Frontiers in Virology

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.

We previously generated a mouse monoclonal antibody, N179, against the SARS-CoV-2 nucleocapsid (N) protein and developed a colloidal gold-based immunochromatographic test strip. This assay achieved a detection limit of 2 ng/mL and displayed 98% concordance with RT-qPCR results. However, the precise epitope recognized by mAb N179 had not been defined. Using a panel of GST-fused N protein truncation fragments, we mapped the linear B-cell epitope recognized by mAb N179 to the flexible C-terminal tail of the N protein by Western blotting and ELISA. The minimal binding motif required for mAb N179 recognition was identified as 390QTVTLL395. Multiple sequence alignment of 11 representative SARS-CoV-2 lineages, including Alpha, Beta, Gamma, Delta, and Omicron subvariants BA.1, BA.2, and BA.3.2, revealed that this epitope was completely conserved across all variants analyzed. Stringent local pairwise alignment analysis using EMBOSS WATER further showed that the 390QTVTLL395 motif achieved a perfect 6/6 match exclusively in SARS-CoV-2; no identical sequence was detected in the seven common human coronaviruses, four influenza viruses, or five bat coronaviruses examined. Structural prediction analyses indicated that this region is surface-exposed and possesses a strong linear B-cell epitope propensity. Together, these findings identify 390QTVTLL395 as a specific molecular signature of SARS-CoV-2 among the viruses analyzed. Our results provide an epitope-level explanation for the sustained diagnostic reliability of the mAb N179-based assay against emerging variants, clarify the molecular basis for its lack of cross-reactivity, and may inform the rational design of SARS-CoV-2 diagnostics targeting conserved, mutation-resistant epitopes. ImportanceWe identified the exact nucleocapsid protein epitope recognized by monoclonal antibody N179, a diagnostic antibody used in a colloidal gold rapid assay. The identified 390QTVTLL395 motif at residues 390 to 395 was conserved among the SARS-CoV-2 variants analyzed and was not present as an identical continuous sequence in the related respiratory viruses examined. This work supports precise epitope mapping as a useful strategy for evaluating and revalidating diagnostic antibodies as respiratory viruses evolve.

MotivationInfluenza is an infectious disease associated with excess human deaths. For influenza A virus (IAV), the surface antigens hemagglutinin (HA) and neuraminidase (NA) define the specific subtype. The high mutation rate of IAV can make clinical testing and assigning subtype after sequencing challenging. Accurate subtype classification is important for tracking circulating IAV strains that may impact human health. ResultsWe analyzed a large IAV protein sequence dataset with known HA and NA subtypes. Using logistic regression and random forest approaches, we identified a small set of subtype-associated amino acids, which were then used to develop a subtype characterization method with near-optimal accuracy. Further analysis indicated that signal sequence peptide variation and indels in HA and NA explained the unique combination of subtype-associated amino acids.

Enveloped plant viruses are rare, and accumulating evidence suggests deep evolutionary ties to arthropod hosts. Brevipalpus-transmitted viruses (BTVs), which include cileviruses, higreviruses, and dichorhaviruses, are transmitted exclusively by Brevipalpus mites, causing localized infections in plants. Some cileviruses and dichorhaviruses can also replicate within vector cells. Despite their enveloped virions, the involvement of virion structural proteins mediating the interaction with the vector remains unresolved. Here, we investigated the putative glycoproteins P61 of citrus leprosis virus C (CiLV-C; Cilevirus, Kitaviridae) and G of clerodendrum chlorotic spot virus (ClCSV; Dichorhavirus, Rhabdoviridae) to define their interaction networks in the Brevipalpus yothersi vector. Using membrane-based yeast two-hybrid screening with a B. yothersi cDNA library, we identified 73 interactors for P61 and 162 for G, including a shared core set enriched for membrane-associated proteins involved in intracellular trafficking, ER-Golgi dynamics, protein synthesis and folding, and signal transduction. Numerous hypothetical membrane proteins emerged as strong candidates for viral receptors or co-receptors. Three proteins (ARF1, SERP2, and a hypothetical transmembrane protein) were validated by BiFC and Co-IP in Spodoptera frugiperda (Sf9) cells, confirming interactions with both viral proteins in an arthropod-like environment. Together, this work provides the first comprehensive interactome of BTV glyco-like proteins with those of its natural vector and reveals partially convergent host-interaction strategies between phylogenetically distinct viruses, establishing a mechanistic basis for dissecting BTV transmission. IMPORTANCEBTVs damage economically important crops worldwide, and citrus leprosis (CL) is a significant disease in the Brazilian citrus belt. Brazil is responsible for over 75% of global sweet orange juice production and widely cited estimates indicate that CL causes >US$69 million in annual yield losses and acaricide-based control of Brevipalpus yothersi, the vector of CiLV-C and several other BTVs. Despite this impact, the molecular basis of BTV acquisition, persistence, and transmission by Brevipalpus spp. remains largely unexplored. Here, we provide the first systematic map of mite proteins interacting with glycoproteins from two BTVs belonging to distinct viral families. These interactomes reveal key components of the cellular machinery targeted by cileviruses and dichorhaviruses in their arthropod vector, offering mechanistic insight into how these viruses establish and maintain infections in mites. This work establishes a foundational framework for future studies aimed at disrupting BTV transmission and developing sustainable control strategies.

