Viruses

17 pages, 3328 KB

Open AccessArticle

Optimization of a High-Throughput Human Papillomavirus Neutralizing Antibody Assay Based on Pseudotyped Viruses for the 15-Valent Human Papillomavirus Vaccine Types

by Huan Liu, Haiyang Qin, Lingling Nie, Yanru Shen, Jiayi Li, Pengcheng Xiu, Shasha Wang, Meng Wang, Youchun Wang, Jianhui Nie, Weijin Huang and Li Zhang

Viruses 2025, 17(9), 1164; https://doi.org/10.3390/v17091164 (registering DOI) - 26 Aug 2025

Abstract

Vaccination is highly effective in preventing human papillomavirus (HPV) infection, but traditional pseudovirion-based neutralization assays (PBNA) are technically demanding, labor-intensive, and costly, limiting their use in multivalent vaccine studies. We developed and validated an automated, high-throughput PBNA in a 384-well format that quantifies [...] Read more.

Vaccination is highly effective in preventing human papillomavirus (HPV) infection, but traditional pseudovirion-based neutralization assays (PBNA) are technically demanding, labor-intensive, and costly, limiting their use in multivalent vaccine studies. We developed and validated an automated, high-throughput PBNA in a 384-well format that quantifies neutralizing antibodies against 15 HPV types using triple-color pseudotyped viruses. Non-interfering type triplets were defined from cross-neutralization assays of serum against pseudotyped viruses, enabling simultaneous detection of three fluorescence signals per well. The workflow integrates a cap-decapper, semi-automatic sample addition and dilution, and a microplate stacker with automated imaging to reduce hands-on time. The 384-well method showed strong concordance with the conventional 96-well PBNA while increasing daily sample throughput by approximately 6.7-fold, reducing assay duration (including ~4-fold faster imaging), and lowering reaction volume by ~5-fold. Analytical validation demonstrated acceptable specificity, accuracy, repeatability, linearity and robustness for high-throughput use. Serostatus cutoff values were established in an age-appropriate female population to support classification of positive versus negative sera. This platform provides a scalable tool for evaluating neutralizing antibodies after natural infections or vaccination and is well suited for large clinical trials and the development of next-generation and multivalent HPV vaccines. Full article

(This article belongs to the Section Viral Immunology, Vaccines, and Antivirals)

21 pages, 1090 KB

Open AccessArticle

The Impact of COVID-19 and Related Public Health Measures on Hepatitis C Testing in Ontario, Canada

by Yeva Sahakyan, Samantha S. M. Drover, Zoë R. Greenwald, William W. L. Wong, Alexander Kopp, Richard L. Morrow, Naveed Z. Janjua and Beate Sander

Viruses 2025, 17(9), 1163; https://doi.org/10.3390/v17091163 - 26 Aug 2025

Abstract

The COVID-19 pandemic disrupted progress towards global HCV elimination goals by interrupting essential health services in Canada and globally. We aimed to evaluate the effect of the pandemic on hepatitis C virus (HCV) testing rates in a population-based cohort study in Ontario using [...] Read more.

The COVID-19 pandemic disrupted progress towards global HCV elimination goals by interrupting essential health services in Canada and globally. We aimed to evaluate the effect of the pandemic on hepatitis C virus (HCV) testing rates in a population-based cohort study in Ontario using health administrative data. All residents with records of either HCV antibody or ribonucleic acid (RNA) tests were included. Monthly testing rate per 1000 population were compared during the pre-pandemic (01/01/2015–29/02/2020) and pandemic (01/03/2020–31/12/2022) periods using interrupted time series models, stratified by sex, homelessness, human immunodeficiency virus (HIV), and immigration status, and people who inject drugs (PWID). The HCV testing rate followed a statistically significant upward trend before the pandemic, dropping at its onset with 1.38/1000 fewer individuals initiating testing monthly. Compared to counterfactual estimates, the observed monthly number of people tested per 1000 population was lower by 1.41 (95% CI: 1.18–1.64) in 2020 (May–Dec), 1.17 (95% CI: 0.99–1.36) in 2021, and 1.41 (95% CI: 1.22–1.59) in 2022, corresponding to relative reductions of 47%, 34%, and 41%, respectively. Testing rates remained below expected levels across all subgroups throughout 2020–2022, with the greatest absolute declines observed among people co-infected with HIV, people experiencing homelessness, and PWID. Tailored, equity-focused interventions are needed to address these persistent gaps in HCV testing, without which Canada’s progress toward its 2030 elimination targets remains at risk. Full article

(This article belongs to the Section Coronaviruses)

14 pages, 2588 KB

Open AccessArticle

Wild Citrus CTV Genomic Data Provides Novel Insights into Its Global Transmission Dynamics

by Xiang Li, Jun Zhou, Aijun Huang and Long Yi

Viruses 2025, 17(9), 1162; https://doi.org/10.3390/v17091162 - 26 Aug 2025

Abstract

Citrus tristeza virus (CTV) is an important pathogen threatening the global citrus industry, but its evolution and transmission mechanism in wild citrus has not been clarified. Most of the existing studies are based on CTV-specific gene fragments, lacking genome-wide analysis. There is especially [...] Read more.

