Multiplex PCR and nanopore sequencing reveal the genetic stability of Hantaan virus in Xi'an, China.

Hantaan virus (HTNV), a negative-sense RNA virus, is the primary causative agent of Hemorrhagic Fever with Renal Syndrome (HFRS) and is predominantly endemic in Eurasia, with the highest incidence reported in China. In this study, we applied a multiplex PCR amplification strategy combined with Oxford Nanopore GridION sequencing to analyze 33 HTNV-positive samples collected from Xi’an City, Shaanxi Province, China, between 2010 and 2022. The sample set included four cell culture isolates and 29 rodent lung tissue specimens. A total of 29 primer pairs targeting the S, M, and L genomic segments were used for whole-genome amplification. All samples were successfully sequenced, achieving > 98.2% genome coverage across all three segments (S, M, and L). Sensitivity evaluation demonstrated stable and uniform amplification at RNA concentrations ≥ 10⁴ copies/mL. Phylogenetic analysis revealed that all 33 HTNV genomes clustered within the H4 lineage and shared 98.1%–99.9% nucleotide identity with strains previously reported in Tianjin and Guizhou Provinces. The selection pressure analysis indicated that purifying selection is the dominant force across all four proteins. The Xi’an strains demonstrated sustained genetic stability over the surveillance period. This study establishes a rapid, sensitive, and reliable whole-genome sequencing approach for HTNV, offering a valuable tool for molecular surveillance, epidemiological tracking, and the evaluation of vaccine efficacy in HFRS-endemic regions.