US20050032041A1 - Rupestris stem pitting associated virus nucleic acids, proteins, and their uses - Google Patents

Rupestris stem pitting associated virus nucleic acids, proteins, and their uses Download PDF

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US20050032041A1
US20050032041A1 US10/803,063 US80306304A US2005032041A1 US 20050032041 A1 US20050032041 A1 US 20050032041A1 US 80306304 A US80306304 A US 80306304A US 2005032041 A1 US2005032041 A1 US 2005032041A1
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Dennis Gonsalves
Baozhong Meng
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Cornell Research Foundation Inc
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/26011—Flexiviridae
    • C12N2770/26022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S424/00—Drug, bio-affecting and body treating compositions
    • Y10S424/801—Drug, bio-affecting and body treating compositions involving antibody or fragment thereof produced by recombinant dna technology

Definitions

  • the present invention relates to Rupestris stem pitting associated virus (“RSPaV”) proteins, DNA molecules encoding these proteins, and diagnostic and other uses thereof.
  • RSPaV Rupestris stem pitting associated virus
  • the major virus diseases are grouped into: (1) the grapevine degeneration caused by the fanleaf nepovirus , other European nepoviruses, and American nepoviruses, (2) the leafroll complex, and (3) the rugose wood complex (Martelli, ed., Graft Transmissible Diseases of Grapevines, Handbook for Detection and Diagnosis , FAO, UN, Rome, Italy (1993)).
  • RW complex is a term to describe a group of graft-transmissible diseases which are important and widespread on grapevines grown world-wide. Symptoms of RW are characterized by pitting, grooving, or distortion to the woody cylinder of the grapevine scion, rootstock, or both. Based on symptoms developed on different indicator plants after graft inoculation, RW complex can be divided into four components: Kober 5BB stem grooving (KSG), LN 33 stem grooving (LNSG), grapevine corky bark (GCB), and Rupestris stem pitting (RSP) (Martelli, “Rugose Wood Complex,” in Graft - Transmissible Diseases of Grapevines.
  • KSG Kober 5BB stem grooving
  • LNSG LN 33 stem grooving
  • GCB grapevine corky bark
  • RRP Rupestris stem pitting
  • the disease was defined by Goheen as follows: after graft inoculation with a chip bud from an infected grapevine, the woody cylinder of the indicator plant Vitis rupestris Scheele St. George (“St. George”) develops a narrow strip of small pits extending from the inoculum bud to the root zone. Grafted St. George plants were checked for wood symptoms 2 to 3 years after inoculation. In contrast to GCB, which elicits pitting and grooving on St. George and LN 33, RSP does not produce symptoms on the latter (Goheen, “ Rupestris Stem Pitting,” in Compendium of Grape Diseases , p. 53, Pearson and Goheen, eds., American Phytopathological Society Press, St. Paul, Minn., USA (1988)).
  • RSP is probably the most common component of the RW complex on grapevines.
  • Surveys in California revealed a high disease incidence in many grapevine cultivars imported from Western Europe and Australia (Goheen, “Rupestris Stem Pitting,” in Compendium of Grape Diseases , p. 53, Pearson and Goheen, eds., American Phytopathological Society Press, St. Paul, Minn., USA (1988)).
  • An examination of indexing records in California compiled over 23 years revealed RSP infection in 30.5% of 6482 grapevine selections introduced from around the world (Golino and Butler, “A Preliminary Analysis of Grapevine Indexing Records at Davis, California,” in Proceedings of the 10 th Meeting of the ICVG , pp.
  • RSP is the most frequently detected component of the RW complex in Italy (Borgo and Bonotto, “Rugose Wood Complex of Grapevine in Northeastern Italy: Occurrence of Rupestris Stem Pitting and Kober Stem Grooving,” in Extended Abstracts of the 11 th Meeting of the International Council for the Study of Viruses and Virus Diseases of the Grapevine ( ICVG ), pp.
  • RSP is the most difficult grapevine disease to diagnose.
  • Serological or molecular methods are not available for diagnosing RSP.
  • Biological indexing on St. George has remained the only approach to diagnose RSP. Biological indexing is labor intensive, time consuming (i.e., often requiring up to about three years to obtain results), and, by its very nature, subjective.
  • symptoms on St. George can be variable and not exactly as those defined by Goheen.
  • 359 bp was isolated from 21 of 31 grapevine cultivars, all of which were previously indexed on St. George and considered to be infected with RSP (Monette et al., “Double-Stranded RNA from Rupestris Stem Pitting-Affected Grapevines,” Vitis 28:137-44 (1989)).
  • the present invention relates to an isolated protein or polypeptide corresponding to a protein or polypeptide of a RSP virus.
  • the encoding RNA molecule or DNA molecule in either isolated form or incorporated in an expression system, a host cell, or a transgenic Vitis scion or rootstock cultivar, are also disclosed.
  • Another aspect of the present invention relates to a method of imparting RSP virus resistance to Vitis scion or rootstock cultivars by transforming them with a DNA molecule encoding the protein or polypeptide corresponding to a protein or polypeptide of a RSP virus.
  • the present invention also relates to an antibody or binding portion thereof or probe which recognizes proteins or polypeptides of the present invention.
  • Still another aspect of the present invention relates to diagnostic tests which involve methods for detecting the presence of a RSP virus in a sample.
  • the methods include the use of an antibody or binding portion of the present invention (i.e., in an immunoassay), or a nucleic acid probe obtained from a DNA molecule of the present invention (i.e., in a nucleic acid hybridization assay or gene amplification detection procedure).
  • the antibody or binding portion thereof, or nucleic acid probe is introduced into contact with the sample, whereby the presence of Rupestris stem pitting virus in the sample is detected using an assay system.
  • RSP virus resistant transgenic variants of the current commercial grape cultivars and rootstocks allows for more complete control of the virus while retaining the varietal characteristics of specifics cultivars. Furthermore, these variants permit control over RSP virus transmitted by infected scions or rootstocks. Moreover, the diagnostic tests offer significant improvement over conventional diagnostic means currently employed, namely, rapid results and greater accuracy.
  • FIG. 1 is a photograph of St. George indicators which comparatively display the symptoms of RSP.
  • the St. George indicator (a) has been graft-inoculated with infected bud wood from a grapevine accession, resulting in the indicator displaying pitting below the inoculum bud, as indicated by an arrow.
  • This RSP symptom was defined by Goheen, “ Rupestris Stem Pitting,” in Compendium of Grape Diseases , p. 53, Pearson and Goheen, eds., American Phytopathological Society Press, St. Paul, Minn., USA (1988), which is hereby incorporated by reference.
  • the St. George indicator (b) was not graft-inoculated and represents a normal appearance.
  • FIGS. 2A and 2B are photographs which respectively display the results of dsRNA analysis and Northern hybridization for dsRNA. Together the photographs may be used to correlate the dsRNA analysis of FIG. 2A with the Northern hybridization (for dsRNA isolated from grapevines indexed positive for Rupestris stem pitting (RSP)) of FIG. 2B .
  • RSP Rupestris stem pitting
  • Hind III digested lambda DNA maker lane 1, Aminia; lane 2, Bertille Seyve 5563; lane 3, Canandaigua; lane 4, Colobel 257; lane 5, Couderc 28-112; lane 6, Freedom; lane 7, Grande Glabre; lane 8, M 344-1; lane 9, Joffre; lane 10, Ravat 34; lane 11, Seyval; lane 12, Seyve Vinard 14-287; lane 13, Verdelet; lane 14, Pinot Noir (positive control); lane 15, Verduzzo 233A (negative control for RSP as judged by indexing on St. George); lane 16, insert of clone RSP149. Arrows indicate the position of the 8.7 kb dsRNA.
  • the two dsRNA bands are larger or smaller than the 8.7 kb dsRNA associated with RSP and they did not hybridize with the RSP specific probe in Northern analysis. Thus, they are not specific to RSP.
  • FIG. 3A is an illustration which depicts the strategy for obtaining the complete nucleotide sequence of RSPaV-1.
  • the overlapping regions of the nucleotide sequences of the sequenced clones and RT-PCR-amplified cDNA fragments are as follows: 52-375 for RSPA/RSP28; 677-1474 for RSP28/RSP3; 3673-3766 for RSP3/RSPB; 40094320 for RSPB/RSP94; 5377-5750 for RSP94/RSPC; 5794-6537 for RSPC/RSP95; 6579-6771 for RSPC/RSP140; and 8193-8632 for RSP140/TA5.
  • FIG. 3B is an illustration which comparatively depicts the genome structures of RSPaV-1, ASPV, PVM, and PVX. Boxes with the same patterns represent the comparable ORFS.
  • FIG. 4A is a comparative sequence listing of amino acid sequences of region 1 (aa 1-372) of RSPaV-1 ORF1 with the corresponding sequences of carlavirus PVM and ASPV.
  • the methyltransferase motif is underlined.
  • Capital letters indicate consensus residues.
  • FIG. 4B is a comparative sequence listing of amino acid sequences of region II (aa 1354 to end) of RSPaV-1 ORF1 with the corresponding regions of ASPV and PVM carlavirus.
  • the NTP binding motif is underlined at (A) and the GDD containing sequence is underlined at (B).
  • capital letters indicate consensus residues
  • the symbol * indicates identical amino acid residues between RSPaV-1 and ASPV
  • the symbol # indicates identical amino acid residues between RSPaV-1 and PMV.
  • FIGS. 5 A-D are comparative sequence listings of amino acid sequences for ORF2, ORF3, ORF74, and a C-terminal part of ORF5 (CP) of RSPaV-1, respectively, with ASPV and PVM carlavirus.
  • FIG. 5A the NTP binding motif, located near the C terminus of ORF2, is underlined.
  • FIG. 5D the conserved motif (RR/QX—XFDF), located in the central region of the coat proteins and proposed to be involved in the formation of a salt bridge structure, is underlined.
  • capital letters indicate consensus residues.
  • FIG. 6A is a comparative sequence listing of DNA nucleotide sequences for the 3′ untranslated region (UTR) of RSPaV-1 and ASPV.
  • FIG. 6B is a comparative sequence listing of DNA nucleotide sequences for the 3′ untranslated region (UTR) of RSPaV-1 and PVM.
  • Clustal method of MegAlign was used to generate sequence alignments. The 21 identical consecutive nucleotides between RSPaV-1 and PVM are indicated as shadowed letters.
  • FIGS. 7 A-B are photographs comparing the results of RT-PCR of grapevines using RSP 149 primers ( FIG. 7A ) and Southern blot hybridization of RT-PCR amplified cDNA fragments to RSPaV-1 specific probe ( FIG. 7B ).
  • MMLV-RT Promega was used in reverse transcription.
  • Taq DNA polymerase Promega was used in PCR.
  • FIG. 8 is a schematic representation of the identical genome organization among RSPaV-1 (the type strain), RSP47-4, and RSP158.
  • the number of amino acid residues of the comparable ORFs (boxes shaded with the same pattern) among these three strains are the same (note: ORFI and ORF5 of RSP47-4 and RSP158 are incomplete).
  • the comparable ORFs also have high nucleotide and amino acid sequence identities, which are indicated on the bottom. Only the C-terminal portion of the ORF1 of RSPaV-1 is shown in this diagram.
  • FIG. 9 is a comparative alignment of nucleotide sequences of seven other clones with the comparable region of RSPaV-1. Shaded areas indicate identical nucleotide sequences, whereas white boxes represent different nucleotide sequences.
  • FIG. 10 is a schematic representation of a plant transformation vector containing the RSPaV-1 coat protein gene.
  • This vector is designated pGA482G/RSPaV-1CP, which has the double CaMV 35S enhancers, the 35S promoter, the leader sequence of AIMV, and the 35S terminator sequence.
  • RB right border
  • LB left border
  • Tet tetracycline resistance gene
  • Gent gentamycin resistance gene.
  • the present invention relates to isolated DNA molecules encoding for the proteins or polypeptides of a Rupestris stem pitting associated virus. Since the nucleotide sequence was derived from cDNA clones of the dsRNA that was associated with RSP, the viral agent has been designated as Rupestris stem pitting associated virus (“RSPaV”). RSP is likely caused by one or a number of viral strains. The genome of each RSPaV has a plurality of open reading frames, each containing DNA molecules in accordance with the present invention. The complete genome of one strain has been sequenced and the strain is designated RSPaV-1. Substantial portions of the genomes of two other RSPaV strains have also been sequenced. These strains are designated by their clone names, RSP47-4 and RSP158.
  • the DNA molecule which constitutes the complete RSPaV-1 genome comprises the nucleotide sequence corresponding to SEQ. ID. No. 1 as follows: CGATAAACAT AACAACAGAA TGTGCATTGC AGTAATATTC CTTGAATATA ATTGCAACGC 60 AATGGCCCTC TCTTATAGGC CTGCTGTTGA AGAGGTGCTC GCAAAATTCA CCTCTGATGA 120 ACAATCCAGG GTTTCTGCTA CAGCTCTCAA GGCATTAGTA GACTTAGAGG AAAGTCAGCA 180 CAATTTGTTC TCTTTCGCAT TGCCTGATAG AAGCAAAGAA AGGCTGATAT CTTCTGGCAT 240 TTACTTAAGT CCTTACAGTT TCAGACCCCA CTCACATCCA GTTTGTAAAA CTTTAGAAAA 300 TCACATTTTG TACAATGTTT TACCTAGTTA TGTTAATAAT TCATTTTACT TTGTAGGAAT 360 CAAGGATTTT AAGCTGCAGT TCTTGAAAAG GAG GA
  • RSPaV-1 ORF 1 Another DNA molecule of the present invention (RSPaV-1 ORF 1) includes nucleotides 62-6547 of SEQ. ID. No. 1.
  • the DNA molecule of RSPaV-1 ORF1 encodes for a RSPaV-1 replicase and comprises a nucleotide sequence corresponding to SEQ. ID. No.
  • the RSPaV-1 replicase has a deduced amino acid sequence corresponding to SEQ. ID. No. 3 as follows: Met Ala Leu Ser Tyr Arg Pro Ala Val Glu Glu Val 1 5 10 Leu Ala Lys Phe Thr Ser Asp Glu Gln Ser Arg Val 15 20 Ser Ala Thr Ala Leu Lys Ala Leu Val Asp Leu Glu 25 30 35 Glu Ser Gln His Asn Leu Phe Ser Phe Ala Leu Pro 40 45 Asp Arg Ser Lys Glu Arg Leu Ile Ser Ser Gly Ile 50 55 60 Tyr Leu Ser Pro Tyr Ser Phe Arg Pro His Ser His 65 70 Pro Val Cys Lys Thr Leu Glu Asn His Ile Leu Tyr 75 80 Asn Val Leu Pro Ser Tyr Val Asn Asn Ser Phe Tyr 85 90 95 Phe Val Gly Ile Lys Asp Phe Lys Leu Gln Phe Leu 100 105 Lys Arg Arg Asn Lys Asp Leu Val Ala
  • RSPaV-1 ORF2 Another DNA molecule of the present invention (RSPaV-1 ORF2) includes nucleotides 6578-7243 of SEQ. ID. No. 1.
  • the DNA molecule of RSPaV-1 ORF2 encodes for a first protein or polypeptide of an RSPaV-1 triple gene block and comprises a nucleotide sequence corresponding to SEQ. ID. No.
  • the first protein or polypeptide of the RSPaV-1 triple gene block has a deduced amino acid sequence corresponding to SEQ. ID. No. 5 as follows: Met Asn Asn Leu Val Lys Ala Leu Ser Ala Phe Glu 1 5 10 Phe Val Gly Val Phe Ser Val Leu Lys Phe Pro Val 15 20 Val Ile His Ser Val Pro Gly Ser Gly Lys Ser Ser 25 30 35 Leu Ile Arg Glu Leu Ile Ser Glu Asp Glu Asn Phe 40 45 Ile Ala Phe Thr Ala Gly Val Pro Asp Ser Pro Asn 50 55 60 Leu Thr Gly Arg Tyr Ile Lys Pro Tyr Ser Pro Gly 65 70 Cys Ala Val Pro Gly Lys Val Asn Ile Leu Asp Glu 75 80 Tyr Leu Ser Val Gln Asp Phe Ser Gly Phe Asp Val 85 90 95 Leu Phe Ser Asp Pro Tyr Gln Asn Ile Ser Ile Pro 100 105 Lys Glu Ala His Phe Ile Lys
  • RSPaV-1 ORF3 Another DNA molecule of the present invention (RSPaV-1 ORF3) includes nucleotides 7245-7598 of SEQ. ID. No. 1.
  • the DNA molecule of RSPaV-1 ORF3 encodes for a second protein or polypeptide of the triple gene block and comprises a nucleotide sequence corresponding to SEQ. ID. No.
  • the second protein or polypeptide of the RSPaV-1 triple gene block has a deduced amino acid sequence corresponding to SEQ. ID. No. 7 as follows: Met Pro Phe Gln Gln Pro Ala Asn Trp Ala Lys Thr 1 5 10 Ile Thr Pro Leu Thr Val Gly Leu Gly Ile Gly Leu 15 20 Val Leu His Phe Leu Arg Lys Ser Asn Leu Pro Tyr 25 30 35 Ser Gly Asp Asn Ile His Gln Phe Pro His Gly Gly 40 45 Arg Tyr Arg Asp Gly Thr Lys Ser Ile Thr Tyr Cys 50 55 60 Gly Pro Lys Gln Ser Phe Pro Ser Ser Gly Ile Phe 65 70 Gly Gln Ser Glu Asn Phe Val Pro Leu Met Leu Val 75 80 Ile Gly Leu Ile Ala Phe Ile His Val Leu Ser Val 85 90 95 Trp Asn Ser Gly Leu Gly Arg Asn Cys Asn Cys His 100 105 Pro Asn
  • the second protein or polypeptide of the RSPaV-1 triple gene block has a molecular weight of about 10 to 15 kDa, preferably 12.8 kDa.
  • RSPaV-1 ORF4 includes nucleotides 7519-7761 of SEQ. ID. No. 1.
  • the DNA molecule of RSPaV-1 ORF4 encodes for a third protein or polypeptide of the RSPaV-1 triple gene block and comprises a nucleotide sequence corresponding to SEQ. ID. No.
  • the third protein or polypeptide of the RSPaV-1 triple gene block has a deduced amino acid sequence corresponding to SEQ. ID. No. 9 as follows: Met Tyr Cys Leu Phe Gly Ile Leu Val Leu Val Gly 1 5 10 Ile Val Ile Ala Ile Gln Ile Leu Ala His Val Asp 15 20 Ser Ser Ser Gly Asn His Gln Gly Cys Phe Ile Arg 25 30 35 Ala Thr Gly Glu Ser Ile Leu Ile Glu Asn Cys Gly 40 45 Pro Ser Glu Ala Leu Ala Ser Thr Val Lys Glu Val 50 55 60 Leu Gly Gly Leu Lys Ala Leu Gly Val Ser Arg Ala 65 70 Val Glu Glu Ile Asp Tyr His Cys 75 80
  • the third protein or polypeptide of the RSPaV-1 triple gene block has a molecular weight of about 5 to 10 kDa, preferably 8.4 kDa.
  • Still another DNA molecule of the present invention includes nucleotides 7771-8550 of SEQ. ID. No. 1.
  • the DNA molecule of RSPaV-1 ORF5 encodes for a RSPaV-1 coat protein and comprises a nucleotide sequence corresponding to SEQ. ID. No.
  • the RSPaV-1 coat protein has a deduced amino acid sequence corresponding to SEQ. ID. No. 11 as follows: Met Ala Ser Gln Ile Gly Lys Leu Pro Gly Glu Ser Asn Glu Ala Phe 1 5 10 15 Glu Ala Arg Leu Lys Ser Leu Glu Leu Ala Arg Ala Gln Lys Gln Pro 20 25 30 Glu Gly Ser Asn Ala Pro Pro Thr Leu Ser Gly Ile Leu Ala Lys Arg 35 40 45 Lys Arg Ile Ile Glu Asn Ala Leu Ser Lys Thr Val Asp Met Arg Glu 50 55 60 Val Leu Lys His Glu Thr Val Val Ile Ser Pro Asn Val Met Asp Glu 65 70 75 80 Gly Ala Ile Asp Glu Leu Ile Arg Ala Phe Gly Glu Ser Gly Ile Ala 85 90 95 Glu Ser Val Gln Phe Asp Val Ala Ile Asp Ile Ala Arg His Cys Ser 100
  • the DNA molecule which constitutes the substantial portion of the RSPaV strain RSP474 genome comprises the nucleotide sequence corresponding to SEQ. ID. No. 12 as follows: GGCTGGGCAA ACTTTGGCCT GCTTTCAACA CGCCGTCTTG GTTCGCTTTG CACCCTACAT 60 GCGATACATT GAAAAGAAGC TTGTGCAGGC ATTGAAACCA AATTTCTACA TTCATTCTGG 120 CAAAGGTCTT GATGAGCTAA GTGAATGGGT TAGAGCCAGA GGTTTCACAG GTGTGTGTAC 180 TGAGTCAGAC TATGAAGCTT TTGATGCATC CCAAGATCAT TTCATCCTGG CATTTGAACT 240 GCAAATCATG AGATTTTTAG GACTGCCAGA AGATCTGATT TTAGATTATG AGTTCATCAA 300 AATTCATCTT GGGTCAAAGC TTGGCTCTTT TGCAATTATG AGATTCACAG GTGAGGCAAG 360 CACCTTCCTA TT
  • ORF1 and ORF5 are only partially sequenced.
  • RSP47-4 is 79% identical in nucleotide sequence to the corresponding region of RSPaV-1.
  • the amino acid sequence identities between the corresponding ORFs of RSP47-4 and RSPaV-1 are: 94.1% for ORF1, 88.2% for ORF2, 88.9% for ORF3, 86.2% for ORF4, and 92.9% for ORF5.
  • the nucleotide sequences of the five potential ORFs of RSP47-4 are given below.
  • Another DNA molecule of the present invention includes nucleotides 1-768 of SEQ. ID. No. 12. This DNA molecule is believed to code for a polypeptide portion of a RSP47-4 replicase and comprises a nucleotide sequence corresponding to SEQ. ID. No.
  • the polypeptide has a deduced amino acid sequence corresponding to SEQ. ID. No. 14 as follows: Met Arg Tyr Ile Glu Lys Lys Leu Val Gln Ala Leu Lys Pro Asn Phe 1 5 10 15 Tyr Ile His Ser Gly Lys Gly Leu Asp Glu Leu Ser Glu Trp Val Arg 20 25 30 Ala Arg Gly Phe Thr Gly Val Cys Thr Glu Ser Asp Tyr Glu Ala Phe 35 40 45 Asp Ala Ser Gln Asp His Phe Ile Leu Ala Phe Glu Leu Gln Ile Met 50 55 60 Arg Phe Leu Gly Leu Pro Glu Asp Leu Ile Leu Asp Tyr Glu Phe Ile 65 70 75 80 Lys Ile His Leu Gly Ser Lys Leu Gly Ser Phe Ala Ile Met Arg Phe 85 90 95 Thr Gly Glu Ala Ser Thr Phe Leu Phe Asn Thr Met Ala Asn Met Leu 100 105 110
  • Another DNA molecule of the present invention (RSP47-4 ORF2) includes nucleotides 857-1522 of SEQ. ID. No. 12. This DNA molecule codes for a first protein or polypeptide of an RSP47-4 triple gene block and comprises a nucleotide sequence corresponding to SEQ. ID. No.
  • the first protein or polypeptide of the RSP47-4 triple gene block has a deduced amino acid sequence corresponding to SEQ. ID. No. 16 as follows: Met Asn Asn Leu Val Lys Ala Leu Ser Ala Phe Glu Phe Val Gly Val 1 5 10 15 Phe Cys Val Leu Lys Phe Pro Val Val Val His Ser Val Pro Gly Ser 20 25 30 Gly Lys Ser Ser Leu Ile Arg Glu Leu Ile Ser Glu Asp Glu Ala Phe 35 40 45 Val Ala Phe Thr Ala Gly Val Pro Asp Ser Pro Asn Leu Thr Gly Arg 50 55 60 Tyr Ile Lys Pro Tyr Ala Pro Gly Cys Ala Val Gln Gly Lys Ile Asn 65 70 75 80 Ile Leu Asp Glu Tyr Leu Ser Val Ser Asp Thr Ser Gly Phe Asp Val 85 90 95 Leu Phe Ser Asp Pro Tyr Gln Asn Val Ser Ile Pro Arg Glu Ala His 100 105 110 Phe Ile Lys
  • Another DNA molecule of the present invention (RSP47-4 ORF3) includes nucleotides 1524-1877 of SEQ. ID. No. 12. This DNA molecule codes for a second protein or polypeptide of the RSP47-4 triple gene block and comprises a nucleotide sequence corresponding to SEQ. ID. No.
  • the second protein or polypeptide of the RSP47-4 triple gene block has a deduced amino acid sequence corresponding to SEQ. ID. No. 18 as follows: Met Pro Phe Gln Gln Pro Ala Asn Trp Ala Lys Thr Ile Thr Pro Leu 1 5 10 15 Thr Ile Gly Leu Gly Ile Gly Leu Val Leu His Phe Leu Arg Lys Ser 20 25 30 Asn Leu Pro Tyr Ser Gly Asp Asn Ile His Gln Phe Pro His Gly Gly 35 40 45 His Tyr Arg Asp Gly Thr Lys Ser Ile Thr Tyr Cys Gly Pro Arg Gln 50 55 60 Ser Phe Pro Ser Ser Gly Ile Phe Gly Gln Ser Glu Asn Phe Val Pro 65 70 75 80 Leu Ile Leu Val Val Thr Leu Val Ala Phe Ile His Ala Leu Ser Leu 85 90 95 Trp Asn Ser Gly Pro Ser Arg Ser Cys Asn Cys His Pro Asn Pro Cys 100 105
  • RSP47-4 ORF4 Another DNA molecule of the present invention (RSP47-4 ORF4) includes nucleotides 1798-2040 of SEQ. ID. No. 12. This DNA molecule codes for a third protein or polypeptide of the RSP47-4 triple gene block and comprises a nucleotide sequence corresponding to SEQ. ID. No.
  • the third protein or polypeptide of the RSP47-4 triple gene block has a deduced amino acid sequence corresponding to SEQ. ID. No. 20 as follows: Met Arg Tyr Leu Phe Gly Ile Leu Val Leu Val Gly Val Ala Ile Ala 1 5 10 15 Ile Gln Ile Leu Ala His Val Asp Ser Ser Ser Gly Asn His Gln Gly 20 25 30 Cys Phe Ile Arg Ala Thr Gly Glu Ser Ile Val Ile Glu Asn Cys Gly 35 40 45 Pro Ser Glu Ala Leu Ala Ala Thr Val Lys Glu Val Leu Gly Gly Leu 50 55 60 Lys Ala Leu Gly Val Ser Gln Lys Val Asp Glu Ile Asn Tyr Ser Cys 65 70 75 80
  • the third protein or polypeptide of the RSP47-4 triple gene block has a molecular weight of about 5 to 10 kDa., preferably 8.3 kDa.
  • RSP474 ORF5 includes nucleotides 2050-2680 of SEQ. ID. No. 12. This DNA molecule codes for a partial RSP47-4 coat protein or polypeptide and comprises a nucleotide sequence corresponding to SEQ. ID. No.
  • the polypeptide has a deduced amino acid sequence corresponding to SEQ. ID. No. 22 as follows: Met Ala Ser Gln Val Gly Lys Leu Pro Gly Glu Ser 1 5 10 Asn Glu Ala Tyr Glu Ala Arg Leu Lys Ala Leu Glu 15 20 Leu Ala Arg Ala Gln Lys Ala Pro Glu Val Ser Asn 25 30 35 Gln Pro Pro Thr Leu Gly Gly Ile Leu Ala Lys Arg 40 45 Lys Arg Val Ile Glu Asn Ala Leu Ser Lys Thr Val 50 55 60 Asp Met Arg Glu Val Leu Arg His Glu Ser Val Val 65 70 Leu Ser Pro Asn Val Met Asp Glu Gly Ala Ile Asp 75 80 Glu Leu Ile Arg Ala Phe Gly Glu Ser Gly Ile Ala 85 90 95 Glu Asn Val Gln Phe Asp Val Ala Ile Asp Ile Ala 100 105 Arg His Cys Ser Asp Val Gly
  • the DNA molecule which constitutes a substantial portion of the RSPaV strain RSP158 genome comprises the nucleotide sequence corresponding to SEQ. ID. No. 23 as follows: GAAGCTAGCA CATTTCTGTT CAACACTATG GCTAACATGT TGTTCACTTT TCTGAGATAT 60 GAACTGACGG GTTCAGAGTC AATAGCATTT GCAGGGGATG ATATGTGTGC TAATAGAAGG 120 TTGCGGCTTA AAACGGAGCA TGAGGGTTTT CTGAACATGA TCTGCCTTAA GGCCAAGGTT 180 CAGTTTGTTT CCAACCCCAC ATTCTGTGGA TGGTGCTTAT TTAAGGAGGG AATCTTCAAG 240 AAACCTCAAC TAATTTGGGA GCGAATATGC ATAGCCAG AGATGGGCAA TCTGGAAC 300 TGTATTGACA ATTATGCGAT AGAAGTGTCC TATGCATATA GATTGGGTGA GCTATCAATT 360 GAAATGATGA CAGAAGAAGA AG
  • ORF1 and ORF5 are only partially sequenced.
  • the nucleotide sequence of RSP158 is 87.6% identical to the corresponding region of RSPaV-1 (type strain).
  • the numbers of amino acid residues of corresponding ORFs of RSP158 and RSPaV-1 (type strain) are exactly the same.
  • the amino acid sequences of these ORFs have high identities to those of RSPaV-1: 99.3% for ORF1, 95% for ORF2, 99.1% for ORF3, 88.8% for ORF4, and 95.1% for ORF5.
  • the nucleotide and amino acid sequence information of the RSP158 ORFs are described below.
  • RSP158 incomplete ORF1 Another DNA molecule of the present invention (RSP158 incomplete ORF1) includes nucleotides 1-447 of SEQ. ID. No. 23. This DNA molecule is believed to code for a polypeptide portion of a RSP158 replicase and comprises a nucleotide sequence corresponding to SEQ. ID. No.
  • the polypeptide encoded by the nucleotide sequence of SEQ. ID. No. 24 has a deduced amino acid sequence corresponding to SEQ. ID. No. 25 as follows: Glu Ala Ser Thr Phe Leu Phe Asn Thr Met Ala Asn 1 5 10 Met Leu Phe Thr Phe Leu Arg Tyr Glu Leu Thr Gly 15 20 Ser Glu Ser Ile Ala Phe Ala Gly Asp Asp Met Cys 25 30 35 Ala Asn Arg Arg Leu Arg Leu Lys Thr Glu His Glu 40 45 Gly Phe Leu Asn Met Ile Cys Leu Lys Ala Lys Val 50 55 60 Gln Phe Val Ser Asn Pro Thr Phe Cys Gly Trp Cys 65 70 Leu Phe Lys Glu Gly Ile Phe Lys Lys Pro Gln Leu 75 80 Ile Trp Glu Arg Ile Cys Ile Ala Arg Glu Met Gly 85 90 95 Asn Leu Glu Asn Cy
  • RSP158 ORF2 Another DNA molecule of the present invention (RSP158 ORF2) includes nucleotides 506-1171 of SEQ. ID. No. 23. This DNA molecule codes for a first protein or polypeptide of the RSP158 triple gene block and comprises a nucleotide sequence corresponding to SEQ. ID. No.
  • the first protein or polypeptide of the RSP158 triple gene block has a deduced amino acid sequence corresponding to SEQ. ID. No. 27 as follows: Met Asn Asn Leu Val Lys Ala Leu Ser Ala Phe Glu 1 5 10 Phe Ile Gly Val Phe Asn Val Leu Lys Phe Pro Val 15 20 Val Ile His Ser Val Pro Gly Ser Gly Lys Ser Ser 25 30 35 Leu Ile Arg Glu Leu Ile Ser Glu Asp Glu Ser Phe 40 45 Val Ala Phe Thr Ala Gly Val Pro Asp Ser Pro Asn 50 55 60 Leu Thr Gly Arg Tyr Ile Lys Pro Tyr Ser Pro Gly 65 70 Cys Ala Val Gln Gly Lys Val Asn Ile Leu Asp Glu 75 80 Tyr Leu Ser Val Gln Asp Ile Ser Gly Phe Asp Val 85 90 95 Leu Phe Ser Asp Pro Tyr Gln Asn Ile Ser Ile Pro 100 105 Gln Glu Ala His Phe Ile Ly
  • RSP158 ORF3 Another DNA molecule of the present invention (RSP158 ORF3) includes nucleotides 1173-1526 of SEQ. ID. No. 23. This DNA molecule codes for a second protein or polypeptide of the RSP158 triple gene block and comprises a nucleotide sequence corresponding to SEQ. ID. No.
  • the second protein or polypeptide of the RSP158 triple gene block has a deduced amino acid sequence corresponding to SEQ. ID. No. 29 as follows: Met Pro Phe Gln Gln Pro Ala Asn Trp Ala Lys Thr 1 5 10 Ile Thr Pro Leu Thr Ile Gly Leu Gly Ile Gly Leu 15 20 Val Leu His Phe Leu Arg Lys Ser Asn Leu Pro Tyr 25 30 35 Ser Gly Asp Asn Ile His Gln Phe Pro His Gly Gly 40 45 Arg Tyr Arg Asp Gly Thr Lys Ile Thr Tyr Cys Gly 50 55 60 Pro Lys Gln Ser Phe Pro Ser Ser Gly Ile Phe Gly 65 70 Gln Ser Glu Asn Phe Val Pro Leu Met Leu Val Ile 75 80 Gly Leu Ile Ala Phe Ile His Val Leu Ser Val Trp 85 90 95 Asn Ser Gly Leu Gly Arg Asn Cys Asn Cys His Pro 100 105 Asn Pro Cy
  • RSP158 ORF4 Another DNA molecule of the present invention (RSP158 ORF4) includes nucleotides 1447-1689 of SEQ. ID. No. 23. This DNA molecule codes for a third protein or polypeptide of the RSP158 triple gene block and comprises a nucleotide sequence corresponding to SEQ. ID. No.
