Characterisation of mosaic virus and development of

microarrays for detecting viruses in

This thesis is presented to Murdoch University for the degree of Doctor of Philosophy

June 2008

by

Craig Graham Webster B.Sc. Biotechnology and Chemistry Honours Molecular Biology

State Agricultural Biotechnology Centre School of Biological Sciences and Biotechnology Murdoch University Perth, Western Australia

DECLARATION

Declaration

I declare that this is my own account of my research and contains as its main content work which has not previously been submitted for a degree at any tertiary education institute.

Craig Graham Webster

ii ABSTRACT

Abstract

A virus causing chlorosis and leaf distortion in the Western Australian endemic legume Hardenbergia comptoniana was detected by biological indexing to Chenopodium quinoa and Nicotiana benthamiana. Enzyme linked immuno-sorbent assay (ELISA) using general Potyvirus antiserum and amplification by reverse transcription polymerase chain reaction (RT- PCR) with degenerate primers indicated that it was a of Potyvirus. It was confirmed as an unknown member of the genus Potyvirus by comparing its coat protein sequence with those of other potyviruses. The name Hardenbergia mosaic virus (HarMV) is proposed for this new virus species. Isolates of HarMV were collected from 13 sites, covering much of the natural range of its host. An experimental host range was determined using nine virus isolates tested against plants from 11 species in three families. Its infectivity on three leguminous species important in agriculture (Lupinus angustifolius, L. luteus and Trifolium subterraneum) was established.

The nucleotide (nt) sequences of the coat proteins (CP) of 28 isolates determined there was 24.1- 27.6% diversity with the closest known relative, Passion fruit woodiness virus (PWV). Studies of the nucleotide sequences of the CP showed that there was considerable intra-species divergence (mean 13.5%, maximum 20.5%) despite its relatively small geographical distribution and single known natural host. The observed broad diversity strongly suggests long genetic isolation and that HarMV evolved in the region where it was collected. An examination of its phylogeny showed that 28 isolates clustered into eight clades with high bootstrap support (6.2-20.5% inter-clade diversity). Isolates collected at locations distant to the Perth metropolitan area (Margaret River and Seabird) diverged more from isolates collected in the metropolitan area (15.4-21.1% nucleotide sequence diversity). This virus represents the first endemic species to be characterised from Western Australia.

Differences in pathogenicity and symptoms induced on key host species were seen between isolates belonging to different phylogenetic clades. Phylogenetic analysis confirmed the inclusion of HarMV within the Bean common mosaic virus group of the potyviruses and also defined a previously unreported subgroup of six previously described Potyvirus species (Clitoria virus Y, Hibbertia virus Y, PWV, Siratro 1 virus Y, and Siratro 2 virus Y), from Australia, which is further evidence for a prolonged period of genetic isolation.

Both in relation to detection of strains of HarMV, and considering the broader issues of biosecurity and parallel detection of viruses, a microarray based detection system was established. To optimise conditions for the development of microarrays for virus detection

iii ABSTRACT poly-L-lysine (PLL) coated microscope slides produced in the laboratory were compared to commercially produced PowerMatrix slides (Full Moon BioSystems). Variables tested for PLL slide production were: choice of printing buffer, probe concentration, method of immobilisation and slide blocking; and in particular the print buffer and immobilisation method had the greatest effect on the quality of PLL microarray slides. Slides printed on PLL surfaces in a high salt buffer (3x Saline sodium citrate) supplemented with 1.5M betaine and immobilised at 42oC overnight retained the highest amounts of probe DNA of the methods tested. Qualitative comparisons of the two showed more probe was retained on PowerMatrix slides which were also more reliable and consistent than the PLL slides.

Probes were designed for eight different virus species and six distinct strains of HarMV to test the potential to use microarrays to distinguish between them. Probes were designed to detect potyviruses at the genus, species and strain levels. Although there was evidence of non-specific hybridisation, the Potyvirus array was used to identify six strains of HarMV by hybridisation to species specific probes. Additionally the array was used to identify three other species of Potyvirus: Bean yellow mosaic virus, PWV and Passiflora foetida virus Y, following amplification with polyvalent PCR primers.

