Virology

Most adenovirus (Ad) vectors are based on the genome of human Ad type 5 (Ad5), which targets their entry to cells that express the Coxsackie and Adenovirus Receptor (CAR or CXADR). However, certain human Ads do not use CAR, for example Ad35 interacts with cell-surface CD46 and Ad3 uses desmoglein 2 (DSG2) for cell entry. In this study, a comparison of different Ad receptors using transcriptomic and proteomic databases showed that CD46 is widely expressed across human cells and tissues whereas CAR and DSG2 are more restricted to epithelial cells. We have used a hybrid virus, Ad5F35, that comprises an Ad5 genome in which the Ad5 fibre was replaced with that of Ad35, thus retargeting the virus from CAR- to CD46-expressing cells and enabling transduction of primary human NK and T cells. However, lymphocytes required approximately 10 to 20-fold more Ad5F35 particles per cell (ppc) compared to A549 epithelial cells to achieve a similar level of transduction. Consistent with this, quantitation of the cell-surface density of CD46 molecules revealed approximately 100 CD46 molecules per {micro}m2 in primary NK cells compared with approximately 2000 CD46 per {micro}m2 in HeLa cells, a 20-fold difference. Cell-surface CD46 density was reduced by approximately 95% by RNA interference in HeLa cells to levels that approximate those found on NK cells. Lower CD46 density reduced transduction by Ad5F35 but this could be compensated for with increased MOI. Our results identify the density of cell surface CD46 as a critical determinant of Ad5F35 transduction and demonstrate that Ad5F35 is an efficient vector for gene delivery in primary human NK cells.

Although controversial, limited publications support the notion that HIV-1 can infect CD4-negative cells. The objective of this study was to provide a comprehensive investigation of a universally available CD4-negative cell line model system that can be infected with X4 and R5 HIV-1 to generate integrated proviral DNA and serve to study latent viral reservoirs. The reason that HIV-1 infection of CD4-negative cells has become less investigated is due to a lack of a fully characterized model for the study of this unusual pathway. To address this critical need, human osteosarcoma (HOS) cells, engineered to express either CD4, CCR5 or CXCR4, and easily available from a commercial source were used. CD4 expression was examined using western immunoblot, flow cytometry, anti-CD4 blocking antibody and mRNA expression. Cells were infected with HIV-1 pseudo-enveloped viruses bearing either JR-FL (R5-tropic) or HXB2 (X4-tropic) envelopes, constructed on NL4-3 luciferase/GFP backbone. Infection was monitored by luciferase readout and visualized by GFP immunofluorescence. Raltegravir was used to inhibit integration, and AMD3100 and maraviroc used to block chemokine coreceptors, CXCR4 and CCR5, respectively. Productive versus latent infection was quantified by dual-fluorescence readouts using HI.fate.E. We confirmed that HOS cells lack CD4. HOS cells expressing only CCR5 or CXCR4 supported HIV-1 infection, although infection was significantly lower than in matched CD4-positive controls. Raltegravir treatment blocked proviral integration in all instances. Coreceptor antagonism and envelope-deficient viruses revealed that infection of CD4-negative CXCR4 cells remained CXCR4-dependent, whereas CD4-negative CCR5 cells showed evidence of CCR5-independent infection. Dual-reporter HI.fate.E assays indicated that CD4-negative cells could support both productive and latent infection. These studies establish a universally available cell line model for the study of CD4-negative HIV-1 infection. This cell line model will provide insight into the question of how CD4-negative cells can be infected with HIV-1 and whether CD4-negative cells can provide latent viral reservoirs in HIV/AIDS. Author summarySince the first description of HIV/AIDS in 1981 and the recognition that CD4 was a primary receptor for HIV-1 in 1983, a limited number of reports have suggested that cells lacking CD4 could be infected with HIV-1. These reports continued even when it was shown in 1996 that co-receptors, CXCR4 and CCR5, were also required for HIV-1 infection of CD4 T-helper cells. Indeed, crystallography studies showed that CD4 was required to interact with the HIV-1 envelope gp120 in order to cause conformational changes in the envelope to expose the binding motif for chemokine co-receptor engagement, required for additional conformational changes to expose the gp41 fusion protein, allowing for entry and infection. However, reports continued that cells lacking CD4 could be infected which raised questions as to how this can happen. To address this critical gap, we have identified a cell line, HOS, that is commercially available, having expression of CD4, CXCR4 and/or CCR5. Using these HOS cell lines, we have been able to confirm that HIV-1, either X4 or R5 enveloped viruses, can infect CD4-negative cells. We have also confirmed that infection is productive and allows for latent proviral integration. Our findings provide a system for further studies of the mechanism(s) of HIV-1 infection of CD4-negative cells using a consistent model and may aid in elucidating establishment of viral reservoirs.

