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.

Sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1) is a dNTPase that depletes intracellular dNTP pools in nondividing cells such as human monocyte-derived macrophages (MDMs). These low dNTP levels suppress HIV-1 reverse transcription kinetics in MDMs. In contrast, viral protein X (Vpx), an accessory protein encoded by human immunodeficiency virus type 2 (HIV-2) and some simian immunodeficiency viruses (SIVs), induces degradation of SAMHD1, thereby increasing cellular dNTP pools and promoting a more permissive infection in MDMs. Our previous study demonstrated that the central polypurine tract (cPPT) facilitates completion of HIV-1 reverse transcription, particularly when reverse transcription is kinetically delayed due to dNTP limitation in nondividing human lung fibroblasts. However, the role of cPPT during HIV-1 replication in macrophages where SAMHD1 establishes low dNTP pools and kinetically restricts viral reverse transcription remains untested. To address this question, we performed time-course analyses of transduction efficiency and fluorescence intensity in MDMs transduced with two distinct HIV-1 vector systems containing or lacking the cPPT sequence, in the presence or absence of Vpx. Our data show that while either cPPT insertion or Vpx treatment alone modestly increases HIV-1 vector transduction efficiency, the combined presence of cPPT and Vpx results in a pronounced enhancement of both transduction efficiency and fluorescence intensity in MDMs. Overall, these findings demonstrate that cPPT and Vpx act cooperatively to enhance HIV-1 vector transduction in nondividing MDMs, supporting an interplay between cPPT function and Vpx-mediated SAMHD1 degradation in macrophages.

Pseudomonas aeruginosa (P. aeruginosa) is widely distributed in the environment. As an opportunistic pathogen, it commonly causes infections in immunocompromised individuals, including respiratory tract infections and burn wound infections. P. aeruginosa possesses multiple antibiotic resistance mechanisms, including efflux pumps, resistance genes, and population dynamics. Phage therapy is a potential approach for addressing drug-resistant P. aeruginosa infections; however, clinical experience and standardized guidelines for its application in severe pneumonia remain limited. A 14-month-old infant was hospitalized for pneumonia. Four days later, he developed acute pneumonia and was sent to the ICU for 38 days of antibiotic therapy; nonetheless, P. aeruginosa remained detectable in the patient's respiratory secretions. During the clinical course, phage zjk6 was detected from a longitudinal P. aeruginosa isolate in the absence of phage therapy. This finding documents the coexistence of a naturally detected phage and MDR P. aeruginosa during prolonged pneumonia, but does not establish that the phage mediated bacterial clearance or clinical recovery. We performed whole-genome sequencing on P. aeruginosa isolates from patients to ascertain if they were infected by the same infection and assessed their antibiotic resistance using drug sensitivity testing. We isolated phages using the drip technique and double-layer plate method, examined their appearance by transmission electron microscopy, and assessed their biological properties through one-step growth curve analysis and lysis spectrum detection. Genome sequencing and comparative genomic analyses were performed to characterize phage zjk6 and representative bacterial isolates and to evaluate phage-host genomic relatedness. P. aeruginosa was isolated repeatedly during 49 days of treatment. Comparative genomic analysis of representative longitudinal isolates revealed multiple strain backgrounds, including distinct ST508 and ST266 lineages and a closely related ST836 lineage. Phage zjk6 was isolated from the fifth clinical isolate, which served as the propagation/reference host. This phage possesses an elongated tail and a limited lysis spectrum, which is capable of gradually lysing the fifth isolated P. aeruginosa strain. Genomic analysis showed that zjk6 formed plaques and displayed slow lytic behavior under the tested conditions, while also carrying lysogeny-associated regulatory modules, indicating temperate potential rather than a strictly lytic lifestyle. A naturally detected slow lytic Pseudomonas phage may coexist with MDR P. aeruginosa during prolonged infection. These findings support further study of phage-bacterium interactions in clinical infections, while the therapeutic significance of zjk6 requires additional validation.

The family Spiciviridae, belonging to the order Ghabrivirales, is currently composed of only one officially recognized genus, Spicivirus. However, metagenomic studies have revealed an increasing diversity of related viruses, suggesting that the current classification may underestimate their evolutionary complexity. In this context, the aim of the present study was to detect and characterize new viruses related to the Spiciviridae in the family Culicidae mosquitoes. A total of nineteen mosquito pools were sequenced and analyzed using metagenomic approaches and bioinformatics tools. Among these, two novel viruses were identified in four pools (S4, S11, S18, and S19), provisionally named Sabethes virus 1 (SV1) and Sabethes virus 2 (SV2). Phylogenetic inference revealed clades with strong statistical support and genetic divergences greater than 20% in the RNA-dependent RNA polymerase (RdRp) protein. These findings contribute to a detailed analysis of the genomic diversity associated with Spiciviridae. Although Spicivirus is currently the only genus officially recognized, our results suggest the presence of viruses that may represent new taxonomic groupings not yet described by the ICTV.