In further microarray tests, using labelled first strand cDNA of Potato virus X (PVX) and Potato virus Y (PVY) on an array, PVX was strongly detected in leaves known to be infected, but PVY was only weakly detected in infected leaves. Three methods of pre-amplification of virus nucleic acid before hybridisation to the array were investigated to improve the sensitivity of the assay. Two of the methods, Klenow amplification and randomly primed PCR, amplified the target virus; as confirmed by real time PCR. Of the methods tested only randomly primed PCR improved the sensitivity of the microarray. The best amplification method used genus-specific primers with adaptor sequences. This method when tested by real time PCR showed a 3.7Ct reduction for PVX and 16.8Ct for PVY. The microarray correctly identified both viruses.

In this work the first virus (HarMV) endemic to Western Australia was identified, and microarray methods were developed both to identify HarMV and other plant viruses of economic importance. The microarray approach, with further development, may be applicable as a means of identifying incursions of new viruses in a biosecurity situation.

iv TABLE OF CONTENTS

Table of Contents

Declaration...... ii Abstract...... iii Table of Contents ...... v Presentations and Publications ...... x Publications...... x Oral Presentations...... x Poster Presentations...... xi List of abbreviations...... xii Viruses and viroids ...... xii Other abbreviations used in the text...... xv Code for degenerate oligonucleotides ...... xvi Acknowledgements...... xvii Chapter 1: Introduction and literature review...... 1 1.1 Introduction to viruses ...... 1 1.2 Genomic structure of plant viruses...... 2 1.2.1 Nucleic acid ...... 2 1.2.2 Genome size...... 2 1.2.3 Genome organization...... 2 1.2.4 Satellite viruses, satellite RNA’s and viroids...... 3 1.2.5 The Potyviridae...... 3 The Potyvirus coat protein gene...... 5 Classification of Potyvirus species...... 5 The BCMV group ...... 7 1.3 Economic importance...... 9 1.4 Diagnostic methods...... 10 1.4.1 Visual detection and indicator hosts...... 10 Electron microscopy ...... 10 1.4.2 Serological procedures...... 10 Enzyme-linked immunosorbent assay (ELISA) ...... 11 Tissue blot immunoassay (TBIA) ...... 11 Lateral flow devices (LFDs) ...... 12 Quartz crystal microbalance (QCM) immunosensors...... 12 Nanowire field effect transistors...... 13 1.4.3 Matrix Assisted Laser Desorption Ionisation Time of Flight Mass Spectrometry (MALDI-TOF MS) ...... 13 1.4.4 Nucleic acid procedures ...... 14 Dot Blots ...... 14 Reverse transcription-Polymerase Chain Reaction (RT-PCR) and PCR ...... 14 Multiplex RT-PCR...... 15 Nested PCR...... 15 Polyvalent PCR ...... 16 Immunocapture PCR (IC-PCR)...... 18 Fluorescence Real Time-PCR using Taqman technology...... 18 Competitive fluorescence PCR (CF-PCR)...... 19 Restriction fragment length polymorphism (RFLP)...... 19 Rolling circle amplification (RCA)...... 19 Loop mediated isothermal amplification (LAMP)...... 20 Macroarrays...... 21 1.4.5 Advantages and disadvantages of current methods...... 21 1.5 Microarrays...... 22 v TABLE OF CONTENTS

