Phylogenetic Analysis Of Coxsackievirus A6 In Hand,Foot And Mouth Disease In Qingdao In 2016-2018

Objective:In this study,we analyzed the etiology of hand-foot-mouth disease and Coxsackie virus A6 genome in Qingdao from 2016 to 2018,to understand the epidemiological characteristics of the hand-foot-mouth disease pathogenic spectrum and the genetic variation characteristics of CA6 and laid the foundation for prevention and control of HFMD.Methods:There are sentinel surveillance sites for HFMD pathogens in 10 districts and cities in Qingdao.All samples are collected and registered by sentinel hospitals in accordance with the "Hand,Foot and Mouth Disease Prevention and Control Guidelines(2014 Edition)" issued by the Ministry of Health.Fluorescence quantitative PCR method was used to detect Enterovirus(EV)in hand-foot-mouth disease pharyngeal swab specimens in Qingdao City from 2016 to 2018,and EV positive specimens were typed and identified for common enteroviruses(including enteroviruses 71 genotype(EV71),Coxsackievirus A16,A6,and A10 genotype),inoculate enterovirus fluorescent RT-PCR detection CA6 positive specimens on RD cells for virus isolation,and collect the supernatant for nucleic acid extraction after 7 days and reverse transcription-polymerase chain reaction(RT-PCR),select the CA6 strain with high viral load(CT value less than 25)for VPl gene full-length and whole genome amplification,and use Sequencher software for sequence splicing of the sequencing results.After ClustalX 2.0 aligned sequences,import Mega 7.0 software for sequence alignment and phylogenetic analysis.The CA6 reference sequence used to establish the phylogenetic tree was selected from the representative strains of enterovirus genotypes and subtypes in GenBank to establish the phylogenetic tree,analysis of sequence evolution characteristics.Results:A total of 2682 cases of Qingdao HFMD were collected from 2016 to 2018.Among the pathogenic spectrum,EV71,CVA16,CA6 and other types accounted for 23.5%(630 /2682)and 18.0%(484 /2682)of the total enteroviruses,respectively.15.5%(416 /2682)and 23.4%(628 /2682),after amplification,132 CA6 VP1 sequences and 10 complete gene sequences were obtained.Phylogenetic studies showed that based on the CA6 VP1 gene evolution tree,VP1 sequences can be divided into Five genotypes(A-E),only one of the CA6 strains measured in Qingdao from 2016 to 2018 is the 2017 strain in the D3 b branch(1/132),and the rest are in the D3 a branch.This branch has been in recent years The most popular genotype in mainland China;based on the evolutionary tree of the CA6 complete gene sequence,the three-year-old CA6 epidemic strains in Qingdao are all in the D3 branch,of which 90%(9/10)of the complete gene sequences are distributed in the D3 genotype On branch A.Whole gene homology analysis showed that the nucleotide homology between Qingdao CA6 strains in this study was greater than 93.9%.Recombination analysis revealed that a CA6 hand,foot and mouth virus strain had genetic recombination with the CA4 genotype 3D region in 2017.Through the analysis of amino acid variation in the VP1 region,there are several VP1 region mutations in Qingdao that deserve attention,including T5 A,A30V,V174 I,I242V,T283 A,S137N,and A255 T.Conclusions:CA6 has become an important pathogen causing hand-foot-mouth disease in Qingdao;CA6 isolates in Qingdao from 2016 to 2018 are located in the D3 a branch of the evolutionary tree.The independent branch of CV6 on D3 a in Qingdao in 2018 may be an imported strain.A large-scale epidemic of CA6 occurred in Qingdao in 2018,and there may be multiple virus transmission chains.30,137,174,242,255 and other VP1 sites are prone to mutations.These mutations may also affect the virulence of the virus,leading to widespread spread and even outbreaks.In 2017,an isolate in the 3D region may be recombined with the CVA4 genotype currently popular in mainland China.It should be concerned about the role of the 3D region as a donor in the recombination of enteroviruses.