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Equine Adenovirus: Molecular and Host Cell Receptor Characterisation

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Equine Adenovirus: Molecular and Host Cell Receptor Characterisation EQUINE ADENOVIRUS: MOLECULAR AND HOST CELL RECEPTOR CHARACTERISATION Ayalew Berhanu Mekonnen ORCID ID: 0000-0003-1686-4201 Submitted in total fulfilment of the requirements of the degree of Doctor of Philosophy April, 2017 Faculty of Veterinary and Agricultural Sciences The University of Melbourne ABSTRACT Equine adenoviruses (EAdV) are members of the family Adenoviridae. While EAdV-1 causes respiratory and ocular infections, EAdV-2 has been associated with gastrointestinal infections. Little is currently known about the way these viruses interact with the host cell, and how the genetic composition of EAdV-2 compares to other adenoviruses. There is also a need for more sensitive detection methods to understand the role of EAdV-2 in foals with diarrhea. The aims of this research project were to characterise the full genome of EAdV2-385/75.4, to develop a quantitative PCR (qPCR) for detection of this virus in faecal samples, and to characterise the putative EAdV receptor molecules on the surface of equine foetal kidney (EFK) cells. By applying next generation sequencing, the size of the EAdV-2 genome was determined to be 33,010 bp in length, with a GC content of 48%. Comparative genome analysis showed EAdV-2 to be a member of the Mastadenovirus genus but has a distinct arrangement and genetic content in the genome termini, which placed the virus in a separate cluster within the genus Mastadenovirus. A high throughput qPCR assay using primers targeting a 109 bp region of the hexon gene and capable of differentiating EAdV-2 from EAdV-1 were also designed and optimised. The assay was specific and sensitive for the detection of EAdV-2. The detection limit of the assay was 27 genome copies per reaction. The performance of the qPCR assay was assessed using archived faecal samples and was shown to exclusively amplify the targeted EAdV-2 hexon fragment. To test the possible role of the fibre knob domain in the infectious cycle of both EAdV- 1 and EAdV-2, a recombinant baculovirus was constructed for the expression of the EAdV fibre knob proteins from both serotypes. A soluble EAdV-2 fibre knob protein was successfully expressed and was immunogenic when analyzed by Western blot. Polyclonal antibodies against purified EAdV-1 and EAdV-2 virion were also produced. Sensitive neutralisation assay has shown heterologous neutralisation of both virus serotypes, suggesting the presence of some cross neutralisation antibodies in these sera. i An immune-fluorescent infectivity assay was developed to characterise potential host cell surface receptors for infection of EAdV-1 and EAdV-2 in EFK cells. EAdV-2 infection was significantly inhibited by NaIO4, neuraminidase, wheat germ agglutinin and Sambucus nigra agglutinin treatments, suggesting a sialic acid molecule with an α(2,6) linkage is involved in the attachment and infection of EAdV-2 to the EFKs. Integrin blocking experiment results using vascular cell adhesion molecule 1(VCAM-1) also showed EAdV-2 utilised α4β1 integrins for subsequent internalisation. Similarly, heparan sulfate (HS), heparinase I and III, NaIO4 and neuraminidase significantly inhibited EAdV-1 infection, suggesting HS plays crucial role in promoting EAdV-1 infection and the possible involvement of sialic acid as a secondary receptor. Future studies using cells which express little or no sialic acid containing molecules would be helpful to unravel whether the sialic acids and integrins are used by EAdV-1 and EAdV-2, respectively, as secondary receptors. This study complements new knowledge on the full genome structure of EAdV-2 and has elucidated some of the mechanisms by which EAdVs interact with their receptor, advancing our knowledge of the EAdV-host interactions. ii DECLARATION This is to certify that: i. The thesis comprises only my original work towards the PhD except where indicated in the preface, ii. Due acknowledgment has been made in the text to all other material used iii. The thesis is fewer than 100,000 words in length, exclusive of tables, maps, bibliographies and appendices. Ayalew Berhanu Mekonnen April, 2017 iii PREFACE The work presented in this thesis was performed at the Centre for Equine Infectious Disease, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, between November 2012 and January 2017. The author was a recipient of a Melbourne Research Scholarships and the work was carried out under the supervision of Prof. James Gilkerson, Dr Carol Hartley and A/Prof. Joanne Devlin. Scientific papers associated with thesis that have been presented are: 1. Ayalew M. Berhanu, Carol A. Hartley, Sally J. Symes, James R. Gilkerson. Development and validation of a serotype specific quantitative PCR assay for detection