Isolation And NS_5B Gene Sequencing Of BVDV In Shaanxi Province

Bovine viral diarrhea-mucosal disease (BVD-MD) caused by the Bovine viral diarrhea virus (BVDV) is a serious disease for the cattle , this disease showed many clinical type, infects a large proportion of cattle worldwide and causes a number of clinical forms of the disease ranging from transiently detectable mild clinical symptoms to a fatal diarrheic condition known as mucosal disease.BVD-MD mainly infects cattle, caprine, deer, swine et. The common symptoms shows fever, diarrhea, mucosal anabrosis, aleucocytosis, milk-reduction, abortion or featus anomalies.BVDVcauses persistence infection, immune tolerance,breeding disable, plate-reduction. Since BVD-MD was firstly reported in 1980 in China, it was reported in many other province in the later years. In 1994, Chen Mao-sheng detected this disease by serology in 13 counties of Shannxi province, the results showed that the positive proportion is 12.7%. Recently years, this disease is spreading in Shannxi, but there is not any BVDV strain separated in Shannxi. In 2007, the cattles of one of the scale cattle nursery showed refractoriness diarrhea, and these disease was suspected to be caused by BVDV or RV. Therefore, the cattle intestine absorbent gland was separated and two pairs of primers were designed. The first pair of primers , P1:5′-TGG AGA TCT TTC ACA CAA TAG C- 3′, P2:5′-GCT GTT TCA CCC AGT TAT ACA T- 3′,are used for the amplification of NS5b gene of BVDV, and the other pair of primers P3:5′-AGA ATT TCC GTT TGG CTA-3′;P4:5′-CGG TCA CAT CAT ACA ACT CT-3′was designed according to conventional sequence of VP1 gene of RV.The total RNA was isolated from the intestine absorbent gland according to the procotals of TRIZOL LS? Reagent(Invitrogen), and the results of the RT-PCR show that the 360bp fragments of NS5 gene of BVDVwas succssfully amplyfied,VP1 gene of RV was not amplified.The above results suggest that this disease is Bovine viral diarrhea-mucosal disease. The supernatant of the intestine absorbent gland was innoculated to MDBK cell line, afer 4 passages, the cells showed classical and regular cytopathic effect. The virus was purified by the plaque technic, and the RNA was isolated and detected by RT-PCR, the fragments of NS5b gene were purified by Gel Extraction Mini Kit W5212 and successfully cloned into the pMD18-T vector, the recommbanant plasmid was identified by incision enzyme digestion and sequenced by Sangon CO.Ltd. The determined sequences share great homolgy(99%) with VEDEVAC strain. The supernatant of cell culture infected by the isolated BVDV was serially diluted and innoculated to 96 well plate, the results of the detection by Reed-Muench of TCID50 ranged is 10-3.62/0.1mL...