Viruses doi: 10.3390/v18050554

Authors: Virginie Mortier Laurent Debaisieux Deborah De Geyter Marie-Luce Delforge Melissa Depypere Géraldine Dessilly Benoît Kabamba-Mukadi Khalid El Moussaoui Samy Mzougui Ben Serrien Karolien Stoffels Dominique Van Beckhoven Ellen Van Cutsem Dorien Van den Bossche Sigi Van den Wijngaert Fien Vanroye Elizaveta Padalko Chris Verhofstede Kristel Van Laethem

The international spread of HIV-1 sub-subtype A6 raises concerns due to its association with contraindications for long-acting injectable formulations of cabotegravir (LA-CAB) and rilpivirine (LA-RPV). This study investigated its increasing proportion in Belgium, assessing transmission dynamics and potential migration links. Additionally, genotypic drug resistance in the Belgian HIV-1 sub-subtype A6 population were analyzed and four automatic subtyping tools were compared. A dataset of 4764 HIV-1 protease and reverse transcriptase (RT) sequences from newly diagnosed, treatment-naïve individuals in Belgium (2013–2022) was analyzed. A combination of phylogenetic analysis and online subtyping tools identified 136 sub-subtype A6 sequences. The increase in the proportion of HIV-1 sub-subtype A6 observed in Belgium since 2020 reflects changing transmission patterns, especially among Belgium-born men having sex with men, and cannot be solely linked to the recent influx of Ukrainian migrants. Of these sub-subtype A6 sequences, less than 10% showed LA-CAB + LA-RPV resistance, mainly due to E138A within RT. HIVdb and ANRS reliably assessed resistance in this therapy-naïve cohort, and HIVdb, COMET, and SmartGene® produced concordant subtyping results. While algorithm choice has little impact at low resistance prevalence, further research is necessary and HIVdb and ANRS remain more suitable for ongoing clinical and research use.

Viruses doi: 10.3390/v18050553

Authors: Feihu Guan Jie Zhang Ye Tian Bofan Fu Ji Liu Yafen Song Aoyang Yan Bing Zhang Ling Chen Min Zhang Pengfei Du Lei Wang Xiaoyue Yang Sifan Guo Chenghuai Yang Hui Zhang Qianyi Zhang

The gut microbiota, often referred to as the “forgotten organ”, plays an indispensable role in maintaining host physiological metabolism, immune function, and nutrient absorption. Moreover, the gut microbiome serves as a critical biological barrier against viral infections and is increasingly recognized as a potential target to augment antiviral therapies. Recent studies have revealed that microbial ligands and metabolites derived from the gut microbiota are pivotal in modulating respiratory immune responses, providing compelling evidence of the complex interaction network between microorganisms and the host, particularly the signaling pathways linking the gut to distal organs such as the lungs. This review examines the communication and regulatory mechanisms between the gut microbiota and pulmonary mucosal surfaces during influenza virus infection, emphasizing how gut microbial communities and probiotics influence host immune responses, promote the production of immune-related molecules, and enhance antiviral defenses. The aim is to provide comprehensive insights into the gut–lung axis and its implications for respiratory health.

Viruses doi: 10.3390/v18050552

Authors: Carolina Filipponi David C. Bloom Carlo Gambotto Callen T. Wallace Jadranka Milosevic Simon C. Watkins Shane Buckley Maribeth A. Wesesky Vishwajit L. Nimgaonkar Leonardo D’Aiuto

Paraspeckles are subnuclear ribonucleoprotein condensates that regulate host stress responses, including those triggered by viral infection. In vitro studies using non-neuronal cells have shown the involvement of specific paraspeckle components in facilitating the replication of certain viruses, including Herpes Simplex Virus 1 (HSV-1), but these processes have not been investigated in human neuronal cells, which represent a relevant target of the virus. We employed human neural precursor cells (NPCs), neurons, and brain organoids derived from hiPSCs to investigate the previously unexplored dynamics of paraspeckle components in HSV-1-infected human neuronal cells. Our results reveal cell-type-specific differences in the expression of paraspeckle genes in response to HSV-1 infection. Unlike other viruses, HSV-1 orchestrates a previously unreported redistribution of paraspeckle proteins, leading to their accumulation in viral replication compartments (VRCs). Importantly, the expression of the paraspeckle proteins NONO and SFPQ correlates with HSV-1 permissiveness in human neuronal cells and may be required to establish a nuclear environment favoring viral transcription/replication. This enhances our understanding of how stress-response pathways in cells can be exploited by viruses in a cell-type-specific manner.

Viruses doi: 10.3390/v18050551

Authors: Krishanu Ray

Significant advances in microscopy methods for biomedical applications have been made in the past two decades, and these methods are now being actively pursued for virus research [...]

Viruses doi: 10.3390/v18050550

Authors: Vanessa M. Scarfone Collin Pachow Pauline U. Nguyen Anita Lakatos Jamie-Jean De La Torre Alisa Xie Kellie Fernandez Charlene Collado Kaitlin Murray Roberto Tinoco Craig M. Walsh Trevor Owens Agnieszka Wlodarczyk Thomas E. Lane

This study investigated the role of microglia-derived insulin-like growth factor 1 (IGF-1) in modulating host defense and disease progression in a viral model of neuroinflammation and demyelination. Intracranial infection of susceptible mice with the glial-tropic JHM strain of mouse hepatitis virus (JHMV) induces acute encephalomyelitis, followed by an immune-mediated demyelinating disease that mimics many clinical and histologic features of multiple sclerosis (MS). Utilizing an inducible fractalkine receptor (Cx3cr1) promoter-driven Cre-loxP recombinant system, we performed timed ablation of Igf1 in microglia to assess its impact on the central nervous system (CNS) response to JHMV. While the loss of microglial IGF-1 did not impair the control of viral replication, it significantly exacerbated spinal cord demyelination. CyTOF and imaging mass cytometry analysis of spinal cords indicated increased myelin damage was associated with increased accumulation of CD8+Ly6C+ effector T cells and reduced expression of TREM2 that impaired transition into a disease-associated microglia (DAM) phenotype capable of sensing and potentially mitigating myelin damage. Collectively, these findings argue that microglial IGF-1 is a non-redundant coordinator of the CNS immune responses that occur in response to CNS viral infection.

Viruses doi: 10.3390/v18050549

Authors: Juan C. Rueda Ana María Santos Ignacio Angarita Ingris Peláez-Ballesta Alfonso Gastelum Igor Rueda Jaime Cortés-Ramos Cristian Astudillo Daniel Rincón-Sierra Karina Guzmán Jesús Giovanny Ballesteros Juan Manuel Bello John Londono

This study applied a syndemic framework to chikungunya virus (CHIKV) infection during the 2014–2015 Colombian epidemic, integrating biological and social determinants. Methods: A community-based cohort of 279 serologically confirmed adults from six cities was analyzed. Clinical, sociodemographic, and cytokine data were evaluated using multilevel and multivariate statistical approaches. Results: Among 279 patients, 141 (50. 5%) met World Health Organization (WHO) criteria for acute CHIKV infection. The cohort was predominantly female and of lower socioeconomic status (SES). The most frequent manifestations were arthralgia (91%), fatigue (58%), fever (50.5%), myalgia (45.9%), and rash (45.2%). Multivariate models identified IL-15, IL-17A, IL-12p40, MCP-1, and MIP-1α as significant correlates of fever, rash, and myalgia. Socioeconomic and ethnic factors influenced cytokine expression; Caucasian patients showed higher proinflammatory cytokine levels than Afro-American patients. Lower SES was associated with greater symptom burden. Network analyses revealed distinct immune signatures linking biological responses with clinical and demographic variables. Conclusion: Immune responses, clinical manifestations, and social disadvantages interact significantly in CHIKV infection. These findings support a syndemic model in which socioeconomic vulnerability amplifies disease impact, highlighting the need for integrated biosociological public health strategies, particularly targeting populations with low socioeconomic status.

Viruses doi: 10.3390/v18050548

Authors: Niloufar A. Boghdeh-Olson Michael D. Barrera Clayton M. Britt David K. Schaffer Jacquelyn A. Brown John P. Wikswo Aarthi Narayanan

Infection by neurotropic alphaviruses such as the Eastern equine encephalitis virus (EEEV) causes extensive inflammation in the central nervous system and tissue damage, including disruption of the blood–brain barrier (BBB). Neuroinflammation and BBB disruption following infection are critical pathological considerations for the development of robust countermeasure strategies. Encephalitic disease resulting from EEEV infection currently lacks FDA-approved therapeutic intervention strategies, thus exposing a major capability gap in the ability to address the global health burden that could result from alphavirus infections. In this manuscript, we present a gravity-flow Neurovascular Unit (gNVU) model of the human BBB that may be used for modeling EEEV-induced neuropathology and evaluating countermeasures. The data generated using this model show that EEEV infection causes a time-dependent disruption of BBB integrity and increases the inflammatory load in a manner that correlates with an increase in the viral load. The data also show that the route of introduction of the pathogen has an impact on the pathology measured, with infection through the brain side eliciting a greater inflammatory outcome than infection through the vascular route. Overall, the included data support the utility of this organ-on-a-chip (OOC) platform of the human BBB in understanding encephalitic disease caused by neurotropic viruses and evaluation of therapeutic intervention strategies.

Viruses doi: 10.3390/v18050547

Authors: Ting Zhang Yihui Fang Jie Liu Ao Guo Bin Yuan Yanan Zhang Lihua Ding Qinong Ye

The hemagglutinin (HA) of influenza A/H3N2 virus evolves rapidly, with glycosylation driving immune evasion. However, how host microenvironmental cues influence this process remains poorly understood. We identified a novel N-linked glycosylation site at position 110 (N110) in contemporary H3N2 viruses (NSS genotype) that enhances viral fitness by increasing receptor-binding signal, HA cleavage, and replication. Remarkably, hypoxia, which mimics the respiratory tract microenvironment, significantly augments N110 glycosylation. Mechanistically, we identified the B4GAT1-B4GALT1 complex as the key mediator of this modification. Hypoxia upregulates their expression and strengthens their interaction with HA. In ferret models, N110-glycosylated viruses exhibit heightened pathogenicity and evade ancestral antibodies. Furthermore, immunization with N110-containing HA confers broad-spectrum protection, whereas reciprocal immunization is ineffective. Our findings reveal hypoxia-driven glycosylation as a previously unrecognized mechanism of H3N2 adaptation, providing critical insights for vaccine efficacy and highlighting the importance of integrating microenvironmental factors into future antiviral strategies.

Viruses doi: 10.3390/v18050546

Authors: Shiyuan Hou Xing Shen Danni Sun Yulin An Yuxuan Zhou Xing Sun Shuhan Wang Xinyue Liu Mengting Zhu Shuai Zhao Ziyu Liu Xingan Wu Rongrong Liu

The innate immune system serves as the primary barrier against viral invasion, utilizing pattern recognition receptors (PRRs) to orchestrate a rapid defense. Among these, the nucleotide-binding domain and leucine-rich repeat (NLR) containing proteins function as central signaling scaffolds, assembling into multiprotein complexes known as inflammasomes. These complexes drive the maturation of pro-inflammatory cytokines IL-1β and IL-18, and initiate gasdermin D (GSDMD)-mediated pyroptosis, a lytic cell death pathway that eliminates intracellular replication niches. This comprehensive review synthesizes the diversified landscape of inflammasome activation during viral infections, extending beyond the canonical NLRP3 inflammasome to include specialized sensors such as NLRP6, NLRP9, NLRP1, NLRP12, and NLRC4. We critically evaluate the evolutionary “arms race” between host defenses and viral pathogens, detailing the sophisticated immune evasion strategies employed by viruses—ranging from the expression of decoy proteins and direct proteolytic cleavage of immune sensors to the manipulation of post-translational modifications (PTMs). Furthermore, we discuss the dual nature of inflammasome activation, which balances protective viral clearance against pathological hyperinflammation, and provide an exhaustive analysis of novel therapeutic strategies, including direct NLR inhibitors and downstream cytokine blockers, currently navigating clinical transition.

Viruses doi: 10.3390/v18050545

Authors: Collins Oduor Owino Balakrishnan Chakrapani Narmada Gian Yi Lin Pauline Poh Kim Aw Nivrithi Ganesh Jovi Tan Siying Marie-Laure Plissonnier Thangavelu Thangavelu Matan Niranjan Shirgaonkar Pablo Bifani Massimo Levrero Giridharan Periyasamy Seng Gee Lim Ramanuj DasGupta

Background and Aims: Understanding regulatory interactions between hepatitis B virus (HBV) and host factors is essential for the development of next generation host-directed antiviral therapies and the achievement of a functional HBV cure. Here, we investigated HBV-induced alterations in host gene expression in primary human hepatocytes (PHH) to identify host factors exploited by the virus for replication and persistence. Whole-transcriptome sequencing (WTS) of HBV-infected PHH identified host pathways with potential roles in the HBV life cycle. RNA interference-based functional screening of dysregulated candidate genes identified cyclin L1 (CCNL1) as a key host factor. RNAi-mediated knockdown of CCNL1 reduced HBV gene expression, including hepatitis B surface antigen (HBsAg). Mechanistically, CCNL1 regulates phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (RNAPII) at serine 2 (S2), consistent with a role in transcriptional regulation. CCNL1 knockdown further reduced the binding of total and phospho- (Ser2/Ser5) RNAPII, pan-acetylated histone H3 (H3ac), and H3K27ac to HBV covalently closed circular DNA (cccDNA), indicating impaired cccDNA-dependent transcription. In addition, CCNL1 expression was elevated in chronic hepatitis B patients compared with those with resolved infection. Collectively, these data demonstrate that CCNL1 promotes HBV transcription and replication through modulation of RNAPII phosphorylation and chromatin-associated transcriptional activity, identifying CCNL1 as a potential host susceptibility factor for HBV. Importance: Hepatitis B virus infection remains a major threat to human health in areas with high prevalence. There is need to fully understand the complex interactions between the virus and human host factors/processes to support ongoing efforts to develop anti-HBV therapies that can be used with existing therapies to achieve a better cure. HBV relies on host cellular factors and biological processes to establish and maintain efficient infection, making host–virus interactions attractive targets for therapeutic intervention. Thus, identifying host factors that support and/or restrict HBV infection is essential for understanding the molecular basis of chronic HBV infection and for developing host-targeting anti-HBV drugs. This study identifies cyclin L1 (CCNL1) as a host susceptibility factor that promotes HBV transcription and replication through regulation of RNA polymerase II activity and or post-transcriptional mechanisms.

Viruses doi: 10.3390/v18050544

Authors: Md Ashiqur Rahman Rebecca Abraham David J. Hampson Sam Abraham Jasim M. Uddin

Phage therapy has enormous potential in combating bacterial resistance in food animals. However, its application via the oral route remains limited due to challenges associated with the gastrointestinal tract (GIT) environment and a lack of rigorous clinical trial evidence. Therefore, we systematically searched in Google Scholar, PubMed, Scopus, and Web of Science databases following PRISMA guidelines and finally identified 111 articles on oral phage therapy in food animals from where we summarized the key physiological and chemical factors of the gut environment hindering the effectiveness of oral phage therapy (OPT), examined the methods used to evaluate phage stability in the GI environment, and highlighted potential strategies to mitigate these challenges. In addition, we performed quantitative analysis to visualize in vitro pH and thermal stability patterns of phages targeting bacteria isolated from food animals and variability in buffer and incubation period across stability studies. The GIT consists of several anatomically and functionally distinct segments, where complex interactions occur among digestive enzymes, gastric acids, electrolytes, commensal microbiota, and mucosal immune components. The acidic pH of the stomach is a major barrier to successful oral phage delivery. According to our analysis of pH stability testing data from the reviewed studies, most phages targeting antimicrobial-resistant bacteria in food animals remained stable at pH 5–9 and inactivated under highly acidic (pH ≤ 2) or highly alkaline (pH ≥ 11) conditions. In addition, phages are susceptible to high temperatures (above 60 °C), digestive enzymes (e.g., pepsin, trypsin, lipases), bile salts, and host immune responses. Several in vitro laboratory techniques are available to assess phage stability under simulated GI conditions, but variations occur in the assessment protocols. Microencapsulation using alginate and chitosan has been used to protect phages from the adverse GI environment. Additionally, enteric-coated capsules, antacids, co-encapsulation with acid-neutralizing agents, consumption of alkaline water, and daily phage administration are suggested to improve phage survival and efficacy. For the successful clinical implementation of OPT in food animals, future research should focus on elucidating the molecular and physicochemical determinants of phage stability, understanding the humoral immune response to OPT, standardizing laboratory protocol for assessing phage viability, improving the scalability of encapsulation methods, and exploring other potential delivery techniques.

Viruses doi: 10.3390/v18050543

Authors: Broglia Canale Vandelli Tartaglia Delli Ponti

RNA viruses encode multiple layers of regulatory information within their genomes, extending beyond their protein-coding sequences. Through local secondary structures and long-range RNA–RNA interactions, viral RNAs control essential steps of the viral life cycle, including translation, replication, genome cyclization, packaging, and evasion of host defenses. Over the last two decades, chemical probing approaches—particularly Selective 2′-Hydroxyl Acylation analyzed by a primer extension (SHAPE) and its high-throughput derivatives—have transformed our ability to investigate these structures at a single nucleotide resolution and on a genome-wide scale. These technologies have revealed that viral genomes are highly structured and contain numerous functional RNA elements within untranslated regions as well as coding sequences. In this review, we summarize the main experimental strategies used to profile viral RNA architecture, with a focus on SHAPE-based methodologies and complementary approaches. We then discuss the major classes of functional RNA structures identified across diverse viral families, focusing on elements involved in translation and replication, such as internal ribosome entry sites (IRES) and cyclization elements, as well as other functional structures, including XRN1-resistant and frameshifting elements. Finally, we examine how structure-guided analyses are opening new avenues for antiviral intervention, including antisense oligonucleotides, small molecules, and RNA-degrading chimeras. Together, these advances highlight the viral RNA structure as both a key determinant of virus biology and a promising target for therapeutic innovation.

Viruses doi: 10.3390/v18050542

Authors: Genna E. Moldovan Gabriel P. Faber Marta Rodriguez-Garcia

HIV is primarily acquired in women at the female genital mucosa through heterosexual contact. Mucosal immune cells reside adjacent to, within, below, and distant from the epithelium that lines the surface of the female genital tract (FGT) mucosa. Innate immune cells play dual roles in HIV acquisition, both poised to rapidly recognize and respond to HIV, but are also capable of promoting HIV infection locally and distantly in the lymph nodes. In this review we emphasize recent human research on the roles of specific innate immune cells in HIV pathogenesis in the FGT, including dendritic cells, macrophages, neutrophils and innate lymphoid cells. We review how FGT mucosal dynamics, including anatomical compartmentalization, menstrual cycle regulation, reproductive history, menopause and chronological aging contribute to tissue conditioning of these cells and changes in HIV susceptibility in women throughout their lives.