  • the third protein or polypeptide of the RSP158 triple gene block has a deduced amino acid sequence corresponding to SEQ. ID. No. 31 as follows: Met Tyr Cys Leu Phe Gly Ile Leu Val Leu Val Gly 1 5 10 Ile Ala Ile Ala Ile Gln Ile Leu Ala His Val Asp 15 20 Asn Ser Ser Gly Ser His Gln Gly Cys Phe Ile Arg 25 30 35 Ala Thr Gly Glu Ser Ile Leu Ile Glu Asn Cys Gly 40 45 Pro Ser Glu Ala Leu Ala Ser Thr Val Arg Glu Val 50 55 60 Leu Gly Gly Leu Lys Ala Leu Gly Ile Ser His Thr 65 70 Thr Glu Glu Ile Asp Tyr Arg Cys 75 80
  • the third protein or polypeptide of the RSP158 triple gene block has a molecular weight of about 5 to 10 kDa., preferably 8.4 kDa.
  • RSP158 ORF5 includes nucleotides 1699-2009 of SEQ. ID. No. 23.
  • This DNA molecule codes for a partial RSP158 coat protein or polypeptide and comprises a nucleotide sequence corresponding to SEQ. ID. No.
  • the polypeptide has a deduced amino acid sequence corresponding to SEQ. ID. No. 33 as follows: Met Ala Ser Gln Val Gly Lys Leu Pro Gly Glu Ser Asn Glu Ala Phe 1 5 10 15 Glu Ala Arg Leu Lys Ser Leu Glu Leu Ala Arg Ala Gln Lys Gln Pro 20 25 30 Glu Gly Ser Asn Thr Pro Pro Thr Leu Ser Gly Val Leu Ala Lys Arg 35 40 45 Lys Arg Val Ile Glu Asn Ala Leu Ser Lys Thr Val Asp Met Arg Glu 50 55 60 Val Leu Lys His Glu Thr Val Val Ile Ser Pro Asn Val Met Asp Glu 65 70 75 80 Gly Ala Ile Asp Glu Leu Ile Arg Ala Phe Gly Glu Ser Gly Ile Ala 85 90 95 Glu Ser Ala Gln Phe Asp Val 100
  • the following seven cDNA clones are located at the central part of the ORF1 of RSPaV-1 and all have high identities (83.6-98.4%) in nucleotide sequence with the comparable regions of RSPaV-1.
  • the universal primers BM98-3F/BM98-3R (SEQ. ID. Nos. 51 and 52, infra) were designed based on the conserved nucleotide sequences of this region.
  • Portions of the genome from yet other strains of Rupestris stem pitting associated viruses have also been isolated and sequenced. These include strains designated 140/94-19 (T7+R1), 140/94-24 (T7+R1), 140/94-2 (T3+F1), 140/94+42 (T7+R1), 140/94-64 (T+R1), 140-94-72 (T7+R1), and 140/94-6 (T3+BM98-3F+F2).
  • the nucleotide sequence of 140/94-19 corresponds to SEQ. ID. No. 34 as follows: GCAGGATTGA AGGCTGGCCA CTGTGTGATT TTTGATGAGG TCCAGTTGTT TCCTCCTGGA 60 TACATCGATC TATGCTTGCT TATTATACGT AGTGATGCTT TCATTTCACT TGCCGGTGAT 120 CCATGTCAAA GCACATATGA TTCGCAAAAG GATCGGGCAA TTTTGGGCGC TGAGCAGAGT 180 GACATACTTA GAATGCTTGA GGGCAAAACG TATAGGTATA ACATAGAAAG CAGGAGGTTT 240 GTGAACCCAA TGTTCGAATC AAGACTGCCA TGTCACTTCA AAAAGGGTTC GATGACTGCC 300 GCTTTCGCTG ATTATGCAAT CTTCCATAAT ATGCATGACT TTCTCCTGGC GAGGTCAAAA 360 GGTCCTTTGG ATGCCGTTTTTTGGTTTCCAGT TTTGAGGAGA AAA
  • the nucleotide sequence of 140/94-24 corresponds to SEQ. ID. No. 35 as follows: ATTAACCCAA ATGGTAAGAT TTCCGCCTTG TTTGATATAA CCAATGAGCA CATAAGGCAT 60 GTTGAGAAGA TCGGCAATGG CCCTCAGAGC ATAAAAGTAG ATGAGTTGAG GAAGGTTAAG 120 CGATCCGCCC TTGATCTTCT TTCAATGAAT GGGTCCAAAA TAACCTATTT TCCAAACTTT 180 GAGCGGGCTG AAAAGTTGCA AGGGTGCTTG CTAGGGGGCC TAACTGGTGT CATAAGTGAT 240 GAAAAGTTCA GTGATGCAAA ACCCTGGCTT TCTGGTATAT CAACTGCGGA TATAAAGCCA 300 AGAGAGCTAA CTGTCGTGCT TGGCACTTTT GGGGCTGGAA AGAGTTTCTTTCTT GTATAAGAGT 360 TTCATGAAGA GATCTGAGGG AAAATTTGTA ACTTTTGTTT
  • the nucleotide sequence of 140/94-2 corresponds to SEQ. ID. No. 36 as follows: CATTTTTAAA ATTTAATCCA GTCGACTCAC CAAATGTGAG CGTAAGCTGT TTCATCCCAA 60 AGTAGGACTG GACTATTTTC TTCTCCTCAA AACTAGAAAC CAGAATGGCA TCCAAAGGAC 120 CTTTTGACCT TGCCAGGAGG AAATCATGCA TATTGTGGAA AATGGCATAA TCAGCAAAGG 180 CAGCAGTCAT TGTACCCTTT TTGAAGTGAC ATGGCAGTCG AGATTCAAAC ATTGGGTTCA 240 CAAATCTTCT GCTTTCTATG TTGTACCTAT ACGTCTTGCC TTCAAGTATT TTGAGTATGT 300 CACTCTGCTC AGCGCCCAAA ATCGAT CTTTTTGTGA GTCATATGTG CTCTGACATG 360 GGTCACCAGC AAGTGAAATG AAAGCATCAC TACGTATAAT AAG
  • the nucleotide sequence of 140/94-42 corresponds to SEQ. ID. No. 37 as follows: GTGGTTTTTG CAACAACAGG CCCAGGTCTA TCTAAGGTTT TGGAAATGCC TCGAAGCAAG 60 AAGCAATCTA TTCTGGTTCT TGAGGGAGCC CTATCCATAG AAACGGACTA TGGCCCAAAA 120 GTTCTGGGAT CTTTTGAAGT TTTCAAAGGG GATTTCAACA TTAAAAAAAT GGAAGAAAGT 180 TCCATCTTTG TAATAACATA CAAGGCCCCA GTTAGATCTA CTGGCAAGTT GAGGGTCCAC 240 CAATCAGAAT GCTCATTTTC TGGATCCAAG GAGGTATTGC TGGGTTGTCA GATTGAGGCA 300 TGTGCTGATT ATGATATTGA TGATTTCAAT ACTTTCTTTG TACCTGGTGA TGGTAATTGC 360 TTTTGGCATT CAGTTGGTTT CTTACTCAGT ACTGACGGA
  • the nucleotide sequence of 140/94-6 corresponds to SEQ. ID. No. 38 as follows: GTCTAACTGG CGTTATAAGT GATGAGAAAT TCAGTGATGC AAAACCTTGG CTTTCTGGTA 60 TATCTACTAC AGATATTAAG CCAAGGGAAT TAACTGTTGT GCTTGGTACA TTTGGGGCTG 120 GGAAGAGTTT CTTGTACAAG AGTTTGATGA AAAGGTCTGA GGGTAAATTC GTAACCTTTG 180 TTTCTCCCAG ACGTGCTTTA GCAAATTCAA TCAAAAATGA TCTTGAAATG GATGATAGCT 240 GCAAAGTTGC CAAAGCAGGT AGGTCAAAGA AGGAAGGGTG GGATGTAGTA ACTTTTGAGG 300 TCTTCCTCAG AAAAGTTGCA GGATTGAAGG CTGGCCACTG TGTGATTTTT GATGAGGTCC 360 AGTTGTTTCC TCCTGGATAC ATCGATCT
  • the nucleotide sequence of 140/94-64 corresponds to SEQ. ID. No. 39 as follows: ATGTTCACCA AATCCAAATT ATGGCTGAAG CGAGATAAAG CAGTAAGCCA CCGCCGATCA 60 TCTGTGTGAA AGGAATCATG TGATATGAGA ATTCCCCCAT TTTTGAAATT CAACCCAGTT 120 GATTCACCAA ATGTGAGTGT GAGCTGTTTC ATTCCAAAGT AGGACTGGAC TATCTTTTTC 180 TCCTCAAAAC TGGAAACCAA AACGGCATCC AAAGGACCTT TTGACCTCGC CAGGAGAAAG 240 TCATGCATAT TATGGAAGAT TGCATAATCA GGGAAAGCGG CAGTCATTGA GCCCTTTTTG 300 AATTGACATG GCAGTCTTGA TTCGAACATT GGATTCACAA ACCTCCTGCT TTCAATGTTA 360 TACCTATACG TCTTGCCCTC AAGCAGTCTA AGTATGTCAC
  • the nucleotide sequence of 140-94-72 corresponds to SEQ. ID. No. 40 as follows: AGAATGCTTA TGCTGAGAAT GAGATGATTG CATTATTTTG CATCCGGCAC CATGTAAGGC 60 TTATAGTAAT AACACCGGAA TATGAAGTTA GTTGGAAATT TGGGGAAAGT GAGTGGCCCC 120 TATGTGGAAT TCTTTGCCTG AGGTCCAATC ACTTCCAACC ATGCGCCCCG CTGAATGGTT 180 GCATGATCAC GGCTATTGCT TCAGCACTTG GGAGGCGTGA GGTTGATGTG TTAAATTATC 240 TGTGTAGGCC TAGCACTAAT CACATCTTTG AGGAGCTGTG CCAGGGCGGA GGGCTTAATA 300 TGATGTACTT GGCTGAAGCT TTTGAGGCCT TTGACATTTG TGCAAAGTGC GACATAAATG 360 GGGAAATTGA GGTCATTAAC CCAAATGGCA AGATT
  • fragments of the DNA molecules of the present invention are constructed by using appropriate restriction sites, revealed by inspection of the DNA molecule's sequence, to: (i) insert an interposon (Felley et al., “Interposon Mutagenesis of Soil and Water Bacteria: A Family of DNA Fragments Designed for in vitro Insertion Mutagenesis of Gram-negative Bacteria,” Gene 52:147-15 (1987), which is hereby incorporated by reference) such that truncated forms of the RSP virus polypeptide or protein, that lack various amounts of the C-terminus, can be produced or (ii) delete various internal portions of the protein.
  • the sequence can be used to amplify any portion of the coding region, such that it can be cloned into a vector supplying both transcription and translation start signals.
  • Suitable DNA molecules are those that hybridize to a DNA molecule comprising a nucleotide sequence of at least 15 continuous bases of SEQ. ID. No. 1 under stringent conditions characterized by a hybridization buffer comprising 0.9M sodium citrate (“SSC”) buffer at a temperature of 37° C. and remaining bound when subject to washing with SSC buffer at 37° C.; and preferably in a hybridization buffer comprising 20% formamide in 0.9M saline/0.9M SSC buffer at a temperature of 42° C. and remaining bound when subject to washing at 42° C. with 0.2 ⁇ SSC buffer at 42° C.
  • SSC sodium citrate
  • Variants may also (or alternatively) be modified by, for example, the deletion or addition of nucleotides that have minimal influence on the properties, secondary structure and hydropathic nature of the encoded protein or polypeptide.
  • the nucleotides encoding a protein or polypeptide may be conjugated to a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or post-translationally directs transfer of the protein.
  • the nucleotide sequence may also be altered so that the encoded protein or polypeptide is conjugated to a linker or other sequence for ease of synthesis, purification, or identification of the polypeptide.
  • the protein or polypeptide of the present invention is preferably produced in purified form (preferably, at least about 80%, more preferably 90%, pure) by conventional techniques.
  • the protein or polypeptide of the present invention is isolated by lysing and sonication. After washing, the lysate pellet is re-suspended in buffer containing Tris-HCl. During dialysis, a precipitate forms from this protein solution. The solution is centrifuged, and the pellet is washed and re-suspended in the buffer containing Tris-HCl. Proteins are resolved by electrophoresis through an SDS 12% polyacrylamide gel.
  • the DNA molecule encoding the RSP virus protein or polypeptide of the present invention can be incorporated in cells using conventional recombinant DNA technology. Generally, this involves inserting the DNA molecule into an expression system to which the DNA molecule is heterologous (i.e., not normally present). The heterologous DNA molecule is inserted into the expression system or vector in proper sense orientation and correct reading frame. The vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences.
  • Recombinant genes may also be introduced into viruses, such as vaccinia virus.
  • Recombinant viruses can be generated by transfection of plasmids into cells infected with virus.
  • Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gt11, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKCO101, SV 40, pBluescript II SK +/ ⁇ or KS+/ ⁇ (see “Stratagene Cloning Systems” Catalog (1993) from Stratagene, La Jolla, Calif., which is hereby incorporated by reference), pQE, pIH821, pGEX, pET series (see Studier et.
  • viral vectors such as lambda vector system gt11, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC
  • Suitable vectors are continually being developed and identified. Recombinant molecules can be introduced into cells via transformation, transduction, conjugation, mobilization, or electroporation.
  • the DNA sequences are cloned into the vector using standard cloning procedures in the art, as described by Maniatis et al., Molecular Cloning: A Laboratory Manual , Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1982), which is hereby incorporated by reference.
  • host-vector systems may be utilized to express the protein-encoding sequence(s).
  • the vector system must be compatible with the host cell used.
  • Host-vector systems include but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); and plant cells infected by bacteria or transformed via particle bombardment (i.e. biolistics).
  • the expression elements of these vectors vary in their strength and specificities. Depending upon the host-vector system utilized, any one of a number of suitable transcription and translation elements can be used.
  • mRNA messenger RNA
  • telomere a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis.
  • the DNA sequences of eukaryotic promoters differ from those of procaryotic promoters.
  • eukaryotic promoters and accompanying genetic signals may not be recognized in or may not function in a procaryotic system, and, further, procaryotic promoters are not recognized and do not function in eukaryotic cells.
  • SD Shine-Dalgarno
  • Promoters vary in their “strength” (i.e., their ability to promote transcription). For the purposes of expressing a cloned gene, it is desirable to use strong promoters in order to obtain a high level of transcription and, hence, expression of the gene. Depending upon the host cell system utilized, any one of a number of suitable promoters may be used. For instance, when cloning in E.
  • promoters such as the T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the P R and P L promoters of coliphage lambda and others, including but not limited, to lacUV5, ompF, bla, Ipp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacV5 (tac) promoter or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.
  • trp-lacV5 (tac) promoter or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.
  • Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promoter unless specifically induced.
  • the addition of specific inducers is necessary for efficient transcription of the inserted DNA.
  • the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside).
  • IPTG isopropylthio-beta-D-galactoside.
  • Specific initiation signals are also required for efficient gene transcription and translation in procaryotic cells. These transcription and translation initiation signals may vary in “strength” as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively.
  • the DNA expression vector which contains a promoter, may also contain any combination of various “strong” transcription and/or translation initiation signals. For instance, efficient translation in E. coli requires a Shine-Dalgarno (“SD”) sequence about 7-9 bases 5′ to the initiation codon (“ATG”) to provide a ribosome binding site. Thus, any SD-ATG combination that can be utilized by host cell ribosomes may be employed.
  • Such combinations include but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DNA or other techniques involving incorporation of synthetic nucleotides may be used.
  • Suitable host cells include, but are not limited to, bacteria, virus, yeast, mammalian cells, insect, plant, and the like.
  • the present invention also relates to RNA molecules which encode the various RSP virus proteins or polypeptides described above.
  • the transcripts can be synthesized using the host cells of the present invention by any of the conventional techniques.
  • the mRNA can be translated either in vitro or in vivo.
  • Cell-free systems typically include wheat-germ or reticulocyte extracts. In vivo translation can be effected, for example, by microinjection into frog oocytes.
  • One aspect of the present invention involves using one or more of the above DNA molecules encoding the various proteins or polypeptides of a RSP virus to transform grape plants in order to impart RSP resistance to the plants.
  • the mechanism by which resistance is imparted in not known.
  • the transformed plant can express the coat protein or polypeptide, and, when the transformed plant is inoculated by a RSP virus, such as RSPaV-1, the expressed coat protein or polypeptide surrounds the virus, thereby preventing translation of the viral DNA.
  • the subject DNA molecule incorporated in the plant can be constitutively expressed.
  • expression can be regulated by a promoter which is activated by the presence of RSP virus.
  • Suitable promoters for these purposes include those from genes expressed in response to RSP virus infiltration.
  • the isolated DNA molecules of the present invention can be utilized to impart RSP virus resistance for a wide variety of grapevine plants.
  • the DNA molecules are particularly well suited to imparting resistance to Vitis scion or rootstock cultivars.
  • Scion cultivars which can be protected include those commonly referred to as Table or Raisin Grapes, such as Alden, Almeria, Anab-E-Shahi, Autumn Black, Beauty Seedless, Black Corinth, Black Damascus, Black Malvoisie, Black Prince, Blackrose, Bronx Seedless, Burgrave, Calmeria, Campbell Early, Canner, Cardinal, Catawba, Christmas, Concord, Dattier, Delight, Diamond, Dizmar, Duchess, Early Muscat, Emerald Seedless, Emperor, Exotic, Anthony de Lesseps, Fiesta, Flame seedless, Flame Tokay, Gasconade, Gold, Himrod, Hunisa, Hussiene, Isabella, Italia, July Muscat, Khandahar, Katta, Kourgane,
  • Rootstock cultivars which can be protected include Couderc 1202, Couderc 1613, Couderc 1616, Couderc 3309, Dog Ridge, Foex 33 EM, Freedom, Ganzin 1 (A ⁇ R #1), Harmony, Kober 5BB, LN33, Millardet & de Grasset 41B, Millardet & de Grasset 420A, Millardet & de Grasset 101-14, Oppenheim 4 (SO 4 ), Paulsen 775, Paulsen 1045, Paulsen 1103, Richter 99, Richter 110, Riparia Gloire, Ruggeri 225, Saint-George, Salt Creek, Teleki 5A, Vitis rupestris Constantia, Vitis California , and Vitis girdiana.
  • Plant tissue suitable for transformation include leaf tissue, root tissue, meristems, zygotic and somatic embryos, and anthers. It is particularly preferred to utilize embryos obtained from anther cultures.
  • the expression system of the present invention can be used to transform virtually any plant tissue under suitable conditions.
  • Tissue cells transformed in accordance with the present invention can be grown in vitro in a suitable medium to impart RSPaV resistance.
  • Transformed cells can be regenerated into whole plants such that the protein or polypeptide imparts resistance to RSPaV in the intact transgenic plants.
  • the plant cells transformed with the recombinant DNA expression system of the present invention are grown and caused to express that DNA molecule to produce one of the above-described RSPaV proteins or polypeptides and, thus, to impart RSPaV resistance.
  • the DNA construct in a vector described above can be microinjected directly into plant cells by use of micropipettes to transfer mechanically the recombinant DNA.
  • the genetic material may also be transferred into the plant cell using polyethylene glycol. Krens, et al., Nature, 296:72-74 (1982), which is hereby incorporated by reference.
  • One technique of transforming plants with the DNA molecules in accordance with the present invention is by contacting the tissue of such plants with an inoculum of a bacteria transformed with a vector comprising a gene in accordance with the present invention which imparts RSPaV resistance.
  • this procedure involves inoculating the plant tissue with a suspension of bacteria and incubating the tissue for 48 to 72 hours on regeneration medium without antibiotics at 25-28° C.
  • Bacteria from the genus Agrobacterium can be utilized to transform plant cells. Suitable species of such bacterium include Agrobacterium tumefaciens and Agrobacterium rhizogenes. Agrobacterium tumefaciens (e.g., strains C58, LBA4404, or EHA105) is particularly useful due to its well-known ability to transform plants.
  • Heterologous genetic sequences can be introduced into appropriate plant cells, by means of the Ti plasmid of A. tumefaciens or the R1 plasmid of A. rhizogenes .
  • the Ti or R1 plasmid is transmitted to plant cells on infection by Agrobacterium and is stably integrated into the plant genome. J. Schell, Science, 237:1176-83 (1987), which is hereby incorporated by reference.
  • the transformed plant cells After transformation, the transformed plant cells must be regenerated.
  • Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts or a petri plate containing explants is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced in the callus tissue. These embryos germinate as natural embryos to form plants.
  • the culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is usually reproducible and repeatable.
  • the expression cassette After the expression cassette is stably incorporated in transgenic plants, it can be transferred to other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
  • transgenic plants of this type are produced, the plants themselves can be cultivated in accordance with conventional procedure so that the DNA construct is present in the resulting plants.
  • transgenic seeds are recovered from the transgenic plants. These seeds can then be planted in the soil and cultivated using conventional procedures to produce transgenic plants.
  • particle bombardment also known as biolistic transformation
  • particle bombardment also known as biolistic transformation
  • this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and to be incorporated within the interior thereof.
  • the vector can be introduced into the cell by coating the particles with the vector containing the heterologous DNA.
  • the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle.
  • Biologically active particles e.g., dried bacterial cells containing the vector and heterologous DNA
  • a grape plant tissue is transformed in accordance with the present invention, it is regenerated to form a transgenic grape plant.
  • regeneration is accomplished by culturing transformed tissue on medium containing the appropriate growth regulators and nutrients to allow for the initiation of shoot meristems. Appropriate antibiotics are added to the regeneration medium to inhibit the growth of Agrobacterium and to select for the development of transformed cells. Following shoot initiation, shoots are allowed to develop tissue culture and are screened for marker gene activity.
  • the DNA molecules of the present invention can be made capable of transcription to a messenger RNA that does not translate to the protein. This is known as RNA-mediated resistance.
  • RNA-mediated resistance When a Vitis scion or rootstock cultivar is transformed with such a DNA molecule, the DNA molecule can be transcribed under conditions effective to maintain the messenger RNA in the plant cell at low level density readings. Density readings of between 15 and 50 using a Hewlet ScanJet and Image Analysis Program are preferred.
  • a portion of one or more DNA molecules of the present invention as well as other DNA molecules can be used in a transgenic grape plant in accordance with U.S. patent application Ser. No. 09/025,635, which is hereby incorporated herein by reference.
  • the RSPaV protein or polypeptide can also be used to raise antibodies or binding portions thereof or probes.
  • the antibodies can be monoclonal or polyclonal.
  • Monoclonal antibody production may be effected by techniques which are well-known in the art. Basically, the process involves first obtaining immune cells (lymphocytes) from the spleen of a manunal (e.g., mouse) which has been previously immunized with the antigen of interest either in vivo or in vitro. The antibody-secreting lymphocytes are then fused with (mouse) myeloma cells or transformed cells, which are capable of replicating indefinitely in cell culture, thereby producing an immortal, immunoglobulin-secreting cell line. The resulting fused cells, or hybridomas, are cultured, and the resulting colonies screened for the production of the desired monoclonal antibodies.
  • lymphocytes immune cells
  • a manunal e.g., mouse
  • myeloma cells or transformed cells which are capable of replicating indefinitely in cell culture, thereby producing an immortal, immunoglobulin-secreting cell line.
  • the resulting fused cells, or hybridomas are cultured
  • Colonies producing such antibodies are cloned, and grown either in vivo or in vitro to produce large quantities of antibody.
  • a description of the theoretical basis and practical methodology of fusing such cells is set forth in Kohler and Milstein, Nature, 256:495 (1975), which is hereby incorporated by reference.
  • Mammalian lymphocytes are immunized by in vivo immunization of the animal (e.g., a mouse) with the protein or polypeptide of the present invention. Such immunizations are repeated as necessary at intervals of up to several weeks to obtain a sufficient titer of antibodies. Following the last antigen boost, the animals are sacrificed and spleen cells removed.
  • Fusion with mammalian myeloma cells or other fusion partners capable of replicating indefinitely in cell culture is effected by standard and well-known techniques, for example, by using polyethylene glycol (“PEG”) or other fusing agents.
  • PEG polyethylene glycol
  • This immortal cell line which is preferably murine, but may also be derived from cells of other mammalian species, including but not limited to rats and humans, is selected to be deficient in enzymes necessary for the utilization of certain nutrients, to be capable of rapid growth, and to have good fusion capability. Many such cell lines are known to those skilled in the art, and others are regularly described.
  • Procedures for raising polyclonal antibodies are also well known. Typically, such antibodies can be raised by administering the protein or polypeptide of the present invention subcutaneously to New Zealand white rabbits which have first been bled to obtain pre-immune serum.
  • the antigens can be injected at a total volume of 100 ⁇ l per site at six different sites. Each injected material will contain synthetic surfactant adjuvant pluronic polyols, or pulverized acrylamide gel containing the protein or polypeptide after SDS-polyacrylamide gel electrophoresis.
  • the rabbits are then bled two weeks after the first injection and periodically boosted with the same antigen three times every six weeks. A sample of serum is then collected 10 days after each boost.
  • Polyclonal antibodies are then recovered from the serum by affinity chromatography using the corresponding antigen to capture the antibody. Ultimately, the rabbits are euthanized with pentobarbital 150 mg/Kg IV. This and other procedures for raising polyclonal antibodies are disclosed in Harlow et. al., editors, Antibodies: A Laboratory Manual (1988), which is hereby incorporated by reference.
  • binding portions of such antibodies can be used.
  • binding portions include Fab fragments, F(ab′) 2 fragments, and Fv fragments.
  • Fab fragments fragments
  • F(ab′) 2 fragments fragments that can be made by conventional procedures, such as proteolytic fragmentation procedures, as described in Goding, Monoclonal Antibodies: Principles and Practice , New York: Academic Press, pp. 98-118 (1983), which is hereby incorporated by reference.
  • the present invention also relates to probes found either in nature or prepared synthetically by recombinant DNA procedures or other biological procedures.
  • Suitable probes are molecules that bind to RSP viral antigens identified by the polyclonal antibodies of the present invention or bind to the nucleic acid of RSPaV.
  • Such probes can be, for example, proteins, peptides, lectins, or nucleic acids.
  • the antibodies or binding portions thereof or probes can be administered to RSPaV infected scion cultivars or rootstock cultivars. Alternatively, at least the binding portions of these antibodies can be sequenced, and the encoding DNA synthesized.
  • the encoding DNA molecule can be used to transform plants together with a promoter which causes expression of the encoded antibody when the plant is infected by an RSPaV. In either case, the antibody or binding portion thereof or probe will bind to the virus and help prevent the usual stem pitting response.
  • Antibodies raised against the proteins or polypeptides of the present invention or binding portions of these antibodies can be utilized in a method for detection of RSPaV in a sample of tissue, such as tissue from a grape scion or rootstock.
  • Antibodies or binding portions thereof suitable for use in the detection method include those raised against a replicase, proteins or polypeptides of the triple gene block, or a coat protein or polypeptide in accordance with the present invention. Any reaction of the sample with the antibody is detected using an assay system which indicates the presence of RSPaV in the sample.
  • assay systems such as enzyme-linked immunosorbent assays, radioimmunoassays, gel diffusion precipitin reaction assays, immunodiffusion assays, agglutination assays, fluorescent immunoassays, protein A immunoassays, or immunoelectrophoresis assays.
  • the RSPaV can be detected in such a sample using the DNA molecules of the present, RNA molecules of the present invention, or DNA or RNA fragments thereof, as probes in nucleic acid hybridization assays for detecting the presence of complementary virus DNA or RNA in the various tissue samples described above.
  • the nucleotide sequence is provided as a probe in a nucleic acid hybridization assay or a gene amplification detection procedure (e.g., using a polymerase chain reaction procedure).
  • the nucleic acid probes of the present invention may be used in any nucleic acid hybridization assay system known in the art, including, but not limited to, Southern blots (Southern, E.
  • the isolated DNA molecules of the present invention or RNA transcripts thereof can be used in a gene amplification detection procedure (e.g., a polymerase chain reaction). Erlich, H. A., et. al., “Recent Advances in the Polymerase Chain Reaction,” Science 252:1643-51 (1991), which is hereby incorporated by reference. Any reaction with the probe is detected so that the presence of RSP virus in the sample is indicated. Such detection is facilitated by providing the DNA molecule of the present invention with a label. Suitable labels include a radioactive compound, a fluorescent compound, a chemiluminescent compound, an enzymatic compound, or other equivalent nucleic acid labels.
