Study On Variations Of Amino Acids Among Different Virulent EV71

Enterovirus 71(EV71) is one of major causative agents of hand-foot-and-mouth disease(HFMD) particularly among children under 5 years old. The clinical characterizations of HFMD are prodromal fever followed by pharyngitis, mouth herpes and rash on the hands and feet. Most of patients recovered within a week while a few severe cases suffered from neurological complications such as convulsion, ataxia, brain-stem encephalitis, poliomyelitis-like paralysis and even death. Unfortunately, the mechanism of how EV71 infected lead to CNS inflammatory lesions is not clear till now. It has been reported that the severity of the HFMD is co-determined by both the host's immune state and viral genetic sequence affecting virulence. For instance, researchers had found that the pathogenicity of EV71 can be enhanced or weakened by a single mutation of its genome. Previously, our group had isolated six EV71 strains from patients with HFMD of Jinan between 2008 and 2010. Those EV71 strains, which were identified to C4 genotype, were classfied into two classes(heavy strain and lowly strain) according to its fatality to one-day-old mice. Sequences analysis demonstrated 8 substitutions in open reading frame(ORF) between heave strains and light strain. And in this study, we focus on searching for virulence determinants of EV71 and trying to uncover the molecular mechanism underlying pathogenic difference. To this end, reverse genetics approach were used to establish 8 mutant rescued viruses with single substitution in amino acid, then the replication ability of these viruses were compared with un-mutanted rescued virus and wild-type(WT strain) of EV71. Interestingly, only the one with a mutant in the 69 th residue of 3Cpro presented profoundly difference campared with WT strain. Tt's well known that EV71 3Cpro plays a significant role in both virus replication and virus-host interaction. Our results have indicated that the mutant in the 69 th residue of EV71 3Cpro would result in distinct reduction of viral replication which will in hence attenuate virulence of the virus. In order to figure out the reason why 3Cpro N69 D mutant is associated with virulence, we carried out the structural analysis of 3Cpro N69 D and enzymic activity assay to reveal dramatic structural and functional changes of 3Cpro N69 D compared with native 3Cpro which may well explain the molecular mechanism of reduced virulence of EV71 light strian.Purpose: To figure out that whether the 8 mutant residues existing in ORF of EV71 between mild and severe strains have relationships with viral virulence, and to figure out the mechanism of mild and severe HFMD causing by slight differences in genomic sequence.Methods: 1. With Quikchange method, mutant infectious clones of M1-M8 were constructed based on A12 plasmid and been rescued in RD cells. Rescued viruses were detected by RT-PCR, western blot and indirect immunofluorescence. One-step growth curves of M1-M8 rescued mutants were made in order to observe these mutants' replication in RD cells. 2. M7 rescued strain which had mutant residue in 3Cpro showed obvious replicational and infectious difference compared with others and wild-type EV71. Peptide substrates with fluorescent of 3Cpro were synthesised and used to detect the efficiency of mutant 3C-N69 D protease.Results: 1. Mutant infectious clones of M1-M7 were constructed successfully by one-step site-directed mutagenesis Quikchange method. Results showed that mutant viruses grew as well as the wild type of EV71 but the intracellular RNA of M7 amplified weakly and tend to a decline as time goes on. M7'RNA transcript could express viral proteins but failed to assemble into a complete virus particle. Q-RT PCR results showed that viral RNA didn't increase in RD cells. All above data indicated that the single substitution in M7 might cause great changes in pathogenicity of EV71 virus. 2. Bioinformatic analysis showed that the substitution of M7 located in the 69 th residue of 3C protease, which is very close to the activity center of 3C protease, and this mutant may be affect the activity of 3C protease. According to the experiment result, the N69 D mutation deprived one of the substrate binding switches and presented a overturning conformation in its active site. Furthermore, the catalytic activity of 3C-N69 D protease had activity decreased. Its ability to cleave the substrates was significantly decreased 5-10 times compared with 3C-wildtype protein.Conclusions: According to all of the above experment data, nucleotide 5591 mutant was the main cause of two groups EV71 strains with different virulence. Mutant residue inhibited viral replication by affecting 3C protease' activity. Nt5591 might be involved in virulence of EV71.