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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.

SUBMITTER: Gye HJ

PROVIDER: S-EPMC7878751 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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

Gye Hyun Jung HJ Nishizawa Toyohiko T

Scientific reports 20210211 1

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 bindin ...[more]

PMID: 33574489

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Project description:MicroRNAs (miRNAs) could regulate various biological processes. Nervous necrosis virus (NNV) is one of the primary germs of the Humpback grouper (Cromileptes altivelis), a commercial fish of great importance for Asian aquaculture. However, there is limited available information on the host-virus interactions of C. altivelis. miRNAs have been shown to play key roles in the host response to infection by a variety of pathogens. To better understand the regulatory mechanism of miRNAs, we constructed miRNA transcriptomes and identified immune-related miRNAs of C. altivelis spleen in response to NNV infection. Reads from the three libraries were mapped onto the Danio rerio reference genome. As a result, a total of 942 mature miRNAs were determined, with 266 known miRNAs and 676 novel miRNAs. Among them, thirty-two differentially expressed miRNAs (DEmiRs) were identified compared to the PBS control. These DEmiRs were targeted on 895 genes, respectively, by using miRanda v3.3a. Then, 14 DEmiRs were validated by qRT-PCR and showed consistency with those obtained from high-throughput sequencing. In order to study the relationship between viral infection and host miRNA, a cell line from C. altivelis brain (CAB) was used to examine the expressions of five known DEmiRs (miR-132-3p, miR-194a, miR-155, miR-203b-5p, and miR-146) during NNV infection. The results showed that one miRNA, cal-miRNA-155, displayed significantly increased expression in response to the virus infection. Subsequently, it was proved that overexpression of cal-miR-155 enhanced cell apoptosis with or without NNV infection and inhibited virus replication in CAB cells. Oppositely, the cal-miRNA-155 inhibitor markedly suppressed apoptosis in CAB cells. The results of the apoptosis-related genes mRNA expression also showed the regulation of cal-miR-155 on the apoptosis process in CAB cells. These findings verify that miR-155 might exert a function as a pro-apoptotic factor in reply to NNV stimulation in CAB cells and help us further study the molecular mechanisms of the pathogenesis of NNV in C. altivelis.

Project description:Background and Aim:Viral nervous necrosis (VNN) is a serious disease of several marine fish species. VNN causes 100% mortality in the larval stages, while lower losses have been reported in juvenile and adult fish. This study aimed to detect the occurrence of VNN while identifying its associated risk factors and the genotypes of its causative agent in a hybrid grouper hatchery in Malaysia. Materials and Methods:A batch of newly hatched hybrid grouper fry (Epinephelus fuscoguttatus × Epinephelus lanceolatus) were followed from the larval stage to market size. Samples of the hybrid groupers, water, live feed, and artificial fish pellets were collected periodically from day 0 to 180 in the hybrid grouper hatchery. Reverse transcription-polymerase chain reaction (RT-PCR) and nested PCR amplifications were carried out on VNN-related sequences. The phylogenetic tree including the sampled causative agent of VNN was inferred from the coat protein genes from all known Betanodavirus species using Molecular Evolutionary Genetics Analysis (MEGA). Pearson's correlation coefficient values were calculated to determine the strength of the correlation between the presence of VNN in hybrid grouper samples and its associated risk factors. Results:A total of 113 out of 146 pooled and individual samples, including hybrid grouper, water, and artificial fish pellet samples, demonstrated positive results in tests for the presence of VNN-associated viruses. The clinical signs of infection observed in the samples included darkened skin, deformation of the backbone, abdominal distension, skin lesions, and fin erosion. VNN was present throughout the life stages of the hybrid groupers, with the first detection occurring at day 10. VNN-associated risk factors included water temperature, dissolved oxygen content, salinity, ammonia level, fish size (adults more at risk than younger stages), and life stage (age). Detection of VNN-associated viruses in water samples demonstrated evidence of horizontal transmission of the disease. All the nucleotide sequences found in this study had high nucleotide identities of 88% to 100% to each other, striped jack nervous necrosis virus (SJNNV), and the reassortant strain red-spotted grouper NNV/SJNNV (RGNNV/SJNNV) isolate 430.2004 (GenBank accession number JN189932.1) (n=26). The phylogenetic analysis showed that quasispecies was present in each VNN-causing virus-positive sample, which differed based on the type of sample and life stage. Conclusion:This study was the first to confirm the existence of a reassortant strain (RGNNV/SJNNV) in hybrid groupers from Malaysia and Southeast Asia. However, the association between the mode of transmission and the risk factors of this virus needs to be investigated further to understand the evolution and potential new host species of the reassortant strain.

Project description:Nervous necrosis virus (NNV) is a devastating pathogen of cultured marine fish and has affected more than 40 fish species. NNV belongs to the betanodaviruses of Nodaviridae and is a nonenveloped icosahedral particle with 2 single-stranded positive-sense RNAs. To date, knowledge regarding NNV entry into the host cell remains limited, and no NNV-specific receptor protein has been published. Using grouper fin cell line GF-1 and purified NNV capsid protein in a virus overlay protein binding assay (VOPBA), grouper heat shock cognate protein 70 (GHSC70) and grouper voltage-dependent anion selective channel protein 2 (GVDAC2) were investigated as NNV receptor protein candidates. We cloned and sequenced the genes for GHSC70 and GVDAC2 and expressed them in Escherichia coli for antiserum preparation. Knockdown of the expression of GHSC70 and GVDAC2 genes with specific short interfering RNAs (siRNAs) significantly downregulated viral RNA expression in NNV-infected GF-1 cells. By performing an immunoprecipitation assay, we confirmed that GHSC70 interacted with NNV capsid protein, while VDAC2 did not. Immunofluorescence staining and flow cytometry analysis revealed the presence of the GHSC70 protein on the cell surface. After a blocking assay, we detected the NNV RNA2 levels after 1 h of adsorption to GF-1 cells; the level was significantly lower in the cells pretreated with the GHSC70 antiserum than in nontreated cells. Therefore, we suggest that GHSC70 participates in the NNV entry of GF-1 cells, likely functioning as an NNV receptor or coreceptor protein.Fish nodavirus has caused mass mortality of more than 40 fish species worldwide and resulted in huge economic losses in the past 20 years. Among the four genotypes of fish nodaviruses, the red-spotted grouper nervous necrosis virus (RGNNV) genotype exhibits the widest host range. In our previous study, we developed monoclonal antibodies with high neutralizing efficiency against grouper NNV in GF-1 cells, indicating that NNV-specific receptor(s) may exist on the GF-1 cell membrane. However, no NNV receptor protein has been published. In this study, we found GHSC70 to be an NNV receptor (or coreceptor) candidate through VOBPA and provided several lines of evidence demonstrating that GHSC70 protein has a role in the NNV entry step of GF-1 cells. To the best of our knowledge, this is the first report identifying grouper HSC70 and its role in NNV entry into GF-1 cells.

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

Publications

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

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