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Volume 14
Issue 11
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Viruses, Volume 14, Issue 11 (November 2022) – 275 articles

Cover Story (view full-size image): Bacteriophages are viruses of bacteria, for which molecular mechanisms of host-hijacking remain poorly explored. In this work, we apply omics technologies—transcriptomics and proteomics—to temporally resolve transcription and protein synthesis during the T4 phage infection of E. coli on a molecular level. For E. coli, we observed degradation of its transcripts and the preservation of the proteome. For T4 phage, the correlation between the transcriptome and proteome reveals specific T4 phage mRNAs and proteins that are temporally decoupled, suggesting post-transcriptional and translational regulation mechanisms. Our tool, POTATO4, provides access to these first comprehensive insights into the gene expression patterns of T4 phage infection of E. coli. View this paper
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26 pages, 4539 KiB  
Review
Heterologous RNA Recombination in the Cystoviruses φ6 and φ8: A Mechanism of Viral Variation and Genome Repair
by Paul Gottlieb and Aleksandra Alimova
Viruses 2022, 14(11), 2589; https://doi.org/10.3390/v14112589 - 21 Nov 2022
Cited by 3 | Viewed by 2134
Abstract
Recombination and mutation of viral genomes represent major mechanisms for viral evolution and, in many cases, moderate pathogenicity. Segmented genome viruses frequently undergo reassortment of the genome via multiple infection of host organisms, with influenza and reoviruses being well-known examples. Specifically, major genomic [...] Read more.
Recombination and mutation of viral genomes represent major mechanisms for viral evolution and, in many cases, moderate pathogenicity. Segmented genome viruses frequently undergo reassortment of the genome via multiple infection of host organisms, with influenza and reoviruses being well-known examples. Specifically, major genomic shifts mediated by reassortment are responsible for radical changes in the influenza antigenic determinants that can result in pandemics requiring rapid preventative responses by vaccine modifications. In contrast, smaller mutational changes brought about by the error-prone viral RNA polymerases that, for the most part, lack a replication base mispairing editing function produce small mutational changes in the RNA genome during replication. Referring again to the influenza example, the accumulated mutations—known as drift—require yearly vaccine updating and rapid worldwide distribution of each new formulation. Coronaviruses with a large positive-sense RNA genome have long been known to undergo intramolecular recombination likely mediated by copy choice of the RNA template by the viral RNA polymerase in addition to the polymerase-based mutations. The current SARS-CoV-2 origin debate underscores the importance of understanding the plasticity of viral genomes, particularly the mechanisms responsible for intramolecular recombination. This review describes the use of the cystovirus bacteriophage as an experimental model for recombination studies in a controlled manner, resulting in the development of a model for intramolecular RNA genome alterations. The review relates the sequence of experimental studies from the laboratory of Leonard Mindich, PhD at the Public Health Research Institute—then in New York City—and covers a period of approximately 12 years. Hence, this is a historical scientific review of research that has the greatest relevance to current studies of emerging RNA virus pathogens. Full article
(This article belongs to the Special Issue Discovery, Classification, and Early Research on the Lipid-Containing, Double-Stranded RNA Bacteriophage φ6)
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<p>Schematic organization (<b>A</b>), replication cycle (<b>B</b>), and genome organization (<b>C</b>) of the φ6 virion particle. (<b>A</b>) The inner layer, procapsid (PC), includes the shell composed of 120 copies of the P1 protein or 60 nonsymmetric dimers of P1A/P1B. Inside the shell are proteins P2 and P7 localized at the fivefold axis of symmetry portal and three double-stranded RNA segments (dsRNA) which have low-symmetry quasi-concentric shell organization. The P4 proteins form a hexameric ring around the fivefold axis. The nucleocapsid (NC) includes the PC and a matrix composed of protein P8. The completed virion has an envelope derived from the cellular bilayer lipid membrane and incorporates proteins P3, P6, P9, and P5 randomly distributed. (<b>B</b>) The replication cycle initiates when P3 protein attaches to the host pili followed by pili retraction (1–2). The viral envelope fuses to the host outer membrane (3). The NC enters the periplasmic space and the cytoplasm (4). The P8 matrix disassembles, and transcription begins (5). The PC self-assembles from P1, P2, P4, and P7 proteins (6) and packages the three genome transcripts accompanied by PC expansion (7). ssRNA is replicated to dsRNA (8), and the P8 matrix loosely assembles around the filled PC (9). The cell-derived bilipid membrane is placed around the NC particle mediated by the nonstructural protein P12 (10). This step is followed by cell lysis and virion release. (<b>C</b>) The three ds-RNA genome segments and genes encoding the viral proteins (shown in light-blue color). The packaging <span class="html-italic">pac</span> signal for each strand located at the 5′ end of ds-RNA is visualized as colored rectangles. The replication signal is located at the 3′ end.</p> Full article ">Figure 2
<p>Recombination events facilitated by introduction of kan cassette structurally organized as a hairpin loop. The φ6K1 recombinant virus contains the M-segment with a kan cassette. Isolation of clear plaques from unstable transducing virus confirmed the recombination between M and S or L segments (<b>A</b>,<b>C</b>). This stable isolated virus φ1697 (<b>A</b>,<b>B</b>) did not have the kan cassette in the M-segment. The 3′ end of M-segment of φ6K1 (3754–5303 bp) was recombined heterologically with the 3′ end of the S segment (2858–2948 bp). Only 6 bp of similarity existed between two segments. The φ 1703 (<b>C</b>,<b>D</b>) virus did not have a kan cassette, as did φ1697, and lacked the P13 gene. The φ1703 is a recombination product between the 3′ end of the M-segment of φ6K1 and L-segment of wildtype φ6. There were only two identical base pairs observed. Recombination was also observed in an S segment of CS that was derived from φ6K1 (<b>E</b>,<b>F</b>). The S segment demonstrated an internal deletion of 189 nucleotides within the 3′ noncoding region. The segment was incorporated into φ1712. The clear plaque variant φ1713 produced by the carrier culture was a recombinant between the segment M from native φ6 and the 3′ end of segment S from φ1712 that contained the internal deletion described above. Only six identical base pairs were observed. Based on Mindich et al. (1992).</p> Full article ">Figure 3
<p>Effect of the hairpin secondary structure in noncoding region of M segment on the stability and homogeneity of the virus. Photograph of plaques from bacteriophage isolates where lacH is inserted in the noncoding region of the M segment and bordered with poly-G and poly-C homopolymer arms (<b>B</b>) or lacking one of the arms (<b>A</b>). Plaques lacking the one of the homopolymer arms are stable and form uniform blue plaques on LB X-Gal substrate (<b>A</b>). The lacH gene bordered by homopolymer arms is unstable, producing the white and blue mixed plaque population (<b>B</b>). Reproduced from Onodera et al. (1993).</p> Full article ">Figure 4
<p>The recombination facilitated by homopolymer base hairpin conformations resulted in an alteration in size of the M dsRNA segment. The agarose electrophoresis gel of dsRNA extracted from recombinant plaques shows a whole variety of sizes of recombinant M segment. Line a shows the dsRNA extracted from φ6. Lines b–h are from recombinant plaques. Upper case denotes dsRNA segments. Reproduced from Onodera et al. (1993).</p> Full article ">Figure 5
<p>Predicted ssRNA secondary structure of terminal 75 bases at 3′ end for three genomic segments. The region of similarity covers the last 75 nucleotides at the 3′ end and starts with AAGU for all three strands. The structurally identical regions are boxed in red outlines. Note that even nonidentical nucleotides kept a similar secondary structure. The calculation was performed using the <span class="html-italic">forna</span> online RNA secondary structure visualization tool (<a href="http://rna.tbi.univie.ac.at/forna/" target="_blank">http://rna.tbi.univie.ac.at/forna/</a>, accessed on 17 August 2022). The sequence identity does not significantly affect recombination.</p> Full article ">Figure 6
<p>Copy choice model for repair of the M segment. (<b>A</b>) The new 3′ end is derived from the l segment when it is used as a template, and the continuation of synthesis shifts to the m plus strand. L synthesis then commences once again and continues to completion. (<b>B</b>) Pathway B is more complex in that transcription of the new plus strands displaces the chimeric minus strand from its original m template. The original template would then reinitiate minus-strand synthesis, which proceeds normally. Reproduced from Onodera et al. (1993).</p> Full article ">Figure 7
<p>Alteration of the secondary structure of the 3′ end of the M segment. Introduced deletions of the 3′ end caused changes in the predicted secondary structure. The deleted nucleotides are encircled in blue in (<b>A</b>). (<b>B</b>) The blue outlined arm shows the difference in structure between the wildtype M segment and mutated ones. Changes in the blue outlined arms caused many recombinants in the M segments, but the replication rate of the M segment was nearly the same as of wildtype M. (<b>C</b>) The structure of the red outlined hairpin on these three mutants was different from the second arm in wildtype M. These mutations caused a significant reduction in M replication rate (&lt;5), and all the produced phages were recombinant. Based on from Mindich et al. (1994). The secondary structure was calculated using the <span class="html-italic">forna</span> online RNA secondary structure visualization tool (<a href="http://rna.tbi.univie.ac.at/forna/" target="_blank">http://rna.tbi.univie.ac.at/forna/</a>, accessed on 17 August 2022).</p> Full article ">Figure 8
<p>Model showing that the packaging process of ssRNA segment with strong hairpin near the 3′ end can block the process. The exposed hairpin structure becomes vulnerable to RNase I digestion. Reproduced from Qiao et al. (1995).</p> Full article ">Figure 9
<p>Recombinant phages can acquire the missing genes from the donor plasmid expressed in Pseudomonas host cells. (<b>A</b>) Transcripts of partial segment of L (pLM1009), isolated l segment from mutant carrying amber mutation from φ6 sus351, and complete ssRNA from m and s segments of φ6 are in vitro packaged in PC and coated with P8, transducing the spheroplasts of HB10Y with plasmid pLM1003, coding the missing 2, 4, and 7 genes and MCS of pUC8 (<b>B</b>). The phage φ1980 carries at least two L segments and M and S segments (<b>C</b>,<b>D</b>). On the dsRNA agarose gel picture reproduced from Onodera et al. (1995) (<b>C</b>), the pattern of φ1980 dsRNA shows a higher intensity of the L band compared to any of the dsRNA bands from the control φ6 dsRNA pattern. If the φ1980 was plated on HB10Y host without donor plasmid pLM1003, the extremely rare lytic plaques were all recombinants. (<b>E</b>) The phages packed an extra segment bearing missing 2, 4, and 7 genes and the replication signal from either the M or the S segment. The recombinants had minimal homology (the homology regions are shown as bold black symbols, whereas the recombined RNA origins are shown as light green for the L segment, light pink for S, and blue for pUC8 MCS). Based on Onodera et al. (1995).</p> Full article ">Figure 10
<p>The cDNA copy of φ8 M and the tester phage 21 with lacα in place of genes F and G. The complemented plasmid transcript was of variable size compared to the 3b gene. Reproduced from Onodera et al. (2001).</p> Full article ">Figure 11
<p>Proposed model for the process of template switching recombination in φ8. Normally, the 3′ end of the template plus strand (green) enters the polymerase and is arrested near the site of nascent minus-strand chain formation (<b>A</b>); as the minus strand (red) is synthesized the dsRNA leaves the polymerase through the exit pore (<b>B</b>). The 3′ end of the nascent chain can be displaced from the template (<b>C</b>). It can reanneal to the template, or it can anneal to an empty template (blue) (<b>E</b>). A plus-strand RNA that does not have a proper 3′ end can enter the polymerase, but the template is not arrested at the catalytic site (<b>D</b>). Instead, it passes through the polymerase and can scan backward and forward. When a nascent minus strand anneals to the empty template and the template moves back into the polymerase, the nascent chain can act as a primer to start minus-strand synthesis on the empty template (<b>F</b>). The result is a recombinant minus strand. Reproduced from Onodera et al. (2001).</p> Full article ">Figure 12
<p>Furin site insertion “PRRAR” into SARS-CoV-2 from Bat_RaTG13. Based on Gallaher (2020).</p> Full article ">
10 pages, 1181 KiB  
Article
SARS-CoV-2 Variants Identification; A Fast and Affordable Strategy Based on Partial S-Gene Targeted PCR Sequencing
by Antonio Martínez-Murcia, Adrian Garcia-Sirera, Aaron Navarro and Laura Pérez
Viruses 2022, 14(11), 2588; https://doi.org/10.3390/v14112588 - 21 Nov 2022
Cited by 2 | Viewed by 1718 | Correction
Abstract
A considerable number of new SARS-CoV-2 lineages have emerged since the first COVID-19 cases were reported in Wuhan. As a few variants showed higher COVID-19 disease transmissibility and the ability to escape from immune responses, surveillance became relevant at that time. Single-nucleotide mutation [...] Read more.
A considerable number of new SARS-CoV-2 lineages have emerged since the first COVID-19 cases were reported in Wuhan. As a few variants showed higher COVID-19 disease transmissibility and the ability to escape from immune responses, surveillance became relevant at that time. Single-nucleotide mutation PCR-based protocols were not always specific, and consequently, determination of a high number of informative sites was needed for accurate lineage identification. A detailed in silico analysis of SARS-CoV-2 sequences retrieved from GISAID database revealed the S gene 921 bp-fragment, positions 22784–23705 of SARS-CoV-2 reference genome, as the most informative fragment (30 variable sites) to determine relevant SARS-CoV-2 variants. Consequently, a method consisting of the PCR-amplification of this fragment, followed by Sanger’s sequencing and a “single-click” informatic program based on a reference database, was developed and validated. PCR-fragments obtained from clinical SARS-CoV-2 samples were compared with homologous variant-sequences and the resulting phylogenetic tree allowed the identification of Alpha, Delta, Omicron, Beta, Gamma, and other variants. The data analysis procedure was automatized and simplified to the point that it did not require specific technical skills. The method is faster and cheaper than current whole-genome sequencing methods; it is available worldwide, and it may help to enhance efficient surveillance in the fight against the COVID-19 pandemic. Full article
(This article belongs to the Special Issue SARS-CoV-2 Genomics)
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<p>Phylogenetic tree displaying the relationships between the SARS-CoV-2 lineages based on the selected S-gene 921 bp fragment database. It also shows the phylogenetic affiliation of the four clinical samples (green) assayed.</p> Full article ">Figure 2
<p>Agarose gel of the preparative RT-epPCR products from SARS-CoV-2-positive RNA clinical samples. The Ct obtained by qPCR (GPS™ SARS-CoV-2 dtec-RT-qPCR) assay has been indicated below each lane.</p> Full article ">
14 pages, 3560 KiB  
Article
RSAD2 Is an Effective Target for High-Yield Vaccine Production in MDCK Cells
by Zilin Qiao, Yuejiao Liao, Mengyuan Pei, Zhenyu Qiu, Zhenbin Liu, Dongwu Jin, Jiayou Zhang, Zhongren Ma and Xiaoming Yang
Viruses 2022, 14(11), 2587; https://doi.org/10.3390/v14112587 - 21 Nov 2022
Cited by 4 | Viewed by 2570
Abstract
Increasingly, attention has focused on improving vaccine production in cells using gene editing technology to specifically modify key virus regulation-related genes to promote virus replication. In this study, we used DIA proteomics analysis technology to compare protein expression differences between two groups of [...] Read more.
Increasingly, attention has focused on improving vaccine production in cells using gene editing technology to specifically modify key virus regulation-related genes to promote virus replication. In this study, we used DIA proteomics analysis technology to compare protein expression differences between two groups of MDCK cells: uninfected and influenza A virus (IAV) H1N1-infected cells 16 h post infection (MOI = 0.01). Initially, 266 differentially expressed proteins were detected after infection, 157 of which were upregulated and 109 were downregulated. We screened these proteins to 23 genes related to antiviral innate immunity regulation based on functional annotation database analysis and verified the mRNA expression of these genes using qPCR. Combining our results with published literature, we focused on the proteins RSAD2, KCNN4, IDO1, and ISG20; we verified their expression using western blot, which was consistent with our proteomics results. Finally, we knocked down RSAD2 using lentiviral shRNA expression vectors and found that RSAD2 inhibition significantly increased IAV NP gene expression, effectively promoting influenza virus replication with no significant effect on cell proliferation. These results indicate that RSAD2 is potentially an effective target for establishing high-yield vaccine MDCK cell lines and will help to fully understand the interaction mechanism between host cells and influenza viruses. Full article
(This article belongs to the Section Viral Immunology, Vaccines, and Antivirals)
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<p>Proteomic - analysis of MDCK cells infected with Influenza A virus (IAV) H1N1 relative to uninfected MDCK cells. (<b>A</b>) The volcano plot represents the significance and magnitude of protein level changes in uninfected MDCK cells (MDCK mock) and IAV H1N1-infected MDCK cells 16 h post infection (hpi) (MDCK H1N1 16 hpi). Green dots represent downregulated differentially expressed proteins, red dots represent upregulated differentially expressed proteins, and “None” represents proteins with no significant expression differences between the two groups of cells. (<b>B</b>) The heat map represents significant protein differences between MDCK mock and MDCK H1N1 16 hpi cells. The color scale indicates the relative protein abundance, with darker shades representing the greatest difference in protein abundance between the two groups; red indicates up-regulation and blue indicates downregulation. The student’s <span class="html-italic">t</span>-test was used to identify statistical significance.</p> Full article ">Figure 2
<p>Screening of proteins related to the immune response of MDCK cells after IAV H1N1 infection. (<b>A</b>) Preliminary GO results for all 266 differentially expressed proteins, which are classified according to biological process, cellular component, and molecular function. (<b>B</b>) The GO results of 42 differentially expressed proteins after screening for proteins related to cellular innate immunity and viral response, which are classified according to biological process, cellular component, and molecular function. (<b>C</b>) KEGG analysis of 42 differentially expressed proteins after screening for proteins related to cellular innate immunity and viral response, and the different signaling pathways involved in the regulation of these proteins, were classified.</p> Full article ">Figure 3
<p>Validation of mRNA expression. A: Proteins were found to be differentially expressed between uninfected MDCK mock and infected MDCK H1N1 16 hpi cells based on proteomics screening. Of these proteins, 23 are involved in regulation of the host antiviral innate immune response. qPCR was used to validate these proteomics results at the mRNA level, using the 2<sup>−ΔΔCt</sup> method and GAPDH as the internal reference gene. * Was used to determine statistically significant differences between groups; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001.</p> Full article ">Figure 4
<p>Verification of protein level expression of differential proteins. (<b>A</b>) The protein expression of RSAD2, IDO1, ISG20, and KCNN4 in infected MDCK H1N1 16 hpi cells was analyzed using western blot with GAPDH as the loading control. (<b>B</b>) Image software used for the gray value analysis of immunoblotting. * Was used to determine statistically significant differences between groups; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 5
<p>Knockdown of RSAD2 promotes influenza virus proliferation. (<b>A</b>) RSAD2 gene expression was detected in MDCK cells infected with IAV H1N1 at 0, 12, 24, 36, 48, and 60 h post infection (hpi) using qPCR. (<b>B</b>) RSAD2 protein expression was detected in MDCK cells infected with IAV H1N1 at 0, 24, 48, and 60 hpi using western blot. (<b>C</b>) Representative images of sh-RSAD2 with a GFP tag using a 40× fluorescence microscope are shown. (<b>D</b>) Protein expression of RSAD2 in sh-RSAD2 MDCK cells was measured using western blot to verify successful knockdown. (<b>E</b>) Gene expression of RSAD2 in sh-RSAD2 (#1, #2, #3) MDCK cells was measured using qPCR to verify successful knockdown. (<b>F</b>) The proliferation of sh-control and sh-RSAD2 cells was detected using the digestion technique. (<b>G</b>) Influenza A/Puerto Rico/8/34 (A/PR/8/34) H1N1 virus, A/Texas/50/2012 (H3N2) NYMCX-223A, B/Colorado/06/2017-like virus B (Victoria lineage), and B/Phuket/3073/2013-like virus B (Yamagata/16/88 lineage) NP gene expression were measured in sh-RSAD2 MDCK cells using qPCR. (<b>H,I</b>) Influenza A/Puerto Rico/8/34 (A/PR/8/34) H1N1 virus, A/Texas/50/2012 (H3N2) NYMCX-223A, B/Colorado/06/2017-like virus B (Victoria lineage), and B/Phuket/3073/2013-like virus B (Yamagata/16/88 lineage) NP protein expression were measured in sh-RSAD2 MDCK cells using western blot. (<b>J</b>,<b>K</b>) Growth kinetics of the influenza A/Puerto Rico/8/34 (A/PR/8/34) H1N1 virus, A/Texas/50/2012 (H3N2) NYMCX-223A, B/Colorado/06/2017-like virus B (Victoria lineage), and B/Phuket/3073/2013-like virus B (Yamagata/16/88 lineage) vaccine strain by TCID<sub>50</sub> method. (<b>L</b>) Antiviral factor gene expression was measured in sh-RSAD2 MDCK cells using qPCR. GAPDH was used as the reference gene for qPCR and GAPDH was used as the loading control for western blot. * Was used to determine statistically significant differences between groups; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">
9 pages, 2231 KiB  
Case Report
Prenatal Diagnosis of Congenital Lymphocytic Choriomeningitis Virus Infection: A Case Report
by Fanny Tevaearai, Laureline Moser and Léo Pomar
Viruses 2022, 14(11), 2586; https://doi.org/10.3390/v14112586 - 21 Nov 2022
Cited by 4 | Viewed by 2088
Abstract
Lymphocytic choriomeningitis virus (LCMV) is an emerging neuroteratogen which can infect humans via contact with urine, feces, saliva, or blood of infected rodents. When the infection occurs during pregnancy, there is a risk of transplacental infection with subsequent neurological or visual impairment in [...] Read more.
