Spatial Clustering Analysis Of Hand,Foot And Mouth Disease In Mainland China From 2008 To 2017 And The Genetic Characteristics And Evolutionary Analysis Of Coxsackievirus A Group 5

Research backgroundHand-foot-mouth disease（HFMD）is a common global acute infectious disease caused by a variety of human enterovirus（EV）in children.It mainly occurs in children under 5 years old.Severe complications such as encephalitis,pulmonary edema,pulmonary hemorrhage,heart and lung failure occur in a few patients.The condition of some severe patients progresses rapidly.It even causes death,so it is one of the important public health problems facing our country.Since the introduction of the statutory reporting infectious disease management system in 2008,the incidence,severity and mortality of HFMD have remained high in the statutory reporting infectious diseases ranking.For example,the incidence of HFMD in China continued to rank first among the statutory infectious diseases in 2010-2017.Since 2008,China has carried out a multi-level epidemiological descriptive analysis of HFMD.The spatial autocorrelation analysis method in spatial statistics is a supplement to traditional statistical methods,and explores the spatial distribution characteristics of diseases and other variables by using spatial data and attributes.At present,China has carried out a lot of spatial analysis on HFMD,including spatial statistical analysis at different levels,but there are few studies on HFMD-related spatial aggregation across the country and over a long time span.This paper uses spatial autocorrelation analysis to analyze the monitoring data of HFMD in mainland China from 2008 to 2017,clarifies the spatial distribution characteristics of HFMD in China,and provides theory and science for HFMD prevention and control.Research objective1.To explore the patialclustering and evolution aryprocessof hand,foot and mouth disease（HFMD）in China from 2008 to 2017.2.Spatial autocorrelation analysis was used to study the spatial distribution patterns of reported incidence,the rate of severe cases and reported mortality of HFMD in mainland China from 2008 to 2017 in provinces（Autonomous Regions and Municipalities Directly under the Central Government）to identify the hot spots of HFMD,and to provide scientific data for formulating strategies for prevention and control of hand-foot-mouth disease,determining key control areas,and rationally allocating medical resources,（such as vaccine delivery）.Research findings1.Since 2009,the reported incidence of hand,foot and mouth disease has increased every other year and reached a high point in 2014,but the rate of severe cases and mortality showed an downward trend collectively.2.Global Moran’s I coefficient results shows that the reported incidence of HFMD in 2008～2017,the rate of severe cases of HFMD in 2008～2009,and mortality of HFMD in 2010,2012,2015～2017,were clustered respectively.3.Local autocorrelation analysis showed that the areas in whichthe incidence persisted（or for most of the time）in the first quadrant（HH）include Hainan,Guangdong,Guangxi,Hunan,Zhejiang,Fujian,and Shanghai in China;rate of severe cases of HFMD was in the first quadrant（HH）or fourth quadrant（HL）occurred in Hainan,Guangdong,Yunnan,Guizhou,Hunan,Henan inChina;mortality of HFMD was in the first quadrant（HH）were Hainan,Guangdong,Chongqing,Guizhou,and Hunan inChina;above three rates were similar in the third quadrant（LL）were Sichuan,Southwest Tibet,North-west（excluding Shaanxi）and North-east region.Research conclusion1.The global spatial distribution patterns of reported incidence,severity and mortality of HFMD in mainland China from 2008 to 2017 were not insimilar pattern.What is more,local autocorrelation suggested that Hainan,Guangdong and Hunan were all in HH accumulation area,Guizhou was in HH accumulation area of the rate of severe cases and mortality,and the above provinces were the top priority for prevention and control.2.Besides,spatial analysis combined with traditional methods can help to better understand the regularty of epidemic and to guide prevention and control of HFMD.Stable spatial distribution of disease will need follow-up data and to be tracted.Background:HFMD is a worldwide infectious disease,especially in China.Although Enterovirus A71（EV-A71）and