  • probes having nucleotide sequences that correspond with conserved or variable regions of the ORF or UTR For example, to distinguish RSPaV from other related viruses (as described herein), it is desirable to use probes which contain nucleotide sequences that correspond to sequences more highly conserved among all RSPaV strains. Also, to distinguish between different RSPaV strains (e.g., RSPaV-1, RSP47-4, RSP158), it is desirable to utilize probes containing nucleotide sequences that correspond to sequences less highly conserved among the RSP virus strains.
  • Nucleic acid (DNA or RNA) probes of the present invention will hybridize to complementary RSPaV-1 nucleic acid under stringent conditions. Less stringent conditions may also be selected. Generally, stringent conditions are selected to be about 50° C. lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH. The T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • Nonspecific binding may also be controlled using any one of a number of known techniques such as, for example, blocking the membrane with protein-containing solutions, addition of heterologous RNA, DNA, and SDS to the hybridization buffer, and treatment with RNase.
  • suitable stringent conditions for nucleic acid hybridization assays or gene amplification detection procedures are as set forth above. More or less stringent conditions may also be selected.
  • dsRNA preparations from Colobel 257, Ravat 34, Couderc 28-112, and Seyval were pooled and used as templates for cDNA synthesis.
  • dsRNA bands were excised from low melting temperature agarose gels after electrophoresis and recovered by extraction with phenol and chloroform (Sambrook et al., Molecular Cloning: A LaboratorEy Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
  • dsRNA was denatured with 20 mM methyl mercuric hydroxide and cDNAs were synthesized using slightly modified methods of Jelkmann et al., “Cloning of Four Viruses from Small Quantities of Double-Stranded RNA,” Phytopathology, 79:1250-53 (1989), which is incorporated herein be reference.
  • the cDNA fragments were first blunt-ended with T4 DNA polymerase at 12° C. T4 DNA ligase was used to add EcoR I adapters to both ends of the cDNAs.
  • Plaque hybridization was used to screen cDNA clones by transferring recombinant cDNA plaques to nylon membranes and hybridizing to 32 P-labeled first-strand cDNA probes generated from the dsRNA according to manufacturer's recommendations (Du Pont, 1987). Clones with strong hybridization signals were converted into pBluescript SK through in vivo excision (Stratagene, 1991). After digestion of the resulting plasmids with EcoR I, 20 clones were selected and further analyzed in Southern hybridization with radio labeled first strand cDNA probes synthesized from the dsRNA. The specificity of two selected clones to the dsRNA was confirmed by Northern analysis using 32 P labeled inserts of the two clones.
  • RSP3-RSP94 primer 1 (sense, nt 3629-3648) has a nucleotide sequence corresponding to SEQ. ID. No. 41 as follows: GCTTCAGCAC TTGGAAGGCG 20
  • RSP3-RSP94 primer 2 (antisense, nt 4350-4366) has a nucleotide sequence corresponding to SEQ. ID. No. 42 as follows: CACACAGTGG CCAGCCT 17 After gel electrophoresis, PCR amplified cDNA bands were excised from gels and recovered with the phenol/chloroform method (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), which is hereby incorporated by reference).
  • RSP94 RSP95 primer 1 (sense, nt 5272-5291) has a nucleotide sequence corresponding to SEQ. ID. No. 43 as follows: GGAGGTGCGT TGTGGTTATG 20
  • RSP94-RSP95 primer 2 (antisense, nt 6791-6808) has a nucleotide sequence corresponding to SEQ. ID. No. 44 as follows: CCGTGGCACT GCACACCC 17
  • a primer (sense, nt 8193-8210) having a nucleotide sequence corresponding to SEQ. ID. No. 45 as follows: GGAGGTGACC ACATTACG 18
  • the dsRNA was first tagged with poly (A) using yeast Poly (A) polymerase (USB) (Pappu et al., “Nucleotide Sequence and Organization of Eight 3′ Open Reading Frames of the Citrus tristeza Closterovirus Genome,” Virology 199:35-46 (1994), which is hereby incorporated by reference) and then used as templates to generate cDNA fragments by RT-PCR using (dT) 18 primer and primer (antisense, nt 429-449) having a nucleotide sequence corresponding to SEQ. ID. NO. 46 as follows: CATCACGACT TGTCACAAAC C 21
  • RT-PCR Reverse Transcription-Polymerase Chain Reaction
  • Primer RSP95F1 an antisense strand primer, has a nucleotide sequence corresponding to SEQ. ID. NO. 47 as follows: TGGGCCTCCA CTTCTTC 17
  • Primer RSP95R1 a sense strand primer, has a nucleotide sequence corresponding to SEQ. ID. No. 48 as follows: GGGGTTGCCT GAAGAT 16
  • Primer RSP149F1 an antisense strand primer, has a nucleotide sequence corresponding to SEQ. ID. No. 49 as follows: ACACCTGCTG TGAAAGC 17
  • Primer RSP149R1 a sense strand primer
  • RSP95F1/R1 has a nucleotide sequence corresponding to SEQ. ID. No. 50 as follows: GGCCAAGGTT CAGTTTG 17
  • RSP95F1/R1 were used in RT-PCR to test samples collected in 1994.
  • RSP149R1/F1, alone or together with RSP95F1I/R1 were used to test samples collected in 1995 and 1996.
  • blind tests were conducted for samples from Canada in 1995 and 1996. The indexing results of these samples were kept untold until the RT-PCR tests were complete.
  • dsRNAs were denatured with methylmercuric hydroxide (CH4HgOH) and reverse transcribed into cDNAs with Moloney murine leukemia virus (MMLV) or Avian Myeloblastosis Virus (AMV) reverse transcriptases (Promega) at 42° C. for 1 to 3 h.
  • MMLV Moloney murine leukemia virus
  • AMV Avian Myeloblastosis Virus
  • Five of 20 ⁇ l of the RT reactions were added to PCR mix and amplified in thermal cycler (HYBAID OmniGene, National Labnet Company) with Taq DNA polymerase (buffer B, Promega) using the following parameters: initial denaturation at 94° C. for 5 min, 40 cycles of amplification at 94° C. for 45 s, 52° C. for 1 min, and 72° C.
  • PCR products were analyzed by electrophoresis on 1% agarose gels containing ethidium bromide. Hae III digested Phix 174 fragments were used as molecular weight markers.
  • DNA fragments amplified by PCR from cDNA clone RSP149 with primers RSP149F1/R1 were labeled with 32 P by random priming and used as probes.
  • Products of RT-PCR of randomly selected grapevines including 26 positives and 6 negatives by RT-PCR were electrophoresed on an 0.8% agarose gel, transferred to nylon membranes, and hybridized to the probes following manufacturer's instructions (Du Pont).
  • FIG. 1A illustrates these typical RSP symptoms.
  • FIG. 2A illustrates twelve grapevine accessions with typical RSP symptoms revealed a dsRNA of ca. 8.7 kb with gel electrophoresis.
  • a smaller dsRNA of about 6.6 kb was observed in Colobel 257 and Seyval.
  • Verduzzo 233A which was indexed free of RSP on St. George
  • the two dsRNA species isolated from Verduzzo 233A were not observed in other healthy grapevines such as Cabernet Franc and LN 33. TABLE 1 St.
  • dsRNA The yield of dsRNA was low and varied significantly among different accessions.
  • phloem tissue 14 g for Bertille Seyve 5563 and Couderc 28-112; 18.5 g for the others
  • phloem tissue 14 g for Bertille Seyve 5563 and Couderc 28-112; 18.5 g for the others
  • Bertille Seyve 5563, Couderc 28-112, Joffre, and Verdelet showed weak bands after staining with EtBr, as shown in FIG. 2A .
  • Bertille Seyve 3408 and Seyve Villard 3160 were analyzed in separate experiments and dsRNA bands of the same size were observed.
  • a total of 182 clones were selected after plaque hybridization. Eighty clones with strong hybridization signals were subcloned into pBluescript SK through in vivo excision. Resulting plasmids were shown to have inserts ranging from 0.3 to 3.0 kb. A total of 20 clones with inserts of ca. 0.8 kb or larger were selected. Southern analysis of these 20 clones to radio labeled first strand cDNA probes derived from the dsRNA resulted in 15 clones with strong hybridization signals. Several of these clones were used to determine the genome sequence of the dsRNA: RSP3, RSP28, RSP94, RSP140, RSP95, and TA5. Another clone (RSP149), which was 97% similar in nucleotide sequence to RSP95, was used as one of the two probes in Northern hybridization.
  • ORF1 (nt 62 to 6547 of SEQ. ID. No. 1) has a nucleotide sequence corresponding to SEQ. ID. NO. 2.
  • ORF1 believed to encode a protein or polypeptide having a molecular weight of about 244 kDa and an amino acid sequence corresponding to SEQ. ID. No. 3. According to Lutcke et al., “Selection of AUG Initiation Codons Differs in Plants and Animals,” Eur. Mol. Biol.
  • ORF2 (nt 6578 to 7243 of SEQ. ID. No. 1) has a nucleotide sequence corresponding to SEQ. ID. No. 4.
  • ORF2 is believed to encode a protein or polypeptide having a molecular weight of about 24.4 kDa and an amino acid sequence corresponding to SEQ. ID. NO. 5. The first two ORFs were separated by an intergenic region of 30 nts.
  • ORF3 (nt 7245 to 7598 of SEQ. ID. NO.
  • ORF3 is believed to encode a protein or polypeptide having a molecular weight of about 12.8 kDa and an amino acid sequence corresponding to SEQ. ID. NO. 7.
  • ORF4 (nt 7519 to 7761 of SEQ. ID. NO. 1), which overlapped with ORF3 by 80 nts, has a nucleotide sequence corresponding to SEQ. ID. No. 8.
  • ORF3 is believed to encode a protein or polypeptide having a molecular weight of about 8.4 kDa and an amino acid sequence corresponding to SEQ. ID. No. 9.
  • ORF5 Nine nucleotides downstream of ORF4 was the start of ORF5 (nt 7771 to 8550 of SEQ. ID. No. 1), which has a nucleotide sequence corresponding to SEQ. ID. No. 10.
  • ORF5 is believed to encode a protein or polypeptide having a molecular weight of about 28 kDa and an amino acid sequence corresponding to SEQ. ID. No. 11.
  • Downstream of ORF5 was the 3′ end LJTR of 176 nts.
  • RSPaV-1 ORF 1 When the total amino acid sequence of RSPaV-1 ORF 1 was used for comparison, it showed 39.6% and 37.6% identities with the replicases of ASPV and PVM respectively (Table 2). These homologies were mainly found in regions I (aa 1 to 372) and II (aa 1354-2161), which are at the N and C terminal portions of the putative replicase, respectively, shown at FIGS. 4A and 4B . Within region I, the identities of RSPaV-1 with ASPV and PVM were 49.2% and 47.2%, respectively (Table 2).
  • a NTP binding motif “GXXXXGKS/T” (aa 1356 to 1363) (“X” stands for any amino acid residue), which is conserved in helicase proteins and helicase domains of eukaryotic positive strand RNA viruses (Gorbalenya et al., “A Novel Superfamily of Nucleotide Triphosphate-Binding Motif Containing Proteins which are Probably Involved in Duplex Unwinding in DNA and RNA Replication and Recombination,” FEBS Letters, 235:16-24 (1988), which is hereby incorporated by reference), was found in the beginning of region II ( FIG. 4B ).
  • amino acid sequences of this motif in ASPV and PVM were identical to that of RSPaV-1 except for one position. Furthermore, amino acid sequence surrounding the GDD motif, which is conserved in all RNA dependent RNA polymerases of positive strand RNA viruses (Koonin, “The Phylogeny of RNA-Dependent RNA Polymerases of Positive-Strand RNA Viruses,” J. Gen. Virology 72:2197-2206 (1991), which is hereby incorporated by reference), was located near the C terminus of the RSPaV-1 replicase protein and showed high identities to those of ASPV and PVM ( FIG. 4B ).
  • the triple gene block is a common feature of several groups of plant viruses including carlaviruses, potexviruses, and ASPV.
  • Comparison of RSPaV-1 ORF2 with those of PVM and ASPV showed evenly distributed homologies in amino acid sequence: 38.0% identity to ASPV and 34.8% to PVM (Table 2).
  • the N terminal region of the 24.4K protein (ORF2) contained the consensus sequence “GXGKS S/T” (aa 31 to 36) ( FIG.
  • the 12.8K protein of RSPaV-1 encoded by ORF3 had 39.3% and 31.2% identities with its counterparts in ASPV and PVM respectively (Table 2). However, most of the matching occurred in a region from aa 29 to 62, among which 18 aa were fully conserved in all three viruses ( FIG. 5B ). These 12-13K proteins may function in membrane binding (Morozov et al., “Nucleotide Sequence of the Open Reading Frames Adjacent to the Coat Protein in Potato Virus X Genome,” FEBS Letters 213:438-42 (1987), which is hereby incorporated by reference).
  • the 8.4K protein encoded by RSPaV-1 ORF4 showed much lower identities: 27.1% with that of ASPV and 19.0% with that of PVM (Table 2). However, four residues “TGES” (aa 38 to 41) were conserved in all three viruses ( FIG. 5C ).
  • a sequence similarity search in a DNA database revealed identities between the putative protein encoded for by RSPaV-1 ORF5 to the coat proteins (CPs) of several groups of plant viruses, indicating that RSPaV-1 ORF5 may code for the coat protein.
  • MegAlign analysis revealed that RSPaV-1 ORF5 had 31.3% and 21.2% identities with the CPs of ASPV and PVM, respectively (Table 2). Most of the identities were found in the C terminal portion of the coat proteins (aa 142 to 245 for RSPaV-1), while the N terminal portions were quite variable in the numbers and sequences of amino acid residues.
  • MegAlign analysis shown in FIGS. 6A and 6B , revealed that the 3′ UTR of RSPaV-1 is more similar to that of PVM than to that of ASPV. For example, in a 75 nts stretch, RSPaV-1 had 68% identity with PVM. Within this region, 21 consecutive nucleotides were identical between these two viruses. The significance of this conservation in nucleotide sequence remains to be explored. In contrast, the 5′ UTR of RSPaV-1 did not reveal significant similarities with those of PVM and ASPV.
  • RSPaV-1 The complete nucleotide sequence of RSPaV-1 was determined from overlapping cDNA clones and RT-PCR-amplified cDNA fragments generated from the dsRNA.
  • the RSPaV-1 genome has five ORFs coding for the putative replicase (ORF1), the triple gene block (ORF24), and the CP(ORF5). The existence of these ORFs and their potential to code for structural and non-structural viral proteins were further supported by the identification of conserved motifs which are the signatures of various viral proteins.
  • RSPaV-1 has the most similarities to ASPV, which has not yet been grouped into a virus genus. Both viruses have the same genome organization and their ORFs code for putative proteins of similar sizes, except that the coat protein of ASPV is significantly larger (44 kDa) than that of RSPaV-1 (28 kDa). Comparisons of RSPaV-1 with PVM carlavirus show some similarities in genome organization except that RSPaV-1 lacks ORF6 which is located at the 3′ end of PVM genome. Although the genome organization of RSPaV-1 is similar to PVX potexvirus, the latter has a much smaller putative replicase. RSPaV-1 has no relation to grape viruses whose genomes have been sequenced so far.
  • GVA Garman virus A: Nucleotide Sequence, Genome Organization, and Relationship in the Trichovirus Genus,” Arch. Virology 142:417-23 (1997), which is hereby incorporated by reference
  • GVB Sydarelli et al., “The Nucleotide Sequence and Genomic Organization of Grapevine Virus B,” J. General Virology 77:2645-52 (1996), which is hereby incorporated by reference
  • Symptoms induced by RSP on the woody cylinder of St. George after graft inoculation with chip-buds can be divided into two types.
  • the first type is called “specific”, that is, pits and/or grooves being restricted to the area on the woody cylinder below the inoculation sites.
  • the other is called “nonspecific”, that is, pits and/or grooves being present above, around, and below the inoculation sites.
  • RSPaV-1 was detected not only from grapevines which indexed positively for RSP but also from some of the grapevines indexed negatively for RSP, a search for more healthy materials for RT-PCR tests became necessary. As the majority of plant viruses do not pass on through seeds, grapevine seedlings are probably free of RSPaV-1. Based on this assumption, six seedlings from five Vitis species were included in RT-PCR (see Table 7). None of them produce cDNA of expected size in RT-PCR using RSP149R1/F1 primers (SEQ. ID. Nos. 49 and 50).
  • RSPaV-1 is closely associated with RSP and that it is likely the causal agent of RSP.
  • RT-PCR detected RSPaV-1 specific sequences from most of the RSP-infected grapevines collected from a wide range of viticultural regions of the world. Among the 93 grapevine accessions indexed positively for RSP on St. George, 85% were positive in RT-PCR (see Table 5). The data also suggests that RT-PCR has the potential to be used as a standard method for diagnosing RSP. This method is advantageous over the biological indexing on indicator St. George, because it is simpler, quicker, and more sensitive.
  • RT-PCR did not detect RSPaV-1 sequences from 14 of the grapevine accessions indexed positively for RSP (see Table 6).
  • the discrepancy between RT-PCR and indicator indexing can be attributed to the existence in grapevines of different viruses or strains of the same virus which may all induce similar pitting and/or grooving symptoms on St. George upon graft-inoculation. It is believed these agents are only slightly different from RSPaV-1 at the level of their nucleotide sequences, but significant enough to hinder them from being detected by RT-PCR using RSPaV-1 specific primers.
  • RSPaV strains have genomes with nucleotide sequences that are highly similar to the nucleotide sequence of the RSPaV-1 genome.
  • Evidence that supports this hypothesis includes the finding of a highly conserved region of ca. 600 bps among the nucleotide sequences of RSPaV-1 (type strain) and seven other cDNA clones, as shown in FIG. 9 .
  • the nucleotide sequence identities of these strains to RSPaV-1 (type strain) range from 83.6% to 98.4%.
  • oligonucleotides are chosen which are conserved among all these strains (i.e., with one or only a few mismatches), then the oligonucleotides should function as universal primers, allowing all of the strains to be detected by RT-PCR.
  • a primer pair (BM98-3F/BM98-3R) can be designed to amplify a DNA fragment of 320 bps from all these clones.
  • BM98-3F has a nucleotide sequence corresponding to SEQ. ID. No. 51 as follows: GATGAGGTCCAGTTGTTTCC 20
  • BM98-3R has a nucleotide sequence corresponding to SEQ. ID. No. 52 as follows: ATCCAAAGGACCTTTTGACC 20
  • Primers BM98-3F/BM98-3R can be used in RT-PCR to test further some of the grapevine samples which were negative for RSPaV in RT-PCR using RSP95F1/RSP95R1 primers (SEQ. ID. Nos. 47 and 48, respectively) or RSP149F1/RSP149R1 primers (SEQ. ID. Nos. 49 and 50, respectively). Results show that 6 of the 9 samples included were positive for RSPaV in RT-PCR using BM98-3F/BM98-3R primers. This indicates that these universal primers can be used to achieve even higher detection rates.
  • BM98-1F/BM98-1R Another pair of primers (BM98-1F/BM98-1R) can be designed in a way that they can amplify DNA of 760 bps from RSPaV-1, RSP47-4, and RSP158.
  • BM98-1F has a nucleotide sequence corresponding to SEQ. ID. No. 53 as follows: CTTGATGAGTACTTGTC 17
  • BM98-1R has a nucleotide sequence corresponding to SEQ. ID. No. 54 as follows: GCAAGGATTTGGATGGC 17
  • Other “universal primers” can be designed manually or with computer programs (such as PrimerSelect) in the same way so that they contain conserved regions of nucleotide sequences for different strains of RSPaV-1.
  • RT-PCR detected RSPaV-1 sequences from 54% of grapevines negative for RSP as judged by indexing on St. George (see Tables 3 and 4).
  • Several possibilities may account for this discrepancy.
  • RT-PCR is much more sensitive than indicator indexing. Virus(es) of extremely low concentration may not induce visible symptoms on St. George within the standard indexing period, while they can be detected by RT-PCR.
  • judging indexing results can, in some cases, be very subjective. For example, it is very difficult to reach a conclusion on whether a grapevine is infected with RSP when only one or a few small pits are present on the woody cylinder of St. George.
  • Third, uneven distribution of virus(es) within grapevines and the relatively limited number of replicates of St. George indicators may result in the failure to detect RSP-infection.
  • RSP seems to be widespread in different types of grapevines including V. vinifera , hybrids, V. riparia , and rootstocks. It occurs in a wide range of geographic regions including North America, Europe, Australia, and possibly many other countries as well. Testing grapevines from other areas of the world using RSPaV-1 specific primers will provide definitive information on the exact distribution of RSP throughout the world. It is also interesting to investigate whether RSP is transmitted by any vectors in nature.
  • RSP is a disease under quarantine in Washington and New York of the USA. Since this work and the work of others (Golino and Butler, “A Preliminary Analysis of Grapevine Indexing Records at Davis, California,” in Proceedings of the 10 th Meeting of the ICVG , pp.
  • RSP is a disease which induces, after graft-inoculation with a chip bud from an infected grapevine, a row of small pits on the woody cylinder of St. George below the point of inoculation. This definition may not be comprehensive. Indexing record indicated that two types of stem pitting (specific vs. nonspecific) were often observed on the woody cylinder of St. George upon graft inoculation with chip buds.
  • RT-PCR detected RSPaV-1 sequences from a wide range of grapevines collected from a number of major grapevine growing countries.
  • the data clearly suggest that RSPaV-1 is closely associated with Rupestris stem pitting of grapevines and that it is likely the causal virus of RSP.
  • Use of “universal” primers which can detect multiple agents which are highly similar to RSPaV-1 in nucleotide sequences would improve the detection rate by RT-PCR
  • antibodies produced against bacteria-expressed coat proteins of RSPaV-1 will help in finding the viral particles from RSP infected grapevines and in rapid detection of RSP.
  • Southern blot hybridization was conducted using 32 P labeled probe specific to RSPaV-1. As shown in FIG. 7 , the Southern blot hybridization confirmed the results of the RT-PCR in each of the tested samples. Specifically, cDNA fragments amplified by RT-PCR from 16 selected RT-PCR positive samples hybridized with the probe.
  • the coat protein gene (SEQ. ID. No. 10) of RSPaV-1 was cloned into the EcoRI and HindIII sites of the polylinker region of a protein expression vector pMAL-c2 which, upon induction by inducer IPTG, produces a fusion protein containing maltose binding protein (MBP) and the coat protein of RSPaV-1.
  • the fusion protein of expected size (ca. 71 KDa) was produced in E. coli bacteria after induction with IPTG. This fusion protein was purified through affinity chromatography using an amylose column. Purified fusion protein was used as an antigen to immunize a rabbit (by subcutaneous injection along the back) with the following scheme:
  • the antibodies produced against the expressed RSPaV-1 coat protein are useful in the identification of the particles associated with RSP disease of grapevines, in the purification of the particles of RSPaV-1, and in the development of a serological diagnosis for RSP in grapevine.
  • the use of the antibodies is suitable for detecting different strains of RSPaV-1. Because the coat proteins for strains RSP47-4 and RSP158 have high amino acid identities with the coat protein of RSPaV-1, it is very likely that the antibodies raised against RSPaV-1 coat protein will also detect other strains.
  • Antibodies can be used in an ELISA to assay rapidly a large number of samples, thus making commercial development and utilization of diagnostic kits possible.
  • the DNA molecule coding for the RSPaV-1 coat protein (e.g., SEQ. ID. No. 10) was cloned into a pEPT8 plant expression vector that contains the double 35S enhancer at restriction sites SalI and BamHI.
  • the resulting recombinant plasmid designated pEPT8/RSPaV-1 coat protein, was then cloned into the plant transformation vector pGA482G, which has resistance genes to gentamycin and tetracycline as selection markers.
  • the resultant pGA482G containing pEPT8/RSPaV-1 CP was used to transform grapevines using the Agrobacterium method.
  • the rootstock Vitis rupestris Scheele St. George was used in genetic transformation. Anthers were excised aseptically from flower buds. The pollen was crushed on a microscope slide with acetocarmine to observe the cytological stage (Bouquet et al., “Influence du Gentype sur la Production de cals: Dembryoides et Plantes Entieres par Culture Danthers in vitro dans le Genre Vitis,” C. R. Acad. Sci. Paris III 295:560-74 (1982), which is hereby incorporated by reference). This was done to determine which stage was most favorable for callus induction.
  • Anthers were plated under aseptic condition at a density of 40 to 50 per 9 cm diameter Petri dish containing MSE. Plates were cultured at 28° C. in the dark. After 60 days, embryos were induced and transferred to hormone-free medium (HMG) for differentiation. Torpedo stage embryos were transferred to MGC medium yo promote embryo germination. Cultures were maintained in the dark at 26-28° C. and transferred to fresh medium at 3-4 week intervals. Elongated embryos were transferred to rooting medium (5-8 embryos per jar). The embryos were grown in a tissue culture room at 25° C. with a daily 16 h photoperiod (76 ⁇ mol. s) to induce shoot and root formation. After plants developed roots, they were transplanted to soil in the greenhouse.
  • HMG hormone-free medium
  • embryogenic calli were transferred to initiation MSE medium containing 25 mg/l kanamycin plus the same antibiotics listed above. All plant materials were incubated in continuous darkness at 28° C. After growth on selection medium for 3 months, embryos were transferred to HMG or MGC without kanamycin to promote elongation of embryos. They were then transferred to rooting medium without antibiotics. Non-transformed calli were grown on the same media with and without kanamycin to verify the efficiency of the kanamycin selection process.
  • the X-gluc (5-bromo-4-chloro-3-indoyl- ⁇ -glucuronidase) histochemical assay was used to detect GUS ( ⁇ -glucuronidase) activity in embryos and plants that were transformed with constructs containing the GUS gene that survived kanamycin selection. All propagated plants were screened using an enzyme linked immunoabsorbent assay (ELISA) system (5 Prime-3 Prime, Boulder, Co.) to detect the NPTII (neomycin phosphotransferase II) protein in leaf extracts. ELISA tests with respective coat protein (CP)-specific antibodies were used to assay for CP. ELISA results were read on an SLT Spectra ELISA reader (Tecan U.S. Inc., Research Triangle Park, N.C.) 15-60 minutes after the substrate was added.
  • ELISA enzyme linked immunoabsorbent assay
  • Genomic DNA was isolated from transformed and non-transformed grape plants according to the method of Lodhi et al., “A Simple and Efficient Method for DNA Extraction from Grapevine Cultivars and Vitis Species,” Plant Mol. Biol. Rpt. 12:6-13 (1994), which is hereby incorporated by reference.
  • Primer sets included those of specific primers to the transgene.
  • DNA was initially denatured at 94° C. for 3 minutes, then amplified by 35 cycles of 1 minute at 94° C. (denaturing), 1 minute at 52° C. (annealing), and 2 minutes at 72° C. (polymerizing). Reaction samples were directly loaded and electrophoresed in 1.5% agarose gels.
  • Southern analysis of transformants was accomplished by extracting genomic DNA from young leaves of transformed and non-transformed plants (3309C) as described above. DNA (10 ⁇ g) was digested with the restriction enzyme Bgl II, electrophoresed on a 0.8% agarose gel in TAE buffer and transferred to a Genescreen Plus membrane by capillary in 10 ⁇ SSC. A probe was prepared by random primer labeling of a PCR amplified gene coding sequence with radioisotope 32 P-dATP (Dupont, NEN). Pre-hybridization and hybridization steps were carried out at 65° C. following the manufacturer's instruction. The autoradiograph was developed after overnight exposure.

