Viruses

SARS-CoV-2 replication depends on a tightly coordinated series of intracellular processes that remain incompletely quantified. Here, we integrated high-resolution time-resolved measurements of viral RNA, protein expression, and infectious virion production with mechanistic mathematical modeling to obtain a quantitative description of the viral replication cycle in human lung cells. Using transcriptomic, proteomic, and infectivity data collected over the first 24 hours of infection, we calibrated an ordinary differential equation model that captures genomic and subgenomic RNA synthesis, viral protein production, virion assembly, and virus release. The model accurately reproduced the observed replication dynamics and enabled estimation of kinetic parameters that are difficult to measure experimentally. Sensitivity analysis identified viral RNA replication and non-structural protein maturation as dominant determinants of viral replication efficiency. To assess predictive power, the model was challenged with independent antiviral perturbation experiments using remdesivir, nirmatrelvir, and montelukast. Model predictions closely matched experimentally observed treatment responses and correctly reproduced drug interaction effects during combination therapy. Furthermore, comparison of alternative mechanistic hypotheses supported NSP5 rather than NSP1 as the primary antiviral target of montelukast. Together, these results establish a predictive framework for dissecting intracellular coronavirus replication and evaluating antiviral intervention strategies.

BackgroundHuman T-lymphotropic virus type 1 (HTLV-1) is an oncogenic retrovirus which is transmitted via cell-containing blood fluids or from mother to child via breastfeeding, leading to severe diseases such as adult T-cell leukemia/lymphoma (ATL) and neuroinflammation. Most studies focus on virus detection in peripheral blood due to limited access of tissue material, especially in infants. Thus, our understanding of viral distribution in organs, in particular along the oral route of transmission, is still a critical gap in HTLV-1 research. Methodology/Principal FindingsHere, we present an analysis of tissues from a non-human primate (NHP) colony (olive baboon: Papio anubis) naturally infected with the closely related counterpart of HTLV-1, simian T-lymphotropic virus type 1 (STLV-1). Various organs and tissues of the oropharyngeal and gastrointestinal tract including tonsils, stomach, small intestine and colon were analyzed for the presence or absence of STLV-1. Beside TaqMan qPCR measuring relative copy numbers, we established a highly sensitive and precise droplet digital (dd) PCR protocol to measure absolute copy numbers of viral Tax DNA. Tax DNA was detectable in the tonsils in two NHPs, but to a greater extent in stomach in four NHPs. We also found Tax in parts of the small intestine, i.e. duodenum and Peyers patches, in a NHP with high blood proviral load. Conclusion/SignificanceThese data provide a quantitative analysis of STLV-1 Tax in the gastrointestinal tract and are, to our knowledge, the first indication of STLV-1 detection in stomach tissue of naturally STLV-1-infected asymptomatic NHPs. Although it is unclear how infection occurred - from mother-to-child, sexual or via animal bites - our study suggests that these parts of the gastrointestinal tract might either serve as site of virus transmission or as viral reservoir. Author summaryHuman T-lymphotropic virus type 1 (HTLV-1) is a human oncogenic retrovirus being transmitted via cell-containing body fluids such as breast milk, blood, or semen. The estimated number of infected people is around 10 to 20 million. To study the viral distribution and persistence of HTLV-1 in different organs in the oropharyngeal and gastrointestinal tract, a suitable in vivo model is essential. In this study, tissues of non-human primates (NHPs, baboon: Papio anubis) naturally infected with the closely related simian counterpart of HTLV-1, simian T-lymphotropic virus type 1 (STLV-1), were analyzed for the presence or absence of the viral gene Tax. We identified the presence of STLV-1 Tax in the stomach, duodenum and Peyers patches by using two different detection methods: TaqMan-based qPCR and the more sensitive droplet digital PCR (ddPCR). Tax could be detected in tonsils in two NHPs only, but in stomach in four NHPs. Together, this is the first time that STLV-1 Tax was detected in stomach tissue of STLV-1-infected asymptomatic NHPs, highlighting the importance of investigating viral persistence and/or viral reservoirs of primate T-lymphotropic viruses independently of the entry route.

