Analysis Of The VP1 And 5'Untranslated Region Of Human Enterovirus 71

Enterovirus 71 (EV71) is the major cause of Hand, foot and mouth disease (HFMD) in children. EV71 infection can be accompanied by a series of syndromes with or without central nervous system involvement, including aseptic meningitis and neurogenic pulmonary edema, et al. During 2008, a large HFMD epidemic, caused mostly by EV71 infection, occurred in China, resulting in many severe cases and deaths. Since May 2008, HFMD has been managed as the type C infectious disease by the Ministry of Health. EV71 outbreaks have been and will continue to be an important public health issue in China.EV71 is a single stranded, positive-sense RNA virus. It belongs to the human Enterovirus A species of the Enterovirus genus within the family Picornaviridae. As one of the four capsid proteins, VP1 is the most external and important immunodominant and it's genetic diversity correlates with viral serotype closely. So VP1 has been used as the basic sequence for phylogenetic analyses and genotype. Beyond the 5'side of the ORF, the 5'untranslated region (5'UTR) which contains the internal ribosome entry site (IRES) and other structural elements play an important role in RNA replication and translation. Study on the exact location of IRES in the EV715'UTR may provide a potential specific target site for antiviral treatment.Objectives1. To analyze the nucleotide sequences of VP1 and 5'UTR of EV71 with different clinical outcomes,2. To study the genotype of the strains isolated from Zhegnzhou,2010.2. To predict and analyze the RNA secondary structure of the 5'UTR of EV71 strains with different clinical outcomes.3. To Find out the exact location of IRES in the 5'UTR.Methods1. Specimens:Stool specimes (n=46) were obtained from the HFMD patients at the 6th People's Hospital of Zhengzhou.2. Extraction of RNA:Viral RNA was extracted from supernatants of stool suspensions.3. RT-PCR with EV71-specific primers to detect the EV71 positive samples.4. RT-PCR and nucleotide sequencing of the VPl of EV71.5. RT-PCR, gene cloning and nucleotide sequencing of the 5'UTR of EV716. Phylogenetic analysis:A phylogenetic dendrogram was constructed using the neighbor-joining method of the MEGA5 program.7. RNA draw 1.1 was used to predict the RNA secondary structure of the 5'UTR of EV71.Results1. The complete nucleotide sequences of VP1 and 5'UTR of EV71 were obtained.2. The VP1 of 11 EV71 strains isolated were over 97.6%homologous at the nucleotide level and the VP1 of 5 mild cases were over 97.8%homologous to that of 6 severe cases. At the amino acid level, these strains were almost 100%homologous to one another. As for the 5'UTR region of 9 EV71 strains, they were over 96.9% homologous at the nucleotide level and the 5'UTR of 4 mild cases were over 96.9% homologous to that of 5 severe cases.3. The VP1 of 11 EV71 strains this study were 97.4%homologous to members of the C4a subgenogroup averagely. It was indicated that these EV71 isolates belonged to genotype C4a. The phylogenetic tree constructed based on 5'UTR was similar to the tree made based on VP1.4. Compared with the EV71 strains isolated from mild cases, the secondary structures of EV71 strains isolated from severe and dead cases were more complicated and compact.5. The IRES located between 101-592 of the 5'UTR of the EV71.Conclusions1. The homology of VP1 and 5'UTR nucleotide sequences of the EV71 strains in this study was rather high. And all these strains shared the same VP1 amino acid sequence. The neurovirulence determinant sites at VP1 and 5'UTR were not found among these strains.2. The EV71 strains in this study belonged to C4a genotype.3. The Phylogenetic tree constructed based on 5'UTR was similar to the tree made based on VP1.4. The IRES located between 101-592 of the 5'UTR of the EV71.