Classification and identification of pestivirus RNA based on secondary structures

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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