Molecular Characterization Of Coxsackievirus A10 And Transcriptome Analysis Of Coxsackievirus A10-infected RD Cells

Objective（s）:In this study,the characteristics of CV-A10 VP1 gene in China,including Coxsackivirus A10（CV-A10）isolated in our laboratory,were analyzed to understand the variation of CV-A10.The P148 virus strain isolated from severe patients belong to the main epidemic genotype in China was selected as the representative strain of CV-A10,and the genetic recombination analysis was performed on the VP1 region,P1 region,P2 region and P3 region to further explore the epidemiological characteristics of CV-A10.Finally,the differentially expressed gene profiles of Rhabdomyosarcoma（RD）cells infected with CV-A10 representative strain P148 was found by RNA sequencing（RNA-Seq）technology,and the pathogenic mechanism of CV-A10 infection was explored.It provides basic data for the diagnosis,treatment,prevention and control of CV-A10.Method（s）:1.The full-length amplification and sequencing of VP1 were performed on11 CV-A10 virus isolates obtained from clinical isolates in the laboratory,and homology analysis was performed with representative CV-A10 virus isolates worldwide retrieved from Gen Bank.2.According to the results of the homology analysis,the representative strains of CV-A10 were selected,and the full-length gene was amplified and sequenced.Based on the sequence information of the VP1 region,P1 region,P2region,and P3 region,the phylogenetic tree was constructed with the prototype strain of enterovirus group A and the sequence with the close genetic relationship after Blast.3.The representative strain of CV-A10 was used to infect RD cells with a multiplicity of infection（MOI）of 0.1,and a different infection time was designed.Then the TCID₅₀ method was used to detect the virus titer in the cell culture supernatant at different time points,and then the optimal time for CV-A10 to infect RD cells was determined.4.The cell samples of the infected group and the control group were collected at the optimal infection time point,and the total RNA of the cells was extracted by the kit,and the purity and concentration were detected,and then sent to the company for high-throughput sequencing.The transcript information of RD cells under different treatment conditions was obtained,and the differentially expressed genes were screened by bioinformatics analysis.Finally,GO（Gene ontology）functional annotation and KEGG（Kyoto encyclopedia of genes and genomes）pathway analysis were performed based on differentially expressed genes to explore the pathogenic mechanism of CV-A10 infection.Result（s）:1.The nucleotide sequence homology of the VP1 region of 11 CV-A10strains isolated in our laboratory from 2009 to 2013 was 94.9%～100%,and the amino acid sequence homology was 98.3%～100%.A phylogenetic tree was constructed based on the full-length VP1 sequences of 11 CV-A10 isolates preserved in the laboratory and 54 representative CV-A10 isolates worldwide retrieved from Gen Bank.Based on the nucleotide homology of the full-length VP1 sequence,the 65 CV-A10 isolates were divided into six genotypes:A,B,C,D,E,and G.After 2009,the main epidemic genotype in China was genotype C,and the 11 CV-A10 isolates in this study belonged to genotype C.The average genetic distance of nucleotides within the group of C genotypes was 5.09%,and the average genetic distance among the groups of other genotypes was16.41%～29.06%.2.The P148 strain isolated from critically ill patients was selected as the representative strain of CV-A10,and its full-length gene was amplified and sequenced.The total length of the whole gene sequence of the P148 isolate after splicing was 7411 bp,and the G+C%content was 47.73%.The phylogenetic tree was constructed based on the sequence information of the VP1 region,P1region,P2 region and P3 region of the P148 isolate and the information of prototype strain of enterovirus group A and strains with the close genetic relationship after Blast.The P2 region of the P148 isolate was found to be in the same evolutionary branch as the CV-A5 isolate（Gen Bank:HQ728261）from Shandong in 2009,and the nucleotide homology was 66.0%.The P3 region of the P148 isolate was closely related to CV-A4 isolates from Hunan（Gen Bank:MK391073）and Shandong（MH086032）in 2016,with nucleotide homology of86.2%and 86.3%,respectively.It is suggested that the P2 region of the P148isolate may have a recombination relationship with CV-A5.There may be recombination with CV-A4 in the P3 region.However,the specific restructuring time and relationship need to be further verified.3.The representative strain of CV-A10 was used to infect RD cells at a multiplicity of infection（MOI）of 0.1.After detecting the TCID₅₀ of the cell supernatant at multiple time points,it was found that the virus titer in the cell supernatant reached a high level at 12 h and 24 h after CV-A10 isolate P148infected RD cells.Therefore,the optimal infection time of CV-A10 isolate P148infected RD cells at an MOI of 0.1 was 12 h and 24 h.4.RNA-Seq technology was used to analyze the transcriptome of RD cells infected with CV-A10 isolate P148 for 12 h and 24 h,and compared with the control group（uninfected RD cells at the same time point）,2610 and 2766differentially expressed genes were screened respectively.Further Venn diagram analysis showed that there were 603 overlapping genes in the differential gene set of infected RD cells at 12 h and 24 h.Further GO and KEGG enrichment analysis of differentially expressed genes showed that the differentially expressed genes were mainly enriched in 308 signaling pathways after 12 h of CV-A10 infection of RD cells.After 24 h of CV-A10 infection,the differentially expressed genes were mainly enriched in 301 signaling pathways.In the process of CV-A10 infection of RD cells,the key pathways with significant enrichment are mostly related to signal transduction,antiviral immunity,cell cycle,apoptosis,autophagy,and other processes,such as m TOR signaling pathway,IL-17 signaling pathway,Wnt signaling pathway and so on.Subsequently,we used GADPH as an internal reference gene to verify the 8 differentially expressed genes randomly selected by RT-q PCR.The results showed that the changing trend of differentially expressed genes was consistent with the RNA-Seq results.Conclusion（s）:1.After 2009,the main epidemic genotype of CV-A10 in China was genotype C.The 11 CV-A10 clinical isolates in this study belong to genotype C,which is in line with the epidemic trend of CV-A10 in China and can be used as a representative strain of CV-A10 in China.2.The P2 region of CV-A10 representative strain P148 may have a recombination relationship with CV-A5;recombination with CV-A4 may occur in the P3 region.3.In the process of CV-A10 infection of RD cells,IL-17 signaling pathway,m TOR signaling pathway and Wnt signaling pathway were significantly enriched,suggesting that RD cells may resist CV-A10 infection through immune regulation,autophagy,ubiquitin system degradation protein and other ways.CV-A10 may achieve immune escape by up-regulating PIM1expression and inhibiting IFN production.