Viruses

11 pages, 794 KB

Open AccessArticle

Analysing Antibodies Against Respiratory Viruses in Breast Milk: A Pilot Study

by Sindre H. Hauan, Camilla H. Nundal, Sarah Lartey Jalloh, June Skudal, Elin Ekornes Håskjold, Sigrid Christiansen Bøe, Camilla Tøndel, Linn Marie Sørbye, Rebecca J. Cox and Karl A. Brokstad

Viruses 2026, 18(6), 593; https://doi.org/10.3390/v18060593 (registering DOI) - 24 May 2026

Abstract

Background. Lower respiratory tract infections remain a major cause of morbidity and mortality in infants worldwide. Newborns possess an immature immune system but acquire passive immunity through maternal antibodies transferred via the placenta (IgG) and breast milk (IgA). Maternal vaccination may enhance this [...] Read more.

Background. Lower respiratory tract infections remain a major cause of morbidity and mortality in infants worldwide. Newborns possess an immature immune system but acquire passive immunity through maternal antibodies transferred via the placenta (IgG) and breast milk (IgA). Maternal vaccination may enhance this protection. This study aimed to quantify antibody levels against respiratory viruses in serum and breast milk from lactating women. Methods. Serum and breast milk samples were collected from 26 lactating mothers. Antibody levels were measured using an indirect enzyme-linked immunosorbent assay (ELISA) targeting seven viral antigens: influenza A (A/Thailand, A/California), influenza B (B/Phuket, B/Austria), SARS-CoV-2 (Spike and receptor-binding domain, RBD) and RSV F pre-fusion protein. Antibody isotypes IgG, IgA and IgM were analysed. Results. Virus-specific IgG and IgA antibodies were detected in all samples. Breast milk showed the highest levels of IgA, whereas serum contained higher IgG levels. A moderate positive correlation was observed between serum and milk IgG. No correlation was found between serum IgG and milk IgA, but both levels were elevated. Conclusions. Breast milk and serum contain relatively high levels of antibodies against the tested respiratory viruses. The elevated levels of serum IgG and milk IgA indicate a coordinated defence between systemic and mucosal immunity in response to infections. The levels and correlation of specific isotypes point to the source of the antibodies: milk IgG probably originates from the blood, whereas milk IgA is produced locally. Full article

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

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23 pages, 3572 KB

Open AccessArticle

Cascade Semantic Segmentation by a Convolutional Neural Network in Combination with Image Super-Euclidean Pixels Processing for SARS-CoV-2 Microscopy Images

by Santiago Tello-Mijares, Francisco Flores and Fomuy Woo

Viruses 2026, 18(6), 592; https://doi.org/10.3390/v18060592 (registering DOI) - 24 May 2026

Abstract

Although SARS-CoV-2 has been extensively studied from clinical, virological, and diagnostic perspectives, the problem of accurate automatic semantic segmentation of SARS-CoV-2 particles in electron microscopy images remains inadequately explored. Existing studies have largely focused on virus detection, classification, morphometry, or conventional image analysis, [...] Read more.

Although SARS-CoV-2 has been extensively studied from clinical, virological, and diagnostic perspectives, the problem of accurate automatic semantic segmentation of SARS-CoV-2 particles in electron microscopy images remains inadequately explored. Existing studies have largely focused on virus detection, classification, morphometry, or conventional image analysis, while comparatively little attention has been paid to pixel-level delineation of viral structures using specialised deep learning segmentation frameworks. To address this gap, we propose here a deep learning system based on convolutional neural networks (CNNs) combined with image processing techniques to establish semantic segmentation tools for the automatic identification of SARS-CoV-2. Our approach utilises the super-Euclidean pixels method as an intermediate layer within the CNN for semantic segmentation. We then compare its performance against the gradient vector flow (GVF) and Poisson inverse gradient (PIG) segmenters. The proposed CNN model surpassed the traditional GVF and PIG segmentation models, achieving the following metrics (mean ± variance): Dice similarity coefficient (DSC) = 0.9345 ± 0.0006; intersection over union (IoU) = 0.8782 ± 0.0018; sensitivity/true positive rate (TPR) = 0.9373 ± 0.0018; specificity/true negative rate (SPC) = 0.9517 ± 0.0012; accuracy = 0.9449 ± 0.0004; area under the ROC curve (AUC) = 0.9446 ± 0.0431; and Cohen’s Kappa = 0.9137 ± 0.0011. This method enables virologists to employ an automatic CNN-based segmentation tool for detecting SARS-CoV-2 and demonstrates superiority over GVF and PIG. Full article

(This article belongs to the Section Coronaviruses)

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19 pages, 1771 KB

Open AccessArticle

Avian Metapneumovirus Subtype B at the Wildlife–Poultry Interface in Egypt: Molecular and Serological Insights into Cross-Ecological Transmission

by Omar S. Saeed, Sara A. Shabana, Mahmoud Gamal, Basem M. Ahmed, Ayman H. El-Deeb and Haitham M. Amer

Viruses 2026, 18(6), 591; https://doi.org/10.3390/v18060591 (registering DOI) - 24 May 2026

Abstract

Avian metapneumovirus (aMPV) is a major respiratory pathogen of poultry with a significant economic impact; however, its epidemiology at the wildlife–poultry interface remains poorly understood, particularly within Afro–Eurasian migratory systems. This cross-sectional study (December 2024–April 2026) investigated aMPV occurrence in wild birds across [...] Read more.

