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Volume 15
Issue 11
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Viruses, Volume 15, Issue 11 (November 2023) – 144 articles

Cover Story (view full-size image): Influenza A virus (IAV) causes severe respiratory infections and substantial global morbidity and mortality. Innate immunity, cell death, and inflammation are key in driving severe disease in response to IAV. ZBP1 is an innate immune sensor for IAV infection that triggers NLRP3 inflammasome activation and formation of a PANoptosome complex. Activation of the PANoptosome leads to inflammatory cell death, PANoptosis. However, the role of specific pore-forming molecules downstream of the PANoptosome and potential redundancies between them remain unclear. In this issue, Malireddi et al. show that IAV-induced lytic cell death, PANoptosis, activates MLKL but is not dependent on MLKL. These findings suggest that functional redundancies in pore-forming molecules drive IAV-induced PANoptosis. View this paper
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23 pages, 4404 KiB  
Article
Revealing Novel-Strain-Specific and Shared Epitopes of Infectious Bronchitis Virus Spike Glycoprotein Using Chemical Linkage of Peptides onto Scaffolds Precision Epitope Mapping
by Samantha Sives, Sarah Keep, Erica Bickerton and Lonneke Vervelde
Viruses 2023, 15(11), 2279; https://doi.org/10.3390/v15112279 - 20 Nov 2023
Cited by 1 | Viewed by 1939
Abstract
The avian coronavirus, infectious bronchitis virus (IBV), is an economically important infectious disease affecting chickens, with a diverse range of serotypes found globally. The major surface protein, spike (S), has high diversity between serotypes, and amino acid differences in the S1 sub-unit are [...] Read more.
The avian coronavirus, infectious bronchitis virus (IBV), is an economically important infectious disease affecting chickens, with a diverse range of serotypes found globally. The major surface protein, spike (S), has high diversity between serotypes, and amino acid differences in the S1 sub-unit are thought to be responsible for poor cross-protection afforded by vaccination. Here, we attempt to address this, by using epitope mapping technology to identify shared and serotype-specific immunogenic epitopes of the S glycoprotein of three major circulating strains of IBV, M41, QX, and 4/91, via CLIPS peptide arrays based on peptides from the S1 sub-units. The arrays were screened with sera from chickens immunised with recombinant IBV, based on Beau-R backbone expressing heterologous S, generated in two independent vaccination/challenge trials. The screening of sera from rIBV vaccination experiments led to the identification of 52 immunogenic epitopes on the S1 of M41, QX, and 4/91. The epitopes were assigned into six overlapping epitope binding regions. Based on accessibility and location in the hypervariable regions of S, three sequences, 25YVYYYQSAFRPPNGWHLQGGAYAVVNSTN54, 67TVGVIKDVYNQSVASI82, and 83AMTVPPAGMSWSVS96, were selected for further investigation, and synthetic peptide mimics were recognised by polyclonal sera. These epitopes may have the potential to contribute towards a broader cross-protective IBV vaccine. Full article
(This article belongs to the Special Issue RNA Viruses and Antibody Response, 2nd Edition)
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<p>Recognition of IBV S1 peptide arrays with a panel of IBV mAbs. Representative example of epitope mapping of four anti-IBV S1 monoclonal antibodies, (<b>a</b>) CVI-IBV-69.3, (<b>b</b>) CVI-IBV-48.1, (<b>c</b>) CVI-IBV-62.8, and (<b>d</b>) CVI-IBV-51.2, screened against all three IBV S1 peptide arrays. Individual IBV S1 peptide arrays are labelled according to the specific S1 sequence from which the CLIPS peptides were derived. The x-axis is the position of the last residue of a peptide with respect to the appropriate IBV S1 sequence. Optical density signals obtained via CCD measurement are given in milli-absorbance units (mAU) for all CLIPS peptides. All intensity profiles were smoothened using moving average function with window of three, and are depicted for all four construct types displayed on each S1 array, namely, coil, helical, linear and loop, which are colour coded as depicted in the key for construct.</p> Full article ">Figure 2
<p>Antibody binding of IBV S1 peptide arrays. Representative examples of epitope mapping of experimentally derived polyclonal serum against the three IBV S1 peptide arrays: (<b>a</b>) Mock/Mock = non-vaccinated/non-challenged, (<b>b</b>) Mock/M41 = non-vaccinated/M41 challenge, (<b>c</b>) Mock/QX = non-vaccinated/QX challenge, (<b>d</b>) two individual serum samples from the homologous vaccinated/challenged chickens, and (<b>e</b>) three individual serum samples from the heterologous vaccinated/challenged chickens. The three IBV S1 peptide arrays are labelled according to the specific S1 sequence from which the CLIPS peptides were derived. The x-axis is the position of the last residue of a peptide with respect to the appropriate IBV S1 sequence. Optical density signals obtained via CCD measurement are given in milli-absorbance units (mAU) for all CLIPS peptides. The intensity traces are depicted for all four construct types (coil, helical, linear, and loop) displayed on each individual S1 peptide array, colour coded as depicted.</p> Full article ">Figure 3
<p>Recognition of selected putative epitopes across all three IBV S1 peptide arrays. Polyclonal sera classified according to recombinant IBV vaccination trial: (<b>a</b>) homologous vaccination/challenge and (<b>b</b>) heterologous vaccination/challenge. Shaded boxes indicate the recognition of peptide sequence by serum from the specified vaccinated group. Vaccination and challenge virus received are depicted in italics in the table vertically in the “Serum” column as follows: Homologous vaccination (Vaccination–Challenge) and Heterologous vaccination (Vaccination–Boost–Challenge). Epitopes were assigned to epitope binding regions (termed “A–F”, split by 100aa due to overlapping individual epitope sequences), and the amino acid position was given at the start and end of the peptide sequence.</p> Full article ">Figure 4
<p>Heat maps showing the recognition of peptide sequences by individual 14 dpc serum samples by binding to the three IBV S1 peptide arrays; QX, 4/91, and M41. Panel (<b>a</b>) shows individual sera from homologous vaccinated/challenged chickens (<span class="html-italic">n</span> = 20) binding to serotype-specific epitopes across all three IBV S1 arrays. Panel (<b>b</b>) displays the profiles of individual sera from heterologous vaccinated/challenged chickens (<span class="html-italic">n</span> = 38) binding to IBV S1 peptide arrays. All serum samples were collected at 14 dpc. Putative epitope sequences are shared across IBV serotypes. Dendrograms between serum samples were calculated using Pearson correlation, based on the average pairwise distance between all points of each data set and scaled to the intensity of the rows. The x-axis represents an individual serum sample. The y-axis represents a single peptide sequence.</p> Full article ">Figure 5
<p>IBV spike structural modelling. (<b>a</b>) A surface model representation of the IBV M41 spike trimer is shown in light pink. Epitope regions (A–F) of one of the S1 monomers are coloured as per key. (<b>b</b>) Structural domains of IBV M41 spike highlighted on one monomer, S1-NTD (19–237aa), S1-CTD (269–414aa), SD1 (248–269aa, 414–492aa), and SD2 (237–248aa, 492–567aa). (<b>c</b>) Trimeric surface and (<b>d</b>) structural model of IBV M41 spike with three selected epitopes highlighted with colour coding as per Epitope 1 (Blue), Epitope 2 (Red), and Epitope 3 (Black). The model was built using structural homology based on the cryo-EM of IBV M41 spike and structural domains annotated as stated in [<a href="#B14-viruses-15-02279" class="html-bibr">14</a>].</p> Full article ">Figure 6
<p>Conservation of identified peptide epitopes on the spike glycoprotein of IBV strains, M41, QX, and 4/91. Logo analyses are shown for the frequency of each amino acid within each of the three epitopes in spike from selected IBV strains, (<b>a</b>) Epitope 1, (<b>b</b>) Epitope 2 and (<b>c</b>) Epitope 3. The height of the letter indicates the frequency of the individual amino acid residue at that site. The residue position in the block is shown on the X-axis, and the information content is shown on the Y-axis. The default colour scheme displaying different amino acids according to their different chemical properties is as follows: polar amino acids (G, S, T, Y,) coloured with green, (C, Q, N) coloured with pink, basic (K, R, H) with blue, acidic (D, E) with red, and hydrophobic (A, V, L, I, P, W, F, M) with black.</p> Full article ">Figure 7
<p>Heat maps showing the recognition of selected peptide sequences by individual 14 dpc serum samples from heterologous vaccinated/challenged chickens. All heatmaps show the selected epitope sequence displayed on multiple constructs across the IBV S1 peptide arrays: panel (<b>a</b>) Epitope 1 and (<b>b</b>) Epitope 3. The y-axis represents a single construct-specific peptide, displaying the selected epitope sequence across all three IBV S1 peptide arrays. The x-axis represents an individual serum sample. Serum samples included individual mapped and unmapped (classified with background deemed too high to identify clear peaks in intensity traces) samples collected at 14 dpc. Dendrograms between serum samples were calculated using Pearson correlation, based on the average pairwise distance between all points of each data set and scaled to the intensity of the rows.</p> Full article ">Figure 8
<p>Recognition of synthetic peptides (representing epitopes 1–3) by sera from heterologous vaccinated/challenged chickens. Serum anti-peptide titres were assessed by a peptide-based ELISA, (<b>a</b>) Peptide/Epitope 1, (<b>b</b>) Peptide/Epitope 2 and (<b>c</b>) Peptide/Epitope 3. The sera from heterologous vaccinated, Mock/QX, and Mock/Mock chickens were diluted 1:40 and corrected for background absorbance before the determination of the recognition of peptides. Individual values and the mean (±standard error of mean) from each group (<span class="html-italic">n</span> = 10) were corrected for background absorbance, and include three technical replicates/bird. The dashed line indicates the cut-off threshold which was calculated on the mean value obtained for the Mock/Mock group. Statistical differences between groups were evaluated using Kruskal–Wallis with correction for Dunns’s multiple comparison tests to Mock/Mock, where ** denotes a significant difference, <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">
15 pages, 2720 KiB  
Review
Whitefly-Transmitted Viruses of Cucurbits in the Southern United States
by Ragunathan Devendran, Saritha Raman Kavalappara, Alvin M. Simmons and Sudeep Bag
Viruses 2023, 15(11), 2278; https://doi.org/10.3390/v15112278 - 20 Nov 2023
Cited by 8 | Viewed by 2451
Abstract
Cucurbits are economically important crops that are widely cultivated in many parts of the world, including the southern US. In recent years, higher temperatures have favored the rapid build-up of whiteflies in the fall-grown cucurbits in this region. As a result, whitefly-transmitted viruses [...] Read more.
Cucurbits are economically important crops that are widely cultivated in many parts of the world, including the southern US. In recent years, higher temperatures have favored the rapid build-up of whiteflies in the fall-grown cucurbits in this region. As a result, whitefly-transmitted viruses (WTVs) have severely impacted the marketable yield of cucurbits. In this review, we discuss three major groups of WTVs negatively impacting cucurbit cultivation in the southern US, including begomoviruses, criniviruses, and ipomoviruses. Here, we discuss the available information on the biology, epidemiology and advances made toward detecting and managing these viruses, including sources of resistance and cultural practices. Full article
(This article belongs to the Special Issue Plant Virus Epidemiology and Control 2023)
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<p>Symptoms caused by cucurbit leaf crumple virus (CuLCrV). Squash is the most affected crop by CuLCrV (<b>A</b>–<b>D</b>). Symptoms begin as diffuse yellow spots (<b>A</b>) and progress to crumpling (<b>B</b>,<b>C</b>) and stunting (<b>C</b>) in severe cases. The fruits on infected squash display green streaks and bumps (<b>D</b>). Symptoms of CuLCrV in zucchini (<b>E</b>) are milder than in squash and include mild chlorosis and crumpling. Photo Credit: S.R.K. and S.B.</p> Full article ">Figure 2
<p>Symptoms of criniviruses on cucurbits. Chlorosis on the crown region of cantaloupe (<b>A</b>) and cucumber (<b>B</b>) in commercial fields due to mixed infection of cucurbit chlorotic yellows virus (CCYV) and cucurbit yellow stunting disorder virus (CYSDV). Initial symptoms of CCYV infection as yellow spots on cucumber (<b>C</b>,<b>D</b>) and squash (<b>E</b>,<b>F</b>). Severe chlorosis is on the lower leaves of squash (<b>G</b>) and interveinal chlorosis is on wild radish infected with CCYV (<b>H</b>). Photo Credit: S.R.K. and S.B.</p> Full article ">Figure 3
<p>Symptoms of watermelon vine decline disease caused by squash vein yellow virus. The vine declined at the time of harvest (<b>A</b>), with discolored blotch and necrotic symptoms on the rind (<b>B</b>) and dark red color on the flesh (<b>C</b>) on a commercial watermelon field in Georgia during the Fall of 2023. Photo Credit: S.B. and S.R.K.</p> Full article ">
16 pages, 2680 KiB  
Article
Engineering a Novel Modular Adenoviral mRNA Delivery Platform Based on Tag/Catcher Bioconjugation
by Kexin Geng, Paul J. Rice-Boucher, Elena A. Kashentseva, Igor P. Dmitriev, Zhi Hong Lu, S. Peter Goedegebuure, William E. Gillanders and David T. Curiel
Viruses 2023, 15(11), 2277; https://doi.org/10.3390/v15112277 - 20 Nov 2023
Cited by 1 | Viewed by 1967
Abstract
mRNA vaccines have attracted widespread research attention with clear advantages in terms of molecular flexibility, rapid development, and potential for personalization. However, current mRNA vaccine platforms have not been optimized for induction of CD4/CD8 T cell responses. In addition, the mucosal administration of [...] Read more.
