Whole-genome analysis of bovine alphaherpesvirus 1 associated with abortion in a vaccinated U.S. cattle herd.

First detection and molecular characterization of influenza D virus in livestock from Kazakhstan reveals circulation of the D/Yama2019 lineage

Influenza D virus (IDV) is an emerging Orthomyxovirus primarily associated with cattle and increasingly recognized as a pathogen of veterinary and zoonotic relevance. Despite growing evidence of its circulation across Asia, Europe, and North America, no data have previously been available from Kazakhstan or Central Asia. This study investigated the presence of IDV in domestic livestock samples collected during active surveillance between 2023 and 2026. A total of 867 biological samples were obtained from cattle ( n  = 611), camels ( n  = 175), Maral Deer ( n  = 51), and pigs ( n  = 30) across multiple regions of Kazakhstan. Viral RNA was screened by reverse transcription polymerase chain reaction (RT-PCR) targeting the HEF , PB1 , PB2 , and P3 genes using a newly designed primer panel. Positive amplicons were subjected to Sanger sequencing and phylogenetic analysis. IDV RNA was detected in 11 animals, corresponding to an overall prevalence of 1.27%. Positive samples were identified in several geographically distinct regions, indicating wider circulation than previously recognized. Complete HEF gene sequences were successfully obtained from two representative cattle strains, designated D/bovine/Zhetysu/KAZ/678/2025 and D/bovine/Zhetysu/KAZ/680/2025. Phylogenetic analysis demonstrated that both strains clustered within the D/Yama2019 lineage and shared >98.83% nucleotide identity with contemporary Asian strains. To our knowledge, this is the first report of IDV detection in Kazakhstan. These findings expand the known geographic range of IDV and suggest possible transboundary transmission routes across Central Asia. Continued molecular surveillance is warranted to clarify epidemiology, host range, and potential economic impact.

Isolation and identification of a potential new bovine viral diarrhea virus genotype associated with severe outbreaks in yaks (Bos grunniens) in China

Bovine viral diarrhea virus (BVDV) is a globally significant pestivirus currently classified into three species: BVDV-1 (Pestivirus A), BVDV-2 (Pestivirus B), and BVDV-3 (HoBi-like, Pestivirus H). Here, we report the isolation and identification of a potential new BVDV genotype associated with severe outbreaks in yaks in China. Since 2024, an epidemic characterized by respiratory and gastrointestinal symptoms has been spreading across Sichuan and Qinghai Provinces. From January 2024 to October 2025, we collected 260 samples from 167 affected yaks from 14 farms. Among these, 55.69% of the yaks and all sampled farms tested positive for BVDV. Sequencing of the 5′ untranslated region (5′ UTR) and Npro fragments from clinical samples revealed that the dominant epidemic strains may represent a novel BVDV lineage. We isolated four cytopathic strains and successfully sequenced two genomes of strains SC-H03 and QH-B04, which share less than 77.8% nucleotide identity across the complete coding sequence (CDS) with well-defined BVDV-1/2/3 strains. Phylogenetic analyses of the complete CDS and individual genes placed SC-H03 and QH-B04 in a distinct monophyletic clade with strong bootstrap support. Pairwise genetic distances confirmed substantial divergence, and cross-neutralization assays revealed significant antigenic differences from BVDV-1 and BVDV-2 reference strains. In accordance with ICTV Pestivirus classification standards, these strains satisfy the criteria for a new Pestivirus species, which we propose to name BVDV-4 (Pestivirus T). Given its wide prevalence in yaks and association with high morbidity and mortality, our findings underscore the urgent need for targeted prevention and control measures.

Isolation and partial 5′UTR-based molecular characterization of bovine enterovirus F from non-diarrheic cattle in Türkiye

Isolation and Genomic Characteristics of the First Bovine Viral Diarrhea Virus Subgenotype 2b Isolate from Buffalo in Guangxi Province, China

