Complete Sequence Of Two Rabies Virus Strains And Development Of Reverse Genetics System For Rabies Virus Strain AG

Rabies is a serious zoonosis with worldwide distribution that can cause severe encephalitis inthe central nerve system. Once infected, the morality is nearly100%. At present, vaccination is themost effective method to prevent rabies. The rabies virus aG strain is the vaccine strain employedin China. Although part of the sequence of the genome has been reported, the whole genomesequence is still unknown. As a result, the molecular character of the strain aG, including thewhole length of the genome, organization of the genome, basic structural of the genome, therelation between the structure and functions, the genetic relationship with other rabies strain are allknown. In this study the genome of aG was sequenced and analyzed, cDNA covering the wholegenome, and three helper plasmids, PCI-NEO-N, PCI-NEO-P, PCI-NEO-L have also beenconstruced. Then reverse genetic system was established for aG. The results are as follows:1. The complete genome of aG was sequenced and compared with other rabies vaccinestrains available in GenBank. The results indicated that the genome of aG is11925nt in lengthand encodes five major structural proteins, that is nucleoprotein (N), phosphoprotein (P), matrixprotein (M), glycoprotein (G) and the large (L) RNA-dependent RNApolymerase, in the order3ˊ-N-P-M-G-L-5ˊ. The leader sequence is58nt long while the trailer sequence is70nt inlength, which is similar to that of other rabies strains. The intergenic regions between N-P, P-M,M-G, G-l are2nt、5nt、5nt and24nt respectively. Exact complementarity is found between the firstand the last11nt of the genome, and the region may play an important role in the life cycle ofrabies virus.2. The complete genome sequence of rabies virus CVS-24was sequenced, the resultsindicated that the the whole genome of CVS-24was11927nt, containing5open reading frame,which encodes nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) andthe large (L) RNA-dependent RNA polymerase. The leader sequence is58nt long while the trailersequence is70nt in length, the virus show showed classic rabies virus genome character.3. Primers were designed according to the sequence genome of aG and three segmentscovering the complete genome were amplified, then the full-length cDNA clone of rabies virusvaccine strain aG was assembled from the five subgenomic cDNA fragments and cloned in PCIvector, named PCI-aG. The full-length viral cDNA was flanked by hammerhead ribozyme(HamRz) and hepatitis delta virus ribozyme (HdvRz) sequences, resulted in synthesis ofantigenomic RNA with exact termini, the sequence of HamRz and HdvRz were confirmed bysequencing. Also, in order to recover virus from in vitro, three helper plasmids expressingnucleoprotein, phosphoprotein and large (L) RNA-dependent RNA polymerase were constructed,and confirmed by enzyme digestion and sequencing, named pCINEON、pCINEOP、pCINEOL.4. Cotransfection of pCINEO-N、pCINEO-P、pCINEO-L and PCI-aG into BHK-21with acertain ratio according to the manufactures’ instructions. Five days post-transfection, thesupernatants and transfected cells were subject to RT-PCR, the segments of expected size were obtained.5. The rescued virus has been diluted by10series, and injected Balb/c mice by intracerebralinoculation. After15days, the potency of the rescued virus has been calculated, that is6.2LogLD50.The results indicated the reverse genetics system has been constructed. The system offeredplatform to study molecular pathogenicity mechanism and vector expressing foreign genes.