mRNA vaccines have attracted widespread research attention with clear advantages in terms of molecular flexibility, rapid development, and potential for personalization. However, current mRNA vaccine platforms have not been optimized for induction of CD4/CD8 T cell responses. In addition, the mucosal administration of mRNA based on lipid nanoparticle technology faces challenges in clinical translation. In contrast, adenovirus-based vaccines induce strong T cell responses and have been approved for intranasal delivery. To leverage the inherent strengths of both the mRNA and adenovirus platforms, we developed a novel modular adenoviral mRNA delivery platform based on Tag/Catcher bioconjugation. Specifically, we engineered adenoviral vectors integrating Tag/Catcher proteins at specific locales on the Ad capsid proteins, allowing us to anchor mRNA to the surface of engineered Ad viruses. In proof-of-concept studies, the Ad-mRNA platform successfully mediated mRNA delivery and could be optimized via the highly flexible modular design of both the Ad-mRNA and protein bioconjugation systems. Full article
(This article belongs to the Special Issue 15th International Adenovirus Meeting)
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<p>Ad-piggyback mRNA binder. (<b>A</b>) Schematic illustration of the construction process of Ad-piggyback mRNA binder via Catcher/Tag molecular glue. (<b>B</b>) Model of Ad-piggyback mRNA binder system generated by 3ds Max software 2022.</p> Full article ">Figure 2
<p>Construction process of the “Ad-piggyback protamine” complex. (<b>A</b>) Quality evaluation of SpyCat-SnoopCat intermediate linker. SnoopTag-MBP, SpyCat-SnoopCat, and SpyTag-MBP were incubated at room temperature for 2 h with a molar ratio of 1:1:1. (<b>B</b>) Formation of SpyCat-SnoopCat-SnoopTag-protamine conjugate. SnoopTag-protamine fused peptide was incubated with SpyCat-SnoopCat at room temperature for 2 h with a molar ratio of 1:1 and 2:1. (<b>C</b>) Anchoring of SpyCat-SnoopCat to hexon locales of Ad capsid and the formation of Ad.hexon.SpyTag-SpyCat-SnoopCat complex. Top: SpyCat or SpyCat-SnoopCat was incubated with wild-type Ad (non-SpyTag integrated) at room temperature for 2 h with a molar ratio of 2:1. Bottom: SpyCat or SpyCat-SnoopCat was incubated with Ad.hexon.SpyTag (SpyTag-integrated) at room temperature for 2 h with a molar ratio of 2:1. (<b>D</b>) Formation of “AdPro” or Ad.hexon.SpyTag-SpyCat-SnoopCat-SnoopTag-protamine complex. Ad.hexon.SpyTag, SpyCat-SnoopCat, and SnoopTag-protamine were incubated at room temperature for 2 h with a molar ratio of 1:2:4.</p> Full article ">Figure 3
<p>Ad-piggyback protamine mediated effective mRNA delivery in vitro. Engineered Ad with SpyTag incorporated into the hexon locales on the capsid protein serves as an anchoring platform for the protamine/mRNA complex to attach. Ad encodes the GFP gene, and the piggybacked mRNA encodes mCherry fluorescent protein gene. The contribution of each component to effective mRNA delivery is examined by setting the following groups: Group 1: non-treatment; Group 2: mRNA 0.5 <math display="inline"><semantics> <mrow> <mi mathvariant="sans-serif">μ</mi> </mrow> </semantics></math>g, SnoopTag-protamine/mRNA = 8:1(mass ratio); Group 3: Ad.hexon.SpyTag 2 × 10<sup>−8</sup> vp, mRNA 0.5 <math display="inline"><semantics> <mrow> <mi mathvariant="sans-serif">μ</mi> </mrow> </semantics></math>g; Group 4: Ad.hexon.SpyTag 2 × 10<sup>−8</sup> vp, mRNA 0.5 <math display="inline"><semantics> <mrow> <mi mathvariant="sans-serif">μ</mi> </mrow> </semantics></math>g, SnoopTag-protamine/mRNA = 8:1 (mass ratio); Group 5: Ad.hexon.SpyTag 2 × 10<sup>−8</sup> vp, SpyCat-SnoopCat/Ad.hexon.SpyTag = 2:1 in molar ratio, mRNA 0.5 <math display="inline"><semantics> <mrow> <mi mathvariant="sans-serif">μ</mi> </mrow> </semantics></math>g, SpyTag-protamine/mRNA = 8:1 (mass ratio). * <span class="html-italic">p</span> ≤ 0.05. (<b>A</b>) Qualitative analysis of gene transfer as visualized by fluorescent images. (<b>B</b>) Assessment of mRNA condensation level by RiboGreen assay. (<b>C</b>) Representative flow cytometry density plot from a sample in Group 5. (<b>D</b>) Quantitative analysis of mRNA delivery efficiency by flow cytometry. Indicated groups were analyzed using a standard <span class="html-italic">t</span>-test.</p> Full article ">Figure 4
<p>“AdPro” system mediated circRNA delivery in vitro. Engineered Ad with SpyTag incorporated into the hexon locales on the capsid protein serves as an anchoring platform for the protamine/circRNA complex to attach. Ad encodes the GFP gene, and the piggybacked circRNA encodes mCherry fluorescent protein gene. The contribution of each component to effective circRNA delivery is examined by setting the following groups: Group 1: Non-treatment; Group 2: circRNA 0.5 <math display="inline"><semantics> <mrow> <mi mathvariant="sans-serif">μ</mi> </mrow> </semantics></math>g, SnoopTag-protamine/circRNA 8:1(mass ratio); Group 3: Ad.hexon.SpyTag 2 × 10<sup>−8</sup> vp, circ RNA 0.5 <math display="inline"><semantics> <mrow> <mi mathvariant="sans-serif">μ</mi> </mrow> </semantics></math>g, SnoopTag-protamine/mRNA = 8:1(mass ratio); Group 4: Ad.hexon.SpyTag 2 × 10<sup>−8</sup> vp, SpyCat-SnoopCat/Ad.hexon.SpyTag = 2:1 (in molar ratio), circ RNA 0.5 <math display="inline"><semantics> <mrow> <mi mathvariant="sans-serif">μ</mi> </mrow> </semantics></math>g, SpyTag-protamine/mRNA = 8:1(mass ratio). (<b>A</b>) Assessment of circRNA condensation level by RiboGreen assay. (<b>B</b>) Representative flow cytometry density plot from a sample in group 4. (<b>C</b>) Quantitative analysis of circRNA delivery efficiency by flow cytometry. Indicated groups were analyzed using a standard <span class="html-italic">t</span>-test.</p> Full article ">Figure 5
<p>The “AdRALA” system achieved higher gene transfer. (<b>A</b>) Assessment of mRNA condensation level by RiboGreen assay. (<b>B</b>,<b>C</b>) Quantitative analysis of gene transfer efficiency by flow cytometry. (<b>D</b>) Assessment of circRNA condensation level by RiboGreen assay. (<b>E</b>,<b>F</b>) Quantitative analysis of gene transfer efficiency by flow cytometry. Statistical comparisons were obtained via one-way ANOVA with Tukey’s multiple comparisons test.</p> Full article ">Figure 5 Cont.
<p>The “AdRALA” system achieved higher gene transfer. (<b>A</b>) Assessment of mRNA condensation level by RiboGreen assay. (<b>B</b>,<b>C</b>) Quantitative analysis of gene transfer efficiency by flow cytometry. (<b>D</b>) Assessment of circRNA condensation level by RiboGreen assay. (<b>E</b>,<b>F</b>) Quantitative analysis of gene transfer efficiency by flow cytometry. Statistical comparisons were obtained via one-way ANOVA with Tukey’s multiple comparisons test.</p> Full article ">
9 pages, 4491 KiB  
Brief Report
Aerosol Delivery of Palivizumab in a Neonatal Lamb Model of Respiratory Syncytial Virus Infection
by Hasindu S. Edirisinghe, Anushi E. Rajapaksa, Simon G. Royce, Magdy Sourial, Robert J. Bischof, Jeremy Anderson, Gulcan Sarila, Cattram D. Nguyen, Kim Mulholland, Lien Anh Ha Do and Paul V. Licciardi
Viruses 2023, 15(11), 2276; https://doi.org/10.3390/v15112276 - 19 Nov 2023
Cited by 1 | Viewed by 1773
Abstract
(1) Background: Palivizumab has been an approved preventative monoclonal antibody for respiratory syncytial virus (RSV) infection for over two decades. However, due to its high cost and requirement for multiple intramuscular injections, its use has been limited mostly to high-income countries. Following our [...] Read more.
(1) Background: Palivizumab has been an approved preventative monoclonal antibody for respiratory syncytial virus (RSV) infection for over two decades. However, due to its high cost and requirement for multiple intramuscular injections, its use has been limited mostly to high-income countries. Following our previous study showing the successful lung deposition of aerosolised palivizumab in lambs, this current study evaluated the “proof-of-principle” effect of aerosolised palivizumab delivered as a therapeutic to neonatal lambs following RSV infection. (2) Methods: Neonatal lambs were intranasally inoculated with RSV-A2 on day 0 (day 3 post-birth) and treated with aerosolised palivizumab 3 days later (day 3 post-inoculation). Clinical symptoms, RSV viral load and inflammatory response were measured post-inoculation. (3) Results: Aerosolised therapeutic delivery of palivizumab did not reduce RSV viral loads in the nasopharynx nor the bronchoalveolar lavage fluid, but resulted in a modest reduction in inflammatory response at day 6 post-inoculation compared with untreated lambs. (4) Conclusions: This proof-of-principle study shows some evidence of aerosolised palivizumab reducing RSV inflammation, but further studies using optimized protocols are needed in order to validate these findings. Full article
(This article belongs to the Section Human Virology and Viral Diseases)
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<p>Study design and sampling timeline using an RSV lamb model. Neonatal lambs were infected with the RSV-A2 strain intranasally on day 0. Nasopharyngeal (NP) swabs, blood, and bronchoalveolar lavage fluid (BALF) were collected at baseline (day 0) and then on day 2, 4, 6, 8 and 10 post-inoculation. Lambs were euthanised either on day 6 (peak infection) or day 10 (recovery) post-inoculation according to the study group (refer to <a href="#viruses-15-02276-t001" class="html-table">Table 1</a>). Image was created using BioRender.com.</p> Full article ">Figure 2
<p>Clinical parameters of newborn lambs given aerosol treatment with palivizumab. (<b>A</b>) Nebuliser setup with face mask; (<b>B</b>) body weight; (<b>C</b>) body temperature; and (<b>D</b>) oxygen saturation for each group of lambs. The normal physiological range is shaded in (<b>C</b>,<b>D</b>). Red circles = control group 0 (<span class="html-italic">n</span> = 2); green squares = RSV/untreated group 1 (<span class="html-italic">n</span> = 6); blue triangles = RSV/palivizumab group 2 (<span class="html-italic">n</span> = 6). Data are presented as the mean ± SEM.</p> Full article ">Figure 3
<p>Comparison of RSV load in NP swabs and BALF samples on day 6 post-inoculation (time of peak-infection). (<b>A</b>) Nasopharyngeal (NP) and (<b>B</b>) bronchoalveolar lavage fluid (BALF) viral load are displayed as averages of all lambs in each group. Red circles = control group 0 (<span class="html-italic">n</span> = 2); green squares = RSV/untreated group 1 (<span class="html-italic">n</span> = 12); blue triangles = RSV/palivizumab group 2 (<span class="html-italic">n</span> = 11). Data are represented as the median ± IQR.</p> Full article ">Figure 4
<p>Comparison of inflammation/congestion scores in lambs. (<b>A</b>–<b>C</b>) Representative images (H&amp;E stain, day 6 post-inoculation) in (<b>A</b>) control group lambs; (<b>B</b>) RSV/untreated group 1a lambs; and (<b>C</b>) RSV/palivizumab group 2a lambs. Scale bar = 100 μm. (<b>D</b>) Analysis of inflammation/congestion scores in each group at day 6 post-inoculation (H&amp;E stain, median ± IQR). Red circles = control group 0 (<span class="html-italic">n</span> = 2); green squares = RSV/untreated group 1a (<span class="html-italic">n</span> = 6); blue triangles = RSV/palivizumab group 2 (<span class="html-italic">n</span> = 6).</p> Full article ">Figure 5
<p>Goblet cell (GC) counts in cartilaginous and non-cartilaginous airways of the lambs. Representative images (AB-PAS stain, day 6 post-inoculation) of cartilaginous airways and non- cartilaginous airways in (<b>A</b>) control group 0 lambs; (<b>B</b>) RSV/untreated group 1a lambs; and (<b>C</b>) RSV/palivizumab group 2a lambs. Scale bar = 100 μm. (<b>D</b>) Mean number of GC counts per 100 μm in each lamb from each group at day 6 post-inoculation (AB-Pas stain, median ± IQR). Red circles= control group 0 (<span class="html-italic">n</span> = 2); green squares = RSV/untreated group 1a (<span class="html-italic">n</span> = 6); blue triangles = RSV/palivizumab group 2a (<span class="html-italic">n</span> = 6).</p> Full article ">
17 pages, 1551 KiB  
Review
African Swine Fever: Transmission, Spread, and Control through Biosecurity and Disinfection, Including Polish Trends
by Małgorzata Juszkiewicz, Marek Walczak, Grzegorz Woźniakowski and Katarzyna Podgórska
Viruses 2023, 15(11), 2275; https://doi.org/10.3390/v15112275 - 19 Nov 2023
Cited by 14 | Viewed by 4310
Abstract
African swine fever is a contagious disease, affecting pigs and wild boars, which poses a major threat to the pig industry worldwide and, therefore, to the agricultural economies of many countries. Despite intensive studies, an effective vaccine against the disease has not yet [...] Read more.
African swine fever is a contagious disease, affecting pigs and wild boars, which poses a major threat to the pig industry worldwide and, therefore, to the agricultural economies of many countries. Despite intensive studies, an effective vaccine against the disease has not yet been developed. Since 2007, ASFV has been circulating in Eastern and Central Europe, covering an increasingly large area. As of 2018, the disease is additionally spreading at an unprecedented scale in Southeast Asia, nearly ruining China’s pig-producing sector and generating economic losses of approximately USD 111.2 billion in 2019. ASFV’s high resistance to environmental conditions, together with the lack of an approved vaccine, plays a key role in the spread of the disease. Therefore, the biosecurity and disinfection of pig farms are the only effective tools through which to prevent ASFV from entering the farms. The selection of a disinfectant, with research-proven efficacy and proper use, taking into account environmental conditions, exposure time, pH range, and temperature, plays a crucial role in the disinfection process. Despite the significant importance of ASF epizootics, little information is available on the effectiveness of different disinfectants against ASFV. In this review, we have compiled the current knowledge on the transmission, spread, and control of ASF using the principles of biosecurity, with particular attention to disinfection, including a perspective based on Polish experience with ASF control. Full article
(This article belongs to the Special Issue Strategies for Preventing Viral Diseases of Domestic Animals)
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<p>The occurrences of ASF in Poland between 2014 and 2022; red—outbreaks in pigs, purple—outbreaks in wild boars [<a href="#B38-viruses-15-02275" class="html-bibr">38</a>].</p> Full article ">Figure 2
<p>The distribution of restricted zones and ASF outbreaks in pigs (red dots) and wild boars (yellow dots) in Poland in 2023 (state as of July, 2023) [<a href="#B82-viruses-15-02275" class="html-bibr">82</a>].</p> Full article ">
12 pages, 3199 KiB  
Article
Challenges for Precise Subtyping and Sequencing of a H5N1 Clade 2.3.4.4b Highly Pathogenic Avian Influenza Virus Isolated in Japan in the 2022–2023 Season Using Classical Serological and Molecular Methods
by James G. Komu, Hiep Dinh Nguyen, Yohei Takeda, Shinya Fukumoto, Kunitoshi Imai, Hitoshi Takemae, Tetsuya Mizutani and Haruko Ogawa
Viruses 2023, 15(11), 2274; https://doi.org/10.3390/v15112274 - 18 Nov 2023
Cited by 1 | Viewed by 2510
Abstract
The continuous evolution of H5Nx highly pathogenic avian influenza viruses (HPAIVs) is a major concern for accurate diagnosis. We encountered some challenges in subtyping and sequencing a recently isolated H5N1 HPAIV strain using classical diagnostic methods. Oropharyngeal, conjunctival, and cloacal swabs collected from [...] Read more.
