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Molecular cloning of blueberry shock ilarvirus (BSIV) RNA 3 containing the coat protein gene and identification… Bremsak, Irene Jane 1994

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MOLECULAR CLONING OF BLUEBERRY SHOCK ILARVIRUS (BSIV)RNA 3 CONTAINING THE COAT PROTEIN GENEANDIDENTIFICATION OF A VIRUS ASSOCIATEDWITH BSIV-INFECTED BLUEBERRY BLOSSOMSbyIRENE JANE BREMSAKB.Sc. (Hons.), Carleton University, 1989A THESIS SUBMITTED IN PARTIAL FULFILMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIES(Department of Plant Science)We accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAAugust 1994© Irene Jane Bremsak, 1994In presenting this thesis in partial fulfillment of therequirements for an advanced degree at the University of BritishColumbia, I agree that the Library shall make it freely availablefor reference and study. I further agree that permission forextensive copying of this thesis for scholarly purposes may begranted by the head of my department or by his or herrepresentatives. It is understood that copying or publication ofthis thesis for financial gain shall not be allowed without mywritten permission.(Signature)Department of__________________The University of British ColumbiaVancouver, CanadaDate S2fi1IJJ& alc?9LY.AbstractBlueberry shock ilarvirus (BSIV) was cloned using random primers. ComplementaryDNA (cDNA) was ligated into Bluescript II and used to transform into DH5a Escherichiacoil. The plasmids were screened for BSIV RNA 3 cDNA by Northern, Southern and dotblots. Seven clones were restriction mapped. A 412 base pair region of three clones wassequenced and compared to tobacco streak, citrus variegation, apple mosaic and prune dwarfilarvirus coat proteins (cp) at the nucleotide and amino acid levels. The BSIV cp amino acidsequence was 61% homologous with that of apple mosaic virus cp. BSIV virions werelocalized in ‘Bluetta’ and ‘Rancocas’ blueberry pollen by immunoelectron microscopy.The identity of a virus co-isolated with BSIV from blueberry blossoms wasdetermined. It’s host range and symptomatology differed significantly from BSIV. Purifiedvirions were isometric, Ca. 32 nm in diameter, with electron dense centres. The cp subunithad a molecular weight (Mw) of 30 kD. The genome was single-stranded and tripartite withM’ s of 1.19, 1.01, 0.74 and 0.37 x 106 D and when the double-stranded RNA profile wascompared to that of cucumber mosaic virus (CMV) isolated from primula, they wereidentical. Polyclonal antiserum and monoclonal antibodies were produced. The polyclonalantiserum reacted with CMV serotypes I and II; all the monoclonal antibodies reacted withCMV-II, although some monoclonal antibodies reacted with CMV-I. The virus, calledCMV-B since it was isolated from blueberry blossoms, was an isolated of CMV-II.A relationship between BSIV and CMV in blueberry plants was investigated. Noneof the 650 BSIV infected blueberry tissue samples tested in 1993 were infected with CMV.Vegetation within and near the BSIV-infected field, was surveyed and seven of nineteenspecies tested were infected with CI\4V. It was concluded that CMV-infected pollendeposited by pollinators on the blueberry blossoms was the source and that blueberry wasprobably not a host of CMV.11Table of Contents• 11• 111AbstractTable of ContentsList of Figures.List of TablesList of AbbreviationsAcknowledgementsCHAPTER I - GENERAL INTRODUCTION1 BlueberriesCHAPTER II - CLONING THE COAT PROTEIN GENE OF BSIV.1 Ilarviruses1.1 BSIV1.2 Research objectives2 Materials and methodsvivii’ixxiv116612141515151718192021212222232425252626272.1 BSIV Propagation and host reactions to infection2.1.1 Electron microscopy2.2 BSIV Purification2.2.1 Sodium dodecyl sulphate polyacrylamide gel electrophoresis2.3 Virion RNA extraction2.3.1 Methylmercuric hydroxide agarose gel electrophoresis ofRNA2.4 BSIV Cloning strategies2.4.1 First-strand eDNA synthesis2.4.2 Second-strand eDNA synthesis2.4.3 Isolation of cDNA from unincorporated nucleotides2.4.4 Ligation of cDNA into a vector2.4.5 Transformation of competent E. coil cells2.5 Screening eDNA clones2.5.1 ‘Alkaline lysis method’ of plasmid isolation2.5.2 Restriction enzyme digestion of mini-prep DNA2.5.3 Random primed cDNA probes2.5.4 Sap dot blots using Zetaprobe membrane111272829• 29• 29303030313132333 Results3.13.23.33.43.53.63.73.8Host reactions to BSIV infectionElectron microscopyPurificationRNA ExtractionCloning BSIV RNA 3Dot blots with BSIV-infected sapNorthern, Southern and dot blots with plasmid DNANucleotide and protein sequence analysis4 Discussion 58CHAPTER III - CMV ISOLATED FROM BLUEBERRY FLOWERS641 Cucumoviruses1.1 CMV1.2 Research objectives2 Materials and methods2.1 Characterization of an unknown virus2.1.1 Source of virus and propagation2.1.2 Mechanical transmission2.1.3 Purification2.1.3.1 SDS-PAGE2.1.4 Virion RNA extraction2.1.5 Double-stranded viral RNA extraction2.2 Serology2.2.1 Preparation of Polyclonal antiserum2.2.1.1 Purification and conjugation of y-globulin2.2.1.2 Double antibody sandwich ELISA2.2.1.3 Relationship to ilarvirus group2.5.5 Zetaprobe’ membrane hybridization and washing conditions2.6 Northern analysis using formamide-formaldehyde gels2.6.1 Transfer of RNA from agarose gels to Zetaprobe membrane2.7 Identifying and mapping BSIV cDNA clones2.7.1 Dot blots using plasmid DNA2.7.2 Restriction enzyme analysis2.7.3 Southern blots using Zetaprobe membrane2.8 Sequencing2.8.1 Preparation of subclones2.8.2 Sequencing with Sequenase®2.8.3 Sequencing gels2.8.4 Sequence analyses34343637374444464964656670707070717272737474757677iv2.2.1.4 Polyclonal antiserum specificity 772.2.2 Preparation of monoclonal antibodies 792.2.2.1 Screening McAb 792.2.2.2 Monoclonal antibody specificity 802.3 Survey of CMV-B in BSIV-infected blueberry plants 812.3.1 Collection of blueberry samples 812.3.2 DAS-ELISA to test blueberry samples 812.3.3 Collection of nearby vegetation 822.3.4 DAS- and TAS-ELISA to test vegetation 822.3.5 Mechanical transmission of CMV-B infected buttercupsamples 823 Results 833.1 Identification and characterization of second virus . . 833.2 Polyclonal Antibodies 893.3 Monoclonal antiserum 923.4 Survey of CMV-B in BSIV-infected blueberry plants 954 Discussion 99CHAPTER IV - SUMMARY AND CONCLUSIONS 104Bibliography 106vList of FiguresFigure 1A: N clevelandii 7 days after mock inoculation with phosphate buffer 35Figure 1B: N clevelandii 7 days after inoculation with BSIV 35Figure 2A-B: Thin section of healthy ‘Bluecrop’ pollen decorated with BSIV PcAsand labelled with protein A gold 38Figure 3A-B: Thin section of BSIV-infected ‘Bluetta’ and’Rancocas’ pollendecorated with BSIV PcAs and labelled with protein A gold 40Figure 4: Purified BSIV 42Figure 5: SDS-polyacrylamide gel of BSIV and CMV-B cp subunits 43Figure 6: Methylmercuric hydroxide gel of BSIV and CMV-B RNA 45Figure 7A-B: Nucleic acid hybridization of sap dot blots with BSIV probes 47Figure 8A-B: Formamide-formaldehyde gel and Northern blot of BSIV RNA . . . 48Figure 9A-D: Detection of BSIV clones by nucleic acid hybridization 50Figure 10: Partial restriction maps of BSIV RNA 3 and 4 eDNA clones 52Figure 11: Partial nucleotide sequence of BSIV RNA 3 and 4 53Figure 12: Alignment of BSIVseq and ApMV cp 57Figure 13: N clevelandii 7 days after inoculation with an unknown virus 68Figure 14: Purified BSIV and an unknown virus decorated with BSIV PeAs . . 69Figure 15A-C:N tabacum ‘Xanthi’ and ‘Harrownova’ and Cucumis sativus 16 daysafter inoculation with CMV-B 86Figure 16: Purified CMV-B 88viFigure 17: DsRNA profiles of CMV-B. 91viiList of TablesTable 1: Members of the ilarvirus group 7Table 2: Known ilarvirus-vector-host interactions 8Table 3: Genomic data of ilarvirus subgroups 10Table 4: Results from Northern, Southern and plasmid dot blots 51Table 5: Comparison of nucleic acid sequences of BSIVseq and ilarviruses . 55Table 6: Comparison of amino acid sequences of BSIVpro and ilarviruses . 56Table 7: Ilarvirus antisera used to determine the serological reactivity of theunknown virus 78Table 8: Symptoms on indicator plants produced 16 days after inoculation withCMV-B 84Table 9: Estimated M and size of CMV ssRNA 90Table 10: Serological reactivity of CMV-B PcAs to cucumovirus serotypes . . 93Table 11: Serological reactivity of CMV-B McAb’s to cucumovirus serotypes 94Table 12: Survey for CMV-B in BSIV-infected blueberry fields 96Table 13: Survey for CMV-B in vegetation within and around BSIV-infectedblueberry fields 97Table 14: Extensive survey for CMV-B in species testing positive within andaround BSIV-infected blueberry fields 98viiiList of AbbreviationsA (mA) - amperes (milliamperes)A - absorbance at x nmaa - amino acidAAT - a selection medium for growing hybridoma cultures including adenine,aminopterin and thymidineA1MV - alfalfa mosaic virusAmp - ampicillinAPLPV - American plum line pattern virusApMV - apple mosaic virusAPS - ammonium persulphateATP - adenosine triphosphateAVII - asparagus virus IIBALB/c - a mouse line used for producing hybridomasBBScV - blueberry scorch virusB.C. - British ColumbiaBE - 40 mM borate, 1 mM EDTA buffer, pH 8.213-mer - 3-mercaptoethanolbp (kb) - base pair (kilobase pairs)BSA - bovine serum albuminBSIV - blueberry shock ilarvirusC - CelsiusCARNA 5 - CMV associated RNA 5cDNA - complementary DNACH3HgOH - methylmercuric hydroxideCi (j.iCi) - curies (microcuries)CiCLV - citrus crinkly leaf virusCIP - calf intestinal phosphataseCLRV - citrus leaf rugose virusixCMV - cucumber mosaic virusCO2 - carbon dioxidecp - coat proteincpm - counts per minuteCVV - citrus variegation virusD (kD) - dalton (kilodalton)DAS-ELISA - double antibody sandwich enzyme-linked immunosorbent assaydATP - deoxyadenosine triphosphatedCTP - deoxycytosine triphosphateddA - dideoxy adenosine triphosphateddC - dideoxy cytidine triphosphateddG - dideoxy guanosine triphosphateddT - dideoxy thymidine triphosphateDEPC-H20- diethylpyrocarbonate-treated waterdGTP - deoxyguanosine triphosphateDIG - digoxigeninDIG-dUTP - digoxigenin-labelled deoxyuridine triphosphateDMEM Dulbecco’s Modified Eagle MediumDNA - deoxyribonucleic acidDNAse - deoxyribonucleasedNTP - combination of DATP, DCTP, DGTP and dTTPdpi - days post inoculationdsrna - double-stranded RNADTT - dithiothreitolDTTP - deoxythymidine triphosphateEDTA - ethylenediaminetetraacetateELISA - enzyme-linked immunosorbent assayEMoV - elm mottle virusEtBr - ethidium bromideEtOH - ethanolxf - frameFcIV - Fragaria chiloensis ilarvirusFCS - fetal calf serumFOX-NY - a myeloma cell line used to produce hybridomasg (kg, mg, fig, ng) - gram (kilogram, milligram, microgram, nanogram)GAM - goat-anti-rabbitHC1 - hydrochloric acidHg - mercuryHJV - Humulus japonicus virus1120 - waterHOAc - acetic acidhr - hourhrp - herbicide resistance proteinHydMV - Hydrangea mosaic virusKC1 - potassium chloride1 (ml, tl) - litre (millilitre, microlitre)LB - Luria brothLiC1 - lithium chlorideLRMV - Lilac ring mottle virusm (cm, mm, .tm, nm) - metre (centimetre, millimetre, micrometre, nanometre)M (Mm) - molar, millimolarMcAb - monoclonal antibodyMeOH - methanolmm - minuteMnC12 - manganese chlorideMOPS - 3-(N-morpholino)propanesulfonic acidmRNA - messenger RNAM - molecular weightNaC1 - sodium chlorideNaCLO4 - sodium perchioratexiNa2HPO4- dibasic sodium phosphateNaOAc - sodium acetateNaOH - sodium hydroxidenc - non-coding sequenceNR - not reportednt - nucleotideNT - not testedParMV - Parietaria mottle virusPAT - L-phosphinothricin-N-acetyltransferase genepBKS - Plasmid Bluescript II KSPBS - phosphate buffered salinePcAs - polyclonal antiserumPDV - Prune dwarf virusPEG - polyethylene glycolPLRV - potato leaf roll virusPNRSV - Prunus necrotic ringspot viruspro - proteinPSV - peanut stunt virusPVP - polyvinylpyrrolidone, M 44,000RAM - rabbit-anti-mouseRNA - ribonucleic acidRNAse - ribonucleaserpm - revolutions per minuteRT - room temperatureSDI - serological differentiation indexSDS - sodium dodecyl sulphateSDS-PAGE - sodium dodecyl sulphate polyacrylamide gel electrophoresissec - secondSIVIP- skim milk powderSPLV - spinach latent virusxiiSSC - standard salt citratessrna - single-stranded RNASTE - 100 Mm NaC1, 50 Mm Tris-Ci, 1 mM EDTA bufferSTE-EtOH - STE buffer with 16% EtOHSuperscript - Moloney Murine Leukemia Virus RNase H- Reverse TranscriptaseTAE - 40 mM Tris-HC1, pH 8.0, 40 mM glacial acetic acid, 2 mM EDTA bufferTAMV - Tulare apple mosaic virusTAS-ELISA - triple antibody sandwich enzyme-linked immunosorbent assayTAV - tomato aspermy virusTBE - 0.89 Tris-HC1 M, 0.89 borate M, 20 mlvi EDTA bufferTCA - trichioroacetateTE - 100 mM Tris-Ci, pH 7.5, 10 mM EDTA bufferTEMED - tetramethylethylenediamineTMV - tobacco mosaic virusTris-Cl - tris(hydroxymethyl)aminomethane - hydrochloric acidTRSV - tomato ringspot virusTSV - tobacco streak ilarvirusTSWV - tomato spotted wilt virusTw - TweenU - unitsUA - uranyl acetateV - voltsv, vol - volumeW-wattsw - weightX-gal - 5-bromo-4-chloro-3-indolyl--D-galactosidexl”AcknowledgementsI would like to extend my sincere appreciation to my graduate research supervisor,Dr. R. R. Martin for his guidance, financial support and opportunity to work in his lab. Iwould also like to thank the members of my graduate committee, Dr. Brian Ellis andDr. James Hudson, for their helpful advice and suggestions in these studies.A special thank you is extended to Dr. Dean L. Struble, director of the VancouverResearch Station, Agriculture Canada, for providing the research facilities for my work. Iwish to thank the staff of the VRS for their help and support especially Dr. Stephan Winterfor assistance with nucleic acid hybridization, Dr. Fran Leggett and Mr. Frank Skelton fortheir patience in teaching transmission electron microscopy techniques, Dr. Tim Sit for hisexpertise in sequencing, Mrs. Jenny Vermuellen for her help in the monoclonal lab, Ms.Anita Quail for her helpful discussions with respect to technical assistance, Dr. MorvenMcLean and Jim Moody who collected samples for ELISA tests.This study was supported in part by the Natural Sciences and Engineering ResearchCouncil in the form of postgraduate scholarships, by the VRS and grants from the B.C.Blueberry Council, Oregon Blueberry Commission and Washington Blueberry Commissionheld by Dr. R. R. Martin.Finally, I want to express my sincerest appreciation to John Lee and my family inOttawa for their understanding, patience and encouragement.xivCHAPTER I - GENERAL INTRODUCTION1 BlueberriesBlueberries are an important small fruit crop in British Columbia, which is the secondlargest producer of blueberries in Canada. It is virtually the only province to producehighbush blueberries. Since 1979, land used for blueberry production has increased from2,500 acres to 5,000 acres. As the acreage increased, so too did the yield. In 1979, eightmillion pounds of blueberries were harvested, whereas in 1993 fifteen million pounds wereharvested. By the year 2000, BC may produce as much as thirty million pounds per year(Bains, 1994).Blueberries are members of the heath family Ericaceae (Eck and Childers, 1966).Several species are grown commercially in North America. These include highbushblueberry, Vaccinium corymbosum L. and V australe Small, lowbush blueberry, V.angustfolium Ait., and rabbiteye blueberry V. ashei Reade. Highbush blueberries areperennial, deciduous, woody shrubs. They grow to a height of 3 m. Berries are harvestedby hand or by machine. Lowbush blueberries grow wild mainly in central and easternCanada and in the Northeastern states of the U.S.A. (Strik, 1993). They are usually only 0.5m tall. They are hand picked locally.A large initial capital investment is required to establish a blueberry planting. Plantsare vegetatively propagated from hardwood cuttings called whips. They are nurtured inprotective frames for 2-3 years before being sold to farmers (Eck and Childers, 1966). Itmay take up to six years before the plants are in full production.1Pollination is very important since blueberry plants can set 100% of their blossoms.Most commercial blueberry crops require 80% pollination for a good yield. Other fruitcrops, such as apples and peaches need only 20%. The blueberry’s floral structure facilitatescross-pollination rather than self-pollination. When a pollinating insect touches the stamen,it is showered with pollen. Cross-pollination also has the side benefit of increasing berrysize (Eck and Childers, 1966).The maj or viral diseases affecting blueberries in the Pacific Northwest include tomatoringspot virus (TRSV), blueberry scorch virus (BBScV) and blueberry shock ilarvirus(BSIV). TRSV-infected blueberries are found only in Washington and Oregon. Diseasedplants exhibit circular chlorotic spots, 2-5 mm in diameter, on malformed leaves, stems andtwigs, while young leaves show leaf-strapping and mottling. TRSV is transmitted by thenematode Xiphinema americanum Cobb. Alternatively, it is also spread by vegetativepropagation, grafting and mechanical inoculation with infected sap (Ramsdell, 1987).BBScV, transmitted by aphids, causes blossom and leaf blight (Strik, 1993). Blossoms turngreyish-black in spring. If not pruned off, the blossoms may turn silver-grey, but do notdrop off. Leaves may become orange-brown. Infection may initially occur in only a fewbranches. Over the next 1-3 years, the entire bush will become infected. BSIV will bydiscussed in detail in Chapter II. All three diseases can be detected by enzyme linkedimmunosorbent assay (ELISA) and bioassays, during which it is important to test severaldifferent leaves on the bush, since the distribution of the virus may not be uniform.The measures commonly taken to prevent and curb virus diseases are imperfect.Growers are encouraged to plant only certified, virus-free, planting stock. While breeders2have tried to breed natural resistance against viruses or their vectors into the crop, it isessentially a prohibitive investment of time and labour due to the plasticity of plant viralgenomes and the presence of resistance-breaking virus isolates (Wilson, 