Citrus tristeza virus (CTV) is an important pathogen threatening the global citrus industry, but its evolution and transmission mechanism in wild citrus has not been clarified. Most of the existing studies are based on CTV-specific gene fragments, lacking genome-wide analysis. There is especially a lack of understanding of CTV transmission dynamics in wild citrus, which needs further investigation. In this study, wild citrus samples from three provinces of China were collected, virus genome data were obtained by high-throughput sequencing (HTS) technology and combined with public database data, and Bayesian phylogeographic inference was used to analyze virus composition characteristics in wild citrus, as well as the population genetic structure, temporal dynamic evolution, and spatial transmission mode of CTV. The results showed that Yunnan wild citrus samples contained the most abundant virus components, including CTV, Citrus Exocortis Viroid (CEVd), Citrus associated Ampelovirus 1 (CaAV-1), and Citrus Virus B (CiVB), while Jiangxi and Hunan samples only contained CTV and CEVd, with all samples showing mixed infection. Phylogenetic analysis showed that nine wild citrus CTV isolates were scattered in different evolutionary clades, and only 9.27% of genetic variation existed between the populations, while 90.72% of genetic variation existed within the populations, indicating little effect of geographic isolation on gene flow. The time to the most recent common ancestor (tMRCA) of CTV was estimated at 1360 CE, with subsequent divergence into two lineages, with population size stabilizing after a rapid increase in 1980–1990. Asia has been identified as the central source of CTV’s global spread, with key migration events including Asia to North America (1746), Asia to Oceania (1829), and Asia to South America (1965), coinciding with global maritime trade and the expansion of the citrus industry. Full article

(This article belongs to the Section Viruses of Plants, Fungi and Protozoa)

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17 pages, 1420 KB

Open AccessArticle

Genomic Evolution of SARS-CoV-2 Variants of Concern Under In Vitro Neutralising Selection Pressure Following Two Doses of the Pfizer-BioNTech BNT162b2 COVID-19 Vaccine

by Kerri Basile, Jessica E. Agius, Winkie Fong, Kenneth McPhie, Danny Ko, Linda Hueston, Connie Lam, David Pham, Sharon C.-A. Chen, Susan Maddocks, Matthew V. N. O’Sullivan, Dominic E. Dwyer, Vitali Sintchenko, Jen Kok and Rebecca J. Rockett

Viruses 2025, 17(9), 1161; https://doi.org/10.3390/v17091161 - 25 Aug 2025

Abstract

We aimed to explore SARS-CoV-2 evolution during in vitro neutralisation using next generation sequencing, and to determine whether sera from individuals immunised with two doses of the Pfizer-BioNTech vaccine (BNT162b2) were as effective at neutralising the variant of concern (VOC) Delta (B.1.617.2) compared [...] Read more.

We aimed to explore SARS-CoV-2 evolution during in vitro neutralisation using next generation sequencing, and to determine whether sera from individuals immunised with two doses of the Pfizer-BioNTech vaccine (BNT162b2) were as effective at neutralising the variant of concern (VOC) Delta (B.1.617.2) compared to the earlier lineages Beta (B.1.351) and wild-type (A.2.2) virus. Using a live-virus SARS-CoV-2 neutralisation assay in Vero E6 cells, we determined neutralising antibody titres (nAbT) against three SARS-CoV-2 strains (wild type, Beta, and Delta) in 14 participants (vaccine-naïve (n = 2) and post-second dose of BNT162b2 vaccination (n = 12)), median age 45 years [IQR 29–65]; the median time after the second dose was 21 days [IQR 19–28]. The determination of nAbT was based on cytopathic effect (CPE) and in-house quantitative reverse transcriptase real-time quantitative polymerase chain reaction (RT-qPCR) to confirm SARS-CoV-2 replication. A total of 110 representative samples including inoculum, neutralisation breakpoints at 72 h, and negative and positive controls underwent genome sequencing. By integrating live-virus neutralisation assays with deep sequencing, we characterised both functional antibody responses and accompanying viral genetic changes. There was a reduction in nAbT observed against the Delta and Beta VOC compared with wild type, 4.4-fold (p ≤ 0.0006) and 2.3-fold (p = 0.0140), respectively. Neutralising antibodies were not detected in one vaccinated immunosuppressed participant and the vaccine-naïve participants (n = 2). The highest nAbT against the SARS-CoV-2 variants investigated was obtained from a participant who was vaccinated following SARS-CoV-2 infection 12 months prior. Limited consensus level mutations occurred in the various SARS-CoV-2 lineage genomes during in vitro neutralisation; however, consistent minority allele frequency variants (MFV) were detected in the SARS-CoV-2 polypeptide, spike (S), and membrane protein. Findings from countries with high COVID-19 incidence may not be applicable to low-incidence settings such as Australia; as seen in our cohort, nAbT may be significantly higher in vaccine recipients previously infected with SARS-CoV-2. Monitoring viral evolution is critical to evaluate the impact of novel SARS-CoV-2 variants on vaccine effectiveness, as mutational profiles in the sub-consensus genome could indicate increases in transmissibility and virulence or suggest the development of antiviral resistance. Full article

(This article belongs to the Special Issue Emerging Concepts in SARS-CoV-2 Biology and Pathology 2.0)

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13 pages, 3035 KB

Open AccessArticle

Topography and Nanomechanics of the Tomato Brown Rugose Fruit Virus Suggest a Fragmentation-Driven Infection Mechanism

by Péter Puskás, Katalin Salánki, Levente Herényi, Tamás Hegedűs and Miklós Kellermayer

Viruses 2025, 17(9), 1160; https://doi.org/10.3390/v17091160 - 25 Aug 2025

Abstract

Tomato brown rugose fruit virus (ToBRFV) has been causing severe agricultural damage worldwide since its recent discovery. While related to tobacco mosaic virus, its properties and infection mechanisms are poorly understood. To uncover their structure and nanomechanics, we carried out atomic force microscopy [...] Read more.

