Viruses doi: 10.3390/v18101135

Authors: Min Zhu Zhou Sun Ke Xu Ling Ye

Human immunodeficiency virus type 1 (HIV-1) replication depends on a complex interplay between viral proteins and host cellular factors, which represent promising therapeutic targets. However, systematic integration of results from genome-wide CRISPR screens for HIV host factors remains limited. Here, we performed a systematic review and meta-analysis of pooled genome-wide CRISPR-Cas9 screens to identify host dependency, restriction, and latency-promoting factors in HIV-1 infection. Following PRISMA guidelines, we identified 11 eligible studies from which we extracted 26 gene lists. These were classified into three phenotypic categories, including dependency (5 lists), restriction (10 lists), and latency-promoting (7 lists), and integrated using meta-analysis by information content (MAIC). GO over-representation analysis and Gene Set Enrichment Analysis were performed on MAIC-ranked gene lists to characterize pathway architectures. MAIC successfully recovered well-established host factors, including the entry receptors CD4, CCR5, and CXCR4; the integration cofactor PSIP1 (LEDGF/p75); and the restriction factors TRIM5, TRIM22, PML, G3BP1, and EIF2AK2. RNF114 emerged as the top-ranked restriction factor, supported by four independent publication-level categories. Additional high-confidence hits included TRIM56, BIRC3, DTX3L, and components of the ubiquitin–proteasome system across the dependency and latency datasets, further supporting the robustness of the MAIC framework. Beyond these canonical factors, we identified multiple undercharacterized genes and pathways, including coagulation and IL-17 signaling in dependency factors, SUMOylation and DNA damage response in restriction factors, and the Integrator complex and proteasome-granule co-enrichment in latency-promoting factors. Leading-edge analysis confirmed that most of these non-canonical enrichments are driven by coherent gene modules rather than a small set of pleiotropic hubs. These findings reveal distinct pathway architectures across the three infection states and highlight previously unrecognized host processes that may serve as targets for antiviral therapy. Our analysis provides a comprehensive resource for guiding future mechanistic studies of host-HIV interactions and developing strategies to enhance immunity, block viral replication, and reverse latency.

Viruses doi: 10.3390/v18101134

Authors: Aleksandra Majewska Klara Weronika Siejda Maciej Kowalec Karolina Aleksandrowicz Yuliia Panchuk Małgorzata Marta Rogala Karolina Pianka Aleksandra Minich Weronika Jarocka Maciej Walędziak Katarzyna Komorowska Andrzej Kwiatkowski Sylwia Rydzik Tomasz Syryło Agnieszka Bajek Michał Wiłkojć Leszek Gryszko Grzegorz Suwalski Jakub Mróz Michał Zembala Jacek Doniec Klaudia Klarysa Brodaczewska Leszek Lisowski

Vectors based on adeno-associated viruses (AAVs) are widely used as a delivery system in gene therapy. However, preexisting immunity against AAV remains a major limitation. This study examines the prevalence of AAV genomes and anti-AAV2 immunity in a Polish cohort of adult patients undergoing planned surgical interventions, which serves as a random sample of the general Polish population. The presence of AAV capsid sequences was analyzed in tissue samples using PCR and endogenous AAVs were detected in 33.95% of samples. Anti-AAV2 IgG antibodies were measured by a single point ELISA and were present in 80.93% of patients. Neutralizing activity was evaluated using an AAV2 transduction inhibition assay in HEK293T cells, revealing a 60% reduction in functional activity by patients’ sera. Tissue AAV PCR-positivity was associated with stronger neutralizing activity although the correlation with seropositivity was not as strong. These results suggest that pre-existing humoral immunity against AAV is common in the Polish adult population, indicating frequent exposure to naturally occurring AAVs. The findings provide important data on pre-existing anti-AAV immunity—a key factor considered in clinical trials and gene therapy development. Furthermore, the identification of naturally occurring AAV variants in human tissues points to the potential use of new sources of capsids with potentially distinct biological properties, which could contribute to the advancement of gene therapies.

Viruses doi: 10.3390/v18101133

Authors: Jian Zhang Jason Murray Wenli Mu Surya Venugopal Ella Yim Teresa Einhaus Stefan Radtke Christopher W. Peterson Hans-Peter Kiem Dong Sung An

Despite significant medical progress, HIV-1 remains a critical global health crisis. While combination antiretroviral therapy (cART) effectively suppresses viremia, it is not curative due to the persistence of latent viral reservoirs. Autologous hematopoietic stem and progenitor cell (HSPC) gene therapy offers a promising avenue toward sustained, drug-free remission. In this study, we investigated the feasibility and safety of a multifunctional “defend and attack” HSPC gene therapy strategy in the pigtail macaque (Macaca nemestrina) model. The species-optimized lentiviral vector, PM-MNDU-anti-HIV-1-huEGFRt, carries four therapeutic payloads: a pigtail macaque-specific CCR5 shRNA (PM-CCR5sh1005), a CD4-based chimeric antigen receptor (D1D2CAR), a C46 fusion inhibitor peptide, and a truncated human epidermal growth factor receptor (huEGFRt) serving as a cell-surface tracking marker and anti-EGFR antibody Cetuximab (CTX)-mediated negative selection marker. Following busulfan conditioning, autologous transplantation of vector-modified HSPCs in two pigtail macaques resulted in safe hematopoietic recovery. Both animals achieved sustained, multilineage gene marking in peripheral blood and lymphoid tissues without adverse effects. In one animal, upon Simian–Human Immunodeficiency Virus (SHIV) challenge, vector-modified cells underwent robust, antigen-driven multilineage expansion across both lymphoid and myeloid compartments, which contracted during cART-induced viral suppression and rapidly rebounded following treatment interruption. Administration of CTX demonstrated partial, transient reduction in huEGFRt+ cells, accompanied with a reciprocal increase in plasma viral load. These findings support the feasibility of evaluating a multi-pronged anti-HIV HSPC gene therapy strategy with a negative selection marker in a non-human primate (NHP) model.

Viruses doi: 10.3390/v18101132

Authors: Cheng-An Lyu Emma F. Harding Zhaobo Zhang Yao Shen Aris Katzourakis Peijun Zhang

Endogenous retroviruses (ERVs) are the molecular remnants of ancient retroviral infections and constitute a substantial fraction of many vertebrate genomes. Although most ERV loci have lost coding capacity and are epigenetically silenced, some remain biologically relevant because they have been co-opted for host physiology. Recent advances in structural biology are beginning to clarify how these elements have evolved from infectious retroviruses into genomic components with diverse molecular and cellular functions. In this review, we organise current structural insights by retroviral functional modules: group-specific antigen (Gag), polymerase (Pol) and envelope (Env). In the Gag section, we describe capsid-related assemblies of human endogenous retrovirus K (HERV-K), including mature-like CA assemblies reconstituted in vitro and immature lattices resolved in intact virus-like particles, as well as structural studies of the endogenous lentiviruses RELIK and PSIV. In the Pol section, we discuss HERV-K reverse transcriptase, biochemical studies of protease, and emerging structural evidence for integrase. In the Env section, we review prefusion and postfusion structures of HERV-K Env and receptor-bound structures of domesticated Env proteins, including syncytin-1, syncytin-2 and suppressyn. Together, these studies show how structural approaches can connect ERV evolution with molecular function, while also defining major unresolved questions regarding conformational dynamics, host interactions, and physiological relevance.

Viruses doi: 10.3390/v18101131

Authors: Michele Ghidini Giuseppe Sberna Luigi Pio Guerrera Fabrizio Maggi Federica Novazzi

The gastrointestinal tract contains a dense multi-kingdom ecosystem in which bacteria, fungi, archaea, viruses, the mucosa, and the immune system interact. Although cancer microbiome research has focused mainly on bacteria, the gut virome dominated by bacteriophages may influence gastrointestinal carcinogenesis by reshaping bacterial communities, transferring functional genes, modulating inflammation, and altering treatment response. The strongest evidence comes from colorectal cancer, where multiple cohorts show altered phage diversity, temperate-phage enrichment, disrupted phage–bacteria networks, and reproducible stool-based diagnostic signals. Emerging studies in pancreatic cancer, esophageal adenocarcinoma, hepatocellular carcinoma, and precursor lesions suggest that virome disruption extends beyond colorectal cancer, although evidence remains uneven. Classic oncogenic viruses, including Epstein–Barr virus and hepatitis viruses, provide complementary examples of direct viral carcinogenesis in digestive organs. Anelloviruses are widespread in healthy individuals and are not established causes of cancer, but their abundance and composition may reflect immune status, inflammation, tissue injury, and altered host viral ecology. Virome-related interventions remain clinically immature: fecal microbiota transplantation has reached small phase I/II studies in gastrointestinal cancers, whereas phage, fecal-filtrate, and fecal virome approaches remain preclinical or non-oncology; direct oncolytic viruses have reached late-phase testing. Major barriers include viral dark matter, specimen and assay heterogeneity, disease-related confounding, limited external validation, and weak causal evidence. Standardized, mechanistically anchored, multi-kingdom studies are needed to establish clinical utility.

Viruses doi: 10.3390/v18101130

Authors: Laura Morais Nascimento Silva Andreina de Carvalho Araujo Talita Dayane Pereira e Silva Andrew Y. Cho Juliana A. Sinhorini Luiz Alfredo Martins Lopes Baptista Terezinha Knöbl Guilherme Pereira Scagion Bruna Larotonda Telezynski Lopes Larissa Mayumi Bueno Luciano Matsumiya Thomazelli Alessandra Greatti Meriane Demoliner Micheli Fillipi Vyctoria Malayhka de Abreu Góes Pereira Jansen de Araújo Sun-Hak Lee Clarice Weis Arns Edison Luiz Durigon Maria Clorinda Soares Fioravante Fernando Rosado Spilki Dong-Hun Lee Kiril Dimitrov Helena Lage Ferreira

Pigeon paramyxovirus 1 (PPMV-1) comprises pigeon-associated variants of Newcastle disease virus (NDV), predominantly belonging to genotypes VI, XX, and XXI. These viruses have been detected in feral pigeons in Brazil since at least 2014, and an NDV outbreak associated with a pigeon-origin genotype VI virus was reported in commercial broilers in 2024. Here, we characterized two PPMV-1 strains detected in pigeons samples from geographically distinct regions of Brazil. Virus BR/105/21 was detected in Midwestern Brazil, whereas BR/34/22 was detected in Southeastern Brazil. Genome sequencing yielded a complete genome sequence for BR/105/21 (15,192 nt; 100% reference-genome coverage; mean depth, 3334×) and a near-complete genome sequence for BR/34/22 (15,133 nt; 99.5% reference-genome coverage; mean depth, 20.8×). Both viruses possessed the F-protein cleavage-site motif ^113RQKR↓F^117, which is consistent with the molecular criterion for virulent NDV. Phylogenetic analysis based on the complete F-gene coding sequence grouped both viruses with PPMV-1 previously detected in Brazil between 2014 and 2019. Analysis of VI.2.1.2 sequences identified three well-supported phylogenetic groups with between-group nucleotide distances of 6.1–8.0%. Based on established NDV classification criteria, two groups are proposed as VI.2.1.2.1, comprising viruses from Nigeria detected between 2007 and 2015, and VI.2.1.2.2, comprising Brazilian viruses detected between 2014 and 2022 and one virus detected in Argentina in 1997. These findings expand the available genetic data for pigeon-associated NDV in South America and reveal substantial genetic heterogeneity and phylogenetic structure within VI.2.1.2, reinforcing the importance of genomic surveillance of NDV in pigeons.

Viruses doi: 10.3390/v18101129

Authors: Andrea D. Ordoñez Edward B. Chuong

Transposable elements (TEs) occupy roughly half of the genomic content across mammals and have long been recognized for their contributions to immune gene regulation as co-opted enhancers and promoters. Less appreciated is their role in diversifying the immune transcriptome itself. TE sequences embedded in introns and gene-flanking regions can provide alternative splice sites, transcription start sites, and polyadenylation signals that generate structurally distinct isoforms of host immune genes. Here we review the mechanisms by which TEs contribute to alternative transcription and the cases with experimental evidence that TE-derived isoforms introduce alternative regulation or function to immune signaling pathways. We give particular attention to the species-specific nature of these events, as each vertebrate lineage carries a distinct TE repertoire and elements shared between species are not always used in the same way, making TE exonization a source of immune regulatory variation both across and within species. These events also contribute to cancer, where epigenetic dysregulation can aberrantly activate TE-derived transcripts with potential as biomarkers and therapeutic targets. Finally, we discuss how advances in long-read sequencing, bioinformatic tools, and proteomics are enabling more systematic characterization of TE-derived isoforms, and outline priorities for cataloguing the full scope of TE contributions to immune transcript diversity.

Viruses doi: 10.3390/v18101128

Authors: Igor Babkin Viktor Panov Sergey Tkachev Valeria Fedorets Nina Tikunova

CRESS DNA viruses belong to a widely distributed and highly diverse group of viruses, which are often found during the analysis of viromes. However, data on their genetic diversity are still incomplete. The intestinal viromes of widespread vole species—northern red-backed vole and tundra vole—were sequenced. Based on the Rep protein sequence, 22 sequences of various CRESS DNA viruses and alphasatellites were identified; 14 of them were complete genomes. According to the genome organization ten isolates were assigned to CRESS DNA viruses of known families of the Cressdnaviricota phylum, and one isolate to the Alphasatellitidae family. Three complete genomes were unique and possibly belong to new viral families. Most sequences were assigned to the Vilyaviridae family of the Cirlivirales order. An unusual genome from the clade of regular CRESS DNA viruses but without the cp gene was identified, which suggests that it is a possible alphasatellite. This fact raises the question of revising the current paradigm of dividing Circular Rep-Encoding DNA sequences into viruses and alphasatellites. Two pairs of similar CRESS DNA genomes were detected in different rodent species, which indicates the need for further study of diversity and host specificity of CRESS DNA viruses.

Viruses doi: 10.3390/v18101127

Authors: Jiecai Li Xin Huang Wenfang Luo Junhui Zhou Xiang Zhang Siqiong Tang Wei He Jianjun Xu

Viral diseases threaten protected tomato production in Xinjiang, China, yet the virus spectrum in symptomatic crops and the local sequence context of Tomato brown rugose fruit virus (ToBRFV) remain incompletely characterized. We used pooled small-RNA sequencing for virus discovery and individual reverse transcription PCR (RT-PCR)/Sanger confirmation in 116 symptomatic tomato samples and compared nine Xinjiang ToBRFV coding-region genomes with 435 curated public GenBank sequences. Nine viruses were identified in the symptomatic tomato samples, with ToBRFV being the most frequently detected (113/116, 97.4%). Virus co-detections occurred in 115 samples (99.1%), with four-virus combinations being the most common. In the 444-sequence ToBRFV coding-region dataset, the nine Xinjiang genomes obtained in this study represented distinct exact coding-sequence types and occupied multiple phylogenetic placements; 139 biallelic segregating sites were observed among them. These results document complex multi-virus co-detection in symptomatic protected-tomato samples and provide a local ToBRFV sequence resource. Because sampling was symptom-targeted and the genome set was small, representative sampling and epidemiological metadata are required to estimate regional prevalence, ToBRFV diversity and transmission history.

Viruses doi: 10.3390/v18101126

Authors: Svetlana V. Svyatchenko Natalia I. Goncharova Ivan M. Susloparov Natalia N. Vasiltsova Natalia P. Kolosova Marina L. Egorova Tatyana V. Tregubchak Natalia V. Danilchenko Tatyana N. Ilyicheva Vasiliy Y. Marchenko

In this study, we aimed to analyze the antiviral susceptibility of clade 2.3.4.4b H5N1 and H5N5 influenza viruses detected in Russia during 2021–2022. During this period, we investigated 40 avian influenza outbreaks in wild and domestic birds, isolating 109 H5N1 and 9 H5N5 strains. Using a genetic approach, we analyzed the presence of markers associated with reduced susceptibility to baloxavir and M2 blockers, as well as markers of reduced inhibition by neuraminidase inhibitors (NAIs). Additionally, the isolated strains were tested in a functional assay to evaluate the inhibitory activity of oseltamivir, zanamivir, and peramivir against viral neuraminidase. None of the isolates harbored genetic markers of reduced susceptibility to M2 blockers and baloxavir or reduced inhibition by NAIs. Functional assay showed that the median IC50 values of oseltamivir, zanamivir, and peramivir were 1.73, 0.49, and 0.16 nM for the H5N1 subtype, and 0.65, 1.13, and 0.12 nM for the H5N5 subtype, respectively. None of the tested strains demonstrated a ≥10-fold increase in NAI IC50 values compared to the corresponding subtype medians, and were thus classified as having a normal level of inhibition. The median IC50 values of the NAIs determined for the tested avian viruses were comparable to those of a susceptible seasonal A(H1N1)pdm09 strain, except for a slight reduction in oseltamivir activity against H5N1 viruses (4.3-fold) and zanamivir activity against H5N5 viruses (3.4-fold). Our study revealed a group of H5N1 viruses whose NA belonged to the ea5.1 genetic subclade, characterized by NA-Y253H and T452I amino acid substitutions, which exhibited a 28.8-fold decrease in oseltamivir IC50 values compared to the subtype median. These strains were detected in the European and Ural regions of Russia between May 2022 and January 2023, with no subsequent detections in the available surveillance data. To the best of our knowledge, such strains have not been identified outside Russia.

Viruses doi: 10.3390/v18101125

Authors: Marielle Elen Volkwein Judith Köster Ernst Großmann Nathalie Hafner Thomas Miller Andreas Salditt Petra Benz Martin Beer Angele Breithaupt Kerstin Wernike

Shamonda virus (SHAV), which has recently emerged in Central Europe and has caused disease in numerous cattle herds, was detected in brain tissue from two horses that showed clinical signs, including neurological disorder, and underwent pathological examination. The horses reportedly exhibited seizure-like episodes (Horse A) as well as fever and ataxia (Horse B). In both horses, severe lymphohistiocytic meningoencephalitis with hemorrhages was observed in various parts of the central nervous system (CNS), while outside the CNS no abnormalities were found. The virus genome could be detected by RT-qPCR in all examined parts of the CNS of both horses. In a postmortem cerebrospinal fluid sample of one horse, the virus could only be detected with a very low genome load; however, postmortem contamination cannot be completely ruled out. Consistent with the histological findings, no SHAV genome was detected in either the blood or organs outside the CNS. Postmortem, CNS material—especially brainstem tissue—is suitable for detection of histological lesions and virus genomes via RT-qPCR, while intravital diagnostics of a SHAV infection in horses exhibiting neurological signs is at the moment only possible to a very limited extent. The epidemiological role of horses in the SHAV outbreak is currently unclear.

Viruses doi: 10.3390/v18101124

Authors: Aleksandr Zurochka Mariya Dobrynina Vladimir Zurochka Viktor Gritsenko Aleksandr Semenov

We assessed the antiviral activity of a synthetic GM-CSF active-center peptide against a number of RNA- and DNA-containing viruses, both enveloped and nonenveloped. We evaluated the antiviral activity in vitro and in vivo against parainfluenza virus type 3 (strain Vok) and adenovirus serotype 3 (strain 3/Voronezh/2174/82) in a continuous culture of human laryngeal carcinoma HEp-2 cells. Virucidal activity was evaluated against human adenovirus type 5, Coxsackievirus B3, Echovirus 30 virus, influenza virus A/Puerto Rico/8/34 (H1N1), human parainfluenza virus type 3, herpes simplex virus type 1, OC43 coronavirus, and human cytomegalovirus propagated in various permissive cell lines. The peptide exhibited a stronger prophylactic effect than the ZP2 peptide preparation and showed preferential activity against enveloped RNA viruses. We detected no cytotoxic effect of ZP2 on HEp-2 cells in vitro.