Lamprey reddening syndrome (LRS) is an emerging disease affecting pouched lamprey (Geotria australis; kanakana/piharau), a culturally and ecologically significant species in Aotearoa New Zealand. Characterised by skin haemorrhaging and elevated mortality, the aetiology of LRS has remained unresolved despite previous investigations. We used a metatranscriptomic approach to characterise viral communities in 28 lamprey from New Zealand and Tasmania, Australia, comparing diseased and presumably healthy individuals. This analysis revealed eight fish-infecting RNA viruses, seven of which were novel, including two highly divergent coronaviruses. One of these coronaviruses possessed a bi-segmented genome structure, and three lamprey were co-infected with both coronaviruses. While these coronaviruses were detected in both healthy and diseased individuals, lamprey with reddening exhibited markedly higher viral abundance, driven by elevated RNA transcripts of both viruses. This pattern suggests that increased coronavirus replication in diseased individuals may be influenced by host stress to environmental factors or co-infection with other pathogens, rather than acting as a sole causative agent of disease. Beyond identifying candidate viral associations, this study expands the known virosphere of an ancient vertebrate lineage and demonstrates the utility of genomics-informed diagnostics for investigating disease in threatened wildlife.

We report the detection of a novel hantavirus in the lung tissue of two diseased Australian dolphins with histopathological changes. Phylogenetic analysis placed this virus within the genus Mobatvirus. This highlights the ability of hantaviruses to infect non-terrestrial mammals and the potential role of marine mammals as one health sentinels.

The Rosaceae family comprises thousands of species across over 100 genera, including Salmonberry (Rubus spectabilis), a Pacific Northwest native within the diverse Rubus genus. Its berries and leaves are used for food and medicinal purposes, and ecologically it functions as a pioneer species that supports biodiversity and limits erosion. Although many Rubus genomes were sequenced and analyzed, salmonberry remains undercharacterized: despite a recently sequenced genome, no publicly available annotation or gene expression analysis currently exists. Here, we used RNA sequencing to characterize the salmonberry leaf transcriptome and examine its phylogenetic relationship within Rubus. The assembled 63,285 unique transcripts included 1,389 high-confidence lncRNA transcripts expressed in salmonberry leaves, 218 of which are conserved across Rubus. Phylogenetic analysis indicates that salmonberry is closely related to Rubus arcticus. In addition, we detected a novel species of virus associated with salmonberry. These findings provide foundational genomic resources for R. spectabilis and offer new insights into its evolutionary relationships and endogenous viral integrations.

XIAP-associated factor 1 (XAF1) is a proapoptotic protein known to be involved in tumor suppression and regression whose gene expression has been reported to be dysregulated in a wide variety of tumor malignancies by different molecular mechanisms. Using a sterile alpha and TIR motif containing 1 (SARM1) knockout mouse model, we previously showed that XAF1 could be a candidate gene for protection against neurotropic virus infection. Here, using a CRISPR-knockout XAF1 mouse model, we show that XAF1 knockout mice are more susceptible to disease after VSV infection, a well-known neurotropic virus in mice. Interestingly, VSV-increased sensitivity in XAF1 knockout mice was not accompanied by differences in viral replication in the central nervous system (CNS). Nevertheless, infection of XAF1 knockout mice resulted in an increased pro-inflammatory response and immune cell infiltration into the CNS compared to that in wild-type mice. Similarly, XAF1 knockout mice showed slight increase to disease after infection with a different neurotropic virus, West Nile Virus (WNV). However, no differences in viral disease due to the absence of XAF1 were found upon infection with a respiratory virus such as influenza A virus (IAV). In vitro, XAF1-deficient cells showed a significant increase in interferon-stimulated genes (ISGs) expression upon stimulation with IFN and with different PAMPs, such as Poly(I:C), HT-DNA and LPS. Consistently, ectopic overexpression of XAF1 decreased IFN-signaling in a dose-dependent manner. Altogether, the data presented here suggest that the host factor XAF1 has a protective role in viral-induced neuropathogenesis due to excessive IFN responses. Author summaryWe previously identified XIAP-associated factor 1 (XAF1) as a candidate cell factor involved in viral phenotypes attributed to SARM1 deficiency. Even though the role of this factor has been studied in the cancer field as a proapoptotic tumor suppressor, its relevance in the context of viral infections has remained unclear. Here, we show that XAF1-deficient mice show increased susceptibility upon neurotropic virus infection and augmented levels of proinflammatory cytokines. We observe higher immune cell infiltration into the brain and disease exacerbation upon infection in mice lacking XAF1. This increased pathology is restricted to the brain, since no morbidity was observed upon infection with a respiratory virus, such as influenza virus. Gene expression analysis unveiled an unbalanced immune response in XAF1-deficient mice resulting in an elevated proinflammatory response and diminished capacity to restore homeostasis. Our data demonstrates the protective capacity of XAF1 and provides new insights into the host response against virus infections.

