The Study Of Viral Replication Capacity Of The Differernt Clinical Phenotype EV71 Isolates

Entero virus 71 (EV71) is a major neurotropic virus that causes hand-foot-and-mouth disease(HFMD) in infants and young children after poliovirus. HFMD is usually a mild disease, however, HFMD may be a severe neurogenic disease and even death, occasionally. The genome of EV71 contains a single open reading frame (ORF) flanked by 5’UTR and 3’UTR. Currently, the pathogenic mechanism of EV71 is not clear yet, meanwhile, vaccines and antiviral therapies are not available. The different clinical phenotype EV71 isolates can lead to diverse clinical outcomes, the mechanism of which was poorly understood. The capacity of EV71 replication plays an important role in the infection of EV71. The current study was designed to detect the replication of different clinical phenotype EV71 isolates. Further, we investigated whether the 5’UTR was responsible for the different replication of different virulent strains of EV71 using the reverse genetics method.Objective1. Growth curve of experiments were conducted to compare the replication capacity of different virulent strains of EV71.2. The replicon of EV71 was used to investigated the impact of UTR on the replication and expression of EV71.3. The recombinant EV71 was conducted to investigated whether the 5’UTR was responsible for the different replication of different virulent strains of EV71.Methods1. RD was infected with different virulent strains of EV71 and cultured at 37℃ and 39.5℃, respectively. Growth curve of EV71 was detected by qRT-PCR.2. The complete 5’UTR/3’UTR region of the SDLY 107 strain and GFP gene were amplified. The replicon of EV71-pMD-19T-5’UTR-EGFP-3’UTR was constructed by using restrict enzymes and ligases. Transcription in vitro was performed to generate S’UTR-EGFP-S’UTR RNA. The transcripted RNA was transfected into RD cells. The expression of GFP was observed through the fluorescent microscope and the replication of RNA was detected by qRT-PCR.3. The full-length EV71 infectious DNA clones was amplified by RT-PCR, and sequencing test was undertaken to validate the infectious DNA clones. Transcription in vitro was performed to generate the full-length EV71 infectious RNA. The transcripted RNA was transfected into RD cells. When more than 90% of the cells showed a typical cytopathic effect (CPE), the virus solution was harvested by freeze-thawing three times and the rescued virus was generated after some blind passages. Titers of the rescued viruses were determined by the Reed and Muench formula. Growth curve of the rescued EV71 was detected by qRT-PCR.4. The recombinant viruses (SDLY1+107) was constructed by replacing 5’ UTR of SDLY107 with that of SDLY1. Growth curve of the recombinant viruses (SDLY1+107) and rescued EV71 were detected by qRT-PCR.Result1. The replication capacity of severe EV71 strains (SDLY 107 and SDLY52) were higher than mild EV71 strains (SDLY11 and SDLY1) at high temperature (39.5℃) rather than at optimal temperature (37℃).2. The EV71 replicon RNA could express well in RD cell, while only EGFP RNA could not express in RD cell. The result revealed that the UTR region of EV71 was crucial for expression of EV71. However, the EV71 replicon RNA could not replicate in RD cell, which suggested that an important element might be exsisting in ORF region of EV71 besides UTR region of EV71.3. When three blind passages were done, the RD cells infected with rescued viruses appeared to show typical CPE, such as cell rounding, detachment, and floatation. The growth curves of the rescued SDLY 107 showed proliferation rates similar to wild-type SDLY107, which demonstrated that the rescue of EV71 was successful.4. The replication curves showed that the replication capacity of rescued SDLY107 were higher than the recombinant SDLY1+107 at high temperature (39.5℃) rather than at optimal temperature (37℃). The result suggested the 5TJTR might be responsible for the different replication of different virulent strains of EV71.Conclusion1.The capacity of replication was different in different virulent strains of EV71 at high temperature, which might be explaining why different EV71 strains caused different clinical outcomes in humans.2.The replication capacity of the severe EV71strains declined after 5’UTR replacement occurred in the severe EV71 strains, suggesting that 5’UTR was responsible for the different replication of different virulent strains of EV71...