Avian metapneumovirus (aMPV) is a major respiratory pathogen of poultry with a significant economic impact; however, its epidemiology at the wildlife–poultry interface remains poorly understood, particularly within Afro–Eurasian migratory systems. This cross-sectional study (December 2024–April 2026) investigated aMPV occurrence in wild birds across eleven Egyptian governorates representing key ecological zones along major migratory flyways. A total of 1280 samples were collected from 800 wild birds representing migratory waterfowl and synanthropic species, including 800 oropharyngeal swabs tested by real-time RT-qPCR for aMPV subtypes A and B and 480 serum samples analyzed using indirect ELISA. aMPV RNA was detected in 28/800 samples (3.5%), with all positives identified as subtype B and confined to the Nile Delta, Middle Egypt, and Canal Region. In contrast, serological analysis revealed a high seroprevalence of 58.3% (280/480), indicating widespread prior exposure with significant spatial and species-level variation (p < 0.05). The marked disparity between low molecular detection and high seroprevalence supports transient infection with cumulative exposure. The exclusive detection of subtype B may reflect epidemiological connectivity between poultry and wild bird populations within shared ecological interfaces; however, the directionality of transmission and the possibility of independent wildlife maintenance could not be determined within the scope of the present cross-sectional study. Future studies incorporating whole-genome sequencing, longitudinal surveillance, and broader flyway-scale sampling are needed to resolve transmission pathways and distinguish field strains from potential vaccine-derived viruses within wildlife–poultry interfaces. Full article

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

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10 pages, 397 KB

Open AccessArticle

Transmission Dynamics of Gilts Persistently Infected with Atypical Porcine Pestivirus

by Alexandra C. Buckley, Bailey L. Arruda, Juan-Carlos Mora-Díaz, Ronaldo L. Magtoto, Luis G. Gimemez-Lirola and Shollie M. Falkenberg

Viruses 2026, 18(6), 590; https://doi.org/10.3390/v18060590 (registering DOI) - 23 May 2026

Abstract

Atypical porcine pestivirus (APPV) is a pestivirus that infects swine and has been associated with congenital tremor (CT) in piglets in the field, as well as those born to experimentally infected sows. However, APPV has also been detected in swine of various ages [...] Read more.

Atypical porcine pestivirus (APPV) is a pestivirus that infects swine and has been associated with congenital tremor (CT) in piglets in the field, as well as those born to experimentally infected sows. However, APPV has also been detected in swine of various ages without clinical signs. Experimental and field studies have demonstrated prolonged detection of viral RNA in serum, secretions, and tissues. For this work, nine gilts from a longitudinal APPV field study were selected based on birth in CT-positive litters and evidence of prolonged detection of virus in the sera. These six-month old animals were transported to a research facility for further sampling, breeding, and necropsy. The gilts were placed in contact with naïve animals for approximately one month at two different timepoints, prior to and during gestation, to assess transmission. After farrowing, litters were monitored for CT and tested for APPV. Following arrival, serum samples were PCR-negative for APPV; however, the gilts consistently tested positive in oral fluids and had detectable APPV in the cerebellum months later at necropsy. The gilts had a delayed humoral immune response, with the majority not seroconverting until approximately ten months of age. There were PCR-positive tissues and evidence of seroconversion in animals from both contact groups; however, none of the litters tested PCR-positive for APPV. These findings improve our understanding of the temporal dynamics and transmission potential of APPV infection and can help guide control measures to reduce viral spread. Full article

(This article belongs to the Section Animal Viruses)

18 pages, 5317 KB

Open AccessArticle

Molecular Characterization of H5N1 Clade 2.3.4.4B Virus in Vaccinated Layer Chickens

by Ahmed H. Salaheldin, Mustafa Ozan Atasoy, Juliane Lang, Ann Kathrin Ahrens, Anne Pohlmann, Mohammed A. Rohaim, Hatem S. Abd El-Hamid and Elsayed M. Abdelwhab

Viruses 2026, 18(6), 589; https://doi.org/10.3390/v18060589 - 22 May 2026

Abstract

The global emergence of the avian influenza virus (AIV) H5N1 clade 2.3.4.4B since 2016 has caused substantial losses in wild bird and poultry populations, along with heightened risks of transmission to humans and other mammals. Vaccination of poultry has been a key strategy [...] Read more.

The global emergence of the avian influenza virus (AIV) H5N1 clade 2.3.4.4B since 2016 has caused substantial losses in wild bird and poultry populations, along with heightened risks of transmission to humans and other mammals. Vaccination of poultry has been a key strategy to curb the virus’s spread and mitigate its socioeconomic impact. This report describes an outbreak of high pathogenicity avian influenza virus (HPAIV) H5N1 clade 2.3.4.4B in a flock of 15,000 brown layer chickens (170 days old), all of which had received a four-dose vaccination regimen with H5N1/H5N8 commercial vaccines at 17, 50, 100, and 125 days of age. Despite this vaccination history, H5N1 infection was confirmed approximately seven weeks post-vaccination. H5N1 infection was confirmed by RT-qPCR, virus isolation, and full genome sequencing covering all eight gene segments, followed by phylogenetic and molecular analyses. Clinical signs included reduced feed intake, decreased egg production, and a cumulative mortality rate of 35% over 52 days. Hemagglutination inhibition (HI) testing with various H5 antigens revealed inconsistent antibody titers (geometric mean: 4.0 to 9.1 log2). Genetic analysis of the full-length HA and NA gene sequences further revealed strong similarity to contemporaneous H5N1 clade 2.3.4.4B strains circulating in Egypt, with multiple mutations in the HA head domain, particularly near immunogenic epitopes and receptor binding sites. These findings highlight the limitations of current vaccination strategies under conditions of antigenic mismatch and complex immunization schedules, emphasizing the need for improved vaccine matching and continuous molecular surveillance. To improve outbreak management in poultry, enhanced vaccination protocols, stringent biosecurity measures, and rigorous monitoring practices are critical. Full article

