Early endocytosis pathways in SSN-1 cells infected by dragon grouper nervous necrosis virus

Liu, Wangta; Hsu, Chi‐Hsin; Hong, Yi‐Ren; Wu, Shao-Chun; Wang, Chun-Hsiung; Wu, Yimin A.; Chao, Chia-Ben; Lin, Chan-Shing

doi:10.1099/vir.0.81021-0

Cited by 52 publications

(40 citation statements)

References 31 publications

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“…The different replication behaviour of r247 + 270 in both E-11 and sole neurons could be explained by an interaction with different cell receptors. Sialic acid seems to be involved in the binding of betanodavirus to SNN-1 cells [38] and therefore also to E-11, which are a clone of SSN-1 [17]. However, in other cell lines, other NNV receptors have been identified.…”

Section: Discussionmentioning

confidence: 99%

Betanodavirus infection in primary neuron cultures from sole

Souto

Olveira

Vázquez-Salgado

et al. 2018

Vet Res

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Nervous necrosis virus (NNV), G. Betanodavirus, is the causative agent of viral encephalopathy and retinopathy, a disease that causes mass mortalities in a wide range of fish species. Betanodaviruses are neurotropic viruses and their replication in the susceptible fish species seems to be almost entirely restricted to nerve tissue. However, none of the cell lines used for NNV propagation has a nervous origin. In this study, first we established a protocol for the primary culture of neurons from Senegalese sole, which made it possible to further study virus-host cell interactions. Then, we compared the replication of three NNV strains with different genotypes (SJNNV, RGNNV and a RGNNV/SJNNV reassortant strain) in sole neuron primary cultures and E-11 cells. In addition, to study how two amino acid substitutions at the c-terminal of the capsid protein (positions 247 and 270) affect the binding to cell receptors, a recombinant strain was also tested. The results show that sole neural cells enabled replication of all the tested NNV strains. However, the recombinant strain shows a clearly delayed replication when compared with the wt strain. This delay was not observed in virus replicating in E-11 cells, suggesting a viral interaction with different cell receptors. The establishment of a sole primary neuronal culture protocol provides an important tool for research into betanodavirus infection in sole.

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Section: Discussionmentioning

confidence: 99%

Betanodavirus infection in primary neuron cultures from sole

Souto

Olveira

Vázquez-Salgado

et al. 2018

Vet Res

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“…Recently, SSN-1 fish cells (Frerichs et al 1996) were shown to have receptor-like molecules for RGNNV on their surfaces (Lu et al 2003). The sialic acid moiety on the receptor-like molecule is required for the binding of SSN-1 cells to virus particles (Liu et al 2005). Because SSN-1 cells are susceptible to all the 4 types of betanodaviruses (Iwamoto et al 1999), the putative receptor should be able to bind the 4 kinds of virus particles and induce viral entry into the cells.…”

Section: Discussionmentioning

confidence: 99%

Variable region of betanodavirus RNA2 is sufficient to determine host specificity

Ito

Okinaka

Mori³

et al. 2008

Dis. Aquat. Org.

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Betanodaviruses, the causative agents of viral nervous necrosis in marine fish, have bipartite positive-sense RNA genomes. The viruses have been classified into 4 distinct types based on nucleotide sequence similarities in the variable region (the so-called T4 region) of the smaller genomic segment RNA2 (1.4 kb). Betanodaviruses have marked host specificity, although the primary structures of the viral RNAs and encoded proteins are similar among the viruses. We have previously demonstrated, using reassortants between striped jack nervous necrosis virus (SJNNV) and redspotted grouper nervous necrosis virus (RGNNV), that RNA2, which encodes the coat protein, strictly controls host specificity. However, because RNA2 is large, we were unable to propose a mechanism underlying this RNA2-based host specificity. To identify the RNA2 region that controls host specificity, we constructed RNA2 chimeric viruses from SJNNV and RGNNV and tested their infectivity in the original host fish, striped jack Pseudocaranx dentex and sevenband grouper Epinephelus septemfasciatus. Among these chimeric viruses, SJNNV mutants containing the variable region of RGNNV RNA2 infected sevenband grouper larvae in a manner similar to RGNNV, while RGNNV mutants containing the variable region of SJNNV RNA2 infected striped jack larvae in a manner similar to SJNNV. Immunofluorescence microscopic studies using anti-SJNNV polyclonal antibodies revealed that these chimeric viruses multiplied in the brains, spinal cords and retinas of the infected fish, as in infections by the parental viruses. These results indicate that the variable region of RNA2 is sufficient to control host specificity in SJNNV and RGNNV.

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“…NNV-specific monoclonal antibodies with high neutralizing titers were developed (16), suggesting that NNV-specific receptors exist on the host cell membrane. NNV has been reported to infect cells through receptor-mediated endocytosis and macropinocytosis, and the sialic acid residue is the binding target for NNV during viral binding in susceptible SSN-1 cells (17). However, no NNVspecific receptor protein has been reported.…”

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confidence: 99%

GHSC70 Is Involved in the Cellular Entry of Nervous Necrosis Virus

Chang

Chi

2015

J Virol

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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. IMPORTANCEFish 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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Early endocytosis pathways in SSN-1 cells infected by dragon grouper nervous necrosis virus

Cited by 52 publications

References 31 publications

Betanodavirus infection in primary neuron cultures from sole

Betanodavirus infection in primary neuron cultures from sole

Variable region of betanodavirus RNA2 is sufficient to determine host specificity

GHSC70 Is Involved in the Cellular Entry of Nervous Necrosis Virus

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