Virus Research

Enterovirus D68 (EV-D68) is a non-polio picornavirus that has caused increasing rates of severe respiratory illness and acute flaccid myelitis in children worldwide this century. There are no approved vaccines or antivirals for EV-D68. Thus, we conducted a crystallographic fragment screening (CFS) and a high-throughput screening (HTS) biochemical assay against the EV-D68 RNA-dependent RNA polymerase 3D (3Dpol) to identify ligandable sites and non-nucleoside compounds that can spearhead anti-enteroviral drug discovery. The CFS, involving 650 fragments, identified 68 hit compounds (~10% hit rate) distributed across 3Dpol, including the functionally relevant sites RNA template channel, Active site, and RNA primer channel, and the previously unknown "Thumb site II" and "Index-middle finger pocket". Inhibition assays confirmed that compounds binding to each site can inhibit EV-D68 3Dpol activity. The HTS, a fluorescence-based PicoGreen biochemical assay, permitted screening 50,000 compounds of the ChemBridge Premium Library (0.77% hit rate). After a second-round dose-response screening, we identified 5-aminoindazole as a promising scaffold that inhibits EV-D68 3Dpol, including hit-to-lead compound 727590, which displayed an IC50 value of 25 M and preliminary structure-activity relationships. These hits offer amenable starting points for discovery and development of non-nucleoside inhibitors and provide opportunities for structure-based drug design against enteroviruses. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/737532v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@14a54a6org.highwire.dtl.DTLVardef@fb6621org.highwire.dtl.DTLVardef@ee2e2aorg.highwire.dtl.DTLVardef@118f91d_HPS_FORMAT_FIGEXP M_FIG Created with biorender.com and PyMOL Molecular Graphics System, version 2.5.0. Schrodinger, LLC. C_FIG

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.

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.

Chronic hepatitis B virus (HBV) infection contributes to hepatocellular carcinoma by disrupting host transcription, cell-cycle control, and apoptotic signaling. Isochlorogenic acid A (ICAA), a natural compound with antiviral and hepatoprotective properties, was previously shown to inhibit HBV replication by interfering with multiple steps of the viral life cycle. Because chronic HBV often reflects an imbalance between proliferation and cell death, we investigated how ICAA affects gene expression related to these processes in the presence or absence of HBV. We performed transcriptome analysis using RNA sequencing (RNA-seq) in HepAD38 cells (a HepG2-derived stable HBV-expressing line) and HepG2 control cells (HBV-negative) treated with ICAA or DMSO. HBV caused major differences in gene expression in HepAD38 cells compared with HBV-negative HepG2 cells. Principal component analysis showed that ICAA significantly altered HBV-dependent expression patterns, resulting in 189 differentially expressed genes (DEGs) that were regulated in opposite directions by both HBV and ICAA. Functional enrichment analysis highlighted pathways in viral carcinogenesis, apoptosis, MAPK signaling, and p53 signaling. Annexin V/propidium iodide assays showed apoptotic cells in both treated and untreated HepAD38 cultures, with only minor pattern changes. Mechanistically, in untreated HBV-positive cells caspase-9 cleavage failed to activate PARP, suggesting that induction of intrinsic apoptosis is followed by blocked execution. In contrast, ICAA inhibits caspase-9 cleavage in a dose-dependent manner, while activating PARP. Consistent with this, ICAA treatment increased apoptotic DNA fragmentation in HepAD38, reflecting the proapoptotic potential of ICAA under these conditions facilitating the elimination of HBV-positive cells by apoptosis. These findings highlight the potential therapeutic relevance of this compound in processes associated with HBV pathogenesis, together with its antiviral effect. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=185 SRC="FIGDIR/small/733975v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@38d107org.highwire.dtl.DTLVardef@235a13org.highwire.dtl.DTLVardef@ee988aorg.highwire.dtl.DTLVardef@60cb13_HPS_FORMAT_FIGEXP M_FIG C_FIG

