RT-PCR-based Differential Diagnosis For BVDV 1 And 2 And The Development Of A Hybridoma Cell Strain Secreting Monoclonal Antibody Against BVDV

Bovine viral diarrhea disease is caused by bovine viral diarrhea virus (BVDV), classified as one member of the genus Pestivirus, family Flaviviridae, which is very important in the bovine infections.Based on the difference in the 5'-untranslated region (5'-UTR) of BVDV genome, it can be divided into two genotypes, genotype 1 (BVDV1) and genotype 2 (BVDV2). BVDV1 with many classical strains and commercial vaccine strains has been widely studied, and the pathogenic strains can cause disease such as fever, diarrhea, and acute/chronic form of mucosal disease; whereas BVDV2, first reported by research groups in North America in 1989, can cause the acute diseases which are characterized by fever, diarrhea, thrombocytopenia, hemorrhages, respiratory disorders, and high abortion and mortality rates. What is more, it can also lead to the abortus, death of the fetus, and the immunosuppressant and abnormality for newborn calves when mother cows were infected during the late stage of the pregnancy.So far, there is not any BVDV2-related case reported in China, how to differenciate BVDV2 from BVDV1, the precondition for that is to set up the diagnosis as soon as possible.Based on the 5'-UTR sequences known in the GenBank from both BVDV1 and BVDV2 isolates, total three specific primer pairs were designed for 5'-UTR amplification using RT-PCR, each one pair for only BVDV1, only BVDV2, or both BVDV1 and BVDV2, respectively, RT-PCR-based methods were set up to differentiate the two genotypes of BVDV, BVDV1 from reference strain NADL and BVDV2 from reference strain 890, which provides a basis of BVDV infection investigation and further molecular epidemiology of BVDV.Of the four structure proteins (nucleocapsid protein C, envelop proteins Erns, E1 and E2) from BVDV, E2 glycoprotein is an immunodominant antigen inducing the neutralizing antibodies after natural infection or following immunization with live or killed vaccines. Therefor, E2 glycoprotein were focused and the BALB/c mice were immunized with the recombinant plasmid pEC143 encoding a secreted form of E2 according to the immune protocol for making monoclonal antibody. Using lymphocyte hybridoma technique, the mouse spleen cells were fused with myelomas cells. A strain of stable hybridoma cell strain, which secretes monoclonal antibody against BVDV1, was obtained by indirect ELISA using the coated antigen from the purified expression products of recombinant pET28a-BE2, and the hybridoma cell strain was stably established, named 3D8, after limited dilution for three times, with immunoglobulin subtype IgM, and the ascites ELISA titer of 2×107. The above preliminary data also makes a basis of serological diagnosis for BVDV.