Viruses doi: 10.3390/v18050541

Authors: Rosheen Sungeni Mthawanji Matthew Baylis Maya Wardeh Marcus S. C. Blagrove

Mosquito-borne diseases are a major global health concern, infecting up to 700 million people annually and causing over one million deaths. Of the several genera of biting mosquitoes, species of Anopheles are mostly studied for their ability (vector competence) to transmit Plasmodium protozoan parasites, some species of which cause malaria. More than 480 species of Anopheles have been described worldwide, and about 70 of these are responsible for Plasmodium spp. transmission. However, the focus on Anopheles as vectors of Plasmodium has led to a relative lack of study about the ability of Anopheles to transmit viruses. Some Anopheles species have been experimentally confirmed as competent for various arboviruses. In most cases, they are secondary vectors, with relatively low competence, contributing to overall transmission while other species of mosquito or other vectors are responsible for sustained transmission. Although secondary vectors may contribute less to transmission, they may play important epidemiological roles by extending transmission seasons and/or providing a means of overwintering viruses. Here, we conducted a review of scientific repositories to build a database of known Anopheles competence for arboviruses. After exclusions, we retained 427 laboratory-confirmed studies from 7343 papers reviewed. Our analysis suggests some Anopheles spp. could contribute to arbovirus overwintering in temperate regions.

Viruses doi: 10.3390/v18050540

Authors: Indrani Choudhuri Jocelyn G. Olvera Avik Biswas Allan Haldane Ronald M. Levy Dmitry Lyumkis

HIV-1 integrase (IN) strand transfer inhibitors (INSTIs) are central to modern antiretroviral therapy (ART) because of their high potency and durable effect on viral suppression. However, drug resistance mutations (DRMs) within HIV-1 IN emerge, which can compromise long-term treatment efficacy. Many distinct DRMs that arise under INSTI therapy have been extensively tabulated in public repositories and literature. However, the timelines over which they emerge, accumulate, and consolidate in patients have not been systematically integrated across clinical and experimental studies. In this review, we synthesize current evidence on the temporal evolution of DRMs within HIV-1 IN by examining mutational kinetic data from viruses derived from people living with HIV/AIDS (PLWH) and from in vitro selection experiments. We compare experimental timelines to recent computational predictions derived from Potts-based fitness landscapes coupled with kinetic Monte Carlo simulations and identify reproducible kinetic classes that distinguish fast-, intermediate-, and slow-emerging DRMs. Rapidly emerging DRMs such as E92Q and N155H typically appear early under drug pressure and often represent low-barrier adaptive responses, whereas the most clinically consequential mutations, such as Q148H/K/R, G140A/S, and E138K, arise only after extended therapy and generally require compensatory mutational backgrounds to persist. Although absolute emergence times vary substantially between in vivo and in vitro systems, consistent temporal trends across datasets support the existence of underlying epistatic constraints that shape drug resistance evolution. Understanding DRM timelines is clinically relevant because it provides a framework for interpreting resistance detected at virological failure, informs optimal timing of resistance testing, and may enable earlier identification of high-risk evolutionary trajectories before durable resistance is established.

Viruses doi: 10.3390/v18050539

Authors: Betty Sebati Anthony Brown

Men who have sex with men (MSM) are among of the key population groups that have been disproportionately affected by the HIV epidemic globally. Hence, MSM living with HIV may experience unique challenges leading to their disengagement from and re-engagement with care. This study aimed to identify factors associated with ART re-engagement among MSM in selected districts of South African provinces. A retrospective observational study design was followed, utilising MSM routine programme data from 1 January 2018 to 31 December 2022. The programme enrolled 3337 MSM aged 16 years or older who resided in the selected provinces/districts. Descriptive statistics characterised participants’ baseline profiles. Binary logistic regression identified factors associated with re-engagement with ART. Data analysis was done using SPSS version 31.0.1.0; p < 0.05 was considered statistically significant. The district was the only statistically significant predictor of re-engagement among MSM, wherein eThekwini district had lower odds of re-engagement (aOR = 0.248, 95% CI: 0.144–0.428, p < 0.001). This represented a 75% reduction in the likelihood of re-engagement compared to the City of Tshwane. There was no association between being re-initiated on ART and HIV testing modality (aOR = 7.299, 95% CI: 0.567–94.037, p = 0.127). Future studies should longitudinally and qualitatively investigate specific programme and contextual factors driving district-level variation in re-engagement, while incorporating individual-level factors.

Viruses doi: 10.3390/v18050538

Authors: Liping Jia Ri De Zeng Li Zhenzhi Han Liying Liu Huijin Dong Shunqiao Feng Rong Liu Linqing Zhao

It has been reported that chronic infection of human norovirus (HuNoV) may potentially serve as a reservoir for viral variants with the possibility to evade population immunity or alter the binding sites of HBGA receptors. In this study, a child diagnosed with Burkitt lymphoma and positive for HuNoV determined by real-time PCR (qPCR) firstly in 15 August 2016, was followed up until 20 March 2018, and 26 fecal specimens and one vomitus were collected to trace the evolutionary characteristics of HuNoV by phylogenetic analysis, meta-genomics next-generation sequencing (mNGS), and temporal evolutionary analysis of VP1 among 23 specimens positive for HuNoV. There were 15 specimens with partial RdRp gene sequences forming an independent cluster with sequences of GII.P31, 14 with the region C sequences and 11 with P domain sequences of VP1 gene clustered together with HuNoV GII.4 Sydney_2012. All these sequences showed that mutations accumulated nearly in a time order, and more mutations were shown in the key epitopes A–E or near the binding sites for HBGA in subdomain P2 with higher evolutionary rates. Analysis of NGS data identified intra-host viral quasi-species, and two genome sequences of the same length from mNGS were assembled from N705, with mutations located in the region of subdomain P2 (1171 nt–1202 nt) which led to five amino acid mutations. In conclusion, the accumulated mutations of HuNoV, especially in subdomain P2, were explored in a child with Burkitt lymphoma, and the sequencing of HuNoV from immunocompromised individuals was proven critical for monitoring intra-host quasi-species evolution and potential variant emergence, providing basic data for clinical infection control.

Viruses doi: 10.3390/v18050537

Authors: Jae Young Kim Sun Young Park Gyeongmin Lee Seung-A Hwangbo Giyoun Cho Jong-Hyeon Park Young-Joon Ko

The Republic of Korea has implemented routine vaccination against foot-and-mouth disease virus (FMDV) in livestock using a bivalent vaccine comprising serotypes O and A following the massive FMD outbreak in 2010, while antigens for the remaining serotypes are maintained in overseas antigen banks. The recent geographic expansion of FMDV Southern African Territories 1 (SAT1) beyond Africa underscores the need for enhanced preparedness in previously unaffected regions. In this study, we evaluated the SAT1 BOT-R strain as a candidate vaccine seed for potential domestic vaccine production by optimizing antigen production conditions, assessing scalability, determining virus inactivation parameters, and examining immunogenicity in pigs. Optimal antigen yield was achieved at 20 h−24 h post infection with a multiplicity of infection of 0.005−0.01, with production remaining stable under mildly alkaline conditions. Antigen productivity was consistently maintained during scale-up from shake flasks to a bioreactor, yielding up to 9.5 μg/mL. Complete virus inactivation was achieved using binary ethylenimine at 2 mM for 24 h at 26 °C. Vaccines formulated from both flask- and bioreactor-derived antigens elicited comparable neutralizing antibody responses in pigs, reaching a median titer of 1:500 following booster immunization. Collectively, these findings demonstrate that the SAT1 BOT-R strain is a viable and scalable candidate for SAT1 antigen banking and future domestic vaccine production, providing a practical framework for strengthening national preparedness against potential incursions of FMDV SAT1.

Viruses doi: 10.3390/v18050536

Authors: Frazer J. T. Buchanan Markella Loi Charlotte Chim ShuXian Zhou Rebekah Penrice-Randal Leandro X. Neves Maximilian Erdmann Edward Emmott

The caliciviruses include important human and animal pathogens such as norovirus, sapovirus and feline calicivirus. Viral reverse genetics is performed to understand the fundamental biology of these viruses, as well as a potential route to generate live-attenuated vaccines. Calicivirus reverse genetics systems have typically relied on either the production of in vitro-transcribed RNA or plasmid-based rescue, either from a mammalian promoter or through supplementing with helper enzymes through means of a helper virus. Here, we present a novel system integrating vaccinia capping enzymes D1R and D12L encoded on plasmids as part of a system for murine norovirus (MNV) reverse genetics. The addition of D1R, D12L and T7 RNA polymerase-expressing plasmids increases the viral titres of rescued MNV in both BSR-T7 cells and transgenic BSR-T7 cells expressing murine CD300LF (BSR-T7CD300LF), and viral protein abundance. When the murine norovirus receptor is expressed in BSR-T7CD300LF, viral titres increased 100–1000-fold compared to standard BSR-T7 cells. This system offers a robust, increased throughput means of assessing viral mutants over parallel in vitro transcription and capping reactions for multiple mutants, without requiring a helper virus.

Viruses doi: 10.3390/v18050535

Authors: Krzysztof Skowron Justyna Bauza-Kaszewska Anna Budzyńska Natalia Wiktorczyk-Kapischke Julia Czuba Ewa Wałecka-Zacharska Kacper Wnuk Mariusz Zapadka Krzysztof Kasprzyk Katarzyna Grudlewska-Buda

The recent SARS-CoV-2 pandemic—which had significant worldwide health, economic, and other effects—indicated the need to monitor zoonotic viruses with pandemic potential. The aim of this review is to assess bat-borne viruses as a potential pandemic risk, with a particular focus on Europe. The presence and activity of bats, as well as diseases emerging in humans in various regions of the world, point to their importance in the context of a possible outbreak of future epidemics. The rate of genetic change observed among viruses requires constant scrutiny on all continents, including Europe. Bats are a considerable source of many zoonotic viruses, including coronaviruses, filoviruses and paramyxoviruses. Among viruses associated with bats, RNA viruses are the dominant ones, characterized by high pathogenicity and often leading to interspecies transmission. The majority (about 80%) of RNA viruses were identified in bats from three families: Vespertilionidae, Rhinolophidae and Pteropodidae. Understanding how viruses are transmitted in the environment and the role of reservoir organisms and intermediate hosts is crucial to determining the level of epidemic risk. This review discuses viruses identified in bats globally, with a special focus on Europe, and evaluates their potential to cause epidemics.

Viruses doi: 10.3390/v18050534

Authors: Hidetomo Iwano Jumpei Fujiki Tomohiro Nakamura

Rising antimicrobial resistance has revived global interest in phage therapy, yet its transition to standard clinical practice remains slow. This challenge is not solely due to a lack of efficacy. Instead, we face a fundamental conceptual barrier caused by an “evaluation mismatch.” Traditional regulations treat phages as static chemical molecules—like taking a “snapshot.” However, biologically, phages are dynamic, evolving populations—more like a living “movie.” In this review, we use Schrödinger’s cat metaphor to explain this reality: phage variability is not a defect, but an essential feature. To bridge this gap, we propose a Controlled Evolutionary Platform. By distinguishing between a fixed “Safety Core” and a fluctuating “Adaptive Periphery,” we can manage viral evolution rather than trying to stop it. Ultimately, to integrate phages into modern medicine, we must redefine “consistency”: shifting our focus from preserving a fixed genetic sequence to ensuring the reliable performance of population dynamics.

Viruses doi: 10.3390/v18050533

Authors: Michaela Lano Barry Milavetz

The Polyomaviridae family contains members known for achieving high seroprevalence within their target species despite a limited genomic economy. Minimalism, by definition, allows for the clarification and streamlining of purpose via the removal of unnecessary or distracting components. Among viruses, Simian Vacuolating Virus 40 (SV40) and other polyomaviruses are master minimalists, achieving efficient replication and persistence with compact genomes of approximately 5 kb in length. This review examines how polyomaviruses employ limited genetic material and simple structure to participate in complex functions and interactions, highlighting minimalism as both an evolutionary and functional advantage. Polyomaviruses make the most of their compact genomes in each stage of the viral lifecycle through the production of multifunctional early proteins and cis-regulatory elements, utilization of alternative splicing and host infrastructure, and organization of compact structural proteins. This allows for the successful replication and proliferation of virions while also reducing evolutionary pressure and promoting host immune evasion. Examination of the implications of polyomaviral minimalism illustrates that genome economy is not a constraint, but rather a driver of biological sophistication.

Viruses doi: 10.3390/v18050532

Authors: Ramesh Kumpatla Vinaya Kruthi Vitala Arunasree M. Kalle

Antimicrobial resistance (AMR) has become a major concern in the treatment of bacterial infections, and bacteriophage therapy has emerged as a promising alternative to antibiotics. Bacteriophages are highly specific to their bacterial hosts; hence, isolating phages indigenous to a specific region offers a significant advantage against various pathogen strains. We have isolated a cocktail of bacteriophages against pathogenic E. coli from sewage water at a primary healthcare centre. Characterisation of the isolated phages demonstrated their stability across a broad pH and temperature range, strong lytic activity, and effective biofilm degradation, with no cross-reactivity with Staphylococcus aureus (S. aureus). Genomic analysis and phylogenetic studies indicated that the largest phage (by genome size) in the cocktail belongs to the genus Vequintavirus (myoviruses, rV5-like phages), and its genome sequence has been deposited in NCBI (Accession ID: PX741096). The phage genome was linear, with headful (PAC) packaging, encoded lysis proteins, and lacked antibiotic-resistant or major lysogeny-associated genes, collectively suggesting a lytic lifestyle. These findings emphasize the therapeutic potential of rV5-like phages and underscore the critical need to establish phage banks in India to improve disease management.

Viruses doi: 10.3390/v18050531

Authors: Siti Aishah Rashid Sakshaleni Rajendiran Nurul Farehah Shahrir Nurul Athirah Naserrudin Terence Tan Yew Chin Janice Chan Sue Wen Imanul Hassan Abdul Shukor Nurul Amalina Khairul Hasni

Dengue fever, with a high proportion of asymptomatic infections, poses a major global public health challenge that traditional surveillance systems frequently underestimate. Wastewater-based epidemiology (WBE) has emerged as a promising approach to monitoring infectious diseases beyond enteric viruses. Dengue virus is shed in urine, feces, and saliva, providing a biological basis for wastewater detection alongside clinical surveillance. This systematic review and meta-analysis synthesize current evidence on dengue virus (DENV) detection in wastewater and evaluate methodological factors influencing detection success in WBE. A systematic literature search using selected databases and predetermined keywords, followed by eligibility screening, resulted in ten studies being included, covering community surveillance and experimental trials. DENV ribonucleic acids (RNA) were most consistently detected and enriched in wastewater solids, indicating this matrix as the most reliable for surveillance. Among concentration methods, ultrafiltration achieved the highest viral recovery efficiency, while reverse transcription digital polymerase chain reaction (RT-dPCR) demonstrated superior sensitivity and precision compared to those of reverse transcription quantitative polymerase chain reaction (RT-qPCR), particularly at low viral concentrations. Storage at −80 °C was critical for preserving RNA integrity. The meta-analysis yielded a pooled DENV positivity rate of 24% (95% CI: 20–28%) after exclusion of outliers. Overall, solid-phase analysis combined with RT-dPCR represents the most sensitive methodological approach across the included studies. Harmonized protocols are needed to support future translation of dengue WBE into community surveillance as current evidence mainly demonstrates methodological feasibility and provides a technical foundation for future public health integration. Therefore, further longitudinal and multi-site validation is required to establish its broader applicability for dengue surveillance.

Viruses doi: 10.3390/v18050530

Authors: Ghizlane El-Amin Naima El Hafidi Soumia Benchekroun Mahraoui Chafiq Amal Zouaki Nora Touyar Najat Bouihat Salma Ech-Cherif El Kettani Saad Harrak Larbi Ed-Dafali Aziza Bentalha Mustapha Alilou Hamza El Hamzaoui Amina Barkat Ilham Elouardighi Tarek Dendane Khalid Abidi Jihane Bel Ayachi Naoufal Madani Redouane Abouqal Hicham Harmouche Mouna Maamar Rachid El Jaoudi Mourad Feindiri Myriam Seffar Mohamed Bouskraoui Hakima Kabbaj

Respiratory syncytial virus (RSV) imposes a substantial burden of severe acute respiratory infection (SARI), especially in young children and the elderly. Methods: We describe RSV epidemiology among hospitalized SARI patients at the Ibn Sina University Hospital Center (Rabat, Morocco) from 1 January 2021, to 31 December 2025, using multiplex PCR (BioFire® RP2.1plus or Xpert® SARS-CoV-2/Flu/RSV). Results: Among 4604SARI samples, RSV prevalence was 16.1% (739/4604), predominantly pediatric (88.6%, p < 0.001), with peak burden in infants <6 months (70.4% of cases, p < 0.001). Pediatric prevalence was 28.3% (655/2316) vs. 3.8% (84/2204) in adults (p < 0.001), with predominance in the elderly ≥60 years (51/1041, 4.9%). Co-infections occurred in 46.7% (310/665) of FilmArray-tested positives (total = 665), led by rhinovirus/enterovirus (198/310, 63.9%), and were significantly higher in children (48.5%, p < 0.001). RSV peaked in winter (51.6%), except for summer dominance in 2021 (52.5%), reflecting COVID-19 non-pharmaceutical intervention effects. Conclusions: These data establish Morocco’s first comprehensive RSV surveillance baseline, highlighting post-pandemic epidemiological shifts. As maternal vaccines and monoclonal antibodies emerge, these data inform optimal implementation in low- and middle-income countries (LMICs).

