Viruses

13 pages, 1448 KB

Open AccessArticle

The Diagnostic Value of Signal-to-Cutoff Ratios in Architect and Alinity HIV Screening Assays: A 10-Year Experience in a Pandemic-Affected, Low-Prevalence Setting

by İmran Sağlık, Melda Payaslıoğlu, Hatice Ortaç and Hülya Ayma Rüzgar

Viruses 2025, 17(9), 1179; https://doi.org/10.3390/v17091179 - 29 Aug 2025

Abstract

Early and accurate diagnosis of HIV remains a cornerstone of public health strategies. This study aimed to evaluate the predictive value of signal-to-cutoff (S/CO) ratios from two fourth-generation HIV screening assays (Abbott Architect and Alinity) and to analyze diagnostic trends across pre-pandemic, pandemic, [...] Read more.

Early and accurate diagnosis of HIV remains a cornerstone of public health strategies. This study aimed to evaluate the predictive value of signal-to-cutoff (S/CO) ratios from two fourth-generation HIV screening assays (Abbott Architect and Alinity) and to analyze diagnostic trends across pre-pandemic, pandemic, and post-pandemic periods in a low-prevalence setting. We retrospectively analyzed 197,642 unique HIV screening tests conducted at Bursa Uludağ University Hospital from 2015 to 2024. Receiver operating characteristic (ROC) analysis was used to determine optimal S/CO thresholds for distinguishing true-positive results. Of the 197,642 samples screened, the overall HIV prevalence was 0.5%, with 196 cases (0.1%) confirmed as new diagnoses. The Architect assay showed an optimal S/CO threshold of ≥11.8 (sensitivity 98.3%, specificity 97.3%). The Alinity assay demonstrated 100% sensitivity and specificity at an S/CO threshold of ≥19.1. Although a temporary decline in test volume occurred in 2020, there was no statistically significant difference in confirmation rates across years. During the pandemic, newly diagnosed individuals were significantly older and had lower CD4 counts, indicating delayed diagnosis (p = 0.026 and 0.008, respectively). Men who have sex with men (MSM)-related transmission significantly increased post-pandemic (p = 0.032). S/CO ratio–guided interpretation enhances diagnostic accuracy and may reduce unnecessary confirmatory testing, especially in low-prevalence and resource-limited regions. Selecting the optimal threshold can help to ensure a timely diagnosis and optimize HIV screening algorithms. Full article

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

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46 pages, 2436 KB

Open AccessReview

Antiviral Strategies Targeting Enteroviruses: Current Advances and Future Directions

by Michelle Felicia Lee, Seng Kong Tham and Chit Laa Poh

Viruses 2025, 17(9), 1178; https://doi.org/10.3390/v17091178 - 28 Aug 2025

Abstract

Enteroviruses, a diverse genus within the Picornaviridae family, are responsible for a wide range of human infections, including hand, foot, and mouth disease, respiratory disease, aseptic meningitis, encephalitis, myocarditis, and acute flaccid paralysis. Despite their substantial global health burden and the frequent emergence [...] Read more.

Enteroviruses, a diverse genus within the Picornaviridae family, are responsible for a wide range of human infections, including hand, foot, and mouth disease, respiratory disease, aseptic meningitis, encephalitis, myocarditis, and acute flaccid paralysis. Despite their substantial global health burden and the frequent emergence of outbreaks, no specific antiviral therapies are currently approved for clinical use against non-polio enteroviruses. This review provides a comprehensive overview of the current landscape of antiviral strategies targeting enteroviruses, including direct-acting antivirals such as capsid binders, protease inhibitors, and viral RNA polymerase inhibitors. We also examine the potential of host-targeting agents that interfere with virus–host interactions essential for replication. Emerging strategies such as immunotherapeutic approaches, RNA interference, CRISPR-based antivirals, and peptide-based antivirals are also explored. Furthermore, we address key challenges, including viral diversity, drug resistance, and limitations in preclinical models. By highlighting recent advances and ongoing efforts in antiviral development, this review aims to guide future research and accelerate the discovery of effective therapies against enterovirus infections. Full article

(This article belongs to the Special Issue Picornavirus Evolution, Host Adaptation and Antiviral Strategies)

