@prefix vivo: . @prefix edm: . @prefix ns0: . @prefix dcterms: . @prefix dc: . @prefix skos: . vivo:departmentOrSchool "Land and Food Systems, Faculty of"@en ; edm:dataProvider "DSpace"@en ; ns0:degreeCampus "UBCV"@en ; dcterms:creator "Rampitsch, Christof"@en ; dcterms:issued "2009-03-17T17:35:15Z"@en, "1996"@en ; vivo:relatedDegree "Doctor of Philosophy - PhD"@en ; ns0:degreeGrantor "University of British Columbia"@en ; dcterms:description """A triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) with a monoclonal antibody was developed and evaluated for the detection of prune dwarf ilarvirus (PDV) in sweet cherry trees {Prunus avium). A reverse transcribed polymerase chain reaction test was also developed to establish the incidence of PDV in 40 sweet cherry trees and to confirm the absence of virus in 15 control trees. Trees with two-thirds of their leaves positive for PDV by TAS-ELISA would be identified with 99% probability by testing four leaves per tree. The monoclonal antibody did not cross- react with Prunus necrotic ringspot ilarvirus in the TASELISA. The nucleotide sequence of PDV RNA1 was determined. The RNA consists of 3374 nucleotides and encodes a single open reading frame of 3168 nucleotides. The putative translation product is 1055 amino acids in length with a calculated molecular mass of 118.9 kDa. Both the nucleic acid and the translated amino acid sequences show stronger homology to RNA1 and the corresponding translation product (ORF1) of alfalfa mosaic alfamovirus (AMV) than' to citrus leaf rugose ilarvirus, the only other ilarvirus for which RNA1 sequence data is available. There is extensive sequence homology in the 3'-untranslated regions of PDV RNA1 and the 3'-regions of other ilarvirus and AMV RNAs. The reported sequence and its single open reading frame conform to the genomic organization typical of the Bromoviridae genus. Clones representing sequence from the 5'and 3'-end of RNA1 were used to construct a deletion-type defective interfering particle and its ability to replicate in vivo was assessed."""@en ; edm:aggregatedCHO "https://circle.library.ubc.ca/rest/handle/2429/6137?expand=metadata"@en ; dcterms:extent "6661533 bytes"@en ; dc:format "application/pdf"@en ; skos:note "THE COMPLETE NUCLEOTIDE SEQUENCE OF PRUNE DWARF ILARVIRUS RNA1 AND VIRUS DETECTION BY REVERSE TRANSCRIPTION PCR AND TRIPLE-ANTIBODY SANDWICH ELISA by CHRISTOF RAMPITSCH B.Sc, University of the Witwatersrand, 1988 M.Sc, University of the Witwatersrand, 1990 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES Department of Plant Science We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH September 1996 ®Christof Rampitsch, COLUMBIA 1996 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of PL/h^T S d ^ J c ^ , The University of British Columbia Vancouver, Canada Date DE-6 (2/88) ABSTRACT A t r i p l e antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) with a monoclonal antibody was developed and evaluated for the detection of prune dwarf i l a r v i r u s (PDV) i n sweet cherry trees {Prunus avium). A reverse transcribed polymerase chain reaction test was also developed to e s t a b l i s h the incidence of PDV i n 40 sweet cherry trees and to confirm the absence of virus i n 15 control trees. Trees with two-thirds of t h e i r leaves p o s i t i v e for PDV by TAS-ELISA would be i d e n t i f i e d with 99% p r o b a b i l i t y by testing four leaves per tree. The monoclonal antibody did not cross-react with Prunus necrotic ringspot i l a r v i r u s i n the TAS-ELISA. The nucleotide sequence of PDV RNA1 was determined. The RNA consists of 3374 nucleotides and encodes a single open reading frame of 3168 nucleotides. The putative t r a n s l a t i o n product i s 1055 amino acids i n length with a calculated molecular mass of 118.9 kDa. Both the nucleic acid and the translated amino acid sequences show stronger homology to RNA1 and the corresponding t r a n s l a t i o n product (ORF1) of a l f a l f a mosaic alfamovirus (AMV) than' to c i t r u s leaf rugose i l a r v i r u s , the only other i l a r v i r u s for which RNA1 sequence data i s available. There i s extensive sequence homology i n the 3'-untranslated regions of PDV RNA1 and the 3'-regions of other i l a r v i r u s and AMV RNAs. The reported sequence and i t s single open reading frame conform to the genomic organization Page i i t y p i c a l o f t h e Bromoviridae g e n u s . C l o n e s r e p r e s e n t i n g s e q u e n c e f r o m t h e 5 ' - a n d 3 ' - e n d o f R N A 1 w e r e u s e d t o c o n s t r u c t a d e l e t i o n - t y p e d e f e c t i v e i n t e r f e r i n g p a r t i c l e a n d i t s a b i l i t y t o r e p l i c a t e in vivo w a s a s s e s s e d . P a g e i i i TABLE OF CONTENTS Abstract i i Table of Contents i v L i s t of Tables v i i L i s t of Figures v i i i L i s t of Abbreviations ix Acknowledgements x i Foreword x i i 1 . 0 INTRODUCTION 1 1.1 Diseases and spread of prune dwarf virus 1 1. 2 Detection of prune dwarf virus 4 1.3 Genetics of prune dwarf virus 7 1. 4 Defective i n t e r f e r i n g RNA 11 1.5 The nucleotide sequence of PDV RNA1 12 1. 6 Objectives of t h i s study 14 2 . 0 MATERIALS AND METHODS 15 2.1.0 Pur i f i c a t i o n procedures 15 2.1.1 Virus ori g i n 15 2.1.2 Virus p u r i f i c a t i o n 15 2.1.3 Coat protein electrophoresis 17 2.1.4 Genomic RNA is o l a t i o n 18 2.1.5 Extraction of dsRNA from infected leaves 19 2.2.0 Antibody production 21 2.2.1 Production of polyclonal antibodies i n chickens 21 2.2.2.0 Production of monoclonal antibodies ... 23 2.2.2.1 Immunization with a f t e r cyclophosphamide treatment 23 2.2.2.2 Determination of the immune response by TAS-ELISA 24 2.2.2.3 Fusion mediated by polyethylene glycol 25 2.2.3 Single c e l l cloning 28 2.2.4 Cryogenic storage of hybridomas 29 2.2.5 Isolation of antibody from tissue culture supernatant and isotyping 2 9 2.2.6 Western blot 31 2.2.7 Conjugation of PDA-3C to al k a l i n e phosphatase 32 2.2.8 Production of F(ab') 2 fragments from PDA-3C..34 2.2.9 Detection of PDV by TAS-ELISA, RT-PCR and bioassay i n sweet cherry 35 Page i v 2.3.0 cDNA 1 ibrary of PDV 3 7 2.3.1 Preparation of cDNA: f i r s t - s t r a n d synthesis..37 2.3.2 Second-strand synthesis 38 2.3.3 End polishing reaction 3 8 2.3.4 Blunt-end l i g a t i o n 39 2.3.5 Preparation of competent E. coli 40 2.3.6 Screening colonies for inserts by PCR 42 2.3.7 Identifying inserts using Northern blots 42 2.3.8 Preparation for sequencing 44 2.3.9 Exo III deletions 45 2.3.10 Sequence alignment 4 7 2.3.11 cDNA cloning of PDV RNA1 47 2.3.12 The sequence of the 5' end of PDV RNA1 48 2.3.13 Cloning the 3' region of PDV RNA1 49 2 . 3 .14 Sequence comparisons and phylogeny 51 2.3.15 RT-PCR assay 52 2.4.0 Preparation of a defective i n t e r f e r i n g particle...53 2.4.1 Preparation of a snapback-type DI p a r t i c l e by PCR 53 2.4.2 Preparation of a snapback-type DI p a r t i c l e with synthetic oligonucleotides 56 2.4.3 Preparation of a deletion-type DI particle...59 2.4.4 Replication of a DI p a r t i c l e in vivo 59 3.0 RESULTS 62 3.1.0 Virus p u r i f i c a t i o n 62 3.1.1 V i r a l RNA separation 64 3.2.0 Antibody production 66 3.3.0 Primer pairs #1 and #2 i n RT-PCR 68 3.3.1 Detection of PDV by RT-PCR i n sweet cherry...71 3.4.0 Results of the f i e l d survey 71 3.4.1 I d e n t i f i c a t i o n of PDV-infected trees 71 3.4.2 Detection of PDV by TAS-ELISA 75 3.5.0 Alternate trapping antibodies 78 3.6.0 The p a r t i a l nucleotide sequence of PDV RNA3 81 3.7.0 The complete nucleotide sequence of PDV RNA1 81 3.7.1 Phylogenetic relationships of PDV to other Bromoviridae, based on RNA1 9 0 3.8.0 Replication of the DI RNA 90 4 . 0 DISCUSSION AND CONCLUSION 94 4.1.0 Virus i s o l a t i o n and nucleic acid analysis 94 4.2.0 Monoclonal antibody production 95 4.3.0 T r i p l e antibody sandwich EL ISA 96 4.4.0 Alternate trapping antibodies 98 4.5.0 The p a r t i a l nucleotide sequence of RNA3 99 Page v 4.6.0 The RT-PCRassay 99 4.7.0 The complete nucleotide sequence of RNA1 100 4.8.0 Phylogenetic relationships among Bromoviridae based on RNA1 102 4.9.0 Production and r e p l i c a t i o n of the a r t i f i c i a l DI RNAs 103 4.10.0 Concluding remarks 105 BIBLIOGRAPHY 106 Page v i LIST OF TABLES Table 1. Oligonucleotide cassettes for DI p a r t i c l e s 57 Table 2. TAS-ELISA results using monoclonal antibody PDA-3C..69 Table 3. Summary of f i e l d indexing results 73 Table 4. Summary of RT-PCR, bioassay TAS-ELISA r e s u l t s 76 Table 5. Summary of PDV TAS-ELISA results 77 Table 6. ELISA with alternate trapping antibodies 80 Page v i i LIST OF FIGURES Figure 1. Plasmid used to construct DI template GO Figure 2. Virus p u r i f i c a t i o n , SDS PAGE res u l t s 63 Figure 3. Agarose gel electrophoresis of PDV RNA 65 Figure 4 . Western blot with PDA-3C 67 Figure 5. RT-PCR t r i a l s with primer pairs #1 and #2 70 Figure 6. RT-PCR assay results of sweet cherry trees 72 Figure 7. SDS-PAGE analysis of F(ab') 2 fragments 79 Figure 8. Alignment of PDV RNA3 clones with PDV RNA3 82 Figure 9. Alignment of clones used to sequence RNA1 84 Figure 10. The complete nucleotide sequence of PDV RNA1 85 Figure 11. Comparison of the 3' ends of i l a r - and AMV RNA1. . .89 Figure 12. Phylogenetic relationships of PDV & Bromoviridae..91 Figure 13. Replication of the DI RNA i n pumpkins 92 Page v i i i LIST OF ABBREVIATIONS AMV a l f a l f a mosaic alfamovirus ApMV apple mosaic i l a r v i r u s AP alkaline phosphatase /3ME beta mercaptoethanol BMV brome mosaic bromovirus bp base pairs BSA bovine serum albumin CiLRV c i t r u s leaf rugose i l a r v i r u s CIP c a l f i n t e s t i n a l phosphatase CMV cucumber mosaic cucumovirus DAS-ELISA double antibody sandwich ELISA DI defective i n t e r f e r i n g DMEM Dulbecco's modified Eagle medium DNase deoxyribonuclease DTT d i t h i o t h r e i t o l dsRNA double stranded RNA EDTA ethylenediaminetetra-acetic acid EL ISA enzyme-linked immunosorbent assay FCS foe t a l c a l f serum HAP hydroxylapatite HAT hypoxanthine aminopterin thymidine supplement IgY hen egg yolk antibodies IPTG isopropyl - / 3-D- thiogalactoside kDa kil o d a l t o n kb kilobase MEA 2-mercato ethylamine Me-HgOH methylmercuric hydroxide NC n i t r o c e l l u l o s e ORF open reading frame PAGE polyacrylamide gel electrophoresis PBS Phosphate buffered saline PCR polymerase chain reaction, PEG polyethylene gl y c o l PDA-3C a murine anti-PDV monoclonal IgG1 antibody PDA-3C-AP alkaline phosphatase conjugate of PDA-3C PDV prune dwarf i l a r v i r u s PNRSV Prunus necrotic ringspot i l a r v i r u s Pr Probabi l i t y of detection of PDV RACE rapid amplification of cDNA ends RBDV raspberry bushy dwarf idaeovirus RdRp RNA-dependent RNA polymerase RNase ribonuclease RT-PCR reverse t r a n s c r i p t i o n PCR SDS sodium dodecyl sulphate SSC standard saline c i t r a t e TAS-ELISA t r i p l e antibody sandwich ELISA TBS T r i s buffered saline TDT terminal deoxynucleotidyl transferase TCS tissue culture supernatant Page ix TSV tobacco streak i l a r v i r u s UTR untranslated region X-gal 5-bromo-4 -chloro - 3 - indolyl - / 3-D-galactoside Page x ACKNOWLEDGEMENTS I would l i k e to extend my sincere gratitude to my research supervisor, Dr. Ken Eastwell, for his guidance and for the opportunity of working i n his laboratory. I would also l i k e to thank the dir e c t o r of the Summerland Research Centre for providing the research f a c i l i t i e s . F i nancial assistance i n the form of a one year scholarship from the Austrian Federal Chamber of Commerce was greatly appreciated as was further (and far greater) f i n a n c i a l help from my parents. F i n a l l y I would l i k e to thank members of my lab, my friends at UBC and my supervisory committee for helpful advice and suggestions. Page x i FOREWORD Some of the r e s u l t s i n section 3.3.1 to 3.4.2 have been published i n the following a r t i c l e : Rampitsch, C , Eastwell, K.C. and H a l l , J. (1995) Setting confidence l i m i t s for the detection of prune dwarf virus i n Prunus avium with a monoclonal antibody based t r i p l e antibody-sandwich ELISA. Annals of Applied Biology 126: 485-491 Dr. K. Eastwell's role i n t h i s publication was i n helping to plan the o v e r a l l experiment and i n giving advice and c r i t i c i s m during i t s execution. Dr. J. H a l l also gave advice during the planning of the experiment, but was involved p r i n c i p a l l y i n carrying out the s t a t i s t i c a l analysis of the r e s u l t s . My involvement was i n doing a l l of the laboratory work, including the production of the monoclonal antibody, the f i e l d indexing by TAS-ELISA, the bioassay and the RT-PCR analysis and i n a s s i s t i n g with the s t a t i s t i c a l analysis Results of the nucleotide sequence of RNA1 (sections 3.7.0 and 3.7.1) have been submitted under the t i t l e : Page x i i R a m p i t s c h , C. and E a s t w e l l , K.C. (1996) The complete n u c l e o t i d e sequence o f prune dwarf i l a r v i r u s RNA1. S u b m i t t e d t o the Archives of Virology Dr E a s t w e l l ' s r o l e i n t h i s p u b l i c a t i o n was i n g i v i n g a d v i c e and s u g g e s t i o n s t h r o u g h o u t the d u r a t i o n o f the c l o n i n g and s e q u e n c i n g . My i n v o l v e m e n t was i n c a r r y i n g out the c l o n i n g , s e q u e n c i n g and the a n a l y s i s o f the sequence. The sequence o f RNA1 has been d e p o s i t e d w i t h the GenBank and a s s i g n e d the a c c e s s i o n number U57648. (C. Rampitsch) .\\K.C. E a s t w e l l ) TJ. H a l l ) Page x i i i 1.0 INTRODUCTION 1.1 Diseases and spread of prune dwarf virus The i l a r v i r u s diseases of cherry trees are caused by prune dwarf i l a r v i r u s (PDV) and Prunus necrotic ringspot virus (PNRSV). Strains and combinations of these i l a r v i r u s e s are responsible for at least 11 diseases i n sweet cherry {Prunus avium L.) and sour cherry (P. cerasus L.) (Mink & Jones 1996). This review l i s t s seven diseases on cherry c u l t i v a r s caused by (PNRSV) and six caused by (PDV) with two apparently caused by both. Nemeth (1986) l i s t s a further two i n peach (P. persica L.), three i n apricot (P. armeniaca L.), s i x i n plum (P. domestica L.) and two i n almond (P. dulcis L.). However, some of these are caused by co-infection of PDV and PNRSV, and one by co-infection of PNRSV with apple c h l o r o t i c leafspot t r i c h o v i r u s (Nemeth 1986). Such dual infections usually cause more severe symptoms than single i n f e c t i o n s . F r u i t trees infected with PDV or PNRSV show two phases of symptom expression (Gilmer et al. 1975). An acute, shock symptom appears i n the f i r s t season a f t e r i n f e c t i o n , or i f the i n f e c t i o n occurred early i n spring, shock symptoms may appear i n the same season. T y p i c a l l y these are seen as leaf discolouration, necrotic lesions and may include f r u i t damage. The second phase occurs i n the next season and w i l l reappear annually. These chronic symptoms may be quite mild, Page 1 even absent i n some c u l t i v a r s . Chronic symptoms are di f f e r e n t from acute and may include any or a l l of several d i s t i n c t symptoms including c h l o r o t i c lesions, decreased tree-size, reduced percentage of bud-take, delayed ripening and usually a reduced y i e l d . Y i e l d can be reduced by 50% or more i n sour cherry yellows disease and t h i s i s one of the most serious diseases of sour cherry i n North America (Davidson & George 1965). Although PDV does not affe c t f r u i t quality, i t i s a problematic virus for f r u i t growers since Prunus are propagated vegetatively and the presence of PDV greatly reduces the percentage of bud-take (Gilmer et al. 1975) . Ila r v i r u s e s are spread i n three ways. A l l are spread r e a d i l y by gr a f t i n g infected tissue, some can be spread through seed (Casper 1977) and a few have been shown to be pollen transmissible. Western flower thrips (Frankliniella spp.) are l i k e l y vectors for the pollen transmissible i l a r v i r u s e s (Greber et a 2 . 1991; Sdoode & Teakle 1993). With these three modes of 'transmission, PDV and PNRSV can e a s i l y be introduced and spread rapidly through orchards causing losses i n f r u i t y i e l d within a few years (Uyemoto et al. 1992). The most frequent source of new infect i o n s i n orchards i s the introduction of infected material, since f r u i t trees are vegetatively propagated. It i s very important that a l l bud-wood and seedlings d i s t r i b u t e d by nurseries are c e r t i f i e d v i r u s - f r e e . Seedlings may be Page 2 infected i f a maternal tree was p o l l i n a t e d with infected pollen (Casper 1977). It i s thought that PDV and PNRSV w i l l not i n f e c t the maternal tree during f e r t i l i z a t i o n and i t remains v i r u s - f r e e unless there i s physical damage at the s i t e where infected pollen lands. By analogy to virus transmission to herbaceous hosts, feeding behaviour by thrips appears to provide s u f f i c i e n t damage to allow the virus to i n f e c t the tree (Greber et al. 1992). It has s t i l l not been shown that thrips are vectors for PDV and PNRSV i n Prunus spp. but based on experiments with herbaceous plants, the circumstantial evidence i s over-whelming. Western flower thrips (Frankliniella occidentalis and Thrips madronii) were implicated i n raspberry bushy dwarf virus (RBDV, a pollen transmitted idaeovirus) transmission i n raspberries (Bulger et al. 1991) and Microcephalothrips abdominalis spread tobacco streak i l a r v i r u s (TSV) through tobacco f i e l d s i n Queensland, A u s t r a l i a (Greber et al. 1991). More importantly F. occidentalis are able to i n f e c t cucumber cotyledons dusted with PDV and PNRSV infected pollen (Greber et al. 1992). The experiments indicated that virus was transferred from the pollen to the plant during feeding presumably entering the plant through c e l l s damaged by the feeding insects. Plants dusted with pollen alone, or caged with thr i p s alone f a i l e d to develop symptoms. The authors stressed that i t was u n l i k e l y that the thrips were responsible for transporting the infected pollen from tree to Page 3 tree, because they clean themselves c a r e f u l l y before f l y i n g . This transport role has been assigned to honey bees which p o l l i n a t e the trees i n spring (Uyemoto et al. 1992). Greber et al. (1991) also stressed the importance of weed populations i n tobacco f i e l d s acting as a reservoir and alternate host for th r i p s . Further evidence, using enzyme-linked immunosorbent assays (ELISA), Digiaro et al. (1992) found PNRSV and PDV both on the surface and within pollen grains although they could not detect apple mosaic i l a r v i r u s (ApMV) i n association with pollen. Kryczynski et a l . (1992) have noted a c o r r e l a t i o n between flowering i n t e n s i t y and the spread of PNRSV through sour cherry experimental orchards. 