Viruses

12 pages, 1094 KB

Open AccessArticle

Human-Derived H3N2 Influenza A Viruses Detected in Pigs in Northern Italy

by Laura Soliani, Ada Mescoli, Irene Zanni, Laura Baioni, Giovanni Alborali, Ana Moreno, Silvia Faccini, Carlo Rosignoli, Giorgia De Lorenzi, Laura Fiorentini, Camilla Torreggiani, Benedetta Cordioli, Alice Prosperi, Andrea Luppi and Chiara Chiapponi

Viruses 2025, 17(9), 1171; https://doi.org/10.3390/v17091171 - 27 Aug 2025

Abstract

In recent years, the four main swine influenza A virus (IAV-S) subtypes circulating in swine in the EU have been H1avN1, H1huN2, H1N1pdm09, and H3N2. The latter emerged in 1984 from a reassortment event between a human seasonal H3N2 and H1avN1, and is [...] Read more.

In recent years, the four main swine influenza A virus (IAV-S) subtypes circulating in swine in the EU have been H1avN1, H1huN2, H1N1pdm09, and H3N2. The latter emerged in 1984 from a reassortment event between a human seasonal H3N2 and H1avN1, and is currently detected at low prevalence in swine in Italy. Here, we describe nine H3N2 IAV-S isolates belonging to three novel genotypes, first detected in Italy in 2021, likely resulting from reassortment events between swine and human IAVs. The first genotype was characterized by a hemagglutinin (H3 HA) of human seasonal origin, a neuraminidase (N2 NA) derived from H1huN2 strains circulating in Italian swine, and an avian-like internal gene cassette (IGC). The second genotype differed in its IGC constellation: PB2, PB1, PA and NP segments were of pandemic origin (pdm09), while NS and M segments derived from the Eurasian avian-like lineage. The third genotype combined a human-derived H3, a Gent/84-derived N2, and a pdm09-origin IGC, as well as an avian-like NS. This study aimed to characterize the genetic features of these novel H3huN2 and assess their epidemiological relevance, with implications for surveillance and control, improving preparedness and mitigating the risks posed by zoonotic influenza viruses. Full article

(This article belongs to the Special Issue Influenza and Other Respiratory Viruses: Prevention, Diagnosis, Treatment: 2nd Edition)

25 pages, 3800 KB

Open AccessArticle

An Anti-HIV Drug Is Highly Effective Against SARS-CoV-2 In Vitro and Has Potential Benefit for Long COVID Treatment

by Saken Khaidarov, Abdul Bari Hejran, Aizhan Moldakaryzova, Slu Izmailova, Bayan Nurgaliyeva, Aizhan Beisenova, Aigul Mustafaeva, Kuanysh Nurzhanova, Yelena Belova, Elmira Satbayeva, Askar Aidarov, Saniya Ossikbayeva, Yerlan Kukubassov, Jandos Amankulov, Tatyana Goncharova, Banu Yeszhan, Edan Tulman, Karlygash N. Tazhibayeva, Assel Sadykova, Nurlan Kozhabergenov and Yerbol Burashev Show full author list Hide full author list

Viruses 2025, 17(9), 1170; https://doi.org/10.3390/v17091170 - 27 Aug 2025

Abstract

The persistent evolution of SARS-CoV-2 necessitates novel antiviral strategies. This study evaluated the anti-HIV prodrugs tenofovir disoproxil fumarate (TDF) and tenofovir alafenamide (TAF) for repurposing against SARS-CoV-2, assessing key pharmacological indices (CC₅₀, EC₅₀, cytostatic effect, and therapeutic window). In [...] Read more.

