Vaccination with Recombinant Baculovirus Expressing Ranavirus Major Capsid Protein Induces Protective Immunity in Chinese Giant Salamander, Andrias davidianus.

The Chinese giant salamander iridovirus (CGSIV), belonging to the genus Ranavirus in the family Iridoviridae, is the causative agent of an emerging infectious disease causing high mortality of more than 90% and economic losses in Chinese giant salamanders in China. In this study, a recombinant baculovirus-based vaccine expressing the CGSIV major capsid protein (MCP) was developed and its protective immunity in Chinese giant salamanders was evaluated. The recombinant Autographacalifornica nucleopolyhedrosis virus (AcNPV), expressing CGSIV MCP, designated as AcNPV-MCP, was generated with the highest titers of 1 × 108 plaque forming units/mL (PFU/mL) and confirmed by Western blot and indirect immunofluorescence (IIF) assays. Western blot analysis revealed that the expressed MCP reacted with mouse anti-MCP monoclonal antibodies at the band of about 53 kDa. The results of IIF indicated that the MCP was expressed in the infected Spodoptera frugiperda 9 (Sf9) cells with the recombinant baculovirus, and the Chinese giant salamander muscle cells also transduced with the AcNPV-MCP. Immunization with the recombinant baculovirus of AcNPV-MCP elicited robust specific humoral immune responses detected by ELISA and neutralization assays and potent cellular immune responses in Chinese giant salamanders. Importantly, the effective immunization conferred highly protective immunity for Chinese giant salamanders against CGSIV challenge and produced a relative percent of survival rate of 84%. Thus, the recombinant baculovirus expressing CGSIV MCP can induce significant immune responses involving both humoral and cell-mediated immunity in Chinese giant salamanders and might represent a potential baculovirus based vaccine candidate for Chinese giant salamanders against CGSIV.

[Prokaryotic expression and antiserum preparation for major antigenic epitope region of major capsid protein of Chinese giant salamander (Andrias davidianus) iridovirus].

Objective To express the fusion protein of major antigenic epitope region of major capsid protein (MCP) of Chinese giant salamander (Andrias davidianus) iridovirus (CGSIV) and prepare the rabbit antiserum. Methods Using the genomic DNA of CGSIV Lueyang strain (CGSIV-LY) as a template, the gene fragment of major antigenic epitope region of MCP was amplified by PCR and cloned into the prokaryotic vector pET-21a(+) to construct the prokaryotic expression recombinant plasmid pET-21a-MCP. The recombinant plasmid was transformed into Escherichia coli BL21(DE3). His-tagged fusion protein was induced by IPTG. After identified by SDS-PAGE and Western blot analysis, the recombinant protein was purified by nitrilotriacetic acid (Ni-NTA) agarose resin. New Zealand rabbits were immunized with the purified recombinant protein to generate antiserum. Specificity and titer of the antiserum were determined by Western blotting and indirect ELISA, and then the antiserum was used to detect the CGSIV in the infected EPC cells by indirect immunofluorescence assay. Results The recombinant protein with the relative molecular mass of 29 000 was expressed. The prepared rabbit antiserum had a good specificity and a high titer. Indirect immunofluorescence assay showed that the antiserum could recognize CGSIV in the infected EPC cells. Conclusion The fusion protein of major antigenic epitope region of MCP of CGSIV is successfully expressed and the rabbit antiserum with a high titer and a good specificity been prepared.