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15 pages, 3579 KB

Open AccessArticle

Pathogenicity of SARS-CoV-2 Omicron Subvariants JN.1, KP.2, and EG.5.1 in K18-hACE2 Transgenic Mice

by Lila D. Patterson, Amany Elsharkawy, Hamid Reza Jahantigh, Zainab Nabi, Shannon Stone and Mukesh Kumar

Viruses 2025, 17(9), 1177; https://doi.org/10.3390/v17091177 - 28 Aug 2025

Abstract

The emergence of the SARS-CoV-2 JN.1 lineage in late 2023 marked a major shift in viral evolution. By January 2024, it had displaced XBB variants to become the dominant strain worldwide. JN.1 and its descendants are antigenically distinct from earlier Omicron subvariants, with [...] Read more.

The emergence of the SARS-CoV-2 JN.1 lineage in late 2023 marked a major shift in viral evolution. By January 2024, it had displaced XBB variants to become the dominant strain worldwide. JN.1 and its descendants are antigenically distinct from earlier Omicron subvariants, with approximately 30 additional spike mutations compared to XBB-derived viruses. The combination of these features alongside growing evidence of considerable immune evasion prompted the FDA to recommend that vaccine formulations be updated to target JN.1 rather than XBB.1.5. The continued dominance of JN.1-derived variants necessitates the characterization of viral infection in established animal models to inform vaccine efficacy and elucidate host–pathogen interactions driving disease outcomes. In this study, transgenic mice expressing human ACE2 were infected with SARS-CoV-2 subvariants JN.1, KP.2, and EG.5.1 to compare the pathogenicity of JN.1-lineage and XBB-lineage SARS-CoV-2 viruses. Infection with JN.1 and KP.2 resulted in attenuated disease, with animals exhibiting minimal clinical symptoms and no significant weight loss. In contrast, EG.5.1-infected mice exhibited rapid progression to severe clinical disease, substantial weight loss, and 100% mortality within 7 days of infection. All variants replicated effectively within the upper and lower respiratory tracts and caused significant lung pathology. Notably, EG.5.1 resulted in neuroinvasive infection with a significantly high viral burden in the brain. Additionally, EG.5.1 infection resulted in a significant increase in CD8⁺ T cell and CD11b⁺ CD11c⁺ dendritic cell populations in infected lungs. Full article

(This article belongs to the Special Issue Multiple Hosts of SARS-CoV-2, 3rd Edition)

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18 pages, 5170 KB

Open AccessArticle

APOBEC3B Promotes SARS-CoV-2 Through Activation of PKR/eIF2⍺ and AMPD2 Dysregulation

by Benjamin Fixman, Lavanya Manjunath, Philip Sell, Shanshan Wang, Tamara Margaryan, Connor Qiu, Hanjing Yang, Rémi Buisson and Xiaojiang S. Chen

Viruses 2025, 17(9), 1176; https://doi.org/10.3390/v17091176 - 28 Aug 2025

Abstract

APOBEC3B (A3B) has been implicated in host–virus interactions, but its role in SARS-CoV-2 infection is unclear. Here, we demonstrate that A3B is overexpressed in bronchoalveolar lavage fluid (BALF) cells from severe COVID-19 patients compared to those with mild disease. A3B knockdown in Caco-2 [...] Read more.

APOBEC3B (A3B) has been implicated in host–virus interactions, but its role in SARS-CoV-2 infection is unclear. Here, we demonstrate that A3B is overexpressed in bronchoalveolar lavage fluid (BALF) cells from severe COVID-19 patients compared to those with mild disease. A3B knockdown in Caco-2 cells significantly reduces SARS-CoV-2 infectivity, likely through attenuation of the PKR-mediated integrated stress response, a pathway proposed to promote SARS-CoV-2. Single-cell RNA sequencing (scRNA-seq) data suggest that BALF cells from severe COVID-19 patients exhibit a repressed state for cellular translation, potentially mediated by eIF2α phosphorylation. However, in A549-ACE2 cells, SARS-CoV-2 does not activate PKR, but A3B knockdown still reduces SARS-CoV-2 infectivity, suggesting an alternative mechanism of action in different cellular contexts. To further investigate A3B’s role in severe COVID-19, we employed Geneformer, a transformer-based machine learning model, which predicted that A3B knockout would perturb AMPD2 (adenosine monophosphate deaminase 2), a key enzyme in purine metabolism and immune regulation. We validated this prediction using bulk RNA-seq and clinical scRNA-seq data, confirming that AMPD2 expression is downregulated in severe COVID-19 but restored upon A3B knockdown. Together, these findings suggest that A3B plays a proviral role in SARS-CoV-2 infection by modulating translational control and immune regulatory networks, warranting further studies to elucidate the underlying mechanistic details. Full article