1993). Onceidentified, virus-infected blueberry plants and those in surrounding rows with possible latentinfections, are rogued out and destroyed. This is a costly solution, since the grower hasalready invested a large amount of capital into the purchase of plants, irrigation equipment,fertilizers, chemical insecticides and fungicides. The financial losses incurred by growers,make blueberry plants prime candidates for novel strategies such as genetic engineering.Cross-protection has been used to protect crops when no other source of resistanceor control measure was available. Plants are infected first with a protecting or mild strainof virus. When later superinfected by a challenging or severe strain of the virus, plantsexhibit fewer or no symptoms. Cross-protection does not protect the inoculated plant againstall viruses, but only against closely related viruses. There are also other disadvantages. Forexample, the protecting strain may cause small, but significant crop losses. Secondly, it mayspread to other crops on which its effect is severe. The protecting strain may possiblymutate to a severe form, eventually leading to crop losses. Finally, the crop may developa synergistic reaction to an unrelated virus i.e. potato virus Y and potato virus X (Buck,1991).Transforming plants with virus genes may be a way of imitating cross-protection(Mayo, 1992). Virus gene-mediated resistance could protect crops grown annually from seedor perennial woody plants i.e. orchards, without the harmful side effects of cross-protection(Buck, 1991).3Coat protein-mediated protection was first reported by Powell-Abel and co-workersin 1986. Agrobacterium tumefaciens was used to mediate the transfer of the coat proteingene from tobacco mosaic virus (TMV) placed behind the 35S constitutive promoter fromcauliflower mosaic virus. Transgenic plants expressing cp exhibited symptoms much laterthan susceptible plants. Today, transgenic dicotyledons, including tobacco, potato, sugar beetand canola, have shown varying degrees of resistance to more than twenty positive-sense,single-stranded RNA (ssRNA) viruses from at least ten different taxonomic groups (Wilson,1993). Transgenic plants can be protected against heterologous viruses if the amino acidsequences of the coat proteins are appreciably similar (Mayo, 1992).Virus resistance is defined as the ability of a plant to either delay symptomdevelopment or prevent it. Resistance is caused by a decrease in the number of infectedcentres, a slower systemic spread, or both. The overall effect results in reduced virusmultiplication, and consequently decreased virus accumulation in infected tissue (Wilson,1993). Usually, the degree of protection varies inversely with the concentration of theinoculum. In some cases, the level of protection correlates with the concentration of coatprotein synthesized in transgenic cells (Mayo, 1992). Coat protein-mediated resistance ismeiotically stable over several generations (Buck, 1991).In general, resistance falls into one of two categories: resistance to inoculation withvirus particles, but not to RNA, or resistance to both (Mayo, 1992). Both patterns can beexplained by either of the following theories. First, protection can be the result of an excessof coat protein, which prevents uncoating or re-encapsidates the challenging virus’s genome.Alternatively, it can be due to the inhibitory interactions between sense and antisense RNA’ s4of the two competing viruses (Wilson, 1993). A challenging, positive-strand RNA virusinitiates replication by synthesising a negative-strand of RNA, which in turn hybridizes withmRNA from the transgene. This hybridization prevents the challenging virus from actingas a template for synthesis of positive strands (Buck, 1991).5CHAPTER II- CLONING THE COAT PROTEIN GENE OF BSIV1 IlarvirusesIlarviruses are defined as a group of plant viruses with isometric labile particlesusually producing ringspot symptoms mainly in woody hosts (Fulton, 1983). Its membersare listed in Table 1. Some have wide host ranges, infecting plants in ten different families,while others have narrow host ranges infecting only a few plant species.Complete invasion of woody hosts often requires more than one year, spreadingslowly from one or a few infected branches throughout the bush. Systemic invasion ofherbaceous hosts is faster. Necrotic shock affecting both leaves and flowers may occur inthe early stages of infection, then disappear after a few weeks. The leaves of infected plantsmay become chiorotic or exhibit bright yellow discolouration in the form of blotching,stippling, ring spotting or oak leaf and line patterns. If necrotic shock symptoms persist,they may return in cycles, either seasonally or yearly. Plants often recover, but remainassymptomatic, although the leaves still contain virus.One mechanism for natural transmission has been proposed (Sdoodee and Teakie,1993). Pollinators carry infected pollen to healthy plants. There, thrips inoculate the plantsby feeding on both the infected pollen and the healthy flower tissue. This means oftransmission has recently been proven experimentally for three ilarvirus subgroups (Table2).Ilarviruses are both pollen- and seed-borne (Fulton, 1983). Mechanical transmissionfrom woody to herbaceous hosts depends on judicious selection of source tissue. Since the6Table 1: Members of the ilarvirus groupSubgroup Virus Reference1 Tobacco streak IVHydrangea mosaic II2 Asparagus virus II IVCitrus crinidy leaf IICitrus variegation IVElm mottle IVTulare apple mosaic IVLilac ring mottle IIElm mosaic IFragaria chiloensis V3 Apple mosaic IVBlueberry shock ilarvirus IIIHumulus japonicus IVPrunus necrotic ringspot IV4 Prune dwarf IV5 American plum line pattern IV6 Spinach latent IV7 Parietaria mottle IITentative Elm mosaic IMembers Pear ringspot IPelargonium zonate spot ISunflower ringspot II - Brunt et al., 1990II - Francki, 1985III - MacDonald and Martin, 1991IV - Mink, 1992V - Speigel et al., 19937Table2:Knownilarvirus-vector-hostinteractions00I-Fulton,1983II-GreberetaL,1991aIII-Greberetal.,199lbIV-GreberetaL,1992V-Mink,1992VI-SdoodeeandTeakie,1993NR-datanotreportedSubgroup,VirusSourceofpollenVectorHostReferenceTobaccostreakLycopersiconesculentumThripstabaciChenopodiumVIamaranticolorTobaccostreakNicotianaclevelandii7:tabaciC.amaranticolorVITobaccostreakNicandraphysalodes7:tabaciC.amaranticolorVITobaccostreakAgeratumhoustonianumthripsC.amaranticolorIITobaccostreakNRFrankliniellaoccientalisNRINRVTobaccostreakNRMicrocephalothrisNRIIabdominalis3PrunusnecroticPrunusaviumFoccidentalisCucumissativusIVringspotPrunusnecroticP.cerasusFoccidentalisC.sativusIVringspotPrunusnecroticPlummixof7:imaginis,7:tabaciC.sativusIIIringspotand7:australis4PrunedwarfP.aviumF.occidentalisC.sativusIVPrunedwarfP.cerasusFoccidentalisC.sativusIVvirus is often found in low concentrations in leaf tissue, young leaves or flower tissuecontaining infected pollen should be used (Francki, 1985). Two other laboratory methodsused when mechanical inoculation fails, are vegetative propagation and grafting (Fulton,1983).Purified virus preparations are unstable. They remain infectious in vitro between0.1-6.0 days and longer in the presence of ethylenediaminetetraacetate (EDTA) (Francki,1985). Particles are often fixed with glutaraldehyde if used to produce antibodies.The particles of a single isolate may vary in shape and size (Table 3). Someilarviruses such as BSIV consist of identical particles with a diameter of Ca. 30 nm. Otherssuch as Fragaria chiloensis ilarvirus (FCIV) consist of three or more types of quasi-isometricto bacilliform particles ranging in width from 12-3 5 nrn and in length from 20-70 nm (Mink,1992, Spiegel et al., 1993). Centrifugation through sucrose gradients separates particles onthe basis of size and density.Ilarviruses have single-stranded, tripartite, positive-sense RNA genomes. RNA 1 andRNA 2 are separately encapsidated, one genomic species per virus particle, whereas RNA3 and its subgenomic messenger, RNA 4, are found together in a separate particle. Eachparticle has approximately the same proportion of nucleic acids to protein, 13-24% (Francki,1985).Capsids are made up of coat protein (cp) subunits with a molecular weight (Mw) of19-30 kD (Table 3). The arrangement of cp subunits in the capsomere however, is not easilyseen (Fulton, 1983). Virions are easily dissociated in strong salt solutions or in solutionscontaining sodium dodecyl sulphate (SDS) and therefore probably held together by9Table3:GenomicdataofilarvirussubgroupsRNA(106 D)CoatSubgroupVirusProteinReference1234(kD)1tobaccostreak1.1-1.30.9-1.10.7-0.90.328III2asparagusvirusII,1.0-1.10.9-1.00.70.319-28IIcitruscrinklyleaf,citrusvariegation,lilacringmottleTulareapplemosaic3applemosaic,1.23-1.30.89-1.000.67-0.690.27-0.3125Iprunusnecroticringspot4prunedwarf1.260.950.760.6824I5Americanplumline1.1-1.30.9-1.10.7-0.9NRNRII6spinachlatent1.31.180.910.3528INR-datanotreportedII-Mandahar,1989I-Francki,1985III-Loesch-FriesetaL,1977electrovalent bonds (Francki, 1985). In addition, the capsid is susceptible to ribonucleases(RNAses) and to trypsin. This indicates that the virion may also be stabilized by proteinRNA interactions (Mink, 1992).RNA species 1, 2, and 3 alone are not infectious. All of these species and eitherRNA 4 or the cp are needed for infectivity. The requirement of RNA 4 or the cp forinfectivity may be fulfilled by RNA 4 or the cp of homologous or heterologous ilarvirusesor alfalfa mosaic virus (A1MV). For example, citrus leaf rugose virus (CLRV) RNA 1, 2and 3 can be activated by the addition of citrus variegation virus (CVV) RNA 4, the roseisolate of prunus necrotic ringspot (PNRSV) RNA 4, CVV cp or tobacco streak ilarvirus(TSV) cp (Gonsalves and Fulton, 1977; Gonsalves and Garnsey, 1975; Van Vloten-Doting,1975). CLRV, a member of subgroup 2, can be activated by members of its own subgroup,i.e. CVV and CLRV, and also by members of subgroup 1, TSV, and 3, rose isolate ofPNRSV. In addition, the cp or RNA 4 of A1MV, which is not a member of the ilarviruses,can also be used to activate CLRV. The cp’s of TSV and A1MV, bind to the 3’ termini ofthe RNA species, may activate replication and therefore, infectivity. Although, there is nohomology between either the nucleic acid or amino acid sequences of TSV and A1IvIV, bothhowever, exhibited stable hairpin structures flanked by the repeating tetra-nucleotidesequence AUGC (Cornelissen et aL, 1984).Most positive-sense, single-stranded RNA (5sRNA) plant viruses cause cytologicalchanges in the host cell (Lesemann, 1991; Martelli, 1991). Ilarviruses can be found scatteredthroughout the cytoplasm, in amorphous bodies, in paracrystalline arrays or in single rowswithin incomplete tubular structures (Martelli and Russo, 1985). Virus inclusions may be11the result of viral replication, altered host cell metabolism or modification of the host cell’sorganelles (Lesemann, 1991).1.1 BSIVSymptoms of BSIV were first observed in Clark County, Washington in 1980. Sincethen, it has been found in coastal regions of Washington and the Willamette Valley ofOregon, but not in B.C. BSIV infects all varieties of highbush blueberry and the one varietyof rabbiteye which has been tested (Martin and Bristow, 1994). Once detected, infectedplants should be destroyed to prevent further spread.Once infected with BSIV, blueberry plants do not exhibit necrotic shock symptoms,such as blossom and leaf blight, until the following spring. Symptoms will vary, reoccurring for 1-5 years after initial infection. Most plants recover and become asymptomatic(MacDonald and Martin, 1991).BSIV can be transmitted to herbaceous hosts by mechanical inoculation with infectedpollen. A partial host range includes Nicotiana clevelandii A. Grey, N tabacum ‘Havana425’ and ‘Samson’, N benthamiana L. and N sylvestis Speg. & Comes (MacDonald andMartin, 1991). The plant of choice for propagation, Nclevelandii, maintains the virus at ahigh titre from which it can be easily purified.BSIV exhibits many characteristics common to ilarviruses. Purified preparations areunstable. The virus is completely degraded if stored in phosphate or citrate buffer, pH 7.0,at 4°C for one week. Stability increases with the addition of EDTA (Anita Quail, personalcommunication). The virions are isometric, Ca. 27 nm in diameter, composed of cp subunits,12M of 27.3 kD, whose arrangement cannot be distinguished by electron microscopy. BSIVparticles were found in cytoplasm and the tubules attached to the plasmodesmata of ribosomedenatured N clevelandii leaf cells (MacDonald and Martin, 1991). Similar to otherilarviruses, the tripartite, single-stranded RNA genome species have of 1.03, 0.84 and0.57 x 106 D plus a subgenomic 0.3 x 106 D (MacDonald and Martin, 1991). It is evenserologically related to two ilarviruses, PNRSV and apple mosaic virus (ApMV), but not tocucumo-, bromo- or nepoviruses (MacDonald and Martin, 1991). These characteristicsprobably place it within subgroup 3 of the ilarvirus group.Since BSIV is pollen-borne, chemical or biological controls are not effective inpreventing spread. Transgenic blueberry plants resistant to BSIV could eliminate the highfinancial losses caused by this virus. Until this year, little was known about the molecularorganization of ilarviruses. Although, the TSV RNA 3 sequence was published in 1984 byCornelissen and co-workers, the cp sequences of prune dwarf virus (PDV) and ApMV werenot published until 1994 (Bachman et at. and Sanchez-Navarro and Pallas respectively). Thecp genes of TSV, PDV and ApMV are found on the 3’ half of RNA 3. Ilarvirus cp genesare translated from the subgenomic messenger RNA 4 (Bol et al., 1985). Therefore, a fulllength complementary DNA clone of either BSIV RNA 3 or RNA 4 would tentatively carrythe cp gene. Transformed tobacco plants, expressing TSV cp, were highly resistant tomechanical inoculation with TSV virions (van Dun et a!., 1988). Similarly, it should bepossible to make blueberry plants resistant to BSIV by inserting the viral cp gene into theplant’s genome.131.2 Research objectivesThe main objective of this portion of the study was to generate a BSIV conpiementaryDNA (cDNA) library including the cp gene. A portion of the cp gene would then becompared to the nucleotide and amino acid sequences of other ilarvirus cp’s. The secondaryobjective was to locate BSIV particles in blueberry pollen by immunodetection.142 Materials and methods2.1 BSIV Propagation and host reactions to infectionThe original source of BSIV was isolated from blueberry plants in Whatcom county,Washington. Pollen was obtained from blossoms that were air dried, filtered first throughan 18 mesh, then through a 25 mesh screen and stored at -70°C. The frozen pollen was usedas inoculum when blueberry blossoms or BSIV-infected N clevelandii were not available.N clevelandii were inoculated mechanically from one of three sources: BSIV-infectedblueberry blossoms, pollen or N clevelandii. The inoculum was homogenized with mortarand pestle in 0.05 M phosphate buffer, pH 7.0 (Sørensen’s 0.1 M phosphate buffer = 78.4ml of 1.0 M monobasic potassium phosphate, 121.6 ml of 1.0 M dibasic sodium phosphate(Na2HPO4)and 800 ml of water) and 2% (w/v) polyvinylpyrrolidone, M 44, 000 (PVP).Four to six week old plants, dusted with medium grade carborundum, were mechanicallyinoculated with inoculum soaked sponges, then rinsed with water. Propagation plants weremaintained in a greenhouse or growth chamber with 16 hr of light at 21°C and 8 hr of darkat 17°C.2.1.1 Electron microscopyImmature stamens from BSIV-infected blueberry bushes were cut into 1 piecesand fixed in 4% (v/v) glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2 (0.1 M sodiumcacodylate adjusted to pH 7.2 with hydrochloric acid (HC1)), with shaking for 2-4 hr at roomtemperature (RT) followed by three 10 mm washes in 0.1 M cacodylate buffer. Tissue was15post-fixed in 1% (v/v) osmium tetroxide in 0.1 M cacodylate buffer for 1 hr followed bythree 10 mm washes in water. The stamens were dehydrated by rinsing in 30% (v/v) ethanol(EtOH), then twice for 10 mm in 50%, 70%, 95% and absolute EtOH. The pieces weretransferred to propylene oxide for 10 mm, then for 16 hr to a 1:1 solution of propylene oxideand Epon (5.0 g Epon, 2.23 g nadic methyl anhydride, 1.88 g dodecenyl succinic anhydrideand 0.18 g DMP-30). The stamens were flat embedded in pure Epon, allowing it to set at60°C for 2 days. Embedded tissue was sectioned using a Reichart U2 Ultramicrotome(Germany). Thin sections, between 60-120 nm, were transferred to parlodion-coated, carbon-sprayed 100 mesh copper grids. Sections were stained with 4% (w/v) uranyl acetate (UA)for 20 mm, washed extensively with water, then stained with Reynold’s Lead Citrate Stain(3.9 mM lead nitrate, 6.8 mM sodium citrate dihydrate dissolved in 500 ml water and 100 ml1 M sodium hydroxide (NaOH)) diluted 1:5 with 0.01 M NaOH for 10 mm, washedextensively with water and blotted dry (Fran Leggett, personal communication).Colloidal protein A gold, 5-10 nm in diameter, was synthesized by the followingprocedure (Hayat, 1981). Solution A (1 ml of 1% (w/v) gold trichioride acid) and solutionB (a 20 ml solution containing 4 ml of 1% (w/v) trisodium citrate and 50 .il of 1% (w/v)tannic acid) were heated separately to 60°C, then mixed. Once red, the solution was heatedto boiling, then cooled at RT for 16 hr.Thin sections were immunolabelled in the following procedure (Hayat, 1981 withmodifications by Fran Leggett). Grids with thin sections were floated on drops of thesesolutions: 0.0 1% (wlv) lysine in phosphate-buffered saline (PBS; lx PBS = 127 mM sodiumchloride (NaC1), 2.6 M potassium chloride (KC1), 8.5 mM Na21-1P04, 1.1 mM potassium16dihydroxyphosphate) for 5 mm, 6 drops of 1% (w/v) bovine serum albumin (PBS-BSA) for5 mm, BSIV polyclonal antiserum (PcAs) diluted 1:100 in PBS-BSA for 5 mm, 10 tg/mlof 5-10 urn protein A gold, diluted 1:15 or 1:25 in PBS-BSA, for 30 mm, 6 drops of PBSfor 5 mm, 4% glutaraldehyde for 5 mm, 6 drops of water for 5 mm, then stained with