Nervous necrosis virus (NNV) is a pathogenic fish virus belonging to family Nodaviridae. The objective of this study was to analyze stabilities of NNV surface protrusion and free coat protein (CP) conformational structures by analyzing changes of NNV infectivity and antigenicity after incubation at moderate-low temperatures. When cultured NNV suspension was incubated at 45 °C, its infectivity declined gradually but its antigenicity maintained. In contrast, both infectivity and antigenicity of purified NNV declined after incubation at 45 °C. After heat-treatment, surface protrusions of NNV particles disappeared completely, although viral particle structures maintained. Therefore, the reduction in NNV infectivity appeared to specifically occur as a result of heat-denaturation of virus surface protrusions. The loss of NNV infectivity in the presence of fetal bovine serum (FBS) was delayed compared to virus heated in the absence of FBS, demonstrating that FBS could function as a stabilizer for conformational structures of NNV surface protrusions. Moreover, the stabilizing function of FBS changed depending on salt concentration. Continued maintenance of antigenicity for heated cultured NNV suspension containing free-CPs may suggest that conformational structures corresponding to protrusion-domain of free-CP are more heat-stable than those of surface protrusions on NNV particles.