Nucleotide sequence characterization and phylogenetic analysis of hantaviruses isolated in Shandong Province, China

China is the most severe endemic area of hemorrhagic fever with renal syndrome (HFRS) in the world with 30,000-50,000 cases reported annually, which accounts for more than 90% of total number of cases worldwide. The incidence rate of the syndrome in Shandong Province is one of the highest in China, which has ever reached 50 per 100,000 persons per year. However, the molecular characteristics of hantaviruses (HV) epidemic in Shandong Province remain unclear. Therefore it is useful to clarify nucleotide sequence and phylogenetic characteristics of HV isolated in Shandong Province in order to provide better advices to control and prevent HFRS.

RNAs were extracted from sera of clinically diagnosed patients and positive rodent lungs that were detected by indirect immunofluorescent assay (IFA). Partial M segments of HV were amplified from the RNAs with reverse transcription nested polymerase chain reactions (nested PCR) using hantavirus genotype specific primers. The nested PCR products were sequenced and compared with those from previously epidemic isolates in Shandong and with other representative HV sequences from GenBank. Phylogenetic tree analyses were performed based on the sequences of the M genes.

Thirty-four HV isolates in Shandong showed 67.1%-100% nucleotide identities. The nucleotide homologies among 6 Hantaan viruses (HTNV) isolates in Shandong were 78.1%-98.7%, while the homologies among 28 Seoul virus (SEOV) isolates in Shandong were 93.7%-100%. There were at least 3 subtypes HTNV (H2, H5, H9) and 2 subtypes SEOV (S2, S3) in Shandong Province.

In Shandong Province, the homologies of HTNV were lower and there were no predominant subtypes, while the homologies of SEOV were higher and S3 was the predominant subtype. The homologies of SEOV from rodents were higher than those from patients. The distribution of subtypes in Shandong was similar to that of the adjoining provinces. Phylogenetic analyses of the sequences showed geographic clustering of HV in Shandong.