Peste des petits ruminants (PPR) is a highly contagious viral disease of small ruminants caused by the peste des petits ruminants virus (PPRV), which is classified into four distinct genetic lineages (I-IV). A critical concern in the recent epidemiological history of PPRV is the rapid and widespread expansion of lineage IV (LIV) across West Africa over the past decade. This dominance suggests a potential adaptive advantage of circulating LIV strains in the regions current epidemiological context. In this study, we obtain the genome sequence of 26 new PPRV samples, including historical (pre-2000) and many recent African LIV isolates, offering the first opportunity to investigate the evolutionary history of LIV in Africa and identify genetic events potentially associated with its recent spread. Phylogenomic analyses implemented on a dataset of 167 curated PPRV genome sequences reveal that the most ancestral LIV group comprises strains circulating in Sub-Saharan Africa (designated clade LIVssa), providing robust evidence for an African origin of lineage IV. Our results further indicate that PPRV strains linked to the recent West African expansion of LIV belong to a specific LIVssa subgroup, termed NigB. We identified multiple signatures of selection pressure within the LIVssa sublineage, particularly in the NigB cluster. Several amino acid substitutions unique to LIVssa or NigB were detected, some of which may impact protein function and warrant prioritised investigation. Additional genomic data are required to confirm the association between the NigB group and the ongoing spread of LIV in West Africa. The evolutionary adaptations observed in LIVssa - potentially enhancing transmission efficiency, host range or pathogenicity - could undermine current disease control strategies in regions where PPR poses significant threats to food security and local economies. Author SummaryPeste des petits ruminants virus (PPRV) infects sheep and goats across Africa, Middle East, Asia and Europe, causing disease with major impact on global economy and food security. One genetic lineage of PPRV, called lineage IV (LIV), is at the origin of most recent expansion of the distribution of the disease, including replacement of other lineages in areas of African where PPRV is historically present. Here, we generated genome sequences from PPRV LIV isolates from different dates and places to study the evolution of this genetic lineage and explore whether its recent spread can be associated with the appearance of new mutations in the virus genome. Our results provide evidence that the PPRV LIV originated in Sub-Saharan Africa and identify mutations present only virus isolates currently spready in new regions of Africa. Further research should investigate the impact of these mutations on protein functions and capacity of transmission of PPRV.

The Sarthroviridae are a family of highly compact satellite RNA viruses comprising one recognised species, extra small virus (XSV). Macrobrachium rosenbergii nodavirus (MrNV) is the associated helper virus of XSV and their co-infection has been linked to white tail disease in freshwater prawns globally, although the role of XSV is remains unclear. Here, we describe the discovery and characterisation of ten novel, highly divergent sarthrovirus species from a range of hosts and environments within a small geographical region in Australia. These comprise novel sarthroviruses associated with marine sponges, seal and dingo faeces, environmental marine sediment samples and Indo-Pacific geckos (Hemidactylus garnotii). All the novel viruses possess only a capsid protein, consistent with the genome of XSV, yet exhibit substantial sequence divergence. Notably, some sarthrovirus variants seem to utilise different replication systems despite being genetically identical and present in the same host species. Sequences from nodaviruses, which could plausibly act as helpers, were associated with some, but not all, the sarthroviruses identified here. Phylogenetic analyses support the expansion of the Sarthroviridae into multiple distinct lineages, comprising at least seven genera. Collectively, these findings reveal a broader ecological distribution and evolutionary diversity of sarthroviruses and highlight the possibility of alternative replication strategies and tissue tropism in diverse animal host. SignificanceSarthoviruses are small ([~]800 nucleotides) satellite RNA viruses associated with a nodavirus of crustaceans that acts as a helper. To date, the only known sarthovirus is extra small virus (XSV), which also represents the sole species within the Sarthroviridae. Here, we report the detection of ten divergent sarthroviruses sampled from diverse animal hosts, including vertebrates, that expand the family to 11 species and at least seven genera. These viruses were detected from various host taxa and environmental samples from a confined geographical region in eastern Australia, suggesting that they are ecologically connected. Notably, we did not detect nodaviruses in all samples containing sarthroviruses, suggesting that different viruses may act as helpers for sarthovirus replication.

