NGS and WGS in RNA virus pathogenesis

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1 Serology & NGS and WGS in RNA virus pathogenesis Ki Wook Kim, Sonia Isaacs, Sacha Stelzer-Braid, Jessica Horton, Junipearl Cheng, William D. Rawlinson, and Maria E. Craig. UNSW Virology Research Laboratory Prince of Wales Hospital School of Women s and Children s Health, UNSW

2 Current NGS VRL 1. WGS of enteroviruses 2. Virome Capture Sequencing Hober and Sauter Nature Reviews Endocrinology 2010

3 Q. Why study EV? Prime environmental trigger of type 1 diabetes.

4 Type 1 diabetes (T1D) Immune-mediated destruction of pancreatic β-cells No cure/prevention Life-long dependence on exogenous insulin One of the most common chronic autoimmune diseases of childhood >16 million 11 th ~140,000

5 11th Since 1980s, incidence of childhood T1D has DOUBLED In children (0-14 yrs): i. 7th highest prevalence ii. 6th highest incidence Craig Pediatric Diabetes 2014 Australian Institute of Health and Welfare

6 International Diabetes Federation. IDF Diabetes Atlas, 7th edn. Brussels, Belgium: International Diabetes Federation,

7 Genetic factors in T1D > 40 genetic loci associated with T1D Highly polymorphic genes in the Human Leukocyte Antigen (HLA) region account for ~50% of genetic risk

8 Caucasians (n = 462) diagnosed with T1D before age 18 between High-risk HLA Low-risk HLA Fourlanos Diabetes Care 2008 Vehik Diabetes Care 2008 Gillespie Lancet 2004

9 Increasing role of the environment T1D from high-risk HLA population unchanged Rapid T1D growth from low-risk HLA population Significant geographical variation Seasonal variation Monozygotic twins discordant for T1D T1D healthy

10 Role of viruses in T1D T1D and enterovirus B infection: i. EV and islet autoimmunity (odds ratio 3.7) ii. EV and clinical T1D (odds ratio 9.8) Detection of EV RNA and protein in pancreas biopsies of recent T1D onset patients coxsackievirus B group implicated and examined most extensively Yeung BMJ 2011 Krogvold Diabetes 2014

11 coxsackievirus B Enterovirus B species Non-enveloped (+) ssrna Six genotypes CVB1-CVB6 Specifically infects pancreatic β-cells Insulin VP1 Hober and Sauter, Nature Reviews Endocrinology 2010

12 Enteroviral pathogenesis of T1D Craig Pediatric Diabetes 2013

13 Persistent infection Persistent infection demonstrated in vitro and in vivo: i. human pancreatic β-cells ii. mice model IA/T1D can develop many years following initial EV infection Persistent infection with low-level replication leads to prolonged inflammation and development of IA In rare cases EV causes massive cell lysis Fulminant diabetes

14 Mutations in VP1 and VP2 capsid protein 5 UTR deletions Alidjinou Discov Med 2014

15 Project 1: WGS of full-length EV Aims: 1. Characterise the genetic make-up of EV isolated from children with IA and T1D using NGS 2. Examine intra-host evolution of EVs following infection of human insulin-producing cells EV isolates from IA+/recent T1D onset children from birth cohort EVs from infected human pancreatic islets at multiple postinfection timepoints

16 Full-length EV Genome RT-PCR 2-step Nested PCR using EV-specific primers: 5 UTR 3 UTR R1 R2 VPg VP1 VP2 3C 3D AAAAAAA R2 R1 7 8 kb kb

17 Figure 2. HTS sequence analysis workflow performed on Geneious v9.0.2.

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19 Preliminary Data 30 full-length EV genome amplicons sequenced All clinical samples EVB: CVB & ECHOvirus most common Reference-based assembly resulted in near-complete coverage for most Some contained large gaps After 10 days of passage in human pancreatic islets: i. Two EVs from IA+: single AA substitution in 2C non-structural protein encoding virus helicase ii. One EV from IA+: five AA differences in VP4 important for EV-mediated induction of IFN-α

20 Project 2: Virome Capture Sequencing

21 Human virome Collection of all viruses Virome sequencing decade-long aspiration Now achievable through advances in NGS & VirCapSeq-VERT Rasmussen mbio 2015

22 Singleplex PCR Multiplex PCR

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24 NGS without virus enrichment Several novel viruses discovered Sensitivity too low for diagnostic application ~99% sequence reads: Bacteria Host DNA & Ribosomal RNA Unknown/no reference Majority of viral reads: Bacteriophage Plant viruses