Tomato brown rugose fruit virus (ToBRFV) has been causing severe agricultural damage worldwide since its recent discovery. While related to tobacco mosaic virus, its properties and infection mechanisms are poorly understood. To uncover their structure and nanomechanics, we carried out atomic force microscopy (AFM) measurements on individual ToBRFV particles. The virions are rod-shaped with a height and width of 9 and 30 nm, respectively. Length is widely distributed (5–1000 nm), with a mode at 30 nm. ToBRFV rods displayed a 22.4 nm axial periodicity related to structural units. Force spectroscopy revealed a Young’s modulus of 8.7 MPa, a spring constant of 0.25 N/m, and a rupture force of 1.7 nN. In the force curves a step was seen at a height of 3.3 nm, which is related to virion wall thickness. Wall thickness was also estimated by predicting coat protein structure with AlphaFold, yielding a protein with a length of 7.3 nm. Accordingly, the structural element of ToBRFv is a right circular cylinder with an equal height and diameter of ~22 nm and a wall thickness between 3.3 and 7.3 nm. Thus, at least four to nine serially linked units are required to encapsidate a single, helically organized RNA genome. Fragmentation of ToBRFV into these cylindrical structural units may result in a facilitated release of the genome and thus efficient infection. Full article

(This article belongs to the Section Viruses of Plants, Fungi and Protozoa)

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22 pages, 8472 KB

Open AccessReview

Epitranscriptomic Regulation of Hepatitis B Virus by RNA 5-Methylcytosine: Functions, Mechanisms, and Therapeutic Potential

by Xuliu Zhou, Yanling Huang, Xueyan Zhang, Wuxiang Guan, Fang Zhang and Haojie Hao

Viruses 2025, 17(9), 1159; https://doi.org/10.3390/v17091159 - 24 Aug 2025

Abstract

Hepatitis B virus (HBV) remains a major global health challenge, with over 296 million people chronically infected worldwide. Despite the availability of antiviral therapies, a functional cure is rarely achieved, highlighting the need for novel therapeutic strategies. RNA 5-methylcytosine (m⁵C) is [...] Read more.

Hepatitis B virus (HBV) remains a major global health challenge, with over 296 million people chronically infected worldwide. Despite the availability of antiviral therapies, a functional cure is rarely achieved, highlighting the need for novel therapeutic strategies. RNA 5-methylcytosine (m⁵C) is a pivotal epitranscriptomic mark implicated in RNA stability, transport, and translation. Emerging evidence shows that m⁵C is conserved within HBV RNA and plays critical roles in the viral life cycle. This review provides a comprehensive overview of the molecular mechanisms governing m⁵C deposition and recognition, summarizes recent advances in m⁵C biology, and highlights the emerging role of epitranscriptomic m⁵C regulation in HBV infection. We discuss the identification of HBV-specific m⁵C sites, the functions of key regulatory enzymes, and their interplay in viral RNA stabilization and evasion of innate immune responses. Interplay between m⁵C and other RNA modifications—particularly N6-methyladenosine (m⁶A)—is examined alongside virus-specific m⁵C regulation in EV71, HIV, HCV, EBV, and SARS-CoV-2. Potential links between m⁵C dysregulation and HBV-induced hepatocarcinogenesis are outlined, and emerging therapeutic strategies targeting the m⁵C machinery are highlighted. Together, these insights position the epitranscriptomic landscape as a promising avenue for innovative antiviral strategies. Full article

(This article belongs to the Special Issue Epigenetic Modifications in Viral Infections, Volume II)

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11 pages, 442 KB

Open AccessArticle

Virological Effectiveness of Dolutegravir Plus Darunavir in People with Multi-Drug-Resistant HIV: Data from the PRESTIGIO Registry

by Filippo Lagi, Michele Bellomo, Riccardo Lolatto, Filippo Ducci, Seble Tekle Kiros, Vincenzo Spagnuolo, Rebecka Papaioannu Borjesson, Tommaso Clemente, Leonardo Calza, Marcello Feasi, Emanuele Focà, Andrea Giacomelli, Roberto Gulminetti, Barbara Menzaghi, Antonella Castagna and on behalf of the PRESTIGIO Study Group

Viruses 2025, 17(9), 1158; https://doi.org/10.3390/v17091158 - 24 Aug 2025

Abstract

Background: Data on the use of dolutegravir (DTG) plus boosted darunavir (DRV/b) in people with 4-class drug-resistant HIV (4DR-PWH) are limited. This study assessed the virological effectiveness of DTG + DRV/b in this population using real-world data from the PRESTIGIO Registry. Methods: We [...] Read more.

Background: Data on the use of dolutegravir (DTG) plus boosted darunavir (DRV/b) in people with 4-class drug-resistant HIV (4DR-PWH) are limited. This study assessed the virological effectiveness of DTG + DRV/b in this population using real-world data from the PRESTIGIO Registry. Methods: We compared three regimen groups: dual DTG + DRV/b (DODA), DTG + DRV/b plus an additional antiretroviral drug (DODA + Other), and regimens excluding DTG + DRV/b (NO-DODA). Virological failure (VF) was defined as ≥2 HIV-RNA values ≥ 50 copies/mL or 1 ≥ 1000 copies/mL. Mixed-effects logistic regression was used to assess VF, adjusting for antiretroviral therapy (ART) duration, age, number of fully active drugs, sex at birth, and nadir CD4+. Individuals could switch regimens during follow-up. Results: Among 249 4DR-PWH (median follow-up: 8.7 years), 844 ART regimens were analyzed: 72 (8.5%) DODA, 264 (31.3%) DODA + Other, and 508 (60.2%) NO-DODA. Compared to NO-DODA, the odds of VF were 77% and 35.9% lower with DODA and DODA + Other, respectively. Notably, in the DODA group, DTG and DRV/b were fully active in only 63.9% and 47.2% of the cases, respectively. Conclusions: DTG + DRV/b regimens were associated with a significantly lower risk of virological failure, even when drug activity was partial. This strategy remains a valuable option for managing multi-drug-resistant HIV. Full article

