Clinical Enterovirus Type 71 VP1 Gene Research Of Hand,Foot,and Mouth Disease

Objective:Hand, foot ,and mouth disease (HFMD ) is a common infectious disease caused by intestinal virus in childhood, clinical with hand, foot and mouth of different level herpes as the main symptoms, accompanied by fever, sore throat and diarrhea, good hair at 5 years old or so children. Related studies have shown that HFMD have a late spring early summer seasonal and pandemic of the disease subtype of space-time distribution and regional difference, and affected by temperature, humidity and influence factors such as age, mainly through the way such as gastrointestinal,respiratory and close contact. Most pathogens for HFMD are enterovirus 71 (EV71)and coxsackie virus type A 16 (CA16) and other intestinal virus subtype group(enterovirus, EVs), EV71 infection often can cause severe central nervous system(CNS) complications, such as acute encephalitis, encephalitis, encephalomyelitis,paralytic paralysis, etc. EV71 has A, B and C three genotypes, and its plasmid encoding VP 1 protein is the most pathogenic , also one of the most important structural protein, its change is a major determinant of EV71 immunogenicity and it can be used for the identification and evolutionary analysis of the virus.This study of clinical HFMD EV71 VP1 gene sequence analysis of isolated strains, from the molecular level, to explore the molecular genetic evolution of HFMD change for screening HFMD susceptible population, risk assessment, provide a scientific basis for clinical prevention and treatment.Methods:1 The object of study: collect during January 2015 to December 2016 in Yan’an Hospital Affiliated to Kunming Medical University clinical suspected HFMD patients 590 cases, among which 355 cases were male, female 235 cases aged 0 to 36,5 years of age or less 470 cases, 118 cases aged 5 to 18, 18 years old or more in 2 cases.Suspected patients mainly because of the palm of the hand, foot and mouth sores which appear for the first symptoms or accompanied by fever, diarrhea and and other diseases in our hospital department of Dermatology and Department of Pediatrics and other clinical departments.2 Specimen collection and preservation: refer to the People’s Republic of China Ministry of Health issued "HFMD prevention and control guidelines (2008 Edition)".The principle of informed consent in the object of study, collecting HFMD patients within 7 days of onset of 5-8g/ stool specimens, used for pathogen detection and after being taken into the sterile tube immediately for the stool sampling inspection, not timely detection of - below 20 ℃ low temperature freezing, avoid repeated freezing and thawing in the detection and storage process.3 EV71 detection: using real-time fluorescence PCR method is adopted for the detection of EV71 virus nucleic acid, using the Enterovirus 71 Viral RNA (EV71)Qualitative Diagnostic Kit (PCR Fluorescence Probing), the kit is provided by Jiangsu biological technology co.,LTD, the specific testing steps following the kit operating instructions.4 Extraction of viral RNA: the virus positive cases EV71 detection of clinical stool samples of EV71 virus RNA was extracted ,using Nucleic Acid Isolation Kit(Spin Column Method),kit provided by of da an gene co.,LTD., zhongshan university ,the specific operation steps according to the kit operating instructions.5 Reverse transcriptase-polymerase chain reaction (RT-PCR): EV71 virus RNA was extracted as a template for reverse transcription polymerase chain reaction,the reverse transcription kit is provided by TaKaRa company in Japan, the specific steps according to the kit operating instructions.6 PCR reaction for amplification purpose of VP1 gene: specific primers were designed according to Primer Premier 5.0 software, and by the Beijing Bomaide Technology Development Co. Ltd. for the biosynthesis of VP1 gene amplification,VP1 gene was amplified by PCR reaction.7 Agarose gel electrophoresis of PCR products: the product was amplified by PCR after 1% agarose gel electrophoresis. According to the results of agarose gel