Epstein-Barr virus (EBV) is an oncogenic virus which is responsible for various malignant as well as non-malignant diseases and leads to about 200,000 deaths each year. Despite efforts, there are no FDA-approved drugs targeting EBV. Reactivation of EBV plays a critical role in the transition from latency to lytic cycle, leading to viral replication and disease progression, and is primarily regulated by the transactivator BZLF1. In this study, we combined computational screening with experimental validation to identify repurposing drugs that inhibit EBV reactivation and replication. FDA-approved compounds predicted using in-house AI/ML-based model (Anti-EBV) and miRNA-seq and RNA-seq analyses, were selected for further evaluation. Molecular docking against BZLF1, supported by in silico alanine scanning to identify critical DNA-binding residues, led to the selection of seven candidate drugs. Among these, an antimalarial drug, dihydroartemisinin (DHA), showed the strongest inhibitory activity in vitro, with an IC99 of 1 {micro}M and an SI Index of 113.5. DHA reduced both EBV viral copy number and the expression of early and late lytic genes. Molecular docking and simulation studies demonstrated stable binding of DHA within the BZLF1 DNA-binding pocket, inhibiting the key residues involved in BZLF1 activation and DNA binding. Analysis at the gene level confirmed its inhibitory effect on EBV replication, while expression analysis at the transcriptional and protein levels, along with immunofluorescence analysis, indicated its inhibitory effect on EBV reactivation and virion assembly. These findings suggest DHA as a promising repurposing antiviral candidate targeting EBV lytic proteins and offers an effective target-based therapeutic strategy. ImportanceThis study identifies a repurposed small-molecule inhibitor of EBV reactivation and replication. Here, we proposed target-based therapy, integrating computational and experimental approaches to target the EBV lytic transactivator BZLF1. Since early lytic EBV protein BZLF1 plays a critical role in viral reactivation and replication, inhibition of its activation and DNA-binding function represents a promising therapeutic approach to prevent EBV infection. Molecular docking and simulation studies revealed stable binding of DHA within the BZLF1 DNA-binding pocket. Furthermore, in vitro analyses demonstrated significant inhibition of viral gene copy number and reduced mRNA and protein levels of key lytic proteins. Thus, this study demonstrated DHA as a safe and effective repurposed therapeutic candidate against EBV infection.

Background: RSV is a major cause of severe lung infections in young children, with over 95% of deaths occurring in poorer countries. Bangladesh has high rates of RSV illness in children but lacks genetic data from after the COVID-19 pandemic. New vaccines and antibody treatments are now available, making local genetic information essential. Objectives: We sequenced complete RSV genomes from Bangladeshi patients to study virus types, genetic changes, and protein mutations, and shared our data openly. Methods: From August 2024 to December 2025, we took 59 RSV-positive samples with high virus levels from hospital patients and sequenced their full genomes using Oxford Nanopore technology. Results: Among 11,874 patients, 1,390 (11.7%) had RSV, mostly RSV-A (94.6%). We obtained 49 good-quality full genomes from the 59 samples (83% success): 43 RSV-A (ON1 type, five sub-lineages) and 6 RSV-B (BA9 type). We found S276N in 35% of RSV-A and S389P in all RSV-B, but neither stops current antibody treatments. All RSV-A viruses gained a new sugar attachment site on their F protein, and most RSV-B viruses gained one too. We uploaded all 49 genomes to GISAID for public use. Conclusion: This work shows we can do full RSV genome sequencing in Bangladesh. The viruses here still match the targets of new vaccines and antibodies, which is reassuring. Our findings provide a foundation for planning RSV prevention in Bangladesh and South Asia.

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.

Zika virus (ZIKV) is an arthropod-borne flavivirus of international public health impact. ZIKV has a positive-sense, single-stranded RNA genome and remodels intracellular membranes to form replication complexes (RCs). The objective of this study was to isolate and characterize the RCs from ZIKV-infected cells and to identify host-cell components recruited to participate in viral replication. Here, we isolated the RCs from ZIKV-infected Vero cells by detergent treatment and flotation centrifugation. Fractional flotation analysis demonstrated that ZIKV proteins NS2B, NS3, and NS5, and ZIKV RNA were present in the detergent-resistant membranous fraction. In contrast, the ER-resident protein calnexin and a mitochondrial protein were present in the detergent-soluble fractions. The isolated RCs were functional for ZIKV RNA synthesis, as shown by quantitative PCR. To determine the components of the RCs, we conducted mass spectrometry analysis and identified numerous cellular proteins. Among them is the replication factor C subunit 2 (RFC2), an accessory protein of DNA polymerase. RFC2 is involved in ATP binding and hydrolysis and may promote cell survival. ZIKV infection increased the RFC2 protein level and induced its relocation to the cytoplasm. RNAi-mediated silencing of RFC2 reduced ZIKV replication. Together, our results provide insights into ZIKV replication and virus-cell interaction.

