应用套式RT-PCR对山东省近年流行的汉坦病毒基因分型的初步研究

PRELIMINARY STUDY ON THE GENOTYPING OF HANTAVIRUS PREVAILING IN SHANDONG PROVINCE

目的 研究山东省近年流行的汉坦病毒的基因型别 ,为山东省汉坦病毒的防治提供依据。方法 使用McAb -IFA ,HI分型 ,RT -PCR分型技术及核苷酸序列测定技术 ,对来源于山东省各主要HFRS疫区的 98份病人血清进行基因分型 ,对部分标本PCR产物纯化 ,采用双脱氧链末端终止法进行核苷酸序列测定 ,最后用DNASTAR对核苷酸序列进行分析比较。结果 2 6份恢复期血清中McAb -IFA分型 2 2份为SEO型 ,HI分型 2 4份SEO型 ;72份急性期病人血清标本中 65例RT -PCR分型为SEO型 ,获得了来自不同地市汉坦病毒 5个样品M片段 ( 134 3～ 1630nt)序列 ;序列比较分析发现 ,山东省内流行的SEO型汉坦病毒核苷酸的同源性较高 ,型间差异一般不超过 7% ,从种系发生树看这些病毒分布在一个分支上 ,表现出明显的地理聚集现象。结论 近年流行的汉坦病毒仍以SEO型为主 ,且病毒间同源性较高 。

Objective To investigate the genotyping of hantavirus prevailing in Shandong province in recent years and to provide theoretical basis for controlling HFRS Methods McAb-IFA, HI and nested RT-PCR were used to genotype of 98 samples of HFRS patients sera from Shandong Province Selected pars of the samples' PCR products were purified for sequencing Then, RNA sequence analysis was performed by the dideoxynucleotide chain termination method Finally, phologenetic analysis of the sequencing data was performed with Megalign and Editseq software of DAN STAR Results Of all the 26 IFA positive samples 22 were determined as SEO subtype by McAb-IFA and 24 SEO subtype by HI Of all the 72 earlier patients' sera,65 were confirmed sa SEO subtype by nested RT-PCR using three pairs of primers With sequencing PCR products,M segment sequences (1343～1630nt) of 5 samples from different regions in Shandong province were gotten Analysis of homology and phylogenic tree based on the sequences showed that nucleotide sequence difference among SEO subtype viruses is very small (<7%), and phylogenic tree indicated hantaviruses from Shandong Province belong to one branch and showed geographical clustering Conclusion SEO subtype prevailed in Shandong Province in recent years and homology of nucleotide were very high, so gene reassortment is not the reason for HFRS to break out