Lymphocytic choriomeningitis virus (LCMV) is an emerging neuroteratogen which can infect humans via contact with urine, feces, saliva, or blood of infected rodents. When the infection occurs during pregnancy, there is a risk of transplacental infection with subsequent neurological or visual impairment in the fetus. In this article, we describe a case report of congenital LCMV infection, including fetal imaging, confirmed by positive LCMV IgM in fetal blood and cerebrospinal fluid. Full article
(This article belongs to the Special Issue Emerging Virus Infections in Adverse Pregnancy Outcomes II)
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<p>Fetal neurosonography at 22 wg. (<b>A</b>) Trans-ventricular axial plane with severe bilateral ventriculomegaly, lateral ventricles (LV) at 23 mm and 17 mm, and disruption of the cavum septi pellucidi (CSP). (<b>B</b>) Mid-sagittal plane with laminated corpus callosum (CC), dilated third ventricle (3 V), thick tectum (T), hypoplasia of the vermis (V), and brainstem (BS). (<b>C</b>) Trans-cerebellar axial plane with dysplasia of a cerebellar hemisphere (blue arrow). (<b>D</b>) Para-sagittal plane with enlarged LV, periventricular hyperechogenicity, thin cortical mantle (CM), irregular cortical ribbon (CR), enlarged peri cerebral spaces, dysmorphic nuclei caudate with calcifications (blue arrows).</p> Full article ">Figure 2
<p>Fetal ultrasounds at 25 and 28 wg. (<b>A</b>) Trans-orbital axial plane with unilateral right microphthalmia (25 wg), (<b>B</b>) axial plane of the optic chiasm showing a global hypoplasia (1 = diameter of the optic chiasm at 5 mm) affecting the right tracts (2 = diameter of the right optic nerve at 2 mm; 5 = diameter of the right posterior tract at 1.8 mm, 28 wg), (<b>C</b>) cardiac four-chamber plane, with cardiomegaly and pericardial effusion (25 wg), (<b>D</b>) parasagittal plane of the fetal liver showing a hepatomegaly (diameter of the right liver lobe at 4.3 cm, 25 wg).</p> Full article ">Figure 3
<p>Fetal MRI at 29 wg (T2 weighted sequences). (<b>A</b>) Trans-ventricular axial plane with severe bilateral ventriculomegaly, lateral ventricles (LV) at 27 mm and 21 mm, disruption of the cavum septi pellucidi (CSP), subependymal nodules (dot arrow), and occipital polymicrogyria (arrow). (<b>B</b>) Mid-sagittal plane with laminated corpus callosum (CC), dilated third ventricle (3 V), thick tectum (T), hypoplasia of the vermis (V), and brainstem (BS). (<b>C</b>) Trans-cerebellar axial plane with dysplasia of a cerebellar hemisphere (blue arrow). (<b>D</b>) Para-sagittal plane with enlarged LV, subependymal nodules (dot arrow), atrophy of the cortical mantle (CM), irregular cortical ribbon (CR), suggestive of occipital polymicrogyria (arrow).</p> Full article ">Figure 4
<p>Post-mortem cerebral and ocular examination, hematoxylin and eosin staining, original magnification x10. (<b>A</b>) Right occipital cortical plate with polymicrogyria and extreme thinning of the subplate. (<b>B</b>) Right temporal polymicrogyria with lamina dissecans, heterotopic neurons (arrow), and periventricular nodule (blue circle). (<b>C</b>) Chorioretinal scars (arrows) with hyperpigmentation suggestive of chorioretinitis on the right optic disc.</p> Full article ">
20 pages, 5596 KiB  
Article
Dual-RNAseq Analysis Unravels Virus-Host Interactions of MetSV and Methanosarcina mazei
by Finn O. Gehlert, Till Sauerwein, Katrin Weidenbach, Urska Repnik, Daniela Hallack, Konrad U. Förstner and Ruth A. Schmitz
Viruses 2022, 14(11), 2585; https://doi.org/10.3390/v14112585 - 21 Nov 2022
Cited by 5 | Viewed by 2220
Abstract
Methanosarcina spherical virus (MetSV), infecting Methanosarcina species, encodes 22 genes, but their role in the infection process in combination with host genes has remained unknown. To study the infection process in detail, infected and uninfected M. mazei cultures were compared using dual-RNAseq, qRT-PCRs, [...] Read more.
Methanosarcina spherical virus (MetSV), infecting Methanosarcina species, encodes 22 genes, but their role in the infection process in combination with host genes has remained unknown. To study the infection process in detail, infected and uninfected M. mazei cultures were compared using dual-RNAseq, qRT-PCRs, and transmission electron microscopy (TEM). The transcriptome analysis strongly indicates a combined role of virus and host genes in replication, virus assembly, and lysis. Thereby, 285 host and virus genes were significantly regulated. Within these 285 regulated genes, a network of the viral polymerase, MetSVORF6, MetSVORF5, MetSVORF2, and the host genes encoding NrdD, NrdG, a CDC48 family protein, and a SSB protein with a role in viral replication was postulated. Ultrastructural analysis at 180 min p.i. revealed many infected cells with virus particles randomly scattered throughout the cytoplasm or attached at the cell surface, and membrane fragments indicating cell lysis. Dual-RNAseq and qRT-PCR analyses suggested a multifactorial lysis reaction in potential connection to the regulation of a cysteine proteinase, a pirin-like protein and a HicB-solo protein. Our study’s results led to the first preliminary infection model of MetSV infecting M. mazei, summarizing the key infection steps as follows: replication, assembly, and host cell lysis. Full article
(This article belongs to the Special Issue Phage–Host Interactions: From Communities to Single Particles)
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<p>Ultrastructural analysis of MetSV-infected <span class="html-italic">M. mazei</span> cells at 180 min p.i. (<b>A</b>) Infected cell with MetSV particles randomly scattered through the cytoplasm (block arrows) or attached along the cell surface (arrows). White arrows point to less electron-dense virus particles, possibly representing particles with incomplete DNA packaging. (<b>B</b>) Part of the same cell imaged at higher resolution. The S-layer and cell membrane can be resolved at the surface of the cell. (<b>C</b>) Infected cell with a higher viral load. (<b>D</b>) Occasionally, surface-attached viral particles were observed in membrane pockets (dashed line). (<b>E</b>,<b>F</b>) Membrane remains representing lysed cells as strongly suggested by virus particles associated with the membrane either at the cytoplasmic (block arrow) or the extracellular side (arrow). Enlarged particles to the right of the main images. Scale bar, (<b>A</b>) 1 μm, (<b>B</b>) 100 nm, (<b>C</b>,<b>D</b>) 200 nm, and (<b>E</b>,<b>F</b>) 500 nm.</p> Full article ">Figure 2
<p>Summary of dual-RNAseq analysis of MetSV-infected <span class="html-italic">Methanosarcina mazei</span> cells. Total RNA of MetSV-infected <span class="html-italic">M. mazei</span> cultures was isolated at defined time points post-infection (p.i) (t0, uninfected; t30 and t180, 30 and 180 min p.i., respectively), sequenced, and analyzed based on READemption (version 1.0.5), DESeq2 (version 1.34.0) and eggNOG-mapper-2.1.7 output. READemption output was normalized by the transcript per million method (tpm). Here, DESeq2 was used for analysis of differentially transcribed genes in comparisons between conditions (t30 vs. t0 and t180 vs. t0). (<b>A</b>) Principal component analysis (PCA) showed similarity of two biological replicates and differences in time points based on the DESeq2 model. Here, light blue is untreated cells; blue is virus-treated cells at 30 min p.i.; dark blue is virus-treated cells 180 min p.i. (<b>B</b>) Dataset contained, in total, 3800 genes (3778 <span class="html-italic">M. mazei</span> ORFs and 22 MetSV ORFs), which were categorized into the following groups of RNA types based on their annotations: CDS/ORFs (<span class="html-italic">M. mazei</span> or MetSV), sRNAs, tRNAs, rRNAs, and not further classified ncRNAs (<span class="html-italic">M. mazei</span>). (<b>C</b>) Overview about abundances of defined clusters of orthologous genes (COG) categories of <span class="html-italic">M. mazei</span> and MetSV genes within the dataset based on eggNOG-mapper-2.1.7 and NCBI database results (Database download on 04/2022). Categories “Chromatin &amp; Dynamics”, “Energy production &amp; conversation”, “Cell cycle control &amp; cell division”, “Cell wall, membrane &amp; envelope biogenesis”, “Lipid metabolism”, “Secondary structure”, “Cell motility”, “Intracellular trafficking, secretion &amp; vesicular transport”, and “inorganic ion transport &amp; metabolism” were grouped in “others”. (<b>D</b>) Changes in the percentages of transcript per million (tpm) normalized read counts for virus (red) and host (gray) over time per analyzed sample. (<b>E</b>) Volcano plots showing regulation of transcripts in the comparisons t30 vs. t0 and t180 vs. t0. The <span class="html-italic">y</span>-axes represent log10-transformed <span class="html-italic">p</span>-values and the <span class="html-italic">x</span>-axis shows Log2FoldChanges (Log2FC). Significance threshold <span class="html-italic">p</span>-value = 0.05; thresholds for regulation (Log2FC) is &lt;−2.5 and &gt;2.5. For <span class="html-italic">M. mazei</span> genes, gray is not significant, and black is significant; for MetSV genes, red is not significant, and purple is significant.</p> Full article ">Figure 3
<p>MetSV infection induced significant changes to the <span class="html-italic">M. mazei</span> transcriptome. (<b>A</b>) Selection of significant regulated transcripts (DESeq2 results, with a <span class="html-italic">p</span>-value = 0.05; thresholds for regulation (Log2FC) of &lt;−2.5 and &gt;2.5) for comparisons of t30-t0 (2 genes) and t180-t0 (285 genes) visualized in a circular heatmap based on log-transformed transcript per million (tpm) normalized read counts using the R package “circlize” (version 0.4.14) and clustered by regulation using the “stats” (version 4.2.0) package (functions were hclust() and dist()). Black arrows are highlighting significantly regulated sRNAs. (<b>B</b>) and (<b>C</b>) are zooming into nodes and show clustered bar plots of tpm-normalized read counts (<span class="html-italic">y</span>-axis) and gene IDs (<span class="html-italic">x</span>-axis). Clustering was performed as described in (<b>A</b>), and was supplemented with clusters of orthologous genes (COG) categories (EggNOG-mapper version 2.1.7 and NCBI database). All represented genes (285) were significantly regulated in the comparisons between t180-t0, and there was only one significant differentially transcribed gene in the comparison between t30-t0, as indicated by stars (two-way ANOVA; <span class="html-italic">p</span>-values were ≤0. 0001 = ****).</p> Full article ">Figure 4
<p>Impact of MetSV infection on transcription of ncRNAs, spRNAs, and asRNAs. Significant regulated sRNAs (comparison t180 vs. t0) are summarized (DESeq2 results with a <span class="html-italic">p</span>-value = 0.05; thresholds for regulation (Log2FC) are &lt;−2.5 and &gt;2.5). The sRNAs were not significantly regulated in a comparison of t30 vs. t0. Colors represent different types of sRNAs, as follows: not further classified sRNAs (light green), small protein encoding sRNAs (spRNAs, red color) and antisense RNAs (asRNAs, blue color).</p> Full article ">Figure 5
<p>qRT-PCR analysis verifying the transcript regulation of host and virus genes. (<b>A</b>) Absolute transcript numbers based on qRT-PCR analysis of virus ORFs separated by time point and normalized to serial dilution of the pRS1332 plasmid. (<b>B</b>) Log2FCs of the comparisons t30 vs. t0 and 180 vs. t0 of selected <span class="html-italic">M. mazei</span> genes are depicted, and corresponding clusters of orthologous gene (COG) categories can be found in the lower panel (EggNOG-mapper version 2.1.7 and NCBI database). Significances are as follows: based on ttwo-way ANOVA (<span class="html-italic">p</span>-values are ≤ 0.05 = *, ≤ 0.01 = **, and ≤ 0. 0001 = ****).</p> Full article ">Figure 6
<p>Hypothetical infection model summarizing general findings. Graphical summary of the involvement of host genes in combination with viral ORFs in MetSV infection processes. Host-encoded proteins and belonging processes are highlighted in blue; MetSV-derived proteins and belonging processes are highlighted in green. Gradient-filled boxes represent processes using viral and host proteins. Viral DNA is transferred into the host cell via an unknown process (<b>a</b>). <span class="html-italic">M. mazei</span> transcription–translation machinery is expressing viral ORFs (<b>b</b>). Viral proteins and protein complexes are folded and assembled under host chaperones e.g., GroEL/GroES (<b>c</b>). The MetSVORFs with a potential role in viral replication are held in solution via CDC48-mediated disaggregation (<b>d</b>). Linear replication is mediated by using hosts’ SSB RepA<sub>1</sub> and dNTP production by the <span class="html-italic">nrdHDG</span> operon (<b>e</b>). Regulatory or structural viral proteins are used after folding and potential disaggregation to mediate viral particle formation, but these further influence cellular processes via calcium homeostasis (<b>f</b>–<b>h</b>). Induction of host-encoded proteins e.g., the membrane-associated cysteine proteinase (<b>i</b>), and the accumulation of ROS (<b>j</b>), could lead to cell lysis and virus particle release (<b>k</b>).</p> Full article ">
16 pages, 10278 KiB  
Article
Characterization of Three Variants of SARS-CoV-2 In Vivo Shows Host-Dependent Pathogenicity in Hamsters, While Not in K18-hACE2 Mice
by Gabriela Toomer, Whitney Burns, Liliana Garcia, Gerelyn Henry, Anthony Biancofiori, Albert George, Ciera Duffy, Justin Chu, Morgan Sides, Melissa Muñoz, Kelly Garcia, Anya Nikolai-Yogerst, Xinjian Peng, Landon Westfall and Robert Baker
Viruses 2022, 14(11), 2584; https://doi.org/10.3390/v14112584 - 21 Nov 2022
Cited by 6 | Viewed by 3349
Abstract
Animal models are used in preclinical trials to test vaccines, antivirals, monoclonal antibodies, and immunomodulatory drug therapies against SARS-CoV-2. However, these drugs often do not produce equivalent results in human clinical trials. Here, we show how different animal models infected with some of [...] Read more.
