Nervous necrosis virus (NNV) is a pathogenic fish-virus belonging to the genus Betanodavirus (Nodaviridae). Surface protrusions on NNV particles play a crucial role in both antigenicity and infectivity. We exposed purified NNV particles to different physicochemical conditions to investigate the effects on antigenicity and infectivity, in order to reveal information regarding the conformational stability and spatial relationships of NNV neutralizing-antibody binding sites and cell receptor binding sites. Treatment with PBS at 37 °C, drastically reduced NNV antigenicity by 66-79% on day one, whereas its infectivity declined gradually from 107.6 to 105.8 TCID50/ml over 10 days. When NNV was treated with carbonate/bicarbonate buffers at different pHs, both antigenicity and infectivity of NNV declined due to higher pH. However, the rate of decline with respect to antigenicity was more moderate than for infectivity. NNV antigenicity declined 75-84% after treatment with 2.0 M urea, however, there was no reduction observed in infectivity. The antibodies used in antigenicity experiments have high NNV-neutralizing titers and recognize conformational epitopes on surface protrusions. The maintenance of NNV infectivity means that receptor binding sites are functionally preserved. Therefore, it seems highly likely that NNV neutralizing-antibody binding sites and receptor binding sites are independently located on surface protrusions.