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The Epidemiology, Classification and Evolution of Human Rhinoviruses Chloé Leanne McIntyre A thesis submitted for the degree of Doctor of Philosophy The University of Edinburgh March 2013 Abstract Human rhinoviruses (HRV) are extremely common human respiratory pathogens, most commonly associated with mild upper respiratory tract infections. The three known species of HRV (HRV-A, -B and –C) are members of the family Picornaviridae and genus Enterovirus. In contrast to the enterovirus (EV-A-D) species that commonly infect the gut, HRV are generally thought to be acid labile with replication restricted to the respiratory tract. Investigations of the clinical correlations of HRV infections detected on diagnostic screening of respiratory specimens demonstrated no specific association between HRV variant and clinical presentation. For example, similar species distributions were observed in patients admitted to the ITU and those discharged with minor illness. Unexpectedly, screening of stool specimens for HRV showed a prevalence of 10% with viral loads similar to EV infections. These findings suggested that a reappraisal of HRV tropism and disease associations may be warranted. HRV-A and -B isolates were originally classified into 100 serotypes by serological neutralisation properties. As HRV-C is difficult to isolate, no attempt had been previously made to classify the wealth of available HRV-C sequences. To facilitate definition of novel HRV types and classification of HRV-C, a system was devised to divide HRV sequences into genotypically defined types. Pairwise VP1 nucleotide p-distance analysis revealed distinct thresholds between inter- and intra- type divergence and available sequences were classified into 77 HRV-A, 29 -B and 51 -C types. This provides a standardised basis for type definition and identification, allowing consistency in studies of genetic diversity, epidemiology and evolution. It has been adopted by the ICTV Picornavirus Study Group for classification of HRV. i Abstract Although the occurrence of recombination has been documented within the coding region of EV, analysis of dated HRV sequences revealed an overall lack of intra-species recombination between three coding regions of HRV-B and -C. In contrast, full HRV-A type groups appeared to have been subject to a large number of recombination events, suggesting extensive recombination during the period of its diversification into types. Putative recombination breakpoints localised to the non-structural region. Within HRV-A and HRV-B, recombination within the 5ˈUTR was infrequent. However, over 60% of analysed HRV-C strains grouped within the HRV-A clade and two recombination hotspots were identified. An additional interspecies recombination event was detected between HRV-A/C in the 2A coding region, with putative breakpoints mapping to the boundaries of the C-terminal domain of the proteinase. The studies within this thesis provide evidence for a broadened understanding of the clinical significance of HRV. In addition, the assignment of HRV sequences into genotypically defined types allowed description of the observed genetic diversity and completion of analysis which reaffirmed the sporadic nature of recombination within the coding region of HRV. ii Declaration of Originality I declare that the work recorded within this thesis was completed entirely by myself at the Centre for Infectious Diseases and Roslin Institute of the University of Edinburgh between September 2009 and March 2013. In cases where others have made a contribution to the results obtained, this is clearly stated within the text. This work has not been submitted for any other degree or professional qualification. In cases where portions of the work contained within this thesis have been published in academic journals, this is indicated within the text and copies of papers are given as appendices. Where required by license agreement, permission to include published papers has been sought from publishers. Chloé McIntyre iii Acknowledgements A hundred people have contributed in a thousand unique ways to the completion of this PhD and there is not enough paper in the world to thank them all as much as they deserve. For my supervisor, Professor Peter Simmonds. Thank you for giving this opportunity to a young surgical trainee with little experience. Your unending patience, enthusiasm and dedication over the last 4 years has taken me from a clueless newcomer to a fledgling scientist able and willing to think for herself. I cannot thank you enough. For the others who have been