Rabbit haemorrhagic disease virus (RHDV) and myxoma virus (MYXV), are two pathogens that have harmful effect on rabbit breeding and population decline of European rabbits in their native range, causing rabbit haemorrhagic disease (rabbit fever) and myxomatosis, respectively. The capsid protein VP60 of the RHDV represents the major antigenic protein. To develop a recombinant bivalent vaccine candidate that can simultaneously prevent these two diseases, we used the nonessential gene TK (thymidine kinase) of MYXV as the insertion site to construct a recombinant shuttle vector p7.5-VP60-GFP expressing the RHDV major capsid protein (VP60) and the selectable marker GFP. Then the shuttle vector p7.5-VP60-GFP was transfected into rabbit kidney cell line RK13 which was previously infected with MYXV. After homologous recombination, the recombinant virus expressing GFP was screened under a fluorescence microscope and named as rMV-VP60-GFP. Finally, the specific gene-knock in and expression verification of the vp60 and gfp genes of the recombinant virus was confirmed by PCR and Western blotting. The results showed that these two genes were readily knocked into the MYXV genome and also successfully expressed, indicating that the recombinant MYXV expressing the vp60 of RHDV was generated. Protection against MYXV challenge showed that the recombinant virus induced detectable antibodies against MYXV which would shed light on development of the effective vaccine.
Animals ; Blotting, Western ; Caliciviridae Infections/veterinary* ; Hemorrhagic Disease Virus, Rabbit/immunology* ; Rabbits ; Vaccines, Synthetic/immunology* ; Viral Structural Proteins/genetics*

Rabbit hemorrhagic disease (RHD) is contagious and highly lethal. Commercial vaccines against RHD are produced from the livers of experimentally infected rabbits. Although several groups have reported that recombinant subunit vaccines against rabbit hemorrhagic disease virus (RHDV) are promising, application of the vaccines has been restricted due to high production costs or low yield. In the present study, we performed codon optimization of the capsid gene to increase the number of preference codons and eliminate rare codons in Spodoptera frugiperda 9 (Sf9) cells. The capsid gene was then subcloned into the pFastBac plasmid, and the recombinant baculoviruses were identified with a plaque assay. As expected, expression of the optimized capsid protein was markedly increased in the Sf9 cells, and the recombinant capsid proteins self-assembled into virus-like particles (VLPs) that were released into the cell supernatant. Rabbits inoculated with the supernatant and the purified VLPs were protected against RHDV challenge. A rapid, specific antibody response against RHDV was detected by an ELISA in all of the experimental groups. In conclusion, this strategy of producing a recombinant subunit vaccine antigen can be used to develop a low-cost, insect cell-derived recombinant subunit vaccine against RHDV.
Animals ; Antigens, Viral/genetics/metabolism ; Caliciviridae Infections/prevention & control/*veterinary/virology ; Capsid Proteins/*genetics/metabolism ; Cell Culture Techniques/*methods ; Codon/genetics/metabolism ; Enzyme-Linked Immunosorbent Assay/veterinary ; *Gene Expression Regulation, Viral ; Hemorrhagic Disease Virus, Rabbit/*genetics/immunology ; *Rabbits ; Recombinant Proteins/genetics/metabolism ; Sf9 Cells ; Spodoptera ; Viral Structural Proteins/*genetics/metabolism ; Viral Vaccines/genetics/immunology