Interaction of Free-living protozoa with water-borne human pathogenic viruses and protection from disinfection Thesis submitted for the degree of Doctor of Philosophy at the University of Leicester By Mohammad A. Alotaibi BSc & MSc Department of Infection, Immunity and Inflammation University of Leicester March, 2011. Acknowledgments First of all, I wish to thank my supervisor Dr. Simon Kilvington for introducing me to the world of research. It was only due to his valuable guidance, cheerful enthusiasm that I was able to complete my research work in a respectable manner. I also extend my appreciation and gratitude to Dr. Roger James for his valuable advice and following writing. I also would like to extend my appreciation to Dr. Wayne Heaselgrave for his valuable experience that enriched me with priceless information and to Kimberly Earland, Niran Patel and Nale Yaqubi for their time and help. I dedicate this work to my wife who was very supportive and very patient during my study to finish and also to my kids the candle of my life Saud, Noory, Bander and Faisal who were waiting for me to come back home. Finally I would like to express my gratitude and appreciation to Kuwait Institute for Scientific Research for sponsoring me to study and complete my PhD degree in the UK and more appreciation to my government for the financial support I had. Finally an appreciation is directed to the Kuwait embassy in UK and the cultural Attaché who were supportive and following my study in UK. ii Abstract Acanthamoeba causes Granulomatous Amoebic Encephalitis (GAE) and Amoebic Keratitis (AK) in humans and in its cystic form is resistant to extreme environmental conditions. Both human pathogenic water-borne viruses and free-living protozoa share the same aquatic environment. This study set out to test the ability of both Acanthamoeba and Tetrahymena to internalise and protect enteric viruses; coxsackievirus (B3, B5), poliovirus (PV) and rotavirus (RV) following co-culture. Viral uptake was assessed by infection of cultured mammalian cells, by indirect immunofluorescence (IF), and by reverse transcriptase-polymerase chain reaction (RT- PCR). The results showed that none of the free suspended viruses were internalised in Acanthamoeba or Tetrahymena. However, both coxsackievirus B3N and rotavirus Wa could be detected within Acanthamoeba by IF and confirmed by RT-PCR when the amoebae were co-cultured (fed) with virally infected mammalian cells. The co-cultured amoeba was allowed to encyst but following this procedure no viruses were detected either by cell culture or RT-PCR. In a second series of experiments, the efficacy of solar disinfection (SODIS) against viruses either alone or when co-cultured with Acanthamoeba was assessed. SODIS reduced the viral infectivity by over 3log10 after 1 h for CVB3N and over 2log10 for PV after 2 h. Repeating these experiments in the presence of riboflavin, a 6log10 reduction was observed for CVB3N after 1 h of light exposure and 6log10 after 6 h for all other viruses tested. The results suggest that Acanthamoeba does not internalise or protect viruses in suspension. However, if a virus is located with an infected mammalian cell then it may be internalised; a new potential mechanism for virus dissemination in the environment. Secondly, solar disinfection is an effective treatment method for water contaminated with viruses which is further enhanced by the addition of riboflavin. This study provides a practical example of low technology methods which could be utilised to provide safe drinking water in various circumstances. iii Statement of Originality This accompanying thesis submitted for the degree of PhD thesis is based on work conducted by the author at the University of Leicester mainly during the period between January 2005 and December 2010. All the work recorded in this thesis is original unless otherwise acknowledge in the text or by references. None of the work has been submitted for another degree in this or any other university. Mohammad Alotaibi iv Table of Contents Acknowledgments ........................................................................................................... ii Abstract ........................................................................................................................... iii Table of Contents ............................................................................................................ v List of Tables ................................................................................................................ viii List of Figures ................................................................................................................. ix Abbreviations ................................................................................................................. xi Chapter One: Introduction ............................................................................................ 1 Introduction ..................................................................................................................... 2 1.0 Purpose of the study ......................................................................................................................... 2 History and Background ................................................................................................ 5 1.1 History............................................................................................................................................... 5 1.2 Classification of protozoa ................................................................................................................. 5 1.3 Free-living protozoa ......................................................................................................................... 6 1.4 Discovery of Acanthamoeba ............................................................................................................. 7 1.5 Acanthamoeba biology and morphology ........................................................................................... 7 1.6 Acanthamoeba Biology – the cyst ..................................................................................................... 8 1.7 Acanthamoeba distribution ............................................................................................................. 10 1.8 Acanthamoeba classification .......................................................................................................... 11 1.9 Acanthamoeba growth .................................................................................................................... 15 1.10 Acanthamoeba pathogenicity ........................................................................................................ 16 1.10.1 Acanthamoeba Keratitis (AK) ................................................................................................ 16 1.10.2 Granulomatous Amoebic Encephalitis (GAE) ........................................................................ 17 1.11 Acanthamoeba interaction with other microorganisms ................................................................ 17 1.12 Protozoa and their interaction with viruses .................................................................................. 23 1.13 Viruses in the aquatic environment ............................................................................................... 26 1.14 Viruses........................................................................................................................................... 30 1.14.1 Coxsackievirus ....................................................................................................................... 31 1.14.2 Poliovirus ............................................................................................................................... 33 1.14.3 Picornaviruses replication cycle ............................................................................................ 33 1.14.4 Rotavirus ............................................................................................................................... 34 1.14.5 Rotavirus replication ............................................................................................................. 36 1.15 Inactivation of viruses ................................................................................................................... 37 1.16 Solar disinfection (SODIS) ............................................................................................................ 39 1.17 Aims............................................................................................................................................... 44 v 1.18 Objectives ...................................................................................................................................... 44 Chapter Two: Materials and Methods ........................................................................ 45 2.1 Materials ......................................................................................................................................... 46 2.1.1 Source of Viruses ..................................................................................................................... 46 2.1.2 Acanthamoeba ........................................................................................................................ 46 2.1.3 Mammalian cell lines ..............................................................................................................