A U.S.-linked HSV-2 clinical isolate from China reveals global transmission and informs region-specific vaccine design

The persistent global burden of herpes simplex virus type 2 (HSV-2) requires region-specific therapeutic strategies, yet the limited number of characterized clinical isolates from China has hindered accurate evaluation of local viral evolution and vaccine efficacy. To address this gap, we isolated and comprehensively characterized HSV-2/KM-1, a novel clinical strain obtained from a genital herpes patient in Kunming, China. Whole-genome sequencing showed 99.92% nucleotide identity with contemporary U.S. strains (MH790606, PP099973), which was markedly higher than its homology to China's reference strain HJ12 (128 amino acid differences). This unexpected phylogeographic similarity challenges existing models of geographically restricted HSV-2 evolution and suggests global viral gene flow facilitated by human mobility. Cell tropism analyses revealed accelerated replication in human foreskin fibroblasts (HFF-1), with early viral protein expression at 12 hpi, and high-titer production (7.0 log10 TCID 50 /mL) in Vero cells at a low MOI (0.001). Comparative genomics identified 27 amino acid substitutions in virulence determinants (ICP4, UL52, UL36, etc.) compared with the U.S strain (PP099973), 14 of which altered residue polarity, potentially influencing viral-host interactions, as suggested by previous studies on these proteins' roles. As a phylogenetically U.S.-linked clinical isolate from China, HSV-2/KM-1 helps to fill a gap in regional pathogen resources and provides a critical tool for assessing globally circulating strains and developing targeted interventions. • A Chinese HSV-2 isolate phylogenetically clustered with U.S. strains (99.92% identity), suggesting potential global transmission. • Ultra-low MOI (0.001) achieves 7 log TCID 50 /mL viral yields in Vero cells, enabling scalable antiviral screening. • A total of 27 conserved amino acid substitutions cluster in virulence proteins (ICP4, UL36, UL52 etc.), suggesting its functional adaptations.