Animal models are used in preclinical trials to test vaccines, antivirals, monoclonal antibodies, and immunomodulatory drug therapies against SARS-CoV-2. However, these drugs often do not produce equivalent results in human clinical trials. Here, we show how different animal models infected with some of the most clinically relevant SARS-CoV-2 variants, WA1/2020, B.1.617.2/Delta, B.1.1.529/Omicron, and BA5.2/Omicron, have independent outcomes. We show that in K18-hACE2 mice, B.1.617.2 is more pathogenic, followed by WA1, while B.1.1.529 showed an absence of clinical signs. Only B.1.1.529 was able to infect C57BL/6J mice, which lack the human ACE2 receptor. B.1.1.529-infected C57BL/6J mice had different T cell profiles compared to infected K18-hACE2 mice, while viral shedding profiles and viral titers in lungs were similar between the K18-hACE2 and the C57BL/6J mice. These data suggest B.1.1.529 virus adaptation to a new host and shows that asymptomatic carriers can accumulate and shed virus. Next, we show how B.1.617.2, WA1 and BA5.2/Omicron have similar viral replication kinetics, pathogenicity, and viral shedding profiles in hamsters, demonstrating that the increased pathogenicity of B.1.617.2 observed in mice is host-dependent. Overall, these findings suggest that small animal models are useful to parallel human clinical data, but the experimental design places an important role in interpreting the data. Importance: There is a need to investigate SARS-CoV-2 variant phenotypes in different animal models due to the lack of reproducible outcomes when translating experiments to the human population. Our findings highlight the correlation of clinically relevant SARS-CoV-2 variants in animal models with human infections. Experimental design and understanding of correct animal models are essential to interpreting data to develop antivirals, vaccines, and other therapeutic compounds against COVID-19. Full article
(This article belongs to the Special Issue SARS-CoV-2 and Animal Models)
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<p>B.1.617.2 (Delta) showed enhanced lethality in infected K18-hACE2 mice. (<b>A</b>) Schematic diagram of SARS-CoV-2 infection and experimental design. Interim Sac = scheduled interim sacrifice/euthanasia. Sac. = final euthanasia. Created with BioRender.com. Animals (<span class="html-italic">n</span> = 12) were intranasally inoculated with SARS-CoV-2 on day 0. Body weights before challenge were used to establish baseline. Survival and body weight change (BWC) were observed or collected daily. Lungs were collected at the interim and terminal euthanasia. (<b>B</b>) Survival in mice model after 14 days postchallenge with 1 × 10<sup>3</sup> TCID<sub>50</sub> of WA1, 1 × 10<sup>1</sup> TCID<sub>50</sub> of B.1.617.2 (Delta), and 9 × 10<sup>6</sup> TCID<sub>50</sub> of B.1.1.529/Omicron BA1. (<b>C</b>) Body weight changes comparison between 1 × 10<sup>3</sup> TCID<sub>50</sub> of WA1, 1 × 10<sup>1</sup> TCID<sub>50</sub> of B.1.617.2 (Delta), and 9 × 10<sup>6</sup> TCID<sub>50</sub> of B.1.1.529/Omicron BA1 for 14 days postchallenge. (<b>D</b>) Viral titers in lungs of infected mice at 3 days postchallenge with 1 × 10<sup>3</sup> TCID<sub>50</sub> of WA1 (<span class="html-italic">n</span> = 7), 1 × 10<sup>1</sup> TCID<sub>50</sub> of B.1.617.2 (Delta, <span class="html-italic">n</span> = 7), and 9 × 10<sup>6</sup> TCID<sub>50</sub> of B.1.1.529/Omicron BA1 (<span class="html-italic">n</span> = 4). No statistical differences were found (<span class="html-italic">p</span> = 0.1553). (<b>E</b>) Histopathological analysis with Hematoxylin and Eosin stain (H&amp;E) of the lungs from uninfected and B.1.617.2 (Delta) of SARS-CoV-2-infected mice at 6 days postchallenge under 100× (<span class="html-italic">n</span> = 2). TCID<sub>50</sub> is a quantitation of virus infectivity and stands for the median <b><span class="underline">T</span></b>issue <b><span class="underline">C</span></b>ulture <b><span class="underline">I</span></b>nfectious <b><span class="underline">D</span></b>ose (TCID<sub>50</sub>) defined as the dilution of a virus required to infect <b><span class="underline">50%</span></b> of a given cell culture.</p> Full article ">Figure 2
<p>Host-dependent pathogenicity of B.1.617.2 (Delta), WA1/2020 and B1.1.529 (BA.1) and BA.5 Omicron in hamster. (<b>A</b>) Schematic diagram of SARS-CoV-2 infection and experimental design. Interim Sac = scheduled interim sacrifice/euthanasia. Sac. = final euthanasia. Created with BioRender.com. Animals were intranasally inoculated with 5 × 10<sup>3</sup> TCID<sub>50</sub>/animal of SARS-CoV-2 WA1 or B.1.617.2/Delta or 6.5 × 10<sup>5</sup> TCID<sub>50</sub>/animal of BA.1 or BA.5 on day 0. Body weights before challenge were used to establish baseline. Survival, body weight changes (BWC), oral swabs, or nasal washes were observed or collected daily. Lungs were collected at the interim and terminal euthanasia on days 3 and 6 for WA1 and day 4 for B.1.617.2 (Delta), and BA.1 and BA.5 (Omicron). (<b>B</b>) Body weight change comparisons between WA1, B.1.617.2 (Delta), BA.1, and BA.5 for 4 dpi (and 5 dpi for WA1). (<b>C</b>) Viral titers estimated as genomic RNA copy number in lungs of infected mice at 3, 4, or 6 days postchallenge with 5 × 10<sup>3</sup> TCID<sub>50</sub> of WA1 (<span class="html-italic">n</span> = 7), 5 × 10<sup>3</sup> TCID<sub>50</sub> of B.1.617.2 (Delta, <span class="html-italic">n</span> = 7), or 6.5 × 10<sup>5</sup> TCID<sub>50</sub> (<span class="html-italic">n</span> = 8) of BA.1 or BA.5 (<span class="html-italic">n</span> = 8). (<b>D</b>) Viral shedding profiles were estimated by RNA viral copy number in oral swabs collected from day 0 to day 5 dpi. Dpi = days postinfection. TCID<sub>50</sub> = <b><span class="underline">50%</span></b> of the<b><span class="underline">T</span></b>issue <b><span class="underline">C</span></b>ulture <b><span class="underline">I</span></b>nfectious <b><span class="underline">D</span></b>ose (TCID<sub>50</sub>).</p> Full article ">Figure 3
<p>Reduced pathogenicity in B.1.1.529/Omicron (BA.1)-infected mice. (<b>A</b>). Schematic diagram of SARS-CoV-2 infection and the experimental design. Created with BioRender.com. Animals were intranasally inoculated with 9 × 10<sup>6</sup>, 1 × 10<sup>5</sup>, 1 × 10<sup>4</sup>, or 1 × 10<sup>3</sup>, in K18-hACE2 mice and 9 × 10<sup>6</sup>, 1 × 10<sup>5</sup>, 1 × 10<sup>4</sup> in C57BL\6J. of B.1.1.529 variant of SARS-CoV-2 on day 0. Body weights before challenge were used to establish baseline. Survival, body weight, and oral swabs were observed or collected daily. Lungs were collected at the interim and terminal euthanasia. (<b>B</b>) Percentage of body weight changes (BWC) in K18-hACE2 mice inoculated with 9 × 10<sup>6</sup>, 1 × 10<sup>5</sup>, 1 × 10<sup>4</sup>, or 1 × 10<sup>3</sup>. All groups started with <span class="html-italic">n</span> = 10, <span class="html-italic">n</span> = 5 were scheduled for interim euthanasia at 3 dpi. From 4 dpi to 14 dpi, <span class="html-italic">n</span> = 5. (<b>C</b>) Percentage of BWC in WT mice (C57BL\6J) inoculated with 9 × 10<sup>6</sup>, 1 × 10<sup>5</sup> or 1 × 10<sup>4</sup>. All groups started with <span class="html-italic">n</span> = 6, and <span class="html-italic">n</span> = 3 were scheduled for interim euthanasia at 3 dpi. From 4 dpi to 14 dpi, <span class="html-italic">n</span> = 3. (<b>D</b>) Viral load as RNA genome copy number per mg of lung tissue of K18-hACE2 mice infected with 9 × 10<sup>6</sup>, 1 × 10<sup>5</sup>, 1 × 10<sup>4</sup>, or 1 × 10<sup>3</sup> of B.1.1.529 SARS-CoV-2 after 3 dpi (<span class="html-italic">n</span> = 4 in technical duplicates). (<b>E</b>) Viral load by TCID<sub>50</sub> assay in all WT and K18-hACE2 mice infected with B.1.1.529. (<b>F</b>) Virus shedding as viral genome copy number (by RT-qPCR) in oral swabs taken at 2, 3, and 4 dpi of K18-hACE2 and WT mice intranasally inoculated with 9 × 10<sup>6</sup> TCID<sub>50</sub>/animal (groups 3 and 7 only). Asterisk (*) represent statistical differences by ANOVA <span class="html-italic">p</span> ≤ 0.05.</p> Full article ">Figure 4
<p>Omicron infection increases CD4+ AIM+ Tem population and promotes cytokine production in AIM+ CD8+ T cells in K18-hACE2 mice. (<b>A</b>) CD8 and CD4 AIM+ T cell populations in two mouse models after Omicron challenge. Graphs on the left are Omicron-peptide-stimulated samples unadjusted for control wells, graphs on the right have unstimulated control wells subtracted from Omicron-peptide-stimulated wells. (<b>B</b>) CD8+ AIM+ and CD4+ AIM+ memory phenotype was analyzed by gating naïve (Tn; CD45RA+ CCR7+), central memory (Tcm; CD45RA− CCR7+), effector memory (Tem; CD45RA− CCR7−), and terminally differentiated effector memory (Temra; CD45RA+ CCR7−) cells. (<b>C</b>) CD8+ AIM+ T cells producing IFN-gamma, TNF-alpha, or Granzyme B by intracellular cytokine staining. (<b>D</b>) Multifunctional activity profiles of CD8+ AIM+ T cells evaluated from IFN-gamma, TNF-alpha, and Granzyme B. Asterisks represent statistical significance by ANOVA test where (*) <span class="html-italic">p</span> ≤ 0.05; (**) <span class="html-italic">p</span> ≤ 0.01; (****) <span class="html-italic">p</span> ≤ 0.0001; if (ns) <span class="html-italic">p</span> &gt; 0.05 not shown.</p> Full article ">Figure 5
<p>Omicron infection increases CD4+ Tem population and promotes cytokine production in CD8+ T cells in K18-hACE2 mice. (<b>A</b>) CD8+ and CD4+ memory phenotype was analyzed by gating naïve (Tn; CD45RA+ CCR7+), central memory (Tcm; CD45RA− CCR7+), effector memory (Tem; CD45RA− CCR7−), and terminally differentiated effector memory (Temra; CD45RA+ CCR7−) cells. (<b>B</b>) CD8+ T cells producing IFN-gamma, TNF-alpha, or Granzyme B by intracellular cytokine staining. (<b>C</b>) Multifunctional activity profiles of CD8+ T cells evaluated from IFN-gamma, TNF-alpha, and Granzyme B. Asterisks represent statistical significance by ANOVA test where (**) <span class="html-italic">p</span> ≤ 0.01; (****) <span class="html-italic">p</span> ≤ 0.0001; if (ns) <span class="html-italic">p</span> &gt; 0.05 not shown.</p> Full article ">
14 pages, 306 KiB  
Article
A Retrospective Study of Viral Molecular Prevalences in Cats in Southern Italy (Campania Region)
by Maria Grazia Amoroso, Francesco Serra, Gianluca Miletti, Lorena Cardillo, Claudio de Martinis, Luisa Marati, Flora Alfano, Gianmarco Ferrara, Ugo Pagnini, Esterina De Carlo, Giovanna Fusco and Serena Montagnaro
Viruses 2022, 14(11), 2583; https://doi.org/10.3390/v14112583 - 21 Nov 2022
Cited by 14 | Viewed by 2284
Abstract
From 2019 to 2021, a retrospective molecular study was conducted in the Campania region (southern Italy) to determine the prevalence of viral diseases in domestic cats. A total of 328 dead animals were analyzed by Real-Time PCR for the presence of feline panleukopenia [...] Read more.
From 2019 to 2021, a retrospective molecular study was conducted in the Campania region (southern Italy) to determine the prevalence of viral diseases in domestic cats. A total of 328 dead animals were analyzed by Real-Time PCR for the presence of feline panleukopenia virus (FPV), feline leukemia virus (FeLV), feline enteric coronavirus (FCoV), rotavirus (RVA), feline herpesvirus type 1 (FHV-1), and feline calicivirus (FCV). The possible presence of SARS-CoV-2 was also investigated by Real-Time PCR. The cats included in this study were specifically sourced and referred by local veterinarians and local authorities to the Zooprofilactic Experimental Institute of Southern Italy (IZSM) for pathological evaluation. The samples consisted of owners, catteries, and stray cats. Results revealed: 73.5% positive cats for FPV (189/257), 23.6% for FeLV (21/89), 21.5% for FCoV (56/266), 11.4% for RVA (16/140), 9.05% for FeHV-1 (21/232), and 7.04 for FCV (15/213). In contrast, SARS-CoV-2 was never detected. FPV was more prevalent in winter (p = 0.0027). FCoV FHV-1, FCV, and RVA predominated in autumn, whereas FeLV predominated in summer. As expected, viral infections were found more frequently in outdoor and shelter cats than in indoor ones, although no statistical association was found between animal lifestyle and viral presence. The study showed a high prevalence of FPV, FeLV, and FCoV and a moderate prevalence of RVA, FHV-1, and FCV. Moreover, the prevalence of these pathogens varied among the cat populations investigated. Full article
(This article belongs to the Special Issue Enteric and Respiratory Viruses in Animals 3.0)
12 pages, 2845 KiB  
Article
Effects of Klebsiella pneumoniae Bacteriophages on IRAK3 Knockdown/Knockout THP-1 Monocyte Cell Lines
by Bryce Dylan Schubert, Heng Ku, Mwila Kabwe, Trang Hong Nguyen, Helen Irving and Joseph Tucci
Viruses 2022, 14(11), 2582; https://doi.org/10.3390/v14112582 - 21 Nov 2022
Cited by 2 | Viewed by 2418
Abstract
Bacterial sepsis characterised by an immunosuppressive and cytokine storm state is a challenge to treat clinically. While conventional antibiotics have been associated with exacerbating the cytokine storm, the role that bacteriophages may play in immune modulation of sepsis remains unclear. Bacteriophages are bacterial [...] Read more.
Bacterial sepsis characterised by an immunosuppressive and cytokine storm state is a challenge to treat clinically. While conventional antibiotics have been associated with exacerbating the cytokine storm, the role that bacteriophages may play in immune modulation of sepsis remains unclear. Bacteriophages are bacterial viruses that have the capacity to lyse specific bacteria and hence provide a natural alternative to antibiotics. K. pneumoniae is known to cause sepsis in humans, and in this study we isolated two lytic bacteriophages against this pathogen, one of which was a novel jumbo bacteriophage. We employed THP-1 monocyte cell lines, with different functional phenotypes for the interleukin-1 receptor associated kinase 3 (IRAK3- a cytoplasmic homeostatic mediator and prognostic marker of inflammation), to evaluate the role of the K. pneumoniae bacteriophages in modulating the immune response in-vitro. We showed for the first time that bacteriophages did not stimulate excessive production of tumour necrosis factor alpha, or interleukin-6, in THP-1 monocyte cell lines which displayed varying levels of IRAK3 expression. Full article
(This article belongs to the Section Bacterial Viruses)
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<p>Characterisation of bacteriophages KPN7 and KPN8. (<b>A</b>,<b>C</b>): Electron micrographs of bacteriophage KPN7 (<b>A</b>) (Capsid dimensions: diameter = 62.3 ± 1 nm, Tail length = 15.3 ± 3 nm, Tail width = 15.6 ± 1 nm) showing a typical Podovirus morphology and bacteriophage KPN8 (<b>C</b>) (Capsid dimensions: diameter = 130.2 ± 3 nm, Tail length = 196.1 ± 4 nm, Tail width = 53.7 ± 11 nm) shown with a typical Myovirus morphology. (<b>B</b>,<b>D</b>): Genomic representation of bacteriophages KPN7 (<b>B</b>) and KPN8 (<b>D</b>), shown in circular form for ease of visualisation. Putative functions of genes are denoted by purple (putative packaging protein), green (putative head or capsid structural protein), blue (putative tail structural genes), red (putative lysin proteins), brown (putative metabolic proteins), and pink (putative DNA manipulation proteins). Hypothetical proteins are shown in yellow.</p> Full article ">Figure 2
<p>Phylogenetic and proteomic analysis of the bacteriophages KPN7 and KPN8 (highlighted in red and blue rectangles, respectively). Phylogenetic tree construction carried out with 100 bootstraps for putative capsid protein (<b>A</b>) putative terminase protein (<b>B</b>) and putative DNA polymerase proteins (<b>C</b>). To ascertain genomic viral taxa, viral protein analysis was constructed using VIPTree webserver (<b>D</b>).</p> Full article ">Figure 3
<p>Effects of PBS, LPS (1 µg/mL) and bacteriophage treatments on cytokine production by wild-type (WT), IRAK3 knockdown and IRAK3 knockout THP-1 monocytes. (<b>A</b>) Interleukin 6 production (<b>B</b>) Tumour necrosis factor alpha production. *** <span class="html-italic">p</span> value &lt;0.001; **** <span class="html-italic">p</span> value &lt; 0.0001.</p> Full article ">
12 pages, 760 KiB  
Article
Assessing the Pre-Vaccination Anti-SARS-CoV-2 IgG Seroprevalence among Residents and Staff in Nursing Home in Niigata, Japan, November 2020
by Keita Wagatsuma, Sayaka Yoshioka, Satoru Yamazaki, Ryosuke Sato, Wint Wint Phyu, Irina Chon, Yoshiki Takahashi, Hisami Watanabe and Reiko Saito
Viruses 2022, 14(11), 2581; https://doi.org/10.3390/v14112581 - 21 Nov 2022
Cited by 2 | Viewed by 2134
Abstract
An outbreak of coronavirus disease 2019 (COVID-19) occurred in a nursing home in Niigata, Japan, November 2020, with an attack rate of 32.0% (63/197). The present study was aimed at assessing the pre-vaccination seroprevalence almost half a year after the COVID-19 outbreak in [...] Read more.
An outbreak of coronavirus disease 2019 (COVID-19) occurred in a nursing home in Niigata, Japan, November 2020, with an attack rate of 32.0% (63/197). The present study was aimed at assessing the pre-vaccination seroprevalence almost half a year after the COVID-19 outbreak in residents and staff in the facility, along with an assessment of the performance of the enzyme-linked immunosorbent assay (ELISA) and the chemiluminescent immunoassay (CLIA), regarding test seropositivity and seronegativity in detecting immunoglobulin G (IgG) anti-severe acute respiratory syndrome 2 (SARS-CoV-2) antibodies (anti-nucleocapsid (N) and spike (S) proteins). A total of 101 people (30 reverse transcription PCR (RT-PCR)-positive and 71 RT-PCR-negative at the time of the outbreak in November 2020) were tested for anti-IgG antibody titers in April 2021, and the seroprevalence was approximately 40.0–60.0% for residents and 10.0–20.0% for staff, which was almost consistent with the RT-PCR test results that were implemented during the outbreak. The seropositivity for anti-S antibodies showed 90.0% and was almost identical to the RT-PCR positives even after approximately six months of infections, suggesting that the anti-S antibody titer test is reliable for a close assessment of the infection history. Meanwhile, seropositivity for anti-N antibodies was relatively low, at 66.7%. There was one staff member and one resident that were RT-PCR-negative but seropositive for both anti-S and anti-N antibody, indicating overlooked infections despite periodical RT-PCR testing at the time of the outbreak. Our study indicated the impact of transmission of SARS-CoV-2 in a vulnerable elderly nursing home in the pre-vaccination period and the value of a serological study to supplement RT-PCR results retrospectively. Full article
(This article belongs to the Special Issue RNA Viruses and Antibody Response)
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<p>Epidemic histogram of SARS-CoV-2 outbreak in nursing care home in Niigata, Japan, November 2020. (<b>A</b>) Epidemic histogram of reverse transcription PCR (RT-PCR) positives at the time of the outbreak (<span class="html-italic">n</span> = 58). (<b>B</b>) Epidemic histogram of RT-PCR positives who participated in this study (<span class="html-italic">n</span> = 29). Daily counts of confirmed cases by RT-PCR tests are described as a function of the day of illness onset. Note that five persons whose date of illness onset by active epidemiological investigations was unknown were excluded in (<b>A</b>). Yellow and green bars correspond to staff and residents, respectively.</p> Full article ">Figure 2
<p>IgG antibody responses of SARS-CoV-2 in nursing care home in Niigata, Japan, April 2021 (<span class="html-italic">n</span> = 101). (<b>A</b>) Responses of severe acute respiratory syndrome 2 (SARS-CoV-2) in staff’s anti-nucleocapsid (N) IgG antibodies by DENKA (Tokyo, Japan) and Abbott (Chicago, IL, USA). (<b>B</b>) Responses of SARS-CoV-2 in staff’s anti-spike (S) IgG antibodies by DENKA and Abbott. (<b>C</b>) Responses of SARS-CoV-2 in resident’s anti-N IgG antibodies by DENKA and Abbott. (<b>D</b>) Responses of SARS-CoV-2 in resident’s anti-S IgG antibodies by DENKA and Abbott. The percentage of positive cases (%) is described and the red dotted line indicates the threshold for positivity (i.e., positive cutoff index).</p> Full article ">
9 pages, 2000 KiB  
Article
Evaluation of Rapid Dot-Immunoassay for Detection Orthopoxviruses Using Laboratory-Grown Viruses and Animal’s Clinical Specimens
by Nikita Ushkalenko, Anna Ersh, Alexander Sergeev, Pavel Filatov and Alexander Poltavchenko
Viruses 2022, 14(11), 2580; https://doi.org/10.3390/v14112580 - 21 Nov 2022
Cited by 3 | Viewed by 1673
Abstract
The aim of the work was an experimental evaluation of the characteristics of the kit for the rapid immunochemical detection of orthopoxviruses (OPV). The kit is based on the method of one-stage dot-immunoassay on flat protein arrays using gold conjugates and a silver [...] Read more.
The aim of the work was an experimental evaluation of the characteristics of the kit for the rapid immunochemical detection of orthopoxviruses (OPV). The kit is based on the method of one-stage dot-immunoassay on flat protein arrays using gold conjugates and a silver developer. Rabbit polyclonal antibodies against the vaccinia virus were used as capture and detection reagents. The sensitivity of detection of OPV and the specificity of the analysis were assessed using culture crude preparations (monkeypox virus, vaccinia virus, rabbitpox virus, cowpox virus, and ectromelia virus), a suspension from a crust from a human vaccination site as well as blood and tissue suspensions of infected rabbits. It has been shown that the assay using the kit makes it possible to detect OPV within 36 min at a temperature of 18–40 °C in unpurified culture samples of the virus and clinical samples in the range of 103–104 PFU/mL. Tests of the kit did not reveal cross-reactivity with uninfected cell cultures and viral pathogens of exanthematous infections (measles, rubella and chicken pox). The kit can be used to detect or exclude the presence of a virus threat in samples and can be useful in various aspects of biosecurity. The simplicity of analysis, the possibility of visual accounting the and interpretation of the results make it possible to use the test in laboratories with a high level of biological protection and in out-of-laboratory conditions. Full article
(This article belongs to the Special Issue Diagnostics, Antiviral Therapy and Vaccination against Orthopoxvirus Infections)
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<p>Design of a kit for dot-immunoassay of orthopoxviruses. (the <b>A</b>)-the basic elements of the kit for detection of orthopoxviruses: 1-protein arrays, 2-analytical baths, 3-perforator, 4-liquid components of the developing system. (<b>B</b>)-the scheme of capture reagents immobilization on the protein array: T-test zone (a/POX-IgG 1:100), C−-negative control (IgG RNS 1:100) and C+-positive control (VACV (LIVP) 10<sup>5</sup> PFU/mL).</p> Full article ">Figure 2
<p>The scheme of the rapid dot-immunoassay of orthopoxvirus using conjugate of colloidal gold with polyclonal antibodies; enhanced optical signal by silver developer and stabilized staining with alkaline solution of thiourea. The top lines show the multiplicity and duration (min) of operations. <span class="html-fig-inline" id="viruses-14-02580-i001"><img alt="Viruses 14 02580 i001" src="/viruses/viruses-14-02580/article_deploy/html/images/viruses-14-02580-i001.png"/></span>—antibodies,<span class="html-fig-inline" id="viruses-14-02580-i002"><img alt="Viruses 14 02580 i002" src="/viruses/viruses-14-02580/article_deploy/html/images/viruses-14-02580-i002.png"/></span>—antigens,<span class="html-fig-inline" id="viruses-14-02580-i003"><img alt="Viruses 14 02580 i003" src="/viruses/viruses-14-02580/article_deploy/html/images/viruses-14-02580-i003.png"/></span>—the multiplicity of washing.</p> Full article ">Figure 3
<p>Comparative assay of the dilution series: (<b>A</b>)-suspension from the crust from the pustule at the site of vaccination; (<b>B</b>)-VACV(LIVP) preparation (initial titer 8.5 × 10<sup>6</sup> PFU/mL). The numbers below indicate the multiplicity of dilutions.</p> Full article ">Figure 4
<p>The results of dot-immunoassays of clinical samples from an infected rabbit. Type of protein arrays after the assays: (<b>A</b>)-blood serum and (<b>B</b>)-blood cells (the numbers under the arrays indicate the days from the moment of infection); (<b>C</b>)-washes from the nasal mucosa (<b>a</b>–<b>c</b>) and homogenates of samples skin areas with papular rashes (<b>d</b>–<b>f</b>) every rabbit.</p> Full article ">
13 pages, 1477 KiB  
Article
Impact of Hepatitis B Virus Infection, Non-alcoholic Fatty Liver Disease, and Hepatitis C Virus Co-infection on Liver-Related Death among People Tested for Hepatitis B Virus in British Columbia: Results from a Large Longitudinal Population-Based Cohort Study
by Jean Damascene Makuza, Dahn Jeong, Mawuena Binka, Prince Asumadu Adu, Georgine Cua, Amanda Yu, Héctor Alexander Velásquez García, Maria Alvarez, Stanley Wong, Sofia Bartlett, Mohammad Ehsanul Karim, Eric M. Yoshida, Alnoor Ramji, Mel Krajden and Naveed Zafar Janjua
Viruses 2022, 14(11), 2579; https://doi.org/10.3390/v14112579 - 21 Nov 2022
Cited by 3 | Viewed by 3728
Abstract
Data on the contribution of hepatitis B virus (HBV) infection and related comorbidities to liver-related mortality in Canada are limited. We assessed the concurrent impact of HBV infection, non-alcoholic fatty liver disease (NAFLD), and hepatitis C virus (HCV) coinfection on liver-related deaths in [...] Read more.
