Getah virus (GETV) is a mosquito-borne alphavirus (family Togaviridae) that poses a significant threat to the swine industry by causing reproductive disorders in sows and neurological symptoms in piglets. In this study, a GETV strain, designated HN-2025, was isolated from a commercial pig farm in Hunan Province, China, during a clinical outbreak. The isolate induced typical cytopathic effects (CPE) in BHK-21 cells, including cell rounding, shrinkage, and detachment. The complete genome of HN-2025 was determined to be 11,689 nucleotides (nt) in length and exhibited the canonical genomic organization of GETV: 5' untranslated region (UTR), nonstructural proteins (nsP1-nsP4), structural proteins (C, E3, E2, 6 K, E1), and 3' UTR. Sequence homology analysis revealed that HN-2025 shared the highest nucleotide identity (98.6%-99.2%) with contemporary GIII genotype GETV strains circulating in Chinese pig herds. Phylogenetic analysis based on the complete genome and E2 gene sequences confirmed that HN-2025 clustered within the GIII genotype and was closely related to porcine GETV strains previously identified in central and southern China. These findings expand the molecular epidemiological understanding of GETV in Hunan Province and provide a valuable viral isolate for future studies on pathogenesis, diagnostics, and vaccine development.

Insect-specific viruses represent a promising yet underexplored resource for the biological control of locust pests. In this study, we performed metatranscriptomic sequencing of three dominant locust species-Calliptamus italicus, Calliptamus barbarus, and Oedaleus decorus decorus-collected from field populations in Xinjiang, China. Bioinformatic analysis identified 32 RNA viruses, 137 DNA viruses, and 33 unclassified viruses. Notably, this study reports the first detection of a novel insect-specific virus in locusts. Phylogenetic and homology analyses revealed that the newly discovered virus belongs to the family Iflaviridae (order Picornavirales), and we provisionally name it Xinjiang Calliptamus italicus Iflavirus (XCiIV). Using transcriptome sequencing and rapid amplification of cDNA ends (RACE), we obtained the complete genome sequence, totaling 9,996 base pairs. The XCiIV genome contains a single predicted open reading frame (ORF) encoding a polyprotein of 2,891 amino acids, which is proteolytically processed into four functional proteins: capsid proteins, RNA helicase, 3C protease, and RNA-dependent RNA polymerase (RdRp). To our knowledge, this is the first report of an Iflavirus identified in C. italicus from the Xinjiang region. The complete genome sequence and comparative analysis of XCiIV expand the known diversity of insect Iflaviruses and provide a reference for future studies on the molecular evolution and host adaptation of locust-associated viruses.

Zucchini yellow mosaic virus (ZYMV) is a major pathogen that negatively affects cucurbit crops. To elucidate the molecular characteristics and evolutionary dynamics of ZYMV in China, ZYMV was isolated from Cucurbita and Luffa samples collected from plants exhibiting typical yellow mosaic symptoms in Tai'an, Shandong Province, and its complete genome sequence was determined. Using reverse transcription PCR combined with rapid amplification of cDNA ends, the full-length genome sequences of two isolates obtained from Luffa and Cucurbita (ZYMV-sg, GenBank accession no. PV007922; ZYMV-ng, GenBank accession no. PV007923) were determined. Both genomes were 9,592 nt in length (excluding the poly(A) tail). The genomes encoded a polyprotein of 3,080 amino acids and exhibited a genomic organization typical of members of the family Potyviridae. Phylogenetic analysis revealed that both isolates were most closely related to Chinese isolates and formed a distinct subclade within the major group B-I. Recombination analysis identified specific recombination events in ZYMV-ng, indicating that genetic recombination contributed to the molecular evolution of ZYMV. Further analyses suggested that purifying selection played a dominant role in shaping ZYMV evolution. These findings provide important molecular insights into the genomic structure, phylogenetic relationships, and sequence evolution of ZYMV isolates from Cucurbita and Luffa in China and provide a basis for further research on virus-host interactions and the development of targeted disease control strategies.