Viruses doi: 10.3390/v18050529

Authors: Yasemin Ay Altintop Esma Saatçi

Hepatitis C virus (HCV) infection is still a major worldwide health concern. It is distinguished by a high degree of genetic variation that affects the course of the illness and the effectiveness of treatment. The epidemiological profile of HCV is prone to rapid change in areas where there is significant human migration, like Turkey. The purpose of this study was to evaluate the impact of long-term migration on local viral diversity by analyzing the distribution and temporal trends of HCV genotypes among Turkish citizens and asylum seekers in Kayseri, Turkey, over an eight-year period. From January 2014 to December 2021. 1173 HCV RNA-positive patients at Kayseri City Training and Research Hospital were the subject of a retrospective analysis. Genotypes were determined using the Abbott RealTime HCV Genotype II assay and Montania 4896 assay (Anatolia Geneworks, Türkiye). The most prevalent genotypes were Genotype 1b (48.3%, 95% CI: 45.5–51.2%), Genotype 4 (25.0%, 95% CI: 22.5–27.5%), and Genotype 1a (10.3%, 95% CI: 8.6–12.1%). Turkish patients exhibited the highest prevalence of Genotype 1b (98.2%), while asylum seekers demonstrated greater relative burdens of Genotype 4 (8.5% of total GT4) and Genotype 5 (83.3% of total GT5). Genotype 3a emerged in 2018, with a predominance in males (73.9%). The Cochran–Armitage trend test revealed statistically significant increasing trends for Genotype 3 (Z = 3.572, p = 0.0004) and Genotype 3a (Z = 2.600, p = 0.009). This eight-year retrospective study demonstrates that the HCV genotype distribution in Kayseri has undergone significant changes in the context of migration and demographic shifts. The statistically significant increasing trends of Genotypes 3 and 3a, particularly among younger male populations, suggest evolving transmission dynamics. These findings underscore the necessity of demographically targeted and culturally appropriate screening and treatment strategies for both resident and migrant populations to achieve HCV elimination goals.

Viruses doi: 10.3390/v18050528

Authors: Aleksandra Milenkovic Simon Nikolic Jelena Aritonovic Pribakovic Branislava Radovic Aleksandra Ilic Milica Stevanovic Sara Kovacevic Kristina Bulatovic Jelena Milovanovic Arijeta Kostic Aleksandra Janicevic

The aim of this study was to evaluate the association between baseline clinical and CT characteristics and to identify factors associated with intensive care unit (ICU) admission in hospitalized COVID-19 patients. This retrospective study included 176 adult patients with laboratory-confirmed SARS-CoV-2 infection hospitalized at the COVID Hospital of the Clinical Hospital Center Kosovska Mitrovica during 2021–2022 (Delta and Omicron variants). Patients were divided into two groups according to intensive care unit requirement: those treated in a general inpatient ward (No ICU) and those requiring ICU admission (ICU group). Demographic and clinical characteristics, lifestyle factors, CT findings, CT severity score (CTSS) values, and therapeutic interventions were compared between groups. Of the total cohort, 113 patients (64%) were hospitalized in a general inpatient ward, while 63 (36%) required intensive care unit admission. Independent predictors of ICU admission identified in the multivariate logistic regression analysis were obesity (B = 2.96, p < 0.001), dyspnea (B = 1.51, p = 0.041), higher CT severity score (B = 0.68, p < 0.001), and lower glucose levels (B = −0.27, p = 0.014). Furthermore, for each one-point increase in the CTSS, the odds of ICU admission nearly doubled (OR = 1.97). Total CT score values above the cut-off point (15.0) demonstrated significant reliability in discriminating the need for ICU transfer in patients with COVID-19. These findings suggest that combined clinical and radiological assessment at hospital admission may facilitate early identification of patients at high risk of requiring ICU care, with the CT severity score representing the strongest radiological predictor.

Viruses doi: 10.3390/v18050527

Authors: Tsuguto Fujimoto Miki Ogi Kazuhiro Kitakawa Takako Sano Yorihiro Nishimura Kouichi Kitamura Minami Kikuchi Ueno Minetaro Arita

Molecular typing of enteroviruses (EVs) is essential for surveillance of hand, foot, and mouth disease (HFMD). Conventional reverse-transcription polymerase chain reaction (RT-PCR) targeting the VP4–VP2 region can be insufficiently sensitive, reducing the detectability of Enterovirus A (EV-A). We developed a single-round RT-PCR assay using a modified reverse primer design (C3R) for rapid EV detection and genotyping. Sensitivity was evaluated using EV-A71 and poliovirus type 1 reference strains, across 60 EV-positive clinical specimens. The C3R-based assay showed ~1000-fold higher sensitivity for EV-A71 than for conventional assays (limit of detection: 6.6 copies/reaction). The assay detected 98.3% (59/60) of clinical specimens in a single-round format, whereas the conventional assay detected only 45.0% (27/60) and showed a marked decline in detection at higher Ct values. The C3R-based assay maintained complete detection for clinical specimens with Ct values below 40. The majority of the amplified products yielded high-quality sequences suitable for genotyping. This C3R-based RT-PCR overcomes sensitivity limitations of existing protocols and provides reliable genotyping from low-viral-load specimens, supporting its use in routine diagnostics and large-scale HFMD surveillance.

Viruses doi: 10.3390/v18050526

Authors: Balazs Sax Adam Koppanyi Katalin Kristof Akos Kiraly Gyula Prinz Istvan Hartyanszky Gergely Gyorgy Nagy Istvan Nemet Fanni Temesvari-Kis Balazs Kiss Bela Merkely

Percutaneous cable infection of left ventricular assist device (LVAD) patients is a significant source of morbidity, often caused by biofilm-producing or multidrug-resistant bacteria. We hypothesized that bacteriophage viruses can be identified from biological samples of patients with active driveline infection. Six patients with local percutaneous lead infections were enrolled. Microbiological samples were collected from the infected wound and other skin regions. The isolated viral strains and phages from wastewater samples were then tested against the pathogen bacterial cultures in vitro. Biofilm disruption assay and genetic analysis of the strains were also performed. Bacteriophages with lytic activity could be identified from samples of two patients. One patient contained four strains showing strong efficacy against his own Staphylococcus epidermidis. Furthermore, this bacterium was susceptible to phages identified from another patient and strains from wastewater samples. Genomic analysis suggested lysogenic lifestyle of the phages. However, none of them have shown any microbiological signs of lysogeny. In conclusion, we have been able to prove in vitro lytic activity of bacteriophages originating from the same LVAD patient. We also found effective phages in biological samples of other patients and wastewater samples, suggesting that patients implanted in the same center may share bacteriophage flora.

Viruses doi: 10.3390/v18050524

Authors: Julia Cyrielle Andeko Sonia Etenna Lekana-Douki Gabriel Falque Nadine N’dilimabaka Jean-Bernard Lekana-Douki

SARS-CoV-2, the causative agent of COVID-19, emerged in late 2019 and rapidly developed into a global health crisis. In this study, we analysed 173,194 SARS-CoV-2 genomes from the GISAID database to explore the intra-continental dynamics and distribution of variants across Africa between 2020 and 2024. We have identified 1377 distinct lineages, which were classified by clade to assess associations with infection and mortality rate. So, we conducted a Shannon entropy analysis to confirm the diversity and we applied a Correspondence Analysis (CA). Our findings revealed that one of the deadliest in Africa during the Delta wave, lineage AY.45 predominated in the South Africa cluster, whereas AY.34.1 drove transmission in the Atlantic West Africa cluster, underscoring regional heterogeneity. Furthermore, early in the pandemic, men exhibited a 39% higher risk of infection compared to women (aOR: 1.39, 95% CI [1.34–1.45]), particularly in association with clade G. By contrast, later stages were dominated by clade GRA, which disproportionately affected the elderly (≥70 years; aOR: 1.39, 95% CI [1.33–1.45]) and children (0–9 years; aOR: 1.26, 95% CI [1.20–1.33]). Our analysis highlighted that the pandemic on the African continent unfolded as a mosaic of epidemics shaped by diverse variants and regional epidemiological contexts. These findings emphasize the importance of genomic surveillance to capture local epidemic signatures and inform region-specific public health strategies.

Viruses doi: 10.3390/v18050525

Authors: Estefania Benedetti Maria Carolina Artuso Alex Byrne Maria de Belen Garibotto Martín Avaro Luana Piccini Ariana Chamorro Marcelo Sciorra Vanina Marchione Mara Russo Maria Elena Dattero Erika Macias Machicado Monica Galiano Nicola Lewis Andrea Pontoriero

The H5N1 subtype of highly pathogenic avian influenza (HPAI) poses a major zoonotic threat due to its high fatality rate and capacity for cross species transmission. In early 2025, Argentina detected a novel triple reassortant A(H5N1) virus in Chaco Province, combining Eurasian, North American, and South American lineage segments. Genomic analyses of subsequent outbreaks in Buenos Aires and Entre Ríos confirmed persistence of this reassortant and additional HA substitutions (T204K, P251S) potentially linked to increased mammalian receptor affinity. Although PB2 sequences lacked canonical mammalian-adaptive markers (E627K, Q591K, D701N), all contained I292M, a mutation associated with human adaptation. Phylogenetic analyses revealed distinct genotypes and increasing divergence. These findings indicate ongoing viral evolution and adaptation within Argentina, emphasizing the urgent need for sustained genomic surveillance, timely data sharing, and integrated One Health strategies to mitigate zoonotic and socioeconomic risks associated with H5N1 spread in South America.

Viruses doi: 10.3390/v18050523

Authors: Linda Mpofu-Dobo Kebaneilwe Lebani Jason T. Blackard Sikhulile Moyo Motswedi Anderson Simani Gaseitsiwe

Hepatitis B virus (HBV) remains a leading cause of chronic liver disease worldwide, contributing to cirrhosis and hepatocellular carcinoma. Sub-Saharan Africa accounts for an estimated 68% of incident HBV infections, where co-infection with human immunodeficiency virus (HIV) is common and associated with poorer clinical outcomes. In Botswana, limited HBV screening and the absence of established HBV management guidelines persist despite reported HIV-HBV co-infection rates ranging from 1.1% to 10.6%. This scoping review aimed to summarise existing research on HBV and HIV-HBV co-infection in Botswana and assess clinical and policy implications. Following PRISMA methodology, searches were conducted across PubMed, Google Scholar, Semantic Scholar, and Consensus databases. Thirty eligible peer-reviewed studies were identified and evaluated for prevalence data, virological characteristics, genotypes, mutations, treatment outcomes, vaccination programs, and the availability of guidelines. Findings indicate intermediate-to-high HBV and HIV-HBV disease burden, substantial occult HBV infection, and gaps in diagnostic and preventive practices. The lack of routine screening, deficient infant birth-dose and adult vaccination, and established treatment pathways likely increase the risk of HBV-associated morbidity and mortality. Strengthened public health interventions, including expanded testing, enhanced vaccination coverage, and prevention of mother-to-child transmission strategies, are recommended to improve disease control and clinical outcomes in Botswana.

Viruses doi: 10.3390/v18050522

Authors: Jinyu Sui Suchun Wang Zihao Pan Kaicheng Wang

Bovine adenovirus (BAdV) is associated with respiratory and enteric diseases in cattle. In this study, the complete genomic sequence of a novel BAdV strain (named BAdV/LN/CHN/2023) was sequenced and annotated using the next-generation sequencing (NGS) technology. The viral genome comprises 32,391 base pairs with a GC content of 44.93%, encoding 33 predicted open reading frames (ORFs), consistent with the genomic organization of mastadenoviruses. Comparative genomic analysis confirmed that BAdV/LN/CHN/2023 contains conserved structural and functional motifs characteristic of the genus Mastadenovirus. Phylogenetic analysis revealed that BAdV/LN/CHN/2023 shares low similarity with all currently recognized bovine mastadenoviruses classified by the International Committee on Taxonomy of Viruses (ICTV). In addition, an open reading frame (ORF) encoding the 146R protein was annotated in this strain; this feature has not been identified in any previously recognized bovine mastadenoviruses. This study presents the first full-length genomic sequence of a putative BAdV-11 strain, and based on ICTV criteria, we propose that this strain represents a novel mastadenovirus species, supported by phylogenetic distance and genomic divergence. Our findings expand the known genetic diversity of BAdVs and contribute to a better understanding of their evolutionary relationships.

Viruses doi: 10.3390/v18050521

Authors: Yuxin Lin Haoting Lin Donggeng Yu Kevin Welsher

The epithelium represents the first line of defense against viral infection, yet the precise mechanisms by which viruses penetrate this complex barrier remain incompletely understood. Single-virus tracking (SVT) has emerged as a powerful fluorescence microscopy approach to directly visualize viral dynamics with nanometer spatial precision and millisecond temporal resolution. In this review, we survey recent progress in SVT methodologies, from image-based approaches to active feedback techniques, and assess their capacity to resolve viral behavior in physiologically relevant epithelial models. We further evaluate advances in virus labeling strategies—including fluorescent proteins, organic dyes, and nanoparticles—that enable prolonged observation while preserving infectivity. By integrating developments in optical instrumentation and molecular labeling, SVT is increasingly capable of capturing critical processes, including extracellular diffusion, receptor engagement, internalization, and trans-epithelial transport. Finally, we discuss current challenges, including limited penetration depth, photobleaching, and the complexity of 3D epithelial tissues, and outline future opportunities to extend SVT towards in situ and tissue-level studies. Together, these advances position SVT as a transformative tool to illuminate virus–epithelium interactions and guide therapeutic strategies.

Viruses doi: 10.3390/v18050517

Authors: Luke D. Griffith Samir Dervisevic Penny P. Powell

The COVID-19 pandemic placed unprecedented pressure on diagnostic services worldwide. The first cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the UK were confirmed on 31 January 2020, prompting National Health Service (NHS) laboratories to scale diagnostic procedures. The demand for testing rapidly exceeded historical norms for respiratory virus diagnostics, necessitating substantial government investment in consumables, assay development, and workforce expansion. This review presents a retrospective evaluation of SARS-CoV-2 diagnostic platforms deployed within the Norfolk and Norwich University Hospital (NNUH) trust and compares them with those implemented by other regional laboratories during the pandemic. It examines the molecular mechanisms, performance, scalability, and specificity of the multiple molecular testing approaches to optimise workflow based on the evolving technology. The integration of complementary platforms through a stratified testing strategy enabled high-throughput population screening while preserving diagnostic resolution for complex respiratory cases, substantially improving laboratory efficiency and resilience. The emerging diagnostic methodologies, RT-LAMP and CRISPR-based assays, are described, and we discuss their potential roles in future outbreaks. We critically evaluate the overall preparedness of UK health services for the COVID-19 pandemic and highlight key priorities for future pandemic preparedness at both local and national levels.

Viruses doi: 10.3390/v18050519

Authors: Adijat Ozohu Jimoh Anika Chicken Brandon Maust Colin Feng Seth Rakoff-Nahoum Jo-Ann S. Passmore Brian R. Kullin Simona Kraberger Fatima Aysha Hussain Heather B. Jaspan Arvind Varsani Anna-Ursula Happel

Lactobacillus crispatus is widely associated with optimal sexual and reproductive health outcomes. While L. crispatus genomes commonly harbor prophages, little is known about their genomic diversity and potential inducibility by clinically relevant compounds. We induced and characterized four bacteriophages from four L. crispatus strains isolated from vaginal secretions of South African adolescents. Sequenced viral DNA from induced phages was assembled, and their respective genomes were annotated and compared to bacteriophage reference genomes. All the phage genomes range in size from 42.9 to 48.3 kbp. Of the four phages, UC101 and UC164 shared <90% pairwise intergenomic similarity to reference phages, suggesting that they represent new species. To explore factors potentially associated with prophage activation, L. crispatus strains were exposed to physiological concentrations of copper ions and tenofovir, selected based on their common use by women in Africa and reported associations with altered vaginal bacterial community composition. The presence of phage-like particles following exposure to copper ions (2.0 × 10−6 M–3.0 × 10−6 M) and tenofovir (500 ng/mL) was observed by transmission electron microscopy, suggesting possible prophage activation under these conditions. This study provides new insights into the genomic diversity of inducible L. crispatus phages and presents hypothesis-generating evidence regarding their potential inducibility using copper ions and tenofovir.

Viruses doi: 10.3390/v18050520

Authors: Marcin Horecki Karol Serwin Miłosz Parczewski

The evolution and spatial dissemination of SARS-CoV-2 Omicron subvariants have been characterized by rapid lineage replacement and complex transmission dynamics influenced by regional connectivity. This study presents a comprehensive discrete phylogeographic analysis of 90,136 SARS-CoV-2 sequences collected in Poland from 2022 to 2024 to reconstruct the dispersal dynamics of major Omicron lineages, including BA.1, BA.2, BA.5, CH.1, XBB.1, and JN.1. Utilizing Bayesian statistical frameworks, we identified significant viral transitions between the 16 Polish voivodeships and established variant-specific dominance windows ranging from 2 to 4 months. Our findings reveal a highly dynamic epidemic landscape with shifting regional epicenters. The initial BA.1 wave was primarily driven by the Mazovian voivodeship, accounting for 36.1% of outward migration events. This pattern shifted dramatically with the rise in BA.2, which was centered in the industrial Silesian region in the south-west, a densely populated area with strong economic ties to neighboring countries, potentially reflecting a different introduction or transmission dynamic. Furthermore, the epidemic landscape continued to reconfigure during the BA.5 wave, marked by the emergence of new transmission hubs in eastern border regions such as Lublin. Subsequent lineages exhibited distinct geographic signatures: BA.5 spread broadly along the Baltic-central corridor, CH.1 was centered in the north-east, XBB.1 re-emerged in the west-central region of Greater Poland, and JN.1 was driven overwhelmingly by Lesser Poland. These transitions highlight that regional transmission hubs are transient and influenced by local factors such as population density, cross-border mobility, and socio-economic connectivity. This study underscores the critical value of dense genomic surveillance in identifying evolving dispersal routes to inform adaptive, region-specific public health interventions.

Viruses doi: 10.3390/v18050518

Authors: Daria O. Neymysheva Galina V. Ilyinskaya Viktoria A. Sarkisova Elena A. Mukhina Sofia A. Romanen-kova Peter M. Chumakov

Cancer remains the leading cause of death in domestic dogs. Conventional therapeutic approaches, including surgery, chemotherapy, and radiotherapy, frequently fail to achieve sustained remission or stabilization. Oncolytic virotherapy, a rapidly advancing therapeutic modality in human oncology, is emerging as a novel strategy in veterinary medicine. This systematic review summarizes current knowledge on the application of oncolytic viruses (OVs) in canine cancer treatment, focusing on their mechanisms of action, safety profiles, and clinical efficacy. We evaluate diverse OV platforms, including myxoma virus, reovirus, vesicular stomatitis virus, canine adenoviruses, vaccinia virus, Sendai virus, and Newcastle disease virus, across preclinical and clinical studies in dogs with various malignancies. While several OVs have demonstrated favorable tolerability and modest antitumor activity, key challenges such as pre-existing immunity, optimization of dosing regimens, and rational combination strategies remain to be addressed. This review emphasizes the translational significance of canine studies for both veterinary and human oncology, underscoring the critical need for rigorously designed clinical trials to refine virotherapy protocols and expand therapeutic options for canine cancer patients.