(This article belongs to the Special Issue Viral Resistance)

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16 pages, 2432 KB

Open AccessArticle

Licoflavone B Suppresses Influenza A Virus by Targeting the Viral RNA-Dependent RNA Polymerase (RdRp)

by Pu Fan, Peng Lv, Sen Zhang, Zheng Zhu, Kewen Qian, Jin Han, Yue Cui, Ye Feng, Zeya Li, Li Qiang, Yunzhu Dong, Ting Fang, Tao Jiang, Changming Yu and Xiangyang Chi

Viruses 2025, 17(9), 1157; https://doi.org/10.3390/v17091157 - 24 Aug 2025

Abstract

Influenza A virus pandemics pose a persistent global health threat, and emerging antiviral resistance underscores the critical importance of developing novel broad-spectrum therapeutic agents. Building on licorice’s (Glycyrrhiza spp.) historical use in traditional Chinese medicine for respiratory infections—as documented in the Chinese [...] Read more.

Influenza A virus pandemics pose a persistent global health threat, and emerging antiviral resistance underscores the critical importance of developing novel broad-spectrum therapeutic agents. Building on licorice’s (Glycyrrhiza spp.) historical use in traditional Chinese medicine for respiratory infections—as documented in the Chinese Guidelines for Diagnosis and Treatment of Influenza—and its demonstrated anti-SARS-CoV-2 activity, we identified licoflavone B as a potent anti-influenza agent, bridging ethnopharmacological knowledge with mechanistic validation. In this study, we identified licoflavone B, a natural flavonoid derived from licorice (Glycyrrhiza spp.), as a potent inhibitor of diverse influenza viruses, including multiple influenza A subtypes and type B virus. Mechanistic studies revealed that licoflavone B selectively targets the viral RNA-dependent RNA polymerase (RdRp), effectively suppressing viral replication. The compound exhibits a favorable selectivity index (SI = 14.9–29.9), indicating a promising therapeutic window. Molecular docking simulations identified potential binding interactions between licoflavone B and regions of the RdRp complex, which were further validated by dose-dependent inhibition of viral nucleoprotein (NP) and polymerase subunit PB2 expression in Western blot and immunofluorescence assays. In addition, licoflavone B maintained broad-spectrum antiviral activity against multiple influenza strains, including H1N1 (A/Puerto Rico/8/34), H3N2 (A/Darwin/9/2021), and a clinical influenza B isolate (B/Beijing/ZYY-B18/2018). These findings position licoflavone B as a promising lead compound for developing next-generation, broad-spectrum antiviral therapies against influenza and potentially other viruses. Full article

(This article belongs to the Special Issue Antiviral Agents to Influenza Virus 2025)

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14 pages, 1331 KB

Open AccessArticle

Comparative Evaluation of Modified Vaccinia Ankara as a Surrogate Virus for Disinfectant Efficacy Testing Against AIV, FMDV, and ASFV

by Sok Song, Su-Jeong Kim, Kyu-Sik Shin, So-Hee Park, Yong Yi Joo, Bokhee Han, Cho-Yeon Lee, Gong-Woo Park, Hyun-Ok Ku, Wooseog Jeong and Choi-Kyu Park

Viruses 2025, 17(9), 1156; https://doi.org/10.3390/v17091156 - 23 Aug 2025

Abstract

Surrogate viruses provide a safe and scalable alternative for evaluating disinfectant efficacy when access to high-risk pathogens is restricted. This study evaluated the potential of Modified Vaccinia Ankara (MVA) virus, which can be handled under BSL-1/2 conditions, as a surrogate for avian influenza [...] Read more.

Surrogate viruses provide a safe and scalable alternative for evaluating disinfectant efficacy when access to high-risk pathogens is restricted. This study evaluated the potential of Modified Vaccinia Ankara (MVA) virus, which can be handled under BSL-1/2 conditions, as a surrogate for avian influenza virus (AIV), foot-and-mouth disease virus (FMDV), and African swine fever virus (ASFV). A total of 64 commercially available disinfectants—classified into four major chemical groups: quaternary ammonium compounds, oxidizing agents, PPMS-based formulations, and organic acids—were tested in suspension assays using a ≥4 log reduction as the efficacy criterion. MVA showed the strongest predictive performance for FMDV (r = 0.671, AUC = 0.83), supporting its use for both binary classification and approximate quantitative prediction. Although its correlation with ASFV was weaker (r = 0.175), the classification performance remained moderate (AUC = 0.78), indicating conditional applicability. While MVA exhibited no meaningful correlation with AIV, its higher chemical resistance meant that disinfectants effective against MVA were consistently effective against AIV. These results support the use of MVA as a conservative exclusion tool for fragile viruses. Overall, the findings demonstrate that MVA can serve as a practical surrogate virus for disinfectant efficacy testing against FMDV, ASFV, and AIV, with application strategies tailored to each virus’s characteristics. Full article

(This article belongs to the Section Animal Viruses)

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13 pages, 1683 KB

Open AccessArticle

Physiological Impacts on the Mosquito Vector Hosts Refine Vectorial Capacity Estimates of Mayaro Virus Transmission Risk

by Luis A. Alonso-Palomares, John F. Williams, Edwin R. Burgess IV, John A. Lednicky and Rhoel R. Dinglasan

Viruses 2025, 17(9), 1155; https://doi.org/10.3390/v17091155 - 23 Aug 2025

Abstract

Mayaro virus (MAYV) is an alphavirus transmitted by mosquito vectors. Among the three MAYV genotypes (D, L, and N), genotype D has the broadest geographical distribution in Latin America and the Caribbean. The virus can be transmitted by the Aedes, Anopheles, [...] Read more.