1.5.1 Diagnostic applications of microarrays ...... 23 1.5.2 Probes for microarrays...... 27 Nucleic acid probes ...... 27 cDNA probes ...... 27 Oligonucleotide probes ...... 28 Probe length...... 28 Antibody/protein probes ...... 29 1.5.3 Slide surface and immobilisation methods...... 30 Arraying of probes...... 30 Slide surfaces...... 31 Covalent binding of probes ...... 31 1.5.4 Methods for labelling and hybridisation...... 32 Fluorescent labelling of DNA...... 32 Hybridisation and washing slides ...... 33 1.6 Conclusions ...... 34 1.7 Project Aims...... 35 Chapter 2: General materials and methods ...... 36 2.1 Introduction ...... 36 2.2 Viruses and Inoculations...... 36 2.3 RNA Extractions...... 36 2.3.1 Qiagen RNeasy Plant Mini Extractions...... 36 2.3.2 Microarray RNA Extractions...... 37 2.4 Reverse Transcription...... 37 2.5 PCR Protocol...... 38 2.5.1 High fidelity PCR...... 38 2.5.2 Lower-fidelity PCR ...... 39 2.5.3 Real time PCR ...... 40 2.6 Agarose Gel Electrophoresis...... 40 2.7 Purification of DNA solutions...... 41 2.7.1 Ethanol Precipitation...... 41 2.7.2 Purification by QiaQuick PCR cleanup kit...... 41 2.8 Quantifying DNA...... 41 2.9 Cloning of PCR products ...... 41 2.9.1 Ligation and Transformation using TOPO-Zero kit...... 41 2.9.2 Ligation and Transformation using pGEM-T easy™ ...... 42 2.10 Screening for recombinant plasmids ...... 42 2.11 Plasmid Extraction ...... 42 2.11.1 QIAprep Spin Miniprep Kit...... 43 2.11.2: Sambrook Plasmid Extractions ...... 43 2.12 Restriction Digest of Plasmid...... 44 2.13 Storing clones as Glycerol Stocks...... 44 2.14 DNA Sequencing...... 44 2.15 Coating Slides with Poly-L-lysine...... 45 2.16 Printing microarray slides ...... 45 2.17 Quality control of printed slides...... 46 2.18 Summary ...... 46 Chapter 3: Bioinformatics for primer and probe design ...... 47 3.1 Introduction ...... 47

vi TABLE OF CONTENTS

3.2 Materials and methods...... 48 3.2.1 Sequence retrieval from online databases ...... 48 3.2.2 Sequence alignments...... 48 ClustalW alignments...... 48 MEGA alignments ...... 48 3.2.3 Comparisons of nucleotide sequences to international database ...... 48 3.2.4 Oligonucleotide design ...... 49 Primer design ...... 49 Oligonucleotide probe design...... 50 3.2.5 Phylogenetic analysis of virus isolates ...... 52 3.3 Bioinformatics results...... 53 3.3.1 Sequences of legume specific PCR primers ...... 53 3.3.2 Design of oligonucleotide probes ...... 53 3.4 Discussion ...... 57 Chapter 4: Characterisation of an undescribed Potyvirus in the Australian native plant species Hardenbergia comptoniana ...... 58 4.1 Introduction ...... 58 4.2 Method and materials ...... 61 4.2.1 Plant Samples and Virus Isolates ...... 61 4.2.2 Virus Inoculations...... 63 4.2.3 Enzyme Linked Immuno-Sorbent Assay (ELISA)...... 63 4.2.4 Incidence, host range and seed transmission studies...... 63 4.2.5 RT-PCR, Cloning and Sequencing...... 64 4.2.6 Phylogenetic Analysis of Isolates...... 65 4.3 Results...... 67 4.3.1 Symptoms of infection...... 67 4.3.2 Virus incidence ...... 68 4.3.3 Alternative hosts of HarMV ...... 68 4.3.4 Host range inoculations...... 71 4.3.5 Aphid and seed transmission tests ...... 71 4.3.6 Amplification of virus genome fragments ...... 72 Sequencing of coat protein genes and 3’ UTRs ...... 72 Amplification and sequencing of full length Nuclear Inclusion B gene...... 73 4.3.7 Hardenbergia mosaic virus specific primers...... 74 4.3.8 The CP genes – phylogenetic inferences ...... 76 4.3.9 Phylogenetic analysis of the 3’ UTR sequence ...... 85 4.3.10 Phylogeny of the HarMV NIb Gene...... 88 4.3.11 Evidence for recombination between HarMV isolates...... 91 4.4 Discussion ...... 95 4.4.1 Phylogeny studies based on the CP sequence...... 95 Experimental host range and designation of virus strains ...... 96 An Australian only subgroup to the BCMV group of Potyviruses...... 97 4.4.2 Phylogeny of the 3’ untranslated region...... 98 4.4.3 Phylogeny of the NIb gene...... 98 4.4.4 Determinants of virus symptoms and host range...... 99 4.4.5 Phylogeny of Potyviruses infecting Passiflora species ...... 100 4.4.6 Recombination in Potyviruses ...... 102 Recombination in MU-2A ...... 102 Recombination in other isolates...... 103 Detecting mixed infections ...... 104 Methods of detecting recombination ...... 104 4.4.7 Surveys of Western Australia...... 105 4.4.8 Surveys of Eastern Australia...... 106 4.4.9 Full genome sequencing ...... 107 4.4.10 Conclusions ...... 108 Chapter 5: Optimisation of microarray printing methods...... 109 5.1 Introduction ...... 109 vii TABLE OF CONTENTS