Viruses doi: 10.3390/v18050516

Authors: Federica Cerini Loreta A. Kondili Chiara Masellis Agostino Cosenza Carmelo Selvaggio Maria Grazia Rumi

National HCV screening, a cost-effective program in Italy, is supported by the Italian Ministry of Health. Free-of-charge screening was approved for people who use drugs, prisoners and the 1969–1989 general population birth cohort. However, the benefits of extending hospital-based screening irrespective of age, in terms of case detection and linkage to care, remain unclear. We assessed a streamlined in-hospital pathway at San Giuseppe Hospital (Milan), between 2021 and 2024, with anti-HCV screening for all admissions and HCV-RNA confirmation when indicated. Anti-HCV seroprevalence among 18,218 screened patients was 1.9% (medical wards 3.8%, surgical 1.4%). Viremia was detected in 23/75 tested (31%); 78% linked to care and 30% initiated treatment. Notably, 91% of viremic patients were born before 1969 indicating hospital-based screening effectively identifies HCV infections in older cohorts missed by national screening programs. Systematic screening implementation across hospital wards represents a pragmatic micro-elimination strategy for Italy and similar epidemiological contexts, advancing 2030 elimination targets.

Viruses doi: 10.3390/v18050515

Authors: Stefano D’Anna Lorenzo Piermatteo Alessia Magnapera Ilaria Grossi Caterina Tramontozzi Antonella Olivero Leonardo Duca Giulia Torre Elisabetta Teti Andrea Di Lorenzo Vincenzo Malagnino Marco Iannetta Sandro Grelli Pierpaolo Paba Ada Bertoli Francesca Ceccherini-Silberstein Leonardo Baiocchi Simona Francioso Ilaria Lenci Michele Milella Annalisa Saracino Alessia Ciancio Giuseppina Brancaccio Loredana Sarmati Pietro Lampertico Mario Rizzetto Gian Paolo Caviglia Romina Salpini Valentina Svicher

Background & Aim: The entry of Hepatitis D Virus (HDV) depends on HBV surface proteins (HBsAg) composed of three isoforms: large-, middle, and small HBsAg. Here, we investigate the levels of total HBsAg and HBsAg isoforms and their correlations with HDV-RNA, HBcrAg, and transaminases in the setting of untreated chronic hepatitis D (CHD). Methods: This study includes 316 HBeAg-negative patients: 192 CHD and 124 with chronic hepatitis B (CHB) as a control group. HBsAg isoforms were quantified by ad hoc-designed ELISAs. Results: The composition of HBsAg isoforms varied between the two groups of patients, with remarkably higher small HBsAg, middle-HBsAg, and large HBsAg in CHD than in CHB. This data was confirmed by multivariable analysis (p < 0.0001). Among CHD, HBsAg isoforms positively correlated with HDV-RNA (p < 0.0001) and HBcrAg (p < 0.0001) but not with HBV-DNA. The results were confirmed by stratifying patients according to HDV-RNA (< or >1000 IU/mL) and HBcrAg (< or >3 logU/mL). Furthermore, CHD patients with ALT > upper limit of normal presented significantly higher S-HBsAg and M-HBsAg levels. Conclusions: CHD is characterized by a more elevated HBsAg isoform production, paralleling HDV-RNA and HBcrAg release. This may suggest a preferential recruitment of HBsAg isoforms in HDV virions at the expense of HBV virions. The association of HBsAg isoforms with higher ALT also suggests their potential contribution in supporting HDV-induced pro-inflammatory stimuli.

Viruses doi: 10.3390/v18050513

Authors: Sarah C. Macon-Foley Meghan E. Hermance

Tick-borne viruses (TBVs) are a diverse group of arthropod-borne pathogens maintained in complex transmission cycles involving both tick vectors and vertebrate hosts. Among the known TBV transmission routes, co-feeding transmission, in which virus is transferred from an infected tick to an uninfected tick feeding on the same vertebrate host even in the absence of a detectable viremia, represents an important route that contributes to viral maintenance in nature. Although co-feeding transmission has been demonstrated across multiple vector, host, and virus combinations, the mechanisms governing this transmission route remain poorly defined. This review synthesizes current understanding of co-feeding transmission and highlights the importance of ecological and immunological factors that shape this process in nature. Specifically, we emphasize the role of the cutaneous microenvironment at the tick co-feeding site, where localized viral replication and tick salivary factors create conditions favorable for virus transfer between co-feeding ticks. We also highlight the requirements for co-feeding transmission to occur in nature and across seasons. Together, these insights support a model in which localized skin infection is a central feature of co-feeding transmission while underscoring key gaps in our understanding of the cellular and molecular mechanisms that govern this process.

Viruses doi: 10.3390/v18050514

Authors: Hao Zheng Tong Zhao Meixian Fu Zirui Niu Yifan Xing Xia Cai Jian-Er Long

Enteroviruses are positive-sense single-stranded RNA viruses and common pathogens that are responsible for diverse public health diseases. To facilitate the study of the virus biology and pathogenesis of enterovirus, we developed a rapid method for construction of the enteroviral cDNA clones including enterovirus A71 (EV-A71) and coxsackievirus B5 (CVB5). As described for EV-A71, the full-length cDNA of CVB5 was amplified by long-distance PCR and cloned into a T7 promoter-containing plasmid using directional seamless cloning technology. The virus was successfully rescued by single transfection into cells stably expressing T7 polymerase and exhibited characteristics similar to the parental virus. Next, through systematic construction and the optimization of the EV-A71 and CVB5 reporter viruses, we successfully generated two novel reporter virus panels with high virus titers, rapid replication, and relatively stable genetic inheritance across passages using the new fluorescence proteins mScarlet3-H and the smallest miRFP670nano3. Analysis of critical determinants for the reporter virus construction revealed that reporter gene sizes, genomic insertion sites, and the usage of protease recognition sites are crucial parameters. The EV-A71 and CVB5 reporter viruses enable antiviral drug evaluation, as demonstrated by our identification of gemcitabine as a broad-spectrum inhibitor of both viruses. These systems also facilitate the functional interrogation of host factors, exemplified by our discovery that METTL3 promotes EV-A71 and CVB5 replication. These reverse genetic tools, including infectious cDNA clones and reporter viruses, will advance basic enterovirus biology and accelerate antiviral drug discovery.

Viruses doi: 10.3390/v18050512

Authors: Ruxandra-Cristina Marin Radu Dumitru Moleriu Gabriela S. Bungau Delia Mirela Tit Călin Muntean

(1) Background: Despite the success of combination antiretroviral therapy (cART), immune recovery in treated HIV infection remains heterogeneous, and discordant immune–virologic responses persist in a substantial proportion of people living with HIV (PLWH). These patterns may reflect ongoing immune dysregulation despite effective viral suppression. This study aimed to characterize discordant treatment classifications, evaluate immune imbalance using the CD4/CD8 ratio, identify associated clinical predictors, and assess opportunistic infection burden in a Romanian cohort of people living with HIV receiving long-term cART. (2) Methods: A retrospective cross-sectional study was conducted in 462 adults with HIV-1 infection receiving cART at the “Prof. Dr. Matei Balș” National Institute of Infectious Diseases, Bucharest (2018–2021). PLWH were classified as concordant responders (CR), immunological discordant responders (ID), or virological discordant responders (VD) based on plasma HIV-1 RNA and CD4+ T-cell count thresholds. Immune dysregulation was assessed using the CD4/CD8 ratio. Multinomial logistic, logistic, and negative binomial regression models were used to identify predictors of discordant responses, severe CD4/CD8 ratio inversion, and opportunistic infection burden. (3) Results: Discordant responses were observed in 30.7% of PLWH (14.5% ID, 16.2% VD). CD4/CD8 ratio inversion occurred in 71.2% and severe inversion in 40.0%. Significant differences across clinical classification groups were found for CD4+T-cell counts (H = 153.62, p < 0.001, ε2 = 0.33) and CD4/CD8 ratio (H = 115.10, p < 0.001, ε2 = 0.25), while CD8+ counts were similar (p = 0.571). Male sex was associated with both ID and VD, and severe CD4/CD8 inversion was strongly associated with ID. Opportunistic infection burden was associated with duration of HIV infection and CDC stage. (4) Conclusions: Discordant immune–virologic responses remain frequent during long-term cART and are characterized by persistent immune imbalance reflected by CD4/CD8 ratio inversion. The CD4/CD8 ratio may provide clinically relevant information on immune recovery beyond CD4+ T-cell counts.

Viruses doi: 10.3390/v18050511

Authors: Ruth S. Bako Colleen F. Kelley

The rectal mucosa houses a large number of viruses with important roles in shaping the local microbial communities and modulating immune responses, which could influence host susceptibility to infection and other diseases. Unique composition of the gut microbiome, including the predominance of clinically significant eukaryotic viruses like herpesviruses, cytomegalovirus, and human papillomavirus, has been described in both people with HIV (PWH) and men who have sex with men (MSM) vulnerable to HIV. Despite these insights, the rectal virome and the clinical implications of virome–bacteriome–immune interactions in the rectal mucosa remain poorly understood. In this review, we synthesize existing data on the composition of the rectal virome, its interactions with the bacteriome and the immune system, and implications on clinical outcomes in people living with or vulnerable to HIV. We also highlight the gaps and research needed to further explore and unravel these relationships.

Viruses doi: 10.3390/v18050510

Authors: Omar Hozayen Joseph Hozayen Benjamin J. Behers Anas Abu Jad Bashar Roumia Matthew W. Miller Christoph A. Stephenson-Moe Nicolas Riveros Manuel Rosario Karen M. Hamad

HIV-associated cardiomyopathy is a significant cause of morbidity and mortality among people living with HIV, contributing to heart failure, arrhythmia, and sudden cardiac death. Despite its clinical importance, its individual-patient clinical spectrum has not been systematically synthesized. We conducted a systematic review of published English-language case reports and small case series describing cardiomyopathy in HIV-infected individuals. Etiologies were classified using a framework distinguishing cardiomyopathy arising from uncontrolled HIV from that occurring despite virologic control. Stratified analyses examined temporal trends and geographic differences. We identified 99 patients (75 male, 20 female, 4 unspecified) from 27 countries (80% high-income). Median age was 35 years (IQR 28–45). Among 52 patients with CD4 data, median was 154 cells/µL (IQR 84–391); 52% had CD4 < 200. Systolic dysfunction was present in 94% with echocardiographic data. Uncontrolled HIV phenotypes predominated (64%), but controlled phenotypes (21%)—including drug-induced cardiomyopathy (n = 19, predominantly zidovudine-associated) and autoimmune or inflammatory mechanisms (n = 13)—were substantial. Mortality declined across eras: 65% pre-ART, 32% early ART, 21% modern ART. Recovery occurred in 58%. HIV-associated cardiomyopathy is heterogeneous with improving outcomes across treatment eras. Systematic etiologic evaluation is warranted in all affected patients. The near absence of data from sub-Saharan Africa represents a critical gap.

Viruses doi: 10.3390/v18050509

Authors: Lina Shalaby Yaman Al-Haneedi Alaa Abdelhamid Hadi Yassine Mohamed M. Emara

Dengue virus (DENV) and SARS-CoV-2 are emerging viral pathogens that share overlapping clinical features, including fever, fatigue, and respiratory symptoms, complicating differential diagnosis in endemic regions. Their co-circulation has increased the risk of co-infections, which may result in unpredictable disease progression, increased morbidity, and mortality. This overlap presents a significant challenge in managing outbreaks, as both viruses pose a major public health threat. Vaccines and direct-acting antivirals may be rendered ineffective by viral mutations, making it difficult to address evolving strains. Host-directed antivirals offer a promising alternative, potentially maintaining efficacy against a multitude of variants. Both DENV and SARS-CoV-2 rely on host proteases for viral maturation and entry, with furin playing a crucial role in viral glycoprotein cleavage. In DENV, furin cleaves the prM protein, facilitating virion maturation, while in SARS-CoV-2, the polybasic furin cleavage site in the spike protein enhances viral entry. This makes furin a compelling pan-viral target, where inhibiting furin could reduce viral fitness without relying on viral mutations. This review highlights the therapeutic rationale for targeting furin and discusses luteolin, a furin inhibitor showing antiviral activity against both viruses. Furin-targeted therapies may offer a durable antiviral strategy effective across DENV serotypes, SARS-CoV-2 variants, and co-infection settings.

Viruses doi: 10.3390/v18050508

Authors: Adriace Chauwa Samuel Bosomprah Bernard Phiri Natasha M. Laban Dhvani H. Kuntawala Dennis Ngosa Harriet Ng’ombe Fraser Liswaniso Chaluma C. Luchen Mutinta Muchimba Innocent Mwape Bertha T. Nzangwa Sekayi F. Tigere Kennedy Chibesa Suwilanji Silwamba Michelo Simuyandi Nyuma Mbewe Roma Chilengi Caroline Chisenga

During Zambia’s 2023–2024 cholera outbreak, reliance on single-pathogen diagnostics risked overlooking co-circulating enteric pathogens. This study estimated the prevalence of rotavirus and described co-detected enteropathogens and rotavirus genotypes among patients admitted with suspected cholera. A sub-analysis was conducted on diarrhoeal stool specimens collected from patients who met the syndromic suspected cholera case definition. Samples were tested using the Bosphore® Gastroenteritis Panel v2, a multiplex PCR enteric panel, to detect rotavirus and other gastrointestinal pathogens. Rotavirus-positive specimens with sufficient viral load were further genotyped by RT-PCR targeting of the VP7 and VP4 genes. Among 319 suspected cholera admissions, rotavirus was detected in 18 patients (5.6%; 95% CI 3.4–8.8%), predominantly in children aged <5 years (27.8%, 5/18) and 6–17 years (27.8%, 5/18). Co-infection was common, with 17/18 (94.4%) of rotavirus-positive samples showing co-infection with at least one additional enteric pathogen, most frequently Campylobacter. Genotyping was successful in five samples and revealed heterogenous circulating strains, including G1P[8], G2P[4], G3P[6], G12P[6], and G1P[6]. Rotavirus accounted for a modest proportion of suspected cholera admissions and was frequently detected in mixed enteric infections, underscoring the value of multi-pathogen diagnostics and continued molecular surveillance during outbreak response.

Viruses doi: 10.3390/v18050507

Authors: Ricarda Plümers Jens Dreier Attila Mandl Cornelius Knabbe Tanja Vollmer

Hepatitis E virus (HEV) is the leading cause of hepatitis globally and poses particular risks for immunocompromised individuals. Mandatory screening of blood donations for HEV RNA and retrospective individual testing of previous donations (lookback investigations) following a reactive result have been implemented in several countries to protect these patients. This includes Germany, where a sensitivity limit of 2000 IU/mL applies to index donations. In total, 334 HEV RNA-positive blood donations were detected at our blood donation service between 2018 and 2024. Lookback testing was applied in 211 cases, revealing previous HEV RNA-positive donations in 23.1% of donors (n = 48, 76 donations). Although 16 of these retrospectively tested HEV RNA-positive donations have already been transfused, no transfusion-transmitted HEV infection has been reported. The HEV RNA viral load in the lookback donation was below 50 IU/mL in 72.4% of cases. Routine testing effectively prevents highly viremic blood products entering the supply, significantly reducing the infection risk. While the administration of virus particles with low-viremic products cannot be ruled out, the remaining risk appears to be minimal and has been deemed so far acceptable for the safety of blood products. The lookback strategy further supports the screening strategy by retrospectively identifying blood products from low-viremic donations and enabling appropriate risk management.

Viruses doi: 10.3390/v18050506

Authors: Jagriti Kakar Liba Sokolovska Maksims Zolovs Modra Murovska Ingrīda Čēma

The presence of human herpesviruses is frequently detected in the oral cavity, yet their disease-specific role in chronic inflammatory oral mucosal disorders remains uncertain. This comparative observational study investigated Epstein–Barr virus (EBV) and human herpesvirus-7 (HHV-7) genomic sequences in saliva and virus-specific antibodies in serum among patients with oral lichen planus (OLP; n = 35), aphthous stomatitis (AS; n = 31), and healthy controls (n = 34). Salivary viral loads were quantified using real-time PCR, while EBV and HHV-7-specific IgG and IgM antibodies were measured using ELISA-based assays. EBV and HHV-7 DNA in saliva were commonly detected across all groups, demonstrating high baseline shedding and marked interindividual variability. Although EBV IgG levels were higher in OLP compared with AS in univariate analysis, multivariate regression revealed that age, rather than disease status, was the primary determinant of EBV IgG levels. After adjustment for age, sex, and discomfort, neither EBV nor HHV-7 salivary loads showed independent associations with OLP or AS. HHV-7 salivary loads were uniformly distributed among groups. These findings suggest that salivary detection of EBV and HHV-7 reflects widespread latent infection rather than disease-specific activity in OLP or AS. Longitudinal and tissue-based studies integrating immunological profiling are warranted to clarify whether herpesvirus reactivation contributes to disease severity in defined patient subgroups.

Viruses doi: 10.3390/v18050505

Authors: Missiani Ochwoto Danielle K. Offerdahl Edwin O. Ogola Barbara C. Weck Dan Long Greg A. Saturday Marshall E. Bloom

The detailed mechanism and sequence by which tick-borne flaviviruses (TBFVs), such as Langat virus (LGTV), infect and disseminate in arthropod hosts remain undefined. To begin characterizing these processes, we used artificial membrane feeding chambers to feed adult Ixodes scapularis ticks with blood containing LGTV. At 24, 48, 72, and 96 hours (h) after attachment, we removed and dissected the partially fed ticks to obtain the midgut and salivary glands. Histology confirmed infection in cells of the digestive epithelium lineage; infection was noted in midgut generative cells and the more differentiated functional digestive cells over the course of feeding. The viral envelope (E) protein, nonstructural protein 3 (NS3), and double-stranded RNA (dsRNA) were readily detected in these cells by 48 h after infection. Parallel analysis indicated that cells in salivary gland acini were also infected by 48 h, where virus target cells appeared to be the granular cells in acini types II and III. Thus, both salivary glands and midgut showed direct evidence of infection by 48 h. Although viral staining was not observed at 24 h, when organs were removed at 24 h and individually cultured ex vivo, the virus was detected. Taken together, our results provide evidence of LGTV infection in both the salivary glands and midgut within the first 24 h of a blood meal. The findings should prompt a reevaluation of the systemic dissemination of TBFV in infected ticks.