The continuous evolution of H5Nx highly pathogenic avian influenza viruses (HPAIVs) is a major concern for accurate diagnosis. We encountered some challenges in subtyping and sequencing a recently isolated H5N1 HPAIV strain using classical diagnostic methods. Oropharyngeal, conjunctival, and cloacal swabs collected from a dead white-tailed eagle (Haliaeetus albicilla albicilla) were screened via real-time RT-PCR targeting the influenza A virus matrix (M) gene, followed by virus isolation. The hemagglutination inhibition test was applied in order to subtype and antigenically characterize the isolate using anti-A/duck/Hong Kong/820/80 (H5N3) reference serum or anti-H5N1 cross-clade monoclonal antibodies (mAbs). Sequencing using previously reported universal primers was attempted in order to analyze the full-length hemagglutinin (HA) gene. Oropharyngeal and conjunctival samples were positive for the M gene, and high hemagglutination titers were detected in inoculated eggs. However, its hemagglutination activity was not inhibited by the reference serum or mAbs. The antiserum to a recently isolated H5N1 clade 2.3.4.4b strain inhibited our isolate but not older strains. A homologous sequence in the previously reported forward primer and HA2 region in our isolate led to partial HA gene amplification. Finally, next-generation sequencing confirmed the isolate as H5N1 clade 2.3.4.4b HPAIV, with genetic similarity to H5N1 strains circulating in Japan since November 2021. Full article
(This article belongs to the Special Issue Avian Respiratory Viruses, Volume III)
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<p>Molecular detection of AIV genes via RT-PCR. PCR products were subjected to agarose gel electrophoresis. (<b>A</b>) Subtype-specific RT-PCR for HA subtyping revealing specific amplification of the H5 gene (545 bp) for the oropharyngeal (Or) and conjunctival (Cj) swabs of WTE/Jp (H5N1), similar to the positive control (PC). All the other RT-PCR tests for subtypes H1–H4 and H6–H15 resulted in no amplification. (<b>B</b>) Amplification of the HA gene via RT-PCR using Hoffmann’s primers. PCR of the WTE/Jp (H5N1) samples resulted in the amplification of an approximately 658-bp product (highlighted by the red box), contrary to the expected size of approximately 1807 bp for the full-length HA gene, which was confirmed using the positive controls. M: DNA size marker; PC H1: A/Puerto Rico/8/1934 (H1N1); PC H5: A/duck/Hong Kong 820/80 (H5N3).</p> Full article ">Figure 2
<p>Phylogenetic tree of the HA gene of H5Nx viruses in clade 2.3.4.4. The nucleotide sequence of the HA gene of WTE/Jp (H5N1) and other sequences belonging to different 2.3.4.4 subclades obtained from GenBank and GISAID were used in the phylogenetic analysis. The tree was rooted by A/whooper swan/Hamanaka/2011 (H5N1) belonging to clade 2.3.2.1. Strains in red, brown, and green indicate the H5N1 HPAIV strain isolated in this study, the isolate used to produce an anti-H5 serum that successfully inhibited our isolate, and a representative strain of this phylogenetic cluster previously used to investigate pathogenicity against chickens, respectively. The evolutionary history was inferred using the maximum likelihood method and Tamura–Nei model. The tree was drawn to scale, with branch lengths measured in the number of substitutions per site. Evolutionary analyses were conducted using MEGA11 software version 11.0.13.</p> Full article ">Figure 3
<p>Multiple nucleotide alignment of the HA gene of the WTE/Jp (H5N1) isolate (highlighted in black) with (i) other H5N1 HPAIV strains in clade 2.3.4.4b detected in 2021–2023, (ii) H5Nx strains detected in/before November 2021, and (iii) nonGs/GD-lineage H5 low-pathogenic AIVs, in descending order of the year of occurrence. The gene region covered by Hoffmann’s HA forward primer is indicated under the alignment. The highlighted nucleotides in the two brown boxes, including one sequence in the non-coding region covering the last eight nucleotides in the HA forward primer and the other in the HA2 region, had similar sequences. The black arrow indicates a conserved nucleotide “T” that was added to the modified forward primer described in this paper.</p> Full article ">Figure 4
<p>Amplification of the HA genes of H5Nx AIV strains. (<b>A</b>) PCR using Hoffmann’s original primers. (<b>B</b>) PCR using the modified Hoffmann HA forward primer. 1: A/duck/HongKong/820/80 (H5N3); 2: A/whistling swan/Shimane/499/83 (H5N3); 3: A/chicken/Miyazaki/K11/07 (H5N1); 4: A/Chicken/Yamaguchi/7/04 (H5N1); 5: A/white-tailed eagle/Japan/OU-1/2022 (H5N1); M: DNA size marker.</p> Full article ">
20 pages, 1056 KiB  
Article
Pathogenicity in Chickens and Turkeys of a 2021 United States H5N1 Highly Pathogenic Avian Influenza Clade 2.3.4.4b Wild Bird Virus Compared to Two Previous H5N8 Clade 2.3.4.4 Viruses
by Mary J. Pantin-Jackwood, Erica Spackman, Christina Leyson, Sungsu Youk, Scott A. Lee, Linda M. Moon, Mia K. Torchetti, Mary L. Killian, Julianna B. Lenoch, Darrell R. Kapczynski, David E. Swayne and David L. Suarez
Viruses 2023, 15(11), 2273; https://doi.org/10.3390/v15112273 - 18 Nov 2023
Cited by 6 | Viewed by 3047
Abstract
Highly pathogenic avian influenza viruses (HPAIVs) of subtype H5 of the Gs/GD/96 lineage remain a major threat to poultry due to endemicity in wild birds. H5N1 HPAIVs from this lineage were detected in 2021 in the United States (U.S.) and since then have [...] Read more.
Highly pathogenic avian influenza viruses (HPAIVs) of subtype H5 of the Gs/GD/96 lineage remain a major threat to poultry due to endemicity in wild birds. H5N1 HPAIVs from this lineage were detected in 2021 in the United States (U.S.) and since then have infected many wild and domestic birds. We evaluated the pathobiology of an early U.S. H5N1 HPAIV (clade 2.3.4.4b, 2021) and two H5N8 HPAIVs from previous outbreaks in the U.S. (clade 2.3.4.4c, 2014) and Europe (clade 2.3.4.4b, 2016) in chickens and turkeys. Differences in clinical signs, mean death times (MDTs), and virus transmissibility were found between chickens and turkeys. The mean bird infective dose (BID50) of the 2021 H5N1 virus was approximately 2.6 log10 50% embryo infective dose (EID50) in chickens and 2.2 log10 EID50 in turkeys, and the virus transmitted to contact-exposed turkeys but not chickens. The BID50 for the 2016 H5N8 virus was also slightly different in chickens and turkeys (4.2 and 4.7 log10 EID50, respectively); however, the BID50 for the 2014 H5N8 virus was higher for chickens than turkeys (3.9 and ~0.9 log10 EID50, respectively). With all viruses, turkeys took longer to die (MDTs of 2.6–8.2 days for turkeys and 1–4 days for chickens), which increased the virus shedding period and facilitated transmission to contacts. Full article
(This article belongs to the Section Animal Viruses)
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<p>Survival curves of chickens inoculated with the H5 HPAIVs and the contact-exposed birds. Birds received low, medium, and high doses of the viruses. Contact-exposed birds were added to isolators one day post-inoculation.</p> Full article ">Figure 2
<p>Survival curves of turkeys inoculated with the H5 HPAIVs and the contact-exposed birds. Birds received low, medium, and high doses of the viruses. Contact-exposed birds were added to isolators one day post-inoculation.</p> Full article ">Figure 3
<p>Virus shedding for chickens inoculated with different doses of the H5 HPAIVs: (<b>A</b>) AMWI/SC/21; (<b>B</b>) TUDU/Denmark/16; (<b>C</b>) GYRF/WA/14. Virus titers from oropharyngeal (OP) and cloacal (CL) swabs were determined by qRT-PCR. The dotted lines indicate the limit of detection for each virus.</p> Full article ">Figure 4
<p>Virus shedding for turkeys inoculated with the H5 HPAIVs: (<b>A</b>) AMWI/SC/21; (<b>B</b>) TUDU/Denmark/16; (<b>C</b>) GYRF/WA/14. Virus titers from oropharyngeal (OP) and cloacal (CL) swabs were determined by qRT-PCR. The dotted lines indicate the limit of detection for each virus.</p> Full article ">
16 pages, 2605 KiB  
Article
Human Metapneumovirus-Induced Host microRNA Expression Impairs the Interferon Response in Macrophages and Epithelial Cells
by Iván Martínez-Espinoza, Anang D. Bungwon and Antonieta Guerrero-Plata
Viruses 2023, 15(11), 2272; https://doi.org/10.3390/v15112272 - 18 Nov 2023
Cited by 2 | Viewed by 1850
Abstract
Human metapneumovirus (HMPV) is a nonsegmented, single-stranded negative RNA virus and a member of the Pneumoviridae family. During HMPV infection, macrophages play a critical role in defending the respiratory epithelium by secreting large amounts of type I interferon (IFN). MicroRNAs (miRNAs) are small, [...] Read more.
Human metapneumovirus (HMPV) is a nonsegmented, single-stranded negative RNA virus and a member of the Pneumoviridae family. During HMPV infection, macrophages play a critical role in defending the respiratory epithelium by secreting large amounts of type I interferon (IFN). MicroRNAs (miRNAs) are small, noncoding, single-stranded RNAs that play an essential role in regulating gene expression during normal cellular homeostasis and disease by binding to specific mRNAs, thereby regulating at the transcriptional and post-transcriptional levels with a direct impact on the immune response and other cellular processes. However, the role of miRNAs in macrophages and respiratory viral infections remains largely unknown. Here, we characterized the susceptibility of THP-1-derived macrophages to HMPV infection and the effect of hsa-miR-4634 on these cells. Transfection of an miRNA mimic and inhibitor demonstrated that hsa-miR-4634 regulates the IFN response in HMPV-infected macrophages, suggesting that HMPV induces the expression of the miRNA as a subversion mechanism of the antiviral response. This effect was not limited to macrophages, as a similar effect was also observed in epithelial cells. Overall, our results demonstrate that hsa-miR-4634 is an important factor in regulating the IFN response in macrophages and epithelial cells during HMPV infection. Full article
(This article belongs to the Section Human Virology and Viral Diseases)
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<p>Susceptibility of macrophages to HMPV infection. THP-1-derived macrophages and LLC-MK2 cells were infected with HMPV at different MOIs. (<b>A</b>) The percentage of infected cells was determined by the expression of HMPV P protein. (<b>B</b>) Cell death was determined by 7-AAD staining. All samples were analyzed by flow cytometry and Flow Jo V10.9. Representative data of contour plot analyses are included. Graph bars represent the mean of 3 independent experiments ± SEM. Statistical differences were calculated using ANOVA followed by a post hoc Tukey’s multiple comparison test. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> ≤ 0.001, **** <span class="html-italic">p</span> ≤ 0.0001.</p> Full article ">Figure 2
<p>Susceptibility of macrophages to HMPV infection at different time points. (<b>A</b>) THP-1-derived macrophages were infected with HMPV at MOI 1.0. (<b>B</b>) LLC-MK2 cells were included as permissive reference cells. The percentage of infected cells was determined by the expression of HMPV P protein at different time points. All samples were analyzed by flow cytometry and Flow Jo V10.9. Graph bars represent the mean of 3 independent experiments ± SEM. Statistical differences were calculated using ANOVA followed by a post hoc Tukey´s multiple comparison test. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> ≤ 0.001, **** <span class="html-italic">p</span> ≤ 0.0001.</p> Full article ">Figure 3
<p>miRNA expression in macrophages infected with HMPV. Macrophages were infected with HMPV at an MOI of 1 for 24 h. miRNA expression was assessed by miRNA RT-qPCR and normalized to RNU6-1. Bar graphs represent mean of 3 independent experiments ± SEM. Statistical differences between infected and uninfected cells were calculated using unpaired Student <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, **** <span class="html-italic">p</span> ≤ 0.0001.</p> Full article ">Figure 4
<p>Effect of hsa-miR-4634 on interferon and proinflammatory cytokines expression. THP-1-derived macrophages were transfected by electroporation with hsa-miR-4634 mimic (100 nM) or inhibitor (50 nM). After 24 h of culture, macrophages were infected with HMPV at an MOI of 1 for 24 h. Total RNA was extracted to assess gene expression using RT-qPCR. (<b>A</b>) IFN-β expression of macrophages transfected with mimic hsa-miR-4634 (upper panel) or inhibitor hsa-miR-4634 (lower panel). (<b>B</b>) Expression of inflammatory cytokines in macrophages transfected with mimic or inhibitor hsa-miR-4634. Bar graphs represent the mean of three independent experiments ± SEM. Statistical differences between infected and uninfected cells were calculated using ANOVA, followed by Sidak’s multiple comparison test. **** <span class="html-italic">p</span> &lt; 0.0001.</p> Full article ">Figure 5
<p>Effect of hsa-miR-4634 on interferon-stimulated genes in macrophages. THP-1-derived macrophages were transfected by electroporation with (<b>A</b>) hsa-miR-4634 mimic (100 nM) or (<b>B</b>) inhibitor (50 nM) (see Methods). After 24 h of culture, macrophages were infected with HMPV at an MOI of 1.0 for 24 h. Total RNA was extracted to assess gene expression of IFIT1, IFIT2, IFIT3, and MX1 by using RT-qPCR. Graph bars represent the mean of four independent experiments ± SEM. Statistical differences between infected and uninfected cells were calculated using ANOVA, followed by Sidak’s multiple comparison test. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 6
<p>Effect of hsa-miR-4634 on interferon expression in epithelial cells. (<b>A</b>) A549 cells were infected at MOI 0.5, 1.0, and 3.0, and the percentage of HMPV-positive cells and cell death was quantified by flow cytometry. (<b>B</b>) A549 cells were infected with HMPV at an MOI of 3.0 for 24 h, and the expression of hsa-miR-4634 was assessed by RT-qPCR. (<b>C</b>–<b>E</b>) A549 cells were transfected by electroporation with mimic-4634 (100 nM) or inhibitor-4634 (50 nM). After 24 h of culture, cells were infected with HMPV at an MOI of 3.0 for 24 h. Total RNA was extracted to assess the expression of IFN-α2, IFN-β, and IFN-λ2/3 by qPCR. Bar graphs represent mean of three independent experiments ± SEM. Statistical differences were calculated using the Student <span class="html-italic">t</span>-test (<b>B</b>) or ANOVA followed by Sidak’s multiple comparison test (<b>A</b>,<b>C</b>–<b>E</b>). * <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> ≤ 0.0001.</p> Full article ">Figure 7
<p>Effect of hsa-miR-4634 on viral replication in macrophages and epithelial cells. Viral copies of the HMPV N gene were quantified by RT-qPCR. Cells were transfected by electroporation with mimic (100 nM) and inhibitor (50 nM). (<b>A</b>) Macrophages and (<b>B</b>) epithelial cells were infected for 24 h with HMPV at MOI 1 or MOI 3, respectively. Bar graphs represent the mean of two independent experiments with similar results ± SEM. Statistical differences were calculated using the Student <span class="html-italic">t</span>-test. * <span class="html-italic">p</span> &lt; 0.05, *** <span class="html-italic">p</span> &lt;0.001.</p> Full article ">
16 pages, 1554 KiB  
Review
Does the Human Gut Virome Contribute to Host Health or Disease?
by Grazia Pavia, Nadia Marascio, Giovanni Matera and Angela Quirino
Viruses 2023, 15(11), 2271; https://doi.org/10.3390/v15112271 - 17 Nov 2023
Cited by 6 | Viewed by 2445
Abstract
The human gastrointestinal (GI) tract harbors eukaryotic and prokaryotic viruses and their genomes, metabolites, and proteins, collectively known as the “gut virome”. This complex community of viruses colonizing the enteric mucosa is pivotal in regulating host immunity. The mechanisms involved in cross communication [...] Read more.
