| Objective:To investigate the genetic characteristics of the whole genome sequence gene of Coxsackievirus A16(CVA16)strains associated with hand,foot,and mouth disease(HFMD)in China and provide scientific basis for vaccine development and prevention of HFMD.Methods:All complete genome sequences of CVA16 strains associated with HFMD in China and the original group A enterovirus strains,were obtained from the GenBank public database on the National Center for Biotechnology Information(NCBI)PubMed website,then we analysed the genetic characteristics of the whole genome sequence gene of CVA16 strains associated with HFMD in China with a series of bioinformatics software.First,Molecular Evolutionary Genetics Analysis software(MEGA;ver.6.0)was used to analyze the deduced amino acid sequence variation sites in the P1 region of the CVA16 strains.BioEdit(ver.7.0.9.0)was used to perform the homology identity analysis of the nucleotides and deduced amino acid sequences of CVA16 strains in different gene regions.The entropy value of each amino acid site on the structural protein-encoding regions(P1)of Chinese CVA16 strains were also calculated with BioEdit(ver.7.1.9.0).To assess the influence of recombination on the CVA16 diversity,we analyzed the complete genome sequences with the program RDP4(ver.3.5)among Chinese CVA16 strains and the original group A enterovirus strains.The potential glycosylation sites of VP1-VP4 of CVA16 were predicted by using NetNGlyc 1.0 Server glycosylation site predictor.At the same time,we forecasted the B lymphocyte antigen epitopes of the structural protein encoded by P1 gene with the Protean module in DNA Star software and ABCpred network server;T lymphocyte antigen epitopes on the structural protein were predicted by CTLPred and SYFPEITHI online software.Results:1.Compared with the CVA16 prototype strain,some great variations had happened among Chinese CVA16 strains.At the complete genome sequence gene level,the nucleotide identity between the Chinese CVA16 strains and the CVA16 prototype strain was 77%-99.8%.While the nucleotide identity of the Chinese CVA16 strains genotyped as B1 strains was more than 87.6%,except for Fuyang strain in Anhui Province.CVA16 was classified into genotypes A and B1 based on the VP1 gene;the B1b and B1a subgenotypes were the major CVA16 strains and predominated in the coastal areas of China.2.Among the phylogenetic trees constructed with different gene fragments,most of Chinese CVA16 strains have the same genotyping results,but some CVA16 strains were obvious heterogeneity.3.The amino acid sequences of the P1 coding region in Chinese CVA16 strains were highly conserved,although some amino acid variations had happened.Further analysis showed that:there were 57,64,46,22 amino acid mutation sites were observed in the VP1,VP2,VP3,and VP4 structural proteins,respectively;while some amino acid changes were observed with high frequency in the P1 protein-encoding regions:VP1-L23M(13.9%),E145V/A/Q(7.6%),E241K(7.6%),T248A/I(7.6%),N14S(5.1%),N102D(5.1%);VP2-T226A(26.6%),I217V(13.9%),P39R/L(6.3%),V252I(6.3%),P2S(5.1%),T45A/S(5.1%);VP3-N41S/G/P(7.6%),Y185H(6.3%),H41R(5.1%);VP4-V68L(97.5%),M46T(5.1%),K51R/D(5.1%),I61M/F(5.1%).4.Chinese CVA16 strains were not affected by obvious positive evolutionary pressure,most genes were under purifying selection.However,some CVA16 strains might show inter-and intra-typic recombination among group A enteroviruses.The recombination mainly occurred in P2 and P3 non-structural protein coding regions and 5UTR non-protein coding region.5.Potential glycosylation site analysis show that there were 11 potential glycosylation sites in the structural protein encoded by P1.Compared with vaccine candidate strains,there were no significant change in the glycosylation sites among most Chinese CVA16 strains.There were 9 potential B cell surface antigen epitope regions in the structural proteins encoded by P1 region were forecasted by using the Protean module in DNAStar software and ABCpred network server:6-12,41-48,119-125,328-333,399-403,412-420,619-625,803-809,840-847;and we have forecasted 11 T lymphocyte antigen epitopes on the structural protein by using CTLPred and SYFPEITHI online software:474-483,697-706,552-561,382-391,748-757,820-827,808-815,159-168,306-315,636-645,492-497.Conclusions:1.CVA16 was classified into genotypes A and B1 based on the VP1 gene;the B1b and Bla subgenotypes were the major CVA16 strains.The amino acid sequences of the P1 coding region in Chinese CVA16 strains were highly conserved,although some amino acid variations had happened.Therefore,it is necessary to pay close attention to the epidemic information of CVA16 of HFMD.2.Chinese CVA16 strains were not affected by obvious positive evolutionary pressure,some CVA16 strains might show inter-and intra-typic recombination among group A enteroviruses,which mainly occurred in the non-structural protein coding regions and non-protein coding regions,such as P2,P3 and 5UTR regions.3.There were 11 glycosylation sites were found in the structural proteins encoded by P1 coding region sequences of Chinese CVA16 strains,while the number and location of glycosylation sites on the structural proteins of most Chinese CVA16 strains had not changed.More likely to become B cell antigen epitope section in the structural protein of CVA16 were 9 amino acid segments,and 11 T lymphocyte antigen epitopes on the structural protein were predicted by CTLPred and SYFPEITHI online software,which provides scientific basis for the development of HFMD vaccine. |
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