Geminiviruses are plant DNA viruses that replicate in the nucleus of the host cell and rely on the host nucleocytoplasmic transport machinery to complete their infection cycle. While various geminiviral proteins have been reported to interact with plant transport factors, the contribution of nuclear pore complex components to geminivirus infection remains largely unexplored. Here, we identify nucleoporin 50a (NUP50a) as a previously unreported host factor that contributes to bhendi yellow vein mosaic virus (BYVMV) infection. Affinity purification coupled with mass spectrometry isolated NUP50a as a potential interactor of the BYVMV pathogenicity determinant C4, which was further validated by pull-down and co-immunoprecipitation assays. Yeast two-hybrid assays, bimolecular fluorescence complementation, and colocalization analysis demonstrated that BYVMV C4 directly associates with NUP50a predominantly in the nucleus. Virus-induced gene silencing of NbNUP50a significantly delayed symptom development and reduced viral DNA accumulation, suggesting that NUP50a is required for efficient BYVMV infection. Silencing NbNUP50a did not influence the subcellular localization of BYVMV C4, indicating that the role of NUP50a extends beyond determining C4 steady-state localization. Notably, NUP50a was found to associate with C4 proteins from three additional geminiviruses, supporting the possibility that targeting NUP50a represents a characteristic strategy among geminiviruses. Together, our findings provide evidence of a nuclear pore complex member involved in geminivirus pathogenesis. These results establish a framework for further study of the potential transport-dependent and/or transport-independent functions of NUP50a during viral infection.

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.

Advances in air-based surveillance of pathogen genetic material are hindered by reliance on centralized, time-consuming molecular techniques. Point-of-care (POC) diagnostic platforms, like the Cepheid(R) GeneXpert(R), offer rapid, simplified testing in clinical settings but have not been evaluated for use with air samples. Here, we paired the ThermoFisherTM AerosolSenseTM air sampler with the Xpert(R) Xpress SARS-CoV-2/Flu/RSV Plus test to evaluate near-real-time air surveillance. To assess analytical sensitivity, we spiked collection substrates with inactivated viruses and performed overnight sampling using the air sampler. As few as 10 copies of influenza A/B (IAV/IBV) and RSV applied to the substrate were detectable by GeneXpert, while SARS-CoV-2 required at least 100 copies for detection. Longitudinal air surveillance was conducted across congregate settings in Columbia, Missouri, and Madison, Wisconsin, in 2024-2025, collecting 281 air samples. SARS-CoV-2 was detected most frequently, followed by IAV. To assess concordance, 191 samples with paired GeneXpert and RT-qPCR results were analyzed across multiple Ct value cutoffs. Agreement between GeneXpert and RT-qPCR for SARS-CoV-2 was fair to moderate (K = 0.306-0.443). Optimal GeneXpert Ct cutoffs for the best balance between sensitivity and specificity, determined using analyses such as Youden's index, were site-specific: 45 for Wisconsin (67% sensitivity, 83% specificity) and 41 for Missouri (76% sensitivity, 62% specificity), reflecting differences in laboratory protocols. For IAV, agreement was moderate (K = 0.56) with GeneXpert Ct cutoff of 40, achieving 85% sensitivity and 81% specificity. Further studies across diverse settings and viral targets are needed to establish GeneXpert's role in routine air surveillance.

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.

