Pathogen Spectrum Research Of Enteroviruses Associated With Hand,Foot And Mouth Disease In Areas Vaccinated With EV-A71 In Jiangsu,Hubei Province,2019

Hand,foot and mouth disease(HFMD)is an acute infectious disease caused by a variety of enteroviruses(EV),which occurs mostly in children under five years old.Clinical symptoms include fever and rash in hands,feet,mouth and buttocks.In 2008,the outbreak of HFMD in Fuyang City,Anhui Province aroused the close attention of the Chinese government.On May 2 of the same year,HFMD was included in the management of Class C infectious diseases stipulated in the Law of the People’s Republic of China on the Prevention and Control of Infectious Diseases.In China,the main pathogens causing HFMD before 2012 are Enterovirus 71(EV-A71)and Coxsackievirus A16(CV-A16).In recent years,the number of HFMD caused by Coxsackievirus A6(CV-A6)and Coxsackievirus A10(CV-A10)has been increasing continuously,which have become the main pathogen of HFMD,posing a greater challenge to the prevention and control of HFMD.Performed in parallel with a Phase IV clinical trial of EV-A71 vaccine developed and produced by Wuhan Institute of Biological Products Co.,Ltd.,in this study the molecular epidemiology of HFMD,Etiological characteristics and spectrum of pathogens after EV-A71 vaccine intervention are investigated,providing data supporting for the monitoring and prevention measurement of HFMD,the research and development of multivalent hand-foot-mouth vaccine and the phase IV clinical trial of EV-A71 vaccine.We collected837 rectal swab samples of children with diagnosed HFMD in Qianjiang City and Gucheng County of Hubei Province,Peixian County and Changzhou City of Jiangsu Province.All rectal swab samples were preliminarily typed by type-specific fluorescence quantitative PCR,and then viral RNAs of some representative samples were extracted to amplify the VP1 gene fragments,and serotypes/genotypes of associated enteroviruses were further determined based on the VP1 sequences.Finally,we detected 795 positive samples of enteroviruses,and the positive rate was 94.98%.A total of 520 samples of HFMD(259 in Gucheng County and 261 in Qianjiang City)were collected in Hubei Province,among which 487 samples were positive for enteroviruses confirmed in laboratory.Among them,242 enterovirus positive samples were detected in Gucheng County,the positive rate was93.43%and 6 enterovirus serotypes were detected.The proportions of several main pathogens were 84.30%,2.48%,7.44% for CV-A6,CV-A16 and CV-A10,respectively.Two hundred fourty eight enterovirus positive samples were detected in Qianjiang City,the positive rate was 95.02%and 4 enterovirus serotypes were detected,with the proportions of CV-A6,CV-A16,CV-A5,CV-A8 were 54.44%,40.73%,4.44% and 0.40%,respectively.A total of 317 samples of hand,foot and mouth disease were collected in Jiangsu province,among which 305 samples were positive for enteroviruses confirmed in laboratory,and the positive rate of enterovirus was 96.21%.Seven serotypes of enteroviruses were detected,among which the proportions of several main pathogens were 68.18%(CV-A6),17.53%(A16)and 9.42%(A10).In this study,all HFMD samples were isolated and cultured on RD and Vero cells,and 22 strains of viruses were isolated,which provided experimental materials for studies on the related enteroviruses,and was of great significance for the development of HFMD related vaccines.Phylogenetic analysis based on VP1 sequences of main pathogens in Hubei and Jiangsu provinces shows that CV-A6 prevalent in these two places belongs to D3 subtype,which is consistent with CV-A6 subtype prevalent in other areas in China in recent years.The prevalent strains of CV-A16 in Jiangsu province belong to B1 a subtype,and those in Qianjiang city,Hubei province belong to B1 b subtype,while B1 a and B1 b subtypes of CV-A16 are co-circulating in Gucheng county,Hubei province which is consistent with trend of epidemics in other areas in China in recent years.CV-A10,which is prevalent in Hubei and Jiangsu provinces,belongs to C gene subtype.A case of EV-A71 in Jiangsu belongs to C4 subtype,which is consistent with the subtype prevalent in China in recent years.With the intervention of EV-A71 vaccine,the spectrum of HFMD pathogens showed that enteroviruses of various serotypes and genotypes were co-prevalent,among which CV-A6 is the dominant pathogen.Only one case of HFMD caused by EV-A71 was found,and the child was not vaccinated with EV-A71 vaccine.