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

Abstract

BackgroundEnterovirus 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.MethodsTwenty-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.ResultsAmong 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 × 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.ConclusionsThis 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.

Ongoing change of severe hand, foot, and mouth disease pathogens in Yunnan, China, 2012 to 2016

“…Recombination plays a very important role in the evolution of viruses and often occurs in EVs. Some isolated EVs have been identified as recombinant strains, such as EV‐A71 and CV‐A16 . Many different EVs cocirculate in outbreaks and sporadic cases of HFMD.…”

Genetic characterization of enterovirus A71 isolates from severe neurological cases in Poland

“…Viral RNA was extracted from 140 µl of infected cell supernatants using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. The complete VP1 coding region and 455 bp region of 3D gene was amplified with nested reverse transcription PCRs using Superscript III (Invitrogen, Carlsbad, CA), primers specific for EV‐A71 and PCR cycling times and temperature as previously described …”

Genetic Diversity of Enterovirus A71, India

“…Bessaud et al proposed 2 new genogroups in sub-Saharan Africa (genogroup E) and Madagascar (genogroup F) ( 12 ). Other new subgenogroups proposed recently include C4a, C4b, C6, C7, and B6 ( 9 , 13 ). We detected 14 EV-A71 among nonpolio enterovirus (NPEV) isolates from persons with AFP, HFMD, and encephalitis reported in Mumbai and surrounding areas during 2008–2012.…”