The persistent evolution of SARS-CoV-2 necessitates novel antiviral strategies. This study evaluated the anti-HIV prodrugs tenofovir disoproxil fumarate (TDF) and tenofovir alafenamide (TAF) for repurposing against SARS-CoV-2, assessing key pharmacological indices (CC₅₀, EC₅₀, cytostatic effect, and therapeutic window). In vitro screening in Vero E6 cells measured cytotoxicity (via CCK-8/MTT assays) and antiviral activity against Kazakh B.1 and Wuhan strains. TDF (50 µg/mL) reduced high viral loads (MOI 2) by ~2 log₁₀ (100% inhibition), with minimal cytotoxicity (≥75% viability). TAF achieved near-complete suppression (100% inhibition) at 50 µg/mL, exhibiting dose-dependent inhibition (68–100%) at lower viral loads (MOI 0.01). Both prodrugs showed enhanced antiviral activity with prolonged exposure (96 h). Synergy assessments demonstrated favourable combination indices (CI < 1). Electron microscopy confirmed virion integrity post-treatment. These findings highlight TDF and TAF as promising candidates against SARS-CoV-2, with particular potential for targeting lymphoid reservoirs—sites implicated in persistent viral reservoirs that may contribute to long COVID pathogenesis. Further clinical validation is warranted. Full article

(This article belongs to the Special Issue Natural, Semisynthetic, and Synthetic Antiviral Drugs Targeting HIV and SARS-CoV-2)

24 pages, 11100 KB

Open AccessArticle

ATRX Promotes Transcription Initiation of HSV-1 Immediate Early Genes During Early Lytic Infection

by Laura E. M. Dunn, Mackenzie M. Clark and Joel D. Baines

Viruses 2025, 17(9), 1169; https://doi.org/10.3390/v17091169 - 27 Aug 2025

Abstract

Herpes simplex virus 1 (HSV-1) transcribes its genome using host RNA polymerase II (Pol II) in a temporally regulated cascade. We previously proposed a model of Transient Immediate Early gene Mediated Repression (TIEMR), in which early repression of immediate early (IE) genes is [...] Read more.

Herpes simplex virus 1 (HSV-1) transcribes its genome using host RNA polymerase II (Pol II) in a temporally regulated cascade. We previously proposed a model of Transient Immediate Early gene Mediated Repression (TIEMR), in which early repression of immediate early (IE) genes is relieved to initiate the cascade. Given the rapid association of promyelocytic leukaemia nuclear body (PML-NB) components with incoming HSV-1 genomes, we sought to investigate their roles in TIEMR. siRNA knockdown revealed that depletion of ATRX, but not PML, significantly reduced nascent transcription from viral IE promoters at 1.5 hpi, while DAXX knockdown increased transcription. ChIP-Seq showed ATRX localizes to both transcriptionally active IE genes and restricted non-IE genes, suggesting diverse functions. Notably, ATRX occupancy at active IE promoters correlated with G-quadruplex (G4) motifs, and G4 stabilization mimicked ATRX knockdown by reducing transcription initiation. These findings uncover a previously unrecognized pro-transcriptional role for ATRX at IE genes and suggest that ATRX promotes escape from TIEMR by facilitating transcription initiation and preventing G4-mediated repression. Full article

(This article belongs to the Special Issue Herpesvirus Transcriptional Control)

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14 pages, 964 KB

Open AccessArticle

Predicting COVID-19 Sepsis Outcomes: Roles of IL-6, Cardiac Biomarkers, Clinical Factors, and Vaccination Status and Exploratory Analysis of Tocilizumab Therapy in an Eastern European Cohort

by Diana-Maria Mateescu, Adrian-Cosmin Ilie, Ioana Cotet, Camelia-Oana Muresan, Ana-Maria Pah, Marius Badalica-Petrescu, Stela Iurciuc, Maria-Laura Craciun, Adrian Cote and Alexandra Enache

Viruses 2025, 17(9), 1168; https://doi.org/10.3390/v17091168 - 27 Aug 2025

Abstract

(1) Background: COVID-19 sepsis, marked by hyperinflammation and cardiac injury, poses significant challenges in high-comorbidity populations. This prospective cohort study evaluates the prognostic value of IL-6, troponin, NT-proBNP, and radiological findings for mortality and unfavorable outcomes in a post-2022 Eastern European cohort. (2) [...] Read more.