Polinton-like viruses (PLVs) are among the most abundant eukaryotic DNA viruses in aquatic environments. Despite their extensive diversity, broad host range and variable gene content, they are commonly treated as a single group, which obscures their evolutionary relationships and complicates their classification. Through analysing thousands of viral genomes from aquatic ecosystems and public metagenomic datasets, we clarify the evolutionary structure encompassed by the term PLV. Using sensitive profile Hidden Markov Model (HMM) comparisons, phylogenies of conserved capsid morphogenetic genes and gene content analysis, we show that viruses referred to as PLVs are distributed across multiple deep lineages spanning at least three currently recognised viral classes. These include the Gosseviruses, aquatic viruses related to Maverick-Polintons in animal genomes. They also include a continuum of related viruses from 15 kb PLVs to the 45 kb Mriyaviruses and more broadly, to the Nucleocytoviricota, potentially representing extant relatives of giant viruses. Our findings suggest that PLVs do not fit neatly within existing taxonomic boundaries, reflecting a complex history of horizontal gene transfer and diversification of life strategies. To support future discovery, we provide a curated set of HMMs representing the known capsid diversity of PLVs, Maverick-Polintons, and virophages. This toolkit enables sensitive detection and identification of PLVs across metagenomic and eukaryotic genome datasets. Our study provides an evolutionary framework for interpreting PLV diversity and a foundation for future refinement of their classification.

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.

BackgroundHuman Metapneumovirus (HMPV) is a major contributor to acute respiratory tract infections. Currently, no approved vaccines or specific antiviral therapies are available worldwide. Genome-scale metabolic models (GEMs), when integrated with Viral Biomass Objective Functions (VBOFs), provide a robust computational framework for identifying host metabolic dependencies essential for viral replication. This approach enables systematic prioritization of potential antiviral drug targets. ResultsThe first comprehensive VBOF for HMPV was constructed by integrating stoichiometric data from the viral genome, including structural proteins with defined copy numbers, amino acid residues per virion, envelope lipids, and glycan modifications. The reconstructed VBOF was incorporated into the human bronchial epithelial cell model, iHBEC1, to analyze metabolic changes between uninfected and infected host cells. Knockout analysis identified two selective antiviral gene targets: PGM3 (phosphoacetylglucosamine mutase) and GNPNAT1 (N-acetylglucosamine-6-phosphate acetyltransferase), as well as seven selective reaction targets primarily within the hexosamine biosynthesis and nucleotide sugar pathways. Knockout of PGM3 or GNPNAT1 completely abolished viral production while preserving complete host cell viability. Additionally, guanylate kinase (GUK1/GK1) emerged as a highly selective target for HMPV, confirming findings from Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) studies and suggesting a conserved vulnerability across respiratory viruses. ConclusionsUDP-GlcNAc, the vital end-product of the hexosamine biosynthesis pathway (HBP), is computationally predicted as a critical metabolic hub. This pathway represents the primary metabolic vulnerability of HMPV, due to the extensive glycosylation requirements of the HMPV attachment protein. The host-directed antiviral targets PGM3 and GNPNAT1 are high-priority candidates for experimental validation and may provide novel strategies to combat this respiratory infection.