Background and AimsHepatitis E virus (HEV) infections are a growing threat to global public health. To obtain an in-depth understanding of HEV infections in untreated and ribavirin-treated rats, we characterized the early HEV viral kinetics using rat HEV (rHEV) as a surrogate model and using two routes of virus inoculation: intravenous (I.V.) or oral infection. Approach and ResultsWe frequently collected feces, serum, and tissue samples up to 60 days after infection in both infection models to characterize the rHEV viral kinetics. A ~2-week delay in quantifiable RNA levels in feces was observed in the oral versus the I.V. infection model. Early rHEV viral kinetics in feces were found to be multiphasic and showed good concordance with those in the various tissue compartments studied. Comparison of the viral kinetics in these samples also revealed that the liver may serve as the initial site of rHEV replication, followed by replication in the intestine and spleen. While a dosage of 60 mg/kg/day ribavirin was found optimal to maintain rHEV RNA levels at (nearly) undetectable in feces, levels were detectable in the liver and increased both in feces and liver after treatment discontinuation. ConclusionsWe found that the two rHEV infection models share similar multiphasic viral kinetics with the liver as the main site of viral replication. Additionally, the rHEV RNA load in feces could be used as a reliable proxy for that in the liver, spleen, and intestine. We also show that ribavirin at 60 mg/kg/day was partially effective in preventing viral rebound. These findings may aid in exploring the correlation between the infection phases and antiviral efficacy, ultimately guiding therapeutic decisions.

Herpes simplex virus type 1 (HSV-1) is a common human pathogen that undergoes lytic replication in epithelial and other permissive cell types and can establish latency in peripheral neurons. ICP22 is a multifunctional HSV-1 immediate-early protein that localizes to the nucleus of infected cells; however, its interactions with host cellular factors remain incompletely understood. Here, ICP22 was demonstrated to interact with the human antisense function 1 protein (ASF1), including both ASF1a and ASF1b, in transfected cells and HSV-1-infected cells, respectively. ICP22 also colocalized with ASF1 in the nucleus. ICP22 amino acids 213 to 340 are important for the interaction of ICP22 with ASF1, whereas amino acids 37 to 153 of ASF1a and ASF1b are critical for their interactions with ICP22. Furthermore, ICP22 expression was associated with reduced ASF1-H3.1 co-immunoprecipitation under the tested conditions. ASF1 knockdown also reduced HSV-1-BAC-Luc luciferase output, indicating that ASF1 contributes to efficient infection-associated reporter activity in this study. Collectively, these results indicate that the interaction of HSV-1 ICP22 with ASF1 might help regulate the transcription of viral or cellular genes during HSV-1 infection. Keywords: HSV-1, ICP22, ASF1, histone H3.

Lumpy skin disease (LSD) is an emerging cattle disease caused by lumpy skin disease virus (LSDV), with major impacts on the industry, being classified as a Category A disease. Although it was historically confined to Africa, LSD has expanded into the Middle East, Asia and Europe. Here, we report two LSDV genomes from the first outbreak detected in Catalonia, Spain, in October 2025. The genomes were assembled from high-throughput sequencing data generated from two homogenized skin nodules. Comparative phylogenetic analyses were performed using all available complete LSDV genomes and rpo30 gene sequences. These analyses placed the LSDV isolates detected in Catalonia within clade 1.2, closely related to the isolates recently reported in Sardinia, Italy. Our findings also support a connection between recent south-western Europe and central African strains, possibly through northern Africa, and highlight the need for more complete genomes to clarify the origin and connections among recent LSDV outbreaks.

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.

Tomato brown rugose fruit virus (Tobamovirus fructirugosum) is an emerging virus that affects tomatoes, capsicum, and chili. Since its first detection in Jordan in 2015, the virus was reported in more than 40 countries across all the continents. In Morocco, the virus was reported for the first time in October 2021. However, its genetic diversity remains unexplored. In this work, we used a collection of tomato fruits from local markets to investigate the variability of the virus in the country. We explored the different pressures acting on the N-terminus of the RNA-dependent RNA polymerase, the movement protein, and the coat protein genes. Then, we used haplotype network analyses to reveal the population structure within the Moroccan isolates and studied their relationships with the ones from the world. We found that genetic diversity is low, which is consistent with the global situation. No signatures of diversifying selection were detected across the analyzed genes. However, the virus sequences from Morocco showed a clear geographic structure, suggesting that geographic factors probably combined with agricultural practices may contribute to shaping the population structure of ToBRFV in Morocco.

