Genomic Sequence Analysis Of Enterovirus Type71Shanghai Strains And Construction Of An Infectious CDNA Clone

Abstract:Enterovirus71(EV71) and coxsackievirus A16(Cox A16) are the main pathogens of the Hand, foot, and mouth disease (HFMD) in infants. EV71infection is more frequently associated with severe diseases such as asepic meningitis, encephalitis, poliomyelitis-like paralysis than Cox A16.EV71, a positive, single-strand RNA virus, is a member of the human enterovirus group. The genome containes approximately7400nucleotides and is flanked by untranslated regions (UTRs) at both ends. A polyprotein of2194amino acids expressed by EV71can be divided into3different regions:P1for capsid proteins (VP1to VP4), P2and P3for non-structural proteins (2A,2B,2C,3A,3B,3C and3D).In this thesis,7virus strains were isolated from stool specimens of HFMD patients in Shanghai region (four patients with severe syndrome and three with mild syndrome). Six pairs of primers were designed for amplification of the full-length genome of EV71. The complete sequence of EV71was obtained from6overlapped DNA segments produced by RT-PCR and3’-RACE. The sequence data were submitted to GenBank (accession numbers from HQ891923to HQ891929).VP1sequences of the seven strains with some reported EV71strains were phylogenetically analyzed. The results showed that all of the Shanghai strains are subgenotype C4, and closer to the strains isolated from Beijing and Fuyang. However, phylogenetic analysis of the full-length genome sequences showed that the Shanghai strains had more relative to strains isolated from Beijing, Fuyang, and Zhejiang. The genomic similarity plot and bootscan analysis of Shanghai strains with other EV71subgenotypes and Cox A16showed that Shanghai strains could recombined with genotype C2sequence from the5’UTR to the2A region (at the position of3600nucleotide), with Cox A16G-10strain from3C (at position of5800nucleotide) to3’UTR.The replication of EV71Shanghai strains in human rhabdomyosarcoma (RD) cells were detected by TCID50and genomic copies of real-time PCR. Among four strains from severe complicated illness (HQ891923, HQ891924, HQ891925and HQ891928) HQ891923strain has no obvious cytopathic effects (CPE) in infected RD cells. Among three strains from mild illness (HQ891926, HQ891927and HQ891929), both HQ891926and HQ891927appear high replication efficiency and CPE. These results showed that the multiplication capability and the cytopathic effects induced by EV71Shanghai strains were not determined by the clinical symptoms.The genome sequences showed no significant differences among seven EV71Shanghai stains. However, the replication and cytopathic effects induced by different virus strains showed significant difference. In order to explore the regulating elements and the main virulence factor, the high replication capability of HQ891927strain was applied to construction of an infectious cDNA clone. The full-length genome of EV71with a poly A tail at3’UTR were cloned into a vector by a strategy of three fragments. RNA transcriptions were transfected into RD cells. The rescued viruses were identified by RT-PCR with EV71-specfic primer and indirect immunofluorecence assay with antibody to VP1. The infectious cDNA clone of shanghai strain (HQ891927) would be applied in the reverse genetics pf EV71.