found (plotted per position and grouped per replicon on the <span class="html-italic">y</span>-axis). In cases where a region with consecutive HMM hits predicted as well by PHASTER was separately faceted.</p> Full article ">
9 pages, 1665 KiB  
Article
Priorities, Barriers, and Facilitators towards International Guidelines for the Delivery of Supportive Clinical Care during an Ebola Outbreak: A Cross-Sectional Survey
by Marie-Claude Battista, Christine Loignon, Lynda Benhadj, Elysee Nouvet, Srinivas Murthy, Robert Fowler, Neill K. J. Adhikari, Adnan Haj-Moustafa, Alex P. Salam, Adrienne K. Chan, Sharmistha Mishra, Francois Couturier, Catherine Hudon, Peter Horby, Richard Bedell, Michael Rekart, Jan Hajek and Francois Lamontagne
Viruses 2019, 11(2), 194; https://doi.org/10.3390/v11020194 - 23 Feb 2019
Cited by 7 | Viewed by 5217
Abstract
During the Ebola outbreak, mortality reduction was attributed to multiple improvements in supportive care delivered in Ebola treatment units (ETUs). We aimed to identify high-priority supportive care measures, as well as perceived barriers and facilitators to their implementation, for patients with Ebola Virus [...] Read more.
During the Ebola outbreak, mortality reduction was attributed to multiple improvements in supportive care delivered in Ebola treatment units (ETUs). We aimed to identify high-priority supportive care measures, as well as perceived barriers and facilitators to their implementation, for patients with Ebola Virus Disease (EVD). We conducted a cross-sectional survey of key stakeholders involved in the response to the 2014–2016 West African EVD outbreak. Out of 57 email invitations, 44 responses were received, and 29 respondents completed the survey. The respondents listed insufficient numbers of health workers (23/29, 79%), improper tools for the documentation of clinical data (n = 22/28, 79%), insufficient material resources (n = 22/29, 76%), and unadapted personal protective equipment (n = 20/28, 71%) as the main barriers to the provision of supportive care in ETUs. Facilitators to the provision of supportive care included team camaraderie (n in agreement = 25/28, 89%), ability to speak the local language (22/28, 79%), and having treatment protocols in place (22/28, 79%). This survey highlights a consensus across various stakeholders involved in the response to the 2014–2016 EVD outbreak on a limited number of high-priority supportive care interventions for clinical practice guidelines. Identified barriers and facilitators further inform the application of guidelines. Full article
(This article belongs to the Special Issue Medical Advances in Viral Hemorrhagic Fever Research)
►▼ Show Figures

Figure 1

Figure 1
<p>Professional affiliations reported by survey respondents.</p> Full article ">Figure 2
<p>Number of active respondents by outbreak period.</p> Full article ">Figure 3
<p>Agreement with supportive care interventions proposed to be embedded within standard practices in Ebola treatment units.</p> Full article ">
13 pages, 2042 KiB  
Article
In Vivo Characterization of Avian Influenza A (H5N1) and (H7N9) Viruses Isolated from Canadian Travelers
by Yao Lu, Shelby Landreth, Amit Gaba, Magda Hlasny, Guanqun Liu, Yanyun Huang and Yan Zhou
Viruses 2019, 11(2), 193; https://doi.org/10.3390/v11020193 - 23 Feb 2019
Cited by 11 | Viewed by 5354
Abstract
Highly pathogenic avian influenza (HPAI) H5N1 and low pathogenic avian influenza (LPAI) H7N9 viruses pose a severe threat to public health through zoonotic infection, causing severe respiratory disease in humans. While HPAI H5N1 human infections have typically been reported in Asian countries, avian [...] Read more.
Highly pathogenic avian influenza (HPAI) H5N1 and low pathogenic avian influenza (LPAI) H7N9 viruses pose a severe threat to public health through zoonotic infection, causing severe respiratory disease in humans. While HPAI H5N1 human infections have typically been reported in Asian countries, avian H7N9 human infections have been reported mainly in China. However, Canada reported a case of fatal human infection by the HPAI H5N1 virus in 2014, and two cases of human illness associated with avian H7N9 virus infection in 2015. While the genomes of the causative viruses A/Alberta/01/2014 (H5N1) (AB14 (H5N1)) and A/British Columbia/1/2015 (H7N9) (BC15 (H7N9)) are reported, the isolates had not been evaluated for their pathogenicity in animal models. In this study, we characterized the pathogenicity of AB14 (H5N1) and BC15 (H7N9) and found that both strain isolates are highly lethal in mice. AB14 (H5N1) caused systemic viral infection and erratic proinflammatory cytokine gene expression in different organs. In contrast, BC15 (H7N9) replicated efficiently only in the respiratory tract, and was a potent inducer for proinflammatory cytokine genes in the lungs. Our study provides experimental evidence to complement the specific human case reports and animal models for evaluating vaccine and antiviral candidates against potential influenza pandemics. Full article
(This article belongs to the Special Issue CSV2018: The 2nd symposium of the Canadian Society for Virology (CSV))
►▼ Show Figures

Figure 1

Figure 1
<p>Survival rate and body weight loss for the AB14 (H5N1) and BC15 (H7N9) strain isolates. The survival rates for (<b>A</b>) AB14 (H5N1) and (<b>C</b>) BC15 (H7N9) as well as body weight changes for (<b>B</b>) AB14 (H5N1) and (<b>D</b>) BC15 (H7N9) were determined in BALB/c mice (<span class="html-italic">n</span> = 6 per group) infected with 10<sup>3</sup> PFU, 10<sup>4</sup> PFU, and 10<sup>5</sup> PFU of the two different strain isolates.</p> Full article ">Figure 2
<p>Lung histopathology of mice after infection with AB14 (H5N1) and BC15 (H7N9) strain isolates. Lung samples were fixed, sectioned, and stained with hematoxylin and eosin. (<b>A</b>–<b>C</b>) Tissues from mock-infected lungs. (<b>D</b>–<b>F</b>) Tissues from mice infected with AB14 (H5N1) showing infiltration of inflammatory cells into the (<b>D</b>) wall of the arteriole, (<b>E</b>) alveolar walls, and (<b>F</b>) bronchiolar epithelium affected by necrosis. (<b>G</b>–<b>I</b>) Tissues from mice infected with BC15 (H7N9) showing necrotic debris and inflammatory cells in the (<b>G</b>) wall of the arteriole, (<b>H</b>) collapsed alveoli, as well as degeneration and necrosis of the (<b>I</b>) bronchiolar epithelium. Scale bar represents 100 µm.</p> Full article ">Figure 3
<p>Viral titration of the mouse lung, spleen, and brain for the AB14 (H5N1) and BC15 (H7N9) strain isolates. Mice were intranasally infected with 10<sup>3</sup> PFU, 10<sup>4</sup> PFU, or 10<sup>5</sup> PFU of AB14 (H5N1) or BC15 (H7N9). The lung, spleen, and brain tissues were collected and homogenized for virus titration by TCID<sub>50</sub> assay. Lung viral titration from (<b>A</b>) AB14 (H5N1) and (<b>B</b>) BC15 (H7N9) infection for all doses. Brain viral titration from (<b>C</b>) AB14 infection (H5N1). Spleen viral titration from (<b>D</b>) AB14 (H5N1) infection. Please note that BC15 (H7N9) was not detected in the spleen and brain of mice infected by all three doses.</p> Full article ">Figure 4
<p>Innate immune receptor RIG-I, as well as cytokine and chemokine gene transcription levels in the lungs of mice infected with the AB14 (H5N1) and BC15 (H7N9) strain isolates. Cytokine and chemokine mRNA levels of RIG-I, IFN-α, IFN-β, IFN-γ, IP-10, TNF-α, IL-6, IL-1β, IL-18, and IL-10 from virus-infected lungs with (<b>A</b>) AB14 (H5N1) and (<b>B</b>) BC15 (H7N9) (<span class="html-italic">n</span> = 3 mice per virus group) were measured by qRT-PCR. Samples were harvested on the indicated d.p.i.; each sample was tested in triplicate.</p> Full article ">Figure 5
<p>Cytokine gene transcription profile in the brains of mice infected with the AB14 (H5N1) and BC15 (H7N9) strain isolates. Cytokine transcription levels of IFN-α, IFN-γ, TNF-α, IL-6, IL-1β, and IL-18 from virus-infected brains with (<b>A</b>) AB14 (H5N1) and (<b>B</b>) BC15 (H7N9) (<span class="html-italic">n</span> = 3 mice per virus group). Samples were harvested on the indicated d.p.i; each sample was tested in triplicate.</p> Full article ">
11 pages, 1224 KiB  
Review
Immune System Modulation and Viral Persistence in Bats: Understanding Viral Spillover
by Sonu Subudhi, Noreen Rapin and Vikram Misra
Viruses 2019, 11(2), 192; https://doi.org/10.3390/v11020192 - 23 Feb 2019
Cited by 105 | Viewed by 16634
Abstract
Bats harbor a myriad of viruses and some of these viruses may have spilled over to other species including humans. Spillover events are rare and several factors must align to create the “perfect storm” that would ultimately lead to a spillover. One of [...] Read more.
Bats harbor a myriad of viruses and some of these viruses may have spilled over to other species including humans. Spillover events are rare and several factors must align to create the “perfect storm” that would ultimately lead to a spillover. One of these factors is the increased shedding of virus by bats. Several studies have indicated that bats have unique defense mechanisms that allow them to be persistently or latently infected with viruses. Factors leading to an increase in the viral load of persistently infected bats would facilitate shedding of virus. This article reviews the unique nature of bat immune defenses that regulate virus replication and the various molecular mechanisms that play a role in altering the balanced bat–virus relationship. Full article
(This article belongs to the Special Issue Viruses and Bats 2019)
►▼ Show Figures

Figure 1

Figure 1
<p>Evolution of tolerance to DNA damage and unique antiviral immune response in bats. Development of flight necessitated the evolution of bats with the ability to modulate the consequences of increased metabolic activity by suppressing inflammation (left). Inflammation was suppressed by dampening the activation of DNA sensors, such as STING, and reducing levels of inflammatory cytokines, such as TNFα (center). These traits were positively selected but a reduced inflammatory response made it advantageous for virus replication (lower right). Increased susceptibility of cells to virus replication was compensated by selection of more effective antiviral measures, such as higher constitutive expression of Interferons or unique ISG expressions (upper right). (Abbreviations used: cGAS—cyclic GMP-AMP synthase, GTP—Guanosine triphosphate, cGMP—cyclic guanosine monophosphate, STING—stimulator of interferon genes, TBK1—TANK binding kinase 1, IRF3—interferon regulatory transcription factor 3, cRel, TNFα—tumor necrosis factor α, RNase-L—ribonuclease L).</p> Full article ">Figure 2
<p>Model showing effect of stress on persistent viral infection. Viruses persistently infect bats due to their reduced inflammation (reduced DNA sensor activation and decreased inflammatory cytokine levels) and their effective antiviral immune response (increased constitutive expression of interferons and unique ISG expressions), as depicted in <a href="#viruses-11-00192-f001" class="html-fig">Figure 1</a>. Stressful events alter the balance between host and virus and lead to an increase in virus replication, thereby leading to viral shedding.</p> Full article ">
13 pages, 780 KiB  
Review
The Virioneuston: A Review on Viral–Bacterial Associations at Air–Water Interfaces
by Janina Rahlff
Viruses 2019, 11(2), 191; https://doi.org/10.3390/v11020191 - 22 Feb 2019
Cited by 16 | Viewed by 7301
Abstract
Vast biofilm-like habitats at air–water interfaces of marine and freshwater ecosystems harbor surface-dwelling microorganisms, which are commonly referred to as neuston. Viruses in the microlayer, i.e., the virioneuston, remain the most enigmatic biological entities in boundary surface layers due to their potential ecological [...] Read more.
Vast biofilm-like habitats at air–water interfaces of marine and freshwater ecosystems harbor surface-dwelling microorganisms, which are commonly referred to as neuston. Viruses in the microlayer, i.e., the virioneuston, remain the most enigmatic biological entities in boundary surface layers due to their potential ecological impact on the microbial loop and major air–water exchange processes. To provide a broad picture of the viral–bacterial dynamics in surface microlayers, this review compiles insights on the challenges that viruses likely encounter at air–water interfaces. By considering viral abundance and morphology in surface microlayers, as well as dispersal and infection mechanisms as inferred from the relevant literature, this work highlights why studying the virioneuston in addition to the bacterioneuston is a worthwhile task. In this regard, major knowledge gaps and possible future research directions are discussed. Full article
(This article belongs to the Section Bacterial Viruses)
►▼ Show Figures

Figure 1

Figure 1
<p>Viral–bacterial dynamics in the surface microlayer (SML) and beyond. DOM = dissolved organic matter, UV = ultraviolet.</p> Full article ">
35 pages, 8902 KiB  
Review
Novel Approaches for The Development of Live Attenuated Influenza Vaccines
by Pilar Blanco-Lobo, Aitor Nogales, Laura Rodríguez and Luis Martínez-Sobrido
Viruses 2019, 11(2), 190; https://doi.org/10.3390/v11020190 - 22 Feb 2019
Cited by 46 | Viewed by 9548
Abstract
Influenza virus still represents a considerable threat to global public health, despite the advances in the development and wide use of influenza vaccines. Vaccination with traditional inactivate influenza vaccines (IIV) or live-attenuated influenza vaccines (LAIV) remains the main strategy in the control of [...] Read more.
Influenza virus still represents a considerable threat to global public health, despite the advances in the development and wide use of influenza vaccines. Vaccination with traditional inactivate influenza vaccines (IIV) or live-attenuated influenza vaccines (LAIV) remains the main strategy in the control of annual seasonal epidemics, but it does not offer protection against new influenza viruses with pandemic potential, those that have shifted. Moreover, the continual antigenic drift of seasonal circulating influenza viruses, causing an antigenic mismatch that requires yearly reformulation of seasonal influenza vaccines, seriously compromises vaccine efficacy. Therefore, the quick optimization of vaccine production for seasonal influenza and the development of new vaccine approaches for pandemic viruses is still a challenge for the prevention of influenza infections. Moreover, recent reports have questioned the effectiveness of the current LAIV because of limited protection, mainly against the influenza A virus (IAV) component of the vaccine. Although the reasons for the poor protection efficacy of the LAIV have not yet been elucidated, researchers are encouraged to develop new vaccination approaches that overcome the limitations that are associated with the current LAIV. The discovery and implementation of plasmid-based reverse genetics has been a key advance in the rapid generation of recombinant attenuated influenza viruses that can be used for the development of new and most effective LAIV. In this review, we provide an update regarding the progress that has been made during the last five years in the development of new LAIV and the innovative ways that are being explored as alternatives to the currently licensed LAIV. The safety, immunogenicity, and protection efficacy profile of these new LAIVs reveal their possible implementation in combating influenza infections. However, efforts by vaccine companies and government agencies will be needed for controlled testing and approving, respectively, these new vaccine methodologies for the control of influenza infections. Full article
(This article belongs to the Special Issue What’s New with Flu?)
►▼ Show Figures