Abstract

The present invention relates to an isolated protein or polypeptide corresponding to a protein or polypeptide of a Rupestris stem pitting associated virus. The encoding DNA molecule, either alone in isolated form, in an expression system, a host cell, or a transgenic grape plant, is also disclosed. Other aspects of the present invention relate to a method of imparting Rupestris stem pitting associated virus resistance to grape plants by transforming them with the DNA molecule of the present invention, and a method of detecting the presence of a Rupestris stem pitting associated virus, such as RSPaV-1, in a sample.

Description

  • This application claims the benefit of U.S. Provisional Patent Applications Ser. No. 60/047,147, filed May 20, 1997, and 60/069,902, filed Dec. 17, 1997.
  • This work was supported by the U.S. Department of Agriculture Clonal Repository—Geneva, Grant Nos. 58-2349-9-01 and 58-2349-9 and U.S. Department of Agriculture Cooperative Agreement Grant Nos. 58-1908-4-023, 58-3615-5-036, and 58-3615-7-060. The U.S. Government may have certain rights in the invention.
    • FIELD OF THE INVENTION
  • The present invention relates to Rupestris stem pitting associated virus (“RSPaV”) proteins, DNA molecules encoding these proteins, and diagnostic and other uses thereof.
    • BACKGROUND OF THE INVENTION
  • The world's most widely grown fruit crop, the grape (Vitis sp.), is cultivated on all continents except Antarctica. However, major grape production centers are in European countries (including Italy, Spain, and France), which constitute about 70% of the world grape production (Mullins et al., Biology of the Grapevine, Cambridge, U.K.:University Press (1992)). The United States, with 300,000 hectares of grapevines, is the eighth largest grape grower in the world. Although grapes have many uses, a major portion of grape production (˜80%) is used for wine production. Unlike cereal crops, most of the world's vineyards are planted with traditional grapevine cultivars, which have been perpetuated for centuries by vegetative propagation. Several important grapevine virus and virus-like diseases, such as grapevine leafroll, corky bark, and Rupestris stem pitting (“RSP”), are transmitted and spread through the use of infected vegetatively propagated materials. Thus, propagation of certified, virus-free materials is one of the most important disease control measures. Traditional breeding for disease resistance is difficult due to the highly heterozygous nature and outcrossing behavior of grapevines, and due to polygenic patterns of inheritance. Moreover, introduction of a new cultivar may be prohibited by custom or law. Recent biotechnology developments have made possible the introduction of special traits, such as disease resistance, into an established cultivar without altering its horticultural characteristics.
  • Many plant pathogens, such as fungi, bacteria, phytoplasmas, viruses, and nematodes can infect grapes, and the resultant diseases can cause substantial losses in production (Pearson et al., Compendium of Grape Diseases, American Phytopathological Society Press (1988)). Among these, viral diseases constitute a major hindrance to profitable growing of grapevines. About 34 viruses have been isolated and characterized from grapevines. The major virus diseases are grouped into: (1) the grapevine degeneration caused by the fanleaf nepovirus, other European nepoviruses, and American nepoviruses, (2) the leafroll complex, and (3) the rugose wood complex (Martelli, ed., Graft Transmissible Diseases of Grapevines, Handbook for Detection and Diagnosis, FAO, UN, Rome, Italy (1993)).
  • Rugose wood (RW) complex is a term to describe a group of graft-transmissible diseases which are important and widespread on grapevines grown world-wide. Symptoms of RW are characterized by pitting, grooving, or distortion to the woody cylinder of the grapevine scion, rootstock, or both. Based on symptoms developed on different indicator plants after graft inoculation, RW complex can be divided into four components: Kober 5BB stem grooving (KSG), LN 33 stem grooving (LNSG), grapevine corky bark (GCB), and Rupestris stem pitting (RSP) (Martelli, “Rugose Wood Complex,” in Graft-Transmissible Diseases of Grapevines. Handbook for Detection and Diagnosis, pp.45-54, Martelli, ed., Food and Agriculture Organization of the United Nations, Rome, Italy (1993)). Because RW can cause severe decline and death to grapevines (Savino et al., “Rugose Wood Complex of Grapevine: Can Grafting to Vitis Indicators Discriminate Between Diseases?”, in Proceedings of the 9th Meetings of the International Council for the Study of Viruses and Virus Diseases of the Grapevine, Anavim, Israel (1989); Credi and Babini, “Effect of Virus and Virus-like Infections on the Growth of Grapevine Rootstocks,” Adv. Hort. Sci., 10:95-98 (1996)), it has been included in healthy grapevine detection schemes used in major grapevine growing countries including Italy, France, and the United States.
  • RSP was discovered in California in the late 1970s (Prudencio, “M. Sc. Thesis: Comparative Effects of Corky Bark and Rupestris Stem Pitting Diseases on Selected Germplasm Lines of Grapes,” University of California, Davis, California, 36 pages (1985); Goheen, “Rupestris Stem Pitting,” in Compendium of Grape Diseases, p. 53, Pearson and Goheen, eds., American Phytopathological Society Press, St. Paul, Minn., USA (1988) (“Goheen”)). The disease was defined by Goheen as follows: after graft inoculation with a chip bud from an infected grapevine, the woody cylinder of the indicator plant Vitis rupestris Scheele St. George (“St. George”) develops a narrow strip of small pits extending from the inoculum bud to the root zone. Grafted St. George plants were checked for wood symptoms 2 to 3 years after inoculation. In contrast to GCB, which elicits pitting and grooving on St. George and LN 33, RSP does not produce symptoms on the latter (Goheen, “Rupestris Stem Pitting,” in Compendium of Grape Diseases, p. 53, Pearson and Goheen, eds., American Phytopathological Society Press, St. Paul, Minn., USA (1988)).
  • RSP is probably the most common component of the RW complex on grapevines. Surveys in California revealed a high disease incidence in many grapevine cultivars imported from Western Europe and Australia (Goheen, “Rupestris Stem Pitting,” in Compendium of Grape Diseases, p. 53, Pearson and Goheen, eds., American Phytopathological Society Press, St. Paul, Minn., USA (1988)). An examination of indexing records in California compiled over 23 years revealed RSP infection in 30.5% of 6482 grapevine selections introduced from around the world (Golino and Butler, “A Preliminary Analysis of Grapevine Indexing Records at Davis, California,” in Proceedings of the 10th Meeting of the ICVG, pp. 369-72, Rumbos et al., eds., Volos, Greece (1990)). Indexing in New York State showed that 66% of 257 grapevines tested on St. George developed typical small pits below the inoculum bud or around the woody cylinder (Azzam and Gonsalves, Abstract: “Survey of Grapevine Stem-Pitting in New York and Isolation of dsRNA from a Grapevine Selection Infected with Stem Pitting,” Phytopathology 78:1568 (1988)). Furthermore, several reports have indicated that RSP is the most frequently detected component of the RW complex in Italy (Borgo and Bonotto, “Rugose Wood Complex of Grapevine in Northeastern Italy: Occurrence of Rupestris Stem Pitting and Kober Stem Grooving,” in Extended Abstracts of the 11 th Meeting of the International Council for the Study of Viruses and Virus Diseases of the Grapevine (ICVG), pp. 61-62, Gugerli, ed., Montreux, Switzerland (1993); Credi, “Differential Indexing Trials on Grapevine Rugose Wood Syndrome,” Extended Abstracts of the 11th Meeting of the International Council for the Study of Viruses and Virus Diseases of the Grapevine (ICVG), p. 63, Gugerh, P., ed., Montreux, Switzerland (1993)).
  • The effect of RSP on growth, yield, and grapevine quality is not well understood and, thus, subject to debate. The reason for this ambiguity is the absence of a rapid and sensitive diagnostic tool. RSP is the most difficult grapevine disease to diagnose. Serological or molecular methods are not available for diagnosing RSP. Biological indexing on St. George, as described above, has remained the only approach to diagnose RSP. Biological indexing is labor intensive, time consuming (i.e., often requiring up to about three years to obtain results), and, by its very nature, subjective. Moreover, symptoms on St. George can be variable and not exactly as those defined by Goheen. In particular, Credi, “Characterization of Grapevine Rugose Wood Sources from Italy,” Plant Disease, 82:1288-92 (1997), recently showed that some RSP infected grapevines induced pitting that is restricted to below the inoculum bud, while others induced pitting around the woody cylinder of inoculated St. George. Thus, the present method of identifying the presence of RSP is not entirely adequate.
  • The etiology of RSP is unknown. Efforts to isolate virus particles from RSP-infected grapevines and to mechanically transfer the causal virus(es) to herbaceous host plants failed (Azzam and Gonsalves, “Detection of in Grapevines Showing Symptoms of Rupestris Stem Pitting Disease and the Variabilities Encountered,” Plant Disease, 75:96-964 (1991)). However, a major dsRNA species of ca. 8.3 kb, accompanied by a smaller dsRNA of ca. 7.6 kb, was consistently isolated from one Pinot Gris and four Pinot Noir clones that had been indexed positive for RSP (Walter and Cameron, “Double-Stranded RNA Isolated from Grapevines Affected by Rupestris Stem Pitting Disease,” Am. J. of Enology and Viticulture, 42:175-79 (1991)). In addition, a third dsRNA of ca. 5.5 kb was observed in three clones. Likewise, an apparently similar dsRNA species of ca. 8.0 and 6.7 kbp was isolated from dormant canes of RSP-infected grapevines collected from California, Canada, and New York (Azzam and Gonsalves, “Detection of dsRNA in Grapevines Showing Symptoms of Rupestris Stem Pitting Disease and the Variabilities Encountered,” Plant Disease, 75:960-64 (1991)). Six of eight Californian and three of five Canadian samples contained these two dsRNA species. However, results of New York samples were not consistent. Among eight RSP infected grapevine selections tested, only one showed these two dsRNAs. Using explants growing in tissue culture as source materials, dsRNA of ca. 359 bp was isolated from 21 of 31 grapevine cultivars, all of which were previously indexed on St. George and considered to be infected with RSP (Monette et al., “Double-Stranded RNA from Rupestris Stem Pitting-Affected Grapevines,” Vitis 28:137-44 (1989)).
  • In view of the serious risk RSP poses to vineyards and the absence of an effective treatment of it, the need to prevent this affliction continues to exist. Moreover, the absence of a rapid and accurate diagnostic assay prevents proper identification of RSP. The present invention is directed to overcoming these deficiencies in the art.
    • SUMMARY OF THE INVENTION
  • The present invention relates to an isolated protein or polypeptide corresponding to a protein or polypeptide of a RSP virus. The encoding RNA molecule or DNA molecule, in either isolated form or incorporated in an expression system, a host cell, or a transgenic Vitis scion or rootstock cultivar, are also disclosed.
  • Another aspect of the present invention relates to a method of imparting RSP virus resistance to Vitis scion or rootstock cultivars by transforming them with a DNA molecule encoding the protein or polypeptide corresponding to a protein or polypeptide of a RSP virus.
  • The present invention also relates to an antibody or binding portion thereof or probe which recognizes proteins or polypeptides of the present invention.
  • Still another aspect of the present invention relates to diagnostic tests which involve methods for detecting the presence of a RSP virus in a sample. The methods include the use of an antibody or binding portion of the present invention (i.e., in an immunoassay), or a nucleic acid probe obtained from a DNA molecule of the present invention (i.e., in a nucleic acid hybridization assay or gene amplification detection procedure). The antibody or binding portion thereof, or nucleic acid probe, is introduced into contact with the sample, whereby the presence of Rupestris stem pitting virus in the sample is detected using an assay system.
  • The characterization of an RSP virus is particularly desirable because it will allow for the determination of whether the virus is associated to the specific (restricted) or nonspecific (nonrestricted) pitting symptoms of RSP, or to both. Also, RSP virus resistant transgenic variants of the current commercial grape cultivars and rootstocks allows for more complete control of the virus while retaining the varietal characteristics of specifics cultivars. Furthermore, these variants permit control over RSP virus transmitted by infected scions or rootstocks. Moreover, the diagnostic tests offer significant improvement over conventional diagnostic means currently employed, namely, rapid results and greater accuracy.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 is a photograph of St. George indicators which comparatively display the symptoms of RSP. The St. George indicator (a) has been graft-inoculated with infected bud wood from a grapevine accession, resulting in the indicator displaying pitting below the inoculum bud, as indicated by an arrow. This RSP symptom was defined by Goheen, “Rupestris Stem Pitting,” in Compendium of Grape Diseases, p. 53, Pearson and Goheen, eds., American Phytopathological Society Press, St. Paul, Minn., USA (1988), which is hereby incorporated by reference. The St. George indicator (b) was not graft-inoculated and represents a normal appearance.
  • FIGS. 2A and 2B are photographs which respectively display the results of dsRNA analysis and Northern hybridization for dsRNA. Together the photographs may be used to correlate the dsRNA analysis of FIG. 2A with the Northern hybridization (for dsRNA isolated from grapevines indexed positive for Rupestris stem pitting (RSP)) of FIG. 2B. M. Hind III digested lambda DNA maker: lane 1, Aminia; lane 2, Bertille Seyve 5563; lane 3, Canandaigua; lane 4, Colobel 257; lane 5, Couderc 28-112; lane 6, Freedom; lane 7, Grande Glabre; lane 8, M 344-1; lane 9, Joffre; lane 10, Ravat 34; lane 11, Seyval; lane 12, Seyve Vinard 14-287; lane 13, Verdelet; lane 14, Pinot Noir (positive control); lane 15, Verduzzo 233A (negative control for RSP as judged by indexing on St. George); lane 16, insert of clone RSP149. Arrows indicate the position of the 8.7 kb dsRNA. With respect to lane 15 of FIG. 2A, the two dsRNA bands are larger or smaller than the 8.7 kb dsRNA associated with RSP and they did not hybridize with the RSP specific probe in Northern analysis. Thus, they are not specific to RSP.
  • FIG. 3A is an illustration which depicts the strategy for obtaining the complete nucleotide sequence of RSPaV-1. The overlapping regions of the nucleotide sequences of the sequenced clones and RT-PCR-amplified cDNA fragments are as follows: 52-375 for RSPA/RSP28; 677-1474 for RSP28/RSP3; 3673-3766 for RSP3/RSPB; 40094320 for RSPB/RSP94; 5377-5750 for RSP94/RSPC; 5794-6537 for RSPC/RSP95; 6579-6771 for RSPC/RSP140; and 8193-8632 for RSP140/TA5. FIG. 3B is an illustration which comparatively depicts the genome structures of RSPaV-1, ASPV, PVM, and PVX. Boxes with the same patterns represent the comparable ORFS.
  • FIG. 4A is a comparative sequence listing of amino acid sequences of region 1 (aa 1-372) of RSPaV-1 ORF1 with the corresponding sequences of carlavirus PVM and ASPV. The methyltransferase motif is underlined. Capital letters indicate consensus residues. FIG. 4B is a comparative sequence listing of amino acid sequences of region II (aa 1354 to end) of RSPaV-1 ORF1 with the corresponding regions of ASPV and PVM carlavirus. In FIG. 4B, the NTP binding motif is underlined at (A) and the GDD containing sequence is underlined at (B). In FIGS. 4A and 4B, capital letters indicate consensus residues, the symbol * indicates identical amino acid residues between RSPaV-1 and ASPV, and the symbol # indicates identical amino acid residues between RSPaV-1 and PMV.
  • FIGS. 5A-D are comparative sequence listings of amino acid sequences for ORF2, ORF3, ORF74, and a C-terminal part of ORF5 (CP) of RSPaV-1, respectively, with ASPV and PVM carlavirus. In FIG. 5A, the NTP binding motif, located near the C terminus of ORF2, is underlined. In FIG. 5D, the conserved motif (RR/QX—XFDF), located in the central region of the coat proteins and proposed to be involved in the formation of a salt bridge structure, is underlined. In each of the figures, capital letters indicate consensus residues. The symbol * indicates identical amino acid residues between RSPaV-1 and ASPV, and the symbol # indicates identical amino acid residues between RSPaV-1 and PMV. In FIG. 5D, numbers which appear in parentheses and precede the sequences indicate the start points of the C-terminal portions of CPs being compared.
  • FIG. 6A is a comparative sequence listing of DNA nucleotide sequences for the 3′ untranslated region (UTR) of RSPaV-1 and ASPV. FIG. 6B is a comparative sequence listing of DNA nucleotide sequences for the 3′ untranslated region (UTR) of RSPaV-1 and PVM. Clustal method of MegAlign (DNASTAR) was used to generate sequence alignments. The 21 identical consecutive nucleotides between RSPaV-1 and PVM are indicated as shadowed letters.
  • FIGS. 7A-B are photographs comparing the results of RT-PCR of grapevines using RSP 149 primers (FIG. 7A) and Southern blot hybridization of RT-PCR amplified cDNA fragments to RSPaV-1 specific probe (FIG. 7B). MMLV-RT (Promega) was used in reverse transcription. Taq DNA polymerase (Promega) was used in PCR. For the RT-PCR and Southern blot hybridization: lane 1, Ehrenfelser PM1 (1169-1A1); lane 2, Cabernet franc 147A; lane 3, Chardonnay 80A; lane 4, Refosco 181A; lane 5, Touriga francesa 313; lane 6, 3309C (330-4A1); lane 7, 420A (1483-4A1); lane 8, Chardonnay 83A; lane 9, Malsavia 153A; lane 10, Aragnonex 350; lane 11, Aminia; lane 12, Chardonnay 127; lane 13, Kober 5BB 100; lane 14, Verduzzo 233A; lane 15, V. riparia; lane 16, V. monticola; lane 17, H2O.
  • FIG. 8 is a schematic representation of the identical genome organization among RSPaV-1 (the type strain), RSP47-4, and RSP158. The number of amino acid residues of the comparable ORFs (boxes shaded with the same pattern) among these three strains are the same (note: ORFI and ORF5 of RSP47-4 and RSP158 are incomplete). The comparable ORFs also have high nucleotide and amino acid sequence identities, which are indicated on the bottom. Only the C-terminal portion of the ORF1 of RSPaV-1 is shown in this diagram.
  • FIG. 9 is a comparative alignment of nucleotide sequences of seven other clones with the comparable region of RSPaV-1. Shaded areas indicate identical nucleotide sequences, whereas white boxes represent different nucleotide sequences.
  • FIG. 10 is a schematic representation of a plant transformation vector containing the RSPaV-1 coat protein gene. This vector is designated pGA482G/RSPaV-1CP, which has the double CaMV 35S enhancers, the 35S promoter, the leader sequence of AIMV, and the 35S terminator sequence. RB, right border; LB, left border; Tet, tetracycline resistance gene; and Gent, gentamycin resistance gene.
    • DETAILED DESCRIPTION OF THE INVENTION
  • The present invention relates to isolated DNA molecules encoding for the proteins or polypeptides of a Rupestris stem pitting associated virus. Since the nucleotide sequence was derived from cDNA clones of the dsRNA that was associated with RSP, the viral agent has been designated as Rupestris stem pitting associated virus (“RSPaV”). RSP is likely caused by one or a number of viral strains. The genome of each RSPaV has a plurality of open reading frames, each containing DNA molecules in accordance with the present invention. The complete genome of one strain has been sequenced and the strain is designated RSPaV-1. Substantial portions of the genomes of two other RSPaV strains have also been sequenced. These strains are designated by their clone names, RSP47-4 and RSP158.
  • The DNA molecule which constitutes the complete RSPaV-1 genome comprises the nucleotide sequence corresponding to SEQ. ID. No. 1 as follows:
    CGATAAACAT AACAACAGAA TGTGCATTGC AGTAATATTC CTTGAATATA ATTGCAACGC   60
    AATGGCCCTC TCTTATAGGC CTGCTGTTGA AGAGGTGCTC GCAAAATTCA CCTCTGATGA  120
    ACAATCCAGG GTTTCTGCTA CAGCTCTCAA GGCATTAGTA GACTTAGAGG AAAGTCAGCA  180
    CAATTTGTTC TCTTTCGCAT TGCCTGATAG AAGCAAAGAA AGGCTGATAT CTTCTGGCAT  240
    TTACTTAAGT CCTTACAGTT TCAGACCCCA CTCACATCCA GTTTGTAAAA CTTTAGAAAA  300
    TCACATTTTG TACAATGTTT TACCTAGTTA TGTTAATAAT TCATTTTACT TTGTAGGAAT  360
    CAAGGATTTT AAGCTGCAGT TCTTGAAAAG GAGGAATAAG GATCTCAGCT TGGTAGCACT  420
    CATAAATAGG TTTGTGACAA GTCGTGATGT TAGTAGGTAT GGGTCTGAGT TCGTTATAAG  480
    TTCTAGTGAC AAATCAAGTC AGGTTGTCAG TAGAAAGGGC ATTGGTGATT CTAACACACT  540
    CCGGAGATTG GTCCCACGTG TAATTTCCAC AGGTGCCAGG AATCTTTTTC TGCATGATGA  600
    GATTCACTAC TGGTCAATTA GTGATCTGAT CAATTTTTTG GACGTTGCCA AGCCAAGCAT  660
    GCTCTTGGCA ACTGCAGTAA TCCCTCCAGA AGTGCTGGTT GGCTCTCCAG AGAGTCTTAA  720
    CCCTTGGGCC TACCAGTATA AAATCAATGG CAACCAACTG CTCTTCGCAC CAGATGGCAA  780
    CTGGAATGAG ATGTACTCAC AACCTTTGTC ATGCAGATAC CTGCTCAAGG CCAGATCTGT  840
    AGTTCTGCCC GATGGCTCAC GCTACTCGGT TGACATCATT CACTCAAAAT TTAGTCACCA  900
    CTTGCTTAGT TTCACCCCTA TGGGTAATCT TTTGACTTCA AACATGCGAT GTTTTTCTGG  960
    CTTCGATGCA ATAGGCATAA AAGATCTTGA ACCTCTAAGC CGCGGCATGC ACAGTTGCTT 1020
    CCCAGTACAT CATGATGTTG TAACTAAGAT ATATCTTTAT TTGAGAACTC TCAAGAAGCC 1080
    AGATAAGGAG TCTGCCGAGG CAAAGCTTCG ACAACTCATA GAAAAACCCA CAGGGAGGGA 1140
    GATAAAGTTT ATCGAGGATT TTTCCTCACT AGTAATAAAT TGTGGGAGGA GTGGCTCTTT 1200
    GCTTATGCCC AACATTTCTA AGTTGGTCAT ATCATTCTTT TGCCGGATGA TGCCAAATGC 1260
    ACTCGCCAGG CTCTCTTCTA GCTTTCGAGA GTGTTCGCTA GATTCATTTG TGTACTCACT 1320
    TGAGCCCTTT AATTTTTCCG TTAATTTAGT GGATATAACT CCTGATTTCT TTGAGCATTT 1380
    ATTTCTCTTC TCCTGCCTAA ATGAGTTGAT CGAGGAGGAC GTTGAAGAGG TCATGGACAA 1440
    TTCTTGGTTT GGACTTGGGG ACTTACAATT CAATCGCCAG AGGGCCCCGT TCTTTCTTGG 1500
    GTCTTCATAT TGGCTCAACT CCAAATTTTC AGTTGAGCAC AAGTTTTCAG GCACCATCAA 1560
    TTCTCAAATC ATGCAAGTTA TTTTATCTTT GATCCCATTT TCTGATGATC CCACTTTTAG 1620
    GCCATCTTCT ACAGAGGTTA ACCTTGCACT ATCAGAGGTT AAGGCTGCGC TAGAAGCTAC 1680
    TGGGCAGTCA AAATTGTTCA GGTTTTTGGT GGACGACTGT GCTATGCGTG AGGTTAGAAG 1740
    TTCCTATAAG GTGGGCCTTT TTAAGCACAT AAAAGCCCTC ACTCATTGCT TTAATTCTTG 1800
    TGGCCTCCAA TGGTTCCTCC TTAGGCAAAG GTCCAACCTC AAATTTCTGA AGGACAGGGC 1860
    ATCGTCCTTT GCTGATCTTG ATTGTGAGGT TATCAAAGTT TATCAGCTTG TAACATCACA 1920
    GGCAATACTT CCTGAGGCTC TGCTTAGCTT GACCAAAGTC TTTGTCAGGG ATTCTGACTC 1980
    AAAGGGTGTT TCCATTCCCA GATTGGTCTC GAGAAATGAG CTAGAGGAAC TAGCTCACCC 2040
    AGCTAATTCA GCCCTTGAGG AGCCTCAATC AGTTGATTGT AATGCAGGCA GGGTTCAAGC 2100
    AAGCGTTTCA AGTTCCGAGC AGCTTGCCGA CACCCACTCT CTTGGTAGCG TTAAGTCATC 2160
    AATTGAGACA GCTAACAAGG CTTTTAACTT GGAGGAGCTA AGGATCATGA TTAGAGTCTT 2220
    GCCGGAGGAT TTTAACTGGG TGGCGAAGAA CATTGGTTTT AAAGACAGGC TGAGAGGCAG 2280
    GGGTGCATCA TTCTTCTCAA AACCAGGAAT TTCATGTCAT AGTTACAATG GTGGGAGCCA 2340
    CACAAGCTTA GGGTGGCCAA AGTTCATGGA TCAGATTCTA AGCTCCACTG GTGGACGTAA 2400
    TTACTACAAT TCATGCCTGG CTCAGATCTA TGAGGAAAAT TCAAAATTGG CTCTTCATAA 2460
    GGATGATGAG AGTTGCTATG AAATTGGGCA CAAAGTTTTG ACTGTTAATT TAATCGGCTC 2520
    AGCAACTTTC ACTATTAGTA AGTCGCGAAA TTTGGTTGGG GGTAATCATT GCAGCCTGAC 2580
    AATTGGGCCA AATGAGTTTT TCGAAATGCC TAGGGGCATG CAATGCAATT ACTTCCATGG 2640
    GGTTTCCAAT TGTACGCCAG GGCGGGTATC GCTGACCTTT AGGCGCCAAA AGTTGGAAGA 2700
    TGATGATTTG ATCTTCATAA ATCCACAGGT GCCCATTGAG CTCAATCATG AAAAGCTTGA 2760
    CCGAAGTATG TGGCAGATGG GCCTTCATGG AATTAAGAAA TCTATTTCTA TGAATGGCAC 2820
    GAGTTTTACC TCAGACCTAT GCTCTTGTTT CTCTTGCCAC AACTTTCATA AATTCAAGGA 2880
    TCTCATCAAT AACTTGAGAT TGGCCCTAGG AGCACAAGGG CTAGGTCAGT GTGACAGGGT 2940
    TGTGTTTGCA ACAACAGGTC CTGGTCTATC TAAGGTTTTA GAAATGCCTC GGAGCAAAAA 3000
    GCAATCAATT TTGGTTCTTG AAGGTGCCCT ATCCATAGAA ACAGATTATG GTCCAAAAGT 3060
    CCTGGGGTCT TTTGAAGTTT TCAAAGGGGA CTTTCACATT AAGAAGATGG AGGAAGGTTC 3120
    AATTTTTGTA ATAACGTACA AGGCCCCAAT TAGATCCACT GGCAGGTTGA GGGTTCACAG 3180
    TTCAGAATGC TCATTTTCCG GATCCAAAGA GGTATTGCTA GGCTGCCAGA TTGAGGCATG 3240
    TGCTGATTAT GATATTGATG ATTTTAACAC TTTCTCTGTG CCTGGTGATG GCAATTGCTT 3300
    TTGGCATTCT GTTGGTTTTT TACTTAGCAC TGATGGACTT GCCCTAAAGG CCGGTATTCG 3360
    ATCTTTCGTG GAGAGTGAGC GCTTGGTAAG TCCAGATCTT TCAGCCCCAG CAATTTCTAA 3420
    ACAATTGGAA GAGAATGCTT ATGCCGAGAA TGAGATGATC GCATTATTCT GCATTCGGCA 3480
    CCACGTAAGG CCTATAGTGA TCACACCAGA ATATGAAGTT AGTTGGAAAT TCGGGGAAGG 3540
    TGAGTGGCCC CTATGTGGAA TTCTTTGCCT TAAATCAAAT CACTTCCAAC CATGCGCCCC 3600
    ACTGAATGGT TGCATGATCA CAGCCATTGC TTCAGCACTT GGAAGGCGTG AAGTTGATGT 3660
    GTTAAATTAT CTGTGTAGAC CCAGCACTAA TCATATTTTT GAGGAGCTTT GTCAGGGAGG 3720
    GGGCCTTAAC ATGATGTATT TAGCTGAAGC TTTTGAGGCC TTTGACATTT GCGCTAAATG 3780
    TGATATAAAT GGAGAGATTG AAGTGATTAA TCCGTGTGGT AAAATTTCTG CATTGTTTGA 3840
    CATAACTAAT GAGCACATAA GGCATGTTGA GAAAATAGGT AATGGCCCTC AGAGCATAAA 3900
    AGTGGATGAA TTGCGGAAGG TCAAGCGATC CGCCCTCGAT TTCCTTTCAA TGAATGGGTC 3960
    TAAAATAACC TACTTCCCAA GCTTTGAGCG GGCTGAAAAG TTGCAAGGAT GTTTGCTAGG 4020
    GGGCCTAACT GGCGTTATAA GTGATGAGAA GTTCAGTGAT GCAAAACCTT GGCTTTCTGG 4080
    TATATCTACT ACTGATATTA AGCCAAGGGA ATTGACTGTC GTGCTTGGTA CATTTGGGGC 4140
    TGGGAAGAGT TTCTTGTACA AGAGTTTCAT GAAAAGGTCT GAGGGTAAAT TCGTAACCTT 4200
    TGTTTCTCCC AGACGTGCTT TAGCAAATTC AATCAAAAAT GATCTTGAAA TGGATGATAG 4260
    CTGCAAAGTT GCTAAAGCAG GTAGGTCAAA GAAGGAAGGG TGGGATGTAG TAACTTTTGA 4320
    GGTTTTCCTT AGAAAAGTTG CAGGATTGAA GGCTGGCCAC TGTGTGATTT TTGATGAGGT 4380
    CCAGTTGTTT CCTCCTGGAT ACATCGATCT ATGCTTGCTT ATTATACGTA GTGATGCTTT 4440
    CATTTCACTT GCTGGTGATC CATGTCAAAG CACATATGAC TCGCAAAAGG ATCGGGCAAT 4500
    TTTGGGCGCT GAGCAGAGTG ACATACTTAG ACTGCTTGAG GGCAAAACGT ATAGGTATAA 4560
    CATAGAAAGC AGGAGGTTTG TGAACCCAAT GTTCGAATCA AGACTGCCAT GTCACTTCAA 4620
    AAAGGGCTCG ATGACTGCCG CTTTCGCTGA TTATGCAATC TTCCATAATA TGCATGACTT 4680
    TCTCCTGGCG AGGTCAAAAG GTCCCTTGGA TGCCGTTTTG GTTTCCAGTT TTGAGGAGAA 4740
    AAAGATAGTC CAGTCCTACT TTGGAATGAA ACAGCTCACA CTCACATTTG GTGAATCAAC 4800
    TGGGTTGAAT TTCAAAAATG GGGGAATTCT CATATCACAT GATTCCTTTC ACACAGATGA 4860
    TCGGCGGTGG CTTACTGCTT TATCTCGCTT CAGCCACAAT TTGGATTTGG TGAACATCAC 4920
    AGGTCTGAGG GTGGAAAGTT TTCTCTCGCA CTTTGCTGGC AAACCCCTCT ACCATTTTTT 4980
    AACAGCCAAA AGTGGGGAGA ATGTCATACG AGATTTGCTC CCAGGTGAGC CTAACTTCTT 5040
    CAGTGGCTTT AACGTTAGCA TTGGAAAGAA TGAAGGTGTT AGGGAGGAGA AGTTATGTGG 5100
    TGACCCATGG TTAAAAGTTA TGCTTTTCCT GGGTCAAGAT GAGGATTGTG AAGTTGAAGA 5160
    GATGGAGTCA GAATGCTCAA ATGAAGAATG GTTTAAAACC CACATCCCCT TGAGTAATCT 5220
    GGAGTCAACC AGGGCCAGGT GGGTGGGTAA AATGGCCTTG AAAGAGTATC GGGAGGTGCG 5280
    TTGTGGTTAT GAAATGACTC AACAATTCTT TGATGAGCAT AGGGGTGGAA CTGGTGAGCA 5340
    ACTGAGCAAT GCATGTGAGA GGTTTGAAAG CATTTACCCA AGGCATAAAG GAAATGATTC 5400
    AATAACCTTC CTCATGGCTG TCCGAAAGCG TCTCAAATTT TCGAAGCCCC AGGTTGAAGC 5460
    TGCCAAACTG AGGCGGGCCA AACCATATGG GAAATTCTTA TTAGATTCTT TCCTATCCAA 5520
    AATCCCATTG AAAGCCAGTC ATAATTCCAT CATGTTTCAT GAAGCGGTAC AGGAGTTTGA 5580
    GGCGAAGAAG GCTAGTAAGA GTGCAGCAAC TATAGAGAAT CATGCAGGTA GGTCATGCAG 5640
    GGATTGGTTA TTAGATGTTG CTCTGATTTT TATGAAGTCA CAACACTGTA CTAAATTTGA 5700
    CAACAGGCTT AGAGTAGCTA AAGCTGGGCA AACCCTTGCT TGCTTCCAAC ATGCTGTTCT 5760
    GGTTCGCTTT GCACCCTATA TGAGATACAT TGAGAAAAAG CTAATGCAAG CTCTGAAGCC 5820
    TAACTTCTAC ATCCATTCAG GGAAAGGTCT GACGAGCTGA AGGAGTGGGT CAGAACTAGA 5880
    GGATTCACTG GAATTTGCAC AGAATCAGAC TACGAAGCCT TTGATGCTTC CCAAGACCAC 5940
    TTCATCCTAG CATTCGAATT GCAGATAATG AAATTTTTGG GGTTACCTGA AGATTTAATT 6000
    TTGGACTATG AATTCATAAA AATTCATTTG GGATCAAAGC TCGGATCATT CTCTATAATG 6060
    AGGTTTACTG GGGAGGCCAG CACATTTCTG TTTAACACTA TGGCTAACAT GTTGTTCACC 6120
    TTTCTGAGGT ACGAACTAAC AGGCTCTGAG TCAATAGCAT TTGCAGGTGA TGACATGTGT 6180
    GCTAATCGAA GGTTGCGGCT TAAAACAGAG CATGAGGGTT TTCTGAACAT GATTTGCCTT 6240
    AAGGCCAAGG TTCAGTTTGT TTCCAATCCC ACATTCTGCG GATGGTGTTT ATTTAAGGAA 6300
    GGGATCTTCA AGAAGCCTCA ATTAATCTGG GAGCGGATAT GCATTGCTAG GGAGATGGGC 6360
    AACCTGGAGA ATTGTATTGA CAATTATGCG ATAGAGGTCT CCTATGCATA CCGACTGGGA 6420
    GAGCTAGCCA TTGAAATGAT GACCGAGGAA GAAGTGGAGG CCCATTATAA TTGTGTTAGA 6480
    TTCTTGGTCA GGAACAAGCA TAAGATGAGA TGCTCAATTT CAGGCCTATT TGAAGCTATT 6540
    GATTAGGCCT TAAGTATTTG GCATTATTTG AGTATTATGA ATAATTTAGT TAAAGCATTG 6600
    TCAGCATTTG AGTTTGTAGG TGTTTTCAGT GTGCTTAAAT TTCCAGTAGT CATTCATAGT 6660
    GTGCCTGGTA GTGGTAAAAG TAGTTTAATA AGGGAGCTAA TTTCCGAGGA TGAGAATTTC 6720
    ATAGCTTTCA CAGCAGGTGT TCCAGACAGC CCTAATCTCA CAGGAAGGTA CATTAAGCCT 6780
    TATTCTCCAG GGTGTGCAGT GCCAGGGAAA GTTAATATAC TTGATGAGTA CTTGTCCGTC 6840
    CAAGATTTTT CAGGTTTTGA TGTGCTGTTC TCGGACCCAT ACCAAAACAT CAGCATTCCT 6900
    AAAGAGGCAC ATTTCATCAA GTCAAAAACT TGTAGGTTTG GCGTGAATAC TTGCAAATAT 6960
    CTTTCCTCCT TCGGTTTTAA GGTTAGCAGT GACGGTTTGG ACAAAGTCAT TGTGGGGTCG 7020
    CCTTTTACAC TAGATGTTGA AGGGGTGCTA ATATGCTTTG GTAAGGAGGC AGTGGATCTC 7080
    GCTGTTGCGC ACAACTCTGA ATTCAAATTA CCTTGTGAAG TTAGAGGTTC AACTTTTAAC 7140
    GTCGTAACTC TTTTGAAATC AAGAGATCCA ACCCCAGAGG ATAGGCACTG GTTTTACATT 7200
    GCTGCTACAA GACACAGGGA GAAATTGATA ATCATGCAGT AAGATGCCTT TTCAGCAGCC 7260
    TGCGAATTGG GCAAAAACCA TAACTCCATT GACAGTTGGC TTGGGCATTG GGCTTGTGCT 7320
    GCATTTTCTG AGGAAGTCAA ATCTACCTTA TTCAGGGGAC AACATCCATC AATTCCCTCA 7380
    CGGTGGGCGT TACAGGGACG GTACAAAAAG TATAACTTAC TGTGGTCCAA AGCAATCCTT 7440
    CCCCAGCTCT GGGATATTCG GCCAATCTGA GAATTTTGTG CCCTTAATGC TTGTCATAGG 7500
    TCTAATCGCA TTCATACATG TATTGTCTGT TTGGAATTCT GGTCTTGGTA GGAATTGTAA 7560
    TTGCCATCCA AATCCTTGCT CATGTAGACA GCAGTAGTGG CAACCACCAA GGTTGCTTCA 7620
    TTAGGGCCAC TGGAGAGTCA ATTTTGATTG AAAACTGCGG CCCAAGTGAG GCCCTTGCAT 7680
    CCACTGTGAA GGAGGTGCTG GGAGGTTTGA AGGCTTTAGG GGTTAGCCGT GCTGTTGAAG 7740
    AAATTGATTA TCATTGTTAA ATTGGCTGAA TGGCAAGTCA AATTGGGAAA CTCCCCGGTG 7800
    AATCAAATGA GGCTTTTGAA GCCCGGCTAA AATCGCTGGA GTTAGCTAGA GCTCAAAAGC 7860
    AGCCGGAAGG TTCTAATGCA CCACCTACTC TCAGTGGCAT TCTTGCCAAA CGCAAGAGGA 7920
    TTATAGAGAA TGCACTTTCA AAGACGGTGG ACATGAGGGA GGTTTTGAAA CACGAAACGG 7980
    TGGTGATTTC CCCAAATGTC ATGGATGAAG GTGCAATAGA CGAGCTGATT CGTGCATTTG 8040
    GTGAATCTGG CATAGCTGAA AGCGTGCAAT TTGATGTGGC CATAGATATA GCACGTCACT 8100
    GCTCTGATGT TGGTAGCTCC CAGAGGTCAA CCCTGATTGG CAAGAGTCCA TTTTGTGACC 8160
    TAAACAGATC AGAAATAGCT GGGATTATAA GGGAGGTGAC CACATTACGT AGATTTTGCA 8220
    TGTACTATGC AAAAATCGTG TGGAACATCC ATCTGGAGAC GGGGATACCA CCAGCTAACT 8280
    GGGCCAAGAA AGGATTTAAT GAGAATGAAA AGTTTGCAGC CTTTGATTTT TTCTTGGGAG 8340
    TCACAGATGA GAGTGCGCTT GAACCAAAGG GTGGAATTAA AAGAGCTCCA ACGAAAGCTG 8400
    AGATGGTTGC TAATATCGCC TCTTTTGAGG TTCAAGTGCT CAGACAAGCT ATGGCTGAAG 8460
    GCAAGCGGAG TTCCAACCTT GGAGAGATTA GTGGTGGAAC GGCTGGTGCA CTCATCAACA 8520
    ACCCCTTTTC AAATGTTACA CATGAATGAG GATGACGAAG TCAGCGACAA TTCCGCAGTC 8580
    CAATAATTCC CCGATTTCAA GGCTGGGTTA AGCCTGTTCG CTGGAATACC GTACTAATAG 8640
    TATTCCCTTT CCATGCTAAA TCCTATTTAA TATATAAGGT GTGGAAAGTA AAAGAAGATT 8700
    TGGTGTGTTT TTATAGTTTT CATTCAAAAA AAAAAAAAAA AAA 8743