The human gastrointestinal (GI) tract harbors eukaryotic and prokaryotic viruses and their genomes, metabolites, and proteins, collectively known as the “gut virome”. This complex community of viruses colonizing the enteric mucosa is pivotal in regulating host immunity. The mechanisms involved in cross communication between mucosal immunity and the gut virome, as well as their relationship in health and disease, remain largely unknown. Herein, we review the literature on the human gut virome’s composition and evolution and the interplay between the gut virome and enteric mucosal immunity and their molecular mechanisms. Our review suggests that future research efforts should focus on unraveling the mechanisms of gut viruses in human homeostasis and pathophysiology and on developing virus-prompted precision therapies. Full article
(This article belongs to the Section Human Virology and Viral Diseases)
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<p>Composition and evolution of human gut virome composition from early life (in utero) to infancy and adulthood.</p> Full article ">Figure 2
<p>The interaction between intestinal epithelial cells, immune cells, microbiota, and metabolites. The strict “connection” between the above protagonists is necessary to create a balance in the protective immune response toward oneself and non-self entities (health status). Conversely, disruption of this equilibrium through genetic and/or environmental factors, such as foods, toxins, drugs, and pathogenetic microorganisms, is associated with intestinal epithelial damage and an altered immune response (disease).</p> Full article ">Figure 3
<p>Bacteriophages and gut mucosal immune system modulation.</p> Full article ">Figure 4
<p>Eukaryotic viruses and gut mucosal immune system modulation.</p> Full article ">
14 pages, 1903 KiB  
Article
Exploring the Susceptibility of C3H Mice to Tick-Borne Encephalitis Virus Infection: Implications for Co-Infection Models and Understanding of the Disease
by Stefania Porcelli, Aurélie Heckmann, Anne-Claire Lagrée, Clémence Galon, Sara Moutailler and Pierre Lucien Deshuillers
Viruses 2023, 15(11), 2270; https://doi.org/10.3390/v15112270 - 17 Nov 2023
Cited by 6 | Viewed by 1819
Abstract
Ticks and tick-borne diseases (TBDs) are increasingly recognized as a critical One Health concern. Tick-borne encephalitis (TBE), a severe neuro infection caused by the tick-borne encephalitis virus (TBEV), has emerged as a significant global public health threat. Laboratory animals, particularly mice, have played [...] Read more.
Ticks and tick-borne diseases (TBDs) are increasingly recognized as a critical One Health concern. Tick-borne encephalitis (TBE), a severe neuro infection caused by the tick-borne encephalitis virus (TBEV), has emerged as a significant global public health threat. Laboratory animals, particularly mice, have played a pivotal role in advancing our understanding of TBD pathogenesis. Notably, BALB/c mice have been employed as models due to their heightened susceptibility to TBEV. However, the use of C3H mice, valued for other tick-borne pathogens, has remained unexplored for TBEV until now. This study aimed to assess the susceptibility of C3H mice to TBEV infection, laying the groundwork for future co-infection models involving TBEV and Borrelia. Experiments revealed that C3H mice are susceptible to TBEV infection through subcutaneous inoculation. While 102 PFU/mouse appeared necessary for full infection, 103 PFU/mouse induced consistent symptoms. However, subsequent assessment of ticks’ acquisition of TBEV from infected mice met with limited success, raising questions about optimal infectious doses for natural infection. These findings suggest the potential of C3H mice for studying TBEV and co-infections with other pathogens, particularly Borrelia. Further exploration of the interplay between these pathogens, their transmission dynamics, and disease severity could enhance prevention and control strategies. Full article
(This article belongs to the Special Issue Animal Models for Virology Research)
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<p>Representation of the experimental design describing the two experiments. (<b>a</b>): Infection of C3H mice with TBEV (first viral production) with 1 PFU/mouse, 10 PFU/mouse, 10<sup>2</sup> PFU/mouse. Blood samples were collected on different days from the infected mice, and mice still alive were sacrificed on day 11 p.i. to collect their brain. Samples were analyzed using real-time RT-PCR. (<b>b</b>): Infection of C3H mice with TBEV (second viral production) with 10<sup>2</sup> PFU/mouse, 10<sup>3</sup> PFU/mouse. The infection was monitored by collecting blood samples on different days and mice still alive were sacrificed on day 15 p.i. to collect their organs. Acquisition of TBEV by ticks was evaluated by placing naive larvae on the backs of mice at different time points. Blood samples and ticks were analyzed using real-time RT-PCR, and organs were analyzed using digital PCR following RT and preamplification steps.</p> Full article ">Figure 2
<p>Experiment B. Survival curve of C3H mice inoculated subcutaneously with two doses of TBEV (strain Hypr): 10<sup>2</sup> PFU (<span class="html-italic">n</span> = 7) and 10<sup>3</sup> PFU (<span class="html-italic">n</span> = 5); and one negative group inoculated with medium (<span class="html-italic">n</span> = 2).</p> Full article ">Figure 3
<p>TBEV RNA quantification in the organs of C3H mice infected with 10<sup>2</sup> and 10<sup>3</sup> PFU using digital PCR following RT and preamplification steps on day 15 p.i. from mice that were euthanized, or between day 10 and day 13 p.i. for mice that died before. Means and standard errors are shown.</p> Full article ">
5 pages, 1534 KiB  
Reply
Reply to Kulić, Ž. Comment on “Subhadra et al. Significant Broad-Spectrum Antiviral Activity of Bi121 against Different Variants of SARS-CoV-2. Viruses 2023, 15, 1299”
by Bobban Subhadra, Ragini Agrawal, Virender Kumar Pal, Agnes-Laurence Chenine, Jeffy George Mattathil and Amit Singh
Viruses 2023, 15(11), 2269; https://doi.org/10.3390/v15112269 - 17 Nov 2023
Viewed by 936
Abstract
We would like to thank Dr. Žarko Kulić [...] Full article
(This article belongs to the Special Issue RNA Viruses: Replication, Assembly and Antivirals)
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<p>Neoilludin B—LC–MS putative identification from Bi121 fractions (F2, F3, F5).</p> Full article ">Figure 2
<p>(<b>A</b>) <span class="html-italic">P. sidoides</span> root powder batch used to prepare Bi121. (<b>B</b>) Prepared Bi121 extract from the <span class="html-italic">Pelargonium</span> root powder.</p> Full article ">Figure 3
<p>Preparation of polyphenol fractions of Bi121 by reversed-phase UHPLC. Fractionation of the polyphenol-enriched fraction was carried out using an Agilent AdvanceBio Column (2.7 µm, 2.1 × 250 mm) with solvent A (10 mM TEABC, pH 8.0) and an Agilent UHPLC 1290 system. The separation was performed by running a gradient of solvent B (10 mM TEABC, pH 8.0, 90% ACN) and solvent A (10 mM TEABC, pH 8.0) at a flow rate of 250 µL/min. The elute fractions were collected into a 96-well plate using a 1260 series auto-sample fraction collector based on the peaks at UV wavelengths between 214 nm and 280 nm. The 96-well plate elute fractions were collected into 1.5 mL tubes according to retention time (12 min per fraction) for a total of 8 fractions, as shown in the LC chromatography. Six UHPLC runs were performed, and the eight fractions were pooled and further evaporated using a speed vacuum.</p> Full article ">
3 pages, 177 KiB  
Comment
Comment on Subhadra et al. Significant Broad-Spectrum Antiviral Activity of Bi121 against Different Variants of SARS-CoV-2. Viruses 2023, 15, 1299
by Žarko Kulić
Viruses 2023, 15(11), 2268; https://doi.org/10.3390/v15112268 - 17 Nov 2023
Cited by 1 | Viewed by 1057
Abstract
The article “Significant Broad-Spectrum Antiviral Activity of Bi121 against Different Variants of SARS-CoV-2” by Subhadra et al. [...] Full article
(This article belongs to the Special Issue RNA Viruses: Replication, Assembly and Antivirals)
16 pages, 4352 KiB  
Article
A Novel Subcluster of Closely Related Bacillus Phages with Distinct Tail Fiber/Lysin Gene Combinations
by Rachel E. Loney, Véronique A. Delesalle, Brianne E. Chaudry, Megan Czerpak, Alexandra A. Guffey, Leo Goubet-McCall, Michael McCarty, Madison S. Strine, Natalie T. Tanke, Albert C. Vill and Greg P. Krukonis
Viruses 2023, 15(11), 2267; https://doi.org/10.3390/v15112267 - 17 Nov 2023
Viewed by 1734
Abstract
Bacteriophages (phages) are the most numerous entities on Earth, but we have only scratched the surface of describing phage diversity. We isolated seven Bacillus subtilis phages from desert soil in the southwest United States and then sequenced and characterized their genomes. Comparative analyses [...] Read more.
Bacteriophages (phages) are the most numerous entities on Earth, but we have only scratched the surface of describing phage diversity. We isolated seven Bacillus subtilis phages from desert soil in the southwest United States and then sequenced and characterized their genomes. Comparative analyses revealed high nucleotide and amino acid similarity between these seven phages, which constitute a novel subcluster. Interestingly, the tail fiber and lysin genes of these phages seem to come from different origins and carry out slightly different functions. These genes were likely acquired by this subcluster of phages via horizontal gene transfer. In conjunction with host range assays, our data suggest that these phages are adapting to hosts with different cell walls. Full article
(This article belongs to the Special Issue Diversity and Evolution of Viruses in Ecosystem)
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<p>Genome map of 056SW001B, chosen because it shares phams with some but not all of the other phages in this subcluster. The ruler shows genome length (in kilobases) with forward and reverse genes shown above and below the ruler, respectively. Functions or putative functions are listed above genes. Due to direct terminal repeats, gene 84 is a duplication of gene 1 (terminase small subunit). Map was created using Phamerator [<a href="#B48-viruses-15-02267" class="html-bibr">48</a>].</p> Full article ">Figure 2
<p>Whole genome maps of the V4 phages, created with Phamerator [<a href="#B48-viruses-15-02267" class="html-bibr">48</a>]. Nucleotide sequence similarity between adjacent genomes is indicated by shading from lavender (most similar) to red (least similar). A lack of shading shows that there is no similarity based on local alignment E score greater than 10<sup>−4</sup>. * marks the WTA-tail fiber/peptidase phams, # marks the lysin/endolysin phams. A total of 96 phams were identified among these seven phages.</p> Full article ">Figure 2 Cont.
<p>Whole genome maps of the V4 phages, created with Phamerator [<a href="#B48-viruses-15-02267" class="html-bibr">48</a>]. Nucleotide sequence similarity between adjacent genomes is indicated by shading from lavender (most similar) to red (least similar). A lack of shading shows that there is no similarity based on local alignment E score greater than 10<sup>−4</sup>. * marks the WTA-tail fiber/peptidase phams, # marks the lysin/endolysin phams. A total of 96 phams were identified among these seven phages.</p> Full article ">Figure 3
<p>Transmission electron microscopy image of phage 019DV002. Image taken at 130,000x using a Phillips CM1000 TEM.</p> Full article ">Figure 4
<p>MUSCLE alignment of 056SW001B gene 20 (Tail_Fiber) and 268TH004 gene 20 (Peptidase), made with [<a href="#B52-viruses-15-02267" class="html-bibr">52</a>,<a href="#B53-viruses-15-02267" class="html-bibr">53</a>].</p> Full article ">Figure 5
<p>MUSCLE alignment of 056SW001B gene 33 (Lysin) and 268TH004 gene 33 (Endolysin), made with [<a href="#B52-viruses-15-02267" class="html-bibr">52</a>,<a href="#B53-viruses-15-02267" class="html-bibr">53</a>].</p> Full article ">
11 pages, 2197 KiB  
Article
Presence and Persistence of Andes Virus RNA in Human Semen
by Roland Züst, Rahel Ackermann-Gäumann, Nicole Liechti, Denise Siegrist, Sarah Ryter, Jasmine Portmann, Nicole Lenz, Christian Beuret, Roger Koller, Cornelia Staehelin, Andrea B. Kuenzli, Jonas Marschall, Sylvia Rothenberger and Olivier Engler
Viruses 2023, 15(11), 2266; https://doi.org/10.3390/v15112266 - 17 Nov 2023
Cited by 1 | Viewed by 1957
Abstract
When infecting humans, Andes orthohantavirus (ANDV) may cause a severe disease called hantavirus cardiopulmonary syndrome (HCPS). Following non-specific symptoms, the infection may progress to a syndrome of hemorrhagic fever combined with hyper-acute cardiopulmonary failure. The case fatality rate ranges between 25–40%, depending on [...] Read more.
When infecting humans, Andes orthohantavirus (ANDV) may cause a severe disease called hantavirus cardiopulmonary syndrome (HCPS). Following non-specific symptoms, the infection may progress to a syndrome of hemorrhagic fever combined with hyper-acute cardiopulmonary failure. The case fatality rate ranges between 25–40%, depending on the outbreak. In this study, we present the follow-up of a male patient who recovered from HCPS six years ago. We demonstrate that the ANDV genome persists within the reproductive tract for at least 71 months. Genome sequence analysis early and late after infection reveals a low number of mutations (two single nucleotide variants and one deletion), suggesting limited replication activity. We can exclude the integration of the viral genome into the host genome, since the treatment of the specimen with RNAse led to a loss of signal. We demonstrate a long-lasting, strong neutralizing antibody response using pseudovirions expressing the ANDV glycoprotein. Taken together, our results show that ANDV has the potential for sexual transmission. Full article
(This article belongs to the Special Issue RNA Viruses and Antibody Response, 2nd Edition)
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<p>Detection of Andes virus RNA in plasma and semen. RNA levels from plasma (circles) and semen (squares) were assessed on the indicated days post infection using in-house quantitative real-time polymerase chain reaction; viral load on day 1 was from a serum sample, and from day 4 onward was from EDTA blood.</p> Full article ">Figure 2
<p>Location of viral material in semen. In total, 1 ml of semen was centrifuged at 600× <span class="html-italic">g</span> for 15 min. The supernatant was transferred into a new tube and the pellet resuspended in 1 ml of PBS. Nucleic acid from 100 μL of input semen, supernatant and resuspended pellet was isolated and analyzed using real-time PCR. Statistical analysis was performed using unpaired the Student’s t-test (***, <span class="html-italic">p</span> &lt; 0.001; **, <span class="html-italic">p</span> &lt; 0.01; *, <span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 3
<p>Analysis of viral material. Nucleic acid from semen was isolated and left untreated (black) or subjected to RNase treatment (white). SNV RNA and a plasmid (not sensitive to RNase treatment) containing the target of the SNV RT-qPCR served as controls. After RNase digestion, no signal could be detected in material derived from semen or control RNA (n.d.; not detected).</p> Full article ">Figure 4
<p>Neutralization of ANDV using patient sera. The neutralizing antibody titer (reciprocal IC<sub>50</sub>) against ANDV in serum samples was assessed using a pseudovirion assay. Half maximal inhibitory concentrations were estimated via a model of nonlinear regression fit with settings for log (inhibitor) vs. normalized response curves using GraphPad Prism v9.</p> Full article ">Figure 5
<p>Phylogenetic analysis of ANDV shows a close relationship to previously sequenced ANDV from Chile and Argentina ((<b>A</b>): segment S, (<b>B</b>): segment M, (<b>C</b>): segment L).</p> Full article ">
12 pages, 1164 KiB  
Article
The Prevalence, Seroprevalence, and Risk Factors of Tick-Borne Encephalitis Virus in Dogs in Lithuania, a Highly Endemic State
by Evelina Simkute, Arnoldas Pautienius, Juozas Grigas, Paulina Urbute and Arunas Stankevicius
Viruses 2023, 15(11), 2265; https://doi.org/10.3390/v15112265 - 17 Nov 2023
Cited by 1 | Viewed by 1809
Abstract
The rising awareness and increasing number of case reports of tick-borne encephalitis (TBE) in dogs indicate that the virus might be an important tick-borne pathogen in dogs, especially in endemic areas. Therefore, the aim of the present study was to investigate the prevalence [...] Read more.
