Development and characterization of monoclonal antibodies against red-spotted grouper nervous necrosis virus and their neutralizing potency in vitro

Zhang, Zhiqi; Xing, Jing; Tang, Xiaoqian; Sheng, Xiuzhen; Chi, Heng; Zhan, Wenbin

doi:10.1016/j.aquaculture.2022.738562

Cited by 7 publications

(9 citation statements)

References 51 publications

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“…7) relevant to virus entry and perhaps subsequent host receptor escape [41]. These novel insights into GNNV structural dynamics should prove useful in facilitating the design of vaccines against NNVs [17,[42][43][44]. Like GNNV, norovirus is a non-enveloped virus with pronounced surface protrusions.…”

Section: Discussionmentioning

confidence: 99%

Cryo-EM inspired NMR analysis reveals a pH-induced conformational switching mechanism for imparting dynamics to Betanodavirus protrusions

Štěrbová,

Wang,

Carillo

et al. 2024

Preprint

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Nervous necrosis virus (NNV), a non-enveloped betanodavirus, causes neuropathies and retinopathies in farmed fish, damaging aquaculture worldwide. NNV has 60 conspicuous surface protrusions comprising the protrusion domain (P-domain) of its capsid protein. Although NNV protrusions play critical roles in infectivity, the underlying dynamics remain unclear. Our cryogenic electron microscopy (cryo-EM)-derived structures of Dragon grouper (Epinephelus lanceolatus) NNV reveal that the protrusions undergo low-pH-induced compaction and movement. We show that the P-domain is monomeric in solution at a pH germane to infection (7.0). Moreover, nuclear magnetic resonance (NMR) structures reveal a peptide (amino acids 311-330) that adopts a flexible loop to form an open pocket. NMR spectral analysis at pH 5.0 aided by molecular dynamics (MD) simulations show that this loop switches to a β-strand under acidic conditions, eliciting pocket closure and P-domain trimerization, highlighting a unique pH-sensing feature. Our docking analysis revealed the N-terminal moiety of sialic acid inserted into and interacting with conserved residues in the pocket. Additionally, a low-pH-induced conformational change in the linker region via peptide bond isomerization conferred malleability on the protrusions. Our work uncovers the protrusion dynamics of a betanodavirus governing its infectivity through a pH-dependent conformational switching mechanism, providing insights into complex virus-host interactions.

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Section: Discussionmentioning

confidence: 99%

Cryo-EM inspired NMR analysis reveals a pH-induced conformational switching mechanism for imparting dynamics to Betanodavirus protrusions

Štěrbová,

Wang,

Carillo

et al. 2024

Preprint

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“…The obtained purified antibody is the mouse anti-grouper IgM polyclonal antibody, named GIgM-Pab [17]. Mouse anti-NNV monoclonal antibody was stored in our laboratory [18].…”

Section: Antibodies and Animalsmentioning

confidence: 99%

“…An absolute fluorescence quantitative PCR standard curve for RNA2 has been established to quantify NNV copies [18]. Total RNA was quantified with Nanodrop 8000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).…”

Section: Quantitative Real-time Polymerase Chain Reactionmentioning

confidence: 99%

Development and Evaluation of a Bicistronic DNA Vaccine against Nervous Necrosis Virus in Pearl Gentian Grouper (Epinephelus lanceolatus × Epinephelus fuscoguttatus)

et al. 2022

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Nervous necrosis virus (NNV) can cause enormous economic losses in mariculture. Vaccines are promising ways to control the disease. In this study: the interferon regulatory factor 3 (IRF3) gene of pearl gentian grouper was cloned and functionally analyzed; then a bicistronic DNA vaccine encoding both capsid protein (CP) and IRF3 was constructed; then the cellular, humoral, and local immune responses in the grouper after immunization were investigated; and then the protective effects after the NNV challenge were investigated. The results showed that the vaccine successfully expressed CP and IRF3. After immunization, the lymphocytes were recruited at the injection site in the muscles. The percentage of sIgM+ lymphocytes in the head, kidney, and spleen significantly increased and peaked at 28.8 ± 3.1% and 42.6 ± 4.2% at the 3rd to 4th weeks. Six immune-related genes were significantly up-regulated. In the meantime, the total antibodies, anti-NNV specific antibodies, and neutralizing antibody titers in serum increased. After the challenge with 105, 106 or 107 TCID50/fish, the relative percent survival rate was 81.25%, 73.91%, and 66.67%, respectively. In 106 TCID50/fish groups, the percentages of sIgM+ lymphocytes, antibodies, and the viral load were investigated. In conclusion, the bicistronic vaccine significantly induced humoral and cellular responses in pearl gentian grouper and provided effective protection against NVV infection.

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“…The monoclonal antibody (3A6) of red-spotted grouper nervous necrosis virus capsid protein (Cp) was produced previously in our laboratory [23] and used in indirect immunofluorescence assay (IIFA).…”

Section: Antibodies and Animalsmentioning

confidence: 99%

“…The genes of B2 protein and Cp were amplified using specific primers listed in Table 1 and in our previous study [23], respectively. The PCR product was purified and inserted into pET-32a prokaryotic expression vector.…”

Section: Antibodies and Animalsmentioning

confidence: 99%

Characterization of Nervous Necrosis Virus (NNV) Nonstructural Protein B2 and Its Enhancement on Virus Proliferation

Zhang

Dong

Xing

et al. 2022

Viruses

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The nerve necrosis virus (NNV), a pathogen of viral nervous necrosis disease in several important mariculture economic fish species, causes economic loss. Its nonstructural protein B2 encoded by the sub-genomic RNA3 affects the amplification of the virus. In this study, the B2 protein was recombinantly expressed, the polyclonal antibodies were produced and the dynamics of the B2 protein and genomes were measured in vivo and in vitro after NNV infection. Then, the effects of the overexpressed B2 protein on virus proliferation were investigated. The results showed that the polyclonal antibodies can recognize the B2 protein in both SSN-1 cells and the brain/eye of the grouper. The RNA3 expression significantly increased at 12 h and kept rising till the end of the experiment; it was 106.9 copies/μL at 120 h. The B2 protein could be first detected at 3 h post-infection, which was earlier than the capsid protein was first detected (12 h post-infection). The B2 protein can be detected in the brain, eye and heart on day 3 and the copy number of genomes reached a maximum at 6 d post-infection. There was a low expression of NNV genomes in the liver, spleen and kidney, and no virus was detected in the gill, stomach and intestine. In the meantime, the B2 protein was successfully expressed in GF-1 cells and significantly enhanced virus proliferation, which produced an earlier cytopathic effect and higher cell death rates after 3 d post-infection than the control. In conclusion, the B2 protein acts as an early expressed protein during virus replication and proliferation and is involved in the early infection of NNV. The results may provide insight into the early stage of virus infection and prevention of the disease.

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Development and characterization of monoclonal antibodies against red-spotted grouper nervous necrosis virus and their neutralizing potency in vitro

Cited by 7 publications

References 51 publications

Cryo-EM inspired NMR analysis reveals a pH-induced conformational switching mechanism for imparting dynamics to Betanodavirus protrusions

Cryo-EM inspired NMR analysis reveals a pH-induced conformational switching mechanism for imparting dynamics to Betanodavirus protrusions

Development and Evaluation of a Bicistronic DNA Vaccine against Nervous Necrosis Virus in Pearl Gentian Grouper (Epinephelus lanceolatus × Epinephelus fuscoguttatus)

Characterization of Nervous Necrosis Virus (NNV) Nonstructural Protein B2 and Its Enhancement on Virus Proliferation

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