[Generation and evaluation of a recombinant myxomavirus expressing the VP60 protein of rabbit haemorrhagic disease virus]

Abstract

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.

Keywords: GFP; VP60 protein; homologous recombination; myxomavirus; rabbit haemorrhagic disease virus; thymidine kinase gene.