    The DNA molecule of SEQ. ID. No. 1 contains at least five open reading frames (e.g., ORF 1-ORF5), each of which encodes a particular protein or polypeptide of RSPaV-1, and a 3′ untranscribed region downstream of ORF5.
  • Another DNA molecule of the present invention (RSPaV-1 ORF 1) includes nucleotides 62-6547 of SEQ. ID. No. 1. The DNA molecule of RSPaV-1 ORF1 encodes for a RSPaV-1 replicase and comprises a nucleotide sequence corresponding to SEQ. ID. No. 2 as follows:
    ATGGCCCTCT CTTATAGGCC TGCTGTTGAA GAGGTGCTCG CAAAATTCAC CTCTGATGAA   60
    CAATCCAGGG TTTCTGCTAC AGCTCTCAAG GCATTAGTAG ACTTAGAGGA AAGTCAGCAC  120
    AATTTGTTCT CTTTCGCATT GCCTGATAGA AGCAAAGAAA GGCTGATATC TTCTGGCATT  180
    TACTTAAGTC CTTACAGTTT CAGACCCCAC TCACATCCAG TTTGTAAAAC TTTAGAAAAT  240
    CACATTTTGT ACAATGTTTT ACCTAGTTAT GTTAATAATT CATTTTACTT TGTAGGAATC  300
    AAGGATTTTA AGCTGCAGTT CTTGAAAAGG AGGAATAAGG ATCTCAGCTT GGTAGCACTC  360
    ATAAATAGGT TTGTGACAAG TCGTGATGTT AGTAGGTATG GGTCTGAGTT CGTTATAAGT  420
    TCTAGTGACA AATCAAGTCA GGTTGTCAGT AGAAAGGGCA TTGGTGATTC TAACACACTC  480
    CGGAGATTGG TCCCACGTGT AATTTCCACA GGTGCCAGGA ATCTTTTTCT GCATGATGAG  540
    ATTCACTACT GGTCAATTAG TGATCTGATC AATTTTTTGG ACGTTGCCAA GCCAAGCATG  600
    CTCTTGGCAA CTGCAGTAAT CCCTCCAGAA GTGCTGGTTG GCTCTCCAGA GAGTCTTAAC  660
    CCTTGGGCCT ACCAGTATAA AATCAATGGC AACCAACTGC TCTTCGCACC AGATGGCAAC  720
    TGGAATGAGA TGTACTCACA ACCTTTGTCA TGCAGATACC TGCTCAAGGC CAGATCTGTA  780
    GTTCTGCCCG ATGGCTCACG CTACTCGGTT GACATCATTC ACTCAAAATT TAGTCACCAC  840
    TTGCTTAGTT TCACCCCTAT GGGTAATCTT TTGACTTCAA ACATGCGATG TTTTTCTGGC  900
    TTCGATGCAA TAGGCATAAA AGATCTTGAA CCTCTAAGCC GCGGCATGCA CAGTTGCTTC  960
    CCAGTACATC ATGATGTTGT AACTAAGATA TATCTTTATT TGAGAACTCT CAAGAAGCCA 1020
    GATAAGGAGT CTGCCGAGGC AAAGCTTCGA CAACTCATAG AAAAACCCAC AGGGAGGGAG 1080
    ATAAAGTTTA TCGAGGATTT TTCCTCACTA GTAATAAATT GTGGGAGGAG TGGCTCTTTG 1140
    CTTATGCCCA ACATTTCTAA GTTGGTCATA TCATTCTTTT GCCGGATGAT GCCAAATGCA 1200
    CTCGCCAGGC TCTCTTCTAG CTTTCGAGAG TGTTCGCTAG ATTCATTTGT GTACTCACTT 1260
    GAGCCCTTTA ATTTTTCCGT TAATTTAGTG GATATAACTC CTGATTTCTT TGAGCATTTA 1320
    TTTCTCTTCT CCTGCCTAAA TGAGTTGATC GAGGAGGACG TTGAAGAGGT CATGGACAAT 1380
    TCTTGGTTTG GACTTGGGGA CTTACAATTC AATCGCCAGA GGGCCCCGTT CTTTCTTGGG 1440
    TCTTCATATT GGCTCAACTC CAAATTTTCA GTTGAGCACA AGTTTTCAGG CACCATCAAT 1500
    TCTCAAATCA TGCAAGTTAT TTTATCTTTG ATCCCATTTT CTGATGATGC CACTTTTAGG 1560
    CCATCTTCTA CAGAGGTTAA CCTTGCACTA TCAGAGGTTA AGGCTGCGCT AGAAGCTACT 1620
    GGGCAGTCAA AATTGTTCAG GTTTTTGGTG GACGACTGTG CTATGCGTGA GGTTAGAAGT 1680
    TCCTATAAGG TGGGCCTTTT TAAGCACATA AAAGCCCTCA CTCATTGCTT TAATTCTTGT 1740
    GGCCTCCAAT GGTTCCTCCT TAGGCAAAGG TCCAACCTCA AATTTCTGAA GGACAGGGCA 1800
    TCGTCCTTTG CTGATCTTGA TTGTGAGGTT ATCAAAGTTT ATCAGCTTGT AACATCACAG 1860
    GCAATACTTC CTGAGGCTCT GCTTAGCTTG ACCAAAGTCT TTGTCAGGGA TTCTGACTCA 1920
    AAGGGTGTTT CCATTCCCAG ATTGGTCTCG AGAAATGAGC TAGAGGAACT AGCTCACCCA 1980
    GCTAATTCAG CCCTTGAGGA GCCTCAATCA GTTGATTGTA ATGCAGGCAG GGTTCAAGCA 2040
    AGCGTTTCAA GTTCCCAGCA GCTTGCCGAC ACCCACTCTC TTGGTAGCGT TAAGTCATCA 2100
    ATTGAGACAG CTAACAAGGC TTTTAACTTG GAGGAGCTAA GGATCATGAT TAGAGTCTTG 2160
    CCGGAGGATT TTAACTGGGT GGCGAAGAAC ATTGGTTTTA AAGAGAGGCT GAGAGGCAGG 2220
    GGTGCATCAT TCTTCTCAAA ACCAGGAATT TCATGTCATA GTTACAATGG TGGGAGCCAC 2280
    ACAAGCTTAG GGTGGCCAAA GTTCATGGAT CAGATTCTAA GCTCCACTGG TGGACGTAAT 2340
    TACTACAATT CATGCCTGGC TCAGATCTAT GAGGAAAATT CAAAATTGGC TCTTCATAAG 2400
    GATGATGAGA GTTGCTATGA AATTGGGCAC AAAGTTTTGA CTGTTAATTT AATCGGCTCA 2460
    GCAACTTTCA CTATTAGTAA GTCGCGAAAT TTGGTTGGGG GTAATCATTG CAGCCTGACA 2520
    ATTGGGCCAA ATGAGTTTTT CGAAATGCCT AGGGGCATGC AATGCAATTA CTTCCATGGG 2580
    GTTTCCAATT GTACGCCAGG GCGGGTATCG CTGACCTTTA GGCGCCAAAA GTTGGAAGAT 2640
    GATGATTTGA TCTTCATAAA TCCACAGGTG CCCATTGAGC TCAATCATGA AAAGCTTGAC 2700
    CGAAGTATGT GGCAGATGGG CCTTCATGGA ATTAAGAAAT CTATTTCTAT GAATGGCACG 2760
    AGTTTTACCT CAGACCTATG CTCTTGTTTC TCTTGCCACA ACTTTCATAA ATTCAAGGAT 2820
    CTCATCAATA ACTTGAGATT GGCCCTAGGA GCACAAGGGC TAGGTCAGTG TGACAGGGTT 2880
    GTGTTTGCAA CAACAGGTCC TGGTCTATCT AAGGTTTTAG AAATGCCTCG GAGCAAAAAG 2940
    CAATCAATTT TGGTTCTTGA AGGTGCCCTA TCCATAGAAA CAGATTATGG TCCAAAAGTC 3000
    CTGGGGTCTT TTGAAGTTTT CAAAGGGGAC TTTCACATTA AGAAGATGGA GGAAGGTTCA 3060
    ATTTTTGTAA TAACGTACAA GGCCCCAATT AGATCCACTG GCAGGTTGAG GGTTCACAGT 3120
    TCAGAATGCT CATTTTCCGG ATCCAAAGAG GTATTGCTAG GCTGCCAGAT TGAGGCATGT 3180
    GCTGATTATG ATATTGATGA TTTTAACACT TTCTCTGTGC CTGGTGATGG CAATTGCTTT 3240
    TGGCATTCTG TTGGTTTTTT ACTTAGCACT GATGGACTTG CCGTAAAGGC CGGTATTCGA 3300
    TCTTTCGTGG AGAGTGAGCG CTTGGTAAGT CCAGATCTTT CAGCCCCAGC AATTTCTAAA 3360
    CAATTGGAAG AGAATGCTTA TGCCGAGAAT GAGATGATCG CATTATTCTG CATTCGGCAC 3420
    CACGTAAGGC CTATAGTGAT CACACCAGAA TATGAAGTTA GTTGGAAATT CGGGGAAGGT 3480
    GAGTGGCCCC TATGTGGAAT TCTTTGCCTT AAATCAAATC ACTTCCAAGC ATGCGCCCCA 3540
    CTGAATGGTT GCATGATCAC AGCCATTGCT TCAGCAGTTG GAAGGCGTGA AGTTGATGTG 3600
    TTAAATTATC TGTGTAGACC GAGCACTAAT CATATTTTTG AGGAGGTTTG TCAGGGAGGG 3660
    GGCCTTAACA TGATGTATTT AGCTGAAGCT TTTGAGGGCT TTGACATTTG CGGTAAATGT 3720
    GATATAAATG GAGAGATTGA AGTGATTAAT CCGTGTGGTA AAATTTCTGC ATTGTTTGAC 3780
    ATAACTAATG AGCACATAAG GCATGTTGAG AAAATAGGTA ATGGCCCTCA GAGCATAAAA 3840
    GTGGATGAAT TGCGGAAGGT CAAGCGATCC GCGCTCGATT TCCTTTCAAT GAATGGGTCT 3900
    AAAATAACCT ACTTCCCAAG CTTTGAGCGG GCTGAAAAGT TGCAAGGATG TTTGCTAGGG 3960
    GGCCTAACTG GCGTTATAAG TGATGAGAAG TTCAGTGATG CAAAACCTTG GCTTTCTGGT 4020
    ATATCTACTA CTGATATTAA GCCAAGGGAA TTGACTGTCG TGCTTGGTAC ATTTGGGGCT 4080
    GGGAAGAGTT TCTTGTACAA GAGTTTCATG AAAAGGTCTG AGGGTAAATT CGTAACCTTT 4140
    GTTTCTCCCA GACGTGCTTT AGCAAATTCA ATCAAAAATG ATCTTGAAAT GGATGATAGC 4200
    TGCAAAGTTG CTAAAGCAGG TAGGTCAAAG AAGGAAGGGT GGGATGTAGT AACTTTTGAG 4260
    GTTTTCCTTA GAAAAGTTGC AGGATTGAAG GCTGGCCACT GTGTGATTTT TGATGAGGTC 4320
    CAGTTGTTTC CTCCTGGATA CATCGATCTA TGCTTGCTTA TTATACGTAG TGATGCTTTC 4380
    ATTTCACTTG CTGGTGATCC ATGTCAAAGC ACATATGACT CGCAAAAGGA TCGGGCAATT 4440
    TTGGGCGCTG AGCAGAGTGA CATACTTAGA CTGCTTGAGG GCAAAACGTA TAGGTATAAC 4500
    ATAGAAAGCA GGAGGTTTGT GAACCCAATG TTCGAATCAA GACTGCCATG TCACTTCAAA 4560
    AAGGGCTCGA TGACTGCCGC TTTCGCTGAT TATGCAATCT TCCATAATAT GCATGACTTT 4620
    CTCCTGGCGA GGTCAAAAGG TCCCTTGGAT GCCGTTTTGG TTTCCAGTTT TGAGGAGAAA 4680
    AAGATAGTCC AGTCGTACTT TGGAATGAAA CAGCTCACAC TCACATTTGG TGAATCAACT 4740
    GGGTTGAATT TCAAAAATGG GGGAATTCTC ATATCACATG ATTCCTTTCA CACAGATGAT 4800
    CGGCGGTGGC TTACTGCTTT ATCTCGCTTC AGCCACAATT TGGATTTGGT GAACATCACA 4860
    GGTCTGAGGG TGGAAAGTTT TCTCTCGCAC TTTGCTGGCA AACCCCTCTA CCATTTTTTA 4920
    ACAGCCAAAA GTGGGGAGAA TGTCATACGA GATTTGCTCC CAGGTGAGCC TAACTTCTTC 4980
    AGTGGCTTTA ACGTTAGCAT TGGAAAGAAT GAAGGTGTTA GGGAGGAGAA GTTATGTGGT 5040
    GACCCATGGT TAAAAGTTAT GCTTTTGCTG GGTCAAGATG AGGATTGTGA AGTTGAAGAG 5100
    ATGGAGTCAG AATGCTCAAA TGAAGAATGG TTTAAAACCC ACATCCGCTT GAGTAATCTG 5160
    GAGTCAACCA GGGCCAGGTG GGTGGGTAAA ATGGCCTTGA AAGAGTATCG GGAGGTGCGT 5220
    TGTGGTTATG AAATGACTCA ACAATTCTTT GATGAGCATA GGGGTGGAAC TGGTGAGCAA 5280
    CTGAGCAATG CATGTGAGAG GTTTGAAAGC ATTTACCCAA GGCATAAAGG AAATGATTCA 5340
    ATAACCTTCC TCATGGGTGT CCGAAAGCGT CTCAAATTTT CGAAGCCCCA GGTTGAAGCT 5400
    GCCAAACTGA GGCGGGCCAA AGCATATGGG AAATTCTTAT TAGATTCTTT CCTATCCAAA 5460
    ATCCCATTGA AAGCCAGTCA TAATTCCATC ATGTTTCATG AAGCGGTACA GGAGTTTGAG 5520
    GCGAAGAAGG CTAGTAAGAG TGCAGCAACT ATAGAGAATC ATGCAGGTAG GTCATGCAGG 5580
    GATTGGTTAT TAGATGTTGG TCTGATTTTT ATGAAGTCAC AACACTGTAC TAAATTTGAC 5640
    AACAGGCTTA GAGTAGCTAA AGCTGGGCAA ACCCTTGCTT GCTTCCAACA TGCTGTTCTG 5700
    GTTCGCTTTG CACCCTATAT GAGATACATT GAGAAAAAGC TAATGCAAGC TCTGAAGCCT 5760
    AACTTCTACA TCCATTCAGG GAAAGGTGTG ACGAGCTGAA CGAGTGGGTC AGAACTAGAG 5820
    GATTCACTGG AATTTGCACA GAATCAGACT ACGAAGCCTT TGATGCTTCC CAAGACCACT 5880
    TCATCCTAGC ATTCGAATTG CAGATAATGA AATTTTTGGG GTTACCTGAA GATTTAATTT 5940
    TGGACTATGA ATTCATAAAA ATTCATTTGG GATCAAAGCT CGGATCATTC TCTATAATGA 6000
    GGTTTACTGG GGAGGCCAGC ACATTTCTGT TTAACACTAT GGCTAACATG TTGTTCACCT 6060
    TTCTGAGGTA CGAACTAACA GGCTCTGAGT CAATAGGATT TGCAGGTGAT GACATGTGTG 6120
    CTAATCGAAG GTTGCGGCTT AAAACAGAGC ATGAGGGTTT TCTGAACATG ATTTGCCTTA 6180
    AGGCCAAGGT TCAGTTTGTT TCCAATCCCA CATTCTGCGG ATGGTGTTTA TTTAAGGAAG 6240
    GGATCTTCAA GAAGCCTCAA TTAATCTGGG AGCGGATATG CATTGCTAGG GAGATGGGCA 6300
    ACCTGGAGAA TTGTATTGAC AATTATGCGA TAGAGGTCTC CTATGCATAC CGACTGGGAG 6360
    AGCTAGCCAT TGAAATGATG ACCGAGGAAG AAGTGGAGGC CCATTATAAT TGTGTTAGAT 6420
    TCTTGGTCAG GAACAAGCAT AAGATGAGAT GCTCAATTTC AGGCCTATTT GAAGCTATTG 6480
    ATTAG 6485
  • The RSPaV-1 replicase has a deduced amino acid sequence corresponding to SEQ. ID. No. 3 as follows:
    Met Ala Leu Ser Tyr Arg Pro Ala Val Glu Glu Val
    1               5                   10
    Leu Ala Lys Phe Thr Ser Asp Glu Gln Ser Arg Val
            15                  20
    Ser Ala Thr Ala Leu Lys Ala Leu Val Asp Leu Glu
    25                  30                  35
    Glu Ser Gln His Asn Leu Phe Ser Phe Ala Leu Pro
                40                  45
    Asp Arg Ser Lys Glu Arg Leu Ile Ser Ser Gly Ile
        50                  55                  60
    Tyr Leu Ser Pro Tyr Ser Phe Arg Pro His Ser His
                    65                  70
    Pro Val Cys Lys Thr Leu Glu Asn His Ile Leu Tyr
            75                  80
    Asn Val Leu Pro Ser Tyr Val Asn Asn Ser Phe Tyr
    85                  90                  95
    Phe Val Gly Ile Lys Asp Phe Lys Leu Gln Phe Leu
                100                 105
    Lys Arg Arg Asn Lys Asp Leu Ser Leu Val Ala Leu
        110                 115                 120
    Ile Asn Arg Phe Val Thr Ser Arg Asp Val Ser Arg
                    125                 130
    Tyr Gly Ser Glu Phe Val Ile Ser Ser Ser Asp Lys
            135                 140
    Ser Ser Gln Val Val Ser Arg Lys Gly Ile Gly Asp
    145                 150                 155
    Ser Asn Thr Leu Arg Arg Leu Val Pro Arg Val Ile
                160                 165
    Ser Thr Gly Ala Arg Asn Leu Phe Leu His Asp Glu
        170                 175                 180
    Ile His Tyr Trp Ser Ile Ser Asp Leu Ile Asn Phe
                    185                 190
    Leu Asp Val Ala Lys Pro Ser Met Leu Leu Ala Thr
            195                 200
    Ala Val Ile Pro Pro Glu Val Leu Val Gly Ser Pro
    205                 210                 215
    Glu Ser Leu Asn Pro Trp Ala Tyr Gln Tyr Lys Ile
                220                 225
    Asn Gly Asn Gln Leu Leu Phe Ala Pro Asp Gly Asn
        230                 235                 240
    Trp Asn Glu Met Tyr Ser Gln Pro Leu Ser Cys Arg
                    245                 250
    Tyr Leu Leu Lys Ala Arg Ser Val Val Leu Pro Asp
            255                 260
    Gly Ser Arg Tyr Ser Val Asp Ile Ile His Ser Lys
    265                 270                 275
    Phe Ser His His Leu Leu Ser Phe Thr Pro Met Gly
                280                 285
    Asn Leu Leu Thr Ser Asn Met Arg Cys Phe Ser Gly
        290                 295                 300
    Phe Asp Ala Ile Gly Ile Lys Asp Leu Glu Pro Leu
                    305                 310
    Ser Arg Gly Met His Ser Cys Phe Pro Val His His
            315                 320
    Asp Val Val Thr Lys Ile Tyr Leu Tyr Leu Arg Thr
    325                 330                 335
    Leu Lys Lys Pro Asp Lys Glu Ser Ala Glu Ala Lys
                340                 345
    Leu Arg Gln Leu Ile Glu Lys Pro Thr Gly Arg Glu
        350                 355                 360
    Ile Lys Phe Ile Glu Asp Phe Ser Ser Leu Val Ile
                    365                 370
    Asn Cys Gly Arg Ser Gly Ser Leu Leu Met Pro Asn
            375                 380
    Ile Ser Lys Leu Val Ile Ser Phe Phe Cys Arg Met
    385                 390                 395
    Met Pro Asn Ala Leu Ala Arg Leu Ser Ser Ser Phe
                400                 405
    Arg Glu Cys Ser Leu Asp Ser Phe Val Tyr Ser Leu
        410                 415                 420
    Glu Pro Phe Asn Phe Ser Val Asn Leu Val Asp Ile
                    425                 430
    Thr Pro Asp Phe Phe Glu His Leu Phe Leu Phe Ser
            435                 440
    Cys Leu Asn Glu Leu Ile Glu Glu Asp Val Glu Glu
    445                 450                 455
    Val Met Asp Asn Ser Trp Phe Gly Leu Gly Asp Leu
                460                 465
    Gln Phe Asn Arg Gln Arg Ala Pro Phe Phe Leu Gly
        470                 475                 480
    Ser Ser Tyr Trp Leu Asn Ser Lys Phe Ser Val Glu
                    485                 490
    His Lys Phe Ser Gly Thr Ile Asn Ser Gln Ile Met
            495                 500
    Gln Val Ile Leu Ser Leu Ile Pro Phe Ser Asp Asp
    505                 510                 515
    Pro Thr Phe Arg Pro Ser Ser Thr Glu Val Asn Leu
                520                 525
    Ala Leu Ser Glu Val Lys Ala Ala Leu Glu Ala Thr
        530                 535                 540
    Gly Gln Ser Lys Leu Phe Arg Phe Leu Val Asp Asp
                    545                 550
    Cys Ala Met Arg Glu Val Arg Ser Ser Tyr Lys Val
            555                 560
    Gly Leu Phe Lys His Ile Lys Ala Leu Thr His Cys
    565                 570                 575
    Phe Asn Ser Cys Gly Leu Gln Trp Phe Leu Leu Arg
                580                 585
    Gln Arg Ser Asn Leu Lys Phe Leu Lys Asp Arg Ala
        590                 595                 600
    Ser Ser Phe Ala Asp Leu Asp Cys Glu Val Ile Lys
                    605                 610
    Val Tyr Gln Leu Val Thr Ser Gln Ala Ile Leu Pro
            615                 620
    Glu Ala Leu Leu Ser Leu Thr Lys Val Phe Val Arg
    625                 630                 635
    Asp Ser Asp Ser Lys Gly Val Ser Ile Pro Arg Leu
                640                 645
    Val Ser Arg Asn Glu Leu Glu Glu Leu Ala His Pro
        650                 655                 660
    Ala Asn Ser Ala Leu Glu Glu Pro Gln Ser Val Asp
                    665                 670
    Cys Asn Ala Gly Arg Val Gln Ala Ser Val Ser Ser
            675                 680
    Ser Gln Gln Leu Ala Asp Thr His Ser Leu Gly Ser
    685                 690                 695
    Val Lys Ser Ser Ile Glu Thr Ala Asn Lys Ala Phe
                700                 705
    Asn Leu Glu Glu Leu Arg Ile Met Ile Arg Val Leu
        710                 715                 720
    Pro Glu Asp Phe Asn Trp Val Ala Lys Asn Ile Gly
                    725                 730
    Phe Lys Asp Arg Leu Arg Gly Arg Gly Ala Ser Phe
            735                 740
    Phe Ser Lys Pro Gly Ile Ser Cys His Ser Tyr Asn
    745                 750                 755
    Gly Gly Ser His Thr Ser Leu Gly Trp Pro Lys Phe
                760                 765
    Met Asp Gln Ile Leu Ser Ser Thr Gly Gly Arg Asn
        770                 775                 780
    Tyr Tyr Asn Ser Cys Leu Ala Gln Ile Tyr Glu Glu
                    785                 790
    Asn Ser Lys Leu Ala Leu His Lys Asp Asp Glu Ser
            795                 800
    Cys Tyr Glu Ile Gly His Lys Val Leu Thr Val Asn
    805                 810                 815
    Leu Ile Gly Ser Ala Thr Phe Thr Ile Ser Lys Ser
                820                 825
    Arg Asn Leu Val Gly Gly Asn His Cys Ser Leu Thr
        830                 835                 840
    Ile Gly Pro Asn Glu Phe Phe Glu Met Pro Arg Gly
                    845                 850
    Met Gln Cys Asn Tyr Phe His Gly Val Ser Asn Cys
            855                 860
    Thr Pro Gly Arg Val Ser Leu Thr Phe Arg Arg Gln
    865                 870                 875
    Lys Leu Glu Asp Asp Asp Leu Ile Phe Ile Asn Pro
                880                 885
    Gln Val Pro Ile Glu Leu Asn His Glu Lys Leu Asp
        890                 895                 900
    Arg Ser Met Trp Gln Met Gly Leu His Gly Ile Lys
                    905                 910
    Lys Ser Ile Ser Met Asn Gly Thr Ser Phe Thr Ser
            915                 920
    Asp Leu Cys Ser Cys Phe Ser Cys His Asn Phe His
    925                 930                 935
    Lys Phe Lys Asp Leu Ile Asn Asn Leu Arg Leu Ala
                940                 945
    Leu Gly Ala Gln Gly Leu Gly Gln Cys Asp Arg Val
        950                 955                 960
    Val Phe Ala Thr Thr Gly Pro Gly Leu Ser Lys Val
                    965                 970
    Leu Glu Met Pro Arg Ser Lys Lys Gln Ser Ile Leu
            975                 980
    Val Leu Glu Gly Ala Leu Ser Ile Glu Thr Asp Tyr
    985                 990                 995
    Gly Pro Lys Val Leu Gly Ser Phe Glu Val Phe Lys
                1000                1005
    Gly Asp Phe His Ile Lys Lys Met Glu Glu Gly Ser
        1010                1015                1020
    Ile Phe Val Ile Thr Tyr Lys Ala Pro Ile Arg Ser
                    1025                1030
    Thr Gly Arg Leu Arg Val His Ser Ser Glu Cys Ser
            1035                1040
    Phe Ser Gly Ser Lys Glu Val Leu Leu Gly Cys Gln
    1045                1050                1055
    Ile Glu Ala Cys Ala Asp Tyr Asp Ile Asp Asp Phe
                1060                1065
    Asn Thr Phe Ser Val Pro Gly Asp Gly Asn Cys Phe
        1070                1075                1080
    Trp His Ser Val Gly Phe Leu Leu Ser Thr Asp Gly
                    1085                1090
    Leu Ala Leu Lys Ala Gly Ile Arg Ser Phe Val Glu
            1095                1100
    Ser Glu Arg Leu Val Ser Pro Asp Leu Ser Ala Pro
    1105                1110                1115
    Ala Ile Ser Lys Gln Leu Glu Glu Asn Ala Tyr Ala
                1120                1125
    Glu Asn Glu Met Ile Ala Leu Phe Cys Ile Arg His
        1130                1135                1140
    His Val Arg Pro Ile Val Ile Thr Pro Glu Tyr Glu
                    1145                1150
    Val Ser Trp Lys Phe Gly Glu Gly Glu Trp Pro Leu
            1155                1160
    Cys Gly Ile Leu Cys Leu Lys Ser Asn His Phe Gln
    1165                1170                1175
    Pro Cys Ala Pro Leu Asn Gly Cys Met Ile Thr Ala
                1180                1185
    Ile Ala Ser Ala Leu Gly Arg Arg Glu Val Asp Val
        1190                1195                1200
    Leu Asn Tyr Leu Cys Arg Pro Ser Thr Asn His Ile
                    1205                1210
    Phe Glu Glu Leu Cys Gln Gly Gly Gly Leu Asn Met
            1215                1220
    Met Tyr Leu Ala Glu Ala Phe Glu Ala Phe Asp Ile
    1225                1230                1235
    Cys Ala Lys Cys Asp Ile Asn Gly Glu Ile Glu Val
                1240                1245
    Ile Asn Pro Cys Gly Lys Ile Ser Ala Leu Phe Asp
        1250                1255                1260
    Ile Thr Asn Glu His Ile Arg His Val Glu Lys Ile
                    1265                1270
    Gly Asn Gly Pro Gln Ser Ile Lys Val Asp Glu Leu
            1275                1280
    Arg Lys Val Lys Arg Ser Ala Leu Asp Phe Leu Ser
    1285                1290                1295
    Met Asn Gly Ser Lys Ile Thr Tyr Phe Pro Ser Phe
                1300                1305
    Glu Arg Ala Glu Lys Leu Gln Gly Cys Leu Leu Gly
        1310                1315                1320
    Gly Leu Thr Gly Val Ile Ser Asp Glu Lys Phe Ser
                    1325                1330
    Asp Ala Lys Pro Trp Leu Ser Gly Ile Ser Thr Thr
            1335                1340
    Asp Ile Lys Pro Arg Glu Leu Thr Val Val Leu Gly
    1345                1350                1355
    Thr Phe Gly Ala Gly Lys Ser Phe Leu Tyr Lys Ser
                1360                1365
    Phe Met Lys Arg Ser Glu Gly Lys Phe Val Thr Phe
        1370                1375                1380
    Val Ser Pro Arg Arg Ala Leu Ala Asn Ser Ile Lys
                    1385                1390
    Asn Asp Leu Glu Met Asp Asp Ser Cys Lys Val Ala
            1395                1400
    Lys Ala Gly Arg Ser Lys Lys Glu Gly Trp Asp Val
    1405                1410                1415
    Val Thr Phe Glu Val Phe Leu Arg Lys Val Ala Gly
                1420                1425
    Leu Lys Ala Gly His Cys Val Ile Phe Asp Glu Val
        1430                1435                1440
    Gln Leu Phe Pro Pro Gly Tyr Ile Asp Leu Cys Leu
                    1445                1450
    Leu Ile Ile Arg Ser Asp Ala Phe Ile Ser Leu Ala
            1455                1460
    Gly Asp Pro Cys Gln Ser Thr Tyr Asp Ser Gln Lys
    1465                1470                1475
    Asp Arg Ala Ile Leu Gly Ala Glu Gln Ser Asp Ile
                1480                1485
    Leu Arg Leu Leu Glu Gly Lys Thr Tyr Arg Tyr Asn
        1490                1495                1500
    Ile Glu Ser Arg Arg Phe Val Asn Pro Met Phe Glu
                    1505                1510
    Ser Arg Leu Pro Cys His Phe Lys Lys Gly Ser Met
            1515                1520
    Thr Ala Ala Phe Ala Asp Tyr Ala Ile Phe His Asn
    1525                1530                1535
    Met His Asp Phe Leu Leu Ala Arg Ser Lys Gly Pro
                1540                1545
    Leu Asp Ala Val Leu Val Ser Ser Phe Glu Glu Lys
        1550                1555               1560
    Lys Ile Val Gln Ser Tyr Phe Gly Met Lys Gln Leu
                    1565                1570
    Thr Leu Thr Phe Gly Glu Ser Thr Gly Leu Asn Phe
            1575                1580
    Lys Asn Gly Gly Ile Leu Ile Ser His Asp Ser Phe
    1585                1590                1595
    His Thr Asp Asp Arg Arg Trp Leu Thr Ala Leu Ser
                1600                1605
    Arg Phe Ser His Asn Leu Asp Leu Val Asn Ile Thr
        1610                1615                1620
    Gly Leu Arg Val Glu Ser Phe Leu Ser His Phe Ala
                    1625                1630
    Gly Lys Pro Leu Tyr His Phe Leu Thr Ala Lys Ser
            1635                1640
    Gly Glu Asn Val Ile Arg Asp Leu Leu Pro Gly Glu
    1645                1650                1655
    Pro Asn Phe Phe Ser Gly Phe Asn Val Ser Ile Gly
                1660                1665
    Lys Asn Glu Gly Val Arg Glu Glu Lys Leu Cys Gly
        1670                1675                1680
    Asp Pro Trp Leu Lys Val Met Leu Phe Leu Gly Gln
                    1685                1690
    Asp Glu Asp Cys Glu Val Glu Glu Met Glu Ser Glu
            1695                1700
    Cys Ser Asn Glu Glu Trp Phe Lys Thr His Ile Pro
    1705                1710                1715
    Leu Ser Asn Leu Glu Ser Thr Arg Ala Arg Trp Val
                1720                1725
    Gly Lys Met Ala Leu Lys Glu Tyr Arg Glu Val Arg
        1730                1735                1740
    Cys Gly Tyr Glu Met Thr Gln Gln Phe Phe Asp Glu
                    1745                1750
    His Arg Gly Gly Thr Gly Glu Gln Leu Ser Asn Ala
            1755                1760
    Cys Glu Arg Phe Glu Ser Ile Tyr Pro Arg His Lys
    1765                1770                1775
    Gly Asn Asp Ser Ile Thr Phe Leu Met Ala Val Arg
                1780                1785
    Lys Arg Leu Lys Phe Ser Lys Pro Gln Val Glu Ala
        1790                1795                1800
    Ala Lys Leu Arg Arg Ala Lys Pro Tyr Gly Lys Phe
                    1805                1810
    Leu Leu Asp Ser Phe Leu Ser Lys Ile Pro Leu Lys
            1815                1820
    Ala Ser His Asn Ser Ile Met Phe His Glu Ala Val
    1825                1830                1835
    Gln Glu Phe Glu Ala Lys Lys Ala Ser Lys Ser Ala
                1840                1845
    Ala Thr Ile Glu Asn His Ala Gly Arg Ser Cys Arg
        1850                1855                1860
    Asp Trp Leu Leu Asp Val Ala Leu Ile Phe Met Lys
                    1865                1870
    Ser Gln His Cys Thr Lys Phe Asp Asn Arg Leu Arg
           1875                 1880
    Val Ala Lys Ala Gly Gln Thr Leu Ala Cys Phe Gln
    1885                1890                1895
    His Ala Val Leu Val Arg Phe Ala Pro Tyr Met Arg
                1900                1905
    Tyr Ile Glu Lys Lys Leu Met Gln Ala Leu Lys Pro
        1910                1915                1920
    Asn Phe Tyr Ile His Ser Gly Lys Gly Leu Asp Glu
                    1925                1930
    Leu Asn Glu Trp Val Arg Thr Arg Gly Phe Thr Gly
            1935                1940
    Ile Cys Thr Glu Ser Asp Tyr Glu Ala Phe Asp Ala
    1945                1950                1955
    Ser Gln Asp His Phe Ile Leu Ala Phe Glu Leu Gln
                1960                1965
    Ile Met Lys Phe Leu Gly Leu Pro Glu Asp Leu Ile
        1970                1975                1980
    Leu Asp Tyr Glu Phe Ile Lys Ile His Leu Gly Ser
                    1985                1990
    Lys Leu Gly Ser Phe Ser Ile Met Arg Phe Thr Gly
            1995                2000
    Glu Ala Ser Thr Phe Leu Phe Asn Thr Met Ala Asn
    2005                2010                2015
    Met Leu Phe Thr Phe Leu Arg Tyr Glu Leu Thr Gly
                2020                2025
    Ser Glu Ser Ile Ala Phe Ala Gly Asp Asp Met Cys
        2030                2035                2040
    Ala Asn Arg Arg Leu Arg Leu Lys Thr Glu His Glu
                    2045                2050
    Gly Phe Leu Asn Met Ile Cys Leu Lys Ala Lys Val
            2055                2060
    Gln Phe Val Ser Asn Pro Thr Phe Cys Gly Trp Cys
    2065                2070                2075
    Leu Phe Lys Glu Gly Ile Phe Lys Lys Pro Gln Leu
                2080                2085
    Ile Trp Glu Arg Ile Cys Ile Ala Arg Glu Met Gly
        2090                2095                2100
    Asn Leu Glu Asn Cys Ile Asp Asn Tyr Ala Ile Glu
                    2105                2110
    Val Ser Tyr Ala Tyr Arg Leu Gly Glu Leu Ala Ile
            2115                2120
    Glu Met Met Thr Glu Glu Glu Val Glu Ala His Tyr
    2125                2130                2135
    Asn Cys Val Arg Phe Leu Val Arg Asn Lys His Lys
                2140                2145
    Met Arg Cys Ser Ile Ser Gly Leu Phe Glu Ala Ile
        2150                2155                2160
    Asp