(1) Background: COVID-19 sepsis, marked by hyperinflammation and cardiac injury, poses significant challenges in high-comorbidity populations. This prospective cohort study evaluates the prognostic value of IL-6, troponin, NT-proBNP, and radiological findings for mortality and unfavorable outcomes in a post-2022 Eastern European cohort. (2) Methods: At “Victor Babes” Hospital, Timisoara, Romania (September 2022–December 2024), 207 adults with COVID-19 sepsis (Sepsis-3 criteria) were enrolled. Baseline IL-6, troponin, NT-proBNP, CRP, PCT, D-dimers, and chest CT lung involvement were measured. Unfavorable outcomes (in-hospital death, ICU transfer, mechanical ventilation, or vasopressor use) were analyzed using logistic and linear regression. (3) Results: Among 207 patients (mean age: 68.7 years, 54.1% male), 52 (25.1%) experienced unfavorable outcomes. Multivariable analysis identified IL-6 (OR 1.016 per pg/mL, p = 0.013), troponin (OR 1.013 per ng/L, p = 0.017), NT-proBNP (OR 1.009 per pg/mL, p = 0.049), >50% lung involvement (OR 1.835, p = 0.011), unvaccinated status (OR 2.312, p = 0.002), and higher BMI (OR 1.112 per kg/m², p = 0.005) as independent predictors of unfavorable outcomes. Tocilizumab use (n = 12) was associated with reduced mortality (p = 0.041). IL-6 (cut-off 39.0 pg/mL, AUC = 0.91) and troponin (cut-off = 111.3 ng/L, AUC = 0.88) showed strong predictive accuracy. (4) Conclusions: Elevated IL-6, troponin, NT-proBNP, severe lung involvement, unvaccinated status, and higher BMI predict adverse outcomes in COVID-19 sepsis. Tocilizumab may offer survival benefits, warranting larger trials. These findings support targeted risk stratification in high-comorbidity populations. Full article

(This article belongs to the Special Issue Viral Sepsis: Pathogenesis, Diagnostics and Therapeutics)

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17 pages, 5538 KB

Open AccessArticle

ACE2-Decoy-Conjugated PLGA-PEG Nanoparticles Loaded with Nafamostat for Potent Antiviral Activity

by Shulin Hou, Yunyun Zhang, Xin Zheng, Ruining Li, Taoran Zhao, Hua Qiao, Xiaozheng Zhang and Zhizhen Liu

Viruses 2025, 17(9), 1167; https://doi.org/10.3390/v17091167 - 27 Aug 2025

Abstract

Angiotensin-converting enzyme 2 (ACE2) is a key mediator of SARS-CoV-2 host cell entry, making it an attractive target for drug delivery strategies. Nafamostat (NM), a multifunctional agent with antiviral and anti-inflammatory properties, holds promise for COVID-19 treatment. In this study, we developed PLGA-PEG [...] Read more.

Angiotensin-converting enzyme 2 (ACE2) is a key mediator of SARS-CoV-2 host cell entry, making it an attractive target for drug delivery strategies. Nafamostat (NM), a multifunctional agent with antiviral and anti-inflammatory properties, holds promise for COVID-19 treatment. In this study, we developed PLGA-PEG nanoparticles encapsulating NM (NM-PP NPs) and further conjugated them with specific ACE2 decoys (CTC-445.2d or SI5α) to generate NM-PP-Pro/Pep NPs. Both unmodified and ACE2-decoy-modified NPs exhibited uniform size distributions (diameter < 200 nm) and negative surface charges, as confirmed by dynamic light scattering and zeta potential measurements. The nanoparticles maintained structural integrity for at least 18 days at 4 °C and room temperature. In vitro release studies revealed sustained and controlled NM release kinetics. Notably, NM-PP-Pro NPs displayed potent antiviral activity, with an IC₅₀ < 0.05 nM against wild-type SARS-CoV-2 and remained effective against the D614G variant (IC₅₀ = 2 nM). These results underscore the potential of NM-PP-Pro NPs as a versatile n;anotherapeutic platform for targeting SARS-CoV-2 and its emerging variants. Full article

(This article belongs to the Section SARS-CoV-2 and COVID-19)

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18 pages, 1918 KB

Open AccessArticle

HPV as a Molecular Hacker: Computational Exploration of HPV-Driven Changes in Host Regulatory Networks

by Massimiliano Chetta, Alessandra Rosati and Nenad Bukvic

Viruses 2025, 17(9), 1166; https://doi.org/10.3390/v17091166 - 27 Aug 2025

Abstract

Human Papillomavirus (HPV), particularly high-risk strains such as HPV16 and HPV18, is a leading cause of cervical cancer and a significant risk factor for several other epithelial malignancies. While the oncogenic mechanisms of viral proteins E6 and E7 are well characterized, the broader [...] Read more.