Data on the contribution of hepatitis B virus (HBV) infection and related comorbidities to liver-related mortality in Canada are limited. We assessed the concurrent impact of HBV infection, non-alcoholic fatty liver disease (NAFLD), and hepatitis C virus (HCV) coinfection on liver-related deaths in British Columbia (BC), Canada. We used data from the BC Hepatitis Testers Cohort (BC-HTC). We used Fine–Gray multivariable sub-distributional hazards models to assess the effect of HBV, NAFLD, and HCV coinfection on liver-related mortality, while adjusting for confounders and competing mortality risks. The liver-related mortality rate was higher among people with HBV infection than those without (2.57 per 1000 PYs (95%CI: 2.46, 2.69) vs. 0.62 per 1000 PYs (95%CI: 0.61, 0.64), respectively). Compared with the HBV negative groups, HBV infection was associated with increased liver-related mortality risk in almost all of the subgroups: HBV mono-infection (adjusted subdistribution hazards ratio (asHR) of 3.35, 95% CI 3.16, 3.55), NAFLD with HBV infection, (asHR 12.5, 95% CI 7.08, 22.07), and HBV/HCV coinfection (asHR 8.4, 95% CI 7.62, 9.26). HBV infection is associated with a higher risk of liver-related mortality, and has a greater relative impact on people with NAFLD and those with HCV coinfection. The diagnosis and treatment of viral and fatty liver disease are required to mitigate liver-related morbidity and mortality. Full article
(This article belongs to the Special Issue Hepatitis B Virus: New Breakthroughs to Conquer an Ancient Disease)
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<p>Study population flowchart for liver-related mortality among a BC population cohort tested for HBV from 1990–2015. BC, British Columbia; HBV, hepatitis B virus; PS: propensity score.</p> Full article ">Figure 2
<p>Cumulative mortality rate for people testing HBV positive vs. those testing negative in BC from 1990–2015. (<b>A</b>) Cumulative liver-related mortality in individuals with and without HBV infection. (<b>B</b>) Cumulative liver-related mortality in individuals with and without HBV infection and co-occurring NAFLD. (<b>C</b>) Cumulative liver-related mortality in individuals with and without HBV infection and HCV coinfection.</p> Full article ">Figure 2 Cont.
<p>Cumulative mortality rate for people testing HBV positive vs. those testing negative in BC from 1990–2015. (<b>A</b>) Cumulative liver-related mortality in individuals with and without HBV infection. (<b>B</b>) Cumulative liver-related mortality in individuals with and without HBV infection and co-occurring NAFLD. (<b>C</b>) Cumulative liver-related mortality in individuals with and without HBV infection and HCV coinfection.</p> Full article ">
18 pages, 1477 KiB  
Review
Understanding the Role of HLA Class I Molecules in the Immune Response to Influenza Infection and Rational Design of a Peptide-Based Vaccine
by A. K. M. Muraduzzaman, Patricia T. Illing, Nicole A. Mifsud and Anthony W. Purcell
Viruses 2022, 14(11), 2578; https://doi.org/10.3390/v14112578 - 21 Nov 2022
Cited by 4 | Viewed by 3943
Abstract
Influenza A virus is a respiratory pathogen that is responsible for regular epidemics and occasional pandemics that result in substantial damage to life and the economy. The yearly reformulation of trivalent or quadrivalent flu vaccines encompassing surface glycoproteins derived from the current circulating [...] Read more.
Influenza A virus is a respiratory pathogen that is responsible for regular epidemics and occasional pandemics that result in substantial damage to life and the economy. The yearly reformulation of trivalent or quadrivalent flu vaccines encompassing surface glycoproteins derived from the current circulating strains of the virus does not provide sufficient cross-protection against mismatched strains. Unlike the current vaccines that elicit a predominant humoral response, vaccines that induce CD8+ T cells have demonstrated a capacity to provide cross-protection against different influenza strains, including novel influenza viruses. Immunopeptidomics, the mass spectrometric identification of human-leukocyte-antigen (HLA)-bound peptides isolated from infected cells, has recently provided key insights into viral peptides that can serve as potential T cell epitopes. The critical elements required for a strong and long-living CD8+ T cell response are related to both HLA restriction and the immunogenicity of the viral peptide. This review examines the importance of HLA and the viral immunopeptidome for the design of a universal influenza T-cell-based vaccine. Full article
(This article belongs to the Special Issue Influenza A Viruses: New Insights in 2022)
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<p>Influenza virus structure and antigenic drift and shift in influenza infections. (<b>A</b>) Typical influenza virus spheroidal form showing all the viral components. HA, NA, and M2 proteins form the envelope, with M1 forming a critical bridge between the viral envelope and the vRNP core, which consists of helical RNP segments containing negative-stranded genomic vRNAs and NP, along with the 3-P complex. (<b>B</b>) Over time, small changes associated with antigenic drift can accumulate and result in viruses that are antigenically different from their ancestors. The current vaccines that induce strain-specific antibodies do not afford complete protection, and as a result, we observe yearly epidemic outbreaks of seasonal influenza. Antigenic shift can result in a new influenza A subtype which is either generated via an intermediate host where there is, for example, gene exchange between human and avian influenza viruses, forming a new strain capable of infecting humans, or, sometimes, influenza strains from other species can directly infect humans by crossing the species barrier (e.g., in the case of H5N1). When antigenic shift occurs, most people have little or no immunity against the new virus, thereby enabling pandemics. Image created with <a href="http://BioRender.com" target="_blank">BioRender.com</a>. (accessed on 14 September 2022).</p> Full article ">Figure 2
<p>T cell recognition of influenza virus-infected cells. Upon infection, respiratory epithelial cells and professional antigen-presenting cells (APCs), such as dendritic cells (DCs), process the viral proteins and present them as peptides in complex with MHC-I on the cell surface. Specific T cell receptors (TCR) expressed on the CD8<sup>+</sup> T cells then recognize these peptide/MHC-I complexes. Upon engagement, the CD8<sup>+</sup> T cells become activated and secrete cytotoxic granules (i.e., granzymes, perforin) targeting the infected cell to induce its death and produce proinflammatory cytokines (i.e., IFNs, TNF, and IL2). Image created with <a href="http://BioRender.com" target="_blank">BioRender.com</a>. (accessed on 14 September 2022).</p> Full article ">
17 pages, 1382 KiB  
Article
Novel RT-PCR Using Sugar Chain-Immobilized Gold-Nanoparticles Correlates Patients’ Symptoms: The Follow-Up Study of COVID-19 Hospitalized Patients
by Takashi Kajiya, Hayate Sawayama, Eriko Arima, Mika Okamoto, Masanori Baba, Masaaki Toyama, Kosuke Okuya, Makoto Ozawa, Nobuhiko Atsuchi, Junichiro Nishi and Yasuo Suda
Viruses 2022, 14(11), 2577; https://doi.org/10.3390/v14112577 - 21 Nov 2022
Cited by 1 | Viewed by 1974
Abstract
Background: The transmissible capacity and toxicity of SARS-CoV-2 variants are continually changing. We report here the follow-up study of hospitalized COVID-19 patients from 2020 to 2022. It is known that the PCR diagnosis for hospitalized patients sometimes causes confusion because of the incompatibility [...] Read more.
Background: The transmissible capacity and toxicity of SARS-CoV-2 variants are continually changing. We report here the follow-up study of hospitalized COVID-19 patients from 2020 to 2022. It is known that the PCR diagnosis for hospitalized patients sometimes causes confusion because of the incompatibility between their diagnosis and symptoms. We applied our sugar chain-immobilized gold-nanoparticles for the extraction and partial purification of RNA from specimens for quantitative RT-PCR assay and evaluated whether the results correlate with patients’ symptoms. Methods and Results: Saliva specimens were taken from hospitalized patients with mild or moderate symptoms every early morning. At the time of RT-PCR diagnosis, two methods for the extraction and partial purification of RNA from the specimen were performed: a commonly used Boom (Qiagen) method and our original sugar chain-immobilized gold nanoparticle (SGNP) method. For symptoms, body temperature and oxygen saturation (SpO2) of patients were monitored every 4 h. Conclusions: It was clear that patients infected with the Delta variant needed more time to recover than those with the Omicron variant, and that the SGNP method showed more realistic correlation with the symptoms of patients compared with the common Qiagen method. Full article
(This article belongs to the Special Issue Nanotechnological Applications in Virology 2023)
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<p>Diagram of copy number/mL of specimen mixed with buffer by SGNP method (<bold>a</bold>) and by Qiagen method (<bold>b</bold>) versus day after onset. Diagram of highest daily body temperature (<bold>c</bold>) and oxygen saturation (<bold>d</bold>) versus day after onset. The average of copy number/mL of specimen mixed with buffer by SGNP method and by Qiagen method, and average of highest daily body temperature versus day after onset (<bold>e</bold>). The average of copy number/mL of specimen mixed with buffer by SGNP method and by Qiagen method, and average of lowest oxygen saturation versus day after onset (<bold>f</bold>).</p> Full article ">Figure 1 Cont.
<p>Diagram of copy number/mL of specimen mixed with buffer by SGNP method (<bold>a</bold>) and by Qiagen method (<bold>b</bold>) versus day after onset. Diagram of highest daily body temperature (<bold>c</bold>) and oxygen saturation (<bold>d</bold>) versus day after onset. The average of copy number/mL of specimen mixed with buffer by SGNP method and by Qiagen method, and average of highest daily body temperature versus day after onset (<bold>e</bold>). The average of copy number/mL of specimen mixed with buffer by SGNP method and by Qiagen method, and average of lowest oxygen saturation versus day after onset (<bold>f</bold>).</p> Full article ">Figure 2
<p>The copy number/mL of specimen mixed with buffer by SGNP method and by Qiagen method, and average of highest daily body temperature versus day after onset. (<bold>a</bold>) Case-25, (<bold>b</bold>) Case-26, (<bold>c</bold>) Case-27, and (<bold>d</bold>) Case-29.</p> Full article ">Figure 2 Cont.
<p>The copy number/mL of specimen mixed with buffer by SGNP method and by Qiagen method, and average of highest daily body temperature versus day after onset. (<bold>a</bold>) Case-25, (<bold>b</bold>) Case-26, (<bold>c</bold>) Case-27, and (<bold>d</bold>) Case-29.</p> Full article ">Figure 3
<p>TEM image of the mixture of Avian influenza (AIV) (<bold>a</bold>) and inactivated SARS-CoV-2 (<bold>b</bold>) with DS-25 immobilized non-magnetized gold nanoparticles.</p> Full article ">
13 pages, 1042 KiB  
Article
Epidemiological Aspects of Equid Herpesvirus-Associated Myeloencephalopathy (EHM) Outbreaks
by Eva Klouth, Yury Zablotski, Jessica L. Petersen, Marco de Bruijn, Gittan Gröndahl, Susanne Müller and Lutz S. Goehring
Viruses 2022, 14(11), 2576; https://doi.org/10.3390/v14112576 - 21 Nov 2022
Cited by 5 | Viewed by 2284
Abstract
Equid Herpesvirus Myeloencephalopathy (EHM) is a multifactorial disease following an EHV-1 infection in Equidae. We investigated a total of 589 horses on 13 premises in Europe in search of risk factors for the development of EHM. We found that fever (p < [...] Read more.
Equid Herpesvirus Myeloencephalopathy (EHM) is a multifactorial disease following an EHV-1 infection in Equidae. We investigated a total of 589 horses on 13 premises in Europe in search of risk factors for the development of EHM. We found that fever (p < 0.001), increasing age (p = 0.032), and female sex (p = 0.042) were risk factors for EHM in a logistic mixed model. Some breeds had a decreased risk to develop EHM compared to others (Shetland and Welsh ponies; p = 0.017; p = 0.031), and fewer EHV-1-vaccinated horses were affected by EHM compared to unvaccinated horses (p = 0.02). Data evaluation was complex due to high variability between outbreaks with regards to construction and environment; viral characteristics and the virus’s transmissibility were affected by operational management. This study confirms earlier suspected host-specific risk factors, and our data support the benefit of high vaccine coverage at high-traffic boarding facilities. Full article
(This article belongs to the Special Issue Equine Viruses in Continental Europe)
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<p>From left to right, 7 distinct breeds (Fjord, Quarter Horse (QH), Warmblood (WB), Arabian, Haflinger, Shetland and Welsh pony) are arranged by decreasing EHM frequency (numbers and percentage as stacked bar plot). The <span class="html-italic">p</span>-values above individual stacked bars compare the proportions of EHM-affected (dark gray) individuals to unaffected (light gray) animals within each breed. A chi-square test (top of the plot) finds a significant relationship between breed and EHM (<span class="html-italic">p</span> = 0.004), suggesting the need of post hoc pairwise comparisons between individual breeds via Fisher’s tests (reason: low numbers per category). The effect of this relationship is small (V<sub>Cramer</sub> = 0.17 [<a href="#B21-viruses-14-02576" class="html-bibr">21</a>]). In contrast to the <span class="html-italic">p</span>-value, the Bayes Factor (BF) provides weak evidence for a relationship between EHM and breed (log(BF01) = 0.28 [<a href="#B22-viruses-14-02576" class="html-bibr">22</a>]), whereas the Bayesian effect size suggests a moderately strong relationship (V<sub>Cramer</sub> = 0.21; 95% Highest Density Intervals 0.22; 0.73). Such disagreement between the frequentist (plot top) and Bayesian methods (plot bottom) might be caused by a low number of observations in several breeds and suggests the need for more data.</p> Full article ">Figure 2
<p>Six breed clusters (1–6) according to similar genetical background are arranged from left to right. EHM frequencies per cluster are shown as percentage in a stacked bar plot (dark grey). The <span class="html-italic">p</span>-values above distinct stacked bars compare the proportions of EHM-affected (dark gray) individuals versus unaffected (light gray) animals inside of every particular breed cluster. The chi-square test finds only a suggestive relationship between breed clusters and EHM (<span class="html-italic">p</span> = 0.05, low evidence against the null hypothesis). Also, the effect of this relationship is small (V<sub>Cramer</sub> = 0.11 [<a href="#B21-viruses-14-02576" class="html-bibr">21</a>]). The Bayes Factor (BF) supports this result by indicating a very strong evidence for the null hypothesis that there is no relationship between EHM and breed (log(BF01) = 4.29 [<a href="#B22-viruses-14-02576" class="html-bibr">22</a>]). The Bayesian effect size also supports this conclusion with a small effect size (V<sub>Cramer</sub> = 0.15; 95% Highest Density Intervals 0.09; 0.20).</p> Full article ">Figure 3
<p>(<b>A</b>) Cluster 1 horses (genetically similar ‘tall’ horses) and 3 (<b>B</b>): all others (<span class="html-italic">not</span>-cluster-1) stratified into animals with EHM (right bar, panel (<b>A</b>) or (<b>B</b>)), or without EHM (left bar, panel (<b>A</b>) or (<b>B</b>)), and further stratified vertically into EHV-unvaccinated (light gray) or EHV-vaccinated (dark gray) animals. The <span class="html-italic">p</span>-values above individual bars compare the proportions of vaccinated versus unvaccinated animals, indicating fewer animals were vaccinated. The chi-square test (results at top of figure) finds a benefit of vaccination in the prevention of EHM (<span class="html-italic">p</span> = 0.02) for cluster 1 horses (panel (<b>A</b>)). Those without EHM (left bar) were more frequently (25%) fully vaccinated when compared to horses with EHM, where only 12% were fully vaccinated. The difference in percentages is small (V<sub>Cramer</sub> = 0.15 [<a href="#B21-viruses-14-02576" class="html-bibr">21</a>]). The Bayes Factor (BF) supports this result by indicating a substantial evidence for the benefit of vaccination in EHM prevention (log(BF01) = −1.29 [<a href="#B22-viruses-14-02576" class="html-bibr">22</a>]). The Bayesian effect size is, however, also small (V<sub>Cramer</sub> = 0.16; 95%; Highest Density Intervals 0.03; 0.28). Findings for <span class="html-italic">not</span>-cluster-1 animal results were not significant, likely due to small sample size. The chi-square test (results: top of figure, panel (<b>B</b>)) finds no relationship between vaccination and EHM (<span class="html-italic">p</span> = 0.45, no evidence against the null hypothesis). The difference in percentages is small (V<sub>Cramer</sub> = 0.0 [<a href="#B21-viruses-14-02576" class="html-bibr">21</a>]). The Bayes Factor (BF) supports the latter result by indicating no evidence for the null or for the alternative hypothesis (log(BF01) = 0.80 [<a href="#B22-viruses-14-02576" class="html-bibr">22</a>]) and by showing a very small Bayesian effect size (V<sub>Cramer</sub> = 0.09; 95% Highest Density Intervals 0; 0.23).</p> Full article ">
19 pages, 18941 KiB  
Review
Immune-Mediated Pathogenesis in Dengue Virus Infection
by Arshi Khanam, Hector Gutiérrez-Barbosa, Kirsten E. Lyke and Joel V. Chua
Viruses 2022, 14(11), 2575; https://doi.org/10.3390/v14112575 - 21 Nov 2022
Cited by 24 | Viewed by 10646
Abstract
Dengue virus (DENV) infection is one of the major public health concerns around the globe, especially in the tropical regions of the world that contribute to 75% percent of dengue cases. While the majority of DENV infections are mild or asymptomatic, approximately 5% [...] Read more.
