Sequence Analysis And Molecular Evolutiou Of The Complete Genomes Of Two Rabies Virus Isolates

Rabies is spreaded all over the world, and has various inquilinous bodies. It isinfected mostly by wild animals in Europe, and infected mostly by cattle in Asia andAfrica. Rabies virus can be divided into seven serotypes now, and several rabiesviruses have not been classified. The number of rabies infections increase these years,because there is no universal investigation in China, so it is unsure about the epidemicsituation of rabies in China. It is essential to study the molecular epidemiology ofstreet rabies virus isolated from the nature in order to prevent rabies infection andunderstand different inquilinous bodies of virus in different areas.This study uss two isolated viruses, the one of which is isolated from a sufferingspotted Deer in a deer factory in Jilin province, and the other one of which is isolatedfrom a suffering mouse somewhere in Henan province. The two isolated viruses aretypical, because they come from different areas and have the trait of differentinquilinous bodies. It is the first time to sequence and analyze the complete genome ofrabies virus in China.In this study, the cDNA is amplified by RT-PCR, and cloned into PMD-18Tvector, the terminal genome is sequenced by 3'-RACE and 5'-RACE. It is designedthat a pair of primers are used in 3'-RACE, two pairs of nesting primers and one 5'phosphated primer are used in 5'-RACE, eight pairs of general primers are used inPCR which cover all the genome of these two isolated rabies viruses. These primersare designed by molecular biology according to the complete genome of rabies viruspublished previously. The products of PCR are purified and is linked to T vector, andthen transferred, cloned. The target vector is identified and sent to sequence in thebiological factory, and the positive coliform added glycerin is conserved by freezing.The result of the complete genome sequence of the two isolated rabies viruses isgained by linking all the fragments using biological tools. The length of completegenome is 11863bp in DRV, and the length of complete genome is 11869bp in MRV.All these two isolated rabies viruses are made up of five genes, the start codon is"ATG", and the stop codon is "TAA" or "TGA" in each gene coding region, and eachposition of their transcriptional signal is precise. Compared with other rabies viruses of serotypeI, the length of the genome is invariant, which shows that there is noinserted or missed gene in the gene coding region of the two isolated rabies viruses.Analyzing the structure of five genes in the two isolated rabies viruses usingcomparing nucleotide method based up on informational theory and data. Thehomology of each gene of the five in the two complete genomes is analyzed, and fivephylogenic trees are made respectively.The result of analysis show that there is several type of mutating includingreplacing, inserting and missing nucleotide in non-coding regions, but somesequences are highly conserved in these regions, which is considered to be associatedwith transcription of gene. There is no inserting or missing nucleotide but individualmutated nucleotide. It is found that there are mutated amino acids in most antibodysites of the two isolated rabies viruses by comparing functional sites of N gene and Ggene, which can affect the virulence and immunity of viruses. It is shown that thesecondary structures of this two isolated viruses are similar and there is similarstructure in 5' non-coding region though their sequences of 5' terminal nucleotide aremutated. There is not obvious variance in the hydrophilic and hydrophobic structureby foreseeing and analyzing their protein secondary structure of the five genes.Comparing the five genes of these two isolated viruses with other rabies virusesin which are typical and selected form GenBank shows that the homology of N, NS,M, G, L genes are 91ï¼…, 91ï¼…, 95ï¼…, 91ï¼…, 91.3ï¼…respectively between DRV and MRV.Compared with DRV, the highest homology of these genes are N gene of 3aG(94ï¼…),NS gene ofNishigahara(95ï¼…), M gene of RH-CL(98ï¼…), G gene of 3aG(95ï¼…), L geneof Ni-CE(98ï¼…) respectively; compared with MRV, the highest homology of thesegenes are N gene of CVS(99ï¼…), NS gene of AV01(98ï¼…), M gene of CVS(99ï¼…), Ggene of CVS-N2C(98ï¼…), L gene of PM1503(99.2ï¼…) respectively.It is shown that G gene has the highest speed during the evolution and has uniqueinquilinous body in order to escape immune pressure from inquilinous body byanalyzing phylogenic trees of five genes in the whole genome. In the evolution of Ggene, DRV and MRV are in different groups. DRV, Chinese vaccine isolated used inhuman and Japanese vaccine isolated used in human are in the same group. MRV,CVS and street viruses isolated from dogs are in the some group. It is shown thatDRV is vaccine isolated compared with phylogenic trees of other genes, and it istestified previous conclusion that unqualified vaccine used for human was used nearthe infected beer cote resulted in spotted deer infected. It is indicated that MRV and street viruses have close phylogenic relation by analyzing the phylogenic tree of eachgene of MRV, and it is short evolutionary time between MRV and DRV by analyzingthe phylogenic tree of L gene. It is illuminated that MRV and street virus in China andAfrica have close phylogenic relation by analyzing phylogenic trees of other genes.Therefore, we can speculate that MRV is a Chinese street rabies virus isolated, whichis a new virus isolated because of variance and evolution of the rabies virus after itinfected mouse.