Construction Of Recombinant Rabbit Hemorrhagic Disease Virus (RHDV)Vaccine Using Myxoma Virus (MV) As A Vector

Rabbit Hemorrhagic disease (RHD) and Myxomatosis are considered the major viral diseases affecting European rabbit population. The RHD which caused by Rabbit Hemorrhagic disease virus (RHDV) is an acute and highly contagious disease in wild and domestic rabbits. Infected rabbits usually die within 48 to 72 h of necrotizing hepatitis. The disease is responsible for high economic losses in rabbitries as well as high mortality rates in wild rabbit population. The disease was first reported in China in 1984 and then also reported in other parts of the world. The disease attracted a wide attention for its high rates of death and transmission. The RHDV is a member of the genus Lagovirus, family Caliciviridae. The virion contains a 7.4Kb single-stranded positive-sense RNA genome. The capsid consists of a major protein component of 60 kDa (Vp60). Myxoma virus (MV) whose genome composes of 163kb nucleotides belongs to the Leporipoxvirus genus of the Poxviridae family. This virus replicates in the cytoplasm of infected cells, the typical characteristic of poxvirus. The virus induces a benign disease in its natural host, Sylviagus rabbits in the Americas. In European rabbits, however, MV causes myxomatosis, a systemic and usually fatal disease. This disease only occurs in wild and domestic rabbits and other animals and human are not susceptible to this virus. The virus is usually transmitted by blood-feeding arthropod vectors such as mosquitoes or fleas.In this study, we use the MV as a recombinant vector for delivery of the structural gene VP60 which serves as epitope for antibody. First, we constructed a transfer vector by flanking the VP60 gene of the RHDV and a selection marker GFP with a non-essential gene thymidine kinase (TK) gene of myxoma poxvirus. In this construction, both VP60 and GFP genes with opposite orientation are under control of the 7.5 promoter of the vaccinia virus to generate the transfer vector pMVP60. An appropriate virulence of MV BE4 was selected as the recombinant virus vector. The RK13 cells initially infected with wild-type BE4 virus were transfected with recombinant plasmid pMVP60. Based on homologous recombination between the TK gene of the wild-type MV and that of the transfer vector, recombinant MVs were selected and picked up in the plaques containing GFP marker using the Fluorescence microscopy after infection of RK13 cell monolayer with the supernatant of transfected RK13 cells with pMVP60. The expression of RHDV VP60 by recombinant virus was confirmed by Western blot. This recombinant virus will be used to immunize the wild rabbits by flea-mediated transmission to protect wild rabbit population against both infection of Myxomatosis and RHD.