Viruses doi: 10.3390/v18101123

Authors: Yue Chen Lin Yuan Wenqiang Wang Dehu Sun Lulu Chen Jie Zhang Zihan Guo Pinnan Yan Yana Chen Junxia Lv Yan Ma Zhenbang Zhu Wei Wen Kegong Tian Xiangdong Li

Porcine epidemic diarrhea virus (PEDV) poses a persistent threat to the global swine industry through the continual emergence of variants driven by mutation and recombination. Here, we report the isolation and genomic characterization of PEDV2−5, an inter-subgenotypic recombinant isolated from clinical samples collected in Jiangxi Province, China. Phylogenetic analysis based on both the complete genome and the full-length S gene placed PEDV2−5 within the GIIb subgenotypes, clustering closely with FL201 and GD-1. Recombination detection using seven independent algorithms in RDP5, corroborated by SimPlot and phylogenetic incongruence analysis, identified PEDV2−5 as a recombinant between a GIIa major parental-like (PEDV-LY) and a GIIb minor parental-like (GD-1), generating a 7931-nucleotide fragment (nt 20060–27990) that spans the complete structural protein cassette (S, ORF3, E, M, N) and the 3′-terminal portion of ORF1b. The recombinant has replication-competent Vero-E6 cells, reaching a peak titer of 5.03 log10 TCID50/mL at 36 h post-infection. Amino acid analysis revealed 98.49% identity between the PEDV2−5 S protein and the GIIb strain AJ1102, together with 13 unique substitutions and a “KKKG” insertion (residues 361–364), including two substitutions within neutralizing epitopes (A517F in COE and Y1374C in 2C10). Relative to AJ1102, the neutralizing epitopes were nearly completely conserved, whereas extensive divergence was observed against the classical GIa strain CV777. These findings highlight the importance of continued whole-genome surveillance for monitoring the emergence and spread of recombinant PEDV lineages and for informing future vaccine antigen-selection strategies.

Viruses doi: 10.3390/v18101122

Authors: Satoshi Kunita Masaharu Takahashi Tominari Kobayashi Kazumoto Murata Hiroaki Okamoto

Hepatitis E virus (HEV) genotypes 3 and 4 are major zoonotic pathogens, with domestic pigs serving as an important reservoir. Vaccination of pigs may therefore help reduce HEV circulation and zoonotic transmission. We evaluated the protective efficacy of an Escherichia coli-produced HEV-3 virus-like particle (VLP), HEV-3_p495, against homologous HEV-3 and heterologous HEV-4 challenge in pigs. Juvenile pigs vaccinated three times with either 80 or 400 μg of HEV-3_p495 remained negative for HEV RNA in serum and feces during prolonged cohabitation with naturally HEV-3-infected pigs, whereas all non-vaccinated controls became infected. At market age, vaccination also provided strong protection against intravenous HEV-3 challenge, with complete protection against detectable infection in the four-dose group and in two of three pigs in the three-dose group. Importantly, HEV-3_p495 conferred cross-protection against HEV-4 following cohabitation exposure. Two-dose vaccination prevented detectable infection in two of three young pigs and delayed fecal virus detection without viremia in the remaining pig. At market age, all vaccinated pigs remained negative for HEV RNA in serum and feces following HEV-4 exposure, whereas all non-vaccinated controls became infected. These findings provide proof-of-concept evidence for homologous and cross-genotype protection by HEV-3_p495 and support its further evaluation as a candidate swine vaccine for reducing HEV infection and shedding.

Viruses doi: 10.3390/v18101121

Authors: Charles R. M. Bangham

HTLV-1 resists clearance from its host by the strong HTLV-1-specific immune response and by drugs. The tactics and strategies by which it does so illustrate the exquisite adaptation to the host that HTLV-1 has made in its tens of thousands of years of stable co-existence with humans. This adaptation contrasts strongly with the biology of HIV-1, which has established widespread infection in humans for only around a century. The mode of persistence of HTLV-1 makes it less pathogenic than HIV-1, despite causing serious malignant and inflammatory diseases in a proportion of people with the virus, but at the same time makes HTLV-1 harder to treat and eradicate. In this article I outline the different means that HTLV-1 has evolved to persist in the host.

Viruses doi: 10.3390/v18101120

Authors: Xiaofeng Qiu Feifei Cao Shi Cheng Jun Li

Coxsackievirus A4 (CV-A4) is a major cause of hand, foot, and mouth disease and herpangina in children, and has been linked to severe complications. However, current detection methods, including commercial RT-qPCR kits, suffer from false-negative and false-positive results. This study aimed to develop a sensitive and specific TaqMan-based RT-qPCR assay for the rapid detection of CV-A4, targeting the conserved VP1 region covering genotypes A–D and subgenotypes C1–C5, with emphasis on the predominant C2 subgenotype in China. The assay was evaluated using a panel of enteroviruses, influenza virus, adenovirus, and rhinovirus, and compared with two commercial kits using 82 CV-A4-positive and 100 negative clinical specimens. The established assay demonstrated high specificity with no cross-reactivity, a detection limit of 2.1 TCID50 equivalents/mL, and excellent reproducibility (CV ≤ 3.03%). In clinical testing, it achieved a 100% detection rate (82/82) with no false positives within 1.5 h, outperforming the two commercial kits, which showed positive rates of 91.5% and 89.0%, and false-positive rates of 0% and 1%, respectively. These findings indicate that our RT-qPCR assay offers superior sensitivity and specificity for CV-A4 detection, particularly for the C2 subgenotype, and represents a promising tool for laboratory diagnosis and epidemiological surveillance.

Viruses doi: 10.3390/v18101118

Authors: Wojciech Socha Michał K. Krzysiak Elżbieta Moniuszko Paweł Kulik Jarosław Tomana Magdalena Larska

Wildlife disease investigations require diagnostic methods capable of detecting diverse and previously unrecognized pathogens. Metagenomic next-generation sequencing (mNGS) was applied to samples from three Dorcas gazelles (Gazella dorcas osiris) affected by sudden mortality and four European bison (Bison bonasus) with respiratory disease or necrotic balanoposthitis (NBP). No dominant bacterial species was detected in gazelles. In European bison, respiratory disease was associated with Pasteurella multocida, whereas NBP samples were dominated by Porphyromonas levii and other anaerobes. Dorcas gazelle samples contained mainly Retroviridae-related sequences of undetermined pathogenic significance. Parapoxvirus-related annotations were not confirmed by PCR, suggesting non-specific or host-related sequence similarity. Macavirus-related sequences were consistent with previous PCR findings and enabled reconstruction of an approximately 51.1 kb fragment of a divergent gammaherpesvirus genome. These findings demonstrate the value of mNGS for wildlife disease investigation and genomic surveillance, while emphasizing the need for independent validation of metagenomic taxonomic assignments.

Viruses doi: 10.3390/v18101119

Authors: Isadora L. J. Canto Clécio A. C. Filho Lethícia R. Henriques João Victor R. P. Carvalho Maurício T. Lima Felipe C. M. Iani Amanda Haisi João P. Araújo Júnior Rodrigo A. L. Rodrigues

A novel chlorovirus was isolated from a freshwater sample collected from a Brazilian urban lake. Members of the family Phycodnaviridae, the chloroviruses have large linear dsDNA genomes (285–410 kbp) that encode hundreds of proteins and tRNAs. Despite their ecological and evolutionary importance, chloroviruses remain poorly studied, especially in South America. This study aimed to expand the understanding of this viral group by characterizing a new chlorovirus found in Brazil within a species-level framework. Following inoculation of algal cultures with environmental samples, plaque formation was observed 72 h after incubation on a Chlorella variabilis lawn. The new isolate was named BR-MG-N02. The virus remained stable across a temperature range of −30 °C to 37 °C, yet was highly sensitive to UV exposure. This virus has a capsid of approximately 160 nm and establishes a large viral factory in the host cytoplasm. Whole-genome hybrid sequencing revealed a 344,634 bp genome, containing 11 tRNAs and 389 protein-coding sequences. Phylogeny and comparative genomic analyses revealed that BR-MG-N02 is a new member of the species Chlorovirus americanus. We show that viruses belonging to this species harbor an extensive DNA methylation machinery, accounting for ~5% of their gene content and suggesting a potential for DNA methylation. Using representatives isolates, we present evidence of intraspecific differences in biological traits of these chloroviruses, as for burst size and plaque morphology. Altogether, our results expand current knowledge about the viral diversity in Brazil and provide valuable insights into the biology of giant algal viruses.

Viruses doi: 10.3390/v18101117

Authors: Sha Xie Lu Zhang Yan-Gang Sun Meixin Shu

Porcine reproductive and respiratory syndrome virus (PRRSV) and other members of the Arteriviridae family cause substantial economic losses in livestock. However, their pathogen-associated molecular patterns (PAMPs) remain poorly characterized. Here, we investigated whether the 3′ untranslated region (UTR) pseudoknot structures of Arteriviruses act as PAMPs and explored the underlying innate immune recognition mechanisms. Bioinformatics predictions (RNAalifold, RNArtist, and RNAComposer) revealed that the 3′ UTR pseudoknot structures are conserved across all four Arteriviruses. In vitro transcribed pseudoknot RNAs significantly upregulated the mRNA expression of type I interferons (IFNα/β) and interferon-stimulated genes (protein kinase R, PKR; interferon-stimulated gene 15, ISG15; interferon-stimulated gene 56, ISG56) in Marc145 cells. Screening of RNA-sensing pattern recognition receptors (retinoic acid-inducible gene I, RIG-I; melanoma differentiation-associated protein 5, MDA5; Toll-like receptor 3/7/8/9, TLR3/7/8/9) in pseudoknot RNA-stimulated cells showed that RIG-I and TLR8 were significantly upregulated, whereas MDA5, TLR3, TLR7, and TLR9 were not. Short hairpin RNA (shRNA)-mediated knockdown of RIG-I or TLR8 significantly reduced pseudoknot-induced expression of IFNβ, PKR, and ISG56, confirming that both receptors are functionally required for this response. Upregulation of myeloid differentiation primary response 88 (MyD88), interferon regulatory factor 7 (IRF7), and signal transducer and activator of transcription 2 (STAT2) suggested that IFNα/β and downstream interferon-stimulated genes (ISGs) were induced via the TLR8-MyD88-IRF7 and RIG-I-IRF7 signaling pathways. Moreover, small-angle X-ray scattering (SAXS) analysis of the PRRSV pseudoknot RNA complexed with the porcine RIG-I regulatory domain was consistent with the formation of a complex between the pseudoknot RNA and RIG-I. In conclusion, this study identified the 3′ UTR pseudoknot-containing region of Arteriviruses as a conserved PAMP that activates RIG-I- and TLR8-dependent type I IFN responses, providing a mechanistic basis and potential targets for antiviral therapy and vaccine adjuvant development against Arterivirus infections.

Viruses doi: 10.3390/v18101116

Authors: Chenhao Jiang Jianhui Nie Li Zhang

Herpes simplex virus (HSV) is a highly prevalent neurotropic DNA virus that causes orolabial herpes, genital herpes, keratitis, neonatal infection, meningitis and encephalitis. HSV-1 and HSV-2 share epithelial entry, lytic replication, immune evasion, lifelong sensory-ganglion latency and periodic reactivation, yet they differ in typical transmission routes, anatomical recurrence and public-health burden. Rather than mechanically separating both viruses throughout, this review emphasizes shared HSV biology and highlights type-specific differences where they affect clinical interpretation or intervention design. We connect glycoprotein-mediated entry, receptor use, axonal transport, immune evasion and latency with mucocutaneous, ocular, neurologic, neonatal and genital disease. We then synthesize lessons from failed glycoprotein-D vaccine trials, multivalent and mRNA platforms, helicase–primase inhibitors, monoclonal antibodies, RNA-based approaches, genome editing and latency-directed strategies. A mechanism-guided HSV strategy should reduce acquisition, shedding and recurrence, prevent ocular, neurologic and neonatal complications, manage antiviral resistance and ultimately address latent infection.

Viruses doi: 10.3390/v18101115

Authors: Zhen Lei Chang Liu Zhihua Yang Xianzheng Yin Wuchao Zhang Jiangwei Song

Feline herpesvirus type 1 (FHV-1) causes respiratory and ocular disease in cats and establishes lifelong latency, but the extent to which vaccination modifies clinical disease, viral shedding, and latency-related outcomes remains uncertain. We systematically reviewed controlled in vivo studies of FHV-1 vaccination in cats, searching PubMed, Web of Science, and the CNKI from inception to 20 May 2026. Risk of bias was assessed with the SYRCLE tool and certainty for core outcomes with the GRADE approach. Exploratory random-effects meta-analyses were undertaken only when outcomes were sufficiently comparable. Of 327 records identified, 23 reports describing 20 independent animal studies (25 cohorts) were included. Fifteen reports evaluated shedding or viral load, 20 assessed immunogenicity, and one companion report examined latency and reactivation. In two comparable challenge studies involving 17 vaccinated and nine control cats, vaccination was associated with fewer post-challenge clinical signs (risk ratio [RR] = 0.64; 95% confidence interval [CI] 0.41 to 0.99). In one monovalent modified-live vaccine study, cumulative clinical scores were lower after vaccination (mean difference [MD] = −36.00; 95% CI −57.25 to −14.75), with Hedges’ g = −1.90 (95% CI −3.52 to −0.27). Estimates for fever, disease-related removal, and post-vaccination sneezing were imprecise. Most virological studies reported lower shedding or viral loads after vaccination, but heterogeneous assays, outcome definitions, and observation windows precluded robust pooling. Antibody responses could not be directly equated with clinical protection. Overall, FHV-1 vaccination may mitigate post-challenge clinical disease, whereas evidence for effects on shedding, post-vaccination reactions, latency, and reactivation remains limited or inconsistent.

Viruses doi: 10.3390/v18101114

Authors: Diana Horta Angels Ramos Raimon Rifà Carme Loras Marcos Jimenez-Sena Agnes Raga Maria Esteve-Comas

Occult hepatitis B infection (OBI) is defined by detectable hepatitis B virus (HBV) DNA in liver tissue in the absence of hepatitis B surface antigen (HBsAg) and remains insufficiently characterized in low-endemic European settings. This study aimed to determine the prevalence of OBI using direct HBV DNA detection in liver tissue in a real-world cohort of patients undergoing diagnostic liver biopsy. This observational study included consecutive HBsAg-negative patients who underwent liver biopsy between 2020 and 2023. HBV DNA in liver tissue was quantified by real-time PCR, and clinical and serological data were collected. A total of 86 patients were included. OBI was identified in 4 patients (4.7%). All OBI cases were anti-HBc-positive, and two were also anti-HBs-positive. No cases of seronegative OBI were detected. OBI was observed across different underlying liver disease etiologies, including viral, metabolic, autoimmune, and cholestatic conditions. In conclusion, OBI was infrequent in this cohort and consistently associated with anti-HBc seropositivity. These findings suggest that anti-HBc may help identify individuals with prior HBV exposure in whom occult infection can be detected in liver tissue in low-prevalence settings.

Viruses doi: 10.3390/v18101113

Authors: Nicolás Bejerman David Huebert Robert Alvarez-Quinto Diego Quito-Avila Humberto Debat

Large-scale mining of public transcriptomic datasets can reveal viral diversity that remains invisible to conventional virus-surveillance approaches, while increasingly powerful structure-prediction methods provide a complementary route to characterizing highly divergent viral proteins. Here, we combine sequence detection, phylogenetic analysis, structural prediction, and host-association analyses to investigate the cryptic diversity and biology of a new clade of plant-associated lispi-like viruses. Analyses of RNA-sequencing datasets identified and enabled assembly of 87 coding-complete lispi-like virus genome sequences associated with 75 plant hosts, expanding the known diversity of this new group by approximately 40-fold. The viruses share a conserved four-cistron genome organization, 3′-N(P1)-P2-P3-L(P4)-5′, in which the first cistron is referred to interchangeably as N or P1 and the fourth as L or P4. Structural analyses generate testable structural hypotheses for the four conserved proteins. N (P1) adopts a canonical negative-strand RNA virus nucleocapsid architecture with conserved RNA-interacting residues and a predicted RNA-packaging configuration. P2 is exceptionally divergent, although a subset of structures resembles the ITPase/HAM1 fold. P3 forms a conserved trimeric coiled-coil architecture reminiscent of a viral fusion-protein stalk, but lacks the family-wide sequence features expected of a canonical membrane glycoprotein. L (P4) contains a structurally resolved Mononegavirales-type RNA-dependent RNA polymerase (RdRp) core with invariant catalytic motifs, including the characteristic GDN signature, whereas its accessory regions are substantially more divergent. Phylogenetic insights form a distinct monophyletic lineage sister to the predominantly invertebrate-associated Lispiviridae, supporting their recognition as the new proposed family Masuviridae, comprising 15 tentative genera. Genus-level clustering is accompanied by marked differences in host association, ranging from strong specialization to broader host ranges. Retrospective screening of public sequencing libraries further identified lispi-like virus sequences in 1536 libraries representing 134 plant species, 11 plant families and 182 geographic locations, highlighting a substantial and geographically widespread cryptic virome. Together, these results establish Masuviridae as a deeply divergent lineage of plant-associated negative-sense RNA viruses and illustrate how the integration of sequence, structural, and large-scale transcriptomic analyses can move viral dark matter from detection towards evolutionary and functional characterization.

Viruses doi: 10.3390/v18101112

Authors: Patrícia Ribeiro de Carvalho França João Pedro Barros de Paiva Amanda Gabriel Valente Alan Silva Minho Renato Sampaio Carvalho Rosangela Ribeiro de Carvalho Luiz Ricardo Berbert Marcel Frajblat Maria Marta Figueiredo Daisymara Priscila de Almeida Marques Jordana Grazziela Alves Coelho-dos-Reis Patricia Dias Fernandes

COVID-19 has been associated with reproductive dysfunction, although the underlying mechanisms remain unclear. Here, we investigated the effects of inactivated SARS-CoV-2 (iSARS-CoV-2) exposure on reproductive outcomes in K18-hACE2 mice. Pregnant females exposed during early gestation showed reduced fetal number, increased resorptions and decreased placental efficiency. These alterations were associated with pulmonary inflammation, characterized by elevated TNF-α, IL-6 and IFN-γ levels and histopathological damage. In males, exposure impaired the sperm count, motility and reproductive performance, and negatively affected pregnancy outcomes in untreated females. Notably, inflammatory responses occurred in the absence of viral replication, indicating that viral components can trigger systemic effects. Together, these findings demonstrate that inactivated SARS-CoV-2 induces inflammation that compromises fertility and gestational success in both sexes.

Viruses doi: 10.3390/v18101111

Authors: Shuwen Guan Yaoying Jian Linzhi Zhang Zongyang Lv Ruiming Hu Huansheng Wu Huali Xie Deping Song Zhen Ding Zheng Chen

Porcine epidemic diarrhea virus (PEDV) is a major pathogen affecting the global swine industry, causing severe diarrhea, dehydration, and high mortality in neonatal piglets. Due to the continuous emergence of viral variants, the protective efficacy of currently available vaccines remains suboptimal, highlighting the urgent need for effective antiviral agents. In this study, we investigated the anti-PEDV activity of muscone in vitro. Our results demonstrated that muscone inhibits PEDV replication in a dose-dependent manner and exerts its antiviral effect primarily during the early stages of viral infection. Further mechanistic studies revealed that muscone acts mainly by interfering with viral entry into host cells. Specifically, Western blot analysis and confocal microscopy showed that muscone treatment led to the degradation of caveolin-1, suggesting that muscone may block PEDV entry by disrupting caveolin-mediated endocytosis. Collectively, our findings provide clear evidence that muscone possesses potent anti-PEDV activity in vitro and offer mechanistic insights into its mode of action, thereby establishing muscone as a promising lead compound for the development of novel antiviral strategies against PEDV.

Viruses doi: 10.3390/v18101110

Authors: Daniel Desmecht Hani Boshra

Zoonotic viral diseases continue to pose major challenges to global health, particularly as environmental, social and economic changes increasingly influence interactions among humans, animals, vectors, and ecosystems. This review explores Rift Valley fever virus (RVFV), Crimean-Congo hemorrhagic fever virus (CCHFV), and hantaviruses (HTVs) within the framework of One Health, emphasizing the ecological and epidemiological factors that govern their emergence and transmission. Despite their distinct transmission cycles, these viruses share important determinants of zoonotic risk. RVFV is primarily maintained through interactions between mosquitoes, livestock, wildlife, and humans; CCHFV is sustained within complex tick–vertebrate systems involving Hyalomma ticks, domestic animals, and wildlife, while HTVs are predominantly maintained through persistent infections in mammalian reservoir hosts, particularly rodents. Climate variability, habitat alteration, agricultural expansion, livestock movement, urbanization, and other anthropogenic pressures can modify these transmission systems and create new opportunities for pathogen spillover. Effective prevention and control therefore require integrated surveillance that encompasses human disease, animal populations, vectors or wildlife reservoirs, and environmental conditions. Advances in molecular diagnostics, genomic epidemiology, ecological modeling, remote sensing, and climate-based forecasting provide valuable opportunities for improving early detection and outbreak preparedness. To reduce the burden of these zoonotic viral diseases, a sustained collaboration among human and veterinary health professionals, ecologists, entomologists, environmental scientists, and public health authorities is required. Therefore, a coordinated One Health approach is essential for anticipating emerging threats, strengthening preparedness, and protecting human, animal, and ecosystem health.