Mayaro virus (MAYV) is an alphavirus transmitted by mosquito vectors. Among the three MAYV genotypes (D, L, and N), genotype D has the broadest geographical distribution in Latin America and the Caribbean. The virus can be transmitted by the Aedes, Anopheles, and Haemagogus mosquitoes. To explore the potential expansion of MAYV across the Atlantic Ocean, we compared MAYV (D) infection kinetics in Floridian Aedes aegypti with New World (Anopheles albimanus) and Old World (Anopheles gambiae) anophelines. The MAYV infection of both An. albimanus and An. gambiae was rapid, resulting in a higher dissemination rate than Ae. aegypti. We detected MAYV in saliva from An. albimanus (16.6% transmission rate) as early as 2 days post-infection (dpi), increasing to 60% after 7 dpi, a phenomenon that has not been described (2 dpi) to date for mosquitoes. We observed similar increases in the MAYV infection of the ovaries and noted marked differences in fecundity for each species tested. Although the MAYV infection in An. gambiae was rapid, mosquito lifespan was significantly reduced as compared with both Ae. aegypti and An. albimanus. We discuss the implications of our observations on the MAYV transmission risk in Africa by An. gambiae and in the Caribbean and Central America by An. albimanus. Full article

(This article belongs to the Special Issue Advances in Alphavirus and Flavivirus Research, 2nd Edition)

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17 pages, 1108 KB

Open AccessArticle

Gene Expression Factors Associated with Rubella-Specific Humoral Immunity After a Third MMR Vaccine Dose

by Lara I. Teodoro, Iana H. Haralambieva, Inna G. Ovsyannikova, Krista M. Goergen, Diane E. Grill, Gregory A. Poland and Richard B. Kennedy

Viruses 2025, 17(9), 1154; https://doi.org/10.3390/v17091154 - 23 Aug 2025

Abstract

Rubella is typically a mild viral illness, but it can lead to severe complications when contracted during pregnancy, such as pregnancy loss or developmental defects in the fetus (congenital rubella syndrome). Therefore, it is crucial to develop and maintain protective immunity in women [...] Read more.

Rubella is typically a mild viral illness, but it can lead to severe complications when contracted during pregnancy, such as pregnancy loss or developmental defects in the fetus (congenital rubella syndrome). Therefore, it is crucial to develop and maintain protective immunity in women of childbearing age. In this study, we assessed the transcriptional factors associated with rubella-specific immune outcomes (IgG binding antibody and avidity, neutralizing antibody, and memory B cell ELISpot response) following a third MMR vaccine dose in women of reproductive age to identify key factors/signatures impacting the immune response. We identified baseline (Day 0) and differentially expressed (Day 28–Day 0) genes associated with several RV-specific immune outcomes, including the transferrin receptor 2 (TFR2), which is an important factor regulating iron homeostasis and macrophage functional activity, and a close functional homolog of TFR1, the cellular receptor of the New World hemorrhagic fever arenaviruses. We also identified enriched KEGG pathways, “cell adhesion molecules”, “antigen processing and presentation”, “natural killer cell-mediated cytotoxicity”, and “immune network for IgA production”, relevant to immune response priming and immune activation to be associated with RV-specific immune outcomes. This study provides novel insights into potential biomarkers of rubella-specific immunity in women of childbearing age. Full article

(This article belongs to the Special Issue Measles, Mumps, and Rubella)

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29 pages, 10646 KB

Open AccessReview

The Triplex-Centric Assembly and Maturation of the Herpesvirus Procapsid

by J. Bernard Heymann

Viruses 2025, 17(9), 1153; https://doi.org/10.3390/v17091153 - 22 Aug 2025

Abstract

Herpesviruses are prevalent infectious agents in humans, with complex structures and life cycles. The viability and detail of a model of capsid assembly and maturation can now be examined against the recently available mature herpesvirus capsids structures. The first large assembly product is [...] Read more.

Herpesviruses are prevalent infectious agents in humans, with complex structures and life cycles. The viability and detail of a model of capsid assembly and maturation can now be examined against the recently available mature herpesvirus capsids structures. The first large assembly product is the icosahedral procapsid with an outer shell composed of major capsid proteins (MCPs) connected by triplexes (heterotrimers composed of one Tri1 protein and two Tri2 proteins), and an inner shell of scaffold proteins. The asymmetric triplexes have specific and conserved orientations, suggesting a key role in assembly. In the mature capsid structures, triplexes bound to three MCPs may represent an assembly unit where, in most cases, the N-terminus of one MCP wraps around the E-loop of another MCP. The model accommodates the incorporation of a portal into capsid, required for genome encapsidation and viral viability. Cleavage of the scaffold triggers maturation of procapsid. Each of the MCPs rotates mostly as a rigid body, except for the flexible peripheral parts that remodel to close the capsid inner surface. Angularization of the capsid shifts the portal outward to a better contact with the capsid shell. Understanding these events in the herpesvirus life cycle to atomic detail could facilitate the development of drugs that uniquely target assembly and maturation. Full article