(This article belongs to the Special Issue Molecular Epidemiology, Evolution, and Transmission of Avian Influenza Viruses: 2nd Edition)

23 pages, 3968 KB

Open AccessArticle

Optimizing HIV-1 Genotypic Resistance Testing for Low- and Middle-Income Countries: High-Impact HIV-1 Mutations Across WHO-Defined Scenarios

by Robert W. Shafer, Kaiming Tao, Tom Loosli, Ana Abecasis, Daniele Armenia, George Bwire, Ricardo Sobhie Diaz, Joseph Fokam, Amalia Giron, Seth Inzaule, Rami Kantor, Cissy Kityo, Roger D. Kouyos, Swarali Kurle, Anne-Genevieve Marcelin, Roger Paredes, Martine Peeters, Victor F. Pimentel, Jonathan M. Schapiro, Kim Steegen, Marco Vitoria, Annemarie M. Wensing, Neil Parkin and Michael R. Jordan Show full author list Hide full author list

Viruses 2026, 18(6), 588; https://doi.org/10.3390/v18060588 - 22 May 2026

Abstract

Introduction: Drug resistance testing may improve the management of people living with HIV (PLWH) in several scenarios in low- and middle-income countries (LMICs). To guide assay development, the WHO published a target product profile (TPP) outlining two priority use cases (scenarios) for genotypic [...] Read more.

Introduction: Drug resistance testing may improve the management of people living with HIV (PLWH) in several scenarios in low- and middle-income countries (LMICs). To guide assay development, the WHO published a target product profile (TPP) outlining two priority use cases (scenarios) for genotypic resistance testing: (1) PLWH with confirmed virological failure (VF) on an integrase strand transfer inhibitor (INSTI)-based regimen, such as tenofovir (TFV) disoproxil fumarate, lamivudine (3TC), and dolutegravir (DTG) and (2) heavily treated PLWH, including infants and young children, with confirmed VF after receiving multiple regimens including a boosted protease inhibitor (PI). An additional potential scenario includes PLWH testing positive for HIV-1 while on pre-exposure prophylaxis (PrEP). Methods: To identify drug-resistance mutations (DRMs) most likely to influence clinical management of PLWH in each WHO TPP scenarios and to inform development of assays that detect individual DRMs and the interpretation of sequence-based assays, we reviewed prevalence and in vitro susceptibility data on HIV-1 DRMs in the Stanford HIV Drug Resistance Database associated with the nucleoside RT inhibitor (NRTI), nonnucleoside RT inhibitor (NNRTI), PI, and INSTI classes and the capsid inhibitor lenacapavir. Results: In the first scenario, the most informative NRTI DRMs were K65R and M184V/I; and the most informative INSTI DRMs were G118R, N155H, Q148H/K/R, and R263K. In the second scenario, a broader spectrum of DRMs is likely to be clinically relevant, including additional NRTI DRMs, the PI DRMs associated with reduced susceptibility to darunavir, and the NNRTI DRMs associated with reduced susceptibility to etravirine and doravirine. In PLWH testing positive for HIV-1 despite PrEP, the most informative NRTI and INSTI DRMs overlap with those in the first scenario, together with the capsid DRMs reported in persons experiencing VF while receiving lenacapavir. Conclusions: As global ART programs increasingly rely on INSTI-based regimens, and as the number of heavily treated individuals and difficult-to-treat pediatric cases grows, many LMICs have begun introducing HIV drug resistance testing for patient management. Although sequence-based assays provide the most comprehensive information for managing individual PLWH, assays that detect individual DRMs are also likely to be highly useful in the three WHO TPP scenarios. Full article

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

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18 pages, 2192 KB

Open AccessArticle

Interactomics of SARS-CoV-2 Macrodomain 1 Reveals Putative Clients of ADP-Ribosyl Hydrolase Activity

by Crissey D. Cameron, Grace Heilmann, Brynn K. Roman and Lars Plate

Viruses 2026, 18(6), 587; https://doi.org/10.3390/v18060587 - 22 May 2026

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has greatly impacted public health due to high rates of transmissibility and mutation during the COVID-19 pandemic. Macrodomain 1 (Mac1) of non-structural protein 3 remained well conserved across variants and is critical to suppression of host [...] Read more.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has greatly impacted public health due to high rates of transmissibility and mutation during the COVID-19 pandemic. Macrodomain 1 (Mac1) of non-structural protein 3 remained well conserved across variants and is critical to suppression of host immune response to infection, making Mac1 a promising target for therapeutic development. Mac1 binds and cleaves the post-translational modification ADP-ribose and is hypothesized to have a downstream effect on the host interferon response, but the exact cellular targets of Mac1 are still unknown. Characterizing the substrates of Mac1 ADP-ribosyl hydrolase activity using a catalytically inactive mutant N40D can reveal critical virus–host interactions to identify protein targets of Mac1 and reveal mechanisms of host interferon suppression. Here, we performed affinity enrichment with WT Mac1 and Mac1 N40D in HEK293T and A549 cells and quantified changes in protein interactions by TMT-multiplexed tandem mass spectrometry. We identified interactions between Mac1 and ADP-ribosylated substrates involved in DNA damage response, cytoskeletal components, and cell cycle regulation. Additionally, several members of the TRiC complex involved in protein folding were selectively enriched with mutant Mac1 from A549 cells. These findings suggest a novel role of Mac1 in regulating host protein folding. Full article

