Utilization of a monoclonal antibody targeting the functional P-domain of Nervous Necrosis virus (NNV) coat protein: Developing an Epitope-blocking ELISA to quantify viral neutralizing antibodies

Nervous necrosis virus (NNV), a member of the family Nodaviridae, infects over 170 fish species, causing substantial economic losses within the aquaculture industry. The increasing outbreaks across diverse fish species underscore the critical necessity for implementing protocols focused on the monitoring of neutralizing antibodies, as these procedures are indispensable for precise serological diagnosis and comprehensive assessment of vaccination efficacy. In this study, we generated a highly neutralizing monoclonal antibody (mAb), 5E11, targeting a conformational epitope on the capsid protein (cp) of NNV. Epitope mapping via escape mutant analysis identified amino acid S316 in the protrusion (P) domain loop (G311-Q321) of NNVcp as a critical residue. The 5E11 mAb effectively neutralized GGNNV and the highly pathogenic RGNNV 283.2009 strains, with 50% inhibitory concentration (IC50) of 8.7 ng/ml and 20.9 ng/ml, respectively. An epitope-blocking enzyme?linked immunosorbent assay (EB-ELISA) using mAb 5E11 and NNVcp virus-like particles (NNVcp-VLP) was developed for the detection of neutralizing antibodies against RGNNV. The assay’s specificity and sensitivity were assessed using NNV-infected sera obtained from Asian sea bass (ASB) and Orange-spotted grouper (OSG). Results from the assay revealed high sensitivity in the detection of serum neutralizing antibody titers against RGNNV as compared to the gold-stranded virus microneutralization (VMN) assay. Overall, our results demonstrated that EB-ELISA is a reliable and rapid diagnostic method for serological monitoring of RGNNV infection. It is also valuable tool for assessing neutralizing antibody responses post-vaccination in various fish species, potentially leading to more effective control and prevention strategies.