    The replicase of SEQ. ID. No. 3 has a molecular weight of about 240 to 246 kDa, preferably about 244 kDa.
  • Another DNA molecule of the present invention (RSPaV-1 ORF2) includes nucleotides 6578-7243 of SEQ. ID. No. 1. The DNA molecule of RSPaV-1 ORF2 encodes for a first protein or polypeptide of an RSPaV-1 triple gene block and comprises a nucleotide sequence corresponding to SEQ. ID. No. 4 as follows:
    ATGAATAATT TAGTTAAAGC ATTGTCAGCA TTTGAGTTTG TAGGTGTTTT CAGTGTGCTT  60
    AAATTTCCAG TAGTCATTCA TAGTGTGCCT GGTAGTGGTA AAAGTAGTTT AATAAGGGAG 120
    CTAATTTCCG AGGATGAGAA TTTCATAGCT TTCACAGCAG GTGTTCCAGA CAGCCCTAAT 180
    CTCACAGGAA GGTACATTAA GCCTTATTCT CCAGGGTGTG CAGTGCCAGG GAAAGTTAAT 240
    ATACTTGATG AGTACTTGTC CGTCCAAGAT TTTTCAGGTT TTGATGTGCT GTTCTCGGAC 300
    CCATACCAAA ACATCAGCAT TCCTAAAGAG GCACATTTCA TCAAGTCAAA AACTTGTAGG 360
    TTTGGCGTGA ATACTTGCAA ATATCTTTCC TCCTTCGGTT TTAAGGTTAG CAGTGACGGT 420
    TTGGACAAAG TCATTGTGGG GTCGCCTTTT ACACTAGATG TTGAAGGGGT GCTAATATGC 480
    TTTGGTAAGG AGGCAGTGGA TCTCGCTGTT GCGCACAACT CTGAATTCAA ATTACCTTGT 540
    GAAGTTAGAG GTTCAACTTT TAACGTCGTA ACTCTTTTGA AATCAAGAGA TCCAACCCCA 600
    GAGGATAGGC ACTGGTTTTA CATTGCTGCT ACAAGACACA GGGAGAAATT GATAATCATG 660
    CAG 663
  • The first protein or polypeptide of the RSPaV-1 triple gene block has a deduced amino acid sequence corresponding to SEQ. ID. No. 5 as follows:
    Met Asn Asn Leu Val Lys Ala Leu Ser Ala Phe Glu
    1               5                   10
    Phe Val Gly Val Phe Ser Val Leu Lys Phe Pro Val
            15                  20
    Val Ile His Ser Val Pro Gly Ser Gly Lys Ser Ser
    25                  30                  35
    Leu Ile Arg Glu Leu Ile Ser Glu Asp Glu Asn Phe
                40                  45
    Ile Ala Phe Thr Ala Gly Val Pro Asp Ser Pro Asn
        50                  55                  60
    Leu Thr Gly Arg Tyr Ile Lys Pro Tyr Ser Pro Gly
                    65                  70
    Cys Ala Val Pro Gly Lys Val Asn Ile Leu Asp Glu
            75                  80
    Tyr Leu Ser Val Gln Asp Phe Ser Gly Phe Asp Val
    85                  90                  95
    Leu Phe Ser Asp Pro Tyr Gln Asn Ile Ser Ile Pro
                100                 105
    Lys Glu Ala His Phe Ile Lys Ser Lys Thr Cys Arg
        110                 115                 120
    Phe Gly Val Asn Thr Cys Lys Tyr Leu Ser Ser Phe
                    125                 130
    Gly Phe Lys Val Ser Ser Asp Gly Leu Asp Lys Val
            135                 140
    Ile Val Gly Ser Pro Phe Thr Leu Asp Val Glu Gly
    145                 150                 155
    Val Leu Ile Cys Phe Gly Lys Glu Ala Val Asp Leu
                160                 165
    Ala Val Ala His Asn Ser Glu Phe Lys Leu Pro Cys
        170                 175                 180
    Glu Val Arg Gly Ser Thr Phe Asn Val Val Thr Leu
                    185                 190
    Leu Lys Ser Arg Asp Pro Thr Pro Glu Asp Arg His
            195                 200
    Trp Phe Tyr Ile Ala Ala Thr Arg His Arg Glu Lys
    205                 210                 215
    Leu Ile Ile Met Gln
                220

    The first protein or polypeptide of the RSPaV-1 triple gene block has a molecular weight of about 20 to 26 kDa, preferably 24.4 kDa.
  • Another DNA molecule of the present invention (RSPaV-1 ORF3) includes nucleotides 7245-7598 of SEQ. ID. No. 1. The DNA molecule of RSPaV-1 ORF3 encodes for a second protein or polypeptide of the triple gene block and comprises a nucleotide sequence corresponding to SEQ. ID. No. 6 as follows:
    ATGCCTTTTC AGCAGCCTGC GAATTGGGCA AAAACCATAA CTCCATTGAC AGTTGGCTTG  60
    GGCATTGGGC TTGTGCTGCA TTTTCTGAGG AAGTCAAATG TACCTTATTC AGGGGACAAC 120
    ATCCATCAAT TCCCTCACGG TGGGCGTTAC AGGGACGGTA CAAAAAGTAT AACTTACTGT 180
    GGTCCAAAGC AATCCTTCCC CAGCTCTGGG ATATTCGGCC AATCTGAGAA TTTTGTGCCC 240
    TTAATGCTTG TCATAGGTCT AATCGCATTC ATACATGTAT TGTCTGTTTG GAATTCTGGT 300
    CTTGGTAGGA ATTGTAATTG CCATCCAAAT CCTTGCTCAT GTAGACAGCA G 351
  • The second protein or polypeptide of the RSPaV-1 triple gene block has a deduced amino acid sequence corresponding to SEQ. ID. No. 7 as follows:
    Met Pro Phe Gln Gln Pro Ala Asn Trp Ala Lys Thr
    1               5                   10
    Ile Thr Pro Leu Thr Val Gly Leu Gly Ile Gly Leu
            15                  20
    Val Leu His Phe Leu Arg Lys Ser Asn Leu Pro Tyr
    25                  30                  35
    Ser Gly Asp Asn Ile His Gln Phe Pro His Gly Gly
                40                  45
    Arg Tyr Arg Asp Gly Thr Lys Ser Ile Thr Tyr Cys
        50                  55                  60
    Gly Pro Lys Gln Ser Phe Pro Ser Ser Gly Ile Phe
                    65                  70
    Gly Gln Ser Glu Asn Phe Val Pro Leu Met Leu Val
            75                  80
    Ile Gly Leu Ile Ala Phe Ile His Val Leu Ser Val
    85                  90                  95
    Trp Asn Ser Gly Leu Gly Arg Asn Cys Asn Cys His
                100                 105
    Pro Asn Pro Cys Ser Cys Arg Gln Gln
        110                 115
  • The second protein or polypeptide of the RSPaV-1 triple gene block has a molecular weight of about 10 to 15 kDa, preferably 12.8 kDa.
  • Yet another DNA molecule of the present invention (RSPaV-1 ORF4) includes nucleotides 7519-7761 of SEQ. ID. No. 1. The DNA molecule of RSPaV-1 ORF4 encodes for a third protein or polypeptide of the RSPaV-1 triple gene block and comprises a nucleotide sequence corresponding to SEQ. ID. No. 8 as follows:
    ATGTATTGTC TGTTTGGAAT TCTGGTCTTG GTAGGAATTG TAATTGCCAT CCAAATCCTT  60
    GCTCATGTAG ACAGCAGTAG TGGCAACCAC CAAGGTTGCT TCATTAGGGC CACTGGAGAG 120
    TCAATTTTGA TTGAAAACTG CGGCCCAAGT GAGGCCCTTG CATCCACTGT GAAGGAGGTG 180
    CTGGGAGGTT TGAAGGCTTT AGGGGTTAGC CGTGCTGTTG AAGAAATTGA TTATCATTGT 240
  • The third protein or polypeptide of the RSPaV-1 triple gene block has a deduced amino acid sequence corresponding to SEQ. ID. No. 9 as follows:
    Met Tyr Cys Leu Phe Gly Ile Leu Val Leu Val Gly
    1               5                   10
    Ile Val Ile Ala Ile Gln Ile Leu Ala His Val Asp
            15                  20
    Ser Ser Ser Gly Asn His Gln Gly Cys Phe Ile Arg
    25                  30                  35
    Ala Thr Gly Glu Ser Ile Leu Ile Glu Asn Cys Gly
                40                  45
    Pro Ser Glu Ala Leu Ala Ser Thr Val Lys Glu Val
        50                  55                  60
    Leu Gly Gly Leu Lys Ala Leu Gly Val Ser Arg Ala
                    65                  70
    Val Glu Glu Ile Asp Tyr His Cys
            75                  80

    The third protein or polypeptide of the RSPaV-1 triple gene block has a molecular weight of about 5 to 10 kDa, preferably 8.4 kDa.
  • Still another DNA molecule of the present invention (RSPaV-1 ORF5) includes nucleotides 7771-8550 of SEQ. ID. No. 1. The DNA molecule of RSPaV-1 ORF5 encodes for a RSPaV-1 coat protein and comprises a nucleotide sequence corresponding to SEQ. ID. No. 10 as follows:
    ATGGCAAGTC AAATTGGGAA ACTCCCCGGT GAATCAAATG AGGCTTTTGA AGCCCGGCTA  60
    AAATCGCTGG AGTTAGCTAG AGCTCAAAAG CAGCCGGAAG GTTCTAATGC ACCACCTACT 120
    CTCAGTGGCA TTCTTGCCAA ACGCAAGAGG ATTATAGAGA ATGGACTTTC AAAGACGGTG 180
    GACATGAGGG AGGTTTTGAA ACACGAAACG GTGGTGATTT CCCCAAATGT CATGGATGAA 240
    GGTGCAATAG ACGAGCTGAT TCGTGCATTT GGTGAATCTG GCATAGCTGA AAGCGTGCAA 300
    TTTGATGTGG CCATAGATAT AGCACGTCAC TGCTCTGATG TTGGTAGCTC CCAGAGTTCA 360
    ACCCTGATTG GCAAGAGTCC ATTTTGTGAC CTAAACAGAT CAGAAATAGC TGGGATTATA 420
    AGGGAGGTGA CCACATTACG TAGATTTTGC ATGTACTATG CAAAAATCGT GTGGAACATC 480
    CATCTGGAGA CGGGGATACC ACCAGCTAAC TGGGCCAAGA AAGGATTTAA TGAGAATGAA 540
    AAGTTTGCAG CCTTTGATTT TTTCTTGGGA GTCACAGATG AGAGTGCGCT TGAACCAAAG 600
    GGTGGAATTA AAAGAGCTCC AACGAAAGCT GAGATGGTTG CTAATATCGC CTCTTTTGAG 660
    GTTCAAGTGC TCAGACAAGC TATGGCTGAA GGCAAGCGGA GTTCCAACCT TGGAGAGATT 720
    AGTGGTGGAA CGGCTGGTGC ACTCATCAAC AACCCCTTTT CAAATGTTAC ACATGAA 777
  • The RSPaV-1 coat protein has a deduced amino acid sequence corresponding to SEQ. ID. No. 11 as follows:
    Met Ala Ser Gln Ile Gly Lys Leu Pro Gly Glu Ser Asn Glu Ala Phe
    1               5                   10                  15
    Glu Ala Arg Leu Lys Ser Leu Glu Leu Ala Arg Ala Gln Lys Gln Pro
                20                  25                  30
    Glu Gly Ser Asn Ala Pro Pro Thr Leu Ser Gly Ile Leu Ala Lys Arg
            35                  40                  45
    Lys Arg Ile Ile Glu Asn Ala Leu Ser Lys Thr Val Asp Met Arg Glu
        50                  55                  60
    Val Leu Lys His Glu Thr Val Val Ile Ser Pro Asn Val Met Asp Glu
    65                  70                  75                  80
    Gly Ala Ile Asp Glu Leu Ile Arg Ala Phe Gly Glu Ser Gly Ile Ala
                    85                  90                  95
    Glu Ser Val Gln Phe Asp Val Ala Ile Asp Ile Ala Arg His Cys Ser
                100                 105                 110
    Asp Val Gly Ser Ser Gln Ser Ser Thr Leu Ile Gly Lys Ser Pro Phe
            115                 120                 125
    Cys Asp Leu Asn Arg Ser Glu Ile Ala Gly Ile Ile Arg Glu Val Thr
        130                 135                 140
    Thr Leu Arg Arg Phe Cys Met Tyr Tyr Ala Lys Ile Val Trp Asn Ile
    145                 150                 155                 160
    His Leu Glu Thr Gly Ile Pro Pro Ala Asn Trp Ala Lys Lys Gly Phe
                    165                 170                 175
    Asn Glu Asn Glu Lys Phe Ala Ala Phe Asp Phe Phe Leu Gly Val Thr
                180                 185                 190
    Asp Glu Ser Ala Leu Glu Pro Lys Gly Gly Ile Lys Arg Ala Pro Thr
            195                 200                 205
    Lys Ala Glu Met Val Ala Asn Ile Ala Ser Phe Glu Val Gln Val Leu
        210                 215                 220
    Arg Gln Ala Met Ala Glu Gly Lys Arg Ser Ser Asn Leu Gly Glu Ile
    225                 230                 235                 240
    Ser Gly Gly Thr Ala Gly Ala Leu Ile Asn Asn Pro Phe Ser Asn Val
                    245                 250                 255
    Thr His Glu

    The RSPaV-1 coat protein has a molecular weight of about 25 to 30 kDa, preferably 28 kDa.
  • The DNA molecule which constitutes the substantial portion of the RSPaV strain RSP474 genome comprises the nucleotide sequence corresponding to SEQ. ID. No. 12 as follows:
    GGCTGGGCAA ACTTTGGCCT GCTTTCAACA CGCCGTCTTG GTTCGCTTTG CACCCTACAT 60
    GCGATACATT GAAAAGAAGC TTGTGCAGGC ATTGAAACCA AATTTCTACA TTCATTCTGG 120
    CAAAGGTCTT GATGAGCTAA GTGAATGGGT TAGAGCCAGA GGTTTCACAG GTGTGTGTAC 180
    TGAGTCAGAC TATGAAGCTT TTGATGCATC CCAAGATCAT TTCATCCTGG CATTTGAACT 240
    GCAAATCATG AGATTTTTAG GACTGCCAGA AGATCTGATT TTAGATTATG AGTTCATCAA 300
    AATTCATCTT GGGTCAAAGC TTGGCTCTTT TGCAATTATG AGATTCACAG GTGAGGCAAG 360
    CACCTTCCTA TTCAATACTA TGGCCAACAT GCTATTCACT TTCCTGAGGT ATGAGTTGAC 420
    AGGTTCTGAA TCAATTGCAT TTGCTGGAGA TGATATGTGT GCTAATCGCA GGTTAAGACT 480
    CAAGACTGAG CACGCCGGCT TTCTAAACAT GATCTGTCTC AAAGCTAAGG TGCAGTTTGT 540
    CACAAATCCC ACCTTCTGTG GATGGTGTTT GTTTAAAGAG GGAATCTTTA AAAAACCCCA 600
    GCTCATTTGG GAAAGGATCT GCATTGCTAG GGAAATGGGT AACTTGGACA ATTGCATTGA 660
    CAATTACGCA ATTGAGGTGT CTTATGCTTA CAGACTTGGG GAATTGTCCA TAGGCGTGAT 720
    GACTGAGGAG GAAGTTGAAG CACATTCTAA CTGCGTGCGT TTCCTGGTTC GCAATAAGCA 780
    CAAGATGAGG TGCTCAATTT CTGGTTTGTT TGAAGTAATT GTTTAGGCCT TAAGTGTTTG 840
    GCATGGTGTG AGTATTATGA ATAACTTAGT CAAAGCTTTG TCTGCTTTTG AATTTGTTGG 900
    TGTGTTTTGT GTACTTAAAT TTCCAGTTGT TGTTCACAGT GTTCCAGGTA GCGGTAAAAG 960
    TAGCCTAATA AGGGAGCTCA TTTCTGAAGA CGAGGCTTTT GTGGCCTTTA CAGCAGGTGT 1020
    GCCAGACAGT CCAAATCTGA CAGGGAGGTA CATCAAGCCC TACGCTCCAG GGTGTGCAGT 1080
    GCAAGGGAAA ATAAACATAC TTGATGAGTA GTTGTCTGTC TCTGATACTT CTGGCTTTGA 1140
    TGTGCTGTTC TGAGACCCTT ACCAGAATGT CAGCATTCCA AGGGAGGCAC ACTTCATAAA 1200
    AACCAAAACC TGTAGGTTTG GTACCAACAC CTGCAAGTAC CTTCAATCTT TTGGCTTTAA 1260
    TGTTTGTAGT GATGGGGTGG ATAAAGTTGT TGTAGGGTCG CCATTTGAAC TGGAGGTTGA 1320
    GGGGGTTCTC ATTTGCTTTG GAAAGGAGGC TGTAGATCTA GCAGTTGCAG ACAATTCTGA 1380
    CTTCAAGTTG CCCTGCGAGG TGCGGGGTTC AAGATTTGAC GTTGTAACGT TATTGAAGTC 1440
    CAGGGATCCA ACTTCAGAAG ATAAGCATTG GTTGTACGTT GCAGCCACAA GGCATCGAAG 1500
    TAAACTGATA ATAATGCAGT AAAATGCCTT TTCAGCAACC TGCCAACTGG GCTAAGACCA 1560
    TAACTCCATT AACTATTGGT TTGGGCATTG GGTTGGTTCT GCACTTCTTA AGGAAATCAA 1620
    ATCTGCCATA TTCAGGAGAC AATATTCACC AGTTCCCACA CGGAGGGCAT TACAGGGACG 1680
    GCACGAAGAG TATAACCTAT TGTGGCCCTA GGCAGTCATT CCCAAGCTCA GGAATATTCG 1740
    GTCAGTCTGA AAATTTCGTA CCTCTAATAT TGGTCGTGAC TCTGGTCGCT TTTATACATG 1800
    CGTTATCTCT TTGGAATTCT GGTCCTAGTA GGAGTTGCAA TTGCCATCCA AATCCTTGCA 1860
    CATGTAGACA GCAGTAGTGG CAACCATCAA GGCTGTTTCA TAAGAGCCAC CGGGGAGTCA 1920
    ATAGTAATTG AGAATTGTGG GCCGAGCGAG GCCCTAGCTG CTACAGTCAA AGAGGTGTTG 1980
    GGCGGTCTAA AGGCTTTAGG GGTTAGCCAA AAGGTTGATG AAATTAATTA CAGTTGTTGA 2040
    GACAGTTGAA TGGCAAGTCA AGTTGGAAAA TTGCCTGGCG AATCAAATGA AGCATATGAG 2100
    GCTAGACTCA AGGCTTTAGA GTTAGCAAGG GCCCAAAAAG CTCCAGAAGT CTCCAACCAA 2160
    CCTCCCACAC TTGGAGGCAT TCTAGCCAAA AGGAAAAGAG TGATTGAGAA TGCACTCTCA 2220
    AAGACAGTGG ATATGCGTGA AGTCTTAAGG CATGAATCTG TTGTACTCTC CCCGAATGTA 2280
    ATGGACGAGG GAGCAATAGA CGAGCTGATT GGTGCCTTTG GGGAGTCGGG CATAGCTGAA 2340
    AATGTGCAGT TTGATGTTGC AATAGACATT GCTCGCCACT GTTCTGATGT GGGGAGCTCT 2400
    CAGAGGTCAA CCCTTATTGG TAAAAGCCCC TTCTGTGAGT TAAATAGGTC TGAAATTGCC 2460
    GGAATAATAA GGGAGGTGAC CACGCTGCGC AGATTTTGCA TGTACTACGC AAAGATTGTG 2520
    TGGAACATCC ATTTGGAGAC GGGAATACCA GCAGCTAATT GGGCCAAGAA AGGATTTAAT 2580
    GAGAATGAAA AGTTTGCAGC CTTTGACTTC TTCCTTGGAG TCACAGATGA AAGCGCGCTT 2640
    GAGCCTAAGG GTGGAGTCAA GAGAGCTCCA ACAAAAGCAG 2680