1.2 Detect ion of prune dwarf v i r u s T r a d i t i o n a l l y , f r u i t trees are tested for PDV and PNRSV in the spring, by budding onto an indicator tree. The most commonly used indicator for these i l a r v i r u s e s i s P. serrulata cv. Shirofugen (Gilmer et al. 1975). This tree responds to i l a r v i r u s i n f e c t i o n by a hypersensitive gumming response at the bud union within four to six weeks when budded with infected wood. Mechanical transmission to cucumber (Cucumis sativus) and squash (Cucurbita maxima) i s also possible with both PDV and PNRSV. Both viruses show l o c a l c h l o r o t i c lesions on the inoculated cotyledons accompanied by a systemic i n f e c t i o n . PNRSV infections often r e s u l t i n apical necrosis. Some squash c u l t i v a r s , such as 'Buttercup', show Page 4 c h l o r o t i c vein c l e a r i n g of the true leaves when infected with PDV and t h i s c u l t i v a r i s frequently used as a source of PDV for p u r i f i c a t i o n . PNRSV w i l l e l i c i t ringspot symptoms i n Chenopodium quinoa and t h i s plant i s sometimes used to d i s t i n g u i s h between the two i l a r v i r u s e s i n a bioassay. Serological techniques have been applied to indexing plant viruses (Clarke & Adams 1977). The most popular i s the ELISA because of i t s f l e x i b i l i t y and scale-up p o t e n t i a l . It i s quicker than the bioassay, y i e l d i n g r e s u l t s within days rather than weeks and i t i s very r e l i a b l e and less expensive, but i t does require the a v a i l a b i l i t y of antiserum against the virus to be tested. Serological assays are also more s p e c i f i c than bioassays and are able to d i f f e r e n t i a t e between PDV and PNRSV. The r e l i a b i l i t y of the assay depends to a large degree on the s e r o l o g i c a l d i v e r s i t y of the virus i n question and on the a b i l i t y of the available serum to recognize these serotypes. Polyclonal and monoclonal antisera can both be used i n ELISA. In general monoclonal antibodies o f f e r a lower background, but t h e i r higher s p e c i f i c i t y may cause a f a i l u r e to react with c e r t a i n virus serotypes. Polyclonal antibodies are generally better suited f o r viruses with many serotypes, e s p e c i a l l y i f f a l s e negative r e s u l t s are highly undesirable. Both mono- and polyclonal antisera s p e c i f i c for PDV and PNRSV are available (Torrance & Dolby 1984; McMorran & Cameron 1983), and have been used i n s e r o l o g i c a l assays to assess the extent of virus Page 5 i n f e c t i o n s i n Washington (Mink 1984) and C a l i f o r n i a (Uyemoto et al. 1989; 1992) by double-antibody sandwich (DAS)-ELISA. Perhaps the biggest drawback to s e r o l o g i c a l techniques applied to indexing i s t h i s problem of serotypes of viruses which the antibodies may not recognize or recognize poorly. PNRSV i s s e r o l o g i c a l l y diverse and i s s e r o l o g i c a l l y related to ApMV, but no d i s t i n c t subgroups of PDV have been found based on serology (Torrance & Dolby 1984; McMorran & Cameron 1983). The problem of s e r o l o g i c a l d i v e r s i t y can be overcome by using a pool of antisera, i f available, or by resorting to alternate techniques such as bioassays, nucleic acid hybridization or polymerase chain reaction (PCR). In a recent survey C r o s s l i n et al. (1992) used r a d i o a c t i v e l y l a b e l l e d PNRSV riboprobes to confirm ELISA res u l t s , since the l a t t e r does not detect the CH3 0 serotype of PNRSV. This serotype was r e a d i l y detected by nucleic acid hybridization. PCR and related assays such as reverse t r a n s c r i p t i o n PCR (RT-PCR) and immuno-capture PCR, are the most sensitive techniques currently available for the routine detection of plant viruses. Wetzel et al. (1991) were able to detect 10 fg of plum pox potyvirus RNA (approximately 2000 virus p a r t i c l e s ) i n a f i e l d indexing t r i a l by RT-PCR. They found t h i s technique to be more sensitive than nucleic acid hybridization with 3 2P l a b e l l e d probes, which had previously been the most sensitive assay for plant virus detection in vivo. Another study comparing monoclonal antibodies, DNA » , Page 6 probes and PCR to detect grapevine yellows disease caused by a phytoplasma, also found that PCR could detect approximately 10 fg of target and was the most sensitive of the three techniques tested (Chen et al. 1993). Similar r e s u l t s were obtained from a study with grapevine fanleaf nepovirus detection i n grapevine (Rowhani et al. 1993) where again the authors were able to detect virus i n the fg range. In addition these studies highlight some of the problems encountered with PCR of woody tissue samples (leaves, roots, bark and shoots) because of i n h i b i t o r y substances present i n the tissue. There have been no large-scale PCR indexing studies involving i l a r v i r u s e s to date. E f f o r t s are now being made to s i m p l i f y and modify the basic PCR procedure, e s p e c i a l l y i n tissue preparation and detection, so that the technique can be used to index large numbers of plants as r a p i d l y and inexpensively as possible without s a c r i f i c i n g s e n s i t i v i t y (eg. Korschineck et al. 1991). 1.3 Genetics of prune dwarf virus PDV i s a member of the Bromoviridae (Rybicki 1995). This genus includes four genera of plant viruses: the bromoviruses, i l a r v i r u s e s , cucumoviruses and alfamoviruses. A l l members are t r i p a r t i t e with icosahedral or quasi-isometric p a r t i c l e s . Their genomic RNAs are positive-sense, single-stranded and each RNA i s encapsidated separately. The genomic RNAs are 3.2-3.6 kb (RNA1), 2.6-3.0 kb (RNA2), 2.1-Page 7 2.2 kb (RNA3) with a 0.8-1.0 kb subgenomic RNA. The genome organization of the bromovirus, cucumovirus and alfamovirus genera i s well established since several members have been sequenced completely (eg. AMV: Cornelissen et a l . 1983a; 1983i>; Barker et al. 1983; Brederode et al. 1980). The genome organization of a l l Bromoviridae studied to date i s i d e n t i c a l with analogous genes encoded by analogous segments. RNA1 and RNA2 are monocistronic, encoding proteins thought to be involved i n r e p l i c a t i o n since RNA1 and RNA2 can re p l i c a t e independently of RNA3 i n protoplasts (Kiberstis et al. 1981; Nassuth et a l . 1981). RNA3 i s b i c i s t r o n i c encoding the movement and coat proteins. The l a t t e r i s translated from the subgenomic RNA. These proteins have been produced i n c e l l - f r e e t r a n s l a t i o n systems (Dougherty & Hiebert 1985); the movement protein (ORF3a) i s required for systemic i n f e c t i o n of AMV i n tobacco leaves (Huisman et a l . 1986). The RNA species of a l l known Bromoviridae have a 5'-m7G5'ppp5'Gp cap but no 3'-poly (A) t a i l . In spite of these s i m i l a r i t i e s at the genome l e v e l , there are important differences i n structure, serology and mode of transmission amongst the genera of the Bromoviridae. TSV i s the type member of the i l a r v i r u s genus and the f i r s t member for which sequence data was reported. Its RNA3 has been sequenced completely (Cornelissen et a l . 1984), and the 180 3'- and 140 3'- nucleotides of RNA1 and RNA2 respectively, have also been sequenced (Koper-Zwarthoff & Bol Page 8 1980). The genome organization of TSV has been i n f e r r e d from c e l l - f r e e t r a n s l a t i o n assays and i s t y p i c a l of the Bromoviridae organization as outlined above: RNA1 and RNA2 encode 120 kDa and 100 kDa proteins, respectively. These RNAs exhibit sequence homology with RNA replicases from other plant and animal viruses as well as phage (Kamer & Argos 1984). RNA3 of TSV i s b i c i s t r o n i c , encoding a 34 kDa movement protein and a 24 kDa coat protein translated from a subgenomic RNA4 (van Tol & van Vloten-Doting 1979). The following i l a r v i r u s sequences are also known: ApMV RNA3 (Al r e f a i et al. 1994; Sanchez-Navarro & Pallas 1994), PNRSV RNA3 (Guo et al. 1995; Hammond & Cr o s s l i n 1995), PDV RNA3 (Bachman et a l . 1994), c i t r u s leaf rugose virus (CiLRV) RNA3 and c i t r u s variegation virus RNA3 (Scott & Ge 1995b), l i l a c r i n g mottle RNA3 (Scott & Ge 1995a) CiLRV RNA2 (Ge & Scott 1994) and CiLRV RNA1 (Scott & Ge 1995c). A l l of these sequences are consistent with the Bromoviridae genome organization t y p i f i e d by AMV. There are at least two known exceptions to t h i s organization: Di T e r l i z z i et a l . (1992) reported an extra subgenomic RNA of unknown o r i g i n associated with PNRSV and Ding et a l . (1994) showed that an extra subgenomic RNA i n cucumber mosaic cucumovirus (CMV) preparations i s derived from RNA2 and contains an ORF of 100 codons. The function of the potential gene product, ORF 2b, i s not known and i t i s not known whether either of these RNAs are translated in vivo. Page 9 The i l a r v i r u s e s and AMV require a l l three genomic RNAs plus eit h e r a copy of the coat protein or the subgenomic RNA4 to i n i t i a t e i n f e c t i o n . The coat protein has been shown to bind s t r u c t u r a l hairpins at the 3'-ends of the RNAs (Houwing & Jaspars 1982) and i t i s thought that the coat protein of AMV i s located i n the replicase complex. The 3'-untranslated region (UTR) of a l l known i l a r v i r u s sequences (and of AMV) shares these s t r u c t u r a l hairpin motifs which are flanked by AUGC. Coat protein a c t i v a t i o n i s not s p e c i f i c and the coat protein of TSV can provide some early functions for the r e p l i c a t i o n of AMV even though the two share l i t t l e sequence homology (Reusken et al. 1995). BMV and CMV do not require coat protein to i n i t i a t e i n f e c t i o n . Species of the bromo- and cucumovirus group have also been sequenced and t h e i r genome organization i s the same as that of AMV. However, the 3'-UTRs f o l d into a tRNA-like structure and can be charged with tyrosine in vitro (Hall et a l . 1972). The function of t h i s aminoacylation i s not known but there are some theories. The 3'-end of these v i r a l RNAs has other tRNA-like functions: when aminoacylated they are able to interact with GTP and a host elongation factor, and i f supplied with ATP can repair incomplete or broken 3' v i r a l CCA termini (the s i t e of aminoacylation of host tRNA). Analogy to tRNA i s str u c t u r a l only and there i s almost no sequence homology with tRNA (Perret et al. 1989). Deletion analysis of the 3'-region of BMV RNA and subsequent Page 10 r e p l i c a t i o n in vitro suggests that the tRNA-like structure has a promoter function, but th i s in vitro r e p l i c a t i o n system has not been perfected and these findings are tentative (Dreher & Ha l l 1988). It i s not known whether BMV RNA i s aminoacylated in vivo; r e p l i c a t i o n of BMV RNA proceeds i n the absence of free tyrosine in vitro. F i n a l l y i t has been suggested that aminoacylation of the 3'-end of v i r a l RNA i s a strategy to protect against exonuclease degradation. 1.4 Defective I n t e r f e r i n g RNA Defective i n t e r f e r i n g (DI) p a r t i c l e s are RNA species derived from the supporting virus and can not r e p l i c a t e i n i t s absence. DI RNAs may be either deletion-type, mosaic-type or snapback-type (Schlesinger 1988). Deletion DIs generally contain the 5'- and 3'- termini of the supporting virus genome which are essential for r e p l i c a t i o n , but have deletions i n the open reading frame, which y i e l d s no functional product. Mosaic DIs share sequence with non-contiguous portions of the supporting virus genome and have the 5'- and 3'- termini of the supporting virus genome. There i s no functional gene product. The snapback type DIs are long palindromes which can form either hairpins or panhandles and do not encode a functional gene product. Both termini are derived from one terminus of the supporting virus genome. DI p a r t i c l e s are believed to arise by the copy-choice mechanism of the RNA-dependent RNA polymerase (RdRp) Page 11 i n a scheme proposed by Lazzarini et al. (1981). Although DI p a r t i c l e s are quite common i n animal viruses, they r a r e l y a r i s e from plant viruses (eg. Finnen & Rochon 1993; Hillman et al. 1987) and there are only two reports of a natural DI p a r t i c l e i n Bromoviridae (Romero et al. 1993; Marsh et al. 1991). However, a r t i f i c i a l DI p a r t i c l e s from two Bromoviridae, BMV and beet necrotic yellow vein cucumovirus and from cymbidium ringspot tombusvirus r e p l i c a t e in vivo and attenuate disease symptoms (Hehn et al. 1994; Marsh et al. 1991; Kollar et al. 1993). These DI p a r t i c l e s are of the deletion-type. Attenuation of disease symptoms and reduction i n the t i t r e of the supporting virus are frequently caused by DI p a r t i c l e s although there are rare cases where symptoms are exacerbated by DI p a r t i c l e s (Romero et al. 1993) . Because of t h i s potential symptom attenuation, because they are unable to r e p l i c a t e i n healthy trees i n the absence of helper virus and because they can be synthesized in vitro from cDNA clones, DI p a r t i c l e s have been investigated for c o n t r o l l i n g plant diseases (Hull & Davies 1991). 1.5 The nucleotide sequence of PDV RNA1 The complete nucleotide sequences of several members of the Bromoviridae have been determined (section 1.3). Among the i l a r v i r u s e s , only CiLRV has been sequenced completely (Scott & Ge 1995b; 1995c; Ge & Scott 1994), and the sequence Page 12 of AMV i s also known (see section 1.3). Since a l l RNA viruses (except retroviruses), i r r e s p e c t i v e of t h e i r host, must encode an RdRp and since t h i s gene i s highly conserved, i t i s used extensively i n phylogenetic analysis among unrelated viruses (Kamer & Argos 1984). The RNA1 segment of the Bromoviridae encodes part of the v i r a l replicase complex, and t h i s RNA, along with RNA2 (which also encodes a replicase protein), should provide useful information to es t a b l i s h the rel a t i o n s h i p of PDV with other Bromoviridae and with other i l a r - and i l a r - l i k e viruses such as AMV, o l i v e latent virus 2 (0LV2, an alfamo-like virus, Grieco et al. 1990) and RBDV. Phylogenetic analyses of the i l a r v i r u s e s and studies of th e i r genetic relationships to other Bromoviridae have been made possible as the RNA3 sequences of eight i l a r v i r u s e s have recently been determined (section 1.3). The i l a r v i r u s e s have been divided into 10 sub-groups based on se r o l o g i c a l assays. These groupings are si m i l a r to phylogenetic groupings observed i n some studies using RNA3 sequence, where a close genetic r e l a t i o n s h i p has been observed between PNRSV and ApMV (subgroup I I I : Guo et al. 1995) and between CiLRV and c i t r u s variegation virus (subgroup II: Scott & Ge 1995b). However, not a l l i l a r v i r u s RNA3 segments have been sequenced and the phylogenetic analyses presented i n these studies are not complete and may be inaccurate. Sequences of i l a r v i r u s RNA1 and RNA2 segments are even scarcer and are also required to form a complete phylogeny of the i l a r v i r u s e s . Closely Page 13 related viruses such as AMV and 0LV2, w i l l have to be included i n a f i n a l phylogenetic analysis. 1.6 Objectives of t h i s study In order to control a plant disease a r e l i a b l e detection method for the causal agent i s e s s e n t i a l . The f i r s t objective was to produce a TAS-ELISA assay based on a monoclonal antibody and to es t a b l i s h a routine diagnostic procedure, determining the r e l i a b i l i t y and accuracy of the ser o l o g i c a l assay to detect PDV i n infected material. At the beginning of t h i s study, there was no sequence data available for PDV and thus there were no means of developing an RT-PCR assay for the detection of PDV. The second objective was to obtain sequence data from RNA3 of PDV to produce a r e l i a b l e RT-PCR test. The RT-PCR would be used to evaluate the TAS-ELISA r e s u l t s . The t h i r d objective was to obtain the complete sequence of RNA1 of PDV. Analogy to other members of t h i s family suggest that RNA1 would encode a putative replicase enzyme. This sequence information would be used to construct a r t i f i c i a l DI- p a r t i c l e s to be used as poten t i a l biocontrol agents for PDV. The sequence would also be used to investigate phylogenetic relationships among the Bromoviridae, e s p e c i a l l y between PDV, CiLRV and AMV. Page 14 2.0 MATERIALS AND METHODS 2.1.0 P u r i f i c a t i o n procedures 2.1.1 Virus o r i g i n A PDV i s o l a t e was o r i g i n a l l y detected i n P. avium cv. Salmo i n 1971 at the Summerland Research Centre. Its i d e n t i t y has been confirmed by ELISA i n the laboratory of Dr. G. Mink (WSU-IAREC Prosser, WA) and by indexing on herbaceous hosts (Cucurbita spp. and C. quinoa) and on the woody i n i c a t o r P. serrulata cv. Shirofugen. The virus was transferred to P. mahaleb where i t i s maintained. This tree has been shown to be free of PNRSV by ELISA (Eastwell, unpublished r e s u l t s ) . PDV was transmitted to pumpkin, C. maxima cv. Buttercup, i n the spring of 1991 by grinding young a p i c a l leaves i n i c e - c o l d 0.05 M phosphate buffer, pH 7 with 1% nicotine and rubbing sap onto pumpkin cotyledons dusted with carborundum powder. Male flowers of t h i s pumpkin plant were used as inoculum for further pumpkin plants. Pollen was also collected, allowed to a i r dry for about 24 hours and stored at -70°C. This frozen pollen was used to i n i t i a t e new infections of pumpkin plants as required. 2.1.2 Virus p u r i f i c a t i o n Approximately 60 pumpkin seedlings were inoculated with PDV from the flowers of an infected pumpkin. After 10-14 days virus was i s o l a t e d from these plants by a modified Page 15 procedure of Fulton (1959). Leaves were homogenized i n 1.2 ml/g tissue i c e - c o l d 30 mM sodium phosphate; 10 mM EDTA, pH 8.0 (PDV buffer) containing 0.14% /3-mercaptoethanol {(3ME) and 7% (w/v) alumina powder, i n a c h i l l e d Waring blender. This homogenate was c l a r i f i e d by centrifugation for 10 minutes at 6000 rpm i n a Beckman JA 14 rotor at 4°C. The supernatant was returned to the blender and homogenized b r i e f l y with c h i l l e d hydrated calcium phosphate (approximately 5 g). This was centrifuged at 8000 rpm for 10 minutes i n a Beckman JA 14 rotor at 4°C. The supernatant was f i l t e r e d through miracloth and centrifuged at 42000 rpm for 2.5 hours i n a Beckman 45Ti rotor at 4°C. P e l l e t s were resuspended i n 200 fil c h i l l e d PDV buffer, pooled and layered onto a 25% (w/v) sucrose cushion prepared i n PDV buffer. This was centrifuged for 2 hours at 50000 rpm i n a Beckman 70Ti rotor at 4°G. The p e l l e t s were resuspended and pooled as before and layered onto a 10% to 40% (w/v) l i n e a r sucrose gradient i n PDV buffer and centrifuged for 2.5 hours at 38000 rpm i n a Beckman SW40Ti rotor at 4°C. The gradient was scanned at 340 nm with an ISCO UA-5 scanner and a large, c e n t r a l l y located band was collected. These fracti o n s were pooled and virus c o l l e c t e d by centrifugation for 1.5 hours at 50000 rpm i n a Beckman 70.1Ti rotor at 4°C. The f i n a l p e l l e t was resuspended i n a minimal volume of PDV buffer ( t y p i c a l l y 100 ill) and stored at 4°C. The amount of virus contained i n these fract i o n s was determined by spectrophotometry assuming Page 16 an e x t i n c t i o n c o e f f i c i e n t of 5.0 for 1 cm of a 0.1% solution at 260 nm (Halk & Fulton 1978). 