UAand Reynold’s Lead Citrate.2.2 BSIV PurificationBSIV was purified as per Macdonald and Martin (1991). All manipulations were doneon ice or at 4°C. N clevelandii leaves exhibiting mosaics or necrotic lesions were harvested10-20 days after inoculation. Infected tissue was homogenized in a Waring blender for 2-3mm in 2 ml of 0.03 M Na2HPO4, pH 8.0, containing 0.02 M ascorbate, 0.02 M f3-mercaptoethanol (13-mer) per gram of fresh weight. The homogenate was expressed throughstretchable nylon cloth and the filtrate centrifuged at 10,000 revolutions per minute (rpm)in a GSA rotor (Sorval, Norwalk, CO) for 20 mm. The pH of the supernatant was adjustedto 5.0 with 6 M HC1, stirred for 2 hr and centrifuged as above. The supernatant was adjustedto 8% (w/v) polyethylene glycol-8000 (PEG-8000) and 1% (w/v) NaCl, stirred for 1 hr andcentrifuged as above. The pellet was resuspended in 0.1 the original vol of 0.05 M citratebuffer, pH 7.0, using a ground glass homogenizer, then shaken at 250 rpm for 16 hr andcentrifuged as above. The supernatant was centrifuged at 35,000 rpm in a 50.2 Ti rotor(Beckman, Irvine, CA) for 2 hr. The pellet was resuspended in 0.01 original vol of 0.05 Mcitrate buffer and layered in 0.5 ml aliquots onto 10 ml 10-40% (w/v) sucrose gradients in0.05 M citrate buffer in 10 ml polyallomer tubes (Beckman). The gradients were centrifuged17at 38,000 rpm in a SW41 rotor (Beckman) for 2.5 hr and scanned at 254 nm with an UA-5absorbance/fluorescence detector (ISCO, Lincoln, NB). Gradient fractions corresponding tothe absorbance peak were collected and concentrated by centrifugation at 60,000 rpm in a70.1 Ti rotor (Beckman) for 1 hr. The pellet was resuspended in either 0.05 M citrate or0.05 M phosphate buffer, pH 7.0. Samples were stored at 4°C or -80°C for later use.The ultraviolet absorbance spectrum, 220-320 nm (A220-3), of purified virus wasmeasured and recorded on a Model 8451A diode array spectrophotometer (Hewlett-Packard,U.S.A.). A260 was used to estimate the concentration of virus. A 1 p.g/jil solution ofilarvirus particles should have a value of 5.1-5.3 at 260 nm (Brunt et a?., 1990). Purity wasdetermined by the 260 nm/280 nm ratio, nucleic acids absorb at 260 nm and proteins at 280nm. Published values for fractionated preparations ranged from 1.43-1.67 (Fulton, 1983).Transmission electron microscopy was also used to judge purity. Purified BSIVpreparations were examined in a Hitachi 7000 transmission electron microscope (Japan).Parlodion coated grids were floated for 1 minldrop in a series of solutions: virus diluted in0.05 M phosphate buffer, pH 7.0, 5 drops of 0.3% (w/v) bacitracin and 1% (w/v) UA (FranLeggett, personal communication).2.2.1 Sodium dodecyl sulphate polyacrylamide gel electrophoresisThe M of the cp subunit was determined by sodium dodecyl sulphate polyacrylamidegel electrophoresis (SDS-PAGE). Electrophoresis was carried out in a Mini-Protein IIElectrophoresis Cell (Bio-Rad, Richmond, CA) using a discontinuous buffer system(Laemmli, 1970). A 10% separating gel prepared from a stock solution of 30% (w/v)18acrylamide and 0.8% (w/v) AN’-bis-methyIene acrylamide in 0.375 M Tris(hydroxymethyl)methylamine-chloride (Tris-Ci), pH 8.8, containing 0.1% (w/v) SDS waspolymerized with 0.05% (v/v) tetramethylethylenediamine (TEMED) and 0.05% (wlv)ammonium persuiphate (APS). The 4% stacking gel was prepared in 0.125 M Tris-Ci, pH6.8, containing 0.1% (w/v) SDS. Samples were dissolved in 62.5 mM Tris-Cl, pH 6.8,containing 2% (w/v) SDS, 10% (v/v) glycerol, 5% (v/v) 13-mer, 0.00125% (w/v)bromophenol blue, boiled for 3 mm and loaded at 0.2-1 i’g protein per lane. The gels wereelectrophoresed in 25 mM Tris-Cl, pH 8.3, containing 192 mM glycine and 0.1% (w/v) SDSat 10 mA per gel until the tracking dye had migrated from the stacking gel into theseparating gel. Then the current was increased to 20 mA per gel until the tracking dyereached the bottom of the gel. The gel was stained in 0.2% (w/v) Coomassie Brilliant BlueR-250, in 40% (v/v) methanol (MeOH) and 10% (v/v) acetic acid (HOAc) for 30 mm thendestained in 40% (v/v) MeOH and 10% (v/v) HOAc. The gels were sandwiched betweenCellophane membrane backing (Bio-Rad) and dried under vacuum in a Model 224 Gel SlabDryer (Bio-Rad) for 1.75 hr, then at RT for 1 hr.2.3 Virion RNA extractionSsRNA was isolated from purified BSIV particles (MacDonald, 1989). Fivehundred jig of virus was diluted in 0.2 M Tris-Cl, pH 7.5, 0.025 M EDTA, 0.3 M NaCI, 2%(w/v) SDS, 25% (wlv) Proteinase K (GIBCO Bethesda Research Labs (BRL), Gaithersburg,MD) in a total vol of 0.5 ml and incubated at 37°C for 30 mm. An equal vol ofphenol:chloroform:isoamyl alcohol (25:24:1) was added and vortex mixed for ca. 1 mm.19The phases were separated by centrifugation at 14,000 rpm in an Eppendorf microfuge for5 mm at 4°C. The aqueous phase was drawn off, extracted a second time as above and athird time using only chloroform:isoamyl alcohol (24:1). RNA contained in the finalaqueous phase was precipitated with either 0.1 vol of 3 M sodium acetate (NaOAc) and 2.5vol of absolute EtOH at -80°C for 16 hr or with 0.1 vol 8 M lithium chloride (LiC1) and 2.5vol of absolute EtOH on ice for 2 hr and centrifuged as above for 30 mm. The pellet waswashed with 70% EtOH, dried under vacuum and suspended in diethylpyrocarbonate treatedwater (DEPC-H20;deionized water adjusted to 0.1% (v/v) DEPC was shaken, incubated at30°C for 16 hr, then autoclaved for 1 hr). RNA samples were stored at -70°C.Virion RNA was quantified spectrophotometrically. The absorbance of an aliquotdiluted to 300 j.il with DEPC-H20was measured at A260. At A260, a 1 ig/jil solution of RNAhas a value of 25 (Sambrook et al., 1989).2.3.1 Methylmercuric hydroxide agarose gel electrophoresis of RNAThe quality of virion RNA was assessed by agarose gel electrophoresis in thepresence of 5 mM methylmercuric hydroxide (CH3HgO ; Sambrook et aL, 1989). BSIVRNA species were fractionated by size in a 1% (w/v) agarose gel containing lx borateEDTA (lx BE = 40 mM borate, pH 8.2, 1 mM EDTA) containing 5 mM CH3HgOH cast ina Minigel Apparatus (Bio-Rad, Richmond, CA). RNA samples were denatured in 3x BEcontaining 15 mM CH3HgOH, 30% (v/v) glycerol, 0.00125% (w/v) bromophenol blue. Upto 10 jig of denatured RNA was loaded per lane and electrophoresed at 80 V per 25 ml gelfor 1 hr in BE buffer. After electrophoresis, gels were stained in 100 ml of deionized water20containing 0.5 jig/mi ethidium bromide (EtBr) and 10 mM 3-mer for 15 mm. Bands werevisualized at 320 nm on a CAMAG Reprostar transilluminator and photographed through aWratten 2A red filter with 667 Polariod film (Kodak, Cambridge, MA).2.4 BSIV Cloning strategies2.4.1 First-strand cDNA synthesisAttempts to synthesize double-stranded DNA complementary to BSIV RNA werebased on the ‘one tube double-strand cDNA method’ by BRL (D’Alessio eta!., 1987). Totaltripartite BSIV ssRNA was used as the template for the synthesis of the first-strand ofeDNA. Approximately 3 jig of RNA and 50 ng of random priming hexamers wereincubated at 65°C for 2 mm, then at RT for 10 mm. DNA complementary to BSIV RNAwas synthesized at 37°C for 1 hr using 400 units (U) of Moloney murine leukemia virusribonuclease (RNAse) H- reverse transcriptase (superscript; BRL, Gaithersburg, MD) in a50 jil reaction mixture containing 50 mM Tris-Cl, pH 8.3, 50 mM potassium chloride (KC1),8 mM magnesium chloride (MgC12), 10 mM DTT, 1 mM each of dATP, dCTP, dGTP anddTTP and 20 U of RNAGuard (Pharmacia, Uppsala, Sweden). Synthesis was monitored ina pilot reaction containing 10 jiCi of c-32P dATP (ICN Biomedicals, Mississauga, Ont.) and5 jil of first-strand mixture. The pilot reaction was terminated by adding 5 jil of 0.5 MEDTA and diluted to 100 jil with water. Ten jil aliquots were spotted onto GF/C glassfilters (Whatman, Clifton, NJ), air dried, washed with 10% (w/v) trichloroacetate (TCA)containing 1% (w/v) sodium pyrophosphate, then with 95% EtOH and air dried. Each filterwas immersed in Aquasol-2 (DuPont NEN Research Products, Boston, MA) scintillation21fluid and its radioactivity measured in a Packard Tri-Carb 4530 scintillation counter (UnitedTechnologies Packard, Downers Grove, IL).2.4.2 Second-strand cDNA synthesisThe non-radioactive first-strand reaction was diluted to a fmal vol of 100 il whichcontained 20 mM Tris-Ci, pH 7.6, 100 mM KC1, 5 mM MgC12, 1 mM each of dATP, dCTP,dGTP, dTTP, 25 U of Eschericia coli DNA Polymerase I (BRL, Gaithersburg, MD), 2 U ofRNAse H (BRL) and 10 iCi of x-32P dATP and incubated at 16°C for 2 hr. This wasfollowed by the addition of 12 U of T4 DNA Polymerase (BRL), incubated at 16°C for 5mm and terminated with il of 0.5 M EDTA. Second-strand synthesis was monitored asper first- strand synthesis.2.4.3 Isolation of cDNA from unincorporated nucleotidesBSIV cDNA was separated from unincorporated nucleotides by agarose gelelectrophoresis followed by gel purification with glass milk (QUIAGEN, West Germany).A 25 ml 1% agarose gel in buffer containing 40 mM Tris-Ci, pH 8.0, containing 40 mMglacial acetic acid, 2 mM EDTA (TAE) was cast in a Minigel Apparatus. ComplementaryDNA was diluted in 5x DNA loading buffer (5x DNA loading buffer = 5x TAE containing30% glycerol, 0.00125% bromophenol blue, 0.00125% cyanol blue), loaded beside a 1 kbDNA M marker (BRL) and electrophoresed at 80 V per 25 ml gel for 45 mm in TAE. Thegel was stained for 15 mm in 100 ml of water containing 0.5 jig/mI EtBr and photographed(Chapter II 2.3.1).22The largest visible inserts were cut out and purified with QIAEX glass milk. Each100 tg gel slice was dissolved in 300 jtl QX1 buffer (3 M sodium iodide, 4 M sodiumperchiorate (NaC1O4), 10 mM Tris-HC1, pH 7.0, 10 mM sodium thiosulfate) containing 10p.1 of QIAEX matrix by incubation at 50°C with intermittent vortexing. The matrix waspelleted by centrifuging at 14,000 rpm for 30 sec. The pellet was washed twice by vortexmixing in 500 j.il of QX2 buffer (8 M NaC1O4, 10 mM Tris-HC1, pH 7.0) followed bycentrifugation for 30 sec at 14,000 rpm and washed twice in QX3 buffer (70% EtOH, 100mM NaCl, 10 mM Tris-HC1, pH 7.5). The pellet was dried under vacuum. DNA bound tothe matrix was released by incubation in 20 p.1 of Tris-EDTA buffer containing 100 mMTris-CI, pH 7.5, 10 mM EDTA (TE) at RT for 5 mm.2.4.4 Ligation of cDNA into a vectorPlasmid Bluescript II KS (pBKS; Stratagene, San Diego, CA) was chosen for itsfacility of selection and screening. It is selected by its ampicillin resistance. The polylinkerregion in the N-terminal portion of the lacZ gene fragment has 21 unique restriction enzymerecognition sites. The Kpn I restriction site is the closest to the lacZ promoter and the SacI site is the farthest. The multiple cloning site is flanked by T3 and T7 promoters. Oncetransformed into bacteria containing the C-terminal portion of the lacZ gene i.e. E. coliDH5c, colonies are screened by their breakdown of 5-bromo-4-chloro-3-indolyl-13-D-galactoside (X-gal; BRL, Gaithersburg, MD). Plasmids without inserts in the polylinkerregion form blue colonies, whereas those with DNA inserts form white colonies.pBKS was prepared for blunt-end ligation. Two jig of pBKS were digested with234.5 U of Sma Tin BRL React 4 buffer (20 mM Tris-Ci, pH 7.4, 5 mM MgC12, 50 mM KCI)in 20 ti at RT for 4 hr. The ends were dephosphorylated with 2 U of calf alkaline intestinalphosphatase (CIP; Boehringer-Mannheim, Laval, Que.) in 2 i1 of lOx CIP buffer (lx CIPbuffer = 0.01 M Tris-Ci, pH 8.3, 1 mM zinc chloride, 1 mM MgC12) at 37°C for 30 mm.The reaction was terminated with 5 l of 3 M EDTA. Linearized and dephosphorylatedpBKS was gel purified with QIAEX (Chapter II, 2.4.3) and resuspended in 40 tl of TE. Itsconcentration was estimated by comparing known amounts of pBKS against differentquantities of DNA molecular weight marker after separation by gel electrophoresis.All of the BSIV eDNA was ligated into Ca. 150 ng of linearized, dephosphorylatedpBKS in 50 mM Tris-Ci, pH 7.6, containing 10 mM MgCI2, 1 mM ATP, 1 mM DTT, 5%(wlv) PEG-8000 and 4 U of T4 DNA Ligase (BRL) in a total vol of 20 i1 at 14.8°C for16 hr.2.4.5 Transformation of competent Escherichia coil cellsChimeric plasmids were used to transform maximum efficiency DH5c E. coil cells(1990; BRL, Gaithersburg, MD). All reactions were done on ice unless indicated otherwise.E. coil cells were thawed and 50 j..tl aliquoted to Falcon Labware 2059 polypropylene tubes(Baxter Diagnostics Corp, Toronto Ont (CAN-Lab)). Five t1 of ligation mixture were gentlypipetted among the cells, incubated on ice for 30 mm, heat shocked in a water bath at 42°Cfor 45 sec, then placed on ice for 2 mm. Transformed cells were shaken at 250 rpm at 37°Cfor 1 hr in either 0.5 ml of Luria Broth (LB) or 0.9 ml of S.O.C. LB was prepared byautoclaving a solution of 1% (wlv) bactotryptone (Difco, Detroit, MI), 0.5% (w/v) yeast24extract (Difco), 100 tg of 1 M NaC1, pH 7.4. S.O.C. was prepared from 10 ml ofautoclaved LB by adding 100 tl of a 1 M KC1 and 100 jil each of filter sterilized solutionsof 2 M glucose and 2 M Mg2 (2 M Mg 2+ = 1 M magnesium chloride hexahydrate, 1 Mmagnesium sulphate heptahydrate). Cultures were spread onto LB plates (LB plate = LBcontaining 1.5% (w/v) agar) containing 80 mg/i filter-sterilized ampicillin (LB plate + Amp;Sigma Chemical Co., St.Louis, MO) and 40 ti of 20 mg/ml X-gal and incubated at 37°C for16 hr.2.5 Screening cDNA clones2.5.1 ‘Alkaline lysis method’ of plasmid isolationRecombinant plasmids were isolated using the ‘alkaline lysis method’ for purifyingplasmids (Sambrook et al., 1989). White colonies were grown in 3 ml of LB containing80 mg/i Amp with shaking at 37°C for 16 hr. Half the volume of each bacterial culture wastransferred to an Eppendorf tube and centrifuged for 1 mill. The pellet was resuspended withvortexing in 150 iil of 50 mM glucose, 10 mM EDTA, 50 mM Tris-Ci, pH 8.0, andincubated at RT for 5 mm. The cells were gently lysed with 350 p1 of 0.2 M NaOH, 1%(w/v) SDS by inverting the tube several times and incubated on ice for 15 mm. Anadditional 250 pl of potassium acetate solution (potassium acetate = 60 ml of 5 M potassiumacetate, 11.5 ml of glacial acetic acid and 28.5 ml of water) were added, mixed by inversionand incubated on ice for 10 mi Chromosomal DNA and cellular debris were pelleted bycentrifugation for 5 mm. The supernatant was transferred to a fresh tube and extracted withone vol of phenol/chloroform (1:1). The aqueous phase was precipitated with 1 vol of25isopropanol on ice for 15 mm and centrifuged for 5 mm. The pellet was washed with 70%EtOH, dried under vacuum and resuspended in 50 p1 of TE and 1 p1 of 1 mg/mi of RNAseA (BRL, Gaithersburg, MD). DNA isolated by the ‘alkaline lysis method’ will be referredto as mini-prep DNA.2.5.2 Restriction enzyme digestion of mini-prep DNAPlasmids were digested with restriction enzymes Xho I and Xba I, which excised thecDNA insert (Sambrook et al., 1989). Digests were done at 37°C in 1.5 ml microfuge tubescontaining 2-5 p1 of mini-prep DNA, Ca. 100 ng, 1.5 il of lOx restriction buffer suppliedby the manufacturer and 5 U of restriction enzyme in a total volume of 15 p.1. Mini-prepDNA was digested for 2 hr, then diluted with 2 p.1 of 5x DNA loading buffer and separatedin a 1% agarose gel made with TAE buffer.2.5.3 Random primed cDNA probesThe Genius Digoxigenin (DIG) DNA Non-Radioactive Labelling and Detection Kit(1992; Boebringer Mamtheim, Laval, Que.) was used to prepare non-radioactive probes tothe plasmids with the largest cDNA inserts corresponding to BSIV RNA 3 and or RNA 4.Approximately 200 ng of BSIV cDNA insert of clone 29, 35 or 112 were denatured at 95°Cfor 10 mm and immediately placed on ice. DNA was incubated at 37°C for 2 hr in a 19 p.1reaction consisting of 2 p.1 of hexanucleotide mixture, 2 p.1 of dNTP labelling mixture (1 mMeach of dATP, dCTP, dGTP, 0.65 mM dTTP, 0.35 mM DIG-dUTP, pH 7.5) and 2 U ofKlenow fragment of DNA polymerase I (BRL, Gaithersburg, MD). The reaction was26terminated with 2 il of 0.5 M EDTA and DIG-labelled DNA precipitated with 2 il of 4 MLiC1 and 60 p.1 of absolute EtOH. The probes were washed, dried under vacuum, rinsedwith 70% EtOH and dissolved in 20 p.1 of TE.Probes were diluted to a concentration of 50 ng!ml in pre-hybridization solutioncontaining 50 mM phosphate buffer, pH 6.8, 5x standard saline citrate (SSC; lx SSC =150 mM NaCI, 15 mM sodium citrate, pH 7.0), 7% (w/v) SDS, 2% (w/v) casein, 5% (w/v)yeast transfer RNA and denatured at 90°C for 10 mm and cooled on ice. Probes werestored at -20°C for up to a year.2.5.4 Sap dot blots using ZetaprobeTM membraneOne gram of BSIV-infected N clevelandii leaves was macerated with 10 ml oflOx SSC between two layers of cheesecloth in a plastic bag. Two vol of sap were dilutedwith 1 vol of 37% (v/v) formaldehyde solution, pH 4.0 (BDH, Toronto, Ont.), incubated at65°C for 15 mm and placed on ice. Sap diluted 1:50, 1:250, 1:750 and 1:1250 (w/v) inlOx SSC containing 4% formaldehyde, was blotted in 15 p.1 aliquots onto Zetaprobemembrane (Bio-Rad, Richmond, CA) prewetted with lOx SSC in a Bio-Dot Apparatus (BRL,Gaithersburg, MD). The membrane was rinsed with lOx SSC, air dried for 10 mm, driedat 37°C for 10 mm, then at 80°C for 30 mm.2.5.5 Zetaprobe membrane hybridization and washing conditionsDot blots prepared from plant sap were hybridized with probes 29 and 35, clones withthe largest cDNA inserts from the first cloning experiment. Hybridization was carried out27as described by Holtke and co-workers (1992) with modifications by S. Winter (personalcommunication). Membranes were prehybridized with continuous rotation in prehybridization solution at 50°C for 1-2 hr. The pre-hybridization solution was replaced withdenatured probe solution (Chapter II, 2.5.3) and incubated at 42°C for 16 hr. Followinghybridization, membranes were washed with 30 ml of 2x SSC, 0.1% SDS at 50°C for 15mi 0.5x SSC, 0.1% SDS at 50°C for 15 mm, twice in 0.lx SSC, 0.1% SDS at 68°C for 15mm, wash buffer (wash buffer maleic acid buffer (0.1 M maleic acid, 0.15 M NaC1, pH7.4) containing 0.3% (vlv) Tween 20 (Tw-20)) at RT for 5 mm, then in maleic acid buffercontaining 2% casein for 30 mm. Membrane-bound probe was labelled with 1 il of anti-DIG alkaline phosphatase, diluted in 10 ml of maleic acid buffer containing 1% casein for30 mm and washed thrice in wash buffer for 10 mm. It was equilibrated in substrate buffer(substrate buffer = 0.1 M Tris-Cl, pH 9.5, containing 0.1 M NaC1, 50 mM MgC12)for 5 mm,then blotted dry on filter paper. The membrane was placed on clear plastic and saturatedwith alkaline phosphatase substrate Luminigent’PPD (Detriot, MI) diluted 1:100 (v/v) insubstrate buffer for 20 mm. The membrane was exposed to x-ray film (Kodak, Cambridge,MA) for 5-30 mm.2.6 Northern analysis using formamide-formaldehyde gelsNorthern blots were used to determine which BSIV RNA species each probe detected.SsRNA was separated in a 1.2 % (w/v) agarose gel containing 1 M 3-(N-morpholino)propanesulfonic acid, pH 7.0 (MOPS) and 2.2 M formaldehyde. RNA samples weredenatured in 0.5x MOPS containing 2.2 M formaldehyde, 50% (v/v) formamide, 0.005%28(w/v) EtBr and sterile 5x DNA loading buffer. Samples were loaded up to 30 jig per laneand electrophoresed at 70 V for 1.5 hr in MOPS buffer. The gel was then photographed.2.6.1 Transfer of RNA from agarose gels to ZetaprobeTM membraneThe gel was rinsed in deionized water, then soaked in 50 mM NaOH for 5 mm.RNA was transferred by vacuum to Zetaprobe membrane prewetted with lOx SSC at 60mm Hg for 60-90 mm. After transfer, the membrane was rinsed with lOx SSC, dried,hybridized with either probes 29, 35 or 112, washed, immunolabelled and detected withluminescent substrate.2.7 Identifying and mapping BSW cDNA clonesAdditional clones corresponding to BSIV RNA 3 and 4 were identified by plasmiddot and Southern blots with three different probes. Mini-prep DNA or these clones weredigested with many restriction enzymes, separated by agarose gel electrophoresis andoriented relative to each other based on location of restriction enzyme sites.2.7.1 Dot blots using plasmid DNA (Plasmid dot blots)Probes 29 and 35 were hybridized with DNA from clones 9, 11, 14, 16, 17, 19, 25,29 and 35. Later probe 112 was hybridized with clones 14, 16, 17, 19, 29, 35, 112, 123,132, 136, 153, 161, 166 and 173. Mini-prep DNA diluted 1:9 with 0.4 M NaC1 and 0.4 MNaOH was blotted onto Zetaprobe prewetted with TE, washed with 0.4 M NaOHcontaining 0.00 125% bromophenol blue, then rinsed with 2x SSC. The membrane was dried29(Chapter II, 2.5.4), hybridized with probes 35 or 112, washed, immunolabelled and detectedwith luminescent substrate.2.7.2 Restriction enzyme analysisMini-preps of clones with cDNA inserts corresponding to RNA 3 or RNA 4 asdetermined by Northern and plasmid dot blots, were digested with restriction enzymes KpnI, Xho I, Hinc II, Acc I, Sal I, Cia I, Hind III, EcoR V, EcoR I, Pst I, BamH I, Xba I, NotI, Sst I and Pvu II (BRL, Gaithersburg, MD) and separated by agarose gel electrophoresisin TAE buffer. Clones were oriented relative to each other based on location of restrictionenzyme sites.2.7.3 Southern blots using ZetaprobeTM membraneClones 35, 101, 112, 115, 117, 123, 132, 136, 153, 158, 160, 161, 166 and 173 weredigested with restriction enzymes and separated by agarose gel electrophoresis were blottedonto Zetaprobe prewetted with lOx SSC and vacuum transferred. The membrane wasrinsed with lOx SSC, dried, hybridized with probe 112, washed, immunolabelled anddetected with luminescent substrate.2.8 SequencingThe region to be sequenced, a portion of RNA 3, lay between the 3’ end of themultiple cloning site and the Xho I site closest to the 3’ end. This region included an AceI site within the insert. It was Ca. 400 bp as determined from restriction enzyme analysis of30plasmids 112, 123 and 132 (see Fig. 10 for region sequenced).2.8.1 Preparation of subclonesMini-prep DNA of plasmids 112, 123 and 132 were digested with Xho I andseparated by agarose gel electrophoresis. Linearized 3.2 kb fragments corresponding to thesum of the pBKS and the 400 bp region to be sequenced were gel purified with QIAEX,religated and used to transform Library Efficiency DH5cL E. coli (BRL, Gaithersburg, MD).Mini-prep DNA from the subclones of 112, 123 and 132 was isolated by the ‘alkaline lysismethod’, digested with Xba I and fractionated by agarose gel electrophoresis to verify thata portion of the multiple cloning site was still intact.Double-stranded mini-prep DNA was used as the template for sequencing (Hsiao,1993). Approximately 5 jig of mini-prep DNA and 10 ng of sequencing primers either T3(University of British Columbia Oligonucleotide Synthesis Laboratory, Vancouver, B.C.) orT7 (U.S. Biochemical Corp.) were denatured in a 7 pl vol containing 1 j.il of 1 mM NaOHat 37°C for 10 mm. NaOH was neutralized with 1 jil of 1 mM HC1 at 37°C for 5 mmallowing primer and template to anneal.2.8.2 Sequencing with SequenaseSequences were determined using reagents provided and described by U.S.Biochemical Corporation (1990). Sequencing reactions were done in a total vol of 10 ilcontaining 2 il of 5x Sequenase buffer (200 mM Tris-Cl, pH 7.5, 100 mM MgC12,250 mMNaC1). Short reactions synthesized complementary labelled strands near the primer, whereas31long reactions synthesized strands labelled 200 bp 3’ from the primer. Short reactionscontained primer-template, 1 tl 0.1 M DTT, 2 iil labelling mix (2.5 jiM of each dGTP,dCTP, dTTP), 10 pCi of35S-dATP and 3 U Sequenase® Version 2.0 (diluted 1:8 in 10 mMTris-CI, pH 7.5, 5 mM DTT, 0.5 mg/mi BSA) incubated at RT for 5 mm. The short reactionmix was divided into four 3.5 p1 aliquots to which was added 2.5 p1 of ddG, ddA, ddC orddT termination mix (ddG = 80 pM of each dGTP, dATP, dCTP, dTTP, 8 pM ddGTP, 50mM NaC1) and incubated at 37°C for 5 mm. Reactions were stopped by the addition of 4p1 of stop solution (95% formamide, 20 mM EDTA, 0.05% bromophoenol blue, 0.05%xylene cyanol). Long reactions consisted of the same reagents as the short reaction with theaddition of 1 p1 manganese buffer (0.15 M sodium isocitrate, 0.1 M manganese chloride)added to the reaction catalysed by Sequenase®. One jii of each termination mix was dilutedwith 1.5 p1 of extension mix (180 pM each dGTP, dATP, dCTP, dTTP, 50 mM NaC1)incubated at 37°C for 10 mm and stopped as above. The samples were stored at -20°C.Before loading, samples were denatured at 90°C for 10 mm and immediately chilled.2.8.3 Sequencing gelsSix per cent polyacrylamide gels were prepared by dissolving 28.6 g of urea in 6 mlof lOx TBE (lx TBE = 0.89 mM Tris-HC1, 0.89 mM borate, 20 mM EDTA), 12 ml of 19%acrylamide containing 1% bis-acrylamide and filter sterilized. The gel was polymerized with40 p1 of TEMED and 20 p1 of 25% APS before pouring into a 21 x 50 x 0.04 cm3Sequencing Gel Apparatus (Bio-Rad, Richmond CA). After polymerization, the gel was preelectrophoresed in TBE at 85 W until the temperature reached 50°C. Samples were loaded,322 il for narrow wells and 3 pi for wide wells and electrophoresed at 45 W for 4-6 hr. Afterelectrophoresis, gels were transferred to 3 MM filter paper (Whatman, Clifton, NJ) and driedunder vacuum for 1.5 hr at 80°C. Autoradiography was done overnight at RT withoutintensifying screens. Autoradiograms were read manually and sequence information storedand sequences compiled using the DNA Analysis program of Gene Works 2.1 .1 software(Intelligenetics, Inc., Mountain View, CA).2.8.4 Sequence analysesThe sequence, from the internal Xho I site to the 3’ multiple cloning region(BSIVseq), was aligned with BSIV RNA 3 (M. M’Lean, unpublished data) using theNALIGN subprogram in PCGene (IntelliGenetics, Inc., Geneva, Switzerland). Then theBSIVseq was compared to TSV, CVV, ApMV, PDV, potato leaf roll virus (PLRV) and Lphosphinothricin-N-acetyltransferase gene (PAT) nucleotide sequences. The BSIV cp genewas similarly aligned.Both the BSIVseq and its complementary strand were each translated in three framesusing the translation subprogram in Genesys (C.S.I.R.O. Division of Plant Industry,Canberra, Australia). The stop codons were deleted from each frame of translated sequenceand aligned with the cp sequences of TSV, CVV, ApMV, PDV, PLRV and the PAT proteinusing the PALIGN subprogram in PCGene. The results were compared to alignments withthe BSIV cp.333 Results3.1 Host reactions to BSIV infectionThe titre of BSIV in blueberries was sensitive to environmental changes. BSIV wasreadily detected by ELISA in blossoms and in young and mature leaves in spring andsummer. Identification, however, was not as reliable if senescent or dormant leaf andblossom buds were collected in the autumn or winter. In addition, BSIV could be movedfrom its natural host to a new host if pollen was the inoculum. Thus, virus titre andtransmission from blueberries were dependent on both the season and the tissue utilized.Symptom development in N clevelandii was also affected by the source of inoculumand the season. Seven days after mechanically inoculating N clevelandii leaves with BSIVinfected blueberry blossoms, the indicator host developed ringspots. Alternatively, hostsinoculated with BSIV-iufected N clevelandii leaves, resulted only in a mild mottle.Irrespective of the source material, twelve days post inoculation (dpi), necrotic areasappeared on the youngest expanding leaves (Fig. 1B). Approximately one month afterinoculation, the infected plants either died or recovered completely. In spring, necroticlesions along the mid-ribs developed after ten days, whereas in winter, symptom appearancewas retarded by 4-12 days. Therefore, only the use of BSIV-infected blueberry pollen as asource of inoculum ensured a high percentage of infected hosts. Using any other inoculumin summer, fall or winter resulted in at least a few healthy escapes.BSIV was difficult to maintain in the greenhouse for three reasons. First, itsherbaceous indicator, N clevelandii, was susceptible to many viral diseases. All BSIV34FigurelA-B:A-Nicotianaclevelandiisevendaysaftermockinoculationwith0.05Mphosphatebuffer,pH7.0,and2%(w/v)polyvinylpyrrolidone,M44,000(PVP).B-Nclevelandiisevendaysafterinoculationwithblueberryshockilarvirusinfectedblueberryblossomshomogenizedin0.05Mphosphatebuffer,pH7.0,and2%(w/v)PVP.ABinfected N clevelandii were contaminated with tomato spotted wilt virus (TSWV) in 1991due to an outbreak of Thrips and the presence of TSWV in an adjacent greenhouse. Thiseffectively eliminated all available sources of BSIV until the spring of 1992. Another lotof BSIV-infected plants unavoidably moved to a growth chamber empty of plants for sixmonths, subsequently became infected with TMV previously propagated there.Secondly, BSIV was temperature sensitive. Initially, all herbaceous hosts werepropagated in a greenhouse where temperatures fluctuated widely in the summer. As thetemperature increased, fewer plants developed symptoms with each transfer until finally theinoculated plants were symptomless. When tested by ELISA, the plants were healthy.Plants, maintained in the stable environment of a growth chamber, became infected withBSIV, but showed milder symptoms.Since blueberry blossoms were only available in spring, a back-up source of inoculumwas necessary. Pollen sacs collected from beehives placed in a BSIV-infected blueberryfield, were not infective. Virus-infected pollen lost its infectivity in less than four monthsif stored at RT, but remained infective if frozen at -70°C. BSIV-infected N clevelandiiplants also had a short period of infectivity. Only the leaves exhibiting necrotic lesions forfewer than six days were infective. Therefore, BSIV inoculum was only available from threesources, frozen pollen stored at -70°C, fresh blueberry blossoms or infected herbaceous hosts.3.2 Electron microscopyThe pollen grains, embedded in Epon, were at various stages of development. Somewere unicellular, whereas others had differentiated into mature tricellular structures36(Esau, 1977). Pollen from healthy ‘Bluecrop’, used as a control was decorated with BSIVPcAs and labelled with protein A gold. The cytoplasm and intine were non-specificallylabelled (Fig. 2A-B). Similar to the controls, pollen from BSIV-infected ‘Rancocas’ and‘Bluetta’ exhibited non-specific labelling even when no PcAs was used. But compared tothe controls, certain cytoplasmic membrane-bound aggregates were preferentially labelled.The aggregates differed between the two cultivars studied. ‘Bluetta’ aggregates had a singlebilayer membrane encompassing spherical particles (Fig. 3A), whereas in ‘Rancocas’, thegold bound to structures almost entirely composed of bilayer membranes (Fig. 3B).Colloidal gold studded both the bilayer membranes and the interiors of these structures.3.3 PurificationBetween 5-40 mg of BSIV was purified per kg of fresh leaf tissue. Fractionatedpreparations of BSIV had an A260/A8 ratio of 1.25-1.7. Particles, stained with UA, wereisometric, Ca. (30.2 ± 1.4) nm in diameter (Fig. 4). Two proteins were detected by SDSPAGE (Fig. 5). The major component, the putative BSIV cp subunit, had a M of (29.5 ±0.9) kD. A minor band had a M of 55 kD (Macdonald and Martin, 1991).3.4 RNA ExtractionNeither hot phenol nor cold phenol extraction methods yielded RNA from purifiedpreparations of BSIV (Sambrook et al., 1989). Using proteinase K, RNA was extracted fromvirions suspended in phosphate, but not in citrate buffer. From 1 mg of purified BSIVparticles, 10-30 ig of ssRNA were extracted. If precipitated with NaOAc and EtOH, the37Figure 2A-B: Thin sections of healthy ‘Bluecrop’ blueberry pollen coated 5 mm with 0.0 1%(w/v) lysine in phosphate-buffered saline (PBS), washed for 5 mm six times with 1% (w/v)bovine serum albumin in PBS (PBS-BSA), decorated for 5 mm with blueberry shockilarvirus polyclonal antiserum diluted 1:100 in PBS-BSA, labelled for 30 mm with 5-10 nmprotein A gold diluted 1:25 in PBS-BSA, washed for 5 mm six times in PBS, 5 mm in 4%(v/v) glutaraldehyde, 5 mm six times in water and stained for 20 mm in 4% (w/v) uranylacetate and 10 mm in Reynold’s lead citrate diluted 1:5 in 0.01 M NaOH and blotted dry.A - Magnification of intine and exine of pollen grain. Note the high background of proteinA gold. x 31,875. - -...II‘IIIF.IIsIIp38Figure 2B: Magnification of pollen cytoplasm. Note the high background of protein A gold.x 56,250.39Figure 3A-B: Thin sections of blueberry shock ilarvirus-infected blueberry pollen. Theywere coated 5 mm with 0.0 1% (wlv) lysine in phosphate-buffered saline (PBS), washed for5 mm six times with 1% (w/v) bovine serum albumin in PBS (PBS-BSA), decorated for 5mm with blueberry shock ilarvirus polyclonal antiserum diluted 1:100 in PBS-BSA, labelledfor 30 mm with 5-10 nm protein A gold diluted 1:15 or 1:25 in PBS-BSA, washed for 5 mmsix times in PBS, then 5 mm in 4% (v!v) glutaraldehyde, then for 5 mm six times in waterand stained for 20 mm in 4% (w/v) uranyl acetate and 10 min in Reynold’s lead citratediluted 1:5 in 0.01 M NaOH and blotted dry. A - Thin sections of ‘Bluetta’ labelled withprotein A gold diluted 1:25 in PBS-BSA. Note the protein A gold concentrated within amembrane zlosed structure. x 15,000.-IFigure 3B: Thin sections of ‘Rancocas’ blueberry pollen labelled with protein A gold diluted1:15 in PBS-BSA. Note the protein A gold concentrated within a membrane enclosedstructure. x 75,000.41Figure 4: Purified blueberry shock ilarvirus diluted in phosphate-buffered saline, washed for1 mm in 0.3% (w/v) bacitracin and stained for 2 mm in 1% (w/v) uranyl acetate. The virushas a diameter of (30.2 ± 1.4) nm. x 155,520.42Figure 5: Purified blueberry shock ilarvirus (BSIV) and cucumber mosaic virus, blueberryisolate (CMV-B) proteins separated by sodium dodecyl sulphate polyacrylamide gelelectrophoresis (II 2.2.1). Lanes A and F are low range molecular weight standard markers(Bio-Rad) composed of 97.4 kD rabbit phosphorylase, 66.2 kD bovine serum albumin, 45kD hen egg white ovalbumin, 31 kD bovine carbonic anhydrase, 21.5 kD soybean trypsininhibitor and 14.4 kD hen egg white lysozyme. Lanes B and C contained 918 and 612 ngrespectively of BSIV proteins. Lanes D and E contained 159 and 318 ng respectively ofCMV-B proteins.97.4 kD66.2 kD45.0 kD31.0 kD21.5 kD14.4 kD43resulting salt pellet interfered with first-strand synthesis. Fewer salts were precipitated whenLiC1 was used. The RNA was fractionated into four species by denaturing agarose gelelectrophoresis. Their Mw’s were (1.19 ± 0.07, 0.85, 0.66 ± 0.02, and 0.31 ± 0.02) x 106 D,which corresponded to (3,500 ± 200, 2,600, 1,940 ± 50 and 910 ± 60) nucleotides asaveraged from four gels containing CH3HgOH (Fig. 6).3.5 Cloning BSIV RNA 3Incorporation ofx-32P dATP was monitored during first- and second-strand synthesisas per D’Alessio et al. (1987). In each experiment, eDNA was separated fromunincorporated nucleotides in a 1% agarose gel made with TAE buffer and stained withEtBr. Radioactive eDNA inserts were identified visually and with a geiger counter, thenexcised and gel purified. Complementary DNA fragments were ligated into Sma I digested,dephosphorylated pBKS and the mixture used to transform competent DH5c E. coil cells.Approximately 80 white colonies were isolated from each cloning experiment. Eachtransformant was isolated and amplified in LB + Amp media. Plasmids from each bacterialculture were mini-prepped by the ‘alkaline lysis method’. Mini-prep DNA was digested withrestriction enzymes, Xho I and Xba I, and the insert’s size determined by agarose gelelectrophoresis.3.6 Dot blots with BSIV-infected sapThe origins of plasmids 29 and 35, produced during the first cDNA synthesis, wereverified. The cDNA inserts were isolated by restriction enzyme digestion, separated by44Figure 6: Purified blueberry shock ilarvirus (BSIV) and cucumber mosaic virus, blueberryisolate (CMV-B) RNA separated by methylmercuric hydroxide agarose gel electrophoresis.The gel was prepared, electrophoresed and stained as described in the test (II 2.3.1). LanesA and D were loaded with 0.24-9.5 kb RNA ladder (GIBCO BRL) composed of 9.5, 7.5,4.4, 2.4, 1.4 and 0.24 kb species. Lane B contained 