Epidemiological Investigation and Analysis of Orthohantavirus hantanense and Orthohantavirus seoulense From Wild Rodents in Liaoning Province, China

Background Liaoning Province possesses distinct ecological characteristics, including high forest cover and developed agriculture, which provide suitable habitats for various rodent species. Over the years, hemorrhagic fever with renal syndrome (HFRS), caused by members of the genus Orthohantavirus , such as Orthohantavirus hantanense (HTNV) and Orthohantavirus seoulense , remains highly prevalent in Liaoning Province, representing a significant public health concern. The transmission of these viruses is sustained by wild rodent reservoirs, necessitating continuous surveillance of infection in these host populations. This study is designed to investigate the distribution and genetic evolution of Orthohantavirus infections within wild rodent reservoir populations in Liaoning Province from 2022 to 2023 and to provide a scientific basis for effective prevention and control strategies. Methods We conducted an epidemiological investigation of 403 wild rodents collected across Liaoning Province. Samples were screened using a combination of metatranscriptomic next‐generation sequencing (mNGS) and specific two‐step RT‐PCR protocols. Results We identified a total of 19 Orthohantavirus ‐positive rodent samples (4.71% prevalence). Initial screening based on national standards (SN/T 2777‐2011) identified 15 positive samples, primarily from the Fushun, Shenyang, and Chaoyang areas. Notably, while standard RT‐PCR failed to detect viral RNA in certain samples from the Dandong area, mNGS revealed HTNV nucleic acid sequences, indicating significant genomic divergence in the primer‐binding regions of the M segment. Consequently, based on the obtained M‐segment sequences, we designed novel, strain‐specific primers (HTN‐DD), which successfully identified an additional four HTNV‐positive samples in the Dandong area. Sequence alignment demonstrated high sequence identity with major strains found in local human infections among these rodent‐derived sequences. Conclusions HTNV and SEOV exhibit a significant prevalence in wild rodents across Liaoning, which confirms the region remains a high‐risk area for HFRS. Our findings suggest that viral genomic divergence can compromise standard molecular detection. Therefore, surveillance protocols must be updated to avoid false negatives in clinical diagnosis and epidemiological investigations.