(1) Background: Bovine viral diarrhea virus (BVDV) is an economically important pathogen affecting cattle worldwide. The genetic diversity of BVDV-2 in buffalo remains poorly understood. This study aimed to elucidate the genomic and antigenic features of GX24, the first BVDV-2b isolate identified from dairy buffalo in China. (2) Methods: The virus was isolated from a rectal swab through three blind passages in MDBK cells and identified through immunofluorescence and RT-PCR. The near-complete genome was sequenced using the Illumina platform and subjected to phylogenetic and recombination analysis. B-cell epitopes and glycosylation sites were predicted using BepiPred-3.0, Epitope1D, DiscoTope-3.0, NetNGlyc-1.0, and NetO-Glyc-4.0. (3) Results: The 12,267-nt GX24 genome (GenBank: PX682047) was classified as BVDV-2b. Recombination analysis detected a putative recombination signal within the NS5A gene involving Chinese BVDV-2b and Italian BVDV-2a strains. Integrative analyses predicted conserved linear and conformational epitope clusters in the N- and C-terminal regions of the E2 protein. Spatial analysis indicated that several epitope residues may be masked by glycan shielding. (4) Conclusions: This study provides the first genomic evidence of a BVDV-2b isolate from dairy buffalo in China, suggesting that BVDV-2b may be present in this population. The putative recombination signal and antigenic characteristics offer valuable insights for the development of diagnostics and vaccine design in the future.

Genetic characterization and zoonotic potential of G1-Lineage H9N2 avian influenza viruses isolated from poultry in Pakistan, 2023.

The H9N2 subtype of avian influenza A virus has been endemic in poultry populations across Asia and the Middle East, posing ongoing economic and public health concerns. Since their initial detection in Pakistan in the 1990s, H9N2 viruses have caused repeated outbreaks in commercial poultry, leading to substantial economic losses and raising concerns about zoonotic transmission. To characterize the recent genetic evolution and zoonotic potential of circulating strains, five H9N2 isolates were obtained from poultry in Pakistan between January and March 2023. Phylogenetic analysis of the haemagglutinin gene revealed that all five isolates belong to the G5.3.2, formerly known as B2, sub-lineage of the G1 Eurasian lineage, in accordance with the revised global classification system for H9 viruses. Comparative genomic analysis confirmed that all eight gene segments were closely related to previously reported Pakistani strains, with no evidence of recent reassortment, suggesting localized persistence and ongoing genetic drift. Notably, several mammalian-adaptive mutations were identified in internal gene segments of the isolates, suggesting a potential risk of cross-species transmission. In addition, all five isolates exhibited dual receptor-binding characteristics, recognizing both α2,3-linked (avian-type) and α2,6-linked (human-type) sialic acid glycans, with a stronger affinity for α2,6-linked glycans. These findings underscore the need for continuous surveillance and risk assessment of H9N2 viruses circulating in Pakistan.

Genomic characterization of avian metapneumovirus subtypes A and B in United States poultry by targeted amplicon sequencing

Avian metapneumovirus (aMPV) subtypes A and B emerged in United States poultry in late 2023 and early 2024, prompting genome-scale surveillance from clinical samples. Here, we developed and optimized targeted amplicon sequencing (TAS) assays for both subtypes and applied them to 104 subtype-positive clinical samples collected from chicken and turkey farms across nine US states between early 2024 and early 2026. TAS recovered 91 genomes suitable for comparative analysis, including 44 aMPV-A and 47 aMPV-B sequences, with successful recovery extending to Ct values of 34.6 for aMPV-A and 31.2 for aMPV-B. Recovered genomes showed near-complete breadth and high mapping efficiency. Phylogenetic analyses of both G-gene and whole-genome datasets showed that US aMPV-A field strains formed a distinct monophyletic lineage within group IV and resolved into three closely related clusters. Cluster 2, first recognized in North Carolina and later detected in Ohio, spread across 30 turkey and chicken farms. Cluster 2 genomes were defined by a concentrated G-protein hotspot within residues 209–275, and most North Carolina Cluster 2 genomes carried 10–11 nonsynonymous substitutions in this region, including multiple proline substitutions suggestive of local structural change. Missouri Cluster 3 remained cohesive but distinct from both Cluster 1 and Cluster 2 in the G-gene and whole-genome trees. In contrast, US aMPV-B field strains remained highly homogeneous across hosts and states, with more than 99% nucleotide identity by both G-gene and WGS analyses. We also identified 12 vaccine-derived genomes on both vaccinated and nonvaccinated farms. These included six genomes related to aMPV-A vaccine and six related to aMPV-B vaccines (VCO3/50 and 1062), all of which retained vaccine-defining markers together with additional substitutions consistent with continued circulation after vaccine use in the field. Selection analyses showed that the G gene had the highest gene-wise dN/dS ratio in both subtypes. Additional elevated signal was observed in SH and M2, and candidate positively or episodically selected codons were concentrated in the subtype A Cluster 2 G-gene hotspot. These findings show that TAS supports direct-from-sample aMPV genomic surveillance and provides genomic context for field clusters, vaccine-derived lineages, and continued adaptive change in aMPV in US poultry.