Classification and identification of pestivirus RNA based on secondary structures

用逆转录聚合酶链式反应检测了20个不同动物来源的细胞系中是否存在鼠疫病毒污染，发现15个细胞系(75%)呈阳性。对鼠疫病毒基因组5‘非翻译区的RT-PCR产物进行测序并进行基因分型。通过5‘端非编码区分析构建的进化树表明，边境病病毒(BDV)与猪瘟病毒(HCV)之间的亲缘关系远比牛腹泻病毒(BVDV)基因间的亲缘关系近。这表明BDV、BVDV和丙型肝炎病毒是同一物种内的宿主变异体，可以通过比较5‘-UTR3个可变区的二级结构来区分，5’-UTR3个可变区的茎环结构提供了受污染的鼠疫病毒的基因分型。被测试的牛细胞系都被BVDV的I、II或III型中的任何一种污染。狗的细胞系FIFI…BVDV的II型污染了更多的Ne和灵长类动物来源的细胞，山羊来源的ChlEs也被BDV污染。因此，建立了一种基于5‘-UTR二级结构分析的简单实用的鼠疫病毒基因分型方法。由计算机预测的3个稳定的茎环结构V1、V2和V3在5‘-非编码区中包含严格保守的一致碱基配对，这些碱基配对是所有疫病病毒基因型所共有的或每种疫病病毒所特有的。根据二级结构水平上的回文核苷酸替换，已在不同的细胞致病和非细胞致病生物型的鼠疫病毒株中鉴定出6种基因型。它们是牛病毒性腹泻病毒的Ia、Ib、Ic和II型，边境病病毒的II型，猪瘟病毒的IV型。稳定的茎环结构由茎区域的回文核苷酸替换所维持，可作为疫病毒种类和/或基因类型分类和鉴定的参考。较少

Twenty cell lines of various animal origin were examined for the presence of pestivirus contamination by the reverse transcription-polymerase chain reaction, and 15 (75%) cell lines were found to be positive. The RT-PCR products of the 5' untranslated region (UTR) of pestivirus genome were sequenced and subjected to genotyping. A phylogenetic tree among pestiviruses constructed by analyzing the 5'-UTR indicated that the genetic relatedness between border disease virus (BDV) and hog cholera virus (HCV) is much closer than those between genotypes of bovine diarrhea virus (BVDV). This suggests that BDV, BVDV and HCV are host variants within a single species, which can be distinguished by comparison of secondary structures at three variable locations in the 5'-UTR.Stem-loop structures at three variable regions in the 5'-UTR render genotyping of the contaminated pestiviruses. Bovine cell lines tested were all contaminated with any of genotypes I, II or III of BVDV.Cell lines of canine, feli … More ne and primate origin were contaminated with genotype II of BVDV.Cell line ChlEs of caprine origin was contaminated with BDV.Thus a simple and practical method for pestivirus genotyping based on analysis of the secondary structures in the 5'-UTR was developed. Three stable stem-loop structures, V1, V2 and V3, predicted by computer in the 5'-UTR, included strictly conserved consensus base-pairings which are shared by all the genotypes of pestivirus or are characteristic to each genotype of pestivirus. On the basis of the palindromic nucleotide substitution at the secondary structural level, six genotypes have been identified among pestivirus strains, irrespective of the cytopathic and non-cytopathic biotypes. They are genotypes Ia, Ib, Ic and II in bovine viral diarrhea virus, genotype III in border disease virus, and genotype IV in classical swine fever virus. The stable stem-loop structures, which were maintained by palindromic nucleotide substitutions in the stem region, may represent references for the classification and identification of pestivirus species and/or genotypes. Less