(This article belongs to the Special Issue Advances in HSV)

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26 pages, 6717 KB

Open AccessArticle

A Pan-H5N1 Multiepitope DNA Vaccine Construct Targeting Some Key Proteins of the Clade 2.3.4.4b Using AI-Assisted Epitope Mapping and Molecular Docking

by Nithyadevi Duraisamy, Abid Ullah Shah, Mohd Yasir Khan, Mohammed Cherkaoui and Maged Gomaa Hemida

Viruses 2025, 17(9), 1152; https://doi.org/10.3390/v17091152 - 22 Aug 2025

Abstract

The presently used vaccines do not offer solid immunity/protection against the currently circulating strains of the H5N1 viruses. We aim to design a pan-H5N1 vaccine that protects birds against the presently circulating clade 2.3.4.4b in chickens. We used AI tools, including epitope mapping, [...] Read more.

The presently used vaccines do not offer solid immunity/protection against the currently circulating strains of the H5N1 viruses. We aim to design a pan-H5N1 vaccine that protects birds against the presently circulating clade 2.3.4.4b in chickens. We used AI tools, including epitope mapping, molecular docking, and immune simulation, to design a multiepitope DNA vaccine including the top-ranked B and T cell epitopes within four major proteins (HA, NA, NP, and M2) of H5N1 clade 2.3.4.4b. We selected the top-ranked 12 epitopes and linked them together using linkers. The designed vaccine is linked to IL-18 as an adjuvant. The molecular docking results showed a high binding affinity of those predicted epitopes from the MHC I and MHC II classes of molecules with chicken alleles. The immune simulation results showed that the designed vaccine has the potential to stimulate the host immune response, including antibody and cell-mediated immunity in chickens and other birds. We believe this vaccine is going to be a universal vaccine that offers good protection against HPAI-H5N1 clade 2.3.4.4b. We are reporting the successful molecular cloning of a recombinant multiepitope-based vaccine spanning some key epitopes within some key proteins of the currently circulating H5N1 clade 2.3.4.4b. These designed vaccines could be a great positive impact on the protection of birds and various species of animals, as well as humans, against the HP-H5N1 influenza virus. Further studies are required to validate this vaccine candidate in chickens. Full article

(This article belongs to the Section Animal Viruses)

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13 pages, 1203 KB

Open AccessArticle

Peste des Petits Ruminants Vaccine: Criteria for Assessing Its Thermotolerance

by Charles S. Bodjo, Hassen Belay Gelaw, Zione D. Luhanga, Yebechaye Degefa Tessema, Jean-De-Dieu Baziki, Cisse R. Moustapha Boukary, Gelagay Ayelet Melesse, Ethel Chitsungo, Nick Nwankpa, Simon Kihu, Felix Njeumi, Satya Parida and Adama Diallo

Viruses 2025, 17(9), 1151; https://doi.org/10.3390/v17091151 - 22 Aug 2025

Abstract

The Peste des Petits Ruminants (PPR) live attenuated vaccines, the PPR virus (PPRV) Nigeria 75/1 strain (lineage II) and PPRV India Sungry 96 strain (lineage IV), currently used for control and eradication programme are very efficient vaccines as they provide the host, sheep [...] Read more.

The Peste des Petits Ruminants (PPR) live attenuated vaccines, the PPR virus (PPRV) Nigeria 75/1 strain (lineage II) and PPRV India Sungry 96 strain (lineage IV), currently used for control and eradication programme are very efficient vaccines as they provide the host, sheep and goats, a lifelong immunity after a single minimum recommended dose of 10^2.5 TCID₅₀/mL. Unfortunately, both live attenuated vaccines are thermolabile and their use requires maintaining the cold chain from the manufactory premises to the field as most PPR-infected regions are facing of hot climate, with poor infrastructure, and the maintenance of an effective cold chain remains a challenge. To address this challenge, efforts have focused on developing thermotolerant (ThT) PPR vaccines using different stabilisers and improving the freeze-drying process. This study aimed to define the criteria for the evaluation of the stability of ThT PPR vaccines. A total of 37 batches of freeze-dried PPR vaccines using the PPRV Nigeria 75/1 strain, including eight (8) and twenty-nine (29) vaccines labelled as ThT and conventional formulations, respectively, were tested to evaluate the stability at temperatures of 40 °C to simulate the field conditions in some hot climate regions. All the vaccine batches included in this study initially showed acceptable levels of residual moisture, below 3%, and titres above the minimum WOAH standard requirement of 10^2.5 TCID₅₀/mL. Following the incubation at 40 °C, 56.7% and 46% of the 37 vaccine batches tested retained titres above 10^2.5 TCID₅₀/mL on day 3 and day 5, respectively. These vaccines use stabilisers such as skimmed milk, lactalbumin–sucrose, trehalose and one unnamed product (which may be protected for patent). The mean of titre loss among the PPR vaccines maintaining titres above 10^2.5 TCID₅₀/mL was 0.78 log10 at day 3 and 0.99 log10 at day 5, suggesting a significant early degradation during the first 3 days. Based on these data, it is proposed that thermotolerant PPR vaccines should maintain a minimum titre of 10^2.5 TCID₅₀/mL for vaccine dose on day 5 post-incubation at 40 °C with a titre loss below 1 log10 per mL. Preliminary immunogenicity test results showed that the PPR ThT vaccine meeting this criterion could be used in the field without maintaining a cold chain for up to 3 weeks, offering a practical solution for vaccination in remote areas. Full article

(This article belongs to the Special Issue Peste Des Petits Ruminants (PPR): Progress and Efforts Toward 2030 Eradication)

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11 pages, 552 KB

Open AccessPerspective

Viral Infections in Type 2 Diabetes: A Dangerous Liaison

by Azizul Haque and Anudeep B. Pant

Viruses 2025, 17(9), 1150; https://doi.org/10.3390/v17091150 - 22 Aug 2025

Abstract

Type 2 diabetes mellitus (T2DM) is increasing in incidence in many parts of the world and is becoming an important global health threat. T2DM results from a disturbance in glucose metabolism and is triggered by a combination of genetic and environmental factors. In [...] Read more.