The rising awareness and increasing number of case reports of tick-borne encephalitis (TBE) in dogs indicate that the virus might be an important tick-borne pathogen in dogs, especially in endemic areas. Therefore, the aim of the present study was to investigate the prevalence rate of TBEV RNA and TBEV-specific antibodies in clinical samples of dogs living in a highly endemic region of Lithuania and to evaluate the main risk factors for severe disease course and death. The blood samples (n = 473) of dogs were collected in two veterinary clinics in central Lithuania. Tick-borne encephalitis virus (TBEV) RNA was detected in 18.6% (88/473; CI 95% 15.2–22.4) and TBEV-specific antibodies were found in 21.6% (102/473; CI 95% 17.9–25.6) of dog blood serum samples after confirmation with a virus neutralization test. The death/euthanasia rate was 18.2% (16/88; CI 95% 10.8–27.8) in PCR-positive dogs. Male dogs were more likely to develop neurological symptoms (p = 0.008). Older dogs (p = 0.003), dogs with the presence of neurological symptoms (p = 0.003), and dogs with the presence of TBEV-specific antibodies (p = 0.024) were more likely to experience worse outcomes of the disease. The results of the present study demonstrate that TBEV is a common and clinically important pathogen in dogs in such endemic countries as Lithuania. Full article
(This article belongs to the Special Issue Usutu Virus, West Nile Virus and Neglected Flaviviruses)
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<p>(<b>A</b>) The prevalence rate (%) of TBEV RNA and TBEV-specific antibodies in blood/blood serum samples of dogs and (<b>B</b>) prevalence rate (%) in different months of sample collection.</p> Full article ">Figure 2
<p>Prevalence of TBEV−specific antibodies. (<b>A</b>) Number of seropositive dogs and levels of detected TBEV−specific antibodies. (<b>B</b>) Q−Q plot. (<b>C</b>) Differences in levels of TBEV−specific antibodies in seropositive dogs. (<b>D</b>) Differences in levels of TBEV−specific antibodies in dogs with potentially TBE−specific symptoms, non−specific symptoms, and dogs with non−TBE−related symptoms. (<b>E</b>) Differences in levels of TBEV−specific antibodies in dogs with other tick−borne diseases and dogs without other concurrently diagnosed tick−borne diseases.</p> Full article ">Figure 3
<p>(<b>A</b>) Comparison of the prevalence rates (%) of TBEV RNA in tick suspensions and third passage isolates in Neuro-2a, Vero, and Marc-145 cell cultures. (<b>B</b>) TBEV RNA prevalence rate (%) changes at each passage in different cell cultures.</p> Full article ">
13 pages, 1747 KiB  
Article
Prophylactic Treatment with Baloxavir Protects Mice from Lethal Infection with Influenza A and B Viruses
by Keita Fukao, Takeshi Noshi, Shinya Shano, Kaoru Baba, Kenji Sato, Masashi Sakuramoto, Naohisa Kitade, Hideki Tanioka, Shinji Kusakabe and Takao Shishido
Viruses 2023, 15(11), 2264; https://doi.org/10.3390/v15112264 - 16 Nov 2023
Cited by 1 | Viewed by 1919
Abstract
Influenza remains a worldwide health concern. Antiviral drugs are considered as one of the useful options for its prevention as a complementary measure to vaccination. Baloxavir acid selectively inhibits the cap-dependent endonuclease of influenza viruses and exhibits marked viral titre reduction in patients. [...] Read more.
Influenza remains a worldwide health concern. Antiviral drugs are considered as one of the useful options for its prevention as a complementary measure to vaccination. Baloxavir acid selectively inhibits the cap-dependent endonuclease of influenza viruses and exhibits marked viral titre reduction in patients. Here, we describe the prophylactic potency of baloxavir acid against lethal infection with influenza A and B viruses in mice. BALB/c mice were subcutaneously administered once with baloxavir acid suspension, or orally administered once daily for 10 days with oseltamivir phosphate solution at human relevant doses. Next, the mice were intranasally inoculated with A/PR/8/34 (H1N1) or B/Hong Kong/5/72 strain at 24 to 96 h after the initial dosing. Prophylactic treatment with the antiviral drugs significantly reduced the lung viral titres and prolonged survival time. In particular, baloxavir acid showed a greater suppressive effect on lung viral titres compared to oseltamivir phosphate. In this model, baloxavir acid maintained significant prophylactic effects against influenza A and B virus infections when the plasma concentration at the time of infection was at least 0.88 and 3.58 ng/mL, respectively. The significant prophylactic efficacy observed in our mouse model suggests the potential utility of baloxavir marboxil for prophylaxis against influenza in humans. Full article
(This article belongs to the Section Viral Immunology, Vaccines, and Antivirals)
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<p>Plasma concentration–time profiles after single subcutaneous administration of baloxavir acid suspension to mice. Four mice were administered baloxavir acid (BXA) subcutaneously. Blood samples were collected at each time point, followed by determination of the concentrations of BXA in plasma. Each plot represents the mean ± SD of 4 mice. The lower limit of quantification (BLQ) is &lt;0.1 ng/mL. The mean values for BLQ were not plotted. The plasma concentrations after treatment with 10 mg of BXA were from another study [<a href="#B20-viruses-15-02264" class="html-bibr">20</a>].</p> Full article ">Figure 2
<p>Effect of prophylactic treatment with baloxavir acid and oseltamivir phosphate on mortality, body weight loss and lung virus titre in mice infected with influenza A or B virus. Ten mice were subcutaneously administered baloxavir acid (BXA) or vehicle 24 h before infection. As a reference, from 24 h before infection, mice were orally administered oseltamivir phosphate (OSP) once a day for 10 days. The mice were then intranasally inoculated with A/PR/8/34 (H1N1) or B/Hong Hong/5/72 strain at 24 h after the initial dosing. Survival (<b>a</b>,<b>b</b>) and body weight (<b>c</b>,<b>d</b>) were monitored daily for 21 days after the virus infection. Lung virus titres were measured at 1, 2, 4, 8 and 10 days after infection (<b>e</b>,<b>f</b>). Triangles indicate the time points of administration of each compound. Survival times among the three groups were analysed by log-rank test. For the pairwise comparison of the virus titres in lungs between two groups at each time point, the one-way analysis of variance model was applied by time point. * and ** denote <span class="html-italic">p</span> &lt; 0.05 and <span class="html-italic">p</span> &lt; 0.0001 vs. the vehicle group. <sup>†</sup> and <sup>††</sup> denote <span class="html-italic">p</span> &lt; 0.05 and <span class="html-italic">p</span> &lt; 0.0001 vs. OSP group.</p> Full article ">Figure 3
<p>Effect of baloxavir acid at each administration timing and dosage on mortality and lung virus titre in mice infected with influenza A virus. Ten mice were subcutaneously administered baloxavir acid (BXA) or vehicle at 72 or 96 h before infection. The mice were then intranasally inoculated with A/PR/8/34 (H1N1) strain at 72 or 96 h after dosing. Survival and body weight were monitored daily for 28 days after the virus infection (<b>a</b>,<b>b</b>). Viral titres in supernatants of lung homogenates were determined (<span class="html-italic">n</span> = 10 per group) on days 1, 2, 4, 6, 8 and 10 after infection by standard TCID<sub>50</sub> assay (<b>c</b>). Triangles indicate the time points of administration of each compound. Survival times among the three groups were analysed by log-rank test. For the pairwise comparison of the virus titres in lungs between two groups each at each time point, the one-way analysis of variance model was applied by time point. ** and *** denote <span class="html-italic">p</span> &lt; 0.01 and <span class="html-italic">p</span> &lt; 0.0001 vs. the vehicle group, respectively.</p> Full article ">Figure 4
<p>Effect of baloxavir acid at each administration timing and dosage on mortality and lung virus titre in mice infected with influenza B virus. Ten mice were subcutaneously administered baloxavir acid (BXA) or vehicle 48, 72 or 96 h before infection. The mice were then intranasally inoculated with B/Hong Hong/5/72 strain at 48, 72 or 96 h after dosing. Survival and body weight were examined daily for 28 days after the virus infection (<b>a</b>–<b>c</b>). Viral titres in supernatants of lung homogenates were determined (<span class="html-italic">n</span> = 10 per group) on days 1, 2, 4, 6, 8 and 10 after infection by standard TCID<sub>50</sub> assay (<b>d</b>). Triangles indicate the time points of administration of each compound. Survival times among the three groups were analysed by log-rank test. For pairwise comparison of the virus titres in lungs between two groups each at each time point, the one-way analysis of variance model was applied by time point. *, ** and *** denote <span class="html-italic">p</span> &lt; 0.05, <span class="html-italic">p</span> &lt; 0.01 and <span class="html-italic">p</span> &lt; 0.0001 vs. the vehicle group, respectively.</p> Full article ">
16 pages, 1454 KiB  
Article
Phenotypic Characterization of Recombinant Marek’s Disease Virus in Live Birds Validates Polymorphisms Associated with Virulence
by Taejoong Kim, Cari J. Hearn, Jody Mays, Deborah Velez-Irizarry, Sanjay M. Reddy, Stephen J. Spatz, Hans H. Cheng and John R. Dunn
Viruses 2023, 15(11), 2263; https://doi.org/10.3390/v15112263 - 16 Nov 2023
Viewed by 1451
Abstract
Marek’s disease (MD) is a highly infectious lymphoproliferative disease in chickens with a significant economic impact. Mardivirus gallidalpha 2, also known as Marek’s disease virus (MDV), is the causative pathogen and has been categorized based on its virulence rank into four pathotypes: mild [...] Read more.
Marek’s disease (MD) is a highly infectious lymphoproliferative disease in chickens with a significant economic impact. Mardivirus gallidalpha 2, also known as Marek’s disease virus (MDV), is the causative pathogen and has been categorized based on its virulence rank into four pathotypes: mild (m), virulent (v), very virulent (vv), and very virulent plus (vv+). A prior comparative genomics study suggested that several single-nucleotide polymorphisms (SNPs) and genes in the MDV genome are associated with virulence, including nonsynonymous (ns) SNPs in eight open reading frames (ORF): UL22, UL36, UL37, UL41, UL43, R-LORF8, R-LORF7, and ICP4. To validate the contribution of these nsSNPs to virulence, the vv+MDV strain 686 genome was modified by replacing nucleotides with those observed in the vMDV strains. Pathogenicity studies indicated that these substitutions reduced the MD incidence and increased the survival of challenged birds. Furthermore, using the best-fit pathotyping method to rank the virulence, the modified vv+MDV 686 viruses resulted in a pathotype similar to the vvMDV Md5 strain. Thus, these results support our hypothesis that SNPs in one or more of these ORFs are associated with virulence but, as a group, are not sufficient to result in a vMDV pathotype, suggesting that there are additional variants in the MDV genome associated with virulence, which is not surprising given this complex phenotype and our previous finding of additional variants and SNPs associated with virulence. Full article
(This article belongs to the Section Animal Viruses)
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<p>The EcoRV digestion of recombinant MDV 686-BAC clones. (<b>A</b>) Agarose gel electrophoresis image of MDV 686-BAC clones. (<b>B</b>) The expected EcoRV digestion pattern of MDV 686-BAC clones. (Lane 1) 686-BAC, (Lane 2) 686-BAC with TRS-TRL deletion (686∆TRS-TRL), (Lanes 3–4) two viruses independent of 686-BAC with nine single-nucleotide modifications (686 mut1, 686 mut2), (M) 1 kb Extend DNA ladder (NEB). The specific changes in fragmentations are on the right (*).</p> Full article ">Figure 2
<p>Survival rates of ADOL 15I<sub>5</sub> × 7<sub>1</sub> chickens infected with v686-BAC, v686∆TRS-TRL, v686 mut1, or v686 mut2. The survival rate was analyzed with the Log-rank (Mantel–Cox) test using Prism 10.0.1. (<b>A</b>) Survival percent of clinical trial 1 with maternal-antibody-negative birds; (<b>B</b>) survival percent of clinical trial 2 with maternal-antibody-positive birds. Statistical differences (<span class="html-italic">p</span>-value &lt; 0.05) between v686∆TRS-TRL, v686 mut1, and v686 mut2 are indicated (n.s. = not significant, * <span class="html-italic">p</span>-value &lt; 0.05, ** <span class="html-italic">p</span>-value &lt; 0.01).</p> Full article ">Figure 3
<p>Survival curve of JM/102W, Md5, 648A, v686∆TRS-TRL, v686 mut1, or v686 mut2 challenged group in non-vaccinated birds. Survival curve of MDV JM/102W, Md5, 648A, v686∆TRS-TRL, v686 mut1, or v686 mut2 challenged group in HVT-vaccinated birds is in <a href="#app1-viruses-15-02263" class="html-app">Figure S6</a> and survival curve of MDV JM/102W, Md5, 648A, v686∆TRS-TRL, v686 mut1, or v686 mut2 challenged group in bivalent-vaccinated birds is in <a href="#app1-viruses-15-02263" class="html-app">Figure S7</a>.</p> Full article ">Figure 4
<p>Proportional distances of the virulence of v686 mut1, v686 mut2, v686∆TRS-TRL, and the reference MDV strains with known pathotypes. The reference MDV strains are JM/102W (vMDV), Md5 (vvMDV), and 648A (vv+MDV).</p> Full article ">
17 pages, 599 KiB  
Review
Tumor Tropism of DNA Viruses for Oncolytic Virotherapy
by Junior A. Enow, Hummad I. Sheikh and Masmudur M. Rahman
Viruses 2023, 15(11), 2262; https://doi.org/10.3390/v15112262 - 16 Nov 2023
Cited by 3 | Viewed by 2650
Abstract
Oncolytic viruses (OVs) have emerged as one of the most promising cancer immunotherapy agents that selectively target and kill cancer cells while sparing normal cells. OVs are from diverse families of viruses and can possess either a DNA or an RNA genome. These [...] Read more.
Oncolytic viruses (OVs) have emerged as one of the most promising cancer immunotherapy agents that selectively target and kill cancer cells while sparing normal cells. OVs are from diverse families of viruses and can possess either a DNA or an RNA genome. These viruses also have either a natural or engineered tropism for cancer cells. Oncolytic DNA viruses have the additional advantage of a stable genome and multiple-transgene insertion capability without compromising infection or replication. Herpes simplex virus 1 (HSV-1), a member of the oncolytic DNA viruses, has been approved for the treatment of cancers. This success with HSV-1 was achievable by introducing multiple genetic modifications within the virus to enhance cancer selectivity and reduce the toxicity to healthy cells. Here, we review the natural characteristics of and genetically engineered changes in selected DNA viruses that enhance the tumor tropism of these oncolytic viruses. Full article
(This article belongs to the Section General Virology)
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<p>Oncolytic DNA virus cellular tropism. Oncolytic DNA viruses are engineered to selectively infect, replicate within, and eliminate cancer cells while sparing normal cells. Notable examples include vvDD (vaccinia virus double-deleted) and HSV-1 lacking ICP6 (infected-cell protein 6).</p> Full article ">
12 pages, 1051 KiB  
Article
An Outbreak of Parvovirus B19 in Israel
by Tal Patalon, Yaki Saciuk, Daniel Trotzky, Gal Pachys, Amir Ben-Tov, Yaakov Segal and Sivan Gazit
Viruses 2023, 15(11), 2261; https://doi.org/10.3390/v15112261 - 16 Nov 2023
Cited by 8 | Viewed by 3793
Abstract
Human parvovirus B19 (B19V) has a wide clinical spectrum, ranging from an asymptomatic infection to a life threatening one. During pregnancy, it can lead to fetal loss and hydrops fetalis. This retrospective study examined the incidence rates of B19V in Israel, analyzing anonymized [...] Read more.