BackgroundPangolins are critically endangered mammals that suffer from high rates of gastrointestinal disease during captivity, yet the role of the gut virome in their health remains unexplored. This study presents the first comprehensive characterization of the gut DNA virome in Malayan (Manis javanica) and Chinese (M. pentadactyla) pangolins across different health states. ResultsMetaviromic sequencing of 16 fecal samples from healthy, diarrheal, pneumonic, free-ranging, and deceased pangolins generated 7.2-11.8 Gb clean data per sample. A total of 12 viral phyla, 26 families, 219 genera, and 1,132 species were identified. Caudovirales phages (Siphoviridae, Myoviridae, and Podoviridae) dominated the gut virome of healthy individuals, with phage content exceeding 90% in most healthy samples. However, diseased and deceased individuals exhibited a significant reduction in phage proportion (94.5% vs. 53.3%, P = 0.02), accompanied by a dramatic increase in eukaryotic viruses--particularly Parvoviridae, which accounted for 66% of the virome in deceased Malayan pangolins. Iridoviridae (38%) and Polydnaviridae (34%) dominated in deceased Chinese pangolins. SIMPER analysis identified Parvoviridae as the primary contributor to virome dissimilarity between healthy and diseased Malayan pangolins, whereas Iridoviridae and Polydnaviridae were the key contributors for Chinese pangolins. LEfSe analysis revealed 12 biomarker viruses in free-ranging pangolins (predominantly Staphylococcus and Escherichia phages), six in healthy Chinese pangolins (predominantly Streptococcus phages and Lactobacillus viruses), and six in deceased Malayan pangolins (predominantly vertebrate viruses). KEGG functional annotation revealed that genes related to DNA replication, repair, and recombination were the most abundant, suggesting frequent genomic recombination within the pangolin gut virome. ConclusionsThe gut virome of pangolins is closely associated with health status. Disease induces a shift from a phage-dominated to a eukaryotic virus-dominated virome, with Parvoviridae and Iridoviridae emerging as candidate pathogenic viruses in Malayan and Chinese pangolins, respectively. These findings provide molecular evidence for virome monitoring in pangolin conservation and highlight the need for targeted surveillance of ImportancePangolins are the most trafficked mammals in the world, and all eight species are critically endangered. During rescue and captive care, these animals suffer from high rates of gastrointestinal disease, yet the role of viruses in their gut health has never been studied. This research provides the first comprehensive map of the gut virus community in pangolins, revealing that healthy individuals carry a virus community dominated by beneficial bacterial viruses called phages, while sick and dying animals show a dramatic shift toward disease-causing viruses. Notably, parvoviruses emerge as potential pathogens in Malayan pangolins, while iridoviruses dominate in deceased Chinese pangolins. These findings offer a new tool for monitoring pangolin health in rescue centers: by tracking the balance between beneficial phages and harmful viruses, veterinarians may be able to detect illness earlier and improve survival rates. The study also highlights the importance of environmental exposure for maintaining a healthy gut virus community, supporting the creation of semi-natural enclosures for rescued pangolins. Beyond conservation, this work contributes to our understanding of how viruses move between wildlife and humans, which is critical for preventing future disease outbreaks.

BackgroundWest Nile virus (WNV) is a vector-borne zoonotic pathogen maintained in an enzootic cycle between birds and mosquitoes which is considered a significant public health concern in Europe, particularly in relation to its recent increase in reported human cases and range expansion. While a comprehensive understanding of the viruss epidemiological dynamics is essential to inform effective prevention and control strategies, to date significant knowledge gaps remain in quantifying interspecific differences within the complex avian communities involved in WNV circulation. Globally, WNV-infection has indeed been documented across more than 300 bird species, however, whether and how inter-specific differences in avian hosts traits affect the spread of WNV is still largely unknown. A substantial body of research has investigated how epidemiological traits, such as the duration of infection and competence, influence WNV dynamics. However, much less is known about the role of avian demography. Methodology/Principal findingsWe therefore investigated through mathematical modelling the role of avian demographic traits in shaping patterns of mosquito WNV infection dynamics in northern Italy (Lombardy Region, 2016-2018). We focused on the effects of annual offspring production, timing and synchrony of breeding which ultimately affect seasonal abundance of competent avian hosts. We highlighted that timing of breeding has the greatest effect on the number of infected mosquitoes, while annual offspring production influences the timing of the infection peak. Our simulations provide evidence that non-corvid species can have a key impact on WNV transmission. Conclusion/SignificanceThese results can support future research by providing priority bird species to direct further studies and by suggesting that the acknowledgment of spatio-temporal variation in the abundance of competent avian hosts plays a key role in the development of effective surveillance strategies and mosquito control actions. Author summaryWest Nile virus (WNV) is endemic in Italy and represents a significant public health threat in Europe, with increasing cases of severe neuroinvasive disease in humans in recent years. Surveillance data reveal marked spatial and temporal variability in infection dynamics, suggesting that key drivers of WNV transmission remain poorly understood. The contribution of different bird species (over 300 are implicated in the WNV cycle) is often overlooked despite evidence that species-specific traits are critical determinants of WNV infection dynamics. Few studies have examined birds demographic traits, despite their well-established importance in shaping infection dynamics across diseases. Given the challenges in collecting detailed wildlife data, we employed mechanistic models to explore transmission scenarios and test whether avian demographic traits influence bird species roles in WNV transmission and maintenance in Lombardy. Our findings demonstrate that brood size, hatching synchrony, and hatching time significantly affect estimated WNV prevalence in mosquitoes.

