Heat-denaturation of conformational structures on nervous necrosis virus for generating neutralization antibodies

Nervous necrosis virus (NNV) belongs to genus Betanodavirus within family of Nodaviridae. It is highly pathogenic to various marine fishes. In this study, we determined conformational structures of NNV epitopes for generating NNV-neutralizing antibodies using several rabbit antisera and mouse monoclonal antibodies (MAbs). Rabbit antiserum against naïve NNV particles (anti-NNV serum) with high NNV-neutralization activity reacted with naïve NNV particles, but not with heat-denatured NNV particles, suggesting that NNV-neutralizing antibodies in the anti-NNV serum could not react with heat-denatured NNV antigens. In contrast, rabbit antiserum against heat-denatured NNV particles (anti-HD-NNV serum) reacted with both naïve and heat-denatured NNV particles. However, no NNV-neutralization activity was observed in this anti-HD-NNV serum, suggesting that heat-denatured NNV particles had no epitope for generating NNV-neutralizing antibodies. Mouse MAbs with NNV-neutralization activity also reacted with naïve NNV particles, but not react with heat-denatured NNV particles. Therefore, epitopes of NNV particles for generating NNV-neutralizing antibodies had conformational structures sensitive to heat-denaturation. NNV-neutralizing antibodies were also generated by immunization with SDS-denatured NNV antigens after dialysis and ultrafiltration, suggesting that epitopes for generating NNV-neutralizing antibodies might have relatively simple conformational structures.