Molecular Epidemiological Study On Enterovirus 71 Of Hand Foot And Mouth Disease’s Pathogen In Zunyi

Objective: To provide a theoretical reference for the prevention and treatment of hand foot and mouth disease(HFMD)caused by enterovirus 71(EV71)in Zunyi by exploring and discussing the genotype,variation,genetic evolution and understand from molecular level the epidemiological characteristics of EV71 in Zunyi area.Methods: From 2013 to 2015,220 cases of throat swabs from HFMD hospitalized children were selected as research samples,of which 187 cases were severe cases,and 33 cases were ordinary cases.EV71 infection samples were screened by identificating the virus type of all throat swabs,EV71 was cultured in vitro,its nucleic acid was extracted and the nucleic acid type was identified by reverse transcription-polymerase chain reaction(RT-PCR).The EV71 nucleic acid was amplified by RT-PCR and Escherichia coli plasmid,the amplified products were sequenced,then the sequence were sequentially edited,stitched,compared,similarity analysis and genetic evolutionary tree constructed by bioinformatics software.Results:(1)The detection of EV71: 220 cases of HFMD swab samples were tested,of which 105 cases were EV71,accounting for 47.7%(105/220).of the 187 cases of severe samples,100 cases were EV71,accounting for 53.5%(100/187).Of the 33 cases of common samples,5 cases were EV71,accounting for 15.2%(5/33).(2)Whole genome analysis of EV71:The whole genome sequence of seven EV71 strains were obtained in this study,the length of the whole genome sequence was 7405 base pair(bp),2193 amino acids were encoded by the whole genome sequence,there were differences in 10 sites of amino acid among seven strains.(3)Genetic and evolutionary analysis of EV71:(1)The similarity of seven EV71 strains were analyzed based on genome wide nucleotide,whole genome encoding amino acids,VP1 nucleotide and VP1 amino acid :The similarity of seven EV71 strains was very high,over 99%.(2)Similarity and phylogenetic tree analysis based on gene sequence of VP1 region between seven EV71 strains and Coxsackievirus A16 and various EV71 reference strains:The similarity and genetic distance between seven EV71 strains and each reference strain were significant.The similarity between seven EV71 strains and the EV71 reference strains was significantly higher than the CVA16 reference strains,and the genetic distance was significantly longer than the CVA16 reference strains.Compared with various EV71 reference strains,seven EV71 strains had the highest similarity and the most distant genetic distance with genotype C,followed by genotype B and genotype A.Among the C genotypes,similarity of seven EV71 strains and the C4 a subtype reference strain was the highest,the genetic distance was the furthest.(3)Similarity and phylogenetic tree analysis based on gene sequence of 5’Untranslated Region between seven EV71 strains and Coxsackievirus A16 and various EV71 reference strains:The similarity between 7 EV71 strains and the EV71 C4 a reference strain was the highest,and the genetic distance was the furthest,the similarity between seven EV71 strains and the EV71 A reference strain was the lowest,and the genetic distance was the closest.(4)Similarity and phylogenetic tree analysis based on whole genome sequence between seven EV71 strains and Coxsackievirus A16 and various EV71 reference strains:The similarity and genetic distance difference based on the whole genome sequence between seven EV71 strains and each reference strain is not as obvious as VP1.The similarity between seven EV71 strains and EV71 reference strains was slightly higher than the CVA16 reference strains,and the genetic distance was slightly distant.Compared with each EV71 reference strains,the similarity of seven EV71 strains with the B and C genotype reference strain was higher than the A genotype reference strain,and the genetic distance was also farther than the A genotype reference strain.About the comparison between seven EV71 strains and the reference strains of B and C gene subtype,the similarity between seven EV71 strains and the C4 a subtype reference strain was the highest,and the genetic distance also was the furtherst,followed by the C4 b subtype reference strain,the similarity and genetic distance between seven EV71 strains and C subtype and B subtype were not regular.Conclusion:EV71 may be the main pathogen of severe HFMD from 2013 to 2015 years in Zunyi area,its genotype is C4 a subtype,which is consistent with EV71 epidemic type in other parts of China,temporarily,no antigen transformation and introduction of new subtype of the virus,but there are sporadic variation;The study of EV71 genetic evolution based on the VP1 gene sequence is the most reasonable and reliable.