Viral infectivity factor (Vif) is an HIV-1 accessory protein best known for its counteraction of APOBEC3 enzymes, interferon-inducible host defenses against viral infection, as well as PPP2R5A-E, which are regulatory subunits of the PP2A cellular phosphatase holoenzyme, resulting in striking Vif-dependent phosphoproteome remodeling. One reported consequence of this remodeling is hyperphosphorylation of several Aurora kinase substrates in HIV-1 infected cells, which is reversed when Vif-deficient virus is used. We previously showed that infection of T cells with Vif-deficient HIV-1 results in significantly accelerated formation of syncytia compared to wild-type HIV-1 infection. More recently, others have shown that application of Aurora kinase B inhibitors during HIV-1 infection in T cells also results in a similar hyperfusogenic phenotype. Both effects were specific to Env-driven cell-cell fusion, and did not influence virus infectivity. We thus hypothesized that Vifs influence on the rate of HIV-1-induced cell-cell fusion was mediated by Aurora kinase activity. To start testing this hypothesis, we have evaluated the effects of a small panel of Aurora kinase inhibitors on HIV-1-induced cell-cell fusion in the presence or absence of Vif. Our results replicate the previously documented increase in cell-cell fusion in the absence of Vif, as well as the increase in cell-cell fusion observed upon inhibition of Aurora kinase B in the presence of Vif. Critically, we now present evidence that Vif deletion significantly blunts the impact of Aurora kinase inhibition on cell-cell fusion, supporting our hypothesis that Vif-mediated regulation of cell-cell fusion depends on Aurora kinase signaling dysregulation, likely because of PPP2R5A-E degradation. Further, we document that the cell-cell fusion regulator downstream of Aurora kinase signaling is likely Ezrin, which we have previously shown to prevent excess HIV-1-induced syncytium formation when in its phosphorylated (activated) state. Taken together, these findings establish a Vif, Aurora kinase, and Ezrin-dependent mechanistic framework for the regulation of HIV-1-induced cell-cell fusion in infected T cells which likely helps preserve optimal cell-to-cell virus transmission.

Viral haemorrhagic septicaemia (VHS) is a severe disease affecting rainbow trout (Oncorhynchus mykiss) and a wide range of wild freshwater and marine fish species. VHSV threatens rainbow trout aquaculture, as it may cause 100% mortality in fry. Previous studies identified a quantitative trait locus (QTL) on chromosome 3 associated with resistance to VHSV waterborne challenge and reduced viral replication in fin explants, although these findings were obtained using limited genetic diversity. The objective of this study was to validate and extend the identification of genomic regions associated with resistance to VHSV in the genetically diverse rainbow trout line designated "synthetic." A genome-wide association study (GWAS) was conducted using whole-genome sequences from parents of progeny classified as resistant or susceptible to a VHSV waterborne challenge. While the QTL on chromosome 3 was not validated in the synthetic line, four novel suggestive SNPs associated with survival following VHSV waterborne challenge were identified on chromosomes 6, 8, 17, and 32. Notably, one SNP on chromosome 17 was located within a gene potentially involved in antiviral defence, a paralog of lrp1 (low-density lipoprotein receptor-related protein 1). To further investigate its role, lrp1 function was analysed in vitro using CRISPR-Cas9 genome editing. Three independent lrp1-/- CHSE-EC cell lines were generated and challenged with VHSV. The results showed that lrp1 is not essential for viral entry but may modulate the inflammatory response during VHSV infection in epithelial cell lines.

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.