5.1.1 Creating arrays on PLL coated slides ...... 109 Printing buffer...... 109 DNA concentration ...... 110 Immobilisation of probes...... 110 Slide Blocking...... 110 5.1.2 Pre-activated slide chemistries...... 111 5.1.3 Probe intensity and morphology...... 111 5.1.4 Gene expression versus diagnostic arrays...... 112 5.1.5 Aims ...... 112 5.2 Methods and materials...... 113 5.2.1 Printing and immobilising PLL slides ...... 113 Immobilising probes on slides...... 114 Blocking microarray slides...... 114 Slide scanning and data analysis...... 114 5.2.2 Printing PowerMatrix slides...... 115 5.2.3 Virus extraction, labelling and hybridisation ...... 116 Fluorescent labelling of PCR amplicons ...... 116 Purification and hybridisation...... 116 5.3 Results...... 117 5.3.1 Optimising PLL slides...... 117 Probe morphology...... 117 DNA retention ...... 117 5.3.2 Printing arrays on Power Matrix slides ...... 120 5.3.3 Detection and discrimination of Potyvirus species...... 121 5.3.4 Probe design...... 123 5.4 Discussion ...... 125 5.4.1 Arrays on PLL slides ...... 125 Printing buffer...... 125 Probe immobilisation...... 127 Probe concentration...... 127 5.4.2 Comparison of slide surfaces...... 128 5.4.3 Virus identification by microarray...... 129 Probe design ...... 130 5.4.4 Conclusions ...... 131 Chapter 6: Optimising methods of nucleic acid amplification for microarray-based virus detection...... 132 6.1 Introduction ...... 132 6.2 Materials and methods...... 135 6.2.1 Microarray slides...... 135 6.2.2 Microarray hybridisation and scanning ...... 135 6.2.3 RNA extraction and precipitation...... 136 6.2.4 Gel Electrophoresis ...... 136 6.2.5 CyScribe Post-Labelling Kit (Indirect Labelling)...... 136 6.2.6 Burton’s random amplification method...... 137 6.2.7 Klenow based random amplification ...... 138 6.2.8 Genus-specific primer amplification...... 139 6.2.9 Real Time PCR ...... 140 6.3 Results...... 142 6.3.1 Comparison of PVX and PVY virus titre...... 142 6.3.2 Burton’s amplification method ...... 144 6.3.3 Amplification using Klenow labelling...... 147 6.3.4 Genus specific primer amplification...... 148 6.4 Discussion ...... 153 6.4.1 CyScript post labelling ...... 153 6.4.2 Burton amplification method ...... 153 6.4.3 Klenow amplification...... 155 6.4.4 Genus specific primers ...... 155 6.4.5 A Multiplex polyvalent PCR ...... 157 viii TABLE OF CONTENTS

6.4.6 Other amplification approaches ...... 158 6.4.7 Conclusions ...... 159 Chapter 7: General discussion ...... 162 7.1 Overview...... 162 7.2 Characterisation of Hardenbergia mosaic virus...... 162 7.3 Development of microarray based methods...... 164 7.3.1 Cost effective methods for microarray slides...... 166 7.3.2 Strain specific microarrays...... 166 7.3.3 Labelling methods for increased sensitivity...... 167 7.4 Future work...... 170 7.5 Conclusions ...... 171 Appendix 1: ...... 172 Appendix 2: ...... 178 References ...... 180

ix PRESENTATIONS AND PUBLICATIONS

Presentations and Publications

Some of the results presented in this thesis have been published and presented at scientific meetings.

