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The aim of this article is to express envelope glycoprotein E2 of bovine viral diarrhea virus (BVDV) in yeastPichia pastorisand its utility as a diagnostic antigen in ELISA. The BVDV E2 gene was cloned into the pPICZαA vector followed by integration into thePichia pastorisstrain X-33 genome for methanol-induced expression. SDS-PAGE and Western blot results showed that the recombinant BVDV E2 protein (72 kDa) was expressed and secreted into the medium at a concentration of 40 mg/L of culture under optimized conditions. An indirect ELISA was then developed by using the yeast-expressed E2 protein. Preliminary testing of 300 field cattle serum samples showed that the E2 ELISA showed a sensitivity of 91.07% and a specificity of 92.02% compared to the reference virus neutralization test. The concordance between the E2 ELISA and VNT was 91.67%. This study demonstrates feasibility of BVDV E2 protein expression in yeastPichia pastorisfor the first time and its efficacy as an antigen in ELISA for detecting BVDV neutralizing antibodies in cattle. [ABSTRACT FROM AUTHOR]

The aim of this study was to develop an indirect ELISA using the helicase domain of bovine viral diarrhoea virus (BVDV) NS3 protein instead of full-length NS3 protein for detection of BVDV and BDV antibodies in sheep and goats and its validation by comparing its sensitivity and specificity with virus neutralization test (VNT) as the reference test. The purified 50 kDa recombinant NS3 protein was used as the coating antigen in the ELISA. The optimal concentration of antigen was 320 ng/well at a serum dilution of 1:20 and the optimal positive cut-off optical density value was 0.40 based on test results of 418 VNT negative sheep and goat sera samples. When 569 serum samples from sheep (463) and goats (106) were tested, the ELISA showed a sensitivity of 91.71% and specificity of 94.59% with BVDV VNT. A good correlation (93.67%) was observed between the two tests. It showed a sensitivity of 85% and specificity of 86.6% with VNT in detecting BDV antibody positive or negative samples. This study demonstrates the efficacy of truncated recombinant NS3 antigen based ELISA for seroepidemiological study of pestivirus infection in sheep and goats. [ABSTRACT FROM PUBLISHER]