Figure 1

Figure 1
<p>Influenza A virus (IAV) virion structure and genome organization. A) Virion structure: IAV is an eight-segmented, negative-sense, single-stranded RNA enveloped virus surrounded by a lipid bilayer that contains three viral glycoproteins: hemagglutinin (HA), responsible for binding to sialic acid receptors, entry into the cell and fusion of the viral envelop with the endosome; neuraminidase (NA), which removes sialic acids, allowing for viral release from infected cells; and, the ion channel matrix 2 (M2) protein, which is responsible for the acidification of the virion following endocytosis, and viral assembly. Under the viral envelope, there is a protein layer that is made of the matrix 1 (M1) protein, which is involved in virion assembly and budding. The nuclear export protein (NEP) is found inside the viral particle and it is required for the nuclear export of the eight viral ribonucleoprotein (vRNP) complexes from the nucleus to the cytoplasm at the late stages of viral replication. The vRNP complexes, which are present in the core of the virion, are made of the negative-sense, single-stranded viral (v)RNAs packed by the viral nucleoprotein (NP) and the three subunits (PB2, PB1, and PA) of the RNA-dependent RNA polymerase (RdRp) complex that are responsible for viral RNA genome replication and gene transcription in the nuclei of infected cells. IAV proteins and their schematic representation are shown at the bottom. B) Genome organization: The IAV genome is made of eight single-stranded, negative-sense, vRNA segments (PB2, PB1, PA, HA, NP, NA, M, and NS). White boxes represent packaging signals that are responsible for the selective packaging of each vRNA segment into the virion. Numbers represent the nucleotide lengths of each of the 3′ and 5′ packaging signals in each of the vRNAs. Each vRNA is flanked by the 3’and 5´ non-coding regions (NCRs, black lines) recognized by the viral RdRp for viral genome replication and gene transcription.</p> Full article ">Figure 2
<p>Production of the inactivated influenza vaccine (IIV): To prepare IIV, 10–11 days old chicken embryonated eggs are infected with two IAVs: the candidate virus recommended by the WHO (top, gray) and a high-growth virus (bottom, black). Reassortant viruses are harvested from the allantoic fluid of infected eggs 2–3 days post-infection and the appropriate reassortant virus to be included in the IIV containing the HA and NA viral segments from the WHO candidate virus (gray) and the six internal segments (PB2, PB1, PA, NP, M, and non-structural (NS)) of the high-growth virus (black) is selected by amplification in the presence of antibodies against the HA and NA glycoproteins of the high-growth virus. Genomic composition of the reassortant virus must be confirmed by sequencing. The selected virus to be used in the IIV is chemically inactivated with β-propiolactone (β-PL) or formalin, concentrated, purified for vaccine production, and then administrated intramuscularly (IM).</p> Full article ">Figure 3
<p>Generation of the live-attenuated influenza vaccine (LAIV): The LAIV is produced by co-infection of 10–11 days old chicken embryonated eggs with the candidate virus recommended by the WHO (top, gray) and the A/Ann Arbor/6/60 H2N2 (bottom, black) Master Donor Virus (MDV). After 2–3 days post-infection, the appropriate reassortant seed virus containing the six internal segments (PB2, PB1, PA, NP, M, and NS) derived from the MDV (black) and the two glycoprotein (HA and NA) segments from the recommended WHO circulating strain (gray) is selected by amplification at low temperatures in the presence of antibodies against the MDV, HA, and NA. The selected LAIV is then administrated intranasally (IN). Amino acid substitutions in the PB2 (N265S), PB1 (K391E, E581G, and A661T) and NP (D34G) viral segments responsible for the attenuated (att), temperature sensitive (ts), and cold-adapted (ca) phenotype of the MDV A/Ann Arbor/6/60 H2N2 are indicated at the bottom.</p> Full article ">Figure 4
<p>IAV vRNA cloning into ambisense/bidirectional plasmids for the generation of recombinant viruses using plasmid-based reverse genetic approaches. (<b>A</b>) Schematic representation of the ambisense/bidirectional rescue plasmid to generate recombinant IAV: Ambisense/bidirectional rescue plasmids containing the human polymerase I promoter (hPol-I, black arrow) and the mouse polymerase I terminator (T, black box) sequences that regulate the synthesis of the negative sense vRNAs are indicated. In the opposite direction to the Pol-I cassette, and from the same cDNA, the polymerase II dependent promoter (Pol-II, white arrow) and the bovine growth hormone polyadenylation termination sequence (aBGH, white box) direct the synthesis of positive sense mRNA to produce viral proteins. Newly synthesized vRNAs generated from the Pol-I cassette are recognized by the viral RdRp subunits (PB2, PB1 and PA) that, together with the viral NP, lead to the formation of vRNP complexes responsible of viral genome replication and gene transcription. Transcription from newly synthesized vRNAs results in mRNA expression and the production of new viral proteins. Replication of newly synthesized vRNAs results in the formation of complementary (c)RNAs for the amplification and synthesis of new vRNAs that will be incorporated into the nascent virions as novel vRNP complexes. (<b>B</b>) Plasmid-based reverse genetics to generate recombinant IAV: FDA-approved cell lines for vaccine production are co-transfected with the eight (PB2, PB1, PA, HA, NP, NA, M, and NS) ambisense/bidirectional IAV plasmids. Viable virus recovered from the tissue culture supernatants 3–4 days after transfection is amplified using fresh FDA-approved cell lines or 10–11 day-old embryonated chicken eggs.</p> Full article ">Figure 5
<p>LAIV based on truncations and/or deletion of the viral NS1: Schematic representation of wild-type, WT (<b>A</b>), NS1 truncated (<b>B</b>), or NS1 deficient (<b>C</b>) recombinant IAV. WT NS vRNA is represented in gray boxes. WT NS1 and NEP open reading frames (ORFs) are represented as dark and light gray boxes, respectively. Modified NS segments and truncated NS1 ORFs are indicated in black boxes. White lines represent stop codons. White boxes indicate the packaging signals located at the 3´and 5´ ends of the NS vRNA. Lines at the end of the NS vRNA indicate the 3´and 5´ NCR. Expression of WT NS1 protein (<b>A</b>) results in inhibition of interferon (IFN) induction and efficient viral replication. NS1 1–126 (top), 1–99 (middle), or 1–73 (bottom) truncations in the NS1 ORF (<b>B</b>) or deletion of the entire NS1 ORF (<b>C</b>) result in the higher induction of IFN and reduced levels of viral replication and, therefore, virus production.</p> Full article ">Figure 6
<p>Codon deoptimization (CD) for the generation of LAIV: (<b>A</b>,<b>B</b>) Schematic representation of WT (<b>A</b>) and CD (<b>B</b>) viral NS segments: WT NS vRNA is represented in gray boxes (<b>A</b>). WT NS1 and NEP ORFs are represented as dark and light gray boxes, respectively (<b>A</b>). CD NS1 (NS1<sub>CD</sub>, top), NEP (NEP<sub>CD</sub>, middle) or both NS1 and NEP (NS<sub>CD</sub>, bottom) proteins as well as their respective NS vRNA segments are indicated with black boxes (<b>B</b>). After infection with a virus encoding a WT NS segment, expression of NS1 results in inhibition of IFN induction, allowing the efficient viral replication (<b>A</b>). Infection with viruses encoding a codon deoptimized NS1 protein (NS1<sub>CD</sub>, top; NS<sub>CD</sub>, bottom) results in reduced NS1 protein expression levels and inefficient inhibition of type I IFN responses, resulting in reduced viral replication and viral production. CD of NEP (NEP<sub>CD</sub>, middle) results in lower expression of NEP, without significantly affecting viral replication. CD of NS1 and NEP (NS<sub>CD,</sub> bottom) results in higher attenuation than viruses containing only NS1 or NEP CD ORFs, correlating with the amount of codon changes introduced in the viral segment. (<b>C</b>,<b>D</b>) Schematic representation of WT and IAV attenuated by codon-pair bias: WT PB1, HA, NP, and NA vRNA segments are indicated in gray boxes (<b>C</b>). Codon-pair deoptimized PB1, HA, and NP (PB1/HA/NP<sup>3F</sup>) (<b>D</b>, top); or, HA and NA (HA/NA<sup>Min</sup>) (<b>D</b>, bottom) proteins are represented in black boxes. During WT viral infection (<b>C</b>), vRNPs mediate viral genome replication and gene transcription, allowing efficient viral protein synthesis and viral production. Likewise, optimal expression of the viral HA and NA results in efficient production of infectious viral progeny. Infection with PB1/HA/NP<sup>3F</sup> virus (<b>D</b>, top), results in reduced levels of viral replication and transcription mediated by lower levels of PB1 and NP expression. The codon-pair deoptimization of HA also affects protein expression levels, contributing to the attenuation of the PB1/HA/NP<sup>3F</sup> virus in mice but with reasonable growth in vitro. Likewise, the reduction in the level of expression of the viral HA and NA during infection with the HA/NA<sup>Min</sup> virus (<b>D</b>, bottom) results in reduced virion formation and therefore lower infectious viral production. White boxes (<b>A</b>–<b>D</b>) indicate the packaging signals that were located at the 3´and 5´ ends of each of the vRNAs. Lines at the end of each vRNA (<b>A</b>–<b>D</b>) indicate the 3´and 5´ NCR.</p> Full article ">Figure 7
<p>Rearrangement of IAV genome for the generation of LAIV: (<b>A</b>,<b>B</b>) Schematic representation of WT (A) and rearranged viral segments 2 (PB1) and 8 (NS) (B): WT segments 2, 4, and 8 (A) or rearranged segment 2 (PB1 and NEP) and segment 8 (NS1) are indicated with dark gray boxes (B) The light gray box indicates the secondary H5 inserted in the NS segment. White boxes (A and B) indicate the packaging signals located at the 3´and 5´ ends of each vRNA. Black boxes (B) indicate the sequence of the foot-and-mouth disease virus (FMDV) 2A autoproteolytic cleavage site. Lines at the end of each vRNA (A and B) indicate the 3´and 5´ NCR. A white line in the NS segment (B) represents a stop codon in the NS1 resulting in a truncated (1-99 amino acids) NS1 protein (black box). Expression of NEP from a single polypeptide downstream of the modified PB1 viral segment results in a reduction on the activity of the PB1and an impaired growth of the rearranged virus (B). The expression of the H5 ORF from a modified segment 8 results in an LAIV expressing two different HA (H9, dark gray; and H5, light gray) and the induction of neutralizing antibodies against the two viral glycoproteins. (<b>C</b>,<b>D</b>) Schematic representation of WT (C) and rearranged segment 4 (HA) and segment 6 (NA) viruses (D): WT (C) and rearranged segment 4 expressing NA-HA (D) are represented in dark gray boxes. Rearranged segment 6 expressing a secondary HA is represented in a light gray box (D). White boxes indicate the packaging signals located at the 3´and 5´ ends of each vRNA (C and D). Black boxes indicate the sequence of the porcine teschovirus (PTV-1)2A autoproteolytic cleavage site (D). Lines at the end of each vRNA indicate the 3´and 5´ NCRs (C and D). While WT virus expresses the HA and NA glycoproteins from the segment 4 and 6 respectively (C), the rearranged virus expresses both the subtype 1 HA and NA glycoproteins from the modified segment 4; and the subtype 3 HA from the modified segment 6, where NA is normally located (D). This rearrangement of the viral genome results in an attenuated recombinant virus able to induce neutralizing antibodies against the two viral glycoproteins (H1 and H3) (D).</p> Full article ">Figure 8
<p>Development of LAIV based on modification of the spliced viral RNA segments 7 (M) and 8 (NS): Schematic representation of WT (A) and modified (B) viral RNA segments 7 (Ms, top), 8 (NS, middle), or both 7 and 8 (Ms/NSs, bottom) in which the overlapping ORFs of the M1 and M2 proteins (Ms, top), NS1 and NEP proteins (NSs, middle), or both (Ms/NSs, bottom) are produced from the same transcript by using the PTV-1 2A autoproteolytic cleavage site. Viral products from the M (M1 and M2) and NS (NS1 and NEP) WT (<b>A</b>) or modified (<b>B</b>) viral segments are indicated in grey boxes. M2 ORF is shown as a lighter grey box. Black boxes (<b>B</b>) indicate the sequence of the PTV-1 2A autoproteolytic cleavage site. The packaging signals located at the 3´and 5´ ends of each vRNA are indicated with white boxes (<b>A</b>,<b>B</b>). Lines at the end of each vRNA indicate the 3´and 5´ NCR in the M and NS viral segments (<b>A</b>,<b>B</b>). During infection with WT virus, the optimal expression of M1 and M2 proteins (M segment), as well as NS1 and NEP (NS segment) from the spliced vRNA segments, results in efficient virus replication and production. Infection with a modified M segment (<b>B</b>, top) results in slightly reduction of virus replication and production at 33 °C; and significant reduction of viral production at high temperatures (37 °C or 39 °C). Modification of the NS vRNA segment (<b>B</b>, middle) results in a slight reduction in virus replication and production that is not temperature dependent. The recombinant virus containing both modified M and NS segments (Ms/NSs) (<b>B</b>, bottom) results in impaired viral replication and production, similar to the recombinant virus containing the modified M segment (Ms) at non permissive temperatures (37 °C or 39 °C).</p> Full article ">Figure 9
<p>Single-cycle infectious IAV (sciIAV) as LAIV: Schematic representation of sciIAV based on deletions in the PB2 (<b>A</b>), PB1 (<b>B</b>), HA (<b>C</b>), or NA (<b>D</b>) viral proteins. SciIAV based on a modified uncleavable HA (<b>E</b>) or a non-functional M2, M2SR (<b>F</b>) are also indicated. The packaging signals located at the 3’and 5’ ends of each vRNA are indicated with white boxes (<b>A</b>–<b>F</b>). Lines at the end of each vRNA indicate the 3’and 5’ NCRs (<b>A</b>–<b>F</b>). Striped boxes represent an internal deletion of the PB2 (<b>A</b>), PB1 (<b>B</b>), HA (<b>C</b>), or NA (<b>D</b>) ORF. A black line represents an amino acid substitution (R325T) in the cleavage site of HA that results in an uncleavable HA protein (<b>E</b>). A black box represents the deletion of the M2 transmembrane domain (amino acids 25 to 53) that together with the insertion of two stop codons (black lines) downstream of M1 ORF abolish M2 expression (<b>E</b>). In the case of ∆PB2 (<b>A</b>), ∆PB1 (<b>B</b>), ∆HA (<b>C</b>), uncleavable HA (<b>E</b>), and M2SR (<b>F</b>) sciIAVs, efficient viral replication is accomplished by complementation, <span class="html-italic">in trans</span>, of the deficient (<b>A</b>–<b>C</b>) or mutated (<b>E</b>–<b>F</b>) viral proteins by constitutively expressing PB2 (<b>A</b>), PB1 (<b>B</b>), HA (<b>C</b>,<b>E</b>), or M2 (<b>F</b>) using stable cell lines. In the case of the ∆NA sciIAV (<b>D</b>), exogenous NA is provided in the tissue culture supernatant for the efficient release of infectious viral particles.</p> Full article ">Figure 10
<p>Generation of LAIV based on replication-incompetent viruses: (<b>A</b>) Introduction of premature termination codons (PTC) into the viral genome: IAV segments PB2, PB1, PA and NP containing four amber codon substitutions for the generation of replication-incompetent viruses are indicated. Packaging signals of each vRNAs located at 3’and 5’ ends are represented in white boxes. Lines at the end of each vRNA indicate the 3’and 5’ NCR. (<b>B</b>) Schematic representation of the orthogonal translation system: Schematic representation of ribosomal incorporation of the orthogonal unnatural amino acid (UAA) and the UAA-tRNA recruitment during the translational elongation event. An UAA is charged onto a tRNA with the required non-sense anticodon by an orthogonal tRNA synthetase. This tRNA then recognizes its corresponding mRNA non-sense codon in the ribosome, leading to the incorporation of the UAA into the protein of interest. (<b>C</b>) Establishment of a virion packaging system compatible with the orthogonal translation machinery: Generation of premature termination codon (PTC) viruses are characterized by replication incompetence in conventional cells (left) but efficient replication in cells that contain the cassettes for the expression of orthogonal tRNA (tRNA<sub>CUA</sub>), tRNA synthase (pylRS), and a gene encoding an amber codon–containing GFP (GFP<sup>39TAG</sup>), leading to viral production (right).</p> Full article ">
15 pages, 717 KiB  
Review
Viruses of Polar Aquatic Environments
by Sheree Yau and Mansha Seth-Pasricha
Viruses 2019, 11(2), 189; https://doi.org/10.3390/v11020189 - 22 Feb 2019
Cited by 33 | Viewed by 7031
Abstract
The poles constitute 14% of the Earth’s biosphere: The aquatic Arctic surrounded by land in the north, and the frozen Antarctic continent surrounded by the Southern Ocean. In spite of an extremely cold climate in addition to varied topographies, the polar aquatic regions [...] Read more.
The poles constitute 14% of the Earth’s biosphere: The aquatic Arctic surrounded by land in the north, and the frozen Antarctic continent surrounded by the Southern Ocean. In spite of an extremely cold climate in addition to varied topographies, the polar aquatic regions are teeming with microbial life. Even in sub-glacial regions, cellular life has adapted to these extreme environments where perhaps there are traces of early microbes on Earth. As grazing by macrofauna is limited in most of these polar regions, viruses are being recognized for their role as important agents of mortality, thereby influencing the biogeochemical cycling of nutrients that, in turn, impact community dynamics at seasonal and spatial scales. Here, we review the viral diversity in aquatic polar regions that has been discovered in the last decade, most of which has been revealed by advances in genomics-enabled technologies, and we reflect on the vast extent of the still-to-be explored polar microbial diversity and its “enigmatic virosphere”. Full article
(This article belongs to the Special Issue Viruses of Microbes V: Biodiversity and Future Applications)
►▼ Show Figures

