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

Institute of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, PR China.
Journal of General Virology (Impact Factor: 3.18). 01/2009; 89(Pt 12):3080-5. DOI: 10.1099/vir.0.2008/003525-0
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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.

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    • "RCV-A1 causes a non-pathogenic infection and is transmitted via the faecal–oral route This study delivers the experimental proof that the viral RNA detected and characterised in previous studies (Strive et al., 2009) indicated the presence of an infectious virus which causes an entirely non-clinical infection in rabbits. Although recent progress was made towards developing reverse genetics system for RHDV (Liu et al., 2008), lagoviruses including RCV-A1 do not readily grow in cell culture and therefore plaque assay titration of infectious virus is not possible. Measuring viral RNA loads was therefore used to study the course of infection and virus shedding. "
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