The characterisation of a nucleopolyhedrovirus infecting the insect Trichoplusia ni by Michael Tobin Thesis submitted in fulfilment of the requirements for the degree Master of Science in Biomedical Sciences In the Faculty of Health and Wellness Sciences at the Cape Peninsula University of Technology Supervisor: Prof Sehaam Khan Co-supervisor: Prof Wesaal Khan Bellville January 2019 CPUT copyright information The dissertation/thesis may not be published either in part (in scholarly, scientific or technical journals), or as a whole (as a monograph), unless permission has been obtained from the University i DECLARATION I, Michael Tobin, declare that the contents of this dissertation/thesis represent my own unaided work, and that the dissertation/thesis has not previously been submitted for academic examination towards any qualification. Furthermore, it represents my own opinions and not necessarily those of the Cape Peninsula University of Technology. Signed Date ii ABSTRACT Background: Baculoviruses have great potential as alternatives to conventional chemical insecticides. The large scale adoption of such agents has however been hampered by the slow killing times exhibited by these bio-insecticides, limitation to single target insect and difficulty of large scale production of these preparations. Trichoplusia ni single nucleopolyhedrovirus (TnSNPV), initially identified in the Eastern Cape region of South Africa, has potential as a biocontrol agent as it possesses a higher speed of kill compared to other baculoviruses. Aims and methods: The main objective of this study was the identification, molecular characterisation and cloning of a structural core gene (polyhedrin) and three auxiliary genes, the inhibitor of apoptosis (iap2 and iap3) and the ecdysteroid UDP-glucosyltransferase (egt) genes, from TnSNPV in order to delineate its phylogenetic relationship to a Canadian isolate of the same virus and to other baculoviruses. In addition, the genes were expressed in an Escherichia coli (E. coli) based system as a prelude to genetic modification to increase the pesticidal property of the virus. Results: The genome size of the South African strain of TnSNPV was estimated at 160 kb and is significantly larger than the Canadian isolate of TnSNPV and may reflect genetic variation as the two strains have adapted to varying environmental conditions. Occlusion bodies of the South African strain of TnSNPV were visualised by Transmission Electron Microscopy and consisted of rod shaped single virions composed of a single enveloped nucleocapsid. Insect bioassays showed that the median lethal time (LT50) of the virus strain averaged 1.8 days which is significantly faster than other baculoviruses. The South African and Canadian strains of TnSNPV share nucleotide similarities greater than 95% for the genes analysed in this study, which indicates that they are closely related. From this analysis, the South African strain of TnSNPV identifies as a Group II NPV with the closest relatives being the Canadian strain of TnSNPV and ChchNPV. The topology of the tree for the polyhedrin protein was better resolved than that of the IAP2, IAP3 and EGT proteins and was comparable to the tree inferred from a concatenated data set consisting of complete polyhedrin/granulin, LEF8, and LEF9 proteins of 48 completely sequenced genomes. For the IAP2, IAP3 and EGT proteins, the separation of the lepidopteran and hymenopteran specific baculoviruses was not evident while the separation of Group I and II Alphabaculoviruses diverged from that observed from the baculovirus core gene polyhedrin as well as the tree inferred from complete polyhedrin/granulin, LEF8, and LEF9 proteins. Five distinct groups relating to IAP-1, 2, 3, 4 and 5 could be distinguished from the tree inferred from all IAP proteins from 48 fully sequenced baculoviruses. From this analysis, the IAP protein from the South African isolate of TnSNPV can be designated as an IAP3 due to sequence homology to other IAP3 proteins. Similarly, the IAP2 can be confirmed as an IAP2 protein as it clusters with other IAP2 proteins. iii RNA transcripts of the four genes were detected by RT-PCR at one hr after induction with L- arabinose in BL21-A1 E. coli and persisted until four hrs post induction. Antisera directed against the C-terminal 6X His tag was able to detect the recombinant proteins at two hours after induction confirming the rapid rise in expression of the proteins which persisted at high levels until four hrs after induction. The discrepancy observed with the predicted molecular mass of the EGT protein and the migration on SDS-PAGE may be due to the absence of post- translational modification in the E. coli expression system and the hydrophobic residues present in the N-terminal signal sequence. Conclusion: Sequence and phylogenetic analysis suggest that the two isolates of TnSNPV have been exposed to similar evolutionary pressures and evolved at similar rates and represent closely related but