Figure 1

Figure 1
<p>Maps indicating the locations of the polar regions described in this review (red stars) that have been sampled for viruses. When multiple different sites were sampled in a region, they are listed in parentheses as follows. (<b>a</b>) A topographic map of Antarctica modified with labels identifying the South Shetland Islands (Livingston Island: Limnopolar Lake and nine freshwater lakes on Byers Peninsula), the Vestfold Hills (Ace, Organic, and Deep Lakes), Palmer Station on the West Antarctica Peninsula, Prydz Bay, and Lake Vostok. (<b>b</b>) A map of the Arctic modified to include the Arctic Archipelago, Baffin Bay, Beaufort Sea, Amundsen Gulf, Svalbard Archipelago highlighting Spitsbergen Island (six freshwater lakes: Linnevatnet, Borgdammane, Tunsjoen, Tenndammen, IR2, and Nordammen; Kongsfjorden; and Storfjorden), Barents Sea, Bothnian Bay, and the Baltic Sea. Image Credits: The Antarctica map was created by Philippe Rekacewicz and Emmanuelle Bournay, UNEP/GRID-Arendal, available at grida.no. The Arctic map was created by Hugo Ahlenius, UNEP/GRID-Arendal, available at grida.no.</p> Full article ">
22 pages, 3378 KiB  
Article
Host Resistance, Genomics and Population Dynamics in a Salmonella Enteritidis and Phage System
by Angela Victoria Holguín, Pablo Cárdenas, Catalina Prada-Peñaranda, Laura Rabelo Leite, Camila Buitrago, Viviana Clavijo, Guilherme Oliveira, Pimlapas Leekitcharoenphon, Frank Møller Aarestrup and Martha J. Vives
Viruses 2019, 11(2), 188; https://doi.org/10.3390/v11020188 - 22 Feb 2019
Cited by 19 | Viewed by 6660
Abstract
Bacteriophages represent an alternative solution to control bacterial infections. When interacting, bacteria and phage can evolve, and this relationship is described as antagonistic coevolution, a pattern that does not fit all models. In this work, the model consisted of a microcosm of Salmonella [...] Read more.
Bacteriophages represent an alternative solution to control bacterial infections. When interacting, bacteria and phage can evolve, and this relationship is described as antagonistic coevolution, a pattern that does not fit all models. In this work, the model consisted of a microcosm of Salmonella enterica serovar Enteritidis and φSan23 phage. Samples were taken for 12 days every 48 h. Bacteria and phage samples were collected; and isolated bacteria from each time point were challenged against phages from previous, contemporary, and subsequent time points. The phage plaque tests, with the genomics analyses, showed a mutational asymmetry dynamic in favor of the bacteria instead of antagonistic coevolution. This is important for future phage-therapy applications, so we decided to explore the population dynamics of Salmonella under different conditions: pressure of one phage, a combination of phages, and phages plus an antibiotic. The data from cultures with single and multiple phages, and antibiotics, were used to create a mathematical model exploring population and resistance dynamics of Salmonella under these treatments, suggesting a nonlethal, growth-inhibiting antibiotic may decrease resistance to phage-therapy cocktails. These data provide a deep insight into bacterial dynamics under different conditions and serve as additional criteria to select phages and antibiotics for phage-therapy. Full article
(This article belongs to the Special Issue Diversity and Evolution of Phage Genomes)
►▼ Show Figures

Figure 1

Figure 1
<p>Population compartment model describing dynamics of two distinct phages infecting a population of bacteria. Colored compartments represent subdivisions of the population, corresponding to state variables in the ordinary differential equation (ODE) model. Bacteria may be susceptible to both phages (blue), resistant to one phage and susceptible to the other (light orange), resistant to both phages (dark orange), or infected with one phage (pink). Free phages are denoted in green compartments. Arrows represent population flow between compartments, with constants governing the rate annotated on each.</p> Full article ">Figure 2
<p>Interaction between <span class="html-italic">S.</span> Enteritidis s25pp and phage <span class="html-italic">φ</span>San23 under controlled conditions. The lower horizontal axis denotes times P (present), C (Contemporary), and F (Future). The upper horizontal axis shows the transfer number. The vertical axis indicates population resistance frequency, on a scale from 0 to 1.00. The plots indicated that no antagonistic co-evolutionary relationship was established. Phages from each transfer isolated from the control where only the phage was able to evolve were shown to be infective against ancestral bacteria, and bacteria isolated from the control where only bacteria was able to evolve were shown to be susceptible to the ancestral phage.</p> Full article ">Figure 3
<p>(<b>A</b>) Bacteria isolated from the control where only bacteria were allowed to evolve were shown to be susceptible to the ancestral phage. (<b>B</b>) Phages from each transfer from the control where only phages were allowed to evolve were shown to be infective against ancestral bacteria.</p> Full article ">Figure 4
<p>Visual comparison of genomic changes in the bacterial evolutionary control and phage resistant mutants over time. Inset (<b>A</b>) Bacterial control genomes; each ring represents a sequenced genome from the control, i.e. bacteria that evolve with no phage pressure. The single black line in the inner circle shows the ancestral genome. Color represents the transfer number in time: fuchsia rings correspond to transfer 1, black rings to transfer 3, and grey rings to transfer 6, white spaces represent mutations. Inset (<b>B</b>) shows de genomes of phage resistant mutants over time. The single black line in the inner circle shows the ancestral genome. Each ring represents a sequenced mutant genome and colors represent the transfer number in time: red rings correspond to transfer 1; turquoise to transfer 2; black to transfer 3; blue to transfer 4; fuchsia to transfer 5; and grey to transfer 6; white spaces represent mutations over time. Multiple point mutations at the prophage location (ca. 2500 bp) were found, in both control and phage resistant mutant bacteria.</p> Full article ">Figure 5
<p>Increase in <span class="html-italic">S.</span> Enteritidis resistance to <span class="html-italic">φ</span>San23 through successive cultures. Each time series represents a different treatment of successively propagated <span class="html-italic">S.</span> Enteritidis cultures. Error bars portray standard error (<span class="html-italic">n</span> = 2). Dotted lines represent corresponding behavior as predicted by the mathematical model.</p> Full article ">Figure 6
<p>Population composition dynamics predicted under different treatment regimes (single phage without antibiotic, single phage with antibiotic, two phages without antibiotic, two phages with antibiotic). Dotted lines denote culture transfers, simulated by multiplying all compartments in the model by a dilution factor <span class="html-italic">v</span>. Note traces with similar behavior overlap each other near the horizontal axis, causing only the peaks of certain populations to be visible, as is the case of bacteria infected with Phage 1 in the two-phage systems (orange line), for example. Graphs have different vertical axis scales in order to better view dynamics.</p> Full article ">Figure 7
<p>Bacterial population total size and resistance to phage predicted under different treatment regimes (single phage without antibiotic, single phage with antibiotic, two phages without antibiotic, two phages with antibiotic). Dotted lines denote culture transfers, simulated by multiplying all compartments in the model by a dilution factor <span class="html-italic">v</span>.</p> Full article ">
9 pages, 863 KiB  
Article
Tomato Chlorotic Spot Virus (TCSV) Putatively Incorporated a Genomic Segment of Groundnut Ringspot Virus (GRSV) Upon a Reassortment Event
by João Marcos Fagundes Silva, Athos Silva de Oliveira, Mariana Martins Severo de Almeida, Richard Kormelink, Tatsuya Nagata and Renato Oliveira Resende
Viruses 2019, 11(2), 187; https://doi.org/10.3390/v11020187 - 22 Feb 2019
Cited by 8 | Viewed by 5174
Abstract
Tomato chlorotic spot virus (TCSV) and groundnut ringspot virus (GRSV) share several genetic and biological traits. Both of them belong to the genus Tospovirus (family Peribunyaviridae), which is composed by viruses with tripartite RNA genome that infect plants and are transmitted by [...] Read more.
Tomato chlorotic spot virus (TCSV) and groundnut ringspot virus (GRSV) share several genetic and biological traits. Both of them belong to the genus Tospovirus (family Peribunyaviridae), which is composed by viruses with tripartite RNA genome that infect plants and are transmitted by thrips (order Thysanoptera). Previous studies have suggested several reassortment events between these two viruses, and some speculated that they may share one of their genomic segments. To better understand the intimate evolutionary history of these two viruses, we sequenced the genomes of the first TCSV and GRSV isolates ever reported. Our analyses show that TCSV and GRSV isolates indeed share one of their genomic segments, suggesting that one of those viruses may have emerged upon a reassortment event. Based on a series of phylogenetic and nucleotide diversity analyses, we conclude that the parental genotype of the M segment of TCSV was either eliminated due to a reassortment with GRSV or it still remains to be identified. Full article
(This article belongs to the Special Issue Plant Virus Ecology and Biodiversity)
►▼ Show Figures

Figure 1

Figure 1
<p>Phylogenetic tree based on <span class="html-italic">NSm</span> gene fragments from groundnut ringspot virus (GRSV), tomato chlorotic spot virus (TCSV), and tomato spotted wilt virus (TSWV) isolates. TCSV sequences are represented by grey triangles next to their accession numbers, while GRSV sequences are represented by black circles. Nodes with bootstrap values above 50%, 70%, and 90% are indicated by one, two, and three asterisks, respectively.</p> Full article ">Figure 2
<p>Genomic organization of TCSV and GRSV. The numbers on the right indicate the length of each segment of isolates SA-05 (GRSV) and BR-03 (TCSV), respectively.</p> Full article ">Figure 3
<p>Phylogenetic trees based on concatenated protein sequences encoded in the S, M, and L RNAs of TCSV, GRSV, and TSWV isolates, and the observed/expected synonymous mutations in the coding regions of TCSV and GRSV isolates. (<b>a</b>) Maximum likelihood trees and protein identity plots of the S, M, and L segments of TCSV, GRSV, and TSWV and (<b>b</b>) suppression of synonymous mutation variability in the concatenated ORFs of TCSV and GRSV using a sliding window of 250 codons.</p> Full article ">
10 pages, 393 KiB  
Article
Chronic Hepatitis E in Rheumatology and Internal Medicine Patients: A Retrospective Multicenter European Cohort Study
by Sven Pischke, Jean-Marie Peron, Moritz von Wulffen, Johann von Felden, Christoph Höner zu Siederdissen, Sophie Fournier, Marc Lütgehetmann, Christoph Iking-Konert, Dominik Bettinger, Gabriella Par, Robert Thimme, Alain Cantagrel, Ansgar W. Lohse, Heiner Wedemeyer, Robert de Man and Vincent Mallet
Viruses 2019, 11(2), 186; https://doi.org/10.3390/v11020186 - 22 Feb 2019
Cited by 36 | Viewed by 4371
Abstract
Objectives: Hepatitis E virus (HEV) infection is a pandemic with regional outbreaks, including in industrialized countries. HEV infection is usually self-limiting but can progress to chronic hepatitis E in transplant recipients and HIV-infected patients. Whether other immunocompromised hosts, including rheumatology and internal medicine [...] Read more.
Objectives: Hepatitis E virus (HEV) infection is a pandemic with regional outbreaks, including in industrialized countries. HEV infection is usually self-limiting but can progress to chronic hepatitis E in transplant recipients and HIV-infected patients. Whether other immunocompromised hosts, including rheumatology and internal medicine patients, are at risk of developing chronic HEV infection is unclear. Methods: We conducted a retrospective European multicenter cohort study involving 21 rheumatology and internal medicine patients with HEV infection between April 2014 and April 2016. The underlying diseases included rheumatoid arthritis (n = 5), psoriatic arthritis (n = 4), other variants of chronic arthritis (n = 4), primary immunodeficiency (n = 3), systemic granulomatosis (n = 2), lupus erythematosus (n = 1), Erdheim–Chester disease (n = 1), and retroperitoneal fibrosis (n = 1). Results: HEV infection lasting longer than 3 months was observed in seven (33%) patients, including two (40%) patients with rheumatoid arthritis, three (100%) patients with primary immunodeficiency, one (100%) patient with retroperitoneal fibrosis and one (100%) patient with systemic granulomatosis. Patients with HEV infection lasting longer than 3 months were treated with methotrexate without corticosteroids (n = 2), mycophenolate mofetil/prednisone (n = 1), and sirolimus/prednisone (n = 1). Overall, 8/21 (38%) and 11/21 (52%) patients cleared HEV with and without ribavirin treatment, respectively. One patient experienced an HEV relapse after initially successful ribavirin therapy. One patient (5%) was lost to follow-up, and no patients died from hepatic complications. Conclusion: Rheumatology and internal medicine patients, including patients treated with methotrexate without corticosteroids, are at risk of developing chronic HEV infection. Rheumatology and internal medicine patients with abnormal liver tests should be screened for HEV infection. Full article
(This article belongs to the Special Issue Hepatitis E Virus)
►▼ Show Figures

