| Bovine viral diarrhea virus (BVDV) is a member of the genus Pestivirus family, which can infect bovine, sheep, pig and deer and cause bovine viral diarrhea/mucosal disease. BVDV infection has resulted in a significant loss in the livestock industry all over the world, especially in USA and New Zealand. According to the difference of their heredity and antigenicity, BVDV can be divided into two genotypes as type I BVDV and type II BVDV. The clinical syndromes caused by type II BVDV acute infection are much more severe than type I BVDV and currently there is no commercial BVDV vaccine in China. Most of overseas BVDV vaccines are against type I BVDV, however, cross immune protection between different genotype strains are not strong enough for animal immunity, all the above making the investigation of type II BVDV vaccine quite urgent .The BVDV JZ05-1 strain isolated from cattle in China can be cultured steadily on MDBK cells and has good immunogenicity and no pathogenicity in host. The analysis of biotype and genotype of JZ05-1 was performed through viral culture, fluorescence observation and RT-PCR genotyping. Entire genome sequence was cloned and sequenced by RT-PCR. In order to investigate the genetic background and characters of the JZ05-1 strain, the genome sequence was analyzed by bioinformatics methods.The BVDV JZ05-1 strain generates cytopathic effects in MDBK cells, thus is a typical cpe BVDV strain. The results of fluorescence antibody detection and RT-PCR genotyping indicate that the BVDV JZ05-1 strain belongs to type II BVDV. BVDV contains a single positive-stranded RNA genome without poly(A) tail at 3'-terminal, thus the traditional method of 3'-RACE cannot be used for the amplification of the 3'-terminal of BVDV genome. When we performed multiple sequence alignment of 14 strains of BVDV-2 entire genome sequences, we found there is consensus sequence GCn at the 3'-terminal of BVDV sequence. According to this consensus sites, we designed palindrome anchorage primer CGend to reverse transcribe and amplify the 3'- terminal of the BVDV JZ05-1 genome. RT-PCR amplification products using primer CGend were much better compared with the random primer control and other downstream gene specific primer. Using this method, the 3'-terminal sequence was cloned and sequenced. This simple and practicable method of reverse transcription and 3'- terminal sequencing might be useful in research of other Pestivirus.Eight overlapped gene fragments amplified by RT-PCR were sequenced, and assembled to get the complete genomic sequence of BVDV strain JZ05-1. According to our analysis, the JZ05-1 genome is composed of 12 285 nucleotides (GenBank accession No. GQ888686), which can be divided into three regions: a 387-nt 5'-untranslated region (UTR), an 11694-nt single large open reading frame encoding a polyprotein, and a 204-nt 3'-UTR.The evolution distribution of BVDV 5'-UTR and genome sequences were analyzed by constructing its phylogenetic tree. We found that theJZ05-1 strain was classified as BVDV type 2a. The BVDV-2 strain JZ05-1 genome showed high similarity to the p11Q isolated in Canada and the XJ-04 isolated in China, with 90% and 91% identity in nucleotide sequence, respectively. Compared with the similarity within the BVDV-2 genotype (96%), the JZ05-1 has low sequence similarity with other BVDV-2 strains. Compared with other BVDV-2 strains, the untranslated regions of JZ05-1 strain have unique secondary structure. Except the variable regions, mutations in untranslated regions are mainly located between stem and loop structures. There are no large insertion or deletion mutations in JZ05-1 genome. Most of the point mutations in JZ05-1 genome are neutral mutation, and most of the amino acid mutations are substitution between polar amino acid sites with short side chains. Most amino acid mutations in RdRp protein localized on protein surface with good accessibility, which could not affect the RdRp protein tertiary structure and active center.
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