(This article belongs to the Special Issue Coronavirus Pathogenesis and Virus-Host Interaction)

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14 pages, 1049 KB

Open AccessArticle

Temporal Trends in the Use of Healthcare Services for Respiratory Infections in the Paediatric Population of Anoia (2017–2024): Primary and Hospital Care

by María José Macías Reyes, Josep Vidal-Alaball, Laia Sola Reguant and Anna Ruiz-Comellas

Viruses 2026, 18(6), 586; https://doi.org/10.3390/v18060586 - 22 May 2026

Abstract

Respiratory infections are among the leading causes of healthcare consultations in paediatric populations. The SARS-CoV-2 pandemic significantly altered both the circulation of respiratory pathogens and the utilisation of healthcare services. This retrospective longitudinal observational study analysed temporal trends in consultations for respiratory infections [...] Read more.

Respiratory infections are among the leading causes of healthcare consultations in paediatric populations. The SARS-CoV-2 pandemic significantly altered both the circulation of respiratory pathogens and the utilisation of healthcare services. This retrospective longitudinal observational study analysed temporal trends in consultations for respiratory infections among children under 15 years of age in the Anoia region between 2017 and 2024. Descriptive analyses and time-series modelling using negative binomial regression were performed. A total of 71,918 consultations were recorded, of which 71.7% occurred in primary care and 28.9% in hospital settings. The mean age of patients was lower in the hospital setting (3.4 years) than in primary care (8.7 years). During the pandemic, consultations decreased by 38% compared with the pre-pandemic period, followed by a rebound in 2022, particularly in hospital care. In the post-pandemic period, hospital consultations remained above pre-pandemic levels, whereas primary care activity tended to stabilise. No increase in bronchiolitis consultations was observed compared with the pre-pandemic period. Full article

(This article belongs to the Special Issue Advances in Respiratory Viruses Research: From Basic Studies to Public Health)

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14 pages, 1848 KB

Open AccessArticle

Genomic Evidence for Novel Introduction and Intra-Host Diversity of DENV-2 in Dar es Salaam, Tanzania

by Silvan Hälg, Frank S. C. Tenywa, Nicole Liechti, Christian Beuret, Sarah J. Moore and Pie Müller

Viruses 2026, 18(5), 585; https://doi.org/10.3390/v18050585 - 21 May 2026

Abstract

Dengue virus (DENV) poses a growing risk in Tanzania, yet its genetic diversity in mosquito populations remains poorly understood. Using Nanopore sequencing, we recovered full coding sequences from six DENV-2 positive mosquito pools collected in Dar es Salaam outside recognized outbreak periods. Phylogenetic [...] Read more.

Dengue virus (DENV) poses a growing risk in Tanzania, yet its genetic diversity in mosquito populations remains poorly understood. Using Nanopore sequencing, we recovered full coding sequences from six DENV-2 positive mosquito pools collected in Dar es Salaam outside recognized outbreak periods. Phylogenetic analysis placed these sequences in a distinct monophyletic clade within genotype II, separate from strains linked to Tanzania’s 2014 outbreak. Instead, they clustered with Asian lineages and showed the closest relatedness to DENV-2 strains from Kenya (2013) and India (2014), with divergence estimated to have occurred around 2010. Variant profiling identified 212 low-frequency intra-pool variants, predominantly non-synonymous changes in the NS3, NS4B, and NS5 coding regions. These results suggest a previously unrecognized introduction of genotype II that is now circulating silently within local mosquito populations. Our findings highlight the value of genomic surveillance in mosquito vectors for early detection of arboviral threats, even in the absence of reported human cases. Full article

(This article belongs to the Special Issue Current Trends in Arbovirus Outbreaks and Research)

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18 pages, 17507 KB

Open AccessArticle

Infectome Landscape of Rodents and Shrews in Guangdong Province Reveals Diverse Pathogens with Zoonotic Potential in Wildlife

by Yukun Lin, Fenxiang Li, Peiyu Liang, Yangzi Zhou, Lihua Zhang, Wudi Zhou, Yufeng Liang, Ruolan Yu, Wei Yang, Zhijian Zhou, Zeliang Wei, Jian He, Jingzhe Jiang and Huacheng Yan

Viruses 2026, 18(5), 584; https://doi.org/10.3390/v18050584 - 21 May 2026

Abstract

Rodents and shrews are important reservoir hosts due to their close association with human activities and their role in carrying various zoonotic pathogens. Recently, meta-transcriptomic sequencing has become a powerful tool for surveilling and screening novel pathogens from wild animals. However, many of [...] Read more.

Rodents and shrews are important reservoir hosts due to their close association with human activities and their role in carrying various zoonotic pathogens. Recently, meta-transcriptomic sequencing has become a powerful tool for surveilling and screening novel pathogens from wild animals. However, many of these studies focused only on the diversity and genetic evolution of viruses from wildlife, while ignoring non-viral pathogens such as bacterial and eukaryotic microorganisms. Here, we performed a comprehensive infectome analysis of 227 tissue samples collected from 42 rodents and 16 shrews across six cities of Guangdong Province, China. We identified 34 viral families, including 23 mammalian viruses. Phylogenetic analysis revealed a henipavirus from the kidneys of shrews closely related to the Langya virus with potential infection risks to humans. Additionally, two potential pathogenic bacteria and 12 eukaryotic pathogens from six genera were found, showing clearer organ tropism than viruses. Interestingly, a moderate positive abundance correlation between Usmuvirus newyorkense and Trichinella suggested a potential virus–parasite association. We used machine learning models to evaluate the zoonotic potential of the obtained viruses, which indicated that 15 of 23 viral species were high risk for human infection. These findings provide important insight into the substantial zoonotic threat posed by pathogens circulating in wild small mammals in southern China and highlight the necessity for persistent wildlife pathogen surveillance. Full article