Figure 1

Figure 1
<p>ALT peak levels (<b>A</b>) and duration of proven HEV viremia (<b>B</b>) in patients with spontaneous clearance, patients with reduction of immunosuppression, patients treated with ribavirin and patients treated with reduction of immunosuppression plus ribavirin.</p> Full article ">
14 pages, 243 KiB  
Review
Co-Infection of Swine with Porcine Circovirus Type 2 and Other Swine Viruses
by Ting Ouyang, Xinwei Zhang, Xiaohua Liu and Linzhu Ren
Viruses 2019, 11(2), 185; https://doi.org/10.3390/v11020185 - 21 Feb 2019
Cited by 128 | Viewed by 9428
Abstract
Porcine circovirus 2 (PCV2) is the etiological agent that causes porcine circovirus diseases and porcine circovirus-associated diseases (PCVD/PCVAD), which are present in every major swine-producing country in the world. PCV2 infections may downregulate the host immune system and enhance the infection and replication [...] Read more.
Porcine circovirus 2 (PCV2) is the etiological agent that causes porcine circovirus diseases and porcine circovirus-associated diseases (PCVD/PCVAD), which are present in every major swine-producing country in the world. PCV2 infections may downregulate the host immune system and enhance the infection and replication of other pathogens. However, the exact mechanisms of PCVD/PCVAD are currently unknown. To date, many studies have reported that several cofactors, such as other swine viruses or bacteria, vaccination failure, and stress or crowding, in combination with PCV2, lead to PCVD/PCVAD. Among these cofactors, co-infection of PCV2 with other viruses, such as porcine reproductive and respiratory syndrome virus, porcine parvovirus, swine influenza virus and classical swine fever virus have been widely studied for decades. In this review, we focus on the current state of knowledge regarding swine co-infection with different PCV2 genotypes or strains, as well as with PCV2 and other swine viruses. Full article
(This article belongs to the Special Issue Porcine Viruses 2019)
7 pages, 203 KiB  
Article
Estimating Disability-Adjusted Life Years (DALYs) in Community Cases of Norovirus in England
by John P. Harris, Miren Iturriza-Gomara and Sarah J. O’Brien
Viruses 2019, 11(2), 184; https://doi.org/10.3390/v11020184 - 21 Feb 2019
Cited by 5 | Viewed by 4061
Abstract
Disability adjusted life years (DALYs) have been used since the 1990s. It is a composite measure of years of life lost with years lived with disability. Essentially, one DALY is the equivalent of a year of healthy life lost if a person had [...] Read more.
Disability adjusted life years (DALYs) have been used since the 1990s. It is a composite measure of years of life lost with years lived with disability. Essentially, one DALY is the equivalent of a year of healthy life lost if a person had not experienced disease. Norovirus is the most common cause of gastrointestinal diseases worldwide. Norovirus activity varies from one season to the next for reasons not fully explained. Infection with norovirus is generally not severe, and is normally characterized as mild and self-limiting with no long-term sequelae. In this study, we model a range of estimates of DALYs for community cases of norovirus in England and Wales. We estimated a range of DALYs for norovirus to account for mixing of the severity of disease and the range of length of illness experienced by infected people. Our estimates were between 1159 and 4283 DALYs per year, or 0.3–1.2 years of healthy life lost per thousand cases of norovirus. These estimates provide evidence that norovirus leads to a considerable level of ill health in England and Wales. This information will be helpful should candidate norovirus vaccines become available in the future. Full article
(This article belongs to the Special Issue Noroviruses)
18 pages, 1104 KiB  
Article
Development and Characterization of a Sin Nombre Virus Transmission Model in Peromyscus maniculatus
by Bryce M. Warner, Derek R. Stein, Bryan D. Griffin, Kevin Tierney, Anders Leung, Angela Sloan, Darwyn Kobasa, Guillaume Poliquin, Gary P. Kobinger and David Safronetz
Viruses 2019, 11(2), 183; https://doi.org/10.3390/v11020183 - 21 Feb 2019
Cited by 16 | Viewed by 6799
Abstract
In North America, Sin Nombre virus (SNV) is the main cause of hantavirus cardiopulmonary syndrome (HCPS), a severe respiratory disease with a fatality rate of 35–40%. SNV is a zoonotic pathogen carried by deer mice (Peromyscus maniculatus), and few studies have [...] Read more.
In North America, Sin Nombre virus (SNV) is the main cause of hantavirus cardiopulmonary syndrome (HCPS), a severe respiratory disease with a fatality rate of 35–40%. SNV is a zoonotic pathogen carried by deer mice (Peromyscus maniculatus), and few studies have been performed examining its transmission in deer mouse populations. Studying SNV and other hantaviruses can be difficult due to the need to propagate the virus in vivo for subsequent experiments. We show that when compared with standard intramuscular infection, the intraperitoneal infection of deer mice can be as effective in producing SNV stocks with a high viral RNA copy number, and this method of infection provides a more reproducible infection model. Furthermore, the age and sex of the infected deer mice have little effect on viral replication and shedding. We also describe a reliable model of direct experimental SNV transmission. We examined the transmission of SNV between deer mice and found that direct contact between deer mice is the main driver of SNV transmission rather than exposure to contaminated excreta/secreta, which is thought to be the main driver of transmission of the virus to humans. Furthermore, increases in heat shock responses or testosterone levels in SNV-infected deer mice do not increase the replication, shedding, or rate of transmission. Here, we have demonstrated a model for the transmission of SNV between deer mice, the natural rodent reservoir for the virus. The use of this model will have important implications for further examining SNV transmission and in developing strategies for the prevention of SNV infection in deer mouse populations. Full article
(This article belongs to the Special Issue Hantaviruses)
►▼ Show Figures

Figure 1

Figure 1
<p>Sin Nombre virus (SNV) replication and shedding in male and female deer mice of different ages. (<b>A</b>) Groups of 1–2-month-old deer mice were inoculated with a VeroE6-adapted SNV. <span class="html-italic">n</span> = 4. (<b>B</b>) and (<b>C</b>) Groups of 10 (intramuscularly (IM) infected) or 12 (intraperitoneally (IP) infected) deer mice were infected with SNV, and S segment copies were detected in (<b>B</b>) pooled lung homogenates from each group for producing viral stocks or (<b>C</b>) each individual mouse to determine the amounts of viral replication and shedding. (<b>B</b>) 1 IM group was tested, and 3 IP groups were tested. (<b>D</b>) and (<b>E</b>) Comparison of replication and shedding seen in (<b>D</b>) male vs. female deer mice 10 days post-infection (circles: 1–2-month-old deer mice; triangles: 5–6-month-old deer mice) or (<b>E</b>) 1–2-month-old (young) vs. 5–6-month-old (old) deer mice 10 days post-infection (circles: male deer mice; triangles: female deer mice). The data shown are the mean + SEM (<b>B</b>) and medians (<b>C</b>–<b>E</b>) for each group. The numbers indicate the p value as assessed by a Mann–Whitney test.</p> Full article ">Figure 2
<p>Induction of heat shock protein 70 (HSP70) expression in deer mice. (<b>A</b>) Levels of HSP70 expression were assessed in either the blood or the lungs as compared with the vehicle-treated mice. <span class="html-italic">n</span> = 3. (<b>B</b>) Mice were given PFL, and HSP70 expression was assessed 24 h later in tissues where SNV persistence has been shown to occur. <span class="html-italic">n</span> = 3. (<b>C</b>) HSP70 expression in various tissues in both male and female mice 24 h following oral gavage of PFL. <span class="html-italic">n</span> = 6. (<b>D</b>) Change in weight following daily doses of PFL for 7 days. <span class="html-italic">n</span> = 12. (<b>E</b>) HSP70 expression levels at different time points in the blood and lungs of deer mice following daily PFL administration. <span class="html-italic">n</span> = 12.</p> Full article ">Figure 3
<p>Replication and shedding of SNV in control and paeoniflorin (PFL)-treated deer mice. The SNV viral copy number was assessed in the tissues and samples listed at the indicated time points post-infection. The numbers indicate the p value as assessed by a Mann–Whitney test.</p> Full article ">Figure 4
<p>Replication and shedding of SNV in castrated male deer mice given testosterone. (<b>A</b>) Serum testosterone levels in deer mice before and 1 month following castration. (<b>B</b>) Serum testosterone levels in deer mice implanted with osmotic pumps containing either testosterone enanthate or the control. (<b>C</b>) The SNV viral copy number was assessed in the tissues and samples listed at the indicated time points post-infection. The numbers indicate the p value as assessed by a Mann–Whitney test.</p> Full article ">
18 pages, 2618 KiB  
Review
Mastomys Species as Model Systems for Infectious Diseases
by Daniel Hasche and Frank Rösl
Viruses 2019, 11(2), 182; https://doi.org/10.3390/v11020182 - 21 Feb 2019
Cited by 19 | Viewed by 5962
Abstract
Replacements of animal models by advanced in vitro systems in biomedical research, despite exceptions, are currently still not satisfactory in reproducing the whole complexity of pathophysiological mechanisms that finally lead to disease. Therefore, preclinical models are additionally required to reflect analogous in vivo [...] Read more.
Replacements of animal models by advanced in vitro systems in biomedical research, despite exceptions, are currently still not satisfactory in reproducing the whole complexity of pathophysiological mechanisms that finally lead to disease. Therefore, preclinical models are additionally required to reflect analogous in vivo situations as found in humans. Despite proven limitations of both approaches, only a combined experimental arrangement guarantees generalizability of results and their transfer to the clinics. Although the laboratory mouse still stands as a paradigm for many scientific discoveries and breakthroughs, it is mandatory to broaden our view by also using nontraditional animal models. The present review will first reflect the value of experimental systems in life science and subsequently describes the preclinical rodent model Mastomys coucha that—although still not well known in the scientific community—has a long history in research of parasites, bacteria, papillomaviruses and cancer. Using Mastomys, we could recently show for the first time that cutaneous papillomaviruses—in conjunction with UV as an environmental risk factor—induce squamous cell carcinomas of the skin via a “hit-and-run” mechanism. Moreover, Mastomys coucha was also used as a proof-of-principle model for the successful vaccination against non-melanoma skin cancer even under immunosuppressive conditions. Full article
(This article belongs to the Special Issue Animal Models for Viral Diseases)
►▼ Show Figures

Figure 1

Figure 1
<p>Wolfgang Pauli and Niels Bohr are watching a spinning top as a model for the spinning electron. Photograph by Erik Gustafson, courtesy of AIP Emilio Segré Visual Archives. Courtesy of the Margrethe Bohr collection, Kopenhagen.</p> Full article ">Figure 2
<p>The multimammate mouse <span class="html-italic">Mastomys coucha</span>. (<b>A</b>–<b>D</b>) Black-eyed <span class="html-italic">Mastomys coucha</span> with different coat colors. (<b>E</b>,<b>F</b>) Chamois-colored red-eyed <span class="html-italic">Mastomys coucha</span> used at the DKFZ (derived from the GRA-Giessen strain). (<b>G</b>) Metaphase chromosome spread obtained from a <span class="html-italic">Mastomys coucha</span> splenocyte (2<span class="html-italic">n</span> = 36; 630× magnification).</p> Full article ">Figure 3
<p>Young <span class="html-italic">Mastomys coucha</span>. (<b>A</b>) Young <span class="html-italic">Mastomys</span> usually huddle together. (<b>B</b>,<b>C</b>) Parental <span class="html-italic">Mastomys</span>, especially the mother, have a strong protective instinct and always stay close to their offspring.</p> Full article ">Figure 4
<p>The <span class="html-italic">Mastomys natalensis</span> papillomavirus. (<b>A</b>) A spontaneous skin tumor (papilloma) near the nose. (<b>B</b>) HE staining of a spontaneous skin tumor (“so-called” keratoacanthoma) shows typical endo-exophytic growth and strong keratinization. (<b>C</b>) EM micrograph of MnPV particles (P) in the most upper layer of a spontaneous MnPV-induced skin lesion. While larger host cell compartments are already degraded during terminal differentiation and desquamation, tonofibrils (T) are still visible. (<b>D</b>) Schematic representation of the genomes of HPV16 (an alpha-type), HPV8 (a beta-type) and MnPV (an iota-type). While the genomes of all PV types harbor an upstream regulatory region (URR) and code for early genes (E1, E2, E4, E6, E7) and late genes (L1, L2), PVs from the genus beta and iota do not contain E5 open reading frames.</p> Full article ">Figure 5
<p>The <span class="html-italic">Mastomys coucha</span> papillomavirus 2. (<b>A</b>) Schematic representation of the McPV2 genome. (<b>B</b>) Condylomas in the anogenital region induced by McPV2. (<b>C</b>) Tongue papillomas induced by McPV2 (these are frequently positive for MnPV as well). (<b>D</b>) HE staining of a condyloma. (<b>E</b>) HE staining of a tongue papilloma.</p> Full article ">Figure 6
<p>UV-induced SCCs in the <span class="html-italic">Mastomys</span> model. (<b>A</b>) A UV-induced KSCC. (<b>B</b>) KSCCs are characterized by the growth of well-differentiated squamous cells and show strong keratinization comparable to spontaneous MnPV-induced tumors (HE staining). (<b>C</b>) A UV-induced nKSCC. (<b>D</b>) HE staining of a poorly differentiated nKSCC.</p> Full article ">
13 pages, 1240 KiB  
Article
Usutu Virus Isolated from Rodents in Senegal
by Moussa Moïse Diagne, Marie Henriette Dior Ndione, Nicholas Di Paola, Gamou Fall, André Pouwedeou Bedekelabou, Pape Mbacké Sembène, Ousmane Faye, Paolo Marinho de Andrade Zanotto and Amadou Alpha Sall
Viruses 2019, 11(2), 181; https://doi.org/10.3390/v11020181 - 21 Feb 2019
Cited by 43 | Viewed by 5511
Abstract
Usutu virus (USUV) is a Culex-associated mosquito-borne flavivirus of the Flaviviridae family. Since its discovery in 1959, the virus has been isolated from birds, arthropods and humans in Europe and Africa. An increasing number of Usutu virus infections in humans with neurological [...] Read more.
Usutu virus (USUV) is a Culex-associated mosquito-borne flavivirus of the Flaviviridae family. Since its discovery in 1959, the virus has been isolated from birds, arthropods and humans in Europe and Africa. An increasing number of Usutu virus infections in humans with neurological presentations have been reported. Recently, the virus has been detected in bats and horses, which deviates from the currently proposed enzootic cycle of USUV involving several different avian and mosquito species. Despite this increasing number of viral detections in different mammalian hosts, the existence of a non-avian reservoir remains unresolved. In Kedougou, a tropical region in the southeast corner of Senegal, Usutu virus was detected, isolated and sequenced from five asymptomatic small mammals: Two different rodent species and a single species of shrew. Additional molecular characterization and in vivo growth dynamics showed that these rodents/shrew-derived viruses are closely related to the reference strain (accession number: AF013412) and are as pathogenic as other characterized strains associated with neurological invasions in human. This is the first evidence of Usutu virus isolation from rodents or shrews. Our findings emphasize the need to consider a closer monitoring of terrestrial small mammals in future active surveillance, public health, and epidemiological efforts in response to USUV in both Africa and Europe. Full article
►▼ Show Figures

Figure 1

Figure 1
<p>A map of Kedougou with the locations of the five captured Usutu virus-positive mammals.</p> Full article ">Figure 2
<p>The comparative genome organization and mapping of amino acid differences between Usutu virus (USUV) sequences, characterized from different hosts. Positions are annotated and aligned to their loci in the USUV polyprotein. Host of origin and strain names can be found to the left and right of the polyprotein sequence maps, respectively. The shared non-synonymous changes from the SAAR-1776 to the rodent USUV strains are shown in the bottom right corner.</p> Full article ">Figure 3
<p>A maximum-likelihood phylogenetic tree estimated using 28 USUV polyprotein sequences. Rodent taxon labels are in bold. The colored circles indicate the host where the USUV sequence originated. The asterisks (*) at major nodes indicate an SH-like support value &gt; 70%. The tree branches are scaled by the nucleotide substitutions per site.</p> Full article ">Figure 4
<p>The in vivo evaluation of mouse mortality and virulence of three Usutu virus strains inoculated in 3-to-4-week-old Swiss mice. Survival (left panels) and changes in body weight (right panels) were observed after three different inoculation methods: intracerebral (IC, panels (<b>A</b>,<b>B</b>)), intraperitoneal (IP, panels (<b>C</b>,<b>D</b>)), and subcutaneous (SC, panels (<b>E</b>,<b>F</b>)) for 20 days, post-infection period. The control mice (red) were injected with PBS in duplicate groups using the same inoculation methods. Error bars show the 95% confidence intervals for each sampled time point. The days of disease-specific symptom presentations (SSP) is shown in the top left corner of panels (<b>B</b>,<b>D</b>,<b>F</b>). The number of days (<b>D</b>) post-infection when disease-specific symptoms are shown for each strain, as well as the proportion of symptomatic mice in each group.</p> Full article ">
14 pages, 3243 KiB  
Article
Global In-Silico Scenario of tRNA Genes and Their Organization in Virus Genomes
by Sergio Morgado and Ana Carolina Vicente
Viruses 2019, 11(2), 180; https://doi.org/10.3390/v11020180 - 21 Feb 2019
Cited by 31 | Viewed by 4329
Abstract
Viruses are known to be highly dependent on the host translation machinery for their protein synthesis. However, tRNA genes are occasionally identified in such organisms, and in addition, few of them harbor tRNA gene clusters comprising dozens of genes. Recently, tRNA gene clusters [...] Read more.
Viruses are known to be highly dependent on the host translation machinery for their protein synthesis. However, tRNA genes are occasionally identified in such organisms, and in addition, few of them harbor tRNA gene clusters comprising dozens of genes. Recently, tRNA gene clusters have been shown to occur among the three domains of life. In such a scenario, the viruses could play a role in the dispersion of such structures among these organisms. Thus, in order to reveal the prevalence of tRNA genes as well as tRNA gene clusters in viruses, we performed an unbiased large-scale genome survey. Interestingly, tRNA genes were predicted in ssDNA (single-stranded DNA) and ssRNA (single-stranded RNA) viruses as well in many other dsDNA viruses of families from Caudovirales order. In the latter group, tRNA gene clusters composed of 15 to 37 tRNA genes were characterized, mainly in bacteriophages, enlarging the occurrence of such structures within viruses. These bacteriophages were from hosts that encompass five phyla and 34 genera. This in-silico study presents the current global scenario of tRNA genes and their organization in virus genomes, contributing and opening questions to be explored in further studies concerning the role of the translation apparatus in these organisms. Full article
►▼ Show Figures