Publications Chapter 1: Webster C.G., Wylie S.J. & Jones M.G.K (2004). “Diagnosis of plant virus pathogens.” Current Science 86: 1604-1607.

Chapter 4: Webster, C. G., Coutts, B. A., Jones, R. A. C., Jones, M. G. K. & Wylie, S. J. (2007). Virus impact at the interface of an ancient ecosystem and a recent agroecosystem: studies on three legume infecting potyviruses in the southwest Australian floristic region. Plant Pathology 56: 729-742.

Wylie, S. J., Webster, C. G., Coutts, B. A., Jones, M. G. K. & Jones, R. A. C. (2007). Hardenbergia mosaic virus, the first indigenous plant virus identified from the Southwest Australian Floristic Region. In ISHS International working group on legume and vegetable viruses: Annual newsletter 2006. Wageningen.

Oral Presentations Chapter 4: Australasian Plant Pathology Early Researchers Seminar Series, Perth, 19 October 2007. “An indigenous plant virus from the South West Australasian Floristic Region.”

Chapter 5: 6th Australasian Plant Virology Workshop (APVW), Gold Coast, Australia, 31 August – 2 September, 2004 “Towards highly parallel tests for plant virus diagnosis.”

Chapter 6: 7th Australian Plant Virology Workshop (APVW), Perth, Western Australia, 8-11 November, 2006. “Identification of unknown viral pathogens using microarrays.”

x PRESENTATIONS AND PUBLICATIONS

Poster Presentations Webster C. G., Coutts B. A., Jones R. A. C., Jones M. G. K., Wylie S. J. (2007) “The first indigenous plant virus identified from the South West Australian Floristic Region,” Proceedings of the CRC for National Plant Biosecurity Science Exchange, Melbourne, p26.

Webster C.G., Coutts B.A., Jones R.A.C., Jones M.G.K. & Wylie S.J. (2006). “The first indigenous plant virus identified from the South West Australian Floristic Region,” Proceedings of the 7th Australasian Plant Virology Workshop, Perth, p12.

Webster C.G., Wylie S.J., Chen G., Kenworthy W. & Jones M.G.K. (2005). “Towards highly parallel plant tests for plant virus diagnosis,” Proceedings of the AusBiotech 2005 National Biotechnology Conference, Perth, p20-State Winner of Student Excellence Award.

Webster C.G., Wylie S.J., Chen G., Kenworthy W. & Jones M.G.K. (2004). “Towards highly parallel plant tests for plant virus diagnosis,” Proceedings of the Combined ASBMB, ANZSCDB and ASPS Annual Meeting, Perth, p110.

xi LIST OF ABBREVIATIONS

List of abbreviations

Viruses and viroids Virus and viroid species names are in italics if approved by the International Committee on of Viruses (ICTV) as listed in the 8th ICTV report (Fauquet et al., 2005) or ICTV database (ICTVdB - The Universal Virus Database, version 4, April 2006, http://www.ncbi.nlm.nih.gov/ICTVdb/ICTVdB/). Unapproved or tentative names are given in Roman text.