involved in my academic and scientific growth, a special thanks. For Professor Paul Sharp who has always been available for advice and support. For Dr Heli Harvala, whose infectious enthusiasm and incredible passion for virology has widened the horizons of my interest and experience far beyond what I initially imagined. For Dr Carol McWilliam-Leitch who took me from having never held a pipette to confidently designing my own PCR protocols far faster than I ever thought possible. For all the PhD students and post-docs in our lab past and present; Elly, Ingrid, Sinead, Nora, Nigel, Jereon, Donald, Jill, Colin, Inga, Nicky, Richard and Carol. Not only have you all been at various times an indispensible source of wisdom, but you have brightened my days and almost never been too busy to meet for a beer and a giggle after hours. To the students whose projects I have been fortunate enough to be involved in, thank you for allowing me to learn how to teach and keeping me smiling with your enthusiasm and humour. For my wonderful parents who have been unfailingly loving and supportive and never questioned my decision to follow my passion. Without you, none of this would have ever been possible. You have always made me feel like a star! I cannot thank you enough. For my best friend, life-partner, handyman, life-coach, chef, therapist, IT-support, removal man, musical hero, brew-master and most of all, amazing husband; Kevin. Thank you for being so endlessly, uncomplainingly, beautifully patient with me; with my long working hours, tiredness, frustration and endless, obsessive fixation on minute details. Your love and support has been the cornerstone of everything I’ve done. And most of all, to our little bun in the oven. I hope that someday you will read this and smile. Mummy loves science and Mummy loves you. iv List of Original Publications Original publications directly associated with this thesis McIntyre CL, McWilliam Leitch EC, Savolainen-Kopra C, Hovi T, Simmonds P. 2010. Analysis of genetic diversity and sites of recombination in human rhinovirus species C. J Virol 84:10297–310. McIntyre CL, Savolainen-Kopra C, Hovi T, Simmonds P. 2013. Recombination in the evolution of human rhinovirus genomes. Arch Virol. [Epub ahead of print] Simmonds P, McIntyre CL, Savolainen-Kopra C, Tapparel C, Mackay IM, Hovi T. 2010. Proposals for the classification of human rhinovirus species C into genotypically assigned types. J Gen Virol 91:2409–19. Harvala H, McIntyre CL, McLeish NJ, Kondracka J, Palmer J, Molyneaux P, Gunson R, Bennett S, Templeton K, Simmonds P. 2012. High detection frequency and viral loads of human rhinovirus species A to C in fecal samples; diagnostic and clinical implications. J Med Virol 84:536–42. McIntyre CL, Knowles NJ, Simmonds P. 2013. Proposals for the classification of human rhinovirus species A, B and C (HRV-A, -B and –C) into genotypically assigned types. Submitted. Original publications to which the author has otherwise contributed Gaunt ER, Harvala H, McIntyre CL, Templeton KE, Simmonds P. 2011. Disease burden of the most commonly detected respiratory viruses in hospitalized patients calculated using the disability adjusted life year (DALY) model. J Clin Virol. 52(3):215-21 McLeish NJ, Witteveldt J, Clasper L, McIntyre CL, McWilliam Leitch EC, Hardie A, Bennett S, Gunson R, Carman WF, Feeney SA, Coyle PV, Vipond B, Muir P, Benschop K, Wolthers K, Waris M, Osterback R, Johannessen I, Templeton K, Harvala H, Simmonds P. 2012. Development and assay of RNA transcripts of enterovirus species A to D, rhinovirus species A to C, and human parechovirus: assessment of assay sensitivity and specificity of real-time screening and typing methods. J Clin Micro 50:2910–7. v List of Original Publications Harvala H, Gaunt E, McIntyre CL, Roddie H, Labonte S, Curran E, Othieno R, Simmonds P, Bremner J. 2012. Epidemiology and clinical characteristics of parainfluenza virus 3 outbreak in a Haemato-oncology unit. J Infect. 65(3):246-54 Harvala H, McIntyre CL, Imai N, Clasper L, Djoko CF, LeBreton M, Vermeulen M, Saville A, Mutapi F, Tamoufé U, Kiyang J, Biblia TG, Midzi N, Mduluza T, Pépin J, Njouom R, Smura T, Fair JN, Wolfe ND, Roivainen M, Simmonds P. 2012. High seroprevalence of enterovirus infections in apes and old world monkeys. Emerg Infect Dis 18(2):283–6. Harvala H, Mcleish N, Kondracka J, Mcintyre CL, Leitch ECM, Templeton K, Simmonds P. 2011. Comparison of Human Parechovirus and Enterovirus Detection Frequencies in Cerebrospinal Fluid Samples Collected Over a 5-Year Period in Edinburgh : HPeV Type 3 Identified as the Most Common Picornavirus Type.