Recombination is a key driver of RNA virus evolution, yet its extent and evolutionary implications in dengue virus (DENV) remain incompletely understood. We conducted a comprehensive, genome-wide recombination screen across 6,905 complete DENV genomes representing all four serotypes, 82 countries, and eight decades of sampling (1944-2023) retrieved from the Bacterial and Viral Bioinformatics Resource Center. Using seven complementary recombination detection methods implemented in RDP5, we identified 66 recombination events across 53 unique recombinant sequences, of which 29 are newly described. Events included intra-genotypic (n = 18), inter-genotypic (n = 32), and inter-serotypic (n = 16) exchanges spanning 14 genotypes and four continents, with no meaningful serotype-level enrichment (Cramers V = 0.054). Recombination was concentrated in non-structural genes, most frequently NS3 (19 events), NS5 (17), and NS2 (12), while the capsid gene contained no recombination events, consistent with strong functional constraint. Single-nucleotide polymorphism analyses confirmed low divergence between recombinants and their inferred parents in both recombinant and non-recombinant regions. Phylogenomic analysis of 6,642 sequences revealed that recombinants cluster significantly closer to their major parents (p = 8.9 x 10-6) and that their removal does not significantly alter tree topology (p = 0.898), suggesting that the short length of recombinant regions limits phylogenetic conflict. We also introduce RECOSIM, an unsupervised machine-learning tool for recombination detection that achieved higher precision than RDP5 on both simulated (93.4% vs. 80.0%) and empirical (98.1% vs. 39.3%) datasets. Collectively, these results establish recombination as a widespread, pan-serotypic phenomenon in DENV with implications for genomic surveillance, vaccine evaluation, and evolutionary inference.

Post-transcriptional chemical modifications of RNA, collectively termed the epitranscriptome, have emerged as critical regulatory layers governing viral replication, pathogenicity, and host-virus interactions. Despite the rapid accumulation of experimental data on viral RNA modifications, no dedicated, freely accessible resource existed for systematically cataloguing these sites across diverse viral species. Here we present ViralEpiBase, a manually curated database of epitranscriptomic modification sites identified in viral RNA genomes and virus-encoded transcripts at single-nucleotide resolution. ViralEpiBase currently integrates seven chemically distinct RNA modification types: N6-methyladenosine (m6A), N1-methyladenosine (m1A), pseudouridine ({Psi}), 5-methylcytosine (m5C), 2'-O-methylation (2'OMe), inosine and N4-acetylcytidine (ac4C); across 12 viral species encompassing both DNA and RNA viruses of clinical and biological significance. Each entry is linked to its primary literature source or deposited dataset and is retrievable by modification type, genomic coordinates, or viral taxonomy. The database is freely accessible through an intuitive web interface and is updated continuously as new experimental evidence becomes available. ViralEpiBase thus provides the first unified platform dedicated exclusively to viral epitranscriptomics and is designed to facilitate mechanistic investigation of RNA modification functions in viral biology.

Free chlorine reacts with viral proteins, but the protein structural determinants of viral resistance to chlorine treatment remain poorly understood. Here, we curated a dataset of 498 icosahedral virus structures, including intact virions and virus-like particles (VLPs), from the Protein Data Bank. Surprisingly, only 6.6% of these structures are associated with published viral chlorine inactivation rate constants (kobs). In these matched cases representing 12 virus families, total and maximum solvent accessible surface areas (SASA) of methionine residues within viral attachment and entry proteins correlated significantly with median kobs (Pearsons r = 0.83 and 0.45, respectively; p < 0.05), suggesting a critical role of methionine exposure in viral resistance phenotypes. Across the full curated dataset, fuzzy c-means clustering upon total and maximum SASA profiles of chlorine-reactive residues demonstrates that the common surrogate panel (MS2, PhiX174, Phi6, PRD1, and PR772) fails to represent the SASA diversity of human viruses. Instead, VLPs and novel phages may serve as better surrogates for chlorine treatment due to SASA profile similarities to human viruses. Our findings highlight that residue SASA features provide a quantitative baseline for screening viral resistance to chlorine and offer a data-driven strategy to select structurally representative virus surrogates for future disinfection studies. SYNOPSISSolvent accessibility of chlorine-reactive residues correlates strongly with viral chlorine resistance, providing a robust quantitative metric to compare chlorine resistance phenotypes across viral capsids. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/732355v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@dd6bd1org.highwire.dtl.DTLVardef@d17c53org.highwire.dtl.DTLVardef@1394528org.highwire.dtl.DTLVardef@eb6378_HPS_FORMAT_FIGEXP M_FIG C_FIG