APLV Andean potato latent virus APMoV Andean potato mottle virus ApVY Apium virus Y ApMV Apple mosaic virus ArMV Arabis mosaic virus BanMMV Banana mild mosaic virus BSV Banana streak virus BaMMV Barley mild mosaic virus BaYMV Barley yellow mosaic virus BCMNV Bean common mosaic necrosis virus BCMV Bean common mosaic virus BYMV Bean yellow mosaic virus BtMV Beet mosaic virus BNYVV Beet necrotic yellow vein virus BBWV-2 Broad bean wilt virus 2 BStMV Broome streak mosaic virus CCFV Cardamine chlorotic fleck virus CaLV Cardamine latent virus CarVY Carrot virus Y CYBV Cassia yellow blotch virus CeMV Celery mosaic virus CerMV Ceratobium mosaic virus ChiVMV Chilli veinal mottle virus CSMV Chloris striate mosaic virus CSVd Chrysanthemum stunt viroid CSFV Classical swine fever virus ClVY Clitoria virus Y CYVV Clitoria yellow vein virus ClYVV Clover yellow vein virus CSV Cocksfoot streak virus CABMV Cowpea aphid-borne mosaic virus CFMoMV Cucumber fruit mottle mosaic virus CGMMV Cucumber green mottle mosaic virus CMV Cucumber mosaic virus CABYV Cucurbit aphid-borne yellows virus CYMMV Cymbidium mosaic virus DsMV Dasheen mosaic virus DCMV Dianella chlorotic mottle virus DiSMV Digitara striate mosaic virus DiVY Diuris virus Y EAPV East Asian passiflora virus EBV Epstein-Barr virus EVY Eustrephus virus Y FHV Florida hibiscus virus FMDV Foot-and-mouth disease virus xii LIST OF ABBREVIATIONS

GFLV Grapevine fanleaf virus GFkV Grapevine fleck virus HarMV Hardenbergia mosaic virus HBV Hepatitis B virus HCV Hepatitis C virus HiVY Hibbertia virus Y HAdV-A Human adenovirus-A CV-A16 Human coxsachievirus A-16 EV-71 Human enterovirus 71 HHV Human herpes virus HIV Human immunodeficiency virus HPV Human papillomavirus HPIV-3 Human parainfluenza virus 3 HRSV Human respiratory syncytial virus HRV-A Human rhinovirus A HRV-B Human rhinovirus B HTLV Human T-cell lymphotrophic virus INSV Impatiens necrotic spot virus FLUVA Influenza A virus JYMV Japanese yam mosaic virus JGMV Johnsongrass mosaic virus KSHV Karposi's sarcoma-associated herpesvirus KVY Kennedya virus Y KYMV Kennedya yellow mosaic virus KGMMV Kyuri green mottle mosaic virus LYSV Leek yellow stripe virus LMV Lettuce mosaic virus LMoV Lily mottle virus LASV Lucerne Australian symptomless virus MDMV Maize dwarf mosaic virus MSSV Maize sterile stunt virus NVMV Nicotiana velutina mosaic virus OMV Oat mosaic virus ONMV Oat necrotic mottle virus ORSV Odontoglossum ringspot virus OYDV Onion yellow dwarf virus PLDMV Papaya leaf distortion mosaic virus PRSV Papaya ringspot virus PSMV Paspalum striate mosaic virus PClV Passiflora chlorosis virus PCV Passiflora crinkle virus PFVY Passiflora foetida virus Y PfMV Passiflora mosaic virus PFRSV Passiflora ringspot virus PaVY Passiflora virus Y PCV Passionfruit crinkle virus PaMV Passionfruit mosaic virus PFMoV Passionfruit mottle virus PFMTV Passion fruit mottle Thailand virus PWV Passion fruit woodiness virus PEMV-1 Pea enation mosaic virus-1 PSbMV Pea seed-borne mosaic virus PMMoV Pepper mild mottle virus PeMoV Peanut mottle virus PStV Peanut stripe virus PSV Peanut stunt virus PepMV Pepino mosaic virus PepMoV Pepper mottle virus PTV Peru tomato mosaic virus PlVY Pleione virus Y PPV Plum pox virus PBRSV Potato black ringspot virus PLRV Potato leafroll virus xiii LIST OF ABBREVIATIONS