Type I Interferons (IFN-I) are cytokines with pleiotropic activities involved in antiviral and antitumor immune responses. They can reduce oncolytic virus replication in tumor cells by inducing expression of interferon stimulated genes (ISG) with antiviral functions. To specifically neutralize the IFN-/-{beta} receptor (IFNAR) on specific cell types, we created novel IFNAR1-targeted antagonists constituted of a high-affinity nanobody targeting a specific cell surface marker conjugated to a low-affinity blocking nanobody targeting IFNAR1. We first show in vitro and in vivo that such an antagonist targeting the mouse CD20 molecule (mCD20) inhibits IFNAR signaling only in B cells among splenocytes. We then showed in vitro that a human CD20 (hCD20)-targeted antagonist blocks IFNAR signaling and induces vesicular stomatitis virus (VSV) oncolytic activity against IFN-11-treated AK7 mesothelioma or B16 melanoma cells only if these cells express hCD20. In vivo, we show that the antagonist binds to hCD20 and enhances VSV replication by inhibiting ISG expression specifically in hCD20+ AK7 mesothelioma tumors. Altogether our results demonstrate the efficient and cell-type specific inhibition of IFNAR signaling through the use of these novel IFNAR1 antagonists, both in vitro and in vivo. These antagonists could have many therapeutic applications given the importance of IFN-I in numerous diseases. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/729496v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@4e76b6org.highwire.dtl.DTLVardef@153c39borg.highwire.dtl.DTLVardef@4f0948org.highwire.dtl.DTLVardef@ea8d85_HPS_FORMAT_FIGEXP M_FIG C_FIG eTOC synopsisIn this study, we created cell-specific IFNAR antagonists that allow to inhibit selectively IFNAR signaling in particular types of cell. We show that this antagonist can be used to target IFNAR at the surface of tumor cells that lead to the inhibition of IFNAR signaling and ISG expression in these cells rendering them more permissive to VSV replication. Beside antitumor virotherapy, these novel antagonist could be useful to study role of IFN-I in normal or pathological context.

We recently reported the identification of endogenous viral elements (EVEs) originating from the Caulimoviridae family within the alfalfa (Medicago sativa L.) genome. Our subsequent identification of ubiquitous rhabdoviral elements in infected and healthy alfalfa tissues by high throughput sequencing prompted us to suggest that the alfalfa genome might be populated with integrated rhabdoviruses as well. Bioinformatics analysis using 26 publicly available alfalfa genomes proved the suggestion accurate. We found multiple non-retroviral segments of the Rhabdoviridae family belonging to the genera Betanucleorhabdovirus and Betacytorhabdovirus that appeared to be stable constituents of the host genome. In that capacity they could potentially acquire functional roles in alfalfas development and response to environmental stresses. We believe this study reveals the first documented case of rhabdoviruses integrated into the alfalfa genome.

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.

Alongshan virus (ALSV) is an emerging tick-borne segmented RNA virus belonging to the Jingmenvirus group and has been reported in humans, ticks, and vertebrates across Asia and Europe. Despite its potential public health relevance, its distribution and genetic diversity remain poorly characterized in several European regions where tick-borne pathogens are endemic. In this study, we developed a specific TaqMan-based real-time RT-PCR assay targeting a conserved region of ALSV segment 2 and used it to investigate the presence of ALSV RNA in Ixodes ricinus ticks collected in northeastern Italy. The assay showed high linearity over a broad dynamic range and no cross-reactivity with related flaviviruses. A total of 212 archival tick samples collected between March 2021 and November 2022 were screened, and 28 samples (13.2%) tested positive for ALSV RNA. Positive ticks were detected in the provinces of Belluno and Vicenza and included individual adult males and nymph pools. A subset of positive samples was further characterized by nested PCR and Sanger sequencing of all four genomic segments. Phylogenetic analyses showed that Italian ALSV sequences clustered within the broader European ALSV diversity and were closely related to strains from Central and Northern Europe, without forming a distinct country-specific lineage. Sequence comparisons suggested purifying selection and revealed differences in predicted structural proteins between European and Chinese strains. These findings provide the first molecular evidence of ALSV circulation in Italy and support further studies to clarify its epidemiology, host range, genetic diversity, and potential clinical relevance. IMPORTANCEAlongshan virus (ALSV) is an emerging tick-borne virus identified in febrile patients in China and subsequently detected in ticks in Russian Federation and several European countries. Although severe disease has not yet been reported in humans, surveillance and elucidation of the virus distribution are essential to assess its pathogenicity and potential public health impact. We developed a specific real-time RT-PCR protocol and detected ALSV in Ixodes ricinus ticks collected in northeastern Italy. Sequence analyses suggested multiple introductions and revealed differences in structural proteins between European and Chinese strains, suggesting potential adaptation and differences in pathogenicity. Since the clinical signs of ALSV infection in humans may overlap with those of tick-borne encephalitis (TBE), differential diagnostic procedures should be developed to improve patient management, particularly in TBE-endemic regions such as northeastern of Italy.