(This article belongs to the Special Issue Host-Mediated Viral Mutations: APOBECs, ADARs, and Beyond)

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12 pages, 1870 KB

Open AccessArticle

A Novel Cogu-like Virus Identified in Wine Grapes

by Jennifer Dahan, Gardenia E. Orellana, Edison Reyes-Proaño, Jungmin Lee and Alexander V. Karasev

Viruses 2025, 17(9), 1175; https://doi.org/10.3390/v17091175 - 28 Aug 2025

Abstract

A new negative-strand RNA virus was identified in grapevines from a 38-year-old ‘Chardonnay’ block in Idaho through high-throughput sequencing (HTS) of total RNA. This virus was tentatively named grapevine-associated cogu-like Idaho virus (GaCLIdV). GaCLIdV has three negative-sense, single-stranded RNA genome segments of ca. [...] Read more.

A new negative-strand RNA virus was identified in grapevines from a 38-year-old ‘Chardonnay’ block in Idaho through high-throughput sequencing (HTS) of total RNA. This virus was tentatively named grapevine-associated cogu-like Idaho virus (GaCLIdV). GaCLIdV has three negative-sense, single-stranded RNA genome segments of ca. 7 kb, 1.9 kb, and 1.3 kb, encoding L protein (RNA-dependent RNA polymerase, RdRP), a movement protein (MP), and a nucleocapsid protein (NC), respectively, identified based on pair-wise comparisons with other cogu- and cogu-like viruses. In phylogenetic analysis based on the RdRP, GaCLIdV grouped within the family Phenuiviridae and was placed in a lineage of plant-infecting phenuiviruses as a sister clade of the genus Laulavirus, clustering most closely with switchgrass phenui-like virus 1 (SgPLV-1) and more distantly related to grapevine-associated cogu-like viruses from the Laulavirus and Coguvirus clades. Both GaCLIdV and SgPhLV-1 are proposed to form a new genus, Switvirus, within the family Phenuiviridae. The presence of GaCLIdV in the original ‘Chardonnay’ samples was confirmed by RT-PCR amplification and Sanger sequencing. This new virus was found in five wine grape cultivars and in six vineyards sampled in Idaho and in Oregon during the 2020–2024 seasons. GaCLIdV may have contributed to the decline observed in the old ‘Chardonnay’ block, although the role of the virus in symptom development awaits further investigation. Full article

(This article belongs to the Special Issue Emerging and Re-Emerging Plant Viruses and Vector Complexes: Advances in Characterization, Surveillance, and Mitigation)

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13 pages, 1565 KB

Open AccessReview

Recent Advances in Therapeutics for Severe Fever with Thrombocytopenia Syndrome Virus

by Huimin Dang, Yuanyuan Wang, Lihong Zhang, Shan Xu, Lei Liu and Yigang Tong

Viruses 2025, 17(9), 1174; https://doi.org/10.3390/v17091174 - 28 Aug 2025

Abstract

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne bunyavirus with a mortality rate of up to 30%. There is no specific treatment for SFTSV. This article systematically reviews the progress of major anti-SFTSV drugs. The nucleotide analogues (favipiravir, 4′-fluorouridine diphosphate prodrug [...] Read more.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne bunyavirus with a mortality rate of up to 30%. There is no specific treatment for SFTSV. This article systematically reviews the progress of major anti-SFTSV drugs. The nucleotide analogues (favipiravir, 4′-fluorouridine diphosphate prodrug VV261) have shown clinical potential. Calcium channel blockers (nifedipine, etc.) block virus invasion by inhibiting calcium influx. Monoclonal antibody (S2A5/SNB02) has achieved targeted therapy, and SNB02 nanoantibody has entered clinical trials. However, many candidate agents predominantly focus on a single target, such as viral RdRp or host calcium channels, which makes it difficult to block the entire viral replication cycle and may accelerate the accumulation of resistant mutations. In addition, the low bioavailability of small-molecule drugs, the obstacles to industrial-scale production of antibody-based therapies, and the lack of Phase III clinical evidence severely restrict their clinical translation. Future research should focus on exploring viral replication mechanisms, developing drugs against key viral proteins, and designing multi-target combination therapies and novel drug delivery systems. Full article