450 ng of CMV RNA. Lane Ccontained 360 ng of BSIV RNA.9.57.54.4 3.5 3.52.982.4 2.182.61.941.41.10.910.2445agarose gel electrophoresis and gel purified with QIAEX. The inserts, 1.9 and 1.2 kbrespectively, were labelled by random priming. The two probes hybridized only with BSIVinfected N clevelandii sap and not with healthy or CMV-infected sap. Probe 29 recognizedviral sequences in a 1/1,250 dilution of BSIV-infected sap (Fig. 7A) and probe 35 in a 1/6,250 dilution (Fig. 7B).3.7 Northern, Southern and dot blots with plasmid DNAProbe 29 recognized BSIV RNA 2 in a Northern blot (Fig. 8B, lanes 2-4), whereasprobe 35 recognized only RNA 3, but not RNA 4 (Fig. 8B, lanes 5-7). The probes wereused to identify other plasmids from the first synthesis containing inserts 600 bp or greaterin dot blots. The plasmids tested were 9, 11, 14, 16, 17, 19, 25, 29 and 35. Probe 29hybridized with plasmids 14, 16, 17, 19 and itself. The most intense reactions were withplasmids 16 and 29. The results of probe 35’s hybridization were difficult to interpret sincethe mini-prep DNA had run between the blots. It seemed to preferentially hybridize withplasmids 14, 17, 19 and 35.Since no full length clones of either RNA 3 or 4 were produced in the first synthesis,a second synthesis experiment was conducted using only 50 ng of primer. Plasmid miniprep DNA was again isolated and the inserts’ sizes determined. The same gel on which theinserts were separated was transferred to a membrane and hybridized with RNA 3 specificprobe 35 in a Southern blot. Plasmids 112, 123, 132 and 136 caused intense reactions. Ofthese four, plasmid 112, with the largest 1.8 kb insert, was used to synthesize another DIGdUTP labelled probe. In Northern analysis, probe 112 hybridized with both BSIV RNA 346Figure 7A-B: Healthy, blueberry shock ilarvirus-infected sap (BSIV) and cucumber mosaicvirus-infected sap from (CMV-B) sap from Nicotiana clevelandii hybridized with eitherprobe 29 or 35. Sap was diluted to 1:50, 1:250, 1:1,250 and 1:6,250 with lOx SSCcontaining 4% formaldehyde. Preparation of the probes and membrane were described inthe text (II 2.5.3 and II 2.5.4) as were the hybridization and detection procedures (II 2.5.5).A - Hybridization with probe 29. Only BSIV-infected sap hybridized with probe 29. B -Hybridization with probe 35. Only BSIV-infected sap hybridized with probe 35.BSIVCMVHealthyBSIVCMV1/50 1/250 1/1,250 1/6,250Healthy47Figure8A-B:Purifiedblueberryshockilarvirus(BSIV)andcucumbermosaicvirus,blueberryisolate(CIvIV-B)RNAseparatedbyformamide-formaldehydeagarosegelelectrophoresisfollowedbyaNorthernblothwithprobes29,35and112.Thegelwasprepared,electrophoresedandstainedasperII2.6.Preparationoftheprobesandmembraneweredescribedinthetext(II2.5.3andII2.5.4)aswerethehybridizationanddetectionprocedures(II2.5.5).A-Formamide-forinaldehydeagarosegel.Lanes1and8eachcontained3jigof CMV-BRNA.Lanes2and7eachcontained3jigofBSIVRNA.Lanes3and6eachcontained1.5tgofBSIVRNA.Lanes4and5eachcontained0.75jigofBSIVRNA.Lane9(notphotographed)contained2jigofBSIVRNA.B-Northernblot.Lanes1-4werehybridizedwithprobe29.Lanes5-7werehybridizedwithprobe35.Lane9washybridizedwithprobe112.B0035 2.982.18RNA31.1RNA4A).91and 4, but not with CMV RNA (Fig. 8B, lane 9). Probe 112 hybridized with plasmids 14,17, 19, 35, 123, 132,136, 161, 173 and itself (Fig. 9C-D). The cumulative results of allNorthern, Southern and plasmid dot blots are summarized in Table 4.Plasmids 14, 17, 19 and 35 from the first synthesis experiment were analyzed byrestriction digests. Their inserts, ranging from 0.6-1.1 kb, all overlapped. They had similar5’ termini with variable 3’ ends. Similarly, plasmids 112, 123 and 132 were analyzed.Plasmids 112 and 132 were almost identical in their digestion profiles, whereas plasmid 123lacked the internal Sst I site (Fig. 10). The orientation of the cDNA inserts relative to theirrestriction digests represented most of the 1.9 kb of BSIV RNA 3 and most of RNA 4.3.8 Nucleotide and protein sequence analysisA portion of the putative BSIV cp gene of clones 112, 123 and 132, whichcorresponded to the region 3’ from the internal Xho I site to the multiple cloning site, wassequenced. The sequence of each of the three clones was identical.This BSIVseq was 412 bp long (Fig. 11). When aligned with BSIV RNA 3, it was96% homologous. There were five difference between the two sequences. The first 259 bp,which corresponded to the 3’ terminus of the BSIV cp gene, contained one addition and onedeletion. The next 153 bp, which corresponded to the RNA 3 3’ non-coding region,contained three replacements.BSIVseq, including both the cp and non-coding regions, was aligned with the cp andnon-coding regions 3’ of the cp genes of TSV, ApMV and PDV. Since the non-codingregion of CVV was not available, BSIVseq was aligned to the CVV cp only. BSIVseq was49Figure9A-D:DetectionofblueberryshockilarvirusplasmidcDNAbynucleicacidhybridizationwithprobes29, 35and112.Preparationofprobesandmembranesandhybridizationweredescribedinthetext(II2.5.3,2.7.1and2.5.5respectively).A-Clones9,11,14,16,17,19,25,29and35werehybridizedwithprobe29.Thefilmwasexposedfor30sec.B-Clones9,11,14,16,17,19,25,29and35werehybridizedwithprobe35.Thefilmwasexposedfor 30sec.C-Clones14,17,19,123,132,136,173,35and112werehybridizedwithprobe112.Thefilmwasexposedfor10mm.D-Clones112,123,136,153,161,166,173,14,17,16,19,29and35werehybridizedwithprobe112.Thefilmwasexposedfor10mm.9291121112317U,14‘“136161615319171612919166352529 3514173Probe29Probe35Probe112Probe112Table4:SummaryofresultsfromNorthern,Southernandplasmiddotblots.LJProbeClone29(RNA2)35(RNA3and4)112(RNA3and4)dotblotdotblotSoutherndotblotdotblot9--NTNTNT11--NTNTNT14weak+NT++16+-NT-NT17weak+NT++19-+NT++25-weakNTNTNT29+-NT+NT35-+NT++101NTNT-NTNT112NTNT+++115NTNT-NTNT117NTNT-NTNT123NTNT+++132NTNT+++136NTNT+++153NTNT--NT158NTNTweakNTNT160NTNT-NTNT161NTNTweakweakNT166NTNT--NT173NTNT-++NT-nottestedFigure10:PartialrestrictionenzymecleavagemapsofBSIVRNA3andRNA4eDNAclonesandtheirlocationsrelativetoRNA3.ThenumbersonthethickRNA3barscorrespondtotheapproximatesizeinkb.Clones14,17,19,35,112,123and132wereobtainedbyrandomhexamerpriming.Thesolidblockonthe3’endwassequenced.RNA3IIIII0.00.51.01.51.94 C1one1121132MSSstIHincIIPitIXhoIXhoIAidMC1.8kbHindIIIMCSPstIXhoJXhoIAccIMCSIIIIICIone35MCSSstIHincIIPstIXhoIMCS1.2kbHindIIIClone17SstIHindIIIS0.9kbIII1Clonel4Il9MCSSstlH,ndIIIMCS0.58kbFigure 11: Partial nucleotide sequence of BSIV RNA 3 and RNA 4. The region betweeninternal Xho I site to the polyclonal linker. Underlined nucleotides are the sequence of XhoI restriction enzyme. Bold nucleotides are the sequence of Acc I restriction enzyme.CTCGAGCCCG ACTGGTCCTG CTGTCCTTAC CCCGAAAAGGGTTTCTTAAG GATCAGGCCA GAGGATGGCA GTGGCTTGCG 80CCGTCCGATT TGGAGTACGA TAAGTTCTCC GAAGAATACGAATTAGTATT CGAATTTAAG TCTGACTACC CGATAGGGTG 160GTCATGACTA GGGATTTGTA CGTGGTGACG TCTAGTCTACCACGGGTACG AATACCCGAT GATCTCTTAT TTGTCGATGA 240AGACTTATTA GAGATATAGA AGTGATTTGA TCACACTTCGATTAGAACCG TGAAGGTTCG ATACCGGTCC TCCGTGAGGA 320TTTACCGGTA GAATCTATTG TATCTCGGCT CCGAGATAGATGATCGATAG ATTTAACAAC CGATATGGTT GATTCCTAAT 400TCCGTGAAGG AA 41253also compared to two controls: the cp gene of PLRV, a luteovirus, and the plant herbicideresistancegene, PAT. The BSIV cp gene was similarly aligned and the percentage homologyfor each comparison was recorded (Table 5). The degree of homology between the BSIVcp gene and the unrelated PLRV cp gene or the PAT gene, 54.2 or 59.1% respectively, weresimilar to values for other ilarvirus cp genes. Results for BSIVseq were similar. There wereno discernable nucleotide homologies between either the BSIVseq or the BSIV cp gene andother ilarvirus cp genes.The BSIV ep sequence was aligned with each of the six translation frames ofBSIVseq visually. The cp sequence moved from BSIVseq frame 2 to 3, then back to frame2. Each frame also had many stop codons in the coding and non-coding regions. The stopcodons were deleted from the protein sequences and each translation frame was comparedto the cp’s of TSV, ApMV, PDV, PLRV and the PAT protein. The second and third framesof BSIVseq had the highest percentage identity with ApMV, 21.1 and 22.3 % respectively(Table 6). The portions of frames 2 and 3 corresponding to the cp were joined resulting inthe C terminal of the BSIV cp (BSIVf2,3). The BSIVf2,3 cp was then compared to eachprotein sequence. BSIVf2,3 had some regions of homology with the cp’s of CVV and PDV,17.6 and 24.7% respectively, but significant regions of homology with ApMV, 61.2% (Fig.12). Likewise, the identity between the cp’s of BSIV and ApMV, was the highest, 46.9%,while those with CVV and PDV were lower, 10 and 14.2% respectively.54L1Table5:Comparisonofthenucleicacidsequencesofblueberryshockilarvirus(BSIV)sequencedinthisreport(BSIVseq)orthecoatproteingene(BSIVcp),sequencedbyM.McLean,torelatedilarviruscoatproteingenesandtwonon-relatedcontrols.TheBSIVseqincludedsectionsofthecpgeneandthe3’non-codingregionofRNA3,thereforeitwascomparedtoboththecpandthenon-codingregions3’ofthecoatproteinsofotherilarviruseswhenavailable.nt-nucleotidescp-coatproteinhrp-herbicideresistanceproteinnc-non-codingsequenceDNA_SequenceIdentity(%)VirusGroupOriginFunctionSizeBSIVseqBSIVcp(nt)(412nt)(686nt)Ilarvirus,1Tobaccostreakcp740-51.9cp+RNA33’nc103061.6-Ilarvirus,2Citrusvariegationcp65455.647.9Ilarvirus,3Applemosaiccp681-62cp+RNA33’nc84970.3-Ilarvirus,4Prunedwarfcp656-51.5cp+RNA33’nc91661.4-LuteovirusPotatoleafrollcp62765.754.2(Plant)L-phosphinothricinhrp55257.259.1N-acetyltransferaseTable6:Comparisonoftheaminoacidsequencesofblueberryshockilarvirus(BSIV)sequencedinthisreport(BSIVseq)orthecoatproteingene(BSIVcp),sequencedbyM.McLean,torelatedilarviruscoatproteingenesandtwonon-relatedcontrols.TheBSIVseqwhichincludedsectionsofthecpgeneandthe3’non-codingregionofRNA3,wastranslatedinallthreeframesforthesequencedandcomplementarystrands.Allstopcodonswereremovedbeforetheaasequenceswerecomparedtootherilarviruscp’sandnon-relatedproteins.Valuesinboldindicateasignificantlevelofhomology.AminoAcid_SequenceIdentity(%)VirusGroupOrigin,FunctionSize(aa)BSIVseqBSIVcp(229aa)flf2f3f4f5f6f2,3Ilarvirus,1Tobaccostreak,cp2374.70.84.63.84.65.38.27Ilarvirus,2Citrusvariegation,cp2173.114.13.161.55.317.610Ilarvirus,3Applemosaic,cp2266.321.122.36.80.81.561.246.9Ilarvirus,4Prunedwarf,cp2173.116.4105.35.49.924.714.2LuteovirusPotatoleafroll,cp2088.65.51.50.84.67.65.94.6(Plant)L-phosphinothricin18372.34.63.80.80.85.96.6N-acetyltransferase,hrpan-aminoaicdsf-framecp-coatproteinhrp-herbicideresistanceproteinFigure12:Alignmentoftwoproteinsequences.Thecoatprotein(cp)portionoftheblueberryshockilarvirussequence(BSIVseq)wastranslated(BSIVpro)andalignedwiththeBSIVcp.Portionsofthecpwerefoundinframes2and3becauseoftheerrorsinsequencing.TheframeswerejoinedresultingintheCteminaloftheBSIVcp(BSIVf2,3).TheBSIVf2,3cpwasthenalignedwiththeapplemosaicvirus(ApMV)cp.BSIVf2,3normalsizedlettersrefertoframe2;boldlettersrefertoframe3.Theiridentitywas61.2%.-50-1-100-1-150-10-200-59-226-85ApMVcpBSIVf2,3ApMVcpBSIVf2,3ApMVcpBSIVf2,3ApMVcpBSIVf2,3ApMVcpBSIVf2,3MVWRICNHTHASGCRSCKKCHPNDALVPLRAQQRAANNPSRSRNPNRVSSS GVGPAIARQPVVKTTWTVRGANVPPRI PKGYVAHNQAEVTTTEAVNYLSIDFTTTLPQLMGQNLTLLTVMVRMNSMS SNGWIGMVEDYKVDQPDGPNALSIIIIIISPTGPAVLTRKGFLKDQPRGWQFEPPSDLDFDTFARTHRVVIEFKTEVPAGAKVLVRDL111111111111liiiIIliiiIIIIII1111111111111111IIIIIIIIIIIRKGFLKDQARGWQWLAPSDLEYDKFSEEYELVFEFKSDYPIGV-VMTRDLYVVVSDLPRVQIPTDVLLVDEDLLEI1111111111111111111111111111111111111111YVVTSSLPRVRIPDDLLFVDEDLLEI4 DiscussionBlueberry blossoms are the best source of BSIV inoculum. This may be due to ahigh concentration of virus as indicated by ELISA values or to a low level of viral inhibitorsin flower tissue compared to leaf tissue or a combination of the two.Using immuno-gold labelling, BSIV virions were localized within blueberry pollengrains embedded in Epon. Thin sections of stamens were labelled with BSIV PcAs, thendecorated with protein A gold. Protein A gold was bound non-specifically to the intine ofhealthy and BSIV-infected tissue, but it was bound specifically to membranous aggregatesfound only in virus-infected pollen. In ‘Bluetta’ pollen, these vesicles were composed of abilayer membrane surrounding isometric particles Ca. 30 nm in diameter. In ‘Rancocas’,these vesicles were composed almost entirely of membrane. Similar vesicles were notpresent in healthy tissue. Immunolabelling of these membrane-bound aggregates suggestedthat BSIV was located in the cytoplasm of infected blueberry pollen. However, evidence ofviral particles on the outer surface or between the intine and the exine was not conclusive.Non-specific labelling of the cell wall is a common artifact in immunocytochemistry (Hayat,1981).No data were available on immunolabelling of other ilarviruses, therefore informationwas inferred from other microscopy studies. Kelly and Cameron (1986) found PDV particlesconcentrated beneath the pores of infected cherry pollen fixed with potassium permanganate.Unfortunately, their analysis indicated that their staining technique was faulty. The presenceof particles near the pores was considered an artifact of the fixing procedure. Particles notfixed in the tissue would subsequently be lost with osmium tetroxide fixation. Kalashjan58(1987) observed vesicles filled with PNRSV particles in the xylem cells of cherry leaves.These reports indicated that ilarviruses may be present within the pollen and leaves of hostplants.Complementary DNA synthesis was affected by many factors including theconcentration and purity of template RNA. Genome purity was compromised in someexperiments. When precipitated with NaOAC and EtOH, RNA was pelleted with SDS saltswhich did not dissolve with 70% EtOH washes or LiC1 and EtOH reprecipitation. The saltsdid not interfere with size fractionation in either non-denaturing TAE gels or denaturingCH3HgOH and formamide gels. They did interfere with first-strand synthesis (BRLinstructions for Superscript, 1991). Only when precipitated first with LiC1 and EtOH, wasthe RNA free of visible salt pellets. The concentration of template was low in somesynthesis experiments, since a large percentage was lost isolating single-stranded and double-stranded RNA 3 and 4. Both the impurities and the isolation procedures contributed todecreased amounts of usable template.Another factor that interfered with cloning was the presence of contaminants,including SDS. Another contaminant was RNAid matrix. Neither high speed centrifugationnor phenol:chloroform extraction eliminated all the matrix from the RNA solution. A thirdchemical which may have reduced the length of eDNA inserts was CH3HgOH. It wassuccessfully used to denature dsRNA before first-strand synthesis of CMV, plum pox,raspberry leaf spot and strawberry mild yellow edge viruses (Jelkmann et a!., 1989). Now,it is no longer commonly used to denature ssRNA temple. IfCH3HgOH was not completelyneutralized with DTT, the first-strand solution remained opaque and failed to yield cDNA.59Therefore, either unneutralized CH3HgOH or an excess of DTT may have limited theefficiency of cDNA synthesis. First-strand synthesis proceeded only when RNA wasprecipitated with LiC1, then denatured by heating.No matter where the primers attach, first-strand synthesis extends in a 3’ to 5’direction relative to the template RNA. Oligo deoxythymidine primers which bind to apolyadenylated 3’ tail, can synthesize full-length clones. Since BSIV did not have apolyadenylated tail, synthesis of a polyadenylated tail and ligation of an oligodeoxyriboadenylated tail were tried. Because neither succeeded, random primers were theonly alternative. When either 50 or 150 ng of random hexamer primer were used to primefirst-strand synthesis, BSIV cDNA was synthesized. The longest RNA 3 cDNA insertproduced was 1.2 kb with 150 ng of primer and 1.8 kb with 50 ng. The RNA 3 clones werenearly full-length.A combination of Northern blots and plasmid dot blots identified nine clonessynthesized from BSIV RNA 3 or 4. Probe 29 hybridized with RNA 2 and with plasmids14, 16, 17, 19 and itself. Differences in the intensity of the reaction with the dot blots mayhave been due to differences in the concentrations of the mini-prep DNA. Probe 35,synthesized from a 1.2 kb insert, hybridized with RNA 3 in a Northern blot and withplasmids 14, 17, 19, 25 and 35. Cross-hybridization of plasmids 14, 17 and 19 to bothprobes 29 and 35 may have been due to homologous sequences between BSIV RNA 2 and3. Plamsids 14, 17 and 19 were mapped since they had had the most intense reactions inplasmid dot blots. Probe 35 identified plasmids 112, 123, 132, 136, 158 and 161 from thesecond eDNA synthesis in a Southern blot. Probe 112, with a 1.8 kb insert, hybridized with60RNA 3 and 4 in a Northern blot. It hybridized with plasmid dot blots of 14, 17, 19, 35, 123,132, 136 and 161 effectively confirming the results from Southern and dot blots with probe35. Probes 29, 35 and 112 hybridized only with BSIV RNA, not with genomic materialfrom healthy plant or CMV-B. In total, nine clones were synthesized from BSIV RNA 3and 4.These clones were partially mapped with restriction enzymes. The three shortest, 14,17 and 19, were nested within plasmid 35. The largest, 112 and 132, could not bedistinguished by enzyme mapping. Clone 123, 1.5 kb, was nested within clones 112 and132. Clones 35 and 112 had a large overlapping sequence. When combined, these twoclones should represent nearly the full-length of RNA 3.Results from Northern blots and restriction mapping suggested the orientation of theclones. Clone 35 may encode sequence from the 5’ end of RNA 3 since it hybridized onlywith RNA 3, not with RNA 4. In contrast, clone 112 might carry a sequence closer to the3’ end of RNA 3 or RNA 4. Since ilarviruses are known to encode the cp on the 3’terminus of RNA 3 (Cornelissen et al., 1984.; Bachman et al., 1994), clone 112 may encodethe cp gene. This would explain the equally intense reactions to RNA 3 and 4.Existing data on ilarvirus cp’s were amalgamated. The M of the cp subunits rangebetween 24-30 kD, with the exception of Tulare