2.1.3 Coat protein electrophoresis The protein content of the f i n a l v i r u s f r a c t i o n was determined by a Bradford dye-binding assay (BioRad: Bradford 1976) with bovine serum albumin (BSA f r a c t i o n V: Sigma Chemical Co) used as standard. The protein content of the virus samples was adjusted to approximately 5 mg/ml and i t was mixed with 4 volumes of loading buffer (62.5 mM Tris-HCl pH 6.8; 10% (v/v) g l y c e r o l ; 2% (w/v) sodium dodecyl sulphate (SDS); 5% (v/v) /8ME; 0.001% (w/v) bromophenol blue). The sample was heated to 95°C for 2 minutes p r i o r to loading. For electrophoresis the method described by Laemmli (1970) using a discontinuous buffer system was used with a BioRad Mini-protean II unit. Samples were electrophoresed on a 12% polyacrylamide gel ov e r l a i d with a 4% stacking gel, at 200 V for 40 minutes i n 0.1% SDS; 25 mM T r i s ; 200 mM glycine; pH 8.3. The following proteins were used as molecular weight markers (Dalton VII set: Sigma Chemical Co.): BSA (66.0 kDa); ovalbumin (45.0 kDa); glucose-3-phosphate dehydrogenase (36.0 kDa); trypsinogen (24.0 kDa); t r y p s i n i n h i b i t o r (20.1 kDa); ot-lactalbumin (14.2 kDa). The protein bands were v i s u a l i z e d by staining the gel with Coomassie b r i l l i a n t blue R-250 or s i l v e r n i t r a t e (Merril 1990). Gels were dried under vacuum and photographed. Page 17 2.1.4 Genomic RNA i s o l a t i o n Genomic PDV RNA was is o l a t e d from p a r t i a l l y p u r i f i e d v i r u s . Pelleted material obtained a f t e r the f i r s t u l t r a c e n t r i f u g a t i o n step (section 2.1.2) was resuspended i n 100 /xl PDV buffer and extracted twice with an equal volume of water saturated phenol heated to 8 0°C. Two extractions using phenol:chloroform:isoamyl alcohol (25:24:1) and a f i n a l extraction with chloroform:isoamyl alcohol (24:1) followed. RNA was pr e c i p i t a t e d from the f i n a l aqueous f r a c t i o n by adding 0.1 volume 3.1 M sodium acetate, pH 5.2, and 3 volumes of i c e - c o l d 95% ethanol. The tube was l e f t at -70°C for at least 1 hour, then centrifuged at 13000 rpm for 45 minutes at 4°C i n a microcentrifuge. The p e l l e t was washed with 70% ethanol, dried under vacuum and dissolved i n 50 fil d i e t h y l pyrocarbonate-treated water. The sample was immediately divided into 5 u.1 portions which were stored at -70°C. One aliquot was used to measure the RNA content by spectro-photometry and to examine i t s q u a l i t y by electrophoresis i n a 1% agarose gel containing 5 mM methylmercuric hydroxide (MeHgOH) and borate buffer (4 0 mM sodium borate; 1 mM EDTA; pH 8.2), as described by Sambrook et a l . (1989). RNA samples were denatured i n 15 mM MeHgOH for 5 minutes at room temperature p r i o r to loading. After electrophoresis the MeHgOH was inactivated with 0.1 M ammonium acetate and the RNA was v i s u a l i z e d by staining with ethidium bromide. Single stranded RNA molecular size standards (Sigma) were co-Page 18 electrophoresed to estimate the sizes of the genomic RNA bands and the gel was photographed to allow measurement of band migration. 2.1.5 Extraction of double-stranded ( r e p l i c a t i v e form) RNA from infected leaves Young leaves were c o l l e c t e d from the P. mahaleb stock tree as soon as they became available i n the spring and stored i n batches of 7 g at -70°C where t h e i r dsRNA was stable for at least a year. To extract dsRNA, 7 g of leaves were ground to a powder i n l i q u i d nitrogen with a pestle and mortar and allowed to thaw i n a 50 ml tube containing 9 ml water saturated phenol, 9 ml GPS buffer (0.2 M glycine; 0.1 M sodium phosphate buffer, pH 9.5; 0.6 M NaCI) and 0.5 ml jSME. On thawing, 0.5 ml 20% (w/v) SDS was added and the tube placed on a rotary shaker for about 45 minutes. The samples were then centrifuged for 5 minutes i n a bench-top centrifuge at 3 000 rpm. The aqueous phase was transferred to a fresh tube on ice and the organic phase was re-extracted with 5 ml GPS buffer. The two aqueous phases were pooled and one t h i r d volume of 10 M L i C l , c h i l l e d to -20°C, was added dropwise whilst vortexing to ensure rapid mixing. A p r e c i p i t a t e was allowed to form overnight at 4°C. The sample was centrifuged i n a bench-top centrifuge for 10 minutes at f u l l speed (3000 rpm). The supernatant was aspirated into a fresh tube and 95% ethanol was added to a Page 19 f i n a l concentration of 18% along with 1.5 g c e l l u l o s e powder (CC41: Whatman). The dsRNA was allowed to bind to the c e l l u l o s e by shaking the tubes on ice for 3 0 minutes on a rotary shaker. The c e l l u l o s e was washed 3 times with STE (100 mM NaCl; 10 mM Tris-HCl; 1 mM EDTA, pH 8.0) containing 18% ethanol and samples were loaded into 20 x 1 cm chromatography columns and each colum washed with 3 00 ml STE containing 18% ethanol at a flow rate of approximately 2 ml/min. After the columns had drained completely, they were purged dry by forcing a i r from a syringe through the column. DsRNA was eluted from the c e l l u l o s e with 3 ml 0.5 mM EDTA, pH 8.0, followed by forced a i r to dry the column. Two more 2 ml portions were passed through the column and the eluants pooled i n a 30 ml Corex tube containing 700 /xl 3 M sodium acetate pH 5.2. Three volumes of ice cold 95% ethanol were added to the tubes and they were placed at -70°C overnight. Samples were centrifuged at 11000 rpm i n a Beckman JS 13.1 rotor at -5°C for 45 minutes to p e l l e t the dsRNA. The supernate was discarded and the p e l l e t dried under vacuum and redissolved i n 200 /xl DNase buffer (0.1 M sodium acetate; 5 mM MgCl2, pH 5.0), transferred to an eppendorf tube and incubated with 10 U deoxyribonuclease I (DNase I: Gibco/BRL) at 37°C for 20 minutes. The samples were centrifuged b r i e f l y to remove fin e c e l l u l o s e p a r t i c l e s c a r r i e d over from the chromatography before adding 20 til 3 M sodium acetate, pH 5.2 and 3 volumes i c e - c o l d 95% ethanol. DsRNA was p r e c i p i t a t e d Page 20 for several hours at -70°C or for 2 0 minutes i n an isopropanol:dry ice bath. The pr e c i p i t a t e was c o l l e c t e d by centrifugation at 13 000 rpm i n a microcentrifuge for 45 minutes at 4°C. The supernate was removed and the p e l l e t washed with 100 /xl 70% ethanol, dried under vacuum and redissolved i n 20 pi TE (10 mM Tris-HCl; 1 mM EDTA, pH 8.0). A 4 fil aliquot was analyzed by agarose gel electrophoresis. DsRNA i s o l a t e d from Nicotiana glauca infected with CMV was used as a molecular weight marker. To remove single stranded RNA (tRNA and rRNA), samples were eithe r digested with 1 unit RNase T - L (Pharmacia) i n STE for 10 minutes at room temperature p r i o r to electrophoresis, or the gel was placed i n a solution of 2X SSC containing 50 /xg/ml RNase A (Pharmacia) for 1 hour at room temperature with shaking a f t e r electrophoresis. 2 . 2 . 0 Antibody production 2 . 2 . 1 Production of polyclonal antibodies i n chickens F i f t e e n week old laying hens (Red Sussex) were purchased from Rump & Sendall, Vernon, BC. When the chicken to be immunized was laying r e l i a b l y , i e . laying at least 5 eggs/ week for 2 weeks, pre-immune eggs were c o l l e c t e d and the chicken was given an intramuscular i n j e c t i o n of approximately 1 mg p u r i f i e d PDV i n Freund's complete adjuvant. After ten days, eggs were c o l l e c t e d i n groups of si x . Antibody (IgY) was i s o l a t e d from them using the following procedure (van Page 21 Regenmortel 1982). Yolks were separated from whites and washed i n a beaker with d i s t i l l e d water. Their volume was measured and 3 volumes of saline buffer (5 mM sodium phosphate, pH 7.2, 0.1 M NaCI) were mixed with the yolks. The yolks were broken and the solution was brought to 3.5% (w/v) ground polyethylene g l y c o l (PEG 6000: Sigma) and s t i r r e d on ice for 30 minutes. The sample was centrifuged i n a Beckman JA 14 rotor at 10000 rpm for 10 minutes at 4°C. The supernatant was f i l t e r e d through moistened cotton wool, brought to 12% (w/v) with PEG 6000 and s t i r r e d at 4°C for 3 0 minutes. The centrifugation was repeated and the supernatant discarded. The p e l l e t was drained well and dissolved i n saline buffer overnight. The solution was c l a r i f i e d by centrifugation as above and antibodies reprecipitated with 12% PEG as before. The f i n a l p e l l e t was dissolved i n a minimal volume of saline buffer containing 0.2% sodium azide and stored at 4°C. This sample was analyzed by SDS polyacrylamide gel electrophoresis (PAGE) (section 2.1.3) and p u r i f i e d further by hydroxyl-apatite (HAP: Boehringer Mannheim) chromatography (cf. section 2.2.5) for use i n ELISA. A 10 ml HAP column was e q u i l i b r a t e d with 10 mM phosphate buffer, pH 6.8. The IgY sample was d i l u t e d ten-fold with running buffer, loaded onto the column, and eluted with a 100 to 500 mM phosphate gradient at pH 6.8 at a rate of approximately 2 ml/min. The eluant was monitored at 280 nm and 0.5 ml f r a c t i o n s were Page 22 c o l l e c t e d . Peaks were pooled and IgY p r e c i p i t a t e d by adding sodium sulphate to 14% (w/v) from a saturated solution (36% (w/v). A f t e r 1 hour at room temperature the IgY were pe l l e t e d by centrifugation at 10000 rpm, 10 minutes i n a Beckman JS 13.1 rotor. The highly soluble p e l l e t was dissolved a minimal volume of saline buffer containing 0.02% (w/v) sodium azide and stored at 4°C. 2.2.2.0 Production of monoclonal antibodies 2.2.2.1 Immunization a f t e r cyclophosphamide treatment Healthy pumpkin extract was prepared by harvesting 130 g healthy pumpkin plants and proceeding with the PDV i s o l a t i o n as d e t a i l e d i n section 2.1.2 u n t i l a f t e r the f i r s t u l t r a -centrifugation step. At t h i s stage, the p e l l e t s were redissolved i n phosphate buffered saline (PBS: Harlow & Lane 1988), c l a r i f i e d by low speed centrifugation and stored at 4°C. The protein concentration of the supernate was determined by the Bradford assay. Cyclophosphamide (Sigma) was dissolved i n PBS (1 g/ 30 ml) and d i l u t e d to 16.5 mg/ml shortly before use. The healthy pumpkin extract was d i l u t e d with PBS to 2.5 mg protein/ml and injected i n t r a p e r i t o n e a l l y into two female Balb/c mice without adjuvant. This was followed by an intraperitoneal i n j e c t i o n of 100 mg cyclophosphamide/kg mouse aft e r 10 minutes, 24 hours and 48 hours (Matthew & Sandrock 1987). After the drug had been allowed to cle a r for 2 weeks, Page 23 the mice were given an intraperitoneal i n j e c t i o n of PDV (250 ag) i n Freund's complete adjuvant. This i n j e c t i o n scheme was repeated a f t e r a further 2 weeks, but using Freund's incomplete adjuvant. A f i n a l intraperitoneal boost of 250 pig PDV was given without adjuvant 3 days before the fusion. 2.2.2.2 Determination of the immune response by TAS-ELISA T a i l bleeds were ca r r i e d out a f t e r the f i n a l boost to determine the immune response of the mice. Approximately 100 / i l of blood was c o l l e c t e d into heparinized c a p i l l a r y tubes (Oxford Labware). Blood c e l l s were p e l l e t e d by centrifugation for 5 minutes at 800 g and the clear supernatant serum was c a r e f u l l y removed and stored at 4°C. The serum was tested by TAS-ELISA using four wells for each mouse. M i c r o t i t r e plate wells (EIA poly-styrene: Nunc) were coated with rabbit serum prepared against PDV s t r a i n 876 (PVAS 290: American Type Culture Collection) d i l u t e d 1:2000 i n PBS and incubated at room temperature for 2 hours. Wells were washed three times with PBS a f t e r t h i s and a l l subsequent steps. Blocking was achieved with 3% BSA, 0.05% Tween 20 i n PBS for 1 hour at room temperature. P u r i f i e d PDV at a concentration of 300 ng/ml i n PBS with 0.5% BSA was applied to the wells to act as antigen and pumpkin cotyledons ground i n PBS served as negative controls. Plates were incubated at 4°C overnight with the antigen. Serum from the Page 24 test-mice was di l u t e d 1:250 to 1:3000 i n 0.5% BSA i n PBS, applied to the wells and incubated for 2 hours at room temperature. Alkaline phosphatase l a b e l l e d goat-anti-mouse antiserum (Gibco/BRL) was added at the recommended concentration (1:3000) i n 0.5% BSA i n PBS and incubated for 2 hours at room temperature. Detection was achieved by the addition of 0.1% (w/v) para-nitrophenol phosphate i n 10% (w/v) diethanolamine, pH 9.5. The micr o t i t r e plate was read i n a micr o t i t e r plate reader (Titertek MCC/340: ICN) at 405 and 620 nm af t e r 2 and 24 hour incubations under subdued l i g h t . A S 2 0 readings were subtracted from A 4 0 5 readings. 2.2.2.3 Fusion mediated by polyethylene g l y c o l A l l work described i n t h i s section was done i n a b i o l o g i c a l containment hood using s t e r i l e technique. Twenty 96-well thymocyte feeder plates were prepared the day before the fusion. Ten 6 week old Balb/c mice were s a c r i f i c e d to provide thymocytes. Thymuses were removed a s e p t i c a l l y from the mice and pooled together i n a p e t r i - d i s h containing 2 0 ml prewarmed DMEM (Dulbecco's modified Eagle medium: Sigma). C e l l s were released by overlaying the organs with several layers of s t e r i l e gauze and crushing them gently with a s t e r i l e syringe plunger. Thymocytes were transferred to a centrifuge tube with a pipette and pe l l e t e d by centrifugation at 800 g for 2 minutes. The p e l l e t was washed once with DMEM and resuspended i n 100 ml DMEM containing 20% fo e t a l c a l f Page 25 serum (FCS: Gibco/BRL) and 50 /xg/ml gentamycin (Sigma). Thymocytes were plated into twenty 96-well plates, 50 jxl per well, and placed into a 37°C incubator with a 10% C02 atmosphere. NS1 myeloma c e l l s were also prepared at least 2 days before the fusion. These had been stored i n l i q u i d nitrogen and were thawed by immersing the storage-cryovial i n tepid water u n t i l the c e l l s had just thawed. C e l l s were immediately d i l u t e d i n 10 ml DMEM, pel l e t e d at 800 g, washed again and resuspended i n 30 ml DMEM containing 10% FCS and 50 /xg/ml gentamycin. C e l l s were seeded into a 15 cm p e t r i dish and placed i n the C02 incubator overnight. The next day, the NS1 c e l l s were resuspended and half (15 ml) transferred to a new p e t r i dish. A further 15 ml DMEM was added to each dish and the c e l l s were allowed to grow to 80% confluency. The fusion was performed e s s e n t i a l l y as described by Harlow & Lane (1988). The mouse with the higher s p e c i f i c immune response, based on the t a i l - b l e e d TAS-ELISA, was s a c r i f i c e d and i t s spleen removed under aseptic conditions. Splenocytes were extracted using the same procedure described for thymocytes. The c e l l s were pe l l e t e d at 800 g i n a c l i n i c a l centrifuge and washed once with DMEM. Special care was taken to remove any blood c l o t s and connective tissue present. The NS1 c e l l s from two p e t r i dishes estimated to be 80% confluent were harvested and washed twice with DMEM Page 26 taking care to remove as much media as possible. The two c e l l types, the splenocytes and the NS1 myelomas, were then mixed i n DMEM and pe l l e t e d together. Care was taken to remove as much of the super-natant as possible. The p e l l e t was resuspended by adding 0.9 ml of a solution of PEG 3000 (Sigma) previously mixed 1:1 with DMEM and warmed to 37°C, over a period of 1 minute with gentle s t i r r i n g . The c e l l s were s t i r r e d for a further minute, followed by the addition of 1 ml DMEM over 1 minute and 9 ml DMEM over 2 minutes with continuous gentle s t i r r i n g . After t h i s , the c e l l s were immediately centrifuged for 5 minutes at 400 g and resuspended i n 10 ml DMEM containing 2 0% FCS and gentamycin. This was mixed with a further 100 ml of the same medium and plated out onto the feeder plates at 50 /xl per well. After approximately 10 hours at 37°C i n the C02 incubator, 25 /xl of 5X HAT sel e c t i o n medium was added to each well, (IX HAT i s 100 ixM hypoxanthine, 16 /xM thymidine, 0.4 /xM aminopterin i n DMEM with 20% FCS and 50 fig/ml gentamycin) . The HAT medium was replaced by HT medium (containing 100 ixM hypoxanthine and 16 /xM thymidine) a f t e r approximately 2 weeks. The hybridomas were l e f t undisturbed i n the C02 incubator for 1 week. After t h i s period, wells were inspected for hybridomas under an inverted microscope and a l l wells containing one or more hybridomas were marked. Wells were screened for anti-PDV antibodies by TAS-ELISA at th i s stage. The assay was the same as described for t a i l bleeds Page 27 (section 2.2.2.2) except that 50 /xl tissue culture supernatant (TCS) was removed a s e p t i c a l l y from each well to be assayed for use i n the mouse-anti-PDV antibody step i n the ELISA. Each well was assayed using PDV-infected and healthy cucumber cotyledons as antigen. After removal of the TCS from the tissue culture plates, i t was immediately replaced with fresh DMEM containing 20% FCS, gentamycin and HAT. Hybridomas i n wells giving p o s i t i v e values by TAS-ELISA against PDV-infected material were transferred to 24-well plates and were single c e l l cloned by l i m i t i n g d i l u t i o n (Harlow & Lane 1988). 2.2.3 Single c e l l cloning Ninety-six well microtitre plates with thymocyte or spleenocytes feeder c e l l s were prepared for single c e l l cloning as described i n section 2.2.2.3. Each plate could accommodate two c e l l l i n e s for single c e l l cloning. Hybridomas to be single c e l l cloned were pipetted into well Al (or El) of a plate. S e r i a l d i l u t i o n s (1:1) were made f i r s t down the column and then across the rows of the plate. The plates were returned to the incubator and l e f t u n t i l hybridoma colonies became v i s i b l e . The numbers of hybridomas per well was marked on the l i d s of the plates. At t h i s point plates were screened by TAS-ELISA as described above and one p o s i t i v e well per plate containing a single hybridoma was subjected to another round of single c e l l cloning. If there Page 28 were no wells with single, p o s i t i v e hybridomas on a plate, the p o s i t i v e well with the fewest hybridomas was single c e l l cloned, u n t i l two rounds had been completed successfully. Clonal hybridomas were grown to high density i n 24-well, then 12-well plates and f i n a l l y i n 50 ml tissue culture f l a s k s . At t h i s stage c e l l s were also frozen i n l i q u i d nitrogen for safe, long-term storage (section 2.2.4). A monoclonal antibody c a l l e d PDA-3C was i d e n t i f i e d at t h i s stage. 2.2.4 Cryogenic storage of hybridomas Hybridomas to be frozen were grown u n t i l 8 0% confluent i n 15-20 ml DMEM supplemented with 20% FCS and 50 ptg/ml gentamycin. C e l l s were harvested by flushing them out of the T-flask with a 25 ml pipette being sure to wash them off the bottom of the fla s k . They were transferred to a centrifuge tube, p e l l e t e d at 800 g for 5 minutes, resuspended i n 1 ml of p r e c h i l l e d 46% DMEM, 46% FCS, 8% dimethyl sulphoxide and transferred to p r e c h i l l e d cryovials (Nunc). These were placed into an insulated box at -70°C to allow them to cool slowly (approximately -l°C/min) overnight. Afte r t h i s they were sealed with cryoflex tubing (Nunc) and transferred to l i q u i d nitrogen for storage. 2.2.5 I s o l a t i o n of antibody from tissue culture supernatant and isotyping Hybridomas were grown i n 15 ml culture flasks u n t i l 80% Page 2 9 confluent. At t h i s stage they were resuspended with a 25 ml pipette and approximately 80% of the volume transferred to a centrifuge tube. The remaining hybridomas were returned to the f l a s k and the media replaced. C e l l s were removed from the c o l l e c t e d media by centrifugation and the supernatant stored at 4°C u n t i l required. The flask was harvested u n t i l about 150 ml of TCS had been coll e c t e d . The monoclonal antibody (PDA-3C) was i s o l a t e d from the TCS by HAP chromatography (Harlow & Lane 1988). A 10 ml HAP column was eq u i l i b r a t e d with 10 mM sodium phosphate, pH 6.8 at a flow rate of 2.5 ml/min. The TCS was loaded i n 3 batches of 50 ml onto a single column and eluted u n t i l a peak, monitored at 2 80 nm, had been removed. Bound antibody was removed from the HAP by e l u t i o n with 200 mM sodium phosphate, pH 6.8. A single peak was c o l l e c t e d . A l l antibody containing fractions were pooled, c h i l l e d to 4°C and the antibody p r e c i p i t a t e d by dropwise addition of an equal volume of c h i l l e d , saturated ammonium sulphate, pH 7. The sample was p r e c i p i t a t e d at 4°C for 1 hour and centrifuged i n a Beckman JA 14 rotor at 10000 rpm for 10 minutes, 4°C. The p e l l e t was redissolved i n a minimal volume (approximately 1 ml) 0.02% sodium azide i n PBS and stored at 4°C. This f r a c t i o n was used for a l l se r o l o g i c a l assays requiring a monoclonal antibody. The column was regenerated by washing with 1 M NaCI and re-e q u i l i b r a t i n g with 10 mM sodium phosphate, pH 6.8. The isotype of PDA-3C was determined using an Page 3 0 erythrocyte agglutination assay k i t (Serotec). One column (8 wells) of a 96-well plate with U-bottom wells was f i l l e d with 30 /xl of the antiserum to be tested d i l u t e d 1:50 with PBS. This was mixed with an equal volume of each s p e c i f i c isotyping reagent to be tested (anti-IgG-,; -IgG 2 a; -IgG 2 b; -IgG3; -IgA; -IgM,.all linked to sheep erythrocytes,) as well as p o s i t i v e and negative control wells. The plates were tapped gently to mix and l e f t covered on a f l a t , stable surface for 1 hour. A small red button at the bottom of the well was considered negative; a p a r t i a l or f u l l carpet of agglutination was considered p o s i t i v e . If a l l wells f a i l e d to agglutinate, the assay was repeated using higher concentrations of the te s t - s o l u t i o n . 