Sequence Analysis And Bioinformatics Characterization Of The Important Viruses Caused Hand, Foot And Mouth Disease

ObjectiveHand, foot and mouth diease is a kind of epidemic diease which is caused byenteroviruses. It is characterized by vesicular lesions on the hands, feet and oral mucosa.Most of them are mild and self-limited. However, they can cause severe complications onthe heart, pulmonary and neurological system. In this study, we detected throat swabsamples which were obtained from the children with HFMD. In order to study thepathogens caused HFMD and the characteristics of the evolution, the degenerate primerswhich were higher specific to enteroviruses and the genotype primers were used to analyzeits VP1region.Human enteroviruses belong to the family Picornaviridae, including Polioviruses(PVs), Coxsackievirus A (CVA), Coxsackievirus B (CVB), ECHO virus (EntericCytopathic Human Orphan virus) and Enteroviruses. Enterovirus71is the most importantcausative agent of HFMD. It has been associated with severe clinical syndromes:asepticmeningitis, brain-stem encephalitis, acute flaccid paralysis mimicking paralyticpoliomyelitis and rapidly fatal pulmonary edema and hemorrhage. The mutation andrecombination are more obvious, so that there is no better prevention and treatment till now.We analysis the sequences of VP1region and complete genome of Enterovirus71, andstudy the rule of mutation and recombination.Coxsackievirus A10and A6are the pathogens caused HFMD besides CoxsackievirusA16and Enterovirus71. In the last work, we study the VP1protein of Coxsackievirus A10with bioinformatics methods, analyze and calculate the physical and chemical properties,the spatial structure and B cell antigenic epitopes. Methods1. Samples collectionThe throat swarbs were collected from the children with HFMD in our hospital. Insertnormal saline in the sward, treat violently to wash down the viruses and cells with viruses,and aspirate the supernatant and insert normal antibiotics.Then put them in the4℃conditons for use.2. RNA extractionThe virus RNA was extracted from specimens by using the Viral RNA Extraction Kit(TIANGEN, Beijing). RNA was dissolved in60ul RNase-free ddH2O to be used forRT-PCR or put them in the－20℃.3. Molecular identificationMolecular identification of enteroviruses was carried by the Real-time RT-PCRNucleic Acid Detect Kit for Enterovirus.4. VP1sequences analysis of enterovirus(1) Primer designSynthesize degenerate primers040-011with high specific for the3＇sequences of VP1region of enterovirus and design diagnosis primers through retrieving the sequences ofVP1region of EV71, CVA16and CVA10in the GenBank.(2) RT-PCR amplificationThe VP1region sequences of EV71, CVA16, CVA6and CVA10were amplifiedthrough RT-PCR.(3)DNA purificationThe amplicons were separated by agarose gel electrophoresis, purified with theTIANgel Midi Purification Kit (TIANGEN, Beijing).(4) Sequences and analysisSequences alignment and phylogenetic tree construction are carried by the bioinfor-matics software: DNAMAN5.2.2and BioEdit7.0.9.0.5. Recombination analysis of EV71genomeAnalyze the recombination of EV71genome with the recombination analysissoftwares: RDP and Simplot_v3.5.1.6. Bioinformatics analysis of the VP1protein of CVA10(1) Secondary structure prediction of the VP1protein of CVA10Secondary structure (Random coil, beta sheets, alpha helix, beta turn) of the VP1protein of CVA10is predicted by R4, HNN, PHD, Predator, SOPMA, etc.(2) Tertiary structure prediction of the VP1protein of CVA10We predict the spatial structure and model the VPl protein using SWISS-MODEL tertiary structure homology modeling in Expasy (http://au.Expasy.org/tools/).(3) Prediction and analysis the hydrophilicity, flexibility, surface probability andantigenic epitope of VP1protein of CVA10We analyze the single-parameter by using Kyte-Doolittle, Karplus, Schultz, Emini,and Jameson-Wolf, hydrophilic amino acids, flexibility, surface the possibility of antigenicindex in Protean of DNAstar software.(4) Comprehensive analysis of antigen epitopes of CVA10VP1proteinUsing ABCpred and DiscoTope server, we predict B cell epitope. Summary the resultsof Single-parameter prediction and identify the random coil in the secondary structure asthe B cell epitopes. And then verify the epitope prediction results by using ABCpred andDiscoTope.Result1. Nucleotide homology is95.7%-99.1%, and amino acid homology is98.7%-100%among the five Jinan EV71isolates. They have a higher homology with subgenotype C4aof EV71. The nucleotide homology is94.7%-98.8%. Therefore, they belong tosubgenotype C4a.2. The nucleotide and amino acid homology between two Jinan CVA16isolates are97.7%and96.8%, respectively. There were highly homology between them and CVA16subgenotype B1b. The nucleotide and amino acid homology are90.5%-95.5%and96.8%-100%, respectively. Compared with most of the strains, there is no amino acidmutation on JN-C07and JN-B11.3. We amplified39specimens, which were enterovirus positive but EV71and CVA16negative, using the primer040-011.We detected that5cases were CVA10and1case wasCVA6. The enterovirus serotypes of the remaining33samples needed to be determined.4. The33undetermined specimens were amplified using the diagnostic primers P16and P11.2. Four of these specimens determined CVA10. The nucleotide homology was94.5%-99.0%and amino acid homology was97.2%-99.5%among them.5. Contrast with BLAST program, JN-D04belong to CVA6genotype. Compared with12CVA6strains during2003to2009. The homology of nucleotide is75.1%-92.3%. Thehighest homology is EU908148which is reported in Taiwan in2008. The homology ofamino acid is85.2%-89.0%. The mutation sites of amino acids were767,773,776,777,792,806,812,824,827,841,843and845.6.2Jinan isolates in2010had a greater simlarity to EV71genotype C in the structuralP1region(>80%similarity in Simplot), but was more similar to the genotype B in the 2B-3B region(>80%). Low similarity of the3＇end of the2isolates genome to thereferernce strains was identified (<75%). However, it had a greater similarity to CVA16G-10in the3C-3＇UTR region (>75%).7. JN-C10is most closely with CVA10D genotypes. The homology of nucleotide andamino acid is91.5%-97.6%and90.4-98.6%, respectively. The length of VPl region whichwas sequenced is732nucleotides, its relative molecular mass is60,580, and theoreticalisoelectric point is5.10. Random coil is the main structure in secondary structure of VPlprotein. There were no transmembrane regions,and it is the extracellular protein. And wealso found that there was a known structure protein which was in the protein database(PDB)(3vbhA) with65%sequence identity with VP1. And we use it as a template to getthe VP1protein tertiary structure model. Finally, We obtained a variety of B cell epitope(12-23,100-110,115-125,169-180,195-215,220-238) by comprehensively analyzed.Conclusion1. Degenerate primers040-011has a high specificity to the3＇end sequences of VP1region of HEVA.2. Besides EV71and CVA16, CVA10and CVA6are the common pathogens toHFMD in our hospital.3. CVA10can cause serious clinical manifestations, such as severe hand, foot andmouth disease, viral encephalitis, epilepsy.4. Between2010and2011, Jinan enterovirus71isolates belong to subgenotype C4a,CVA16isolates belong to B1b and CVA10belong to genotype D, which are thepredominant virus genotype circulating in mainland China in recent years.5. A combination of intertypic and intratypic recombination were found in EV71which were isolated in2010in Jinan.6. CVA10VP1encoded protein contains many antigenic epitope regions,which mightbe potential target antigen for immunodiagnosis, treatment and prevention of CVA10infection.