apple mosaic virus. By extrapolation, thelength of the cp sequence is between 800-1100 bp. The cp gene is transcribed from RNA 3as mRNA or RNA 4 and translated (Francki, 1985). Published sequences of the cp genesof ApMV, PDV and TSV confirmed the presence of the cp gene of the 3’ end of RNA 3(Sanchez-Navarro and Pallas, 1994; Bachman et a!., 1994; Cornelisson et a?., 1984). Since61BSIV RNA 4 Ca. 900 bp long, it is in agreement with a M of (30.2 ± 1.4) kD. Thereforeclones, 112, 123 and 132, which hybridized with BSIV RNA 4 should encode coat proteinsequence.A short region of three overlapping clones was sequenced to confirm the results ofrestriction mapping. The 412 bp sequences of clones 112, 123 and 132, between the internalXho I site and the 3’ multiple cloning region, were identical. The BSIVseq included 259 bpof the BSIV cp gene’s 3’ terminus and 153 bp of the 3’ non-coding region.The degree of homology between the BSIV cp gene and the PAT gene, 59.1%, anunrelated plant herbicide resistance protein, was close to those of TSV or PDV, 61.6 or61.4% respectively. The results for BSIVseq including both cp and non-coding regions ofRNA 3 were similar, 57.2, 51.9 and 51.5% respectively. There was no homology betweenthe BSIVseq and any other ilarvirus at the nucleotide level.When the BSIV cp sequence was compared to ApMV cp, there were long regions ofhomology. Their identity, 46.9%, agreed with serological data which placed both BSIV andApMV in ilarvirus subgroup 3. Identities between BSIV cp and two ilarvirus cp’s, TSV andPDV, were 10 and 14.2% respectively. These were approximately twice the value of thecontrols, PAT and PLRV cp, 6.6 and 4.6%.The addition and deletion of nucleotides in the cp region of BSIVseq resulted inframe shifts and additional stop codons in the protein sequences. The first error, a deletion,caused the protein sequence to shift from the second to the third frame. The second error,a nucleotide addition, returned the protein to the second frame. This explained the 21.2 and22.3% identities of BSIVseq frames 2 and 3 to ApMV. In addition, the presence of non62coding region may also have decreased the degree of alignment. BSIVseq protein sequenceframes 2 and 3 showed a lower degree of identity to ApMV cp than that between BSIV andApMV cp’s. When the protein sequence of frames 2 and 3 that showed homology withApMV were joined and aligned with ApMV cp, a higher level of homology was observed.BSIVf2,3 and ApMV cp had regions of high homology, 61.2%, similar to comparisonsbetween the full length cp’s of BSIV and ApMV, 46.9%. There were also regions of lowhomology between BSIVf2,3 and the cp’s of CVV and PDV, 17.6 and 24.7% respectively.Likewise, the full length cp’ s when compared were 10 and 14.2% respectively. These resultsindicate that there may be greater homology between BSIV and other ilarviruses in the Cterminus.63CHAPTER III - CMV ISOLATED FROM BLUEBERRY FLOWERS1 CucumovirusesAll three members of the cucumovirus group infect an extremely wide range ofherbaceous and woody hosts. Members include cucumber mosaic virus (CMV), peanut stuntvirus (PSV) and tomato aspermy virus (TAV).Historically, the symptoms on indicator plants were used to identify plant viruses.Symptoms are dependant on the interactions between the viral genes, their products, the hostplant’s genome and environmental factors. Therefore, although symptomatology and hostrange can suggest the virus’s identity, they cannot unequivocally prove it (Francki andHatta, 1980). Reactions on indicator plants are now used as one of many techniques toidentify an unknown virus.Serological tests are an indirect comparison of viral cp’ s nucleotide sequences sinceviruses can be differentiated by the epitopes present on the surface of intact particles (Haaseet a!., 1989; Mandahar, 1985). Antibodies made against viral particles can be used todistinguish viruses and their serotypes.In spite of being unstable and a poor immunogen, antibodies have been producedagainst CMV. Its inimunogenicity is enhanced by fixation with either glutaraldehyde orformaldehyde (Francki et aL, 1979). Fixation preserves viral integrity in serological assaysand prolongs antigen survival in the injected animal. PcAb’s are prepared by intramuscular,subcutaneous, intravenous or foot pad injections of rabbits. Antiserum titre is higher ifproduced against fixed, rather than unfixed virus. Titres against fixed CMV range from641/128 to 1/4,096 depending on the rabbit strain and the CMV serotype (Palukaitis et al.,1992).1.1 CMVCMV is found world-wide. It is a collection of isolates, whose properties are toosimilar to distinguish each with a separate name (Brunt et a!., 1990). It is divided into twoserotypes of which CMV-II is found mainly in temperate zones and CMV-I in tropical zones(Haase et a!., 1989). During the summer season in temperate zones, there is a shift fromCMV-II to CMV-I which is more thermostable (Haase et a!., 1989). CMV has the widestknown host range of all plant viruses, infecting more than 775 species. It naturally infectscereals, forages, woody and herbaceous ornamentals, vegetable and fruit crops and weeds(Douine et a!., 1979). Its host range is artificially extended through mechanical inoculation.CMV is best propagated in N c!evelandii (Kaper and Waterworth, 1981).Mosaics are the most commonly produced symptoms, but occasionally blight, fernleaf and ringspots are observed. Some plants remain asymptomatic, whereas for others,infection is fatal (Kaper and Waterworth, 1981). Generally, CMV-I induces more severesymptoms than CMV-II (Haase et a!., 1989). Wahyuni and co-workers (1992) used thesymptoms of three indicator plants, N edwardsonii, N tabacum ‘Xanthi’ and Capsicumfrutescens, to distinguish between the serotypes. In N tabacum ‘Xanthi’, CMV-I infectionresulted in green mosaics, leaf distortion, severe stunting and possible enation, whereasCMV-II caused only chiorotic mosaics and leaf distortion.CMV is naturally pollen- and seed-borne (Kaper and Waterworth, 1981). Infection65can be maintained or passed on by vegetative propagation, grafting and mechanicalinoculation (Kaper and Waterworth, 1981). However, the most thoroughly documentedmode of transmission, is the aphid vector. CMV is spread nonpersistently by more thanseventy-five species of aphid (Palukaitis et al., 1992; Kaper and Waterworth, 1981).CMV can be stained with UA, but is degraded by phosphotungstic acid. The particlesare 28-30 nm in diameter, with electron dense centres (Francki et al., 1979). The cpsubunits, M of 22-26.3 kD, easily disassemble in low concentrations of SDS or highconcentrations of neutral chloride salts.CIvIV has a single-stranded, tripartite, positive-sense RNA genome. RNA 1 and RNA2 are encapsidated in separate particles. RNA 3 and its subgenomic messenger, RNA 4, areencapsidated together in another particle (Kaper and Waterworth, 1981). Virion stabilitymay be due to RNA-protein interactions where the nucleic acid forms an inner layer partiallypenetrating the protein shell (Tolin, 1977).The presence of small satellite ssRNA molecules alters the symptoms associated withCMV. In tabasco peppers and sweet corn, CMV causes necrosis. When these plants areinoculated with CMV and its associated RNA 5 (CARNA 5), they cause a mild chlorosis.Conversely, CMV and CARNA 5 in tomatoes result in lethal necrosis (Kaper andWatersworth, 1981). Other CMV satellites vary in biological properties and nucleotidesequences ranging from 0.1-0.5 kD (Tolin, 1977).1.2 Research objectivesTwo pieces of eveidence indicated that blueberry shock disease may have been caused66by two viruses. First, isolates of BSIV, one maintained for three years in N clevelandii inthe greenhouse and others isolated from N clevelandii inoculated with blueberry blossoms,exhibited symptoms different from published results (Fig. 13). Both isolates when inoculatedonto Cucumis sativus seedlings, a non-host of BSIV, developed chlorotic spots. Secondly,not all the virus particles in a purified preparation of BSIV were imunolabelled with BSIVPcAs (Fig. 14).The objectives were to first identify the unknown virus and secondly to determine theinteraction between it and BSIV in infected blueberry plants.67Figure 13: Nicotiana clevelandii seven days after inoculation with either cucumber mosaicvirus (CMV-B) infected N clevelandii leaves (BSIV mild) or blueberry shock ilarvirusinfected blueberry blossoms (BSIV necrotic) homogenized in 0.05 M phosphate buffer, pH7.0, and 2% (w/v) polyvinylpyrrolidone, M 44,000 (PVP).68Figure 14: Purified blueberry shock ilarvirus (BSIV) decorated with BSIV polyclonalantiserum and stained with uranyl acetate by S. MacDonald. Note the two types of isometricspheres, one decorated, one not. x 62,500.I692 Materials and methods2.1 Characterization of an unknown virusMany factors are used to identify an unknown virus. In this report, the followingcharacteristics were studied: mechanical transmission to indicator plants, virion morphology,M of the cp subunit, genome type, genome and dsRNA profiles and serology.2.1.1 Source of virus and propagationA virus other than BSIV was isolated from BSIV-infected blossoms from a field inWhatcom county, Washington. The blossoms were homogenized with mortar and pestle in0.05 M phosphate buffer, pH 7.0, and 2% PVP. Four to six week old N clevelandii weredusted with medium grade carborundum, mechanically inoculated and later rinsed with water.Plants were maintained in a greenhouse with a 16 hr day and 8 hr night regime and daytimeand nighttime temperature of 22 and 16°C, respectively for more than three years.A second isolate, also from BSIV-infected blueberry pollen, was the only plant amongtwenty to exhibit milder symptoms uncharacteristic of BSIV infection. It caused crinklingand distortion of developing leaves and occasionally a mild mosaic. Additional isolates wereidentified among BSIV-infected plants, but were not propagated.2.1.2 Mechanical transmissionN clevelandii leaves infected with the unknown virus were homogenized and usedto mechanically inoculate four to six plants of seven different families. The plants included70Brassica juncea Czerniak ‘Florida Broadleaf’, Chenopodium amaranticolor Coste & Reyn.,C. quinoa Willd., Citrullus vulgaris Schrad. ‘Sweet Favourite’, Cucumis melo L.‘Delicious 51’, C. sativus L. ‘Straight 8’, Cucurbita pepo L. ‘Small Sugar’, Daturastramonium L., Gomphrena globosa L., Lycopersicon esculentum Mill. ‘Starfire’, Nbenthamiana Domin., N clevelandii L., N glutinosa L., N rustica L., N sylvestris L. Speg.& Comes, N tabacum L. ‘Harrownova’, ‘Havana 425’, ‘Samsun’, ‘White Burley’, ‘Xanthi’,Petunia Juss. calypso, Phaseolus vulgaris L. ‘Top Crop’, Physalis pubescens L. and Vincarosea G. Don. Plants mock inoculated with buffer served as healthy controls. Plants wereobserved for three weeks for symptom development.2.1.3 PurificationA purification protocol for the unknown virus was based on a modified procedure fortomato ringspot virus (Rott, 1989). All manipulations were done on ice or at 4°C.N clevelandii leaf tissue was harvested 10-14 days after inoculation. Using a Waringblender, tissue was homogenized for 3 mm in 2 ml of buffer (0.03 M Na2HPO4,pH 8.0, 0.02M ascorbate, 0.02 M -mer) per gram of tissue. The homogenate was filtered throughstretchable nylon cloth and the filtrate centrifuged at 10,000 rpm in a GSA rotor for 20 mm.The supematant was centrifuged at 35,000 rpm in a 50.2 Ti rotor for 2 hr. The pellet wasresuspended in 0.1 vol of 0.05 M citrate buffer, pH 7.0, using a ground glass homogenizer,then shaken at 250 rpm for 16 hr. The mixture was centrifuged at 10,000 rpm for 20 mm.The supernatant was layered onto 5 ml of 20% sucrose cushions in 0.1 M phosphate buffer,pH 7.0, in 25 ml tubes and centrifuged at 38,000 rpm for 2 hr. The pellets were resuspended71in 10 ml of 0.05 M phosphate buffer, pH 7.0, containing 4.4 g of cesium sulphate. Thegradients were centrifuged at 55,000 rpm in a 70.1 Ti rotor for 16 hr at 10°C. Each of thethree opaque bands were removed using a 20 gauge needle and syringe. Each wasconcentrated by centrifugation at 60,000 rpm in a 70.1 Ti rotor for 1 hr. The pellets wereresuspended in 0.05 M phosphate buffer. Only the middle band contained intact particlesas determined by transmission electron microscopy. Samples were stored at 4°C.The absorbance spectrum, A220-A3 was measured and recorded. Initially theconcentration was determined as for BSIV, but later when identified as an isolate of CMV,it was corrected. A solution of 1 pg/pJ of CMV has an absorbance of 5 at 260 nm. PurifiedCMV had an A260/A8 of 1.7 (Francki et a!., 1979). Purity of the virus was also checkedby transmission electron microscopy.2.1.3.1 SDS-PAGEPurified preparations were diluted in 5x SDS-PAGE sample buffer and boiled for 3mm to denature all proteins. Samples were separated in a denaturing polyacrylamide gel,stained with Coomassie Brilliant Blue R-250 and dried under vacuum (Chapter II, 2.2.1).The M of the cp’s were compared against a low M standard (Bio-Rad, Richmond, CA).2.1.4 Virion RNA extractionSsRNA was extracted from purified virions as per Chapter II, 2.3. The concentrationof RNA was determined by measuring the absorbance at 260 nm. The number of RNAspecies and their Mw’s were determined by electrophoresis in a denaturing CH3HgOH72agarose gel (Chapter II, 2.3.1).2.1.5 Double-stranded viral RNA extractionDsRNA was extracted as per Kurppa and Martin (1986). All manipulations weredone at RT. Twenty gram samples of fresh BSIV-infected blueberry blossoms or Ntabacum varieties ‘Harrownova’, ‘Havana 425’, ‘Samson’, ‘White Burley’ and ‘Xanthi’ werepowdered with a mortar and pestle in liquid nitrogen. Two vol of 2x STE (lx STE = 100mM NaCl, 50 mM Tris-Cl, pH 7.1, 1 mM EDTA), 0.02 vol -mer, 0.5 vol 10% (w/v) SDS,1.5 vol STE saturated phenol, 1.5 vol STE saturated chloroform were added, shaken at 250rpm for 30 mm and centrifuged at 10,000 rpm in a GSA rotor for 20 mm. The aqueousphase adjusted to 16% EtOH and shaken with 2.5 g of CF-il cellulose powder (Whatman,Maidstone, GB) was poured into a 1.5 cm diameter Econo-Column (Bio-Rad, Richmond,CA). The slurry was drained, washed with 80 ml of 16% EtOH in STE (STE-EtOH) andpurged with air. DsRNA was eluted from the column with 15 ml of STE. The eluent wascentrifuged at 5,000 rpm in a Baxter Scientific Megafuge (Can-Lab, Toronto, Ont.) for 10mm to pellet matrix that escaped through the column. Any ssRNA in the supernatant wasdigested with 2 U/ml of RNAse T1 (Sigma Chemical Co., St.Louis, MO) at 37°C for 30 mm.The concentration of MgC12 was adjusted with 0.033 vol of a 1 M solution and DNAdigested with 1 U/ml of deoxyribonuclease I (DNAse I; Sigma Chemical Co.) at 37°C for30 mm. The reaction was stopped with 0.033 vol of 0.5 M EDTA, pH 8.0, and the eluentadjusted to 20% EtOH. It was shaken with 0.3 g of 100 micron Microcrystalline non-ionicmicrocellulose (J.T. Baker, Phillipsburg, NJ) for 20 mm, poured into the column and washed73with 20 nil of STE-EtOH. Nucleic acids were eluted with 1.5 ml of STE, centrifuged for10 mm at high in a microfuge to pellet matrix. DsRNA in the supernatant was precipitatedwith 0.1 vol of 3 M NaOAc and 3 vol of 95% EtOH.DsRNA was dissolved in 5x DNA loading buffer, loaded into a 0.7% agarose gel andelectrophoresed at 3.2 V/ml per 25 ml of gel for 1 hr in TAE (III, 2.4.3). The gel wasstained with EtBr and photographed.2.2 Serology2.2.1 Preparation of Polyclonal antiserumProduction of PcAs against the unknown virus was begun prior to positiveidentification. A New Zealand white rabbit was immunized with purified virus in 0.05 Mphosphate buffer, pH 7.0. The first injection, 150 jig of virus emulsified 1:1 with Freund’scomplete adjuvant, was given subcutaneously. The next three injections, of 100 jigemulsified 1:1 with Freund’s incomplete adjuvant, were administered intramuscularly everythree weeks. Ten days after the last injection, a test bleed of 10 ml was obtained. yGlobulins were purified from the raw antiserum and conjugated with alkaline phosphataseas described below. Immunoglobulins failed to distinguish between healthy and virusinfected N clevelandii sap, but the background was low indicating that the rabbit hadproduced antibodies neither to healthy sap nor to virus. Injections were resumed, but undera different schedule (R.R. Martin, personal communication). Five injections of 200 jig wereadministered intervenously every 2-5 days. Ten days after the final injection, the animal wastest bled. y-Globulins clearly differentiated between healthy and virus-infected sap. Twenty74days after the final injection, the rabbit was sacrificed by doing a total body bleed. Rabbitblood was incubated for 1 hr, the clot separated from the serum, then incubated at 4°C for16 hr. The supematant was centrifuged at 4,500 rpm in a Baxter Scientific Megafuge for10 mm. Antiserum was stored at 4°C with 0.02% NaN3.2.2.1.1 Purification and conjugation of y-globulinImmunoglobulins were purified from antiserum by ammonium sulphate precipitationfollowed by DEAE-cellulose column chromatography (Clark et a!., 1986). All reactionswere done at RT. One vol of PeAs was diluted in 9 vol of water and 10 vol of saturatedammonium sulphate, incubated for 1 hr and centrifuged as above. The precipitate wasdissolved in 2 vol of 0.5x PBS and dialysed three times against 0.5x PBS. Immunoglobulinswere separated on a DEAE-22 Cellulose column (Whatman, Clifton, NJ) washed with 0.5xPBS and detected at 280 nm with an ISCO UA-5 absorbance/fluorescence detector. Theconcentration of 7-globulins was adjusted to approximately 1 mg/mi (1 mg immunoglobulinper ml has an A280 of 1.4; Harlow and Lane, 1988). Inimunoglobulins were stored at 4°Cwith 0.02% NaN3.Alkaline phosphatase was conjugated to 7-globulins. Gluteraldehyde, at 0.16%, wasused to conjugate 200 jig of 7-globulins to 200 jig of EIA Grade alkaline phosphatase(Boehringer-Mannheim, Lava!, Que.) in a total vol of 227 jil at 30°C for 4 hr. Conjugatewas dialysed three times against lx PBS, then diluted to 2 