2.2.6 Western b l o t A sample of PDV coat protein was electrophoresed as described i n section 2.1.3. After electrophoresis, the gel was soaked i n transfer buffer (50 mM Tris-HCl, pH 7.6; 380 mM glycine; 0.1% SDS; 20% methanol). N i t r o c e l l u l o s e (NC: Schleicher & Schuell) was cut to the same size as the gel, wetted i n d i s t i l l e d water and soaked b r i e f l y i n transfer buffer. The NC was overlaid onto the gel and sandwiched between three layers of Whatman 3MM paper, taking care to remove a l l a i r bubbles i n the sandwich. This was placed into an e l e c t r o b l o t transfer apparatus and the proteins were e l e c t r o p h o r e t i c a l l y transferred overnight to the NC at 4°C Page 31 with a constant voltage set to 60 V. After the transfer the gel was removed and stained with Coomassie b r i l l i a n t blue R-250 to v e r i f y transfer and the NC was immersed i n isopropanol for 1 minute before being allowed to dry completely. The NC was then rewetted i n d i s t i l l e d water, blocked i n 3% skim milk powder (Carnation) i n PBS with 0.05% Tween-20 for 20 minutes and transferred to a p l a s t i c containing 2 ml PDA-3C d i l u t e d 1:1000 with 0.5% skim milk powder i n PBS and l e f t at 3 7°C for 1 hour. The NC was then removed from the bag, washed i n three changes of PBS and incubated with al k a l i n e phosphatase-linked goat-anti-mouse antibody (Gibco/ BRL) d i l u t e d 1:1000 (section 2.2.2.2). After three further washes i n PBS, the NC was washed i n PBS, pH 9.5 and transferred to a solution of 0.033% nitro-blue tetrazolium, 0.017% 5-bromo-4-chloro-3-indolyl phosphate i n TBS, pH 9.5 with 5 mM MgCl 2. Colour development was allowed to proceed for at least 20 minutes i n subdued l i g h t . The reaction was terminated by tr a n s f e r r i n g the blot to a solution of 10 mM EDTA, pH 8.0. Results were photographed. 2.2.7 Conjugation of PDA-3C to al k a l i n e phosphatase A maleimide al k a l i n e phosphatase k i t (Pierce) was used to conjugate monoclonal PDA-3C to alk a l i n e phosphatase (AP). PDA-3C, 6.25 mg, was pre c i p i t a t e d with an equal volume of (NH4)2S04, redissolved i n 1 ml TBS and desalted over a beaded Page 3 2 polyacrylamide column (Pierce k i t ) . One-half ml fractions were c o l l e c t e d and t h e i r protein contents determined by the Bradford dye-binding assay. The two fractions with the highest protein contents were pooled to y i e l d 2 mg protein/ml. Six mg 2-mercapto ethylamine (MEA) was added and allowed to react at 3 7°C for 1 hour. Activated AP was prepared by adding 30 / i l 2.9% (w/v) [sulpho succinimidyl 4-(N-maleimidomethyl) cyclohexane-l-carboxylate]) to 5 mg AP. This was allowed to react for 3 0 minutes at room temperature and then desalted over a cross-linked dextran column (Pierce kit) e q u i l i b r a t e d with TBS supplemented with 0.15 M NaCl, 2 mM ZnCl 2, 4 mM MgCl2, pH 7.6. One-half ml fractio n s were co l l e c t e d and t h e i r protein contents determined as before. Mixing 0.5 mg MEA-reduced monoclonal with 2 mg activated AP in supplemented TBS buffer, i n i t i a t e d conjugation which proceeded at room temperature for 1 hour. The r e s u l t i n g conjugate, PDA-3C-AP was stored at 4°C. PDA-3C-AP was assayed by DAS-ELISA. M i c r o t i t e r plates (Linbro EIA) were coated for 2 hours at room temperature with PDA-3C i n PBS at d i l u t i o n s of 1:100 to 1:3000. Plates were blocked and antigen applied as described i n section 2.2.2.2. PDA-3C-AP was applied i n 0.5% BSA, PBS at d i l u t i o n s of 1:100 to 1:3000 and l e f t for hours at room temperature. Afte r three washes with TBS, substrate was applied and the plates read at 405 and 620 nm af t e r 2 hrs and 24 hrs. Page 33 2.2.8 Production of F(ab') 2 fragments from PDA-3C Approximately 1 mg hydroxylapatite-purified PDA-3C i n 225 /xl, was incubated with 2.5 /xl (0.5 /xg) pepsin (porcine mucosa: Sigma) i n 100 mM sodium c i t r a t e buffer, pH 3.5, overnight at 37°C (Harlow & Lane 1988) . The reaction was terminated by the addition of 0.1 volume 3 M Tris-HCl, pH 8.8. The sample was then centrifuged to p e l l e t denatured protein and the supernatant analyzed by SDS PAGE. Native samples, and samples reduced with (SME before loading, were loaded side-by-side on the gel. Electrophoresis was performed as described i n section 2.1.3 and the gels were stained with s i l v e r n i t r a t e . The F(ab') 2 fragments were p u r i f i e d further by diethylaminoethyl (DE-52: Whatman) ion exchange chromatography to remove F c fragments and intact antibodies. A 15 ml column was prepared and equilibr a t e d with 10 mM Tris-HCl, pH 8.5. The digested antibody solution was neutralized with 100 mM Tris-HCl, pH 8.5, d i l u t e d 10-fold with d i s t i l l e d water and loaded onto the column at 1 ml/min. The column was washed with 10 mM Tris-HCl, pH 8.5 and the eluant monitored at 280 nm. When the baseline had returned to zero, a 50 to 500 mM NaCI gradient i n column buffer was applied and a l l peaks collected. F(ab') 2 fragments were concentrated by ammonium sulphate p r e c i p i t a t i o n . The column was regenerated by washing sequentially at a flow rate of 1 ml/min with 3 0 ml each of i M NaCI; 0.5 N HCl; 0.5 N NaOH; then 100 ml 150 mM Tris-HCl, pH 8.5 and f i n a l l y 100 ml 10 mM Page 34 Tris-HCl, pH 8.5. The p u r i f i e d F(ab') 2were assayed for t h e i r a b i l i t y to trap PDV by TAS-ELISA by loading them onto a m i c r o t i t r e plate (1:10-1:1000) i n PBS and allowing them to bind for 2 hours at room temperature. Plates were blocked and antigen applied as described i n section 2.2.2.2. PDA-3C, AP-labelled sheep-anti-mouse (anti-Fc region: Kirkegaard Perry Labs) antibody, d i l u t e d as recommended by the manufacturer, and substrate were added as set out i n section 2.2.2.2 and the absorbances read at 405 and 620 nm a f t e r 2 and 24 hours. 2.2.9 Detection of PDV by TAS-ELISA, RT-PCR and bioassay i n sweet cherry To evaluate the monoclonal PDA-3C for routine f i e l d indexing by TAS-ELISA (triple-antibody sandwich ELISA), 40 sweet cherry trees showing symptoms suggesting a v i r a l i n f e c t i o n (eg. shot-holes, leaf discolouration) were selected for analysis. They were indexed by RT-PCR, TAS-ELISA and the 'Shirofugen' bioassay. The RT-PCR was performed as described i n section 2.3.15; the bioassay was done by T-budding two buds of each sample-tree onto a 'Shirofugen' indicator tree i n the spring. These were inspected a f t e r 4 and 6 weeks for the presence of gumming at the bud union which would indicate the presence of i l a r v i r u s . For the TAS-ELISA, s i x young r leaves were taken at random from each tree and assayed i n Page 3 5 duplicate on separate plates using the assay described i n section 2.2.2.2. In addition, 15 symptomless trees were selected to act as negative controls for the TAS-ELISA. These trees were also tested by RT-PCR and by the bioassay. Since the bioassay cannot d i s t i n g u i s h between PDV and PNRSV, a l l trees which tested p o s i t i v e on the bioassay were also assayed for PNRSV by TAS-ELISA using PVAS-22 (American Type Culture Collection) rabbit serum at a d i l u t i o n 1:500 to trap virus and monoclonal antibody UCB 1332 (a g i f t from D. Opgenorth) d i l u t e d 1:25, and AP-linked goat anti-mouse serum (Gibco/BRL) to detect PNRSV. Buffers and incubation times were the same as described for the PDV TAS-ELISA. The r e s u l t s of the RT-PCR and the bioassay on 'Shirofugen' established that the 15 negative control trees were free from PDV. To es t a b l i s h the threshold value for TAS-ELISA, leaves from these healthy trees were included i n each TAS-ELISA for the test trees. The sum of the mean plus four standard deviations of the healthy control population (xH+4S) was used as the threshold value for a p o s i t i v e reaction i n the test population (Sutula et a l . 1986). To normalize for s l i g h t differences between plates, a l l data were expressed as the r a t i o of the test value to the threshold value associated with that plate. The r a t i o s for duplicate samples were averaged and the values used for further analysis. A Chi-squared dispersion test (Maxwell 1961) was used to test whether the proportion of leaves that Page 3 6 would show a p o s i t i v e reaction by TAS-ELISA was the same for a l l p o s i t i v e trees. 2.3.0 cDNA l i b r a r y of PDV 2.3.1 Preparation of cDNA: f i r s t strand synthesis Single-stranded genomic RNA was i s o l a t e d from p a r t i a l l y p u r i f i e d PDV p a r t i c l e s as described i n section 2.1.4. A 3 /xg sample was heated to 65°C i n the presence of 450 ng random hexamers (pd(N)6) and cooled on i c e . A f i r s t strand reaction mix was prepared separately and added to the RNA to give the following conditions: 50 mM Tris-HCl, pH 8.3; 40 mM KC1; 6 mM MgCl2; 1 mM d i t h i o t h r e i t o l (DTT); 0.1 mg/ml BSA and 0.75 mM each dNTP i n a f i n a l volume of 25 u.1. This was e q u i l i b r a t e d to room temperature and 50 U Superscript II™ reverse transcriptase (Gibco/BRL) was added, mixed gently and allowed to react for 10 minutes at 25°C to allow primer extension. The reaction was then warmed to 48°C, a further 200 U RTase were added and i t ' was incubated for 1 hour. A 1 txl sample of t h i s reaction was removed immediately and added to 0.5 i l l [a32P]dCTP (5 ixCi) and run i n p a r a l l e l with the main reaction. This p i l o t f i r s t strand reaction was terminated by the addition of 0.5 ixl 0.5 M EDTA, pH 8.0. The f i r s t strand products were separated by electrophoresis on an a l k a l i n e agarose gel (Sambrook et a l . 1989) and autoradiographed by wrapping the gel i n Saran wrap and placing i t onto a sheet of Kodak X-omat AR f i l m overnight. Page 3 7 2.3.2 Second-strand synthesis A f t e r 1 hour, the main reaction was c h i l l e d on ice and combined with 13 6 /xl of second strand reaction mix to give the following f i n a l conditions: 40 mM Tris-HCl, pH 7.5; 10 mM MgCl 2; 10 mM (NH4)2S04; 100 mM KCl; 50 /xg/ml BSA; 0.5 mM dNTPs; 5 mM DTT; 0.15 mM |S-NAD+; 40 U E. c o l i DNA polymerase I (Gibco/BRL); 10 U E. c o l i DNA ligase (Gibco/BRL) and 5 U E. c o l i RNase H (Gibco/BRL) . A 10 /xl sample was immediately removed from the reaction and mixed with 1 /xl (10 /xCi) [a32P]dCTP. Both reactions were incubated at 12°C for 1 hour followed by 4 hours at 20°C. The second strand reactions were terminated by the addition of 0.5 M EDTA, pH 8.0, to 20 mM. The products of the main reaction were pre c i p i t a t e d i n ethanol and ammonium acetate, the p i l o t reaction products were analyzed by agarose gel electrophoresis and autoradiography. 2.3.3 End po l i s h i n g reaction A f t e r p r e c i p i t a t i o n , the cDNA p e l l e t was washed with 70% ethanol, dried and redissolved i n 7.8 /xl water and 1 /xl 10X Pfu buffer (IX Pfu buffer i s 10 mM KCl; 6 mM (NH4)2S04; 20 mM Tris-HCl, pH 8.0; 2mM MgCl2; 0.1% Tri t o n X-100 and 10 /xg/ml BSA: Stratagene) , 2 /xl 10 mM dNTP mix and 0.2 /xl Pfu polymerase (0.5 U: Stratagene). This was ov e r l a i d with mineral o i l and incubated at 72°C for 20 minutes. The reaction was terminated by the addition of 0.5 M EDTA, Page 3 8 pH 8.0, to 20 mM and stored at -20°C u n t i l required. The DNA content of t h i s sample was estimated by measuring the emission of a 1 /xl sample i n a fluorometer, using Hoechst Dye 33342 (Hoeffer S c i e n t i f i c Instruments). To select large cDNA molecules, the entire sample was loaded onto a single well i n a 1% agarose gel and electrophoresed for 15 minutes at 60 V. Suitable molecular weight markers (eg. 1 kb ladder: Gibco/BRL) were also run. The gel was stained with ethidium bromide and the region of the gel containing the required size of cDNA was excised from the gel on a long wavelength UV transilluminator. The cDNA was extracted from the gel s l i c e using the Qiaex gel extraction k i t (Qiagen). 2.3.4 Blunt-end l i g a t i o n The r e s u l t s of the autoradiographs of the f i r s t and second strand products were used to estimate the average lengths of the cDNA products and the fluorometer reading was used to calculate the concentration of cDNA. Using t h i s information, a l i g a t i o n reaction containing pBluescript SK+ (Stratagene) or pGem4Z (Promega) cut with EcoRV or Smal respectively and dephosphorylated with c a l f i n t e s t i n a l phosphatase (CIP: Gibco/BRL) was prepared using a 5:1 molar r a t i o of vector:cDNA. The reaction conditions were: 50 mM Tris-HCl pH 7.6; 10 mM MgCl2; 1 mM ATP; 1 mM DTT; 5% (w/v) PEG 8000 and 10 U T4 DNA ligase (Gibco/BRL). The reaction was allowed to proceed overnight at 16°C. The plasmids were Page 3 9 then transformed into competent E. coli DH5a by one of the two methods described below (section 2.3.5) and plated onto 2X TY (1.6% bacto-tryptone, 1% yeast extract, 0.5% NaCI, 2% agar) plates containing 200 /xg/ml a m p i c i l l i n and spread with 800 /xg X-gal (5-bromo-4-chloro-3-indolyl - jS-D-galactoside) and 800 /xg IPTG (isopropylthio-/3-galactoside) . 2 . 3 . 5 Preparation of competent E. coli Competent c e l l s for transformation by electroporation or by heat shock were prepared i n bulk and stored i n small aliquots at -70°C. C e l l s were prepared as follows. E. coli DH5a were inoculated to 2X TY plates overnight. A single colony was transferred into 5 ml 2X TY media and incubated overnight at 37°C i n a rotary shaker. This was used to inoculate 200 ml 2X TY medium i n a 11 flask and the c e l l s grown u n t i l A 6 0 0 of the culture measured 0.6. The flasks were immediately immersed into an ice-water bath and c h i l l e d for 15 minutes. The c e l l s were harvested by centrifugation at 3500 rpm i n a JA14 rotor (Beckman) for 10 minutes at 4°C. The supernatant was discarded, draining away as much of i t as possible. Electroporation-competent c e l l s were resuspended i n 100 ml p r e c h i l l e d d i s t i l l e d , s t e r i l e water; heat-shock competent c e l l s were resuspended i n 40 ml c h i l l e d 3 0 mM potassium acetate, 50 mM MnCl2, 100 mM KCl, 10 mM CaCl 2, 15% g l y c e r o l ( f i l t e r - s t e r i l i z e d ) . Both cell-types were recentrifuged as before and the supernatants again drained Page 40 c a r e f u l l y , keeping them on ice as much as possible. C e l l s for electroporation were suspended gently i n 2 ml d i s t i l l e d , s t e r i l e water and transferred into 1.5 ml microcentrifuge tubes on ice i n 60 /xl aliquots, to be used immediately or stored at -70°C. C e l l s for heat shock-transformation were resuspended i n 8ml c h i l l e d 10 mM Na-morpholinepropane sulphonate, pH 7.0; 75 mM CaCl 2; 10 mM KCl and 15% glycerol ( f i l t e r - s t e r i l i z e d ) a f t e r the second centrifugation and divided into aliquots as described above. The transformation e f f i c i e n c y of the electroporation-competent c e l l s was determined by c a l c u l a t i n g the number of colonies formed a f t e r electroporation of 1 pg, 10 pg and 100 pg pBluescript SK+ using a cuvette with a 1 mm gap at 1800 V (Genepulser™: BioRad). Afte r electroporation the c e l l s were incubated i n 1 ml 2X TY medium supplemented with 10 mM MgCl2, 10 mM MgS04 and 40 mM glucose at 37°C i n a rotary shaker. C e l l s were then spread onto a 2X TY agar plate containing 2 00 /xg/ml a m p i c i l l i n and allowed to form colonies overnight. Colonies were counted the next day and the transformation e f f i c i e n c y calculated as the number of colonies formed per /xg plasmid used. Heat shock-competent c e l l s were transformed with the same plasmid by allowing a 40 /xl aliquot of the c e l l s to thaw on ice i n the presence of the plasmid for 30 minutes with occasional gentle mixing. The tubes were then heated to 3 7°C for 1 minute, returned to ice for 2 minutes and transferred to 1 ml 2X TY medium and placed i n a rotary shaker at 3 7°C Page 41 for 1 hour before being plated as described above. Colonies were counted the next day and the transformation e f f i c i e n c y determined. 2.3.6 Screening colonies for inserts by PCR Af t e r overnight incubation on IPTG/Xgal plates, some white colonies ( t y p i c a l l y 10) were selected at random from each cDNA plate and screened for inserts using PCR. Colonies were picked with a s t e r i l e disposable p i p e t t e - t i p and b r i e f l y immersed i n 20 /xl PCR mix (2 ill 10X Taq buffer (Stratagene) , 2 /xM M13 universal and reverse primers, 0.2 mM each dNTP, 0.2 U Taq DNA polymerase (Stratagene) o v e r l a i d with 20 /xl mineral o i l ) . These were heated to 94°C for 2 minutes and then given 30 cycles of 50°C, 30 sec (annealing) ; 72°C, 1.5 min (extension); 94°C, 30 sec (denaturation), i n a thermocycler (Techen: PHC2). PCR products were v i s u a l i z e d by electrophoresis on a 1% agarose gel run for 1 hour at 6 0 V, stained with ethidium bromide and photographed. Molecular size markers were either the 1 kbp ladder, 100 bp ladder or 3>X 174 RF DNA cut with Hae III ( a l l from Gibco/BRL). 2.3.7 Identifying inserts using northern bl o t s To i d e n t i f y the v i r a l RNA corresponding to cDNA inserts, i t was necessary to hybridize the cDNA to dsRNA i n northern b l o t s . Northern blots were prepared by electrophoresis of PDV dsRNA as described i n section 2.1.5. After Page 42 electrophoresis the gel was denatured by soaking i n 0.4 N NaOH, 1 M NaCl (two 10 minute washes), neutralized i n 1 M Tris-HCl, pH 7.0; 1 M NaCl for 20 minutes and placed i n an el e c t r o b l o t apparatus. The dsRNA was transferred to a nylon membrane (Gene screen™: DuPont) i n 25 mM sodium phosphate buffer, pH 6.5, overnight at 12 V, 4°C. The dsRNA was cross-linked to the membrane by a 5 minute exposure to UV l i g h t . The blot was prehybridized i n 5 ml 50% deionized formamide; IX Denhardt's solution; 50 mM Tris-HCl, pH 7.5; 1.0 M NaCl; 1% (w/v) SDS and 10% dextran sulphate, at 42°C for 1 hour i n a rotary hybridization oven. To prepare probes, PCR products (section 2.3.6) were digested with r e s t r i c t i o n enzymes whose s i t e s c l o s e l y flanked the ins e r t cDNA to be investigated and the ins e r t was gel-p u r i f i e d using Qiaex beads (Qiagen). The p u r i f i e d cDNA fragment was d i l u t e d to approximately 50 ng//xl and l a b e l l e d with [a32P] dCTP by random primer l a b e l l i n g with 2 U Klenow fragment (Sambrook et a l . 1990). The l a b e l l e d cDNA probe was p u r i f i e d with a spin column (Sephadex G-10: Pharmacia), denatured by b o i l i n g and hybridized to dsRNA immobilized on the nylon i n the prehybridization solution overnight at 42°C i n a rotary hybridization oven. The blot was washed twice at room temperature with 2X SSC (standard saline c i t r a t e i s 150 mM NaCl; 5 mM sodium-c i t r a t e , pH 7.0), 0.1% (w/v) SDS for 5 minutes per wash, then twice more with the same but at 65°C and f i n a l l y twice with Page 43 0.5X SSC, 0.1% (w/v) SDS at 65°C. The blot was kept damp, wrapped i n Saran wrap and autoradiographed overnight at room temperature, or at -70°C with an i n t e n s i f y i n g screen i f added s e n s i t i v i t y was required. Kodak X-omat AR f i l m was used. 2.3.8 Preparation for sequencing A l l clones which hybridized to PDV dsRNA were prepared for sequencing by preparing a high q u a l i t y stock of the cDNA in i t s vector using commercially available plasmid miniprep k i t s (eg. Magic™ Minipreps: Promega; Qiagen minipreps: Qiagen). The smallest of these clones, with approximately 300 to 500 bp cDNA inserts, were sequenced f i r s t using the dideoxy chain termination method (Sanger et a l . 