electrophoresis appraisal amplification purpose gene product, take photos and then record the related experimental results at the same time.8 EV71 VP1 gene PCR amplification product sequencing: VP1 gene amplification of enterovirus EV71, the amplified products to Beijing Bomaide Technology Development Co. Ltd. were sequenced and analyzed. According to the determination of EV71 VP1 nucleotide sequence and gene library (GenBank) for comparison and identification of EV71 related reference strains sequence homology analysis and determination it belongs to genotype.9 Constructing phylogenetic trees: inspection EV71 strains compared with EV71 related reference strains in GenBank sequence identification, the sequencing result of application of DNAMAN (5.1.0.0) software (Lynnon Biosoft) gene sequence comparison and homology analysis, the specific operation in accordance with the instructions for use in software. In addition, and using the method of MEGA 4 software (http://www.megasoftware.net/) using neighbor joining (NJ) and Kimura 2-parameter model was used to construct the phylogenetic tree of genetic variation of the species, and the bootstrap value was 1000, to understand the evolutionary characteristics of the VP1 gene sequence and the phylogenetic relationship between the domestic and foreign related strains.Results:1 Check out the situation: January 2015 to December 2016 in Yanan Hospital Affiliated Hospital of Kunming Medical University, 590 cases of suspected HFMD patients in laboratory examination were detected in 50 cases of type EV71, the detection rate was 8.47% (50/590 cases), of which 28 cases were detected in 2015, 22 cases yearly check in 2016. 50 cases<5 years 35 cases, 5-18 years 13 cases, ≥18 years 2 cases, and accounted for 64.00% (32/50 cases) of the men and women, 36.00%(18/50 cases), including 2 cases of adult patients with HFMD in female patients.2 Sequencing analysis: analysis of 50 strains of virus EV71 VP1 gene isolates detected by gene sequencing, sequencing and comparison of gene sequences of reference strains GeneBank( Sequence ID: KU936121.1) and homology analysis, the homology of 97.96%-99.43%, and to determine the inspection strains were EV71 C4 subtype.3 Phylogenetic tree analysis: EV71 strain and Beijing (No.: EV71 strain BJ4211 VP1), Anhui Hefei in 2008 (No.: EV71 isolate 1401-Luan (CHN) -08 VP1;EV71 isolate 1404-Luan (CHN) -08 VP1) and Malaysia Sarawak region EV71 epidemic strain (No.: EV71 isolate SB12007-SAR-03 VP1; EV71 isolate SB12278-SAR-03 VP1; EV71 isolate SB10712-SAR-03 VP1) genotypes were in the same evolutionary lineage, genetic distance is close. And Ningbo in Zhejiang province in 2010 (No.: EV71 strain EV71/Ningbo.CHN/001/2010), Shanghai in 2014(No.: EV71 strain SHAPHC5251/SH/ CHN/14; EV71 strain SHAPHC5330/SH/CHN/14), Guangdong, Shenzhen in 2014 (No.: EV71 isolate EV71/SZ07/CHN/2014; EV71 isolate EV71 /SZ88/CHN/2014), from 2013 to 2014 in Wenzhou Zhejiang province,(No.: EV71 isolate EV71/P156/2013/China; EV71 isolate EV71/P654/2013/China; EV71 strain 15/EV71/Wenzhou /CHN/2014) separated from the clinical epidemic area EV71 strains heredity distance is farther. With the Australian region(No.: EV71 strain 2978-SYD-92 VP1; EV71 strain 7784-SYD-90 VP1; EV71 strain 1182-SYD-91 VP1; EV71 strain 6560-SYD-86 VP1) separation of EV71 strains genetic distance farthest.Conclusions:1 Homology analysis and identification: from January 2015 to December 201650 isolated strains of EV71 VP1 and GeneBank EV71 gene sequences of reference strains of Sequence ID:KU936121.1, the homology of the sequences is 97.96%-99.43%; the sequences are EV71 C4 subtype.2 Phylogenetic analysis: constructing phylogenetic tree showed that strains of EV71 in 2 years without the occurrence of genetic variation, genetic distance of isolated EV71 phylogenetic relationship with the domestic Beijing and central Anhui Hefei and Southeast Asian countries in Malaysia EV71 is near; and the Shanghai area,Zhejiang, Ningbo, Wenzhou and Guangdong Shenzhen area phylogenetic genetic distance is 1.52, and the Australia EV71 strain genetic distance is 2.11.