Flaviviruses contribute significantly to the global disease burden and are known to remodel host organelles extensively to their advantage. Centrosomal microtubule-organizing centres (MTOCs) having established role in cell division and signalling are also targeted by viruses. However, it remains uncertain whether they act as bystanders or actively engage in viral processes. Here, we elucidate the centrosome and cytoskeletal involvement during Japanese Encephalitis Virus (JEV) infection. Virus-free expression studies have identified a centrosome-targeting region within the C-terminal helicase domain that mediates the association of virus-derived structures with host MTOCs. When expressed exogenously, JEV-NS3 formed pericentriolar aggresomes resembling the distribution pattern of helicase-containing viroplasm in infected cells. Centriole depletion assays revealed the proviral role of centrosome facilitating JEV replication, where the viroplasm organization depends on centrosomal MTOCs, and vimentin cages. Additionally, microtubule disruption and dynarrestin blockade assays emphasized the roles of microtubules and dynein in concentrating NS3-containing vesicular packets towards centrosomes, highlighting the centrosome-cytoskeleton axis as a potential target for flaviviral intervention. SummaryThis study demonstrates that JEV helicase is directed to centrosomes via its CTHD domain, using them to initiate pericentriolar viroplasm formation. It also highlights the unappreciated function of centrosomes as a proviral hub, orchestrating cytoskeletal remodelling and viral factory organization. Graphical abstractJEV helicase is targeted to centrosomes to form viroplasm with the aid of microtubule and motor proteins. A) JEV lifecycle from receptor binding (Step 1) to endocytic internalization (Step 2) followed by uncoating and release of viral genome (Step 3) followed by replication within the ER derived Vesicular packets (Step 4). This vesicle packets harbors viral proteins that form replication complex, like helicase and replicase and the replicative intermediate, dsRNA. These vesicle packets with the aid of retrograde motor protein gets targeted towards the centrosome (Step 5). Ultimately multiple vesicle packets accumulate in the pericentrosomal region, where it forms a separate compartment enclosed by vimentin and tubulin cage (Step 6). Inset A, B and C depict the effect of cytoskeletal perturbations on Viroplasm formation. B) Disruption of microtubules using Nocodazole completely abolishes pericentriolar viroplasm organization. C) Centrinone mediated centrosome depletion markedly reduces both the number and size of pericentriolar viroplasm. D) Dynarrestin mediated retrograde microtubule transport significantly decreases the reorganization of vesicle packets to form the viroplasm in the pericentriolar region. O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/740276v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@17a6354org.highwire.dtl.DTLVardef@f208eorg.highwire.dtl.DTLVardef@1224fcaorg.highwire.dtl.DTLVardef@1a48bab_HPS_FORMAT_FIGEXP M_FIG C_FIG

Rodents are an important reservoir of zoonotic viruses and are ubiquitously present in densely populated urban areas. Betacoronaviruses in the Embecovirus lineage are well known to infect both humans and animals and have established rodent reservoirs. Here three Betacoronavirus gravedinis genomes were sequenced and characterized in white footed mice (Peromyscus leucopus, commonly white footed mice) collected in New York City, the second most populous city in North America. The genomes were distinct from mouse hepatitis virus (MHV), the prototype mouse betacoronavirus, and highly similar and identical in one case to previously characterized B. gravedinis sequences from white footed mice in Connecticut. Codon aware evolutionary models were used to identify specific sites under positive selection within the spike protein of B. gravedinis. A novel method was developed to predict the probable geographic distribution of the virus using publicly available data from the Global Biodiversity Information Facility to generate a weighted distribution map highlighting overlapping potential host ranges based on the evolutionary distance using a high resolution cytocrome B (CYTB) phylogeny of rodent species with potentially overlapping ranges. Our models predict three current hotspots of circulation in North America under different possible transmission regimes, and an additional fourth hotspot was predicted to arise in a warming future. This study highlights the continued need for biodiversity-informed surveillance of potential zoonotic pathogens in rodents.

SARS-CoV-2 persistence has been proposed as a potential contributor to the pathogenesis of long COVID, with reservoir tissues potentially serving as sites for viral persistence, intra-host evolution, and intermittent viral shedding. Here, we used experimentally infected Syrian hamsters to investigate long-term SARS-CoV-2 persistence across tissues, viral infectivity, and associated immunological and metabolic alterations. Syrian hamsters (Mesocricetus auratus) were intranasally infected with a SARS-CoV-2 parental strain or Gamma and Delta variants and monitored for up to one year, with samples collected at 3, 15, 30, 90, 150, and 365 days post-infection (dpi). During the acute phase, infected animals exhibited significant weight loss, viral shedding, and marked pulmonary inflammation, accompanied by increased expression of pro-inflammatory cytokines at 3 dpi. Infection was confirmed by seroconversion, with sustained IgG responses and low-titer neutralizing antibodies against Omicron. Viral nucleoprotein was detected in multiple tissues up to 365 dpi, while RT-qPCR identified persistent low-level viral RNA in the lungs, brain, spleen, and thymus throughout the observation period, without evidence of productive viral replication. Immune gene expression displayed organ-specific temporal patterns: acute pulmonary inflammation transitioned into broad late-stage suppression, except for sustained TGF-{beta} expression; the brain exhibited a late chemokine signature at 365 dpi; and the thymus showed a delayed immune activation peak at 150 dpi, particularly in Delta-infected animals. Metabolomic profiling revealed a shared acute-phase metabolic signature across variants that largely resolved by 365 dpi, whereas Delta-infected animals retained distinct residual metabolic alterations. Collectively, these findings establish a model of long-term SARS-CoV-2 tissue persistence characterized by organ-specific immune and metabolic signatures, providing a platform to investigate mechanisms underlying post-acute sequelae and evaluate potential therapeutic strategies.

