Genetic and Phylogenetic Characterization of Influenza D Viruses From South Korean Cattle, 2022-2023.

Influenza D virus (IDV) is an emerging orthomyxovirus with cattle as its principal reservoir, and D/Yama2019-lineage viruses have become dominant in East Asia. Although IDV has been detected in Korean cattle, the genomic identity, phylogenetic placement, and regional evolutionary relationships of circulating Korean strains have not been defined. To address these gaps, nasal swabs were collected from 578 cattle with mild respiratory signs on 157 farms across eight provinces in South Korea during 2022-2023 and screened by RT-qPCR targeting the PB1 gene. Positive samples underwent complete genome sequencing of all seven segments, followed by maximum-likelihood and Bayesian phylogenetic analyses, discrete phylogeographic inference using a Bayesian stochastic search variable selection (BSSVS) model, and positive selection analyses. Six samples from three farms were IDV-positive (sample-level positivity: 1.04%; farm-level positivity: 1.9%), all from 8-to-10-month-old calves. Phylogenetic analysis of all seven genomic segments placed all six Korean strains within the D/Yama2019 lineage with strong bootstrap support (99%-100%), forming a monophyletic cluster more closely related to Chinese than to Japanese D/Yama2019 reference strains. No phylogenetic evidence of reassortment was detected. Bayesian time-scaled analysis estimated the most recent common ancestor of the Korean strains at ~2018.1-2020.1 across all seven segments. HEF-based BSSVS analysis suggested a China-to-South Korea transition within the sampled dataset (posterior probability (PP) = 0.982; Bayes factor (BF) = 163.67), although this result should be interpreted in light of the small number of Korean sequences and the single-segment basis of the phylogeographic analysis. Positive selection analyses revealed limited, method-dependent signals without support from the fixed effects likelihood model, and Korean-associated amino acid substitutions in PB1, P3, NS1, and NS2 were interpreted as lineage-associated molecular signatures rather than evidence of adaptive evolution. These findings provide a whole-genome baseline for IDV surveillance in South Korea and support continued longitudinal monitoring to clarify the persistence and transmission dynamics of D/Yama2019-lineage viruses in the region.