Poster Presentation 8th Australasian Virology Society Meeting and 11th Annual Meeting of the Australian Centre for Hepatitis & HIV Virology Meeting 2015

Investigating enterovirus infection and islet autoimmunity in children using next generation sequencing. (#147)

Sonia R Isaacs 1 , Sacha J Stelzer-Braid 2 3 , Ki Wook Kim 4 , Maria E Craig 4 5 6 , William D Rawlinson 1 2 3
  1. School of Biotechnology and Biomolecular Sciences, Faculty of Science, University of New South Wales, Sydney, NSW, Australia
  2. School of Medical Sciences, Faculty of Science, University of New South Wales, Sydney, NSW, Australia
  3. Virology Research Laboratory, Serology and Virology Division, South Eastern Area Laboratory Services, Prince of Wales Hospital, Sydney, NSW, Australia
  4. School of Women’s and Children’s Health, Faculty of Medicine, University of New South Wales, Sydney, NSW, 2032
  5. Institute of Endocrinology and Diabetes, The Children’s Hospital at Westmead, Sydney, NSW, Australia
  6. Discipline of Paediatrics and Child Health, University of Sydney, Sydney, NSW, Australia

Type 1 diabetes (T1D) is a childhood onset autoimmune disease characterised by immune-mediated destruction of pancreatic β-cells, and is preceded by islet autoimmunity (IA). Human enteroviruses (EV), especially coxsackievirus B species, are strongly associated with T1D and β-cell destruction 1. This study aimed to characterise EVs precipitating IA by comparing EV genomes isolated from stools of 11 children with IA (IA+) and nine without IA (IA-) in the Viruses in the Genetically at Risk longitudinal cohort study. Stool suspensions had been previously cultured in both human islets (HI) and human islet precursor cells (HIPCs), with supernatants collected at days 1, 4, 7 and 10. The first 23 size selected, full genome products including eight EV prototype strains, five IA+ HI-cultured samples from day 10, five IA+ HIPCs-cultured samples from day 10, 2 IA+ pre-culture stool samples and 3 IA- pre-culture stool samples were sequenced using next generation sequencing (NGS), specifically the Illumina MiSeq v3 2x150 bp paired-end run. Paired, short reads were trimmed to increase quality, and mapped to 101 NCBI reference EV sequences using Geneious 8.1.7. Regions of recombination were determined from depth of coverage, and consensus sequences were assembled for each sample. The pilot run produced over 67.6 million reads in total, ranging from 2.1 to 3.6 million reads per sample, with the highest mean sequence output originating from HI-infected samples. All 23 samples were characterised within the EV-B genogroup, including some which were previously classified within EV-A from their 5’UTR and VP1 sequences, highlighting the limitations of single region-based genotyping in comparison with NGS. Further investigation into the impact of sequence variation over time on IA using single nucleotide polymorphism analysis will provide extensive type-specific genetic information that may further elucidate our understanding of the role of EVs in IA, with the ultimate goal of preventing T1D.

  1. Yeung W.C., Rawlinson W.D., Craig M.E. (2011) Enterovirus infection and type 1 diabetes mellitus: systematic review and meta-analysis of observational molecular studies. BMJ. 342, d35.