Coxsackievirus A16（CV-A 16）are the most common pathogens of HFMD in the world recently,the network monitoring system of HFMD laboratories in China has shown that the proportion of other EV has increased,and even have replaced EV-A71 and CV-A 16 in some areas as the dominant pathogens,among which Coxsackievirus A5（CV-A5）has gradually raised concern.At present,although CV-A5 has not caused a large-scale outbreak of HFMD in mainland China,there are reports of the outbreak of HFMD/HA caused by CV-A5 both at home and abroad.It has been found that the recombination of CV-A5 with Coxsackievirus A10（CV-A10）and Coxsackievirus A4（CV-A4）caused the outbreak of HFMD has certain significance for enhancing the virulence of CV-A5 and other EV.So far,the research on macro-epidemiology and molecular epidemiology of CV-A5 is still insufficient.Most CV-A5 studies are mainly related to local/provincial studies of HFMD pathogenicity spectrum,and few etiological and evolutionary analyses of CV-A5 gene characteristics are available.Therefore,based on the National Polio and Measles Laboratory Strain and Specimen Database of the National Institute for Viral Disease Control and Prevention（IVDC）of Chinese Center for Disease Control and Prevention（CDC）,the epidemiology,genetic characteristics,origin and evolution of CV-A5 strains in mainland China from 2008 to 2017 were analyzed comprehensively and systematically in order to provide scientific data for the prevention and control of HFMD and the development of multivalent HFMD vaccine in China.Objective:1.The epidemiological regularity of CV-A5 related-HFMD cases in mainland China from2008 to 2017 was preliminaiy elaborated.2.The genetic characteristics and evolutionary rules of CV-A5,such as genotyping,gene recombination and molecular evolution were expounded.3.Establish RT-PCR and rPCR methods for the determination of complete CV-A5 VP 1sequence and whole genome sequence.4.For the fost time,the etiology of CV-A5 related-HFMD in mainland China was systematically and comprehensively studied,it provides scientific basis for the prevention and control of HFMD and Whether to develop multivalent vaccine.Methods:Based on the management of national infectious disease reporting system of China CDC,the epidemiological and etiological data of HFMD in various provinces（Autonomous Regions and Municipalities Directly under the Central Government）in mainland China were collated.Descriptive epidemiological methods were used to describe the epidemiological characteristics of HFMD caused by CV-A5 in mainland China.Real-time Fluorescent Quantitative Polymerase Chain Reaction（rPCR）and Reverse Transcription-Polymerase Chain Reaction（RT-PCR）were used to detect the complete VP1 coding region nucleosides of 42 representative CV-A5 isolates from National Polio and Measles Laboratory Strain and Specimen Database of IVDC.4representative strains of CV-A5 were sequenced.At the same time,combined with 19 complete VP1 coding sequences of CV-A5 from GenBank database were used to establish the CV-A5 virus information database of worldwide and mainland China.In addition,collating the sequences of other EV-A prototype strains,the current international bioinformatics research soflware/methods（Sequencher,Bioedit,MEGA,Simplot software and Beast software packages）were used to illustrate the genetic characteristics and evolutionary rules of CV-A5 strains in mainland China from 2008 to 2017.Results:1.47 cases of HFMD were collected and caused by CV-A5 from 2008 to 2017 in Mainland China（GenBank database has 5 cases,and the information of epidemic is not clear）.Among them,the onset time was mainly from May to July;the distribution area involved 16 provinces（autonomous regions,municipalities directly under the Central Government）,with the largest number of cases in East China（21 cases）,and the most cases in Shandong Province of East China（8 cases）.There were 44 mild cases and 3 severe cases,22 males and 20 females.The age of onset ranged from 8 months to 6 years and 3 months.2.Based on complete VP1 sequences,CV-A5 was divided into three genotypes.Prototype strain（Swartz strain）in 1950 and Australian strain in 2016 constituted genotypes A and B of CV-A5,respectively.C genotype is further divided into three sub-genotypes of Cl～C3,and Hungarian strain（KC879543/41143/RUS/2011）and