Viruses doi: 10.3390/v18101109

Authors: Honglin Su Kai Zhao Yuqiong Li Zongti Shao Xinglou Yang Jian Wang Shengyao Chen

Mosquitoes and biting midges harbor diverse viruses, but the factors shaping their viromes remain incompletely understood. We analyzed 35 pooled libraries from seven host groups across 15 locations in Yunnan, China, sampled in June 2025, using total-RNA sequencing, viral annotation, phylogenetics, and community analyses. We retained 1295 viral contigs grouped into 221 annotation-defined viral taxonomic units (VTUs) spanning 24 families; 107 VTUs were supported only by protein homology. Banna virus-related sequences, an Aedes-associated flavivirus, and four putative components of a divergent Jingmenvirus-related candidate were identified. Host group was associated with family-level virome composition after adjustment for location and sequencing depth (R2 = 0.213, p = 0.0052), and the association persisted across alternative feature definitions. Culex tritaeniorhynchus showed the greatest cumulative VTU richness, although its ranking changed when library number was equalized. Most VTUs detected in this host occurred at one sampled site, without a significant monotonic distance-decay pattern. This study expands knowledge of mosquito-associated and biting midge-associated viral diversity in Yunnan, identifies representative lineages for further characterization, and documents host-associated differences in the detected viromes.

Viruses doi: 10.3390/v18101108

Authors: Reina Sasaki-Tanaka Tatsuo Kanda Hanako Yamazaki Shun Yamazaki Ryo Jimbo Tomoaki Yoshida Naruhiro Kimura Hiroyuki Abe Takeshi Yokoo Akira Sakamaki Hiroteru Kamimura Kenya Kamimura Hiroaki Okamoto Shuji Terai

Hepatitis A virus (HAV) infection is a major cause of acute viral hepatitis worldwide. However, no effective antiviral therapy is currently available. HAV manipulates host secretory pathways, including the endosomal sorting complex required for transport (ESCRT)-dependent pathways, to facilitate viral propagation. Nevertheless, whether HAV also utilizes alternative secretory pathways remains unclear. Here, we provide evidence suggesting that an ESCRT-independent pathway may contribute to extracellular HAV RNA levels in Huh7 cells infected with HAV genotype IB (HM175-18f) and genotype IIIA (HA11-1299). Treatment with the ESCRT-independent pathway inhibitor GW4869 significantly reduced extracellular HAV RNA levels. GW4869 did not significantly affect HAV subgenomic RNA replication. These results suggest that the neutral sphingomyelinase 2 (nSMase2)-dependent ESCRT-independent pathway may contribute to extracellular HAV RNA levels rather than intracellular HAV RNA replication. Consistent with these findings, knockdown of sphingomyelin phosphodiesterase 3, which encodes SMase2, also significantly reduced extracellular HAV RNA levels. These findings suggest that an nSMase2-dependent ESCRT-independent extracellular vesicle pathway may contribute to extracellular HAV RNA levels.

Viruses doi: 10.3390/v18101107

Authors: Anna Chiara Vittucci Livia Antilici Alessandra Salvatori Domenico Umberto De Rose Rossana Scutari Velia Chiara Di Maio Lelia Rotondi Aufiero Mara Pisani Anna Maria Musolino Andrea Dotta Carlo Federico Perno Alberto Villani

The introduction of universal prophylaxis with nirsevimab for all children in their first epidemic season has substantially reduced the burden of respiratory syncytial virus (RSV). However, a small number of breakthrough infections continue to occur despite immunization, mostly in previously healthy infants, and these cases remain incompletely characterized. We conducted a retrospective study across three epidemic seasons at a third-level hospital, including 536 RSV-positive children under 12 months of age hospitalised for bronchiolitis. Immunised infants with breakthrough infection showed a higher prevalence of prematurity and comorbidities than unimmunised infants, and were more frequently represented in the 9–12-month age group, with a median of 291 days since nirsevimab administration. Immunised infants had significantly lower odds of requiring supplemental oxygen (aOR 0.33, 95% CI 0.19–0.57, p < 0.001). However, once oxygen therapy was required, immunisation status did not independently predict the need for advanced ventilatory support. Severe breakthrough cases requiring invasive mechanical ventilation occurred even among infants without traditional risk factors. These findings confirm the substantial real-world impact of universal nirsevimab prophylaxis while underscoring the need for continued clinical vigilance and further research into the mechanisms underlying breakthrough disease.

Viruses doi: 10.3390/v18101106

Authors: Matteo Riccò Luca Pipitò Claudio Costantino Marco Bottazzoli Paolo Manzoni Marco Falcone Pasquale Gianluca Giuri Antonio Cascio

Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are major causes of respiratory tract infections across different age groups. Although primarily regarded as respiratory pathogens, both viruses have been associated with cardiac complications, whose clinical spectrum remains incompletely characterized. We systematically reviewed published cases of RSV- and hMPV-associated cardiac involvement and compared their clinical characteristics and outcomes. PubMed, Scopus, and EMBASE were systematically searched for reports describing cardiac complications associated with laboratory-confirmed RSV or hMPV infection. Individual patient data were pooled and analyzed using descriptive and univariate analyses. Multivariable logistic regression was used to identify characteristics associated with hMPV versus RSV diagnosis, while multiple correspondence analysis (MCA) was performed to explore multivariate patterns of clinical presentation. Age-stratified sensitivity analyses were additionally performed. Overall, 102 cases were included, comprising 71 RSV (69.6%) and 31 hMPV (30.4%). hMPV cases were significantly older than RSV cases (41.5 years, interquartile range 3.5–63.0 vs. 1.5 years, interquartile range 0.2–31.0; p < 0.001) and had a higher burden of comorbidities. Myocarditis was the most frequently reported cardiac complication in both groups (87.1% hMPV vs. 74.6% RSV), followed by heart failure and pericarditis. ICU admission was more frequent among hMPV cases (96.8% vs. 77.5%; p = 0.034), whereas overall clinical outcomes did not significantly differ between groups. In exploratory multivariable analyses, dyspnea (aOR 16.280, 95% CI 1.370–193.475) and cough (aOR 6.059, 95% CI 1.062–34.552) were associated with hMPV diagnosis, whereas rhinorrhea (aOR 0.031, 95% CI 0.003–0.286) and lethargy/altered mental status (aOR 0.103, 95% CI 0.015–0.712) were associated with RSV diagnosis. ICU admission was also associated with hMPV diagnosis (aOR 11.033, 95% CI 1.125–108.173). These estimates were characterized by wide confidence intervals and should be interpreted cautiously. MCA showed substantial overlap between RSV and hMPV cases, without clear separation of their overall clinical presentation, while most between-group differences were attenuated in age-stratified analyses. In conclusion, both RSV and hMPV can be associated with a broad spectrum of potentially severe cardiac complications, with myocarditis representing the predominant reported manifestation. Despite some differences in demographic and clinical characteristics, their overall cardiac and clinical phenotypes substantially overlap. These findings support greater awareness of potential cardiac involvement during severe RSV and hMPV infections, while highlighting the need for prospective, systematically investigated cohorts to establish the incidence, mechanisms, and long-term outcomes of these complications.

Viruses doi: 10.3390/v18101105

Authors: Çiğdem Mermutluoğlu Saim Dayan Yakup Demir Ayşe Özlem Mete Mustafa Kemal Çelen

Background and Aim: Hepatitis delta virus (HDV) infection is among the most aggressive forms of chronic viral hepatitis: fibrosis advances quickly, and cirrhosis, hepatocellular carcinoma (HCC) and hepatic decompensation develop more often than with other hepatitis viruses. Metabolic dysfunction-associated steatotic liver disease (MASLD) is now the leading chronic liver disorder worldwide, and it may add to liver damage when it occurs alongside viral hepatitis. To date, however, data on hepatic steatosis in patients with chronic HDV infection remain scarce. We therefore aimed to describe the frequency of hepatic steatosis, assessed by the controlled attenuation parameter of transient elastography (FibroScan), in patients with chronic HBV infection and persistent anti-HDV seropositivity, and to examine whether it was associated with liver fibrosis. Methods: A total of 125 patients with chronic HBV infection and anti-HDV antibodies documented for at least six months were enrolled in this cross-sectional study between March and September 2024. HDV-RNA was tested in all participants. FibroScan provided liver stiffness measurement (LSM, kPa) and the controlled attenuation parameter (CAP, dB/m). We used CAP ≥ 248 dB/m to identify steatosis and LSM ≥ 12.5 kPa to identify cirrhosis. Because cardiometabolic risk factors were not systematically recorded, the criteria for MASLD could not be applied, and steatosis is reported as CAP-defined hepatic steatosis. Multivariable logistic regression was used to look for factors linked to LSM-defined advanced fibrosis (LSM ≥ 9.5 kPa), with sensitivity analyses accounting for ALT elevation. Results: The mean age was 39.0 years, and 74.4% (n = 93) of patients were male. HDV-RNA was detectable in 68.0% (n = 85). Of the 85 patients previously treated with pegylated interferon-alpha, 26 (30.6%) had a sustained virological response (SVR). Median LSM was 10.1 kPa (IQR 9.1–10.7) and median CAP 218 dB/m (IQR 211–239); cirrhosis was present in 15.2% (n = 19). CAP-defined steatosis was found in 24.0% (n = 30). Patients with and without steatosis were similar in age, sex, HDV-RNA status, liver stiffness, cirrhosis rate and SVR rate (36.4% vs. 28.6%, p = 0.593). After adjustment, detectable HDV-RNA was associated with advanced fibrosis (adjusted odds ratio [aOR] 4.89, 95% CI 2.13–11.26, p < 0.001), including after further adjustment for ALT. No significant association was found for steatosis (aOR 0.82, 95% CI 0.32–2.12, p = 0.684), although the confidence interval was wide. Conclusions: About one in four anti-HDV-positive patients had CAP-defined hepatic steatosis. Detectable HDV-RNA was associated with LSM-defined advanced fibrosis, whereas no significant association with steatosis was found; the precision of this estimate was limited, and the exploratory cirrhosis analysis was underpowered. Whether steatosis in this setting reflects metabolic dysfunction, and whether it affects long-term outcomes, warrants prospective study with full metabolic phenotyping.

Viruses doi: 10.3390/v18101103

Authors: Julie Andreina Beltrán Yoneira Fabiola Sulbarán Nahir Martínez-Urbina Mario Comegna María Graciela López Lily Soto Carlos Pérez Moraima Hernández Flor Helene Pujol Rossana Celeste Jaspe

Coinfection between hepatitis viruses and human immunodeficiency virus (HIV) significantly increases liver disease and AIDS progression. This retrospective cross-sectional study evaluated the prevalence and genetic diversity of hepatitis B (HBV) and C viruses (HCV) among people living with HIV (PLWH) in Venezuela’s Capital District. A total of 420 serum samples collected between 2022 and 2025 were tested for serological and molecular markers, including whole-genome HBV sequencing via next-generation sequencing (NGS) to characterize viral genotype distribution and mutations. Seroprevalences for HBsAg, anti-HBc, and anti-HCV were 2.9%, 20%, and 4.0%, respectively. HBV exposure was significantly higher in older men, with no differences between treatment-naïve and treated patients. Occult HBV infection (OBI) prevalence decreased compared to 2014 data (seropositive: 7% vs. 16%, seronegative: 0.9% vs. 20%). This decline, along with the higher HBV exposure among older adults, may be partially attributed to the universal HBV vaccination introduced in Venezuela in 1998, as well as modifications in antiretroviral therapy affecting HBV replication. Notably, key vaccine- and diagnostic-escape mutations were detected in OBI cases, highlighting clinical and public health concerns. Increased HCV prevalence appears to be linked to rising intravenous drug use in the country. HBV phylogenetic analysis revealed genotype F3 as predominant, followed by F1b (Cosmopolitan clade), while HCV showed genotype 1a to be the most frequent, followed by 1b and 3a. These findings underscore the success of immunization efforts but highlight the urgent need for continuous molecular surveillance and updated public health policies, including targeted harm-reduction programs, to control viral hepatitis in vulnerable populations.

Viruses doi: 10.3390/v18101104

Authors: David Lupande Mwenebitu Demelash Areda Pascal Adroba Franklin Mwamba Philippe Boribo Kikunda Olivier Mukuku Danstan Bagenda Misaki Wayengera Patrick D. M. Katoto Gaylord Ngaboyeka

In May 2026, the Democratic Republic of the Congo declared an outbreak of Bundibugyo virus disease caused by Bundibugyo ebolavirus. We analysed aggregated official Situation Reports (SitReps) issued from 14 May through 14 July 2026 to describe the reported epidemic trajectory, geographic expansion, mortality, interval-specific growth, and conditional six-month projections. Confirmed cases increased from 8 to 2073 and confirmed deaths reached 796; 45 health zones in five provinces were affected. Average interval-specific growth rates generally declined over the observation period, although cumulative cases and the geographic footprint continued to increase. Gompertz and Richards curves described the observed cumulative trajectory better than logistic and exponential curves according to root mean square error (RMSE), the corrected Akaike information criterion (AICc), and the Bayesian information criterion (BIC); this comparison reflects in-sample fit only. Conditional six-month scenarios varied substantially across assumptions (approximately 2435 to 10,839 cumulative cases by 14 January 2027) and are contingent and highly uncertain; Gompertz fits showed underprediction of the subsequently reported total in four temporal checks. Reported case fatality ratios (CFRs) varied across provinces but are vulnerable to outcome delay, incomplete case ascertainment, small denominators, and retrospective reclassification. The results support continued surveillance and response planning but do not identify the causes of epidemic slowing or establish that transmission was controlled.

Viruses doi: 10.3390/v18101102

Authors: Vinayagamurthy Balamurugan Kirubakaran Vinod Kumar Rakshit Ojha Ponnidurai Malini Ramachandra Deshpande Prajakta Prashant Bokade Annett Helcita Dsouza Shweta Priya Anand Asha Archana Pal Gangarajareddy Deekshitha Mahadevappa Swathi Shakuntala Krishnaiah Harshitha Shanmugam Chandra Sekar Kuralayanapalya Puttahonnappa Suresh GurrappaNaidu Govindaraj Divakar Hemadri Nayakvadi Shivasharanappa Sujit Nayak Njeumi Felix Baldev Raj Gulati Satya Parida

India launched a nationwide Peste des Petits Ruminants (PPR) Eradication Programme in 2023 with the objective of achieving countrywide control and elimination of PPR by 2030, adapting a systematic mass vaccination, surveillance, and seromonitoring strategy in alignment with the WOAH/FAO Global Control and Eradication Strategy (GCES) under the PPR Global Eradication Programme (PPR-GEP) 2030. As part of the first round of mass vaccination, the present study evaluates the effectiveness of the PPR Sungri-96 (Lineage IV) vaccine in sheep and goats in fields across all the states in India, with particular emphasis on the post-vaccination evaluation (PVE) as per WOAH guidelines. A total of 24,542 post-vaccination (30–90 days) serum samples were collected from sheep and goats aged 6–12 months from 2528 epidemiological units (epi-units, villages) across 24 states of India, following GCES guidelines. Sampling was conducted according to state vaccination schedules during January 2023–December 2024, ensuring appropriate post-vaccination immune-response windows. Serum samples were analysed to detect PPR virus (PPRV)-specific antibodies using the native PPR competitive ELISA (c-ELISA) kit. The study demonstrated an overall post-vaccination seroconversion rate of 69.3% (95% CI: 68.7–69.8%) among small ruminants, indicating a substantial vaccine-induced immune response under field conditions in the target age group with desirable vaccine effectiveness. Exceptionally high post-vaccination seroconversion was observed in Telangana (91.6%) and Andhra Pradesh (82.3%), reflecting effective implementation of vaccination campaigns in these two states. Overall, 61% (1553/2528) of the evaluated epi-units recorded 70% seroconversion post vaccination, whereas 22% (510/2287) of epi-units showed ≥70% seroprevalence pre vaccination in sheep and goats aged 6 to 12 months. These findings demonstrate the effectiveness of the PPR Sungri-96 vaccine in inducing protective immunity under field conditions in accordance with GCES guidelines. The observed inter-state variations in immune response may be attributed to differences in vaccination coverage, implementation efficiency, animal demographics, husbandry practices, and local epidemiological conditions. However, some states failed to achieve the desired level of post-vaccination immunity, indicating the need for intensified vaccination efforts and improved campaign implementation in such regions. Continued mass vaccination, strengthened surveillance, real-time disease monitoring, farmer awareness programs, and capacity-building initiatives for veterinary services are critical for improving vaccine effectiveness and accelerating progress towards a PPR-free India. This study provides important field evidence supporting the effectiveness of the PPR Sungri-96 vaccine and contributes significantly to both national and global PPR eradication initiatives.

Viruses doi: 10.3390/v18101101

Authors: Léo Pomar Loïc Epelboin Najeh Hcini

Background: Oropouche virus (OROV), an Orthobunyavirus endemic to Latin America, has recently emerged as a pathogen of concern in pregnancy following reports of vertical transmission, fetal demise, congenital anomalies, and neonatal disease. The unprecedented 2023–2025 outbreaks in Latin America have expanded the geographic distribution of OROV and renewed concerns regarding its potential as an emerging human teratogen. Methods: We conducted a narrative comprehensive review of the literature focusing on epidemiology, maternal–fetal transmission, placental infection, experimental models, fetal and neonatal outcomes, and comparisons with other congenital arboviral infections. Results: Increasing evidence supports the ability of OROV to infect the placenta, cross the maternal–fetal interface, and invade fetal tissues. Human cases have documented miscarriage, stillbirth, fetal demise, microcephaly, ventriculomegaly, cerebral atrophy, corpus callosum abnormalities, posterior fossa defects, arthrogryposis, and neonatal death following maternal infection. Viral RNA and OROV-specific antibodies have been detected in placental, fetal, and neonatal samples, providing direct evidence of congenital infection. Experimental studies using trophoblast cultures, placental organoids, neural progenitor cells, brain organoids, and animal models have confirmed placental susceptibility, vertical transmission, fetal neurotropism, and disruption of neurodevelopment. The congenital phenotype shares important similarities with congenital Zika syndrome and other neurotropic arboviral infections. Conclusions: OROV is newly and increasingly recognized as a vertically transmissible neurotropic arbovirus with potential teratogenic effects. Although biological plausibility is supported by converging clinical and experimental findings, prospective epidemiological studies are urgently needed to quantify risks, identify determinants of fetal injury, and define long-term outcomes among affected children.

Viruses doi: 10.3390/v18101099

Authors: Camilla Costa Silva Karina Carvalho Marques Pablo Fabiano Moura das Neves Daniel Carvalho de Menezes Stanley Soares Xavier Cristiane de Fátima Pimenta da Costa Marcio Clementino de Souza dos Santos Ápio Ricardo Nazareth Dias Mário Augusto Costa Prazeres Pedro Fernando da Costa Vasconcelos Juarez Antônio Simões Quaresma João Sérgio de Sousa Oliveira Luiz Fábio Magno Falcão

Background: Neuromuscular dysfunction in long COVID remains poorly understood, and conventional biomarkers of muscle injury often fail to account for persistent symptoms. This study aimed to compare laboratory biomarkers and neuromuscular outcomes by sex, long COVID duration, and hospitalisation status, and examine the association between neuromuscular outcomes and laboratory biomarkers. Methods: In this cross-sectional study, 153 adults with long COVID underwent clinical, body composition, handgrip strength, surface electromyography, and laboratory assessments, including creatine kinase (CK), lactate dehydrogenase (LDH), ferritin, erythrocyte sedimentation rate (ESR), haemoglobin, sodium, potassium, and calcium. Analyses were stratified by sex, disease duration, and hospitalisation. Results: Significant between-group differences in laboratory and neuromuscular variables were more frequent among men than among women. Hospitalised women had lower muscle mass and higher body fat than non-hospitalised women. Men with longer long COVID duration differed in CK, ferritin, haemoglobin, and vastus lateralis electromyographic activity. Ferritin, calcium, potassium, haemoglobin, and LDH showed consistent, modest multivariate associations with neuromuscular outcomes. The haemoglobin–ferritin–ESR axis correlated most consistently with muscle mass and handgrip strength. Conclusions: These exploratory findings suggest a sex-specific pattern and a consistent haemoglobin–ferritin–ESR axis in long COVID, requiring confirmation in longitudinal studies.

