Molecular Epidemiology Of Non-EV71 And Non-CVA16 Enterovirus Related To HFMD And The Different Severity Mechanism Between CVA10 And EV71

ObjectiveThe aim of this study is to establish protocols that amplify partial and complete VP1 gene of human enterovirus from clinical specimens,analyze the molecular epidemiology of other enteroviruses（non-EV71 and non-CVA16 EVs）related to HFMD in Fujian province,and investigate the molecular mechanism in transformation of pathogen spectrum.Meanwhile,the pathogenesis mechanism of CVA10 was investigated,particularly on the different severity mechanism between CVA10 and EV71.MethodsAmplification of partial and complete VP1 gene by RT-sn PCR:（1）Establish RT-sn PCR method,including viral RNA extraction,first-step RT-PCR amplification,second-step PCR amplification,and primer screening and design;（2）Evaluation of the detection limit,including cell culture,virus isolate identification and virus titer tests.Molecular epidemiology of other EVs related to HFMD in Fujian province:（1）CVA6-and CVA10-specific fluorescent RT-PCR was used to screen CVA6 and CVA10 in clinical specimens;（2）partial VP1 and complete VP1 genes of HEVs were amplified from clinical specimens and sequenced by Sanger chemistry;（3）HEVs isolates were obtained by virus isolation from cell culture;（4）Full-length genome sequences were obtained by an Ion Torrent S5 platform for next generation sequencing.The different severity mechanism between CVA10 and EV71:（1）THP-1 cells were cultured and differentiated to macrophages;（2）THP-1 macrophages were stimulated with CVA10 or EV71,and expression of IL-1βin supernatants were detectedby ELISA;（3）A THP-1 macrophages model was constructed with NLRP3si RNA and NLRP3 inflammasome inhibitor（Glyburide,high concentration of potassium ion）,which inhibite NLRP3 inflammasome.Its inhibition efficiency were evaluated by QRT-PCR and Western blot;（4）High-throughput sequencing of micro RNAs from THP-1 macrophages infected with CVA10 and EV71 was carried out on a Illumina Next Seq 500 next generation sequencing platform;（5）Viral RNA and micro RNAs were detected by q RT-PCR.ResultsRT-sn PCR methods for amplifying and sequencing partial and complete VP1gene of HEVs from clinical specimens were established,and the sensitivity was10TCID₅₀/100μl and 100TCID₅₀/100μl,respectively.Molecular epidemiology of other enteroviruses in Fujian province:（1）In2011-2018,other enteroviruses related to HFMD in Fujian province consisted of 25serotypes of human enterovirus A,B,C and D groups;（2）The gender distribution of CVA6,CVA10 and CVA4 infection cases was consistent with that of EV71 and CVA16,that is,men are more susceptible than women;children under 5 years old were dominant;but the age distribution of CVA6 and CVA10 infection cases was relatively lower than EV71,CVA16 and CVA4;the severity rate ranked from high to low as EV71,CVA10,CVA6 and CVA16;（3）The change process of the pathogen spectrum of HFMD in Fujian province during 2008-2018 consisted of three stages,namely,“EV71-dominant outbreak”in 2008-2010,“EV71 to CVA6 transition in2011-2012”and“CVA6-dominant outbreak”in 2013-2018.Other serotypes of human enterovirus including CVA16,CVA10,CVA4 co-circulated in Fujian during2008-2018,while the mortality rate in severe HFMD cases was declining;（4）The temporal distribution of CVA6 genotypes in Fujian province in 2011-2018,evolved from the previous D2 subtype to the later D3a branch of the D3 subtype;（5）The genotypes and sub-genotypes of CVA10 in Fujian province were stable and classified to C2 subtype during 2011-2018;（6）Although no recombination events was identified in CVA5,CVA10,CVA21 and EV68 strains from Fujian during 2011-2018,the other18 serotypes,such as CVA6,were suspected of intraspecific recombinationwith diverse recombination patternswhich was observed among isolates of the same serotype,as well as isolates among different serotypes in Fujian province,notably,the E30 strains of a viral encephalitis outbreak in Fujian province,participated in the recombination events of multiple group B enteroviruses in Fujian province.The different severity mechanism between CVA10 and EV71:（1）CVA10 infected THP-1 macrophages induced elevated IL-1βsecretion,despite of its lower IL-1βexpression level than that of EV71;（2）Inhibition of NLRP3 inflammasome reduced the level of IL-1beta secretion in THP-1 macrophages infected with CVA10;（3）CVA10 infected THP-1 macrophages possesed lower viral load than EV71;（4）Distinct expression profiles of micro RNAs in THP-1 macrophages infected with CVA10 were observed in comparison with those of EV71;（5）The expression level of micro RNA-146a in THP-1 macrophages infected with CVA10 was lower than that of EV71.Conclusion（1）Have established RT-sn PCR methods that amplify and sequence partial and complete VP1 gene of HEVs from clinical specimens,are applicable to molecular typing and molecular epidemiology of human enterovirus in clinical specimens.（2）The other enteroviruses of HFMD in Fujian province during 2011-2018 were composed of 25 serotypes of human enterovirus A,B,C and D.CVA6 isolates of the D3a branch within the D genotype,contributed to the transformation of HFMD pathogen spectrum in Fujian province during 2011-2018 and current CVA6-dominant outbreak;while the C2 subtype strains of CVA10 that was co-circulating in Fujian province,were relatively stable,with slower mutation rates and no recombination occurs,without large-scale outbreaks.（3）The macrophages infected with CVA10 induced secretion of IL-1βby activating NLRP3 inflammasomes,but the expression level of IL-1βwas lower than that of EV71,which may be one of the important factors that cause the lowered severity rate of CVA10 infection than that of EV71.（4）The expression profiles of micro RNAs in THP-1 mononuclear macrophages infected with CVA10 were different from those of EV71;the low expression of mi R-146a in CVA10 may contribute to the reduced severity rate of CVA10 infection than that of EV71.In summary,this study provides important technical resolutions for the molecular typing and molecular epidemiology of non-EV71 non-CVA16 HEVs,and provides new evidence and clues for the molecular epidemiology and pathogenesis of HFMD-associated HEVs.