Analysis Of The Gene Characteristic Of Hantavirus M Segment Among HFRS Patients In Hebei Province

Objective: To approach the genotype of HV among HFRS patients, analyze the distribution and variation of HV in the population, provide scientific evidence for prevention and control of hemorrhagic fever with renal syndrome in Hebei province.Methods:1 According to epidemiological investigation, serum specimens of patients or suspects were collected in acute period;2 Positive serum specimens were screened by indirect immunofluorescence and HV RNA was extracted;3 The viral nucleic acid were amplified by RT-nested PCR and genotyped;4 The representative HV were sequenced which had been purification reclaiming. The results were compared with previous strain of GENBANK in homology and phylogenetics, the subtype distribution, nucleotide diversity and characteristic were analyzed.Results:1 In 2006, 1060 cases of HFRS were reported and incidence was 0.155/million. According to the incidence situation, the HFRS was widespread in all of the 11 districts of Hebei province and the high incidence districts concentrated in Tangshan, Qinhuangdao, in the Northeast, taking up 63.77% of all cases in Hebei province; the lower ones were Baoding, Shijiazhuang, Xingtai, Cangzhou and Hengshui; Zhangjiakou, Chengde, Langfang was the lowest one. It presented a district characteristic of highly diverging and relative concentration.2 The number of the serum specimens of HFRS was 152, tested by indirect immunofluorescence (IFA), and the positive was 94 and the positive rate was 61.84%.3 Among 69 positive serum specimens, 17specimens were amplificated successfully by RT-PCR and the positive rate is 24.64%, 35 positive serum specimens of the acute period, less than 7 days, got 12 positive results, the positive rate is 34.29%(12/35); 26 positive serum specimens of 8-14 days, got 5 positive results, the positive rate is 19.23%(5/26); 8 positive serum specimens at more than 14 days, got 0 positive results.4 HV positive serum specimens of Hebei were amplified by RT-nested PCR using primers of the type of SEO and HTN. The results indicated that HV, carried by 17 serum specimens, all belonged to SEO type and HTN type haven't been found.5 11 typical specimens were chosen to sequence, the result of comparing of nucleotide homology displayed that they have higher homology, both transition and transversion were found. The nucleotide homology of G1 segment (217-573nt) was high (94.4%~100%) and the variation was not great. Moreover, their nucleotide constitutions were similar with R22 strain (94.7%~98.0%). But when compared with 76-118 the nucleotide homology was low (70.6%~72.0%). The nucleotide homology of G2 segment (2002-2301nt) was high (92.0%~100%). When compared with R22 and 76-118 strains the rates of nucleotide homology was 91.3%~97.7% and 70.3%~72.7% respectively. It can be seen that the variation of HV in the population were small, the genome sequence was relatively conservative for homeotype of M segment.6 The phylogenetic tree of G1 (217-573nt) and G2 (2002-2301nt) segments were constructed by DNASTAR package. The phylogenetic tree of G1 segment displayed that HeB2, HeB15, HeB26, HeB36, HeB45, HeB46, HeB57, HeB60, HeB82 and HeB95 had the closer genetic relationship all belonged to S3 subtype; HeB7 with L99, R22 and HB55 which belonged to S1 subtype had the closer genetic relationship. The phylogenetic tree of G2 segment displayed that except HeB7 with L99, R22 and HB55, which belonged to S1 subtype, had the closer genetic relationship, the others all had closer genetic relationship with SD10, ZT10, which belonged to S3 subtype. According to phylogenetic tree HTN type wasn't founded, this had the same results with RT-nested PCR electrophoresis and nucleotide homology comparison.7 According to the genotyping and phylogenetic trees analysis, we could draw the conclusion that Hebei province is a typical epidemic area of SEOV. The subtype about partial segments of G1 and G2 indicated that HV, which had been sequenced, was S1, S3 and the majority subtype was S3. The results of typing according to G1 segment were similar to that according to G2 segment. The viruses that come from the geographic setting close to each other have a high homology in nucleotide sequence, and their relationship of phyletic evolution was near. It suggests that the distribution of HV in the population has the characteristic of geography cluster in Hebei province.Conclusion:1 It is feasible to amplify the HV from serum specimens of patients in acute period. It can be used for rapid diagnosis in earlier period, but more consummate was needed to improve the detection rate. 2 It had some difficult to extraction HV RNA from serum specimens. However, it was easy to extract RNA from acute phase serum, but with the extension of invasion time the rate of extraction decreased.3 On the basis of genotyping and phylogenetic trees analysis, Hebei province is a typical epidemic area of SEOV. The subtype of S1 and S3 was exit and S3 was the major subtype .The results of classify according to G1 segment were similar with that according to G2 segment and new subtype haven't been discovered.4 The variation of HV in the population were small, the genome sequence was relatively conservative for homeotype of M segment.5 The district which setting close to each other has the high homology of virus genome nucleotide sequence and near phylogenetic evolution relationship. The distribution of HV has the character of geography cluster.