Avian influenza viruses (AIVs) are endemic in the Americas and responsible for outbreaks in both domestic and wild birds that occasionally spill over into humans. We report the first known outbreak of AIV H9N2 in lesser rhea (Rhea pennata), also known as Darwins rhea, in the region of Puno-Peru. The animals in this study lived in an isolated conservation center located in remote highlands above 4,000 m.a.s.l. Between June and July 2025, a total of 46/92 animals were recorded sick, with symptoms including greenish diarrhea (100%), hyporexia (24%), dyspnea (76%), nasal discharge (42%), drowsiness (18%) and isolation from the flock (73%), and 94% later died. Gross pathology exams revealed septicemia characterized by severe hepatitis, pneumonia, tracheitis, enteritis, and encephalitis. Swab and necropsy samples tested positive for Influenza A by PCR and were later identified as H9N2 through whole genome sequencing. We generated complete H9N2 genomes for two individuals. No additional pathogens were found. Phylogenetic analysis across all eight segments revealed that the viruses were low pathogenicity H9N2 AIV strains of North American origin, which indicated this outbreak was a new introduction of the virus into South America. We also performed a comparative mutational analysis and identified multiple mutations previously associated with mammalian host adaptation, increased virulence, increased pathogenicity, and increased virus binding to 2-6 receptors, which may explain the high mortality rates observed despite the supposedly low pathogenicity of the strain. We also identified novel mutations specific to rhea viruses that will need to be experimentally validated. This is the first report of a natural H9N2 systemic infection in an avian host, highlighting a need for increased surveillance efforts for zoonotic influenza viruses with pandemic potential. Author SummaryAvian influenza viruses (AIVs) are endemic in the Americas and cause more than 7,600 infections annually in domestic and wild birds worldwide each year. We report detection of AIV H9N2 in lesser rhea during an outbreak that occurred in June-July 2025 in the Andean highlands of Puno in Peru. Multiple sick animals were reported with symptoms of respiratory and gastrointestinal disease and 94% of them later died. Samples collected tested positive for Influenza A and they were subtyped as H9N2 of low pathogenic origin from North America. This is the third time H9N2 enters South America from North America, presumably through wild birds, some of which migrate along the Pacific Flyway. Comparison with other H9N2 sequences revealed a total of 44 mutations of interest that may explain the elevated death rates observed. Surveillance in wild birds remains patchy at best and needs to be strengthened in order to prevent spillover events into other animals, including humans.

Parasites can manipulate host behavior to increase their fitness while decreasing host fitness, a phenomenon known as an extended phenotype. Nucleopolyhedroviruses (NPVs), baculoviruses that infect Lepidopteran larvae, have been found to induce vertical climbing behavior and hyperactivity in exposed larvae. We quantified variation in the horizontal wandering behavior induced by different naturally-occurring pathogen isolates in the NPV that infects Dione (Agraulis) vanillae Linnaeus (Lepidoptera: Nymphalidae). Lab-raised larvae were infected with a constant dose of one of five different field-collected NPV isolates or a water control (n=98 larvae total), and placed in mazes to measure the horizontal distance wandered away from a food source. Virus-exposed larvae exhibited increased maximum distance of horizontal movement compared to the control, but did not significantly differ in the probability of wandering versus no movement. We also found variation in the distance wandered among the five virus isolates. However, grouping the five isolates into two previously-described viral strains or genogroups did not improve predicted differences in movement, perhaps due to the presence of within-strain genetic variation among isolates in the viral genes involved in controlling host behavior. Further work is needed to determine whether the observed between-isolate variation is the result of adaptive evolution. These results suggest that the NPV infecting D. vanillae manipulates larval behavior to increase horizontal wandering, which could lead to higher pathogen fitness by increasing long-distance dispersal of the virus across the landscape.

