Sequence analyses for full length genomes of human enterovirus 71 strains isolated from children in Beijing from 2008 to 2009

Objective To investigate whether the variation of the pathogenicity of enterovirus71(EV71) was related to the variation of the nucleotide sequencing of the virus of the full length genomes of EV71 strains isolated from pediatric patients with hand,foot and mouth disease (HFMD) presenting different symptoms in Beijing from 2008 to 2009.Methods Five EV71 strains in 2008 and 4 EV71 strains in 2009 isolated from Laboratory of Virology of Capital Institute of Pediatrics were chosen,of which 4 EV71 strains were isolated from severe HFMD children with high fever,continuous convulsion and loss of consciousness. Five EV71 strains were isolated from mild HFMD children.The full length genomes from 9 EV71 strains isolated from children with various clinical presentations were sequenced by amplification with RT-PCR and sequencing of 10 overlapped gene fragments covering full length of the genomes. Their nucleotide and amino acid sequences were aligned and phylogenetic relations of the sequences were analyzed by using DNAStar and MEGA software (version 4.1).Results The full length of 9 EV71 strains isolated from children in Beijing from 2008 to 2009 was 7 405 bp or 7 406 bp (Poly A tail not included) with a deletion in the 5'UTR for 6 out of these 9 strains. The overall nucleotide sequence identities of these 9 EV71 isolates were in the range of 96.3%-99.4%,while amino acid sequence identities were 98.2%-99.6%. The homology of the nucleotide sequences of VP1 of these 9 EV71 isolates was 96.9%-99.9%,while the homology of the amino acid sequences was 98.3%-100.0%. The majority of nucleotide changes were located at the third codon positions which caused silent mutations,thus the deduced amino acid sequence changes among those 9 EV71 isolates were scanty. The residue 144 in the VP2 protein and the residues 140 and 263 in the 3D polymerase (3Dpol) which were T,R and I were substituted by S,K and V,respectively in 3 out of 4 neurovirulent strains. Two bases,G and A at the nucleotides 208 and 254 in the 5'UTR were substituted respectively by A and G in the same 3 of those 4 neurovirulent strains. The nucleotide sequences of 9 EV71 isolates in the 5'UTR,P1,P2,P3 and 3'UTR regions exhibited the highest homology to the sub-genotype C4 of EV71. However,when compared with CV-A16 prototype strain G10,the nucleotide identities within the 3D and 3'UTR regions were higher than those of known genotypes A,B,C1-C3 and C5 of EV71. These 9 EV71 isolates formed a branch with other EV71 isolates circulating in mainland China and Taiwan previously identified as genotype C4 on the two phylogenetic trees.Conclusions The data from this study suggests that mutations at the residue 144 in the VP2 protein (T→S),the residues 140 and 263 in the 3Dpol (R→K and I→V) and the nucleotide 254 in the 5'UTR (A→G) may be associated with the severity of clinical presentations. Based on the entire VP1 gene sequences,the EV71 viruses circulating in Beijing from 2008 to 2009 should be classified as the sub-genotype C4. The non-structural protein encoding genomic regions may play a role in the evolution of EV71.