(This article belongs to the Section Animal Viruses)

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22 pages, 2354 KB

Open AccessArticle

Influence of Sampling Strategies and Disease Prevalence on SARS-CoV-2 Detection Dynamics in Wastewater Surveillance

by Siti Aishah Rashid, Mohd Ishtiaq Anasir, Fadly Syah Arsad, Nurul Farehah Shahrir, Khayri Azizi Kamel, Sakshaleni Rajendiran, Nurul Amalina Khairul Hasni, Mohamad Iqbal Mazeli, Yuvaneswary Veloo, Syahidiah Syed Abu Thahir, Wan Rozita Wan Mahiyuddin, Khor Bee Chin, Alijah Mohd Aris, Redzuan Zainudin, Rafiza Shaharudin and Raheel Nazakat

Viruses 2026, 18(5), 583; https://doi.org/10.3390/v18050583 - 21 May 2026

Abstract

Background: Wastewater-based surveillance (WBS) has emerged as a valuable tool for population-level monitoring of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) transmission, yet the interplay between sampling strategies and disease prevalence in shaping detection performance remains ambiguous. We investigated how grab and composite [...] Read more.

Background: Wastewater-based surveillance (WBS) has emerged as a valuable tool for population-level monitoring of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) transmission, yet the interplay between sampling strategies and disease prevalence in shaping detection performance remains ambiguous. We investigated how grab and composite sampling influence SARS-CoV-2 ribonucleic acid (RNA) detection dynamics and predictive lag times across high- and low-prevalence communities in Selangor, Malaysia. Methods: A 28-week longitudinal study was conducted in Selangor, Malaysia, comparing grab and composite wastewater sampling in communities with high and low Coronavirus disease 2019 (COVID-19) prevalence. SARS-CoV-2 RNA in 348 samples was quantified using digital Reverse Transcription Polymerase Chain Reaction (RT-dPCR), and viral lineages were characterized by Nanopore sequencing. Detection sensitivity and lead times relative to reported cases were evaluated. Results: In low-prevalence settings, grab sampling showed higher detection sensitivity than composite sampling (92.0% vs. 70.0%), whereas both methods achieved similarly high detection in high-prevalence areas (>97.0%). Lag-time analysis indicated that grab sampling in high-prevalence settings was significantly associated with case trends at potential two-week lead (p = 0.024), while composite sampling in low-prevalence settings showed the strongest association at a potential one-week lead (p = 0.0022). Overall, lag structures varied by both sampling strategy and prevalence context. Both sampling approaches captured the replacement of Omicron sublineages (XBB.1.5, XBB.1.9.1, XBB.1.16) and identified additional circulating variants, including EG.5, that were not captured in the available clinical sequencing dataset during the same period. Conclusions: These findings reveal that local transmission intensity is associated with the utility of different sampling designs. Context-specific optimization of WBS sampling strategies enhances sensitivity, reduces detection lag, and strengthens early warning and genomic-tracking capacity in public health surveillance frameworks. Full article

(This article belongs to the Special Issue Wastewater-Based Epidemiology and Viral Surveillance)

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9 pages, 405 KB

Open AccessArticle

Study on the Protective Efficacy of the Japanese Encephalitis Live Attenuated Vaccine SA14-14-2 Against Newly Isolated Genotype I Japanese Encephalitis Viruses

by Shuai Shang, Qikai Yin, Tingyi Che, Xinzhu Wang, Qi Su, Shihong Fu, Hongshan Xu, Yongxin Yu, Qunying Mao, Huanyu Wang and Xinyu Liu

Viruses 2026, 18(5), 582; https://doi.org/10.3390/v18050582 - 21 May 2026

Abstract

Japanese encephalitis virus (JEV) comprises a single serotype but can be classified into five genotypes (genotypes I–V, GI–GV) based on nucleic acid sequences. Historically, genotype III (GIII) was the predominant strain. However, since the 21st century, genotype I (GI) rapidly replaced GIII as [...] Read more.

Japanese encephalitis virus (JEV) comprises a single serotype but can be classified into five genotypes (genotypes I–V, GI–GV) based on nucleic acid sequences. Historically, genotype III (GIII) was the predominant strain. However, since the 21st century, genotype I (GI) rapidly replaced GIII as the major genotype in China, Southeast Asia, and other regions. The live attenuated vaccine (LAV) SA14-14-2, licensed in China in 1988, was successfully exported to 13 countries, with cumulative vaccinations exceeding one billion doses. The vaccine seed virus SA14-14-2 belonged to genotype III. Whether this GIII-based vaccine provided sufficient protection against the currently circulating GI strains warranted systematic investigation. In this study, recent JEV isolates collected from China were subjected to genotypic analysis, followed by comprehensive evaluations including protective efficacy against challenge and serum neutralizing antibody levels. The results indicated that, despite antigenic differences between GIII and GI strains, no significant differences in protective efficacy post-challenge were observed. The SA14-14-2 LAV remained effective in preventing GI strain infection. Full article

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

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28 pages, 1121 KB

Open AccessArticle

Comparative Evaluation of Polymeric Nanocarriers for DNA Vaccine Delivery Against Avian Orthoavulavirus 1 in Chickens

by Ahmed H. Khattab, Mahmoud Bayoumi, Zienab E. Eldin, Basem M. Ahmed and Haitham M. Amer

Viruses 2026, 18(5), 581; https://doi.org/10.3390/v18050581 - 21 May 2026

Abstract

Vaccination represents the cornerstone of Newcastle disease control. Nanotechnology offers a promising approach to improve the effectiveness of DNA vaccines, supporting their use as an alternative to conventional platforms. Herein, the Avian Orthoavulavirus 1 (AOAV-1) fusion (F) gene was cloned into [...] Read more.

