Construction Of Rabbit Hemorrhagic Disease Virus(RHDV) Replicon And Variation Analyses Of Its Capsid Protein

Rabbit hemorrhagic disease (RHD), a kind of acute, lethal infectious disease caused by Rabbit Hemorrhagic Disease Virus(RHDV), usually threats rabbit production all over the world. It is reported that RHDV has showed genetic variation and brought some difficults in controlling the disease. In order to study whether the genome of RHDV has mutated in past 20 years in China, and to understand the genetic relationship between Chinese RHDV isolates and foreign isolates, the capsid protein(VP60) of eight field RHDV isolates from Zhejiang (from 1989 to 2006) were sequenced and analysed. The results showed that the complete sequence of VP60 gene of eight field RHDV isolates all have the same size of 1740nt, encoding 580aa. The VP60 gene of the eight strains shared high sequence homology (from 94.0% to 99.8%) with those of the reference strains. The results of phylogenetic analysis showed that all the RHDV isolates could be divided into two genetic groups (Gâ… and Gâ…¡), which can be divided into two subgroup(A and B;C and D), respectively. Most of Chinese strains located in C subgroup, and showed closer genetic relation with some European strains. Besides, the phylogenetic tree also indicated that RHDV have evolved to different extents, which means RHDV may exist genetic variation during its epidemic and evolution. These results will be helpful to make efficient controlling measures.RHDV is a kind of acute pathogen, and has great infectivity. It will be dangerous to operate RHDV in normal environment. Besides, the genome of RHDV is a single positive RNA molecular, which means that we can't manipulate its genome with recombinant DNA technology. Reverse genetic technology is a newly developed technique, and has been applied widely in RNA virus research. Recently, the infectious cDNA clone of RHDV has been constructed by Liu, which is benefit for studying RHDV, but the recovered virus is still infectious and dangerous to surrounding. In order to resolve the problem, a safe replicon system of RHDV was constructed in this paper. The protocols are as following: firstly, two nucleotide mutations were introduced into the genome of RHDV, which produced two restricted enzyme recognized sites (NarI) flanked Vp60 gene; secondly, the recombinant DNA plasmid was digested by NarI and VP60 gene was deleted, the final recombinant plasmid is called replicon (pBlReplicon), for it retains all cis elements, RdRp and other non-structural protein coding regions. To study on the replication of the replicon, pBlReplicon was linearized with NruI, and replicon RNA was synthesized in vitro with SP6 polymerase system. Then, the transcripts was transfected into RK-13 cells, and typical RHDV CPE could be observed after 24 hour. Results from RT-PCR, IFA and qRT-PCR confirmed that the replicon RNA could efficiently replicated in RK-13cells. Besides, the results also suggested that the capsid protein is the necessary structural protein for RHDV maintaining infectivity. The construction of RHDV replicon will provide an efficient technology platform for studying on the genome function and pathogenesis mechanism of RHDV, and the study also will be helpful for developing a new type RHD vaccine.