Genetic characterization of EV71 isolates from 2004 to 2010 reveals predominance and persistent circulation of the newly proposed genotype D and recent emergence of a distinct lineage of subgenotype C2 in Hong Kong

Background: Enterovirus 71 (EV71) is a common etiological agent of hand, foot and mouth disease (HFMD) in children. EV71 epidemics have been reported in Hong Kong in recent years, and yet the genetic information of EV71 strains circulating in our locality is limited. The objective of this study was to investigate the genetic evolution of these EV71 isolates in Hong Kong over a 7-year period. Methods: Twenty-two EV71 isolates from Hong Kong during 2004-2010 were included for phylogenetic analysis using partial VP2-VP3, 2C and 3D regions. Eight EV71 strains were selected for complete genome sequencing and recombination analysis. Results: Among the 22 EV71 isolates, 20 belonged to subgenotype C4 and 2 belonged to subgenotype C2 based on the phylogenetic analysis of partial VP2-VP3, 2C and 3D gene regions. Phylogenetic, similarity plot and bootscan analyses using complete genome sequences of seven EV71 isolates of subgenotype C4 supported that the double-recombinant strains of subgenotype C4 persistently circulating in Hong Kong should belong to a newly proposed genotype D. Further analysis revealed two clusters, subgenotypes C4b and C4a (proposed genotypes D1a and D1b respectively), with genotype D1b strains being predominant in recent years in Hong Kong. A distinct lineage of EV71 subgenotype C2 has emerged in Hong Kong in 2008. The evolutionary rate of EV71 was 3.1 x 10(-3) nucleotide substitutions per site per year similar to that of other enterovirus, such as EV68, but was relatively lower than those of echovirus 30 and poliovirus. Molecular clock analysis using VP1 gene dated the time to the most recent common ancestor of all EV71 genotypes to 1900s, while the EV71 double-recombinant strains of genotype D were detected as early as 1998. Conclusions: This study provides the molecular basis for proposing a new genotype D of EV71 and assigning a discrete lineage of subgenotype C2. EV71 strains of genotype D have been circulating in Hong Kong for over 7 years, with genotype D1b being predominant.