ml with PBS for a finalconcentration of 0.1 jig/pi.752.2.1.2 Double antibody sandwich ELISAA double-antibody sandwich ELISA (DAS-ELISA) was used to test PcAs against theunknown virus (Clark and Adams, 1977). All reagents were used at 100 $11 per well in flat-bottomed Lindbro or Corning microtiter plates (Flow, Laboratories, Mississauga, Ont. orBaxter Diagnostics Corp, Toronto, Ont) except for blocking steps, which used 200 JIi perwell. Wells were coated with trapping polyclonal immunoglobulins, diluted 0.5-4 .ig/ml incoating buffer (0.05 M sodium carbonate, pH 9.6), and incubated at 4°C for 16 hr or at 30°Cfor 4 hr. Plates were blocked with blocking buffer (PBS with 0.05% Tw-20 and 0.2% skimmilk powder (SMP)) at 30°C for 30 mm. Leaf tissue from infected and healthy plants washomogenized in a sap extractor (Erich Pollähne, Germany) while adding grinding buffereither blocking buffer alone or blocking buffer containing 2% PVP drop-wise onto thebevelled rollers at a rate of 1 ml per 0.1 g leaf tissue. Extracts were diluted to 1 g leaf tissueper 40 ml of grinding buffer and incubated in the wells at 4°C for 16 hr. Plates werewashed three times with tap water. Trapped virus was detected with y-globulin alkalinephosphatase conjugate, diluted 0.5-4 ig/ml in blocking buffer and incubated at 30°C for 4hr. Plates were washed as above and 0.5 ig substrate (p-nitrophenyl-phosphate, Sigma 104-105, Sigma Chemical Co., St. Louis) per ml in 10% (v/v) diethanolamine, pH 9.8, wasincubated at 30°C for 4 hr, then at RT for 16 hr. The absorbance was read at 405 nm (A405)in a Titertek Multiscan MCC plate reader (Flow Laboratories). Subsequent ELISA tests usedCMV-B y-globulins diluted to 2 jig/ml in coating buffer and conjugate diluted 2 .tg/ml inblocking buffer.762.2.1.3 Relationship to ilarvirus groupSerological relationship between the unidentified virus and 12 known ilarviruses wasdetermined by indirect ELISA. Plates were coated with either purified virus, diluted to 1ig/ml with PBS, or healthy N clevelandii sap diluted 1:50 with PBS and incubated at RTfor 2 hr. Plates were then washed with water and blocked with PBS-Tw-SMP at RT for 1hr. Five 5-fold serial dilutions of the various antisera (Table 7) in blocking buffer were donedirectly in the microtiter plates and incubated at 30°C for 2 hr. Plates were washed andgoat-anti-rabbit immunoglobulin (GAM) alkaline phosphatase conjugate (Jackson ImmunoResearch Laboratories, Inc., Bio/Can Scientific, Mississauga, Ont.), diluted 1:2500 (v/v) inblocking buffer, was incubated in the wells at 30°C for 2 hr. The A405 was measured 1 hrafter adding substrate.2.2.1.4 Polyclonal antiserum specificityDAS-ELISA was used to test the unknown virus antiserum against type strains of thecucumovirus family. Wells were coated with CMV-B y-globulins at 2 tg/m1 in eithercoating buffer or PBS and incubated at 4°C for 16 hr. Plates were blocked. Leaves fromhealthy and CMV-I, CMV-II, PST, TAV-infected N tabacum were homogenized 1:40 ineither blocking buffer alone or containing 2% PVP. The sap was incubated in the wells at4°C for 16 hr. The plates were washed. Unknown virus polyclonal ‘y-globulin alkalinephosphatase conjugate at 0.2 ig/m1 in blocking buffer, was incubated at 30°C for 2 hr. TheA405 was measured 5 hr after adding substrate.77Table7:Listofilarvirusantiserausedtodeterminetheserologicalreactivityoftheunknownvirus.00SubgroupVirusAntiserumDonatedbyLocationDilutionTobaccostreak(TSV),TjgW.KaiserPullman,WA1:200TSV,R515R.Stace-SmithVancouver,B.C.1:200TSVR.H.ConverseCorvallis,OR1:100TSVR.FultonMadison,WI1:200TSVR.H.ConverseCorvallis,OR1:2002AsparagusvirusIIR.FultonMadison,WI1:200CitrusvariegationG.I.MinkProsser,WA1:200CitrusleafrugoseG.I.MinkProsser,WA1:200ElmmottleG.I.MinkProsser,WA1:200TulareapplemosaicG.I.MinkProsser,WA1:200LilacringmottleG.LeoneWageningen,Netherlands1:200FragariachiloensisR.R.MartinVancouver,B.C.1:2003Applemosiac(ApIVW),broadhostrangeG.I.MinkProsser,WA1:200ApMV,thimbleberryR.Stace-SmithVancouver,B.C.1:200ApMVR.H.ConverseCorvallis,OR1:200ApMV,birchR.H.ConverseCorvallis,OR1:200ApMV,appleR.H.ConverseCorvallis,OR1:100ApMVR.H.ConverseCorvallis,OR1:100BlueberryshockilarvirusR.R.MartinVancouver,B.C.1:200HumulusjaponicusA.AdamsEastMalling,U.K.1:200Prunusnecroticringspot,roseG.I.MinkProsser,WA1:2004PrunedwarfM.F.ClarkEastMalling,U.K.1:2006SpinachlatentG.I.MinkProsser,WA1:2002.2.2 Preparation of monoclonal antibodiesA BALB/c mouse was immunized with seven injections of purified virus in 0.05 Mphosphate buffer, pH 7.0. The first injection, 75 jig of virus emulsified with Freund’scomplete adjuvant, was given subcutaneously. The remaining injections of 20-50 jig eachwere administered intraperitoneally every 2-5 days following the first injection. Four daysafter the final injection, the mouse was killed by carbon dioxide asphyxiation and the spleenremoved aseptically.FOX-NY myeloma cells (Hyclone Laboratories, Inc., Logan, UT) were cultured inDulbecco’s Modified Eagle Medium (DMEM; BRL, Gaithersburg, MD) supplemented with10% (v/v) fetal calf serum (FCS). Spleen cells were fused with myeloma cells in50% PEG-4000 as described by Kannangara and co-workers (1989). All myelomas andhybridomas were grown at 37 C in an atmosphere of 10% (vlv) CO2. The cell fusionmixture was dispensed into six 96-well culture plates (Nunc, Denmark) and incubatedovernight in nonselective media (DMEM containing 20% FCS) with mouse thymocytes asfeeder cells. Thymocyte cultures were then fed with adenine/aminopterin/thymidine (AAT)selection media (AAT 7.5 x iO M adenine, 8 x iO M aminopterin, 1.6 x iO Mthymidine (Taggart and Samloff, 1983)). After 10 days, culture fluids from the hybridomaswere screened for McAb produced against plant sap and virus by direct ELISA.2.2.2.1 Screening McAbTen days after fusion, culture fluids from the hybridomas were screened for antibodiesproduced against plant sap and CMV-B by ELISA. Wells were coated with sap from healthy79and virus-infected N tabacum ‘Samson’, homogenized and diluted 1:40 in blocking buffercontaining 2% PVP and incubated at 30°C for 4 hr. The wells were washed and blocked.Undiluted hybridoma culture fluid was added and incubated at 4°C for 16 hr. The wellswere washed. GAM alkaline phosphatase conjugate, diluted 1:2500 in blocking buffer, wasincubated at 30°C for 1 hr. Hybridoma cell lines which produced antibodies that testedpositive with CMV-B and negative against healthy N clevelandii were cloned twice bylimiting dilution, grown in cell culture and retested. Sub-clones were tested by ELISA,GAM being replaced by rabbit-anti-mouse (RAM) alkaline phosphatase conjugate (JacksonImmuno Research, Bio/Can Scientific, Mississauga, Ont.). Culture fluid testing positivelyonly to virus-infected tissue was stored at 4 C with 0.02% NaN3 and used for the survey.Positive sub-clones were pelleted, resuspended in DMEM containing 10% (v/v)dimethylsulfoxide and frozen at -80°C.2.2.2.2 Monoclonal antibody specificityThe specificity of McAb prepared against CMV-B to cucumovirus serotypes wastested by indirect ELISA. Wells were coated with sap from healthy and CMV-I, CMV-II,PST, TAV, CMV-B-infected N tabacum homogenized 1:40 in blocking buffer and incubatedat 30 C for 4 hr. The wells were washed and blocked. Undiluted hybridoma culture fluidwas incubated at 4 C for 16 hr. The wells were washed. RAM alkaline phosphataseconjugate was diluted 1:2500 in blocking buffer at 30°C for 1 hr. The A405 was measured1.5 hr after adding substrate.802.3 Survey of CMV-B in BSIV-infected blueberry plants2.3.1 Collection of blueberry samplesSamples were collected by R. Martin and M. M’Lean in Whatcom county,Washington. In May of 1993, flower samples from Ca. 200 ‘Bluetta’ and 200 ‘Jersey’blueberry plants were collected. Samples were placed in labelled plexiglass trays, packedin coolers and brought to Vancouver. In June and September, only ‘Bluetta’ plants weresampled since more than 95% had exhibited disease symptoms and were known to beinfected with BSIV and was the pollen source for the CMV isolate. In total, 659 samplesof blueberry plants were tested for CMV-B infection.2.3.2 DAS-ELISA to test blueberry samplesDAS-ELISA was used to identify blueberry plants infected with CMV-B. TrappingCMV-B y-globulins were diluted to 1 or 2 ig/ml in coating buffer and incubated at 30°Cfor 4 hr or 4°C for 16 hr. The plates were washed and blocked with blocking buffer at 30°Cfor 30 mm. Blueberry tissue was homogenized in 0.1 M borate buffer, pH 8.0, containing0.2% SMP, 2% PVP and 0.5% (v/v) nicotine alkaloid. All other tissue such as healthy andCMV-B infected tobacco, was homogenized in blocking buffer. Sap was incubated at 4 Cfor 16 hr. The plates were washed and CMV-B polyclonal y-globulin alkaline phosphataseconjugate was diluted to 1 or 2 tg/ml in blocking buffer and incubated at 30 C for 4 hr.The A405 was measured 2 and or 16 hr after adding substrate.812.3.3 Collection of nearby vegetationResults indicated that blueberry plants may not be a host for CMV-B (III, 3.4).However, many weeds are known to be hosts of CMV. Other vegetation, including tissuesamples from weeds, shrubs and trees were collected on two different occasions. In total,more than 170 leaf samples were tested either by DAS- or triple antibody sandwich-ELISA(TAS-ELISA) or both for CMV-B infection.2.3.4 DAS- and TAS-ELISA to test vegetationDAS-ELISA was used to detect CMV-B in half of the plates containing samples ofnative vegetation. Three tests were conducted as per III, 2.3.2. In duplicate plates, afterincubation of sap, virus was detected with culture fluid from CMV-B specific hybridomas,either undiluted or diluted 1:1 with blocking buffer and incubated either at 30°C for 4 hr orat 4°C for 16 hr. The plates were washed and RAM alkaline phosphatase conjugate diluted1:2500 in blocking buffer was added and incubated at 30°C for 2 hr. The A405 was measured16 hr after adding substrate.2.3.5 Mechanical transmission of CMV-B infected buttercup samplesTissue from six samples of buttercup with the highest absorbance values in ELISAwere used to mechanically inoculate three N clevelandii and one Cucumis sativus seedling.Plants were monitored for 4 week for symptoms.823 Results3.1 Identification and characterization of second virusFourteen days after inoculation, symptoms developed on some host plants.Descriptions of the disease symptoms are recorded in Table 8. Symptoms that ranged froma mild mosaic to necrotic spots were found in Chenopodium amaranticolor, Cucumis sativus(Fig. 1 5C), Cucurbita pepo, Datura stramonium, Gomphrena globosa L., Lycopersiconesculentum, N clevelandii, N glutinosa, N rustica, N sylvestris, N tabacum ‘Harrownova’(Fig. 15B), ‘Havana 425’, ‘Samson’, ‘White Burley’, ‘Xanthi’ (Fig. 15A), Petunia calypsoand Physalis pubescens.The unknown virus could not be purified using the protocol for BSIV. The virus didnot remain in solution when the pH was lowered to 5.0. Therefore, a new procedure wasdeveloped. After homogenization in the same buffer used for BSIV, the mixture wasclarified through a series of differential centrifugations in citrate buffer, followed by pelletingthrough a sucrose cushion and further concentration in a cesium sulphate gradient. Highspeed centrifugation removed cesium sulphate and the virus was resuspended in phosphatebuffer at a concentration of 0.7-12.3 tg/pi. Virus yields were 2-14 mg per kg of fresh leaftissue. Fractionated preparations of virus had an A260/A8 ratio of 1.79-1.82 (compared to1.7 for CMV reported by Francki and co-workers in 1979). Purified virus was stained withUA, but disrupted with phosphotungstic acid. The virions were isometric, (31.7 ± 0.8) nmin diameter with electron dense centres (Fig. 16).Two proteins, found in the purified preparation, were separated by SDS-PAGE. The83Table8:Comparisonofindicatorplants16daysafterinoculationwithcucumbermosaicvirus,blueberryisolate,infectedNclevelandiileaves(III,2.1.1)topublishedreportsofcucumbermosaicvirussymptoms.Family,Species,CultivarSymptomsfromthisreport(Fig.)Cultivar,PublishedsymptomsRef.AmaranthaceaeGomphrenaglobosachiorosischloroticlesions;yellowmosaic,leafdistortionVApocynaceaeVincaroseasymptomlessBrassicaceae/CruciferaeBrassicajuncea,‘FloridaBroadleafsymptomlessChenopodiaceaeChenopodiumamaranticolornecroticlesionsnotsystemic;chloroticornecroticlocallesionsIIsymptomless;chioroticornecroticlocallesionsIVChenopodiumquinoa(weed)symptomlessnotsystemic;chioroticornecroticlesionsIInotsystemic;locallesions,whitespots1-3mmdiameterVICucurbitaceaeCitrullusvulgarissymptomlesssymptomless;chloroticornecroticlocallesionsIVCucumismelo,’Delicious51’chloroticlesions,stunting‘DoublonCharentais,’symptomless;mildmosaicIVCucumissativus,‘Straight8’chloroticspots,mosaicalongvein,‘vertlongMaraich’chloroticspots,mildmottleIVveinclearing(Fig.15A)systemicmosaic;stuntingII‘Polaris’chloroticlocallesions;mosaicVCucurbitapepo,’SmallSugar’chloroticspotsmosaicI‘Fl.Diamant’chloroticspots;mosaicIV00SolanaceaeDaturastratamoniumLycopersiconesculentum,’Starfire’NicotianabenthamianaNicotianaclevelandiiNicotianaglutinosaNicotianarusticaNicotianasylvestrisNicotianatabacum,’Harrownova’Nicotianatabacum,’Havana425’Nicotianatabacum,’Samson’Nicotianatabacum,’WhiteBurley’Nicotianatabacum,’Xanthi’PetuniacalypsoPhysalispubescensmosaicleafnarrowing;mottlestuntedgrowth;leafdistortionmildmosaic,leafdistortionmosaic,necroticlesions,veinclearing,stuntedgrowthleafdistortion,stuntedgrowthmosaic,pin-pointlesions,stuntedgrowthnecroticlesionsalongveins(Fig.1 5C)ringspotsoninoculatedleaves,mosaic,leafdistortiondownwardcurlingleaves,veinclearing,mildmosaicpin-pointnecroticlesions,mosaicringspots,distortedleaves(Fig.15B)chlorosissymptomlessmildtoseveremosaic;chioroticlesions;leafdistortionmosaic;narrowingofleafwithfernleafsymptomsnaturalhost;mosaic;leafdistortionsymptomless;chloroticspots;mildmosaicmildtosevere,possiblyyellowmosaic;leafdistortionmildtosevere,chloroticmosaic;veinyellowingmosaicandleafmalformationmildtosevere,possiblyyellowmosaic;leafdistortionsymptomless;mildmottlesymptomless,chioroticmottle,etchingmildtosevere,possiblyyellowmosaic,leafdistortionfaintchloroticlocallesions,systemicyellowmosaicwithnecroticspotsetch;mildmottleI-DanielsandCampbell,1992IV-MarrouetaL,1975II-FranckietaL,1979V-RaoandFrancki,1982III-KaperandWaterworth,1981VI-TomlinsonandCarter,1970Family,Species,CultivarSymptomsfromthisreport(Fig.)Cultivar,PublishedsymptomsPdLeguminosae/PapillonaceaePhaseolusvulgaris,’Pinto’symptomless‘Pinto’-symptomless;microscopicornecroticlocallesionsIV0OV II III IV V II III V IV IV V VIIVBFigure 15A-C: Indicator plants 16 days after inoculation with cucumber mosaic virus-infected N clevelandii leaves homogenized in 0.05 M phosphate buffer, pH 7.0, and 2%(w/v) polyvinylpyrrolidone, M 44,000. A - N tabacum ‘Xanthi’. B - N tabacum‘H’ wa’. C - sativus’A_% -.Nicotiafla tabaCumXanthi”4Nicc,tiatla t&bacnimC86C 8”87Figure 16: Purified cucumber mosaic virus, blueberry isolate (CMV-B) in phosphate-buffered saline, washed for 1 mm in 0.3% (w/v) bacitracin and stained for 2 mm in 1%(w/v) uranyl acetate The virus had a diameter of (31.7 ± 0.8) mu. x 155,520.88Imost abundant protein, the cp subunit, had a M of (29.5 ± 0.3) kD (Fig. 5). The proteinwith the higher M was a contaminating healthy plant protein (MacDonald and Martin,1991).Four species of ssRNA were isolated. When separated in a methyl mercurichydroxide gel, RNA 1-4 were (3.49 ± 0.2, 2.98 ± 0.1, 2.18 ± 0.06 and 1.10 ± 0.1) kb inlength with Mw’s of (1.19 ± 0.07, 1.01 ± 0.03, 0.74 ± 0.02 and 0.37 ± 0.03) x 106 D (Fig.6; Table 9). There was no evidence of CARNA 5.DsRNA was purified from N tabacum ‘Harrownova’, ‘Havana 425’, ‘Samson’,‘White Burley’ and ‘Xanthi’. The dsRNA profiles of all five varieties were identical to onestandard, CMV isolated from primula (Fig. 17). The isolate was tentatively named CMV-B,to identify its isolation from blueberry blossoms.3.2 Polyclonal AntibodiesDifficulties in PcAs production arose from its poor immunogenicity. After the firstset of injections failed to produce antibodies, a different approach was used. R. W. Fultonhad produced many successful PcAs by injecting rabbits with purified virus over a shorterperiod than the recommended three weeks (R. R. Martin, personal communication). If thetime between injections was decreased, the animal’s immune system would be exposed toa higher concentration of intact virus. The second set of injections induced an immuneresponse in the rabbit. PeAs, that could be used to distinguish between healthy and virus-infected N clevelandii or N tabacum ‘Samson’, was obtained.The virus was tested against antisera from fourteen different ilarviruses, as well as89Table9:Estimatedandreportedsizesandweightsofcucumbermosaicvirus(CMV)single-strandedRNAspeciesRNASpeciesVirusUnitsofReferenceMeasurement1234CMV-B3490±2102980±1002180±601100±100bpthisreportCMV-I3357305022161031bpPalukaitisetal.,1992CMV-II3389303521971034bpPalukaitisetal.,1992CMV-B1.19±0.071.01±0.030.74±0.020.37±0.03106DthisreportCMV1.271.130.820.35106DFranckietal.,1979CMV1.01-1.350.89-1.210.68-0.930.33-0.38106DLoesch-Friesetal.,1977CMV1.271.130.820.35106DFranckiandHatta,1980CMV1.010.890.680.33106DKaperandDiaz-Ruiz,1977C±-onestandarddeviationFigure 17: Double-stranded RNA (dsRNA) extracted from cucumber mosaic virus (CMV-B)infected tissue, then separated by non-denaturing agarose gel electrophoresis. CMV-BdsRNA was extracted and separated (III 2.1.5) as described in the test. Lanes 1 and 8 wereloaded with 1 kb DNA molecular weight marker (GIBCO BRL). Lane 2 was a CMVdsRNA standard isolated from Primula (donated by Anita Quail). Lanes 3-7 were all CMVdsRNA isolated from Nicotiana tabacum from the respective cultivars ‘White Burley’,‘Xanthi’, ‘Harrownova’, ‘Havana 425’ and ‘Samson’.6.11-12.22kb.506.394.298kbkbkb123456785.094.073.05kbkbkb2.04 kb1.64kb1.02kb91to its homologous antiserum. Although it failed to react with any ilarvirus antiserum inindirect ELISA with antigen coated plates, an A405of 0.88 was obtained with the homologousPcAs, compared to an A405 of 0.14 