Respiratory viral-bacterial co-infections cause severe disease across species, yet the molecular mechanisms underlying enhanced pathogenesis remain poorly understood. This study characterised H3N8 equine influenza A virus (IAV) and Streptococcus equi subspecies zooepidemicus (SEZ) co-infections using complementary ultrastructural and transcriptomic approaches. Transmission electron microscopy demonstrated direct physical binding between spherical (A/equine/Miami/63) and filamentous (A/equine/Sussex/89 and A/equine/Newmarket/5/2003) IAV isolates and SEZ, including when SEZ was heat-inactivated ({theta}SEZ). Lectin staining revealed that SEZ expresses predominantly 2,3-linked sialic acids, the receptor for equine IAV. However, virus-bacteria binding persisted despite neuraminidase treatment. Scanning electron microscopy quantification demonstrated that viral pre-infection significantly enhanced bacterial adherence to cells of the DH82 canine macrophage-like cell line (2-fold increase, p<0.01) but not ExtEqFL (equine lung-derived) cells, revealing cell-type-specific enhancement. RNA-sequencing analysis showed that bacterial infection drove most transcriptional changes during co-infection with little difference in the number of differentially expressed genes (DEGs) between infection with SEZ alone (146 DEGS) or after pre-infection with either A/equine/Sussex/89 (166 DEGS) or A/equine/Newmarket/5/2003 (149 DEGS). Validation of upregulation of selected cytokines by RT-qPCR and ELISA demonstrated that SEZ infection drives dramatic cytokine upregulation compared to mock or {theta}SEZ controls. Viral pre-infection did not alter the SEZ-induced pro-inflammatory cytokine responses (IL-6, IL-8, TNF-) but significantly reduced IFN-{beta} expression compared to SEZ infection alone. These findings suggest that direct virus-bacteria physical interactions may drive cell-type-specific enhancement of bacterial colonisation, fundamentally advancing our understanding of respiratory co-infection pathogenesis.

Flaviviruses like Zika virus (ZIKV), contain RNA tertiary structures within the 3 untranslated region (UTR) that halt the 5-to-3 RNA exonuclease, Xrn1. Halting of Xrn1 at the two RNA structures, termed exonuclease-resistantRNA1 and 2 (xrRNA1 and xrRNA2), results in the formation of subgenomic flavivirus RNAs (sfRNA) that support viral pathogenesis. While the role of the flavivirus xrRNA1 in pathogenesis is well characterized, the role of the flavivirus xrRNA2 structure is not well studied. Using xrRNA crystal structure data, we inserted structure-informed mutations in ZIKV xrRNA2 to disrupt tertiary folding independent of significant sequence changes, evaluate sfRNA production, and define pathogenesis in a murine model of ZIKV infection. Compared to our prior work with ZIKV xrRNA1, we found that ZIKV xrRNA2 is under increased selection pressure to maintain sfRNA production resulting in multiple targeted mutations in xrRNA2 junctional region to induce a stable mutant. Using three targeted xrRNA junctional mutations termed ZIKV X2.L1, we found that the resulting ZIKV clone exhibits attenuated cell death in cultures and decreased viral growth in tissue cultures. In a murine model of ZIKV infection, mice inoculated with ZIKV X2.L1 exhibit significantly decreased symptomatic infection, improved survival, decreased end-organ infection in the brain, and continued robust neutralizing antibody responses to ZIKV. Despite attenuation, serum from ZIKV X2.L1-infected mice or mice vaccinated with ZIKV X2.L1, exhibited 100% protection from lethal ZIKV challenge. These studies show that RNA structure-informed mutations provide a robust model for flavivirus attenuation and vaccine design. Additional studies defining the mechanisms of robust neutralizing antibody responses and flavivirus-specific vaccine development are needed to continue the development of this novel vaccine platform approach for medically important flavivirus infections. Author summaryZika virus is a member of the Orthoflavivirus (referred to as flavivirus) genus that exhibit conserved RNA structures in the 3 untranslated region of the viral RNA genome. Two concerned RNA structures, termed exonuclease-resistant RNA 1 and 2, are important to support the ability of the virus to cause disease. While the first RNA structure is well studied, less is known about the role of exonuclease-resistant RNA 2 in the flavivirus infection. Using reverse genetics, we made mutations in the Zika virus exonuclease-resistant RNA 2 structure and studied how this mutant Zika virus was weakened or attenuated. We found that the mutant Zika virus clone exhibits reduced virus replication, reduced ability to kill cells, and decreased virulence in mouse models of Zika virus disease. Using this mutant virus as a potential vaccine candidate, we found that Zika virus with mutations in the exonuclease-resistant RNA 2 structure provide complete protection from lethal Zika virus challenge. These data suggest that targeting the second exonuclease resistant RNA structure in flaviviruses is a viable platform for the development of vaccine candidates for this important group of viruses.