Since its emergence in the human population, SARS-CoV-2 has continuously evolved to evade immune responses and robustly establish global circulation. In this process, the structural viral membrane (M) protein has accumulated amino acid changes whose impact on viral particle assembly and innate immune evasion remains incompletely understood. Here, we designed a SARS-CoV-2 replicon system lacking M that assesses the influence of transiently transfected M protein variants on viral particle production independently of viral RNA replication. We found that M protein variants have reduced particle assembly while innate immune antagonism functions are strengthened. Notably, the assembly defect is rescued by co-evolving N protein variants, highlighting how SARS-CoV-2 evolution coordinates between two of its structural proteins to optimize viral infection. Our work underscores the complex evolutionary trajectories of SARS-CoV-2 variants across different viral proteins and informs future therapeutic strategies targeting viral assembly and limiting infection.

Nipah virus is a highly lethal, zoonotic paramyxovirus that has caused recurring outbreaks in several South and Southeast Asian countries since its discovery in Malaysia in 1998. Symptoms of infection include severe respiratory and neurological disease, often resulting in death. As no approved vaccines or antivirals are currently available to reduce the burden of this virus, it is classified as a biosafety level 4 pathogen. There is an urgent need for systems that enable research in a lower biocontainment setting, especially since the World Health Organization declared Nipah virus a priority pathogen for pandemic concern. In the past, several minigenome systems have already been developed as safe alternatives to working with infectious virus; however, these systems remain relatively inefficient and lack robustness and reliability for further applications. Therefore, we developed novel optimized RNA polymerase II-driven minigenomes with nanoluciferase or enhanced green fluorescent protein reporter genes. Both systems outperform previously designed Nipah virus minigenomes, are easily operable, and can be implemented for antiviral compound screenings.

Microbial hydrolases are considered to be promising enzymes for pathogen control. Bacterial and viral chitinases of the glycosyl hydrolase (GH) 18 family are important biological macromolecules with antifungal and anti-insect activity. Chloroviruses, nucleocytoplasmic large DNA viruses (NCLDVs) that infect unicellular green algae have a considerable number of genes involved in carbohydrate metabolism, including the chitinase GH18 family. In this study, we investigated the abundance and diversity of chitinases in chlorovirus genomes using a combination of silico and in vitro strategies, and characterized these enzymes at a molecular and biochemical level. Different enzymatic profiles were observed in Chlorovirus subgenera revealing the different viral machinery related to host species. We performed a comprehensive biochemical characterization of three heterologous expressed GH18 domains, which revealed their endo and exochitinase activity and thermostability. Crystallographic analysis of the GH18 domain by X-ray diffraction yielded a structure at 1.0 [A] resolution, representing the highest-resolution structure reported to date for a giant viral protein and showing lower predominancy of residue coevolution compared GH18 chitinases from other organisms. Additionally, our binding site characterization predicted high conservation in betachloroviruses and gammachloroviruses, and less so in alphachloroviruses. Lastly, these enzymes did not inhibit fungal growth of medical and agricultural importance species in vitro but exhibited high inhibitory activity against different algae at nanogram/mL range. Together, our experimental and computational data show that evolutionary events may contribute to maintaining viral chitinases enzymatic activity and specificity. These findings highlight the potential of virus-derived enzymes as promising new biotechnological tools for microbial control against different algal strains.