Viruses doi: 10.3390/v18101100

Authors: Pietro Hiram Guzzi Francesco Branda Pierangelo Veltri Federico Manuel Giorgi

Hantaviruses cause haemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome, which are together the cause of more than 200,000 human cases every year. However, no specific antiviral therapy is approved. Here, we develop a three-stage computational framework for systematic drug repurposing. Transcriptome-wide analysis of hantavirus-infected human endothelial cells showed a response dominated by interferon pathways. A STRING protein–protein interaction network and a composite network centrality score identified 15 hub genes, including ISG15, IRF1, CXCL10, STAT1, and DDX58. Druggability and literature analysis of these hubs provided 12 repurposing candidates across four priority tiers. An ordinary differential equation (ODE) model of type I interferon signalling then evaluated eight drug perturbations. Early IFN-β achieved the fastest viral clearance (3.5 days) with reduced CXCL10 output. Early JAK inhibition delayed clearance (4.9 days) and increased IRF1-driven CXCL10. Our simulation showed that IFN-β combined with an anti-CXCL10 monoclonal antibody produced the best balance between antiviral effect and CXCL10 output. A structural sensitivity analysis showed the antiviral predictions to hold under an alternative network topology, while the chemokine predictions depended on it. This computational pipeline prioritises candidate therapies and phase-specific combinations for preclinical hantavirus studies.

Viruses doi: 10.3390/v18101098

Authors: Ying Zhou Chuanfeng Zhang Qi Sun Tao Qiu Qianjin Fan Jing Lu Haiyang Hu Xiaoqin Xu Dandan Yang Haitao Yang

Background: To provide evidence for HIV-1 DNA genotypic resistance testing (GRT) strategies in low-level viremia (LLV) populations by comparing the amplification efficiency and resistance mutation profiles between HIV-1 DNA GRT and RNA GRT. (2) Methods: Convenience sampling was used for participant enrolment. Plasma HIV-1 RNA and peripheral blood monocyte (PBMC) DNA were extracted separately. The protease–reverse transcriptase (PR-RT) region was amplified via an in-house protocol, followed by Sanger and Nanopore sequencing. The exact McNemar’s test and Fisher’s test were applied to compare amplification success rates. Univariate and multivariable logistic regression analyses implemented in R were conducted to identify factors associated with the detection of HIV-1 drug-resistant strains. BEAST-based molecular clock analysis was performed to calculate the divergence time between RNA-derived and DNA-derived sequences. (3) Results: A total of 209 samples were enrolled, and valid sequences were obtained from 166 samples. Within the viral load range of 50–200 copies/mL, HIV-1 DNA GRT yielded significantly higher amplification success rates than RNA GRT (p < 0.05). The overall resistance detection rate for DNA GRT (20.8%) was lower than that for RNA GRT (33.7%), with discrepancies mainly involving nucleoside reverse transcriptase inhibitor (NRTI)- and non-nucleoside reverse transcriptase inhibitor (NNRTI)-associated mutations. Individuals receiving antiretroviral therapy (ART) for 7–18 months exhibited higher odds of detecting drug-resistant strains using either DNA GRT or RNA GRT. The median evolutionary divergence time between matched RNA- and DNA-derived sequences was 3.13 years. By contrast, the median evolutionary divergence time between paired DNA generated by Sanger and Nanopore sequencing was 0.84 years. (4) Conclusions: DNA GRT serves as a valuable supplementary screening tool in public health scenarios of LLV with failed RNA GRT amplification. However, clinical interpretation should combine RNA GRT findings, longitudinal viral load monitoring and treatment history, rather than relying solely on DNA GRT to modify antiretroviral regimens.

Viruses doi: 10.3390/v18101097

Authors: Shujie Zhao Xiaozhou Liu Xinyu Shi Zhengdong Zhao Yu Sun

Sudden sensorineural hearing loss (SSNHL) is an otological emergency with complex and heterogeneous etiologies, and most cases remain unexplained and are therefore classified as idiopathic. Viral infection has long been considered an important potential cause of SSNHL; however, the mechanisms underlying virus-associated hearing loss and the causal relationship between viral infection and SSNHL have not been fully elucidated. Current evidence suggests that viruses may induce acute hearing loss through direct invasion of the inner ear, reactivation of latent viruses, immune-mediated injury, and cellular stress and inflammatory responses. This review describes the major viruses associated with SSNHL and their epidemiological and clinical characteristics; summarizes evidence from serological testing, molecular virology, imaging, temporal bone histopathology, and animal studies; and discusses recent advances in the pathogenic mechanisms, etiological diagnosis, and antiviral treatment of virus-associated SSNHL. This review aims to systematically clarify the current evidence and controversies surrounding the viral etiology of SSNHL and to provide a reference for etiological identification, patient stratification, and targeted treatment of virus-associated SSNHL.

Viruses doi: 10.3390/v18101096

Authors: Carissa Gaghan Matthew Browning Aakriti Tyagi Pok Man Chan Mohamed Faizal Abdul-Careem Isabel M. Gimeno Raveendra R. Kulkarni

Recombinant herpesvirus of turkey laryngotracheitis (rHVT-LT) and fowlpox virus (rFPV)-LT in-ovoin-ovo vaccines are used for ILT control in broiler chickens. Previously, we showed that in-ovoin-ovo CpG-oligodeoxynucleotide (CpG-ODN) adjuvantation of rHVT-LT can protect broilers against ILT at 28 days of age. Here, we evaluated the immunogenicity and protective efficacy of in-ovoin-ovo CpG-ODN-adjuvanted rHVT-LT and rFPV-LT in broilers when challenged at 14 days of age. Eighteen experimental groups incorporated CpG-ODN-only treatments or those receiving rHVT-LT or rFPV-LT with/without CpG-ODN or non-CpG control ODN in-ovoin-ovo, followed by ILTV challenge at 14 days of age. While none of the vaccinated groups provided significant clinical protection, rHVT-LT+CpG-ODN vaccination showed a numerical reduction (p = 0.0710) in disease severity following ILTV challenge compared with the unvaccinated control. CpG-ODN-only or CpG-ODN-adjuvanted vaccinations followed by challenge resulted in increased (p < 0.05) tracheal and/or splenic TLR21, IL-12, or IFNγ transcription, indicating TLR-mediated antiviral adjuvant effects. Furthermore, CpG-ODN+rHVT-LT/rFPV-LT groups receiving ILTV had increased (p < 0.05) splenic frequencies of macrophages, γδ T cells, B cells, and activated CD4+/CD8+ T cells. Collectively, our findings indicate that in-ovo CpG-ODN adjuvantation with recombinant-vectored ILT vaccines can induce measurable immune activation and a trend toward reduced ILT disease severity following early ILTV challenge in broiler chickens.

Viruses doi: 10.3390/v18101095

Authors: Samantha N. Atkinson Halli E. Miller Jason Jarzembowski Christine Duris Amy Y. Pan Li-Chun Lin Ravit Arav-Boger

Congenital cytomegalovirus (CMV) is the most common congenital infection and a leading infectious cause of childhood hearing loss and neurodevelopmental disability. The cellular organization of the placental response to infection is incompletely defined. We applied Visium CytAssist spatial transcriptomics to formalin-fixed tissue from two CMV-infected and two uninfected late-gestation placentas and annotated the major maternal and fetal cell populations by reference-based label transfer. Across 16,074 spatial spots, the only significant compositional changes were increased T cell spots and decreased syncytiotrophoblast-enriched spots in CMV-infected placentas; the remaining populations showed similar changes but did not reach significance. Differential expression comparing CMV-infected to uninfected samples within each cell type identified two opposing programs. Infected placentas displayed a broad interferon-stimulated gene signature accompanied by induction of CXCR3-ligand chemokines, antigen-processing machinery, and the major histocompatibility complex class I transactivator NLRC5, classical-pathway complement components, and lymphocyte and plasma-cell markers, most pronounced in endothelial, stromal, and syncytiotrophoblast clusters and minimal in extravillous trophoblasts. Concurrently, infected placentas showed downregulation of trophoblast differentiation and syncytialization genes, including PAPPA2, pregnancy-specific glycoproteins, CGA, and GCM1, as well as vascular and hemostatic genes, including FLT1, VEGFA, PECAM1, and TFPI2. These data provide a cell-type-resolved molecular correlation of immune activation and placental dysfunction associated with congenital CMV. Together, the data indicate that congenital CMV infection is associated with a coordinated shift at the maternal–fetal interface, in which a broad interferon-driven immune response accompanies suppression of trophoblast-developmental and vascular programs, providing a cell-type-resolved reference for larger studies of placental CMV transmission. Future studies in larger cohorts will be needed to confirm these findings.

Viruses doi: 10.3390/v18101094

Authors: Kaiyue Wei Xiaofeng Sun Anum Farid Hong Liu

Next-generation sequencing (NGS) has dramatically accelerated the identification of viral genomic sequences from arthropod vectors, uncovering an unprecedented diversity of potential arboviruses. Nevertheless, the majority of these novel viral genomic sequences (NVGS) remain uncharacterised genomic data without corresponding viral isolates, highlighting the discrepancy between sequence detection and biological validation. This gap impedes our understanding of the biological properties, host range, and pathogenic potential of these newly identified viruses. To bridge this gap, the review advocates for integrated strategies, including expanded cell culture screening, novel cell line development, and reverse genetics approaches. These are complemented by artificial intelligence (AI) for predictive viral characterization. In parallel, NGS can be combined with other sequencing strategies to obtain more complete and accurate viral whole-genome sequences. Validating NGS discoveries is essential for effective risk assessment and outbreak preparedness against emerging arboviral threats, with important implications for public health surveillance.

Viruses doi: 10.3390/v18101093

Authors: Junhan Dong Zhiyun Cheng Yong Diao

This work builds AAV-CapNet, a deep learning model integrating Fourier frequency enhancement and cross-attention fusion, to predict the infectivity of 20 AAV capsid variants across 52 cancer cell lines with a self-built multi-omics dataset of 392 validated measurements. Validated via stratified splitting and repeated five-fold cross-validation, it extracts dual-domain capsid features fused with host transcriptomics for joint classification and regression. The model exhibits favourable performance under low-false-positive-rate conditions suitable for high-throughput screening. Attention mapping and ablation tests validate core modules’ necessity. Despite poor generalization to unseen serotypes, this interpretable tool facilitates intraserotype capsid screening and rational engineering.

Viruses doi: 10.3390/v18101092

Authors: Hazel Yetiskin Muhammet Ali Uygut Shaikh Terkis Islam Pavel Busra Kaplan Aykut Ozdarendeli

Background: Continuous antigenic evolution of SARS-CoV-2 has reduced the effectiveness of vaccines based on a single ancestral strain, highlighting the need for multivalent vaccine strategies. We developed a trivalent whole-virion inactivated vaccine containing ancestral (ERAGEM), Delta, and Omicron BA.5 antigens and evaluated its immunogenicity and protective efficacy in K18-hACE2 transgenic mice. Methods: β-propiolactone-inactivated ERAGEM, Delta, and Omicron BA.5 antigens were combined at 1 µg each (3 µg total) with aluminium hydroxide and administered intraperitoneally on days 0 and 21. Variant-specific binding and neutralizing antibody responses were assessed by enzyme-linked immunosorbent assay (ELISA) and microneutralization assays. On day 42, mice were challenged with ERAGEM, Delta, or Omicron BA.5. Survival, body weight, body temperature, infectious viral titers, and viral RNA loads were evaluated. Results: The trivalent vaccine induced binding and neutralizing antibodies against all three vaccine components, and titers rose after the booster dose. Neutralizing responses were highest against the BA.5 component. Vaccinated mice showed 100% survival following challenge with each vaccine-matched variant and maintained stable body weight and temperature throughout the observation period. Unvaccinated controls challenged with Omicron BA.5 showed no mortality, so protection against this component could be assessed only virologically. No infectious virus was detected in the lungs of vaccinated animals, while viral titers and RNA loads in the nasal turbinates were significantly reduced compared with unvaccinated controls. Conclusions: A trivalent whole-virion inactivated SARS-CoV-2 vaccine containing ancestral, Delta, and Omicron BA.5 antigens elicited variant-specific humoral immunity and protected mice against challenge with each vaccine-matched component: against lethal challenge for the ancestral and Delta components, and against viral burden for Omicron BA.5, which was not lethal in unvaccinated controls. These outcomes were obtained at a lower per-antigen dose than in our previously reported monovalent and bivalent formulations; because those were independent studies rather than contemporaneous comparators, this suggests a dose-sparing potential that warrants formal evaluation rather than demonstrating one. Cross-protection against variants not included in the formulation was not assessed.

Viruses doi: 10.3390/v18101091

Authors: Bingmei Dong Jing Cai Haiqi Zhao Pengyan Gao Qingqing Xu Weiqin Meng Na Tang

The innate immune system plays a significant role in inhibiting viral infection and proliferation. To explore the signaling pathway of the innate immune response induced by Newcastle disease virus (NDV) in BHK-21 cells, the mRNA expression levels of pattern recognition receptors such as TLR3, TLR7, TLR8, and NOD2, as well as those of cytokines including TNF-α, IL-6, IL-1β, IL-10, IL-17, TRIF, TRAF3, and NF-κB were determined by transcriptome sequencing, qRT-PCR, and ELISA. All these results indicated that the NDV LaSota strain primarily induces the TLR3-TRIF-NF-κB signaling pathway in BHK-21 cells. Subsequently, an NF-κB inhibitor, a TLR3 inhibitor, and a TRAF3 inhibitor were separately used, and the viral titer was measured during the NDV LaSota strain infection of BHK-21 cells. The results displayed that the inhibition of TLR3 or NF-κB effectively increased the viral titer of NDV. Hence, the CRISPR/Cas9 system was used to create a BHK-TLR3—cell clone in which TLR3 was knocked out, and the cells were assessed for morphology, cell density, cell viability, and genetic stability. Finally, the cells were infected with the NDV LaSota strain. The results showed that the BHK-TLR3—cell line is a stable cell line with markedly enhanced NDV LaSota strain production. Furthermore, the knockout of the TLR3 gene significantly inhibited the expression of IFN-α, IFN-β, IL-1β, and TNF-α, especially that of IL-1β and TNF-α. In summary, the present research revealed the main signaling pathway of the innate immune response induced by the NDV LaSota strain and provides a promising basis for NDV production.

Viruses doi: 10.3390/v18101090

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

The impact of the COVID-19 pandemic and related public health measures on hepatitis C (HCV) treatment uptake in Ontario remains uncertain. We evaluated changes in HCV treatment initiation during the pandemic overall and among priority populations. We conducted a population-based study using Ontario health administrative data from January 2015 to December 2022. The primary outcome was HCV treatment initiation rate, defined as the first antiviral dispensation per 1000 eligible individuals. Interrupted time series analysis estimated changes during the pandemic relative to counterfactual projections based on pre-pandemic trends. We stratified analyses by birth cohort, immigration status, homelessness, people who inject drugs (PWID), and human immunodeficiency virus status. A sensitivity analysis restricted the pre-pandemic period to 2018 onward. Among 63,691 eligible individuals, treatment initiation increased from 2015, peaked in 2017, and declined thereafter, resulting in a flat pre-pandemic trend. Treatment initiation declined at pandemic onset and continued downward through 2022; however, neither level nor slope changes were statistically significant. Results were sensitive to pre-pandemic period specification: restricting analyses to 2018 onward showed a significant pre-pandemic decline, with observed pandemic-period rates exceeding counterfactual projections. Younger cohorts, PWID, and individuals experiencing homelessness showed heterogeneous trends. We found no clear evidence that the COVID-19 pandemic independently altered HCV treatment initiation trends in Ontario, although treatment initiation declined at pandemic onset and remained lower throughout the pandemic period.

Viruses doi: 10.3390/v18101089

Authors: Jan Inge Øvrebø Hilde Sindre Hege Løkslett Torfinn Moldal Monika J. Hjortaas

Salmonid alphavirus (SAV) causes pancreas disease (PD) in Atlantic salmon and is a major viral pathogen affecting salmon aquaculture in Northern Europe. In Norway, PD is associated with genotypes SAV3 and marine SAV2, which have distinct geographical distributions. SAV3 predominates along the southwestern coast, whereas SAV2 has historically been restricted to Mid-Norway. In 2023, SAV2 was detected at four aquaculture sites along the Helgeland coast, north of the established PD zone, representing substantial northward expansion of the virus’ distribution. Whole-genome sequencing (WGS) provides high-resolution data for molecular epidemiological investigations and can distinguish between local viral persistence and recent introductions. In this study, nanopore-based WGS was applied to SAV2-positive samples collected from Norwegian aquaculture sites between 2017 and 2025. Genome sequences were analyzed with previously published Norwegian SAV2 sequences using Bayesian phylogenetic methods. Phylogenetic analyses estimated the most recent common ancestor of the Norwegian SAV2 lineage around 2006, indicating that the virus likely circulated in Norway for several years before its initial detection. SAV2 genomes from Helgeland showed high genetic similarity to genomes from sites within the PD zone, providing strong evidence of aquaculture-related spread. These findings demonstrate the value of WGS for tracing transmission pathways and supporting PD outbreak investigations.

Viruses doi: 10.3390/v18101088

Authors: Haydar Witwit Roaa Khafaji Rachel Y. Sattler Patricia Mingo-Casas Ana-Belén Blázquez Miguel A. Martín-Acebes Juan Carlos de la de la Torre

Mammarenaviruses (MaAvs) cause persistent infections in diverse rodent reservoirs worldwide and several are zoonotic pathogens with an important public-health burden in their endemic regions. Moreover, the globally distributed MaAv lymphocytic choriomeningitis virus (LCMV) is an underrecognized pathogen of clinical significance in congenital infections and immunocompromised individuals. The lack of FDA-approved vaccines or antivirals for MaAv infections underscores the urgent need for novel anti-MaAv therapeutic strategies. Neutral sphingomyelinase 2 (nSMase2) was recently identified as a host factor contributing to LCMV multiplication, and its inhibitor cambinol exhibits dose-dependent antiviral activity against LCMV, but the underlying mechanisms remain undefined. Here, we show that cambinol disrupts multiple stages of the LCMV life cycle. Cambinol inhibits the pH-dependent fusion event mediated by MaAv glycoprotein, a step required for completion of virus cell entry. It also reduces viral ribonucleoprotein (vRNP)-directed genome replication and transcription and impairs the budding activity of the virus matrix Z protein. Cambinol also inhibits sirtuins 1 and 2 (Sirt-1 and Sirt-2), two NAD⁺-dependent protein deacetylases with pleiotropic roles in cellular metabolism and stress responses, raising the question of whether cambinol anti-LCMV activity reflects nSMase2 inhibition alone or also involves sirtuin-dependent pathways. LCMV multiplication was significantly reduced in SIRT1, but not SIRT2, knockout (KO) cells, uncovering a pro-viral role for Sirt-1 in the LCMV life cycle. Consistent with this finding, LCMV vRNP activity and production of infectious progeny were reduced in SIRT1 KO cells. These findings identify Sirt-1 as a host factor required for optimal LCMV multiplication. Sirt-1 inhibitors are in clinical development for oncological and neurological indications, raising the possibility of repurposing Sirt-1 inhibitors as host-directed antivirals (HDAs) against human pathogenic MaAvs.

Viruses doi: 10.3390/v18101087

Authors: Nailya Klivleyeva Tatyana Glebova Assem Baimukhametova Kobey Karamendin Nurbol Saktaganov Nuray Ongarbayeva Mereke Kalkozhayeva Indira Ibragimova Aknur Mutaliyeva Altynay Gabiden Richard Webby

Background/Objectives: Seasonal influenza threatens global public health through rapid mutations. This study evaluated the prevalence and molecular-genetic characteristics of seasonal influenza viruses circulating in Kazakhstan during the 2023–2026 epidemic seasons. Methods: Nasopharyngeal swabs (n = 1756) and serum samples (n = 718) were screened using RT-qPCR, HI, and ELISA assays. Selected A(H1N1)pdm09 isolates underwent full-length HA and NA gene next-generation sequencing, followed by phylogenetic and phenotypic analyses. Results: Influenza A(H3N2) strains dominated in Kazakhstan during the 2023–2024 and 2025–2026 seasons. Conversely, the 2024–2025 season showed a shifting landscape, characterized by co-circulation of influenza B and A(H1N1)pdm09 strains belonging to genetic clade 6B.1A.5a.2a (subclades C.1.9 and C.1.9.3). Mutational mapping revealed ongoing in situ microevolution adjacent to the receptor-binding site (HA-R222K). Genomic screening of the NA gene identified critical I223R and S247N mutations traditionally linked to resistance. In silico profiling predicted that while the majority maintained baseline sensitivity, the solitary A/Shymkent/NRL-1999/2024 isolate harboring the I223R substitution exhibited a multidrug-resistant genotype to oseltamivir, peramivir, and zanamivir. Conclusions: These findings demonstrate dynamic inter-seasonal etiological shifts and the predicted emergence of an independent resistant lineage (NA-I223R). This underscores the necessity for continuous, expanded genomic surveillance of respiratory pathogens across Kazakhstan.

