Incidence and characterisation of mycoviruses from Aspergillus fumigatus By: Atif Jamal A thesis submitted for the degree of Doctor of Philosophy and the Diploma of Membership of Imperial College The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without the prior written consent of the author Department of Life Sciences Division of Biology Imperial College London Imperial College Road London, SW7 2AZ 1 Statement of Originality The material in this thesis has not been previously submitted for a degree in any university, and to the best of my knowledge contains no material previously published or written by another person except where due acknowledgement is made in the thesis itself. 2 For my Parents 3 Acknowledgments First of all I thank Almighty ALLAH, who gave me power to carry out the research. I am heartily thankful to my supervisor, Dr Robert Coutts, for his continuous support. His informed guidance, encouragement and support, from the preliminary to the concluding level, enabled me to develop an understanding of the subject and to complete my thesis work. His constructive criticism and comments during write-up is also highly commendable. I am also greatly indebted to Dr Elaine Bignell for the support she extended during the work related to Aspergillus fumigatus. Her patience and willingness to discuss the minutiae of the different obstacles I encountered while working were invaluable. I also extend my sincere gratitude to the members of my lab, namely, Dr Zisis Kozlakidis, Fiang Lang (Jackie) and especially Muhammad Faraz Bhatti for their support and company. I am also thankful to my friends Akhlas and Niaz for helping me out in the statistical analysis phase. Thanks are also due to all the members of Elaine‟s Bignell group for their support during the completion of my research. I am also thankful to my family and friends who have always supported, encouraged and believed in me in all my endeavours. My wife Aqeela, without whom, this effort would have been worth nothing. Her love, support and constant patience were really crucial for me in the difficult times. I would also like to acknowledge contribution of my daughter Namra, who was the source of happiness and joy throughout this period. Last, but not least, I am grateful to Higher Education Commission of Pakistan (HEC) for their support. 4 Abstract This study investigated the incidence and characterisation of mycoviruses in a range of different fungi including Phytophthora spp. Phlebiopsis gigantea and Aspergillus fumigatus and their effects on their hosts. The investigation developed methodology for rapid molecular characterisation of dsRNA elements from different fungi which were applied in detail to a range of isolates of A. fumigatus. DsRNA elements or mycoviruses are present in almost all major classes of fungi where they lack an extracellular phase and are transmitted intracellulary via anastomosis or spores. Mycoviruses can confer a range of phenotypes on their fungal hosts ranging from symptomless to debilitating and hypovirulence to hypervirulence. In most cases most fungal mycovirus infections are symptomless and the effects of A. fumigatus mycoviruses on fitness and growth of infected isolates were also investigated. A screen of thirty nine clinical isolates of A. fumigatus, which is an opportunistic human pathogen and causes aspergillosis in immunocompromised patients, revealed the presence of dsRNA elements which were hitherto unknown. Two mycoviruses were identified including a chrysovirus (isolate A-56) and an unclassified, triripartite dsRNA- containing mycovirus (isolate A-54). All four dsRNA segments of the A-56 chrysovirus were sequenced in their entirety using the methodologies developed earlier in the investigation and the sequences analysed and compared to other members of the family Chrysoviridae and other characterised mycovirus families. Also the effects of the chrysovirus on the fitness of the host fungus were studied as was transfer of the purified chrysovirus to cured strains of A. fumigatus via protoplast fusion and direct transfection by different methods. It is intended to develop the use of dsRNA elements for gene silencing in A. fumigatus and towards this aim a full-length clone of the smallest A-56 dsRNA has been constructed and characterised and used to produce dsRNA transcripts for infection and amplification in the fungus. 5 Table of Contents Statement of originality 2 Acknowledgments 4 Abstract 5 List of Abbreviations 24 1 Introduction ..................................................................................................................27 1.1 General properties of viruses .......................................................................................27 1.2 History and characteristics of dsRNA mycoviruses .......................................................27 1.3 Transmission and origin of dsRNA mycoviruses ...........................................................28 1.3.1 Origin of dsRNA mycoviruses ...............................................................................30 1.4 Significance of dsRNA viruses......................................................................................31 1.4.1 Symptomless or cryptic infections .........................................................................31 1.4.2 Beneficial interactions ...........................................................................................32 1.4.3 Debilitating effects and hypovirulence ...................................................................32 1.4.4 Effect of mycoviruses on Aspergilli .......................................................................33 1.4.5 Curing fungi of dsRNA infection ............................................................................34 1.5 Replication and gene expression strategy of dsRNA viruses ........................................35 1.6 Mycoviruses as biocontrol agents .................................................................................36 1.7 Taxonomy of dsRNA mycoviruses ................................................................................36 1.7.1 Chrysoviridae........................................................................................................37 1.7.2 Hypoviridae ..........................................................................................................40 1.7.3 Narnaviridae .........................................................................................................41 1.7.4 Partitiviridae ..........................................................................................................42 1.7.5 Reoviridae ............................................................................................................44 1.7.5.1 Mycoreovirus ................................................................................................45 1.7.6 Totiviridae .............................................................................................................46 1.8 Defective and satellite dsRNA viruses ..........................................................................49 1.9 Plant and fungal dsRNA viruses ...................................................................................50 1.9.1 Endornaviridae .....................................................................................................50 6 1.10 Aspergillus ................................................................................................................51 1.10.1 Importance of Aspergillus species ........................................................................51 1.11 Aspergillus fumigatus ...............................................................................................52 1.12 DsRNA mycoviruses in Aspergilli ..............................................................................54 1.13 Aims of the project ....................................................................................................57 1.13.1 Chapter 3: Development of the technology ...........................................................57 1.13.1.1 Identification of dsRNA endornaviruses from Phytophthora ramorum ...........57 1.13.1.2 Characterisation of dsRNA elements present in Aspergillus foetidus ............57 1.13.1.3 Characterisation of Phlebiopsis gigantea dsRNA ..........................................57 1.13.2 Chapter 4: Identification and characterisation of a group of dsRNA elements in one isolate of Aspergillus fumigatus ........................................................................................58 1.13.3 Chapter 5: Phenotypic screening of virus-infected and virus-free Aspergillus fumigatus isolates; curing A. fumigatus isolate A-56 from AfuCV infection and transfection of virus-free isolates of A. fumigatus .................................................................................58 1.13.4 Chapter 6: Construction of cDNA clones of AfuCV dsRNA 4; in vitro transcription of dsRNA from cDNA clones; over-expression of the AfuCV coat protein gene ....................58 1.13.5 Chapter 7: General discussion (Conclusions and future plans) .............................58 2 Materials and Methods .................................................................................................60 2.1 General laboratory practice ..........................................................................................60 2.2 Sterilisation...................................................................................................................60