Study On Pathogen Spectrum Of Severe Hand,Foot,Mouth Disease In Hebei Province From 2017 To 2018 Based On High-throughput Sequencing

Objective:Enterovirus is the pathogen that causes severe hand,foot and mouth disease(HFMD).In recent years,the pathogenic spectrum of severe HFMD has been constantly changed.New technologies,including high-throughput sequencing(HTS),provid help for the identification of the pathogenic spectrum of HFMD.The purpose of this study is to clarify the pathogenic spectrum of severe HFMD in Hebei Province from 2017 to 2018,to study the characteristics of its change,to analyze the causes of the changes in the pathogenic spectrum,and finally clarify the epidemiological characteristics behind the change and the significance of public health prevention and control.Methods:In this study,50 specimens(29 in 2017 and 21 in 2018)of severe HFMD cases in Hebei Province were collected.The virus was isolated from 23 of the samples,and the entire VP1 region sequences were obtained by sanger sequencing method,the remaining 27 specimens were negative in viral isolation,and 17 of them obtained partial or full-length genomic sequences by High-throughput sequencing(HTS)method.The phylogenetic trees based on VP1 region of Coxsackievirus A16(CV-A16),Enterovirus A71(EV-A71),Coxsackievirus A10(CV-A10)and Coxsackievirus A6(CV-A6)were constructed by using RAxML software,and the molecular epidemiological characteristics of enteroviruses(EVs)detected in the samples were clarified.At the same time,8 enteroviruses(6 CV-A6 and 2 CV-A10)were analyzed by using RDP4 and SimPlot software,and the evolutionary characteristics of these sequences were analyzed by using the maximum likelihood tree constructed in the 3D region of the sequences.The global CV-A10 was divided into recombination forms based on the 3D region of CV-A10.Results:1.The results of nucleic acid detection and high-throughput sequencing(HTS)showed that the pathogenic spectrum of severe HFMD patients in Hebei Province has changed greatly in recent years.In 2017,EV-A71 was detected in 10 specimens,CV-A6 in 10 specimens(5 of which were detected by HTS),CV-A16 in 1 specimen and other enteroviruses were detected in 3 specimens.In 2018,CV-A6 in 8 specimens by HTS,CV-A16 was detected in 1 specimen,CV-A10 in 5 specimens(3 of which were detected by HTS),and other enteroviruses were detected in 3 specimens2.Through HTS,the number of specimens collected from severe HFMD patients that can detect pathogens increased from 23 to 40,the number of samples that can detect CV-A10 increased from 2 to 5,and the number of CV-A6 increased from 5 to 18.The proportion of CV-A10 in the pathogenic spectrum of severe HFMD cases increased from 8.7%(2/23)to 12.5%(5/40),and the proportion of CV-A6 increased from 21.7%(5/23)to 45.0%(18/40).Because EV-A71 was sensitive to virus isolation,all the samples were positive for virus isolation,and HTS did not detect more.The proportion of EV-A71 in the pathogenic spectrum decreased from 43.5%(10/23)to 25.0%(10/40).3.The phylogenetic tree based on VP1 region of EV-A71,CV-A16,CV-A6 and CV-A10 constructed by RAxML software shows that EV-A71 belongs to the dominant genotype C4a in China,CV-A16 belongs to the dominant genotype Blb in China,CV-A6 belongs to the dominant genotype D3a in China,and CV-A10 belongs to the dominant genotype C in China.4.The results of recombination analysis by RDP4 and SimPlot shows that three CV-A6 strains recombined with unknown EV serotypes;There were large-area recombination between other three CV-A6 strains and two CV-Al0 strains recombined with circulating CV-A2 and CV-A5 in the 3D region,respectively.5.The phylogenetic tree based on 3D region of CV-A6 and CV-A10 constructed by RAxML software shows that there are three CV-A6 strains belong to the dominant RF-A in China,while the three CV-A6 with recombination in 3D region in Hebei Province belong to RF-K.There are five RFs of CV-A10 in the world,and two recombinant CV-A10 belong to the dominant RF-B in China.Conclusions:1.CV-A6 and CV-A10 account for an increasing proportion in the pathogenic spectrum of severe HFMD cases in Hebei Province.CV-A6 exceeds EV-A71,which has become the dominant type in the pathogenic spectrum.It is suggested that the surveillance of CV-A6 and CV-A10 should be strengthened.2.High throughput sequencing assisted the detection of negative samples of viral isolation,which improved the positive rate of enterovirus detection,indicating that high throughput sequencing has great advantages in enterovirus identification,and it is worth popularizing in the future enterovirus surveillance.3.Through the evolution analysis of VP1 coding region of each enterovirus,it is found that the predominant genotype of each enterovirus serotype is responsible for severe HFMD.4.The recombination of 3D coding region will change the recombination form(RF)of C V-A6,which is no longer the main RFs in China.However,the recombination may increase the virulence of the virus and aggravate the clinical symptoms of severe HFMD.5.Because of the existence of RFs,the results of evolutionary analysis based on the VP1 and 3D coding regions of enteroviruses are different;suggestting that molecular typing based on VP1 region combined with full-length genomic sequencing can provide a clearer understanding of the evolution and variation of enteroviruses,which is conducive to the traceability analysis.