Viruses doi: 10.3390/v18050504

Authors: Doreswamy Kenchegowda Brian D. Carey Joshua Shamblin Collin J. Fitzpatrick Danielle L. Porier Susan Coyne Jeffrey Koehler Candace D. Blancett Christina E. Douglas Cheryl Taylor-Howell Aura R. Garrison Christopher P. Stefan Charles J. Shoemaker Joseph W. Golden

Crimean–Congo hemorrhagic fever virus (CCHFV) and severe fever with thrombocytopenia syndrome virus (SFTSV) are tick-borne pathogens that cause severe illness and high mortality. Early diagnosis is critical, particularly in resource-limited settings, to enable timely intervention. Host gene expression profiling offers a promising approach to identify potential biomarkers for early detection, disease staging, and logical treatment decision-making. Using a transient IFN-α/β receptor-suppressed mouse model, we performed targeted transcriptomic analysis on blood samples collected at 2, 3, and 4 days after CCHFV or SFTSV challenge. A significant increase in viral load and changes in gene expression were observed as early as two days post-challenge. CCHFV induced a progressively evolving interferon-driven response, while SFTSV triggered rapid, sustained immune activation. Affected targets included interferon-stimulated genes, chemokines, cytokines, Toll-like receptors, and genes associated with viral evasion and innate immune response. Despite shared expression patterns, unique genes were identified as potential biomarkers to distinguish between CCHFV and SFTSV infections. Differential gene expression revealed distinct immune response dynamics, with suppression of critical immune regulatory genes suggesting transcriptional signatures associated with viral evasion mechanisms contributing to disease severity. These findings provide a comparative analysis of molecular pathways and gene expression changes, offering critical insights for biomarker discovery, effective triage, and evaluation of appropriate medical intervention.

Viruses doi: 10.3390/v18050503

Authors: Meagan M. Taylor Rosemary W. Roberts Jonathan O. Rayner

Chikungunya virus (CHIKV) is an alphavirus that infects dermal fibroblasts as a primary target cell during natural mosquito-borne transmission. While primary human dermal fibroblasts (hDFs) have been implicated as a key source of type I interferon (IFN-I) during CHIKV infection, the dynamics of this response and its sufficiency for antiviral protection remain incompletely understood. Here, we systematically characterize in vitro CHIKV infection of primary hDFs, evaluating the effects of single-passage viral stock origin (mammalian- vs. mosquito-propagated), donor variability, and multiplicity of infection (MOI) on infection kinetics and innate immune induction. We demonstrate that hDFs support high-titered CHIKV replication at both MOI 1 and 0.01, resulting in universal cell death by 72 hpi despite robust IFNβ transcript induction—reaching up to ~2800-fold over mock—and secretion of pro-inflammatory cytokines, including IFNα2, TNFα, IL-1β, and IL-8. Notably, IFNβ protein levels remained below 10 pg/mL under all infection conditions, revealing a disconnect between transcriptional and translational responses, suggesting CHIKV-mediated translational suppression. Pharmacological inhibition of TBK1/IKKε via amlexanox did not suppress IFNβ transcript induction at any tested concentration, suggesting that canonical PRR signaling through this node—including both RIG-I/MAVS and TLR3/TRIF pathways—is not the major driver of the observed transcriptional response. In contrast, co-inoculation with exogenous IFNβ as low as 20 pg/mL activated IFNAR signaling, robustly upregulated interferon-stimulated genes (ISGs), and fully rescued hDFs from otherwise lethal infection. Together, these findings demonstrate that CHIKV-infected hDFs mount a transcriptionally robust but translationally insufficient innate immune response and that the transcriptional response appears to operate independently of TBK1/IKKε. These results have direct implications for understanding how the skin microenvironment may modulate early CHIKV pathogenesis and suggest that paracrine IFNβ signaling from neighboring cell types may be critical for fibroblast survival during natural infection.

Viruses doi: 10.3390/v18050502

Authors: Deepanker Tewari Manoj K. Sekhwal Chrislyn Nicholson Mary L. Killian Corey Zellers Julia Livengood Kristina Lantz Mia Torchetti Alex Hamberg

The 2022 highly pathogenic avian influenza (HPAI) outbreak of H5N1 clade 2.3.4.4b was one of the major avian influenza outbreaks, leading to multiple spillover events infecting domestic and wild bird flocks, as well as mammals. The sustained spread was a result of viral circulation in wild birds across migratory flyways in North America. Pennsylvania has a significant poultry population that supports both retail and live bird markets. The state also features migratory bird stopovers on the Atlantic flyway, increasing exposure to HPAI infections. This study investigates clinical presentation and sequence data from H5N1 clade 2.3.4.4b viruses during the 2022 outbreak in Pennsylvania. Eight different H5N1 clade 2.3.4.4b genotypes were detected (A1, B1.1, B1.2, B1.3, B2.2, B3.3, B3.5, and one minor genotype) during the first year. The earliest detection was genotype A1, a fully Eurasian virus, in commercial poultry in April 2022. All other genotypes identified were reassortants of A1 with North American avian influenza gene segments (denoted with “B”). Genotype B3.3 was a rare genotype prior to the initial spillover into the live bird market system, but remained predominant among backyard flocks in Pennsylvania and surrounding states until September 2023. Genotype B3.3 has not been detected in migratory waterfowl since, suggesting the genotype has waned and is no longer in circulation. This study sheds light on the genotype diversity of H5N1 during the 2022 outbreak in Pennsylvania poultry, contributing to the understanding of virus evolution and its potential impacts.

Viruses doi: 10.3390/v18050501

Authors: Bruna Alves Ramos Daniel Damous Dias Joaquim Pinto Nunes-Neto José Wilson Rosa Junior Durval Bertram Rodrigues Vieira Valéria Lima Carvalho Ana Lúcia Monteiro Wanzeller Eliana Vieira Pinto da Silva Maria Nazaré Oliveira Freitas Landeson Junior Leopoldino Barros Maissa Maia Santos Jamilla Augusta de Souza Pantoja Ercília de Jesus Gonçalves Ana Claudia da Silva Ribeiro Ana Cecília Ribeiro Cruz Sandro Patroca Silva Carine Fortes Aragão Alexandre do Rosário Casseb Livia Caricio Martins

Mosquitoes are recognized as the arthropod group with the greatest vectorial capacity, and the viruses they transmit constitute a significant concern in the context of global One Health. In addition, these insects act as hosts for a wide diversity of insect-specific viruses (ISVs), which exclusively infect arthropods. Expanding knowledge of ISVs is particularly relevant, given their potential influence on arbovirus replication and their role in elucidating the evolutionary processes that shape virus–vector interactions. In this study, we report the isolation and molecular analysis of three negeviruses associated with different mosquito species of the genera Culex, Coquillettidia, Mansonia, and Ochlerotatus, collected in Belém, Pará State, in the Brazilian Amazon: Loreto virus, Wallerfield virus, and a putative new species, designated Terra firme virus. Eleven pools exhibited cellular alterations consistent with cytopathic effects in invertebrate C6/36 cells but showed no evidence of replication in vertebrate Vero cells. Notably, simultaneous infections by two or three negeviruses were detected in some mosquito pools, indicating the occurrence of multiple viral infections within individual samples. Genomic analyses revealed that the isolated strains share conserved domains with previously described isolates from other countries. Phylogenetic inferences demonstrated that the investigated strains are classified within the clades Nelorpivirus and Sandewavirus. Taken together, these findings expand the currently known diversity of the negevirus group and contribute to a more comprehensive understanding of its host range and geographic distribution.

Viruses doi: 10.3390/v18050500

Authors: Zijia Wang Zongminghan Liu Juntao Zeng Jiwei Li Jiahao Cheng Xiaoxue Qi Jingwen Li Shijie Bai

Viruses are key regulators of marine microbial communities, yet their temporal dynamics in tropical intertidal sediments remain poorly characterized. We conducted a year-long metagenomic survey of sandy intertidal sediments in Sanya Bay (60 monthly samples from five sites) to examine viral taxonomy, community structure, lytic proteins, and auxiliary metabolic genes (AMGs). Within the classifiable fraction, the assemblages were consistently dominated by Assiduviridae. However, NMDS analysis revealed a significant overall seasonal shift, with October–December samples separating from the rest of the year. Co-occurrence network analysis identified five co-occurrence modules with distinct temporal patterns, alongside a concurrent decline in module abundance and lytic proteins in October. Functional annotation showed that cysteine and methionine metabolism, primarily driven by DNA methyltransferases, was identified as a highly represented AMG category among the annotated functions, while other pathways displayed seasonal variability. Collectively, these findings suggest that although characterized by a classifiable fraction dominated by Assiduviridae, the highly complex tropical intertidal viral communities undergo substantial seasonal reorganization in structure and functional potential.

Viruses doi: 10.3390/v18050499

Authors: Daniella Rahamim-Cohen Ayelet Aviva Basson Clara Weil Izana Kaplan-Lavi Odelia Tassa-Liani Yael Topol Gabriel Chodick Bar Cohen Limor Adler Shirley Shapiro Ben David

Objectives: The World Health Organization (WHO) goal of eradicating hepatitis C virus (HCV) infection by 2030 has encouraged healthcare providers to implement proactive strategies to improve diagnosis and treatment. The aims of this retrospective cohort study were to assess a program designed to improve the HCV care cascade and facilitate access to treatment, within a national healthcare provider in Israel, Maccabi Healthcare Services (MHS). Methods: Included were adult patients newly diagnosed with HCV infection before and after the implementation of a screening and care optimization program. Patients diagnosed in 2017 served as the reference group (RG), while those diagnosed in 2019 (following the program implementation) comprised the intervention group (IG). Study outcomes included completion of HCV laboratory testing, time to consultation with gastroenterologist/hepatologist (GE), and initiation of treatment with direct-acting antivirals (DAAs). Results: The study sample included 356 HCV Ab+ patients in the RG (median age = 46 years; 41% females), and 328 in the IG (median age = 48 years; 39% females). Compared to RG, IG demonstrated higher rates of patient visiting GE visit (78.1% vs. 63%) and initiating DAA treatment (66.3% vs. 35.5%). Conclusions: Implementation of a restructured HCV care cascade was associated with a greater proportion of patients receiving expert consultation and higher DAA treatment uptake, important steps towards HCV eradication.

Viruses doi: 10.3390/v18050498

Authors: Leticia F. Baumbach Raquel S. Alves Laura J. Camargo Eduardo O. Sanguinet Leticia S. Santos Lucas Marian Gabriela E. Birlem Roberto Schroeder Fabiano Barreto João Marcos N. Costa Renata A. Casagrande Matheus N. Weber Cláudio W. Canal

Bovine viral diarrhea virus (BVDV) is a major cattle pathogen associated with significant economic losses worldwide. In Brazil, the high genetic diversity of circulating strains represents an additional challenge for disease control. To update the molecular epidemiology of BVDV in southern Brazil, 16,198 bovine serum samples collected in 2020 through a national surveillance program were screened for pestivirus RNA by RT-qPCR. Forty-nine samples (0.36%) were positive and subjected to partial sequencing of the 5′UTR and Npro regions. Phylogenetic analysis identified BVDV-1a (25/49; 51%), BVDV-1b (1/49; 2%), BVDV-1d (7/49; 14%), and BVDV-2b (16/49; 33%), with no detection of HoBiPeV. When compared descriptively with data from 2010 in the same region, BVDV-1a remained the most frequent subgenotype, while BVDV-2b also represented a substantial proportion of detections, contrasting with other regions worldwide. Although the two datasets are not directly comparable, and no statistically significant differences were observed, these findings provide an updated overview of circulating BVDV subgenotypes in Rio Grande do Sul. The absence of HoBiPeV contrasts with reports from other regions of Brazil and suggests a distinct regional pattern of pestivirus circulation. Overall, the results reinforce the importance of continuous genomic surveillance to monitor changes in viral diversity and support control strategies in cattle populations.

Viruses doi: 10.3390/v18050497

Authors: Oula Mansour Artem V. Fadeev Alexander A. Perederiy Andrey D. Ksenafontov Anastasiia Y. Boyarintseva Daria M. Danilenko Dmitry A. Lioznov Andrey B. Komissarov

Human parainfluenza virus type 4 (hPIV4) remains poorly characterized compared with other hPIV serotypes and information on its genomic diversity is particularly limited for Russia and Eastern Europe. In this study, we report the first complete genome sequences of hPIV4 isolates from Russia and place them in the context of global hPIV4 genetic diversity. Eight hPIV4 viruses were isolated in cell culture from respiratory samples collected from hospitalized children in Saint Petersburg between 2017/2018 and 2023/2024. Complete viral genomes were recovered using a metagenomic whole-genome amplification approach based on SMART-9N technology. Phylogenetic analysis of 178 complete hPIV4 genomes showed clear separation into hPIV4a (n = 132) and hPIV4b (n = 46) subtypes. Based on genetic distance approach, hPIV4a formed two major clusters, with the dominant cluster B subdivided into four subclusters (B1–B4); and subcluster B4 further resolved into four genetic lineages. All Russian isolates belonged to the subcluster B4 and were distributed among multiple co-circulating lineages. In contrast, hPIV4b genomes segregated into three distinct clusters, reflecting structured genetic diversity within the subtype. Collectively, this study provides, to the best of our knowledge, the first p-distance-based framework for hPIV4 whole-genome classification and contributes new complete genome sequences for an underrepresented region.

Viruses doi: 10.3390/v18050496

Authors: Peihan Wu Shuai Wang Xu Li

The m6A RNA methylation pathway plays a critical role in host antiviral defense. Host cells employ m6A readers such as YTHDF2 to regulate viral RNA fate through diverse mechanisms, including degradation, translational control, and immune recognition. However, we found that YTHDF2 is essential for SARS-CoV-2 replication, suggesting that a virus may exploit this host machinery to its advantage. Through integrative RNA-proteome analysis, we identified the SARS-CoV-2 nucleocapsid (N) transcript as the most heavily m6A-modified viral transcript and a direct interactor of YTHDF2. The N protein forms a complex with YTHDF2 in the cytoplasm and redirects this host RNA decay machinery toward host antiviral transcripts. N suppresses ISG15, IFIT1, MX1 and pro-inflammatory cytokines in a largely YTHDF2-dependent manner, an effect that is lost in YTHDF2-knockout cells. These findings reveal a viral immune evasion strategy wherein a viral protein actively hijacks an m6A reader to silence antiviral gene expression, establishing the N-YTHDF2 axis as a therapeutic target against SARS-CoV-2 and other coronaviruses.

Viruses doi: 10.3390/v18050495

Authors: Maryline Santerre Jenny Shrestha Charles N. S. Allen Natalia Shcherbik Bassel E. Sawaya

Although neurons are not productively infected by HIV-1, the envelope glycoprotein gp120, detectable in cerebrospinal fluid independently of active viral replication, gains intraneuronal access via lipid raft-mediated endocytosis, macropinocytosis, and retrograde axonal transport, contributing to persistent neurobiological dysfunction within the central nervous system. Once internalized, gp120 is associated with neuronal dysfunction involving convergent pathways, including excitotoxic calcium dysregulation, mitochondrial and metabolic failure, and inflammatory and senescence-associated amplification. These pathways converge on suppression of CREB and BDNF signaling, dismantling the transcriptional and neurotrophic programs required for synaptic maintenance and cognitive resilience. Extracellular vesicle-mediated dissemination and microRNA reprogramming extend gp120-associated neurobiological effects beyond sites of receptor engagement, while gut-derived metabolites, particularly quinolinic acid, lower the excitotoxic threshold through synergistic activation of NMDA receptors. Together, these mechanisms define HAND as a network disorder in which gp120 contributes to persistent neurocognitive dysfunction beyond active viral replication, identifying convergent therapeutic nodes where combination strategies targeting excitotoxicity, mitochondrial dysfunction, and neuroinflammation offer the most promising path toward durable neuroprotection.

Viruses doi: 10.3390/v18050494

Authors: Israel Pagán Rafael de Andrés-Torán Nuria Montes Aurora Fraile Fernando García-Arenal

Plant defences are assumed to evolve in response to the negative effects of virus infection on plant fitness (virulence), and to drive plant–virus coevolution. However, viruses are not always antagonistic symbionts of plants, and the expression of defence traits is environment-dependent. Thus, understanding plant–virus interactions requires analysing the expression of defence traits in the host’s native habitat. Here we analyse the effect of cucumber mosaic virus (CMV) infection, and the expression of resistance and tolerance in the native habitat of a wild Arabidopsis thaliana population. Plants from ten genotypes from that population, which have been shown to differ in resistance and tolerance to CMV in a greenhouse, were inoculated with an Arabidopsis isolate of CMV and transplanted to their habitat. Resistance was rated based on virus accumulation in leaves, and tolerance was rated based on the effect of infection on plant fecundity relative to virus accumulation. Consistent with the greenhouse assays, virulence depended on the host genotype, and polymorphisms for resistance and tolerance were expressed in the field, supporting the validity of the conclusions from the greenhouse assays. Our results also support theoretical predictions on the relationships between pathogen multiplication and virulence and between resistance and tolerance.

Viruses doi: 10.3390/v18050493

Authors: Imaan Muhammad Kaci Craft Shaokai Pei Ruth Cruz-Cosme Qiyi Tang

Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) represent fundamental cellular adaptive mechanisms that maintain protein homeostasis and metabolic balance. Many RNA viruses, particularly flaviviruses such as dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), yellow fever virus (YFV), and Japanese encephalitis virus (JEV), extensively remodel the ER to establish replication compartments and assemble progeny virions. This massive reorganization disrupts ER homeostasis, leading to UPR activation. Emerging evidence reveals that flaviviruses not only trigger but also manipulate the three UPR branches—PERK, IRE1, and ATF6—to optimize viral translation, replication, and egress. In parallel, flavivirus infection profoundly alters host lipid metabolism and promotes dynamic changes in lipid droplets (LDs), key organelles that mediate lipid storage and serve as scaffolds for viral replication and assembly. The UPR intimately connects to LD biogenesis through transcriptional and translational programs mediated by XBP1, ATF4, and ATF6, thereby coupling ER stress responses to lipid remodeling and energy homeostasis. This intricate crosstalk between UPR and LDs creates a metabolic and structural niche favorable for viral replication but detrimental to host cell integrity. This review provides a comprehensive analysis of the molecular mechanisms by which flaviviruses exploit ER stress and the UPR to reprogram lipid metabolism and LD dynamics. We highlight the dual role of UPR signaling in promoting adaptive lipid synthesis and initiating cell death under prolonged stress, discuss recent insights into ER–LD interactions during flavivirus infection, and explore therapeutic opportunities targeting UPR–lipid metabolic pathways as broad-spectrum antiviral strategies. Understanding this interconnected network will advance our knowledge of viral pathogenesis and identify new avenues for host-directed antiviral intervention.