Figure 1

Figure 1
<p>Correlations between tRNA gene number and genome length. (<b>A</b>) Correlation between the total number of tRNA genes in each genome and their length (Spearman’s correlation coefficients: <span class="html-italic">R</span> = 0.5, <span class="html-italic">p</span> = 10<sup>−16</sup>). (<b>B</b>) Correlation between the number of clustered tRNA genes and the genome length of viruses carrying tRNA gene clusters (Spearman’s correlation coefficients: <span class="html-italic">R</span> = −0.49, <span class="html-italic">p</span> = 10<sup>−15</sup>).</p> Full article ">Figure 2
<p>Codon patterns of the tRNA gene clusters. The heatmap shows the tRNA gene copy number (codons and isotypes) of each tRNA gene cluster. The background color of the labels is associated with each tRNA gene cluster group (indicated by the red labels or shown in <a href="#app1-viruses-11-00180" class="html-app">Figure S1</a>). The yellow background labels represent the <span class="html-italic">Cellulophaga</span> phages with the same tRNA gene cluster group. Genomes having identical codon pattern were collapsed, represented by the bold label. A larger version of this figure is provided in <a href="#app1-viruses-11-00180" class="html-app">Figure S4</a>.</p> Full article ">
11 pages, 438 KiB  
Article
The Application and Interpretation of IgG Avidity and IgA ELISA Tests to Characterize Zika Virus Infections
by Fátima Amaro, María P. Sánchez-Seco, Ana Vázquez, Maria J. Alves, Líbia Zé-Zé, Maria T. Luz, Teodora Minguito, Jesús De La Fuente and Fernando De Ory
Viruses 2019, 11(2), 179; https://doi.org/10.3390/v11020179 - 20 Feb 2019
Cited by 15 | Viewed by 4105
Abstract
In the absence of viremia, the diagnostics of Zika virus (ZIKV) infections must rely on serological techniques. In order to improve the serological diagnosis of ZIKV, ZIKV-IgA and ZIKV-IgG avidity assays were evaluated. Forty patients returning from ZIKV endemic areas, with confirmed or [...] Read more.
In the absence of viremia, the diagnostics of Zika virus (ZIKV) infections must rely on serological techniques. In order to improve the serological diagnosis of ZIKV, ZIKV-IgA and ZIKV-IgG avidity assays were evaluated. Forty patients returning from ZIKV endemic areas, with confirmed or suspected ZIKV infections were studied. Samples were classified as early acute, acute and late acute according to the number of days post illness onset. Low avidity IgG was only detected at acute and late acute stages and IgA mostly at the early acute and acute stages. The date of sampling provides useful information and can help to choose the best technique to use at a determined moment in time and to interpret low avidity IgG and IgA results, improving the serological diagnosis of ZIKV. Full article
(This article belongs to the Special Issue New Advances on Zika Virus Research)
►▼ Show Figures

Figure 1

Figure 1
<p>Serological testing in patients from groups I and II. (<b>A</b>) ZIKV IgG avidity results; (<b>B</b>) ZIKV IgA Results.</p> Full article ">
14 pages, 2932 KiB  
Article
Molecular Characterization of a Novel Endornavirus Conferring Hypovirulence in Rice Sheath Blight Fungus Rhizoctonia solani AG-1 IA Strain GD-2
by Li Zheng, Canwei Shu, Meiling Zhang, Mei Yang and Erxun Zhou
Viruses 2019, 11(2), 178; https://doi.org/10.3390/v11020178 - 20 Feb 2019
Cited by 44 | Viewed by 4858
Abstract
The complete sequence and genome organization of a novel Endornavirus from the hypovirulent strain GD-2 of Rhizoctonia solani AG-1 IA, the causal agent of rice sheath blight, were identified using a deep sequencing approach and it was tentatively named as Rhizoctonia solani endornavirus [...] Read more.
The complete sequence and genome organization of a novel Endornavirus from the hypovirulent strain GD-2 of Rhizoctonia solani AG-1 IA, the causal agent of rice sheath blight, were identified using a deep sequencing approach and it was tentatively named as Rhizoctonia solani endornavirus 1 (RsEV1). It was composed of only one segment that was 19,936 bp in length and was found to be the longest endornavirus genome that has been reported so far. The RsEV1 genome contained two open reading frames (ORFs): ORF1 and ORF2. ORF1 contained a glycosyltransferase 1 domain and a conserved RNA-dependent RNA polymerase domain, whereas ORF2 encoded a conserved hypothetical protein. Phylogenetic analysis revealed that RsEV1 was phylogenetically a new endogenous RNA virus. A horizontal transmission experiment indicated that RsEV1 could be transmitted from the host fungal strain GD-2 to a virulent strain GD-118P and resulted in hypovirulence in the derivative isogenic strain GD-118P-V1. Metabolomic analysis showed that 32 metabolites were differentially expressed between GD-118P and its isogenic hypovirulent strain GD-118P-V1. The differential metabolites were mainly classified as organic acids, amino acids, carbohydrates, and the intermediate products of energy metabolism. Pathway annotation revealed that these 32 metabolites were mainly involved in pentose and glucuronate interconversions and glyoxylate, dicarboxylate, starch, and sucrose metabolism, and so on. Taken together, our results showed that RsEV1 is a novel Endornavirus, and the infection of virulent strain GD-118P by RsEV1 caused metabolic disorders and resulted in hypovirulence. The results of this study lay a foundation for the biocontrol of rice sheath blight caused by R. solani AG1-IA. Full article
(This article belongs to the Special Issue Plant Virus Ecology and Biodiversity)
►▼ Show Figures

Figure 1

Figure 1
<p>Genomic organization of a novel dsRNA endornavirus, <span class="html-italic">Rhizoctonia solani</span> endornavirus 1 (RsEV1) in <span class="html-italic">Rhizoctonia solani</span> AG-1 IA. The open reading frame (ORF) and the untranslated regions (UTRs) are indicated by a rectangle and single line, respectively. The shadowing parts indicate the glycosyltransferase 1 (GT1) and RNA-dependent RNA polymerase (RdRp) conserved domains. The corresponding nucleotides in the genome are given above the rectangle, and the amino acid numbers and the molecular masses of the proteins are shown below the rectangle.</p> Full article ">Figure 2
<p>Sequence alignment of RsEV1 RdRp motifs with those of selected viruses in the genus <span class="html-italic">Endornavirus</span>. Horizontal lines, labeled with A to E, above the alignment indicate the five motifs, and the numbers in brackets show the amino acid positions. Asterisks, colons, and dots show the same amino acid residues, conservative, and semi conservative, respectively.</p> Full article ">Figure 3
<p>Phylogenetic analysis of RsEV1 and other endornaviruses based on the deduced amino acid sequences of RdRps using the maximum likelihood (ML) method with 1000 bootstrap replicates. The scale bar indicates a genetic distance of 0.5 amino acid substitutions per site. The green box represents the novel mycovirus RsEV1 in this study. The abbreviations of viruses and their GenBank accession numbers are as follows: RsEV2, <span class="html-italic">Rhizoctonia solani</span> endornavirus 2 (AMM45288.1); RsEV3, <span class="html-italic">Rhizoctonia solani</span> endornavirus 3 (ANR02699.1); RcEV1, <span class="html-italic">Rhizoctonia cerealis</span> alphaendornavirus 1 (YP_008719905.1); HmEV1, <span class="html-italic">Helicobasidium mompa</span> alphaendornavirus 1 (YP_003280846.1); CbEVC, <span class="html-italic">Ceratobasidium</span> endornavirus C (YP_009310111.1); CbEVB, <span class="html-italic">Ceratobasidium</span> endornavirus B (YP_009310114.1); CbEVG, <span class="html-italic">Ceratobasidium</span> endornavirus G (YP_009310116.1); PvEV2, Phaseolus vulgaris alphaendornavirus 2 (ATB20098.1); BPEV-YW, Bell pepper alphaendornavirus YW (AEK22062.1); BPEV-2,Bell pepper alphaendornavirus 2 (BAK52155.1); VfEV-1, Vicia faba endornavirus 1 (YP_438201.1); VfEV-2, Vicia faba endornavirus 2 (CAA04392.1); PEV1, Phytophthora alphaendornavirus 1 (YP_241110.1); GEEV-1, Grapevine endophyte alphaendornavirus 1 (YP_007003829.1); GEEV-2, Grapevine endophyte alphaendornavirus 2 (AFV91541.1); CeEV1, Chalara elegans endornavirus 1 (ADN43901.1); TaEV-2, Tuber aestivum betaendornavirus 2 (ADU64759); TaEV-1, Tuber aestivum betaendornavirus 1 (YP_004123950.1); SsEV1, Sclerotinia sclerotiorum endornavirus1(AJF94392.1); GaBRV-XL1, Gremmeniella abietina type B RNA virus XL1 (YP_529670.1); GaBRV-XL2, Gremmeniella abietina type B RNA virus XL2 (ABD73306.1); PaEV-2, Persea americana alphaendornavirus 2 (AEX28369.1); PaEV-1, Persea americana alphaendornavirus 1 (YP_005086952.1); PvEV1, Phaseolus vulgaris alphaendornavirus 1 (YP_009011062.1); OrEV, Oryza rufipogon alphaendornavirus (YP_438202.1); OsEV, Oryza sativa alphaendornavirus (YP_438200.1). AbV1 (Agaricus bisporus virus 1) and FoCV1 (Fusarium oxysporum chrysovirus 1) were used as outgroups.</p> Full article ">Figure 4
<p>Detection of dsRNAs in viral donor and recipient strains of <span class="html-italic">Rhizoctonai solani</span> AG-1 IA. M: molecular markers (λ DNA digested with <span class="html-italic">Hind</span> III); 1: The presence of dsRNA in derivative isogenic strain GD-118P-V1; 2: The presence of dsRNA in virus-containing donor strain GD-2; 3: The absence of dsRNA in virus-free recipient strain GD-118P.</p> Full article ">Figure 5
<p>Hypovirulence-associated traits in strain GD-118P-V1 of <span class="html-italic">Rhizoctonai solani</span> AG-1 IA. (A) Colony morphology. Culture characteristics of strains GD-118P, GD-118P-V1, and GD-2 on PDA plates at 28 °C for seven days. (B) Pathogenicity. The symptoms on detached rice leaves caused by isogenic strains GD-118P and GD-118P-V1, and GD-2 incubated at 28 °C for 72 h. (C and D) Comparison of average mycelial growth rate and sclerotia dry weight on PDA plates of the strains GD-118P, GD-118P-V1, and GD-2. (E) Average lesion areas caused by the strains GD-118P, GD-118P-V1, and GD-2 on detached rice leaves. In (C), (D), and (E), the data were indicated as arithmetic means ± standard error, and significant differences were assessed using the Student <span class="html-italic">t</span> test. Single asterisk (*) indicates <span class="html-italic">p</span> &lt; 0.05, and double asterisks (**) indicate <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 6
<p>Clustering analysis of the metabolic data of isogenic fungal samples. Notes: Heat map showing the clustering of detected metabolites into three metabolite classes across 16 samples. Each row represents the level changes of a metabolite in all samples. Red colors indicate metabolite levels greater than the median value (yellow colors), and blue colors indicate metabolite levels lower than the median value (yellow colors).</p> Full article ">Figure 7
<p>PCA scores derived from GC-MS spectra from isogenic strains GD-118P and GD-118P-V1 samples. Score plot of PCA: GD-118P (red square) and GD-118P-V1 (blue diamond).</p> Full article ">Figure 8
<p>PLS-DA score plots derived from GC-MS data from isogenic strains GD-118P and GD-118P-V1 samples. A: PLS-DA score plot: GD-118P (red square) and GD-118P-V1 (blue diamond); B: Permutation test plot (100 permutations) derived from GC-MS data of GD-118P and GD-118P-V1 samples.</p> Full article ">Figure 9
<p>Pathway analysis of the differential metabolites from virus-free and -containing isogenic strains. Plots depicting computed metabolic pathways as a function of −log (<span class="html-italic">p</span>) and pathway impact for the key differential metabolites from GD-118P vs. GD-118P-V1.</p> Full article ">
13 pages, 1618 KiB  
Review
GII.4 Human Norovirus: Surveying the Antigenic Landscape
by Michael L. Mallory, Lisa C. Lindesmith, Rachel L. Graham and Ralph S. Baric
Viruses 2019, 11(2), 177; https://doi.org/10.3390/v11020177 - 20 Feb 2019
Cited by 41 | Viewed by 6649
Abstract
Human norovirus is the leading cause of viral acute onset gastroenteritis disease burden, with 685 million infections reported annually. Vulnerable populations, such as children under the age of 5 years, the immunocompromised, and the elderly show a need for inducible immunity, as symptomatic [...] Read more.
Human norovirus is the leading cause of viral acute onset gastroenteritis disease burden, with 685 million infections reported annually. Vulnerable populations, such as children under the age of 5 years, the immunocompromised, and the elderly show a need for inducible immunity, as symptomatic dehydration and malnutrition can be lethal. Extensive antigenic diversity between genotypes and within the GII.4 genotype present major challenges for the development of a broadly protective vaccine. Efforts have been devoted to characterizing antibody-binding interactions with dynamic human norovirus viral-like particles, which recognize distinct antigenic sites on the capsid. Neutralizing antibody functions recognizing these sites have been validated in both surrogate (ligand blockade of binding) and in vitro virus propagation systems. In this review, we focus on GII.4 capsid protein epitopes as defined by monoclonal antibody binding. As additional antibody epitopes are defined, antigenic sites emerge on the human norovirus capsid, revealing the antigenic landscape of GII.4 viruses. These data may provide a road map for the design of candidate vaccine immunogens that induce cross-protective immunity and the development of therapeutic antibodies and drugs. Full article
(This article belongs to the Special Issue Noroviruses)
►▼ Show Figures