Human parvovirus B19 (B19V) has a wide clinical spectrum, ranging from an asymptomatic infection to a life threatening one. During pregnancy, it can lead to fetal loss and hydrops fetalis. This retrospective study examined the incidence rates of B19V in Israel, analyzing anonymized electronic medical records of 2.7 million individuals between January 2015 and September 2023. A generalized linear model with a Poisson distribution was fit to the data, adjusting for potential confounders. A marked increase in B19V was observed in 2023, with an adjusted incidence rate ratio (IRR) of 6.6 (95% CI 6.33–6.89) when comparing 2023 to previous years. When specifically comparing 2023 to COVID-19 years (2020–2022), adjusted IRR climbs to 9.21 (8.66–9.80). Moreover, in 2023, previously existing seasonality has largely disappeared. High SES characterized most infected individuals with a marked discrepancy in social sectors; the Arab population was significantly less likely to be found B19V positive, even when adjusting for SES. Most infections occurred in school-aged children (6–11 years old). Pregnant women experienced the most significant rise in B19V, with an adjusted IRR of 11.47 (9.44–13.97) in 2023 compared to previous years; most cases were diagnosed in the first trimester. This study demonstrates that Israel is currently experiencing the largest and longest reported outbreak of B19V to date. Policymakers should consider setting screening policies in place, at least for populations at risk, while specifically studying and potentially targeting low socioeconomic populations and specific social sectors to avoid health inequalities. Full article
(This article belongs to the Section Human Virology and Viral Diseases)
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<p>Parvovirus B19 incidence rates per 10,000 person days at risk between 2015 and 2023, by month.</p> Full article ">Figure 2
<p>Monthly Parvovirus B19 incidence rates per 10,000 person days at risk between 2015 and 2023, by month, by socioeconomic status.</p> Full article ">Figure 3
<p>Number of Parvovirus B19 serology tests taken each year, and their respective rates per 10,000 person days at risk. To allow for seasonality considerations, months October to November are separated visually, as the corresponding data in 2023 is not included. A linear extrapolation of this quarter is present in 2023 is presented in yellow.</p> Full article ">Figure 4
<p>Monthly Parvovirus B19 incidence rates per 10,000 person days at risk of MHS member under 18 years between 2015 and 2023, by age group.</p> Full article ">Figure 5
<p>Monthly Parvovirus B19 incidence rates per 10,000 person days at risk of pregnant women between 2015 and 2023, by trimester of infection.</p> Full article ">
11 pages, 587 KiB  
Article
Association between Pulmonary Aspergillosis and Cytomegalovirus Reactivation in Critically Ill COVID-19 Patients: A Prospective Observational Cohort Study
by Valeria Caciagli, Irene Coloretti, Marta Talamonti, Carlotta Farinelli, Ilenia Gatto, Emanuela Biagioni, Mario Sarti, Erica Franceschini, Marianna Meschiari, Cristina Mussini, Roberto Tonelli, Enrico Clini, Massimo Girardis, Stefano Busani and Modena COVID-19 Working Group
Viruses 2023, 15(11), 2260; https://doi.org/10.3390/v15112260 - 15 Nov 2023
Cited by 4 | Viewed by 1893
Abstract
COVID-19-associated invasive pulmonary aspergillosis (CAPA) is common and is associated with poor outcomes in critically ill patients. This prospective observational study aimed to explore the association between CAPA development and the incidence and prognosis of cytomegalovirus (CMV) reactivation in critically ill COVID-19 patients. [...] Read more.
COVID-19-associated invasive pulmonary aspergillosis (CAPA) is common and is associated with poor outcomes in critically ill patients. This prospective observational study aimed to explore the association between CAPA development and the incidence and prognosis of cytomegalovirus (CMV) reactivation in critically ill COVID-19 patients. We included all consecutive critically ill adult patients with confirmed COVID-19 infection who were admitted to three COVID-19 intensive care units (ICUs) in an Italian hospital from 25 February 2020 to 8 May 2022. A standardized procedure was employed for early detection of CAPA. Risk factors associated with CAPA and CMV reactivation and the association between CMV recurrence and mortality were estimated using adjusted Cox proportional hazard regression models. CAPA occurred in 96 patients (16.6%) of the 579 patients analyzed. Among the CAPA population, 40 (41.7%) patients developed CMV blood reactivation with a median time of 18 days (IQR 7–27). The CAPA+CMV group did not exhibit a significantly higher 90-day mortality rate (62.5% vs. 48.2%) than the CAPA alone group (p = 0.166). The CAPA+CMV group had a longer ICU stay, fewer ventilation-free days, and a higher rate of secondary bacterial infections than the control group of CAPA alone. In the CAPA population, prior immunosuppression was the only independent risk factor for CMV reactivation (HR 2.33, 95% C.I. 1.21–4.48, p = 0.011). In critically ill COVID-19 patients, CMV reactivation is common in those with a previous CAPA diagnosis. Basal immunosuppression before COVID-19 appeared to be the primary independent variable affecting CMV reactivation in patients with CAPA. Furthermore, the association of CAPA+CMV versus CAPA alone appears to impact ICU length of stay and secondary bacterial infections but not mortality. Full article
(This article belongs to the Special Issue Impact of Co-infections in COVID-19: Predisposing Mechanisms and Interplay between Invasive Viral, Bacterial and Fungal Pathogens)
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<p>Kaplan–Meier curve for cumulative 90-day survival in CAPA population developing CMV blood reactivation or not.</p> Full article ">
19 pages, 3538 KiB  
Article
Effects of Deformed Wing Virus-Targeting dsRNA on Viral Loads in Bees Parasitised and Non-Parasitised by Varroa destructor
by Zoe E. Smeele, James W. Baty and Philip J. Lester
Viruses 2023, 15(11), 2259; https://doi.org/10.3390/v15112259 - 15 Nov 2023
Cited by 5 | Viewed by 2118
Abstract
The Varroa destructor mite is a devastating parasite of honey bees; however the negative effects of varroa parasitism are exacerbated by its role as an efficient vector of the honey bee pathogen, Deformed wing virus (DWV). While no direct treatment for DWV infection [...] Read more.
The Varroa destructor mite is a devastating parasite of honey bees; however the negative effects of varroa parasitism are exacerbated by its role as an efficient vector of the honey bee pathogen, Deformed wing virus (DWV). While no direct treatment for DWV infection is available for beekeepers to use on their hives, RNA interference (RNAi) has been widely explored as a possible biopesticide approach for a range of pests and pathogens. This study tested the effectiveness of three DWV-specific dsRNA sequences to lower DWV loads and symptoms in honey bees reared from larvae in laboratory mini-hives containing bees and varroa. The effects of DWV-dsRNA treatment on bees parasitised and non-parasitised by varroa mites during development were investigated. Additionally, the impact of DWV-dsRNA on viral loads and gene expression in brood-parasitising mites was assessed using RNA-sequencing. Bees parasitised during development had significantly higher DWV levels compared to non-parasitised bees. However, DWV-dsRNA did not significantly reduce DWV loads or symptoms in mini-hive reared bees, possibly due to sequence divergence between the DWV variants present in bees and varroa and the specific DWV-dsRNA sequences used. Varroa mites from DWV-dsRNA treated mini-hives did not show evidence of an elevated RNAi response or significant difference in DWV levels. Overall, our findings show that RNAi is not always successful, and multiple factors including pathogen diversity and transmission route may impact its efficiency. Full article
(This article belongs to the Section Viral Immunology, Vaccines, and Antivirals)
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<p>A mini-frame and mini-hive used in experiments. (<b>a</b>) Two mini-frames adhered together to fit as a single frame inside a hive for acquiring brood. Mini-frames were separated for one mini-frame of larvae to be introduced to the mini-hive. (<b>b</b>) A mini-hive with two plexiglass viewing windows in the front, and two chambers separated by a plexiglass partition to fit a mini-frame in the left chamber and the treatment pouch in the right foraging chamber.</p> Full article ">Figure 2
<p>Relative DWV levels of uncapped bees from sugar, non-specific dsRNA and DWV-dsRNA treated mini-hives. Coloured boxes show different parasitism phenotypes: non-parasitised (pink), parasitised with wing deformities (orange) and parasitised with normal wings (green). Raw data points (i.e., Relative DWV level of individual bees) for each box plot are overlayed. Upper and lower hinges of the boxes show 75% and 25% quantiles, respectively, separated by black lines showing the median. Upper and lower whiskers extend to 1.5* interquartile range.</p> Full article ">Figure 3
<p>Effect of treatment on the proportion of uncapped bees with wing deformities from each mini-hive. Bars show mean proportion of uncapped bees with wing deformities for sugar water (n = 4), non-specific dsRNA (n = 4), and DWV-dsRNA (n = 5) treated mini-hives with error bars showing standard deviation. No significant difference in proportion of bees with deformed wings was found between treatments (df = 2, chi-squared = 1.4251, <span class="html-italic">p</span> = 0.49).</p> Full article ">Figure 4
<p>Multidimensional scaling (MDS) plot of expression profile (TMM normalised gene counts) of candidate RNAi-associated genes for each mite sample. Each triangle denotes an individual mite sample with colours indicating which treatment group the sample belongs to: Non-specific dsRNA (orange), sugar control (green) or DWV-dsRNA (purple). Dimensions one and two are shown and account for 92% of the variation in the data.</p> Full article ">Figure 5
<p>Relative abundance of deformed wing virus loads represented as transcripts per million (TPM) in varroa mite RNA-seq samples from sugar water, non-specific dsRNA and DWV-dsRNA-treated mini-hives. Upper and lower hinges of the boxes show 75% and 25% quantiles, respectively. Whiskers extend to 1.5* interquartile range. Raw data is overlaid to show TPM values for each sample (black points). ANOVA results showed no significant difference in DWV loads in varroa mites between treatment groups (df = 2, F = 2.883, <span class="html-italic">p</span> = 0.09).</p> Full article ">Figure 6
<p>Unrooted Bayesian phylogenetic tree of DWV-A genome sequences, labelled with NCBI accession IDs. Branch labels show posterior probabilities and are coloured by country of isolate sample origin. The DWV contig identified in varroa samples from our study is shown in the green box.</p> Full article ">
12 pages, 1427 KiB  
Article
High Seroprevalence of IgG Antibodies to Multiple Arboviruses in People Living with HIV (PLWHIV) in Madagascar
by Fetra Angelot Rakotomalala, Julie Bouillin, Santatriniaina Dauphin Randriarimanana, Guillaume Thaurignac, Luca Maharavo, Mihaja Raberahona, Lucien Razafindrakoto, Jasmina Rasoanarivo, Mala Rakoto-Andrianarivelo, Danielle Aurore Doll Rakoto, François Xavier Babin, Tahinamandranto Rasamoelina, Eric Delaporte, Luc Hervé Samison, Martine Peeters, Eric Nerrienet and Ahidjo Ayouba
Viruses 2023, 15(11), 2258; https://doi.org/10.3390/v15112258 - 15 Nov 2023
Cited by 4 | Viewed by 1874
Abstract
To estimate the prevalence of IgG antibodies against six arboviruses in people living with HIV-1 (PLWHIV) in Madagascar, we tested samples collected between January 2018 and June 2021. We used a Luminex-based serological assay to detect IgG antibodies against antigens from Dengue virus [...] Read more.
To estimate the prevalence of IgG antibodies against six arboviruses in people living with HIV-1 (PLWHIV) in Madagascar, we tested samples collected between January 2018 and June 2021. We used a Luminex-based serological assay to detect IgG antibodies against antigens from Dengue virus serotypes 1–4 (DENV1–4), Zika virus (ZIKV), West Nile virus (WNV), Usutu virus (USUV), Chikungunya virus (CHIKV), and O’nyong nyong virus (ONNV). Of the 1036 samples tested, IgG antibody prevalence was highest for ONNV (28.4%), CHIKV (26.7%), WNV-NS1 (27.1%), DENV1 (12.4%), USUV (9.9%), and DENV3 (8.9%). ZIKV (4.9%), DENV2 (4.6%), WNV-D3 (5.1%), and DENV4 (1.4%) were lower. These rates varied by province of origin, with the highest rates observed in Toamasina, on the eastern coast (50.5% and 56.8%, for CHIKV and ONNV, respectively). The seroprevalence increased with age for DENV1 and 3 (p = 0.006 and 0.038, respectively) and WNV DIII (p = 0.041). The prevalence of IgG antibodies against any given arborvirus varied over the year and significantly correlated with rainfalls in the different areas (r = 0.61, p = 0.036). Finally, we found a significant correlation between the seroprevalence of antibodies against CHIKV and ONNV and the HIV-1 RNA plasma viral load. Thus, PLWHIV in Madagascar are highly exposed to various arboviruses. Further studies are needed to explain some of our findings. Full article
(This article belongs to the Special Issue Usutu Virus, West Nile Virus and Neglected Flaviviruses)
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<p>Location of sample collection sites. The figure shows the map of Madagascar, with Africa in perspective. Samples were collected in five of the six provinces of Madagascar. The numbers in parentheses represent the sample size from each of the five provinces.</p> Full article ">Figure 2
<p>Seroprevalence of IgG antibodies to multiple arboviruses in people living with HIV-1 from Madagascar. The figure represents graphically the seroprevalence of IgG antibodies to the ten arboviral antigens studied in the five provinces of Madagascar. The data are also detailed in <a href="#viruses-15-02258-t001" class="html-table">Table 1</a>.</p> Full article ">Figure 3
<p>Variation in seroprevalence of antibodies against arboviruses in Madagascar over time. The left axis (blue squares) represents the average rainfall (in millimeters), and the right axis (pink circles) represents the seroprevalence of IgG antibodies against arboviruses (in percentage). The horizontal axis represents the months of sampling averaged over the four years of the study period. A significant correlation was observed between rainfall and seroprevalence (Pearson, r = 0.61, <span class="html-italic">p</span> = 0.036).</p> Full article ">
16 pages, 1742 KiB  
Article
The Circulating miRNA Profile of Chronic Hepatitis D and B Patients Is Comparable but Differs from That of Individuals with HBeAg-Negative HBV Infection
by Daniela Cavallone, Eric David B. Ornos, Gabriele Ricco, Filippo Oliveri, Barbara Coco, Piero Colombatto, Laura De Rosa, Leslie Michelle M. Dalmacio, Ferruccio Bonino and Maurizia Rossana Brunetto
Viruses 2023, 15(11), 2257; https://doi.org/10.3390/v15112257 - 15 Nov 2023
Cited by 2 | Viewed by 1811
Abstract
miRNAs circulating in whole serum and HBsAg-particles are differentially expressed in chronic hepatitis B (CHB) and HBeAg-negative-HBV infection (ENI); their profiles are unknown in chronic hepatitis D (CHD). Serum- and HBsAg-associated miRNAs were analyzed in 75 subjects of 3 well-characterized groups (CHB 25, [...] Read more.