Vaccination represents the cornerstone of Newcastle disease control. Nanotechnology offers a promising approach to improve the effectiveness of DNA vaccines, supporting their use as an alternative to conventional platforms. Herein, the Avian Orthoavulavirus 1 (AOAV-1) fusion (F) gene was cloned into a DNA expression plasmid (pDNA). After validating the constructed pDNA-F and confirming robust intracellular protein expression in vitro, three polymeric nanoparticles (NPs)-based formulations were generated using Chitosan (Cs), poly(lactic-co-glycolic) (PLGA), and poly(amidoamine) (PAMAM)-Dendrimers. Physicochemical characterization, stability assessment, and in vitro release analysis confirmed nanoparticle formation and effective DNA incorporation. In vivo experiments were conducted to comparatively evaluate the immunogenicity, particularly the immune priming capacity, and protective efficacy of nanoparticle-based formulations and naked pDNA-F, all tested in parallel at standardized pDNA doses via intranasal (IN) and intramuscular routes. PAMAM-Dendrimers-pDNA-F IM group demonstrated superior efficacy, with 100% survival, the highest post-challenge anamnestic antibody titers, and a pronounced reduction in viral RNA shedding. PLGA-NPs-pDNA-F IN group demonstrated enhanced efficacy, with 90% survival. Naked pDNA-F surpassed the Cs-NPs-pDNA-F in both immune priming and clinical protection, with Cs-NPs-pDNA-F exhibiting the lowest overall performance. These findings highlight that DNA vaccine performance depends on both carrier type and administration route, with PAMAM dendrimers and PLGA enhancing efficacy, whereas chitosan demonstrated reduced efficacy under the tested conditions. Full article

(This article belongs to the Section Animal Viruses)

24 pages, 1716 KB

Open AccessArticle

Whole Blood Volume-Based Absolute Quantification of HTLV-1 Proviral Load: A Comparative Method Evaluation Study

by Gabriel O. Franco, Andreas Stocker, Eduardo M. Netto, Heliene Pereira and Carlos Brites

Viruses 2026, 18(5), 580; https://doi.org/10.3390/v18050580 - 21 May 2026

Abstract

The proviral load of human T-cell lymphotropic virus type 1 (HTLV-1) is an important biomarker associated with the monitoring and risk stratification of adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). However, the lack of standardized quantification methods limits its broader [...] Read more.

The proviral load of human T-cell lymphotropic virus type 1 (HTLV-1) is an important biomarker associated with the monitoring and risk stratification of adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). However, the lack of standardized quantification methods limits its broader application. This study evaluated a novel absolute quantification approach based on whole blood volume and compared its performance with established protocols. A total of 66 HTLV-1-infected individuals were analyzed using six qPCR-based methodologies, including volumetric quantification (copies/µL) by absolute quantification and the Tamegão-Lopes method, as well as normalization per 1000 cells (whole blood, buffy coat, PBMCs, and CD4+ T cells). Association and agreement were assessed using Pearson’s correlation, Bland–Altman analysis, concordance correlation coefficients (CCCs), and Deming regression. Absolute quantification showed strong correlation with both the Tamegão-Lopes method and CD4+-based quantification (r = 0.93 and 0.84, respectively; p < 0.001) and high agreement (CCC = 0.866 and 0.811, respectively), with modest systematic bias (−0.273 log₁₀ copies/µL and 0.115 log₁₀ copies/10³ cells, respectively). Leukocyte-normalized methods showed greater discrepancies, likely due to dilution by uninfected cells. These findings show that quantification based on total blood volume is a simplified, operationally feasible alternative for assessing HTLV-1 proviral load. Full article

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

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19 pages, 2678 KB

Open AccessArticle

Aerosol Inhalation of a Recombinant H7N9 Hemagglutinin Antigen Elicits Systemic and Mucosal Immune Responses in Mice

by Zhuoran Hou, Han Wang, Bin Zhang, Ruixi Liu, Yuli Zhang, Ye Yang, Jianxin Wu, Xuchen Hou, Xiuguo Ge, Jun Wu and Bo Liu

Viruses 2026, 18(5), 579; https://doi.org/10.3390/v18050579 - 21 May 2026

Abstract

Highly pathogenic avian influenza A (H7N9) remains a threat to poultry health and poses a zoonotic risk, highlighting the need for vaccine antigens capable of inducing both systemic and mucosal immunity. In this study, we evaluated X33CLS-H7, a clarified cell-lysate supernatant derived from [...] Read more.

