Identification and genetic characterization of a distinct genotype of Puumala orthohantavirus in Hebei Province, China

Orthohantavirus infections pose a significant threat to human health, while numerous orthohantaviruses have been identified, suspected viral infections remain undiagnosed in the world, which highlights the need for further identification and characterization of viruses circulating in humans and host animals. In this study, viral metagenomics was utilized to investigate orthohantaviruses present in tissue samples collected from rodents trapped at the Bashang Grassland of Hebei Province, China. A total of 145 wild rodents belonging to six species were captured in the study area, and 725 tissue samples (lung, liver, kidney, spleen, gut) were collected in 2024. A Puumala orthohantavirus (PUUV), named Guyuan strain, was identified in Myodes rufocanus, with a positive rate of 0.69%. The complete genomic sequences of the L, M, and S segments were obtained and confirmed by Sanger sequencing. Phylogenetic analysis of these genomic sequences with those of other orthohantavirus species showed that the L, M, and S segments clustered with PUUV genomic sequences, while sharing a nucleotide sequence similarity of 81.2%, 80.2%, and 84.3% with previously characterized reference viral strains Kitahiyama128L, Tobetsu_04, and Baltic/205 Cg, respectively. Amino acid homology analysis demonstrated that the sequences exhibited the highest identity to PUUV Hokkaido strain at a level of 95.4%, 94.6%, and 97.0% respectively. Viral particles were observed in lung and kidney tissues using transmission electron microscopy, and viral protein antigen was detected in viral RNA-positive lung, liver, and kidney tissues through immunofluorescence assay with antibodies against the PUUV nucleocapsid protein, thereby confirming the virus's multiorgan tropism. The results demonstrated that a distinct genotype of PUUV was circulating in rodents in the study areas, which may have implications for zoonotic transmission surveillance and public health management in Hebei Province.