Dengue virus (DENV) infection is one of the major public health concerns around the globe, especially in the tropical regions of the world that contribute to 75% percent of dengue cases. While the majority of DENV infections are mild or asymptomatic, approximately 5% of the cases develop a severe form of the disease that is mainly attributed to sequential infection with different DENV serotypes. The severity of dengue depends on many immunopathogenic mechanisms involving both viral and host factors. Emerging evidence implicates an impaired immune response as contributing to disease progression and severity by restricting viral clearance and inducing severe inflammation, subsequently leading to dengue hemorrhagic fever and dengue shock syndrome. Moreover, the ability of DENV to infect a wide variety of immune cells, including monocytes, macrophages, dendritic cells, mast cells, and T and B cells, further dysregulates the antiviral functions of these cells, resulting in viral dissemination. Although several risk factors associated with disease progression have been proposed, gaps persist in the understanding of the disease pathogenesis and further investigations are warranted. In this review, we discuss known mechanisms of DENV-mediated immunopathogenesis and its association with disease progression and severity. Full article
(This article belongs to the Special Issue Boosting Flavivirus Research: A Pandengue Net Initiative)
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<p>T cell response during DENV infection. History of exposure to dengue virus (DENV) is one of the factors that drive the T cell response in the evolution of the disease. (<b>A</b>) During primary infection, naïve T cells differentiate into effector T cells, leading to clearance of infection by direct lysis or by releasing antiviral cytokines, such as IFN-γ and TNF-α. (<b>B</b>) During a secondary heterologous infection, a robust immune response is developed by the early activation of cross-reactive serotype T cells, which produce abundant proinflammatory cytokines and chemokines, leading to excessive inflammatory environment, causing endothelial dysfunction that could trigger vascular permeability. (<b>C</b>) During a third or fourth heterologous infection, the activation of cross-reactive serotype T cells and their role in the severity or protection against infection is largely unknown.</p> Full article ">Figure 2
<p>Antibody-dependent enhancement (ADE) in dengue virus infection. ADE is one of the phenomena contributing to the severity of dengue infection during a secondary heterologous infection in a patient with pre-existing non-neutralizing antibodies. The ADE phenomenon has two components: (<b>A</b>) Extrinsic ADE, which contributes to the enhancement of virus entry in the susceptible cells via the interaction of the Fcγ of the antibody–virus complex with the Fcγ receptor in the cell membrane, triggering endocytosis (actin- or clathrin-mediated), facilitating the first steps in viral replication. (<b>B</b>) Intrinsic ADE, which results in increased viral production by targeting different pathways, including downregulation of TLR signaling, inhibition of type I interferon, and a skewed immune activation favoring Th2 response.</p> Full article ">Figure 3
<p>Cytokine storm seen in severe dengue infection. During a secondary heterologous infection, a robust immune response is generated, inducing the production of biological mediators including cytokine, chemokine, and other soluble factors from different immune cells as a consequence of complex interactions between the virus and host factors. These mediators promote vascular permeability resulting in an increase in plasma leakage.</p> Full article ">Figure 4
<p>Exosomes during dengue virus infection. DENV infection induces the secretion of small membrane vesicles (known as exosomes) from infected cells to the extracellular space. These exosomes perform different functions such as the induction of cytokine production, apoptosis, endothelial cell activation, viral dissemination, inhibition of viral entry, and interference with ADE phenomenon depending on its content and cell origin, contributing to disease severity or protection against infection.</p> Full article ">
16 pages, 5382 KiB  
Article
A Characterization and an Evolutionary and a Pathogenicity Analysis of Reassortment H3N2 Avian Influenza Virus in South China in 2019–2020
by Tengfei Liu, Yuhao Huang, Shumin Xie, Lingyu Xu, Junhong Chen, Wenbao Qi, Ming Liao and Weixin Jia
Viruses 2022, 14(11), 2574; https://doi.org/10.3390/v14112574 - 21 Nov 2022
Cited by 3 | Viewed by 2923
Abstract
Seasonal H3N2 influenza virus has always been a potential threat to public health. The reassortment of the human and avian H3N2 influenza viruses has resulted in major influenza outbreaks, which have seriously damaged human life and health. To assess the possible threat of [...] Read more.
Seasonal H3N2 influenza virus has always been a potential threat to public health. The reassortment of the human and avian H3N2 influenza viruses has resulted in major influenza outbreaks, which have seriously damaged human life and health. To assess the possible threat of the H3N2 avian influenza virus to human health, we performed whole-genome sequencing and genetic evolution analyses on 10 H3N2 field strains isolated from different hosts and regions in 2019–2020 and selected representative strains for pathogenicity tests on mice. According to the results, the internal gene cassettes of nine strains had not only undergone reassortment with the H1, H2, H4, H6, and H7 subtypes, which circulate in poultry and mammals, but also with H10N8, which circulates in wild birds in the natural environment. Three reassorted strains were found to be pathogenic to mice, of these one strain harboring MP from H10N8 showed a stronger virulence in mice. This study indicates that reassorted H3N2 AIVs may cross the host barrier to infect mammals and humans, thereby, necessitating persistent surveillance of H3N2 AIVs. Full article
(This article belongs to the Section Animal Viruses)
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Figure 1
<p>Phylogenetic analysis of HA gene of H3Ny and NA genes of H<sub>X</sub>N2 subtypes. Phylogenetic tree of (<b>a</b>) HA gene of H3Ny AIVs; (<b>b</b>) N2 gene of HxN2 AIVs. Phylogenetic analysis was performed three times using the maximum likelihood (ML) method in IQ-TREE under the GTR + F + G4 model with 5000 bootstrap replications. Reference sequences were downloaded from the available databases. The phylogenetic tree of the HA was beautified according to the host, subtype and geographic information and that of NA according to the subtype. The turquoise color represents the isolates in this study.</p> Full article ">Figure 2
<p>Phylogenetic analysis of internal genes of H3N2-subtype AIVs isolated from 2019 to 2020 using the maximum likelihood method. The phylogenetic trees of PB2 (<b>a</b>), PB1 (<b>b</b>), PA (<b>c</b>), NP (<b>d</b>), MP (<b>e</b>) and NS (<b>f</b>) were obtained as a midpoint-root tree. Phylogenetic analysis was performed three times using the maximum likelihood (ML) method in IQ-TREE under the GTR + F + G4 model with 5000 bootstrap replications. Reference sequences were downloaded from the available databases. The turquoise color represents the isolates in this study. Light yellow2, gray, orange red 1, med spring green, gold, tan 1, violet, slate blue 1, coral 1 and snow 1 represent H1, H2, H3N2, H3, H4, H5, H7, H9, H10, and H11, respectively.</p> Full article ">Figure 2 Cont.
<p>Phylogenetic analysis of internal genes of H3N2-subtype AIVs isolated from 2019 to 2020 using the maximum likelihood method. The phylogenetic trees of PB2 (<b>a</b>), PB1 (<b>b</b>), PA (<b>c</b>), NP (<b>d</b>), MP (<b>e</b>) and NS (<b>f</b>) were obtained as a midpoint-root tree. Phylogenetic analysis was performed three times using the maximum likelihood (ML) method in IQ-TREE under the GTR + F + G4 model with 5000 bootstrap replications. Reference sequences were downloaded from the available databases. The turquoise color represents the isolates in this study. Light yellow2, gray, orange red 1, med spring green, gold, tan 1, violet, slate blue 1, coral 1 and snow 1 represent H1, H2, H3N2, H3, H4, H5, H7, H9, H10, and H11, respectively.</p> Full article ">Figure 3
<p>Bayesian Markov chain Monte Carlo method tree of MP gene of isolated H3N2 avian influenza viruses. Blue node bars represent 95% credible intervals of lineage divergence times, and diamonds represent posteriors of every node. The sequence of H3N2 viruses isolated in this study are in red, and viral sequences named in black were downloaded from the databases.</p> Full article ">Figure 4
<p>Nucleotide substitution rates for each of the eight segments shown as mean substitution rate for each gene, with the 95% lower and upper HPD values presented as error bars.</p> Full article ">Figure 5
<p>Five-week-old female (SPF) BALB/C-mice were inoculated with 10<sup>6</sup>EID<sub>50</sub> of the test viruses in a 50 μL volume; (<b>a</b>) survival rates of mice (mice that lost more than 20% of their body weights were regarded as dead). (<b>b</b>) Changes in body weights of mice; (<b>c</b>) horizontal dashed line indicates the lower limit of detection. Each bar represents the virus titer of the four strain replications in the brains, spleens, lungs and kidneys of infected mice.</p> Full article ">
12 pages, 1072 KiB  
Article
Change in Nutritional and Biochemical Status in People Living with HIV-1 on Antiretroviral Therapy
by Ranilda Gama de Souza, Sandra Souza Lima, Andresa Corrêa Pinto, Jacqueline Silva Souza, Tuane Carolina Ferreira Moura, Ednelza da Silva Graça Amoras, Luiz Fernando Almeida Machado, João Farias Guerreiro, Antonio Carlos Rosário Vallinoto, Maria Alice Freitas Queiroz and Ricardo Ishak
Viruses 2022, 14(11), 2573; https://doi.org/10.3390/v14112573 - 20 Nov 2022
Cited by 3 | Viewed by 1972
Abstract
Antiretroviral therapy (ART) improves the quality of life of people living with HIV-1 (PLHIV) and reduces the mortality rate, but some individuals may develop metabolic abnormalities. This study evaluated changes in the nutritional status and biochemistry of PLHIV on antiretroviral therapy in a [...] Read more.
Antiretroviral therapy (ART) improves the quality of life of people living with HIV-1 (PLHIV) and reduces the mortality rate, but some individuals may develop metabolic abnormalities. This study evaluated changes in the nutritional status and biochemistry of PLHIV on antiretroviral therapy in a cohort that had not previously received ART and to follow up these individuals for 24 months after starting treatment. The initial cohort consisted of 110 individuals and ended with 42 people, assessed by a physical examination. A biochemical assay was performed using the colorimetric enzyme reaction technique, the proviral load was detected by qPCR and the quantification of the CD4/CD8 T lymphocytes was conducted by flow cytometry. PLHIV had increased levels of total cholesterol, LDL, triglycerides, ALT, urea and creatinine after 24 months of ART use (p < 0.05). In the assessment of the nutritional status, PLHIV had increased measures of Triciptal Skinfold, body mass index and arm circumference after the use of ART (p < 0.05). The viral load levels decreased and the CD4 levels increased after 24 months of ART use (p < 0.05). The change in the nutritional status in PLHIV on antiretroviral therapy seems to be a slow process, occurring in the long term, therefore, there is the need for a constant evaluation of these people to identify patients who need a nutritional intervention. Full article
(This article belongs to the Special Issue State-of-the-Art HIV and HTLV Research in Latin America)
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Figure 1

Figure 1
<p>Comparison of the biochemical levels (<b>A</b>) total cholesterol, (<b>B</b>) LDL, (<b>C</b>) HDL, (<b>D</b>) triglycerides, (<b>E</b>) CD4<sup>+</sup> T lymphocytes, (<b>F</b>) ALT, (<b>G</b>) AST, (<b>H</b>) urea and (<b>I</b>) creatinine of individuals with HIV1 between the periods evaluated. T0: initial assessment, before starting ART; T1: evaluation after 12 months of ART; T2: evaluation after 24 months of ART. Reference values: total cholesterol: &lt;200 mg/dL; HDL:&gt; 45 mg/dL; LDL: &lt;110 mg/dL; triglycerides: &lt;150 mg/dL; AST: 5 a 37 U/L; ALT: 7 a 41 U/L; urea: 20 a 40 mg/dL; creatinine 0,6 a 1,3 mg/dL.</p> Full article ">Figure 2
<p>Comparison of the nutritional parameters (<b>A</b>) tricipital skinfold – TS, (<b>B</b>) arm circumference – AC and (<b>C</b>) body mass index – BMI of individuals with HIV1 between the periods evaluated. T0: initial assessment, before starting ART; T1: evaluation after 12 months of ART; T2: evaluation after 24 months of ART.</p> Full article ">
27 pages, 8463 KiB  
Article
Discovery, Genomic Sequence Characterization and Phylogenetic Analysis of Novel RNA Viruses in the Turfgrass Pathogenic Colletotrichum spp. in Japan
by Islam Hamim, Syun-ichi Urayama, Osamu Netsu, Akemi Tanaka, Tsutomu Arie, Hiromitsu Moriyama and Ken Komatsu
Viruses 2022, 14(11), 2572; https://doi.org/10.3390/v14112572 - 20 Nov 2022
Cited by 7 | Viewed by 2266
Abstract
Turfgrass used in various areas of the golf course has been found to present anthracnose disease, which is caused by Colletotrichum spp. To obtain potential biological agents, we identified four novel RNA viruses and obtained full-length viral genomes from turfgrass pathogenic Colletotrichum spp. [...] Read more.
Turfgrass used in various areas of the golf course has been found to present anthracnose disease, which is caused by Colletotrichum spp. To obtain potential biological agents, we identified four novel RNA viruses and obtained full-length viral genomes from turfgrass pathogenic Colletotrichum spp. in Japan. We characterized two novel dsRNA partitiviruses: Colletotrichum associated partitivirus 1 (CaPV1) and Colletotrichum associated partitivirus 2 (CaPV2), as well as two negative single-stranded (ss) RNA viruses: Colletotrichum associated negative-stranded RNA virus 1 (CaNSRV1) and Colletotrichum associated negative-stranded RNA virus 2 (CaNSRV2). Using specific RT-PCR assays, we confirmed the presence of CaPV1, CaPV2 and CaNSRV1 in dsRNAs from original and sub-isolates of Colletotrichum sp. MBCT-264, as well as CaNSRV2 in dsRNAs from original and sub-isolates of Colletotrichum sp. MBCT-288. This is the first time mycoviruses have been discovered in turfgrass pathogenic Colletotrichum spp. in Japan. CaPV1 and CaPV2 are new members of the newly proposed genus “Zetapartitivirus” and genus Alphapartitivirus, respectively, in the family Partitiviridae, according to genomic characterization and phylogenetic analysis. Negative sense ssRNA viruses CaNSRV1 and CaNSRV2, on the other hand, are new members of the family Phenuiviridae and the proposed family “Mycoaspirividae”, respectively. These findings reveal previously unknown RNA virus diversity and evolution in turfgrass pathogenic Colletotrichum spp. Full article
(This article belongs to the Special Issue Diversity and Coinfections of Plant or Fungal Viruses)
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Figure 1

Figure 1
<p>Agarose gel electrophoresis of dsRNA isolated from nine Colletotrichum isolates. As a positive control, we used dsRNA from Magnaporthe oryzae chryso-virus 1 (MoCV1) A infected Magnaporthe oryzae isolate. Clear and distinct dsRNA bands were found in two Collectotrichum isolates, MBCT-264 and MBCT-288.</p> Full article ">Figure 2
<p>(<b>A</b>) Schematic representation of the CaPV1 genome structure. ORF1 encodes an RdRp and ORF2 encodes a hypothetical protein (comparable to coat protein, CP). (<b>B</b>) Comparison of the 5′- and 3′-terminal sequences of dsRNA-1, dsRNA-2 and dsRNA-3. (<b>C</b>) Multiple alignment of the sequences of the conserved motifs in the RdRps of CaPV1 and other partitiviruses. The six conserved motifs are indicated by the colored boxes. (<b>D</b>) Several conserved regions were discovered in a multiple sequence alignment of hypothetical proteins encoded by CaPV1 and closely related viruses from the Partitiviridae family. (<b>E</b>) Alignments of ORF4 product of CaPV1 with the hypothetical proteins encoded by dsRNA-3 of CgPV1 (QED88098) and PvLaPV4 (QHD64811).</p> Full article ">Figure 2 Cont.
<p>(<b>A</b>) Schematic representation of the CaPV1 genome structure. ORF1 encodes an RdRp and ORF2 encodes a hypothetical protein (comparable to coat protein, CP). (<b>B</b>) Comparison of the 5′- and 3′-terminal sequences of dsRNA-1, dsRNA-2 and dsRNA-3. (<b>C</b>) Multiple alignment of the sequences of the conserved motifs in the RdRps of CaPV1 and other partitiviruses. The six conserved motifs are indicated by the colored boxes. (<b>D</b>) Several conserved regions were discovered in a multiple sequence alignment of hypothetical proteins encoded by CaPV1 and closely related viruses from the Partitiviridae family. (<b>E</b>) Alignments of ORF4 product of CaPV1 with the hypothetical proteins encoded by dsRNA-3 of CgPV1 (QED88098) and PvLaPV4 (QHD64811).</p> Full article ">Figure 2 Cont.
<p>(<b>A</b>) Schematic representation of the CaPV1 genome structure. ORF1 encodes an RdRp and ORF2 encodes a hypothetical protein (comparable to coat protein, CP). (<b>B</b>) Comparison of the 5′- and 3′-terminal sequences of dsRNA-1, dsRNA-2 and dsRNA-3. (<b>C</b>) Multiple alignment of the sequences of the conserved motifs in the RdRps of CaPV1 and other partitiviruses. The six conserved motifs are indicated by the colored boxes. (<b>D</b>) Several conserved regions were discovered in a multiple sequence alignment of hypothetical proteins encoded by CaPV1 and closely related viruses from the Partitiviridae family. (<b>E</b>) Alignments of ORF4 product of CaPV1 with the hypothetical proteins encoded by dsRNA-3 of CgPV1 (QED88098) and PvLaPV4 (QHD64811).</p> Full article ">Figure 3
<p>Phylogenetic analyses of RdRps (<b>A</b>) and CPs (<b>B</b>) of CaPV1, CaPV2 and related partiti-viruses using the function “build” of ETE3 3.1.2 [<a href="#B39-viruses-14-02572" class="html-bibr">39</a>] as implemented on the GenomeNet (<a href="https://www.genome.jp/tools/ete/" target="_blank">https://www.genome.jp/tools/ete/</a>, accessed on 2 September 2022). Alignment was performed with MAFFT v6.861b with the default options [<a href="#B40-viruses-14-02572" class="html-bibr">40</a>]. ML tree was inferred using IQ-TREE 1.5.5 ran with ModelFinder and tree reconstruction [<a href="#B41-viruses-14-02572" class="html-bibr">41</a>]. Best-fit models, according to BIC, were VT+F+R5 and VT+F+I+G4 for RdRp and CP phylogenetic trees, respectively. Tree branches were tested by SH-like aLRT with 1000 replicates. Different members of the partiti-virus genera, including Alpha-, Beta-, Delta-, Gamma- and the proposed Epsilonpartitivirus were included in the analyses. Viruses found in this study were marked with a circle. Bootstrap values are indicated at the branches. The scale bar (lower left) represents the genetic distance of 0.50 for the phylogenetic tree of partitiviral RdRps (<b>A</b>) or CPs (<b>B</b>). Full names and GenBank accession numbers of the viruses listed in <a href="#app1-viruses-14-02572" class="html-app">Tables S1–S7</a>.</p> Full article ">Figure 3 Cont.
<p>Phylogenetic analyses of RdRps (<b>A</b>) and CPs (<b>B</b>) of CaPV1, CaPV2 and related partiti-viruses using the function “build” of ETE3 3.1.2 [<a href="#B39-viruses-14-02572" class="html-bibr">39</a>] as implemented on the GenomeNet (<a href="https://www.genome.jp/tools/ete/" target="_blank">https://www.genome.jp/tools/ete/</a>, accessed on 2 September 2022). Alignment was performed with MAFFT v6.861b with the default options [<a href="#B40-viruses-14-02572" class="html-bibr">40</a>]. ML tree was inferred using IQ-TREE 1.5.5 ran with ModelFinder and tree reconstruction [<a href="#B41-viruses-14-02572" class="html-bibr">41</a>]. Best-fit models, according to BIC, were VT+F+R5 and VT+F+I+G4 for RdRp and CP phylogenetic trees, respectively. Tree branches were tested by SH-like aLRT with 1000 replicates. Different members of the partiti-virus genera, including Alpha-, Beta-, Delta-, Gamma- and the proposed Epsilonpartitivirus were included in the analyses. Viruses found in this study were marked with a circle. Bootstrap values are indicated at the branches. The scale bar (lower left) represents the genetic distance of 0.50 for the phylogenetic tree of partitiviral RdRps (<b>A</b>) or CPs (<b>B</b>). Full names and GenBank accession numbers of the viruses listed in <a href="#app1-viruses-14-02572" class="html-app">Tables S1–S7</a>.</p> Full article ">Figure 4
<p>(<b>A</b>) Schematic representation of the CaPV2 genome organization. All the genome segments contain a single ORF (rectangular box) except dsRNA-3. dsRNA-1 encoded an RNA-dependent RNA polymerase (RdRp) and dsRNA-2 encoded a capsid protein (CP). The untranslated regions (UTRs) at both termini of dsRNA segments are also shown. (<b>B</b>) Sequence alignment of the respective 5′ UTRs between dsRNA-1 and dsRNA-2 of CaPV2. (<b>C</b>) Predicted secondary structure of the 5′ UTR sequences of CaPV2 dsRNA-1 (left) and dsRNA-2 (right). (<b>D</b>) Multiple sequence alignment analysis of RdRp from different members of the genus <span class="html-italic">Alphapartitivirus</span> revealed that CaPV2 RdRp contains all three conserved motifs (motifs A–C) found in the catalytic palm subdomain and these motifs are well conserved among dsRNA viruses. The conserved motifs are indicated by the colored boxes.</p> Full article ">Figure 4 Cont.