    The RSP47-4 strain contains five open reading frames (i.e., ORF1-5). ORF1 and ORF5 are only partially sequenced. RSP47-4 is 79% identical in nucleotide sequence to the corresponding region of RSPaV-1. The amino acid sequence identities between the corresponding ORFs of RSP47-4 and RSPaV-1 are: 94.1% for ORF1, 88.2% for ORF2, 88.9% for ORF3, 86.2% for ORF4, and 92.9% for ORF5. The nucleotide sequences of the five potential ORFs of RSP47-4 are given below.
  • Another DNA molecule of the present invention (RSP47-4 incomplete ORF1) includes nucleotides 1-768 of SEQ. ID. No. 12. This DNA molecule is believed to code for a polypeptide portion of a RSP47-4 replicase and comprises a nucleotide sequence corresponding to SEQ. ID. No. 13 as follows:
    ATGCGATACA TTGAAAAGAA GCTTGTGCAG GCATTGAAAC CAAATTTCTA CATTCATTCT 60
    GGCAAAGGTC TTGATGAGCT AAGTGAATGG GTTAGAGGCA GAGGTTTCAC AGGTGTGTGT 120
    ACTGAGTCAG ACTATGAAGC TTTTGATGCA TCCCAAGATC ATTTCATCCT GGCATTTGAA 180
    CTGCAAATCA TGAGATTTTT AGGACTGCCA GAAGATCTGA TTTTAGATTA TGAGTTCATC 240
    AAAATTCATC TTGGGTCAAA GCTTGGCTCT TTTGCAATTA TGAGATTCAC AGGTGAGGCA 300
    AGCACCTTCG TATTCAATAC TATGGCCAAC ATGCTATTCA CTTTCCTGAG GTATGAGTTG 360
    ACAGGTTCTG AATCAATTGC ATTTGCTGGA GATGATATGT GTGCTAATCG CAGGTTAAGA 420
    CTCAAGACTG AGCACGCCGG CTTTCTAAAC ATGATCTGTC TCAAAGCTAA GGTGCAGTTT 480
    GTCACAAATC CCACCTTCTG TGGATGGTGT TTGTTTAAAG AGGGAATCTT TAAAAAACCC 540
    CAGCTCATTT GGGAAAGGAT CTGCATTGCT AGGGAAATGG GTAACTTGGA CAATTGCATT 600
    GACAATTACG CAATTGAGGT GTCTTATGCT TACAGACTTG GGGAATTGTC CATAGGCGTG 660
    ATGACTGAGG AGGAAGTTGA AGCACATTCT AACTGCGTGC GTTTCCTGGT TCGCAATAAG 720
    CACAAGATGA GGTGCTCAAT TTCTGGTTTG TTTGAAGTAA TTGTTTA 767
  • The polypeptide has a deduced amino acid sequence corresponding to SEQ. ID. No. 14 as follows:
    Met Arg Tyr Ile Glu Lys Lys Leu Val Gln Ala Leu Lys Pro Asn Phe
    1               5                   10                  15
    Tyr Ile His Ser Gly Lys Gly Leu Asp Glu Leu Ser Glu Trp Val Arg
                20                  25                  30
    Ala Arg Gly Phe Thr Gly Val Cys Thr Glu Ser Asp Tyr Glu Ala Phe
            35                  40                  45
    Asp Ala Ser Gln Asp His Phe Ile Leu Ala Phe Glu Leu Gln Ile Met
        50                  55                  60
    Arg Phe Leu Gly Leu Pro Glu Asp Leu Ile Leu Asp Tyr Glu Phe Ile
    65                  70                  75                  80
    Lys Ile His Leu Gly Ser Lys Leu Gly Ser Phe Ala Ile Met Arg Phe
                    85                  90                  95
    Thr Gly Glu Ala Ser Thr Phe Leu Phe Asn Thr Met Ala Asn Met Leu
                100                 105                 110
    Phe Thr Phe Leu Arg Tyr Glu Leu Thr Gly Ser Glu Ser Ile Ala Phe
            115                 120                 125
    Ala Gly Asp Asp Met Cys Ala Asn Arg Arg Leu Arg Leu Lys Thr Glu
        130                 135                 140
    His Ala Gly Phe Leu Asn Met Ile Cys Leu Lys Ala Lys Val Gln Phe
    145                 150                 155                 160
    Val Thr Asn Pro Thr Phe Cys Gly Trp Cys Leu Phe Lys Glu Gly Ile
                    165                 170                 175
    Phe Lys Lys Pro Gln Leu Ile Trp Glu Arg Ile Cys Ile Ala Arg Glu
                180                 185                 190
    Met Gly Asn Leu Asp Asn Cys Ile Asp Asn Tyr Ala Ile Glu Val Ser
            195                 200                 205
    Tyr Ala Tyr Arg Leu Gly Glu Leu Ser Ile Gly Val Met Thr Glu Glu
        210                 215                 220
    Glu Val Glu Ala His Ser Asn Cys Val Arg Phe Leu Val Arg Asn Lys
    225                 230                 235                 240
    His Lys Met Arg Cys Ser Ile Ser Gly Leu Phe Glu Val Ile Val
                    245                 250                 255
  • Another DNA molecule of the present invention (RSP47-4 ORF2) includes nucleotides 857-1522 of SEQ. ID. No. 12. This DNA molecule codes for a first protein or polypeptide of an RSP47-4 triple gene block and comprises a nucleotide sequence corresponding to SEQ. ID. No. 15 as follows:
    ATGAATAACT TAGTCAAAGC TTTGTCTGCT TTTGAATTTG TTGGTGTGTT TTGTGTACTT 60
    AAATTTCCAG TTGTTGTTCA CAGTGTTCCA GGTAGCGGTA AAAGTAGCCT AATAAGGGAG 120
    CTCATTTCTG AAGACGAGGC TTTTGTGGCC TTTACAGCAG GTGTGCCAGA CAGTCCAAAT 180
    CTGACAGGGA GGTACATCAA GCCCTACGCT CCAGGGTGTG CAGTGCAAGG GAAAATAAAC 240
    ATACTTGATG AGTACTTGTC TGTCTCTGAT ACTTCTGGCT TTGATGTGCT GTTCTCAGAC 300
    CCTTACCAGA ATGTCAGCAT TCCAAGGGAG GCACACTTCA TAAAAACCAA AACCTGTAGG 360
    TTTGGTACCA ACACCTGCAA GTACCTTCAA TCTTTTGGCT TTAATGTTTG TAGTGATGGG 420
    GTGGATAAAG TTGTTGTAGG GTCGCCATTT GAACTGGAGG TTGAGGGGGT TCTCATTTGC 480
    TTTGGAAAGG AGGCTGTAGA TCTAGCAGTT GCACACAATT CTGACTTCAA GTTGCCCTGC 540
    GAGGTGCGGG GTTCAACATT TGACGTTGTA ACGTTATTGA AGTCCAGGGA TCCAACTTCA 600
    GAAGATAAGC ATTGGTTCTA CGTTGCAGCC ACAAGGCATC GAAGTAAACT GATAATAATG 660
    CAGTAA
    666
  • The first protein or polypeptide of the RSP47-4 triple gene block has a deduced amino acid sequence corresponding to SEQ. ID. No. 16 as follows:
    Met Asn Asn Leu Val Lys Ala Leu Ser Ala Phe Glu Phe Val Gly Val
    1               5                   10                  15
    Phe Cys Val Leu Lys Phe Pro Val Val Val His Ser Val Pro Gly Ser
                20                  25                  30
    Gly Lys Ser Ser Leu Ile Arg Glu Leu Ile Ser Glu Asp Glu Ala Phe
            35                  40                  45
    Val Ala Phe Thr Ala Gly Val Pro Asp Ser Pro Asn Leu Thr Gly Arg
        50                  55                  60
    Tyr Ile Lys Pro Tyr Ala Pro Gly Cys Ala Val Gln Gly Lys Ile Asn
    65                  70                  75                  80
    Ile Leu Asp Glu Tyr Leu Ser Val Ser Asp Thr Ser Gly Phe Asp Val
                    85                  90                  95
    Leu Phe Ser Asp Pro Tyr Gln Asn Val Ser Ile Pro Arg Glu Ala His
                100                 105                 110
    Phe Ile Lys Thr Lys Thr Cys Arg Phe Gly Thr Asn Thr Cys Lys Tyr
            115                 120                 125
    Leu Gln Ser Phe Gly Phe Asn Val Cys Ser Asp Gly Val Asp Lys Val
        130                 135                 140
    Val Val Gly Ser Pro Phe Glu Leu Glu Val Glu Gly Val Leu Ile Cys
    145                 150                 155                 160
    Phe Gly Lys Glu Ala Val Asp Leu Ala Val Ala His Asn Ser Asp Phe
                    165                 170                 175
    Lys Leu Pro Cys Glu Val Arg Gly Ser Thr Phe Asp Val Val Thr Leu
                180                 185                 190
    Leu Lys Ser Arg Asp Pro Thr Ser Glu Asp Lys His Trp Phe Tyr Val
            195                 200                 205
    Ala Ala Thr Arg His Arg Ser Lys Leu Ile Ile Met Gln
        210                 215                 220

    The first protein or polypeptide of the RSP47-4 triple gene block has a molecular weight of about 20 to 26 kDa., preferably 24.3 kDa.
  • Another DNA molecule of the present invention (RSP47-4 ORF3) includes nucleotides 1524-1877 of SEQ. ID. No. 12. This DNA molecule codes for a second protein or polypeptide of the RSP47-4 triple gene block and comprises a nucleotide sequence corresponding to SEQ. ID. No. 17 as follows:
    ATGCCTTTTG AGCAACCTGC CAACTGGGCT AAGACCATAA CTCCATTAAC TATTGGTTTG 60
    GGCATTGGGT TGGTTCTGCA CTTCTTAAGG AAATCAAATC TGCCATATTC AGGAGACAAT 120
    ATTCACCAGT TCCCACACGG AGGGCATTAC AGGGACGGCA CGAAGAGTAT AACCTATTGT 180
    GGCCCTAGGC AGTCATTCCC AAGCTCAGGA ATATTCGGTC AGTCTGAAAA TTTCGTACCT 240
    CTAATATTGG TCGTGACTCT GGTCGCTTTT ATACATGCGT TATCTCTTTG GAATTCTGGT 300
    CCTAGTAGGA GTTGCAATTG CCATCCAAAT CCTTGCACAT GTAGACAGCA GTAG 354
  • The second protein or polypeptide of the RSP47-4 triple gene block has a deduced amino acid sequence corresponding to SEQ. ID. No. 18 as follows:
    Met Pro Phe Gln Gln Pro Ala Asn Trp Ala Lys Thr Ile Thr Pro Leu
    1               5                   10                  15
    Thr Ile Gly Leu Gly Ile Gly Leu Val Leu His Phe Leu Arg Lys Ser
                20                  25                  30
    Asn Leu Pro Tyr Ser Gly Asp Asn Ile His Gln Phe Pro His Gly Gly
            35                  40                  45
    His Tyr Arg Asp Gly Thr Lys Ser Ile Thr Tyr Cys Gly Pro Arg Gln
        50                  55                  60
    Ser Phe Pro Ser Ser Gly Ile Phe Gly Gln Ser Glu Asn Phe Val Pro
    65                  70                  75                  80
    Leu Ile Leu Val Val Thr Leu Val Ala Phe Ile His Ala Leu Ser Leu
                    85                  90                  95
    Trp Asn Ser Gly Pro Ser Arg Ser Cys Asn Cys His Pro Asn Pro Cys
                100                 105                 110
    Thr Cys Arg Gln Gln
            115

    The second protein or polypeptide of the RSP47-4 triple gene block has a molecular weight of about 10 to 15 kDa., preferably 12.9 kDa.
  • Another DNA molecule of the present invention (RSP47-4 ORF4) includes nucleotides 1798-2040 of SEQ. ID. No. 12. This DNA molecule codes for a third protein or polypeptide of the RSP47-4 triple gene block and comprises a nucleotide sequence corresponding to SEQ. ID. No. 19 as follows:
    ATGCGTTATC TCTTTGGAAT TCTGGTCCTA GTAGGAGTTG CAATTGCCAT CCAAATCCTT 60
    GCACATGTAG ACAGCAGTAG TGGCAACCAT CAAGGCTGTT TCATAAGAGC CACCGGGGAG 120
    TCAATAGTAA TTGAGAATTG TGGGCCGAGC GAGGCCCTAG CTGCTACAGT CAAAGAGGTG 180
    TTGGGCGGTC TAAAGGCTTT AGGGGTTAGC CAAAAGGTTG ATGAAATTAA TTACAGTTGT 240
    TGA 243
  • The third protein or polypeptide of the RSP47-4 triple gene block has a deduced amino acid sequence corresponding to SEQ. ID. No. 20 as follows:
    Met Arg Tyr Leu Phe Gly Ile Leu Val Leu Val Gly Val Ala Ile Ala
    1               5                   10                  15
    Ile Gln Ile Leu Ala His Val Asp Ser Ser Ser Gly Asn His Gln Gly
                20                  25                  30
    Cys Phe Ile Arg Ala Thr Gly Glu Ser Ile Val Ile Glu Asn Cys Gly
            35                  40                  45
    Pro Ser Glu Ala Leu Ala Ala Thr Val Lys Glu Val Leu Gly Gly Leu
        50                  55                  60
    Lys Ala Leu Gly Val Ser Gln Lys Val Asp Glu Ile Asn Tyr Ser Cys
    65                  70                  75                  80

    The third protein or polypeptide of the RSP47-4 triple gene block has a molecular weight of about 5 to 10 kDa., preferably 8.3 kDa.
  • Yet another DNA molecule of the present invention (RSP474 ORF5) includes nucleotides 2050-2680 of SEQ. ID. No. 12. This DNA molecule codes for a partial RSP47-4 coat protein or polypeptide and comprises a nucleotide sequence corresponding to SEQ. ID. No. 21 as follows:
    ATGGCAAGTC AAGTTGGAAA ATTGCCTGGC GAATCAAATG AAGCATATGA GGCTAGACTC 60
    AAGGCTTTAG AGTTAGCAAG GGCCCAAAAA GCTCCAGAAG TCTCCAACCA ACCTCCCACA 120
    CTTGGAGGCA TTCTAGCCAA AAGGAAAAGA GTGATTGAGA ATGCACTCTC AAAGACAGTG 180
    GATATGCGTG AAGTCTTAAG GCATGAATCT GTTGTACTCT CCCCGAATGT AATGGACGAG 240
    GGAGCAATAG ACGAGCTGAT TCGTGCCTTT GGGGAGTCGG GCATAGCTGA AAATGTGCAG 300
    TTTGATGTTG CAATAGACAT TGCTCGCCAC TGTTCTGATG TGGGGAGCTC TCAGAGGTCA 360
    ACCGTTATTG GTAAAAGCCC CTTCTGTGAG TTAAATAGGT CTGAAATTGC CGGAATAATA 420
    AGGGAGGTGA CCACGCTGCG CAGATTTTGC ATGTACTACG CAAAGATTGT GTGGAACATC 480
    CATTTGGAGA CGGGAATACC ACCAGCTAAT TGGGCCAAGA AAGGATTTAA TGAGAATGAA 540
    AAGTTTGCAG CCTTTGACTT CTTCCTTGGA GTCACAGATG AAAGCGCGCT TGAGCCTAAG 600
    GGTGGAGTCA AGAGAGCTCC AACAAAAGCA G 631
  • The polypeptide has a deduced amino acid sequence corresponding to SEQ. ID. No. 22 as follows:
    Met Ala Ser Gln Val Gly Lys Leu Pro Gly Glu Ser
    1               5                   10
    Asn Glu Ala Tyr Glu Ala Arg Leu Lys Ala Leu Glu
            15                  20
    Leu Ala Arg Ala Gln Lys Ala Pro Glu Val Ser Asn
    25                  30                  35
    Gln Pro Pro Thr Leu Gly Gly Ile Leu Ala Lys Arg
                40                  45
    Lys Arg Val Ile Glu Asn Ala Leu Ser Lys Thr Val
        50                  55                  60
    Asp Met Arg Glu Val Leu Arg His Glu Ser Val Val
                    65                  70
    Leu Ser Pro Asn Val Met Asp Glu Gly Ala Ile Asp
            75                  80
    Glu Leu Ile Arg Ala Phe Gly Glu Ser Gly Ile Ala
    85                  90                  95
    Glu Asn Val Gln Phe Asp Val Ala Ile Asp Ile Ala
                100                 105
    Arg His Cys Ser Asp Val Gly Ser Ser Gln Arg Ser
        110                 115                 120
    Thr Leu Ile Gly Lys Ser Pro Phe Cys Glu Leu Asn
                    125                 130
    Arg Ser Glu Ile Ala Gly Ile Ile Arg Glu Val Thr
            135                 140
    Thr Leu Arg Arg Phe Cys Met Tyr Tyr Ala Lys Ile
    145                 150                 155
    Val Trp Asn Ile His Leu Glu Thr Gly Ile Pro Pro
                160                 165
    Ala Asn Trp Ala Lys Lys Gly Phe Asn Glu Asn Glu
        170                 175                 180
    Lys Phe Ala Ala Phe Asp Phe Phe Leu Gly Val Thr
                    185                 190
    Asp Glu Ser Ala Leu Glu Pro Lys Gly Gly Val Lys
            195                 200
    Arg Ala Pro Thr Lys Ala
    205                 210
  • The DNA molecule which constitutes a substantial portion of the RSPaV strain RSP158 genome comprises the nucleotide sequence corresponding to SEQ. ID. No. 23 as follows:
    GAAGCTAGCA CATTTCTGTT CAACACTATG GCTAACATGT TGTTCACTTT TCTGAGATAT   60
    GAACTGACGG GTTCAGAGTC AATAGCATTT GCAGGGGATG ATATGTGTGC TAATAGAAGG  120
    TTGCGGCTTA AAACGGAGCA TGAGGGTTTT CTGAACATGA TCTGCCTTAA GGCCAAGGTT  180
    CAGTTTGTTT CCAACCCCAC ATTCTGTGGA TGGTGCTTAT TTAAGGAGGG AATCTTCAAG  240
    AAACCTCAAC TAATTTGGGA GCGAATATGC ATAGCCAGAG AGATGGGCAA TCTGGAGAAC  300
    TGTATTGACA ATTATGCGAT AGAAGTGTCC TATGCATATA GATTGGGTGA GCTATCAATT  360
    GAAATGATGA CAGAAGAAGA AGTGGAGGCA CACTACAATT GTGTGAGGTT CCTGGTTAGG  420
    AACAAGCATA AGATGAGGTG CTCAATTTCA GGCCTGTTTG AAGTGGTTGA TTAGGCCTTA  480
    AGTATTTGGC GTTGTTCGAG TTATTATGAA TAATTTAGTT AAAGCATTAT CAGCCTTCGA  540
    GTTTATAGGT GTTTTCAATG TGCTCAAATT TCCAGTTGTT ATACATAGTG TGCCTGGTAG  600
    TGGTAAGAGT AGCTTAATAA GGGAATTAAT CTCAGAGGAC GAGAGTTTCG TGGCTTTCAC  660
    AGCAGGTGTT CCAGACAGTC CTAACCTCAC AGGGAGGTAC ATCAAGCCTT ACTCACCAGG  720
    ATGCGCAGTG CAAGGAAAAG TGAATATACT TGATGAGTAC TTGTCCGTTC AAGACATTTC  780
    GGGTTTTGAT GTACTGTTTT CAGACCCGTA CCAGAATATC AGTATTCCCC AAGAGGCGCA  840
    TTTCATTAAG TCCAAGACTT GTAGGTTTGG TGTGAACACT TGCAAATACC TTTCCTCTTT  900
    CGGTTTCGAA GTTAGCAGCG ACGGGCTGGA CGACGTCATT GTGGGATCGC CCTTCACTCT  960
    AGATGTTGAA GGGGTGCTGA TATGTTTTGG CAAGGAGGCG GTAGATCTCG CTGTTGCGCA 1020
    CAACTCTGAA TTCAAGTTGC CGTGTGAGGT TCGAGGTTCA ACCTTCAATG TGGTAACCCT 1080
    TTTGAAATCA AGAGACCCAA CCCCAGAGGA CAGGCACTGG TTTTACATCG CTGCCACAAG 1140
    ACATAGGAAG AAATTGGTCA TTATGCAGTA AAATGCCTTT TCAGCAGCCT GCTAATTGGG 1200
    CAAAAACCAT AACTCCATTG ACTATTGGCT TAGGAATTGG ACTTGTGCTG CATTTTCTGA 1260
    GAAAGTCAAA TCTACCATAT TCAGGAGACA ACATCCATCA ATTTCCTCAC GGGGGGCGTT 1320
    ACCGGGACGG CACAAAAAGT ATAACTTACT GTGGCCCTAA GCAGTCCTTC CCCAGTTCAG 1380
    GAATATTTGG TCAGTCTGAG AATTTTGTGC CCTTAATGCT TGTCATAGGT CTAATTGCAT 1440
    TCATACATGT ATTGTCTGTT TGGAATTCTG GTCTTGGTAG GAATTGCAAT TGCCATCCAA 1500
    ATCCTTGCTC ATGTAGACAA CAGTAGTGGC AGTCACCAAG GTTGCTTTAT CAGGGCCACT 1560
    GGAGAGTCTA TTTTGATTGA AAATTGTGGC CCAAGCGAGG CCCTTGCATC AACAGTGAGG 1620
    GAGGTGTTGG GGGGTTTGAA GGCTTTAGGA ATTAGCCATA CTACTGAAGA AATTGATTAT 1680
    CGTTGTTAAA TTGGTTAAAT GGCGAGTCAA GTTGGTAAGC TCCCCGGAGA ATCAAATGAG 1740
    GCATTTGAAG CCCGGCTGAA ATCACTGGAG TTGGCTAGAG CTCAAAAGCA GCCAGAAGGT 1800
    TCAAACACAC CGCCTACTCT CAGTGGTGTG CTTGCCAAAC GTAAGAGGGT TATTGAGAAT 1860
    GCACTCTCAA AGACAGTGGA CATGAGGGAG GTGTTGAAAC ACGAAACGGT TGTAATTTCC 1920
    CCAAATGTCA TGGATGAGGG TGCAATAGAT GAACTGATTC GTGCATTCGG AGAATCAGGC 1980
    ATAGCTGAGA GCGCACAATT TGATGTGGC 2009

    The RSP158 strain contains five open reading frames (i.e., ORF1-5). ORF1 and ORF5 are only partially sequenced. The nucleotide sequence of RSP158 is 87.6% identical to the corresponding region of RSPaV-1 (type strain). The numbers of amino acid residues of corresponding ORFs of RSP158 and RSPaV-1 (type strain) are exactly the same. In addition, the amino acid sequences of these ORFs have high identities to those of RSPaV-1: 99.3% for ORF1, 95% for ORF2, 99.1% for ORF3, 88.8% for ORF4, and 95.1% for ORF5. The nucleotide and amino acid sequence information of the RSP158 ORFs are described below.
  • Another DNA molecule of the present invention (RSP158 incomplete ORF1) includes nucleotides 1-447 of SEQ. ID. No. 23. This DNA molecule is believed to code for a polypeptide portion of a RSP158 replicase and comprises a nucleotide sequence corresponding to SEQ. ID. No. 24 as follows:
    GAAGCTAGCA CATTTCTGTT CAACACTATG GCTAACATGT TGTTCACTTT TCTGAGATAT  60
    GAACTGACGG GTTCAGAGTC AATAGCATTT GCAGGGGATG ATATGTGTGC TAATAGAAGG 120
    TTGCGGCTTA AAACGGAGCA TGAGGGTTTT CTGAACATGA TCTGCCTTAA GGCCAAGGTT 180
    CAGTTTGTTT CCAACCCCAC ATTCTGTGGA TGGTGCTTAT TTAAGGAGGG AATCTTCAAG 240
    AAACCTCAAC TAATTTGGGA GCGAATATGC ATAGCCAGAG AGATGGGCAA TCTGGAGAAC 300
    TGTATTGACA ATTATGCGAT AGAAGTGTCC TATGCATATA GATTGGGTGA GCTATCAATT 360
    GAAATGATGA CAGAAGAAGA AGTGGAGGCA CACTACAATT GTGTGAGGTT CCTGGTTAGG 420
    AACAAGCATA AGATGAGGTG CTCAATT 447
  • The polypeptide encoded by the nucleotide sequence of SEQ. ID. No. 24 has a deduced amino acid sequence corresponding to SEQ. ID. No. 25 as follows:
    Glu Ala Ser Thr Phe Leu Phe Asn Thr Met Ala Asn
    1               5                   10
    Met Leu Phe Thr Phe Leu Arg Tyr Glu Leu Thr Gly
            15                  20
    Ser Glu Ser Ile Ala Phe Ala Gly Asp Asp Met Cys
    25                  30                  35
    Ala Asn Arg Arg Leu Arg Leu Lys Thr Glu His Glu
                40                  45
    Gly Phe Leu Asn Met Ile Cys Leu Lys Ala Lys Val
        50                  55                  60
    Gln Phe Val Ser Asn Pro Thr Phe Cys Gly Trp Cys
                    65                  70
    Leu Phe Lys Glu Gly Ile Phe Lys Lys Pro Gln Leu
            75                  80
    Ile Trp Glu Arg Ile Cys Ile Ala Arg Glu Met Gly
    85                  90                  95
    Asn Leu Glu Asn Cys Ile Asp Asn Tyr Ala Ile Glu
                100                 105
    Val Ser Tyr Ala Tyr Arg Leu Gly Glu Leu Ser Ile
        110                 115                 120
    Glu Met Met Thr Glu Glu Glu Val Glu Ala His Tyr
                    125                 130
    Asn Cys Val Arg Phe Leu Val Arg Asn Lys His Lys
            135                 140
    Met Arg Cys Ser Ile
    145
  • Another DNA molecule of the present invention (RSP158 ORF2) includes nucleotides 506-1171 of SEQ. ID. No. 23. This DNA molecule codes for a first protein or polypeptide of the RSP158 triple gene block and comprises a nucleotide sequence corresponding to SEQ. ID. No. 26 as follows:
    ATGAATAATT TAGTTAAAGC ATTATCAGCC TTCGAGTTTA TAGGTGTTTT CAATGTGCTC  60
    AAATTTCCAG TTGTTATACA TAGTGTGCCT GGTAGTGGTA AGAGTAGCTT AATAAGGGAA 120
    TTAATCTCAG AGGACGAGAG TTTCGTGGCT TTCACAGCAG GTGTTCCAGA CAGTCCTAAC 180
    CTCACAGGGA GGTACATCAA GCCTTACTCA CCAGGATGCG CAGTGCAAGG AAAAGTGAAT 240
    ATACTTGATG AGTACTTGTC CGTTCAAGAC ATTTCGGGTT TTGATGTACT GTTTTCAGAC 300
    CCGTACCAGA ATATCAGTAT TCCCCAAGAG GCGCATTTCA TTAAGTCCAA GACTTGTAGG 360
    TTTGGTGTGA ACACTTGCAA ATACCTTTCC TCTTTCGGTT TCGAAGTTAG CAGCGACGGG 420
    CTGGACGACG TCATTGTGGG ATCGCCCTTC ACTCTAGATG TTGAAGGGGT GCTGATATGT 480
    TTTGGCAAGG AGGCGGTAGA TCTCGCTGTT GCGCACAACT CTGAATTCAA GTTGCCGTGT 540
    GAGGTTCGAG GTTCAACCTT CAATGTGGTA ACCCTTTTGA AATCAAGAGA CCCAACCCCA 600
    GAGGACAGGC ACTGGTTTTA CATCGCTGCC ACAAGACATA GGAAGAAATT GGTCATTATG 660
    CAGTAA
    666
  • The first protein or polypeptide of the RSP158 triple gene block has a deduced amino acid sequence corresponding to SEQ. ID. No. 27 as follows:
    Met Asn Asn Leu Val Lys Ala Leu Ser Ala Phe Glu
    1               5                   10
    Phe Ile Gly Val Phe Asn Val Leu Lys Phe Pro Val
            15                  20
    Val Ile His Ser Val Pro Gly Ser Gly Lys Ser Ser
    25                  30                  35
    Leu Ile Arg Glu Leu Ile Ser Glu Asp Glu Ser Phe
                40                  45
    Val Ala Phe Thr Ala Gly Val Pro Asp Ser Pro Asn
        50                  55                  60
    Leu Thr Gly Arg Tyr Ile Lys Pro Tyr Ser Pro Gly
                    65                  70
    Cys Ala Val Gln Gly Lys Val Asn Ile Leu Asp Glu
            75                  80
    Tyr Leu Ser Val Gln Asp Ile Ser Gly Phe Asp Val
    85                  90                  95
    Leu Phe Ser Asp Pro Tyr Gln Asn Ile Ser Ile Pro
                100                 105
    Gln Glu Ala His Phe Ile Lys Ser Lys Thr Cys Arg
        110                 115                 120
    Phe Gly Val Asn Thr Cys Lys Tyr Leu Ser Ser Phe
                    125                 130
    Gly Phe Glu Val Ser Ser Asp Gly Leu Asp Asp Val
            135                 140
    Ile Val Gly Ser Pro Phe Thr Leu Asp Val Glu Gly
    145                 150                 155
    Val Leu Ile Cys Phe Gly Lys Glu Ala Val Asp Leu
                160                 165
    Ala Val Ala His Asn Ser Glu Phe Lys Leu Pro Cys
        170                 175                 180
    Glu Val Arg Gly Ser Thr Phe Asn Val Val Thr Leu
                    185                 190
    Leu Lys Ser Arg Asp Pro Thr Pro Glu Asp Arg His
            195                 200
    Trp Phe Tyr Ile Ala Ala Thr Arg His Arg Lys Lys
    205                 210                 215
    Leu Val Ile Met Gln
                220

