Detections of Rainbow Trout Antibodies to Infectious Pancreatic Necrosis Virus and Viral Hemorrhagic Septicemia Virus via a Recombinant Protein-Based Enzyme-Linked\nImmunosorbent Assay

Abstract

Infectious Pancreatic Necrosis Virus (IPNV) and Viral hemorrhagic septicemia virus (VHSV) cause significant losses in the aquaculture industry. There have been few reports of the use of screening rainbow trout for antibodies against IPNV and VHSV as an epidemiological tool. Several ELISAs using a whole virus or recombinant IPNV and VHSV proteins have been described. In this study, a recombinant protein-based enzyme-linked immunosorbent assay (ELISA) for the detection of IPNV and VHSV antibodies in rainbow trout (Oncorhynchus mykiss) was evaluated. To develop recombinant protein-based enzyme-linked immunosorbent assays, a fragment containing the entire length of the gG gene of VHSV and VP2 of IPNV was amplified by PCR using the viruses genomic RNA and cloned in pET-28a(+) plasmid. Recombinant structural viral proteins (rVP2 and rgG) were expressed in the Escherichia coli BL21 (DE3). The rgG was extracted and purified. 96-well plates were coated with VP2 and gG separately. For VHSV, Assay could detect until 1/15625 dilution in VHSV positive fish serum. For IPNV, Assays could detect until 1/3125 dilution in IPNV positive fish serum. These results show rgG and rVP2, used in ELISA, are more sensitive than virus neutralization tests.