Viruses doi: 10.3390/v18050492

Authors: Subash Chapagain Lauren F. Woodburn Natalie C. J. Strynadka Eric Jan

Viral internal ribosome entry sites (IRESs) are specialized RNA structures that facilitate cap-independent translation as a strategy to usurp the host translational machinery. The Type 6 IRESs are the most streamlined mechanism to date, as they adopt a three pseudoknot RNA structure to initiate factorless translation initiation by directly recruiting the ribosome and drive translation. The Halastavi árva virus (HalV) IRES represents the most minimalistic subclass identified to date, whereby the IRES lacks specific pseudoknot domains that bind to the 40S subunit but instead recruits pre-assembled 80S ribosomes via a mechanism that is not fully understood. Here, we examined cellular conditions that can support HalV IRES translation. We demonstrated that the HalV IRES is translationally active in insect Sf21 lysates and Drosophila S2 cells, but inactive in mammalian RRL and wheat germ extract. Cells treated with heat shock or serum starvation suppressed HalV IRES activity, whereas virus infection robustly enhanced HalV IRES-mediated translation. Finally, the HalV IRES can support viral translation and replication using a heterologous viral replicon. These findings highlight the context-specific cellular conditions that allow ribosome assembly and translation by a factorless minimalist IRES.

Viruses doi: 10.3390/v18050491

Authors: Hélène Boogaerts Janne Tollenaere Kim Bekelaar Els Oris Sarah Resseler Baptist Declerck Dorien Van den Bossche Marjan Van Esbroeck Deborah Steensels

Despite serological evidence of tick-borne encephalitis virus (TBEV) circulation in Belgian animals since 2007, confirmed autochthonous human infection was only first documented in 2020. We review the current national epidemiologic situation and investigate a household cluster of confirmed autochthonous cases identified in 2025. A cohabiting couple experienced a near-simultaneous onset of meningoencephalitis and tested positive for TBEV-specific IgM and IgG, with confirmation by PRNT90. One patient reported a recent tick bite, and both patients reported consumption of unpasteurized milk and goat cheese, suggesting possible alimentary transmission. The identification of Case 2, who lacked neurological symptoms at presentation and was only tested due to the index case, illustrates the risk of missed diagnoses and supports the notion that human TBEV infection is likely underdiagnosed in Belgium. These findings underscore the need to increase clinical awareness, strengthen surveillance, and reinforce prevention strategies. TBE should be considered in the differential diagnosis of patients presenting with non-specific fever or neurological syndromes such as meningoencephalitis, particularly during the spring-to-autumn tick activity season.

Viruses doi: 10.3390/v18050490

Authors: Suresh V. Kuchipudi Shubhada K. Chothe Santhamani Ramasamy James S. Holt Iulia Vann Reginald A. Hoyt Andrew M. Hoffman Melissa Bilec Ananias A. Escalante Jennifer R. Fetter Sarah States Maureen Lichtveld

The inaugural Pennsylvania One Health Consortium Annual Meeting brought together partners from universities, state agencies, public health, veterinary, agriculture, industry, and community organizations to align around a shared One Health agenda. The program highlighted zoonotic threats, antimicrobial stewardship, wildlife and ecosystem health, invasive species, and climate-sensitive hazards. Participants affirmed privacy-preserving data exchange, projects integrating genomic epidemiology with field and environmental surveillance, cross-disciplinary education, and transparent governance, concluding with a phased roadmap for an integrated statewide One Health framework.

Viruses doi: 10.3390/v18050489

Authors: Francesca Romana Tonellato Francesca Poletto Cristina Andolfatto Claudia Maria Tucciarone Giovanni Franzo Mattia Cecchinato Matteo Legnardi

Infectious bursal disease virus (IBDV) is an immunosuppressive pathogen posing a major threat to poultry health worldwide. Its marked phenotypic variability is driven by the rapid evolution of its double-stranded RNA genome, primarily achieved through mutation and reassortment. Although extensive evidence has been generated on molecular determinants of antigenicity and pathogenicity, interpretation is often hindered by heterogeneity and lack of systematicity. This scoping review synthesizes over 35 years of research on amino acid positions influencing IBDV phenotype. A total of 62 studies reporting 107 functionally relevant sites were identified and critically appraised based on evidence type, methodological approach, and ability to infer causality. The results confirmed the central role of VP2, particularly its hypervariable region, while also highlighting the increasingly recognized contribution of other viral proteins. Despite good agreement, comparability across studies was limited by substantial heterogeneity in experimental design and the frequent focus on partial genomic regions. Notably, some molecular markers were context-dependent or inconsistently associated with phenotypic outcomes, underscoring the need for proper interpretation of molecular determinants and for more standardized and comprehensive approaches, including full-genome analyses and reverse genetics. Overall, these findings provide a valuable framework for enhancing molecular diagnostics and supporting the rational design of next-generation vaccines.

Viruses doi: 10.3390/v18050488

Authors: Yuwei Wu Zhifan Li Nuo Xu Zijun Lu Yurui Dong Kunlin Li Ying Bian Chenzhi Huo Tao Qin Sujuan Chen Hui Yang Daxin Peng Xiufan Liu

H5 subtype avian influenza virus (AIV) can infect both chickens and ducks, leading to substantial economic losses. Nevertheless, certain strains cause silent infections in ducks. In this study, a goose-origin clade 2.3.4.4h H5N6 AIV was isolated, which caused high mortality in mixed-gender white leghorn chickens but no deaths in mixed-gender mallard ducks. After independent serial in vitro passage in duck embryo fibroblasts (DEFs) and in vivo passage in specific-pathogen-free (SPF) ducks, the DEF-passage 10 (P10) virus induced markedly higher mortality rates and viral loads in SPF ducks compared to the DEF-P1 virus and the original parental virus prior to passage. Similarly, the in vivo-passaged P3 and P4 viruses exhibited significantly higher mortality rates than the P1 virus in SPF ducks, with 100% mortality and markedly increased viral titers in the organs. A whole-genome SNP analysis identified seven high-frequency mutations in the M1, NA and NS1 proteins. The NS1-F161L substitution virus exhibited significantly increased mortality rates, viral loads in multiple tissues, and a robustly induced innate immune response in ducks. Furthermore, dynamic evolutionary variations in the NS1 protein among global H5 avian influenza viruses revealed that the NS1-F161L substitution became dominant in clade 2.3.4.4b viruses in 2021 and subsequent years. Collectively, our findings demonstrate that host-driven adaptation can rapidly increase the pathogenicity of H5N6 AIVs in ducks and identify NS1-F161L as a critical virulence marker. These results offer novel insights relevant to the molecular surveillance, virulence prediction, and risk assessment of circulating H5 AIVs in waterfowl.

Viruses doi: 10.3390/v18050487

Authors: Thomas B. Voigt Noor Ghosh Brandon C. Rosen Taylor Newbolt Johan J. Louw Aaron Yrizarry-Medina Christakis Panayiotou Jack T. Mauter Giovana de Figueiredo Godoy Joshua Terao Eva G. Rakasz Matthew R. Reynolds Dawn M. Dudley David I. Watkins Michael J. Ricciardi

Henipaviruses (HNVs), including Nipah virus (NiV) and Hendra virus (HeV), are highly pathogenic and often lethal zoonotic viruses with broad species tropism and no approved human vaccines. The emergence of genetically divergent HNVs—including Ghana virus (GhV), Langya virus (LayV), and Mojiang virus (MojV)—emphasizes the need for broadly protective countermeasures. Here, we evaluated the antibody (Ab) responses to sequential mRNA vaccines encoding the membrane-bound attachment glycoprotein (gG) from NiV, GhV, and/or LayV in a pilot study with Indian rhesus macaques. Serum binding Ab responses were quantified by ELISA against five soluble gG antigens (NiV, HeV, GhV, LayV, MojV). Functional activity was assessed by neutralization assays using NiV, HeV, and GhV pseudoviruses, and by receptor-blocking ELISA. Sequential vaccination induced high-titer IgG binding against all five HNV gGs with increasing breadth after each dose. Pan-genus regimens elicited moderate neutralizing Ab titers against NiV, HeV, and GhV, whereas the NiV-only regimen elicited potent but narrow neutralization against NiV and HeV. Conversely, the GhV-LayV-GhV regimen elicited strong binding to GhV, LayV, and MojV gG and robust neutralization of GhV pseudovirus, but limited cross-reactivity to NiV and HeV. In this pilot study, we demonstrated that mRNA vaccination can elicit broadly reactive binding and neutralizing Ab responses across phylogenetically distant HNVs. Additionally, we show GhV pseudovirus neutralization for the first time. Collectively, these data provide a foundation for the development of next-generation pan-genus HNV vaccines capable of mitigating future HNV outbreaks.

Viruses doi: 10.3390/v18050486

Authors: Weizhao Lin Xiaoping Shao Junjie Wang Hongqing Wen Jiahong Liu Can Xiong Zixia Qian Wei Zhao Jun Liu Jiufeng Sun

Determining the characteristics of hepatitis B virus (HBV) infection in the healthy population and evaluating the effectiveness of detection strategies will facilitate the optimization of hepatitis B screening strategies in the community and accelerate the elimination of HBV infection in China by the end of 2030. Hepatitis B surface antigen (HBsAg)-electrochemiluminescence immunoassays (ECLIAs), HBsAg-rapid diagnostic tests (RDTs), and HBV DNA-nucleic acid tests (NATs) were performed on serum samples from 2721 community-based healthy participants in Guangdong Province. The screening performance of the RDT and NAT and the distribution characteristics of HBsAg and HBV DNA were evaluated. The prevalence rates of HBsAg-ECLIA, HBsAg-RDT and HBV DNA-NAT in Guangdong Province were 6.10% (95% CI: 5.26~7.06), 4.96% (95% CI: 4.21~5.84) and 6.55% (95% CI: 5.64~7.49), respectively, and the prevalence rates for the three methods for individuals aged over 30 years were 11.18%, 10.92% and 12.57%, respectively. When the ECLIA was used as the gold standard, the sensitivities of the RDT, NAT and RDT and NAT in parallel were 80.7% (95% CI: 73.9~86.4), 86.7% (95% CI: 80.6~91.5) and 93.4% (95% CI: 88.5~96.6), respectively, and the sensitivity of the RDT and NAT in parallel was greater than that of the RDT alone (p < 0.001). The parallel RDT and NAT revealed an additional cost–benefit ratio (ACBR) < 1 for males and individuals aged over 30 years, which indicated that switching from the RDT screening strategy to the RDT and NAT in parallel is more cost effective. Adults aged over 30 years are the main population with hepatitis B infection in Guangdong Province, China, whose prevalence of HBsAg-ECLIA was 11.18%. Single RDT screening is prone to miss individuals with low levels of HBsAg. It is recommended to implement an RDT and NAT in parallel for individuals older than 30 years.

Viruses doi: 10.3390/v18050485

Authors: Danielle N. Burner Giselle Y. López Justin T. Low Micah A. Luftig

Epstein–Barr virus (EBV)-positive primary central nervous system lymphoma (PCNSL) is a rare entity typically associated with profound immunosuppression, most commonly in transplant recipients or individuals with HIV. We report a case of EBV-positive PCNSL arising in a 75-year-old male with myasthenia gravis receiving chronic mycophenolate mofetil (MMF) therapy outside the transplant setting. The patient presented with progressive neurological deficits, and brain magnetic resonance imaging demonstrated multiple enhancing lesions. Stereotactic biopsy revealed diffuse large B-cell lymphoma of non–germinal center subtype with immunoblastic features and EBV-encoded RNA (EBER) positivity, confirming EBV-positive PCNSL. MMF was discontinued, and the patient was treated with rituximab and high-dose methotrexate, resulting in stable disease. This case highlights that prolonged MMF therapy may confer sufficient immunosuppression to permit EBV-driven lymphoproliferative disease even in non-transplant patients. Early recognition, withdrawal of immunosuppression, and initiation of methotrexate-based chemotherapy can lead to favorable outcomes.

Viruses doi: 10.3390/v18050484

Authors: Miloud Sabri Khaoula Mektoubi Orges Cara Roukia Bougheloum Angelo De Stradis Giuseppe Parrella Toufic Elbeaino

Xanthomonas species are Gram-negative bacterial pathogens responsible for diseases in over 400 plant hosts, including numerous economically important crops such as Brassica species. The limited efficacy and environmental concerns associated with chemical control strategies underscore the need for sustainable and targeted alternatives. In this study, we evaluated the suitability and biocontrol efficacy of phages Phi1 and Phi3 to combat Xanthomonas campestris pv. campestris (Xcc) in broccoli plants. Kill-curve assays demonstrated that both phages effectively suppressed Xcc growth across a range of multiplicities of infection. Transmission electron microscopy further confirmed their lytic activity, revealing pronounced structural damage to Xcc cells following phage treatment, accompanied by the subsequent release of phage progeny. To assess host specificity and biosafety, the phages were tested against 41 bacterial isolates that were isolated and taxonomically characterized from broccoli and cauliflower in this study. Neither Phi1 nor Phi3 exhibited lytic activity against any non-target isolate, indicating high host specificity and minimal risk to the native Brassica-associated microbiota. In planta assays demonstrated that the combined application of Phi1 and Phi3 reduced Xcc-induced symptom severity in broccoli plants by 80%. Collectively, these results demonstrate that phages Phi1 and Phi3 represent effective and biologically precise agents for the control of black rot disease in Brassica crops.

Viruses doi: 10.3390/v18050483

Authors: Katerina Tsioka Smaragda Sotiraki Danai Pervanidou Styliani Pappa Konstantina Stoikou Annita Vakali Chrisovaladou-Niki Kefaloudi Christina Sapanidou Panagiota Ligda Angeliki Liakata Anastasios Saratsis Dimitrios Chatzidimitriou Anna Papa

Ixodid ticks are vectors for a plethora of pathogens, including the Crimean–Congo hemorrhagic fever virus (CCHFV), which causes severe disease in humans. Two autochthonous CCHF human cases were reported in 2025 in Greece. The aim of the present study was to gain a better insight into the geographic distribution and prevalence of CCHFV and the related Aigai virus (AIGV) in ticks in Greece. Therefore, 680 ticks (135 Hyalomma and 545 Rhipicephalus ticks) collected during 2024 from livestock (sheep, goats, cattle) and from the environment were tested for CCHFV and AIGV. AIGV was detected in 12 adult Rhipicephalus bursa ticks (12/511, 2.3%), while all Hyalomma ticks and R. bursa nymphs were negative for both viruses. AIGV-positive ticks were collected in May and June from goats and sheep in two distantly located regional units of Greece. AIGV sequences from partial S RNA segment differ from the prototype AIGV strain (AP-92) by 10.3% and 1.4% at the nucleotide and amino acid level, respectively. Integrated surveillance studies are needed in ticks, humans, wild and domestic animals within a One Health framework to gain a better insight into the epidemiology of CCHF in Greece, while clinical research is needed to elucidate the impact of AIGV in public health.

Viruses doi: 10.3390/v18040482

Authors: Julia C. Frederick Kristine A. Lacek Matthew J. Wersebe Bo Shu Lisa M. Keong Juliana DaSilva Malania M. Wilson Sydney R. Sheffield Jimma Liddell Natasha Burnett Reina Chau Amanda H. Sullivan Yunho Jang Juan A. De La Cruz Elizabeth A. Pusch Dan Cui Yasuko Hatta Sabrina Schatzman Norman Hassell Xiao-Yu Zheng Ha T. Nguyen Larisa Gubareva Rebecca Kondor Han Di Vivien G. Dugan Charles T. Davis Benjamin L. Rambo-Martin Marie K. Kirby

The first influenza A(H5N1) human case associated with the A(H5N1) dairy cattle outbreak in the United States was identified in April 2024. The U.S. CDC response to this outbreak was activated days later and remained active until July 2025. During this time, 70 human cases of influenza A(H5N1) were detected with a range of epidemiological links to sources of exposure. Next-generation sequencing (NGS) of human samples was an effectual mechanism for tracking and analyzing the outbreak evolution throughout the response. Due to the specimens’ importance and their variable physical quality, an assortment of laboratory methods was utilized including influenza segment-specific amplification, enrichment capture, short-read, and long-read sequencing. Combining these methods allowed for high-quality genomic data production with rapid turnaround times—typically 2 days from sample receipt to public database submission. By leveraging replicate sequencing, enrichment capture, and sequencing of diagnostic amplicons, valuable genomic data could be produced directly from human clinical specimens that would have normally been considered too weak for routine virologic surveillance sequencing. The resulting assemblies were characterized and analyzed by CDC and shared with local and state public health authorities to facilitate case investigations and risk assessment. These data were further used for phylogenetic analyses of viruses from human cases to investigate likely animal-to-human transmission events and identify clusters within the outbreak that might indicate trends in the types of exposures. Through the adaptable laboratory workflow and the rapid release of viral genomic data, the public health risk mitigation strategies could be evaluated and adjusted in real time.

Viruses doi: 10.3390/v18040481

Authors: Emese Demián Réka Sáray Asztéria Almási Kata Pogácsás Katalin Salánki

The increasing global movement of plant material and the complexity of viral communities associated with cultivated crops complicate routine plant virus diagnostics. High-throughput sequencing (HTS) has therefore become an important tool for the comprehensive characterization of plant viromes. In this study, symptomatic pepper (Capsicum annuum) samples submitted to our laboratory between 2020 and 2025 were investigated using HTS following unsuccessful routine diagnostic assays, despite the presence of virus-like symptoms. Virome analysis revealed the presence of multiple viruses with distinct biological characteristics. Eggplant mottled dwarf virus (EMDV) sequences were identified, representing, to our knowledge, the first sequence data from Hungary. In addition, sequences related to tobacco vein clearing virus (TVCV) showed highest similarity to endogenous viral element present in Capsicum annuum genome assemblies. Persistent viruses, including bell pepper alphaendornavirus (BPEV) and pepper cryptic virus 2 (PCV2), were also detected. These findings demonstrate the complex viral communities associated with cultivated pepper and highlight the limitations of strictly targeted diagnostic approaches. The results emphasize the value of HTS for comprehensive virome characterization in horticultural crops.