Figure 1

Figure 1
<p>GII.4 VP1 diversity over time. Sequence identity of VP1 (capsid), and known blockade antibody epitopes compared to GII.4 US95/96 (represented by GII.4 1997, AFJ04707.1), the first known GII.4 pandemic strain. GII.4 2002, 2004, 2006b, 2009, and 2012 are sequential pandemic strains, represented by isolates, AAZ31376.2, AFJ04709.1, AEX91909.1, and AFV08794.1, respectively. GII.4 1987 (AAK50355.1) is an endemic GII.4 strain that circulated before GII.4 US95/96 emergence. Overall identity within VP1 is high. Identity within the known evolving blockade antibody epitopes is less well conserved, resulting in the emergence of new pandemic strains refractive to herd immunity shaped by previous GII.4 exposure. Epitopes conserved between GII (epitope nanobody-85) and GI/GII strains (epitope TV20) remain largely unchanged over time in GII.4 strains.</p> Full article ">Figure 2
<p>Mouse and human anti-epitope A monoclonal antibody blockade of time-ordered GII.4 strain virus-like particles (VLP). (<b>A</b>) Monoclonal antibodies to epitope A were generated in response to the immunization of mice with GII.4 VLPs (1987, 2006, 2009, 2012 mAbs) or the infection of humans (NVB mAbs), and their IC<sub>50</sub> for the blockade of VLP-ligand binding declined from highly potent (orange) to no inhibition (blue) as reported in References [<a href="#B25-viruses-11-00177" class="html-bibr">25</a>,<a href="#B26-viruses-11-00177" class="html-bibr">26</a>,<a href="#B38-viruses-11-00177" class="html-bibr">38</a>,<a href="#B39-viruses-11-00177" class="html-bibr">39</a>,<a href="#B60-viruses-11-00177" class="html-bibr">60</a>,<a href="#B61-viruses-11-00177" class="html-bibr">61</a>]. GII.4 2004 data not available. (<b>B</b>) Antigenic drift within epitope A limits the breadth of mAb recognition of epitope A.</p> Full article ">Figure 3
<p>Ligand-blockade antibody epitopes are surface exposed and usually within hypervariable loops within the VP1 P2 subdomain. GII.4 2012 P dimer homology model (4OP7) with blocking antibody epitopes color coded.</p> Full article ">
26 pages, 2256 KiB  
Article
Identification of Broad-Spectrum Antiviral Compounds by Targeting Viral Entry
by Michela Mazzon, Ana Maria Ortega-Prieto, Douglas Imrie, Christin Luft, Lena Hess, Stephanie Czieso, Joe Grove, Jessica Katy Skelton, Laura Farleigh, Joachim J. Bugert, Edward Wright, Nigel Temperton, Richard Angell, Sally Oxenford, Michael Jacobs, Robin Ketteler, Marcus Dorner and Mark Marsh
Viruses 2019, 11(2), 176; https://doi.org/10.3390/v11020176 - 20 Feb 2019
Cited by 43 | Viewed by 8839
Abstract
Viruses are a major threat to human health and economic well-being. In recent years Ebola, Zika, influenza, and chikungunya virus epidemics have raised awareness that infections can spread rapidly before vaccines or specific antagonists can be made available. Broad-spectrum antivirals are drugs with [...] Read more.
Viruses are a major threat to human health and economic well-being. In recent years Ebola, Zika, influenza, and chikungunya virus epidemics have raised awareness that infections can spread rapidly before vaccines or specific antagonists can be made available. Broad-spectrum antivirals are drugs with the potential to inhibit infection by viruses from different groups or families, which may be deployed during outbreaks when specific diagnostics, vaccines or directly acting antivirals are not available. While pathogen-directed approaches are generally effective against a few closely related viruses, targeting cellular pathways used by multiple viral agents can have broad-spectrum efficacy. Virus entry, particularly clathrin-mediated endocytosis, constitutes an attractive target as it is used by many viruses. Using a phenotypic screening strategy where the inhibitory activity of small molecules was sequentially tested against different viruses, we identified 12 compounds with broad-spectrum activity, and found a subset blocking viral internalisation and/or fusion. Importantly, we show that compounds identified with this approach can reduce viral replication in a mouse model of Zika infection. This work provides proof of concept that it is possible to identify broad-spectrum inhibitors by iterative phenotypic screenings, and that inhibition of host-pathways critical for viral life cycles can be an effective antiviral strategy. Full article
(This article belongs to the Section Viral Immunology, Vaccines, and Antivirals)
►▼ Show Figures

Figure 1

Figure 1
<p>Strategy for the identification of broad-spectrum antiviral (BSA) compounds inhibiting Semliki Forest virus (SFV) and dengue virus serotype 2 (DENV-2) infection. (<b>A</b>) Schematic of the screening procedure. Cells in 96 well plates were pre-treated with 10 μM compounds for 45 min and then infected in the presence of compounds for a time sufficient to detect expression of viral proteins (7h for SFV and 24h for DENV-2). Uninfected cells, and infected cells treated with DMSO or with Monensin were included as controls. After fixation, plates were stained and images acquired using a PE Opera LX high-throughput confocal microscope, which images multiple fields in each well, allowing calculation of the percentage of infected cells. (<b>B</b>) Schematic of the screening strategy. In order to identify compounds with BSA activity, all 43 compounds displaying inhibitory activity against SFV (complete list in <a href="#app1-viruses-11-00176" class="html-app">Table S1</a>) were tested for their ability to block infection by DENV-2. Of the 22 compounds blocking both viruses (complete list in <a href="#app1-viruses-11-00176" class="html-app">Table S2</a>), the 12 least toxic, as determined by MTT assay, were selected for further studies (<a href="#app1-viruses-11-00176" class="html-app">Table S3</a>).</p> Full article ">Figure 2
<p>Analysis of compounds modes of action in the virus entry pathway. (<b>A</b>) Percentage of VSV_Blam (blue) or EBOV_Blam (green) pseudotyped VLP fusion events upon compound treatment, measured as cytosolic release of β-lactamase. HeLa Kyoto cells were treated with the indicated compounds for 1 h before infection with pseudotyped Blam VLPs. After 1 h at 37 °C, compounds were removed, the β-lactamase substrate added, and the percentage of cells in which fusion had occurred was quantified by flow cytometry. Data are normalised to Blam signal in DMSO-treated control cells (100%, dotted line). Monensin was used as a positive control. Statistics: one-way analysis of variance (Anova), Fisher’s least significant difference (LSD) test. * = <span class="html-italic">p</span> &lt; 0.05; ** = 0.01 &gt; <span class="html-italic">p</span> &gt; 0.005; *** = <span class="html-italic">p</span> &lt; 0.005. (<b>B</b>) Western blots showing the amount of SFV E1/E2 proteins that remains bound to the surface of infected HeLa Kyoto cells after 1 h compound treatment at 37 °C, and 1 h SFV infection on ice, in the presence of compounds. Untreated samples were included as controls. A Western blot for tubulin was used as a loading control. (<b>C</b>) Western blots showing SFV E1/E2 protein after subtilisin treatment. HeLa Kyoto cells were treated with the indicated compounds for 1 h at 37 °C, and SFV bound for 1 h on ice in the presence of compounds. Next, virus was allowed to internalise at 37 °C for 20 min, before subtilisin treatment on ice to remove surface-bound virus Ice-treated samples (where the virus was not internalised) treated or not with subtilisin, as well as untreated samples incubated at 37 °C (where the virus was internalised) were included as controls. (<b>D</b>) Western blot showing SFV E1/E2 proteins and low pH-induced E1 trimers. HeLa Kyoto cells were treated with the indicated compounds for 1 h at 37 °C, SFV bound 1 h on ice in the presence of compounds, and then internalised at 37 °C for 40 min, before cell lysis. A fraction of each lysate was treated trypsin to verify the identity of the trypsin-resistant E1 trimer (top panel). Monensin and Chloroquine (100 μM), known inhibitors of endosomal acidification were used as positive controls. Untreated samples were included as negative controls. (<b>E</b>) Percentage of DID-labelled SFV hemifusion/fusion events normalised to DMSO treated cells (100%, dashed line). HeLa Kyoto cells were pre-treated with compounds for 1 h at 37 °C before adding DID-SFV for an additional hour on ice. Unbound virus was then washed away and infection left to proceed for 40 min at 37 °C to allow virus internalisation and fusion. Bafilomycin (100 nM), a known inhibitor of viral fusion, was used as positive control. Hemifusion/fusion events were quantified on a PE Opera LX. Averages from three independent experiments are shown. Statistics: one-way Anova, Fisher’s LSD test. * = <span class="html-italic">p</span> &lt; 0.05; ** = 0.01&gt; <span class="html-italic">p</span> &gt; 0.005; *** = <span class="html-italic">p</span> &lt; 0.005.</p> Full article ">Figure 3
<p>Analysis of compounds mode of action after fusion. (<b>A</b>) A Renilla expressing plasmid was transfected in HeLa Kyoto cells. 24 h later, cells were treated with compounds for 8 h and then lysed to measure Renilla activity. Data are normalised to a DMSO treated control (100%). Emetine (10 μM), a known inhibitor of protein synthesis, was used as a control. Averages of three independent experiments are shown. Statistics: one-way Anova, Fisher’s LSD test. * = <span class="html-italic">p</span> &lt; 0.05; ** = 0.01&gt; <span class="html-italic">p</span> &gt;0.005; *** = <span class="html-italic">p</span> &lt; 0.005. (<b>B</b>) Percentage of inhibition of SFV infection following administration of each compound 60 min before infection, or at 30 and 60 min after infection. Cells (HeLa Kyoto) were fixed at 7 hpi and percentages of infection quantified following images acquisition on a PE Opera LX. Data are normalised to pre-treated controls samples (100% inhibition). Averages of two independent experiments are shown. Statistical significance is shown in <a href="#app1-viruses-11-00176" class="html-app">Table S4</a>, as determined with a two-way Anova with Dunnett’s test for multiple comparisons.</p> Full article ">Figure 4
<p>Niclosamide and Tyrphostin A9 impact on endocytosis. (<b>A</b>) Western blot showing SFV E1/E2 protein after subtilisin treatment. HeLa Kyoto cells were treated with 10 μM Niclosamide or Tyrphostin A9 for 1 h at 37 °C, and infected with SFV for 1 h on ice in the presence of compounds. Virus was left to internalise at 37 °C for 20, 40, or 60 min, before subtilisin treatment to remove surface-bound virus. Untreated cells not exposed to subtilisin, were included as controls. (<b>B</b>) HeLa Kyoto cells were treated with 10 μM Niclosamide or Tyrphostin A9 for 1 h before internalisation of Alexa 488-conjugated transferrin for 10, 20, and 30 min. DMSO-treated cells were used as controls. One representative of three independent experiments is shown. Statistics: two-way Anova with Dunnett’s test for multiple comparisons. *** = <span class="html-italic">p</span> &lt; 0.005.</p> Full article ">Figure 5
<p>In vivo activity of Tyrphostin A9, Monensin, and Sofosbuvir. (<b>A</b>) Schematic of the drug treatment and infection regime in AG129 mice. Mice were treated with 5.7 mg/kg of Sofosbuvir, 10 mg/kg of Monensin, or 1mg/kg of Tyrphostin A9 before and after infection with 10<sup>5</sup> pfu of ZIKV, at the indicated intervals. Administration of Tyrphostin A9 was suspended at day 3 due to toxicity. Focus-forming units (ffu, top panels) and ZIKV RNA copies (bottom panels) per ml of serum at day 1, 3, and 7 p.i. upon treatment with Sofosbuvir (<b>B</b>), Monensin (<b>C</b>), or Tyrphostin A9 (<b>D</b>). N.D. (not detected), indicates that no infectious units were recovered. ZIKV RNA copies/ng at day 7 p.i. in the lymph nodes (<b>E</b>), liver (<b>F</b>), and brain (<b>G</b>) upon indicated treatment. Percentages of CD14<sup>+</sup>CD11b<sup>+</sup> macrophages (<b>H</b>), CD14<sup>+</sup>CD11c<sup>+</sup> DC (<b>I</b>), and CD3<sup>+</sup>CD4<sup>+</sup> T cells (<b>J</b>) infected with ZIKV at day 7 p.i. upon indicated treatment. P values from unpaired T tests are displayed for statistically significant comparisons.</p> Full article ">
26 pages, 945 KiB  
Review
Tropism of the Chikungunya Virus
by Giulia Matusali, Francesca Colavita, Licia Bordi, Eleonora Lalle, Giuseppe Ippolito, Maria R. Capobianchi and Concetta Castilletti
Viruses 2019, 11(2), 175; https://doi.org/10.3390/v11020175 - 20 Feb 2019
Cited by 80 | Viewed by 11240
Abstract
Chikungunya virus (CHIKV) is a re-emerging mosquito-borne virus that displays a large cell and organ tropism, and causes a broad range of clinical symptoms in humans. It is maintained in nature through both urban and sylvatic cycles, involving mosquito vectors and human or [...] Read more.
Chikungunya virus (CHIKV) is a re-emerging mosquito-borne virus that displays a large cell and organ tropism, and causes a broad range of clinical symptoms in humans. It is maintained in nature through both urban and sylvatic cycles, involving mosquito vectors and human or vertebrate animal hosts. Although CHIKV was first isolated in 1953, its pathogenesis was only more extensively studied after its re-emergence in 2004. The unexpected spread of CHIKV to novel tropical and non-tropical areas, in some instances driven by newly competent vectors, evidenced the vulnerability of new territories to this infectious agent and its associated diseases. The comprehension of the exact CHIKV target cells and organs, mechanisms of pathogenesis, and spectrum of both competitive vectors and animal hosts is pivotal for the design of effective therapeutic strategies, vector control measures, and eradication actions. Full article
(This article belongs to the Special Issue Chikungunya Virus and (Re-) Emerging Alphaviruses)
►▼ Show Figures

Figure 1

Figure 1
<p>Sites of chikungunya virus replication in the <span class="html-italic">Aedes</span> mosquito vector.</p> Full article ">Figure 2
<p>Examples of tissue and cell tropism of chikungunya virus. Chikungunya virus (CHIKV) infected cells in skin (<b>A</b>); skeletal muscle (<b>B</b>); joint and bone (<b>C</b>). DC—dendritic cell.</p> Full article ">
17 pages, 2168 KiB  
Review
Global Epidemiology of Bat Coronaviruses
by Antonio C. P. Wong, Xin Li, Susanna K. P. Lau and Patrick C. Y. Woo
Viruses 2019, 11(2), 174; https://doi.org/10.3390/v11020174 - 20 Feb 2019
Cited by 253 | Viewed by 32450
Abstract
Bats are a unique group of mammals of the order Chiroptera. They are highly diversified and are the group of mammals with the second largest number of species. Such highly diversified cell types and receptors facilitate them to be potential hosts of [...] Read more.
Bats are a unique group of mammals of the order Chiroptera. They are highly diversified and are the group of mammals with the second largest number of species. Such highly diversified cell types and receptors facilitate them to be potential hosts of a large variety of viruses. Bats are the only group of mammals capable of sustained flight, which enables them to disseminate the viruses they harbor and enhance the chance of interspecies transmission. This article aims at reviewing the various aspects of the global epidemiology of bat coronaviruses (CoVs). Before the SARS epidemic, bats were not known to be hosts for CoVs. In the last 15 years, bats have been found to be hosts of >30 CoVs with complete genomes sequenced, and many more if those without genome sequences are included. Among the four CoV genera, only alphaCoVs and betaCoVs have been found in bats. As a whole, both alphaCoVs and betaCoVs have been detected from bats in Asia, Europe, Africa, North and South America and Australasia; but alphaCoVs seem to be more widespread than betaCoVs, and their detection rate is also higher. For betaCoVs, only those from subgenera Sarbecovirus, Merbecovirus, Nobecovirus and Hibecovirus have been detected in bats. Most notably, horseshoe bats are the reservoir of SARS-CoV, and several betaCoVs from subgenus Merbecovirus are closely related to MERS-CoV. In addition to the interactions among various bat species themselves, bat–animal and bat–human interactions, such as the presence of live bats in wildlife wet markets and restaurants in Southern China, are important for interspecies transmission of CoVs and may lead to devastating global outbreaks. Full article
(This article belongs to the Special Issue Viruses and Bats 2019)
►▼ Show Figures