<p>(<b>A</b>) Schematic representation of the CaPV2 genome organization. All the genome segments contain a single ORF (rectangular box) except dsRNA-3. dsRNA-1 encoded an RNA-dependent RNA polymerase (RdRp) and dsRNA-2 encoded a capsid protein (CP). The untranslated regions (UTRs) at both termini of dsRNA segments are also shown. (<b>B</b>) Sequence alignment of the respective 5′ UTRs between dsRNA-1 and dsRNA-2 of CaPV2. (<b>C</b>) Predicted secondary structure of the 5′ UTR sequences of CaPV2 dsRNA-1 (left) and dsRNA-2 (right). (<b>D</b>) Multiple sequence alignment analysis of RdRp from different members of the genus <span class="html-italic">Alphapartitivirus</span> revealed that CaPV2 RdRp contains all three conserved motifs (motifs A–C) found in the catalytic palm subdomain and these motifs are well conserved among dsRNA viruses. The conserved motifs are indicated by the colored boxes.</p> Full article ">Figure 5
<p>(<b>A</b>) Schematic representation of the CaNSRV1 genome organization. (<b>B</b>) The presence of the six conserved motifs, which included pre-motif A and motifs A–E, was verified by aa alignments in RdRps of CaNSRV1 and other related (-ss) RNA viruses, which match to highly conserved sections of the order Bunyavirales RdRps [<a href="#B34-viruses-14-02572" class="html-bibr">34</a>,<a href="#B47-viruses-14-02572" class="html-bibr">47</a>,<a href="#B48-viruses-14-02572" class="html-bibr">48</a>,<a href="#B49-viruses-14-02572" class="html-bibr">49</a>]. CaNSRV1 comprises the motifs A (DATKWC), B (QGILHYTSS), C (SDD), D (KS) and E (E(F/Y)xS). E is a tetrapeptide motif found in the RdRp of segmented negative-sense RNA viruses. Furthermore, three basic residues in pre-motif A: K, R and R/K, as well as a glutamic acid. The conserved motifs are indicated by the colored boxes. (E) downstream of pre-motif A, were shown to be conserved in CaNSRV1 and related viruses RdRps. (<b>C</b>) N-terminal region of CaNSRV1 had an endonuclease conserved motif that was engaged in cap-snatching, a method adopted by many negative-stranded viruses to translate viral proteins by utilizing capped terminal ends of host mRNAs. The ExT domain conserved in the RdRp of most bunyaviruses is also found in CaNSRV1. (<b>D</b>) Alignment and phylogenetic reconstructions of RdRps of CaNSRV-1 and selected viruses were performed using the function “build” of ETE3 3.1.2 [<a href="#B39-viruses-14-02572" class="html-bibr">39</a>] as implemented on the GenomeNet (<a href="https://www.genome.jp/tools/ete/" target="_blank">https://www.genome.jp/tools/ete/</a>, accessed on 2 September 2022). Alignment was performed with MAFFT v6.861b with the default options [<a href="#B40-viruses-14-02572" class="html-bibr">40</a>]. ML tree was inferred using IQ-TREE 1.5.5 ran with ModelFinder and tree reconstruction [<a href="#B41-viruses-14-02572" class="html-bibr">41</a>], where best-fit model according to BIC was LG+F+R5. Tree branches were tested by SH-like aLRT with 1000 replicates. CaNSRV1 was marked with a circle. CaNSRV1 is a member of a superclade that also comprises the coguvirus clade, which is made up of viruses from the coguvirus genus. The scale bar (lower left) represents the genetic distance of 0.50 for the phylogenetic tree of RdRps.</p> Full article ">Figure 5 Cont.
<p>(<b>A</b>) Schematic representation of the CaNSRV1 genome organization. (<b>B</b>) The presence of the six conserved motifs, which included pre-motif A and motifs A–E, was verified by aa alignments in RdRps of CaNSRV1 and other related (-ss) RNA viruses, which match to highly conserved sections of the order Bunyavirales RdRps [<a href="#B34-viruses-14-02572" class="html-bibr">34</a>,<a href="#B47-viruses-14-02572" class="html-bibr">47</a>,<a href="#B48-viruses-14-02572" class="html-bibr">48</a>,<a href="#B49-viruses-14-02572" class="html-bibr">49</a>]. CaNSRV1 comprises the motifs A (DATKWC), B (QGILHYTSS), C (SDD), D (KS) and E (E(F/Y)xS). E is a tetrapeptide motif found in the RdRp of segmented negative-sense RNA viruses. Furthermore, three basic residues in pre-motif A: K, R and R/K, as well as a glutamic acid. The conserved motifs are indicated by the colored boxes. (E) downstream of pre-motif A, were shown to be conserved in CaNSRV1 and related viruses RdRps. (<b>C</b>) N-terminal region of CaNSRV1 had an endonuclease conserved motif that was engaged in cap-snatching, a method adopted by many negative-stranded viruses to translate viral proteins by utilizing capped terminal ends of host mRNAs. The ExT domain conserved in the RdRp of most bunyaviruses is also found in CaNSRV1. (<b>D</b>) Alignment and phylogenetic reconstructions of RdRps of CaNSRV-1 and selected viruses were performed using the function “build” of ETE3 3.1.2 [<a href="#B39-viruses-14-02572" class="html-bibr">39</a>] as implemented on the GenomeNet (<a href="https://www.genome.jp/tools/ete/" target="_blank">https://www.genome.jp/tools/ete/</a>, accessed on 2 September 2022). Alignment was performed with MAFFT v6.861b with the default options [<a href="#B40-viruses-14-02572" class="html-bibr">40</a>]. ML tree was inferred using IQ-TREE 1.5.5 ran with ModelFinder and tree reconstruction [<a href="#B41-viruses-14-02572" class="html-bibr">41</a>], where best-fit model according to BIC was LG+F+R5. Tree branches were tested by SH-like aLRT with 1000 replicates. CaNSRV1 was marked with a circle. CaNSRV1 is a member of a superclade that also comprises the coguvirus clade, which is made up of viruses from the coguvirus genus. The scale bar (lower left) represents the genetic distance of 0.50 for the phylogenetic tree of RdRps.</p> Full article ">Figure 5 Cont.
<p>(<b>A</b>) Schematic representation of the CaNSRV1 genome organization. (<b>B</b>) The presence of the six conserved motifs, which included pre-motif A and motifs A–E, was verified by aa alignments in RdRps of CaNSRV1 and other related (-ss) RNA viruses, which match to highly conserved sections of the order Bunyavirales RdRps [<a href="#B34-viruses-14-02572" class="html-bibr">34</a>,<a href="#B47-viruses-14-02572" class="html-bibr">47</a>,<a href="#B48-viruses-14-02572" class="html-bibr">48</a>,<a href="#B49-viruses-14-02572" class="html-bibr">49</a>]. CaNSRV1 comprises the motifs A (DATKWC), B (QGILHYTSS), C (SDD), D (KS) and E (E(F/Y)xS). E is a tetrapeptide motif found in the RdRp of segmented negative-sense RNA viruses. Furthermore, three basic residues in pre-motif A: K, R and R/K, as well as a glutamic acid. The conserved motifs are indicated by the colored boxes. (E) downstream of pre-motif A, were shown to be conserved in CaNSRV1 and related viruses RdRps. (<b>C</b>) N-terminal region of CaNSRV1 had an endonuclease conserved motif that was engaged in cap-snatching, a method adopted by many negative-stranded viruses to translate viral proteins by utilizing capped terminal ends of host mRNAs. The ExT domain conserved in the RdRp of most bunyaviruses is also found in CaNSRV1. (<b>D</b>) Alignment and phylogenetic reconstructions of RdRps of CaNSRV-1 and selected viruses were performed using the function “build” of ETE3 3.1.2 [<a href="#B39-viruses-14-02572" class="html-bibr">39</a>] as implemented on the GenomeNet (<a href="https://www.genome.jp/tools/ete/" target="_blank">https://www.genome.jp/tools/ete/</a>, accessed on 2 September 2022). Alignment was performed with MAFFT v6.861b with the default options [<a href="#B40-viruses-14-02572" class="html-bibr">40</a>]. ML tree was inferred using IQ-TREE 1.5.5 ran with ModelFinder and tree reconstruction [<a href="#B41-viruses-14-02572" class="html-bibr">41</a>], where best-fit model according to BIC was LG+F+R5. Tree branches were tested by SH-like aLRT with 1000 replicates. CaNSRV1 was marked with a circle. CaNSRV1 is a member of a superclade that also comprises the coguvirus clade, which is made up of viruses from the coguvirus genus. The scale bar (lower left) represents the genetic distance of 0.50 for the phylogenetic tree of RdRps.</p> Full article ">Figure 6
<p>(<b>A</b>) Schematic representation of the CaNSRV2 genome organization. (<b>B</b>) Sequence alignments of the terminal ends of CaNSRV2 RNA-1 and RNA-2. (<b>C</b>) The multiple alignment of the RdRp sequences of CaNSRV2 and the other related viruses, which indicated four conserved motifs: motif A(SLLLDIEGHNQSMQ), motif B (QLGGIEGWLNPLWTL), motif C (YSDD) and motif D (ADGIRADSTLKRL) and pre-motif A (KEREQKYEARLF). (<b>D</b>) Bipartite nuclear localization signals (NLSs) predicted in the RdRp protein of CaNSRV2. (<b>E</b>) Alignment and phylogenetic reconstructions were performed using the function “build” of ETE3 3.1.2 [<a href="#B39-viruses-14-02572" class="html-bibr">39</a>] as implemented on the GenomeNet (<a href="https://www.genome.jp/tools/ete/" target="_blank">https://www.genome.jp/tools/ete/</a>, accessed on 2 September 2022). Alignment was performed with MAFFT v6.861b with the default options [<a href="#B40-viruses-14-02572" class="html-bibr">40</a>]. ML tree was inferred using IQ-TREE 1.5.5, run with ModelFinder and tree reconstruction [<a href="#B41-viruses-14-02572" class="html-bibr">41</a>]. The best-fit model, according to BIC was LG+F+R5. Tree branches were tested by SH-like aLRT with 1000 replicates. CaNSRV2 was marked with a circle. The scale bar (lower left) represents the genetic distance of 1 for the phylogenetic tree of RdRps.</p> Full article ">Figure 6 Cont.
<p>(<b>A</b>) Schematic representation of the CaNSRV2 genome organization. (<b>B</b>) Sequence alignments of the terminal ends of CaNSRV2 RNA-1 and RNA-2. (<b>C</b>) The multiple alignment of the RdRp sequences of CaNSRV2 and the other related viruses, which indicated four conserved motifs: motif A(SLLLDIEGHNQSMQ), motif B (QLGGIEGWLNPLWTL), motif C (YSDD) and motif D (ADGIRADSTLKRL) and pre-motif A (KEREQKYEARLF). (<b>D</b>) Bipartite nuclear localization signals (NLSs) predicted in the RdRp protein of CaNSRV2. (<b>E</b>) Alignment and phylogenetic reconstructions were performed using the function “build” of ETE3 3.1.2 [<a href="#B39-viruses-14-02572" class="html-bibr">39</a>] as implemented on the GenomeNet (<a href="https://www.genome.jp/tools/ete/" target="_blank">https://www.genome.jp/tools/ete/</a>, accessed on 2 September 2022). Alignment was performed with MAFFT v6.861b with the default options [<a href="#B40-viruses-14-02572" class="html-bibr">40</a>]. ML tree was inferred using IQ-TREE 1.5.5, run with ModelFinder and tree reconstruction [<a href="#B41-viruses-14-02572" class="html-bibr">41</a>]. The best-fit model, according to BIC was LG+F+R5. Tree branches were tested by SH-like aLRT with 1000 replicates. CaNSRV2 was marked with a circle. The scale bar (lower left) represents the genetic distance of 1 for the phylogenetic tree of RdRps.</p> Full article ">Figure 6 Cont.
<p>(<b>A</b>) Schematic representation of the CaNSRV2 genome organization. (<b>B</b>) Sequence alignments of the terminal ends of CaNSRV2 RNA-1 and RNA-2. (<b>C</b>) The multiple alignment of the RdRp sequences of CaNSRV2 and the other related viruses, which indicated four conserved motifs: motif A(SLLLDIEGHNQSMQ), motif B (QLGGIEGWLNPLWTL), motif C (YSDD) and motif D (ADGIRADSTLKRL) and pre-motif A (KEREQKYEARLF). (<b>D</b>) Bipartite nuclear localization signals (NLSs) predicted in the RdRp protein of CaNSRV2. (<b>E</b>) Alignment and phylogenetic reconstructions were performed using the function “build” of ETE3 3.1.2 [<a href="#B39-viruses-14-02572" class="html-bibr">39</a>] as implemented on the GenomeNet (<a href="https://www.genome.jp/tools/ete/" target="_blank">https://www.genome.jp/tools/ete/</a>, accessed on 2 September 2022). Alignment was performed with MAFFT v6.861b with the default options [<a href="#B40-viruses-14-02572" class="html-bibr">40</a>]. ML tree was inferred using IQ-TREE 1.5.5, run with ModelFinder and tree reconstruction [<a href="#B41-viruses-14-02572" class="html-bibr">41</a>]. The best-fit model, according to BIC was LG+F+R5. Tree branches were tested by SH-like aLRT with 1000 replicates. CaNSRV2 was marked with a circle. The scale bar (lower left) represents the genetic distance of 1 for the phylogenetic tree of RdRps.</p> Full article ">
9 pages, 1022 KiB  
Article
Comparisons of Viral Etiology and Outcomes of Hepatocellular Carcinoma Undergoing Liver Resection between Taiwan and Vietnam
by Song-Huy Nguyen-Dinh, Wei-Feng Li, Yueh-Wei Liu, Chih-Chi Wang, Yen-Hao Chen, Jing-Houng Wang and Chao-Hung Hung
Viruses 2022, 14(11), 2571; https://doi.org/10.3390/v14112571 - 20 Nov 2022
Viewed by 1891
Abstract
Epidemiologic data have suggested that etiologic variations of hepatocellular carcinoma (HCC) exist in different geographic areas, and might be associated with different outcomes. We compared the viral etiology, clinicopathological characteristics and surgical outcomes between 706 Taiwanese and 1704 Vietnamese patients with HCC undergoing [...] Read more.
Epidemiologic data have suggested that etiologic variations of hepatocellular carcinoma (HCC) exist in different geographic areas, and might be associated with different outcomes. We compared the viral etiology, clinicopathological characteristics and surgical outcomes between 706 Taiwanese and 1704 Vietnamese patients with HCC undergoing liver resection. Vietnamese patients had a significantly higher ratio of hepatitis B virus (HBV) (p < 0.001) and a lower ratio of hepatitis C virus (HCV) (p < 0.001) and non-B non-C than Taiwanese patients. Among patients with HBV or non-B non-C, the mean age was younger in Vietnam than in Taiwan (p < 0.001, p = 0.001, respectively). The HCC patients in Vietnam had significantly higher serum alpha-fetoprotein (AFP) levels (p < 0.001), larger tumors (p < 0.001), and a higher ratio of macrovascular invasion (p < 0.001) and extrahepatic metastasis (p < 0.001), compared to those in Taiwan. Patients treated in Vietnam had a higher tumor recurrent rate (p < 0.001), but no difference in overall survival was found between both groups. In subgroup analysis, the recurrent rate of HCC was the highest in patients with dual HBV/HCV, followed by HCV or HBV, and non-B non-C (p < 0.001). In conclusion, although the viral etiology and clinicopathological characteristics of HCC differed, postoperative overall survival was comparable between patients in Taiwan and Vietnam. Full article
(This article belongs to the Special Issue Hepatitis-Associated Liver Cancer)
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<p>(<b>A</b>). Recurrence-free survival after resection of HCC, stratified by site of treatment. (<b>B</b>). Overall survival after resection of HCC, stratified by site of treatment.</p> Full article ">Figure 2
<p>Recurrence-free survival after resection of HCC, stratified by viral etiology.</p> Full article ">
12 pages, 571 KiB  
Article
Recent Information on Pan-Genotypic Direct-Acting Antiviral Agents for HCV in Chronic Kidney Disease
by Fabrizio Fabrizi, Federica Tripodi, Roberta Cerutti, Luca Nardelli, Carlo M. Alfieri, Maria F. Donato and Giuseppe Castellano
Viruses 2022, 14(11), 2570; https://doi.org/10.3390/v14112570 - 20 Nov 2022
Cited by 3 | Viewed by 1979
Abstract
Background: Hepatitis C virus (HCV) is still common in patients with chronic kidney disease. It has been recently discovered that chronic HCV is a risk factor for increased incidence of CKD in the adult general population. According to a systematic review with a [...] Read more.
Background: Hepatitis C virus (HCV) is still common in patients with chronic kidney disease. It has been recently discovered that chronic HCV is a risk factor for increased incidence of CKD in the adult general population. According to a systematic review with a meta-analysis of clinical studies, pooling results of longitudinal studies (n = 2,299,134 unique patients) demonstrated an association between positive anti-HCV serologic status and increased incidence of CKD; the summary estimate for adjusted HR across the surveys was 1.54 (95% CI, 1.26; 1.87), (p < 0.0001). The introduction of direct-acting antiviral drugs (DAAs) has caused a paradigm shift in the management of HCV infection; recent guidelines recommend pan-genotypic drugs (i.e., drugs effective on all HCV genotypes) as the first-choice therapy for HCV, and these promise to be effective and safe even in the context of chronic kidney disease. Aim: The purpose of this narrative review is to show the most important data on pan-genotypic DAAs in advanced CKD (CKD stage 4/5). Methods: We recruited studies by electronic databases and grey literature. Numerous key-words (‘Hepatitis C’ AND ‘Chronic kidney disease’ AND ‘Pan-genotypic agents’, among others) were adopted. Results: The most important pan-genotypic combinations for HCV in advanced CKD are glecaprevir/pibrentasvir (GLE/PIB) and sofosbuvir/velpatasvir (SOF/VEL). Two clinical trials (EXPEDITION-4 and EXPEDITION-5) and some ‘real-world’ studies (n = 6) reported that GLE/PIB combinations in CKD stage 4/5 gave SVR12 rates ranging between 86 and 99%. We retrieved clinical trials (n = 1) and ‘real life’ studies (n = 6) showing the performance of SOF/VEL; according to our pooled analysis, the summary estimate of SVR rate was 100% in studies adopting SOF/VEL antiviral combinations. The drop-out rate (due to AEs) in patients on SOF/VEL ranged between 0 and 4.8%. Conclusions: Pan-genotypic combinations, such as GLE/PIB and SOF/VEL, appear effective and safe for HCV in advanced CKD, even if a limited number of studies with small sample sizes currently exist on this issue. Studies are under way to assess whether successful antiviral therapy with DAAs will translate into better survival in patients with advanced CKD. Full article
(This article belongs to the Special Issue Novel Antiviral Agents: Synthesis, Molecular Modelling Studies and Biological Investigation)
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<p>The impact of antiviral therapy on death rate in patients with advanced CKD: pooled adjusted RR according to fixed- and random-effects models.</p> Full article ">Figure 2
<p>Sofosbuvir: metabolism in advanced kidney impairment and structure.</p> Full article ">
12 pages, 4169 KiB  
Article
Effects of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Infection on the Surface Glycoprofiling of Porcine Pulmonary Microvascular Endothelial Cells
by Xiaoxiao Song, Yanmei Wu, Xianping Wu, Ge Hu and Tao Zhang
Viruses 2022, 14(11), 2569; https://doi.org/10.3390/v14112569 - 20 Nov 2022
Cited by 4 | Viewed by 1743
Abstract
Previously, our study has demonstrated that porcine pulmonary microvascular endothelial cells (PPMVECs) were susceptible to highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) and produced a significant non-specific immune response to it. The significance of microvascular endothelial glycocalyx is increasingly attracting attention, [...] Read more.
