Establishment Of RT-PCR Methods For BVDV Detection And Genetic Variation Analysis Of 5'-UTR Of BVDV

Bovine viral diarrhea virus (BVDV) belongs to Flaviviridae Pestivirus. It is a single strand RNA virus with approximately 12-13kb long genome. The genome consists of 5' uncoding region (5'-UTR), a large open reading frame (ORF) and 3' uncoding region (3'-UTR). 5'-UTR gene sequences are highly conserved among different strains of BVDV, hence, the primers for BVDV nucleotide probes and classification of BVDV type were usually designed based on it. According to the difference of 5'-UTR gene sequences, BVDV can be divided into BVDV-I type and BVDV-II type, and BVDV-I type can be further divided into the BVDV-Ia type, BVDV-Ib type.In recent years, BVDV can be found existed in CSFV vaccine and sick pigs occasionally, but BVDV-infected pigs commonly showed no obvious BVD clinical symptoms, whereas some of them appeared fever scene, or similar clinical symptoms and pathological changes just like CSF. BVDV had a high homology with CSFV, so it often easy to make wrong detection for BVDV and CSFV for their similarity. Therefore, it's necessary and very important to act some research on BVDV detection method and analyze the genetic variation of BVDV for controlling BVD prevalences in pigs.This study has been acted based on bovine viral diarrhea virus 5'-UTR sequences available on the GenBank . Considering the sequences differences of different serotypes of BVDV, and identity between CSFV and BVDV, as well as the differences between BVDVⅠtype and BVDVⅡtype strains, the most conservative of a 218bp long region of 5 'UTR of BVDV genomic sequences was selected as expected amplified fragment, and a RT-PCR method for BVDV detection was established based on it. We detected 6 samples of the standard BVDV strain Oregon C24V, CSFV-positive strain, BTV-positive strain, IBRV-positive strain, PRRSV-positive strain and PPV-positive strain, MDBK normal cell aoso were detected, the results manifested all 6 samples and MDBK normal cell showed BVDV negative except Oregon C24V. The sensitivity of this RT-PCR was up to 10-1TCID50. 70 BVDV-suspicious clinical samples from pigs were detected by the established RT-PCR method. The positive detection rate was about 15.7%. 5 domestic and imported calf serum products for cell culture were also detected, 1 domestic one and 1 imported one showed BVDV positive, which suggested the established detection method was BVDV-specific, sensitive, highly effective and could be operated quickly.The 11 tested BVDV-suspicious positive pig clinical samples, and 1 bovine serum were treated properly, then inoculated on MDBK cells, and obtained 12 BVDV isolates. Some of these isolates formed regular CPE after incubating on MDBK cells, and some of virus do not cause CPE, the CPE caused by the above virus were same with CPE made by BVDV standard strain. 12 isolates were detected by the established RT-PCR method, and the results were all positive, while normal cells detected as negative.The fragments of 12 isolates amplified by the established PCR were ligated into PGEM-Teasy vector. The recombined plasmid PGEM-Teasy / 5'-UTR were transformed into DH5αEscherichia coli. The positive plasmids were screened and identified by restriction enzyme digestion detection and PCR detection. The results show that the obtained positive recombinant gene containing 5'-UTR, and is correctly inserted into the vector. The expected fragments were sequenced and then compared with other BVDV sequences available on GenBank using DNAstar software. The analyses results showed that the fragments were BVDV sequences. The obtained strains showed 77.9%- 98.6% nucleotide identity with BVDV I-type, and 56.2% - 66.5% with BVDV-II type. These determined BVDV sequences can be divided into two subgroups, SX12, SX32, TJ287, XY60, 27, NXQ, SX59, SX57, SX27, 13 belong to BVDV-Ia subtype, SX54, XY55 were BVDV-Ib subtype. .This study isolated and identified some BVDV strains from pigs in Shaanxi province and other 4 provinces, the 5'UTR sequences of the obtained isolates were determined and analyzed. The results reflected the current prevalence extent and features of BVDV in pigs in China from molecular level, which provided useful theory basis for better control of BVDV infection and prevalence.