In 2020, highly pathogenic avian influenza (HPAI) isolates from clade 2.3.4.4b emerged in Europe and spread globally, including in bovine hosts in the USA. Viruses from this clade cause minimal disease in dairy cattle, characterized by decreased milk production but low mortality rates. Infections have also occurred in feline hosts. In contrast to cows, infection of cats (and closely related species, including skunks and foxes) can result in severe neurological signs and mortality. Documented feline H5N1 infections from clade 2.3.4.4.b have a mortality rate of approximately 80% following rapid onset of clinical signs. No antiviral compounds have been tested in an experimental feline model; however, anecdotal clinical evidence suggests early treatment with oseltamivir may improve outcomes in felines with HPAI. Here, we show the in vitro efficacy of several influenza inhibitors in feline glial astrocyte (PG-4) and kidney (CRFK) cell culture models using the clade 2.3.4.4.b virus Tx2/24 (H5N1). The neuraminidase inhibitor oseltamivir carboxylate did not effectively inhibit viral replication in either cell line. The cap-dependent endonuclease inhibitor baloxavir exhibited the strongest inhibition of this virus, with EC50 values of 30 nM in PG-4 and 1 M in CRFK cells. Amantadine and rimantadine, M2 ion channel inhibitors, were unable to completely inhibit viral replication in either cell line at any concentration utilized. The broad-spectrum nucleoside analog GS-441524 demonstrated little to no inhibition of viral replication in either cell line. Additionally, the mutagenic NHC analogs EIDD-1931 and EIDD-2801 successfully inhibited viral replication at the maximum tested concentration of 100 M but exhibited significant cytotoxicity. Our findings suggest that baloxavir should be considered by veterinary clinicians as the first-line drug of choice when presented with felines or other species infected with HPAI.

Today, the deformed wing virus (DWV) can be considered as one of the major causes of global elevated western honey bee colony losses (Apis mellifera). Virus transmission may occur horizontally between individuals of the same generation, but also vertically from parents to offspring. The recently defined heritable suppressed in ovo virus infection (SOV) trait describes the absence of viruses in pooled drone eggs of a queen, associated with significant lower DWV prevalence and viral loads in the subsequent developmental offspring stages. By definition, the trait reflects the absence of vertical virus transmission from SOV-positive (SOV+) queens themselves to their offspring. However, the genetic basis influencing this heritable virus resilience has not been identified yet. In this study, we aimed to identify SOV-associated genetic marker(s) or loci in the honey bee genome through genome-wide variant comparison of 44 DWV-positive and 44 DWV-negative drone pupae descendent from an artificially created hybrid SOV+/SOV- colony. After whole genome sequencing (WGS), variant calling, and genotype-phenotype association analysis by means of single marker tests and elastic net regression, one variant in a locus of 241.246 bp on chromosome 7 that contained 17 other highly SOV-associated variants classified 68,2% of the drone phenotypes correctly. These results may support the potential application of marker-assisted selection (MAS) strategies targeting reduced vertical virus transmission in honey bees.

Influenza A virus (IAV) causes significant morbidity and mortality worldwide. Understanding how viral RNAs may regulate host genes through microRNA-like mechanisms can clarify pathogenesis and reveal therapeutic targets. In this study, we screened all eight IAV H3N2 RNA segments (PB2, PB1, PA, HA, NP, NA, M, and NS) using an ab initio computational pipeline; five segments (PB2, PB1, PA, HA, and M) met the VMir scoring threshold for further analysis, while NP, NA, and NS were excluded due to low pre-miRNA scores. Mature miRNAs were identified using MatureBayes, and target genes in the human genome were predicted with the miRDB server. From these targets, we selected two genes per qualifying segment (10 genes total) based on their functional relevance to influenza infection and supporting literature; all selected genes are unique to their respective segment. We identified 10 segment-specific target genes (IFNL1, DDX60, SAMHD1, MAVS, IRF4, BIRC2, AGO1, MAP3K1, NOD1, and TNFAIP1) and one common target across all five analyzed segments (CADM2). Gene Ontology and pathway analyses showed enrichment in interferon signaling, RIG-I-like receptor pathways, antiviral restriction, RNA interference, and inflammatory responses. Literature supports roles for these genes in pulmonary and antiviral innate immunity. Our findings provide a basis for experimental validation and may help the research community better understand influenza virus pathogenesis and identify novel therapeutic candidates. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/725090v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@2b14adorg.highwire.dtl.DTLVardef@5a9b2eorg.highwire.dtl.DTLVardef@81ffc1org.highwire.dtl.DTLVardef@be119b_HPS_FORMAT_FIGEXP M_FIG C_FIG