PMTV Potato mop-top virus PSTVd Potato spindle tuber viroid PVA Potato virus A PVM Potato virus M PVS Potato virus S PVT Potato virus T PVV Potato virus V PVX Potato virus X PVY Potato virus Y PYVV Potato yellow vein virus PDV Prune dwarf virus PNRSV Prunus necrotic ringspot virus PtVY Pterostylis virus Y RhoVY Rhopalanthe virus Y RUBV Rubella virus RGMV Ryegrass mosaic virus SarVY Sarcochilus virus Y ScaMV Scallion mosaic virus SARS-CoV Severe acute respiratory syndrome coronavirus S1VY Siratro 1 virus Y S2VY Siratro 2 virus Y SNMoV Solanum nodiflorum mottle virus SrMV Sorghum mosaic virus SAPV South African passiflora virus SMV Soybean mosaic virus SCMoV Subterranean clover mottle virus SCMV Sugarcane mosaic virus SCSMV Sugarcane streak mosaic virus SPFMV Sweet potato feathery mottle virus SPMMV Sweet potato mild mottle virus TEV Tobacco etch virus TMV Tobacco mosaic virus TMSV Tobacco mosaic satellite virus TSV Tobacco streak virus TVMV Tobacco vein mottling virus TYDV Tobacco yellow dwarf virus TAV Tomato aspermy virus TPMVd Tomato planta macho viroid TSWV Tomato spotted wilt virus TYLCV Tomato yellow leaf curl virus TuMV Turnip mosaic virus VZV Varicella-zoster virus VTMoV Velvet tobacco mottle virus WMV Watermelon mosaic virus WSMV Wheat streak mosaic virus WYMV Wheat yellow mosaic virus WSSV White spot syndrome virus-1 WPMV Wild potato mosaic virus WVMV Wisteria vein mosaic virus YMV Yam mosaic virus ZGMMV Zucchini green mottle mosaic virus ZYMV Zucchini yellow mosaic virus

xiv LIST OF ABBREVIATIONS

Other abbreviations used in the text oC degrees Celsius 3' hydroxyl-terminus of DNA molecule 5' phosphate-terminus of DNA molecule A. A. amino acid a. a. amino allyl ACT Australian Capital Territory ANGIS Australian National Genome Information Service BHQ1 black hole quencher 1 BLAST basic local alignment search tool bp base pairs BSA bovine serum albumin CI cytoplasmic inclusion cDNA complementary deoxyribonucleic acid CF-PCR competitive fluorescence PCR COX cytochrome oxidase CP coat (capsid) protein CSL Central Science Laboratories, York, UK Ct threshold cycle CTAB cetyl-trimethylammonium Bromide C-terminus carboxy terminus cv cultivar Da Dalton DAFWA Department of Agriculture and Food, Western Australia DEPC diethylpyrocarbonate DIG digoxigenin DMSO dimethyl sulphoxide DNA deoxyribose nucleic acid dNTP deoxyribose nuceltodie tri-phosphate (mix of A,C,G +T) ds double stranded DTT dithiothreitol et al. et alia, with others. EDTA ethylene diamine tetra-acetic acid ELISA enzyme-linked immunosorbent assay EtOH ethanol FAM 5' carboxyfluorescein FMB Full Moon Biosystems fs cDNA first strand complementary DNA h hour HCl hydrochloric acid HC-Pro helper component-protease IC-PCR immunocapture PCR ICTV International Committee on the Taxonomy of Viruses ITS internal transcribed spaces IVT in-vitro transcription JOE 6-carboxy 4',5'-dichloro-2',7'-dimethoxy fluorescein label k kilo kb kilobase KCl potassium chloride L litre LAMP loop-mediated isothermal amplification LB medium Luria-Bertani medium LiCl lithium chloride LFD lateral flow device LNA locked nucleic acid µ micro µg microgram µL microlitre µm micrometre M molar MAb monoclonal antibody xv LIST OF ABBREVIATIONS

MALDI-TOF matrix assisted laser desorption ionization time of flight MEGA molecular evolutionary genetics analysis min minute mL mililitre mM millimolar mRNA messenger ribose nucleic acid MS mass spectrometry n nano NaAc sodium acetate NaBH4 sodium borohydride NaCl sodium chloride NaOH sodium hydroxide NaPO4 sodium phosphate ng nanogram

