Development of an indirect ELISA based on the VP1 protein for detection of antibodies against water buffalo Hunnivirus

Abstract

Buffalo hunnivirus (BufHuV) is an important pathogen, which can cause diarrhea in water buffaloes, and as yet, there are no vaccines and drugs for its prevention and control. Here we studied the immunogenicity and predicted the three-dimensional structure of the BufHuV VP1 protein, in order to establish a rapid and efficient serological assay for detection of its antibodies in the host. The N-terminal truncated gene, consisting of amino acids 5–117, was selected and cloned into the prokaryotic expression vector pET-32a (+) to obtain the recombinant plasmid, pET-32a-BufHuV-VP1-1. These were then transformed into BL21 Escherichia coli to obtain BufHuV-VP1-1 recombinant proteins, which were then purified for used as coating antigens for ELISAs. An indirect ELISA was subsequently established by optimizing a series of operational steps. This VP1-1-ELISA had good specificity, sensitivity and repeatability, and the coincidence rate between the detection results and western blotting analysis was 95.8 %. A total of 997 clinical bovine serum samples were assessed by the VP1-1-ELISA, and the positive rate was 7.42 %. Overall, the VP1-1-ELISA established in this study is currently the first reported method to detect BufHuV serologically, and it will provide a powerful tool for the detection and epidemiological surveillance of hunniviruses in water buffaloes.