Previously, our study has demonstrated that porcine pulmonary microvascular endothelial cells (PPMVECs) were susceptible to highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) and produced a significant non-specific immune response to it. The significance of microvascular endothelial glycocalyx is increasingly attracting attention, and its rich carbohydrate components are not only important signaling molecules, but also remarkably influence the signaling of most proteins. Comprehending changes in the carbohydrate chains contributes to understanding cell functions. This study aimed to reveal the effects of HP-PRRSV infection on the surface carbohydrate chains of PPMVECs. PPMVECs were isolated and cultured in vitro and infected with HP-PRRSV HN and JXA1 strains. Scanning electron microscopy analysis indicated that at 48 h post-infection, some broken holes were in their cell membranes, and that the surface fibrous glycocalyx was obviously reduced or even disappeared. Lectin microarray analysis indicated that the fluorescence intensities of 8 and 7 lectin sites were significantly changed by the HP-PRRSV HN and JXA1 strains, respectively, among which there were 6 common lectin sites. The up-regulation of common lectins (RCA-I, LEL, and STL) and the down-regulation of common lectins (LCA, DSA, and PHA-E) were confirmed by lectin fluorescence staining and lectin flow cytometry, respectively. Together, the results show that the HP-PRRSV infection can induce the glycocalyx disruption of PPMVECs and their surface glycoprofiling changes, and that the poly-N-acetyllactosamine and complex N-glycan are the main up-regulated and down-regulated carbohydrate chains, respectively. Our findings may provide insights into revealing the pathogenesis of HP-PRRSV from the perspective of glycobiology. Full article
(This article belongs to the Special Issue State-of-the-Art Porcine Virus Research in China)
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<p>Microscopic morphology and FVIII immunofluorescence staining of PPMVECs. Bar = 50 μm. (<b>A</b>) Microscopic morphology. (<b>B</b>) Positive staining for FVIII. (<b>C</b>) Negative control of FVIII immunofluorescence staining.</p> Full article ">Figure 2
<p>Ultrastructure analysis of HP-PRRSV-infected PPMVECs by scanning electron microscopy. (Bar = 4 μm). (<b>A</b>) Normal control group. (<b>B</b>) HP-PRRSV HN-infected group. (<b>C</b>) HP-PRRSV JXA1-infected group.</p> Full article ">Figure 3
<p>Lectin information in the lectin microarray.</p> Full article ">Figure 4
<p>Scans of the lectin microarray. (<b>A</b>) Normal control group. (<b>B</b>) HP-PRRSV HN-infected group. (<b>C</b>) HP-PRRSV JXA1-infected group. (<b>D</b>) The layout of the lectin microarray (see <a href="#app1-viruses-14-02569" class="html-app">Table S1</a> for full names). Each lectin was spotted in triplicate per block. Yellow frames and white frames marked indicated the lectin sites significantly increased and decreased compared to the normal control group, respectively. The negative controls showed no positive signal. NC: negative control.</p> Full article ">Figure 5
<p>Fluorescence intensity analysis of the lectin microarray. (<b>A</b>) Normalized values of fluorescence signal intensities at positive lectin sites. (<b>B</b>) Heat map and hierarchical clustering of the positive lectins in PPMVECs. Samples are listed in columns and lectins in rows.</p> Full article ">Figure 6
<p>Differentially expressed lectin sites in both HP-PRRSV strain-infected PPMVECs. Statistical analysis was performed by Student’s <span class="html-italic">t</span>-test. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 7
<p>Lectin fluorescence staining of HP-PRRSV HN infected PPMVECs. Bar = 20 μm. (<b>A</b>) Photographs of lectin fluorescence staining. (<b>B</b>) Mean intensities of lectin fluorescence staining. (<b>C</b>) Comparison of the fold change in <sub>-</sub>fluorescence intensities between lectin microarrays and lectin fluorescence staining (IF). Statistical analysis was performed by Student’s <span class="html-italic">t</span>-test. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 8
<p>Lectin flow cytometry of HP-PRRSV JXA1-infected PPMVECs. (<b>A</b>) Flow cytometry histograms. (<b>B</b>) Mean fluorescence intensities. (<b>C</b>) Comparison of the fold changes in fluorescence intensities between lectin microarrays and lectin flow cytometry (FCM). Statistical analysis was performed by Student’s <span class="html-italic">t</span>-test. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">
7 pages, 730 KiB  
Brief Report
Features of Audio-Vestibular Deficit and 3D-FLAIR Temporal Bone MRI in Patients with Herpes Zoster Oticus
by Jiyeon Lee, Jin Woo Choi and Chang-Hee Kim
Viruses 2022, 14(11), 2568; https://doi.org/10.3390/v14112568 - 20 Nov 2022
Cited by 2 | Viewed by 1679
Abstract
Herpes zoster oticus (HZO) is characterized by otalgia and erythematous vesicles in the auricle or external auditory canal. Ramsay Hunt syndrome (RHS) can be diagnosed when facial nerve palsy is accompanied by these symptoms of HZO, and in this case, audio-vestibular symptoms such [...] Read more.
Herpes zoster oticus (HZO) is characterized by otalgia and erythematous vesicles in the auricle or external auditory canal. Ramsay Hunt syndrome (RHS) can be diagnosed when facial nerve palsy is accompanied by these symptoms of HZO, and in this case, audio-vestibular symptoms such as hearing loss or dizziness often develop. Recently, 3D-fluid-attenuated inversion recovery sequence (3D-FLAIR) magnetic resonance imaging (MRI) has been introduced in order to evaluate the inner ear structure pathology. The purpose of this study was to investigate the audio-vestibular characteristics in correlation with temporal bone MRI findings in HZO patients. From September 2018 to June 2022, 18 patients with HZO participated in the study. Thirteen patients (77%) showed high-signal intensity in the inner ear structures in 4 h post-contrast 3D-FLAIR images. In a bithermal caloric test, the lateral semicircular canal showed high signal intensity in 4 h post-contrast 3D-FLAIR images in 75% of patients with abnormal canal paresis. While the cochlea showed high signal intensity in 4 h post-contrast 3D-FLAIR images in 75% of patients with hearing loss, the vestibulo-cochlear nerve showed enhancement in post-contrast T1-weighted images in only 33% of patients with hearing loss. The present study demonstrates that audio-vestibular deficits are well-correlated with increased signal intensity of the inner ear endorgans in 4 h post contrast 3D-FLAIR MRI. Full article
(This article belongs to the Section Human Virology and Viral Diseases)
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<p>Internal auditory canal (IAC) MRI images of patients with Ramsay Hunt syndrome. Post-contrast 3D-FLAIR MRI of patient 12 (<b>a</b>), and post-contrast 3D T1-weighted images of patient 13 (<b>c</b>) and patient 9 (<b>b</b>). (<b>a</b>) High-signal intensities of cochlea (long arrow), posterior semicircular canal (small arrow), lateral semicircular canal (large arrow), and vestibule (arrowhead) were observed in the right ear. (<b>b</b>) The labyrinthine segment of the left facial nerve is enhanced (arrow). (<b>c</b>) In the left ear, contrast enhancement is observed in the vestibulo-cochlear nerve(small arrow) and the dura of the IAC (large arrow), respectively.</p> Full article ">
10 pages, 1384 KiB  
Article
Longitudinal Dynamics of HPV16 Antibodies in Saliva and Serum among Pregnant Women
by Tiina Pirttilä, Stina Syrjänen, Karolina Louvanto and Vuokko Loimaranta
Viruses 2022, 14(11), 2567; https://doi.org/10.3390/v14112567 - 20 Nov 2022
Cited by 2 | Viewed by 1631
Abstract
Oral infections with high-risk (hr)HPV genotypes are associated with a subset of head and neck squamous cell carcinomas. Oral hrHPV infections may result from having oral sex, but also from horizontal infection from mouth to mouth. In such cases, saliva can serve as [...] Read more.
Oral infections with high-risk (hr)HPV genotypes are associated with a subset of head and neck squamous cell carcinomas. Oral hrHPV infections may result from having oral sex, but also from horizontal infection from mouth to mouth. In such cases, saliva can serve as a vehicle for HPV transmission. Still, the prevalence and dynamics of salivary HPV antibodies in healthy non-vaccinated individuals are poorly known and the role of the salivary antibodies in protection from oral HPV infection is unclear. We used an ELISA assay to evaluate the dynamics and correlation of oral HPV16 infection and HPV16L1 and E7 specific antibody levels in saliva and serum samples among 39 women, 13 of which had persistent oral HPV16 infection. The women were mothers-to-be, sampled before delivery and followed up for 36 months postpartum. HPV16L1 IgG and sIgA antibodies were regularly detected in saliva. Antibody levels in serum remained stable during the 36-month follow-up, while antibody levels in saliva fluctuated. There was considerable individual variation in salivary HPV16L1 antibody levels, and some women had persistent oral HPV16 infection but no salivary antibodies. No differences in salivary HPV16L1 levels were found between the women with persistent or transient oral HPV16 infection. Full article
(This article belongs to the Special Issue HPV in the Head and Neck Region 2.0)
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<p>Dynamics of saliva and serum HPV16 L1 antibodies of women who had either persistent (blue lines) or transient (yellow lines) oral HPV16 infection. Mean: solid line, 95% confidence, upper and lower: dashed line. (<b>A</b>) saliva sIgA, (<b>B</b>) saliva IgG, (<b>C</b>) serum IgA, and (<b>D</b>) serum IgG.</p> Full article ">Figure 2
<p>IgA levels in saliva samples that were collected at the time point when HPV16 DNA was detected (positive, <span class="html-italic">n</span> = 73) or not detected (negative, <span class="html-italic">n</span> = 144) in the oral cavity.</p> Full article ">Figure 3
<p>Correlation of serum anti-HPV16L1 IgG (<b>A</b>), anti-HPV16L1 IgA (<b>B</b>), saliva anti-HPV16L1 IgG (<b>C</b>), and anti-HPV16L1 IgA (<b>D</b>) levels of the same individual at different time points. The Ig values were measured from saliva and serum samples collected during pregnancy and compared to values measured from samples collected 36 months after delivery.</p> Full article ">Figure 4
<p>(<b>A</b>–<b>C</b>) Individual saliva and serum (s)IgA/IgG HPV16L1 responses to oral and genital HPV16 infections. The columns represent genital (HPVgen) and oral HPV16DNA (HPVoral) DNA positivity at different time points, and the lines show the levels of serum and saliva antibodies, as indicated.</p> Full article ">
17 pages, 1391 KiB  
Article
Cell Type Variability in the Incorporation of Lipids in the Dengue Virus Virion
by Atitaya Hitakarun, Maia Kavanagh Williamson, Nathamon Yimpring, Wannapa Sornjai, Nitwara Wikan, Christopher J. Arthur, Julien Pompon, Andrew D. Davidson and Duncan R. Smith
Viruses 2022, 14(11), 2566; https://doi.org/10.3390/v14112566 - 19 Nov 2022
Cited by 5 | Viewed by 2323
Abstract
A lipid bilayer produced from the host membrane makes up around 20% of the weight of the dengue virus (DENV) virion and is crucial for virus entry. Despite its significance, the virion’s lipid composition is still poorly understood. In tandem with lipid profiles [...] Read more.
A lipid bilayer produced from the host membrane makes up around 20% of the weight of the dengue virus (DENV) virion and is crucial for virus entry. Despite its significance, the virion’s lipid composition is still poorly understood. In tandem with lipid profiles of the cells utilised to generate the virions, this work determined a partial lipid profile of DENV virions derived from two cell lines (C6/36 and LLC-MK2). The results showed distinctive profiles between the two cell types. In the mammalian LLC-MK2 cells, 30.8% (73/237 identified lipid species; 31 upregulated, 42 downregulated) of lipid species were altered in response to infection, whilst in insect C6/36 cells only 12.0% (25/208; 19 upregulated, 6 downregulated) of lipid species showed alterations in response to infection. For virions from LLC-MK2 cells, 14 lipids were detected specifically in virions with a further seven lipids being enriched (over mock controls). For virions from C6/36 cells, 43 lipids were detected that were not seen in mock preparations, with a further 16 being specifically enriched (over mock control). These results provide the first lipid description of DENV virions produced in mammalian and mosquito cells, as well as the lipid changes in the corresponding infected cells. Full article
(This article belongs to the Special Issue Omics of Virus-Host Interactions)
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<p>Detection of exosome protein markers in purified virus samples. The supernatants from mock and DENV infected C6/36 and LLC-MK<sub>2</sub> cells were separated through a 10/30/60% (<span class="html-italic">w</span>/<span class="html-italic">v</span>) discontinuous sucrose gradient. The exome markers ALIX and Hsp90 were detected in the purified preparations and in lysates of infected cells used for the preparations by western blot analysis.</p> Full article ">Figure 2
<p>Different cell types display distinct lipid profiles. The lipid composition of whole cell lysates from mock and DENV 2 infected C6/36 and LLC-MK<sub>2</sub> cells are shown grouped by LIPIDSMAPS class. Statistical significance determined by Welch’s 2-tailed <span class="html-italic">t</span>-test, * <span class="html-italic">p</span> ≤ 0.05, ** <span class="html-italic">p</span> ≤ 0.01. Data are from three biological replicates. Bars show average + S.D. Cer (Ceramide), SM (Sphingomyelin), S (Sulfoglycosphingolipids), PC (Phosphatidylcholine), LPC (Lyso-PC), PE (Phosphatidylethanolamine), LPE (Lyso-PE), PS (Phosphatidylserine), PG (Phosphatidylglycerol), PI (Phosphatidylinositol), LPI (Lyso-PI), CL (Cardiolipin), TG (Triglyceride).</p> Full article ">Figure 3
<p>Log2 fold change of lipid species in DENV 2 infected compared with mock control cells. Bubbles represent individual species analysed in biological triplicate and normalised by global mean scaling. Bubble size represents significance of the difference as determined by two-tailed Welch’s <span class="html-italic">t</span>-test. Lipid classes are shown as separate colours and arranged in increasing summed chain length from left to right. Total lipids shown C6/36 cells = 208 and LLC-MK<sub>2</sub> cells = 237. Significant changes in C6/36 cells = 25 and LLC-MK<sub>2</sub> cells = 73. Cer (Ceramide), SM (Sphingomyelin), S (Sulfoglycosphingolipids), PC (Phosphatidylcholine), LPC (Lyso-PC), PE (Phosphatidylethanolamine), LPE (Lyso-PE), PS (Phosphatidylserine), PG (Phosphatidylglycerol), PI (Phosphatidylinositol), LPI (Lyso-PI), CL (Cardiolipin), TG (Triglyceride).</p> Full article ">Figure 4
<p>Profile of lipid classes from virion preparations from C6/36 and LLC-MK<sub>2</sub> cells. The amounts of background lipids detected in mock preparations were subtracted from the amounts detected in virion preparations from DENV 2-infected C6/36 and LLC-MK<sub>2</sub> cells. Each lipid was represented as the % of total lipid and then grouped by LIPIDSMAPS class as shown. Classes in which the summed lipid % contribution &lt;0.2 of the total lipid are not shown to simplify the data set. Bars show average + S.D. Statistical significance determined by Welch’s 2-tailed <span class="html-italic">t</span>-test, * <span class="html-italic">p</span> ≤ 0.05. Data from three biological triplicates. Cer (Ceramide), SM (Sphingomyelin), PC (Phosphatidylcholine), PE (Phosphatidylethanolamine), LPE (Lyso-PE), PS (Phosphatidylserine), PI (Phosphatidylinositol).</p> Full article ">
13 pages, 293 KiB  
Conference Report
The Challenges and Opportunities of Next-Generation Rotavirus Vaccines: Summary of an Expert Meeting with Vaccine Developers
by Jessie Chen, Stephanie Grow, Miren Iturriza-Gómara, William P. Hausdorff, Alan Fix and Carl D. Kirkwood
Viruses 2022, 14(11), 2565; https://doi.org/10.3390/v14112565 - 19 Nov 2022
Cited by 8 | Viewed by 2502
Abstract
The 2nd Next Generation Rotavirus Vaccine Developers Meeting, sponsored by PATH and the Bill and Melinda Gates Foundation, was held in London, UK (7–8 June 2022), and attended by vaccine developers and researchers to discuss advancements in the development of next-generation rotavirus vaccines [...] Read more.
The 2nd Next Generation Rotavirus Vaccine Developers Meeting, sponsored by PATH and the Bill and Melinda Gates Foundation, was held in London, UK (7–8 June 2022), and attended by vaccine developers and researchers to discuss advancements in the development of next-generation rotavirus vaccines and to consider issues surrounding vaccine acceptability, introduction, and uptake. Presentations included updates on rotavirus disease burden, the impact of currently licensed oral vaccines, various platforms and approaches for next generation rotavirus vaccines, strategies for combination pediatric vaccines, and the value proposition for novel parenteral rotavirus vaccines. This report summarizes the information shared at the convening and poses various topics worthy of further exploration. Full article
(This article belongs to the Special Issue Viral Gastroenteritis 2022)
14 pages, 4947 KiB  
Article
Identification of Three Viruses Infecting Mulberry Varieties
by Lei Chen, Zi-Long Xu, Pei-Gang Liu, Yan Zhu, Tian-Bao Lin, Tian-Yan Li, Zhi-Qiang Lv and Jia Wei
Viruses 2022, 14(11), 2564; https://doi.org/10.3390/v14112564 - 19 Nov 2022
Cited by 2 | Viewed by 2266
Abstract
Viruses-mediated genome editing in plants is a powerful strategy to develop plant cultivars with important and novel agricultural traits. Mulberry alba is an important economic tree species that has been cultivated in China for more than 5000 years. So far, only a few [...] Read more.
Viruses-mediated genome editing in plants is a powerful strategy to develop plant cultivars with important and novel agricultural traits. Mulberry alba is an important economic tree species that has been cultivated in China for more than 5000 years. So far, only a few viruses have been identified from mulberry trees, and their application potential is largely unknown. Therefore, mining more virus resources from the mulberry tree can pave the way for the establishment of useful engineering tools. In this study, eight old mulberry plants were gathered in seven geographic areas for virome analysis. Based on transcriptome analysis, we discovered three viruses associated with mulberries: Citrus leaf blotch virus isolate mulberry alba 2 (CLBV-ML2), Mulberry-associated virga-like virus (MaVLV), and Mulberry-associated narna-like virus (MaNLV). The genome of CLBV-ML2 was completely sequenced and exhibited high homology with Citriviruses, considered to be members of the genus Citrivirus, while the genomes of MaVLV and MaNLV were nearly completed lacking the 5′ and 3′ termini sequences. We tentatively consider MaVLV to be members of the family Virgaviridae and MaNLV to be members of the genus Narnavirus based on the results of phylogenetic trees. The infection experiments showed that CLBV-ML2 could be detected in the inoculated seedlings of both N. benthamiana and Morus alba, while MaVLV could only be detected in N. benthamiana. All of the infected seedlings did not show obvious symptoms. Full article
(This article belongs to the Special Issue Next-Generation Sequencing in Plant Virology)
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<p>The heatmap shows the distribution of MaVLV, CLBV-ML2, and MaNLV in samples.</p> Full article ">Figure 2
<p>Predicted open reading frames (ORFs, with boxes), conserved motifs, domains, and viral proteins (different colors). (<b>A</b>) MaVLV, (<b>B</b>) CLBV-ML2, and (<b>C</b>) MaNTL. Viral methyltransferase (MT, pfam01660), viral helicase (HEL, pfam01443), and RNA-dependent RNA polymerase_2 (RdRp, pfam00978), 2OG-FeII Oxy superfamily (2OG-F, cl21496), peptidase C23 superfamily (Pep, cl05111), SP24 (pfam16504), coat protein (CP) and movement protein (MP). The unknown sequences of 5′ and 3′ UTRs are shown by black lines at both extremities, An: poly A tail.</p> Full article ">Figure 3
<p>Phylogenetic trees of (<b>A</b>) RdRp proteins constructed for representative members of the family <span class="html-italic">Virgaviridae</span> (40 tobamoviruses; 5 furoviruses; 6 pomoviruses; 2 pecluviruses; 3 tobraviruses and 32 unclassified virgaviridaes). (<b>B</b>) The pairwise identity plots of the RNA-dependent RNA polymerase amino acid sequences aligned by ClustalW and displayed by sequence demarcation using TBtools software. The trees were constructed using the maximum likelihood method, and the statistical significance of the branches was evaluated by bootstrap analysis (1000 replicates). Newly discovered viruses are marked in red. 40 tobamovirus are collapsed together. Relationship: pink background.</p> Full article ">Figure 3 Cont.
