Laboratory Parameters For Forecasting Severe Hand-foot-mouth Disease, Phylogenetic Analysis Of Pathogens Causing HFMD, And The Cellular Disease Modeling

Hand foot and mouth disease?HFMD?,characterized by acute onset and strong infectivity,is caused by enteroviruses which belong to the genus of the Picornavirus family.With the deepening of its clinical features and pathogenic mechanism,the fact that the pathogens that caused severe HFMD included not only EV-A71,that no difference was observed between the mild cases caused by EV-A71 and CV-A16,and that the progression of the clinical features of severe cases caused by CV-A16 was similar to that of EV-A71 make it more urgent for clinicians to identify severe cases than to identify the pathogens.At present,symptomatic treatment is to be given priority for severe HFMD since there is no vaccine for HFMD and anti-inflammatory treatment is not ideal.Therefore,the core strategy of prevention and control of HFMD is to control the sources of infection and to cut off the route of transmission.With the increase of population mobility and the acceleration of disease transmission,it is difficult to achieve complete control of the disease by risk factors the traditional epidemiology have obtained,so the analysis of the clinical epidemiology and molecular genetic evolution is particularly important to prevent and control the disease.In addition,the pathogenesis can help to control the disease.Until now,little about the pathogenesis has been known.As we know,the first step for virus to infect the host cells is binding with the receptor,and the receptor binding to EV-A71 is still unknown,and there is a possibility that the pathogenesis of viral diseases and their potential biological marker for early diagnosis and prognosis can be found with the rapid development of proteomics.Retrospectively,77 severe HFMD cases from Zhengzhou Children?s hospital from April to June between years 2013 to 2015 were collected,with 77 mild HFMD cases in the same area.Firstly,the clinical features and laboratory parameters were analyzed,after screening the important variables using Mann-Whitney U test,the study also matched discriminant analysis?DA?to screen the main laboratory parameters,which can help to provide new sight into early recognition,clinical intervention and therapy of the disease.And then,we aimed to investigate the genetic characteristics of the dominant pathogens causing HFMD.In addition,the glial cell sensitive to EV-A71 is screened out to study the mRNA changes of toll-like receptors,inflammatory factors and so on,and the differentially expressed proteins in response to EV-A71 infection,in order to further understand the mechanism of HFMD,which can provide a basis for exploring the effective prevention and control measures.Objectives1 To screen out laboratory parameters for forecasting severe HFMD,which can help to provide new sight into early recognition,clinical intervention and the therapy of the disease,and provide a new direction for its mechanism.2 To investigate the VP1 gene characteristics of the dominant pathogens causing HFMD in the peaking period between years 2013 to 2015,which can lay the foundation for the surveillance and control of HFMD in Henan Province.3 To study the mRNA changes of toll-like receptors,inflammatory factors and inflammasome-related genes in astrocytes upon infection of EV-A71,which can help to primarily understand its potential neuro-tropism.4 To preliminarily understand the differentially expressed protein of astrocytes in response to EV-A71 infection,which can provide potential target proteins to its neuro-tropism.Methods1 Study design,investigators training and pre-investigation,formal investigation and quality control,and statistical analysis.2 Collection and preservation of stool samples.The disposing of stool samples was conducted as per the laboratory manual for HFMD announced by the national polio and measles laboratory in 2010.The RNA extraction was as per the manual of QIAamp Viral RNA Mini Kit.The enteroviruses were detected by RT-PCR kit produced by Shuoshi biological science and technology company.The positive samples were inoculated on RD cells.According to the QIAGEN One Step RT-PCR Kit instructions,two pairs of primers were used for RT-PCR amplification,respectively.1.5%agarose gel was used for electrophoresis analysis of PCR products,and then do gene sequencing.Sequence analysis.3 Culture of RD cell.Preparation and quantification of EV-A71 strains.Infection model and the determination of time point of pathological changes occurred.Cell collection and RNA extraction.Detection of target gene by Real-time fluorescent quantitative PCR.4 U87 cell in logarithmic growth phase was selected.The suitable time was determined according to preliminary experiments,and then to collect cells and extract cell protein.Determination of the sample concentration.5?L of each sample was used to perform SDS-PAGE.Protein pretreatment.Detection of protein sample by chromatography mass spectrometry,and the original data was obtained.Results1 Laboratory parameters for forecasting severe HFMDCompared with that of the mild group,serum levels of WBC,PLT,PCT,MCV,MCH,LCR,SCR,LCC,GLO,CK-MB,K,S100,and B in the severe group were higher?p<0.05?,while MCR,EOR,BASOR,SCC,MCC,EO,BASO,NA,CL,T,Th,and Th/Ts were lower?p<0.05?.Five indicators including MCR,LCC,Th,CK-MB,and CL were screened out by DA.79.9%of original grouped cases were