miRNAs circulating in whole serum and HBsAg-particles are differentially expressed in chronic hepatitis B (CHB) and HBeAg-negative-HBV infection (ENI); their profiles are unknown in chronic hepatitis D (CHD). Serum- and HBsAg-associated miRNAs were analyzed in 75 subjects of 3 well-characterized groups (CHB 25, CHD 25, ENI 25) using next-generation sequencing (NGS). Overall miRNA profiles were consonant in serum and HBsAg-particles but significantly different according to the presence of hepatitis independently of Hepatitis D Virus (HDV)-co-infection. Stringent (Bonferroni Correction < 0.001) differential expression analysis showed 39 miRNAs upregulated in CHB vs. ENI and 31 of them also in CHD vs. ENI. miRNA profiles were coincident in CHB and CHD with only miR-200a-3p upregulated in CHB. Three miRNAs (miR-625-3p, miR-142-5p, and miR-223-3p) involved in immune response were upregulated in ENI. All 3 hepatocellular miRNAs of MiR-B-Index (miR-122-5p, miR-99a-5p, miR-192-5p) were overexpressed in both CHB and CHD patients. In conclusion, CHD and CHB patients showed highly similar serum miRNA profiling that was significantly different from that of individuals with HBeAg-negative infection and without liver disease. Full article
(This article belongs to the Section Human Virology and Viral Diseases)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Hierarchical clustering in HBsAg-immunoprecipitated particles. A variance-stabilized transformation was performed on the raw count matrix, and 35 genes with the highest variance across samples were selected for hierarchical clustering. Each row represents one gene, and each column represents one sample. The color represents the difference of the count value to the row mean.</p> Full article ">Figure 2
<p>Hierarchical clustering of whole cohort study. The heatmap shows the result of the two-way hierarchical clustering of microRNAs and samples. Each row represents one gene, and each column represents one sample. The color represents the difference of the count value to the row mean.</p> Full article ">Figure 3
<p>MA plot of differentially expressed genes (DEG). Differential expression of serum miRNAs between HBsAg carriers with HBeAg-negative infection (ENI) and CHB patients.</p> Full article ">Figure 4
<p>MA plot of differentially expressed genes (DEG). Differential expression of serum miRNAs between HBsAg carriers with HBeAg-negative infection (ENI) vs. CHD patients.</p> Full article ">Figure 5
<p>miRNAs were differentially expressed in individuals with HBeAg-negative infection (ENI), chronic hepatitis B (CHB), and chronic hepatitis D (CHD) patients; 31 miRNAs were downregulated in ENI when compared to both CHB and CHD. An additional 8 miRNAs were downregulated and 3 were upregulated when comparing ENI to CHB, and 3 were downregulated in the comparison between ENI and CHD.</p> Full article ">Figure 6
<p>Box plot of the MiR-B-Index in the three groups of HBsAg-positive/HBeAg-negative individuals.</p> Full article ">
14 pages, 2304 KiB  
Article
Effects of US7 and UL56 on Cell-to-Cell Spread of Human Herpes Simplex Virus 1
by Jun Wang, Ke Wu, Longquan Ni, Chenxuan Li, Ruoyan Peng, Yi Li, Zhaojun Fan, Feifei Yin, Fei Deng, Shu Shen and Xiaoli Wu
Viruses 2023, 15(11), 2256; https://doi.org/10.3390/v15112256 - 14 Nov 2023
Cited by 2 | Viewed by 1908
Abstract
Human herpes simplex virus (HSV), a double-stranded DNA virus belonging to the Herpesviridae family and alpha herpesvirus subfamily, is one of the most epidemic pathogens in the population. Cell-to-cell spread is a special intercellular transmission mechanism of HSV that indicates the virulence of [...] Read more.
Human herpes simplex virus (HSV), a double-stranded DNA virus belonging to the Herpesviridae family and alpha herpesvirus subfamily, is one of the most epidemic pathogens in the population. Cell-to-cell spread is a special intercellular transmission mechanism of HSV that indicates the virulence of this virus. Through numerous studies on mutant HSV strains, many viral and host proteins involved in this process have been identified; however, the mechanisms remain poorly understood. Here, we evaluated the effect of the membrane protein genes US7 and UL56 on cell-to-cell spread in vitro between two HSV-1 (HB94 and HN19) strains using a plaque assay, syncytium formation assay, and the CRISPR/Cas9 technique. US7 knockout resulted in the inhibition of viral cell-to-cell spread; additionally, glycoprotein I (US7) of the HB94 strain was found to promote cell-to-cell spread compared to that of the HN19 strain. UL56 knockout did not affect plaque size and syncytium formation; however, the gene product of UL56 from the HN19 strain inhibited plaque formation and membrane infusion. This study presents preliminary evidence of the functions of US7 and UL56 in the cell-to-cell spread of HSV-1, which will provide important clues to reveal the mechanisms of cell-to-cell spread, and contributes to the clinical drugs development. Full article
(This article belongs to the Section Human Virology and Viral Diseases)
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<p>Phenotypic difference between HB94 and HN19. (<b>A</b>) Difference in syncytial size. Vero cells (3 × 10<sup>5</sup> cells/well) were infected by 50 PFU HB94 or HN19 strains and cultured for 3 days. The white squares shows the enlarged plaque image (right) with a bar of 1 mm. (<b>B</b>) Difference in syncytia size. Vero cells were infected by HSV-1 at 0.01 MOI cultured for 24 h. The ZO−1 protein indicates the ability of syncytia. (<b>C</b>) One-step growth curve at 5 or 0.01 MOI. The cellular supernatants were harvested at 12, 24, 36, 48, and 72 h.p.i., and the viral titers were detected by plaque assay. (<b>D</b>) Growth curve of the ratio of virions entering cells. Vero cells (3 × 10<sup>5</sup> cells/well) were infected by 120 PFU HB94 or HN19 strains at 4 °C for 1 h. Virus supernatant was replaced by DMEM medium and cultured at 37 °C for viral entry. Then, the medium was replaced by low-PH citrate buffer solution at 0, 10, 20, 30, 45, and 60 min. Virions that did not enter the cell were inactivated by citric acid buffer solution. After that, the number of plaques in the experimental group and the control group was calculated to determine the proportion of virus entry. (<b>E</b>) Growth curve of <span class="html-italic">gD</span> gene copies at 5 MOI. The cellular supernatants were harvested at 2, 6, 12, 18, and 24 h.p.i., and the viral gD gene copies were detected by qPCR. Ns: not significant.</p> Full article ">Figure 2
<p>Analysis of genetic evolution and genome differences of HB94 and HN19. (<b>A</b>) Phylogenetic evolutionary tree. The blue branch represents the Europe-N. America clade, the green branch represents the Africa clade, and the purple branch represents the Asia clade. The HB94 and HN19 strains are marked in red. The (<b>B</b>) Gene annotation and mutation diagrams; black, synonymous mutation; green, nonsynonymous mutation; red, insert. ORFs are indicated by arrows. The direction of the arrows indicates the orientation of gene transcription. (<b>C</b>) <span class="html-italic">US7</span> and <span class="html-italic">UL56</span> gene schematic of HB94 and HN19. The right side represents differences in the US7 gene, and the left side represents differences in the UL56 gene.</p> Full article ">Figure 3
<p>Effects of <span class="html-italic">US7</span> and <span class="html-italic">UL56</span> on HSV-1 plaque formation. (<b>A</b>,<b>B</b>) Analysis of plaque size and area of HB94 strain by deletion and restoration of <span class="html-italic">US7</span> gene. Vero cells (3 × 10<sup>5</sup> cells/well) were infected by 50 PFU HB94, HB94-US7KO, and HB94-HN19US7KI strains and cultured for 3 days. (<b>C</b>,<b>D</b>) Analysis of plaque size and area of exogenous <span class="html-italic">US7</span> gene expression in HB94-US7KO strain. The eukaryotic expression plasmid pUS7-HN19 or pUS7-HB94 was transfected into Vero cells (1 × 10<sup>5</sup> cells/well) and infected with 50 PFU HB94-US7KO after 24 h. (<b>E</b>,<b>F</b>) Analysis of plaque size and area of HB94 strain with deletion of <span class="html-italic">UL56</span> gene. Vero cells (3 × 10<sup>5</sup> cells/well) were infected by 50 PFU HB94 and HB94-UL56KO strains and cultured for 3 days. (<b>G</b>,<b>H</b>) Analysis of plaque size and area of exogenous <span class="html-italic">UL56</span> gene expression in the HB94-UL56KO strain. The eukaryotic expression plasmid pUl56-HN19 or pUl56-HB94 was transfected into Vero cells (1 × 10<sup>5</sup> cells/well) and infected with 50 PFU HB94-Ul56KO after 24 h. Similar data were obtained from at least two independent experiments. Stats: one-way ANOVA with a Tukey multiple comparisons; * <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. ns: not significant.</p> Full article ">Figure 4
<p>Effects of <span class="html-italic">US7</span> and <span class="html-italic">UL56</span> on HSV-1 syncytia. (<b>A</b>) Effects of HB94 strain <span class="html-italic">US7</span> or <span class="html-italic">UL56</span> genes on deletion and restoration of plaques. Vero cells (3 × 10<sup>5</sup> cells/well) were infected with 0.01 MOI HB94 or HN19 strains and cultured for 3 days. Bar, 200 μm. (<b>B</b>) Syncytial ratio. Stats: one-way ANOVA with a Tukey multiple comparisons; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01. (<b>C</b>) Number of nuclei in a single syncytium. ns: not significant.</p> Full article ">
13 pages, 2605 KiB  
Article
Integrase Defective Lentiviral Vector Promoter Impacts Transgene Expression in Target Cells and Magnitude of Vector-Induced Immune Responses
by Sneha Mahesh, Jenny Li, Tatianna Travieso, Danai Psaradelli, Donatella Negri, Mary Klotman, Andrea Cara and Maria Blasi
Viruses 2023, 15(11), 2255; https://doi.org/10.3390/v15112255 - 14 Nov 2023
Cited by 1 | Viewed by 1815
Abstract
Integrase defective lentiviral vectors (IDLVs) are a promising vaccine delivery platform given their ability to induce high magnitude and durable antigen-specific immune responses. IDLVs based on the simian immunodeficiency virus (SIV) are significantly more efficient at transducing human and simian dendritic cells (DCs) [...] Read more.
Integrase defective lentiviral vectors (IDLVs) are a promising vaccine delivery platform given their ability to induce high magnitude and durable antigen-specific immune responses. IDLVs based on the simian immunodeficiency virus (SIV) are significantly more efficient at transducing human and simian dendritic cells (DCs) compared to HIV-based vectors, resulting in a higher expansion of antigen-specific CD8+ T cells. Additionally, IDLV persistence and continuous antigen expression in muscle cells at the injection site contributes to the durability of the vaccine-induced immune responses. Here, to further optimize transgene expression levels in both DCs and muscle cells, we generated ten novel lentiviral vectors (LVs) expressing green fluorescent protein (GFP) under different hybrid promoters. Our data show that three of the tested hybrid promoters resulted in the highest transgene expression levels in mouse DCs, monkey DCs and monkey muscle cells. We then used the three LVs with the highest in vitro transgene expression levels to immunize BALB/c mice and observed high magnitude T cell responses at 3 months post-prime. Our study demonstrates that the choice of the vector promoter influences antigen expression levels in target cells and the ensuing magnitude of T cell responses in vivo. Full article
(This article belongs to the Special Issue Advances in Antiviral Immunity and Virus Vaccines)
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<p>Impact of vector promoter on transgene expression in 293T Lenti-X cells over time. 293T Lenti-X cells were transduced with a multiplicity of infection (MOI) of 1 using the LVs expressing GFP under the indicated promoters. (<b>a</b>) Results of time-course flow cytometric analyses performed at the indicated time points to compare the mean fluorescence intensity (MFI) among the different LVs. (<b>b</b>) MFI normalized by the percentage of transduced cells expressing GFP, to account for differences in transduction efficiency. Histograms show the means of three replicates. Error bars indicate standard error mean (S.E.M).</p> Full article ">Figure 2
<p>Impact of vector promoter on transgene expression in mouse-bone-marrow-derived dendritic cells (BMDCs). Mouse-BMDCs were transduced with a MOI of 2 using the LVs expressing GFP under the indicated promoters. (<b>a</b>) Results of time-course flow cytometric analyses performed at Days 3 and 7 post-transduction, to compare MFIs among the different LVs. (<b>b</b>) MFI normalized by the percentage of transduced cells expressing GFP. Histograms show the means of two replicates. Error bars indicate S.E.M.</p> Full article ">Figure 3
<p>Impact of vector promoter on transgene expression in monkey-monocyte-derived dendritic cells. Monkey-monocyte-derived DCs were transduced with an MOI of 4 using the LVs expressing GFP under the indicated promoters. (<b>a</b>) Results of time-course flow cytometric analyses performed at the indicated time points to compare MFIs among the different LVs. (<b>b</b>) MFI normalized for the percentage of transduced cells expressing GFP. Histograms represent means of two replicates. Error bars indicate S.E.M.</p> Full article ">Figure 4
<p>Impact of vector promoter on transgene expression in human-monocyte-derived dendritic cells (MDDCs). Human MDDCs were transduced with an MOI of 4 using the LVs expressing GFP under the indicated promoters. (<b>a</b>) Results of time-course flow cytometric analyses performed at the indicated time points to compare MFIs among the different LVs. (<b>b</b>) MFI normalized for the percentage of transduced cells expressing GFP. Histograms represent the means of two replicates. Error bars indicate S.E.M.</p> Full article ">Figure 5
<p>Impact of vector promoter on transgene expression in cynomolgus macaque skeletal muscle cells. Monkey skeletal muscle cells were transduced with an MOI of 0.5 using the LVs expressing GFP under the indicated promoters. (<b>a</b>) Results of time-course flow cytometric analyses performed at the indicated time points to compare mean fluorescence intensity (MFI) among the different LVs. (<b>b</b>) MFI normalized by the percentage of transduced cells expressing GFP. Histograms show the means of two replicates. Error bars indicate S.E.M. (<b>c</b>) Fluorescence microscopy images of monkey muscle cells transduced with the indicated LVs at 7 days post-transduction.</p> Full article ">Figure 6
<p>Magnitude of T cell responses in mice immunized with IDLVs expressing GFP under different promoters. (<b>a</b>) A total of 25 BALB/c mice were immunized intramuscularly with 50 ng RT/mouse corresponding to 5 × 10<sup>6</sup> transducing units (TU) of the indicated IDLVs or saline. Spleens were harvested 12 weeks post-immunization to measure T cell responses. (<b>b</b>) Magnitudes of GFP-specific T cell responses induced by the indicated IDLVs at 12 weeks post-immunization, as measured by IFN-γ ELISpot. Data are expressed (left panel) as numbers of specific spot-forming cells (SFCs) per million cells and (right panel) as % of GFP-specific SFCs normalized per ConA-induced SFCs, to account for potential differences in T cell responsiveness to stimuli among samples. Background responses in unstimulated wells (medium only) were subtracted. GFP = green fluorescent protein; ConA = concavalin A.</p> Full article ">
7 pages, 214 KiB  
Opinion
Screening Drugs for Broad-Spectrum, Host-Directed Antiviral Activity: Lessons from the Development of Probenecid for COVID-19
by Ralph A. Tripp and David E. Martin
Viruses 2023, 15(11), 2254; https://doi.org/10.3390/v15112254 - 14 Nov 2023
Cited by 7 | Viewed by 2059
Abstract
In the early stages of drug discovery, researchers develop assays that are compatible with high throughput screening (HTS) and structure activity relationship (SAR) measurements. These assays are designed to evaluate the effectiveness of new and known molecular entities, typically targeting specific features within [...] Read more.