Begomoviruses are one of the most genetically diverse plant-infecting DNA viruses. They significantly limit chilli (Capsicum annuum L.) production in tropical and sub-tropical areas. Their evolution is largely influenced by recombination and mixed infections. However, the relationship between genetic diversity and selection pressure in natural populations of chilli-infecting begomoviruses remains poorly understood. We collected chilli plants showing leaf curl disease symptoms from Bargarh district, Odisha, India, and tested them for begomovirus presence. Molecular analysis showed the presence of chilli leaf curl Ahmedabad virus (ChiLCAV), tomato leaf curl Joydebpur virus (ToLCJoV), and an associated betasatellite, tomato leaf curl Joydebpur betasatellite (ToLCJoB). Recombination analysis revealed multiple statistically significant recombination events in both begomovirus genomes and the betasatellite. To assess broader patterns of genetic diversity and selection, the newly generated sequences were analysed alongside publicly available sequences retrieved from the NCBI virus database. These comparative analyses revealed high haplotype diversity and a high number of segregating sites, whilst most coding regions showed strong purifying selection, with only a small number of sites showing evidence of episodic diversifying selection. These findings indicate that mixed infections can provide opportunities for recombination and the generation of genetic variation, whereas strong purifying selection constrains changes in functionally important coding regions. The combined action of recombination and selection, therefore, appears to shape the genetic diversity and evolutionary stability of chilli-infecting begomoviruses. To the best of our knowledge, this is the first report of a mixed infection involving ChiLCAV, ToLCJoV, and ToLCJoB associated with chilli leaf curl disease in India, providing new insights into the evolutionary dynamics of begomovirus complexes infecting chilli.

Bovine leukemia virus (BLV) is an oncogenic deltaretrovirus that establishes lifelong infection in cattle and may progress from asymptomatic carriage to persistent lymphocytosis (PL) and lymphoma. Although adaptive immune alterations have been widely studied, the stage-specific transcriptional landscape of innate immunity in BLV infection remains incompletely defined. In this study, we performed a targeted innate immune transcriptomic analysis using RNA-seq data derived from 18 Holstein dairy cows selected from a screened population of 130 animals in Querétaro, Mexico. Animals were classified into three groups based on serology, proviral load, and hematological criteria: BLV-seronegative controls (n = 5), asymptomatic BLV-infected cattle (AL), and BLV-infected cattle with PL. Total peripheral blood leukocytes were subjected to RNA extraction and sequencing on the Illumina NovaSeq platform. Differential gene expression analysis was conducted using DESeq2 with significance defined as adjusted P-value (padj) < 0.05 and |log2 fold change|≥ 1. A curated panel of innate immune-related genes encompassing pattern recognition receptors, interferon signaling, inflammatory mediators, inflammasome components, and cytotoxic effectors was analyzed. Comparison between Control and Asymptomatic cattle identified 8 genes that were significantly altered (padj < 0.05). Asymptomatic animals demonstrated upregulation of antiviral and immune-regulatory genes, including EIF2AK2 (log2FC = 1.10, padj = 0.011), KLRK1 (log2FC = 0.98, padj = 0.009), and IL18 (log2FC = 0.77, padj = 0.015), indicating activation of interferon-associated and inflammatory pathways. Marked upregulation of TNFRSF13C (log2FC = 3.25, padj = 0.046) and TNFAIP3 (log2FC = 2.03, padj = 0.049) suggests modulation of B-cell survival signaling and NF-κB regulatory control. In contrast, NCR1 (log2FC = - 1.04, padj = 0.044), LMX1B (log2FC = - 0.80, padj = 0.030), and TNFSF12 (log2FC = - 0.78, padj = 0.044) were downregulated, reflecting selective adjustment of cytotoxic and TNF superfamily pathways. PL animals exhibited broader innate immune transcriptional alterations consistent with sustained inflammatory activation. These findings demonstrate that asymptomatic BLV infection is characterized by controlled activation of antiviral and regulatory innate immune pathways, whereas disease progression toward PL is associated with expanded inflammatory reprogramming. Stage-specific innate immune signatures may therefore contribute to viral persistence and clinical outcome in BLV-infected cattle.

