A generic assay for whole-genome amplification and deep sequencing of enterovirus A71

Le Van Tan,Do Chau Viet,Nguyen Thi Kim Tuyen,Ha Manh Tuan,Nguyen Thi Han Ny,Tran Tan Thanh,Nguyen To Anh,Vu Thi Ty Hang,Do Quang Ha,Kum Thong Wong,Lam Anh Nguyet,Kien Chai Ong,H Rogier Van Doorn,Phan Tu Qui,Tran Thi My Van,Jemma L Geoghegan,David Perera,Tran Thuy Ngan,Guy Thwaites,Edward C Holmes,Hoang Minh Tu Van,Saraswathy Sabanathan,Le Thị Thanh ,Nguyễn Văn Vĩnh Châu

doi:10.1016/j.jviromet.2015.02.011

Abstract

Enterovirus A71 (EV-A71) has emerged as the most important cause of large outbreaks of severe and sometimes fatal hand, foot and mouth disease (HFMD) across the Asia-Pacific region. EV-A71 outbreaks have been associated with (sub)genogroup switches, sometimes accompanied by recombination events. Understanding EV-A71 population dynamics is therefore essential for understanding this emerging infection, and may provide pivotal information for vaccine development. Despite the public health burden of EV-A71, relatively few EV-A71 complete-genome sequences are available for analysis and from limited geographical localities. The availability of an efficient procedure for whole-genome sequencing would stimulate effort to generate more viral sequence data. Herein, we report for the first time the development of a next-generation sequencing based protocol for whole-genome sequencing of EV-A71 directly from clinical specimens. We were able to sequence viruses of subgenogroup C4 and B5, while RNA from culture materials of diverse EV-A71 subgenogroups belonging to both genogroup B and C was successfully amplified. The nature of intra-host genetic diversity was explored in 22 clinical samples, revealing 107 positions carrying minor variants (ranging from 0 to 15 variants per sample). Our analysis of EV-A71 strains sampled in 2013 showed that they all belonged to subgenogroup B5, representing the first report of this subgenogroup in Vietnam. In conclusion, we have successfully developed a high-throughput next-generation sequencing-based assay for whole-genome sequencing of EV-A71 from clinical samples.

Highlights

Enterovirus A71 (EV-A71) belongs to the Enterovirus A species of the family Picornaviridae, and is genetically divided into three genogroups (A, B, and C)
An EV-A71 subgenogroup C4 isolate from a child with HFMD admitted to Hospital for Tropical Diseases (HTD) during the 2011 outbreak, and eight different isolates each belonging to a different subgenogroup (Table 2) obtained from the Department of Biomedical Science, University of Malaya, Malaysia were used for the initial assessment of assay performance
We first tested the assays on RNA derived from culture supernatants of different EV-A71 subgenogroups

Summary

Introduction

Enterovirus A71 (EV-A71) belongs to the Enterovirus A species of the family Picornaviridae, and is genetically divided into three genogroups (A, B, and C). Phylogenetic analyses and molecular clock dating suggest that strain replacement commonly occurs in EV-A71 (Tan et al, 2011), and that emerging subgenogroups may circulate cryptically in the community at low prevalence for years and cause mild infection prior to outbreak emergence (Tee et al, 2010). It is unclear whether viral evolution is the driver of emergence and large outbreaks of HFMD in Southeast Asia, or a consequence of the greater number of infected hosts. Taken together the available data highlight the importance of understanding EV-A71 evolution and population dynamics within and between endemic countries, which may be essential for understanding and controlling this emerging infection, including vaccine development

Methods

Results

Conclusion