Australian strain（KU761262/13164/HUN/2015）formed C2 and C3 sub-genotype independently.Cl genotype includes one Australian strain（2016）.one Spanish strain（2008）,two Russian strains（2007 and 2011）,and all mainland strains and Taiwan strains of China.All the strains of CV-A5 causing HFMD in mainland China belong to Cl sub-genotype.3.Based on the global complete VP1 sequences of CV-A5,the average base substitution rate of CV-A5 was 4.58x10₃/site/year from 1950 to 2017,and 95% HPD was [3.28><10’3,5.96><10"3].The earliest ancestor（Tmrca）could be traced back to 92.61 years ago（95% HPD was68.00-119.16 years ago）,that is,1925;and the average base substitution rate of CV-A5 isolated from mainland China from 2008 to 2017.The replacement rate is 4.79x10-3/site/year;95% HPD is [3.59x10-3,6.13X10-3].Its earliest ancestor（Tmrca）can be traced back to about 15.83 years ago（95% HPD is 12.04-20.46 years ago）,that is.2002.4.The identity of the whole genome sequence bel^veen four CV-A5 strains from mainland China and prototype（Swartz strain）range from 81.00%-81.66%,and the amino acid homology from 96.30% to 96.85%.The identity of four CV-A5 strains from mainland China between was the exception of the prototype（Swartz strain）range from 83.48%～96.29%,and the amino acid homology from 97.44% to 99.18%.In the PI structural protein coding region（except VP4）,the homology of CV-A5 strain and prototype strain was highest,but in the non-structural protein coding region of P2 and P3,the homology of some fragments of CV-A5 strain with other EV-A prototype strains was higher than that of CV-A5 prototype strain.5.The recombination analysis of the whole genome sequence of four CV-A5 strains in mainland China and other EV prototypes in EV-A group showed that there was no obvious recombination hint of 3rUTR,while 5’UTR,CV-A8（Donowanand strain）and CV-A12 prototype strain（Texa-12 strain）could be recombined at position of around 240 bp and 400 bp respectively.In the coding sequence,PI region was conservative,and no recombinant sites were found;P2region suggested that CV-A5 strain could recombine with CV-A16（G10 strain）,CV-A2（Fleetwood strain）at about 3500 bp（2A region）and 4100 bp（2C region）,respectively;in P3 region,CV-A5 strain could recombine with EVA71（BrCr strain）,CV-A2（Fleetwood strain）at about 5400 bp ～5600 bp（3C region）and 5100 BP（3B region）,respectively.Some fragments can be recombined,and the percentage of Permuted Trees can reach to 90%.6.Compared with prototype strains（Swartz strain）,eight CV-A5 strains（sub-genotype Cl）in mainland China had 49,28 and 91 amino acid loci mutations in Pl,P2 and P3 regions respectively,and the frequency of amino acid mutations was 5.70%（49/860）,4.84%（28/578）and 9.43%（71/753）,respectively.Meanwhile,five CV-A5 strains（sub-genotype Cl）in Taiwan of China had 27,17,38 amino acid loci mutations in P2 and P3 regions,respectively.The mutation frequency was 3.14%（27/860）,2.94%（17/578）and 5.05%（38/753）,respectively.CV-A5 strains in mainland and Taiwan strains of China had the same amino acid mutation at most of the mutation sites,or only CV-A5 strains in mainland China had amino acid mutation.Conclusions:1.The distribution of HFMD cases caused by CV-A5 in the mainland of China during2008-2017 is consistent with the overall cases of HFMD in China,while there are more cases in Shandong,Jiangsu in East China and Sichuan provinces in Southwest China.2.At present,based on the global and complete VP1 sequences: CV-A5 was di\ided into three genotypes of A～C,C genotype further divided into three sub-genotypes of Cl ～C3.and Cl genotype includes three lineages.All the cases of HFMD in China caused by CV-A5 were Cl sub-genotypes.3.From 2008 to 2017,the Cl sub-genotype of CV-A5 strain continued to be prevalent in mainland China.It was the dominant sub-genotype of CV-A5 causing HFMD in mainland China,and cycled with Spanish strain,Russian strain and Australian strain.4.CV-A5 of mainland China evolved actively because of its rapid evolution rate（4.79x10"3/site/year）and frequent recombination.Amino acid mutations of Cl sub-subtype C V-A5 causing HFMD in mainland China are very active,especially in the coding regions of P2 and P3,which may contribute to immune escape of CV-A5 strain and cause HFMD outbreak by accumulating susceptible population.