Investigation Of BVDV Infection In Swine In Shandong Province And Sequence Analysis Of E2 Genes Of BVDV

Bovine viral diarrhea virus (BVDV), also known as BVD-MDV is a leading member of the plague virus, belonging to Flaviviridae, Plague virus the same with CSFV and BDV。Now there hasn't any Commercial kits in the market for the diagnosis of BVDV antibody in pig serum . The purified Bovine Viral Diarrhea Virus (BVDV) of NADL and Oregon C24V strains were used as coating antigen, and its cross-reacting determinants between BVDV and CSFV were blocked by the rabbit anti-CSFV positive serum, HRP-labeled goat anti-porcine IgG was used as secondary antibody. Finally, by means of the screening of the optimal reaction conditions, a complex trapping-blocking ELISA, which could be used to detect BVDV specific antibody in pig sera and discriminate CSFV specific antibody from it, was established successfully.And using this method to test 866 swine sera collected from 26 farms that belong to the main pig-industry areas in Shandong province, such as Zibo, Linyi, Rizhao, Jinan, Taian, Weifang and Dezhou , were detected for the specific antibody against BVDV using this assay. The result revealed that the positive rates of anti-BVDV antibody of sow sera and piglet sera were 45.1% (101/224) and 32.3% (209/642) respectively, and 88%(23/26)of pig farms in the Shandong province were positive. Simultaneously, the titer of anti-CSFV antibody was evaluated by positive-indirect hemagglutination in some serum samples. The result indicated that the sera with high titer (1:64~1:256) of anti-CSFV antibody were detected negative for the anti-BVDV antibody, however, the sera with anti-BVDV antibody were low in the titer of anti-CSFV antibody (1:4~1:16). It was suggested that the BVDV infection in swine flocks widespreads in Shandong province, and the generation of anti-CSFV antibody is restrained in swine flocks infected with BVDV.In the result there are five parts showing BVDV is positive when detected BVDV of collected from suspended hog cholera cases in pig farms, through the method of PCR, and in the five parts, four parts of CSFV show positive,one part of PRRSV shows postive,PCV-2,PRV both show negitive。In the study, the BVDV(+) CSFV(-) PRRSV(+) is inoculated into MDBK cells, and get a stable genetic source BVDV named JN-1. It has been sequenced and analyzed. The result showed that, it shares 92.6% identity with ZM-95 of BVDV type 1, and both locate in the same phylogenetic branch. It was demonstrated that the BVDV in sample may belong to BVDV genotype 1, and named it JN-1. We detecting the blood serum of which calf of the testis will be served as primary cell by RT-PCR, on the positive serum for virus isolation .Through the RT-PCR and the gene sequence analysis, a BVDV isolat of genotype 2 named JN-2 was obtained. By plaque assay and TCID50 detection, the JN-2 isolate was purified and it's viral titer rised significantly in MDBK cells.E2 glycoprotein is the major protective antigen. The host naturally infected by the virus or stimulated by the vaccine could quickly produce neutralizing antibodies for E2 protein. The cloning and sequencing results of the E2 of JN-1 and JN-2 showed that, in JN-1 and JN-2 area, nucleotides and amino acids were less conservative, higher rate of protein variability which may lead to the escape, vaccine failure and so on.