Banana streak OL virus (BSOLV) is the primary species of banana streak virus (BSV) affecting banana production in South China. Reverse genetics is a powerful method for studying viral pathogenicity and the interactions between viruses and their hosts; however, no infectious clone of BSOLV has been reported until now. In this study, the BSOLV genome was amplified in overlapping segments to assemble 1.3 times full-length sequence. The resulting construct was then cloned into the yeast-Escherichia. Coli-Agrobacterium shuttle vector pCB301 via yeast homologous recombination, yielding the infectious clone BSOLV-GZ. The complete genome of the cloned BSOLV isolate was 7, 410 bp and shares 99.81% nucleotide sequence identity with the BSOLV isolate 3-S36. The BSOLV-GZ infectious clone was introduced into Brazilian banana seedlings through Agrobacterium-mediated injection. At 30 days post-inoculation (dpi), BSOLV was detected in 19 out of 30 inoculated banana plants by RT-PCR. By 42 dpi, distinct chlorotic streaks appeared in 12 of the 19 RT-PCR-positive plants. This was confirmed by immunocapture PCR (IC-PCR) and Southern blot. Additionally, rod-shaped viruses were observed under transmission electron microscopy in the symptomatic Brazilian banana plants. These findings indicated that the banana plants inoculated with the BSOLV-GZ infectious clone exhibited typical symptoms of infection. The host range of the BSOLV-GZ infectious clone was assessed through Agrobacterium-mediated infiltration; however, none of the tested plants was detected positive by RT-PCR. This research provides a foundational basis for future studies on the activation of endogenous BSOLV and the interactions between the virus and its hosts.

Bovine leukosis continues to impose significant economic losses on the global cattle industry due to its persistent infection and widespread prevalence worldwide. Although control strategies such as test-and-cull programs have been implemented in some countries, an effective and safe vaccine remains unavailable. In this study, an immunoinformatics-based approach combined with reverse vaccinology was applied to design a multi-epitope peptide (MEP) vaccine candidate against Bovine leukemia virus (BLV). Conserved protein regions were analyzed to identify potential immune targets, resulting in the selection of five epitopes, including three cytotoxic T-lymphocyte (CTL) epitopes, one helper T-lymphocyte (Th) epitope, and one linear B-cell epitope derived from the highly conserved BLV Gag and RT-IN proteins. These epitopes were assembled using AAY and GPGPG linkers to generate a 67-amino-acid MEP construct. In silico evaluation indicated that the designed construct possesses favorable immunological properties, including high predicted antigenicity (VaxiJen score: 0.6511, which is notably higher than the baseline score of 0.4784 for the native viral proteins; Pr44: 0.4784, RT-IN: 0.4214), non-allergenic characteristics, and absence of predicted toxic motifs. Homology analysis against the Bos taurus proteome revealed no significant similarity, suggesting a low risk of host cross-reactivity. Structural analyses using PSIPRED and AlphaFold 3 predicted localized secondary structural elements within an overall flexible peptide architecture; this flexibility is highly advantageous for optimal proteasomal cleavage, efficient antigen processing, and MHC presentation. Taken together, these findings provide a rational framework for the development of a BLV MEP vaccine candidate and support further experimental validation to evaluate its predicted immunogenic potential.

In this study, viral metagenomic approach was employed to identify a novel canine papillomavirus (CPV) strain in the lymphoid tissue of a stray dog. The complete circular genome (8,297 bp) of a novel papillomavirus strain, designated canpapil01, was characterized and taxonomically assigned to the genus Taupapillomavirus. The genome encodes five early genes (E1, E2, E4, E6, E7) and two late genes (L1 and L2), lacking the E5 gene. Conserved motifs essential for viral replication and host interaction were identified, including ATP-binding and cyclin interaction sites in E1, DNA-binding domains in E2, and zinc-binding domains in E6 and E7. Notably, the pRb-binding motif (LXCXE) was absent in E7. The long control region (LCR) contained predicted E1 and E2-binding sites, indicating regulatory functions. Phylogenetic analysis based on the L1 gene revealed that canpapil01 clustered with Taupapillomavirus 2 strains, which were isolated from nasal swab or blood samples of dogs and wolves in the Switzerland and the USA, forming a separate branch. Genome-wide comparisons showed 71.4% amino acid identity with the Taupapillomavirus 2 reference strain (NC_040578), confirming it as a novel strain within this species. These findings expand the known genetic diversity of canine papillomaviruses and provide valuable genomic data for future research into CPV-associated diseases.

