Pathogenesis And Molecular Epidemiology Of Other Enteroviruses Associated With Hand,Foot And Mouth Disease In Anhui Province

ObjectiveTo understand the pathogenic spectrum composition and molecular epidemiology of enterovirus related to hand,foot and mouth disease（HFMD）in Anhui Province from2016 to 2020,to understand the genetic characteristics of other enterovirus strains in Anhui Province,to explore the appropriate cell lines for the growth of different types of enterovirus,and to explore the recombination and pathogenesis of other enterovirus in the epidemic process of hand,foot and mouth disease.In order to provide basic data onto HFMD prevention and control and strategy formulation in Anhui Province.Methods1.Source of samples and strains:Throat swab samples of HFMD children sent to the provincial CDC microbiology laboratory from Anhui Provincial Centers for Disease Control and Prevention（CDC）from January 2016 to December 2020 were selected,and480 representative positive samples of other enterovirus nucleic acid were selected.The whole genome sequenced strain was isolated and cultured from the cells of HFMD pathogen monitoring and detection,and was stored in the refrigerator below-70℃.2.Sample identification:sample RNA was extracted for VP1 region gene amplification and whole genome amplification,and the PCR products were analyzed by agarose electrophoresis and gene sequencing.3.Cytotoxicity test:The successfully isolated and identified CV-A6,CV-A10 and CV-A4 strains were infected with RD cells,Vero cells and Hep-2 cells respectively,and the pathogenicity of enterovirus to different cells was analyzed by observing the pathogenicity of enterovirus to different cells and calculating TCID₅₀.4.Bioinformatics analysis:sequence homology analysis was performed by Bio Edit7.2.5software;MEGA6.0 software was used to cut and compare the study sequences with the representative sequences,and construct the VP1 region and the whole genome evolutionary tree.Arc GIS10.2 software to draw incidence map;RDP4 software detects the recombination signal;Simplot 3.5 software verifies the recombination event;Graph Pad Prism 9 software maps virus infection titers.Results1.Epidemiological analysis showed that a total of 471,995 cases of HFMD were reported in Anhui Province from 2016 to 2020,including 949 severe cases and 11deaths,with an average annual incidence of 151.4 per 100,000.The morbidity was still at a high level,but the number of severe cases and deaths decreased.The prevalence of other enteroviruses dominated in the five years,and scattered children under the age of five were the main infected population,accounting for 76.48%of all cases,with a sex ratio of 1.50∶1.The incidence is seasonal with two peaks.Cases were reported in all cities in the past five years.Northern Anhui was the area with the highest incidence of the disease,followed by central Anhui,and southern Anhui had the lowest incidence.The top three cities with the highest number of reported cases were Fuyang City（82,695）,Hefei（81,763）and Bozhou City（45,835）.2.Genetic evolution analysis showed that other enteroviruses dominated the epidemic during 2016-2020.Five genotypes were identified in 245 samples,including 215CV-A6 cases（87.76%）,16 CV-A10 cases（6.53%）,12 CV-A4 cases（4.90%）,and 1CV-A5 case.There was one case of CV-A2,accounting for 0.41%（1/245）.CV-A6accounted for the largest proportion and was the main pathogen causing hand-foot-mouth disease in Anhui Province.The evolutionary analysis of VP1 region showed that the epidemic type of CV-A6 in Anhui Province was D3a,the epidemic type of CV-A10 was C2,and the epidemic type of CV-A4 was C2.All of them were dominant epidemic types in mainland China,and there were different transmission chains.All of them were far away from the original strains and close to the strains in other parts of China.The results showed that the Anhui strain co-evolved with the domestic strain,and the epidemic type changed greatly in the past five years.3.Cytopathic experiments showed that CPE occurred to different degrees in different cell lines of the three types of enterovirus.The virus lesions of CV-A4 and CV-A10appeared more thoroughly in RD cells,while CV-A6 did not occur significantly in the three cell lines.4.Recombination analysis suggested that both CV-A6 and CV-A10 in Anhui Province might be derived from the recombinant strains,and CV-A5 and EV-A92 might be the recombinant parents of CV-A6 in Anhui Province.CVA-10 showed small fragment recombination in P3,and CV-A5 may be a recombinant donor or a close relative.Recombination has certain influence on the virulence and transmissibility of enterovirus.Conclusions1.The incidence of hand,foot and mouth disease was still at a high level in Anhui Province,but the number of severe cases and deaths decreased.In the past five years,the prevalence of other enteroviruses dominated,and scattered children under the age of five were the main affected population.The disease showed seasonal distribution,with a high incidence in summer and autumn,and there were two peaks of one big and one small.Most cases occurred in northern Anhui Province.2.Other enteroviruses have become important pathogens causing HFMD in Anhui Province,among which CV-A6 dominates.Three other enterovirus genotypes monitored were endemic in mainland China and were still evolving.3.For cell lines suitable for replication of different types of enterovirus,further studies should be carried out after expanding the sample size and cell types to help improve the isolation rate of each type of virus.RD cells are still the appropriate cell lines for isolation of enterovirus at present.4.The CV-A6 and CV-A10 strains circulating in Anhui Province may be derived from the recombinant strains,and the breakpoints in the P2 and P3 regions suggest that CV-A5and EV-A92may be one of the sequence recombinant donors,and the recombinant strains may have certain effects on the virulence and transmissiveness of enterovirus.