The Evolutionary Dynamics of Norovirus
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The evolutionary dynamics of norovirus John-Sebastian Eden Bachelor of Medical Science (Hons) A dissertation submitted for the fulfilment of the requirements for the degree Doctor of Philosophy Submitted 2012 Originality statement ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’ 29th July 2012 Signed ………………………………………………………… Date ……………………………………… ii Copyright statement ‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.' 29th July 2012 Signed ………………………………………………………… Date ……………………………………… Authenticity statement ‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’ 29th July 2012 Signed ………………………………………………………… Date ……………………………………… iii Abstract Norovirus (NoV) is the leading cause of both outbreak and sporadic community –acquired acute gastroenteritis. The overall incidence of NoV infection has grown dramatically since the emergence of epidemic NoV strains of the GII.4 lineage in the mid-1990s that have been associated with five pandemics and account for 80% of NoV infections. This thesis aimed to describe the mechanisms of evolution that facilitate the emergence of epidemic GII.4 variants and to elucidate factors that contribute to their higher epidemiological fitness. Two molecular epidemiological studies were performed to characterise the NoV strains linked to epidemics in New South Wales, Australia and those in circulation globally between 2007 and 2010. The pandemic GII.4 variant 2006b was identified as the cause of the 2007 and 2008 epidemics and the GII.4 variant New Orleans 2010 was the aetiological agent of the epidemics of 2009 and 2010. Each variant demonstrated antigenic drift in the capsid P2 domain that likely contributed to their epidemic potential. These studies also highlighted the role that recombination played in the emergence of New Orleans 2010. A number of factors were identified that may have contributed to the higher epidemiological fitness of the pandemic NoV GII.4 variants. Firstly, by comparing the enzymatic properties of different NoV polymerases, including replication efficiency and fidelity, it was shown that GII.4 variants have higher replication and mutation rates. It was also shown that polymerases from more prevalent genotypes, such as GII.4 and GII.b, are phosphorylated by an important cellular kinase, Akt, at a residue (Thr33) that decreases the de novo polymerase activity. Using next-generation sequencing technology, patterns of intra-host evolution were compared between acute and chronic NoV infections. Extensive heterogeneity and toggling at antigenic sites of the viral capsid was observed in the chronic patient, which suggests that immune-compromised individuals with chronic NoV infections could be a source for novel antigenic variants. In the same study, a transmission cluster iv was also examined and a strong genetic bottleneck was identified at the point of transmission. Overall, this thesis suggests that a complex pattern of mutation, recombination and adaptation drive NoV evolution in response to herd immunity. v Acknowledgements Peter White – You have been an insightful and generous supervisor. Thank you for all the coffees, the conferences and the fishing stories and most recently, I thank you for giving me a job. I don’t know where I’ll eventually end up but I hope we will continue to collaborate together. Rowena Bull – As I come to the end of my PhD, the more I realise that the whole time, I have just been walking in your footsteps. You have paved the way for everything I have worked on and provided me with sage advice on countless occasions. Thanks for everything. Lab people – Since starting in the lab during my honours year (way back in 2007), I have had the pleasure of working with a number of people who have made my time and the experience all the more worthwhile. I must begin by giving my sincerest thanks to Sean Pham, who has been there with me from the start and hopefully will continue to be a friend and colleague into the future. You have been a great support. To the original lab members, Elise Tu and Jenny Mak, you two were great fun and treated me so well. Thanks for all the dinners and for tolerating my mischief. To more recent lab members including Arthur Chee, Han Fui Lim, Filip Bebek, Auda Eltahla, Kun-Lee Lim and Rouba Ballouk, I want to thank you all for making the lab a fun, productive place to work. Outside of my lab, a number of other people have made significant contributions to my project and none more so that Laura Sharpe. I don’t know what I would have done without all your help. I was forever pestering you but you always put up with me. It was great to work with you and Andrew Brown. I know the progress was a little slow (mostly due to my procrastination) but we got there in the end and can be proud of our paper. A similar thank you must also go to Mark Tanaka for the help on my last paper. You might not feel like you’ve done that much; however from my perspective you’ve been a great support and a wonderful source of knowledge. I would like to thank all my neighbours from the Wilkins and Brown Labs for their generosity and willingness to share equipment and reagents. I would also like to thank vi Bill Rawlinson, Juan Merif and Chris McIver from the Virology Diagnostic Lab at Prince of Wales Hospital for their generous provision of norovirus samples and contributions as co-authors. Co-authors – A big thank you to everyone who contributed to the papers presented in this thesis. Family – To Mum and Dad, you have been extremely supportive and generous. Thank you for putting up with me through all this time spent at university. I know it will be worth the effort and you deserve all my thanks. To my brother Tom, you have been a great mate who has helped me have fun and relax through all the bore of scientific research. To my sisters, Kerrianne and Hannah, thank you both for your support. To all my grandparents, I know my work has made you proud (because you tell me all the time) and I am completely grateful for giving me such support. Amber – To my wonderful girlfriend (of at least five years?), thank you, thank you, thank you from the bottom of my heart. You have been my biggest support and continuously cared for me despite having to put up with my shenanigans. Hopefully now we can now move onto to something better (and I might also get to see you more often). All my love. I feel that the final thank you must go to the guys from the library lawn coffee cart, who have kept me energised throughout my time at university. I have been absolutely spoiled to be able to get some of the best coffee in Sydney, just a two minute walk from the lab. You guys are always great fun and make everyday a bit more interesting. Thank you! vii Table of contents 1 General introduction .......................................................................................... 1 1.1 Acute gastroenteritis .......................................................................................... 1 1.2 Background to norovirus .................................................................................... 4 1.3 Structure and genome organisation .................................................................. 5 1.4 Classification ....................................................................................................... 7 1.5 ORF1-encoding non-structural proteins ............................................................. 9 1.6 ORF2/3-encoding structural proteins ............................................................... 12 1.7 NoV immunology .............................................................................................. 13 1.8 Molecular epidemiology ................................................................................... 14 1.9 Antigenic variation in the GII.4 lineage ...........................................................