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

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22 pages, 4780 KB

Open AccessArticle

Hiding in Plain Sight: Genomic Characterization of a Novel Nackednavirus and Evidence of Diverse Adomaviruses in a Hyperpigmented Lesion of a Largemouth Bass (Micropterus nigricans)

by Clayton Raines, John Odenkirk, Michael Isel, Patricia Mazik, Morgan Biggs and Luke Iwanowicz

Viruses 2025, 17(9), 1173; https://doi.org/10.3390/v17091173 - 28 Aug 2025

Abstract

Largemouth bass (LMB; Micropterus nigricans) are popular both as a sportfish and an aquaculture species. At present, six described viruses are associated with LMB, of which two are typically considered in cases of LMB mortality events. Advances in discovery and diagnostic capabilities [...] Read more.

Largemouth bass (LMB; Micropterus nigricans) are popular both as a sportfish and an aquaculture species. At present, six described viruses are associated with LMB, of which two are typically considered in cases of LMB mortality events. Advances in discovery and diagnostic capabilities using next-generation sequencing have augmented surveillance efforts and subsequently led to the discovery of novel cryptogenic viruses. Here, we present evidence of three novel viruses from a single skin sample collected from a hyperpigmented melanistic lesion of an LMB with blotchy bass syndrome associated with MnA-1 co-infection. These viruses represent recently described groups of viruses (adomaviruses and nackednaviruses) that infect fish. Both are markedly understudied and of unknown significance to fish health. This work highlights the diversity of viruses associated with LMB and further advances our understanding of the LMB virome. Application of de novo sequencing approaches presents an opportunity to explore a new frontier of host–pathogen relationships and microbes associated with changing environments. Full article

(This article belongs to the Special Issue Aquatic Animal Viruses and Antiviral Immunity)

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9 pages, 677 KB

Open AccessArticle

Rapid Detection Assay for Infectious Bronchitis Virus Using Real-Time Reverse Transcription Recombinase-Aided Amplification

by Nahed Yehia, Ahmed Abd El Wahed, Abdelsatar Arafa, Dalia Said, Ahmed Abd Elhalem Mohamed, Samah Eid, Mohamed Abdelhameed Shalaby, Rea Maja Kobialka, Uwe Truyen and Arianna Ceruti

Viruses 2025, 17(9), 1172; https://doi.org/10.3390/v17091172 - 27 Aug 2025

Abstract

The infectious bronchitis virus (IBV) causes a severe infectious disease in poultry, leading to significant financial losses. The prevention and treatment of this disease are extremely challenging due to the virus’s rapid mutation. Therefore, quick diagnosis of IBV infections is crucial for controlling [...] Read more.

The infectious bronchitis virus (IBV) causes a severe infectious disease in poultry, leading to significant financial losses. The prevention and treatment of this disease are extremely challenging due to the virus’s rapid mutation. Therefore, quick diagnosis of IBV infections is crucial for controlling the disease. This study aimed to develop a real-time reverse transcription recombinase-aided amplification (RT-RAA) method for IBV. The most effective primer combination was selected for further validation. To determine the assay’s analytical sensitivity, a serial dilution from 10⁵ to 10⁰ EID₅₀/mL was used, and the limit of detection was calculated. The assay could detect down to 10² EID₅₀/mL. The limit of detection (95% Confidence Interval) was 67 EID₅₀ per reaction. There was no cross-reaction with common poultry diseases. When analyzing 39 clinical samples, RT-RAA and RT-PCR showed 100% diagnostic sensitivity and specificity. In conclusion, the IBV RT-RAA detection method is rapid, sensitive, and specific. This approach can be used to improve IBV diagnosis at the point of need. Full article

(This article belongs to the Section Animal Viruses)

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12 pages, 1526 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 (registering DOI) - 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, except for 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)

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25 pages, 4569 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)

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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, 1446 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)

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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, 1197 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)

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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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