distinct variants of the same virus. The difference in genome size between the two strains is likely to reflect actual genetic differences as the strains have adapted to their local environments and hosts and the extent of the differences will only be apparent as more sequencing results become available. Phylogenetic analysis of the IAP and EGT proteins yields a tree that varies from the phylogenetic reconstruction observed for the polyhedrin gene as well as the concatenated data set consisting of complete polh/gran, LEF8, and LEF9 proteins and highlights the risks inherent in inferring phylogenetic relationships based on single gene sequences. The tree inferred from the concatenated data set of polh/gran, LEF8, and LEF9 proteins was a quick and reliable method of identification particularly, when whole genome data is unavailable and mirrors the accepted lineage of baculoviruses. Expression of the recombinant IAP2, IAP3, EGT and polyhedrin was confirmed by RT-PCR and immunoblot analysis and rose rapidly after induction and persisted at high levels. It is as yet unclear if the expressed proteins are functional particularly as post translation modifications are lacking in this system. KEYWORDS Trichoplusia ni single nucleopolyhedrovirus (TnSNPV), phylogeny, polyhedrin, inhibitor of apoptosis (iap2 and iap3), egt, phylogeny, heterologous expression iv DEDICATION To my father, James Frank Tobin v ACKNOWLEDGEMENTS To my friend, mentor and supervisor, Prof Sehaam Khan. You kept faith in me even as I lost faith in myself. This has been a long time coming. I owe it all to you. Thank you, thank you. To my friend, ex-colleague and supervisor, Prof Wesaal Khan. From CPUT to Stellenbosch. Thank you for hosting me in the Department of Microbiology at Stellenbosch University. Your support and guidance was invaluable. To my mother, Anne and my siblings, Jean and Mandy: this couldn’t happen without you. I am also grateful to my extended family members for their support. To the Supper Club: Grant, Mark, Mark and Johan and Mark - you know what you did. To friends and colleagues in the Biotechnology programme at CPUT. My home away from home. To my colleagues and friends in the research labs at CPUT and Stellenbosch, thank you. With thanks to Cape Peninsula University of Technology and Stellenbosch University for the use of their facility and equipment. vi TABLE OF CONTENTS DECLARATION i ABSTRACT ii DEDICATION iv ACKNOWLEDGEMENTS v LIST OF FIGURES ix LIST OF TABLES x APPENDICES xi GLOSSARY xii CHAPTER ONE 1 LITERATURE REVIEW 1.1 Introduction 1 1.2 Trichoplusia ni, the insect pest 2 1.3 Baculovirus Structure and Taxonomy 4 1.4 Baculovirus Life Cycle 6 1.5 Baculoviruses as Pesticides 11 1.6 Gene Expression Within Baculoviruses 15 1.7 The ecdysteroid UDP-glucosyltransferase (egt) gene 17 1.8 Apoptosis 19 1.8.1 Baculoviruses and apoptosis 20 1.9 OUTLINE OF THESIS 24 REFERENCES 26 vii CHAPTER TWO 2 MATERIALS AND METHODS 2.1 Phenotypic characterisation of TnSNPV 43 2.1.1 Virus isolation 43 2.1.2 Baculovirus propagation in T. ni larvae 43 2.1.3 Extraction of haemolymph from T. ni larvae 44 2.1.4 Viral infection of High Five cells 44 2.1.5 Viral DNA extraction from budded virus 44 2.1.6 Viral DNA extraction from insect larvae and haemolymph 45 2.1.7 Transmission Electron Microscopy 45 2.2 Phylogenetic characterisation of T. ni 46 2.2.1 Cloning of the ecdysteroid UDP–glucosyltransferase (egt) gene 46 2.2.1.1 Identification of the egt gene 46 2.2.1.2 PCR amplification of the egt gene from TnSNPV 46 2.2.1.3 Ligation of the egt gene into the vector pGEM-T Easy 47 2.2.1.4 Screening for egt recombinants 47 2.2.2 Cloning of the polyhedrin (pol) gene 49 2.2.2.1 PCR amplification of the polyhedrin gene 49 2.2.2.2 Ligation of the polyhedrin gene into the vector pGEM-T Easy 49 2.2.2.3 Screening for polyhedrin recombinants 50 2.2.3 Cloning of the iap2 and iap3 genes 50 2.2.3.1 PCR amplification of the iap2 and iap3 genes 50 2.2.3.2 Treatment of the iap inserts and annealing into the vector pIEX1 51 Ek/LIC 2.2.3.3 Transformation of the annealing reaction 52 2.2.4 Phylogenetic analysis 53 2.3 Heterologous Expression in E. coli 54 2.3.1 PCR amplification of the iap2, iap3, egt and polyhedrin genes 54 2.3.2 The TOPO cloning reaction 55 2.3.3 TOPO LR recombination 56 2.3.4 Pilot expression in E. coli 57 2.3.5 RNA extraction 58 2.3.6 Reverse Transcription Polymerase Chain Reaction (RT-PCR) 58 2.3.7 Protein concentration determination 59 2.3.8 Sample preparation 60 2.3.9 SDS PAGE and immunoblot analysis 60 REFERENCES 62 viii CHAPTER THREE 3 RESULTS AND DISCUSSION 3.1 Phenotypic characterisation of TnSNPV 65 3.2 Phylogenetic characterisation of TnSNPV 70 3.2.1 Identification, cloning and sequence analysis of the egt gene 70 3.2.2 Identification, cloning and sequence analysis of the polyhedrin 71 gene 3.2.3 Identification, cloning and sequence analysis of the iap2 and iap3 76 genes 3.2.4 Phylogenetic analysis 78 3.3 Heterologous expression of recombinant proteins in E.