Human adenovirus serotype 4 (Ad4) is used as a replication-competent oral vaccine that safely and effectively prevents Ad4 respiratory illness in US military personnel. Recombinant Ad4 vaccine candidates elicit mucosal and systemic immune responses against respiratory viruses in hamsters, nonhuman primates, and humans. Although evaluation of Ad4 vaccine candidates in mice would be extremely useful given the large number of immunologic tools available, this has been limited by concerns about a lack of viral replication in these animals. Here we generated recombinant Ad4 vectors that express either luciferase (Ad4-Luc) or herpes simplex virus type 2 (HSV-2) glycoprotein D (Ad4-gD2) to identify transgene expression kinetics, the presence of Ad4 vector replication, and HSV-2 immune responses and protection against HSV-2 infection. Local luciferase activity was observed from 7 hours to 20 days after intranasal inoculation of BALB/c and humanized mice. Subsequent inoculations with Ad4-Luc showed reduced luciferase expression in BALB/c mice, but robust expression in humanized mice, suggesting an immune response to the vector in wild-type mice. Ad4 DNA, but not luciferase activity, was reduced in the lungs of BALB/c mice treated with cidofovir before inoculation with Ad4, implying that Ad4 replicated, albeit at a low level, in the lungs. Intranasal vaccination of mice with Ad4-gD2 resulted in HSV-2 neutralizing antibody in the serum, and after HSV-2 intravaginal challenge reduced disease scores, increased survival, and reduced shedding. Overall, the BALB/c mouse model is semi-permissive to Ad4 mucosal infection, but transgene expression is sufficient for the study of Ad4-based vaccine candidates. ImportanceMucosal surfaces serve as the primary site of infection and shedding for many viral pathogens. Immune responses at mucosal sites provide protection, but few mucosal vaccines are licensed. The oral replication-competent adenovirus serotype 4 (Ad4) vaccine is used to prevent respiratory illness in military recruits, has an extraordinary record of safety and efficacy and has been tested as a recombinant platform for other viruses. Further development of this vaccine platform has been partially hindered by the perceived inability to evaluate vaccine candidates in mice. Here we characterize recombinant Ad4 transgene expression kinetics and viral replication after inoculation at various sites and show protection against herpes simplex virus type 2 (HSV-2) genital disease in mice after intranasal vaccination. We show that Ad4 can induce protective efficacy, even in a semi-permissive mouse model, suggesting this is a promising vector for HSV-2 and potentially other viral pathogens.

Japanese encephalitis virus (JEV) causes significant encephalitis across the Asia-Pacific region. Current vaccines target historical genotype III strains, but emerging genotypes,potentially driven by vaccine-mediated selective pressure, threaten vaccine effectiveness through altered envelope protein sequences that may reduce antibody cross-neutralisation. This study employed integrated sequence and structural analyses to identify E protein mutations affecting neutralising antibody binding and protein stability. The study curated JEV polyprotein sequences from NCBI, performed multiple sequence alignment, and used Shannon entropy to pinpoint highly variable positions. Mutations occurring at [&ge;]1% frequency within high-entropy regions were selected for analysis. From 34 initially identified mutations, four candidates were prioritized based on structural stabilization potential. Mutations were evaluated through FoldX stability predictions, molecular docking with antibody 2H4 using HADDOCK3, and molecular dynamics simulations. Binding energies were calculated using MM-GBSA analysis. Results demonstrated that all mutant E-2H4 complexes remained stable during simulations, with root-mean-square deviation plateauing after equilibration and minimal localized changes in root-mean-square fluctuation. These findings suggest that EDIII substitutions represent important candidates for further investigation to understand genotype-specific variations and inform next-generation vaccine development strategies against emerging JEV strains.