Figure 1

Figure 1
<p>Maximum-likelihood phylogeny based on the complete genome sequences of 17 bat CoV species released by ICTV in 2018. A general time-reversible model of nucleotide substitution with estimated base frequencies, the proportion of invariant sites, and the γ distribution of rates across sites were used in the maximum-likelihood analysis. Bootstrap values are shown next to the branches. The scale bar indicates the number of nucleotide substitutions per site. Different colors represent different genera. Red, <span class="html-italic">Alphacoronavirus</span>; blue, <span class="html-italic">Betacoronavirus</span>. Updated subgenera clusters are labelled <span class="html-italic">Setracovirus, Myotacovirus, Rhinacovirus, Colacovirus, Pedacovirus, Decacovirus, Minunacovirus, Nyctacovirus</span> for the <span class="html-italic">Alphacoronavirus</span> and <span class="html-italic">Nobecovirus, Hibecovirus, Sarbecovirus, Merbecovirus</span> for the <span class="html-italic">Betacoronavirus</span>.</p> Full article ">Figure 2
<p>Geographical distribution of bat CoVs from the genera <span class="html-italic">Alphacoronavirus</span> and <span class="html-italic">Betacoronavirus</span>. Each colored region represents the country which reported the discovery of bat CoV. Red regions represent the countries which discovered bat <span class="html-italic">Alphacoornavirus</span>. Green regions represent the countries which discovered bat <span class="html-italic">Betacoronavirus</span>. Red-green striped regions represent the countries which discovered both bat <span class="html-italic">Alphacoronavirus</span> and <span class="html-italic">Betacoronavirus</span>.</p> Full article ">Figure 3
<p>Pie chart showing the relative detection rate of different bat CoVs from different subgenera of <span class="html-italic">Alphacoronavirus</span> and <span class="html-italic">Betacoronavirus</span> in Hong Kong from 2008 to 2017. The potential zoonotic transmission routes of each sub-genus of bat CoV detected are shown. Unclassified <span class="html-italic">Alphacoronavirus</span> represents those without complete genome sequences or genome characterization. Red color represents the sub-genera from <span class="html-italic">Alphacoronavirus</span>; Blue color represents the sub-genera from <span class="html-italic">Betacoronavirus</span>.</p> Full article ">Figure 4
<p>Geographical distribution of different horseshoe bats which were discovered to carry SARS-like BatCoV [<a href="#B114-viruses-11-00174" class="html-bibr">114</a>,<a href="#B115-viruses-11-00174" class="html-bibr">115</a>,<a href="#B116-viruses-11-00174" class="html-bibr">116</a>,<a href="#B117-viruses-11-00174" class="html-bibr">117</a>,<a href="#B118-viruses-11-00174" class="html-bibr">118</a>,<a href="#B119-viruses-11-00174" class="html-bibr">119</a>,<a href="#B120-viruses-11-00174" class="html-bibr">120</a>,<a href="#B121-viruses-11-00174" class="html-bibr">121</a>,<a href="#B122-viruses-11-00174" class="html-bibr">122</a>,<a href="#B123-viruses-11-00174" class="html-bibr">123</a>,<a href="#B124-viruses-11-00174" class="html-bibr">124</a>,<a href="#B125-viruses-11-00174" class="html-bibr">125</a>]. Each colored rectangular box represents the geographical distribution of a specific horseshoe bat species respectively: red box, <span class="html-italic">Rhinolophus affinis</span>; orange box, <span class="html-italic">Rhinolophus blasii</span>; yellow box, <span class="html-italic">Rhinolophus euryale</span>; green box, <span class="html-italic">Rhinolophus ferrumequinum</span>; turquoise box, <span class="html-italic">Rhinolophus hildebrantii</span>; indigo box, <span class="html-italic">Rhinolophus hipposideros</span>; purple box, <span class="html-italic">Rhinolophus macrotis</span>; brown box, <span class="html-italic">Rhinolophus mehelyi</span>; pink box, <span class="html-italic">Rhinolophus pearsonii</span>; gold box, <span class="html-italic">Rhinolophus pusillus</span>; blue-gray box, <span class="html-italic">Rhinolophus rex</span>; black box, <span class="html-italic">Rhinolophus sinicus</span>; lime box, <span class="html-italic">Rhinolophus thomasi</span>. Orange circle represents Yunnan Province; Red circle represents the origin of SARS &amp; SADS outbreaks.</p> Full article ">Figure 5
<p>Geographical distribution of bat CoVs from the genus <span class="html-italic">Betacoronavirus</span>. Each colored region represents the country which reported the discovery of bat CoV from different sub-genera. Navy-blue regions represent the countries which discovered bat CoVs from <span class="html-italic">Sarbecovirus</span>. Yellow regions represent the countries which discovered bat CoVs from <span class="html-italic">Merbecovirus</span>. Purple regions represent the countries which discovered bat CoVs from <span class="html-italic">Nobecovirus</span>.</p> Full article ">
18 pages, 8738 KiB  
Review
Targeting the Viral Polymerase of Diarrhea-Causing Viruses as a Strategy to Develop a Single Broad-Spectrum Antiviral Therapy
by Marcella Bassetto, Jana Van Dycke, Johan Neyts, Andrea Brancale and Joana Rocha-Pereira
Viruses 2019, 11(2), 173; https://doi.org/10.3390/v11020173 - 20 Feb 2019
Cited by 23 | Viewed by 6863
Abstract
Viral gastroenteritis is an important cause of morbidity and mortality worldwide, being particularly severe for children under the age of five. The most common viral agents of gastroenteritis are noroviruses, rotaviruses, sapoviruses, astroviruses and adenoviruses, however, no specific antiviral treatment exists today against [...] Read more.
Viral gastroenteritis is an important cause of morbidity and mortality worldwide, being particularly severe for children under the age of five. The most common viral agents of gastroenteritis are noroviruses, rotaviruses, sapoviruses, astroviruses and adenoviruses, however, no specific antiviral treatment exists today against any of these pathogens. We here discuss the feasibility of developing a broad-spectrum antiviral treatment against these diarrhea-causing viruses. This review focuses on the viral polymerase as an antiviral target, as this is the most conserved viral protein among the diverse viral families to which these viruses belong to. We describe the functional and structural similarities of the different viral polymerases, the antiviral effect of reported polymerase inhibitors and highlight common features that might be exploited in an attempt of designing such pan-polymerase inhibitor. Full article
(This article belongs to the Special Issue Noroviruses)
►▼ Show Figures

Figure 1

Figure 1
<p>(<b>a</b>) Three-dimensional structure and domains of HNoV RdRp (Norwalk virus, GI.1) in complex with RNA template strand, RNA growing strand and 5-nitrocytidine (PDB ID 3BSN, resolution 1.8 Å); (<b>b</b>) HSaV RdRp (Sapporo virus, Hu/SV/Man/1993/UK) in its apo form (PDB ID 2CKW, resolution 2.3 Å); (<b>c</b>) structural superimposition of the two RdRps.</p> Full article ">Figure 2
<p>(<b>a</b>) Domains of the HRV VP1 co-crystallised with an RNA fragment and GTP (PDB ID 2R7X, resolution 2.8 Å); (<b>b</b>) colour-coded motifs A-F in the 2R7X crystal structure, with the catalytic residues Asp631 and Asp632 highlighted (part of the conserved GDD sequence).</p> Full article ">Figure 3
<p>(<b>a</b>) Sequence alignment between HNoV RdRp from the 3BSO crystal structure and HAstV RdRp portion in the Q67726 sequence; (<b>b</b>) final model for HAstV RdRp.</p> Full article ">Figure 4
<p>(<b>a</b>) Sequence alignment between the bacteriophage f29 DNA polymerase and the polymerase domain of HAdV F serotype 40; (<b>b</b>) final model for the AdV Pol, including the nucleic acid and NTP components minimized from the template.</p> Full article ">Figure 5
<p>Similarities between RNA and DNA polymerases of diarrhea-causing viruses. (<b>a</b>) Incoming nucleotide binding pocket of the ternary complex of HNoV RdRp co-crystallised with 5-nitro cytidine triphosphate (PDB ID 3BSN); (<b>b</b>) incoming nucleotide binding pocket of the polymerase homology model obtained for HAdV.</p> Full article ">Figure 6
<p>Structure of reported nucleoside analogues (or pyrophosphate mimics) inhibiting the RdRps of caliciviruses or rotaviruses.</p> Full article ">Figure 7
<p>Nucleoside analogues inhibiting HAdV pol/HAdV replication.</p> Full article ">Figure 8
<p>Potential sugar and nucleobase modifications for broad-spectrum nucleoside inhibitors.</p> Full article ">Figure 9
<p>Predicted binding of one suggested potential broad-spectrum polymerase inhibitor to the HAdV Pol model (<b>a</b>) and to HNoV NTP binding pocket in the 3BSN crystal structure (<b>b</b>).</p> Full article ">Figure 10
<p>Structures of reported non-nucleoside inhibitors of the <span class="html-italic">Caliciviridae</span> RdRp.</p> Full article ">
20 pages, 1512 KiB  
Review
Complexities of Type I Interferon Biology: Lessons from LCMV
by Tamara Suprunenko and Markus J. Hofer
Viruses 2019, 11(2), 172; https://doi.org/10.3390/v11020172 - 20 Feb 2019
Cited by 21 | Viewed by 5921
Abstract
Over the past decades, infection of mice with lymphocytic choriomeningitis virus (LCMV) has provided an invaluable insight into our understanding of immune responses to viruses. In particular, this model has clarified the central roles that type I interferons play in initiating and regulating [...] Read more.
Over the past decades, infection of mice with lymphocytic choriomeningitis virus (LCMV) has provided an invaluable insight into our understanding of immune responses to viruses. In particular, this model has clarified the central roles that type I interferons play in initiating and regulating host responses. The use of different strains of LCMV and routes of infection has allowed us to understand how type I interferons are critical in controlling virus replication and fostering effective antiviral immunity, but also how they promote virus persistence and functional exhaustion of the immune response. Accordingly, these discoveries have formed the foundation for the development of novel treatments for acute and chronic viral infections and even extend into the management of malignant tumors. Here we review the fundamental insights into type I interferon biology gained using LCMV as a model and how the diversity of LCMV strains, dose, and route of administration have been used to dissect the molecular mechanisms underpinning acute versus persistent infection. We also identify gaps in the knowledge regarding LCMV regulation of antiviral immunity. Due to its unique properties, LCMV will continue to remain a vital part of the immunologists’ toolbox. Full article
(This article belongs to the Special Issue LCMV – A Pillar for Immunology Research)
►▼ Show Figures

Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Clustal Omega dendrogram predications generated from the alignments of protein sequences of common laboratory strains of LCMV. The LCMV genome consists of the S and L segments. The S segment encodes the glycoprotein (GP) and the nucleoprotein (NP) and the L segment encodes the L protein (RNA polymerase) and the Z protein. Protein sequences for commonly used laboratory strains LCMV-Armstrong 53b, Clone13, Traub, UBC, WE, Aggressive and Docile were aligned using Clustal Omega and dendrograms generated from the alignments. Sequences were obtained from Genbank<sup>®</sup> and accession numbers used for alignment are indicated. Note, there was no sequence available for the NP of LCMV-WE. The identity between the alignments is as follows; GP: 90.361%, NP: 94.086%, L protein: 81.315%, Z protein: 78.889%.</p> Full article ">Figure 2
<p>TLR-7/-8 signaling in dendritic cells. LCMV ssRNA is detected by the endosomal TLRs; TLR7 and TLR8, expressed by conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs). The association of MyD88 with the receptor results in the association of IRAK1 and IRAK4 through the death domains. Once phosphorylated the IRAK proteins dissociate from the receptor to associate with TRAF6. TRAF6 interacts directly and indirectly with a number of other proteins, including protein kinases such as TAK1. TAK1 can then triggers the activation of transcription factors such as AP-1 via the MAPK pathway or NF-κB through the canonical IKK complex. Upon their activation, the transcription factors translocate to the nucleus and induce production of inflammatory cytokines. In addition, pDCs utilize a unique pathway that allows for the rapid immediate-early production of type I interferons. Similar to above, MyD88 associates with the receptor and a complex including TRAF3, TRAF6, IRAK1, IRAK4 and IKKα is formed. IRAK1 and IKKα phosphorylate the transcription factor IRF7, resulting in its activation and subsequent production of type I interferon (IFN-I). Solid arrows indicate direct steps, dashed arrows indicate multiple steps/additional proteins involved.</p> Full article ">Figure 3
<p>Schematic representation of host detection of LCMV and subsequent type I interferon induction. The production of IFN-Is in response to LCMV infection can be categorized into early and secondary phases. Upon recognition of viral dsRNA by the pattern recognition receptor MDA-5, the adaptor protein MAVS is recruited, initiating a signaling cascade involving multiple factors. This cascade results in the phosphorylation and subsequent activation of the transcription factor IRF3 by the kinases IKKε and TBK1. Simultaneously, the transcription factors NF-κB and AP1 are also activated and translocated to the nucleus. Together these transcription factors initiate the transcription of the initial IFN-I species IFN-β and IFN-α<sub>4</sub>. These early IFN-Is subsequently bind to the IFN-I receptor IFNAR in an autocrine or paracrine fashion and activate canonical IFN-I signaling through the ISGF3 complex. This results in the transcription of interferon-stimulated genes, including the transcription factor IRF7. IRF7 is then activated in a similar fashion to IRF3, resulting in the amplification of the secondary IFN-I response. Solid arrows indicate direct steps, dashed arrows indicate multiple steps/additional proteins involved.</p> Full article ">
17 pages, 592 KiB  
Review
Influenza Virus Infections and Cellular Kinases
by Robert Meineke, Guus F. Rimmelzwaan and Husni Elbahesh
Viruses 2019, 11(2), 171; https://doi.org/10.3390/v11020171 - 20 Feb 2019
Cited by 78 | Viewed by 11042
Abstract
Influenza A viruses (IAVs) are a major cause of respiratory illness and are responsible for yearly epidemics associated with more than 500,000 annual deaths globally. Novel IAVs may cause pandemic outbreaks and zoonotic infections with, for example, highly pathogenic avian influenza virus (HPAIV) [...] Read more.
Influenza A viruses (IAVs) are a major cause of respiratory illness and are responsible for yearly epidemics associated with more than 500,000 annual deaths globally. Novel IAVs may cause pandemic outbreaks and zoonotic infections with, for example, highly pathogenic avian influenza virus (HPAIV) of the H5N1 and H7N9 subtypes, which pose a threat to public health. Treatment options are limited and emergence of strains resistant to antiviral drugs jeopardize this even further. Like all viruses, IAVs depend on host factors for every step of the virus replication cycle. Host kinases link multiple signaling pathways in respond to a myriad of stimuli, including viral infections. Their regulation of multiple response networks has justified actively targeting cellular kinases for anti-cancer therapies and immune modulators for decades. There is a growing volume of research highlighting the significant role of cellular kinases in regulating IAV infections. Their functional role is illustrated by the required phosphorylation of several IAV proteins necessary for replication and/or evasion/suppression of the innate immune response. Identified in the majority of host factor screens, functional studies further support the important role of kinases and their potential as host restriction factors. PKC, ERK, PI3K and FAK, to name a few, are kinases that regulate viral entry and replication. Additionally, kinases such as IKK, JNK and p38 MAPK are essential in mediating viral sensor signaling cascades that regulate expression of antiviral chemokines and cytokines. The feasibility of targeting kinases is steadily moving from bench to clinic and already-approved cancer drugs could potentially be repurposed for treatments of severe IAV infections. In this review, we will focus on the contribution of cellular kinases to IAV infections and their value as potential therapeutic targets. Full article
(This article belongs to the Special Issue Viruses and Cellular Metabolism)
►▼ Show Figures

Figure 1

Figure 1
<p>Host kinases and known roles during IAV infections. Schematic organizing host kinases based on kinase family, signaling pathway involved, specific kinase and effect of inhibition (from innermost to outermost ring; white cone).</p> Full article ">
16 pages, 3341 KiB  
Article
The Three Essential Motifs in P0 for Suppression of RNA Silencing Activity of Potato leafroll virus Are Required for Virus Systemic Infection
by Mamun-Or Rashid, Xiao-Yan Zhang, Ying Wang, Da-Wei Li, Jia-Lin Yu and Cheng-Gui Han
Viruses 2019, 11(2), 170; https://doi.org/10.3390/v11020170 - 20 Feb 2019
Cited by 12 | Viewed by 4911
Abstract
Higher plants exploit posttranscriptional gene silencing as a defense mechanism against virus infection by the RNA degradation system. Plant RNA viruses suppress posttranscriptional gene silencing using their encoded proteins. Three important motifs (F-box-like motif, G139/W140/G141-like motif, and C-terminal conserved region) in P0 of [...] Read more.
Higher plants exploit posttranscriptional gene silencing as a defense mechanism against virus infection by the RNA degradation system. Plant RNA viruses suppress posttranscriptional gene silencing using their encoded proteins. Three important motifs (F-box-like motif, G139/W140/G141-like motif, and C-terminal conserved region) in P0 of Potato leafroll virus (PLRV) were reported to be essential for suppression of RNA silencing activity. In this study, Agrobacterium-mediated transient experiments were carried out to screen the available amino acid substitutions in the F-box-like motif and G139/W140/G141-like motif that abolished the RNA silencing suppression activity of P0, without disturbing the P1 amino acid sequence. Subsequently, four P0 defective mutants derived from a full-length cDNA clone of PLRV (L76F and W87R substitutions in the F-box-like motif, G139RRR substitution in the G139/W140/G141-like motif, and F220R substitution in the C-terminal conserved region) were successfully generated by reverse PCR and used to investigate the impact of these substitutions on PLRV infectivity. The RT-PCR and western blot analysis revealed that these defective mutants affected virus accumulation in inoculated leaves and systemic movement in Nicotiana benthamiana as well as in its natural hosts, potato and black nightshade. These results further demonstrate that the RNA silencing suppressor of PLRV is required for PLRV accumulation and systemic infection. Full article
(This article belongs to the Special Issue Plant Immunity to Virus Infections)
►▼ Show Figures