1977) and the Sequenase™ version 2.0 k i t (Amersham) . Two /xg of the plasmid to be sequenced was denatured with 2 N NaOH and sequenced from the M13 universal and reverse primers following the recommendations of the k i t , but using [a32P] dCTP instead of one of the recommended nucleotides. This necessitated substituting dATP for dCTP i n the l a b e l l i n g mix supplied i n the k i t . A l l other instructions and recommendations of the k i t were followed. Electrophoresis was c a r r i e d out on the same day as the sequencing reactions, since the 3 2 P - l a b e l l e d strands of DNA were unstable. Samples were electrophoresed on a 0.1 mm thick 8% polyacrylamide gel with 8.3 M urea i n IX TBE (0.89 M T r i s ; 1.12 M borate; 25 mM EDTA, pH 8.0) at 50 W for approximately 5 hours (long run) Page 44 and 2.5 hours (short run). After electrophoresis, the gel was transferred to Whatman paper, dried under vacuum and autoradiographed at room temperature overnight using Kodak X-omat AR f i l m . 2.3.9 Exo III deletions cDNA fragments longer than about 500 bp could not be sequenced e n t i r e l y from the M13 universal and reverse primers on the plasmid. A subset of deletion clones was made as follows. Approximately 5 tig of the plasmid to be sequenced was digested with two neighbouring r e s t r i c t i o n enzymes i n the pol y l i n k e r . To ensure u n i d i r e c t i o n a l deletion, an enzyme which creates a 3'-overhang was used adjacent to the primer s i t e and an enzyme creating a 5'-overhang was used adjacent to the insert-cDNA to be deleted. Enzymes which did not cut the cDNA inse r t i n t e r n a l l y were used. On completion of the digest, the l i n e a r i z e d plasmid was extracted with phenol, p r e c i p i t a t e d i n ethanol and redissolved i n 40 til 66 mM Tris-HCl, pH 8.0; 6.6 mM MgCl 2 and allowed to e q u i l i b r a t e to 25°C. Meanwhile 15 tubes containing 2.5 / i l SI nuclease mix (40 mM potassium acetate, pH 4.6; 330 mM NaCl; 1.4 mM ZnCl 2; 6.7% g l y c e r o l and 0.3 U SI nuclease) were prepared and c h i l l e d on i c e . The number of tubes used varied depending on the size of the i n s e r t ; 15 tubes were enough for a 1.5 kb insert, assuming a deletion rate of approximately 100 bp/min. The Exo III reaction was started by adding 500 U Exo III Page 45 (Gibco/BRL) to the plasmid prep at 25°C. The reaction was mixed well and a f t e r an i n i t i a l 20 second lag period, 2.5 ill f r a ctions were removed at 1 minute i n t e r v a l s and transferred to the Sl-mix tubes. When a l l the necessary time points had been taken, the tubes were warmed to room temperature and the SI reaction allowed to proceed for 30 minutes. The reaction was terminated by the addition of 1 til 0.05 M EDTA, pH 8.0 and tubes were heated to 80°C for 10 minutes to denature the SI nuclease. The DNA ends were made fl u s h with Klenow (2 U per tube i n 20 mM Tris-HCl, pH 8.0; 100 mM MgCl 2; 0.125 mM each dNTP) for 5 minutes. This was followed by the addition of 40 il l T4 DNA l i g a t i o n mix (prepared using Gibco/BRL 5X ligase buffer, see section 2.3.4) and 1 U T4 DNA ligase per reaction. Reactions were l e f t at room temperature for 4 hours and then transformed into competent E. coli DH5a (section 2.3.5) and plated onto IPTG/X-gal plates (section 2.3.4) . White colonies were screened for insert size by PCR (section 2.3.6). Colonies with inserts sized approximately 100-200 bp apart were selected so that the smallest insert was <;200 bp i n size and the largest was about 150 bp smaller than the o r i g i n a l cDNA ins e r t . These were sequenced as described above using the primer adjacent to the r e s t r i c t i o n s i t e used to i n i t i a t e the Exo III deletions. Exo III deletion clones were made from both ends so that both strands of each clone could be sequenced. Page 4 6 2.3.10 Sequence alignment Sequence was entered into the computer using the XESEE Version 3.0 programme (Cabot & Beckenbach 1989). This programme was used to ali g n overlapping deletion fragments and clones and to check for mismatches among clones. Care was taken to ensure that a l l regions were sequenced at least twice and that a l l mismatches could be resolved s a t i s f a c t o r i l y . Sequences were also checked against the database maintained by the National Center for Biotechnology Information (NCBI) using the BLASTx and BLASTn programmes. 2.3.11 cDNA c l o n i n g of PDV RNA1 A short cDNA fragment 353 bp i n length, hybridized to. PDV RNA1 on a Northern b l o t . The sequence of t h i s fragment was used to construct the following cDNA synthesis primer s p e c i f i c for RNA1: CGTAATCAACCAAT (position 1244 on RNA1; a l l primers were synthesized by the Core DNA F a c i l i t y , University of Calgary). This was used to prime the f i r s t strand synthesis of cDNA from t o t a l v i r a l RNA i n a reaction s i m i l a r to the one described i n section 2.3.1 but s u b s t i t u t i n g the s p e c i f i c primer for random hexamers. This yielded several large clones of cDNA (ca. 1.3 kb i n s i z e ) . Two of these were sequenced completely as described above and the information used to construct a primer for. RACE PCR (rapid amplification of cDNA ends, section 2.3.12). The.larger of these, 1311 bp i n length was designated pPDV33. Page 47 2.3.12 The sequence of the 5' end of PDV RNA1 A modified procedure of Hirzmann et a l . (1993) was used to determine the sequence of the 5'-terminus of RNA1 up to and including the cap structure. Two primers were synthesized: CGGATCCAGTAAGCGGTGAG (position 316) and CGGGAT(C)10. The former i s complementary to the region approximately 300 bp from the 5'-end of the known sequence on pPDV33. Both primers have a BamHI s i t e (GGATCC) at t h e i r 5' end to f a c i l i t a t e cloning of RACE fragments. The RACE PCR was preceded by a f i r s t - s t r a n d synthesis reaction using randomly primed t o t a l v i r a l RNA exactly as described i n section 2.3.1. After the RTase reaction the f i r s t strand products were extracted with phenol:chloroform and p r e c i p i t a t e d i n ethanol from ammonium acetate. The r e s u l t i n g p e l l e t was resuspended i n 19 til water and 5 til 5X t a i l i n g buffer (5X t a i l i n g buffer i s 0.5 M potassium cacodylate, pH 7.2; 10 mM CoCl 2: Stratagene) and incubated at 37°C with 0.2 mM dGTP and 5 U terminal deoxynucleotidyl transferase (TDT from c a l f thymus: Stratagene) for 20 minutes. The reaction was then placed on ice and 1 til added to a PCR mix containing 2 tiM of each RACE primer, 0.2 /xM each dNTP i n Taq buffer (Stratagene) as well as 0.1 U Taq DNA polymerase. The DNA was amplified using 35 cycles of denaturation at 94°C, annealing at 40°C and extension at 72°C for 3 0 seconds each. The 3 50 bp product was digested with BamHI and p u r i f i e d a f t e r gel electrophoresis (using Qiaex Page 48 beads). The fragment was l i g a t e d overnight at room temperature into BamHI-cut pBluescript SK+ (section 2.3.4) which had been treated with CIP and transformed into competent E. coli DH5a (section 2.3.5). Colonies were selected for inserts on IPTG/X-gal plates as before and white colonies were screened for 350-bp inserts by PCR using the M13 universal and reverse primers (section 2.3.6) . Twelve clones of the correct size were selected and sequenced from the reverse primer (section 2.3.8). Since the l i g a t i o n was not d i r e c t i o n a l , some of these had to be sequenced again from the M13 universal primer to be able to determine t h e i r 5'-sequence. 2.3.13 Cloning the 3' region of PDV RNA1 Another cDNA l i b r a r y was made from random hexamers as described i n sections 2.3.1 - 2.3.3. White colonies from t h i s l i b r a r y were transferred to a 15 cm p e t r i dish marked with a g r i d with space for 150 colonies. These were allowed to grow overnight and transferred to nylon (Genescreen™ Plus: Du Pont) as described by Sambrook et a l . (1989). The transferred colonies were denatured i n 1% (w/v) SDS; 0.5 N NaOH; 1.5 M NaCl, neutralized i n 1 M Tris-HCl, pH 8.0; 1.5 M NaCl. DNA was bound to the nylon by ill u m i n a t i o n with UV l i g h t for 5 minutes. B a c t e r i a l debris was removed by washing the nylon i n several changes of 2X SSC; 0.1% SDS. The nylon was dried and prehybridized as described by Sambrook et a l . Page 4 9 (1989) . A probe was prepared from pPDV33 (section 2.3.11) by-digestion with EcoRV and P s t l . This released a fragment approximately 360 bp i n length from the 3'-end of the clone, which was p u r i f i e d a f t e r gel electrophoresis using Qiaex beads. The EcoRV fragment was l a b e l l e d with [a32P]dCTP as described i n section 2.3.7, hybridized to the c o l o n y - l i f t and autoradio-graphed. Colonies with cDNA which hybridized to the probe were restreaked onto fresh plates and plasmid was p u r i f i e d from them. Two clones containing cDNA inserts 1.5 and 2.0 kb were sequenced completely. This strategy was repeated using a P v u l l / H i n d l l l fragment from the 3'-end of the known sequence to rescreen the cDNA l i b r a r y above. Clones representing RNA1 sequence to pos i t i o n 3165 were obtained and two RACE primers were constructed based on t h i s sequence. The 3'-terminus of RNA1 was amplified by 5'-RACE PCR (section 2.3.12) using denatured dsRNA as a template for the f i r s t strand synthesis (Coffin & Couts 1990) primed with the s p e c i f i c primer CCTATAATGGGAGC TTGC (position 3044) . The f i r s t strand product was amplified by PCR using the nested primer CTGGAGGGGATAATGAATG (position 3118) and CGGGAGT(C) 1 0. The PCR product was blunt-end l i g a t e d into pBluescript (EcoRV-site) and sequenced. The sequence of the 5'-terminus of the negative strand (of the dsRNA) was taken to be the complement of the 3'-end of PDV RNA1. Ten clones were sequenced to ensure that the 3'-end had been reached. The RACE PCR was repeated using the nested Page 50 primer pairs above with (G) 1 2 to amplify poly-(C) t a i l e d f i r s t strand cDNA, to confirm the i d e n t i t y of the terminal nucleotide. 2.3.14 Sequence comparisons and phylogeny The sequence obtained was compared to e x i s t i n g sequences on the GenBank database using the BLASTx programme at the National Center for Biotechnology Information (NCBI). The following RNA1 sequences were obtained from GenBank and used for comparison: CiLRV (accession number U23715); AMV (L00163); CMV (D12537); BMV (X02380) andRBDV (S51557). These were aligned using the PILEUP software i n the Wisconsin Genetics Computer Group package (GCG: Devereux et a l . 1984). RBDV RNA1 encodes a polyprotein (Ziegler et a l . 1992) and only the 5'-region of the RNA and the C terminal of the t r a n s l a t i o n product were used for further analysis. Since PILEUP did not always y i e l d optimal alignments, further e d i t i n g was c a r r i e d out manually with XESEE using r e s u l t s from pairwise alignments (BLASTx and GAP) as a guide. A phylogeny was created from the f i n a l alignment using the Phylip 3.5c software (Felsenstein 1989). Nucleic acids and t h e i r t r a n s l a t i o n products were compared by using SEQBOOT to generate 100 bootstrap rep l i c a t e s which were analysed using DNAPARS (or PROTPARS for amino acid sequences). CONSENSE was used to generate a consensus phylogeny. This analysis was repeated using the sequence of RNA3 and of ORF3a of the Page 51 following viruses: AMV (K02703); ApMV (U15608); BMV (J02042); CiLRV (U17390); CMV (D00385); PNRSV (L38823); PDV (L28145). 2.3.15 RT-PCR assay Two primer pairs were chosen from the RNA3 sequence data. They were: CACGGACTTTCATGGCGTAA and CCCTCCTGCTGGT TTTCTTA (pair #1) as well as ACACCAAAAGCTTTCCTTGTC and AACTTTGAGATTCCCGATTG (pair #2). Pair #1 was chosen from a region covering parts of RNA3 and RNA4 and yielded a product 179 bp i n length. Pair #2 was from RNA3 only y i e l d i n g a product ca. 295 bp i n length. The RT-PCR of leaf tissue was based on the method of Wetzel et a l . (1991). Tissues (leaves, buds or flowers) were ground i n 1 ml d i s t i l l e d s t e r i l e water with 0.1% /3ME i n a p l a s t i c bag l i n e d with cheesecloth using a tissue-X homogenizer (Bioreba AG). The r e s u l t i n g sap was transferred to a microcentrifuge tube with a pasteur pipette and centrifuged b r i e f l y to sediment leaf debris. F i f t y /xl supernatant were transferred to 450 /xl 10% Tr i t o n X-100, heated to 65°C for 10 minutes and then placed on i c e . A 10 /xl sample was removed and denatured by the addition of 2 /xl 4 0 mM MeHgOH. Samples were l e f t at room temperature for 10 minutes and then treated with 1 /xl 260 mM /3ME. A reverse t r a n s c r i p t i o n c o c k t a i l was then added, giving the same reaction conditions as set out i n section 2.3.1, i n a f i n a l volume of 20 /xl. The reaction was incubated at 37°C for 1 hour. It was primed with random hexamers. Page 52 The reaction was brought to 50 /xl by the addition of 17 /xl s t e r i l e water, 2 /xl 10 mM dNTPs, 2 /xl of each primer (2 /iM f i n a l concentration) , 5 /xl 10X Taq buffer (Stratagene) and 0.5 /xl Taq DNA polymerase (Stratagene) . The sample was heated to 95°C for 2 minutes and cycled through 35 cycles of: denaturation, 1 minute at 95°C; primer annealing, 1 minute at 42°C; DNA synthesis, 1 minute at 72°C. There was a f i n a l extension for 4 minutes at 72°C. The products of the reaction were analyzed by agarose gel electrophoresis and stained with ethidium bromide. The presence of PDV was indicated by the appearance of a reaction product corresponding to i t s s p e c i f i c primer pair . In general, each sample was assayed at least twice and every tenth sample was a template-negative control. 2.4.0 Preparation of a defective i n t e r f e r i n g p a r t i c l e 2.4.1 Production of a snapback-type DI p a r t i c l e by PCR One of the RACE clones (#18) containing the 5' end sequence of RNA1 and a Pvu I s i t e at p o s i t i o n 267 was l i g a t e d to pPDV33 (section 2.3.11) using the Pvul s i t e ; t h i s was done as follows. Clone #18 was digested with Pvu I and BamHI and a 265 bp fragment i s o l a t e d from an agarose gel using Qiaex beads. Plasmid pPDV33 was digested with B g l l l and Pvul and a 1030 bp fragment i s o l a t e d from an agarose gel. The concentrations of these fragments was determined by spectrophotometry and they were mixed i n a 1:1:3 molar r a t i o Page 53 with pBluescript SK+ l i n e a r i z e d with BamHI, and incubated with T4 DNA ligase (Gibco/BRL) i n ligase buffer (Gibco/BRL) overnight at room temperature. The l i g a t i o n products were transformed into E. coli DH5a and selection was ca r r i e d out by PCR using the M13 universal and reverse primers (section 2.3.6) to search for clones containing an .insert 1.3 kb i n siz e . This clone would contain the 5'-end of RNA1 and the sequence up to p o s i t i o n 1295; i t was c a l l e d pPDV1300. Three primers were synthesized to construct a snapback type DI p a r t i c l e by PCR. These had the following sequences: DI1: TAAGGATCCTAATACGACTCACTATAGGTTTTACGAACGTGGTTGTTC DI2: TAAGGATCCGCGGTTTTACGAACGTGGTTGTTC DI3: ATGGACAACGGTGGTGAT DI1 features a BamHI s i t e (GGATCC), the T7 promoter (under-lined) and the f i r s t 22 bases of PDV RNA1 (on pPDV1300). DI2 also has a BamHI s i t e , to f a c i l i t a t e cloning, a unique S s t l l s i t e (CCGCGG) to l i n e a r i s e the f i n a l construct before t r a n s c r i p t i o n and the f i r s t 22 bases of pPDV1300. DI3 i s complementary to a region beyond a C l a l (BspDI) s i t e on pPDV1300 about 1 kb downstream from DI1/2. Primer pairs DI1 & DI3 and DI2 & DI3 were used to amplify pPDV1300 i n separate PCR reactions. The PCR reaction was catalyzed by Taq DNA polymerase with the buffer conditions recommended by the manufacturer (Stratagene) using Page 54 each primer at a concentration of 2 0 /xM. The optimal annealing temperature was determined empirically to be 48°C and the following conditions were used for 35 cycles i n a thermocycler: denature 3 0 seconds at 94°C; anneal 4 0 seconds at 48°C; extend 1 minute at 72°C. After PCR, the products were p r e c i p i t a t e d i n ethanol, the p e l l e t dissolved i n 9 /xl NEB buffer 3 (New England Biolabs) and digested with 5 U BspDI and 5 U BamHI (New England Biolabs) for 1 hour at 37°C. The entire digest was loaded onto a 1% agarose gel and the fragments separated e l e c t r o p h o r e t i c a l l y . The desired band was approximately 94 0 bp i n length for each reaction. The band generated by DI1 & DI3 i s 15 bases longer, but i n practise t h i s could not be distinguished from the product of DI2 & DI3. The 94 0 bp bands were extracted from the gel and p u r i f i e d with Qiaex beads (Qiagen). The DNA concentrations were determined spectrophotometrically and the fragments were mixed i n a 1:1:3 molar r a t i o with pUC18 l i n e a r i z e d with BamHI and treated with CIP. The mixture was incubated overnight at room temperature with 1 U T4 DNA ligase i n li g a s e buffer (Gibco/ BRL). Competent E.coli DH5a were transformed with the reaction on the following day. The insert size of white colonies was determined by PCR screening using the M13 universal and reverse primers on pUC18. A band approximately 2.1 kb i n size (1.9 kb insert plus 200 bp polylinker) on an agarose gel would indicate a potential DI template for Page 55 t r a n s c r i p t i o n . Since three d i f f e r e n t l i g a t i o n products would give an inse r t of t h i s size, a l l p o s i t i v e clones also had to be screened by digestion with S s t l l . Only clones with a single S s t l l s i t e would be correct. There i s a unique S s t l l s i t e i n the DI2 primer; pUC18 i s not cut by t h i s enzyme. 