Amplification and next generation sequencing of near full-length human enteroviruses for identification and characterisation from clinical samples

Sonia R Isaacs; Ki Wook Kim; Junipearl X Cheng; Rowena A Bull; Sacha Stelzer-Braid; Fabio Luciani; William D Rawlinson; Maria E Craig

doi:10.1038/s41598-018-30322-y

Amplification and next generation sequencing of near full-length human enteroviruses for identification and characterisation from clinical samples

Sci Rep. 2018 Aug 8;8(1):11889. doi: 10.1038/s41598-018-30322-y.

Authors

Sonia R Isaacs^{1

2}, Ki Wook Kim^{1

2}, Junipearl X Cheng³, Rowena A Bull^{3

4}, Sacha Stelzer-Braid^{2

3}, Fabio Luciani^{3

4}, William D Rawlinson^{1

2

3

5

6}, Maria E Craig^{7

8

9

10}

Affiliations

¹ School of Women's and Children's Health, Faculty of Medicine, University of New South Wales, Sydney, NSW, 2052, Australia.
² Virology Research Laboratory, Prince of Wales Hospital, Sydney, NSW, 2031, Australia.
³ School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, NSW, 2052, Australia.
⁴ Systems Medicine, Inflammation and Infection Research Centre, School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, NSW, 2052, Australia.
⁵ Serology and Virology Division (SAViD), NSW Health Pathology East, Department of Microbiology, Prince of Wales Hospital, Sydney, NSW, 2031, Australia.
⁶ School of Biotechnology and Biomolecular Sciences, Faculty of Science, University of New South Wales, Sydney, NSW, 2052, Australia.
⁷ School of Women's and Children's Health, Faculty of Medicine, University of New South Wales, Sydney, NSW, 2052, Australia. m.craig@unsw.edu.au.
⁸ Virology Research Laboratory, Prince of Wales Hospital, Sydney, NSW, 2031, Australia. m.craig@unsw.edu.au.
⁹ Institute of Endocrinology and Diabetes, The Children's Hospital at Westmead, Sydney, NSW, 2145, Australia. m.craig@unsw.edu.au.
¹⁰ Discipline of Child and Adolescent Health, University of Sydney, Sydney, NSW, 2006, Australia. m.craig@unsw.edu.au.

Abstract

More than 100 different enterovirus (EV) genotypes infect humans and contribute to substantial morbidity. However, current methods for characterisation of full-length genomes are based on Sanger sequencing of short genomic regions, which are labour-intensive and do not enable comprehensive characterisation of viral populations. Here, we describe a simple and sensitive protocol for the amplification and sequencing of near full-length genomes of human EV species using next generation sequencing. EV genomes were amplified from 89% of samples tested, with C_t values ranging between 15.7 and 39.3. These samples included 7 EV-A genotypes (CVA2, 5-7, 10, 16 and EV71), 19 EV-B genotypes (CVA9, CVB1-6, ECHO3, 4, 6, 7, 9, 11, 16, 18, 25, 29, 30, and EV69), 3 EV-C genotypes (CVA19 and PV2, 3) and 1 EV-D genotype (EV70). We characterised 70 EVs from 58 clinical stool samples and eight reference strains, with a minimum of 100X depth. We found evidence of co-infection in four clinical specimens, each containing two distinct EV genotypes (CVB3/ECHO7, CVB3/ECHO18 and ECHO9/30). Characterisation of the complete genome provided conclusive genotyping of EVs, which can be applied to investigate the intra-host virus evolution of EVs, and allows further identification and investigation of EV outbreaks.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Enterovirus / genetics*
Enterovirus Infections / virology
Genome, Viral / genetics
Genotype
High-Throughput Nucleotide Sequencing / methods
Humans
Nucleic Acid Amplification Techniques / methods
Prospective Studies

Abstract

Publication types

MeSH terms

Grants and funding