<p>Phylogenetic trees of (<b>A</b>) RdRp proteins constructed for representative members of the family <span class="html-italic">Virgaviridae</span> (40 tobamoviruses; 5 furoviruses; 6 pomoviruses; 2 pecluviruses; 3 tobraviruses and 32 unclassified virgaviridaes). (<b>B</b>) The pairwise identity plots of the RNA-dependent RNA polymerase amino acid sequences aligned by ClustalW and displayed by sequence demarcation using TBtools software. The trees were constructed using the maximum likelihood method, and the statistical significance of the branches was evaluated by bootstrap analysis (1000 replicates). Newly discovered viruses are marked in red. 40 tobamovirus are collapsed together. Relationship: pink background.</p> Full article ">Figure 4
<p>Phylogenetic trees constructed for representative members of the <span class="html-italic">Citrivirus</span> genus. (<b>A</b>) The whole sequences for phylogenetic trees. (<b>B</b>) The pairwise identity plots of the complete genomic nucleotide sequences aligned by ClustalW and displayed by sequence demarcation using TBtools software. The trees were constructed using the maximum likelihood method, and the statistical significance of the branches was evaluated by bootstrap analysis (1000 replicates). Newly discovered viruses are marked in red; relationship: pink background.</p> Full article ">Figure 5
<p>Phylogenetic trees of (<b>A</b>) RdRp proteins constructed for representative members of the family <span class="html-italic">Narnaviridae</span> (43 unclassified narnaviridaes; 32 unclassified narnaviruses; 2 narnaviruses). (<b>B</b>) The pairwise identity plots of the RNA-dependent RNA polymerase amino acid sequences aligned by ClustalW and displayed by sequence demarcation using TBtools software. The trees were constructed using the maximum likelihood method, and the statistical significance of the branches was evaluated by bootstrap analysis (1000 replicates). Newly discovered viruses are marked in red, unclassified narnaviridaes in black, unclassified narnaviruses in bule, and narnaviruses in green. Relationship: pink background.</p> Full article ">Figure 6
<p>The detection of RdRp protein and UTR sequences. (<b>A</b>) The expression of RdRp proteins of CLBV-ML2, MaVLV, and MaNLV by RT-PCR. (<b>B</b>) RACE amplification of CLBV-ML2. M: Marker. Red boxes: 5’UTR and 3’UTR target strips.</p> Full article ">Figure 7
<p>Symptoms on leaves of <span class="html-italic">N. benthamiana</span> and <span class="html-italic">Morus alba</span> infected by MaVLV, CLBV-ML2, and virus detection. (<b>A</b>) No symptoms on leaves of <span class="html-italic">N. benthamiana</span> and <span class="html-italic">Morus alba</span>. (<b>B</b>) RT-PCR detection of the infected leaves at 10 dpi. Lane 1 and 2: <span class="html-italic">N. benthamiana</span>, Lane 3: <span class="html-italic">Morus alba</span>, S: sample cDNA.</p> Full article ">Figure 7 Cont.
<p>Symptoms on leaves of <span class="html-italic">N. benthamiana</span> and <span class="html-italic">Morus alba</span> infected by MaVLV, CLBV-ML2, and virus detection. (<b>A</b>) No symptoms on leaves of <span class="html-italic">N. benthamiana</span> and <span class="html-italic">Morus alba</span>. (<b>B</b>) RT-PCR detection of the infected leaves at 10 dpi. Lane 1 and 2: <span class="html-italic">N. benthamiana</span>, Lane 3: <span class="html-italic">Morus alba</span>, S: sample cDNA.</p> Full article ">
11 pages, 1995 KiB  
Article
The Impact of Urbanization and Human Mobility on Seasonal Influenza in Northern China
by Jiao Yang, Xudong Guo, Ting Zhang, Qing Wang, Xingxing Zhang, Jin Yang, Shengjie Lai, Luzhao Feng and Weizhong Yang
Viruses 2022, 14(11), 2563; https://doi.org/10.3390/v14112563 - 19 Nov 2022
Cited by 5 | Viewed by 2278
Abstract
The intensity of influenza epidemics varies significantly from year to year among regions with similar climatic conditions and populations. However, the underlying mechanisms of the temporal and spatial variations remain unclear. We investigated the impact of urbanization and public transportation size on influenza [...] Read more.
The intensity of influenza epidemics varies significantly from year to year among regions with similar climatic conditions and populations. However, the underlying mechanisms of the temporal and spatial variations remain unclear. We investigated the impact of urbanization and public transportation size on influenza activity. We used 6-year weekly provincial-level surveillance data of influenza-like disease incidence (ILI) and viral activity in northern China. We derived the transmission potential of influenza for each epidemic season using the susceptible–exposed–infectious–removed–susceptible (SEIRS) model and estimated the transmissibility in the peak period via the instantaneous reproduction number (Rt). Public transport was found to explain approximately 28% of the variance in the seasonal transmission potential. Urbanization and public transportation size explained approximately 10% and 21% of the variance in maximum Rt in the peak period, respectively. For the mean Rt during the peak period, urbanization and public transportation accounted for 9% and 16% of the variance in Rt, respectively. Our results indicated that the differences in the intensity of influenza epidemics among the northern provinces of China were partially driven by urbanization and public transport size. These findings are beneficial for predicting influenza intensity and developing preparedness strategies for the early stages of epidemics. Full article
(This article belongs to the Special Issue State-of-the-Art Influenza Virus Research in China)
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<p>Bubble charts demonstrating the incidence rate in provinces with different levels of urbanization, transport, and relative humidity (<b>A</b>–<b>F</b>). Provinces with higher max and mean incidence tended to have a higher magnitude of urbanization (<b>A</b>,<b>B</b>), lower relative humidity (<b>C</b>,<b>D</b>), and larger transportation size (<b>E</b>,<b>F</b>).</p> Full article ">Figure 2
<p>Transmission potential and relative humidity predicted the observed differences in the intensity of the influenza epidemics in northern Chinese provinces (<b>A</b>–<b>F</b>). (<b>A</b>,<b>C</b>,<b>E</b>) simulated results of the SEIRS model in three provinces. (<b>B</b>,<b>D</b>,<b>F</b>) fitted results of the GLM model in three provinces.</p> Full article ">Figure 3
<p>Observed versus simulated influenza incidence in all provinces. Gray points show simulated influenza incidence rate. Blue line shows the fitted line between simulated influenza incidence and observed incidence.</p> Full article ">Figure 4
<p>Urbanization, transportation size, and combined index estimated from census data predicted transmission potential, maximum <span class="html-italic">R<sub>t</sub></span>, and mean <span class="html-italic">R<sub>t</sub></span> during peak period of influenza season (<b>A</b>–<b>L</b>). Gray points show transmission potential, maximum <span class="html-italic">R<sub>t</sub></span>, and mean <span class="html-italic">R<sub>t</sub></span> during peak period of influenza season estimated from the influenza incidence rate. Red lines refer to the prediction for transmission potential during peak period of influenza season (<b>A</b>–<b>C</b>). Purple lines refer to the prediction for maximum <span class="html-italic">R<sub>t</sub></span> during peak period of influenza season (<b>D</b>–<b>F</b>). Green lines refer to the prediction for mean <span class="html-italic">R<sub>t</sub></span> during peak period of the influenza season (<b>G</b>–<b>L</b>).</p> Full article ">
14 pages, 4105 KiB  
Article
New Insights into Avian Infectious Bronchitis Virus in Colombia from Whole-Genome Analysis
by Gloria Ramirez-Nieto, Daiana Mir, Diego Almansa-Villa, Geovanna Cordoba-Argotti, Magda Beltran-Leon, Nelida Rodriguez-Osorio, Jone Garai, Jovanny Zabaleta and Arlen P. Gomez
Viruses 2022, 14(11), 2562; https://doi.org/10.3390/v14112562 - 19 Nov 2022
Cited by 5 | Viewed by 2491
Abstract
Infectious Bronchitis (IB) is a respiratory disease caused by a highly variable Gammacoronavirus, which generates a negative impact on poultry health worldwide. GI-11 and GI-16 lineages have been identified in South America based on Infectious Bronchitis virus (IBV) partial S1 sequences. However, [...] Read more.
Infectious Bronchitis (IB) is a respiratory disease caused by a highly variable Gammacoronavirus, which generates a negative impact on poultry health worldwide. GI-11 and GI-16 lineages have been identified in South America based on Infectious Bronchitis virus (IBV) partial S1 sequences. However, full genome sequence information is limited. In this study we report, for the first time, the whole-genome sequence of IBV from Colombia. Seven IBV isolates obtained during 2012 and 2013 from farms with respiratory disease compatible with IB were selected and the complete genome sequence was obtained by NGS. According to S1 sequence phylogenetic analysis, six isolates belong to lineage GI-1 and one to lineage GVI-1. When whole genome was analyzed, five isolates were related to the vaccine strain Ma5 2016 and two showed mosaic genomes. Results from complete S1 sequence analysis provides further support for the hypothesis that GVI-1, considered a geographically confined lineage in Asia, could have originated in Colombia. Complete genome information reported in this research allow a deeper understanding of the phylogenetic evolution of variants and the recombination events between strains that are circulating worldwide, contributing to the knowledge of coronavirus in Latin America and the world. Full article
(This article belongs to the Collection Coronaviruses)
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<p>Geographical location of the farms from which the Infectious Bronchitis Virus (IBV) isolates included in the study were obtained. The lower right image shows the exact location of the farms and the seven IBV isolates (V2, V3, V5, V6, V8, V9, and V10).</p> Full article ">Figure 2
<p>Maximum-likelihood (ML) phylogenetic tree of IBV S1 Colombian sequences (<span class="html-italic">n</span> = 7), plus S1 IBV worldwide gene sequences (<span class="html-italic">n</span> = 1972). The circular band of colors around the tree indicates the genotype and lineage of each clade. Black bands indicate unique variants as designated by Valastro et al. 2016. Red lines in the second layer (outer to inner) indicate the placement of the Colombian samples. The SH-aLRT support values were &gt;99% for all the genotypes and defined lineages. The tree was midpoint rooted and the length of the branches are drawn to scale with the bar at the middle indicating nucleotide substitution per site.</p> Full article ">Figure 3
<p>Maximum-likelihood phylogenetic tree of Infectious Bronchitis Virus (IBV) S1 gene sequences from the GI-1 clade (<span class="html-italic">n</span> = 203). Red labels correspond to IBV Colombian sequences and green labels to vaccine strains. Circles at the nodes denote SH-aLRT branch support values larger than 80%. The tree was midpoint rooted and the length of the branches are drawn to scale with the bar at the middle indicating nucleotide substitution per site.</p> Full article ">Figure 4
<p>Maximum-likelihood phylogenetic tree of IBV complete genomes (<span class="html-italic">n</span> = 438). Red labels correspond to IBV Colombian sequences and green labels to vaccine strains. Circles at the nodes denote SH-aLRT branch support values larger than 80%. The tree was midpoint rooted and the lengths of the branches are drawn to scale with the bar at the middle indicating nucleotide substitution per site. Clusters that contain a) V8 b) V6 and c) V2, V3, V5, V9 and V10 Colombian strains are highlighted and magnified. SH-aLRT branch support values are indicated at key nodes and the branch lengths are drawn to scale with the bar at the top indicating nucleotide substitution per site.</p> Full article ">Figure 5
<p>ORF prediction and recombination analyses. (<b>a</b>) Genomic organization of V3, V10, V6, and V8. Red labels and yellow boxes highlight variation regions in detected ORFs. (<b>b</b>) and (<b>c</b>) Bootscan analysis (Simplot 3.5.1) of V6 and V8 whole genome sequences, respectively (windows size: 500 bp, step size: 100 bp). The dotted lines indicate a bootstrap value of 70%, and gray boxes indicate regions with recombination events.</p> Full article ">
26 pages, 4639 KiB  
Article
Isolation and Characterization of a Phapecoctavirus Infecting Multidrug-Resistant Acinetobacter baumannii in A549 Alveolar Epithelial Cells
by Phitchayapak Wintachai, Komwit Surachat, Ganyalak Chaimaha, Abdi Wira Septama and Duncan R. Smith
Viruses 2022, 14(11), 2561; https://doi.org/10.3390/v14112561 - 19 Nov 2022
Cited by 7 | Viewed by 2619
Abstract
Multidrug-resistant Acinetobacter baumannii (MDR A. baumannii) is an emerging pathogen in the ESKAPE group. The global burden of antimicrobial resistance has led to renewed interest in alternative antimicrobial treatment strategies, including phage therapy. This study isolated and characterized a phage vB_AbaM_ ABPW7 [...] Read more.
Multidrug-resistant Acinetobacter baumannii (MDR A. baumannii) is an emerging pathogen in the ESKAPE group. The global burden of antimicrobial resistance has led to renewed interest in alternative antimicrobial treatment strategies, including phage therapy. This study isolated and characterized a phage vB_AbaM_ ABPW7 (vABPW7) specific to MDR A. baumannii. Morphological analysis showed that phage vABPW7 belongs to the Myoviridae family. Genome analysis showed that the phage DNA genome consists of 148,647 bp and that the phage is a member of the Phapecoctavirus genus of the order Caudovirales. A short latent period and a large burst size indicated that phage vABPW7 was a lytic phage that could potentially be used in phage therapy. Phage vABPW7 is a high-stability phage that has high lytic activity. Phage vABPW7 could effectively reduce biofilm formation and remove preformed biofilm. The utility of phage vABPW7 was investigated in a human A549 alveolar epithelial cell culture model. Phage vABPW7 was not cytotoxic to A549 cells, and the phage could significantly reduce planktonic MDR A. baumannii and MDR A. baumannii adhesion on A549 cells without cytotoxicity. This study suggests that phage vABPW7 has the potential to be developed further as a new antimicrobial agent against MDR A. baumannii. Full article
(This article belongs to the Special Issue Viruses versus Bacteria—Novel Approaches to Phage Therapy as a Tool against Multidrug-Resistant Pathogens)
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<p>Isolation and biological characterization of phage vABPW7. (<b>a</b>) Plaques of phage vABPW7 in an MDR <span class="html-italic">A</span>. <span class="html-italic">baumannii</span> lawn. (<b>b</b>) Transmission electron micrograph of phage vABPW7. (<b>c</b>) Adsorption rate of phage vABPW7 to the host bacterial strain. (<b>d</b>) One-step growth curve showing the latent period and burst size of phage vABPW7. (<b>e</b>) Reduction in bacterial growth by phage vABPW7 at different MOIs. Experiments were undertaken independently in duplicate with duplicate assay. The data show the mean ± SEM (* <span class="html-italic">p</span> value &lt; 0.05).</p> Full article ">Figure 2
<p>Morphology of MDR <span class="html-italic">A. baumannii</span> cells under FE-SEM. (<b>a</b>,<b>c</b>) Untreated MDR <span class="html-italic">A. baumannii</span> cells. (<b>b</b>,<b>d</b>) MDR <span class="html-italic">A. baumannii</span> infected with phage vABWU7. The cells were observed at magnifications of ×1000 and ×20,000.</p> Full article ">Figure 3
<p>Stability of phage vABPW7 under different conditions. (<b>a</b>) Thermal stability of phage vABPW7 incubated at different temperatures for 2 h. (<b>b</b>) pH stability of phage vABPW7 incubated at different pHs for 2 h. (<b>c</b>) Stability of phage vABPW7 under varied concentrations of glycerol at −20 °C and −80 °C for 30 days (<b>d</b>) UV stability of phage vABPW7. Experiments were undertaken independently in duplicate with duplicate assay. The data show the mean ± SEM (* <span class="html-italic">p</span> value &lt; 0.05).</p> Full article ">Figure 4
<p>Phage genome characterization. (<b>a</b>) Circular genome map of phage vABPW7. The graphical map of the genome was generated using GC viewer server<sup>Beta</sup>. (<b>b</b>) Phylogenetic tree analysis of the whole-genome sequence of phage vABPW7 and related phages. The phylogeny was assessed by the VICTOR program. <span class="html-italic">Staphylococcus</span> phage JD419 (QOI66744) was used as an outgroup.</p> Full article ">Figure 5
<p>Genomic analysis of phage vABPW7. (<b>a</b>) A synteny plot displaying the genome structure of phage vABPW7 compared to <span class="html-italic">Escherichia</span> phage BI-EHEC, which is the most closely related phage to phage vABPW7. (<b>b</b>) A phylogenetic tree of the tail fiber protein of phage vABWU7. The multiple amino acid sequences were aligned by MUSCLE and the maximum-likelihood phylogenetic tree (JTT matrix-based model) was constructed in MEGA-X using 1000 bootstrap replicates. The tail fiber of <span class="html-italic">Staphylococcus</span> phage JD419 (QOI66744) was used as an outgroup.</p> Full article ">Figure 6
<p>Efficacy of phage vABWU7 to prevent biofilm formation and remove preformed biofilm. The efficacy of phage vABWU7 to prevent biofilm formation was evaluated. The biofilm biomass (<b>a</b>) and bacterial cell viability (<b>b</b>) were determined by the crystal violet assay and colony counting method, respectively. The ability of phage vABWU7 to remove preformed biofilm was investigated by quantification of biofilm biomass (<b>c</b>) and cell viability in biofilm formation (<b>d</b>). Experiments were undertaken independently in triplicate with duplicate assay. The data show the mean ± SEM (* <span class="html-italic">p</span> value &lt; 0.05).</p> Full article ">Figure 7
<p>Efficacy of phage vABPW7 in A549 cell culture model. Cytotoxicity of phage vABWU7 towards A549 cells at MOIs of 0.01 to 100 (<b>a</b>) and MDR <span class="html-italic">A. baumannii</span> at MOIs of 0.01 to 100 (<b>b</b>) after incubation for 24 h. (<b>c</b>) Adsorption efficacy of phage vABPW7 to A549 cells. (<b>d</b>) Efficacy of phage vABWU7 at MOIs of 0.01 to 100 to reduce MDR <span class="html-italic">A. baumannii</span> under the A549 cell culture model. Experiments were undertaken independently in duplicate with the duplicate assay. The data show the mean ± SEM (* <span class="html-italic">p</span> value &lt; 0.05).</p> Full article ">Figure 8
<p>Efficacy of phage vABPW7 to reduce the number of planktonic and adhered MDR <span class="html-italic">A. baumannii</span> on A549 cells. Kinetics of the antibacterial activity of phage vABWU7 on planktonic bacteria under the cell culture model (<b>a</b>) was determined in parallel with the production of phage vABWU7 (<b>b</b>). The ability of phage vABWU7 to reduce bacterial attachment to the surface of A549 cells was investigated. For a prophylactic model, A549 cells were treated with phage vABWU7 and then infected with MDR <span class="html-italic">A. baumannii.</span> The number of adherent bacteria was assessed at the times indicated (<b>c</b>). For a therapeutic model, A549 cells were infected with MDR <span class="html-italic">A. baumannii</span> and then treated with phage vABWU7. The number of adherent bacteria was assessed at the times indicated (<b>d</b>).</p> Full article ">Figure 9
<p>Ultrastructural analysis of MDR <span class="html-italic">A. baumannii</span> that attached to the cell surface of A549 cells under FE-SEM. (<b>a</b>,<b>d</b>) A549 cells incubated with only MDR <span class="html-italic">A. baumannii</span>. (<b>b</b>,<b>e</b>) A549 cells were treated with phage vABWU7 and then infected with MDR <span class="html-italic">A. baumannii</span> as a prophylactic application. (<b>c</b>,<b>f</b>) A549 cells were infected with MDR <span class="html-italic">A. baumannii</span> and then treated with phage vABWU7 as a therapeutic application. The cells were observed at magnifications of ×1000 and ×20,000.</p> Full article ">
7 pages, 263 KiB  
Perspective
Molnupiravir: From Hope to Epic Fail?
by Daniele Focosi
Viruses 2022, 14(11), 2560; https://doi.org/10.3390/v14112560 - 19 Nov 2022
Cited by 20 | Viewed by 6275
Abstract
Molnupiravir has been the first oral antiviral authorized for COVID-19 outpatients, reporting extraordinary sales and preserved in vitro efficacy against Omicron sublineages so far. However, it has recently been associated with very poor clinical efficacy, the risk of creating novel SARS-CoV-2 variants of [...] Read more.
Molnupiravir has been the first oral antiviral authorized for COVID-19 outpatients, reporting extraordinary sales and preserved in vitro efficacy against Omicron sublineages so far. However, it has recently been associated with very poor clinical efficacy, the risk of creating novel SARS-CoV-2 variants of concern, and long-term risk for mutagenicity in humans. The latter two are severe concerns, especially in the indicated population, i.e., long-replicating, immunodeficient patients. We conclude that, at this point, alternative antivirals should be preferred over molnupiravir. Full article
(This article belongs to the Special Issue SARS-CoV-2 inside and outside the Respiratory Tract: From Diagnostics to Therapies)
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