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Observational Study
. 2015 Apr:215-216:30-6.
doi: 10.1016/j.jviromet.2015.02.011. Epub 2015 Feb 19.

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

Le Van Tan  1 , Nguyen Thi Kim Tuyen  2 , Tran Tan Thanh  2 , Tran Thuy Ngan  2 , Hoang Minh Tu Van  3 , Saraswathy Sabanathan  4 , Tran Thi My Van  5 , Le Thi My Thanh  5 , Lam Anh Nguyet  2 , Jemma L Geoghegan  6 , Kien Chai Ong  7 , David Perera  8 , Vu Thi Ty Hang  2 , Nguyen Thi Han Ny  2 , Nguyen To Anh  2 , Do Quang Ha  2 , Phan Tu Qui  9 , Do Chau Viet  10 , Ha Manh Tuan  10 , Kum Thong Wong  7 , Edward C Holmes  6 , Nguyen Van Vinh Chau  5 , Guy Thwaites  4 , H Rogier van Doorn  4
Affiliations
Observational Study

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

Le Van Tan et al. J Virol Methods. 2015 Apr.

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.

Keywords: Deep sequencing; Enterovirus A71; Hand foot and mouth disease; Phylogeny; Picornavirus.

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Figures

Fig. 1
Fig. 1
Agarose gel electrophoresis analysis of amplified products from 11 throat swabs from HFMD patients; (A) PCR 1; (B) PCR 2; (C) PCR 3; lanes 1-11: clinical samples, PC: positive control; NC: negative control; amplified products of expected sizes are indicated by arrows.
Fig. 2
Fig. 2
Boxplots showing Cp values generated by an EV-A71 real time RT-PCR of RT-PCR positive and negative groups, Cp value median; range: 28.2; 23.9–32.4 versus 31.2; 29.2–35.5; P < 0.001.
Fig. 3
Fig. 3
ML phylogeny based on complete genomes (in bold) obtained in this study and those of representatives of EV-A71 downloaded from the GenBank. A similar result was obtained when VP1 sequences were analyzed separately (data not shown). The tree is rooted on a single genogroup A sequence (USA/A/1970) and all horizontal branch lengths are drawn to a scale of nucleotide substitutions per site. Bootstraps >70% are also shown. My: Malaysia, TW: Taiwan, NL: the Netherlands, KOR: Korea, CHN: China.
Flowchart 1
Flowchart 1
Chart showing analysis procedure for obtaining the complete genome of EV-A71 from clinical samples.
See this image and copyright information in PMC

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