Rescue And Replication Characteristic Of Rabbit Hemorrhagic Disease Virus With Capsid Protein Deletion

Rabbit hemorrhagic disease virus (RHDV) is the causative agent of a highly contagious disease of wild and domestic rabbit, belonging to the family Caliciviridae, genus Lagovirus. Study on the pathogenesis of RHDV is helpful for better control the disease. Capsid protein is the most important structural protein of RHDV, which not only determines the immunogenicity of RHDV, but also plays crucial role in the process of infection and virus replication. However, so far we know less of the relation between the capsid protein and the infectivity of RHDV. In this paper, we try to explore these scientific questions using revers genetics technology.The results shows that mutant viruses could be rescued from RK13 cells when the mutant RNAs were tranformed into RK-13 cells, and the progeny viruses still keep infectivity to RK13 cells, suggesting that RHDV are infectious without capsid protein. To better understand the relation between capsid protein and the expression, replication and infectivity of RHDV, RT-PCR, IFA and QRT-PCR were used, respectively. The result of RT-PCR showed that both the positive and negative genomic RNA of RHDV can be detected, indicated that the mutant viruses has replicated in RK13 cells. The results of IFA also showed that viral proteins were expressed in the transfected cells. The results of qRT-PCR showed that the genomic copies of RHDV mutant (pBLRHDV-d (5325-6931)) were related with the culture time of virus. After RK13 cells were transfected with RHDVmutant RNA at about 6h, genome copy number was 4.66×10~8 gene copies / mL, at 24h, the virus genome copy number peak (1.11×109gene copies/mL), and then the genomic copies of RHDV gradually decreased. As for the pBLRHDV-dV60, after transfected RK13 cells, the mutant virus showed similar replication curve to that of pBLRHDV-d (5325-6931), at 6h, its genome copy number was 6.23×10~8 gene copies / mL, at 24h, the virus genome copy number reached its peak (8.81×108 genes copies / mL). But the total replication level of pBLRHDV-dV60 was lower than that of pBLRHDV-d (5325-6931), indicating that may there are some virus replication related elements in the coding region of capsid protein.