25 Physical Enrichment Methods, specific removal of non-viral background very Difficult Laborious Expensive Homogenisation Ultra-centrifugation & Filtration Nuclease treatment Assumption that viral NA protected by necleocapsid Ribo-zero Human rrna depletion $$$ DNA/RNA purification 20-30% increase in total viral reads Random PCR amplification NGS Library Preparation Illumina Sequencing

26 VirCapSeq-VERT October 2015 Homogenisation Ultra-centrifugation & Filtration Prof. Ian Center for Infection and Immunology (CII), Columbia Uni Nuclease treatment Ribo-zero Up to 10,000-fold increase in viral reads DNA/RNA purification Sensitivity on par with qpcr Random PCR No special equipment or enzymes NGS Library Preparation All known vertebrate-infecting virus Illumina Sequencing VirCapSeq

27 Sequence Capture Enrichment Roche/NimbleGen Technology Target 200 Mb of sequence Supports 24-multiplex mer biotinylated oligos ~2 million probes against ENTIRE genomes of 207 viral taxa members known to infect vertebrates Briese mbio 2015

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30 Virome analysis of TTVS pre- and posttransfusion samples. Amit Kapoor et al. mbio 2015; doi: /mbio

31 ENDIA virome study Longitudinal stool samples collected from the ENDIA cohort Environmental Determinants of Islet Autoimmunity Nation-wide prospective cohort study At-risk children with first-degree relative(s) suffering T1D followed from pregnancy Hypothesis: Viral infection during pregnancy and first three years of life modifies the risk of IA in children genetically predisposed to T1D

32 ENDIA virome study Virome of which samples? Stools collected: 1. Longitudinally from mothers with/without T1D during pregnancy and perinatal period 2. Longitudinally from at-risk infants in their first year of life

33 ENDIA virome study Key research questions: Q1. Does longitudinal virome differ between T1D and control women during pregnancy? Q2. How does the longitudinal virome of children change in their first year of life? Q3. Do children inherit their mother s virome? Q4. Do children born from T1D mothers have a different virome profile?

34 Why test for ALL viruses? Previous studies used targeted detection Strong bias toward specific EV strains Other viruses may have been missed

35 Virus Main Finding Reference Rubella Diabetes in patients within a congenital rubella cohort Majority of patients with diabetes from [44] classified as having T2D No islet autoantibodies detected in congenital rubella syndrome cohort Epidemiological study showed association between rubella and T1D [44] [33] [34] [37] Mumps Infection during mumps epidemic more common in children who subsequently developed T1D Epidemiological study showed association between mumps and T1D [36] [37] CMV CMV detected more often in patients with T1D than controls Prospective cohort study found no association between CMV infection in infancy and T1D No association between perinatal CMV infection and T1D Rotavirus Majority of children at risk for T1D developed islet autoantibodies following rotavirus seroconversion No association between rotavirus infection and T1D in children No association between rotavirus infection and islet autoimmunity or T1D in children EV Higher levels of CVB antibodies in recently diagnosed diabetes patients Systematic review of 26 serological studies, inconclusive evidence for a role of CVB in T1D CVB1 antibodies more common in children with T1D VP1 present in pancreas samples from patients with T1D CVB4 infection in islets of 3 of 6 patients with T1D, associated with impaired β cell function VP1 colocalises with viral response element PKR in insulin-containing islet cells in patients with T1D Weak association between EV in blood and islet autoantibody detection No association between EV in stool and T1D Significant association between EV infection and T1D, particularly with severe ketoacidosis Meta-analysis of molecular studies determines >10-fold higher rate of EV in infection in T1D compared to controls [38] [40] [39] [41] [42] [43] [45] [46] [47] [48, 49] [50] [51] [52] [53] [54, 55] [56]

36 ENDIA virome workflow 1. Total nucleic acid extraction (DNA & RNA) 2. cdna synthesis 3. NGS Library preparation 4. VirCapSeq-VERT enrichment 5. Illumina Hiseq Metagenomic sequence analysis

37 ENDIA virome workflow 1. Total nucleic acid extraction (DNA & RNA) 2. cdna synthesis UNSW 3. NGS Library preparation 4. VirCapSeq-VERT enrichment 5. Illumina Hiseq 2500 In collaboration with CII 6. Metagenomic sequence analysis

38 Significance First meaningful virome data generated for T1D research Potential to discover new virus associations Facilitate design of an effective vaccine Reference data for future virome investigations Diagnostic application: Affordability of NGS Streamlined single-tube library preparation (3-4hrs)

39 Acknowledgements Prof. Maria Craig & Prof. Bill Rawlinson Sonia Isaacs, Jessica Horton, Junipearl Cheng Dr. Rowena Bull & group Dr. Fabio Luciani & group Everyone involved in ENDIA Collaborators: Baylor & CII Serology &