2.4.2 Preparation of a snapback-type DI p a r t i c l e with synthetic oligonucleotides. An alternate strategy for the production of DI p a r t i c l e s , which required no PCR, was also devised. This strategy r e l i e s on the presence of a BspHI s i t e 40 bp i n from the 5' end of pPDV1300. Four oligonucleotides were synthesized using a Beckman 1000M DNA synthesizer, Table 1. Oligonucleotides 1 & 2 are designed so that when they anneal they produce a HindiII compatible end to f a c i l i t a t e cloning and the T7 promoter immediately adjacent to the f i r s t 4 0 bases of pPDV1300, ending i n a BspHI compatible end. Oligonucleotides 3 & 4 are designed so that when they anneal they produce a Kpnl compatible end for cloning, a unique Xmal s i t e to l i n e a r i z e the construct before t r a n s c r i p t i o n and the complement of the f i r s t 40 bases of pPDV1300 ending i n a BspHI compatible end. After the synthesis, these oligonucleotides were dissolved i n 100 /xl of d i s t i l l e d water and t h e i r concen-tra t i o n s determined by spectrophotometry. Equal molar amounts of o l i g o l & oligo2 and oligo3 & oligo4 were mixed, Page 56 Table 1. Oligonucleotides used to construct templates for snapback and deletion-type DI p a r t i c l e s . Oligos #1 and #2 a l i g n to form a cassette with HindiII and BspHI ends and with the T7 promoter d i r e c t l y adjacent to the 5' end sequence of PDV RNA1. Oligos #3 and #4 a l i g n to form a cassette with Kpnl and BspHI ends and have an Xmal s i t e adjacent to the Kpnl s i t e . When a palindromic cDNA fragment, derived from the 5' end of RNA1, i s l i g a t e d to these cassettes v i a t h e i r BspHI ends a palindromic sequence results which, when transcribed with T7 RNA polymerase, gives r i s e to a snapback DI RNA. Oligo Sequence 1 AGCTTAATACGACTCACTATAGGTTTTACGAACGTGGTTGTTCGTATTTTAAATCAAT ATTATGCTGAGTGATATCCAAAATGCTTGCACCAACAAGCATAAAATTTAGTTAGTAC 2 3 C ATGATTGATTTAAAATACGAACAAC CACGTT CGT AAAAC C C CGGGTA TAACTAAATTTTATGCTTGTTGGTGCAAGCATTTTGGGGCC 4 (D LTl overlayed with parafilm o i l , heated to 80°C for 10 minutes and allowed to cool to room temperature over a period of 2 hours. These were l i g a t e d onto the ends of pUC18 by mixing i n a 3:3:1 molar r a t i o with pUC18, previously l i n e a r i z e d with HindiII and Kpnl, and incubating with 1 U T4 DNA ligase i n ligase buffer (Gibco/BRL) for 4 hours at room temperature. The l i g a t i o n products were separated from free o l i g o -nucleotides by electrophoresis and the 2.7 kb band was extracted from the gel using Qiaex beads (Qiagen). The concentration of the r e s u l t i n g products was determined by spectrophotometry. The bulk of the genetic material for the DI p a r t i c l e was made from pPDV13 00 by digesting the plasmid with Pvul and BspHI. This released a 225 bp fragment which was gel p u r i f i e d using Qiaex beads. The concentration of t h i s DNA was determined and the fragment was mixed i n a 10:1 molar r a t i o with the prepared pUC18 (above) and incubated overnight at room temperature with 2 U T4 DNA ligase i n ligase buffer (Gibco/BRL). Although the prepared pUC18 vector has compatible BspHI ends, neither of these i s phosphorylated and thus c i r c u l a r i z a t i o n without an insert i s prevented. Also, the Pvul fragments can j o i n either v i a the Pvul or the BspHI ends but since they can only l i g a t e to the vector i f they have two BspHI ends, and since either o r i e n t a t i o n w i l l produce the same clone, almost a l l p o s i t i v e transformants would be correct. Page 58 2.4.3 Preparation of a deletion type-DI p a r t i c l e A template for transcribing a deletion-type DI RNA was assembled from two e x i s t i n g clones, pPDV33 (2.3.11) and a RACE clone containing the 3' end of RNA1 (pRACE32) (see Figure 1). The former was digested with EcoRV and BamHI to remove approximately 460 bp from i t s 3' end. pRACE32 was digested with H i n d i and BamHI and the 150 bp fragment, representing the 3'-end of RNA1 was gel p u r i f i e d and l i g a t e d into prepared pPDV33 described above. Transformants were screened for insert size, and a correct clone was i d e n t i f i e d and amplified. This clone was digested with BamHI and BspHI and cloned into pUC18-T7. Plasmid pUC18-T7 was prepared by digesting pUC18 with BamHI and HindiII and l i g a t i n g oligonucleotides 1/2 (see Table 1) into the H i n d l l l s i t e . The r e s u l t i n g plasmid pPDVdil was sequenced to confirm i t s structure. A 1 pig preparation was digested with BamHI and transcribed using T7 RNA polymerase (Ribomax k i t : Promega). After the reaction, DNA was removed by digestion with DNase I and the RNA recovered by phenol extraction and ethanol p r e c i p i t a t i o n . 2.4.4 Replication of a DI p a r t i c l e in vivo Two tig of p u r i f i e d DI RNA i n TE (10 mM Tris-HCl, pH 8.0; 1 mM EDTA) were mechanically inoculated onto pumpkin cotyledons dusted with carborundum (C. maxima cv. Small Sugar). These had been infected with PDV 1 week e a r l i e r . Page 59 Figure 1. The s t r u c t u r e o f p P D V d i l . The c o n s t r u c t was produced by d i g e s t i n g pPDV33 (y e l l o w ) w i t h EcoRV and BspHI and a f u l l l e n g t h RACE c l o n e (pRACE32, b l a c k ) c o n t a i n i n g the 3'-end o f PDV RNA1, w i t h BamHI and H i n d i . These c l o n e s were l i g a t e d v i a t h e i r b l u n t ends and l i g a t e d i n t o pUC18, which c o n t a i n e d the T7 promoter c a s s e t t e d e s c r i b e d i n T a b l e 1, v i a the BspHI and BamHI s i t e s . Page 6 0 Healthy control plants were also inoculated with DI RNA and some PDV-infected plants were mock-inoculated with TE buffer. Plants were allowed to grow for a period of 3 days and harvested. Total nucleic acid was extracted (section 2.1.5), separated by electrophoresis on a nondenaturing agarose gel and transferred to a nylon membrane by a c a p i l l a r y b l o t . The Northern blot was probed with a ra d i o l a b e l l e d cDNA probe, prepared by l a b e l l i n g a Pvul/Ncol fragment of pPDV33 with 32P, using random primer l a b e l l i n g (section 2.3.7). The nylon membrane was autoradiographed to v i s u a l i z e bands. Page 61 3.0 RESULTS 3.1.0 Virus P u r i f i c a t i o n The virus p u r i f i c a t i o n procedure of Fulton (1959) yielded virus of adequate pur i t y for many applications. This procedure was used to provide antigen for TAS-ELISAs which required p u r i f i e d PDV and for the detection of the PDV coat protein by western b l o t t i n g . Virions prepared by t h i s method were found by SDS PAGE to be contaminated by other proteins. Experiments which required purer v i r i o n s were done with virus which had been p u r i f i e d further by l i n e a r sucrose density gradient u l t r a c e n t r i f u g a t i o n , which eliminated most of the plant contaminants (see Figure 2). Fractions taken from the sucrose gradient a f t e r u l t r a c e n t r i f u g a t i o n were analyzed by SDS PAGE to assess the e f f e c t of t h i s step on virus p u r i t y (Figure 2A), and to determine the optimal centrifugation time. Figure 2A shows the gradient u l t r a c e n t r i f u g a t i o n run for 2 hours. When t h i s time was increased to 2.5 hours, the virus moved to the center of the gradient and was separated from protein contaminants, indicated by a single band i n the 25 kDa region i n lane 3, Figure 2B. The Mr of the coat protein of i l a r v i r u s e s has been reported to be t y p i c a l l y 25 kDa (Fulton, 1975) and the presence of a band i n t h i s region, absent i n negative control lanes, was used as an indicator of the presence of PDV. The y i e l d of p u r i f i e d virus, c o l l e c t e d by u l t r a -Page 62 A B Figure 2 . D e n a t u r i n g p o l y a c r y l a m i d e e l e c t r o p h o r e s i s g e l s (SDS PAGE) showing v i r u s p u r i t y a f t e r u l t r a c e n t r i f u g a t i o n t h r o u g h a l i n e a r s u c r o s e g r a d i e n t . The g e l s were s t a i n e d w i t h Coomassie b r i l l i a n t b l u e R-250, m o l e c u l a r weight markers are g i v e n i n kDa. A. G r a d i e n t f r a c t i o n a n a l y s e d a f t e r 2 hours of u l t r a c e n t r i f u g a t i o n ; l a n e 1: m o l e c u l a r s i z e s t a n d a r d s ( d a l t o n V I I s e t : Sigma); l a n e 2: sample l o a d e d onto s u c r o s e g r a d i e n t ( p r e - g r a d i e n t f r a c t i o n ) ; l a n e 3: upper p a r t o f g r a d i e n t ; l a n e 4: c e n t r a l band i n g r a d i e n t ; l a n e 5: l o w e r p a r t o f g r a d i e n t ; (lane 6: empty); l a n e 7: p r e p a r a t i o n from u n i n f e c t e d l e a v e s ( p r e - g r a d i e n t f r a c t i o n c f . l a n e 2 ) . B. U l t r a c e n t r i f u g a t i o n ( s u c r o s e g r a d i e n t ) r u n f o r 2.5 h o u r s ; l a n e I : p r e - g r a d i e n t f r a c t i o n ; l a n e 2: upper p a r t of g r a d i e n t ; l a n e 3: c e n t r a l peak i n g r a d i e n t ; (lane 4: empty); l a n e 5: h e a l t h y p r e p a r a t i o n ( c f . l a n e 7 i n A); l a n e 6: m o l e c u l a r s i z e s t a n d a r d s ( d a l t o n V I I s e t : Sigma). The Mr o f i l a r v i r u s c o a t p r o t e i n i s r e p o r t e d t o be t y p i c a l l y 25 kDa. Samples were not from the same v i r u s p r e p a r a t i o n and, due v a r i a t i o n s i n the p r e p a r a t i o n s , the same amount was not l o a d e d onto each g e l . Page 63 centrifugation of the pooled sucrose gradient peaks, was determined by spectrophotometry for each preparation based on the reported extinction c o e f f i c i e n t for PDV of 1.57 at 260 nm (Halk & Fulton 1978) . Virus recovery was found to be t y p i c a l l y 25 /xg virus/g fresh weight leaf tissue (approxi-mately 2 mg t o t a l per preparation). 3.1.1 V i r a l RNA separation Extraction of genomic ssRNA d i r e c t l y from i s o l a t e d virus p a r t i c l e s using heated phenol was e f f e c t i v e and ensured that the v i r a l RNA was almost free of contaminating plant rRNAs, since ribosomes were mostly eliminated during the virus p u r i f i c a t i o n procedure. Genomic PDV RNA was analyzed by MeHgOH agarose gel electrophoresis to determine the sizes of the three RNA species (Figure 3A). The sizes were: RNA1: 3.6 kb; RNA2: 2.8 kb; RNA3: 2.2 kb. The subgenomic RNA4 has a reported size of 0.88 kb (Bachman et al. 1994) and i s c l e a r l y v i s i b l e i n Figure 3A. Replicative form dsRNA was analyzed by non-denaturing agarose gel electrophoresis using CMV dsRNA as molecular size marker (Figure 3B). The dsRNA was used i n Northern blots to i d e n t i f y the o r i g i n of cDNA fragments. DsRNA preparations were frequently contaminated with rRNA and tRNA but t h i s could be reduced by the addition of RNase TI to the sample or electrophoresis. This s i m p l i f i e d the i d e n t i f i c a t i o n of dsRNA bands. Contamination by plant DNA was e a s i l y eliminated by Page 64 A E Figure 3A. D e t e r m i n a t i o n of the s i z e s o f the PDV RNA s p e c i e s ( l a n e s 1 & 2) by d e n a t u r i n g agarose g e l e l e c t r o p h o r e s i s i n MeHgOH. The g e l was s t a i n e d w i t h e t h i d i u m bromide. The m o l e c u l a r weight s t a n d a r d s ( l a n e 3, Gibco/BRL) a re g i v e n i n kb. The s i z e s of the RNAs were d e t e r m i n e d t o be: RNA1: 3.3 kb; RNA2: 2.6 kb; RNA3: 1.9 kb. RNA4 (0.88 kb) i s v i s i b l e at the bottom o f the l a n e . B. C o n f i r m a t i o n of the presence o f r e p l i c a t i v e form dsRNA a f t e r e x t r a c t i o n from i n f e c t e d c h e r r y l e a v e s ( l a n e 2 ) . The m o l e c u l a r s i z e s t a n d a r d i s CMV dsRNA and s i z e s a r e shown i n kb ( l a n e 1 ) . The g e l was s t a i n e d w i t h e t h i d i u m bromide. Page 65 by allowing RNase A to hydrolyse single-stranded RNA a f t e r digestion with DNasel. 3.2.0 Antibody Production The sera from the t a i l - b l e e d s of two immunized mice were assayed for anti-PDV antibodies. The res u l t s of a TAS-ELISA, expressed as PDV/healthy, were 2.246/0.876 and 2.239/0.891 for a 1:3000 d i l u t i o n s of the sera, i n d i c a t i n g that both mice had an immune response against the vi r u s . The fusion yielded a single stable hybridoma l i n e , c a l l e d PDA-3C, although two other hybridomas were also i d e n t i f i e d that produced anti-PDV antibodies. However, only the PDA-3C hybridoma l i n e survived in vitro and i t was used i n a l l subsequent s e r o l o g i c a l assays. Serological t e s t i n g revealed that t h i s antibody was of the IgG-L subclass. The HAP p u r i f i e d stock had a protein concentration of 2.5 mg/ml and a d i l u t i o n endpoint of 1:15000 by TAS-ELISA. Routine assays were ca r r i e d out with the antibody at a d i l u t i o n of 1:1000 or 2.5 /xg/ml. To v e r i f y that PDA-3C was reacting with the coat protein of PDV, i t was tested i n an immunoblot (Figure 4). The antibody binds to a protein with a Mr of approximately 25 kDa, corresponding to the size of the coat protein t y p i c a l l y reported for members of the i l a r v i r u s group. PDA-3C was evaluated i n a TAS-ELISA to detect PDV i n the flowers and leaves of a PDV-infected sweet cherry and the Page 66 Figure 4 . R e c o g n i t i o n of a 25 kDa band by PDA-3C i n a Western b l o t . The SDS-PAGE used t o make t h i s b l o t was l o a d e d w i t h a s i n g l e w i d e - t o o t h comb. M o l e c u l a r weight markers were r u n but a r e not shown i n the f i g u r e ; however t h e i r p o s i t i o n s and s i z e s a r e i n d i c a t e d i n kDa. The g e l was l o a d e d w i t h PDV p u r i f i e d as d e s c r i b e d i n s e c t i o n 2.1.2. Page 67 P. mahaleb stock tree (Table 2). The assay could routinely detect PDV during A p r i l and May giving absorbance values above the positive-negative threshold for infected trees, but was no longer r e l i a b l e by late June. The positive-negative threshold i n Table 2 was set at xH+4S, (the mean of the healthy controls plus four standard deviation units (Sutula et al. 1986)). When trees infected with PNRSV were assayed by TAS-ELISA using PDA-3C, the absorbance values were below the threshold established from uninfected trees (Table 2). Thus, PDA-3C did not cross-react with PNRSV i n th i s assay. There are no other known i l a r v i r u s e s which nat u r a l l y i n f e c t sweet cherry (Nemeth, 1986). PDA-3C was also tested i n a TAS-ELISA against two is o l a t e s of PDV giving p a r t i c u l a r l y severe symptoms, obtained from, the virus c o l l e c t i o n i n Wenatchee, WA. The re s u l t s of th i s assay, i n Table 2, show that the antibody was able to recognize these strains of PDV. 3.3.0 Primer pairs #1 and #2 i n RT-PCR Once a protocol for the amplification of p u r i f i e d PDV RNA by RT-PCR had been established, two primer pairs (section 2.3.15) were tested for t h e i r a b i l i t y to detect PDV i n the leaves of the P. mahaleb PDV stock tree, a healthy control sweet cherry tree and a tree infected with PNRSV. The RT-PCR procedure gave po s i t i v e results with both primer pairs (Figure 5) for the PDV infected tree only. Amplification Page 68 Table 2 . TAS-ELISA re s u l t s using monoclonal antibody PDA-3C to d i f f e r e n t i a t e between PDV and PNRSV i n infected sweet cherry and P. mahaleb. Three samples were assayed i n each case. Absorbance measurements were made following a 2 hour substrate incubation. E L I S A r e s u l t s ( A 4 0 5 - A 6 2 0 ) Sample P D V - i n f e c t e d PNRSV-i n f e c t e d H e a l t h y B u f f e r b l a n k P. avium Summerland F l o w e r L e a f 0 . 4 2 8 ± 0 . 0 4 1 0 . 0 7 3 ± 0 . 0 2 1 0 . 066 0 . 038 0 . 2 2 2 ± 0 . 0 2 4 0 . 0 7 1 ± 0 . 0 0 9 P . mahaleb Summerland F l o w e r L e a f 0 . 9 9 1 ± 0 . 0 4 8 ND1 0 . 4 6 0 ± 0 . 0 7 6 0 . 0 6 5 ± 0 . 0 0 3 0.054 0.028 P . avium Wenatchee 2 L e a f L e a f 0 . 7 5 1 ± 0 . 0 2 7 ND 0.088 0.101 0 . 6 6 6 ± 0 . 0 4 4 ND 1ND = not determined 2Two is o l a t e s were assayed Page 69 bp U>0-TOO-100 * F i g u r e 5 . R e s u l t s of RT-PCR as s a y of the P. mahaleb p o s i t i v e c o n t r o l t r e e , a h e a l t h y sweet c h e r r y c o n t r o l t r e e and a sweet c h e r r y i n f e c t e d w i t h PNRSV. The g e l i s s t a i n e d w i t h e t h i d i u m bromide. Lane 1: m o l e c u l a r s i z e s t a n d a r d s (100 bp l a d d e r : G ibco/BRL), s i z e s i n d i c a t e d i n bp; l a n e s 2 & 3: t e m p l a t e -n e g a t i v e c o n t r o l f o r p r i m e r p a i r s #1 & #2 r e s p e c t i v e l y ; l a n e 4: the P. mahalejb t r e e w i t h p a i r #1; l a n e 5: h e a l t h y c o n t r o l f o r p a i r #1; l a n e 6: the P. mahaleb t r e e w i t h p a i r #2; l a n e 7: h e a l t h y c o n t r o l w i t h p a i r #2; l a n e 8: PNRSV i n f e c t e d t r e e w i t h p a i r #1; l a n e 9: PNRSV i n f e c t e d t r e e w i t h p a i r #2. Page 70 f a i l e d i n the healthy tree and furthermore, neither p a i r amplified RNA from PNRSV. Thus, either p a i r could be used to detect PDV i n infected cherry. 3.3.1 Detection of PDV by RT-PCR i n sweet cherry Since RT-PCR i s currently one of the most sens i t i v e method available for PDV detection, i t was used to determine the PDV-infection status of sweet cherry trees which were l a t e r assayed by TAS-ELISA and bioassay (section 3.4.2) . F i f t y - f i v e trees were o r i g i n a l l y selected for a survey designed to e s t a b l i s h a r e l i a b l e TAS-ELISA assay, and these were assayed for the presence of PDV by RT-PCR i n l a t e winter using unopened buds and i n spring using flowers and leaves. The r e s u l t s of the RT-PCR using leaves are shown i n Figure 6; the presence of a 197 bp band indicates the presence of PDV i n a tree. It was found that leaves were the most convenient tissue to work with i n terms of ease of processing and hence lower chance of sample cross contamination. 3.4.0 Results of the f i e l d survey 3.4.1 I d e n t i f i c a t i o n of PDV-infected trees Of the 55 trees o r i g i n a l l y tested by RT-PCR, 40 trees were used i n a f i e l d survey to e s t a b l i s h a r e l i a b l e TAS-ELISA (Table 3). The results of the RT-PCR assay performed on the symptomatic trees indicated the PDV i n f e c t i o n status of these samples are summarized i n Table 4. The 15 symptomless trees, Page 71 Figure 6 . Agarose g e l , s t a i n e d w i t h e t h i d i u m bromide, showing RT-PCR r e s u l t s . Leaves were assay e d f o r the presence of PDV i n the s p r i n g , s h o r t l y a f t e r p e t a l - d r o p . Some of t h e s e t r e e s were used t o s e t c o n f i d e n c e l i m i t s f o r the TAS-ELISA. T h i s group was assayed w i t h p r i m e r p a i r #1, and the p r e s e n c e of a 179 bp band i n d i c a t e s the p r e s e n c e of PDV. The m o l e c u l a r s i z e s t a n d a r d s (100 bp l a d d e r : Gibco/BRL) are g i v e n i n bp. Lanes are l a b e l l e d w i t h the t r e e l o c a t i o n , each l a n e r e p r e s e n t s one t r e e . Page 72 Table 3 . Results of the PDV TAS-ELISA, RT-PCR and bioassay and the PNRSV TAS-ELISA performed on f o r t y cherry trees at the Summerland Research Centre. Most of the trees had symptoms in d i c a t i n g a v i r a l i n f e c t i o n . TAS-ELISA res u l t s are indicated by the number of infected leaves out of a possible si x . Tree Symptoms TAS-ELISA Bio-assay RT-PCR PNRSV 13S 22/8 None 5/6 + + + 13S 24/28 None 0/6 - - - -13S 39/51 None 6/6 + + + -2N 36/26 Shot holes 6/6 + + + 2N 34/23 Shot holes 0/6 - + 11-14 Shot holes 0/6 - • • SP 4/3 Shot holes 6/6 + + • -A 2/1 Shot holes, necrosis 2/6 + + + ND* A 2/5 L i t t l e cherry disease 5/6 + + + + A 9/34 None 0/6 - + - + A 9/41 Rugose 6/6 + + + _ A 9/71 Shot holes 6/6 + + + + A 11/3 Red leaves 6/6 + + + A 20/1 Red leaves 6/6 + + + + A 20/2 Red leaves 5/6 + + + + A 20/3 Red leaves 6/6 + + + + A 20/4 Red leaves 6/6 + + + + B 4/2 Shot holes, brown patches 6/6 + + + _ B 4/3 Twisted l e a f , n e c r o t i c midvein 0/6 - _ B 4/27 Shot holes, enations 6/6 + + + • B 4/52 Twisted l e a f , dieback, ringspots 6/6 + + + + B 5/5 White shoulders 0/6 - - - -B 6/24 Mottle, bumpy f r u i t 6/6 + + + + B 6/25 Mottle, shot holes 6/6 + + + + B 8/30 Shot holes 6/6 + + + + B 8/34 Shot holes 6/6 + + + + B 8/38 Red leaves 4/6 + + + -B 9/22 Shot holes 6/6 + + + -B 9/30 Shot holes 4/6 + + + -Page 73 B 9/45 Shot holes 0/6 - + + B 9/47 Shot holes 6/6 + + • ND B 9/48 None 6/6 + + + ND B 9/49 Shot holes, mottle 0/6 - + - + B 9/50 None 6/6 + + + ND B 9/51 None 6/6 + + + ND B 13/12 Short stem 5/6 + + -B 13/22 Shot holes, mottle, yellows 6/6 + + + + SP 2/18 Shot holes 0/6 - + - + B 5/2 Shot holes 1/6 + + + + A 16/1 None 0/6 - + - + * N D = n o t d e t e r m i n e d P a g e 74 to be used as negative controls, were determined to be PDV-free by RT-PCR and i l a r v i r u s - f r e e by the 'Shirofugen' bioassay. PDV and PNRSV are indistinguishable by indexing on 4Shirofugen', necessitating further investigation of f i v e trees that tested p o s i t i v e by the bioassay but negative for PDV by RT-PCR. These trees were found to be infected with PNRSV i n a separate TAS-ELISA (Table 4). 3.4.2 Detection of PDV by TAS-ELISA The r e s u l t s of the TAS-ELISA on the group of 4 0 symptomatic trees to determine the number of leaves which tested p o s i t i v e for PDV by TAS-ELISA out of a possible six for each tree are summarized (Table 5). Average ELISA values, as well as maximum and minimum values for each set, are expressed r e l a t i v e to the threshold to normalize them for each plate. The proportion of leaves that were infected was not the same for every infected tree (x 2 = 87 .7 , df = 30, P<0.001). PDV was detected i n a 4/6 leaves by TAS-ELISA i n 29 of the 31 infected trees. Thus, most infected trees would be i d e n t i f i e d i f the number of leaves to be tested was chosen so that trees with two-thirds or more of t h e i r leaves p o s i t i v e by TAS-ELISA had a high p r o b a b i l i t y , i e . 