Type 2 diabetes mellitus (T2DM) is increasing in incidence in many parts of the world and is becoming an important global health threat. T2DM results from a disturbance in glucose metabolism and is triggered by a combination of genetic and environmental factors. In regions where diabetes is prevalent, viral infections are also common; both conditions can contribute to increased blood sugar levels. We hypothesize that under these conditions, viral infections could accelerate many of the complications of T2DM in predisposed individuals. The high glucose levels may negatively impact blood vessel structure, white blood cell function, and infection-fighting proteins, which may weaken the immune response and, in turn, increase the frequency of viral infections in diabetic patients. Furthermore, viruses can stimulate an immune response, which induces inflammation and cytokine secretion. This perspective article postulates the existence of an axis between T2DM and viral infections and highlights the mechanistic aspects underlying their connection. A better understanding of the mechanisms between viral infections and blood sugar is likely to reveal new therapeutic avenues for the treatment and management of these diseases. Full article

(This article belongs to the Section Human Virology and Viral Diseases)

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18 pages, 6560 KB

Open AccessArticle

Global Phylogenetic Analysis of the CDV Hemagglutinin Gene Reveals Positive Selection on Key Receptor-Binding Sites

by Tuba Çiğdem Oğuzoğlu and B. Taylan Koç

Viruses 2025, 17(9), 1149; https://doi.org/10.3390/v17091149 - 22 Aug 2025

Abstract

Canine distemper virus (CDV) is a multi-host morbillivirus whose evolution and host-switching capacity are largely determined by its hemagglutinin (H) gene. To reconsider the molecular evolution of this critical gene, we performed comprehensive phylogenetic, selection, and structural analyses on a curated dataset of [...] Read more.

Canine distemper virus (CDV) is a multi-host morbillivirus whose evolution and host-switching capacity are largely determined by its hemagglutinin (H) gene. To reconsider the molecular evolution of this critical gene, we performed comprehensive phylogenetic, selection, and structural analyses on a curated dataset of 68 representative global H gene sequences. Our phylogenetic reconstruction confirmed the segregation of sequences into distinct, geographically associated lineages. To provide stronger evidence for viral adaptation, we performed a site-specific selection analysis, which identified 15 amino acid sites in the H protein undergoing significant episodic positive selection. Crucially, the majority of the known SLAM and Nectin-4 receptor-binding residues were found to be among these positively selected sites. We further contextualized these findings by mapping the sites onto a 3D homology model of the H protein, which confirmed their location on the exposed surfaces of the receptor-binding domain. This compilation provides quantitative evidence that the key functional regions of the H protein are direct targets for adaptive evolution, which has significant implications for understanding host tropism and the ongoing challenge of vaccine mismatch. Full article

(This article belongs to the Special Issue Canine Distemper Virus)

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9 pages, 608 KB

Open AccessBrief Report

“Big Events” and HIV Transmission Dynamics: Estimating Time Since HIV Infection from Deep Sequencing Data Among Sex Workers and Their Clients in Dnipro, Ukraine

by François Cholette, Nicole Herpai, Leigh M. McClarty, Olga Balakireva, Daryna Pavlova, Anna Lopatenko, Rupert Capiña, Paul Sandstrom, Michael Pickles, Evelyn Forget, Sharmistha Mishra, Marissa L. Becker and on behalf of the Dynamics Study Team

Viruses 2025, 17(8), 1148; https://doi.org/10.3390/v17081148 - 21 Aug 2025

Abstract

Background: Major geopolitical events and structural shocks are thought to play a significant role in shaping HIV epidemics by influencing individual behaviours, reshaping social networks, and impacting HIV prevention and treatment programs. Here, we describe individual-level measures of estimated time since HIV infection [...] Read more.

Background: Major geopolitical events and structural shocks are thought to play a significant role in shaping HIV epidemics by influencing individual behaviours, reshaping social networks, and impacting HIV prevention and treatment programs. Here, we describe individual-level measures of estimated time since HIV infection (ETI) from viral next-generation sequencing data among female sex workers and their clients in relation to significant geopolitical events in Ukraine. Methods: The Dynamics Study is a cross-sectional integrated biological and behavioural survey conducted among female sex workers and their clients in Dnipro, Ukraine (December 2017 to March 2018). We were able to successfully sequence a portion of the HIV pol gene on dried blood spot specimens among n = 5/9 clients and n = 5/16 female sex workers who tested positive for HIV (total n = 10/25) using an in-house drug resistance genotyping assay. The “HIV EVO” Intrapatient HIV Evolution web-based tool was used to infer ETI from viral diversity. Results: The median ETIs for female sex workers and their clients were 5.4 years (IQR = 2.9, 6.6) and 6.5 years (IQR = 5.4, 10.8), respectively. Nearly all HIV acquisition events (n = 7/10; 70%) were estimated to have occurred between the Great Recession (2008–2009) and the War in Donbas (May 2014–February 2022). In general, ETI suggests that HIV acquisition occurred earlier among clients (2012 [IQR = 2007, 2013]) compared to sex workers (2013 [IQR = 2012, 2016]). Conclusion: Our findings suggest that most HIV acquisition in this small subset of female sex workers and clients living with HIV occurred during periods of economic decline. Molecular studies on timing of HIV acquisition against timing of major geopolitical events offer a novel way to contextualize how such events may shape transmission patterns. Full article