(ΝΗ4)2SO4 ammonium sulphate NIa nuclear inclusion a NIb nuclear inclusion b NSW New South Wales nt nucleotide oligo oligonucleotide O/N overnight pmol pico mole PAb polyclonal antibody PBS phosphate buffered saline PCR polymerase chain reaction pers. comm. personal communication Pfu Pyrococcus furiosus DNA polymerase PLL poly-L-lysine QCM quartz crystal microbalance Qld Queensland RDP Recombination detection program RdRp RNA dependent RNA polymerase RFLP restriction fragment length polymorphism RNA ribose nucleic acid ROX 6-carboxy-X-rhodamine rpm revolutions per minute rPCR random primed polymerase chain reaction RT-PCR reverse transcriptase polymerase chain reaction RTase, RT reverse transcriptase s second ss single stranded SSC sodium chloride/sodium citrate SDS sodium dodecyl sulphate SWAFR southwest Australian floristic region TAE buffer tris- acetic acid-EDTA electrophoresis buffer Taq Thermus aquaticus DNA polymerase TBIA tissue blot immunoassay TBE buffer tris-boric acid-EDTA electrophoresis buffer TEM transmission electron microscopy Tris tris(hydroxymethyl)aminomethane UV ultra violet vRNA viral ribose nucleic acid WA Western Australia WGA whole genome amplification

Code for degenerate oligonucleotides

A Adenosine M AC V ACG C Cytosine R AG H ACT G Guanine W AT D AGT I Inosine S CG B CGT T Thymine Y CT N AGCT U Uracil K GT xvi ACKNOWLEDGEMENTS

Acknowledgements

First I would like to sincerely thank my principle supervisor Dr. Steve Wylie for encouragement and furthering my interest in science and virology. I would like to thank you for always finding the time to give help or advice no matter how busy you were, for your patience and for making thorough corrections to this manuscript. All the support and guidance is much appreciated. Finally thanks for keeping me on track and motivated on the days when the end seemed such a long way off.

I would also like to thank Professor Mike Jones for giving me the opportunity to undertake a PhD and for his assistance and advice during the course of the project. Thank you also for the funding and for encouraging me to visit and work at CSL.

I also thank Dr. Roger Jones (DAWFA, Perth) for the useful advice on all aspects of research, particularly the advice and critical recommendations for manuscript preparation and on virus characterisation, and for providing samples and advice on virus collection. I would also like to thank Brenda Coutts (DAWFA) for carrying out the many of the host range inoculations of HarMV and preparation of some figures and Monica Kehoe (DAWFA) for the testing so many samples by ELISA during the project.

My sincere thanks go to Dr. Neil Boonham at CSL for giving me the opportunity to work with you and your group. The great advice and useful discussions on all aspects of developing microarrays are greatly appreciated. Thank you for sharing you knowledge and resources so freely. Thanks also for all the lifts to and from the gatehouse in the mornings. I would like to thank Jenny Tomlinson and Rachel Glover (CSL, UK) for the training in microarray methods and help with extractions, labelling and scanning.

I thank Violet Peeva (Lotteries State Microarray Facility, Perth) for protocols, information and advice on all the many different aspects of microarray research; Grace Chen for help with training and setting up of the microarray facilities both for printing and scanning of slides; Bill Kenworthy (Murdoch University) for sharing with me the basics of bioinformatics, for advice on how best to tackle phylogenetic reconstructions and on image adjustments and issues with scanning microarray slides.

The National Plant Biosecurity Cooperative Research Centre (NPBCRC) including Dr Kirsty Bayliss and Dr Simon McKirdy provided some financial assistance, training such as a writing

xvii ACKNOWLEDGEMENTS workshop in Adelaide, and information-sharing opportunities, to help complete the thesis and funding for travel to CSL.

Barry Cayford deserves thanks for all the bizarre, and sometime scientific, conversations we’ve had over the last four years and for knowing when to say “stop complaining and just get on with it.” Past and present members of the Plant Biotechnology Research Group including John Fosu- Nyarko, Kerry Ramsay, Linda Maccarone, Zhaohui Wang, Muhammad Saqib, and Sheila Mortimer-Jones for advice, protocols, support and friendship over the past four years. I also thank other friends and students from Murdoch including; Kylie Ireland, Kate Taylor and Monique Sakalidis for the lunches and coffee breaks.

Finally I would like to thank my family for their love, support and encouragement during this time. Without all your understanding and patience while I was completing this work I could not have achieved this.

xviii