with healthy tissue. The PeAs was tested against allcucumovirus members. In an indirect DAS-ELISA, PcAs detected CMV-I and CMV-II indilutions of 1 g infected tissue per 320 ml of coating buffer, but may have been able todetect even lower concentrations (Table 10). The same tests showed that the y-globulinspreferentially reacted with CMV-TI with approximately double the A405 value as that forCIvW-I. When PBS was used as the trapping buffer instead of carbonate buffer, the PeAsfailed to differentiate between the CMV serotypes. Neither PSV or TAV reacted to thepolyclonal y-globulin. Based on these tests, CMV-B was an isolate of CMV.3.3 Monoclonal antiserumHybridomas secreting antibodies specific to CIvIV-B were produced. Sevenhybridomas labelled ‘S’, ‘T’, ‘U’, ‘V’, ‘W’, ‘Y’, ‘Z’, were recloned and tested. Whenretested, hybridoma ‘5’ subclones reacted positively to both healthy and CMV-B infectedplant sap, while subclones of ‘T’, ‘U’, ‘V’, ‘W’, ‘Y’ and ‘Z’ differentiated between healthyand infected sap.The specificity of each subclone was determined in TAS-ELISA against eachcucumovirus. Culture fluid from hybridomas ‘U, W’ and ‘Y’ reacted only to CMV-II.McAb’s from ‘T’ and ‘Z’ resulted in nearly equal absorbances to either CMV serotype. Theculture fluid from hybridoma ‘V’ detected both CMV-I and CMV-IT, but had a higherabsorbance with CMV-II. The subelones were combined into groups based on their reactions92Table10:Serologicalreactivityofpolyclonalantiserummadeagainstcucumbermosaicvirus,blueberryisolate(CMV-B)tocucumoviruses:CMVserotypesIandII,peanutstunt(PSV)andtomatoaspermy(TAV).Microtitreplateswerecoatedwithpolyclonalimmunoglobulinsdilutedineitherphosphate-bufferedsaline(PBS)orcarbonatebuffer,pH9.6.LeaftissuewashomogenizedanddilutedinPBScontaining0.05%(vlv)Tween20and0.2%(w/v)skimmilkpowder(PBS-Tw+SMP).Allproceduresweredescribedinthetext(III2.2.1.4).TheA405wasmeasuredafter5hr.Absorbanceat405nmBufferSapControlsVirusinfectedtissueDilutionCMV-BHealthyCMV-ICMV-IIPSTTAVPBS1:100.640.030.280.380.060.051:200.840.030.340.360.040.041:400.820.030.360.430.040.071:800.900.020.320.270.030.031:1600.900.020.340.240.030.031:3200.910.020.340.330.030.03Carbonate1:100.700.030.340.660.110.141:200.800.020.390.870.060.121:400.810.020.460.790.040.071:800.830.020.460.820.040.061:1600.880.020.430.760.030.041:3200.770.020.380.700.030.03Table11:Serologicalreactivityofmonoclonalantibodies(McAb)madeagainst cucumbermosaicvirus,blueberryisolate(CMVB)tocucumoviruses:CMVseroytpesIandII,peanutstunt(PSV)andtomatoaspermy(TAV).Microtitreplateswerecoatedwithpolyclonalimmunoglobulinsdilutedincarbonatebuffer,pH9.6.LeaftissuewashomogenizedanddilutedinPBScontaining0.05%(v/v)Tween20and0.2%(w/v)skimmilkpowder(PBS-Tw+SMP).Allproceduresweredescribedinthetext(III2.2.2.2).A405wasmeasuredafter1.5hr.Thereactivitygroupnumbersrefertothespecificityofeachgroup.Group1-McAb’sreactingequallytobothCMV-IandCMV-II.Group2-McAb’sreactingonlytoCMV-II.Group3-McAb’sreactingstronglytoCMV-IIandweaklytoCMV-I.Absorbanceat405nmReactivityHybridomasGroup(numberofControlsVirusinfectedtissuesubclonestested)CMV-BHealthyCMV-ICMV-IIPSTTAV1T(5)1.56±0.060.10±0.011.31±0.081.37±0.100.09±0.0050.08±0.01Z(3)1.98±0.230.14±0.011.49±0.041.15±0.040.17±0.020.13±0.012U(5)1.81±0.030.10±0.030.21±0.121.47±0.040.09±0.0040.09±0.004W(6)1.73±0.050.15±0.020.15±0.011.47±0.030.17±0.030.11±0.01Y(3)1.58±0.210.10±0.0040.16±0.010.95±0.140.10±0.010.08±0.0023V(4)1.17±0.070.11±0.010.41±0.080.80±0.060.12±0.010.10±0.01±-onestandarddeviationwith CMV (Table 11). McAb’s in group 1 reacted equally to both CMV serotypes. McAb’sin group 2 reacted strongly to CMV-II only and finally McAb’s in group 3 reacted stronglyto CMV-II and weakly to CMV-I.3.4 Survey of CMV-B in BSIV-infected blueberry plantsOriginally, it was thought that both BSIV and CMV-B infected blueberry plants.BSIV—infected plants were tested for CMV-B five times between April and September of1993. The tissue included unopened and opened blossoms and leaf buds in spring, matureleaves in summer and senescent leaves in autunm. CMV-B was not detected in any of theblueberry plants (Table 12).Since CMV has one of the widest known host range of any plant virus, alternativehosts such as weeds and trees, were tested. Most of the samples tested in September werediscoloured and had necrotic regions. None of the samples had an A405 value comparableto that of the control in TAS-ELISA using monoclonal ‘W’ specific for CMV-II. Tissuesamples from vine maple, Oregon grape, elderberry, salmonberry, sheep sorrel, cats’ ear andclover, had either negative or low A405 values (Table 13). Some plants with low A405 valueswere retested in mid-October. They included buttercup, sheep sorrel and cats’ ear. Of these,only buttercup recorded a low, positive A405 value when detected with PcAs or McAb’s ‘W’and ‘T’ specific for CMV-II (Table 14). N clevelandii and Cucumis sativus mechanicallyinoculated with CMV-infected buttercup remained symptomless after three weeks. Since theindicator plants appeared healthy, they were not tested by ELISA.95Table12:Surveyforcucumbermosaic,blueberryisolate(CMV-B)amongblueberryshock-infectedblueberryplants(BSIV).MicrotitreplateswerecoatedwithpolyclonalimmunoglobulinsmadeagainstCMV-Bdilutedincarbonatebuffer,pH9.6.DoubleantibodysandwichELISA’swereperformedasdescribedinthetext(III2.3.2).A405wasmeasuredafter16hr.IncidenceofviralinfectionsDateTissuetestedNumberofsamplesCMVBSIVApril21,1993blueberrybuds160NTblueberryleaves120NTMay12,1993blueberryblossoms2890230760NTJune23,1993blueberryleaves1760157September9,1993blueberryleaves90084September28,1993vegetationinand1789NTaroundblueberryfieldOctober12,1993vegetationinand7718NTaroundblueberryfieldNT=nottesteda=seeresultsinTable14Table13:Surveyforcucumbermosaic,blueberryisolate(CMV-B)invegetationwithinandaroundblueberryshock-infectedblueberryplantsinWhatcomcounty,Washington.Microtitreplateswerecoatedwithpolyclonalinimunoglobulinsdilutedincarbonatebuffer,pH9.6.Alltissuewashomogenizedinblockingbuffer.TrappedviruswasdetectedwithCMV-IIpolyclonalimmunoglobulinalkalinephosphataseconjugate.DoubleantibodysandwichELISA’swereperformedasdescribedinthetext(III2.2.1.4).TheA405wasmeasuredafter16hr.NegativePositivevegetationLatinNameCommonname____________________________A405#plantsA405#plantsWeedsNicotianatabacumtobacco(control)0.12±0.0331.79±0.674BerberisOregongrape0.06810.43±0.092Stellariamediachickweed0.06±0.028--Epilobiumangustfo1iumfireweed0.05±0.018--Plantagomajorplantain0.11±0.078--Polygomunaviculareknotweed0.05±0.028--Ranunculusacrisbuttercup0.10±0.0516--Solidagooccidentalisgoldenrod0.05±0.0240.251Hypochoerisradicatacat’s-ear0.11±0.068--Trifoliumpratenseclover0.07±0.0270.5691ShrubsAmelanchieralntfoliaserviceberry0.13±0.069--Crataegushawthorn0.15±0.088--Rubusspectabilissalmonberry0.13±0.05140.4611Rubusursinusblackberry0.09±0.058--Sambucusracemosaelderberry-0.4921TreesPopulusdeltoidescottonwood0.12±0.0660.63±0.292Prunusvirginianachokecherry0.11±0.0470.3151Pyrusmalusapple0.14±0.1015--Pyrusfuscacrabapple0.16±0.113--Sorbussitchensismountainash0.0771.-0OTable14:Extensivesurveyforcucumbermosaic,blueberryisolate(CIvIV-B)inspeciestestingpositivewithinandaroundablueberryshock-infectedblueberryfieldinWhatcomcounty,Washington.Microtitreplateswerecoatedwithpolyclonalimmunoglobulinsdilutedincarbonatebuffer,pH9.6.Tissuewashomogenizedinblockingbuffer.TrappedviruswasdetectedwitheitherCMV-IIpolyclonalimmunoglobulinalkalinephosphataseconjugateorcoatedwithhybridomaculturefluid,thendetectedwithgoat-anti-mousealkalinephosphataseconjugate.TripleantibodysandwichELISA’swereperformedasdescribedinthetext(III2.3.4).TheA405wasmeasuredafter16hr.±representsonestandarddeviation.LatinNameCommonnameDetectionNegativePositiveAntibodiesA405#A40#plantsplantsNicotianatabacumtobacco(control)polyclonal0.0310.82±0.022Hypochoerisradicatacat’s-earantiserum0.03±0.0132--Ranunculusacrisbuttercup0.04±0.02150.16±0.084Rumexacetosellasheepsorrel0.03±0.0126--Nicotianatabacumtobacco(control)hybridoma0.0512.6371Hypochaerisradicatacat’s-earculturefluid0.03±0.0132--Ranunculusacrisbuttercupgroup1-T0.13±0.07110.54±0.168Rumexacetosellasheepsorrel(TB12)0.04±0.0326--Nicotianatabacumtobacco(control)hybridoma0.0311.28±0.332Hypochaerisradicatacat’s-earculturefluid0.03±0.0132--Ranunculusacrisbuttercupgroup2-W0.04±0.01180.1591Rumexacetosellasheepsorrel(WG11)0.02±0.0126--4 DiscussionAn inexhaustible variety of symptoms is caused by interactions between the viralgenome, the host plant genome and environmental factors. The symptoms of plants infectedwith the unknown virus were compared to published descriptions of CMV infections. Someplants, such as Brassica juncea, Physalis pubescens and Vinca rosea, remained symptomless,but neither were there any references to these plants being infected with CMV. These threeplants were probably not hosts of CMV. References for the plants, in most cases, coincidedwith the symptoms recorded from this study. For example, Cucumis sativus exhibitedchlorotic spots, a mosaic along its veins and vein clearing. Marrou and co-workers (1975)found chlorotic spots with a mild mosaic, Francki and co-workers (1979) found systemicmosaic and stunting and Rao and Francki (1982) found chlorotic local lesions and mosaicstypical of CMV-infection. The symptoms agreed with a possible CMV infection.Although the three members of the cucumovirus group could be differentiated by theirsymptoms on indicator plants, whether CMV serotypes can be separated solely on the basisof symptomatology is still unresolved. Wahyuni and co-workers (1992) used N tabacum‘Xanthi’ to distinguish between CMV serotypes. CMV-I caused green mosaics, leafdistortion, severe stunting and enation, whereas CMV-II only caused chlorotic mosaics andleaf distortion. CMV-B exhibited a reaction similar to CMV-II in ‘Xanthi’. Furtherserological testing was done to confirm these results.The yield of virus, although pure, was low. Only 0.7-12 mg of CMV-B was purifiedper kg of fresh tissue, significantly less than the average 100-3 00 mg/kg reported by someauthors. The highest yields, 500 mg/kg (Francki et a!., 1979), 800 mg/kg (Kaper and99Waterworth, 1981) or 1 g/kg (Palukaitus et a!., 1992) were recorded by workers who hadstudied CMV extensively. Published purification procedures suggested homogenizing virusinfected tissue in water, citrate or phosphate buffer containing an antioxidant such ascysteine-HC1, f3-mer, sodium diethyldithiocarbamate or thioglycolate (Kaper andWaterworth, 1981). Some strains of CMV also required the addition of EDTA. Clarificationwas achieved by adding bentonite or Triton X-100, adjusting the pH to 6.0, or extractingwith an organic solvent, such as ether, butanol or chloroform, followed by a low speedcentrifugation (Palukaitus et a!., 1992). Virus was then concentrated by high speedcentrifugation or by the addition of PEG. Further concentration by conventional procedurescould include cycles of differential centrifugation or centrifugation through 5-25% sucrosedensity gradients (Francki et a!., 1979). The experimental ratio ofA260/A8 for fractionatedvirus, 1.79-1.82 was in accordance with the published value, 1.7, (Francki et a!., 1979)suggesting that the preparation was free of major contaminants despite using a cesiumsulphate gradient, not suggested in any other protocol.Purified virions were stained with UA, but disrupted with phosphotungstic acid. Theywere isometric, with a diameter of 28-3 0 nm, and electron dense centres similar to publishedphotographs (Francki et a!., 1979).Prior to nucleic acid sequencing, there was wide disparity in the reported weight ofthe cp subunit, ranging from 22-26.3 kD. Based on sequence data, a M of 24.3 kD wasreported for CMV-II (Palukaitis et a!., 1992). This report calculated a M of (29.5 ± 0.3)kD for the cp subunit of CMV-B. The sizes of the RNA genome, calculated from four gels,were 3.49, 2.98, 2.18 and 1.10 kb (Table 9), similar to those obtained from sequenced data100(Palukaitis et al., 1992). The profile of dsRNA isolated from N tabacum ‘Harrownova’,‘Havana 425’, ‘Samson’, ‘White Burley’ and ‘Xanthi’ was identical to the dsRNA found ina CMV isolate from primula.Evidence suggested that the isolate labelled “BSIV” mild, maintained in thegreenhouse for more than three years, was CMV. It was named CMV-B since it was firstisolated from BSIV-infected blueberry blossoms. It may have been propagated from themixture of viruses purified and photographed for the characterization of BSIV (Macdonaldand Martin, 1991). Since BSIV was temperature sensitive, only CMV would have remainedafter a hot summer.Injections of unfixed virus every 3-5 weeks failed to produce PcAs. The low stabilityof CMV-B was overcome by injecting every 2-5 days for four weeks. One month after thefinal injection, PcAs was produced, and, in a far shorter period of time than advocated forpolyclonal production (Harlow and Lane, 1988).Serological tests were used to identify the virus as a member of the cucumovirusesand to determine its serotype. All CMV PcAs contain antibodies which react with serotypesI and II. Therefore, it was not surprising when it reacted with both CMV serotypes, but notwith other cucumoviruses in DAS-ELISA. It also distinguished between the crude sap ofboth CMV serotypes having a higher A405 for CMV-II than for CMV-I. The use of crudesap did not prevent the differentiation between serotypes as reported by Francki and coworkers (1979).McAb’s provided a tool to discriminate between cucumovirus serotypes. They haveseveral advantages over PeAs. McAb can be produced from a small amount of antigen, even101if it is impure. Mouse McAb’s were produced following the same schedule used for rabbitPcAs production. The McAb’s were divided into two groups serologically. Antibodies inhybridoma culture fluid of group 1 recognized only CMV-B and CMV-II, while those ingroup 2 and 3 recognized both serotypes I and II and the CMV-B isolate. In contrast, Portaand co-workers (1989) found that McAb’s made to unfixed CMV-I did not cross-react withCMV-II and were specific for serotype I. Haase et al. (1990) found that McAb’s madeagainst fixed CMV-II were specific, detecting differences between the epitopes of theserotypes.The PeAs and McAb’s made against CMV-B unequivocally verified the identify ofthe isolate. The PcAs confirmed that it was a cucumovirus, specifically CMV. Althoughonly some of the McAb’ s reacted with CMV-I, all reacted with CMV-II. The largest group,group 1, reacted only with CMV-II. Therefore, CMV-TI was probably isolated fromN clevelandii inoculated with BSIV-infected blueberry blossoms. This is in agreement withthe fact that CMV-II is found more commonly found in temperate climates.Although CMV was isolated from blueberry blossoms, extensive serological testingdid not identify a single CMV-infected blueberry host. It may have overwintered in thevegetation in and around the field. In the spring, pollen from CMV-infected vegetation mayhave been transported to BSIV-infected blueberry plants by the wind and pollinators,explaining its presence on blueberry blossoms. Another alternative was that CMV-infectedinsect vectors, present on the blossoms, were inadvertently homogenized with the blossomstested in ELISA. The second possibility is less likely since most insects flew or crawledaway from tissue left in an open plastic tray during collection. Therefore CIvIV infection of102N clevelandii resulted when BSIV-infected blueberry pollen was used as inoculum is likelydue to the presence of CMV-infected pollen from a plant other than blueberry.103CHAPTER IV - SUMMARY AND CONCLUSIONSA eDNA library of the BSIV genome was generated. Nine clones, 14, 17, 19, 35,112, 123, 132, 158 and 161, hybridized with ssRNA 3 and RNA 4. Clones 112 and 132were 1.8 kb, almost the full length of RNA 3, 1.9 kb. Clone 112 hybridized with RNA 4,the cp mRNA. A 412 bp region of clones 112, 123 and 136, 3’ from the internal Xho I siteto the multiple cloning site, was sequenced and found to be homologous. BSIVseq andBSIV RNA 3 were 96% homologous. BSIVseq differed by one deletion and one additionin the 259 bp cp sequence and three replacements in the 153 bp non-coding sequence. Therewas no homology at the nucleotide level between BSIVseq and any ilarvirus cp genes.When translated, BSIVpro frames 2 and 3 had identities of 21.1 and 22.3% with ApMV cp.When BSIVpro frames 2 and 3 that had homology with ApMV were joined and alignedwith the cp of ApMV, BSIVf2,3 had even a higher level of homology, 61.2%, than the fullBSIV cp, 46.9%. The C termini BSIV’s ep may have longer stretches of homology withother ilarviruses such as CVV, PDV and ApMV, than the full length cp.The identity of a virus labelled “BSIV” mild and maintained in the greenhouse forthree years was CMV. Its disease symptoms were similar to those published for CMV.Isometric viral particles, Ca. 30 nm in diameter, with electron dense centres comparedfavourably with published micrographs. The cp subunit, with a M of (29.5 ± 0.3) kD, wasslightly larger than that determined from the sequence of CMV-II, 24.3 kD (Paluikaitis eta?., 1992). The genome was comprised of four strands of ssRNA with M’ s of (1 .19, 1.01,0.74 and 0.37) x 106 D corresponding to 3.49, 2.98, 2.18 and 1.10 kb, similar to published104values. Its dsRNA profile was identical to that of CMV isolated from primula. All theinitial data supported the hypothesis that the isolate from blueberries was CMV. PcAsproduced against CMV-B identified it as an isolate of CMV. McAb against CMV-Bascertained that it was an isolate of CMV-II.Over 650 samples of blueberry tissue were tested to investigate the relationshipbetween blueberry shock diseases and CMV. All blueberry plants tested negative for CMVby ELISA. Highbush blueberry was either not a host of CMV or the titre of CMV was toolow to be detected. Alternative sources of CMV may have been the weeds in and aroundthe field. CMV may have been transmitted to N clevelandii from infected pollen that landedon BSIV-infected blueberry blossoms. 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