(This article belongs to the Section Human Virology and Viral Diseases)

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14 pages, 632 KB

Open AccessArticle

Prevalence and Associated Factors for HPV in People Living with HIV: Are INSTIs Protective Against HPV-16? The GAIA Study

by Omar Hernández-López, Brenda Clara González-Contreras, Ana Luz Cano-Díaz, José Antonio Mata-Marín, Ericka Nelly Pompa-Mera, Javier Vicente Noyola-Gómez, Salma Triana-González, Paola Edith Padilla-Noguera, Alberto Chaparro-Sánchez, Sócrates Alberto García-Gutiérrez, Gustavo Barriga-Angulo and Jesús Enrique Gaytan-Martinez

Viruses 2025, 17(8), 1147; https://doi.org/10.3390/v17081147 - 21 Aug 2025

Abstract

Human papillomavirus (HPV) significantly contributes to anogenital cancers, with elevated risks among people living with HIV (PWH), particularly men who have sex with men (MSM). This study assessed anal HPV prevalence and associated risk factors in PWH in Mexico, focusing on the role [...] Read more.

Human papillomavirus (HPV) significantly contributes to anogenital cancers, with elevated risks among people living with HIV (PWH), particularly men who have sex with men (MSM). This study assessed anal HPV prevalence and associated risk factors in PWH in Mexico, focusing on the role of antiretroviral therapy (ART). Methods: A cross-sectional study at an HIV clinic in Mexico City (October 2023–December 2024) enrolled 214 MSM with HIV. The participants completed a validated risk factor questionnaire and provided anal samples for real-time PCR testing of 28 HPV genotypes. Logistic regression analyzed associations between HPV infection, ART regimens, and clinical/behavioral factors. Results: HPV prevalence was 89.3%, with HPV-16 (20.1%) being the most common high-risk genotype. Integrase inhibitor (INSTI) use was inversely associated with HPV-16 infection (OR: 0.42; 95% CI: 0.21–0.83; p = 0.011), while protease inhibitor use increased HPV-16 (OR: 2.16; 95% CI: 1.09–4.29; p = 0.025) and HPV-6 risks. Higher CD4+ counts (≥500 cells/mm³) and undetectable HIV viral load (<40 copies/mL) were protective against multiple HPV genotypes. Lower education and smoking increased HPV risk. Conclusions: This first Mexican study in the ART and HPV vaccination era highlights high anal HPV prevalence in PWH and suggests that INSTI-based regimens may reduce HPV-16 risk, informing ART selection for HPV prevention. Full article

(This article belongs to the Special Issue Cascade of Care for HIV, Hepatitis and Sexually Transmitted Infections)

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Graphical abstract

14 pages, 1721 KB

Open AccessBrief Report

Serologic Evidence of Human Exposure to Bat-Borne Zoonotic Paramyxoviruses, Cambodia

by Neil Mittal, Spencer L. Sterling, Phireak Hip, Dolyce H. W. Low, Piseth Ly, Menghou Mao, Pidor Ouch, Adrian C. Paskey, Lianying Yan, Alan Hitch, Gavin J. D. Smith, Jeffery Hertz, Andrew G. Letizia, Ian H. Mendenhall and Eric D. Laing

Viruses 2025, 17(8), 1146; https://doi.org/10.3390/v17081146 - 21 Aug 2025

Abstract

Fruit bats in the genus Pteropus are the natural reservoirs for zoonotic paramyxoviruses, notably henipaviruses and pararubulaviruses, which are found across Southeast Asia and Oceania. The genetic and antigenic diversity of viruses in both genera, and region specificity, are ill-defined, limiting health security [...] Read more.

Fruit bats in the genus Pteropus are the natural reservoirs for zoonotic paramyxoviruses, notably henipaviruses and pararubulaviruses, which are found across Southeast Asia and Oceania. The genetic and antigenic diversity of viruses in both genera, and region specificity, are ill-defined, limiting health security measures aimed at minimizing spillover. For example, Nipah virus has been isolated from bats in the Battambang province of western Cambodia, and surveys suggest bat foraging behaviors occur in close proximity to human settlements. However, there have been no historical cases of Nipah virus in Cambodia. Here, we use a multiplex microsphere immunoassay to identify cryptic human exposure to selected henipaviruses and pararubulaviruses in Cambodia. Convalescent human sera from persons presenting with acute respiratory illness were screened to detect the presence or absence of antibodies reactive with attachment glycoprotein antigens from Nipah virus, Hendra virus, Cedar virus, and Ghana virus, and a hemagglutinin-neuraminidase antigen from Menangle virus. In this sero-survey, we detected antibodies that were specifically reactive with Cedar virus and Menangle virus, including one serum sample that neutralized a recombinant Cedar virus. Additionally, we detected a pattern of cross-reactivity with Hendra virus, Cedar virus, and Ghana virus, suggesting previous infection by an antigenically-related henipavirus. We did not detect high antibody reactivity with the NiV glycoprotein. Future studies should expand serological surveillance for these transboundary pathogens, including genetic surveillance to aid in henipavirus discovery, and focused biosurveillance where interfaces with livestock and humans occur. Full article

(This article belongs to the Special Issue Emerging Zoonotic Paramyxoviruses)

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