Figure 1

Figure 1
<p>Suppression of local RNA silencing by the mutants of P0 protein of PLRV (P0<sup>PL</sup>) in F-box-like motif and G139/W140/G141-like motif. (<b>A</b>) Evaluation of suppression of local RNA silencing in wild-type <span class="html-italic">Nicotiana benthamiana</span> leaves co-infiltrated with <span class="html-italic">Agrobacterium</span> harboring pGDG and empty pGD vector, or P0<sup>PL</sup> (wild-type), or P0 mutants, at 3 dpi under long-wavelength UV light. The bottom-right side of leaves shows GFP expression for pGDG plus pGD, the bottom-left side of leaves shows GFP expression for pGDG plus P0<sup>PL</sup>, and both top-right and left side of the leaves show GFP expression for pGDG plus P0<sup>PL</sup> mutants. (<b>B</b>–<b>E</b>) Western blot analyses of proteins extracted from the infiltrated leaf patches at 3 dpi using the specific antibody for GFP (anti-GFP) and flag (anti-flag). Rubisco stained with Coomassie brilliant blue was used as a loading control and is shown in the lower panel.</p> Full article ">Figure 2
<p>Suppression of systemic RNA silencing by the P0<sup>PL</sup> mutants in F-box-like motif and G139/W140/G141-like motif. (<b>A</b>) Evaluation of suppression of systemic RNA silencing in the <span class="html-italic">N. benthamiana</span> 16c line co-infiltrated with <span class="html-italic">Agrobacterium</span> harboring pGDG plus empty pGD vector (negative control), or P0<sup>PL</sup> (wild-type), or P0 mutants, at 14 dpi under long-wavelength UV light. Designations are given at the bottom-right corner of the photographs showing GFP expression in systemic leaves and the bottom numbers of each treatment indicate silencing ratios. (<b>B</b>) Western blot analyses of the proteins extracted from systemic leaves of <span class="html-italic">N. benthamiana</span> 16c plants co-infiltrated with pGDG plus empty pGD vector, wild-type P0<sup>PL</sup>, L76F, W87R, and G139RRR substitutions respectively, at 14 dpi using GFP specific antibody (anti-GFP). Rubisco stained with Coomassie brilliant blue was used as a loading control and is shown in the lower panel.</p> Full article ">Figure 3
<p>Western blot analysis showing accumulation of GFP and flag-fused P0<sup>PL</sup> mutants in <span class="html-italic">N. benthamiana</span> leaves. Proteins were extracted at 3 dpi from the <span class="html-italic">N. benthamiana</span> leaves co-infiltrated with pGDG plus empty pGD vector (negative control), P0<sup>PL</sup> (wild-type), and L76F, W87R, and G139RRR mutants respectively, in the presence or absence of the <span class="html-italic">Tomato bushy stunt virus</span> (TBSV) P19 protein or P0 protein of <span class="html-italic">Potato leafroll virus</span>. Rubisco stained with Coomassie brilliant blue was used as a loading control as is shown in the lower panel. (<b>A</b>) Plus P19; (<b>B</b>) plus P0<sup>PL</sup>; (<b>C</b>) minus P19 or P0<sup>PL</sup>.</p> Full article ">Figure 4
<p>Infectivity analysis of the VSR defective mutants in F-box-like motif, G139/W140/G141-like motif, and the C-terminal conserved region derived from the full-length infectious cDNA clone of PLRV (pCB-PLRV) in <span class="html-italic">N. benthamiana</span>. (<b>A</b>) Symptoms on <span class="html-italic">N. benthamiana</span> leaves agroinfiltrated with pCB empty vector (negative control), wild-type pCB-PLRV (positive control) and mutants pCB-PL-L76F, pCB-PL-W87R, pCB-PL-G139RRR, and pCB-PL-F220R respectively, at 7 dpi. (<b>B</b>) Assessment of the viral RNA and protein accumulation in <span class="html-italic">N. benthamiana</span> leaves inoculated with pCB-PLRV mutants at 3 dpi by RT-PCR amplification and western blot analysis. (<b>C</b>) Assessment of viral RNA and protein accumulation in systemic leaves of <span class="html-italic">N. benthamiana</span> plants inoculated with pCB-PLRV mutants at 14 dpi by RT-PCR amplification and western blot analysis. The upper panel shows the RT-PCR amplification with PLRV-specific primer pair. The middle panel shows the western blot analysis with the PLRV-MP specific antibody (anti-MP<sup>PLRV</sup>). Rubisco stained with Coomassie brilliant blue used as a loading control is shown in the lower panel.</p> Full article ">Figure 5
<p>Infectivity analysis of VSR defective mutants in F-box-like motif, G139/W140/G141-like motif, and C-terminal conserved region derived from the pCB-PLRV in potato plants. (<b>A</b>) Symptoms on potato (cultivar Lalpakri) leaves agroinfiltrated with pCB empty vector (negative control), wild-type pCB-PLRV (positive control), and mutants pCB-PL-L76F, pCB-PL-W87R, pCB-PL-G139RRR, and pCB-PL-F220R, respectively, at 5 dpi. (<b>B</b>) Assessment of the viral RNA and protein accumulation in potato leaves inoculated with pCB-PLRV mutants by RT-PCR amplification and western blot analysis at 3 dpi. (<b>C</b>) Assessment of viral RNA and protein accumulation in systemic leaves of potato plants inoculated with pCB-PLRV mutants by RT-PCR amplification and western blot analysis at 14 dpi. Extracts from the inoculated <span class="html-italic">N. benthamiana</span> leaves were used as a positive control. The upper panel shows the RT-PCR amplification with PLRV-specific primer pair. The middle panel shows the western blot analysis with the PLRV-MP specific antibody (anti-MP<sup>PLRV</sup>). Rubisco stained with Coomassie brilliant blue used as a loading control is shown in the lower panel.</p> Full article ">Figure 6
<p>Infectivity analysis of the VSR defective mutants in F-box-like motif, G139/W140/G141-like motif, and C-terminal conserved region derived from the pCB-PLRV in black nightshade plants. (<b>A</b>) Symptoms on black nightshade leaves agroinfiltrated with pCB empty vector (negative control), wild-type pCB-PLRV (positive control) and mutants pCB-PL-L76F, pCB-PL-W87R, pCB-PL-G139RRR and pCB-PL-F220R respectively, at 5 dpi. (<b>B</b>) Assessment of viral RNA and protein accumulation in potato leaves inoculated with pCB-PLRV mutants by RT-PCR amplification and western blot analysis at 3 dpi. (<b>C</b>) Assessment of viral RNA and protein accumulation in systemic leaves of potato plants inoculated with pCB-PLRV mutants by RT-PCR amplification and western blot analysis at 14 dpi. Extracts from inoculated <span class="html-italic">N. benthamiana</span> leaves were used as a positive control. The upper panel shows the RT-PCR amplification with PLRV-specific primer pair. The middle panel shows the western blot analysis with the PLRV-MP specific antibody (anti-MP<sup>PLRV</sup>). Rubisco stained with Coomassie brilliant blue used as a loading control is shown in the lower panel.</p> Full article ">
11 pages, 946 KiB  
Article
A Single-Nucleotide Polymorphism of αVβ3 Integrin Is Associated with the Andes Virus Infection Susceptibility
by Constanza Martínez-Valdebenito, Jenniffer Angulo, Nicole Le Corre, Claudia Marco, Cecilia Vial, Juan Francisco Miquel, Jaime Cerda, Gregory Mertz, Pablo Vial, Marcelo Lopez-Lastra and Marcela Ferrés
Viruses 2019, 11(2), 169; https://doi.org/10.3390/v11020169 - 20 Feb 2019
Cited by 6 | Viewed by 3758
Abstract
The Andes Orthohantavirus (ANDV), which causes the hantavirus cardiopulmonary syndrome, enters cells via integrins, and a change from leucine to proline at residue 33 in the PSI domain (L33P), impairs ANDV recognition. We assessed the association between this human polymorphism and ANDV infection. [...] Read more.
The Andes Orthohantavirus (ANDV), which causes the hantavirus cardiopulmonary syndrome, enters cells via integrins, and a change from leucine to proline at residue 33 in the PSI domain (L33P), impairs ANDV recognition. We assessed the association between this human polymorphism and ANDV infection. We defined susceptible and protective genotypes as “TT” (coding leucine) and “CC” (coding proline), respectively. TT was present at a rate of 89.2% (66/74) among the first cohort of ANDV cases and at 60% (63/105) among exposed close-household contacts, who remained uninfected (p < 0.05). The protective genotype (CC) was absent in all 85 ANDV cases, in both cohorts, and was present at 11.4% of the exposed close-household contacts who remained uninfected. Logistic regression modeling for risk of infection had an OR of 6.2–12.6 (p < 0.05) in the presence of TT and well-known ANDV risk activities. Moreover, an OR of 7.3 was obtained when the TT condition was analyzed for two groups exposed to the same environmental risk. Host genetic background was found to have an important role in ANDV infection susceptibility, in the studied population. Full article
►▼ Show Figures

Figure 1

Figure 1
<p>Single Nucleotide Polymorphism (SNP) rs5918 genotype distribution within the Chilean population. The TT genotype is the homozygous allele that codes for leucine at the 33rd position of the plexin–semaphorin–integrin (PSI) integrin domain. The CC genotype is the homozygous allele that codes for a proline at the same position, dramatically reducing <span class="html-italic">Andes Orthohantavirus</span> (ANDV) recognition in ex vivo models (14). The SNPs were in the Hardy–Weinberg equilibrium (<span class="html-italic">p</span> &gt; 0.05).</p> Full article ">Figure 2
<p>SNP rs5918 genotype distribution among cases and close-household contacts. The cases and household contacts were grouped according to the SNP rs5918 genotype. The total number of each population was defined as 100%, and the percentage of individuals according to each genotype was indicated (<span class="html-italic">p</span> &gt; 0.05).</p> Full article ">Figure 3
<p>Genotype distribution among subjects with severe or mild diseases. Severe patients comprised four each with the TT or four CT genotype; TT and CT genotypes were present in 33 subjects with a mild disease.</p> Full article ">
25 pages, 828 KiB  
Review
Myeloid Cells during Viral Infections and Inflammation
by Ashley A. Stegelmeier, Jacob P. van Vloten, Robert C. Mould, Elaine M. Klafuric, Jessica A. Minott, Sarah K. Wootton, Byram W. Bridle and Khalil Karimi
Viruses 2019, 11(2), 168; https://doi.org/10.3390/v11020168 - 19 Feb 2019
Cited by 74 | Viewed by 9382
Abstract
Myeloid cells represent a diverse range of innate leukocytes that are crucial for mounting successful immune responses against viruses. These cells are responsible for detecting pathogen-associated molecular patterns, thereby initiating a signaling cascade that results in the production of cytokines such as interferons [...] Read more.
Myeloid cells represent a diverse range of innate leukocytes that are crucial for mounting successful immune responses against viruses. These cells are responsible for detecting pathogen-associated molecular patterns, thereby initiating a signaling cascade that results in the production of cytokines such as interferons to mitigate infections. The aim of this review is to outline recent advances in our knowledge of the roles that neutrophils and inflammatory monocytes play in initiating and coordinating host responses against viral infections. A focus is placed on myeloid cell development, trafficking and antiviral mechanisms. Although known for promoting inflammation, there is a growing body of literature which demonstrates that myeloid cells can also play critical regulatory or immunosuppressive roles, especially following the elimination of viruses. Additionally, the ability of myeloid cells to control other innate and adaptive leukocytes during viral infections situates these cells as key, yet under-appreciated mediators of pathogenic inflammation that can sometimes trigger cytokine storms. The information presented here should assist researchers in integrating myeloid cell biology into the design of novel and more effective virus-targeted therapies. Full article
(This article belongs to the Special Issue Viruses and Inflammation)
►▼ Show Figures

Figure 1

Figure 1
<p>Schematic of myeloid cells highlighting their ability to respond to pulmonary viral infections via the initiation and modulation of anti-viral inflammatory activity. Lung-resident myeloid cells, such as alveolar macrophages, utilize a complex sensory system to integrate disturbances of pulmonary tissues by viruses such as respiratory syncytial virus (RSV) into the activation of local effector leukocytes. (<b>A</b>) RSV enters and infects the lungs. Viral pathogen-associated molecular patterns (PAMPs), such as double-stranded RNA or danger associated molecular patterns (DAMPs), are detected by pattern recognition receptors (PRRs) in or on sentinel cells in the lungs, such as TLR3 in the endosomes of lung-resident macrophages. TLR stimulation activates the NF-κß signaling cascade, resulting in the release of chemokines and inflammatory cytokines. A chemokine gradient forms between the lungs and bone marrow. (<b>B</b>) Homeostatic bone marrow tends to retain CXCR4<sup>+</sup> neutrophils and monocytes through endogenous expression of high levels of CXCL12. However, the release of PAMPs, as well as the secretion of cytokines and chemokines as a consequence of pulmonary RSV infections, is sensed by cells in the bone marrow, which in turn allow recruitment of new neutrophils and monocytes from the bone marrow into the lungs. Specifically, G-CSF downregulates CXCR4 on neutrophils, triggering their release. Similarly, CCL2 is produced in the bone marrow by endothelial cells following TLR signaling in infected lungs, which is crucial for inflammatory monocyte release into the bloodstream. Once in the bloodstream, these cells sense disrupted endothelium from the viral infection, which triggers a complex adhesion cascade. Activated Ly6C<sup>hi</sup> inflammatory monocytes are recruited to the site of infection by a variety of chemokine receptors including CCR1, 5 and 6, as well as CXCR2 binding to their respective ligands. (<b>C</b>) Once at the site of infection, they differentiate into dendritic cells and macrophages that initiate an inflammatory cascade that includes copious amounts of inflammatory cytokines, in particular IL-12 and IFN-γ, which are potent inducers of Th1-biased immune responses. Once these dendritic cells and macrophages acquire viral antigens, they home to lymph nodes via chemokine receptors, including CCR7. Monocyte-derived dendritic cells that home to lymph nodes present viral antigens to naïve CD4<sup>+</sup> and CD8<sup>+</sup> T-cells that are required to kill infected cells. (<b>D</b>) The basic neutrophil function of clearing an inflamed area by removing killed pathogens and host cells contributes to reduced inflammation and wound debridement. Neutrophils are also capable of promoting tissue repair and increased angiogenesis. Further, monocytes can suppress lymphocytes in various clinical scenarios. In lungs, myeloid cells are able to inhibit pro-inflammatory tissue-resident leukocytes through direct cell-to-cell contact through galectin9/TIM3 and the effect of TGF-β on NKp30 in order to regulate T-cells and NK cells, respectively. Myeloid cells can also exert suppressive functions through secretion of soluble factors such as IL-10, arginase-1 and indoleamine 2,3-dioxygenase. (<b>E</b>) We speculate that disruption of the cellular sensing of type I IFN responses can result in excessive production of pro-inflammatory cytokines, including IFN-γ, IL-1, IL-6, and TNF-α, leading to a toxic cytokine storm. The fatal outcome of severe lung infections is shown to be correlated with the early persistent production of inflammatory cytokines and chemokines that recruit neutrophils and monocytes. While inflammatory cytokines and chemokines are essential for effective control of viral infections, they can also contribute to the severity of disease and tissue damage.</p> Full article ">
Show export options expand_more Show export options expand_less
Select all
Export citation of selected articles as:
 
Displaying articles 1-107
Previous Issue
Next Issue
Back to TopTop