Development and characterization of monoclonal antibodies against red-spotted grouper nervous necrosis virus and their neutralizing potency in vitro

Nervous necrosis virus (NNV) has caused severe diseases in global aquaculture industry with tremendous economic and environmental damages. While red-spotted grouper NNV (RGNNV) is the most catastrophic genotype because of plentiful susceptible hosts and expansive distribution ranges. As the only structural protein, capsid protein enables automatic assembly into virus-like particles (VLPs) remaining native infectivity and immunogenicity. In this study, abundant RGNNV VLPs were artificially manufactured and immunized in BALB/c mice, followed by development and characterization of three monoclonal antibodies (mAbs, 2B7, 3A6 and 6E11) subtyped as IgG with a titer of 1:32,000. These mAbs precisely examined two bands (approximately 40 kDa and 120 kDa) of capsid protein by western blot, demonstrating the recognition targets as linear epitopes instead of conformational epitopes. The results of immunofluorescence, immunohistochemistry, immunoprecipitation and immunoelectron microscopy exhibited that all mAbs not only reacted specifically with intracellular viruses, but also exactly labeled free virions, suggesting promising prospects in the detection, localization and tracking of RGNNV. In addition, mAbs 2B7 and 3A6 possessed modest capacity to neutralize RGNNV infection. The pre-incubation of mAbs 2B7 and 3A6 with RGNNV served to suppress the multiplication of virus with 27.99% and 34.37% inhibition ratio, delay the development of cytopathic effect and enhance cell viability by 28.60% and 31.35%, respectively. Taken together, this article proposes several mAbs, which aims at sharing powerful tools for the establishment of potent diagnostics for RGNNV and further investigation into potential pathogenic mechanism. While mAbs 2B7 and 3A6 may advance the intensive development of antibody-based therapeutic countermeasures for RGNNV infection in fish.