BackgroundHerpesviruses are a group of double-stranded DNA viruses known to infect a wide range of vertebrates and establish life-long latent infections. While bats serve as natural reservoir hosts for numerous viral families, relatively few bat herpesviruses have been successfully isolated. In this study, we report the isolation and characterization of two novel alphaherpesvirus strains obtained from Rousettus aegyptiacus bats in Coastal Kenya. MethodsThe samples of oral and rectal swabs were collected from three different species of bats from coastal Kenya between October 2024 and April 2025; the bat species collected include Hipposideros spp., Coleura afra, and Rousettus aegyptiacus. Virus isolation was performed by inoculation of samples in Vero E6 cells and subsequent monitoring for cytopathic effects (CPE). Total nucleic acids were extracted from CPE positive cultures and subjected to library preparation to enable unbiased detection of both RNA and DNA viruses. The libraries were sequenced using next-generation sequencing with Illumina MiSeq platform. Subsequently, bioinformatic analysis was carried out to identify the virus, generate consensus genomes as well as phylogenetic analysis to determine the placement of identified viruses. ResultsTwo samples from R. aegyptiacus (KIK_460_O and KIK_465_O) induced typical CPE within five days. Sequencing and assembly yielded partial consensus sequences of approximately 60 kb (KIK_460_O) and 70 kb (KIK_465_O), representing extended genomic data for a bat-associated alphaherpesvirus. This virus has a genome of about 140kb, indicating that our partial assemblies account for about 43-50% of the total genome. Both isolates were found to be closely related to Dzifa herpesvirus, an alphaherpesvirus previously identified in Kilifi, Kenya. Alphaherpesvirus was identified based on partial sequencing of UL19 (3,787bp) and UL30 (2,846bp) genes. The two isolates were found to be identical at the UL19 gene, showing that they belonged to the same virus strain. Phylogenetic analysis showed that the novel alphaherpesvirus belongs to primate alphaherpesviruses under the subfamily Alphaherpesvirinae. ConclusionThis study reports the isolation and genomic characterization of a novel fruit bat alphaherpesvirus from Kenyan Rousettus aegyptiacus bats. The partial genome assembly (60-70 kb) represent the first extended genomic data for this virus, covering approximately 43-50% of the estimated 140 kb complete genome. The phylogenetic placement of this alphaherpesvirus near primate viruses, especially Pteropodid alphaherpesvirus 1, suggests bat-association and needs further investigation into its zoonotic potential.

We have used synthetic biology recombination methods in yeast to build herpes simplex virus type-1 (HSV-1) and human cytomegalovirus (HCMV) genomes from multiple fragments. The genomes were built using transformation-associated recombination (TAR) in yeast, by virtue of overlapping sequences between the different fragments. This study demonstrates the successful assembly of the Epstein-Barr virus (EBV) genome. We used as the model genome, the Akata Burkitts lymphoma genome, specifically the BX1 genome which encodes a neomycin selectable marker and a GFP expression cassette in the BXLF1 region. The 171.3 kb genome was first deconstructed into 11 fragments in silico, each having 80 bp overlapping sequence between the fragments. The 11 fragments (TAR 1 to TAR 11) were cloned using TAR in yeast, analyzed by restriction enzyme analyses and Nanopore sequencing to validate the cloned fragment. The EBV genome was built in two stages: TAR fragments 1 to 6 and TAR fragments 7 to 11 were assembled to generate two half-genomes. The whole genome (TAR 1-11) was then assembled by joining TAR 1-6 with TAR 7-11. Complete EBV genomes were examined by PCR assays and restriction enzyme analyses and then transfected into HEK-293 cells to generate virus producer cell lines. The HEK-293 cell clones were tested for virus production following lytic induction using baculovirus transduction of Zta, Rta and glycoprotein B (BALF4). The supernatants from these induced cells were harvested and used to infect Raji cells. This analysis revealed a significant number of cells displaying strong GFP fluorescence indicative of infectious virus. We used this supernatant virus to infect primary B cells and were able to derive lymphoblastoid cell lines (LCL) indicative of the ability of this virus to transform B cells. We tested this method for engineering different mutations. Two mutations were made, one in Zta and the other in the small capsid protein (BFRF3). Mutations were engineered in the TAR plasmid in which the genes reside and after sequence validation, assembled into the TAR 1-6 half genome and then the TAR 1-11 genome, which was used to generate HEK-293 cell clones. For the {Delta}Zta cell lines, we could detect virus in the supernatants only if baculovirus expressing Zta in trans was included, this {Delta}Zta EBV virus could transform B cells. The small capsid protein (BFRF3) decorates the capsid shell and is required for capsid assembly in a self-assembly system. When the HEK-293 cell clones were induced using co-expression of Zta, Rta and gB, no virus was detected in the culture supernatants. However, if we provided BFRF3 in trans using baculovirus expressing this protein, virus was detected in the supernatants. This provides the first report of the essential role of the small capsid protein in EBV-infected cells.

We prepared siRNA libraries against the H5N2 virus NP gene, and the PA, PB1 and PB2 genes that express the proteins that form the virus polymerase complex. The antiviral activity of the siRNA libraries in H5N2 virus infected cells was initially assessed by using qPCR to measure the corresponding mRNA levels in the siRNA-treated cells. In this way siRNA molecules within each library were identified that exhibited to a greater than 70% reduction in levels of each target mRNA. A selection of these siRNA molecules was further evaluated for their antiviral activity in a multi-cycle H5N2 MDCK cell model. The siRNA molecules identified were successful in blocking virus transmission and lead to a reduction in influenza virus progeny virus production. This antiviral activity correlated with both the inhibition of nuclear export of the newly formed RNP complexs that arise from the transcriptional activity of the input virus, and the inhibition of the polymerase activity of the newly formed virus polymerase complexes. This study highlights the potential use of siRNA as a strategy to block virus transmission by targeting the avian influenza virus polymerase complex.

