Development and evaluation of an indirect ELISA for detecting Ovine Rotavirus antibodies based on VP6 protein

Background Ovine rotavirus (ORV) infection is a viral diarrheal disease caused by rotavirus, primarily affecting lambs aged 1 to 7 days. Clinical manifestations include diarrhea, anorexia, dehydration, and depression. The disease can lead to significant mortality in sheep populations. Therefore, the development of a highly sensitive and accurate diagnostic method is crucial for effective control in livestock production. Results The VP6 protein, a highly conserved nucleocapsid protein of ovine rotavirus, exhibits broad antigenic cross-reactivity. A His-tagged recombinant VP6 (rVP6) protein was successfully expressed and purified using an Escherichia coli ( E.coli ) expression system. The specificity of the purified rVP6 was confirmed by Western blot analysis. An indirect ELISA (iELISA) method was developed based on rVP6 protein, which detected serum antibodies at a maximum dilution of 1:800. Both intra- and inter-assay coefficients of variation were below 10%. The assay reacted positively with sera positive for ovine rotavirus, but not with sera positive for other diarrheal pathogens in sheep, indicating high sensitivity, reproducibility, and specificity. When compared with an indirect immunofluorescence assay (IFA) for the detection of 125 clinical ovine serum samples, the iELISA showed a concordance rate of 99.2% and a Kappa value of 0.884, demonstrating strong agreement between the two methods. Conclusions The VP6 protein, the most abundant nucleocapsid protein of ovine rotavirus, has been identified as a key antigen for the detection of rotavirus infection. In this study, an iELISA method was successfully developed using rVP6 protein. This assay exhibits high sensitivity, specificity, and reproducibility. The innovative indirect ELISA established herein shows great promise as an effective tool for serological surveillance and epidemiological investigation of ovine rotavirus.