Viruses doi: 10.3390/v18101086

Authors: Michele Wyler Karin Darpel Rémy Berset Sandra Renzullo Helen Huber Jessica Bauer Judith Peter-Egli Claudia Bachofen Jakub Kubacki

In July 2026, an outbreak of a cattle illness characterized by high fever, diarrhea and reduced milk production, often affecting whole herds, was detected across several regions of northern Switzerland. To investigate the cause of the outbreak, a broad range of diagnostic methods was applied, including pathogen-specific RT-qPCRs, ELISA, serum neutralization test, virus isolation and metagenomic next-generation sequencing. A Shamonda virus (SHAV) was detected in samples from affected cattle, followed by successful virus isolation and complete genome sequencing of all three segments. Viral RNA was also detected in tissue samples from aborted calves, including the brain and spleen, supporting the need for further investigation of the virus in the context of reproductive disease. These findings document the first detection and molecular characterization of a SHAV in Switzerland and demonstrate its widespread circulation in cattle during the 2026 outbreak.

Viruses doi: 10.3390/v18101085

Authors: Liangpeng Suo Haizhen Fan Qiong Wu Xingyang Wei Hanxi Xiao Beibei Fu Xuhu Mao Xiaoyuan Lin

Zika virus (ZIKV), a mosquito-borne member of the Flaviviridae family, is primarily transmitted via the bite of Aedes aegypti mosquitoes but can also be spread through sexual contact and vertical transmission. Infection with ZIKV causes Zika fever and Guillain–Barré syndrome (GBS); more importantly, maternal infection during pregnancy can result in severe neurodevelopmental disorders, such as fetal microcephaly. Although the World Health Organization declared the Zika outbreak a Public Health Emergency of International Concern (PHEIC) in 2016, and extensive research has since been conducted, no approved vaccines or therapeutic drugs are currently available. Recent studies have shown that various ZIKV-encoded proteins participate in host-barrier breaching, regulation of tissue tropism, interference with neurodevelopment, mediation of neurotoxicity, and evasion of type I interferon (IFN-I) immune responses, thereby advancing the development of vaccines and antiviral drugs targeting viral proteins. However, the complete mechanisms by which viral proteins synergistically mediate pathogenesis, the clinical significance of antibody-dependent enhancement (ADE) and its prevention and control strategies, and the practical barriers to clinical translation of protein-targeted interventions have not been systematically elucidated. This review is structured around viral proteins as a central framework: it summarizes the molecular mechanisms by which multiple ZIKV proteins synergistically regulate pathogenic processes; reviews the current research basis and response strategies for flavivirus ADE, and outlines unresolved scientific questions; and discusses progress in vaccines and antiviral drugs targeting viral proteins, with emphasis on evidence gaps and clinical translation barriers. By integrating existing findings and unresolved scientific questions, this review provides a research-gap-oriented theoretical reference for basic ZIKV research and clinical translation of prevention and control strategies.

Viruses doi: 10.3390/v18101084

Authors: Fatma Nur Gazeyoglu Abid Ullah Shah Maged Gomaa Hemida

Avian metapneumovirus (aMPV) is an emerging viral pathogen causing many outbreaks in chickens all over the world. There are several subtypes of the MPV circulating in chickens in the US. The current circulating subtypes of the virus in the US chicken are A, B, and C. Despite the availability of some aMPV vaccines in the US, most of these vaccines are based on foreign strains, particularly European aMPV-A/B strains. Their protective efficacy against contemporary U.S. aMPV isolates has not been fully established. The main goal of this study is to integrate the most recent aMPV genome sequencing data and the machine learning tools to design a novel aMPV. The machine learning tools such as epitope mapping, molecular docking, and immune simulation were used to design the multiepitope DNA vaccine based on the top-ranked epitopes of two major surface proteins of the virus (F and G). The top-ranked seventeen epitopes representing B cells, CD4 and CD8 epitopes were linked using linkers, with IL-18 added as an adjuvant. The selected epitopes showed high antigenicity, no toxicity and no allergenicity values among the screened epitopes. The molecular docking analysis of the designed vaccine construct showed a high binding affinity to the MHC class I and II epitopes to chicken alleles. The immune simulation analysis of this vaccine provides an in silico proof of concept assessment through modeling of the vaccine potential to elicit humoral and cell-mediated immunity. Further functional studies are required to test the immunogenicity and the efficacy of this novel vaccine before applications using chickens and turkey.

Viruses doi: 10.3390/v18101083

Authors: Jennifer L. Cannon Christine Kim Duncan Mahood Brooke A. Bollman Guha Asthagiri Arunkumar Benjamin A. Lopman Katherine B. Carlson

Norovirus is a leading cause of acute gastroenteritis (AGE) worldwide, with significant morbidity and mortality, particularly in young children and older adults. This study characterizes the global prevalence and diversity of norovirus genotypes among sporadic cases. A review of English-language studies (published 2002–2026) that characterized norovirus genotype distribution among sporadic cases was conducted, excluding outbreak and asymptomatic detections. Pooled data were analyzed with stratifications by age group, region, and year of sample collection. A total of 20,588 cases were identified from 72 publications. There were 30 genotypes identified, with GII.4, GII.3, GII.2, GII.17, and GII.6 and each accounting for ≥5% of detections. GII.4 was the predominant genotype globally, accounting for 54% of cases and up to 80% among older adults across all years, but periodic fluctuation was seen, coinciding with fluctuations in the prevalence of other genotypes, especially GII.3, GII.2, and GII.17. Among GI genotypes, GI.3 was most prevalent (46%), particularly among adults. Genotype diversity was highest among children in low- and middle-income countries and in Asia/Oceania. Findings indicate that a small number of genotypes account for most cases. As genotyping characterizes the major capsid protein, the primary target of antibody recognition, findings have important implications for vaccine development and public health interventions.

Viruses doi: 10.3390/v18101082

Authors: Lisumi Hewa Ranchigoda Kankanamge Amal Remtulla Maedeh Naghibosdat Emma Wilson Robert Kozak

Monkeypox virus (MPXV) is an ongoing global public health concern. Historically associated with zoonotic infection, in 2022, a major epidemiological shift occurred when sustained transmission was identified across multiple countries that previously had not had endemic circulation of the virus. While mpox typically presents with characteristic clinical features, infection can also lead to a spectrum of less common manifestations involving multiple organ systems. The extent of viral dissemination and disease severity appear to be modulated by host factors such as immune status, vaccination history, co-infections, and potentially the viral clade. Although complications associated with mpox are infrequent, they can lead to significant morbidity and mortality. Moreover, the underlying pathogenesis and to what extent the viral clade may be playing a role remain incompletely understood. This review summarizes the less common clinical complications of mpox, explores the underlying mechanisms of viral pathogenesis, and discusses what insights can be gained from cell culture and animal models. Additionally, we highlight knowledge gaps that can guide future research priorities.

Viruses doi: 10.3390/v18101081

Authors: Jillian C. Green Greyson A. Moore Matthew D. Irwin Jonathan D. Joyce Andrea S. Bertke

Herpes simplex virus 1 (HSV-1) establishes latency in both sensory and sympathetic neurons. Stress is strongly correlated with HSV-1 reactivation. The acute stress hormone epinephrine initiates HSV-1 reactivation in vitro upon activating at least two adrenergic receptors (ARs), including α2, β1, and/or β2-AR. To determine if blocking these receptors could reduce HSV-1 recurrences, we infected guinea pigs vaginally with HSV-1 (17+) and, after latency was established, treated them daily with atipamezole (specific α2-AR inhibitor), propranolol (nonspecific β-AR inhibitor), or atipamezole/propranolol combined. Clinical recurrences decreased in the treatment groups compared to untreated: atipamezole (29.3%, p = 0.123), propranolol (43.6%, p = 0.021), and atipamezole/propranolol (39.1%, p = 0.021). No significant differences were observed between groups in HSV-1 viral load in pelvic ganglia, or in transcription of the latency-associated transcript (LAT) in guinea pigs that were asymptomatic when euthanized, suggesting AR inhibitors did not affect latency. In guinea pigs euthanized during recurrences, viral DNA was similar between groups in sensory ganglia but reduced in sympathetic ganglia in treatment groups compared to untreated, suggesting that reactivation still occurred in sensory but was limited in sympathetic neurons in AR inhibitor-treated animals. The use of adrenergic beta blockers may be a useful therapeutic to reduce HSV-1 clinical recurrences.

Viruses doi: 10.3390/v18101080

Authors: Natalia Jędruchniewicz Tomasz Klimek Hanna Klimza Krystyna Dąbrowska Małgorzata Wierzbicka

Recurrent respiratory papillomatosis (RRP), caused mainly by human papillomavirus types 6 and 11 (HPV6/11), can persist despite the absence of recognized systemic immunodeficiency. We reanalyzed three transcriptomic datasets using the same predefined immune-gene programs: paired papilloma and autologous laryngeal mucosa from 12 patients, single-cell RNA sequencing from papillomas of four RRP donors and pharyngopalatine mucosa from three controls, and paired papilloma samples from 17 patients before and after INO-3107, a DNA immunotherapy encoding HPV6/11 antigens and interleukin-12. In untreated tissue, cluster of differentiation 8 (CD8)- and natural killer (NK)-associated cytotoxic program scores were lower and the transforming growth factor beta (TGF-β) suppressive program score was higher, reproducing findings from the same cohort. The lower cytotoxic pattern was not reproduced within the recovered combined T-cell and NK-cell population, although no single-cell comparison remained significant after correction for multiple testing. After INO-3107, CD8- and NK-associated cytotoxic, T helper 1 (Th1)/interferon gamma (IFN-γ), and C-X-C motif chemokine receptor 3 (CXCR3)-associated trafficking program scores increased significantly, whereas TGF-β and M2-like suppressive programs showed no comparable statistically supported change. Together, these findings show that different components of the local immune response do not change in parallel and support a model of selective treatment-associated immune remodeling. This pattern may inform future biomarker and combination-treatment studies.

Viruses doi: 10.3390/v18101079

Authors: Daniel A. Mora Trisha Gryder Lauren L. Lessor Jordyn Michalik-Provasek Michael J. Satlin Thomas J. Walsh Jason J. Gill

The emergence and spread of Gram-negative pathogens that are resistant to multiple antibiotic classes pose a global risk to public health. Among such pathogens, carbapenem-resistant strains of Klebsiella pneumoniae (CR-KP) are of concern due to their resistance to treatment by most antibiotics and the mobility of their drug resistance determinants. Bacteriophages that target K. pneumoniae are a promising alternative antimicrobial option. Here, we characterize 30 lytic bacteriophages from various environmental sources using whole-genome sequencing, bioinformatic analysis, virulence assays, and assessment of the effects of media on phage sensitivity. Bioinformatic analysis revealed a diverse collection of phages that span 9 ICTV Caudoviricetes families and 13 genera with genome sizes ranging from 39 kbp to 349 kbp, representing all three tailed phage morphotypes. The phages were all found to be strictly lytic and able to inhibit bacterial growth, and no virulence or antibiotic resistance genes were detected within the phage genomes. Host range testing showed that the culture medium used for plating can have a significant effect on observed susceptibility of some isolates to phages, and phages infecting multiple strains showed greater variation between media. Analysis of phage tail fiber proteins indicated that phages in the collection are predicted to encode from one to nine receptor-binding proteins, with 17/30 phages encoding at least one tail spike with predicted capsular depolymerase activity. The lack of detectable depolymerase domains in 13/30 phages suggests a large portion of the phage collection may recognize non-capsule receptors. This study describes a collection of candidate phages for further development as potential antimicrobial agents against CR-KP and the complexity in understanding phage–host dynamics of phages that recognize non-capsule receptors in K. pneumoniae.

Viruses doi: 10.3390/v18101078

Authors: Anna K. Anderson André Lieber Chang Li

Ex vivo hematopoietic stem cell (HSC) gene therapy strategies have proved effective for the treatment of various diseases, with recent advances leading to the clinical approval of several cell products. To overcome issues associated with the cost and technical complexity of ex vivo strategies, we turn towards in vivo strategies that will allow us to avoid myeloablative transplantation and broaden access. Advances in the engineering of adenoviruses have made them a promising platform for the targeted in vivo delivery of gene therapy vectors to HSCs. This narrative review highlights host barriers to in vivo HSC gene therapy using helper-dependent adenovirus vectors including innate and adaptive immune responses in the blood, virus uptake and sequestration by non-target cells and tissues, difficulty accessing the bone marrow niche, and the inefficient targeting of quiescent HSCs. We present recent advances aimed at overcoming these barriers including immune modulation and vector engineering strategies, improved predictive models, novel HSC targeting strategies, and enrichment of modified HSCs. This forward-looking review highlights a path towards achieving the clinical translation of predictable, safe and accessible in vivo adenovirus-mediated HSC gene therapies.

Viruses doi: 10.3390/v18101077

Authors: Ning Zhu Jincheng Zhong Zengjun Lu Jing Zhang

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important pathogens affecting the global swine industry. This review summarizes current knowledge of PRRSV-induced suppression of adaptive immune responses, with particular emphasis on impaired T cell activity, dysregulated B cell responses, delayed and insufficient neutralizing antibody production, and viral strategies that limit effective immune clearance. In addition, recent advances and limitations in vaccine development and antiviral strategies are presented. By integrating insights from viral pathogenesis and host immune responses, this review provides a comprehensive perspective on current challenges and emerging strategies for effective PRRSV control.

Viruses doi: 10.3390/v18101076

Authors: Ellie Mainou Stella J. Berendam Veronica Obregon-Perko Emilie A. Russell Caroline T. Phan George M. Shaw Katharine J. Bar Mithra R. Kumar Emily J. Fray Janet M. Siliciano Robert F. Siliciano Guido Silvestri Sallie R. Permar Genevieve G. Fouda Janice M. McCarthy Ann Chahroudi Cliburn Chan Jessica M. Conway

Viral dynamics of acute HIV infection and HIV rebound following suspension of antiretroviral therapy may be qualitatively similar but must differ given, for one, the development of adaptive immune responses. Understanding the differences between acute HIV infection and viral rebound dynamics in pediatric populations may provide insights into the mechanisms of viral control with potential implications for vaccine design and the development of effective targeted therapeutics for infants and children. Mathematical models have been a crucial tool to elucidate the complex processes driving viral infections within the host. Traditionally, acute HIV infection has been modeled with the standard model of viral dynamics, initially developed to explore viral decay during treatment, while viral rebound has necessitated extensions of that standard model to incorporate explicit immune responses. Previous efforts to fit these models to viral load data have underscored differences between the two infection stages, such as increased viral clearance rate and increased death rate of infected cells during rebound. However, these findings have been predicated on viral load measurements from disparate adult individuals. In this study, we aim to bridge this gap, in infants, by comparing the dynamics of acute infection and viral rebound within the same individuals by leveraging an infant non-human primate Simian/Human Immunodeficiency Virus (SHIV) infection model. Ten infant Rhesus macaques (RMs) orally challenged with SHIV.C.CH505.375H.dCT were given ART at 8 weeks post-infection. These infants were monitored for up to 60 weeks post-infection to ensure full suppression, then underwent analytic treatment interruption (ATI), resulting in viral rebound. We use the HIV standard viral dynamics model fit to viral load measurements in a nonlinear mixed effects framework to identify primary mechanisms distinguishing acute and rebound infection dynamics. We find that the primary difference between acute infection and viral rebound is the increased death rate of infected cells during the rebound phase. These findings allow us to formulate hypotheses about the effects of adaptive immune responses. Specifically, we propose that delayed viral rebound is characterized by a stronger CD8+ T-cell response. Our analysis provides evidence to support this notion and helps elucidate the observed outcomes.

Viruses doi: 10.3390/v18101075

Authors: Sihan Xia Xueqin Shen Guangyu Chen Ruifang Liu Xin Yang

Chronic hepatitis B virus (HBV) infection remains a leading cause of hepatocellular carcinoma (HCC) worldwide, particularly in Asia. Although nucleos(t)ide analogs suppress viral replication and reduce HCC incidence, residual risk persists, highlighting the need to better understand HBV-driven hepatocarcinogenesis. HBV alters host transcription, DNA repair, lipid metabolism, and the hepatic immune microenvironment. These changes help sustain liver injury, fibrogenesis, and malignant transformation. Ferroptosis, an iron-dependent form of programmed cell death driven by excessive lipid peroxidation, has emerged as an important mechanism in this context, because the liver is central to iron and lipid metabolism. Here, we propose that HBV-driven lipid remodeling shapes ferroptosis susceptibility in a stage-dependent, double-edged manner. During chronic infection, oxidative stress and enrichment of peroxidation-prone lipid substrates may promote ferroptosis-associated hepatocyte injury, immune dysregulation, and fibrosis. After malignant transformation, HBV-related oncogenic programs may instead favor ferroptosis evasion through altered lipid composition and strengthened antioxidant defenses, thereby supporting tumor survival and progression. In this review, we summarize mechanisms linking HBV, lipid metabolic remodeling, and ferroptosis while distinguishing direct HBV-related evidence from findings inferred from other liver disease or cancer models. This HBV-ferroptosis framework may help refine risk stratification and suggest new therapeutic opportunities in HBV-related HCC.

Viruses doi: 10.3390/v18101074

Authors: Bernhard A. Kramer Janin Schokolowski Huib H. Rabouw

Virus infections unfold through spatially organized events whose timing and progression vary among individual cells. Imaging complements population-based approaches by preserving spatial context and, in live experiments, temporal progression at single-cell and tissue scales. This review examines how imaging has advanced our understanding of virus infection, using influenza A virus (IAV) as the principal example. The review is organized around three complementary perspectives: visualization of viral components throughout infection, analysis of virus-induced host-cell and tissue remodeling, and investigation of heterogeneous antiviral responses. Together, these perspectives show how imaging preserves the spatial, temporal, and cell-to-cell context needed to relate viral processes to host-cell responses across scales. Because imaging approaches differ in what they directly measure, this review considers their complementary strengths and limitations and distinguishes descriptive observations from mechanistic conclusions.

Viruses doi: 10.3390/v18101073

Authors: Leyla Genç Hakan Karaca Mehmet Bulut

Hepatitis C virus (HCV) genotype distribution varies across geographic regions and populations and may change over time. This study examined the current distribution and temporal trends of HCV genotypes, and their patterns by demographic characteristics and nationality, at a tertiary care hospital in Istanbul, Türkiye. We retrospectively evaluated 188 patients who underwent HCV genotyping between June 2019 and December 2024. Genotype distribution was assessed according to age, sex, and year, while temporal trends were analyzed using the Cochran–Armitage trend test and binomial logistic regression. GT1b was the predominant subtype (54.8%), followed by GT3 (19.1%), GT1a (10.1%), and GT4 (5.9%); mixed-genotype results were detected in 5.9% of cases. Genotype distribution differed significantly by age group and sex (Monte Carlo p = 0.044 and p = 0.002, respectively), but not by year (p = 0.128). After Holm adjustment, GT1a was more frequent among males, whereas GT1b was more frequent among females. No significant linear temporal trends were observed for GT1a, GT1b, GT3, GT4, or mixed-genotype results. Genotype patterns also varied descriptively across nationality groups. These findings underscore the importance of up-to-date regional data and continued genotype surveillance for monitoring changes in HCV epidemiology.

Viruses doi: 10.3390/v18101072

Authors: Wen Yin Xiaoxu Guo Jie Zhou Guobin Mao Yingxin Ma

Quantum dots (QDs) provide rapid, continuous, and prolonged imaging of individual molecules and viruses, offering more comprehensive dynamic data than traditional fluorophores. However, their application in visualizing viral infections is limited, mostly due to the absence of a mild, efficient, and applicable labeling technique. This study presents a simple and gentle technique for labeling enveloped viruses with QDs, requiring only a single brief incubation phase. This method relies on the inherent attraction of the viral lipid envelope to cholesterol (chol), which is shown on Cadmium telluride (CdTe) QDs via Chol-DNA functionalization. The QDs were synthesized in a single step by the coordination of Cd2+ ions with phosphorothioate-modified DNA strands, maintaining high fluorescence efficiency and small particle size while enabling DNA modification. This labeling technique attains great efficiency in about one hour while maintaining viral structural integrity and infectivity. Employing this lipid-specific technique, this study established dual-color fluorescent SARS-CoV-2 spike-pseudotyped virions by labeling the envelope with QDs and the nucleic acid with Ru[(phen)2(dppz)]2+, facilitating real-time monitoring and analysis of infection dynamics. The established lipid-specific QD labeling technique provides an efficient and versatile tool for visually monitoring viruses and analyzing their infection paths, thus promoting broader utilization of single-virus identification and improving comprehension of complex infection mechanisms.