Viruses doi: 10.3390/v18040480

Authors: Johnbosco U. Osuagwu Julia M. Smith Scott C. Merrill

Foot-and-mouth disease (FMD) poses a significant threat to the United States dairy industry. This study evaluates the effectiveness of regional zoning, enhanced detection, and biosecurity in controlling FMD spread, focusing on the New England milkshed, using the InterSpread Plus (ISP+) model. We adapted a baseline ISP+ configuration incorporating United States dairy farm data, movement networks, cattle dealers, markets, and slaughterhouses, with milk processing plants as a model addition. Four hypotheses were tested to understand the impact of different biosecurity strategies: (H1) regional zoning limits the interregional spread of FMD post-detection; (H2) earlier detection in New England via increased passive surveillance reduces the overall outbreak impact; (H3) reduced indirect transmission through enhanced biosecurity measures improves FMD outbreak control; (H4) the combination of regional zoning and earlier detection provides synergistic reduction in FMD impact beyond either strategy alone. The four hypotheses were tested using three geographically distinct infection seed sets; 100 iterations of each scenario were simulated over 210 days and compared to the baseline. Key impact metrics included the daily number of infected premises, the outbreak duration, and the total number of infected premises across the outbreak scenarios. Results suggest shorter outbreak durations and reduced total infected premises under the hypothesized scenarios, compared to the baseline scenario. Kruskal–Wallis H tests confirmed significant differences across the baseline, regional zoning, early detection, enhanced biosecurity, and the combination of heightened passive surveillance with regional zoning scenarios in terms of total infected premises. Post hoc Dunn’s tests indicated that enhanced biosecurity outperformed other control strategies tested. These findings demonstrate that layered interventions may substantially curtail both the national amplification and local spread of FMD, and thus protect the consumer milk supply and reduce cascading economic shocks from an outbreak.

Viruses doi: 10.3390/v18040479

Authors: Yuichiro Shindo Yi Piao Mark Berry Heribert Ramroth Manami Yoshida

Early initiation of remdesivir (RDV) is recommended to improve COVID-19 outcomes, but real-world studies describing patterns of RDV use and related outcomes among Japanese COVID-19 patients at high-risk of severe outcomes or death are limited. This claims-based cohort study included 60,165 high-risk patients hospitalized with COVID-19 between October 2021 and June 2023 using the DeSC Healthcare claims database. Patients were categorized into early-RDV (within 2 days of hospital admission), late-RDV (between day 3 and day 7), and no-RDV groups based on RDV initiation timing. Descriptive analyses were performed according to RDV groups. Of the study patients, ≥85% were very elderly (≥75 years). Approximately 39% of patients received early RDV, 2% received late RDV, and 59% received no RDV. By day 28, the proportion of alive discharge for early-, late-, and no-RDV groups was 74.9%, 63.1%, and 71.8%, respectively. The mortality for early-, late-, and no-RDV groups was 7.7%, 8.8%, and 8.4%, respectively. Future hypothesis-driven studies with an appropriate adjustment for confounders are needed to formally evaluate the impact of RDV initiation timing on clinical outcomes in this high-risk, predominantly late-elderly population in Japan.

Viruses doi: 10.3390/v18040478

Authors: Berenice Munguía-Ramírez Giovani Trevisan Paul Morris Gustavo S. Silva Danyang Zhang Chong Wang Rodger Main Jeffrey Zimmerman

The continued global spread of WOAH-listed pathogens via trade, transport, and travel calls for the implementation of biosecurity measures to protect the health of our national livestock industries, plus ongoing surveillance to verify that such measures are operative. Despite this urgency, surveillance must be practical and affordable. Herein, we evaluated the performance and cost of participatory surveillance, a nontraditional surveillance design, using the U.S. swine industry as an example. In this context, “participatory” meant that herd veterinarians and/or producers collected and submitted samples from the herd to accredited laboratories for testing. To create an infected population (Phase 1), we simulated the introduction and spread of an unspecified notifiable pathogen within the 48 contiguous U.S states (66,637 swine farms, within 8,080,470 km2) using the USDA Animal Disease Spread Model software (v3.5.10.0). In Phase 2, we calculated the probability of detecting ≥1 infected farm as a function of producer participation, farm-level sensitivity, farm-level prevalence, and sampling frequency. The participatory design was effective: ≥90% probability of detecting the notifiable pathogen at 0.05% farm prevalence (33 positive farms among 66,637 farms) when farm-level sensitivity was ≥20% and producer participation was ≥40%. Depending on the specimen collected, the shipment method, and the test selected, costs ranged from $0.03 to $0.07 USD (€0.02 to €0.06) per pig in inventory. Thus, a surveillance design based on collecting and testing specimens from a few targeted pigs on each of many farms would be both affordable and effective at a national level.

Viruses doi: 10.3390/v18040477

Authors: Raisa Eloise Barbu Mariana Daniela Ignat Roxana Elena Bogdan Goroftei Alexia Anastasia Ștefania Baltă Valerii Lutenco Valentin Bulza Valerian Ionuț Stoian Simona Claudia Cambrea Elena Dumea Liliana Baroiu

This review critically examines the inhibition of viral entry as an emerging disease-modifying strategy in chronic hepatitis B (HBV) and delta (HDV) virus infection, with particular emphasis on bulevirtide, the first-in-class of the sodium taurocholate cotransporting polypeptide entry inhibitor. This paper summarizes the analysis of 7 clinical trials that either underpinned the registration of bulevirtide or are important European real-life trials. We synthesize virological, pathophysiological and clinical evidence, highlighting the impact of this novel bulevirtide-based therapy on virological control, liver inflammation, fibrosis dynamics and long-term prognosis, as well as the limitations of this therapy. The observation of these trials is a greater than 2 log decrease from baseline in hepatitis D virus ribonucleic acid (HDV RNA) in 54–92% of patients and normalization of alanine transaminase (ALT) in 48.8–74% of patients after 23–144 weeks of treatment, and a significant decrease in liver fibrosis, as quantified by Fibroscan, at 12 months of treatment. The conclusion of the study is that this therapy represents an important leap in the etiological approach to chronic HDV infection and in improving the prognosis of these patients, but future clinical studies are needed to define the criteria for discontinuation of therapy, the long-term impact, as well as studies targeting new therapies that can intervene in other stages of the HDV and HBV life cycle not only to achieve HDV RNA negativity but also HBsAg clearance.

Viruses doi: 10.3390/v18040476

Authors: Jiaying Lu Congyi Li Wenzhe Cui Yining Du Jiayi Geng Wenyan Zhang

Enteroviruses represent important human pathogens, posing a substantial disease burden, particularly in children under 5 years of age. Enteroviruses are the primary causative agents of hand-foot-and-mouth disease (HFMD) and are strongly associated with acute flaccid myelitis (AFM), with severe cases potentially resulting in significant neurological complications. Inactivated vaccines against EV-A71 based on the C4 genotype are currently available. However, there are no licensed direct antiviral agents for severe cases. By focusing on viral proteins and host factors, researchers have made great strides in the creation of antiviral medications that target enteroviruses. However, several viral candidates failed to progress in clinical development due to limited efficacy or side effects. This review discusses key findings in enterovirus antiviral research, analyzes the advantages and limitations of each drug target, and highlights knowledge gaps that need to be addressed to advance further development in this field.

Viruses doi: 10.3390/v18040475

Authors: Vlad Vuta Maria Gradinaru Mihnea Hurmuzache Florica Bărbuceanu Lenuta Zamfir Răzvan Moțiu Laura Schmid Dirk Höper Sten Calvelage Thomas Müller Conrad M. Freuling

Rabies is a fatal zoonotic disease once clinical symptoms develop. In Europe, sustained animal rabies control programs have led to a marked decline in animal rabies and subsequently human rabies cases; however, sporadic infections continue to occur. In July 2025, a fatal case of autochthonous (locally acquired) human rabies was confirmed in Romania following a stray dog bite in a patient who did not receive post-exposure prophylaxis (PEP). Here, we report the first molecular characterization of a human rabies virus (RABV) strain isolated in Romania and place it in the context of contemporaneously circulating animal-derived RABV strains. Rabies virus infection was confirmed intra vitam by fluorescent antibody testing and both conventional and real-time RT-PCR on cerebrospinal fluid and saliva, with postmortem confirmation on skin and brain tissue. Whole-genome sequencing was performed on the human isolate and on 22 animal-derived RABV strains collected in northern Romania in 2025. Phylogenetic analyses revealed that all recent Romanian sequences clustered within the North-East European (NEE) rabies virus phylogenetic group and segregated into two geographically distinct genetic clusters: a north-western cluster, closely related to strains from Slovakia and Poland, and a larger north-eastern cluster, linked to viruses circulating in eastern Romania and the Republic of Moldova. The human-derived RABV genome was grouped within the north-eastern cluster and showed the highest genetic similarity to animal viral strains from the same geographical area, supporting a local transmission event. This demonstrates the importance of integrating human viral genomic data into the national rabies surveillance framework.

Viruses doi: 10.3390/v18040474

Authors: Burim Ismajli Zsuzsanna N. Galbács Lilla Dorottya Péri György Pasztor András Péter Takács Éva Várallyay

Weed control of solanaceous weeds growing with solanaceous crops is a constant challenge. Infected by viruses, they can also act as virus reservoirs, complicating this problem further. Viromes of annual Solanum nigrum, Datura stramonium, and Solanum dulcamara, a perennial climbing shrub, were investigated using RNA sequencing and validated using RT-PCR, revealing infection with nine viruses. Broad bean wilt virus 1 (BBWV1), cucumber mosaic virus (CMV), and potato virus M (PVM) were found to infect S. nigrum. Investigating only 46 plants revealed infection with Solanum dulcamara yellow fleck virus (SDYFV) not only in S. dulcamara but in a new host, D. stramonium, which also represents a new host of turnip yellows virus (TuYV). We described the first presence of a potato virus H (PVH)-like, and Oxybasis rubra mitovirus 1 (OxruMV1)-like virus in Europe, in S. dulcamara as a new host. Our results highlight the unexpected complexity of the viromes of solanaceous weeds, which should be considered during reliable and efficient plant protection strategies, in order to alleviate the virus reservoir role of the weeds.

Viruses doi: 10.3390/v18040473

Authors: Xin-Zhou Sun Yan Liu

Liver diseases, ranging from chronic hepatitis and metabolic dysfunction to cirrhosis and hepatocellular carcinoma, represent a major global public health burden. As an immune-privileged organ, the liver harbors a unique and intricate immune microenvironment, which plays a dual role in pathogen clearance and chronicity. Kupffer cells (KCs), the primary resident macrophages in the liver, constitute the first line of defense in liver innate immunity and play complex and important roles in pathogen recognition, phagocytosis, and the regulation of liver inflammation and immune responses. The complement receptor of the immunoglobulin superfamily (CRIg) is a membrane receptor that is specifically expressed on KCs. It serves not only as a sentinel for the liver against pathogen invasion but also as a sophisticated regulator for maintaining immune homeostasis. As a key component of the liver’s immune system, CRIg can efficiently mediate the clearance of complement-opsonized particles, thereby playing multidimensional roles in pathogen clearance, antigen cross-presentation, and the establishment of immune tolerance, functioning as both a “pathogen catcher” and an “immune brake.” This review focuses on the CRIg molecule, detailing its mechanisms in the recognition and phagocytic clearance by KCs, and its subsequent impact on hepatic immune responses. Furthermore, we explored the potential involvement of CRIg in the pathological progression of diverse liver diseases, including persistent inflammation, fibrosis, and hepatocarcinogenesis. This synthesis provides novel insights into the immunopathology of liver diseases and establishes a theoretical foundation for developing CRIg-targeted therapeutic strategies.

Viruses doi: 10.3390/v18040472

Authors: Subhash Thuluva Subbareddy Gunneri Siddalingaiah Ningaiah Vijay Yerroju Rammohan Reddy Mogulla Chirag Dhar Kamal Thammireddy Raju Esanakarra Pradeep Nanjappa Niranjana S. Mahantshetti

Biological E’s BEVAC® is a recombinant DNA hepatitis B vaccine that has been used in India for more than a decade in routine early-life immunization and has recently been prequalified by the World Health Organization (WHO). This multicentre, single-blind, parallel-group, randomized phase IV trial, conducted at seven study sites in India, compared the immunogenicity and safety of BEVAC® with a licensed comparator vaccine (GeneVac-B®, Serum Institute of India Pvt. Ltd, Pune, India.) in healthy term neonates and infants. Participants received three 0.5 mL doses administered intramuscularly at birth (within 24 h), 6 weeks of age, and 14 weeks of age. The primary endpoint was seroprotection (anti-HBs IgG ≥10 mIU/mL) at 28 days after the third dose (Day 126), compared using a non-inferiority margin of −10%. Secondary endpoints included safety and tolerability outcomes through Day 126. A total of 468 neonates were randomized (234 per group), of whom 44% were female. At Day 126, seroprotection rates were 98.2% (95% CI: 95.39, 99.50) with BEVAC® and 99.1% (95% CI: 96.78, 99.89) with the comparator; the between-group difference was −0.9% (95% CI: −3.09, 1.24), meeting the prespecified non-inferiority criterion. Solicited adverse events within 7 days after any dose occurred in 29.1% (95% CI: 23.3, 35.3) of BEVAC® recipients and 35.0% (95% CI: 28.9, 41.5) of comparator recipients, most commonly pyrexia, injection-site pain, and swelling; all were mild-to-moderate. No serious adverse events were reported. BEVAC® demonstrated non-inferior immunogenicity to the licensed comparator and a comparable safety profile.

Viruses doi: 10.3390/v18040471

Authors: Wen-Kai Zhang Man Teng Lu-Ping Zheng Bin Shi Wei-Dong Wang Gui-Xi Li Yong-Xu Zhao Zhen Yang Zu-Hua Yu Jun Luo

Marek’s disease (MD), caused by pathogenic Marek’s disease virus serotype 1 (MDV-1), is one of the most important avian immunosuppressive and neoplastic diseases and has led to huge economic losses to the poultry industry worldwide. Rapid and accurate clinical diagnosis is of great significance for efficient control of the disease. Herein, we have established a multiplex PCR (mPCR) method to simply differentiate all of the three types of MDV, using five specific primers targeting to MDV-1 oncogene meq or MDV-2 and MDV-3/HVT gB genes. Simultaneously, it can detect any type of virulent or vaccine MDV strains in one PCR reaction, with amplicons of the short (S) and long (L)-meq of MDV-1 strains, and the gB of MDV-2 and HVT vaccine strains. Non-specific amplifications of avian leukosis virus (ALV), reticuloendotheliosis virus (REV), or fowl adenovirus virus 4 (FAdV-4) were not observed, indicating a good specificity of this method. A total of 522 clinical samples of tumor-bearing or suspected diseased birds collected from 30 poultry farms were detected. The results demonstrated that the newly developed mPCR method accurately detected and differentiated epidemic MDV-1 infections and vaccine strains, and provided nearly 100% consistency for detecting clinical wild-type infections compared with conventional PCR amplification of the meq gene. Collectively, our data has provided a highly efficient method for early differential diagnosis of MD clinical cases, virus identification and future evaluation of vaccination efficacy in healthy chicken flocks, which would be meaningful for efficient control of the disease.

Viruses doi: 10.3390/v18040470

Authors: Arianna Spellman-Kruse Jodi L. Bubenik Tathiana Ferreira Sa Antunes Alexander J. Lawrence Maurice S. Swanson Ying Wang Svetlana Y. Folimonova

The 5′-proximal region of the citrus tristeza virus (CTV) RNA genome is a hub where several elements involved in different facets of the virus cycle reside, including the sequences driving the production of the viral long non-coding RNA (lncRNA) LMT1. The sequence of this region is one of the most divergent genome areas, allowing for strain differentiation. Beyond its use in assessing viral population diversity, the region provides a valuable model for studying the conservation of RNA structure and function despite sequence variation. Here, we integrated comparative in silico analysis of the LMT1 region from variants of eight CTV strains with selective 2′-hydroxyl acylation, analyzed by primer extension and mutational profiling (SHAPE-MaP) probing of in vitro–generated LMT1 RNAs from two divergent strains, T36 and T68. The predicted consensus structures revealed 19 putative, conserved stem-loops. The SHAPE-MaP reactivity data supported and substantiated the thermodynamics-based predictions for the 15 previously uncharacterized stem-loops and two functional elements identified earlier. The strong structural conservation across strains highlights that the LMT1 RNA structure contributes to its function during CTV infection. These results provide the first experimentally supported structure of this viral lncRNA and lay the foundation for defining how individual RNA motifs influence CTV biology.

Viruses doi: 10.3390/v18040469

Authors: Emanuele Castagno Carola Aschieri Irene Ferri Sara El Khbazi Lorenzo Milani Rosanna Irene Comoretto Irene Raffaldi Irene Tardivo Marco Spada Claudia Bondone Franca Fagioli

Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis in children < 24 months and a major public health concern, causing high rates of Emergency Department (ED) visits, hospitalizations, and Pediatric Intensive Care Unit (PICU) admissions. Nirsevimab is a recombinant monoclonal antibody recommended for all infants and high-risk children < 24 months. A retrospective single-center cohort study was conducted to evaluate the impact of nirsevimab introduction on bronchiolitis epidemiology in an Italian tertiary pediatric ED, accounting for 40,000 admissions/year. All children < 24 months who presented to our ED with bronchiolitis during two consecutive RSV seasons (first season: 1 October 2023 to 30 April 2024; second season: 1 October 2024 to 30 April 2025) were included. Descriptive and multivariate analyses are reported. Overall, 484 patients were analyzed (336 in 2023–2024; 148 in 2024–2025), with immunization coverage reaching 87.5% by April 2025. Compared with the previous season, RSV positivity decreased significantly (32.4% vs. 47.9%; p = 0.003) and was lower in immunized children (16.2% vs. 51.5%; p < 0.001). Immunization was associated with a reduced risk of RSV-positive swab in the second season (OR = 0.159, 95% CI: 0.059–0.397). Among RSV-negative patients, other respiratory viruses increased (p < 0.001), while co-infections increased in RSV-positive cases (p = 0.021). Hospitalization rates remained stable, though absolute admissions were halved. In conclusion, nirsevimab immunization reduced RSV burden, supporting its inclusion in universal prevention programs and the need for multicenter prospective studies to assess long-term outcomes.

Viruses doi: 10.3390/v18040468

Authors: Ze Tong Shiting Ni Jiaqi Liu Pingxuan Liu Daisheng Shi Guosheng Chen Xin Zong Yaning Lv Renhang Xiao Chen Tan

Porcine circovirus type 2 (PCV2) is a pathogen of major importance in swine that is characterized by ongoing genetic evolution. To provide an updated epidemiological assessment for China, our study analyzed 1051 clinical samples collected from 27 provincial-level regions between 2023 and 2024. The overall PCV2 positivity rate was 65.18%, with detection rates showing significant seasonal variation, with higher rates in spring and summer. Genotypic analysis of 379 open reading frame 2 (ORF2) sequences identified PCV2d as the dominant genotype (78.89%), and no significant geographic clustering was observed. Coinfection with porcine reproductive and respiratory syndrome virus (PRRSV) is common, yet statistical tests have revealed an epidemiologically independent relationship between the two viruses. Notably, analysis of the capsid (Cap) protein revealed that high-frequency amino acid mutations were concentrated in immunodominant loop regions. These mutations resulted in genotype-specific substitutions within key neutralizing epitopes. This study provides the latest large-scale national baseline data on PCV2 in China for 2023–2024. It systematically analyzes the epidemiological characteristics of the dominant PCV2d genotype in the post-African Swine Fever era, the patterns of antigenic epitope mutations in the Cap protein, and their potential impact on vaccine efficacy. The study fills a gap in recent national epidemiological data on PCV2 in China and provides a basis for the targeted prevention and control of PCV2 and the updating of vaccine strains.

