Abstract:In order to explore virulence determinants of enterovirus type 71(EV71) and develop novel vaccines,a full-length genomic cDNA clone was constructed in this study. Full-length cDNA of EV71 strain FJ08149 was obtained by RT-PCR and subsequently cloned into vector TOPO-XL-PCR. Two positive cDNA clones,pFJ08149T5 and pFJ08149T25,respectively,were obtained and linearized for RNA synthesis in vitro using T7 polymerase. The synthesized RNA was transfected into RD cells and typical CPE of enterovirus was observed on 72 hours post-transfection,the rescued virus was identified by indirect immunofluorescence(IFA),RT-PCR,sequencing and immunoelectron microscopy,demonstrated successful recovery of infectious EV71 viruses from transfected cells. The construction of infectious EV71 cDNA clone would facilitate ongoing research on pathogenesis of EV71.
〔1〕Lin TY,Twu SJ,Ho MS,et al.Enterovirus71outbreaks,Tai-wan:occurrence and recognition〔J〕.Emerg Infect Dis,2003,9:291-293.
〔2〕世界卫生组织.脊髓灰质炎实验室手册〔R〕.4版.中国疾病预防控制中心:北京,2004:78-96.
〔3〕Racaniello VR,Balti more D.Cloned poliovirus complementary DNAis infectious in Mammalian cells〔J〕.Science,1981,214(11):916-919.
〔4〕Arita M,Shi mizu H,Nagata N,et al.Temperature-sensitive mutants of enterovirus71show attenuation in cynomolgus mon-keys〔J〕.J Gen Virol,2005,86:1391-1401.
〔5〕Arita M,Ami Y,Wakita T,et al.Cooperative effect of the at-tenuation determinants derived from poliovirus sabin1strain is essential for attenuation of enterovirus71in the NOD/SCID mouse infection model〔J〕.J Virol,2008,82(4):1787-1797.
〔6〕Sarnow P.Role of3’-end sequences in infectivity of poliovirus transcripts made in vitro〔J〕.J Virol,1989,63(1):467-470.
〔7〕Spector DH,Balti more D.Requirement of3’-terminal poly(ade-nylic acid)for the infectivity of poliovirus RNA〔J〕.Proc Natl Acad Sci,1974,71:2983-2987.
〔8〕黄莺,贾丽丽,孙志伟,等.流行性乙型脑炎病毒(JEV)全长感染性克隆的制备及恢复病毒的获得〔J〕.病毒学报,2003,19(4):313-319.