In the early stages of drug discovery, researchers develop assays that are compatible with high throughput screening (HTS) and structure activity relationship (SAR) measurements. These assays are designed to evaluate the effectiveness of new and known molecular entities, typically targeting specific features within the virus. Drugs that inhibit virus replication by inhibiting a host gene or pathway are often missed because the goal is to identify active antiviral agents against known viral targets. Screening efforts should be sufficiently robust to identify all potential targets regardless of the antiviral mechanism to avoid misleading conclusions. Full article
(This article belongs to the Special Issue Novel and Repurposed Antiviral Agents)
25 pages, 2536 KiB  
Review
Comparative Analysis of Alpha and Beta HPV E6 Oncoproteins: Insights into Functional Distinctions and Divergent Mechanisms of Pathogenesis
by Josipa Skelin and Vjekoslav Tomaić
Viruses 2023, 15(11), 2253; https://doi.org/10.3390/v15112253 - 14 Nov 2023
Cited by 4 | Viewed by 2904
Abstract
Human papillomaviruses (HPVs) represent a diverse group of DNA viruses that infect epithelial cells of mucosal and cutaneous tissues, leading to a wide spectrum of clinical outcomes. Among various HPVs, alpha (α) and beta (β) types have garnered significant attention due to their [...] Read more.
Human papillomaviruses (HPVs) represent a diverse group of DNA viruses that infect epithelial cells of mucosal and cutaneous tissues, leading to a wide spectrum of clinical outcomes. Among various HPVs, alpha (α) and beta (β) types have garnered significant attention due to their associations with human health. α-HPVs are primarily linked to infections of the mucosa, with high-risk subtypes, such as HPV16 and HPV18, being the major etiological agents of cervical and oropharyngeal cancers. In contrast, β-HPVs are predominantly associated with cutaneous infections and are commonly found on healthy skin. However, certain β-types, notably HPV5 and HPV8, have been implicated in the development of non-melanoma skin cancers in immunocompromised individuals, highlighting their potential role in pathogenicity. In this review, we comprehensively analyze the similarities and differences between α- and β-HPV E6 oncoproteins, one of the major drivers of viral replication and cellular transformation, and how these impact viral fitness and the capacity to induce malignancy. In particular, we compare the mechanisms these oncoproteins use to modulate common cellular processes—apoptosis, DNA damage repair, cell differentiation, and the immune response—further shedding light on their shared and distinct features, which enable them to replicate at divergent locations of the human body and cause different types of cancer. Full article
(This article belongs to the Special Issue HPV-Associated Cancers)
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<p>Differences in genome organization, viral life cycle, and malignant progression between α- and β-HPVs. (<b>A</b>) HR-HPVs infect mucosal epithelia via micro-wounds, and they manipulate differentiating cells to efficiently propagate new virions, with viral gene expression (light blue—oncoproteins, orange—other early proteins and capsid proteins) coinciding with the stages of keratinocyte maturation (middle panel). As basal cells become dysplastic, the viral gene expression becomes more disordered, while E6/E7 acquire the primacy in expression over other proteins. This process leads to a reduced productive infection, resulting in the release of fewer virions (right panel). Premalignant stages are also indicated (LSIL—low-grade squamous interepithelial lesion; HSIL—high-grade squamous interepithelial lesion). (<b>B</b>) The natural reservoir for HPV8 and many other β-types is considered to be cutaneous epithelia, including hair follicles. Their life cycle is also dependent on keratinocyte differentiation, with orchestrated expression of viral genes delaying keratinocyte maturation and enabling the production of new virions (middle panel). β-HPV genomes do not integrate into the host genome and are considered to act as cofactors in tumor initiation but are not necessary for tumor maintenance. The viral load is higher in premalignant states than in cSCC, and it is currently unknown whether β-HPVs can complete their life cycle in transformed cells (right panel). Expanded and adapted from [<a href="#B23-viruses-15-02253" class="html-bibr">23</a>].</p> Full article ">Figure 2
<p>The most important binding partners and target substrates of E6 oncoproteins. α-E6 exclusive interacting partners are indicated in the blue circle, β-E6 specific ones in the green circle, and common ones in the yellow overlap. * denotes that the protein is a partner of the β2 species, <sup>+</sup> refers that the protein is a partner of the β3 species, and <sup><span>$</span></sup> refers that the protein is the partner of β4 species.</p> Full article ">Figure 3
<p>Strategies employed by E6 oncoproteins in manipulating p53 signaling. α- and β-E6 oncoproteins mostly have distinct ways of affecting p53. The ones typical for α-E6 are indicated in the blue box, and the ones typical for β-E6 in the green one. The mechanism shared between both E6 oncoproteins is indicated by the overlapping area. * denotes that the claim refers to β2 species, <sup>+</sup> that it refers to β3 and <sup><span>$</span></sup> that it refers to β4.</p> Full article ">
2 pages, 190 KiB  
Correction
Correction: Escudero-Flórez et al. Dengue Virus Infection Alters Inter-Endothelial Junctions and Promotes Endothelial–Mesenchymal-Transition-like Changes in Human Microvascular Endothelial Cells. Viruses 2023, 15, 1437
by Manuela Escudero-Flórez, David Torres-Hoyos, Yaneth Miranda-Brand, Ryan L. Boudreau, Juan Carlos Gallego-Gómez and Miguel Vicente-Manzanares
Viruses 2023, 15(11), 2252; https://doi.org/10.3390/v15112252 - 13 Nov 2023
Cited by 1 | Viewed by 1059
Abstract
Ryan L [...] Full article
(This article belongs to the Special Issue Boosting Flavivirus Research: A Pandengue Net Initiative)
13 pages, 1603 KiB  
Article
Identification of Host Factors for Rift Valley Fever Phlebovirus
by Velmurugan Balaraman, Sabarish V. Indran, Yonghai Li, David A. Meekins, Laxmi U. M. R. Jakkula, Heidi Liu, Micheal P. Hays, Jayme A. Souza-Neto, Natasha N. Gaudreault, Philip R. Hardwidge, William C. Wilson, Friedemann Weber and Juergen A. Richt
Viruses 2023, 15(11), 2251; https://doi.org/10.3390/v15112251 - 13 Nov 2023
Cited by 1 | Viewed by 2747
Abstract
Rift Valley fever phlebovirus (RVFV) is a zoonotic pathogen that causes Rift Valley fever (RVF) in livestock and humans. Currently, there is no licensed human vaccine or antiviral drug to control RVF. Although multiple species of animals and humans are vulnerable to RVFV [...] Read more.
Rift Valley fever phlebovirus (RVFV) is a zoonotic pathogen that causes Rift Valley fever (RVF) in livestock and humans. Currently, there is no licensed human vaccine or antiviral drug to control RVF. Although multiple species of animals and humans are vulnerable to RVFV infection, host factors affecting susceptibility are not well understood. To identify the host factors or genes essential for RVFV replication, we conducted CRISPR-Cas9 knockout screening in human A549 cells. We then validated the putative genes using siRNA-mediated knock-downs and CRISPR-Cas9-mediated knock-out studies. The role of a candidate gene in the virus replication cycle was assessed by measuring intracellular viral RNA accumulation, and the virus titers were analyzed using plaque assay or TCID50 assay. We identified approximately 900 genes with potential involvement in RVFV infection and replication. Further evaluation of the effect of six genes on viral replication using siRNA-mediated knock-downs revealed that silencing two genes (WDR7 and LRP1) significantly impaired RVFV replication. For further analysis, we focused on the WDR7 gene since the role of the LRP1 gene in RVFV replication was previously described in detail. WDR7 knockout A549 cell lines were generated and used to dissect the effect of WRD7 on a bunyavirus, RVFV, and an orthobunyavirus, La Crosse encephalitis virus (LACV). We observed significant effects of WDR7 knockout cells on both intracellular RVFV RNA levels and viral titers. At the intracellular RNA level, WRD7 affected RVFV replication at a later phase of its replication cycle (24 h) when compared with the LACV replication, which was affected in an earlier replication phase (12 h). In summary, we identified WDR7 as an essential host factor for the replication of two different viruses, RVFV and LACV, both of which belong to the Bunyavirales order. Future studies will investigate the mechanistic role through which WDR7 facilitates phlebovirus replication. Full article
(This article belongs to the Special Issue Emerging Highlights in the Study of Rift Valley Fever Virus)
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Figure 1
<p>Schematics of GeCKO-A549 cells generation, selection, NGS, and data analysis. A549 cells were transduced with the lentivirus-CRISPR-Cas9 library to generate GeCKO-A549 cells. Then, the GeCKO-A549 cells were subjected to three rounds of infection with the RVFV MP-12 (1 MOI) virus. The genomic DNA of round 0 GeCKO-A549 cells, round 1, and round 3 GeCKO-A549cells were sequenced using the Illumina NextSeq 550 platform. The output NGS data were analyzed using the MaGeCK program to generate the list of genes involved in RVFV replication.</p> Full article ">Figure 2
<p>Validation of gene hits via siRNA gene knockdown study. A549 cells were transfected with 50 nM of siRNAs. At 48 h post-transfection, the cells were infected with RVFV MP-12 virus at 0.1 MOI. At 24 h post-infection, the supernatant was collected and titered using plaque assay. NTC-non-target control siRNA; si46N-anti-RVFV siRNA; and <span class="html-italic">WDR7</span>, <span class="html-italic">SLC35B2</span>, <span class="html-italic">EXOC4</span>, <span class="html-italic">LRP1</span>, <span class="html-italic">EMC3</span>, <span class="html-italic">CT47A1</span> gene-specific siRNAs were transfected. Each bar represents the average virus titer (pfu/mL) along with the corresponding standard deviation. Statistical analysis was performed on two independent experiments with four replicates for each, using the Mann–Whitney U test and independent Student’s <span class="html-italic">t</span>-test (** <span class="html-italic">p</span>-value &lt; 0.005, *** <span class="html-italic">p</span>-value &lt; 0.001).</p> Full article ">Figure 3
<p>Effect of <span class="html-italic">WDR7</span> gene knockout (KO) on virus production of bunyaviruses: (<b>A</b>) A549 cells CT (non-knockout control) cells and <span class="html-italic">WDR7</span> gene KO cell lines 1 and 2 were analyzed for WDR7 protein expression via Western blot using a WDR7-specific polyclonal antibody. (<b>B</b>–<b>E</b>) CT cells and WDR7 KO A549 cells were infected with RVFV MP-12 vaccine strain (<b>B</b>), with the wild-type RVFV Kenya 128B-15 strain (<b>C</b>), or with La Crosse encephalitis virus (<b>D</b>,<b>E</b>) at 0.1 MOI. The supernatant was collected at 6, 12 or 24 h post-infection (h pi) and titered using plaque assay (RVFV) or TCID<sub>50</sub>-CPE assay (LACV). RVFV MP-12 testing on A549 CT cells and WDR7 KO lines 1 or 2, involved three to five independent experiments with three to four technical replicates each. RVFV Kenya 128B-15 testing involved independent experiments with three technical replicates each. LACV testing was performed in two independent experiments with eight technical replicates each. Statistical analysis was performed using the Mann–Whitney U test and independent Student’s <span class="html-italic">t</span>-test (* <span class="html-italic">p</span>-value &lt; 0.05, ** <span class="html-italic">p</span>-value &lt; 0.005, *** <span class="html-italic">p</span>-value &lt; 0.001).</p> Full article ">Figure 4
<p>Viral RNA accumulation at various time points post-infection in WDR7 knockout (KO) cells. CT and WDR7 KO 1 cells were infected with (<b>A</b>) RVFV MP-12 vaccine strain or (<b>B</b>) LACV, both at 0.1 MOI. Total cellular RNA was harvested at various hours post-infection (h pi). One-step RT-qPCR was performed to detect the level of viral RNA using the <span class="html-italic">PGK1</span> gene as an internal control. CT and WDR7 KO 1 cells were utilized. Each bar graph represents the average fold change in viral RNA expression, along with the corresponding standard deviation. Statistical analysis was performed on three independent experiments with two to three technical replicates for each, using the Mann–Whitney U test and independent Student’s <span class="html-italic">t</span>-test (* <span class="html-italic">p</span>-value &lt; 0.05, *** <span class="html-italic">p</span>-value &lt; 0.001, ns, non-significant).</p> Full article ">
13 pages, 2306 KiB  
Article
Longitudinal Analysis of Antibody Response Following SARS-CoV-2 Infection Depending on Disease Severity: A Prospective Cohort Study
by Christina Zirou, Sentiljana Gumeni, Ioannis Bellos, Ioannis Ntanasis-Stathopoulos, Aimilia D. Sklirou, Tina Bagratuni, Eleni Korompoki, Filia Apostolakou, Ioannis Papassotiriou, Ioannis P. Trougakos and Evangelos Terpos
Viruses 2023, 15(11), 2250; https://doi.org/10.3390/v15112250 - 13 Nov 2023
Cited by 1 | Viewed by 1654
Abstract
Objective: Severe coronavirus disease 19 (COVID-19) is characterized by a dysregulated inflammatory response, with humoral immunity playing a central role in the disease course. The objective of this study was to assess the immune response and the effects of vaccination in recovered individuals [...] Read more.
Objective: Severe coronavirus disease 19 (COVID-19) is characterized by a dysregulated inflammatory response, with humoral immunity playing a central role in the disease course. The objective of this study was to assess the immune response and the effects of vaccination in recovered individuals with variable disease severity up to one year following natural infection. Methods: A prospective cohort study was conducted including patients with laboratory-confirmed COVID-19. Disease severity was classified as mild, moderate, and severe based on clinical presentation and outcomes. Anti-RBD (receptor binding domain) and neutralizing antibodies were evaluated at multiple timepoints during the first year after COVID-19 diagnosis. Results: A total of 106 patients were included; of them, 28 were diagnosed with mild, 38 with moderate, and 40 with severe disease. At least one vaccine dose was administered in 58 individuals during the follow-up. Participants with mild disease presented significantly lower anti-RBD and neutralizing antibodies compared to those with moderate and severe disease up to the 3rd and 6th months after the infection, respectively. After adjusting for covariates, in the third month, severe COVID-19 was associated with significantly higher anti-RBD (β: 563.09; 95% confidence intervals (CI): 257.02 to 869.17) and neutralizing (β: 21.47; 95% CI: 12.04 to 30.90) antibodies. Among vaccinated individuals, at the 12th month, a history of moderate disease was associated with significantly higher anti-RBD levels (β: 5615.19; 95% CI: 657.92 to 10,572.46). Conclusions: Severe COVID-19 is associated with higher anti-RBD and neutralizing antibodies up to 6 months after the infection. Vaccination of recovered patients is associated with a remarkable augmentation of antibody titers up to one year after COVID-19 diagnosis, regardless of disease severity. Full article
(This article belongs to the Special Issue COVID-19 Diagnostics in Clinical Applications and Pandemic Controls 2024)
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Figure 1

Figure 1
<p>Comparison of neutralizing and anti-RBD antibodies among patients with mild, moderate, and severe COVID-19 at admission and days 7 and 30 after infection. ns: non-significant; ** <span class="html-italic">p</span>-value &lt; 0.01; *** <span class="html-italic">p</span>-value &lt; 0.001. ns: not significant Comparisons were performed with the Kruskal–Wallis test.</p> Full article ">Figure 2
<p>Comparison of neutralizing and anti-RBD antibodies among individuals with history of mild, moderate, and severe COVID-19 in the 3rd month (unvaccinated) and 6th and 12th months (vaccinated) following natural infection. ns: non-significant; *** <span class="html-italic">p</span>-value &lt; 0.001. Comparisons were performed with the Kruskal –Wallis test.</p> Full article ">
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