Highly pathogenic avian influenza A (H7N9) remains a threat to poultry health and poses a zoonotic risk, highlighting the need for vaccine antigens capable of inducing both systemic and mucosal immunity. In this study, we evaluated X33CLS-H7, a clarified cell-lysate supernatant derived from glycoengineered Pichia pastoris expressing H7 hemagglutinin, in BALB/c mice following intramuscular(i.m.) injection, nebulized inhalation, or intranasal instillation. H7 expression and hemagglutination activity were confirmed by Western blotting and hemagglutination assay, respectively. Serum HA7-specific IgG and IgA responses, hemagglutination inhibition(HI) activity, H7N9 pseudovirus neutralization, bronchoalveolar lavage fluid (BALF) antibodies, and safety readouts were assessed. After two i.m. immunizations, X33CLS-H7 induced the strongest systemic antibody responses, with an HI geometric mean titer of 1:1622 95% CI, 1:1108–1:2348 and a mean log10 NT₅₀ of 4.62. Respiratory immunization also elicited antibody responses. After four doses, high-dose nebulized delivery produced the strongest responses among the respiratory delivery regimens, with serum IgG and IgA titers of 1.02 × 10⁵ and 2.24 × 10³, respectively, an endpoint HI GMT r of 1:457 95% CI, 1:211–1:971, and a mean log10 NT₅₀ of 3.77 compared with 2.02 in saline controls. High-dose nebulized delivery also generated detectable HA7-specific IgG and IgA responses in bronchoalveolar lavage fluid. No overt local or systemic toxicity signals were observed under the tested conditions. These findings indicate that X33CLS-H7 retains HA7-associated antigenicity and can induce systemic and respiratory mucosal antibody responses, supporting its further evaluation as a simplified and scalable H7N9 vaccine antigen candidate. Full article

(This article belongs to the Special Issue Animal Models in Emerging/Re-Emerging Infectious Diseases)

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17 pages, 1028 KB

Open AccessArticle

Validated Quantification of HHV-8 DNA Using Inter-Convertible Plasmid and Cell-Derived Calibrators: Optimization of a Whole-Blood qPCR Assay

by Celeste Luján Pérez, Carlos Ochoa Gamboa, Mónica Tous, Julián Hazan, Marcelo Rodríguez, Daniela Feliciotti, Lucía Irazu and Carlos Zala

Viruses 2026, 18(5), 578; https://doi.org/10.3390/v18050578 - 21 May 2026

Abstract

Human herpesvirus 8 (HHV-8) is the etiologic agent of Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), multicentric Castleman disease (MCD), and KS-associated immune reconstitution inflammatory syndrome (IRIS-KS). Quantifying HHV-8 DNA in whole blood is clinically relevant, yet laboratory practices remain heterogeneous. Here, we [...] Read more.

Human herpesvirus 8 (HHV-8) is the etiologic agent of Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), multicentric Castleman disease (MCD), and KS-associated immune reconstitution inflammatory syndrome (IRIS-KS). Quantifying HHV-8 DNA in whole blood is clinically relevant, yet laboratory practices remain heterogeneous. Here, we developed and validated an in-house quantitative PCR (qPCR) assay targeting ORF26, optimized for whole blood. Assay calibration used plasmid, BCBL-1 cell–derived, and commercial HHV-8 DNA standards. Analytical validation was performed following the Clinical and Laboratory Standards Institute (CLSI) guidelines and the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines and showed a 95% limit of detection of 65.7 copies/reaction, efficiencies of 90–101% (R² > 0.99), and intra/inter-assay coefficients of variation < 6.5%. Strong correlations were observed among the three calibrators (R² > 0.97).Clinical validation against a composite reference yielded 100% sensitivity, specificity, PPV, and NPV. Viral loads (log₁₀ copies/mL) varied by clinical condition: classic KS and transplant-associated KS showed the lowest medians (2.30–2.23), MCD HIV− and PEL intermediate values (2.83–3.72), and epidemic KS, MCD HIV+, and IRIS-KS the highest (4.12, 4.86, and 5.03, respectively). Viremia > 5 log₁₀ copies/mL was associated with uncontrolled E-KS, MCD HIV+, and IRIS-KS. Longitudinal follow-up revealed viral load decline paralleled clinical improvement. This validated assay provides a robust, affordable tool for HHV-8 quantification in whole blood and supports its integration into diagnostic workflows and patient monitoring. Full article

(This article belongs to the Special Issue Herpesviruses and Associated Diseases, 2nd Edition)

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16 pages, 4259 KB

Open AccessArticle

The Melon Sterol Transporter Niemann-Pick C1 Protein Is a New Interactor of Cucumber mosaic virus Movement Protein

by Núria Real, Irene Villar, Bin Liu, Manale Gajjout, Weina Hou and Ana Montserrat Martín-Hernández

Viruses 2026, 18(5), 577; https://doi.org/10.3390/v18050577 - 20 May 2026

Abstract

Plant viruses need to use many host factors to establish infection. During the viral cycle, intracellular transport is fundamental to reach the plasmodesmata to enable cell-to-cell transport. Cucumovirus CMV (cucumber mosaic virus, CMV) can infect plants from most economically important crops. To identify [...] Read more.

Plant viruses need to use many host factors to establish infection. During the viral cycle, intracellular transport is fundamental to reach the plasmodesmata to enable cell-to-cell transport. Cucumovirus CMV (cucumber mosaic virus, CMV) can infect plants from most economically important crops. To identify additional host proteins involved in CMV movement in melon, we used the MP as a bait to screen a Yeast two-hybrid cDNA library from CMV-infected plants and identified a Niemann-Pick C1 (NPC1) protein as a novel MP interactor. NPC1 is a transmembrane protein involved in cholesterol transport in animal cells, but also in the infection by several viruses of different families. The identified clone from the melon NPC1 gene spans from exons 25 to 28 and includes two introns. Notably, deletion of the two introns and exon 28 does not impair the interaction capacity of the remaining peptide. The identified CmNPC1 gene maps to chromosome 11. In addition, the melon genome encodes a second copy of NPC1 in chromosome 7 (CmNPC1-C7), highly similar. Functional assays revealed that the interaction domain of CmNPC1-C7 also interacts with CMV MP, suggesting that both genes could have a role in CMV infection. This study represents the first report linking NPC1 to the infection process of a plant virus, expanding our understanding of plant–virus interactions. Full article