RationaleRecent viral metagenomic studies have identified a plethora of enzyme-encoding genes in Phycodnaviridae viruses that are not strictly required for viral replication. These enzymes hold an unexpected metabolic potential during the infection process with their specific green algae host. As neither their role in the infection process nor the subcellular localization of these proteins has been experimentally characterized, comparative sequences, structural and biochemical in silico analyses can help generate functional and localization hypotheses. MethodsIn a recent viral metagenomic dataset, we identified a collection of viral homologs involved in bilin biosynthesis: heme oxygenase (vHMOX1) and Phycocyanobilin:Ferredoxin oxidoreductase (vPcyA). Viral and algal homologues were compared through sequence analyses and AlphaFold3 structural predictions. Predicted biochemical properties were analyzed for their compatibility with subcellular compartments. Active site architecture and putative substrate binding were compared between viral and algal proteins using AlphaFold3 and experimentally resolved structures. ResultsViral HMOX1 and PcyA sequences are truncated compared to algal homologs, lacking the N-terminal extension associated with chloroplast targeting. However biochemical properties, including isoelectric point and surface charge distribution, are compatible with localization in chloroplast stroma. Structural comparisons reveal modifications in the viral HMOX1 active site, including partial substrate reorientation and substitutions of key residues, consistent with modified heme-binding properties. In contrast, vPcyA models show no significant differences to their algal counterparts. ConclusionsActive site remodeling in vHMOX1 protein models suggests that these viral homologues may have evolved distinct heme-binding properties. Unlike vPcyA, vHMOX1 homologs appear to have diverged more substantially from their algal counterparts, potentially reflecting functional specialization in the viral infection context. One sentence summary of key findingsOur bioinformatic analyses expand the repertoire of auxiliary metabolic genes in Phycodnaviridae by identifying a conserved heme degradation pathway, non-canonical vHMOX1/PcyA targeting and structural rearrangements surrounding the catalytic sites of viral HMOX1.

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.

Classical swine fever (CSF) is a highly contagious disease caused by Classical swine fever virus (CSFV), posing a serious threat to the global swine industry. This study aimed to investigate the effect of CSFV on differential genes of histone lactylation at the H3K18 site in the PI3K-AKT signaling pathway. The site with the most significant change in histone lactylation antibody level was screened by Western blot. Omics analysis was performed using CUT&Tag technology to identify differential genes in the PI3K-AKT pathway between the CSFV-infected group and the mock group, followed by validation using RT-qPCR. Functional analysis of significantly differential proteins was conducted, and the protein expression level of THBS4 was detected by Western blot. The results showed that after CSFV infection of 3D4/21 cells, the H3K18la site exhibited the most significant difference in antibody level. A total of 8,859 differential genes at the H3K18la site were identified by CUT&Tag analysis, including 6,349 up-regulated genes and 2,510 down-regulated genes. Further focusing on the PI3K-AKT signaling pathway, 10 differential genes were identified, comprising 6 up-regulated genes and 4 down-regulated genes. Compared with the control group, the mRNA expression levels of CD19, LAMA1, PDGFRA, BDNF, ANGPT4, and THBS4 were up-regulated in the CSFV-infected group, while FOXO3 and NRTN were down-regulated. Western blot results showed that the protein expression level of THBS4 increased after CSFV infection. These findings lay an important foundation for understanding the molecular mechanisms regulating viral replication and immune evasion, and have significant scientific implications and potential application value.

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.

Hospital-based wastewater surveillance may complement community and clinical surveillance data in important ways, and may be useful in jurisdictions without community-based wastewater surveillance. From May 2024-April 2026, we analyzed weekly samples (n=190) from three hospitals in New York City using digital PCR to evaluate the sensitivity, specificity, and positive predictive value (PPV) of wastewater viral detection against facility SARS-CoV-2 and influenza A/B inpatient caseloads. Sensitivity was 38-42% for SARS-CoV-2 and 36-49% for influenza A/B, while specificity exceeded 72% for all pathogens. During respiratory seasons, sensitivity reached 81% for SARS-CoV-2 and 81% for influenza A; both had 100% sensitivity during peak case weeks. Notably, off-peak influenza detections occurred in hospital wastewater at all three hospitals in summer 2024 without corresponding hospital case detection, suggesting the presence of undiagnosed cases. These findings underscore the potential utility of hospital-based wastewater monitoring for tracking respiratory virus activity.

Positive-sense RNA viruses extensively exploit host membrane trafficking pathways to establish intracellular replication organelles required for efficient viral replication. However, the contribution of endosomal trafficking proteins to infection by Drosophila C virus (DCV), a natural dicistrovirus of Drosophila melanogaster, remains poorly understood. Here, we investigated the role of Myopic (Mop), a conserved ESCRT-associated endosomal trafficking protein, during DCV infection. Knockdown of mop in cultured S2 cells significantly increased DCV RNA and viral protein accumulation without affecting viral binding or entry, indicating that Mop restricts DCV replication at a post-entry stage. In addition, depletion of mop altered the intracellular distribution of viral proteins, suggesting that Mop may influence the organization of DCV-associated intracellular membrane compartments. Unexpectedly, fat body-specific knockdown of mop in adult flies produced the opposite phenotype, resulting in reduced viral accumulation and prolonged survival following systemic DCV infection. This antiviral phenotype was not associated with enhanced activation of the STING or JAK-STAT pathways. Instead, transcriptomic and proteomic analyses revealed extensive remodeling of gene and protein expression, including altered abundance of proteins associated with autophagy and RNA interference. Together, our findings identify Mop as a previously unrecognized regulator of DCV infection and reveal distinct cell-autonomous and systemic functions of this conserved trafficking protein. These results highlight the importance of endosomal trafficking in antiviral defense and demonstrate that host membrane trafficking factors can exert fundamentally different effects on virus infection at the cellular and organismal levels.

