Epidemiology Of Hand,foot,and Mouth Disease And Genetic Characteristics Of Coxsackievirus A10 In Taiyuan City,shanxi Province From 2016 To 2020

Objective:In recent years,the number of hand,foot,and mouth disease(HFMD)cases caused by enteroviruses other than enterovirus A71(EV-A71)and coxsackievirus A16(CVA16)has obviously increased,resulting in great changes in the pathogen spectrum of HFMD.In particular,coxsackievirus A10(CVA10)is widespread in mainland China.In this study,we analyzed the epidemiological regularity of HFMD and the genetic characteristics of coxsackievirus A10(CVA10)in Taiyuan City,Shanxi Province,from2016–2020,so as to provide the basis for the development of HFMD vaccine and disease prevention and control measures of HFMD in Taiyuan City.Methods:The epidemiological data from 2016 to 2020 in Taiyuan City was retrieved from the China Information System For Disease Control and Prevention and the population data came from the Taiyuan Statistical Yearbook.In ten hospitals in different districts and counties of Taiyuan City,specimens were collected from clinically diagnosed HFMD cases within three days after the onset of disease,at least five specimens every month,and sent to the virus microbiology laboratory of Taiyuan Center for Disease Control and Prevention(Taiyuan CDC)by ten districts and counties CDC.A total of 2,701 clinically diagnosed HFMD throat swab specimens were collected in Taiyuan City from 2016 to2020.The epidemiological characteristics of HFMD in Taiyuan City were analyzed based on the monthly distribution,age and population category of the disease.Data were statistically analyzed by Chi-square test.Viral RNA was extracted and the real-time quantitative polymerase chain reaction(PCR)kit was used to identify enterovirus types.The specimens identified as CVA10 were amplified by reverse transcription PCR for the entire VP1 region.MEGA11.0.11 and Bio Edit7.2.5 software were used to analyze the gene evolutionary relationship,homology analysis and amino acid sequence alignment between the above CVA10 VP1 sequence and the representative strains downloaded from Gene Bank database after sequencing and sequence splicing.Results:A total of 22,044 clinical HFMD cases were reported in Taiyuan from 2016 to 2020.The average annual HFMD incidence rate was 86.45 per 100,000 population(range20.68–148.49).The average annual incidence in males(101.10 per 100,000 population)was higher than in females(71.18 per 100,000 population)and this difference was statistically significant.The majority(99.35 %)of them were scattered children(49.33%),kindergarten children(41.74%)and school going students(8.27%).Of all cases,81.65%were children aged 1–5.Although HFMD cases occurred throughout the year,two seasonal peaks were observed from June to August and from October to November during 2016–2019,respectively.Due to the impact of COVID-19 pandemic,the peak of2020 was in November.Of the 2,701 specimens collected,1,439(53.28%)were positive for human enterovirus(EV).The positivity rates of EV-A71,CVA16,and other EVs were15.22%(219/1439),28.77%(414/1439),and 56.01%(806/1439),respectively.31 specimens were detected as CVA10.The CVA10 sequences could be assigned to seven genotypes(A,B,C,D,E,F,and G),and genotype C was further divided into C1 and C2 subtypes.Of the 31 CVA10 strains identified in this study,only one belonged to subtype C1 and the remaining 30 belonged to C2.Subtype C2 can be further divided into three evolutionary branches(C2a,C2 b and C2c).All the CVA10 strains detected in 2016 belonged to C2 a.One CVA10 strain detected in 2018 and two CVA10 strains detected in 2019 belonged to C2 b,whereas the remaining CVA10 strains detected between 2018 and 2020 belonged to C2 c.The VP1 nucleotide sequences among CVA10 strains shared 92.6% to 100% nucleotide identity,corresponding to a 96.9% to 100% amino acid identity.A similar analysis with the prototype strain Kowalik(genotype A)showed a 75.9% to 77.0% nucleotide identity,corresponding to a 91.2% to 93.6% amino acid identity.The highest nucleotide similarity with the genotype C downloaded from the Gen Bank was 91.7% to 100% and amino acid similarity of 96.9% to 100%.Compared with the prototype strain,the amino acid sequence in VP1 region had multiple specific mutations.The amino acid sequence analysis of CVA10 genotypes showed that the linear neutralizing epitope of 162-176 residues was highly conservative.Conclusion:The number of male cases was higher than female cases in the same year and the difference of male-to-female incidence was statistically significant.The majority of them were scattered children,kindergarten children and school going students.Of all cases,81.65% were children aged 1–5.Although HFMD cases occurred throughout the year,two peaks were observed from June to August and from October to November during2016–2019,respectively.The phylogenetic analysis showed that all the CVA10 sequences could be assigned to seven genotypes(A,B,C,D,E,F,and G),genotype C was further divided into C1 and C2 subtypes.CVA10 VP1 sequences identified in this study had the highest nucleotide and amino acid homology with genotype C and Taiyuan strains belonged to genotype C,only one belonged to subtype C1 and the remaining 30 belonged to C2.Subtype C2 can be further divided into three evolutionary branches(C2a,C2 b and C2c).Our study identified Taiyuan CVA10 VP1 sequences have multiple amino acid mutation sites and the amino acid sequence was highly conservative in 162-176 residues,which may be the focuses of subsequent research.It is necessary to conduct the continuous surveillance of HFMD pathogen spectrum.