Molecular Epidemiogical Investigation Of Enterovirus 71 And Study On MicroRNA Inhibiting Enterovirus71 Replication In Vitro

In the spring of 2008, an EV71-caused HFMD outbreak occurred in Fuyang city of Anhui Province, China,and spread to neighbor provinces and even across south China area. As a result, HFMD was classified as a category C notifiable infectious disease by the Ministry of Health of China since 2nd May 2008.Hand, foot, and mouth disease (HFMD) is a viral illness commonly occurring in early childhood, which can lead to a distinct clinical presentation of fever, blister-like eruptions in the mouth and/or skin rash on distal extremities. HFMD is commonly caused by members of the Enterovirus genus, namely, human Enterovirus 71 (EV71) or coxsackievirus A16 (CAV16). Usually, HFMD appears mild symptoms and resolves spontaneously within 10-14 days.However, some HFMD cases caused by EV71 can lead to severe neurological complications and even deaths.Objective:This study was to get up a multiplex real-time PCR with TaqMan-LNA probe which can rapidly identify the pathogen of hand, foot and mouth disease (HFMD). The purpose of this study was to assess the epidemiology of HFMD infection in Zhenjiang City of Jiangsu Province and to analyze molecular epidemiology of HEV71 in Jiangsu Province.The purpose of this study was to find some miRNA which differential expressed after EV71 infect the SK-N-SH cell.Then,we want to find the miRNA which can have antiviral activity against EV71.Methods:(1) A multiplex real-time PCR with TaqMan-LNA probe which can rapidly identify EV71 and universal enterovirus(EV-U) simultaneously was developed. A serial dilutions of RNA standard was prepared by transcription in vitro and used for generating standard curve. The specificity, sensitivity, reproducibility of the real time RT-PCR assay were estimated and 234 clinical samples were tested.(2) The HFMD cases were reported in every month, but the peak incidence occurred in Apr to Jun, which accounted for accounted for 56.2% of the total cases. The small peak incidence occurred in Nov to Dec, which accounted for accounted for 17.4% of the total cases.The infected children were predominantly male. The age-specific peak incidence occurred from 1 to 3 years old, the children less than 3 years old accounted for 46.3% of the total cases. The infected children were predominantly scattered inhabiting children and the children in kindergarten accounted for 95.6%of the objects. The incidence between urban area and county was differern too.(3) The HFMD cases in Zhenjiang, Jiangsu, were investigated. Some throat specimens were randomly selected from different patients, and nucleotide sequences of EV71 VP1 regions were successfully determined by RT-nested-PCR and sequencing. EV71 genotypes were characterized by phylogenetic analyses.(4) Differentially expressed miRNAs between the SK-N-SH cell after EV71 infection and normal cell were screened with a miRNAs chip. The miRNAs differentially expressed were searched from those miRNAs. Then the microRNA with differential expression were proved using real-time PCR method.(5) Those miRNAs related to EV71 infection were reverse transfected with miRNA mimics or inhibitors using RNAiMAX in a 96-well plate format. Then, the cells were infected with EV71. Supernatant from the infected cells was collected afer 72 hours, the viral titer, the RNA and protein of the target genes were measured. Target genes regulated by the miRNAs were searched with bioinformaties method.Results:(1) The results showed 100% specificity for the selected panel. The assay met the sensitivity of 102 copies/μL. Analysis demonstrated high reproducibility with a coefficient of variation (CV) of 0.97%-2.02% for EV71 and 0.89%-2.13% for EV-U.234 clinical samples were detected by real-time RT-PCR.(2) The incidence rate of HFMD was highest in the period of Apr-Jun and in the 1-3 years old age groups. Intriguingly, there was a slight predominance for boys and for children living in rural areas in HFMD infection. (3) Phylogenetic analyses indicated that all Jiangsu EV71 strains and most China strains belonged to subgenotype C4a.(4) There were 116 miRNAs differentially expressed between the normal SK-N-SH cell and the cell infected by EV71,including 46 up-regulated miRNAs and 70 down-regulated miRNAs.There was a significant concordance between the qRT-PCR results and the microarray analysis.(5)Using model of SK-N-SH cell infected by EV71, the antiviral effect of the miRNA minics and inhibitior on EV71 was evaluated in the level of mRNA,protein and viral titer (TCID50) in vitro.The results show that the RNA level of EV71 has not changed, the VP1 protein level and the viral titer (TCID50) have been down-regulated after the miR-27a or miR-23b minics treatment.Conclusion:(1) As a result of its high specificity, sensitivity and reproducibility, the TaqMan-LNA assay is suitable for rapid clinical diagnosis of EV71 and EV-U to diagnose HFMD.(2) Routine HFMD surveillance should be focused on the period of Apr to Jun, and more prevention efforts should be aimed at 1-3 years old children. Moreover, government efforts are urgently needed to improve public health condition and medical service quality in rural areas.(3) The C4a was the most prominent EV71 subgenotype circulating in China.(4) There were 116 miRNAs differentially expressed between the normal SK-N-SH cell and the cell infected by EV71.Those miRNAs maybe were closely related with the infection and replication of EV71.(5) miR-27a and miR-23b exert an antiviral function to EV71 upon over-expression. The regulation occurs post-transcriptionally.