三株克里米亚刚果出血热病毒中国分离株糖蛋白基因的全序列测定与比较分析

Sequencing and comparative analysis of the complete glycoprotein gene of three Crimean-Congo hemor-rhagic fever virus Chinese isolates

目的 测定克里米亚 刚果出血热病毒 (CCHFV)中国分离株 (即新疆出血热病毒 ,XH FV)BA6 6 0 19、BA840 2及BA8816 6糖蛋白 (M)基因的全部序列并分析各毒株之间的相互关系。方法 病毒RNA在只与其保守末端互补的特异引物PCM Tag和随机引物的共同引发下逆转录成cDNA ,以单一PCM Tag和高保真DNA聚合酶扩增出完整的M基因。扩增产物纯化后作为模板进行序列测定 ,并将所得序列经计算机辅助分析其种系发生与编码策略。结果 XHFV代表株BA6 0 19的M基因与国际原型株IbAr10 2 0 0的M基因比较多 5个碱基 ,为 5 36 5bp ;比BA840 2、BA8816 6多 4个碱基 ,即5 36 4bp。 3株XHFV长ORF起始于M基因的第 78位碱基 ,比IbAr10 2 0 0株提前 15bp。编码的前体蛋白共 16 89个氨基酸 ,比IbAr10 2 0 0株多 6个。 4株病毒之间核苷酸序列的同源性分别为 :80 9%(IbAr10 2 0 0 BA6 6 0 19) ,80 2 % (IbAr10 2 0 0 BA840 2 ) ,80 2 % (IbAr10 2 0 0 BA8816 6 ) ,83 7% (BA6 6 0 19 BA840 2 ) ,83 6 % (BA6 6 0 19 BA8816 6 ) ,99 0 % (BA840 2 BA8816 6 ) ;对应的氨基酸序列的同源性分别为85 1% ,86 3 % ,86 6 % ,87 8% ,88 0 % ,98 8%。它们与Dugbe病毒在核苷酸和氨基酸的同源性较低 ,大约是 5 5 %和 37 7%。

Objective To determine the complete sequences of glycoprotein(M)gene of Crimean Congo hemorrhagic fever virus(CCHFV)Chinese isolates(Xinjiang hemorrhagic fever virus,XHFV)BA66019,BA8402 BA88166 and to compare and analyze the relationships among the viruses.Methods Based on the conserved reverse complementarity of the CCHFV genomic termini,a primer PCM Tag was designed and used together with random hexmers to initiate reverse transcription of viral RNA to synthesize cDNA.The latter was amplified with single primer PCM\|Tag using the proof\|reading DNA polymerase to produce the complete M gene.The PCR products were gel\|purified and the whole M segment was sequenced and the comparison and analysis were performed aided by computer for the phylogenesis and coding strategy.Results Comparison of the whole M gene sequences of XHFV reference strain BA66019 with international prototype CCHFV IbAr10200 showed an excess of 5 base pairs in length,resulting in 5 365bp;while BA8402 and BA8816 are 5 364bp long,4 more base pairs than IbAr10200.The first start codons of long ORFs were located at the 78th base pair in M gene of BA66019,BA8402 and BA88166,which were 15 base pairs prior to that of IbAr10200.The precursors encoded by these M genes were totaled 1 689 amino acids(AA),6 AAs more than that of IbAr10200.The similaries between CCHFV M genes in nucleotide level were:80.9%(IbAr10200 BA66019),80.2%(IbAr10200 BA8402),80.2%(IbAr10200 BA88166),83\^7%(BA8402 BA66019),83\^6%(BA88166 BA66019),and 99\^0%(BA8402 BA88166),respectively.The corresponding similarities in amino acid level were 85\^1%,86\^3%,86.6%,87.8%,88.0%,and 98\^8%,respectively.The similarities of M genes of CCHFVs and Dugbe virus in both nucleotide and amino acid levels were around 55% and 37%,which were much lower than those among CCHFVs.Conclusion The M genes of XHFVs and IbAr10200 formed respective independent phylogenetic branches genetically and the human\|origin isolate BA88166 might be a variant of tick\|borne BA8402,suggesting that there was only one kind virus prevailing in Xinjiang epdemic areas in 1980s.