Generation of monoclonal antibodies against heterologously expressed major capsid protein of infectious spleen and kidney necrosis virus (ISKNV)

Abstract

Infectious spleen and kidney necrosis virus (ISKNV) is one of the detrimental viruses causing economic losses in several fish species including Asian sea bass (Lates calcarifer). In this study, monoclonal antibodies specific to major capsid protein (MCP) of ISKNV were generated. The MCP encoding gene of ISKNV genotype II was cloned into pGEX-6P-1 and transformed into E. coli. After induction, a glutathione-S-transferase (GST)-MCP fusion protein was produced and used for immunization of Swiss mice for monoclonal antibody (MAb) production. Five MAbs specific to MCP were obtained after selection against naturally ISKNV-infected Asian sea bass by dot blotting, Western blotting and immunohistochemistry. All MAbs showed no cross-reaction with fish tissues, fish viruses including tilapia lake virus (TiLV), nervous necrosis virus (NNV), scale drop disease virus (SDDV) and eight bacterial species commonly found in diseased fish. The detection sensitivity of MAbs was inferior to that of one step PCR. However, a combination of two MAbs enhanced the detection limit about two-fold. The intense immunoreactivity staining could be observed in liver, kidney, gill, intestine, stomach, and spleen tissues from ISKNV-infected fish by immunohistochemical analysis. All five MAbs can be used to detect naturally ISKNV-infected fish from two different farms and phylogenetic tree analysis based on MCP gene sequences demonstrated ISKNV genotype I infection in diseased fish. In addition, all MAbs displayed immunoreactivity to red sea bream iridovirus (RSIV) in the GF cell culture fluid by dot blotting. Therefore, the generated MAbs could be used for detection of ISKNV infection by various immunological assays and might be able to detect RSIV infection as well. © 2022