Human Papillomavirus (HPV), particularly high-risk strains such as HPV16 and HPV18, is a leading cause of cervical cancer and a significant risk factor for several other epithelial malignancies. While the oncogenic mechanisms of viral proteins E6 and E7 are well characterized, the broader effects of HPV infection on host transcriptional regulation remain less clearly defined. This study explores the hypothesis that conserved genomic motifs within the HPV genome may act as molecular decoys, sequestering human transcription factors (TFs) and thereby disrupting normal gene regulation in host cells. Such interactions could contribute to oncogenesis by altering the transcriptional landscape and promoting malignant transformation.We conducted a computational analysis of the genomes of high-risk HPV types using MEME-ChIP for de novo motif discovery, followed by Tomtom for identifying matching human TFs. Protein–protein interactions among the predicted TFs were examined using STRING, and biological pathway enrichment was performed with Enrichr. The analysis identified conserved viral motifs with the potential to interact with host transcription factors (TFs), notably those from the FOX, HOX, and NFAT families, as well as various zinc finger proteins. Among these, SMARCA1, DUX4, and CDX1 were not previously associated with HPV-driven cell transformation. Pathway enrichment analysis revealed involvement in several key biological processes, including modulation of Wnt signaling pathways, transcriptional misregulation associated with cancer, and chromatin remodeling. These findings highlight the multifaceted strategies by which HPV may influence host cellular functions and contribute to pathogenesis. In this context, the study underscores the power of in silico approaches for elucidating viral–host interactions and reveals promising therapeutic targets in computationally predicted regulatory network changes. Full article

(This article belongs to the Special Issue Human and Animal Papillomavirus: Infections, Genetics, and Vaccines)

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16 pages, 1379 KB

Open AccessArticle

Versatile and Scalable Nanoparticle Vaccine as a Scaffold Against Newly Emerging Influenza Viruses

by Alessandro Pardini, Dominik A. Rothen, Pascal S. Krenger, Anne-Cathrine Vogt, Romano Josi, Xuelan Liu, Kaspars Tars, Manfred Kopf, Monique Vogel and Martin F. Bachmann

Viruses 2025, 17(9), 1165; https://doi.org/10.3390/v17091165 - 26 Aug 2025

Abstract

Influenza remains a major health threat due to its high contagiousness and global spread, affecting not only humans but also agricultural livestock and wild animals through transmission via migratory birds. Despite over 70 years of vaccination, influenza still creates epidemics and pandemics, and [...] Read more.

Influenza remains a major health threat due to its high contagiousness and global spread, affecting not only humans but also agricultural livestock and wild animals through transmission via migratory birds. Despite over 70 years of vaccination, influenza still creates epidemics and pandemics, and the ongoing use of vaccination is an essential but currently insufficient strategy. In this study, we assessed the immunogenicity and efficacy of an AP205 virus-like particle (VLP) carrying the HA head domain of the A/PR8/H1N1 strain, administered intranasally and subcutaneously in mice. For this purpose, the entire head region of A/PR8/H1N1 was genetically integrated into a sterically improved version of AP205, which exhibits capsid monomers fused into a dimer, thereby offering inexpensive and scalable production processes. The vaccine induced strong systemic anti-HA IgG and IgA antibodies via both routes, with no significant difference in the levels of IgG. Both immunisation strategies induced protection against a lethal challenge with H1PR8 mouse-adapted influenza virus. The findings demonstrate the potential of the AP205 VLP platform for HA1-based influenza vaccines and its applicability for controlling influenza in both humans and livestock. Full article

(This article belongs to the Special Issue Influenza and Other Respiratory Viruses: Prevention, Diagnosis, Treatment: 2nd Edition)

17 pages, 3503 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 - 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)

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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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