Classification and identification of pestivirus RNA based on secondary structures

用逆转录-聚合酶链反应检测了20株不同动物来源的细胞株是否存在鼠疫病毒污染，其中15株（75%）细胞株呈阳性。对鼠疫病毒基因组5′非翻译区（UTR）的RT-PCR产物进行测序和分型。通过5′-UTR分析构建的鼠疫病毒系统进化树表明，边境病病毒（BDV）与猪瘟病毒（HCV）的遗传亲缘关系比牛腹泻病毒（BVDV）基因型之间的亲缘关系要近得多。这表明BDV、BVDV和HCV是单一物种内的宿主变异，可以通过比较5'-UTR中三个可变位置的二级结构来区分。在5'-UTR的三个可变区域的茎环结构显示了被污染的鼠疫病毒的基因分型。检测的牛细胞系均被BVDV基因型I、II或III中的任何一种污染。犬类、猫科动物和灵长类动物的细胞系感染了BVDV基因型。羊源性ChlEs细胞系被BDV污染。由此建立了一种简单实用的基于5′-UTR二级结构分析的鼠疫病毒基因分型方法。在5′-UTR中，计算机预测了3个稳定的茎环结构V1、V2和V3，其中包含严格保守的一致碱基对，这些碱基对为所有疫病毒基因型所共有或为每种疫病毒基因型所特有。根据在二级结构水平上的回文核苷酸替换，在鼠疫病毒株中确定了6种基因型，而不考虑细胞病变和非细胞病变生物型。它们分别是牛病毒性腹泻病毒的Ia、Ib、Ic和II基因型，边境病病毒的III基因型和猪瘟病毒的IV基因型。这种稳定的茎环结构可为鼠疫病毒种类和/或基因型的分类和鉴定提供参考。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