Identification Of An Antigen Epitope On The P80 Protein With Monoclonal Antibody And Isolation And Characterization Of The Virus Strain LJ36/14 For Bovine Viral Diarrhea Virus

Bovine viral diarrhea virus can cause bovine viral diarrhea/mucosal disease. BVDV infection includes some clinical signs, for example depression, feed intake decrease, salivating, diarrhea, congestive ulcer of oral mucosa. Pregnant cows may abort or output malformation fetus, and newborn calves often have lethal diarrhea. At present, BVDV were distributed worldwide, and cause huge economic losses. BVDV is the most common exotic virus for the tissue or cell culture and biologicals. According to the difference of BVDV 5’UTR, BVDV can be divided into two genotypes: BVDV-1 and BVDV-2. Different genotypes contain different subgenotypes. According to whether or not BVDV produces visible changes in cell cultures, it can be divided into cytopathogenic type(CP) and non cytopathogenic type(NCP). If the pregnant cows were infected with NCP type BVDV, it will lead to fetal immune tolerance. The fetus continues to develop or abort. If the fetus was born smoothly, it will become persistently infected(PI). The PI calf will continue to shed virus to susceptible animals. So far, there is no commercial kit and vaccine available in China.The p80 protein is one of the BVDV non structural proteins, highly conserved in the genus Pestivirus, is an immunodominant protein and has important diagnostic value. In this study, we used prokaryotic expression system to express the recombinant protein p80, which can react specifically to rabbit polyclonal antibody against BVDV in Western blot analysis. At the same time, six and eight-week-old female Balb/c mice were immunized with concentrated BVDV cultures. After three immunizations, the spleen cells of the mice were fused with SP2/0 cells. Hybridomas were screened by indirect ELISA coated with purified p80 protein. The hybridomas were tested with indirect immunofluorescence assay(IFA) after three subcloning. The results showed the recombinant p80 protein had a good response. And eight hybridomas(3E3, 2A4, 8H9, 3G3, 2B3, 3G2, 2G2 and 4A11) producing monoclonal antibodies(MAbs) were obtained, and the ascites titer ranged from 1×105-1×107.The MAbs owned a good reactivity and specificity in IFA. All of the eight MAbs subtypes were IgM/κ. Theses prepared MAbs against the recombinant p80 protein might be used to establish a diagnostic method to test BVDV. Using a prokaryotic expression vector pGEX-6p-1, a series of overlapping short peptides of p80 protein were successfully expressed. We identified an antigen epitopes in p80 protein 446 to 558 amino acids by Western blot analysis.BVDV contained many different subgenotypes in different regions, which brought great challenges to the prevention and control of BVDV. To investigate the pathogens related with bovine respiratory disease, forty bovine nasal swabs collected from feedlot cattle showing respiratory disease were inoculated into MDBK cell monolayers for virus isolation from Heilongjiang Province in 2014. A bovine viral diarrhea virus strain was isolated and named as LJ36/14. The virus was further characterized. LJ36/14 was NCP and could not cause cytopathic effects(CPE) in MDBK cells. Typical virions were observed under transmission electron microscope after negative staining. Specific immunofluorescence was observed in LJ36/14 inoculated MDBK cells stained with monoclonal antibody to BVDV p80 protein. The 50% tissue culture infective doses(TCID50) was 105.9/0.1mL for LJ36/14. The phylogenetic reconstructions were created using the 5’UTR and Npro gene sequences of LJ36/14 and other reference BVDV strains. The phylogenetic tree analysis showed that LJ36/14 formed a separate group and was a new subgenotype, and was typed as 1v.In summary, eight MAbs of BVDV were prepared successfully and had good specificity and reactivity in indirect immunofluorescence assay. The MAbs might be used to establish a diagnostic method for detecting BVDV antigen. We identified an antigen epitopes in p80 protein 446 to 558 amino acids. This result laid the foundation for research on the structure and biological function of BVDV p80 protein. At the same time, a BVDV strain LJ36/14 was successfully isolated and typed as NCP and BVDV-1v subgenotype for the first time. This result was also useful for understanding the geographical distribution and epidemic situation and BVDV vaccine development.