Research On Molecular Epidemiological Dynamics Of Enterovirus 71 And Epidemiology Of Hand,foot And Mouth Disease

Hand,foot and mouth disease?HFMD?is an acute infectious disease common in infants and children under 5 years old.A number of patients develop as severe as encephalitis,brainstem encephalitis,pulmonary edema,acute flaccid paralysis and even death.Over the past two decades,frequent outbreaks of HFMD have become one of the most important public health issues worldwide,especially in the Asia-Pacific region.More than 20 serotypes of enterovirus?EV?can cause HFMD,among which EV-A71,CV-A16,CV-A6,CV-A10 are the major pathogens that cause HFMD outbreaks and epidemics.EV-A71 infections are prone to induce severe illness and death and becomes the top priority target for HFMD vaccine designed.The monovalent EV-A71 inactivated vaccine was approved in mainland China in December 2015,and has gradually promoted vaccination.In the first part of this study,we reconstructed the spatial dynamics of EV-A71virus in mainland China and other worldwide regions.Databases were build based on EV-A71 sequences obtained from Genbank with clustering-based Bayesian methods,systems geographical methods and population dynamics analysis.The results showed that Europe is the origin and core output ground of EV-A71 in history,with a total migrate rate of 5.89 transition/year.East Asia plays a pivotal role in the epidemic and evolution of EV-A71,with a total migrate rate of 8.33 transition/year.The result elucidated the evolution and transmission history of EV-A71.The dynamic history analysis showed that the population expanded significantly four times in world EV-A71 history,with peaks in 1996,2003,2008,and 2012,respectively.EV-A71 in mainland China began to spread around 1996,and the population expansion occurred at the same time.After the expansion events,the effective population size?Ne?of EV-A71 in mainland China increased from 20.58 before 2003 to 249.84 in 2012.The Ne of the EV-A71 in mainland China remained at a relatively high level in 2019?Ne:206.61?.The epidemic surveillance should be strengthened to prevent outbreaks.The positive selection sites of each gene in the epidemic strain from 2016 to2019 were screened and the 2A-102 amino acid site was found to be the under potential positive selection site.The epidemic strain in China is mainly 2A^102Q strain.The mutant in this strain could stabilize the C-terminal conformation of 2A protease by enhancing hydrogen bonding with 142W and 145E,thereby promoting virus replication.The correlation between 2A^102Q strain and the continued epidemic of EV-A71 in mainland China requires further studies.By comparing the important immunogenicity,antigenicity,and virulence sites of EV-A71,VP1T143P and VP1K218R mutations were found in the important linear neutralization epitope PEP27 and SP70 in strains epidemic after 2015,which should be payed close attention to the probability of causing vaccine escape.No mutations were found in the antigenic and virulence sites,and no recombination was found in the structural and nonstructural regions.In the second part of the study,HFMD pathogenic spectrum research were conducted in 5 clinical centers in Guangdong?Gaozhou and Yangchun?and Jiangsu?Ganyu,Sheyang and Xinyi?.The results showed that more than 96%of the cases were positive for CV-A16?63.69⁷2.43%?and CV-A6?24.81³2.38%?,followed by CV-A10 and EV-A71.The seroepidemiological survey of 2⁸3-month-old infants and children at the same clinical centers indicated that unlike EV-A71,CV-A16,and CV-A6,the maternal antibodies positive rates of CV-A10 was as high as 70.87-80.37%in the 2-month-old group,and did not decrease significantly in 6¹2-months-old groups,showing a overall trend of steady increase,which suggested the local mothers and younger infants were highly infected.The neutralizing antibodies positive rates of CV-A10 and CV-A6 in 5-year-old children were 95.31⁹5.61%and 85.16⁸9.76%,respectively,which indicated that the prevalence of CV-A10 and CV-A6 in child population were extremely high.The genetic variations and epidemic trend of them need to be closely monitored.The first serological study of CV-A2,CV-A4 and CV-A5 was carried out among infants and children population.The neutralizing antibody positive rates of CV-A2,CV-A4 and CV-A5 in 5-year-old children were found to be lower than those of CV-A6 and CV-A10,but higher than EV-A71 and CV-A16,suggesting that these three pathogens had spread widely and should be paid more attention.The genotypes of EV-A71,CV-A16,CV-A6 and CV-A10 strains detected in this study were analyzed by constructing the maximum likelihood phylogenetic tree?ML tree?,and epidemic trends were analyzed by Bayesian method.The results showed that the strains in this study are EV-A71 C4 subgenotype,CV-A16B1/B2 subgenotype,CV-A6 D3 subgenotype and CV-A10 F3 subgenotype,which were also the main subgenotypes epidemic in mainland China in the past decade.The population size of EV-A71 and CV-A10 had been stable in recent years,while the population of CV-A16 and CV-A6 had been in a contraction period.However,the Ne of the four EVs were still at high levels in 2019?95.89²08.36?,which indicated potential risk of outbreak.The EV-A71 vaccination rates in Guangdong and Jiangsu populations were 16.44%and 1.77%,respectively.EV-A71 vaccine should be included in National Planned Immunization to establish an immune barrier in infants and children population.In order to evaluate the protective effect of C4 subgenotype inactivated EV-A71 vaccine in mainland China on epidemic strains in other countries and regions worldwide,the cross-neutralization study was performed with clinical immune serum and 10 EV-A71 strains of different subgenotypes.The results showed that the immune serum of C4 subgenotype vaccine could produce broad cross-neutralizing effects on B0^B4,C1,C2,C4JP,C4YA,and C5 subgenotypes strains,which were similar to natural infection.EV-A71 vaccine produced in mainland China could protect infants and children worldwide against different subgenotypes of B-genotype and C-genotype EV-A71 strains,which provide a basis for EV-A71 vaccine made in mainland China to go global.The population size of EV-A71 in mainland China is still at a relatively high level,while the vaccination rate is low,and EV-A71 vaccine should be included in National Planned Immunization.The EV-A71 2A¹02Q02Q site may be related to the continued epidemic of C4 subgenotype strains in mainland China.The mutations of VP1^T143P and VP1^K218R in epidemic strains after 2015 may affect the immune effect of EV-A71 vaccine.Futher studied on these mutation sites should be carried out.CV-A16 and CV-A6 are the most important pathogens causing HFMD.CV-A16 has a lower infection rate and a higher incidence,while CV-A10 has a higher infection rate and a lower incidence,which indicates EV-A71/CV-A16/CV-A6 trivalent vaccine should be the primary goal of the research and development of multivalent HFMD vaccine.CV-A2,CV-A4 and CV-A5 have a high prevalence in infants and children population,and the prevalence should be monitored.The C4 subgenotype EV-A71vaccine showed good cross-neutralizing effect with different EV-A71 subgenotypes,and can help to protect children in other countries or regions against EV-A71 infection.The results provide the basis for the effective application of EV-A71 vaccine,and lay the foundation for the research and development strategy of multivalent HFMD vaccine,which are significant for comprehensive prevention and control of HFMD.