Isolation And Characterization Of Hantavirus In Epidemic Areas Of Hebei Province

Objective: Lung samples were selected as research subjects and hantavirus(HV) were isolated in epidemic areas of Hebei province. To identify their genotype, analyze the S segment nucleotide acid sequence and clarify the type and gene characteristic of Hantavirus in HeBei province, guide the prevention and control of hemorrhagic fever with renal syndrome (HFRS).Methods:1 Lung samples were screened by immunofluorescence assay (IFA). Antigen-positive lung samples were processed and inoculated onto cultures of confluent Vero E6 cells.2 Total RNA from Vero E6 cells infected with HV were extracted.3 The isolated viruses were classified by RT-nested PCR with the special typing primers.4 The entire S segments were amplified by RT-PCR, PCR amplification products were purified. The amplified products were cloned into pMD19-T vector. Plasmids were purified and sequenced.5 The molecular evolution method and software were used to calculate the nucleotide and amino acid homology and phylogenetic trees were constructed.Results:1 Four lung tissues were homogenized and inoculated onto confluent Vero E6 cells. After the third-fourth passage, the characteristic hantavirus antigen particle could be seen by IFA and cytopathic effect(CPE) was not found in the cells.2 At last, all four strains were isolated successfully and designated 06HBB34,06HBB38,06HBC78 and 06HBC79 respectively.3 The cell culture supernatants were used to infect fresh Vero E6 cell cultures. After the first-second passage, the characteristic hantavirus antigen particle could be seen by IFA. At last, all six strains were recovered successfully.4 Nested RT-PCR was carried out using type-specific primers, which indicated that all ten strains belonged to SEO type HV.5 The recombinant plasmid of 93HBX12 was constructed and sequenced, the result showed that S-segment sequence of 93HBX12 was determined to be 1,772nt in length, the percent of A,T,C,G was 31.72%, 23.02%, 25.96%, 15.30% respectively. A+T and C+G accounted for 57.67% and 43.33% respectively. S segment consisted of 3 sections: 5'non-coding region (NCR), one open-reading frame (ORF) and 3'NCR. The nucleotide length of 5'NCR and 3'NCR was 42nt and 440nt respectively. The S segment contained a single ORF (nt 43 to 1,332) which encodes the N protein of 429 amino acids (aa). As a member of HV, 93HBX12 had the distinctive terminal complementary nucleotides that promote the folding of the viral genomic segments into "panhandle" hairpin structures.6 The result which obtained by comparing 93HBX12 nucleotide and amino acid with other type hantavirus showed that SEO type was the closest relative of 93HBX12, secondly HTN, DOB followed. The homology with PUU and HPS related virus (SN, NY, BAY, BCC, ORN, AND) in America was very low.7 The phylogenetic tree based on the complete S-segment indicated that 3 branches were formed in Hantavirus in accordance with the host animals of nurinae, microtinae, and cricetinae rodents respectively. 93HBX12 lay within the lineage of the SEOV species. BjHD01 was the closest relative in all the Hantavirus strains. Phylogenetic analysis of complete coding sequences showed the result consistent with the complete S-segment analysis.Conclusion:1 SEO type was the dominant genotype carried by host animals in epidemic areas in Hebei and it was widespread in Hebei province.2 SEOV didn't produce cytopathic effects in cell culture. However, if appropriate methods were adopted, the isolating rate of SEOV could be raised.3 As long as HV strain was stored appropriately, it could be in good condition for a very long time at low temperature. 4 In China, Hantavirus carried by Microtus fortis was not HPS related virus but SEO type virus. Microtus fortis was an alternative host for SEOV.5 SEOV was the closest relative of 93HBX12, the S-segment expression product of 93HBX12 could not only be used as diagnosis antigen for SEOV but also for HTNV.