99%, of being i d e n t i f i e d . Using the formula n = log (1-P)/log (1-Pr) where n i s the number of leaves to be tested, P i s the desired p r o b a b i l i t y of detection (0.99) and Pr i s the proportion of infected leaves (0 .667) , i t was determined Page 75 Table 4. Summary of the res u l t s of the RT-PCR, bioassay and PNRSV TAS-ELISA c a r r i e d out on 40 symptomatic test-trees and 15 symptomless negative control trees to e s t a b l i s h the incidence of PDV, i l a r v i r u s e s and PNRSV respectively. Symptomatic trees Assay Results Number of trees RT-PCR Bioassay PNRSV ELISA + + ND* 30 - - ND* 4 - + + 5 + - - 1 Symptomless trees - - - 15 *ND = not determined Page 76 Table 5 . Summary of the TAS-ELISA res u l t s of trees infected with PDV; s i x leaves were assayed from each tree i n duplicate assays. To normalize the data between plates mean, minimum and maximum values are expressed as a r a t i o of absorbance and threshold for each plate. # Positive leaves per tree (out of 6) # Trees ELISA Results (Sample value/threshold) Mean Minimum Maximum 0 0 - - -1 1 1. 885 1.885 1. 885 2 1 1.273 1.191 1.283 3 0 - - -4 2 1. 973 1. 620 2 .300 5 4 1.508 1. 093 2 .326 6 23 1. 931 1. 006 4 . 071 Tot a l : 31 Page 77 that four leaves from each tree must be tested i n order to achieve t h i s l e v e l of accuracy i n the TAS-ELISA. Trees with more than two-thirds of t h e i r leaves infected would have a higher p r o b a b i l i t y of detection while trees with fewer infected leaves would have a lower p r o b a b i l i t y of detection. 3.5.0 Alternate trapping antibodies. The F(ab') 2 fragments produced by pepsin digestion of PDA-3C were analyzed by SDS PAGE (Figure 7). Samples reduced by /3ME and unreduced samples were analysed. The presence of a band with an Mr of 110 kDa, indicated the presence of unreduced F(ab') 2 fragments. These were used i n place of the PVAS-290 rabbit serum to trap PDV i n a TAS-ELISA. Chicken IgY antibodies were also assessed for t h e i r a b i l i t y to trap PDV i n a TAS-ELISA and monoclonal PDA-3C was used with a l k a l i n e phosphatase conjugated PDA-3C i n a DAS-ELISA. The re s u l t s of these assays are summarised i n Table 6. Page 78 Figure 7 . R e s u l t s o f SDS PAGE used t o a n a l y s e F ( a b ' ) 2 fragments. D i g e s t i o n of monoclonal PDA-3C w i t h p e p s i n y i e l d e d a band w i t h Mr of 1 1 0 kDa, r e s i s t a n t t o d i g e s t i o n a f t e r 1 0 hours ( l a n e 1) and 24 hours ( l a n e 3 ) . T h i s band i s e l i m i n a t e d by r e d u c t i o n of the sample w i t h / ? M E b e f o r e l o a d i n g ( l a n e 2: 1 0 h sample; l a n e 4: 24 h sample). The 1 1 0 kDa band i s absent i n l a n e 5 (unreduced PDA-3C, I g G j and i n l a n e 6 (reduced PDA-3C). T h i s band i s the c o r r e c t s i z e r e p o r t e d f o r F ( a b ' ) 2 fragments (Harlow & Lane 1 9 8 8 ) . P e p s i n used f o r the d i g e s t was r u n i n l a n e 7 . T h i s 7% S D S - p o l y a c r y l a m i d e g e l was s t a i n e d w i t h s i l v e r n i t r a t e . M o l e c u l a r weight markers (High MW s e t : Sigma, l a n e 8 ) are shown i n kDa. Page 7 9 Table 6 . Results of ELISAs using d i f f e r e n t trapping and detecting antibodies. The numbers represent ELISA values (A 4 0 5 - A 6 2 0) of the optimal d i l u t i o n scheme for each assay-format . Trapping antibody ELSIA value Detecting antibody PDA-3C-AP1 (1:200)2 PDA-3C (l:1000)with AP-conjugate 3 1) PDA-3C (1:400) PDV4: 2.183 Healthy 5: 1.482 ND6 2) F(ab') 2 (1:800) ND PDV: 0.621 Healthy: 0.261 3) ATCC PVAS 290, (1:2000) PDV: 0.672 Healthy: 0.078 4) IgY (1:625) PDV: 0.210 Healthy: 0.089 1PDA-3C-AP = alk a l i n e phosphatase-linked PDA-3C, i n a DAS-ELISA. Concentration giving optimal r e s u l t s . 3AP conjugate = sheep anti-mouse (F c region) a l k a l i n e phosphatase-linked polyclonal (KPL) for detecting Ab 2. 4PDV sample was a PDV-infected sweet cherry leaf, except for IgY assay, where an infected cucumber cotyledon was used. 5Healthy sample was an uninfected sweet cherry leaf, except for IgY assay, where a healthy cucumber cotyledon was used. 6ND = not done. Page 80 3.6.0 The p a r t i a l sequence of PDV RNA3 The p a r t i a l sequence for PDV RNA3 (from clones PDV3a and PDV3b) and t h e i r alignment to the reported sequence of RNA3 (Bachman et al. 1994) i s shown i n Figure 8. The p o s i t i o n of the two primer pairs used for RT-PCR and the ORFs are shown. There are seven differences indicated at the nucleotide l e v e l . The predicted ORF1 & ORF2 products of the published sequence were compared to the tr a n s l a t i o n products of PDV3a and PDV3b (results not shown). There were three differences at the amino acid l e v e l : leucine to valine (position 906 i n the published nucleotide sequence of RNA3, i n the movement protein); asparagine to lysine (position 1584, coat protein); proline to arginine (position 1590, coat protein). For amino acid comparison, the sequence of the RNA3 clones was kept i n -frame with the published sequence i f nucleotide insertions or deletions occurred, such as at pos i t i o n 905. 3.7.0 The complete nucleotide sequence of PDV RNA1 A t o t a l of eight clones was sequenced i n both directions to obtain the complete sequence of RNA1. A map of the positions of these clones i s shown i n Figure 9. A further 12 RACE clones were also sequenced to obtain the sequence of the ends of the RNA (clones not shown i n Figure 9). The complete nucleotide sequence of PDV RNA1 and i t s putative t r a n s l a t i o n product are shown i n Figure 10. The Page 81 guuuuaauuaaccaagagaacugaauaaauuugagauuuaucucguuuauucgugcuaag 6 0 cuguggaaguugacagacaugcgguucucuauaaacccucaagaaauugaguuacugcaa 12 0 gguuucuugcaaagugugaagaagacaaauuugugacguuugagauuuaucucacuaauc 180 agauuuguuccuauucauuaaccucuuugaucucauugagugaaacaauucgguuguaga 24 0 uuuaucuccugaauugaauAUGGCAUUCUCUGGUGUUUCCAGGACCAUUACCGGACAGAC 3 0 0 GUCCGAAGCCAAUGCCAGUUCGGCAUTJUGAAGUUUCCGCUGAAGAUUGGAAUAAAAUACU 3 6 0 CAGCGAGGUGGAUGAUUUCUAUUCCCAAACUAUGAUGAAGAACCUUCCAACUAAGAAAUG 4 2 0 UTTTJUUCuTJUACAGUUGA 4 8 0 AUCUAGAAGUGCUUUAGCAAGAUTrAUCCGCUAAAGCUAAAGGUCAUGUUUAUGUGCAUCA 540 UAGUAUAAUUUACUUGUUGUACAUUCCGACCAUUCUGGAAACAACUAGUGGGGUAUUGAC 600 C AUUGAAACUTJUTJCAAUGUGAAUACUGGU^ 66 0 GAAUGAAGCGGCUAUCUUCGUUGGAAGAUGGCCAAGAGCAGUUCACGCCGAUGACGGUGA 72 0 CGGUAUAUGUTJUATJUAGCAUCAGCCGUAAGUGUAGACGCAAAACAUGCGUCAATJUGUUGG 780 AACUGUUUACCCCUUUUGGGAUGACUCAUUGCAUAAGAAGAAACCAUAUGAGAAAAUGUA 840 CCCAACUUUGAGATJUCCCGATJUGAGAAAUCGGAAGCCCUUGCUGCUGUAGAUGAUGUGAA 90 0 AAUA CUCCAAACAUUCGCCAAAUCGCGUTJUGGUAAUUGGGAGUAGUGGAAAGGUCGAUA 95 9 . . . . G. . . A UCAAUCCCAGACUUAUUGAAAUUAAGUCUGAUGAAUCAAAGAAAGCCUUAACGGUCGAUU 1019 UCAAGAAUGUUGACGUACCUAUAAAAGGUAAAUCUUCCCUUGAGAAAUUCAAGGAAGCCG 107 9 AGUCUGUUCCUCUCAAAGAAGAGAAGUCCGACAAGGAAGCUUUUGGUGUAACGAUUGGUU 113 9 AAcucacuuugugaguuaauagcucguuuuguuuaccaauuuacuuccaacuuucgacug 1199 uuuguucucucaaaAUGUCUGGGAAAGCCAUUAAAUCUGGAAAGCCUACUACCCGAUCAC 1259 AAAGCUTJUGCUUUAGCUCGGAAGAAUAAUAAUACUACCCCUCCUGCUGGUUUUGUUAA 1319 AACAAUUCCCAAGCGGAAGCUCGAAGUCUAUUUCCGAGUGGAUGCUUCACGGACCAAAUG 13 79 UGCCCGUAAAAAGUUUUUCCGGUAUGAUAUCUCGUACCGAGAACCUGACAGUCAAUUCGA 143 9 CUGCUUCCGGUGUAUAUUACACCAUGAAAGUCCGUGAACUGUUUAAGGACUUUGCUGUUG 14 99 ( Page 82 AUACCAAGGUGUACGGAAUUGUCUUCCGUUACUGCCUUGAUGTJUUCUAAUGGUGUCUACG 155 9 GACUCATJUAAAGGUUUCGAUGUGAAUGCGCCUGUGGCGCCUAAUCCCCUACAACGUAGGA 1619 - . .U.G AGTJUCACAGCGAAACAAGCCAGUGGGGUGCAAAUUCTJUGCUCCUACUGGUAUGACCGUUG 167 9 GGGAUAUACCAGAUGAUCUCUGGUUUGUCAUAAAAUAUGACAACGCUUUUCAGCCCAACG 173 9 UUCCGGUGUGGUUUUGUACUCAGUACCUCCAACACUCGAUGCCCAAGAGAGUUGAGGUCC 1799 CUGATJUCAGUUTJUAUACGCUGAGAGGGACACUGCCCUUAUGGAUGCGAUGGAUAAAAUAG 1859 UCAGUGGAUGAcuauaugauccaucauuugauugugcuuccacuaugaguauuccuagga 1919 auauucguaguuggaaaugcugcuuuugcaacagaauccaccauucagaguuugucacug 197 9 aauguuaaauccuuuugguuaaccugcacuaagugcguaaaagguuaagaugaaaaugcc 2 03 9 cauuguauccugaauggaugacacuuuucauugccuacaaauuuuguacaugcccucacc 2 099 guaaggugaggaugccccuuuaagggaugc 212 9 Figure 8. The published sequence (top) of PDV RNA3 (Bachman et al. 1994) aligned with two fragments of PDV RNA3 sequence obtained (position 545 to 1323 and 1444 to 1819). Mismatches between the sequences are indicated by (-) or by the nucleotide. The two open reading frames (position 260 to 1141 and 1214 to 1868) of the published sequence are shown i n cap i t a l s , UTRs are i n lowercase l e t t e r s . The positions of PCR primer pairs #1 (position 844 and 1109) and #2 (position 1296 and 1456) are underlined. I n i t i a t i o n and termination codons are shown i n boldface. Page 83 0 1 kb 2 kb 3 kb 3374 25 (pPDV33) 1336 1 I 31 1257 2773 3244 I I I I 921 1940 2492 3278 I I I I 876 1 1371 2843 I : i 1659 2088 I I 1740 2947 I I Figure 9. Map of RNA1 (1=1) i n d i c a t i n g the r e l a t i v e positions of nine clones, eight of which were sequenced completely, to obtain the sequence of RNA1. The RACE clones are not shown. A l l numbers are i n bp measured from the 5'-terminus of RNA1 unless otherwise indicated. Page 84 GGTTTTACGAACGTGGTTGTTCGTATTTTAAATCAATCATGACTTCTTCCGAGATCACTG 6 0 M T S S E I T 7 CTGCCAATGTCCATGAACTTTTGGTTAAAGTTCTGGAAAAGCAATGCGCTGACGAGACTA 120 A A N V H E L L V K V L E K Q C A D E T 27 CTACCGTCGGTAAGGCTTTCTCTGAGAAAGCGAAACAGTCTTTGAATAAGACATTCGGAC 180 T T V G K A F S E K A K Q S L N K T F G 47 TAAATGATGAGTCCAAGCAACTGAAGATTTCTTTTGATTTGACGGCTGAACAGCAGACGT 240 L N D E S K Q L K I S F D L T A E Q Q T 67 TACTCAAGAGACATTTTCCGGGTCGATCGGTGATTTTTTCAAATTCATCGAGTTCCTCAC 3 00 L L K R H F P G R S V I F S N S S S S S 87 ACAGTTTCGCGGCGGCTCACCGCTTACTGGAGACAGACTTTATATACCAGTGTTTTGGTA 360 H S F A A A H R L L E T D F I Y Q C F G 107 ACACTGATGAAACAATACTTGATTTGGGTGGAAATTATATTTCTCACCTAAAACAAAGGA 420 N T D E T I L D L G G N Y I S H L K Q R 127 GGTACAACGTGCATTGTTGTTGCCCACTTCTTGACGTGAGAGACTGTGCCCGCCATACTG 48 0 R Y N V H C C C P L L D V R D C A R H T 147 AGCGTCTCATGCAGTACACTACCTACAAGACTAGCAGACCTGATGAAGTTCACGAACCAA 540 E R L M Q Y T T Y K T S R P D E V H E P 167 ATTTTTGTGAGAACACATTCCAGGACTGCTCCTTGCAAGGTAAGTATGCCATGGCAATCC 600 N F C E N T F Q D C S L Q G K Y A M A I 187 ATTCCACTTCGGATTTACCCTTAGGTGAACTCTGTGAGAGTTTAAGGAAGAAAGGAGTGA 660 H S T S D L P L G E L C E S L R K K G V 207 TGAAGTTTATATGTTCTGTTATGATCGATCCCGAAATGTACATTAAAGACAGGGGTCACA 72 0 M K F I C S V M I D P E M Y I K D R G H 227 TAGATCATTTCAATCTGGATTGGCATGTGGACAAGGACAAAGACAGAATTTATTTTGACT 78 0 I D H F N L D W H V D K D K D R I Y F D 247 TTGTCGATGCACCCTGTTTAGGGTATGACCATAAGTATTCTACACTTATGGAGTATTTGC 840 F V D A P C L G Y D H K Y S T L M E Y L 267 ATTACAATGCTGTTGATCTAGGTGATGCCGCCTTTCGTGTCGAGCGGAAAACCGATTTTC 900 H Y N A V D L G D A A F R V E R K T D F 287 ATGGGGTCATGATTATCGATATCACCTATTGCTCCGGGTATAAACCTGGAATTGAGTTGA 960 H G V M I I D I T Y C S G Y . K P G I E L 307 ATGCAGGAAGATCCTGTGCCTGGTTGACCAAATTGAAGTCAAAAACTTTGGTCATGGCTA 102 0 N A G R S C A W L T K L K S K T L V M A 327 CTGATATTACGTCAGTAGTACACCCTTCTTTAGAAGCGGTGTCCAGAAGACACATACTGG 1080 T D I T S V V H P S L E A V S R R H I L 347 TTGACACGAAGGTCTTATCCAGAGTGTGTGAGGCTTCATTCCGACAGTACAAACCTAATG 1140 V D T K V L S R V C E A S F R Q Y K P N 367 TCGATGCGCAAAGTGCTATTCAGAGTATTTGCACGATGCTTTCTTCAGCTACTAACCATT 12 00 V D A Q S A I Q S I C T M L S S A T N H 387 GTATAATCAATGGTGTTACCATGATCGCAGGTACTCCCCTCAAATTGGTTGATTACGTAC 1260 C I I N G V T M I A G T P L K L V D Y V 407 Page 85 CTGTTGCCACCACTATTTATTATAGGGTGAAGAAGATCTATGATGCCATTCCAAGGTCAT 132 0 P V A T T I Y Y R V K K I Y D A I P R S 427 TGGGAATGATCAACAATCTGAGAACAACTGGGGAAATGTTGGATTATGCCACCAAACAGA 13 80 L G M I N N L R T T G E M L D Y A T K Q 447 AGGGTGGTATTCCTGATGATAGGAAACTGTTTTCCGACTATGCCTTTGAACCCTTGCGAT 1440 K G G I P D D R K L F S D Y A F E P L R 467 GTTTGCTTTCGTATGTTGGTTCTACTCCCACTCGTGTGGAAACCTACACTCGTGATGATG 1500 C L L S Y V G S T P T R V E T Y T R D D 487 GTTCAATTGAGCAATGTGCTCTTTATGAACGTTGGGGCAATTCCTGGAACCTTTTTAAGG 1560 G S I E Q C A L Y E R W G N S W N L F K 507 GTTTTTTGTCGGGATATATGGAAGTCGAAGGGTTTCTTGTCTCCGATCCACAATTCTTCG 162 0 G F L S G Y M E V E G F L V S D P Q F F 527 TTCCACTTACTGGAGTTCTTCATATGAAGAAATTGATAAGTGATGCTGGGAAGGTCCTTA 1680 V P L T G V L H M K K L I S D A G K V L 547 GTGTTAAGGAATTGCTCGAGGAACAACGCGCTCTTGTTGCCTTAAAGATGCGCGAGCAGA 174 0 S V K E L L E E Q R A L V A L K M R E Q 567 TTGCTGAAAGAGAAAAAGCTGAAAAGAGTCGCCGAGAGTATGAAAAGGCGATTATTCAAT 1800 I A E R E K A E K S R R E Y E K A I I Q 587 TGGCTGCTTGGACCAAAGCACATCCTGATGCTAAGGTTCCAAAGGGACTTTCCGTGGAAG 1860 L A A W T K A H P D A K V P K G L S V E 607 AACCATTGATGCCGGACGTTGTCAAGAAAGTGACGGCCGATGAAGTAGTACCAGATTGCA 1920 E P L M P D V V K K V T A D E V V P D C 627 ACCCTTATTCGGATGCTATATCTGAAGCCATCGACTATTTAAGGTCGACAGCTGAAATTT 1980 N P Y S D A I S E A I D Y L R S T A E I 647 CAAAAAGCAGGTTACAACAACTTGGTGAACATTGCAGGTGGAAGAAATATGGGTTCTCAA 2 040 S K S R L Q Q L G E H C R W K K Y G F S 667 CAGTTTGGGCTGGAGATGAATCCAGAAGAATTTTTCTACCTAAGGAGAATAGATGGGTAG 2100 T V W A G D E S R R I F L P K E N R W V 687 GACCCACATCTACTCGCCAAGTTGGTCCCAAAGCTCAATATGAGAGAGGTTATACCGTTA 2160 G P T S T R Q V G P K A Q Y E R G Y T V 707 ACGGTTATGTGAATTTCACGTGGGATGATGCCGGAAATGTTTCCGATGCCTGCGTACGAT 2220 N G Y V N F T W D D A G N V S D A C V R 727 CTCTCAGAGAATACGAAATCGTCATTGTTGATGATTCCTGTGTTTTCTCATCAGTGGAGA 2280 S L R E Y E I V I V D D S C V F S S V E 747 AGGTAATACCTTCACTGGAAAAAGCTTTGAAGATGAACTGTGATTTTTCAATCACAATTA 2340 K V I P S L E K A L K M N C D F S I T I 767 TGGACGGTGTTGCTGGTTGTGGAAAAACTACCAAGATTAAGTCTATTGCCTCTATGGTTG 2400 M D G V A G C G K T T K I K S I A S M V 787 GAGATGATATAGACTTACTCCTAACTTCCAACAGATCCTCAGCAATTGAGTTGAAAGAAG 2460 G D D I D L L L T S N R S S A I E L K E 807 CTGTTGAAGGGTCCCAGTTAGTTAAAAGTAGGTTCATTCGAACTTGCGATTCCTATCTGA 2 520 A V E G S Q L V K S R F I R T C D S Y L 827 Page 86 TGACAAACAATGCTCCTAAAGCAAAGAAAATGTTGTTTGATGAGTGTTTCATGCAACATG 2580 M T N N A P K A K K M L F D E C F M Q H 847 CTGGGGTGATATATGCTGCTGCCACAATTGCCGGTGTGTCAGAGGTTATAGCCTTTGGTG 2640 A G V I Y A A A T I A G V S E V I A F G 867 ACACTGAACAAATACCATTCATTTCCAGGAATGATATGTTTCTCCTGAAGCACCATGTTT 2 700 D T E Q I P F I S R N D M F L L K H H V 887 TGAAAGGTGACCATGTAAAACAAACAATTACATACCGAAATCCTGCTGATACGGTATATG 276 0 L K G D H V K Q T I T Y R N P A D T V Y 907 CTTTGTCTAAGTTCTTCTATAGAAAGAAGACGCCTGTTAAGACGAAAAGACACATTCTTA 282 0 A L S K F F Y R K K T P V K T K R H I L 927 GGTCTATTAAAGTTAAACCTATAAATGCTCTATCTCAGGTTGAGGTGGATGCCTCCGCTG 2880 R S I K V K P I N A L S Q V E V D A S A 947 TGTATGTTACGCATACTCAAGCTGAGAAGGCCAGTTTATTGGCTACTCCGAGTTTCAAAT 2 940 V Y V T H T Q A E K A S L L A T P S F K 967 CTTGTAAGATTTATACAACTCATGAGGTTCAAGGGGGTAGTTTTGACAAAGTTATATTTG 3 000 S C K I Y T T H E V Q G G S F D K V I F 987 TCAGACTTACTAGAACCAGTAATCATTTATACTCTGGTAAGCACCCTATAATGGGAGCTT 3 060 V R L T R T S N H L Y S G K H P I M G A 1007 GCCATGGACTCGTGGCTTTGTCAAGACACAAGTCGGAATTCATTTATTACACCCTAGCTG 3120 C H G L V A L S R H K S E F I Y Y T L A 1027 GAGGGGATAATGATGATATTCTTTTGAAAGCCTGTCAATACGCTGAAAGAGCGGATGACA 318 0 G G D N D D I L L K A C Q Y A E R A D D 1047 GTGATATTGTCAAACATTATGTTTGACCGTTCAGATTTTGTCACTGGACGTAAAAATCCT 324 0 S D I V K H Y V * * 1055 TTTGGTTAACTCGTACTGCGTACTTTTTGAGTTAAGATAAAAATGCCCATTGTATCCTGA 33 00 * ATGGATGACACTTTTTATTGCCTACAAATTTGTAGATGCCCTCACCGTAAGGTGAGGATG 3360 * CCCCTTAAGGATGC 33 74 Figure 10. The complete nucleotide sequence of PDV RNA1 and i t s putative t r a n s l a t i o n product. Numbers indicate nucleotide or amino acid position. * Indicates in-frame stop codons i n the 3'-UTR. Page 87 sequence i s 3374 nucleotides i n length and could encode a single protein of 1055 amino acids with a calculated molecular mass of 118.9 kDa. The length of the RNA sequence i s i n good agreement with the predicted length of 3.4 kb, estimated by denaturing agarose gel electrophoresis of t o t a l genomic PDV RNA (Figure 3). The calculated size of the protein i s s i m i l a r to that of AMV (128 kDa) and CiLRV 118.3 kDa). The i n i t i a t i o n codon for the 118.9 kDa protein i s at p o s i t i o n 3 9 and the f i r s t in-frame termination codon occurs at 3202. There are no other ORFs longer than 93 amino acids on the RNA. The 3'-UTR of RNA1 i s 171 bases long and shares extensive sequence homology with other i l a r - and alfamovirus 3'-UTRs (Figure 11A), including f i v e U/AUGC motifs which appear i n the 3'-UTRs of a l l known i l a r v i r u s sequences. In each AMV RNA these AUGC motifs flank short sequences which can form stem-loop structures (Houser-Scott et al. 1994). Although the intervening sequences are not s t r i c t l y conserved between PDV and AMV RNA1, the r e l a t i v e positions of the AUGC motifs are i n good agreement, and the 3'-end of PDV RNA1 can form e s s e n t i a l l y the same structure as the 3'-end of AMV RNA1 (Figure 11B). Page 88 C i L R V l UACAAACGUAGAUGCCUAUAUUUUCUCUCCUGAGAAAAUAUAGAUGCCUCCCAAGGAGAUGC TSV1 CUGAUGCUGUUUAUAUCUAAUGAUAUAAACAAUGCCUCCUUAAAGGAGAXGX AMV1 CGUGCUUAUGCACGUAUAUAAAUGCUCAUGCUAAATJUGCAUGAAUGCCCCUAAGGGAUGC PDV1 UAATJUGCCUACAAAUUUGUAGAUGCCCUCACCGUAAGGUGAGGAUGCCCCUUAAGGAUGC PDV3 UUGCCUACAAAAUUUUGUAGAUGCCCUCACCGUAAGGUTJGAGGAUGCCCCTJUUAAGGGAUGC U C A C U U G G U - A U - A A - U C=G C=G c A6 G=C U - A U A j C G A A - U A U C=G A - U A U C=G A - U A - U A - U U A A - U C=G C=G U A A - U A - U U - A C A U - A U - A C=G C=G A - U C=G C=G C=G A A G U U G C A U G C A U G C A U G C Figure 1 1 . Comparison of the 3'-ends of some i l a r v i r u s RNAs and AMV RNA1. A. The conserved U/AUGC motifs are underlined to demonstrate potential relationships. These motifs flank conserved sequences which could form stem-loop structures. B The f o l d i n g of the 3' end of PDV RNA1 i s very s i m i l a r to the 3' stem-loop structure proposed by Koper-Zwartoff & Bol ( 1 9 8 0 ) for the 3' end of AMV. Page 89 3.7.1. Phylogenetic relationships of PDV to other Bromoviridae, based on RNA1 The phylogenetic relationship of members of the Bromoviridae and RBDV i s predicted on the basis of the nucleic acid sequence of t h e i r RNAls and of t h e i r t r a n s l a t i o n products (Figure 12A). The numbers at the forks indicate the number of times the group to the right of the fork occurred out of a possible 100 trees. This p a i r i n g was reproducible and occurred regardless of whether the RNA or the amino acid sequence was analysed. Since there were two clear blocks of homology i n the PILEUP alignments of the s i x sequences, the SEQBOOT analysis was repeated using either the N-terminal or h e l i c a s e - l i k e domain (Candresse et al. 1990) blocks of homology i n d i v i d u a l l y . The r e s u l t i n g phylogeny was the same as i n Figure 12A. A s i m i l a r phylogeny r e s u l t s from parsimony analyses of bootstrap r e p l i c a t e s made with RNA3 and 0RF3a (the putative movement protein; Figure 12B). 