Sites responsible for infectivity and antigenicity on nervous necrosis virus (NNV) appear to be distinct

Abstract

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 10^7.6 to 10^5.8 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.

Figure 1

Alteration in antigenicity and infectivity of purified NNV particles after incubation at 37 °C in PBS. Purified NNV particles were incubated in PBS at 37 °C or at 25 °C, and in DIW at 37 °C for 10 days. (A) Alteration of NNV antigenicity detected with ELISA using anti-NNV PAb, (B) ELISA detection using anti-NNV MAb, (C) Alteration of NNV infectivity. (1) Results of a single set of experiments using the same sample on the same day. (2) The same experiments were conducted five times on different days, but limited to 0, 1 and 10 days incubation. Error bars indicate standard deviation (SD). *: Significant difference (ρ < 0.05) compared to the values on day 0.

Figure 2

Alteration in antigenicity and infectivity of purified NNV particles after treatment with Tris-HCl or carbonate buffers. Purified NNV particles were treated with 15 mM Tris-HCl buffers (pH 8.0–9.5) or 100 mM carbonate buffers (pH 8.5–10.0) at 25 °C for 24 h. (A) Alteration of NNV antigenicity detected with anti-NNV PAb, (B) detection with anti-NNV MAb, (C) alteration of NNV infectivity. (1) Results of a single set of experiments using the same sample on the same day. (2) The same experiments were conducted five more times on different days, but limited to treatments with carbonate buffers at pH 8.5 and 9.5 and DIW. Error bars indicate SD. *: Significant difference (ρ < 0.05) compared to the values for the DIW treatment. NT: not tested due to out of an effective buffer range.

Figure 3

Alteration in antigenicity and infectivity of NNV particles following treatments at different urea concentrations. Purified NNV particles were treated with urea solutions at 25 °C for 24 h. (A) Alteration of NNV antigenicity detected with anti-NNV PAb, (B) detection with anti-NNV MAb, (C) alteration of NNV infectivity. (1) Results of a single set of experiments using the same sample on the same day. (2) The same experiments were conducted five times on different days, but limited to treatments at 0 and 2.0 M urea. Error bars indicate SD. *: Significant difference (ρ < 0.05) compared to the values for 0 M urea treatment.

Figure 4

Comparison of declination patterns among NNV antigenicity and infectivity. The declination rate against control (treatment with DIW) were recalculated and are shown in semi-logarithmic graphs. (A) NNV incubated in PBS at 37 °C, (B) NNV treated with carbonate buffers at different pHs ranging from 8.5 to 9.5, (C) NNV treated with different concentration of urea (0 to 2.0 M). (1) Based on the results of a single set of experiments, (2) based on the reproducibility tests with five separate experiments.