Tonate virus (TONV) is a neglected mosquito-borne alphavirus of the Venezuelan equine encephalitis complex associated with febrile illness, encephalitis, and fetal central nervous system abnormalities. Yet, host pathways that sense TONV infection and restrict its replication remain poorly defined. Here, we investigated the interaction between TONV and the type I interferon (IFN-I) system in human cells. We show that TONV infection led to the accumulation of cytosolic double-stranded RNA and a robust IFN response. RIG-I and MDA5 depletion as well as that of MAVS and IRF3 strongly reduced TONV-induced IFN response. Disruption of RIG-I, MDA5, MAVS, or IRF3 resulted in an increase of dsRNA-positive cells and a higher viral RNA accumulation. Finally, we found that treatment with exogenous IFN-I strongly inhibits TONV replication reducing both viral RNA loads and infectious particle production. Thus, together, our results identify the RIG-I/MDA5-MAVS-IRF3 axis as a major pathway sensing TONV infection and establishing an IFN-I-dependent antiviral state, providing the first molecular characterization of TONV innate immune sensing in human cells.

We examined transmission of the human metapneumovirus (HMPV) in LLC-MK2 cell monolayers using a low multiplicity of infection (moi). In this low moi infection model HMPV transmission initially occurred by localised cell-to-cell transmission, and the virus infectivity remained largely cell associated. At the later stages of infection more widespread virus transmission occurred and was associated with the presence of cell-free virus. The appearance of the cell-free virus correlated with changes in plasma membrane integrity and increased membrane permeability in the cell monolayers. Imaging analysis of HMPV infected cells at the early stages of infection showed the presence of numerous virus filaments attached to the surface of HMPV-infected cells. At the later stages of infection both virus filaments and virus particles with a spherical morphology that was attached to the distal ends of the virus filaments was noted. A proportion of these spherical particles detached from the virus filaments and attached to adjacent non-infected cells at the later stages of infection. The activation of the JNK and MAPKp38 signalling pathways in HMPV-infected cells correlated with increased HMPV replication and appearance of the cell-free virus infectivity. In addition, after the initial phase of STAT1 activation in HMPV-infected cells, both reduced expression of the STAT1 protein and the activated STAT1 protein occurred as the infection proceeded. Collectively, these data provide evidence for a biphasic mode of HMPV transmission involving different virus particle morphologies, a localised virus transmission by virus filaments followed by widespread virus transmission involving cell-free virus particles.

A rapid expansion of influenza A virus (IAV) genome sequencing has transformed global surveillance but has also created major challenges for interpreting the biological significance of viral mutations, particularly amino acid replacements associated with host adaptation. Resources have been created to support mutation annotation and phylogenetic analysis, but there is a need for a tool that integrates experimentally derived phenotypic evidence with evolutionary context in a framework suitable for users without prior training in bioinformatics. Here, we present the Flu Mutation Explorer, an interactive web application that combines large-scale influenza phylogenies with a manually curated database of reported mammalian adaptation mutations, to enable the exploration and interpretation of IAV genetic variation. The underlying database comprises over 1.5 million publicly available IAV sequences and over 1000 mutations associated with mammalian adaptation. The Flu Mutation Explorer enables users to query protein sequences, visualise amino acid distributions across viral lineages, examine host-specific conservation patterns, and identify adaptation mutation with links to supporting literature. We include case studies which demonstrate the platforms use in assessing amino acid conservation at sites of interest and in rapidly identifying candidate mammalian adaptation mutations during the ongoing H5N1 panzootic. By integrating genomic, phylogenetic, and functional information into an intuitive interface, the Flu Mutation Explorer lowers the barriers to interpreting influenza sequences for specialists and non-specialists alike.

The pathogenic mammarenaviruses, Machupo (MACV) and Junin (JUNV), are under evolutionary pressure to leverage human transferrin receptor 1 (hTfR1) for cellular entry while evading host immune responses during zoonosis. We now structurally and functionally investigate cryptic, computationally hybridized JUNV-MACV GP1 sequence variants. We then evaluate the ability of those variants to facilitate internalization of pseudotyped virus-like particles (PVs) into human cells and their recognition by neutralizing antibodies, including plasma from JUNV convalescent patients. We further compare the cryoEM structures of hTfR1-bound MACV GP1 to those of two functional hTfR1-bound MACV-JUNV hybrid GP1 variants that enable robust PV internalization and are also recognized by cross-neutralizing antibodies. Immunization with these variants demonstrates they and other hybrid GP1 sequences can elicit cross-reactive, neutralizing antibodies, supporting a model in which sequence adaptation within GP1 balances receptor recognition with immune evasion. This may inform the rational design of broadly neutralizing GP1-targeted antiviral therapies.