Sequence analysis for the 3' end of genomes from enterovirus 71 isolated in Beijing during 2008-2009

Abstract

Objective To investigate the sequences of the 3' end of genomes from enterovirus 71 isolated from pediatric patients with different symptoms in Beijing during 2008-2009.Methods Clinical specimens were collected from pediatric patients suffering from hand,foot and mouth disease(HFMD)and/or with neurological complications who visited the affiliated Children's Hospital during the epidemic seasons of 2008 and 2009 in Beijing.The samples were inoculated into the Vero cell line,and the virus isolates were further identified by nested reverse transcription-nested PCR(RT-nPCR)assay using universal enterovirus primers,type specific primers for EV71 and CA16.The 3' end of genomes(including 3D and 3' UTR regions)from 10 EV71 strains derived from various clinical presentations were amplified and sequenced.Results Analysis of the nucleotide sequences of the amplified fragments showed that the 3' end of genomes of 10 EV71 isolates include the 3D region of 1386 nucleotides(nt)encoding the 3D polymerase(3Dpol,462 amino acids),the terminator codon TGA and 3' UTR of 81 nt.Nucleotide sequence identities among 3D regions from these 10 EV71 isolates were in the range of 95.8%-99.6%,while the nucleotide sequence identities for 3' UTR were 96.3%-100%.The majority of nucleotide changes were located at the third codon positions which caused silent mutations,thus the amino acid sequence changes of 3Dpol among those 10 EV71isolates were scanty.The residues 140 and 263 which were R and 1 were substituted by K and V,respectively in 3 of 4 neurovirulent strains,whereas only 1 of 6 strains from mild cases had these 2 amino acid changes.The sequences of the 3D and 3' UTR regions of 10 EV71 isolates were compared to the representative strains of known genotypes from the GenBank.The nucleotide and amino acid sequences of 10 EV71 isolates in the 3D region exhibited highest homology to the subgenotype C4 of EV71(92.7%-94.2%and 96.8%-97.6%.respectively).However,3' UTR of 10 EV71 isolates shared the highest nucleotide identity with CA16/G10(88.9%-91.4%).The phylogenetic analysis based on the 3D regions demonstrated that 10 EV71 isolates had the closest genetic relationship with the representative isolate of CA sub-genotype of EV71 and shared more homology with CA16/G10 than other known genotypes of EV71.Conclusion Genetic analysis of the 3' end of genomes from 10 EV71 strains indicated that the 3' end of genome may play a role in the evolution of EV71. Key words: Hand,foot and mouth disease; Enterovirus 71; 3' End; Sequence analysis