Positive-strand RNA viruses remodel host endoplasmic reticulum (ER) membranes into replication organelles (ROs) that support genome replication. Members of the genus Orthoflavivirus exploit host lipid pathways, particularly the phosphatidylinositol 4-kinase (PI4K)-phosphatidylinositol 4-phosphate (PI4P)-oxysterol-binding protein (OSBP) axis, yet they display marked mechanistic divergence. Specifically, it raised a central and unresolved question: is local PI4P enrichment an indispensable determining factor for viral vesicle biogenesis, or is it merely a contributing factor to a more fundamental, underlying mechanical requirement for high membrane curvature? Zika virus (ZIKV) shows strong dependence on PI4P enrichment. ZIKV nonstructural protein 1 (NS1) binds negatively charged lipids, including PI4P, via a positively charged membrane-binding surface that includes Arg31. This interaction supports NS1-mediated ER remodeling and subsequent OSBP-mediated cholesterol delivery. In contrast, dengue virus (DENV) and West Nile virus (WNV) largely bypass strict PI4P dependence. Their RO biogenesis relies more heavily on viral membrane proteins (NS4A/NS4B), host reticulon RTN3.1A, alternative lipid-shape remodeling (including phospholipase A2-generated lysophosphatidylcholine in WNV, sterol availability, and fatty-acid synthesis. Beyond the PI4P-OSBP axis, virus-specific exploitation of lipid peroxidation (ALOX12 pathway), cholesteryl-ester-enriched lipid droplets (via SOAT1/SOAT2), nuclear lipid droplets, and glycerophospholipid/ceramide remodeling further differentiates these viruses. These distinctions have direct implications for host-targeted antivirals. PI4K and OSBP inhibitors show preferential activity against ZIKV, whereas SOAT1/2 inhibition exhibits broader anti-orthoflaviviral potential, and other targets (FASN, NAAA, ALOX12) display virus-weighted efficacy. Recognition of this mechanistic spectrum is essential for accurate interpretation of lipid-perturbation studies and for the rational design of broad-spectrum or virus-selective antiviral strategies.

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 suggested 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 nonretroviral 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 alfalfa development and response to environmental stresses. We believe this study reveals the first documented case of rhabdoviruses integrated into the alfalfa genome.

Epstein-Barr virus (EBV) BamHI-A rightward frame 1 (BARF1) is a viral oncogene implicated in epithelial malignancies, but the determinants of its transforming activity remain incompletely defined. We generated BARF1 truncation constructs and targeted mutants to examine the contribution of residues 35-38 and adjacent Ser39 in BALB/c 3T3 fibroblasts using serum deprivation, tumorigenicity, BCL-2 expression, and molecular docking assays. Deletion of residues 35-38 in full-length BARF1 reduced viable cell maintenance under severe serum deprivation, whereas Ser39-to-Ala substitution in the BARF1 1-56 background markedly reduced the viable cell advantage of this fragment. Neither mutant produced detectable tumors during the 28-day observation period. Full-length BARF1 induced tumors in all animals, whereas BARF1 1-56 retained partial tumor-forming activity. BARF1 1-46 maintained higher viable cell numbers under serum-limiting conditions but produced no detectable tumors, suggesting a contribution of residues 47-56 to tumor formation. BCL-2 remained detectable in both non-tumor-forming mutants. Exploratory docking predicted a more favorable score and distinct positioning for the wild-type 35-39 peptide relative to the catalytic subunit of cAMP-dependent protein kinase A (PKA) than for the mutant peptides; however, this analysis does not demonstrate Ser39 phosphorylation or PKA-dependent BARF1 activity. Overall, these findings support a functional contribution of the BARF1 region encompassing residues 35-39 to viable cell maintenance under serum deprivation and tumor formation. Because construct-level BARF1 protein abundance, stability, secretion, and localization were not assessed, the observed phenotypes cannot be attributed solely to the altered residues.

