Sequencing And Genetic Analysis Of The Complete Genome Of13Street Strain Of Rabies Viruses

Rabies is an important zoonotic infectious disease caused by rabiesvirus (Rabies virus, RABV), infect almost all warm-blooded animals and human withnearly100%of mortality rate after the onset of the disease. The incidence of reportedrabies deaths in China seconds only to India among the world’s second seriousthreat to citizens’ lives, causing very heavy psychological and economic burden tothe people. The RABVs have many host animalsleading the conversion of the virus between different hosts, and a varietyof infected animals also cause the migration of virus in different regions.The molecular epidemiology of RABVs is about determination of the genomesequence of different strains by RT-PCR, studies on the relation between mutation ofRABV gene and virulence, antigenicity, host-specificity on the molecular level with avariety of bioinformatic software for comparison and analysis todetermine the strain source and the evolutionary relationship between different strainsand master virus phylogenyand its variation systemematically to establish thefoundation for the development of rabies control strategies and the option ofappropriate vaccines for animals and human.The object of the study is13strains of street RABVs, the main contents areas follows:1. Isolation and identification of the RABV street strainsRABVs were isolated from13kinds of samples by intracerebralinoculation of suckling mice test and neuroblastoma cell separation test. Brain tissuesand cells were identified by using direct immunofluorescence and RT-PCR.The results show that13street RABVs were isolated successfully.2. The complete genome sequencing and comparative analysis of the RABVstreet strainsPrimers used in PCR for each segmant which cover all the genome of theisolated street RABVs were designed. The products of PCR were cloned andsequenced, and the segmants were assembled and edited by DNAStar and Clustalx for analysis of the structure and composition of the whole genome and multiple sequencealignment and homology analysis of five structural genes. Genome nucleotidesequences of13street RABVs were obtained, and the full length genome was11921-11924nts. The five structural gene open reading frame (ORF) included1575nts of the N gene,894nts of the P gene,609nts of the M gene,1575nts ofthe G gene and6386nts of the L gene. The length of N-P-M-G-L intergenic regionswere respectively2nts,5nts,5nts,422nts.The length of Leaderand Trailer were50nts and70nts. In this study, the whole genome sequence of the13street RABVs are in line with the characteristics of the rhabdovirus.Division rabies virus on the structure and composition. The homology of amino acidsencoded by structural gene was higher than the similarity of the nucleotide sequence.The homology of structural gene nucleotide sequence and amino acid sequenceamong GXQZD01, GDMMD57and HuNPB3strain were highly.The rest10viruses showed low homology relatively, while the10viruses showed higherhomology with each other. The length of amino acids deduced by five structuralgenes remained unchanged, variations appeared on the amino acid sequences,but the main function site of the structural proteins were not mutated.3. Phylogenetic analysis of the RABV street strainsThe structural genes and whole genome of the13strains weredrawn a phylogenetic tree using MEGA in this study for phylogenic analysis. Theresults showed that the13viruses belong to genotype1, with farther evolutionarydistance away from foreign RABVs and unique geographical features in China. So the13viruses are likely to be the inherent street strains in nature. TheN gene phylogenetic tree is basically consistent with the tree of the complete genome,and the other trees based on different genes are much the same. Therelationship between CDMMD57, GXQZD01and HuNPB3among the13viruses arecloser and they and the ferret badger isolate are in a separate evolutionary branch.The other10viruses showed closer between each other, and are in a separateevolutionary branch with the domestic dog street RABVs. There is acertain evolutionary distance between the two evolutionary branch.