(This article belongs to the Special Issue Plant Virus Resistance—2nd Edition)

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11 pages, 1615 KB

Open AccessData Descriptor

From Discovery to Cure—Where Are We Now? Mortality Trends in Chronic Hepatitis C: An Analysis of CDC WONDER Database (1999–2023)

by Ashraf Ullah, Hina Wazir, Abdullah Sultany, Khalil Ur Rehman, Mohammad Ibrahim Sultani, Naeem Ahmed Khan, Saeed A. Khan, Mati Ullah Dad Ullah and Amlish Gondal

Viruses 2026, 18(5), 576; https://doi.org/10.3390/v18050576 - 20 May 2026

Abstract

Background: Hepatitis C virus (HCV) remains a major cause of preventable liver-related mortality in the United States despite highly effective direct-acting antivirals (DAAs). Contemporary assessment of mortality trends and disparities is essential for elimination efforts. Methods: Using CDC WONDER multiple cause-of-death data (1999–2023), [...] Read more.

Background: Hepatitis C virus (HCV) remains a major cause of preventable liver-related mortality in the United States despite highly effective direct-acting antivirals (DAAs). Contemporary assessment of mortality trends and disparities is essential for elimination efforts. Methods: Using CDC WONDER multiple cause-of-death data (1999–2023), we identified HCV-related deaths using ICD-10 codes for acute and chronic HCV (B17.1, B18.2) and calculated age-adjusted mortality rates (AAMRs) per 100,000 (2000 US standard). Rates were stratified by sex, race/ethnicity, census region, and 2013 NCHS urban–rural classification. Joinpoint regression quantified temporal inflection points and annual percent changes (APCs). Results: Overall HCV-related AAMR increased from 1.8 (1999) to a peak of 5.0 (2014), then declined to 2.3 (2023), with a marked post-2014 decrease (APC −8.2%). Mortality was consistently higher in males than females (2023 rate ratio 2.57). In 2023, American Indian/Alaska Native individuals had the highest mortality (AAMR 8.7; rate ratio 3.48 vs. non-Hispanic White), followed by non-Hispanic Black individuals (AAMR 6.2; rate ratio 2.48). Mortality remained highest in the West and was higher in non-metropolitan than metropolitan counties (AAMR 2.8 vs. 2.3; rate ratio 1.22), with a slower post-2014 decline in non-metropolitan areas. Conclusions: Our findings indicate that while the DAA era has been associated with a substantial reduction in HCV-related mortality at the national level, this progress has not been uniform across all populations. Persistent excess mortality among Native American and non-Hispanic Black individuals may reflect inequities in the HCV care cascade, including screening, confirmatory testing, linkage to specialty care, insurance-related restrictions, and the high cost of antiviral therapy. These results highlight the need for policies and public health strategies that improve equitable and affordable access to curative HCV treatment. Full article

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

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16 pages, 4424 KB

Open AccessArticle

USP17L13 Enhances Influenza a Virus Replication by Mediating the Degradation of RIG-I and MDA5

by Yaping Zhang, Chen Qin, Yichao Zhuang, Lei Chen, Xianying Zeng, Li Jiang, Chengjun Li, Hualan Chen and Huihui Kong

Viruses 2026, 18(5), 575; https://doi.org/10.3390/v18050575 - 20 May 2026

Abstract

The innate immune system, particularly the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) signaling pathway, is a major early defense barrier against influenza A virus infection. However, excessive immune responses can trigger lethal cytokine storms and severe immune-mediated pathology. In this study, we [...] Read more.

The innate immune system, particularly the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) signaling pathway, is a major early defense barrier against influenza A virus infection. However, excessive immune responses can trigger lethal cytokine storms and severe immune-mediated pathology. In this study, we performed a genome-wide CRISPR/dCas9 gene activation screen in human lung epithelial (A549) cells by using an A/Puerto Rico/8/1934 (H1N1) reporter virus, and identified the ubiquitin-specific protease USP17L13 as a novel negative regulator of innate immunity that promotes influenza virus replication. Overexpression of USP17L13 significantly enhanced the replication of multiple subtypes of influenza viruses in A549 cells, including a human pandemic H1N1 virus, seasonal H3N2 viruses, as well as a globally circulating clade, 2.3.4.4b, of the highly pathogenic avian H5N1 virus. Transcriptomic analysis demonstrated that USP17L13 suppresses host antiviral defenses by downregulating nuclear factor kappa B (NF-κB) signaling and arachidonic acid metabolism, while upregulating pathways associated with ribosomal translation and oxidative phosphorylation to facilitate viral production. Mechanistically, USP17L13 attenuates the host interferon (IFN) response by promoting the degradation of the key viral RNA sensors, RIG-I, and melanoma differentiation-associated protein 5 (MDA5). Further analysis revealed that USP17L13 is inducible by type I and type II interferons as well as inflammatory cytokines, suggesting that it may act as a negative-feedback regulator to limit excessive inflammation. Collectively, our findings identify USP17L13 as a previously unrecognized proviral host factor and provide new insight into how host deubiquitinases shape influenza virus-host interactions, with potential implications for host-directed approaches to controlling excessive inflammation during viral infection and improving influenza vaccine production. Full article

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

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