Study On The Large-scale Outbreaks Of Sub-genotype D3 CV-A6 That Caused Hand,Foot And Mouth Disease In China

Background:Human enterovirus(EV),which belongs to the genus Enterovirus,family Picornaviridae,is a small non-enveloped,positive sense,single-stranded RNA virus.Human EVs are classified into 4 species:EV-A,EV-B,EV-C,and EV-D;the EV-A species now consists of 25 serotypes:Coxsackievirus group A(CV-A;serotypes 2-8,10,12,14,and 16);newly identified EVs(serotypes EV-A71,A76,A89-92,A114,A119-121);simian enteroviruses SV19,SV43,and SV46;and baboon enterovirus A13.Hand,foot,and mouth disease(HFMD)is a common epidemic disease that affects young children and causes a series of symptoms.More than 90%of HFMD cases are caused by EV-A,surveillance data showed that from 2008 to 2012,two major pathogens EV-A71 and CV-A16 spread alternativelty.However,since 2013,the large-scale HFMD outbreaks which caused by CV-A6 has been reported.In some provinces and cities,CV-A6 has even exceeded EV-A71 and CV-A16 as the main pathogens leading to HFMD,and gradually become HFMD pathogens which are as important as EV-A71 and CV-A16,thus,this virus has become a significant public health threat.CV-A6 epidemics in China has two important characteristics,one is the repeated large-scale HFMD outbreaks in same area,and the other one is that CV-A6-associated HFMD is likely to lead to severe HFMD among children,the reason of such characteristics has been the main problems that calls urgently for scientists to solve.At present,many researchers have studied the genetic characteristics of CV-A6 in local epidemic,but research in China mainly focused on small-scale outbreaks,the geographical distribution is narrow and the time range is small,yet a systematic analysis of the relationship between CV-A6 Evolution and pathogenicity in recent years is nonexist.CV-A6 adapts to the host by mutation,but its variation often leads to changes in its pathogenicity,epidemiological characteristics and clinical manifestations,which leads to some difficulties in HFMD clinical diagnosis,treatment,vaccine development and prevention and control.Responding to CV-A6 variability is one of the great challenges facing HFMD prevention.Objective:To clarify the prevalence of CV-A6 in China,to systematically master its evolution and epidemic situation,to improve its genetic characteristics,genotyping,transmission patterns and genetic evolution of CV-A6 in mainland of China and trying to analyze the relationship between CV-A6 and severe HFMD,trying to reveal the public health significance of CV-A6 in the past five years from many perspectives.Methods:520 CV-A6 VP1 sequences obtained from our laboratory were compiled and isolates with partial VP1 sequences were completed.287 CV-A6 sequences from GenBank database were also collected,finally,the database of all the CV-A6 was established.We analyzed the genome characteristics and evolutionary trend by bioinformatics methods such as the homology analysist and the construction of phylogenetic tree also studied the origin and evolutional rate by using the Bayesian MCMC method.Results:1.The results revealed that CV-A6 strains can be categorised into 4 genotypes designated as A,B,C,and D according to previous data and can be further subdivided into B1-B2,C1-C2,and D1-D3 sub-genotypes.2.D2 and D3 sub-genotypes were co-circulated in mainland of China and D3 is the predominant sub-genotype.Further research split sub-genotype D3 CV-A6 into 2 evolutionary branches,which were designated as D3a and D3b.The data revealed viruses from D3a evolution branch were responsible for the recent CV-A6 outbreaks and D3 sub-genotype may have stronger transmission ability.3.The CV-A6 nucleotide sequence of D2 and D3 subgenotypes was analyzed by Bayesian MCMC method.The results showed that the CV-A6 nucleotide substitution rate in our country was slower than that in the world.The D2 and D3 Subgenotypes of common ancestors can be traced back to about 1996,D3 subtgenoype CV-A6 population in 2013 significantly expanded.4.The whole genome sequence analysis of 50 CV-A6 strains in China suggested that CV-A6 presents 6 recombinant forms,and three recombinant forms CV-A6 recombinants has extensive genetic exchanges with other EV-A circulated in China,which suggested the evolution is relatively active.5.The 2C and 3D coding regions sequences of 14 severe(death)HFMD-associated CV-A6 and 19 mild HFMD-associated CV-A6 were further analyzed.The results suggested that recombination with other circulating EV-A happened in the 3D region may lead to a higher risk of severe HFMD.6.The amino acid mutations in VP1,2C and 3D coding regions of CV-A6 were analyzed.The mutations in amino acid polarity may have some relationships with the virulence.