Antibodies exhibit species-specific sequence and structural features that influence their antigen-recognition properties. Although several studies have investigated porcine antibodies, their repertoire and structural characteristics remain less well characterized than those of several other mammalian species. In this study, we analyzed public porcine heavy-chain repertoire sequencing data together with available antibody structural data to identify characteristic features of porcine antibodies. We found several residues enriched in porcine antibody framework regions, particularly at the base of heavy-chain complementarity-determining region 3 (CDR-H3). In particular, Arg101 and Glu123 were closely positioned in available structures and may influence CDR-H3 conformation at its base, whereas Pro120 may help constrain local backbone conformation. We also observed non-canonical cysteine usage in both framework region 1 and CDR-H3, which may contribute to structural diversity in the porcine repertoire. Finally, we evaluated the humanization potential of a porcine antibody using a human antibody language model and found that human-likeness increased after model-guided substitutions, although the resulting sequences did not exceed the T20 score threshold. Overall, these results indicate that porcine antibodies possess distinct sequence and structural features that may influence CDR-H3 properties and should be considered in future antibody analysis and engineering.

H5N1 clade 2.3.4.4b avian influenza A viruses pose a significant threat to wild animal populations, domesticated animals, and potentially, the human population. For H5N1s to infect and transmit among mammalian species, mutations for improved utilization of mammalian receptors and enhanced replication at the lower temperatures of the upper respiratory tract need to be acquired. A human H1N1pdm09-like virus was compared to H5N1 genotypes B3.13 and D1.1 for replication at 33{o}C, 37{o}C, and 39{o}C - temperatures consistent with the upper and lower respiratory tract in humans, and dairy cow udder tissue. All H5N1 viruses had increased plaque sizes on MDCK cells at 37{o}C and 39{o}C compared to H1N1pdm09. In primary, differentiated human nasal and bronchial epithelial cultures, all H5N1 viruses show restricted infectious virus production compared to H1N1 at 33{o}C. While H5N1 D1.1 also showed restricted replication at 37{o}C and 39{o}C, the H5N1 B3.13 replicated to nearly equivalent titers as H1N1pdm09. All H5N1 viruses demonstrated similar cell tropism in cells from the upper and lower respiratory tract, infecting more ciliated than non-ciliated cells relative to H1N1pdm09. H1N1, H5N1 B3.13 D1.1 infection induced similar innate immune factors, with nasal epithelial cells producing higher levels compared to bronchial epithelial cells. These data suggest that genotype B3.13 and D1.1 H5N1 viruses show different temperature dependent replication patterns compared to H1N1pdm09.

High Plains wheat mosaic virus (HPWMoV) is a wheat and maize-infecting virus of phytosanitary concern due to its potential for seed transmission. Recent phytosanitary restrictions have required sweet corn seed lots to test negative for HPWMoV prior to import into certain countries. To inform the design of more sensitive and broad-spectrum diagnostic primers for seed health testing and phytosanitary certification, we performed deep sequencing of HPWMoV-positive tissue collected from fields in two major sweet corn seed production regions in the Pacific Northwest, the Columbia Basin and Treasure Valley. Virus-like particle enrichment prior to Illumina sequencing facilitated near complete genome coverage (>95%) for the 21 HPWMoV isolates sequenced. De novo assembly of the eight viral genome segments revealed high levels of diversity for each segment, with at least two variants identified for each RNA and three variants for RNA3, RNA6, and RNA8. Within each sample, only one variant per RNA segment was usually present, with the notable exception of RNA3, sorting each isolate into what we designated type A and type B isolates. All but one previously sequenced HPWMoV isolate can be sorted into these two types. Two samples contained at least two variants for every RNA, totaling 17 genome segments, potentially representing a co-infection of type A and type B isolates. Despite this variability, we successfully designed two primer and probe sets for reverse transcription-quantitative polymerase chain reactions (RT-qPCR) that detected all 20 isolates tested in a duplex diagnostic assay, making the assay suitable for seed health testing for HPWMoV.