Functional Research On Rabies Virus M Gene And Polymorphism Analysis Of L Gene Polymerase Activity Module

Rabies is one of nerves infectious diseases caused by rabies virus with 100%mortality rate.Human being and warm-blood animal are susceptible to rabies.Rabies distributes all over the world,mostly in developing countries of Asia,Africa,Europe and America,especially in China and India.Statistical data of WHO indicated that rabies were found in 100 of 145 countries around the world,60000 people died of rabies each year,and became serious threat to human health.The main reasons of rabies prevalence are incorrect consciousness of prevention,low immunized density of domestic animal and failure of immunotherapy immediately after exposure.This study was carried out base on a constructed infectious rRC-HL cDNA clone from a vaccine strain RC-HL used for animals in Japan.The M gene of infectious rRC-HL cDNA clone was replaced with that of rabies virus GX01 isolate epidemic in Guangxi of China.A chimeric GX01 M gene infectious cDNA clone was constructed and a recombinant rabies virus rRC-HL(GX01M)was rescued in BSR cell using reverse genetics technique.Comparing the biological characters of recombinant virus rRC-HL(GX01M)with that of parental rRC-HL,the recombinant rabies virus rRC-HL(GX01M)showed lower virus titer,RNA transcription,protein expression and cell-to-cell spread ability than that of parental rRC-HL virus.To further map the functional domain of GX01 M gene,the deduced amino acid sequence of rabies GX01 M gene was contrasted with that of RC-HL.According to amino acid divergence,4 potential functional domains M1,M2,M3 and M4 of GX01 M gene were chosen and substituted to the corresponding domains of rRC-HL cDNA,respectively.Finally,4 recombinant rabies viruses rRC-HL(GX01M1),rRC-HL(GX01M2),rRC-HL(GX01M3)and rRC-HL(GX01M4)were rescued.In comparison with the biological character of 4 recombinant viruses such as virus titer,virus RNA and protein synthesis,virus plaque and fluorescence intensity,rRC-HL(GX01M2)were found significantly lower than that of the other 3 recombinant viruses rRC-HL(GX01M1),rRC-HL(GX01M3)and rRC-HL(GX01M4),as well as parental rRC-HL virus,demonstrating that M2 domain of GX01 M gene is associated with rabies virus replication,transcription and virus cell-to-cell spread.The L gene polymerase activity module of rabies virus isolates from Guangxi were cloned and sequenced.The sequences were contrasted overseas rabies fixed strains with rabies-related virus strains.The results showed homologies of L gene polymerase activity module nucleotide sequences and deduced amino acid sequences were 88.7%—99.7%and 96.5%—100%between Guangxi strains,84.8%—87.5%and 94.5%—99%between Guangxi strains and fixed strains,75.5%—79.2%and 93.5%—97%between Guangxi strains and rabies-related strains.Phylogenetic tree analysis displayed Guangxi strains could be divided to three groups,group I including GX08,GX091,GX09,GX014,GX195,GX260,GXHX,GXWX,GXLA and GXSL;group II consisting of GX304,GX01,GX074,GX219,GXBM and GXPX;group III just containing GXN119.The amino acid specific mutations of Guangxi strain L gene polymerase activity module were on the positions of 660,754 and 771,especially the position 754 of all Guangxi strains was arginine,while that position of other rabies virus strains was serine,leucine or histidine.