Genomic Sequencing And Proteomic Analysis Of Wild Type Rabies Virus Isolates From Guangxi

Rabies is a highly pathogenic zoonotic disease caused by rabies virus（RABV）with a 100%mortality rate.Guangxi is an old rabies epidemic area.The prevalence of RABV in Guangxi has showed certain regional characteristics.an epidemiological survey of RABV epidemic reveals the trend of rabies prevalence in Guangxi and therefore is still necessary.In this study,two dogs’brains which one from a rabid dog bite human in Shanglin County and another one from a rabid dog bite cattle in Yizhou City were detected by RT-PCR.Both samples were diagnosed as RABV positive.The two wild rabies isolates were obtained and named as GXSL2018 and GXYZ2018,respectively.To the full-length genome sequence,The primers for fragments of full-length genome were designed and used to amplify the corresponding fragments.Finally,the complete sequences of two RABV genomes were obtained,and the lengths of both isolates were identified as same as 11923 nt.The genetic evolution analysis based on the whole genomes and the five viral genes of the GXSL2018 and GXYZ2018 isolates showed that the two isolates belonged to Group II of Guangxi.The Group II concluded the isolates epidemic from Guangxi and also concluded some isolates epidemic from the other Chinese provinces,or from the countries of Southeast and Southeast Asia.Totally,the isolates of Group II displayed highly stable genetic characteristics.When the amino acid sequence of whole genome were aligned,found that the sizes of structural gene and the important functional sites of each RABV isolate were highly conserved,but the 3’UTR of both GXSL2018 and GXYZ2018 exhibited a nucleotide deletion in the 20^th position.RABV invade brain tissues of human and animals and damaged the brain tissues.In order to explore the mechanism of pathology caused by RABV,in this study we had attempt to perform proteomic analysis of RABV infected mouse brain tissue.Based on the GXSL2018 and GXYZ2018 have the highly similarity of 99.1%nucleotides.Therefore,the GXSL2018 isolate were choose as the research objective to conduct proteomic analysis.we set up four groups:the DMEM as blank control group,r RC-HL attenuated control group,CVS-24classical virulent control group and GXSL2018 isolate group.All differential expressed proteins（DEPs）containing the up-regulated and down-regulated proteins were performed GO annatation（Biological process,BP;Molecular Function,MF;Cellular Component,CC）and KEGG annatation.Also,the proteins-interaction analysis of differential expressed proteins（DEPs）was carried out.In r RC-HL vs DMEM group,BP enrichment indicated that DEPs mainly participated in the process of immune response,MF enrichment indicated that DEPs mainly played the role of antigen binding and immunoglobulin receptor binding,CC enrichment indicated that DEPs were mainly component of intermediate filament and phagocytic vesicle membrane.The enrichment of KEGG pathway found that DEPs of r RC-HL vs DMEM were mainly concentrated in systemic lupus erythematosus and herpes simplex infection.In CVS-24 vs DMEM group,BP enrichment indicated that DEPs mainly participated in the process of immune response,MF enrichment indicated that DEPs mainly played the role of identical protein binding and nucleic acid binding,CC enrichment indicated that DEPs were mainly component of blood microparticle and extracellular space.The enrichment of KEGG pathway found that DEPs of CVS-24 vs DMEM were mainly concentrated in antigen processing and presentation.In GXSL2018 vs DMEM group,BP enrichment indicated that DEPs mainly participated in the process of immune response,MF enrichment indicated that DEPs mainly played the role of identical protein binding and nucleic acid binding,CC enrichment indicated that DEPs were mainly component of intermediate filament and extracellular space.The enrichment of KEGG pathway found that DEPs of GXSL2018 vs DMEM were mainly concentrated in herpes simplex infection and RIG-Ⅰ-like receptor signaling pathway.Finally,three proteins namely SAA1,GRN and Lgals3 with up-regulated expression by proteomic analysis were screened to confirm by q RT-PCR（m RNA）.The final results verified that the transcription levels of SAA1,GRN and Lgals3 in the mouse brain of RABV infection were closely similar with that by proteomic analysis.Especially,the infection of RABV virulent strain could significantly increase the expression levels of the three genes.We further examined the phosphorylation level of Tau protein.Since the Tau protein is a phospho-glycoprotein closely related to the assembly of microtubules in cells.Its main function is to promote microtubule assembly and maintain the stability of microtubules.The mice brains were infected with RABV r RC-HL,CVS-24,GXSL2018 and GXYZ2018,and those brain tissues were collected and specific Tau protein phosphorylation were measured using specific phosphorylation site antibody.The results indicate that the virulent RABV CVS-24,GXSL2018 and GXYZ2018 infection could cause phosphorylation of Tau protein at Thr181,Ser416,Ser396 and Ser214 position,which may destroy the structure and function of neurons and lead to neurological disorders.However,the mechanism of the Tau protein phosphorylation remains to be explored.In this study we have carried out the complete genome sequencing of two wild RABV isolates from Guangxi and harvested a sum of novel epidemiological data.These data will be helpful or reference for rabies control and prevention in Guangxi.Furthermore,a lately isolate RABV GXSL2018was choose to be the researching objective and used to injected mouse brain for preteomic analysis.A batch of DEPs of mouse brains infected with RABV were obtained.These data will be useful and provide important refereces for further studying the pathogenic mechanism of RABV.