Viruses doi: 10.3390/v18101071

Authors: Sanda Škrbina Dominik Ljubas Ivana Valenčak Ivana Ferenčak Irena Tabain Srđan Roglić Goran Tešović

Background: Human bocavirus is a non-enveloped DNA virus of the Parvoviridae family, first identified in 2005 and is recognized as a respiratory pathogen across pediatric age groups, with the highest prevalence reported in children aged 6–24 months. Interpretation of HBoV infection is complicated by frequent viral co-detection and the lack of a reliable method to distinguish active infection from prolonged viral sheding. Methods: We analyzed epidemiological trends and viral co-detection rates, along with demographic, clinical, laboratory, and radiological features of patients aged 0–18 years infected with HBoV during a four-year period (2022–2025) at a tertiary pediatric center in Croatia. Results: Of 27,361 respiratory samples collected, 1143 (4.2%) were positive for HBoV. Annual detection rates remained stable (3.6–4.8%), with predominant winter circulation. Viral co-detection was frequent (71.0–82.7%), with adenovirus and rhinovirus being the most commonly co-detected viruses. Pneumonia was diagnosed in 23.7% of patients, with age > 24 months (aOR 2.32; p = 0.012) and higher leukocyte count (aOR 1.13; p < 0.001) recognized as associated factors. Severe disease was uncommon: seven patients required oxygen support, and four were admitted to the PICU. Around one-quarter had gastrointestinal manifestations. Prolonged HBoV detection was observed, with the longest interval being 291 days. Conclusions: This is the largest single-center study of HBoV infection in the general pediatric population in Croatia. HBoV was predominantly detected in toddlers, with frequent viral co-detection, although HBoV-related pneumonia was more common in older children. Further studies are needed to clarify the role of HBoV in respiratory illness.

Viruses doi: 10.3390/v18101070

Authors: Gabriel Schuab Carla de Oliveira Carolina Cardoso dos Santos Laise de Moraes Cintia Damasceno dos Santos Desiree dos Santos Nunes Juan Carlos Proença Moura Felipe Gomes Naveca Fernanda de Bruycker-Nogueira Ana Maria Bispo de Filippis Marta Giovanetti

Dengue virus (DENV) transmission in Brazil is characterized by recurrent epidemics involving the introduction, persistence, and replacement of viral lineages. However, temporal gaps in genomic surveillance limit our understanding of how these processes shape successive epidemics in Rio de Janeiro. Here, in a retrospective observational molecular epidemiology study, we combined archived genomic data with publicly available sequences and epidemiological surveillance data to investigate the temporal dynamics of DENV serotypes and lineages in Rio de Janeiro. Archived DENV-positive samples were curated and subjected to whole-genome amplicon sequencing, and the resulting consensus sequences were analyzed together with publicly available sequences using time-scaled phylogenetic reconstruction and epidemiological data. We recovered 35 DENV genomes from samples collected in 2013, 2018, 2020, and 2021, including DENV-1 genotype V, DENV-2 genotypes II and III, and DENV-4 genotype II. Analysis of the combined datasets identified multiple Rio de Janeiro-associated clades with distinct temporal patterns. Publicly available sequences supported the broader clade structure and long-term temporal patterns independently of most genomes generated here. The newly generated genomes made genotype-specific contributions: the five DENV-1 genotype V genomes placed archived 2020–2021 samples within previously represented Rio de Janeiro-associated groups, while the single DENV-2 genotype II genome provided phylogenetic placement of a travel-associated 2018 case. In contrast, the 20 DENV-2 genotype III genomes identified a Rio de Janeiro-associated cluster not represented among the public sequences, and the nine DENV-4 genotype II genomes increased representation of the 2013 epidemic. Thus, the principal contribution of retrospective sequencing was to fill specific temporal and geographic gaps rather than to redefine the broader historical phylogenetic structure of all genotypes.

Viruses doi: 10.3390/v18101069

Authors: Caleb Paslay Akhtar Ali

Rose rosette virus (RRV), a member of the genus Emaravirus, is responsible for rose rosette disease (RRD), which has significant impacts on the rose industry and ornamental landscapes across the United States (US). To better understand the genetic diversity and population structure of RRV, infected rose leaf samples were collected from garden centers, nurseries, retail stores, and wild rose populations. Total RNA was extracted and analyzed using Sanger sequencing and Illumina-based high-throughput sequencing (HTS), resulting in nearly complete genome sequences of 17 RRV isolates. Phylogenetic and population genetic analyses incorporating >100 publicly available RRV isolates revealed a genetically differentiated lineage comprising eight isolates, seven of which were characterized in this study. This lineage, designated the OK Clade, was consistently supported across the concatenated coding-sequence dataset and several individual genomic segments of RRV. Evolutionary modeling estimated that the divergence between the OK Clade and all other RRV isolates likely occurred more than a century ago. Population genetic analyses further supported substantial differentiation between the OK Clade and other RRV isolates. RRV exhibited greater nucleotide diversity than previously reported, indicating that the genetic diversity of the virus may be broader than previously recognized. Potential recombination breakpoints were also identified in RNA1 and RNA5, although additional analyses are required to validate these events. Collectively, these findings identify a geographically associated and genetically distinct RRV lineage in Oklahoma and provide new insights into the population structure and evolutionary diversity of RRV.

Viruses doi: 10.3390/v18101068

Authors: Pan Wang Yuxi Zhao Yuqiu Yang Juan Zhang Junfang Wu Jiandong Wang

Coronaviruses replicate in membrane-rich cytoplasmic niches while confronting innate immune systems whose activation, signal propagation, and termination are extensively controlled by ubiquitin and autophagy. These two regulatory systems are often discussed separately. Yet they form a coupled information-and-disposal network: ubiquitin chains encode the identity and fate of immune proteins or viral cargo, whereas selective autophagy receptors read these signals and route substrates to lysosomes. Coronaviruses exploit this coupling at several levels. Papain-like proteases remove ubiquitin and ISG15 from antiviral factors; structural, nonstructural, and accessory proteins redirect host ubiquitin ligases or destabilize immune adaptors; and viral proteins remodel autophagosome biogenesis, mitophagy, lysosome function, and selective virophagy. These activities do more than globally suppress immunity. They delay early interferon production, reshape inflammatory signaling, preserve replication organelles, adjust mitochondrial quality control, and protect viral proteins from cargo-selective degradation. Importantly, the outcome is context dependent. Autophagy initiation can support membrane supply or suppress mitochondrial antiviral signaling, whereas completion of autophagic flux can eliminate viral material and restrain damaging inflammation. Likewise, K48-linked ubiquitination may remove antiviral proteins, but nondegradative K63- or M1-linked chains can either assemble antiviral signalosomes or promote pathological NF-κB activity. Here, we synthesize evidence from human and animal coronaviruses to propose that viral immune evasion is governed by spatially and temporally selective rewiring rather than indiscriminate inhibition. We distinguish mechanisms demonstrated during infection from those inferred from ectopic expression, identify points of convergence among RIG-I-like receptor, cGAS-STING, NF-κB, inflammasome, and autophagy pathways, and discuss why apparently contradictory observations often reflect differences in flux, cell type, infection stage, and viral lineage. This integrated view highlights viral proteases, host E3 ligase-substrate interfaces, autophagy receptors, and organelle contact sites as therapeutic nodes, while emphasizing that interventions must preserve the homeostatic functions of ubiquitin and autophagy.

Viruses doi: 10.3390/v18101067

Authors: Mohamed A. Farrag Reem M. Aljowaie Ibrahim M. Aziz Abdulaziz Abdullah Almosa Basel Mohammed Alnafjan Najat A. Y. Marraiki

Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic pathogen that causes severe disease in livestock and humans, with epidemic potential and a capacity for geographic expansion beyond its African endemic range. The 2000–2001 outbreak in Saudi Arabia represented the first confirmed introduction of RVFV outside Africa, yet comprehensive multi-segment genomic characterization of the circulating strains has remained incomplete. Here, we performed an integrated evolutionary and molecular analysis of the three complete-genome Saudi RVFV strains (Saudi 2000-10911, Saudi 200010901, and SA01-1322) using publicly available sequences. Whole-genome comparisons revealed substantial nucleotide and amino acid diversity, with the L segment exhibiting the highest mutation burden (182 nucleotide substitutions, 17 amino acid changes), while the NSs region of the S segment accumulated 52 nucleotide and 11 amino acid mutations compared to the highly conserved nucleocapsid domain. Maximum-likelihood phylogenetic analyses of all three segments consistently placed the Saudi strains within Lineage C (bootstrap = 100/100), clustering with East African strains from Kenya (2006–2007), Tanzania (2007), and South Africa (2008–2010), with congruent topologies across segments confirming the absence of reassortment. Bayesian phylogeographic analysis of 118 L-segment sequences estimated a mean substitution rate of 1.697 × 10−4 substitutions/site/year (95% HPD: 1.5–1.9 × 10−4) and a time to the most recent common ancestor of approximately 1857 (95% HPD: 1835–1876). Selection-pressure analyses using four complementary methods (MEME, FEL, FUBAR, and SLAC) demonstrated pervasive purifying selection across all three segments, with only sporadic episodic positive selection detected at a limited number of codons (six in L, seven in M, and one in S). In silico glycosylation prediction identified conserved N- and O-glycosylation sites on the M segment, with the Saudi strains displaying a Lineage C-typical profile lacking geographically restricted markers. However, the absence of canonical signal peptides in all sequences warrants cautious interpretation of these predictions. Collectively, our findings are consistent with a single introduction of Lineage C RVFV from East Africa among the sampled strains, with no subsequent reassortment, and that the virus has evolved under strong functional constraints with only rare, lineage-restricted episodes of positive selection. The identified Saudi-specific and lineage-defining mutations provide molecular markers for surveillance, while the high conservation of core proteins supports the feasibility of broadly protective vaccines.

Viruses doi: 10.3390/v18101066

Authors: Carlos Andres Rodríguez-Salazar Delia Piedad Recalde-Reyes Santiago Linares-Jiménez Carolina López-Cardoso María Isabel Giraldo

Zika virus (ZIKV) remains a public health concern because of its association with neurological complications and congenital disease, yet no specific antiviral therapy has been approved. Since the envelope (E) protein mediates viral attachment and early entry-associated membrane events, it represents an attractive antiviral target. Here, we used a protein–protein interaction-guided strategy to design peptides to interfere with predicted interfaces between the ZIKV E protein and cellular receptors involved in attachment. After in silico filtering and preliminary screening, six peptides (PZ1–PZ6) were selected. These peptides showed heterogeneous physicochemical profiles, low cytotoxicity and hemolytic activity under the tested conditions, and docking-predicted interactions with functional E protein regions. In HTR-8/SVneo cells, antiviral activity depended on the timing of exposure, with greater reductions in infectious titers during co-treatment and post-treatment. All peptides also reduced titers in anti-adsorption assays, supporting interference with viral attachment or early entry-associated events. Consistently, DiOC18 fluorescence signals were reduced in a peptide-specific manner, particularly for PZ3 and PZ6. Nuclease protection assays showed no extensive exposure of the viral genome, whereas transmission electron microscopy revealed ultrastructural alterations without generalized particle disruption. Overall, PZ1–PZ6 are promising protein–protein interaction-guided anti-ZIKV peptide candidates with activity against early viral entry-associated events.

Viruses doi: 10.3390/v18101065

Authors: Kara L. Gordon Christine G. Rines Laura-Maria Oja Steve Gilmore Lilian Harrison Alyson Smith Randall Ingermanson Vez Repunte-Canonigo Jefferey H. Price Patrick M. McDonough Pietro Paolo Sanna Babu L. Tekwani

HIV-associated neurocognitive disorders (HAND) can persist despite effective ART and are associated with persistent central nervous system (CNS) viral reservoirs in microglia, chronic neuroinflammation, synaptic dysfunction, and neuronal injury. Thus, human-relevant multicellular CNS models are needed to elucidate neuroHIV pathogenesis and ART neurotoxicity. Here, we establish and characterize a fully human induced pluripotent stem cell (hiPSC)-derived CNS tri-culture model comprising isogenic neurons, astrocytes, and microglia that remains viable and functionally active for up to 32 days in vitro. Using a microglia-tropic fluorescent HIV reporter virus, GFP-HIV-AD8, we demonstrate infection of IBA1+ microglia with no detectable infection of IBA1− cells or TUBB3+ neurons. HIV replication was quantified over 14 days post-infection and effectively suppressed by the first-generation integrase strand transfer inhibitor (INSTI)-containing regimen elvitegravir/tenofovir disoproxil fumarate/emtricitabine (EVG/TDF/FTC), and the second-generation INSTI-containing regimens dolutegravir/tenofovir disoproxil fumarate/emtricitabine (DTG/TDF/FTC) and bictegravir/tenofovir alafenamide/emtricitabine (BIC/TAF/FTC), at concentrations based on reported plasma Cmax values. HIV infection reduced microglial viability by approximately 70%, partially rescued by lower in vitro ART concentrations but worsened by the supratherapeutic 3× Cmax condition of DTG-containing regimens. In uninfected cultures, DTG induced microglial morphological changes consistent with a reactive or dysfunctional state. Calcium imaging revealed ART-specific neuronal effects: EVG/TDF/FTC reduced neuronal activity, whereas BIC/TAF/FTC enhanced it. In HIV-infected cultures, DTG shortened neuronal spike duration under viral suppression, identifying an HIV–DTG-associated electrophysiological phenotype. Overall, BIC/TAF/FTC showed the most favorable profile across the cellular and electrophysiological endpoints assessed in this donor line. Together, these findings establish an hiPSC-derived CNS tri-culture model for controlled studies of neuroHIV virology, ART effects, and neuronal function.

Viruses doi: 10.3390/v18101064

Authors: Armando Busqueta-Medina Ramiro Ávalos-Ramírez Diana Elisa Zamora-Ávila Víctor Eustorgio Aguirre-Arzola Juan Francisco Contreras-Cordero Domenica Cedillo-Rosales Sibilina Cedillo-Rosales

The occurrence of acute hemorrhagic diarrhea in dogs has prompted investigation of emerging enteric viruses, including Bocaparvovirus carnivoran 1 (CBoV-1), which has been associated with respiratory, gastrointestinal, and reproductive disorders in dogs. In this study, CBoV-1 was successfully isolated in MDCK cells from a clinical sample obtained from a dog with acute hemorrhagic diarrhea. A CBoV-1 genome sequence (4973 bp) was obtained from isolate MX-ABM30 and deposited in GenBank under accession number PQ683622. Phylogenetic analysis revealed a close genetic relationship between MX-ABM30 and CBoV-1 sequences from the United States, with 98% nucleotide identity. A comparative analysis identified six missense mutations across the genome, resulting in four amino acid substitutions in NS1 (K176R, K182N, P669S, and I680L) and two in VP2 (S200G, S536A). This study represents the first report of isolation and characterization of CBoV-1 in Mexico, providing preliminary data on the genetic diversity of this virus in the region. These findings highlight the need for continued surveillance of CBoV-1 and other emerging enteric viruses in canine populations.

Viruses doi: 10.3390/v18101063

Authors: Jia Yi Jessica Morel Mickaël Costallat Nathalie Lejal Bernard Delmas Christian Muchardt Eric Batsché

The influenza A virus hijacks host cell machinery by “snatching” molecular caps from cellular RNAs to prime its own transcription. While this process is typically associated with the beginning of genes, this study reveals that the viral RNA polymerase exerts a far-reaching influence on host gene expression. Using genome-wide mapping and comparative pathway analyses, we show that viral polymerase (FluPol) recruitment is decoupled from general host transcriptional activity; instead, FluPol is markedly enriched at a distinct subset of promoters and enhancers associated with defense-related pathways. Beyond the initiation site, FluPol remains associated with host transcripts throughout elongation, extending into downstream-of-gene (DoG) regions. It further accumulates at gene ends, potentially contributing to widespread defects in transcription termination. By remaining linked to host transcripts across the entire gene body, FluPol could extend its opportunity for cap-snatching and impair the recycling of cellular transcription machinery. Together, these findings demonstrate how FluPol accumulation across key cellular defense loci spans from initiation through to termination, driving host transcriptional attenuation.

Viruses doi: 10.3390/v18101062

Authors: Shilpa Deshpande Kaistha Barry T. Rouse L. C. Rodrigues Junior

Aging is accompanied by numerous interlinked cellular, genetic, and molecular changes, one of the most consequential being inflammaging: a chronic, low-grade systemic inflammatory state linked to metabolic dysregulation and pleiotropic effects on immune function. Although diverse intrinsic and extrinsic factors are known to drive inflammaging, the contribution of persistent viral infection remains incompletely understood. Here, we evaluate evidence that human herpesviruses, including cytomegalovirus (CMV), Epstein–Barr virus (EBV), herpes simplex viruses (HSV-1/2), and varicella zoster virus (VZV), may act as chronic antigenic and inflammatory stimuli capable of amplifying inflammaging and shaping the trajectory of aging. We review the molecular, cellular, and systems-level mechanisms by which herpesvirus latency and periodic reactivation sustain innate and adaptive immune activation, promote cellular senescence, and contribute to immune dysregulation and exhaustion. We further examine epidemiological evidence linking herpesvirus infection to dementia and other age-associated outcomes and highlight emerging data suggesting that antiviral treatment and vaccination, such as against VZV, may mitigate these effects. We conclude that herpesviruses are best understood as adjuvant contributors, rather than primary drivers, of inflammaging, and propose that persistent herpesvirus infection be considered an addressable risk factor in strategies aimed at promoting healthy aging.

Viruses doi: 10.3390/v18101061

Authors: Patcharin Siringan Panukar Malawan Kanhatey Kong

Bacterial plant diseases caused by the genus Pantoea have emerged as severe threats to global rice cultivation, causing destructive conditions such as bacterial leaf blight and grain rot. Due to increasing antibiotic resistance and the environmental hazards of chemical pesticides, bacteriophage-based biocontrol offers a promising, eco-friendly alternative. In this study, we isolated and characterized novel bacteriophages from infected rice paddies in Thailand, using a newly identified host, Pantoea sp. isolate 2 based on 16S rRNA gene sequencing. Transmission electron microscopy revealed that the phages belong to the class Caudoviricetes: phage vB_PS-10 displays a siphovirus-like morphotype, whereas phage vB_LX1 exhibits a myovirus-like morphotype. One-step growth analysis of vB_PS-10 uncovered a biphasic propagation pattern with a latent period of 30 min and a burst size of 10 PFU/cell. In vitro lytic assays demonstrated that vB_PS-10 significantly suppressed bacterial growth, achieving a 4 log CFU/mL reduction in host cells after 12 h. Stability assays revealed that vB_PS-10 is highly stable across a broad pH range (6.0–10.0) and resilient at tropical field temperatures (35–45 °C), though sensitive to UV-C radiation and direct sunlight. Finally, a detached leaf assay validated its in planta biocontrol efficacy, showing that phage vB_PS-10 exhibited a significant reduction in disease severity, restricting lesion development to just 13.8% ± 4.37%, whereas the pathogen-only control group showed near-total lesion coverage after 7 days. These findings demonstrate that phage vB_PS-10 has robust potential as a sustainable biological control agent to manage emerging Pantoea diseases in rice production.

