Recognition of Linear B-Cell Epitope of Betanodavirus Coat Protein by RG-M18 Neutralizing mAB Inhibits Giant Grouper Nervous Necrosis Virus (GGNNV) Infection

Abstract

Betanodavirus is a causative agent of viral nervous necrosis syndrome in many important aquaculture marine fish larvae, resulting in high global mortality. The coat protein of Betanodavirus is the sole structural protein, and it can assemble the virion particle by itself. In this study, we used a high-titer neutralizing mAB, RG-M18, to identify the linear B-cell epitope on the viral coat protein. By mapping a series of recombinant proteins generated using the E. coli PET expression system, we demonstrated that the linear epitope recognized by RG-M18 is located at the C-terminus of the coat protein, between amino acid residues 195 and 338. To define the minimal epitope region, a set of overlapping peptides were synthesized and evaluated for RG-M18 binding. Such analysis identified the 195VNVSVLCR202 motif as the minimal epitope. Comparative analysis of Alanine scanning mutagenesis with dot-blotting and ELISA revealed that Valine197, Valine199, and Cysteine201 are critical for antibody binding. Substitution of Leucine200 in the RGNNV, BFNNV, and TPNNV genotypes with Methionine200 (thereby simulating the SJNNV genotype) did not affect binding affinity, implying that RG-M18 can recognize all genotypes of Betanodaviruses. In competition experiments, synthetic multiple antigen peptides of this epitope dramatically suppressed giant grouper nervous necrosis virus (GGNNV) propagation in grouper brain cells. The data provide new insights into the protective mechanism of this neutralizing mAB, with broader implications for Betanodavirus vaccinology and antiviral peptide drug development.

Nodaviruses in Wild Fish Population Collected Around Aquaculture Cage Sites from Coastal Areas of Tunisia

“…The evolutionary distances were computed using the Kimura 2-parameter method [26] and are in the units of the number of base substitutions per site [27][28][29][30]. GenBank accession numbers and references for nucleotide sequences are as follow: EU826138.1 [31], EF617326 [32], NC_013461 [33], KF386164 [34], KY354698.1 [35], GU826692.1 [36], HE796793.1 [37], KY354694.1 [35], HE796789.1 [37], HE796797.1 [37], HE796788.1 [37], HE796791.1 [37], HE796790.1 [37], HE796785.1 [37], HE796798.1 [37], HE796799.1 [37], HE796794.1 [37], HE796786.1 [37], AJ698093.1 [38], AF318942.1 [39], AF499774.1 [40], HE796795.1 [37], HE796796.1 [37], HE796787.1 [37], HE796784 [37], KM588181.1 [41], KP455642.1 [42], AY284968.1 [43], HE796792.1 [37], JN190021.1 [44].…”

Characterization of Hagfish (Eptatretus burgeri) Variable Lymphocyte Receptor–Based Antibody and Its Potential Role in the Neutralization of Nervous Necrosis Virus

“…The ability of Abs to neutralize virus is usually confirmed by the lack of a CPE in infected cells, and treatment of the neutralizing Abs can decrease the numbers of cytopathic cells, compared with the untreated conditions (25,(31)(32)(33). In general, the capacity of Abs to neutralize a virus in vitro is 1 3 10 2 TCID 50 in a neutralization test; however, the VLRBs produced in this study were able to inhibit the viral infection at ,13 10 2 TCID 50 (data not shown).…”

“…Nervous necrosis virus (NNV), also known as Betanodavirus, causes viral encephalopathy and retinopathy, which is an infectious disease responsible for high levels of mortality in more than 40 species of marine and freshwater fish. Infection by this virus leads to abnormal behavior and visual dysfunction, such as spiral swimming along with extensive vacuolation and neuronal degeneration (24)(25)(26). Studies cited in the previous sentence have mAbs from seabream (Sparus aurata) and giant grouper (Epinephelus lanceolatus), but our mAb against NNV was produced from hagfish (Eptatretus burgeri).…”

Betanodavirus and VER Disease: A 30-year Research Review

“…Types I and II-IFN are present in fish [211,212] and both have been detected in individuals infected with betanodavirus [163]. I-IFN transcription has been reported to be up-regulated in sea bass [213] and grouper [212], II-IFN production increased in experimentally infected turbot [214], whereas both I and II IFN were induced in infected zebra fish [215]. IFN up-regulation has also been described in in vitro assays, in barramundi brain (BB), grouper brain (GB) and FHM cells [216][217][218].…”