High Pathogenicity Avian Influenza (HPAI) H5N1 clade 2.3.4.4b has spread beyond birds to affect seals across the Southern Ocean and sub-Antarctic region, with southern elephant seals (Mirounga leonina) particularly devastated. The virus, likely introduced via spillover from infected migratory birds, has killed tens of thousands of adult seals and pups throughout most of their range, though Macquarie Island remains unaffected so far. We used twenty years of elephant seal movement data from the southern Indian and Pacific oceans to assess whether seal-to-seal transmission could spread HPAI H5N1 between breeding colonies, despite the vast distances separating them (Marion Island, Iles Crozet, Iles Kerguelen, and Macquarie Island). There was substantial overlap in seals' at-sea distributions during their winter post-moult trips, when seals travel for weeks at average speeds of 3.5 km/h. Two transmission pathways were examined: (1) terrestrial "stepping stone" routes, where infected seals could pass the virus between colonies during short intervals to remain infectious were feasible from Marion Island to Kerguelen but not from Kerguelen to Macquarie Island; and (2) at-sea encounters between seals, which occurred frequently enough to enable transmission. The findings suggest that once established at Macquarie Island, the virus could potentially spread further to New Zealand's sub-Antarctic islands and mainland New Zealand. While seal-to-seal transmission appears possible, we conclude this is unlikely. Nonetheless, understanding at-sea contact rates enhances knowledge of H5N1 epidemiology and demonstrates the value of combining long-term population monitoring with movement data to understand wildlife disease dynamics.

The therapeutic efficacy of oncolytic viruses is often limited by the presence of tumor cells that resist virus-mediated killing. Here, we investigated the molecular mechanisms underlying resistance to Vesicular Stomatitis Virus (VSV) in PC3 cells, an aggressive metastatic prostate cancer (PrCa) cell line, using the VSV-sensitive LNCaP cell line as a comparator. RNA sequencing revealed that, relative to untreated cells, VSV-infected PC3 cells upregulated both pro-apoptotic genes, including BIM, PUMA, and NOXA, and anti-apoptotic and antiviral genes, including A20 and RIG-I. In addition, genes associated with antiviral and pro-survival pathways, including NF{kappa}B and PI3K-Akt signaling, were more highly expressed in PC3 cells than in LNCaP cells. At baseline, PC3 cells also exhibited elevated expression of multiple pro-survival genes, including BCL-xL, MCL1, and CK2, compared with LNCaP cells. Complementary proteomic analyses identified enhanced activation of NF{kappa}B, PI3K-Akt, and MSK1 signaling in VSV-infected PC3 cells relative to infected LNCaP cells. Furthermore, pharmacological inhibition of BCL-2 family proteins or NF{kappa}B signaling restored sensitivity to VSV-induced cell death in PC3 cells. Collectively, these findings identify NF{kappa}B-centered pro-survival signaling networks as key contributors to the resistant phenotype of PC3 cells and suggest that combining oncolytic virotherapy with targeted inhibitors may improve therapeutic efficacy in resistant prostate cancers.

Background: The gut environment is hostile to life, yet the human virome, dominated by bacteriophages, persists. Adaptations to the major capsid protein (MCP) may explain this. Phage MCPs conserve the HK97 fold, ideal for detecting convergent features across phage populations. Prior capsid stability research focused on individual phages, limiting broader pattern identification. Methods: MCPs from the Gut Phage Database (GPD) (n=8,478) and INPHARED (n=4,905) were predicted using ProtPhage + Phold and clustered using MMseqs2 (GPD=902 vs INPHARED=606). Structural predictions, conservation analysis, and capsomere modeling were used to characterize cysteine environments. Results: Biochemical analysis identified cysteine enrichment in GPD MCPs. Phylogenetic mapping was consistent with convergent evolution of high-cysteine MCPs. Over 50% of cysteines were [&ge;]90% conserved within and between clusters. Simulated capsomeres showed 83% of cysteines are buried (RSA <10%). Conclusions: These findings suggest gut phages may have convergently evolved cysteine-based capsid stabilization, with implications for engineering therapeutic phages.