Viruses doi: 10.3390/v18101060

Authors: Anton Gerilovych Ivan Rusev Ihor Korovin Anastasiia Rusanova Gregory E. Glass

Wild migratory birds are the principal natural reservoir of influenza A viruses (IAVs) and play a central role in their evolution and global dissemination. Ukraine lies along the Black Sea–Mediterranean Flyway, making surveillance of avian influenza viruses in wild birds essential within a One Health framework, although genomic data from the region remain limited. Between October 2025 and January 2026, 604 cloacal and oropharyngeal swabs and environmental fecal samples were collected from migratory birds and mixed-species flocks in the Tuzlivski lymany National Nature Park and surrounding areas, together with samples from nearby domestic poultry. Influenza A virus was detected by real-time RT-PCR targeting the matrix gene, followed by H5/H7 screening, Oxford Nanopore sequencing of complete hemagglutinin genes, and phylogenetic analysis. Influenza A virus RNA was identified in 30/604 samples (4.97%), whereas all samples were negative for H5 and H7 subtypes. Complete hemagglutinin sequences revealed three low-pathogenic subtypes (H1, H4, and H6) that clustered within established Eurasian lineages closely related to contemporary avian viruses circulating across Europe, Asia, and North America. No influenza A virus or Newcastle disease virus was detected in domestic poultry. These findings provide an important genomic baseline for avian influenza surveillance in Ukraine and demonstrate the value of portable nanopore sequencing and integrated One Health surveillance for monitoring viral diversity and supporting early detection of emerging influenza threats.

Viruses doi: 10.3390/v18101059

Authors: Xiangtao Zhu Hongyu Yan Wanhui Zhou Haiyu Su Xiumei Huang Chongxian Deng Yuxing Li Yali Wang Yanyan Chang Wenping Yang Haixue Zheng

African swine fever virus (ASFV) is a highly pathogenic agent that poses a severe threat to the global swine industry. The biological function of A859L, an ASFV-encoded RNA helicase, remains largely elusive. Here, we show that A859L is highly conserved across diverse ASFV isolates and belongs to the SF2 superfamily of RNA helicases, possessing characteristic Walker A (GKT) and Walker B (DECH) motifs. Transcriptomic profiling at multiple time points post-infection further revealed that A859L is a late-expressed gene. Functional assays demonstrated that siRNA-mediated knockdown of A859L significantly inhibited progeny virus production in both susceptible macrophages (PAMs and BMDMs) and WSL cells. This knockdown also markedly reduced the expression of viral proteins p30 and p72, as well as the intracellular mRNA levels of other viral helicase genes (QP509L, Q706L, D1133L, and B962L), underscoring the vital role of A859L in the viral life cycle. Conversely, A859L overexpression enhanced viral propagation. Notably, pharmacological inhibition of viral helicase activity effectively suppressed ASFV replication. Collectively, our data establish A859L as a critical determinant of ASFV replication and a promising candidate for antiviral development.

Viruses doi: 10.3390/v18101058

Authors: Xiaoxuan Xu Dian Wang Guiwen Guan Guochao Wei Linfeng Wu Heng Liu Ran Li Haobiao Wang Tingting Chen Fengmin Lu Yuanye Xue Xiangmei Chen Jinfeng Zeng

Background: Blood transfusion is a major route of Human T-lymphotropic virus (HTLV) transmission. In this study, we conducted a cross-sectional study of HTLV seroprevalence among blood donors in Shenzhen, with secondary investigations covering signal-to-cutoff (S/CO) predictive value, longitudinal follow-up, epidemiological characteristics, and possible familial clustering. Methods: Retrospective analysis was conducted on 512,200 Shenzhen Blood Center donors (2014–2022, with partial 2013 data). ELISA was utilized for initial HTLV screening, with MP, INNO-LIA and qPCR for confirmation. Confirmed cases received questionnaire follow-up and retesting. Some relatives of confirmed cases also received HTLV-1/2 testing. Results: Among 512,200 donors, 203 were screening-reactive, with 18 confirmed-positive cases (3.51 per 100,000 donors; 95% CI: 2.08–5.55) and 10 indeterminate cases. The screening-reactive rate declined significantly over the decade (p < 0.001), whereas the confirmed-positive rate remained low and showed no significant temporal trend (p = 0.363). Among initially screening-reactive donors, high S/CO values strongly predicted confirmatory positivity, whereas low-level reactivity was mostly transient. Questionnaire data were available for 9 of the 18 confirmed-positive donors (50.0%) and 51 control donors. In this exploratory analysis, only invasive medical procedures occurring in Fujian remained significantly associated with infection (adjusted p = 0.010). Two of the 10 tested family members were confirmed-positive, suggesting possible familial clustering. Conclusions: The confirmed prevalence of HTLV among Shenzhen blood donors was extremely low, and screening reactivity declined significantly over the ten-year period. Screening S/CO values strongly predicted confirmatory results. Fujian-related invasive medical procedures and possible familial clustering are exploratory findings that require confirmation in larger studies. Continued donor screening and blood safety surveillance remain warranted.

Viruses doi: 10.3390/v18101057

Authors: Mustafa Usanmaz Özgür Günal Burcu Özdemir Esra Arslan Aksu Savaş Gegin Muhammed Abdullah İkra Nur Gergin Furkan Ata Merve Meryem Öztürk Meltem Karslıoğlu Esmeray Mutlu Yılmaz Ayşe Şenbaba Kara Ayşe Banu Ceviz Levent Özdemir

Pandemic non-pharmaceutical interventions disrupted seasonal respiratory virus circulation, but longer-term pathogen-specific patterns in hospitalized adults remain incompletely defined. We evaluated detections across three nonconsecutive respiratory seasons from early relaxation of pandemic measures to five years after pandemic onset. This retrospective, observational, single-center cohort study included 2249 adults hospitalized with pneumonia, chronic obstructive pulmonary disease exacerbation, or asthma exacerbation who underwent multiplex polymerase chain reaction testing in September 2021–May 2022, September 2022–May 2023, or September 2025–May 2026. Influenza A positivity peaked in the second period (12.6%) and decreased to 6.8% in the third (p < 0.001). SARS-CoV-2 likewise peaked in the second period (9.2%) and declined to 1.5% in the third (p < 0.001). Respiratory syncytial virus increased from 0.5% in the first period to 4.9% and 3.8% in the second and third periods, respectively (p < 0.001). Positivity for other respiratory viruses did not differ (16.0%, 16.9%, and 19.1%; p = 0.308). Viral co-detection was highest in the second period (6.1% vs. 2.6% and 2.8%; p < 0.001). Respiratory virus epidemiology remained dynamic five years after pandemic onset, supporting sustained multi-pathogen surveillance.

Viruses doi: 10.3390/v18101056

Authors: Yhiya Amen Marwa Elsbaey George Mpekoulis Evangelia Christou Helena Tsimpili Niki Vassilaki Kuniyoshi Shimizu Grigoris Zoidis

Natural products remain indispensable scaffolds in antiviral drug discovery, particularly against high-burden viral pathogens. In this study, two major sesquiterpene lactones, dehydrocostus lactone (1) and costunolide (2), were isolated from Laurus nobilis L. leaves and evaluated for their antiviral potential against hepatitis C virus (HCV) and dengue virus (DENV). The structures of both compounds were confirmed by HRMS and 1D NMR spectroscopic analyses. In subgenomic replicon assays, the molecules inhibited HCV replication in the low-micromolar range (EC50=2.34 μM for 1 and 2.06 μM for 2) with favorable selectivity indices. In full-length HCV reporter systems, costunolide (2) displayed enhanced anti-HCV potency, reduced viral RNA and NS5A protein levels, and enhanced the suppressive effect of the clinically approved NS5A inhibitor, daclatasvir. Furthermore, costunolide (2) exhibited cross-flaviviridae activity, suppressing DENV replication (EC50=5.89 μM in replicon cells; EC50=1.27 μM post-infection in infectious virus assays), and demonstrated cooperative suppression of viral propagation along with reduced glutathione. Mechanistically, these results highlight the critical role of the sesquiterpene lactone core, and the observed enhanced combined activity between costunolide and reduced glutathione is consistent with a possible contribution of the NRF2/glutathione antioxidant axis to its anti-DENV action. Overall, this study highlights L. nobilis as a previously underexplored source of antiviral sesquiterpene lactones and establishes costunolide (2) as a promising lead against HCV and DENV, warranting further investigation across additional Flaviviridae members.

Viruses doi: 10.3390/v18101055

Authors: Xiaorong Chen Zhishui Zhang Hanyu Cheng Siyuan Qi Pengfei Wang

The global dissemination of coronaviruses continues to pose formidable threats to public health and socioeconomic development, making these viruses a major research priority. Porcine deltacoronavirus (PDCoV), an emerging porcine enteric virus identified over the past two decades, has resulted in substantial economic losses to the swine industry owing to its high mortality and persistent circulation across the globe. Beyond swine, PDCoV possesses a broad host spectrum and is capable of infecting avian species, mice, calves, and other animals. Most alarmingly, its zoonotic potential was highlighted in 2021 through the discovery of PDCoV genomic RNA in pediatric samples from Haiti, highlighting its potential to become the eighth coronavirus species known to infect humans. Despite these dual veterinary and public health threats, licensed vaccines and specific human therapeutics remain unavailable. These facts highlight an urgent demand for comprehensive and in-depth studies on PDCoV to mitigate its potential risks and forestall future outbreaks. This comprehensive review summarizes the latest research advances in PDCoV in four aspects: biological characteristics, epidemiology, pathogenic mechanisms, and antibody development. It is expected that this review can provide a robust theoretical basis for understanding PDCoV infection dynamics and accelerating the development of next-generation, broad-spectrum antiviral strategies to mitigate future interspecies spillover events.

Viruses doi: 10.3390/v18101054

Authors: Nicolas E. Reid Xiangqun Ju Joanne Hedges Sneha Sethi Lisa M. Jamieson

Background: Human papillomavirus (HPV) infects skin and mucous membranes, and high-risk types are associated with oropharyngeal and other cancers. Longitudinal evidence on oral HPV among Aboriginal and Torres Strait Islander peoples remains limited, particularly across geographic settings. Methods: A longitudinal cohort study was conducted among 1011 Indigenous Australian adults in South Australia recruited through Aboriginal Community Controlled Health Organisations. Participants provided saliva samples and completed questionnaires at baseline (2018–2019) and at 12- and 24-month follow-up. Oral HPV was detected using PCR-based assays. Incidence, persistence, and clearance were estimated, and multivariable regression examined associations with behavioural, sexual health, and socioeconomic factors. Results: Oral HPV dynamics differed between metropolitan and non-metropolitan participants. Approximately one-quarter developed an incident infection, with geographic variation in persistence and clearance. Smoking, sexual behaviours, and socioeconomic factors were associated with HPV outcomes. Older age was associated with persistence among non-metropolitan participants, while smoking was associated with higher incidence among metropolitan participants. Conclusions: Oral HPV detection dynamics varied by geographic setting. Culturally appropriate, targeted public health strategies addressing behavioural and social determinants are needed to reduce HPV-related disease burden.

Viruses doi: 10.3390/v18101053

Authors: Chaoran Zhuo Dongdong Cao Fangling Li

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an important human gamma-herpesvirus, particularly with a significant incidence rate among AIDS patients and other immunosuppressed populations. This review introduces the basic virological characteristics of KSHV and elucidates the core molecular mechanisms underlying KSHV tumorigenesis. Clinical management primarily focuses on KSHV-related diseases, utilizing antiretroviral therapy, chemotherapy, radiotherapy, and therapies targeting specific targets. However, there still lack of specific anti-KSHV drugs in terms of treatment. Current research is increasingly directed toward novel therapeutic strategies, including those that sustain viral latency or promote lytic reactivation, alongside antigen-based immunotherapeutic approaches. Despite significant breakthroughs in research, the precise regulatory network of KSHV latent infection, the complex interactions between the virus and the host microenvironment (especially inflammatory and immune cells), as well as the development of highly effective and low-toxicity specific antiviral drugs, remain major challenges for the future. A deep understanding of the pathogenic mechanisms of KSHV will lay a solid foundation for the development of more effective prevention, diagnosis, and treatment methods.

Viruses doi: 10.3390/v18101052

Authors: Rujun Ye Ruihan Shi Haijun Jiang Chang Liu Mengzhu Dong Hairong Tian Tingwen Zheng Yu Zhou

In this study, we evaluated the replication kinetics and protective efficacy of the gene-deleted Marek’s disease (MD) vaccine strain SC9-1 in specific-pathogen-free (SPF) chickens immunized with different doses. Chickens were divided into two groups: one group received a high dose (5000 PFU) and the other a low dose (500 PFU) of the vaccine at 1 day of age, with a non-vaccinated group serving as the control. Vaccine viral copy numbers in the spleen and feather tips were detected by quantitative PCR (qPCR) at different days post-vaccination (dpv) (3, 5, 7, 11, and 14 dpv). Meanwhile, a challenge trial was conducted where chickens were challenged with the very virulent Md5 strain (1000 PFU/bird) at 5 dpv and observed until 70 days of age. Results from the vaccination-only experiment revealed that viral replication in the spleen was detectable in 100% of chickens as early as 3 dpv, reaching its peak at 5 dpv, with the high-dose group exhibiting significantly higher viral loads than the low-dose group. In feather tips, replication was detectable in 100% of chickens by 5 dpv and peaked at 7 dpv, but no significant difference was observed between the two dose groups. The unvaccinated control group exhibited 100% mortality with characteristic gross and histopathological lesions of Marek’s disease. In stark contrast, both vaccinated groups (high and low dose) demonstrated 100% survival until the end of the observation period (70 days). No clinical signs or pathological abnormalities were observed in these groups. Furthermore, vaccination with SC9-1 did not adversely impact growth performance and the development of major immune organs, even following subsequent Md5 challenge. The results indicated that a higher initial vaccination dose of the SC9-1 vaccine was associated with elevated peak early in vivo replication level, whereas vaccination dose may not govern viral maturation and shedding levels of SC9-1 in feather-tip tissues. However, peak replication level was not a direct determinant of final protective efficacy. Notably, even the lowest tested dose (500 PFU) afforded full protection against early challenge with a very virulent MDV strain. Collectively, these observations contribute to our understanding of dose-dependent protection induced by gene-deleted MD vaccines and inform rational vaccination strategies.

Viruses doi: 10.3390/v18101051

Authors: Victor J. Vera Ponce Jhosmer Ballena-Caicedo Julio David Sagastegui Jauregui Nelly Del Carmen Villegas Ampuero Fiorella E. Zuzunaga-Montoya

Dengue transmission has expanded across the Americas, but the spatiotemporal distribution of mortality recorded in death certificates and its ecological relationship with climate remain insufficiently characterized. Objective: To evaluate dengue-associated mortality recorded in Peruvian death certificates during 2017–2024, characterize geographic emergence across fixed provinces, estimate associations with local climate anomalies, and triangulate findings with epidemiological surveillance. Methods: We conducted a retrospective ecological spatiotemporal study combining the National Death Information System, national dengue surveillance, official population projections, gridded climate data, and a fixed 196-province framework. The primary outcome was an A90 or A91 code in any certificate position; province-month was the principal unit. Poisson models included a population offset, provincial fixed effects, calendar month, linear time, a 2020–2021 indicator, and temperature and precipitation anomalies at lags 0–3 months. Results: The updated snapshot contained 987 certificates with A90/A91 during 2017–2024; 968 were territorially eligible. Thirty-six provinces were classified as emergent. Surveillance contributed 772,387 notified cases, and department-year deaths and cases were correlated (Spearman ρ = 0.772). In the corrected primary model, the cumulative ecological mortality rate ratio comparing the 90th with the 50th percentile was 1.75 (95% CI: 1.41–2.17) for mean-temperature anomalies and 2.69 (95% CI: 1.97–3.67) for precipitation anomalies. Estimates remained positive under position-A, code-specific, syndromic, and temporal sensitivity definitions. Conclusions: Recorded dengue-associated mortality was concentrated during recent outbreaks and appeared in provinces without baseline-period mortality. Local climate anomalies preceded higher province-month mortality rates, but these ecological associations are compatible with indirect transmission and health-system pathways and do not establish direct individual-level effects.

Viruses doi: 10.3390/v18101050

Authors: Lia Nadareishvili Nata Nakaidze Mariam Dadiani Dea Nizharadze Elisabed Zaldastanishvili Nana Balarjishvili Lika Leshkasheli Mzia Kutateladze

Cystic fibrosis (CF) is an autosomal recessive genetic disorder that disrupts ion transport across epithelial surfaces, impairing mucociliary clearance and predisposing patients to chronic, often refractory bacterial infections. At the Eliava Phage Therapy Center (EPTC) in Tbilisi, Georgia, we utilize bacteriophage preparations to treat chronic pulmonary infections caused by various bacterial pathogens, including Staphylococcus aureus, Pseudomonas aeruginosa, Achromobacter xylosoxidans, and Burkholderia cepacia. This case report describes the successful treatment of a CF-related infection in a male patient with a ΔF508 mutation using Pyophage and Intestiphage preparations, as well as customized phages developed against specific P. aeruginosa isolates. The efficacy of phage therapy was assessed by evaluating clinical improvement, pulmonary function, inflammatory markers, and bacterial burden. We observed improvement across all assessed parameters, including a complete eradication of P. aeruginosa in sputum culture tests. This case highlights the potential of phage therapy as a primary antimicrobial approach for the long-term management of pulmonary infections in patients with CF.

Viruses doi: 10.3390/v18101049

Authors: Raíssa Martins Deodato Mayara Torquato Lima da Silva Diego Allonso Erik Machado Ferreira Julia Trece Marques Possas Jurema Corrêa da Mota Livia Melo Villar

Hepatitis C virus (HCV) infection poses a major challenge to global public health, highlighting the need for accessible and reliable diagnostic tools to support large-scale screening and early detection. In this study, a recombinant multi-epitope HCV protein was designed, expressed, and evaluated as a diagnostic antigen for the detection of anti-HCV antibodies. The synthetic gene was constructed using immunodominant regions (core, NS3, NS4, NS5A, and NS5B) representative of HCV genotypes 1a, 1b, and 3a. The recombinant protein was expressed in Escherichia coli and purified by affinity chromatography, yielding a soluble protein with an approximate molecular weight of 43 kDa and antigenicity confirmed by Western blot analysis. An enzyme-linked immunosorbent assay (ELISA) was developed in-house using the recombinant protein as the capture antibody. Diagnostic performance was evaluated using a panel of 260 human serum samples, including 127 anti-HCV reactive and 133 anti-HCV non-reactive specimens, and receiver operating characteristic (ROC) curve analysis. The assay demonstrated excellent performance with an area under the curve (AUC) of 0.968, sensitivity of 86.6%, specificity of 98.5%, and overall accuracy of 92.7%. Diagnostic performance was evaluated using receiver operating characteristic (ROC) curve analysis. The assay demonstrated excellent performance in serum samples, with an area under the curve (AUC) of 0.968, a sensitivity of 86.6%, and a specificity of 98.5%. These results demonstrate that the recombinant multi-epitope antigen represents a robust and scalable biomolecular platform for HCV serodiagnosis, contributing to strategies that expand access to hepatitis C screening.

Viruses doi: 10.3390/v18091048

Authors: Evelyn Van de Perre Brieuc Van Nieuwenhuyse Jean Cyr Yombi

While typically associated with benign infectious mononucleosis, Epstein–Barr virus (EBV) is now recognized as a pivotal driver of complex immunopathological disorders. By shuttling between lytic replication and strategic latency programs, EBV reprograms B-cell biology to evade immune surveillance and disrupt homeostasis. Central to this pathogenesis is the viral protein EBNA1, which triggers autoimmunity through molecular mimicry with host antigens, including GlialCAM in multiple sclerosis (MS), keratin in rheumatoid arthritis, and nuclear proteins in systemic lupus erythematosus. These autoimmune responses are further exacerbated by bystander activation and epitope spreading, fueling chronic inflammation in Crohn’s disease, type 1 diabetes, and IgA nephropathy. EBV can also promote the development of lymphoproliferative disorders, whether in immunocompetent hosts or in the context of immunosuppression; however, the emergence of EBV-positive forms within diseases that are usually EBV-negative is closely linked to an underlying state of immunosuppression. Distinct from these chronic states, EBV-associated hemophagocytic lymphohistiocytosis (HLH) represents a critical hyperinflammatory emergency in which defective cytotoxicity leads to an uncontrolled T-cell- or NK-cell-driven cytokine storm; together with chronic active EBV disease (CAEBV), HLH disproportionately affects children, and both receive particular attention in this review. More recently, emerging but not yet consolidated evidence has also suggested a possible association between EBV reactivation and long COVID. Elucidating these diverse molecular interactions is essential for developing the next generation of targeted therapies and vaccine strategies aimed at mitigating the systemic impact of this virus.

