Inclusion of an Arg-Gly-Asp receptor-recognition motif into the capsid protein of rabbit hemorrhagic disease virus enables culture of the virus in vitro.

The fact that rabbit hemorrhagic disease virus (RHDV), an important member of the Caliciviridae family, cannot be propagated in vitro has greatly impeded the progress of investigations into the mechanisms of pathogenesis, translation, and replication of this and related viruses. In this study, we have successfully bypassed this obstacle by constructing a mutant RHDV (mRHDV) by using a reverse genetics technique. By changing two amino acids (S305R,N307D), we produced a specific receptor-recognition motif (Arg-Gly-Asp; called RGD) on the surface of the RHDV capsid protein. mRHDV was recognized by the intrinsic membrane receptor (integrin) of the RK-13 cells, which then gained entry and proliferated as well as imparted apparent cytopathic effects. After 20 passages, the titers of RHDV reached 1 × 104.3 50% tissue culture infectious dose (TCID50)/ml at 72 h. Furthermore, mRHDV-infected rabbits showed typical rabbit plague symptoms and died within 48-72 h. After immunization with inactivated mRHDV, the rabbits survived wild-type RHDV infection, indicating that mRHDV could be a candidate virus strain for producing a vaccine against RHDV infection. In summary, this study offers a novel strategy for overcoming the challenges of proliferating RHDV in vitro Because virus uptake via specific membrane receptors, several of which specifically bind to the RGD peptide motif, is a common feature of host cells, we believe that this the strategy could also be applied to other RNA viruses that currently lack suitable cell lines for propagation such as hepatitis E virus and norovirus.

A DNA-launched reverse genetics system for rabbit hemorrhagic disease virus reveals that the VP2 protein is not essential for virus infectivity.

Rabbit hemorrhagic disease virus (RHDV), a member of the family Caliciviridae comprising positive-stranded RNA viruses, is a highly virulent pathogen of rabbits. Until recently, studies into the molecular mechanisms of RHDV replication and pathogenesis have been hindered by the lack of an in vitro culture system and reverse genetics. This study describes the generation of a DNA-based reverse genetics system for RHDV and the subsequent investigation of the biological role of the RHDV VP2 protein. The full-length RHDV genome was assembled as a single cDNA clone and placed under the control of the eukaryotic human cytomegalovirus promoter. Transfection of cells with the DNA clone resulted in a clear cytopathic effect and the generation of infectious progeny virions. The reconstituted virus was stable and grew to titres similar to that of the parental virus. Although previous reports have suggested that the minor structural protein (VP2) of other caliciviruses is essential for the production of infectious virions, using the DNA-launch-based RHDV reverse genetics system described here it was demonstrated that VP2 is not essential for RHDV infectivity. Transfection of cells with a cDNA clone of RHDV lacking VP2 resulted in the generation of infectious virions. These studies indicate that the presence of VP2 could reduce the replication of RHDV, suggesting that it may play a regulatory role in the life cycle of RHDV.