of equine adenovirus 2 (2015). Oral presentation. Australian Society of Microbiology Annual Scientific Meeting. July12 -15, Canberra, Australia. iv ACKNOWLEDGEMENTS First and for most, I would like to extend the deepest gratitude to my supervisor Prof James Gilkerson. Thank you for giving me the opportunity to do this research and develop my skill in molecular virology, over all support and guidance. Many thanks go to my co-supervisors, Dr. Carol Hartley, A/Prof. Joanne Devlin. It has been a privilege to work under your supervision. Especially thank you Dr. Carol Hartley for broadening my practical skills, your invaluable comments and unlimited contributions to seemingly endless troubleshooting. To everyone at Centre for Equine Infectious Disease, Microbiology and Asia Pacific Centre for Animal Health (APCAH) research groups, I am so grateful for providing me the support, help and guidance whenever needed and your treasured scientific conversations. Sally Symes, your friendly approach and supervision was tremendous. I greatly appreciate it. Also thank you Kirsten Bailey, for providing me faecal samples, support and nice coaching at the beginning of this study. I am also greatly indebted to Prof. Mark Stevenson, for your advice and assistance on the statistical analysis. I would also like to thank Mr. Nino Ficorilli for his excellent technical assistance, advice, support, optimistic encouragement for any issues regarding cell culture. Many special thanks to Dr. Mauricio Coppo for his expert advice on protein expression and many appreciations to all my fellow colleagues, Dr. Olushola Martin, James Adamu, Dr. Zelalem Mekuria, Paola Vaz, Alistair Legoine, Mesula Korsa, thank you all for the direct and indirect input you made to this work and making a friendly working environment. A special thank also goes to Melbourne International Research Scholarship and Melbourne International Fee Remission Scholarship for providing me financial support during my candidature. Lastly, to my lovely wife, Haregewoin Mihretie the sacrifices you made and the pain you passed through during those difficult times to get the PhD done were unforgettable but at the same time inspirational. I am greatly indebted to you. To my son and daughter, Yonathan and Selina, I am so blessed to have you and you are truly source of too much needed laughter during this journey. v TABLE OF CONTENTS ABSTRACT ...................................................................................................................... i DECLARATION ............................................................................................................. iii PREFACE ........................................................................................................................ iv ACKNOWLEDGEMENTS ............................................................................................. v TABLE OF CONTENTS ................................................................................................ vi LIST OF FIGURES ....................................................................................................... xiii LIST OF TABLES ....................................................................................................... xvii ABBREVIATIONS ..................................................................................................... xviii CHAPTER ONE ............................................................................................................... 1 LITERATURE REVIEW ................................................................................................. 1 Adenoviruses ................................................................................................. 1 1.1.1 Taxonomic classification ............................................................................... 1 1.1.2 Adenovirus biology ....................................................................................... 3 1.1.3 Adenovirus virion architecture and physicochemical composition ............... 6 Cell receptors for adenovirus attachment .................................................... 10 1.2.1 Coxsackie virus and adenovirus receptor (CAR) ........................................ 10 1.2.2 Sialic acid..................................................................................................... 11 1.2.3 CD46 ............................................................................................................ 12 1.2.4 CD80 and CD86 .......................................................................................... 12 1.2.5 Heparan sulfate glycosaminoglycans (HS-GAGs) ...................................... 13 1.2.6 Integrins as secondary receptors .................................................................. 13 Adenovirus replication cycle ......................................................................
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