Viruses doi: 10.3390/v18091047

Authors: Julia Gąsiorowska Maciej Mateusz Hanypsiak Natalia Jendrzejaczyk Artur Ams Jan Rafał Falana Karol Zagórski Katarzyna Rekiel Kinga Brawańska-Maśluch Pola Nowacka Wiktoria Żabińska Grzegorz Mikita Piotr Paweł Chmielewski Krzysztof Data Paul Mozdziak Bartosz Kempisty Paweł Dąbrowski

Objective: This review aims to describe the macroscopic methods used to detect poliomyelitis in human skeletal remains and to highlight the limitations of current diagnostic frameworks. Materials: The reviewed materials consist of published paleopathological literature and case studies analyzing human skeletal remains for evidence of the disease. Methods: We evaluated existing studies that focus on the macroscopic analysis of secondary skeletal alterations caused by paralysis, such as unilateral limb atrophy, bone shortening, pronounced asymmetry, and compensatory hypertrophy. Results: Current diagnostic frameworks rely almost exclusively on macroscopic markers; notably, none of the reviewed studies utilized molecular or genetic techniques. Consequently, definitively confirmed cases of ancient poliomyelitis remain exceptionally rare. Conclusions: The paleopathological diagnosis of poliomyelitis currently suffers from a substantial methodological gap due to an overreliance on morphological assessments. Transcending these traditional techniques is essential to definitively confirm ancient cases. Significance: Analyzing skeletal evidence provides crucial insights into how this debilitating viral infection afflicted human populations in antiquity. It also underscores the importance of rigorous differential diagnosis to distinguish poliomyelitis from other paralytic etiologies, such as cerebral palsy.

Viruses doi: 10.3390/v18091046

Authors: Kyla Nel Maria Antonia Papathanasopoulos Adriaan Erasmus Basson

Islatravir is a novel nucleoside reverse transcriptase translocation inhibitor recently approved for the treatment of HIV-1 infection in virologically suppressed PLWH. While we previously documented high-level phenotypic resistance to islatravir (ISL) in HIV-1 subtype C variants harboring combinations of type 2 thymidine analogue mutations (TAM2) along with the M184V resistance-associated mutation, the primary contributor to the observed phenotype remains unclear. This report dissects the relative contribution of M184V to ISL resistance in the context of TAM2-containing variants through systematic reversion analysis. We show that reverting mutant 184V to wildtype M184 resulted in substantial sensitization to ISL, with fold-change reductions ranging from 8.1 to 13.4-fold across three TAM2 genetic backgrounds. Contrary to the well-documented antagonism between M184V and TAMs in zidovudine resistance, these two resistance pathways exhibit cooperative rather than antagonistic interactions in the context of ISL resistance. These findings provide critical evidence for refinement of genotypic resistance interpretation algorithms for ISL and have direct implications for the future clinical management of ISL-based antiretroviral therapy in people living with HIV. Importantly, elucidating the mechanistic basis of M184V-TAM2 cooperation would establish a more predictive framework for understanding how complex resistance mutation patterns impact ISL susceptibility and guide treatment decisions in virologically experienced individuals. Considering the diminished use of TAM2-seletive antiretroviral drugs in contemporary ART regimens, islatravir is likely to be an effective treatment option.

Viruses doi: 10.3390/v18091045

Authors: Alhassan Alrafaie Nasser Alqurainy

Enterobacter cloacae (E. cloacae) is an important healthcare-associated pathogen with increasing antimicrobial resistance. Prophages and phage-inducible chromosomal islands (PICIs) are integral components of the E. cloacae genome, yet their contribution to resistance, virulence, and anti-phage defense remains poorly understood. To address this gap, we analyzed 35 complete E. cloacae genomes, identifying 139 intact prophages (10.8–85.7 kb) with an average GC content (~52%) close to that of the host (54–55%). Most prophages (86.6%) were assigned to the families Peduoviridae and Drexlerviridae within the class Caudoviricetes, and proteome-based clustering showed strong relatedness to phages infecting Enterobacteriaceae. The E. cloacae genomes harbored multiple antimicrobial resistance genes, notably fosA (97%), oqxA/oqxB (89%) and blaCMH-3 (77%). However, on genomic-context verification none were genuinely prophage-encoded; they were instead located on the chromosome or plasmids. Virulence profiling revealed universal presence of ompA and high prevalence of csgG (94%), while the only confirmed prophage-encoded virulence determinants were the Shiga toxin genes stxA and stx1B. Additionally, 16 phage satellites were identified, containing conserved integration, replication, and packaging modules together with diverse accessory and anti-phage defense systems. Overall, prophages and phage satellites drive genome diversification in E. cloacae, with satellites notably serving as reservoirs of anti-phage defense, whereas resistance and virulence genes are located outside prophage and satellite regions.

Viruses doi: 10.3390/v18091044

Authors: Jiakai Li Shuo Li Jie Han Rui Zhou Zili Li

Porcine reproductive and respiratory syndrome virus (PRRSV) has the characteristics of a fast evolution rate and high genetic heterogeneity. PRRSV-1 and PRRSV-2 are co-circulating, and the threat they pose to the health of pigs worldwide continues to rise. In this study, two PRRSV strains with significantly different genetic backgrounds, namely SHEU-2022 (PRRSV-1, full length 15,050 nt) and SHCH-2023 (PRRSV-2, full length 15,019 nt), were simultaneously isolated from a single serum sample of a co-infected sow in Shanghai, China, and their whole-genome characteristics were analyzed. SHEU-2022 clustered with indigenous PRRSV-1 isolates in China, harbored a unique 15-nt deletion in the ORF3-ORF4 overlapping region, carried an additional N-glycosylation site on GP5, and replicated only in porcine alveolar macrophages (PAMs) under the conditions used. SHCH-2023 is a novel recombinant strain with a CH-1a-like (lineage 8) genome backbone and two NADC30-derived fragments—one in Nsp2 (sublineage 1.8) carrying the characteristic 131-amino acid deletion and one in the GP2/E region; it replicated in both PAMs and Marc-145 cells, though this may partly reflect the enrichment procedure. No PRRSV-1/PRRSV-2 recombination was detected between the two co-infected strains.

Viruses doi: 10.3390/v18091042

Authors: Ana Karina Nisperuza Vidal Chenxiao Wang Michaela J. Allen Xiaofeng Ding Jefferson Fernandes Evangelista Yilin Chen Mst Shamima Khatun Micaela R. Boxer Wachler Calder R. Ellsworth Mohammad Islamuddin Robert Blair Jay K. Kolls Derek A. Pociask Xuebin Qin

Influenza A virus (IAV) remains a major cause of respiratory morbidity and mortality worldwide. However, the precise role of the RNA sensor Toll-like receptor 7 (TLR7) and its downstream signaling mediator, interferon regulatory factor 7 (IRF7), during IAV infection remains elusive. To address this gap, we utilized single-cell RNA sequencing (scRNA-seq) alongside Tlr7-deficient and Irf7-deficient mouse models infected with the mouse-adapted IAV strain PR8. Pulmonary scRNA-seq of PR8-infected wild-type mice revealed robust upregulation of Tlr7 and interferon (IFN) pathway-associated genes specifically in dendritic cells (DCs) and B cells, whereas Irf7 induction occurred globally across all major lung compartments. Following PR8 infection, Irf7-deficient but not Tlr7-deficient mice exhibited significantly greater daily weight loss, increased mortality, and more severe bronchial epithelial hyperplasia compared to wild-type controls. Furthermore, infected Irf7-deficient mice displayed diminished early IFN-α and IFN-γ levels. Interestingly, both Irf7- and Tlr7-deficient strains demonstrated impaired anti-hemagglutinin (HA) antibody production. Notably, Tlr7 deficiency did not alter Irf7 protein expression or the production of IFNs and NF-κB in response to IAV, indicating that Tlr7 does not modulate Irf7 activation during infection. Collectively, these findings demonstrate that Irf7 protects against severe IAV pathogenesis independently of Tlr7 recognition in mice, whereas both Irf7 and Tlr7 are required for optimal anti-HA antibody production. Importance: Influenza A virus is a respiratory pathogen that remains a major threat to global health as a seasonal disease and a source of periodic pandemics. The outcomes of the infection can range from mild illness to severe pneumonia and death, particularly in vulnerable populations, yet the reasons why some individuals develop more severe disease are not fully understood. Early immune defenses in the lungs are critical for controlling the virus, but they can also contribute to harmful inflammation if not properly regulated. Key sensors that detect viral genetic material, such as TLR7, and the signaling pathways that activate antiviral responses, such as IRF7, play an essential role in shaping these outcomes. However, whether TLR7 is essential or redundant in IAV remains debated, and the independent role of IRF7 in IAV has not been directly demonstrated in mouse models. The significance of our study lies in defining how these early immune mechanisms influence the course of influenza A infection, providing insight that may guide the development of improved therapies for influenza and related respiratory viruses.

Viruses doi: 10.3390/v18091043

Authors: Diane Ducret Tatjana Mijatovic

The neurological and neuropsychiatric manifestations of long COVID remain incompletely understood. Reactivation of latent herpesviruses has been proposed as a contributing mechanism, but human herpesvirus 7 (HHV-7) has received limited attention despite its neurotropic potential. We conducted a retrospective analysis of HHV-7 polymerase chain reaction (PCR) results in 30 patients (18 women and 12 men; age range, 21–55 years) with a neurological/neuropsychiatric long COVID phenotype tested within 24 months of SARS-CoV-2 infection at R.E.D. Laboratories SA, Belgium. HHV-7 positivity was compared with a historical pre-COVID-19 laboratory reference cohort comprising 320 diagnostic tests performed from 2012 to 2019. HHV-7 positivity was 29.1% (93/320; 95% CI, 24.4–34.3%) in the historical reference cohort and 76.7% (23/30; 95% CI, 59.1–88.2%) in the neurological/neuropsychiatric long COVID cohort. The between-cohort difference was significant using a two-sided Fisher’s exact test (p < 0.000001). All six patients tested within the first 12 months were HHV-7-positive. HHV-7-positive patients frequently reported neuropsychiatric symptoms, and in one longitudinal index case, viral load tracked self-reported symptom severity. In this patient-initiated retrospective study, HHV-7 positivity was substantially more frequent in the long COVID cohort than in the historical reference cohort. These findings are hypothesis-generating and support prospective investigation of HHV-7 in neurological and neuropsychiatric long COVID.

Viruses doi: 10.3390/v18091041

Authors: Wenzhuo Ran Xiaozhou He Deng Luo

Respiratory syncytial virus (RSV) is a leading cause of pediatric respiratory infections, yet the frequency and composition of viral codetections in affected children have not been extensively profiled using unbiased sequencing approaches. Here, we performed viral metagenomic sequencing on nasopharyngeal swabs collected from 42 pediatric patients with confirmed RSV infection to characterize the codetection landscape. RSV was detected and genotyped in all specimens. Beyond RSV, we identified nine additional human respiratory viruses coexisting across the cohort; several of these fall outside the scope of routine diagnostic panels, although their clinical significance remains uncertain. Codetections were the rule rather than the exception: 30 of 42 samples harbored at least three viruses, with up to seven distinct viruses detected in a single case. These findings underscore the taxonomic breadth and polymicrobial complexity of respiratory infections in children. Our results describe the viral codetection landscape in RSV-infected children, revealing a broader spectrum of co-occurring viruses than typically captured by targeted diagnostics, although the clinical significance of most codetections remains uncertain. These findings highlight the polymicrobial complexity of RSV infections and underscore the need for prospective studies to determine which codetections are clinically meaningful.

Viruses doi: 10.3390/v18091040

Authors: Mehmet Tekinsoy Osman Merdan Rabia Rusen Catharina Christa Katharina Müller Alina Priller Hrvoje Mijocevic Hedwig Roggendorf Otto Zelger Kathrin Tinnefeld Samuel D. Jeske Duluur Vasilev Sarah Yazici Johanna Erber Dieter Hoffmann Percy A. Knolle Ulrike Protzer

Objectives: The VaCoMRI study tracked a cohort of healthcare workers from 2020 through June 2024 to characterize the long-term sustainability of hybrid and vaccine-induced SARS-CoV-2 immunity. Methods: An initial anti-nucleocapsid (N) IgG screening of 4554 health-care workers in early 2020 identified participants who had been infected during the first wave. Anti-N-positive and anti-N-negative individuals then received BNT162b2 through the institutional vaccination program, yielding groups with hybrid and vaccine-induced immunity. Both groups were monitored regularly for nearly four years for breakthrough infections (BTI). Serum collected regularly after vaccination and after BTI was analyzed for anti-N, quantitative anti-spike (anti-S) IgG, and surrogate viral neutralizing antibody (sVNT) titers. Results: Over a median follow-up of 1180 days, 142 healthcare workers contributed longitudinal samples; 66.2% experienced one BTI and 14.1% a second. In initially seronegative individuals, anti-N titers decreased 2.3-fold between 3 and 6 months after the first BTI, with seropositivity dropping from 82% to 40.4% (median time to seronegativity: 179 days). Prior anti-N seropositivity was associated with 4.5-fold higher anti-N IgG levels after BTI, with elevated levels persisting through 9 months. In both groups, the third vaccine dose significantly enhanced anti-S antibody persistence after 9 months compared to the second dose. In infection-naïve individuals, anti-S IgG titers remained 4.2-fold and sVNT titers 7.2-fold higher. Hybrid immunity was similarly durable: two vaccine doses after prior anti-N seropositivity yielded anti-S IgG and sVNT levels comparable to three-dose vaccination. Conclusions: This study demonstrates a rapid waning of anti-N IgG that severely limits its reliability for retrospective serosurveillance of SARS-CoV-2 exposure in occupational settings. The combination of vaccination and infection induced robust and durable spike-directed antibody responses. However, these were determined using assays based on the ancestral spike protein. This may limit the prediction of protection from SARS-CoV-2 variants, which usually cause BTIs.

Viruses doi: 10.3390/v18091039

Authors: Lisa Orfe Raymond W. Nims

Akabane virus is an arbovirus of the Bunyaviridae family that is endemic in certain tropical and subtropical regions, including parts of Asia, Africa, the Middle East, and Australia. Since Australian-sourced fetal bovine serum (FBS) is a preferred reagent for use in regulated cell culture applications (including but not limited to manufacturing processes for human and veterinary biologics), the importation of FBS from Australia represents a risk to United States livestock in the form of possible introduction of Akabane virus. This risk is currently mitigated, per United States Department of Agriculture (USDA) mandate, through importation testing and/or barrier treatment of the incoming serum by gamma irradiation at ≥30 kGy. Gamma irradiation, while effective for inactivating any infectious Akabane virus that might be present in the serum pools or finished serum product, is not a practical solution for all types of imported serum products. The testing that is currently required by the USDA for Akabane virus and exotic bluetongue viruses involves the use of virus-naïve sheep, is not characterized (validated) for limit of detection, and is not high-throughput, resulting in delays in satisfying import requirements for importation of serum. This opinion paper discusses these topics in detail and proposes alternative testing methodologies that should overcome the various limitations of the current testing protocol. Following development and validation of the proposed methods for limit of detection at the ultimate USDA testing site, decisions regarding the suitability of these methods to replace the current sheep antibody production assay may be made by the USDA.

Viruses doi: 10.3390/v18091038

Authors: Wiktoria Majewska Barbara Poniedziałek Lidia B. Brydak Andrzej Fal Piotr Rzymski

Respiratory syncytial virus (RSV), influenza viruses, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain major causes of seasonal respiratory morbidity, yet their contemporary temporal relationships and age-specific dynamics are insufficiently characterized. We analyzed nationwide Polish surveillance data for the 2024/25 and 2025/26 epidemic seasons, reporting the weekly infection burden per 100,000 population, both overall and by age group (0–4, 5–19, 20–59, and ≥60 years). The seasonal magnitude, peak timing, peak width, normalized temporal trajectories, and age-specific lead–lag patterns were compared. In both seasons, respiratory virus circulation followed the same temporal sequence: COVID-19 activity increased first and peaked in September, followed by influenza in January/February and RSV infections in February/March. Mean weekly burden was lower in 2025/26 for all three infections, by 22.0% for influenza, 19.5% for COVID-19, and 16.0% for RSV, with the between-season difference reaching statistical significance only for influenza (p = 0.0024). Age-stratified analyses likewise showed significantly lower influenza burden in 2025/26 across all age groups, whereas for COVID-19, a significant reduction was observed only among adults aged ≥60 years; no significant between-season differences were found for RSV in any age group. Despite the lower mean influenza burden, the 2025/26 season produced a higher, narrower influenza peak, while the COVID-19 and RSV peaks were lower. Temporal trajectories were highly concordant between seasons for influenza (Spearman’s Rs = 0.975) and RSV infections (Rs = 0.956) and to a lesser extent for COVID-19 (Rs = 0.883). Age-specific temporal trajectories were strongly concordant across age groups, particularly for influenza (pairwise same-week correlations (Rs = 0.972–0.987 in 2024/25 and Rs = 0.963–0.992 in 2025/26). Influenza activity was largely synchronous across age groups, with pediatric peaks occurring approximately 1 week earlier, whereas COVID-19 and, to a lesser extent, RSV showed more consistent pediatric leads relative to adults aged ≥60 years (approximately 1–2 weeks). However, for all three infections, the ascending phase became apparent earlier in children aged 0–4 years than in adults, suggesting that peak timing alone may underestimate the pediatric lead. These findings support integrated, age-stratified surveillance and indicate that preventive strategies, particularly updated COVID-19 vaccination, should be timed to precede the early-autumn rise in transmission.

Viruses doi: 10.3390/v18091037

Authors: Xuejia Wen Qilin Zhao Wenqiang Wang

Feline coronaviruses (FCoVs) are evolutionarily dynamic alphacoronaviruses whose diversification has been driven by extensive genetic exchange and long-term adaptation. However, how genome plasticity is balanced with the evolutionary constraints required to maintain viral fitness remains poorly understood. Here, we analyzed 127 complete FCoV genomes collected worldwide over more than five decades to investigate the evolutionary processes governing long-term FCoV diversification. Genome-wide phylogenetic analyses resolved three major evolutionary lineages and demonstrated that the two recombinant lineages originated independently through recombination between feline and canine coronaviruses, highlighting repeated interspecies recombination as a recurrent source of genomic innovation. In contrast, gene-level evolutionary analyses revealed substantial discordance with whole-genome phylogenies, indicating that individual genomic regions have followed distinct evolutionary trajectories. Recombination and positive selection were concentrated within a limited subset of genes, particularly NSP3, NSP12, NSP2, and the spike gene, suggesting that evolutionary innovation preferentially targets proteins involved in virus–host interactions. Despite extensive genome restructuring and adaptive diversification, overall dinucleotide composition remained highly conserved across all lineages, with persistent CpG suppression and continued CpG depletion during the long-term evolution of the predominant GI lineage, revealing strong compositional constraints acting on FCoV genomes. Together, these findings support a multi-scale model of coronavirus evolution in which recombination reorganizes genome architecture, adaptive evolution refines genes at the virus–host interface, and long-term genomic constraints preserve fundamental compositional features. This work provides a conceptual framework for understanding how genome plasticity and evolutionary constraint are balanced during the long-term evolution of FCoVs and offers broader insights into the evolutionary organization of alphacoronavirus genomes.

Viruses doi: 10.3390/v18091036

Authors: Almagul Kushugulova Nazira Kamzayeva Samat Kozhakhmetov Elizaveta Vinogradova Zharkyn Jarmukhanov Nurislam Mukhanbetzhanov Artur Kovenskiy Alibek Kossumov Aidana Rakhmankulova Talshyn Ukybassova

The cervicovaginal virome remains poorly characterized in relation to human papillomavirus (HPV) infection and bacterial community structure. We performed shotgun metagenomic sequencing of 311 cervicovaginal specimens from a Kazakhstani cohort spanning seven groups defined by HPV status and cervical cytology. Viral community composition differed according to HPV status, with increasing representation of the oncogenic Alphapapillomavirus 9 clade across more abnormal cytological categories. Predicted phage functional profiles also differed between NILM HPV-positive and NILM HPV-negative women. Integrase, excisionase, transcriptional repressor, anti-repressor Ant, and amidase annotations showed differential prevalence after false-discovery-rate correction. Auxiliary metabolic and host-interaction genes associated with nucleotide metabolism, DNA modification, anti-restriction functions, and toxin–antitoxin systems were also differentially represented. Stratification by bacterial community state type revealed contrasting predicted functional repertoires, with toxin–antitoxin-associated annotations enriched in Lactobacillus crispatus-dominated communities and anti-restriction-associated annotations enriched in Gardnerella vaginalis-dominated communities. These findings identify associations between HPV status, bacterial community structure, and cervicovaginal viral composition and predicted phage functions. Longitudinal and experimental studies are required to determine the directionality and biological activity of these associations.