3.8.0 Replication of a DI RNA Figure 13A shows the DI RNA (transcription product) on a non-denaturing agarose ge l . The DI RNA i s approximately 1 kb i n size and was transcribed from pPDVdil by T7 RNA polymerase. Figure 13B shows results of a Northern blot, using t o t a l RNA i s o l a t e d from plants inoculated with DI RNA i n the presence and absence of PDV as the target and a Pvul/Ncol fragment of pPDV33 as the probe. Bands which Page 90 ,00-Q i—lOO--10 0-1—100-AMV PDV CiLRV CMV BMV RBDV B -85— -99-i—lOO-H 00A oo-Q •AMV 1—PDV ApMV PNRSV CMV BMV CiLRV Figure 12. Phylogenetic relationships i n f e r r e d by parsinomy analysis with Phylip 3.4 using PROTPARS and DNAPARS of 100 Bootstrap r e p l i c a t e s generated by SEQBOOT. Nucleic acid and protein alignments were generated by PILEUP (GCG) and edited using XESEE. A. Relationships between RNA1 and ORF1 of at least one member of each genera of the Bromoviridae and RBDV. RBDV RNA1 encodes a polyprotein and only the C-terminal region was used i n the analyses. Fork numbers indicate the number of times the virus group to the right of that fork occured i n a t o t a l of 100 trees. B. Relationships between RNA3 and movement proteins (0RF3a) using the same analyses as i n A. Page 91 (A) ( B ) Figure 13A. Agarose g e l e l e c t r o p h o r e s i s of the p r o d u c t of in vitro t r a n s c r i p t i o n o f p D I l . B: N o r t h e r n b l o t a n a l y s i s o f p l a n t t o t a l RNA, probed w i t h a P v u l / N c o l fragment o f pPDV33 (complementary t o the 5'-end of RNA1). Lane 1: DI RNA t r a n s c r i p t ( p o s i t i v e c o n t r o l ) ; l a n e 2: RNA from p l a n t i n o c u l a t e d w i t h PDV and DI RNA; l a n e 3: RNA from p l a n t i n o c u l a t e d w i t h PDV o n l y ; l a n e 4: RNA from p l a n t i n o c u l a t e d w i t h DI o n l y ; l a n e 5: RNA from u n i n o c u l a t e d p l a n t ( n e g a t i v e c o n t r o l ) . P l a n t s (pumkins) were i n o c u l a t e d w i t h DI RNA 1 week a f t e r t h e y had been i n o c u l a t e d w i t h PDV and l e a v e s were h a r v e s t e d 3 days l a t e r . Page 92 hybridized are v i r a l RNA1 r e p l i c a t i n g i n the leaves. The DI RNA f a i l e d to r e p l i c a t e in vivo using PDV- infected pumpkins as a herbaceous model system and there were no bands at approximately 1 kb (the pos i t i o n expected for the DI RNA) which hybridized to the probe. Furthermore the presence of inoculated DI RNA did not appear to influence the copy number of PDV RNA1. Page 93 4.0 DISCUSSION AND CONCLUSION 4.1.0 Virus i s o l a t i o n and nucleic acid analysis An e x i s t i n g i s o l a t i o n technique, developed by Fulton (1959), was modified to include a sedimentation v e l o c i t y u l t r a c e n t r i f u g a t i o n step. This removed many of the protein contaminants present i n e a r l i e r fractions and resulted i n a single major band corresponding to the coat protein of PDV when analyzed by SDS PAGE. This p u r i f i e d v i r u s was used to immunize animals to produce polyclonal and monoclonal antibodies. The virus preparation was also found to be r e l a t i v e l y free of plant ribosomes and was thus suitable for i s o l a t i n g single-stranded genomic v i r a l RNA which was r e l a t i v e l y free of plant rRNA. The v i r a l RNA was of good quality, since the virus capsid protects the RNA to some extent against degradation by RNases i n the early stages of the p u r i f i c a t i o n . This single-stranded RNA was used for reverse t r a n s c r i p t i o n and the preparation of a cDNA l i b r a r y . It was also possible to i s o l a t e dsRNA from infected leaves using c e l l u l o s e chromatography. This dsRNA was contaminated to some extent by plant rRNA but was used only i n Northern blots and for RACE PCR where plant rRNA contamination posed no problems. Since dsRNA i s resistant to degradation by many RNases under conditions encountered during i s o l a t i o n , i t was also of good qua l i t y . A disadvantage of the dsRNA i s evident from Figure 3: whereas Page 94 the single stranded RNA bands representing RNA1 and RNA2 are present i n approximately equal amounts, there i s s i g n i f i c a n t l y more RNA3 and RNA4 (Figure 3A). The reason for th i s i s that the coat protein (on RNA4) and movement protein (on RNA3) are required i n higher quantities than the replicase components on RNA1 and RNA2. In contract, i n the dsRNA preparations RNA1 and RNA2 are not v i s i b l e when stained with ethidium bromide, whereas RNA3 i s a clear band (Figure 3b) . 4.2.0 Monoclonal antibody production A single hybridoma secreting antibody was obtained from the fusion. This monoclonal antibody (PDA-3C) does not cross react with PNRSV but recognizes a l l strains of PDV tested. PDV i s s e r o l o g i c a l l y conserved and i t was not anticipated that s e r o l o g i c a l variants would be found. PDA-3C was shown to recognize the coat protein of PDV by an immunoblot. The hybridoma l i n e producing PDA-3C i s stable, divides r e a d i l y i n tissue culture and adequate amounts of antibody could be i s o l a t e d d i r e c t l y from spent TCS obviating the need for ascites f l u i d production. It i s not clear from t h i s study whether the immunization protocol, using cyclophosphamide to manipulate the immune response, was advantageous. Page 95 4.3.0 T r i p l e antibody sandwich ELISA A r e l i a b l e TAS-ELISA using PDA-3C was developed to detect the presence of PDV i n infected sweet cherry leaves. Although the t i t r e of i l a r v i r u s e s i s high i n pollen, most of the assays presented here were performed using young, apical leaves. The reason for thi s i s that leaves were available for a longer period, they were available on a l l trees regardless of age and they generally gave lower background values by TAS-ELISA than flowers. The re s u l t s of the TAS-ELISA performed on the test trees were compared to the results of an independent RT-PCR assay and to a bioassay on 'Shirofugen'. The RT-PCR re s u l t s were taken to be more r e l i a b l e than the bioassay r e s u l t s . Although the bioassay i s sensitive to a l l known strains of PDV, the gumming reaction i s not always unequivocal. For th i s reason, the RT-PCR alone was used to i d e n t i f y infected trees. There was a single tree i n the sample which was deemed infected by RT-PCR but f a i l e d to y i e l d a p o s i t i v e r e s u l t with the bioassay (see Table 4). A possible reason for t h i s discrepancy i s that the t i t r e of PDV may have been too low to e l i c i t a gumming reaction i n the 'Shirofugen' assay but was nevertheless high enough to be detected by the more sensi t i v e RT-PCR assay. A common concern of the use of monoclonal antibody-based te s t i n g for plant viruses i s the p o s s i b i l i t y of obtaining fal s e negatives when a s e r o l o g i c a l l y d i s t i n c t s t r a i n i s Page 96 encountered. Strains of PDV appear to be s e r o l o g i c a l l y conserved. This i s evident from t h i s work where out of 31 trees p o s i t i v e for PDV by RT-PCR, a l l but one were detected by TAS-ELISA, and from s i m i l a r findings by other groups using monoclonal sntibodies (McMorran & Cameron 1983; Torrance & Dolby 1984) who were able to detect by ELISA a l l strains of PDV tested. Also, two severe strains of PDV obtained from the virus c o l l e c t i o n i n Wenatchee, WA and unavailable i n the virus c o l l e c t i o n of the Summerland Research Centre, were detected by PDA-3C i n the TAS-ELISA. PDA-3C did not recognize PNRSV i n the TAS-ELISA, and t h i s i s the only other i l a r v i r u s known to i n f e c t sweet cherry. The s t a t i s t i c a l analysis indicates that the d i s t r i b u t i o n of PDV i s i r r e g u l a r within the trees used i n t h i s study. Although the majority of the infected trees harboured detectable l e v e l s of PDV i n at least two-thirds of t h e i r leaves, the proportion varied. This i s contrary to the uniform d i s t r i b u t i o n of virus reported by Torrance & Dolby (1984) . This incongruity may be a r e f l e c t i o n of the number of growing seasons since the i n i t i a l virus i n f e c t i o n . The consequences of an uneven virus d i s t r i b u t i o n on the TAS-ELISA resu l t s can be p a r t i a l l y a l l e v i a t e d by s e l e c t i n g sample leaves from limbs which display c h a r a c t e r i s t i c PDV symptoms. Thus, when indexing a mature'orchard with a history of PDV, where older infections are expected, the value of Pr could be raised, and fewer leaves collected. In foundation plantings Page 97 of v i r u s - f r e e trees, where recent infections would be more prevalent, one would lower the value of Pr and test a greater number of leaves to detect PDV more r e l i a b l y . Since leaf samples from the same tree can be pooled for TAS-ELISA (Torrance & Dolby 1984), and loaded into the same well i n the m i c r o t i t r e plate, assaying more leaves does not s i g n i f i c a n t l y increase the scale of the assay. The TAS-ELISA could detect PDV with 99% p r o b a b i l i t y i n trees with two-thirds of t h e i r leaves infected, i f four leaves were assayed at random. 4.4.0 Alternate trapping antibodies To ensure an i n d e f i n i t e supply of antibodies for the ELISA assay, i t was necessary to attempt to design an assay which does not r e l y on the a v a i l a b i l i t y of polyclonal antibodies. Alkaline phosphatase was linked d i r e c t l y to PDA-3C and t h i s conjugate was used i n a DAS-ELISA format. Using another approach, the rabbit polyclonal antibodies were replaced with chicken IgY as trapping antibodies. Although the IgY antibodies are polyclonal and hence i n l i m i t e d supply, can be produced i n very large quantities. The most promising assay design used F(ab') 2 fragments to trap PDV i n a TAS-ELISA. These are the product of a peptic digest of PDA-3C. This assay gave higher background values than the rabbit polyclonal antibodies used i n the TAS-ELISA for t h i s study (Table 6). Page 98 4.5.0 The p a r t i a l nucleotide sequence of RNA 3 The p a r t i a l nucleotide sequence obtained from PDV RNA3 clones i s i n strong agreement with the published sequence with only seven differences at the nucleotide and three at the amino acid l e v e l . Some of these differences may have been due to sequencing errors, e s p e c i a l l y nucleotide insertions and deletions which would have put the clones out of frame with the published sequence. Not a l l regions of RNA3 were sequenced from overlapping clones. The sequence was used to obtain primers for RT-PCR. One of the primer pairs was chosen from within ORF3b (the coat protein) since t h i s sequence i s also found on RNA4 and i s therefore present i n a higher copy number which would make RT-PCR with t h i s p a i r more sensitive. However, both primer pairs were capable of detecting PDV under a l l tested conditions and the extra copy-number was not an advantage i n t h i s study. The p a r t i a l sequence was not used for phylogenetic analysis a f t e r the f u l l length sequence became available. 4.6.0 The RT-PCR assay The RT-PCR assay, using one of two primer pairs, was found to be more sensitive than either the TAS-ELISA or the bioassay. Both primer pairs were tested against PNRSV and neither amplified DNA from RNA template from t h i s v i r u s . Tissue preparation for RT-PCR was more complex because Page 99 i t was more prone to cross-contamination. This was e s p e c i a l l y problematic when assaying flower tissue. It i s possible that the high t i t r e of virus within the pollen raised the chances of_cross-contamination. Assaying unopened buds i n winter was also possible but required even more extensive tissue preparation and was not optimal for assaying large numbers of samples. Again, leaves were used for most purposes because of a v a i l a b i l i t y and ease of preparation. When high s e n s i t i v i t y was required, or when re s u l t s of alternate assays were ambiguous, t h i s was the assay of choice and RT-PCR re s u l t s were taken to be more accurate than either bioassay or TAS-ELISA r e s u l t s . As with s e r o l o g i c a l assays, there i s a concern with PCR based assays that c e r t a i n minor changes, even point mutations, i n the target might lead to fals e negative re s u l t s , i f they cause the primer to f a i l to anneal. Using two primer pairs reduces t h i s r i s k and false negatives from RT-PCR were not encountered i n t h i s study. 4.7.0 The complete nucleotide sequence of RNA1 The complete nucleotide sequence or RNA1 was obtained by sequencing eight overlapping clones and 12 RACE PCR clones to obtain the end sequences. The sequence of RNA1 i s i n good agreement with other published Bromoviridae RNA1 sequences and the genome organization appears to be the same: RNA1 i s monocistronic and the gene product shows extensive sequence homology at the Page 100 amino acid level' to RNA1 products of other Bromoviridae ( v i r a l replicase proteins). The AUGC motifs i n the 3'-UTR are also hallmarks of i l a r v i r u s sequences, although these motifs are also found i n AMV. The r e l a t i v e positions of the AUGC motifs are similar, and the 3'-end of PDV RNA1 can form e s s e n t i a l l y the same structure as the 3' end of AMV RNA1 (Figure l i b ) . In AMV these structures are involved i n coat protein binding during genome r e p l i c a t i o n and contain at least two binding s i t e s for the coat protein. (Reusken et al. 1994). The coat protein of the i l a r - and alfamovirus genera i s required to i n i t i a t e r e p l i c a t i o n . The coat protein of TSV i s able to i n i t i a t e r e p l i c a t i o n of AMV RNA i n the absence of AMV coat protein (Reusken et al. 1995). This implies that the mechanism of r e p l i c a t i o n i n i t i a t i o n i n i l a r v i r u s e s and AMV i s s i m i l a r and that the s t r u c t u r a l elements i n the 3'-UTR of PDV may also play an important role i n coat protein recognition and i n the i n i t i a t i o n of r e p l i c a t i o n . However, there i s no experimental evidence available for t h i s . The bromo- and cucumoviruses do not require coat protein to i n i t i a t e r e p l i c a t i o n and t h e i r 3'-UTRs f o l d into a tRNA-l i k e structure (Perret et al. 1989). These viruses do not have the AUGC motifs and there i s no sequence s i m i l a r i t y with the i l a r v i r u s e s or with AMV i n the 3'-UTR. Page 101 4.8.0 Phylogenetic relationships among Bromoviridae based on the sequence of RNA1 The closer p a i r i n g of PDV with AMV than with CiLRV was unexpected, since the l a t t e r i s an i l a r v i r u s whereas AMV i s the only member of the Alfamovirus group. Arguments for the in c l u s i o n of AMV i n the i l a r v i r u s group have appeared p e r i o d i c a l l y i n the l i t e r a t u r e , but AMV remains the only member of the alfamovirus group because of differences i n p a r t i c l e morphology and mode of transmission. The p a i r i n g of AMV with PDV was reproducible and occurred regardless of whether the RNA or the amino acid sequences were analyzed. This indicates that there i s a closer phylogenetic relationship between PDV RNA1 and AMV RNA1 than between the RNA1 sequences of PDV and CiLRV. This re l a t i o n s h i p was not observed using sequence data from RNA3 (Guo et al. 1995). The same phylogeny as i n Figure 12A occurs using parsimony analyses of bootstrap r e p l i c a t e s made with RNA3 and ORF3a (the putative movement protein; Figure 12B) but not with 0RF3b (the coat protein; r e s u l t s not shown). Analysis with 0RF3b sequences alone yielded a s l i g h t l y d i f f e r e n t consensus tree, but with the fork numbers <50 at some branch points. Grieco et al. (1995) have done a more comprehensive analysis on the phylogenetic relationships of Bromoviridae coat proteins but t h e i r analysis did not include PDV. If v i r a l replicase protein(s) are more r e l i a b l e than Page 102 coat protein(s) for determining phylogenetic relationships among plant viruses, then according to the r e s u l t s presented here, PDV i s more c l o s e l y related to AMV than to CiLRV. It should be noted that RNA2 of Bromoviridae also encodes a protein with a replicase function (Cornelissen et al. 1983£>, Ge & Scott 1994) and contains a GDD motif which i s highly conserved amongst a l l RNA viruses (Kamer & Argos 19 84). The sequence of PDV RNA2, which i s currently unavailable, w i l l help to c l a r i f y the phylogeny. 4.9.0 Production and r e p l i c a t i o n of the a r t i f i c i a l DI RNAs It was not possible to synthesize the snapback-type DI RNA as set out i n section 2.4.2 and 2.4.3. The PCR primer designed to incorporate the T7 promoter into the template for t r a n s c r i p t i o n was highly prone to form secondary structures by self-annealing. Since there was no choice i n the sequence of t h i s primer, the only alternative was to optimize the PCR conditions. Even under optimal conditions, the y i e l d of the required bands was low and there were contaminating bands which could not be eliminated. The subsequent l i g a t i o n of the two PCR products into pUC18, to form a template for the t r a n s c r i p t i o n of a snapback-type DI RNA was not f e a s i b l e . The reasons for t h i s are not clear, but i t i s possible to speculate that the palindromic DNA sequence i n the plasmid was unstable and could not be maintained. This l i g a t i o n also f a i l e d when i t was attempted with Page 103 oligonucleotide cassettes attached to pUC18 (sections 2.4.3 and 2.4.4) . Since the l i g a t i o n s were very s i m i l a r i t i s l i k e l y that they f a i l e d for the same reason. Preparation of a deletion-type DI RNA proved to be much simpler, but the DI RNA f a i l e d to r e p l i c a t e in vivo i n a herbaceous model system. The reasons for t h i s are not known. The Northern blot (Figure 13b) shows the absence of a band i n the 1 kb region and shows that l e v e l s of v i r a l genomic RNA1 were not affected by the presence of the DI t r a n s c r i p t . The a b i l i t y of v i r a l RNA to r e p l i c a t e in vivo i s sequence dependent. The sequence at the 5'-end i s e s p e c i a l l y important for infectious v i r a l t r a nscripts (reviewed by Boyer & Haenni 1994) and the presence of any non-viral nucleotides usually reduces i n f e c t i v i t y . The oligonucleotide cassette at the 5'-end of the insert i n pPDVdil was designed to bring the T7 promoter d i r e c t l y adjacent to the f i r s t 5' nucleotide of PDV RNA1 so that there would be no nonviral nucleotides at the 5'-end. The sequence of the template (pPDVdil) was confirmed before t r a n s c r i p t i o n to ensure that the ends were unchanged during the plasmid construction. The t r a n s c r i p t i o n of an RNA molecule of the correct size by T7 RNA polymerase was confirmed by denaturing agarose gel electrophoresis. The presence of r e p l i c a t i n g PDV genomic RNA i n the plants was confirmed on the autoradiograph by the presence of an RNA1 band which hybridized to the probe. In a s i m i l a r study with BMV, Marsh et al. (1991) found that the Page 104 length of a r t i f i c i a l BMV DI RNA did not greatly a f f e c t the l e v e l of i t s own r e p l i c a t i o n although they showed that a smaller DI RNA reduced the synthesis of RNA1 and RNA2 more e f f i c i e n t l y than a larger DI RNA. It i s therefore u n l i k e l y that a DI RNA from PDV of a d i f f e r e n t size (ie. a deletion clone of pPDVdil) would r e p l i c a t e i n pumpkins. 4.10.0 Concluding remarks Two r e l i a b l e assays to detect PDV i n infected f r u i t trees have been developed. One i s a TAS-ELISA which i s based on a monoclonal antibody and can detect PDV with 99% confidence i n trees which have 2/3 or more of t h e i r leaves infected. The assay i s suitable for indexing large numbers of trees and the monoclonal antibody reacts against a l l str a i n s of PDV tested. An RT-PCR assay was also developed and t h i s was found to be more sensitive than the TAS-ELISA and could be used for a longer period during the growing season. This assay was more complex than the TAS-ELISA and not as well suited to indexing large numbers of trees. The complete nucleotide sequence of PDV RNA1 was also determined. This data was used to investigate the phylogenetic relationship of PDV to AMV and CiLRV. It was shown that, based on t h i s sequence, PDV i s more c l o s e l y related to AMV than to CiLRV. The sequence was also used to construct DI RNA. A deletion-type DI p a r t i c l e was made but f a i l e d to r e p l i c a t e in vivo using a herbaceous host. Page 105 BIBLIOGRAPHY A l r e f a i , R.H., Shiel, P.J., Domier, L.L., D'Arcy, C.J., Berger, P.H. & Korban, S.S. (1994). The nucleotide sequence of apple mosaic virus coat protein gene has no s i m i l a r i t y . with other Bromoviridae coat protein genes. Journal of General Virology 75: 2847-2850 Bachman, E.J., Scott, S.W., Xin, G. & Vance, V.B. (1994). The complete nucleotide sequence of prune dwarf virus RNA 3: implication for coat protein a c t i v a t i o n of genome r e p l i c a t i o n i n i l a r v i r u s e s . Virology 201: 127-131 Barker, R.F., Javis, N.P., Thompson, D.V., Loesch-Fries, L.S. & H a l l , T.C. (1983). Complete nucleotide sequence of a l f a l f a mosaic virus RNA3. 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