Viruses doi: 10.3390/v18040467

Authors: Chet R. Ojha Samuel W. Rovito Balaji Banoth Hyunsuh Kim Jeremy C. Jones Mohamad-Gabriel Alameh Po-Ling Chen Richard J. Webby Drew Weissman Charles J. Russell

While current influenza vaccines often lack broad protection against antigenically drifted strains, some modified hemagglutinin (HA) protein antigens have shown promise in eliciting broadly neutralizing antibodies against conserved epitopes. During infection, the mildly acidic environment of the late endosome triggers irreversible HA conformational changes resulting in a post-fusion structure with altered antigenicity. While enhancing the stability of other structural class I viral fusion protein antigens has been instrumental in improving the effectiveness of COVID-19 and RSV vaccines, the role of HA stability in influenza vaccine immunogenicity is relatively unclear. Here, we used the nucleoside-modified mRNA-LNP platform to test engineered HA antigens with specific acid-stabilizing mutations (E47K, K58I, R106K, and K153E) in the HA stalk. All mutations increased HA acid stability, but E47K and R106K did not increase immunogenicity. K153E and K58I, but not E47K and R106K, enhanced the cell-surface expression of the HA protein in vitro. In mice, K153E- and K58I-containing mRNA-LNP vaccines elicited increased neutralizing antibody titers against homologous virus. K153E conferred greater protection than wild-type vaccine against lethal heterologous A/PR/8/34 challenge at low doses (0.5–1.0 µg), despite the absence of neutralizing antibodies against the challenge strain. K153E also elicited greater expansion of antigen-specific antibody-secreting cells (ASCs) in the bone marrow, as well as cross-reactive T follicular helper (Tfh) cells in the spleen. For the vaccines studied, increased HA expression was a stronger correlate of mRNA-LNP enhancement than increased HA stability.

Viruses doi: 10.3390/v18040466

Authors: Xiaolan Ji Donglin Bi Mingqi Liu Xiangru Du Zhiqi Wang Haiqing Li Jinluan Wang Yiyang Fan Hao Gao Derong Zhang Jialin Bai Qiongyi Li

Encephalomyocarditis virus (EMCV) belongs to the genus Cardiovirus of the family Picornaviridae. It is a non-enveloped, positive-sense, single-stranded RNA virus and an important pathogen causing encephalomyocarditis (EMC). Tripartite motif 13 (TRIM13) is a member of the tripartite motif (TRIM) family and serves as an important effector molecule in antiviral innate immunity. However, its antiviral activity and underlying molecular mechanisms during EMCV infection remain unknown. In this study, we identified TRIM13 as a regulator of NF-κB activation. TRIM13, dependent on its E3 ubiquitin ligase activity, directly binds to IκBα and dose-dependently increases its phosphorylation level. To determine the chain type of IκBα polyubiquitination, antibodies specific for K48-linked and K63-linked ubiquitin were used. Our data indicated that IκBα was subjected to polyubiquitination independent of K48 and K63 linkages. This interaction promotes non-K48/K63-linked polyubiquitination of IκBα, thereby inducing NF-κB nuclear translocation. Subsequently, nuclear NF-κB activates the secretion of pro-inflammatory cytokines, exacerbating inflammatory responses and ultimately facilitating EMCV infection.

Viruses doi: 10.3390/v18040465

Authors: Murat Oz Wassim Chehadeh Omamah Alfarisi Farhan S. Cyprian

Objective: The COVID-19 pandemic has strained healthcare systems and has been associated with substantial morbidity and mortality. Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) enters host cells by binding to angiotensin-converting enzyme 2 (ACE2), implicating dysregulation of the renin–angiotensin system (RAS) in COVID-19 pathophysiology. Measurement of circulating RAS components, including ACE and ACE2, may therefore provide an insight into disease severity and underlying mechanisms. Subjects and Methods: In this retrospective cohort study, 224 adults with PCR-confirmed COVID-19 were stratified by World Health Organization disease-severity criteria into asymptomatic, mild, mild-pneumonia, severe, and critical groups. Plasma ACE and ACE2 concentrations were quantified by ELISA. Demographic, clinical, and laboratory data were extracted from electronic medical records. Results and Conclusions: Increasing disease severity was associated with higher mortality, elevated body mass index, and higher viral load estimates. Severe and critical illness was characterized by leukocytosis with neutrophilia, marked lymphopenia, anemia, elevated inflammatory and coagulation markers, renal dysfunction, and hypoalbuminemia. Plasma ACE2 levels declined progressively with increasing severity and were significantly lower in patients with mild-pneumonia, severe, or critical illness compared with asymptomatic or mild cases, showing a strong inverse correlation with severity. In contrast, plasma ACE levels increased significantly with disease severity. The resulting increase in the ACE/ACE2 ratio indicates a progressive shift toward the pro-inflammatory arm of the RAS, providing mechanistic insight into the COVID-19 pathophysiology.

Viruses doi: 10.3390/v18040464

Authors: Talia J. Byrne-Haber Kylee N. Pham Arianna Joob Samantha M. Pinto Oshani C. Ratnayake Ryan Thompson Joel Rovnak Rushika Perera

Located on the traditional and ancestral homelands of the Arapaho, Cheyenne, and Ute Nations, Colorado State University’s Mountain Campus hosted the 25th Annual Rocky Mountain Virology Association meeting. The three-day event, held from 26 September to 28 September 2025, welcomed 152 participants focused on the following topics: viruses, prions, immunology, transmission, structural biology, and vector biology. This year’s Randall Jay Cohrs Keynote Presentation summarized ongoing research on viral glycoproteins in relation to viral entry and assembly. Understanding the role of viral glycoproteins is essential in vaccine and antiviral development for enveloped RNA viruses. Alongside rigorous scientific discourse and networking, attendees made the most of their time by hiking amidst beautiful fall colors, wildlife, and young aspens starting the forest anew. On behalf of the Rocky Mountain Virology Association, this report summarizes select presentations from the 25th annual meeting.

Viruses doi: 10.3390/v18040463

Authors: Robert Meineke Martin Ludlow Albert D. M. E. Osterhaus Guus F. Rimmelzwaan

CX3CR1 is a chemokine receptor expressed on respiratory epithelial and immune cells and has been identified as a host factor important for infections with respiratory syncytial virus (RSV). In this review, we discuss the roles CX3CR1 plays in the pathogenesis of RSV infections as a viral entry receptor and regulator of immune cell trafficking. The conserved CX3C motif of the RSV G glycoprotein binds to CX3CR1 to mediate viral attachment and entry into respiratory epithelial cells. Furthermore, soluble G protein (sG) can bind to CX3CR1 and competitively interfere with cell signaling induced by the chemokine CX3CL1, resulting in inhibition of immune cell recruitment to the site of infection. In addition, sG engages TLR2 on epithelial cells, activating MyD88-NF-κB signaling and priming the NLRP3 inflammasome, which enhances viral dissemination through pyroptotic cell death. CX3CR1 signaling should be viewed as one of several overlapping host factors that—along with developmental changes in interferon and STAT3 signaling, airway anatomy, inflammasome activity, and tissue-resident memory responses—contribute to differential disease outcomes of RSV infection. A more complete molecular understanding of RSV-CX3CR1 interactions and downstream host responses may enable the development of improved prevention and treatment strategies.

Viruses doi: 10.3390/v18040462

Authors: Álex Gómez Idoia Glaria Irati Moncayola Leonor Puzol Laura Arriazu Ainhoa Calero Ignacio de Blas Mikel Nazábal Itziar Hualde Benhur Lee Lluís Luján Ralf Amann Irache Echeverría Ramsés Reina

Orf virus (ORFV) is a globally distributed zoonotic parapoxvirus that causes a highly contagious mucocutaneous disease in small ruminants. Despite the urgent demand for vaccination-based control, no licensed vaccines are currently available universally. In this study, we generated two recombinant Sendai virus (SeV) vectors expressing ORFV 011 (rSeV-GFP-B2L) and ORFV 059 (rSeV-GFP-059) genes and evaluated their ability to stimulate antiviral responses in vitro. Following the transduction, we assessed transgene expression, innate immune activation, induction of interferon-stimulated genes (A3Z1, OBST2, SAMHD1), and antiviral activity. Both vectors significantly upregulated pattern recognition receptors (TLRs, RIG-I) and type I interferon (IFN-β) genes, with rSeV-GFP-059 inducing the strongest response. Remarkably, OBST2 was robustly upregulated, suggesting a potential role in restricting ORFV replication. Antiviral activity assays revealed a marked reduction in ORFV DNA copies and a mild decrease in ORFV RNA transcription in rSeV-GFP-059-transduced cells, particularly at later time points, accompanied by complete abrogation of the typical cytopathic effect. Collectively, these results demonstrate that SeV-based vectors, particularly rSeV-GFP-059, efficiently prime antiviral immunity and suppress ORFV replication, establishing a promising platform for further in vivo vaccine evaluation in sheep.

Viruses doi: 10.3390/v18040461

Authors: Hiroyuki Inoue

Oncolytic viruses represent a paradigm-shifting approach to cancer immunotherapy, functioning as in situ vaccines that convert immunologically “cold” tumors into “hot” tumors through induction of immunogenic cell death (ICD). Despite the clinical success of checkpoint inhibitors targeting programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), many patients exhibit primary or acquired resistance due to insufficient tumor immunogenicity and exclusion of tumor-infiltrating lymphocytes. Oncolytic viruses address this limitation by selectively replicating in tumor cells, inducing robust ICD characterized by four cardinal hallmarks: calreticulin exposure, ATP secretion, HMGB1 release, and type I interferon production. This review systematically examines the molecular mechanisms underlying virus-induced ICD, compares DNA virus platforms (Vaccinia, HSV-1, Adenovirus) with RNA virus platforms (Coxsackieviruses A21, A11, and B3), and analyzes clinical trial data demonstrating synergistic efficacy when combined with checkpoint inhibitors. Notably, RNA viruses generate higher type I interferon responses compared to DNA viruses, correlating with superior clinical outcomes. Coxsackievirus A21 combined with pembrolizumab achieved a 47% objective response rate in melanoma in the CAPRA trial, representing notable efficacy exceeding either monotherapy. Coxsackievirus A11 demonstrates exceptional selectivity for thoracic cancers through ICAM-1-dependent receptor tropism and potent immunogenic cell death induction. Japanese researchers have pioneered microRNA-targeted Coxsackievirus B3, achieving cardiac safety attenuation while preserving complete oncolytic potency and ICD-inducing capacity. This comprehensive analysis synthesizes molecular mechanisms, platform comparisons, clinical efficacy data, and translational challenges to guide future development of oncolytic virotherapy as a cornerstone of cancer immunotherapy.

Viruses doi: 10.3390/v18040460

Authors: Lizhu Chen Haobin Li Huiyi Guo Jinlong Liang Yingyuan Zhong Xucheng He Wenjiao Wu Shuwen Liu

The infection cycle of the Influenza A Virus (IAV) typically requires host factors to regulate replication and proliferation. However, the roles of these factors remain undiscovered. This study focuses on Sorting Nexin 10 (SNX10), which is involved in regulating membrane trafficking and endosomal stabilization. Our previous study identified that SNX10 facilitates the replication of human coronavirus OC43 through enhancing clathrin-mediated endocytosis. In our present study, we found that SNX10 significantly promoted IAV infection in host cells. The conditional knockout of Snx10 in mice lungs prolonged survival following IAV challenge. Mechanistically, SNX10 facilitated the production of acidic endosomal vesicles and promoted the accumulation of pro-viral autophagic structures, a process supported by the specific interaction between SNX10 and the viral NP and M2 protein of IAV. Blocking SNX10-mediated acidic endosomal vesicles and autophagosome formation demonstrated antiviral effects. Moreover, IAV infection increased SNX10 protein levels by suppressing its ubiquitination, suggesting that SNX10 could serve as a potential host-derived antiviral drug target.

Viruses doi: 10.3390/v18040459

Authors: Cong Tang Bai Li Qing Huang Yun Yang Wenhai Yu Yanan Zhou Daoju Wu Hao Yang Haixuan Wang Junbin Wang Shuaiyao Lu

Chikungunya virus (CHIKV) pathogenesis research has long been constrained by the lack of suitable immunocompetent rodent models. Through serial passaging in A129 and C57BL/6 mice, we obtained an adapted strain (CHIKV-Adapt) harboring an E2-K200R substitution along with non-structural protein mutations. Phenotypic analysis in C57BL/6 mice, BALB/c mice, and hamster models demonstrated that compared to the wild-type virus CHIKV-Adapt induced significantly higher and more prolonged viremia, broader tissue tropism, and more severe internal joint inflammation, without exacerbating external swelling. Notably, the K200R mutation did not alter the viral replication kinetics in vitro and was predicted not to affect its binding pattern to the MXRA8 receptor. Furthermore, mice challenged 160 days after primary infection exhibited nearly complete protective immunity. These findings indicate that E2-K200R is a critical adaptive mutation that, together with accompanying non-structural mutations, significantly enhances CHIKV replication capacity and pathogenicity in immunocompetent rodents without changing its in vitro replication ability or predicted receptor-binding mode. The acquisition of this adapted strain provides a new tool for CHIKV pathogenesis research and vaccine evaluation.

Viruses doi: 10.3390/v18040458

Authors: Supriya Mahajan Saurabh Mahajan Nidhi Kaushik

Long COVID (LC), also referred to as post-acute sequelae of SARS-CoV-2 infection, is characterized by persistent symptoms originating 3 months following acute COVID-19, lasting for at least two months and frequently affecting individuals who initially experienced mild to moderate disease. The clinical spectrum is heterogeneous, involving respiratory, cardiovascular, neurological, renal, gastrointestinal, and endocrine systems, thereby posing substantial diagnostic and therapeutic challenges. Despite extensive investigation, the precise immunopathogenic mechanisms underlying LC remain incompletely defined. Accumulating evidence suggests that LC is driven by a multifactorial interplay of persistent viral antigen reservoirs, chronic immune activation, dysregulated innate and adaptive immune responses, autoimmunity, endothelial dysfunction, microvascular injury, and aberrant tissue repair. These systemic immune perturbations manifest variably across different organs, contributing to the diverse clinical phenotypes observed. However, mechanistic clarity is hindered by heterogeneity in study designs, limited longitudinal data, and the absence of standardized immunological profiling. This narrative review provides integrative insights into the immunopathogenesis of LC, synthesizing current evidence on systemic immune dysregulation and organ-specific immunological mechanisms. A conceptual framework is proposed to facilitate a structured understanding of this complex syndrome and to guide future research toward targeted immunomodulatory strategies.

Viruses doi: 10.3390/v18040457

Authors: Andrew McMinn Yantao Liang Ziyue Wang Min Wang

Viruses are abundant and widespread in extreme marine environments, such as sea ice, hydrothermal vents, and ocean trenches. They occur at temperatures up to 122 °C and down to −30 °C and pressures exceeding 100 MPa. Their distribution in these environments is closely correlated with that of their extremophile hosts, which are mostly bacteria, archaea, and microeukaryotes. Viruses have been shown to be capable of long-term survival in conditions simulating interstellar conditions. However, for them to reproduce, they would still need a host. Many recent astro-biological investigations have focused on habitability, specifically the ability of a planet to support the activity of at least one lifeform. The most likely candidates for extraterrestrial habitability in our solar system are the sea ice moons of Jupiter and Saturn, namely Europa and Enceladus. These are both thought to contain subsurface oceans of liquid water and potentially access to the necessary elements for microbial growth. If microorganisms were to be detected in these extraterrestrial environments, viruses might also be found coexisting with their host cells.

Viruses doi: 10.3390/v18040456

Authors: Clairine I. S. Larsen Rhiannon R. Abrahams Kinjal Majumder

The eukaryotic genome is organized into distinct structural units dictated by architectural proteins. The major host architectural protein CCCTC-binding factor (CTCF) is usurped by DNA viruses to regulate viral gene expression. This review will discuss the major ways large (EBV, HSV, HCMV) and small (HPV, HBV, AAV) DNA viruses mimic eukaryotic genome topology using CTCF to regulate viral gene expression. We will further discuss how changes in genome topology can drive virally induced oncogenic progression. Knowledge gained from studying viral genome folding mechanisms will inform the development of targeted anti-viral agents and inform the modification of viruses to serve as gene therapy vectors.

Viruses doi: 10.3390/v18040455

Authors: Betul Gungor Serin Bilal Esat Temiz Haticegul Tuncer Muhammed Onur Atakul Ali Can Gunes Taylan Onat Utku Akgor Derman Basaran Zafer Selcuk Tuncer Murat Gultekin

Background: Persistent cervical human papillomavirus (Human papillomavirus) infection remains a significant public health concern, as it is the primary etiological factor in the development of cervical cancer and its precursor lesions. While prophylactic vaccination and standard screening programs are cornerstones of prevention, a substantial proportion of women with established infection are managed conservatively, often with prolonged follow-up and associated psychological burden. Interest has therefore grown in supportive interventions that may facilitate viral clearance during routine clinical management. Methods: This retrospective cohort study included 239 women with confirmed cervical Human papillomavirus infection followed at a tertiary referral center between February 2023 and August 2025. Participants were classified into a treatment group receiving oral Papivir/Pavirona® twice daily for six months (n = 119) and a control group managed with routine clinical follow-up alone (n = 120). Human papillomavirus DNA testing and cervical cytology were evaluated at baseline and at 6 and 12 months. Results: Human papillomavirus clearance rates were significantly higher in the Papivir/Pavirona® group compared with controls at both 6 and 12 months. Cytological regression was also more frequent in the treatment group at both time points. In multivariate logistic regression analysis, Papivir/Pavirona® use emerged as the only independent predictor of both Human papillomavirus clearance and cytological regression, while demographic, reproductive, behavioral, and virological baseline characteristics were not significantly associated with outcomes. Conclusions: Papivir/Pavirona® supplementation was associated with increased Human papillomavirus clearance and cytological regression rates in women with cervical Human papillomavirus infection, suggesting a potential supportive role alongside standard clinical follow-up.