Cocadviroid rimocitri (citrus bark cracking viroid, CBCVd) diagnostic surveys were carried out from July 2022 to August 2024 in major pistachio (Pistacia vera L.) growing locations of Southeast Anatolia region (Türkiye). Dormant shoots, green twigs, and leaves were collected from a total of 223 symptomatic and asymptomatic trees in 51 pistachio orchards and tested by RT-PCR to investigate the presence of the viroid. CBCVd incidence was about 19.7% out of all tested samples. Full length sequences of CBCVd-pistachio isolates ranged from 282 to 286 nucleotides. They had considerable sequence variation, with 14 distinct haplotypes, resulting in a haplotype diversity of 0.925. Overall, the estimated average number of nucleotide differences (k) and nucleotide diversity (π) were 5.16 and 0.019, respectively. These findings indicate that the CBCVd in tested pistachio trees comprised a heterogeneous population. The nucleotide sequence homology was 96-100% among themselves, and they were most closely related to isolate W11 from pistachio (MF198463). The pathogenicity of these CBCVd-pis variants was demonstrated in cucumber seedlings, and viroid transmission to four healthy rootstocks (P. vera cv. 'Siirt,' P. atlantica, P. khinjuk, and UCB1). Symptom expression on cucumber seedlings was recorded after 20-day post-inoculation. Graft uptake was significantly reduced when viroid-infected buds were grafted onto all rootstocks.

Nipah virus (NiV) is a highly pathogenic zoonotic RNA virus belonging to the genus Henipavirus within the family Paramyxoviridae, representing a continuing global health concern due to its high case fatality rate and potential for epidemic expansion in the era of increasing international connectivity. The virus demonstrates strong evolutionary adaptability driven by the absence of proofreading mechanisms during RNA replication, enabling genetic diversification that may influence host range, virulence, and transmission dynamics. Molecular pathogenesis of NiV is primarily mediated through interaction of viral glycoproteins with ephrin-B2 and ephrin-B3 receptors, facilitating host cell entry, endothelial damage, and neuroinvasion. Immune evasion facilitated by the action of accessory proteins encoded by the P gene (P, V, W, and C) acts to suppress innate antiviral immunity through the inhibition of interferon induction and JAK/STAT signaling. Human-to-human transmission of Nipah virus remains limited, with epidemiological evidence indicating basic reproduction numbers generally below unity; however, respiratory involvement and healthcare-associated exposure may enhance cluster outbreaks. Global travel, ecological disruption, and fragmented surveillance systems contribute to spillover risk, particularly in South and Southeast Asia where fruit bats of the genus Pteropus serve as natural reservoirs. Despite advances in vaccine technology, including subunit, viral vector, mRNA-based platforms, and monoclonal antibody therapies, no licensed prophylactic or therapeutic agent is currently available for human use. Global preparedness remains challenged by the scarcity of high-containment biosafety facilities, limited research funding, and absence of integrated One Health surveillance networks. Ethical considerations surrounding wildlife population control further complicate disease mitigation strategies. Emerging genomic surveillance, artificial intelligence-assisted predictive modeling, and regional data-sharing frameworks are essential for early detection and response. Strengthening molecular research on viral-host interactions and transmission determinants will be critical for preventing future Nipah virus outbreaks in an increasingly interconnected world.

Rotavirus infection is a major cause of paediatric gastroenteritis and has increasingly been associated with neurological complications, although direct evidence of central nervous system involvement remains limited. In this study, Rotavirus A was detected in both cerebrospinal fluid and stool samples from a child presenting with encephalopathy and seizures, and infectious virus was successfully propagated in mammalian cell lines. Genomic analysis revealed a Wa-like genotype constellation, G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. Although lateral flow immunoassay yielded negative results, molecular diagnostic approaches proved valuable for identifying atypical RVA infections.

The study presents the first-time detection of one of the rare clades (clade9 strain) of varicella zoster virus (VZV) from Pakistan. The next-generation sequencing confirmed wild-type clade9 strain through clade-specific markers at C5827A, T33722C, T33725C, T33728C, T38055C, G69424A, C87841T and T95241C and restriction profile of PstI+BgII+SmaI-. The rarely reported SNPs (22/134) were detected along with 12/42 rare amino-acid mutations. However, the mutations at C77Y, Q43H, D613E and A2V were predicted to be not-tolerated hence might affect protein function. The VZV (PV934234) strain clustered with clade9 strains upon phylogenetics. Thus, the first-time detection of clade9 raises concern of limited genomic surveillance of VZV in Pakistan. This necessitates genomic surveillance and continuous clinical vigilance in Pakistan to avoid any potential silent surge in the country.