Thorburn, Fiona (2016) The use of next generation sequencing in the diagnosis and typing of viral infections. PhD thesis. http://theses.gla.ac.uk/7838/ Copyright and moral rights for this work are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This work cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Glasgow Theses Service http://theses.gla.ac.uk/ [email protected] The Use of Next Generation Sequencing in the Diagnosis and Typing of Viral Infections Fiona Thorburn M.B.Ch.B., M.R.C.P. (UK), D.T.M&H Submitted in fulfilment of the requirements for the Degree of Doctor of Philosophy MRC – University of Glasgow Centre for Virus Research College of Medical, Veterinary and Life Sciences University of Glasgow May 2016 i Abstract Viral respiratory infections are associated with substantial mortality, morbidity, and a vast economic and healthcare burden. The diagnosis of such infections has been revolutionised by the introduction of molecular methods such as RT-PCR. This has resulted in high levels of sensitivity and specificity along with a rapid turnaround time in comparison to previous methods. As a product of this success, the diagnosis of respiratory infections makes up a large proportion of the workload in most diagnostic laboratories. The development of next generation sequencing (NGS) may be the next revolution in the field of virus diagnostics. This allows a metagenomic approach to specimen processing whereby target independent sequencing of all genetic material is carried out. The research presented in this thesis initially sought to examine if NGS would be feasible in the field of respiratory virus diagnostics. The aim was to apply NGS to clinical specimens in parallel with the current diagnostic RT-PCR assays employed by the West of Scotland Specialist Virology Centre (WoSSVC) to determine if NGS could give the same level of results as RT-PCR and whether the sequence information generated in the process could be used in further characterising the detected pathogens. Further to this, the NGS method was then applied to the detection of norovirus from faecal specimens to demonstrate the utility in other areas of viral diagnostics. The results show that multiple viral pathogens can be detected from clinical specimens without specific virus targeting. The method was less sensitive than RT-PCR but sequence data generated during the process was utilised viral detection, subtyping and phylogenetic analysis. We also demonstrated that a single workflow could be applied to multiple specimen types in the detection of RNA viral pathogens. ii Publications Thorburn, F., Bennett, S., Modha, S., Murdoch, D., Gunson, R., and Murcia, P. R. (2015) The use of next generation sequencing in the diagnosis and typing of respiratory infections. Journal of Clinical Virology, 69, pp. 96-100. (doi:10.1016/j.jcv.2015.06.082) Nickbakhsh, S., Thorburn, F., Von Wissmann, B., McMenamin, J., Gunson, R. N., and Murcia, P. R. (2016) Extensive multiplex PCR diagnostics reveals new insights into the epidemiology of viral respiratory infections. Epidemiology and Infection, (doi:10.1017/S0950268816000339) iii Abbreviations ARTI Acute respiratory tract infection CPE Cytopathic effect Ct Cycle threshold DIF Direct immunofluorescence DNA Deoxyribonucleic acid ELISA Enzyme-linked Immunosorbent assay EV Enterovirus HA Haemagglutinin hBoV Human bocavirus hCoV Human coronavirus hMPV Human metapneumovirus hRV Human rhinovirus LRTI Lower respiratory tract infection MERS Middle East respiratory syndrome NA Neuraminidase NGS Next generation sequencing PCR Polymerase chain reaction RNA Ribonucleic acid RSV Respiratory syncytial virus RT-PCR Reverse transcription polymerase chain reaction SARS Severe acute respiratory syndrome URTI Upper respiratory tract infection VTM Viral transport medium WoSSVC West of Scotland Specialist Virology Centre iv Table of contents Abstract……………………………………………………………………………...……..i Publications………………………………………………………………..……….…..…ii Abbreviations……………………………………………………………..……….……...iii Table of Contents………………………………………………………..……….………iv List of Tables…………………………………………………………………….………..xi List of Figures………………………………………………………...……….…..……..xii Acknowledgements………………………………………………………….....….……xiv Author Declaration………………………………………..…….………………....…….xv 1 Introduction ....................................................................................................... 1 1.1 Importance of Acute Respiratory Tract Infections ...................................... 3 1.2 Clinical Presentation .................................................................................. 4 1.3 Aetiology of ARTI ....................................................................................... 7 1.3.1 Viral Causes of ARTI ........................................................................... 8 1.3.2 Influenza Viruses ................................................................................. 9 1.3.2.1 Influenza A .............................................................................................. 9 1.3.2.2 Influenza B ............................................................................................ 10 1.3.2.3 Influenza C ............................................................................................ 11 1.3.3 Influenza Epidemiology ..................................................................... 11 1.3.4 Clinical Presentation .......................................................................... 12 1.3.4.1 Complicated Infection .......................................................................... 12 1.3.5 Paramyxoviridae................................................................................ 13 1.3.5.1 Respiratory Syncytial Virus ................................................................. 13 1.3.5.1.1 Structural Proteins ................................................................... 13 1.3.5.1.2 Classification ............................................................................ 14 1.3.5.1.3 Clinical Presentation ................................................................ 15 1.3.5.2 Human Parainfluenza Viruses ........................................................... 15 1.3.5.2.1 Structural Proteins and Non-Structural Proteins ...................... 16 1.3.5.2.2 Clinical Presentation ................................................................ 16 1.3.5.3 Human Metapneumovirus ................................................................... 16 1.3.5.3.1 Classification ............................................................................ 17 1.3.5.3.2 Clinical Presentation ................................................................ 17 1.3.6 Picornaviridae ................................................................................... 18 1.3.6.1 Human Rhinovirus ............................................................................... 18 1.3.6.1.1 Structural Proteins ................................................................... 18 v 1.3.6.1.2 Classification ............................................................................ 19 1.3.6.1.3 Clinical Presentation ................................................................ 19 1.3.7 Adenoviridae ..................................................................................... 20 1.3.8 Coronaviridae .................................................................................... 21 1.3.8.1 Classification ......................................................................................... 23 1.3.8.2 Clinical Presentation ............................................................................ 23 1.3.9 Human Bocavirus .............................................................................. 24 1.3.10 Polyomaviruses ................................................................................. 24 1.4 Management of Viral Acute Respiratory Infections .................................. 25 1.4.1 Anti-viral Drugs .................................................................................. 26 1.4.1.1 Adamantane derivatives ..................................................................... 26 1.4.1.2 Neuraminidase Inhibitors .................................................................... 27 1.4.1.3 Nucleoside/nucleotide analogues ...................................................... 27 1.4.2 Symptomatic Management ................................................................ 28 1.4.3 Vaccines and Immunisations ............................................................. 29 1.5 Diagnosis of Infection............................................................................... 31 1.5.1 Historical aspect of viral diagnostics .................................................. 31 1.5.2 Diagnosis of Viral Infections .............................................................. 32 1.5.2.1 Case definitions .................................................................................... 32 1.5.2.2 Culture and Virus Isolation ................................................................. 33 1.5.2.3 Direct Detection Assays .....................................................................