correctly classified,and 77.9%of cross-validated grouped cases were correctly classified.2 Genetic characteristics of the dominant pathogens causing HFMDThe nucleotide homology and amino acid homology among the 24 EV-A71strains was 93.0%¹00.0%,96.6%¹00.0%,respectively.To compare the 24 EV-A71strains of henan EV-A71 with the known genotypes,gene subtype of representative strains and analyze the nucleotide homology between them,the homology between the 24 strains and the representative strains of A,B1,B2,B3,B4,B5 genotype was82.2%⁸3.8%,82.9%⁸4.1%,83.0%⁸4.3%,82.9%⁸4.7%,82.6%⁸4.0%and82.3%⁸3.9%,respectively,the homology of the 24 strains with C1,C2,C3 and C5representative strains of C gene subtype was 88.2%⁹0.3%,87.5%⁸9.5%,86.6%⁸8.5%and 86.1%⁸9.6%,the homology with C4b was 89.5%⁹1.5%,the homology with C4a was 95.3%⁹7.9%,the homology with CV-A16 was the lowest with the homology of 62.0%⁶3.4%.Phylogenetic tree based on the partial VP1genes was built between the 24 EV-A71 strains and the 31 EV-A71 strains of representative subtypes and CV-A16 G-10,showing EV-A71 strains belonged to the C4a subgenogroup.The nucleotide homology and amino acid homology among the 24 CV-A16 VP1was 88.5%⁹9.6%,98.6¹00.0%,respectively.To compare the 24 CV-A16 strains of Henan with the known U05876 and analyze the nucleotide homology between them,the homology between them was 75.4%⁷7.2%,the homology of the 24 strains with B1a was 91.4%⁹3.8%,the homology with B1b was 88.4%⁹6.5%,the homology with B1c was 91.4%⁹3.1%,the homology with B2 was 88.5%⁹0.6%,and the homology with EV-A71 was the lowest with the homology of 65.3%⁶6.4%.Phylogenetic tree based on the partial VP1 genes was built between the 24 CV-A16strains and the 12 CV-A16 strains of representative subtypes and 1 EV-A71 strain,showing CV-A16 strains belonged to the B1a and B1b sub-genogroup.3 Gene expression changes of U87 cell in response to EV-A71 infectionTLR1-9 mRNA expression:RT-PCR suggested that compared with that of the control,there was no statistical significance of TLR1 mRNA expression at each time point after EV-A71 infection?p>0.05?.TLR2 mRNA expression at 6 hpi was higher in the EV-A71 group?p<0.05?,while there were no statistical significance at the other four time points?p>0.05?.TLR3 mRNA expression at 8 hpi was lower in the EV-A71 group?p<0.05?,while there were no statistical significance at the other four time points?p>0.05?.There was no statistical significance of TLR6 mRNA expression at each time point after EV-A71 infection?p>0.05?Inflammasome-related gene expression:RT-PCR indicated that compared with that of the control group,both IL-1 beta and NLRP3 mRNA were elevated at 8hpi and10hpi?p<0.05?,while there were no statistical significance at the other time points between the two groups?p>0.05?.Gene expression of inflammation factors:RT-PCR showed that compared with that of the control group,IL-6 mRNA and IL-8 mRNA were higher at 6 hpi,8 hpi,10hpi?p<0.05?,and both showed time dependence.Gene expression of nerve growth factor and its receptors:RT-PCR indicated that there were no statistical significance at 2hpi,4hpi and 6hpi between the two groups?p>0.05?,and then NGF mRNA expression decreased,there were statistical significance at 8hpi and 10hpi?p<0.05?,and presented the time dependence.There were no statistical significance of BDNF mRNA expression at each time point between the two groups?p>0.05?,p75^NTR,the low affinity receptor showed the peak at 8hpi?p<0.05?,while there were no statistical significance at the rest time points?p>0.05?.TrkA,the high affinity receptor,can not be detected sometimes,and the Ct value can reach more than 40 sometimes,which implied that TrkA might be suppressed after EV-A71 infection.4 Differentially expressed proteins of U87 cell in response to EV-A71 infection 31 different proteins between EV-A71 group and the control group were recognized by software analysis,including 9 proteins increased?PIAS1,MRC2,TNPO2,PMM2,ZNFX1,APPL2,EDF1,RPA1,MYO5A?whereas the other22?NUDT21,PDCD6,CDKN1A,TRIM41,MRPL18,PRKCSH,KPNA6,ASL,RPL35,RBM42,UACA,ABHD10,AXL,S100A10,XRN1,NAPG,GORASP2,UTP6,SUCLG1,EIF3M,SYAP1,AP3D1?decreased.GO annotation analysis of 31differentially expressed proteins between the two groups showed that the CC mainly included cell,extracellular region,and membrane.Biological processes of the 31differentially expressed proteins mainly involved metabolic process.Molecular functions of the 31 differentially expressed proteins mainly including binding,nucleic acid binding,catalytic activity and molecular structure.KEGG pathway analysis of31 proteins differentially expressed between the two groups showed that these proteins can be involved in a total of 25 signal transduction pathways,among which the metabolic pathways and the TCA cycle were of the highest matching rate.Conclusions1 The findings were that common laboratory parameters were effective to distinguish the mild HFMD cases and severe HFMD cases by DA.2 Phylogenetic analysis showed that EV-A71 strains belonged to the C4a subgenogroup and CV-A16 strains were mainly B1b,complementary with B1a.3 TLR2/TLR3 may play an important role in the immune response to EV-A71infection.IL-6,IL-8 and inflammasome may participate in the immune response to EV-A71 infection,NGF,p75^NTR,and TrkA may also play an important role in the mechanism of EV-A71 neuro-tropism.4 31 differentially expressed proteins were found,and they may provide new sight to EV-A71 neuro-tropism.