Genomic Sequencing For BVDV-2 Isolate XJ-04 And The Development Of Monoclonal Antibody Against BVDV

Bovine-viral diarrhoea (BVD) is a worldwide distributed infectious disease of cattle caused by the bovine viral diarrhoea virus (BVDV), causing a wide range of clinical presentations and considerable economic losses in cattle farming. BVDV is subdivided into two genotypes, BVDV-1 and BVDV-2. Since the end of 1980s, BVDV-2 was first identified in Canada and the USA. BVDV-2 infections involve mainly diarrhea, persistent infection, immunotolerance, congenital defects and a range of reproductive problems including low fertility, abortions and dead fetus. BVDV-2 infection with highly virulent isolates may lead to thrombocytopaenia and fatal haemorrhagic syndrome. BVDV-1 infection has involved in cattle industry for many years, nevertheless, BVDV-2 was first isolated from cattles in China by our laboratory in 2009. Therefore, in this study, we focus on the following two parts: first, preparing the monoclonal antibody against BVDV and developing a double monoclonal antibody-mediated sandwich ELISA combined with biotin-avidin system. Second, sequencing and identification of genome of the XJ-04 isolate of BVDV-2 is undertaken and analyzed. It is to build the foundation of molecular epidemiology research and diagnostic methods for BVDV in China.Study one : RT-PCR strategy was applied to obtain 18 fragments of the sequence of the XJ-04 strain, respectively,i.e. A-Q and the part of 5'and 3'-UTR. The amplified 17 DNA fragments overlapped each other for the most complete genome sequence of XJ-04 except the part of 5'and 3'-UTR. The DNA fragment of the part of 5'and 3'-UTR was obtained by the method of RNA ligation and RT-PCR. The entire nucleotide of XJ-04 was assembled by manual splicing of the validated sequences of all 18 fragments. The complete genome obtained was registered and denoted in GenBank with accession number FJ527854. The genomic RNA of the isolate was 12,284 nucleotide long and contained short 5'- untranslated region (5'-UTR), 3'-non-coding regions (3'-NCR), and one open reading frame (ORF) encoding a large polyprotein of 3,895 amino acids with 20 potential N-glycosylation sites. The phylogram constructed with entire genome sequences and 5'-UTR regions revealed that the isolate belonged to BVDV-2a subtype. The classification was supported by the utmost similarity of XJ-04 to BVDV-2 reference strain 890 at both nucleotide and amino acid level for autoprotease (Npro) gene and structural genes (C, Erns, E1, E2).Study two: Monoclonal antibody(mAb) against BVDV is prepared. Monoclonal antibodies are typically made by fusing SP2/0 cells with the spleen cells of BALB/c mice immuned with the concentrated and purified 890 strain of BVDV-2. Hybridoma cell 3F9 secreting mAb against BVDV-E2 protein were obtained. It belonged to IgG with ascites ELISA titers of 1:216. The monoclonal antibody has no cross reaction with Classical Swine Fever Virus,type 1 Bovine Herpes Virus and Bovine Rota Virus. The ascites were precipitated out using ammonium sulfate to further select for antibody labeling. A double monoclonal antibody-mediated sandwich ELISA was developed using mAb 3D8 as capture antibody and applying standard biotin label methods to label purified 3F9. In a check-board analysis, the working concentration of the capture mAb 3D8 was 2ug/mL, and the Biotin-3F9 was 60ng/mL. With this system, it was possible to detect as low as 84ng/mL of BE2. Furthermore, we detected 35 clinical sera samples through sandwich ELISA method, in which 13 samples were positive. Comparing this result with RT-PCR data, we find 94.25% of them were overlapped. It is suggested that the double mAbs-mediated sandwich ELISA was a valuable method for highly sensitive and specific detection of the BVDV.