A Study On The Molecule Evolution Of Coxsackievirus A6 Based On Bayesian Method

Background and Objective: Hand,Foot and Mouth Disease(HFMD)is a global epidemic of acute infectious diseases characterized by the appearance of vesicles on the hands,soles,and perioral vesicles of children under 5.Most patients have mild symptoms and can use their own immune system to heal themselves.However,if they do not pay attention,they can easily cause complications such as meningitis and pulmonary edema,and are life-threatening.In recent years,the incidence of HFMD has been increasing year by year,and its mortality rate has far exceeded that of Class C infectious diseases in China.The disease has seriously jeopardized children's life and health,and this has also attracted the attention of the Chinese health department.For a long time,human enterovirus 71(EV71)and Coxsackievirus A16(CVA16)were widely considered to be the main pathogens of HFMD.In recent years,the number of cases of HFMD induced by Coxsackievirus A6(CVA6)gradually increased.Due to the development of second-generation sequencing technology,the research of viruses at the molecular level continues to deepen.The development of computers has prompted the development of bioinformatics technology.The use of computer methods to analyze biological problems has become the current trend.The maximum clade credibility tree constructed by Bayesian method is more accurate and more efficient than other methods.It makes the model of the phylogenetic tree more accurate by defining the most suitable nucleotide substitution model,while calculating the posterior probability to normalize the resulting data.Based on the advantages of the Bayesian approach,researchers have used it for phylogenetic analysis of viruses.In the case of enteroviruses,gene recombination is widespread.The virus adapts to the environment through the transfer of genetic information and generates new genotypes.Therefore,gene recombination is an important cause of virus mutation and evolution.At present,the vaccine for EV71,the main pathogen of hand-foot-and-mouth disease,has been used clinically.CVA16 vaccine is also intense development.However,research and development on CVA6 vaccine is rarely reported.Therefore,this study deeply understands the evolutionary trends and genetic characteristics of CVA6,which not only has practical value in the prevention and control of HFMD,but also provides a theoretical basis for the development of vaccines and screening of drug targets.Materials and Methods: A total of 57 samples of hand,foot and mouth disease were collected from Changchun City in 2015.The virus samples were subjected to molecular typing by reverse transcription-PCR(RT-PCR),and 11 CVA6 VP1 genes were amplified and sequenced.The sequencing results were uploaded to Genbank.From the Genbank,353 CVA6 VP1 gene sequences with well-defined separation times and locations were downloaded.Excess sequences were cut with MEGA for recombination detection,saturation detection,and selection of the most suitable nucleotide substitution model.The pre-processed sequences were used to establish the maximum clade credibility tree(MCC)by Bayesian method,and the Bayesian skyline plot was drawn to analyze the evolutionary characteristics of CVA6 VP1 gene.The 148 CVA6 whole genome sequences were downloaded from Genbank and compared with MEGA.RDP4 and Simplot were used for recombination analysis to further explore the evolutionary trends and genetic characteristics of CVA6.Result : In this study,eleven strains of CVA6 were isolated from 57 samples of HFMD.Compared with the original strains,the nucleotide mutation rate of the 11 CVA6 VP1 genes obtained from sequencing was 16.8%-17.6%,and the amino acid mutation rate was 3.3%-6.7%.The maximum clade credibility tree constructed according to the Bayesian method showed that CVA6 was divided into five genotypes of GI-GV from 1992-2015,GIV genotype was further divided into GIV-1 and GIV-2 genotypes.GV genotype was further divided into GV-1 and GV-2 genotypes.Of the 11 CVA6 isolates isolated in this study,10 were GV-2 and one was GIV-2.The CVA6 that has become popular in China in recent years was GIV-2 and GV-2,and mainly GV-2.The CVA6 VP1 gene polymorphism curve showed a clear upward trend in 1999 and 2006,and it was relatively stable during 2002-2005 and 2011-2015.The evolutionary rate of the CVA6 VP1 gene was an average of 4.7982E-3 substitution events per site per year.The mutation rate of the three codons of the amino acid encoded by the CVA6 VP1 gene were 0.303,0.105,and 2.599,respectively.According to the recombination analysis of the CVA6 genome,CVA6 recombined to varying degrees within the VP1,VP2,VP3,and VP4 regions of the structural protein,whereas in the non-structural protein region,gene recombination took place in regions 2A,2B,and 3A.Of the 22 recombinant strains,15 were recombined in the 2B region(68.18%).Conclusion:From 1992 to 2015,the global CVA6 was divided into five genotypes of GI-GV by Bayesian method.During 2002-2005 and 2011-2015,the genetics of CVA6 were relatively stable and no new genotypes appeared.The CVA6 isolated from changchun area was GIV-2 gene subtype(1/11,9.09%)and GV-2 gene subtype(10/11,90.90%).The result of recombination analysis of the CVA6 genome revealed that intra-type recombination of CVA6 occurred in both structural and non-structural protein coding regions,but recombination was more prevalent in non-structural protein coding regions.