Norovirus is a major causative agent of pediatric acute gastroenteritis, whereas GII.8[P8] represents a rare, non-dominant genotype with limited publicly available whole-genome data. We characterized a GII.8[P8] norovirus strain, Harbin-Nov-076-2022, detected through routine surveillance of pediatric acute gastroenteritis in Harbin, China. The strain was obtained from a stool sample of a 15-month-old female patient and subjected to whole-genome sequencing using the DNBSEQ-T7 platform. Integrated analyses included phylogenetic reconstruction, amino acid entropy profiling, selection pressure assessment, homology modeling of the capsid P protein, and B-cell epitope prediction. The near-complete genome of Harbin-Nov-076-2022 is 7486 nt in length, with a GC content of 53.81%, and displays the canonical three-open-reading-frame organization of human noroviruses. The sequence was deposited in GenBank under accession number PV416744. Phylogenetic analysis assigned the strain to the GII.8[P8] lineage, with the highest nucleotide identity of 97.52% to the Thai 2018 strain GII/Hu/TH/2018/GII.8[P8]/B6213 (ACCN: OR546393). Amino acid variability was highest in the VP2 region, with three variable sites (residues 15, 122, and 229) identified among the aligned GII.8[P8] VP2 sequences, whereas selection pressure analysis indicated that the genome was largely constrained by purifying selection. Same-template P-domain modeling predicted localized sequence-associated variations in the P2 subdomain and HBGA-binding-associated surface loops, with 59 predicted B-cell epitopes. These findings expand whole-genome evidence for rare GII.8[P8] norovirus in Northeast China and provide regional genomic data for molecular surveillance, evolutionary assessment, and antigenic characterization of non-dominant norovirus genotypes.

Newcastle disease virus (NDV) represents a major threat to the worldwide poultry industry, and the absence of approved antiviral therapeutics highlights the need for alternative intervention strategies. In this study, an integrated computational and experimental methodology to assess the interaction of selected plant-derived flavonoids against NDV. Molecular docking was conducted to evaluate the interactions of karanjin, mammeigin, and 3, 5-dihydroxy-4',7-dimethoxyflavone (DHDM) with multiple NDV proteins, comprising hemagglutinin neuraminidase (HN), fusion protein (F), matrix protein (M), nucleoprotein (N), and phosphoprotein (P). Based on docking predictions, the NDV phosphoprotein was selected for experimental validation of biophysical interaction studies. Among the tested compounds, karanjin consistently exhibited the strongest binding affinities, with particularly favorable interactions observed for the phosphoprotein, a key regulator of viral transcription and replication. In silico pharmacokinetic analysis further supported the drug-likeness and oral absorption potential of karanjin. Based on docking predictions, the NDV phosphoprotein was selected for experimental validation. Recombinant phosphoprotein was expressed and purified, and flavonoid interactions were examined using UV-Vis absorption spectroscopy, fluorescence quenching, and isothermal titration calorimetry analysis. Karanjin displayed a clear and specific binding profile characterized by moderate-to-strong affinity and an enthalpy-driven interaction, whereas mammeigin and DHDM showed weak or nonspecific interactions. Spectroscopic analyses corroborated these findings, indicating stable complex formation without major structural perturbation. Collectively, these results identify karanjin as a promising flavonoid candidate targeting the NDV phosphoprotein.

The family Amalgaviridae comprises double-stranded RNA viruses with two partially overlapping open reading frames (ORFs), in which the RNA-dependent RNA polymerase is expressed through programmed ribosomal frameshifting. Antonospora locustae virus 1 (AnloV1), the founding member of the proposed genus "Anlovirus", represents a poorly characterized viral lineage because only a limited number of genome sequences have been reported. Here, we systematically mined publicly available transcriptome datasets and identified 13 AnloV1-like genome sequences from four insect species and one mammalian fecal sample. Pairwise sequence comparisons indicated that these genomes represent nine putative viral species, substantially expanding the diversity of this lineage. Comparative genomic analyses showed that all newly identified viruses possess two overlapping ORFs and a highly conserved UUU_CNN motif at the ORF1/ORF2 overlap, further supporting this sequence as the consensus +1 programmed ribosomal frameshifting motif of the proposed genus "Anlovirus". Phylogenetic analysis recovered a well-supported AnloV1-like clade distinct from the recognized genera of the family Amalgaviridae, providing additional evidence for the proposed genus "Anlovirus". Transcriptome datasets from all four insect species also contained transcripts with high similarity to microsporidian proteins, suggesting that at least some AnloV1-like viruses may infect microsporidian parasites rather than the sampled animals. These findings substantially expand the genomic resources available for AnloV1-like viruses and provide an evolutionary framework that may facilitate the future establishment and classification of the proposed genus "Anlovirus".