Complete Genomic Sequence Analysis Of A Sparrows Tembusu Virus And Passaged Study

This study successfully isolated a tembusu virus(TMUV) strain from sparrows inShandong and named Tembusu virus SDS strain.The virulence of this virus was determinedby Reed-Muench method.The complete genomic sequence of this virus was amplified byoverlap RT-PCR to investigate its molecular structure and genetic characteristics. The resultsshowed that full-length genome of this TMUV is10,990nt, containing an open reading frame,encoding a polyprotein of3,425amino acids in length which cleavaged into11viral proteins,and a94nt5'non-coding region and a618nt3' non-coding regions.The nucleotidehomology of genomic sequences between SDS strain and other TMUV isolates ranged from98.2?to99.5?. The aminoacid homology of genomic sequences between SDS strain andother TMUV isolates ranged form98.1?to99.4?. NetNGlyc1.0Server online softwarewas uesd to forecast the potential glycosylation sites. The result showed that the SDS has13potential glycosylation sites in five viral proteins.The results of this study provides animportant theoretical basis for epidemiology of Tembusu virus.The viruses were passaged of50generations.1-40generation of viruses were subculturedwith duck embryo,41-50generation of viruses were subculture alternating with duck embryoand chick subculture.The average death time and median lethal dose for different generationsof viruses was determined. The results show that the10th generation of virus MDT on duckembryo is about84h, the50th generation of virus MDT is101h. The10th generation to50thgeneration on the half duck embryo lethal dose is from10-2.9to10-2.6, did not changesignificantly. Thus,with serial passage of virus, the virulence of virus is slightly attenuated.Different generations virus RNA were extracted,full-length E gene fragment was amplified,the10th generation to40th generation of base substitutions and amino acid mutations is notobvious.40th generation to50th generation appeared more base substitutions and amino acidsmutation,had7base substitution and5amino acid mutations. Thus, more variation may occuralternately subculture on chick and duck embryo than just subculture on the duck embryo inthe same passage. Research indicates that E protein have three Structural domains, domain I,domain II, domain III,50generations virus of E protein three Structural domains havemutations, which distribution I (2), II (1), III (2). Different generations virus's E protein antigen index was predicted by biological information software, result showed that10-40generation virus prediction result is identical,50generation virus predicted result is differentwith10-40generation. that the E protein at439-444position antigenic index is so high whichis different with10-40generation.50th generation of the E protein at441position has aminoacid mutations, the mutation is glycine to aspartic acid. Although high antigen index does notmean that become dominant epitopes,but there may be a theoretical foundation of thestructure and function of proteins and virulence locus studies. About Tembusu virus E proteinamino acids virulence locus is unclear,the change of E gene is the cause of the changevirulence attenuated, but not the only reason. Studies showed that the variation of other genesand non-coding region, deletion or insertion can cause reduced virulence of flaviviruses. Theresult can provides theoretical support to further attenuated mechanism studies and infectiouscDNA cloning technology research.