title: A generic assay for whole-genome amplification and deep sequencing of enterovirus A71.
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.
authors.affiliation: Oxford University Clinical Research Unit, Ho Chi Minh City, Viet Nam. Electronic address: tanlv@oucru.org.
authors.forename: Le Van
authors.initials: le V
authors.lastname: Tan
authors.affiliation: Oxford University Clinical Research Unit, Ho Chi Minh City, Viet Nam.
authors.forename: Nguyen Thi Kim
authors.initials: NT
authors.lastname: Tuyen
authors.affiliation: Oxford University Clinical Research Unit, Ho Chi Minh City, Viet Nam.
authors.forename: Tran Tan
authors.initials: TT
authors.lastname: Thanh
authors.affiliation: Oxford University Clinical Research Unit, Ho Chi Minh City, Viet Nam.
authors.forename: Tran Thuy
authors.initials: TT
authors.lastname: Ngan
authors.affiliation: Oxford University Clinical Research Unit, Ho Chi Minh City, Viet Nam; Children's Hospital 2, Ho Chi Minh City, Viet Nam.
authors.forename: Hoang Minh Tu
authors.initials: HM
authors.lastname: Van
authors.affiliation: Oxford University Clinical Research Unit, Ho Chi Minh City, Viet Nam; Centre for Tropical Medicine, Nuffield Department of Medicine, University of Oxford, Oxford, UK.
authors.forename: Saraswathy
authors.initials: S
authors.lastname: Sabanathan
authors.affiliation: Hospital for Tropical Diseases, Ho Chi Minh City, Viet Nam.
authors.forename: Tran Thi My
authors.initials: TT
authors.lastname: Van
authors.affiliation: Hospital for Tropical Diseases, Ho Chi Minh City, Viet Nam.
authors.forename: Le Thi My
authors.initials: le TM
authors.lastname: Thanh
authors.affiliation: Oxford University Clinical Research Unit, Ho Chi Minh City, Viet Nam.
authors.forename: Lam Anh
authors.initials: LA
authors.lastname: Nguyet
authors.affiliation: Mahir Bashir Institute for Infectious Diseases & Biosecurity, Charles Perkins Centre, School of Biological Science and Sydney Medical School, The University of Sydney, Sydney, Australia.
authors.forename: Jemma L
authors.initials: JL
authors.lastname: Geoghegan
authors.affiliation: University of Malaya, Kuala Lumpur, Malaysia.
authors.forename: Kien Chai
authors.initials: KC
authors.lastname: Ong
authors.affiliation: Institute of Health and Community Medicine, Universiti Malaysia Sarawak, Sarawak, Malaysia.
authors.forename: David
authors.initials: D
authors.lastname: Perera
authors.affiliation: Oxford University Clinical Research Unit, Ho Chi Minh City, Viet Nam.
authors.forename: Vu Thi Ty
authors.initials: VT
authors.lastname: Hang
authors.affiliation: Oxford University Clinical Research Unit, Ho Chi Minh City, Viet Nam.
authors.forename: Nguyen Thi Han
authors.initials: NT
authors.lastname: Ny
authors.affiliation: Oxford University Clinical Research Unit, Ho Chi Minh City, Viet Nam.
authors.forename: Nguyen To
authors.initials: NT
authors.lastname: Anh
authors.affiliation: Oxford University Clinical Research Unit, Ho Chi Minh City, Viet Nam.
authors.forename: Do Quang
authors.initials: do Q
authors.lastname: Ha
authors.affiliation: Oxford University Clinical Research Unit, Ho Chi Minh City, Viet Nam; Hospital for Tropical Diseases, Ho Chi Minh City, Viet Nam.
authors.forename: Phan Tu
authors.initials: PT
authors.lastname: Qui
authors.affiliation: Children's Hospital 2, Ho Chi Minh City, Viet Nam.
authors.forename: Do Chau
authors.initials: do C
authors.lastname: Viet
authors.affiliation: Children's Hospital 2, Ho Chi Minh City, Viet Nam.
authors.forename: Ha Manh
authors.initials: HM
authors.lastname: Tuan
authors.affiliation: University of Malaya, Kuala Lumpur, Malaysia.
authors.forename: Kum Thong
authors.initials: KT
authors.lastname: Wong
authors.affiliation: Mahir Bashir Institute for Infectious Diseases & Biosecurity, Charles Perkins Centre, School of Biological Science and Sydney Medical School, The University of Sydney, Sydney, Australia.
authors.forename: Edward C
authors.initials: EC
authors.lastname: Holmes
authors.affiliation: Hospital for Tropical Diseases, Ho Chi Minh City, Viet Nam.
authors.forename: Nguyen Van Vinh
authors.initials: NV
authors.lastname: Chau
authors.affiliation: Oxford University Clinical Research Unit, Ho Chi Minh City, Viet Nam; Centre for Tropical Medicine, Nuffield Department of Medicine, University of Oxford, Oxford, UK.
authors.forename: Guy
authors.initials: G
authors.lastname: Thwaites
authors.affiliation: Oxford University Clinical Research Unit, Ho Chi Minh City, Viet Nam; Centre for Tropical Medicine, Nuffield Department of Medicine, University of Oxford, Oxford, UK.
authors.forename: H Rogier
authors.initials: HR
authors.lastname: van Doorn
mesh_terms: D002675:Child, Preschool; D029821:Enterovirus A, Human / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D014644:Genetic Variation; D016679:Genome, Viral; D006232:Hand, Foot and Mouth Disease / Q000821:virology*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D014744:Vietnam
keywords: Deep sequencing; Enterovirus A71; Hand foot and mouth disease; Phylogeny; Picornavirus