    The first protein or polypeptide of the RSP158 triple gene block has a molecular weight of about 20 to 26 kDa., preferably 24.4 kDa.
  • Another DNA molecule of the present invention (RSP158 ORF3) includes nucleotides 1173-1526 of SEQ. ID. No. 23. This DNA molecule codes for a second protein or polypeptide of the RSP158 triple gene block and comprises a nucleotide sequence corresponding to SEQ. ID. No. 28 as follows:
    ATGCCTTTTC AGCAGCCTGC TAATTGGGCA AAAACCATAA CTCCATTGAC TATTGGCTTA  60
    GGAATTGGAC TTGTGCTGCA TTTTCTGAGA AAGTCAAATC TACCATATTC AGGAGACAAC 120
    ATCCATCAAT TTCCTCACGG GGGGCGTTAC CGGGACGGCA CAAAAAGTAT AACTTACTGT 180
    GGCCCTAAGC AGTCCTTCCC CAGTTCAGGA ATATTTGGTC AGTCTGAGAA TTTTGTGCCC 240
    TTAATGCTTG TCATAGGTCT AATTGCATTC ATACATGTAT TGTCTGTTTG GAATTCTGGT 300
    CTTGGTAGGA ATTGCAATTG CCATCCAAAT CCTTGCTCAT GTAGACAACA GTAG 354
  • The second protein or polypeptide of the RSP158 triple gene block has a deduced amino acid sequence corresponding to SEQ. ID. No. 29 as follows:
    Met Pro Phe Gln Gln Pro Ala Asn Trp Ala Lys Thr
    1               5                   10
    Ile Thr Pro Leu Thr Ile Gly Leu Gly Ile Gly Leu
            15                  20
    Val Leu His Phe Leu Arg Lys Ser Asn Leu Pro Tyr
    25                  30                  35
    Ser Gly Asp Asn Ile His Gln Phe Pro His Gly Gly
                40                  45
    Arg Tyr Arg Asp Gly Thr Lys Ile Thr Tyr Cys Gly
        50                  55                  60
    Pro Lys Gln Ser Phe Pro Ser Ser Gly Ile Phe Gly
                    65                  70
    Gln Ser Glu Asn Phe Val Pro Leu Met Leu Val Ile
            75                  80
    Gly Leu Ile Ala Phe Ile His Val Leu Ser Val Trp
    85                  90                  95
    Asn Ser Gly Leu Gly Arg Asn Cys Asn Cys His Pro
                100                 105
    Asn Pro Cys Ser Cys Arg Gln Gln
        110                 115

    The second protein or polypeptide of the RSP158 triple gene block has a molecular weight of about 10 to 15 kDa., preferably 12.9 kDa.
  • Another DNA molecule of the present invention (RSP158 ORF4) includes nucleotides 1447-1689 of SEQ. ID. No. 23. This DNA molecule codes for a third protein or polypeptide of the RSP158 triple gene block and comprises a nucleotide sequence corresponding to SEQ. ID. No. 30 as follows:
    ATGTATTGTC TGTTTGGAAT TCTGGTCTTG GTAGGAATTG CAATTGCCAT CCAAATCCTT  60
    GCTCATGTAG ACAACAGTAG TGGCAGTCAC CAAGGTTGCT TTATCAGGGC CACTGGAGAG 120
    TCTATTTTGA TTGAAAATTG TGGCCCAAGC GAGGCCCTTG CATCAACAGT GAGGGAGGTG 180
    TTGGGGGGTT TGAAGGCTTT AGGAATTAGC CATACTACTG AAGAAATTGA TTATCGTTGT 240
    TAA 243
  • The third protein or polypeptide of the RSP158 triple gene block has a deduced amino acid sequence corresponding to SEQ. ID. No. 31 as follows:
    Met Tyr Cys Leu Phe Gly Ile Leu Val Leu Val Gly
    1               5                   10
    Ile Ala Ile Ala Ile Gln Ile Leu Ala His Val Asp
            15                  20
    Asn Ser Ser Gly Ser His Gln Gly Cys Phe Ile Arg
    25                  30                  35
    Ala Thr Gly Glu Ser Ile Leu Ile Glu Asn Cys Gly
                40                  45
    Pro Ser Glu Ala Leu Ala Ser Thr Val Arg Glu Val
        50                  55                  60
    Leu Gly Gly Leu Lys Ala Leu Gly Ile Ser His Thr
                    65                  70
    Thr Glu Glu Ile Asp Tyr Arg Cys
            75                  80

    The third protein or polypeptide of the RSP158 triple gene block has a molecular weight of about 5 to 10 kDa., preferably 8.4 kDa.
  • Yet another DNA molecule of the present invention (RSP158 ORF5) includes nucleotides 1699-2009 of SEQ. ID. No. 23. This DNA molecule codes for a partial RSP158 coat protein or polypeptide and comprises a nucleotide sequence corresponding to SEQ. ID. No. 32 as follows:
    ATGGCGAGTC AAGTTGGTAA GCTCCCCGGA GAATCAAATG AGGCATTTGA AGCCCGGCTG 60
    AAATCACTGG AGTTGGCTAG AGCTCAAAAG CAGCCAGAAG GTTCAAACAC ACCGCCTACT 120
    CTCAGTGGTG TGCTTGCCAA ACGTAAGAGG GTTATTGAGA ATGCACTCTC AAAGACAGTG 180
    GACATGAGGG AGGTGTTGAA ACACGAAACG GTTGTAATTT CCCCAAATGT CATGGATGAG 240
    GGTGCAATAG ATGAACTGAT TCGTGCATTC GGAGAATCAG GCATAGCTGA GAGCGCACAA 300
    TTTGATGTGG C 311
  • The polypeptide has a deduced amino acid sequence corresponding to SEQ. ID. No. 33 as follows:
    Met Ala Ser Gln Val Gly Lys Leu Pro Gly Glu Ser Asn Glu Ala Phe
    1               5                   10                  15
    Glu Ala Arg Leu Lys Ser Leu Glu Leu Ala Arg Ala Gln Lys Gln Pro
                20                  25                  30
    Glu Gly Ser Asn Thr Pro Pro Thr Leu Ser Gly Val Leu Ala Lys Arg
            35                  40                  45
    Lys Arg Val Ile Glu Asn Ala Leu Ser Lys Thr Val Asp Met Arg Glu
        50                  55                  60
    Val Leu Lys His Glu Thr Val Val Ile Ser Pro Asn Val Met Asp Glu
    65                  70                  75                  80
    Gly Ala Ile Asp Glu Leu Ile Arg Ala Phe Gly Glu Ser Gly Ile Ala
                    85                  90                  95
    Glu Ser Ala Gln Phe Asp Val
                100
  • The following seven cDNA clones are located at the central part of the ORF1 of RSPaV-1 and all have high identities (83.6-98.4%) in nucleotide sequence with the comparable regions of RSPaV-1. When their nucleotide sequences are aligned with MegAlign (DNAStar), a highly conserved region of ca. 600 nucleotides was found. The universal primers BM98-3F/BM98-3R (SEQ. ID. Nos. 51 and 52, infra) were designed based on the conserved nucleotide sequences of this region.
  • Portions of the genome from yet other strains of Rupestris stem pitting associated viruses have also been isolated and sequenced. These include strains designated 140/94-19 (T7+R1), 140/94-24 (T7+R1), 140/94-2 (T3+F1), 140/94+42 (T7+R1), 140/94-64 (T+R1), 140-94-72 (T7+R1), and 140/94-6 (T3+BM98-3F+F2).
  • The nucleotide sequence of 140/94-19 (T7+R1) corresponds to SEQ. ID. No. 34 as follows:
    GCAGGATTGA AGGCTGGCCA CTGTGTGATT TTTGATGAGG TCCAGTTGTT TCCTCCTGGA 60
    TACATCGATC TATGCTTGCT TATTATACGT AGTGATGCTT TCATTTCACT TGCCGGTGAT 120
    CCATGTCAAA GCACATATGA TTCGCAAAAG GATCGGGCAA TTTTGGGCGC TGAGCAGAGT 180
    GACATACTTA GAATGCTTGA GGGCAAAACG TATAGGTATA ACATAGAAAG CAGGAGGTTT 240
    GTGAACCCAA TGTTCGAATC AAGACTGCCA TGTCACTTCA AAAAGGGTTC GATGACTGCC 300
    GCTTTCGCTG ATTATGCAAT CTTCCATAAT ATGCATGACT TTCTCCTGGC GAGGTCAAAA 360
    GGTCCTTTGG ATGCCGTTTT GGTTTCCAGT TTTGAGGAGA AAAAGATAGT CCAGTCCTAC 420
    TTTGGAATGA AACAGCTCAC ACTCACATTT GGTGAATCAA CTGGGTTGAA TTTCAAAAAT 480
    GGGGGAATTC TCATATCACA TGATTCCTTT CAGACAGATG ATCGGCCGGT GGCTTACTGC 540
    TTTATCTCGC TTCAGCCACA ATTTGGATTT GGTGAACATT ACAGGTCTGA GGGTGGAAAG 600
    TTTCCTCTCG CACTTTGCTG GCAAACCCCT CTACCATTTT TTAACAGCCA AAAGTGGGGA 660
    GAATGTCATA GGAGATTTGC TCCCAGGTGA GCCTAACTTC TTCAGTGGCT TTAACGTTAG 720
    CATTGGAAAG AATGAAGGTG TTAGGGAGGA GAAGTTATGT GGTGACCCAT GGTTAAAAGT 780
    CATGCTTTTC CTGGGTCAAG ATGAGGATTG TGAAGTTGAA GAGATGGAGT CAGAGTGCTC 840
    AAATGAAGAA TGGTTTAAAA CCCACATTCC CCTGAGTAAT CTGGAGTCAA CCAGGGCTAG 900
    GTGGGTGGGT AAAATGGCTT TGAAAGAGTA TCGGGAGGTG CGTTGTGGTT ATGAAATGAC 960
    TCAACAATTC TTTGATGAGC ATAGGGGTGG AACTGGTGAG CAACTGAGCA ATGCATGTGA 1020
    GAGGTTTGAA AGCATTTACC CAAGGCATAA AGGAAATGAT TCAATAACCT TCCTTATGGC 1080
    TGTCCGAAAG CGTCTCAAAT TTTCGAAGCC CCAGGTTGAA GCTGCCAAAC TGAGGCGGGC 1140
    CAAACCATAT GGGAAATTCT TATTAGACTT TCCTATCCAA AATCCCATTG AAAGCCAGTC 1200
    ATAATT 1206
  • The nucleotide sequence of 140/94-24 (T7+R1) corresponds to SEQ. ID. No. 35 as follows:
    ATTAACCCAA ATGGTAAGAT TTCCGCCTTG TTTGATATAA CCAATGAGCA CATAAGGCAT 60
    GTTGAGAAGA TCGGCAATGG CCCTCAGAGC ATAAAAGTAG ATGAGTTGAG GAAGGTTAAG 120
    CGATCCGCCC TTGATCTTCT TTCAATGAAT GGGTCCAAAA TAACCTATTT TCCAAACTTT 180
    GAGCGGGCTG AAAAGTTGCA AGGGTGCTTG CTAGGGGGCC TAACTGGTGT CATAAGTGAT 240
    GAAAAGTTCA GTGATGCAAA ACCCTGGCTT TCTGGTATAT CAACTGCGGA TATAAAGCCA 300
    AGAGAGCTAA CTGTCGTGCT TGGCACTTTT GGGGCTGGAA AGAGTTTCTT GTATAAGAGT 360
    TTCATGAAGA GATCTGAGGG AAAATTTGTA ACTTTTGTTT CCCCTAGACG AGCCTTGGCA 420
    AATTCAATCA AAAATGATCT TGAAATGGAT GATGGCTGCA AAGTTGCCAA AGCAGGCAAA 480
    TCAAAGAAGG AAGGGTGGGA TGTAGTGACC TTTGAAGTTT TCCTTAGAAA AGTTTCTGGT 540
    TTGAAAGCTG GTCATTGTGT GATTTTTGAT GAGGTTCAGT TGTTTCCCCC TGGATACATC 600
    GATCTGTGTT TACTTGTCAT ACGAAGTGAT GCTTTCATTT CACTTGCTGG TGATCCATGC 660
    CAGAGGACAT ATGATTCACA GAAGGATCGA GCAATTTTGG GAGCTGAGCA GAGTGACATA 720
    CTCAGACTGC TTGAAGGAAA GACATATAGG TACAACATAG AAAGCAGACG TTTTGTGAAC 780
    CCAATGTTTG AATCTAGACT ACCATGTCAC TTCAAAAAGG GTTCAATGAC TGCAGCCTTT 840
    GCTGATTATG CAATCTTCCA CAATATGCAT GACTTCCTCC TGGCGAGGTC AAAAGGCCCC 900
    TTGGATGCTG TTCTAGTTTC CAGTTTTGAG GAGAAGAAAA TAGTCCAATC CTACTTTGGG 960
    ATGAAGCAAC TCACTCTCAC ATTTGGTGAA TCAACTGGGT TGAACTTCAA AAATGGAGGA 1020
    ATTCTCATAT CACATGACTC CTTTCATACT GACGATCGAC GGTGGCTTAC TGCTTTATCT 1080
    CGATTCAGCC ATAATTTGGA TTTGGTGAAC ATCACAGGTC TTGAGGGTGG AAAGTTTTCT 1140
    CTCACATTTT GCTGGTAAAC CCCTTTACCA CTTTTTGACG GCTTAAAAGT GGAGAGAATG 1200
    TCATACGAGA CCTGCTTCAG GTGAGCCTAA CTTCTTTTAG GGGTTCAATG TCAGCATTGG 1260
    AAAAAAATGG AAGGGGTTAG AGAA 1284
  • The nucleotide sequence of 140/94-2 (T3+F1) corresponds to SEQ. ID. No. 36 as follows:
    CATTTTTAAA ATTTAATCCA GTCGACTCAC CAAATGTGAG CGTAAGCTGT TTCATCCCAA 60
    AGTAGGACTG GACTATTTTC TTCTCCTCAA AACTAGAAAC CAGAATGGCA TCCAAAGGAC 120
    CTTTTGACCT TGCCAGGAGG AAATCATGCA TATTGTGGAA AATGGCATAA TCAGCAAAGG 180
    CAGCAGTCAT TGTACCCTTT TTGAAGTGAC ATGGCAGTCG AGATTCAAAC ATTGGGTTCA 240
    CAAATCTTCT GCTTTCTATG TTGTACCTAT ACGTCTTGCC TTCAAGTATT TTGAGTATGT 300
    CACTCTGCTC AGCGCCCAAA ATCGCCCGAT CTTTTTGTGA GTCATATGTG CTCTGACATG 360
    GGTCACCAGC AAGTGAAATG AAAGCATCAC TACGTATAAT AAGCAAACAT AGATCGATGT 420
    ATCCAGGGGG AAACAACTGG ACCTCATCGA AAATTACACA GTGACCAGCT TTTAGACCTG 480
    CAACTTTTCT AAGGAAGACT TCAAAAGTCA CAACATCCCA TCCTTCCTTC TTTGACCTGC 540
    CTGCTTTGGC AACTTTGCAG CTATCATCCA TTTCAAGATC ATTTTTGATT GAATTCGCTA 600
    GAGCCCGTCT GGGGGAAACA AAAGTTACGA ATTTACCCTC AGATCTTTTC ATAAAGCTCT 660
    TGTACAAAAA GCTTTTTCCG GCTCCAAATG TGCCAAGCAC AACAGTTAGC TCCCTCGGCT 720
    TAATGTCAGT AGTTGATATA CCAGAAAGCC AGGGCTTTGC ATCACTGAAC TTCTCATCAC 780
    TTATGACACC AGTTAGGCCT CCTAGCAGAC ACCCTTGCAA CTTTTCAGCC CGCTCAAAAC 840
    TTGGGAAGTA GGTTACCTTG GACCCATTAA TTGAAAGAAG ATCAAGGGCG GATCGCTTGA 900
    CCTTTCGCAA TTCATCTACT TTAATGCTCT GAGGGCCATT ACCTATCTTT TCAACATGCC 960
    TTATGTGCTC ATTAGTTATG TCAAACAGAG CGGAAAACTT GCCATGTGGA TTAATCACCT 1020
    CAATTTCCCC ATTTATGTCA CACTTAGCGC AAATGTCAAA AGCCTCAAAG GCTTCAGCTA 1080
    AGTTACATCA TGTTGAGCCT CCCCCTTGGC AAAGCTCCTC AAAAATGTGG TTAGTGCTAG 1140
    GCCTGCACAA TAATTAACAC ATCAACTTCA CCCTGCCAAT GCTGAACAAT ACTGTTATCA 1200
    TGCAACCATC CATGGGGCAC ATGGTTGGAA TTGATTGATT TAAGGCAAAA ATCCCCACAG 1260
    GGGGCATCCC CTTCCCCAAT TTCCACTGAT TCATACTCTG GCGTTATCAT ATCAACCCAA 1320
    TGTGTCAAAT ACAAATAATG CAATCTCTCA TCTCCGATAA CATTTCCCCC ATTTTTTAAA 1380
    ATGGTGGGG TGAAAATTGG AA 1402
  • The nucleotide sequence of 140/94-42 (T7+R1) corresponds to SEQ. ID. No. 37 as follows:
    GTGGTTTTTG CAACAACAGG CCCAGGTCTA TCTAAGGTTT TGGAAATGCC TCGAAGCAAG 60
    AAGCAATCTA TTCTGGTTCT TGAGGGAGCC CTATCCATAG AAACGGACTA TGGCCCAAAA 120
    GTTCTGGGAT CTTTTGAAGT TTTCAAAGGG GATTTCAACA TTAAAAAAAT GGAAGAAAGT 180
    TCCATCTTTG TAATAACATA CAAGGCCCCA GTTAGATCTA CTGGCAAGTT GAGGGTCCAC 240
    CAATCAGAAT GCTCATTTTC TGGATCCAAG GAGGTATTGC TGGGTTGTCA GATTGAGGCA 300
    TGTGCTGATT ATGATATTGA TGATTTCAAT ACTTTCTTTG TACCTGGTGA TGGTAATTGC 360
    TTTTGGCATT CAGTTGGTTT CTTACTCAGT ACTGACGGAC TTGCTTTGAA GGCCGGCATT 420
    CGTTCTTTCG TGGAGAGTGA ACGCCTGGTG AGTCCAGATC TTTCAGCCCC AACCATTTCT 480
    AAACAACTGG GGGAAAATGC TTATGCCGAG AATGAGATGA TTGCATTATT TTGTATTCGA 540
    CACCATGTGA GGCTGATAGT GATTACGCCA GAGTATGAAG TCAGTTGGAA ATTTGGGGAA 600
    GGTGAATGGC CCCTGTGCGG AATTCTTTGC CTTAAATCAA ATCACTTCCA ACCATGTGCC 660
    CCATTGAATG GTTGCATGAT TACAGCTATT GCTTCAGCAC TTGGTAGGCG TGAAGTTGAT 720
    GTGCTTAATT ATCTGTGCAG GCCTAGCACT AACCACATTT TTGAGGAGCT TTGCCAAGGG 780
    GGAGGCCTCA ACATGATGTA CTTAGCTGAA GCCTTTGAGG CTTTTGACAT TTGCGCTAAG 840
    TGTGACATAA ATGGGGAAAT TGAGGTGATT AATCCACATG GCAAGTTTTC CGCTCTGTTT 900
    GACATAACTA ATGAGCACAT AAGGCATGTT GAAAAGATAG GTAATGGCCC TCAGAGCATT 960
    AAAGTAGATG AATTGCGAAA GGTCAAGCGA TCTGCCCTTG ATCTTCTTTC AATTAATGGG 1020
    TCCAAGGTAA CCTACTTCCC AAGTTTTGAG CGGGCTGAAA AGTTGCAAGG GTGTCTGCTA 1080
    GGAGGCCTAA CTGGTGTCAT AAGTGATGAG AAAGTCAGTG ATGCAAAGCC CTGCTTTTTG 1140
    GTATATCAAC TACTGACATT AAGCCGAGGG AGCTAACTGT TGTGCTTTGG CACATTTGGA 1200
    GCCCGGAAAA AGCCTTTTGT ACCAAGAGCT TTATTG 1236
  • The nucleotide sequence of 140/94-6 (T3+BM98-3F+F2) corresponds to SEQ. ID. No. 38 as follows:
    GTCTAACTGG CGTTATAAGT GATGAGAAAT TCAGTGATGC AAAACCTTGG CTTTCTGGTA 60
    TATCTACTAC AGATATTAAG CCAAGGGAAT TAACTGTTGT GCTTGGTACA TTTGGGGCTG 120
    GGAAGAGTTT CTTGTACAAG AGTTTGATGA AAAGGTCTGA GGGTAAATTC GTAACCTTTG 180
    TTTCTCCCAG ACGTGCTTTA GCAAATTCAA TCAAAAATGA TCTTGAAATG GATGATAGCT 240
    GCAAAGTTGC CAAAGCAGGT AGGTCAAAGA AGGAAGGGTG GGATGTAGTA ACTTTTGAGG 300
    TCTTCCTCAG AAAAGTTGCA GGATTGAAGG CTGGCCACTG TGTGATTTTT GATGAGGTCC 360
    AGTTGTTTCC TCCTGGATAC ATCGATCTAT GCTTGCTTAT TATACGTAGT GATGCTTTCA 420
    TTTCACTTGC CGGTGATCCA TGTCAAAGGA CATATGATTC GCAAAAGGAT CGGGCAATTT 480
    TGGGCGCTGA GCAGAGTGAC ATACTTAGAA TGCTTGAGGG CAAAACGTAT AGGTATAACA 540
    TAGAAAGCAG GAGGTTTGTG AACCCAATGT TCGAATCAAG ACTGCCATGT CACTTCAAAA 600
    AGGGTTCGAT GACTGCCGCT TTCGCTGATT ATGCAATCTT CCATAATATG CATGACTTTC 660
    TCCTGGCGAG GTCAAAAGGT CCTTTGGATG CCGTTTTGGT TTCCAGTTTT GAGGAGAAAA 720
    AGATAGTCCA GTCCTACTTT GGAATGAAAC AGCTCACACT CACATTTGGT GAATCAACTG 780
    GGTTGAATTT CAAAAATGGG GGAATTCTCA TATCACATGA TTCCTTTCAC ACAGATGATC 840
    GGCGGTGGCT TACTGCTTTA TCTCGCTTCA GCCACAATTT GGATTTGGTG AACATTACAG 900
    GTCTGAGGTG GAAAGTTTCC TCTCGCACTT TGCTGGCAAA CCCCTCTACC ATTTTTTAAC 960
    AGCCAAAAGT GGGGAGAATG TCATACGAGA TTTGCTCCCA GGTGAGCCTA ACTTCTTCAG 1020
    TGGCTTTAAC GTTAGCATTG GAAAGAATGA AGGTGTTAGG GAGGAGAAGT TATGTGGTGA 1080
    CCCATGGTTA AAAGTCATGC TTTTCCTGGG TCAAGATGAG GATTGTGAAG TTGAAGAGAT 1140
    GGAGTCAGAG TGCTCAAATG AAGAATGGTT TAAAACCCAC ATTCCCCTGA GTAATCTGGA 1200
    GTCAACCAGG GCTAGGTGGG TGGGTAAAAT GGCCTTGAAA GAGTATCGGG AGGTGCGTTG 1260
    TGGTTATGAA ATGACTCAAC AATTCTTTGA TGACAT 1296
  • The nucleotide sequence of 140/94-64 (T7+R1) corresponds to SEQ. ID. No. 39 as follows:
    ATGTTCACCA AATCCAAATT ATGGCTGAAG CGAGATAAAG CAGTAAGCCA CCGCCGATCA 60
    TCTGTGTGAA AGGAATCATG TGATATGAGA ATTCCCCCAT TTTTGAAATT CAACCCAGTT 120
    GATTCACCAA ATGTGAGTGT GAGCTGTTTC ATTCCAAAGT AGGACTGGAC TATCTTTTTC 180
    TCCTCAAAAC TGGAAACCAA AACGGCATCC AAAGGACCTT TTGACCTCGC CAGGAGAAAG 240
    TCATGCATAT TATGGAAGAT TGCATAATCA GGGAAAGCGG CAGTCATTGA GCCCTTTTTG 300
    AATTGACATG GCAGTCTTGA TTCGAACATT GGATTCACAA ACCTCCTGCT TTCAATGTTA 360
    TACCTATACG TCTTGCCCTC AAGCAGTCTA AGTATGTCAC TCTGCTCAGC GCCCAAAATT 420
    GCCCGATCCT TTTGCGAATC ATATGTGCTT TGACATGGAT CACCGGCAAG TGAAATGAAA 480
    GCATCACTAC GTATAATAAG CAAGCATAGA TCGATGTATC CAGGAGGAAA CAACTGGACC 540
    TCATCGAAAA TCACACAGTG GCCAGCCTTC AATCCTGCAA CTTTTCTGAG GAAAACCTCA 600
    AAAGTTACTA CATCCCACCC TTCCTTCTTT GACCTACCTG CTTTAGCAAC TTTGCAGCTA 660
    TCATCCATTT CAAGATCATT TTTGATTGAA TTTGCTAAAG CACGTCTGGG AGAAACAAAG 720
    GTTACGAATT TACCCTCAGA CCTTTTCATG AAACTCTTGT ACAAGAAACT CTTCCCAGCC 780
    CCAAATGTAC CAAGCACGAC AGTCAACTCC CTTGGCTTAA TATCAGTAGT AGATATACCA 840
    GAAAGCCAAG GTTTTGCATC ACTGAACTTC TCATCACTTA TAACGCCAGT TAGGCCCCCT 900
    AGCAAC 907
  • The nucleotide sequence of 140-94-72 (T7+R1) corresponds to SEQ. ID. No. 40 as follows:
    AGAATGCTTA TGCTGAGAAT GAGATGATTG CATTATTTTG CATCCGGCAC CATGTAAGGC 60
    TTATAGTAAT AACACCGGAA TATGAAGTTA GTTGGAAATT TGGGGAAAGT GAGTGGCCCC 120
    TATGTGGAAT TCTTTGCCTG AGGTCCAATC ACTTCCAACC ATGCGCCCCG CTGAATGGTT 180
    GCATGATCAC GGCTATTGCT TCAGCACTTG GGAGGCGTGA GGTTGATGTG TTAAATTATC 240
    TGTGTAGGCC TAGCACTAAT CACATCTTTG AGGAGCTGTG CCAGGGCGGA GGGCTTAATA 300
    TGATGTACTT GGCTGAAGCT TTTGAGGCCT TTGACATTTG TGCAAAGTGC GACATAAATG 360
    GGGAAATTGA GGTCATTAAC CCAAATGGCA AGATTTCCGC CTTGTTTGAT ATAACTAATG 420
    AGCACATAAG GCATGTTGAG AAGATCAGCA ATGGCCCTCA GAGCATAAAA ATAGATGAGT 480
    TGAGGAAGGT TAAGCGATCC CGCCTTGACC TTCTTTCAAT GAATGGGTCC AAAATAACCT 540
    ATTTTCCAAA CTTTGAGCGG GCTGAAAAGT TGCAAGGGTG CTTGCTAGAG GGCCTGACTG 600
    GTGTCATAAG TGATGAAAAG TTCAGTGATG CAAAACCTTG GCTTTCTGGT ATATCAACTG 660
    CGGATATTAA GCCAAGAGAG CTAACTGTCG TGCTTGGCAC ATTTGGTGCT GGAAAGAGTT 720
    TCTTGTATAA GAGTTTCATG AAGAGATCTG AAGGAAAATT TGTAACTTTT GTTTCCCCTA 780
    GGCGAGCTTT GGCCAATTCG ATCAAGAATG ATCTTGAAAT GGATGATGGC TGCAAAGTTG 840
    CCAAAGCAGG CAAGTCAAAG AAGGAAGGGT GGGATGTGGT AACATTTGAG GTTTTCCTTA 900
    GAAAAGTTTC TGGTTTGAAG GCTGGTCATT GTGTGATTTT CGATGAGGTT CAGTTGTTTC 960
    CCCCTGGATA TATCGATCTA TGTTTACTTG TCATACGCAG TGATGCTTTT ATTTCACTTG 1020
    CCGGTGATCC ATGCCAGAGC ACATATGATT CACAAAAGGA TCGGGCAATT TTGGGAGCTG 1080
    AGCAGAGTGA CATACTCAGA TTGCTTGAAG GAAAGACGTA TAGGTACAAC ATAGAAAGCA 1140
    GACGTTTTGT GAACCCAATG TTTGAATTTA GACTACCATG TCACTTCAAA AAAGGGTTCA 1200
    ATGACTGCTG CCTTTGCTGA TTATGCAATC TT
  • Also encompassed by the present invention are fragments of the DNA molecules of the present invention. Suitable fragments capable of imparting RSP resistance to grape plants are constructed by using appropriate restriction sites, revealed by inspection of the DNA molecule's sequence, to: (i) insert an interposon (Felley et al., “Interposon Mutagenesis of Soil and Water Bacteria: A Family of DNA Fragments Designed for in vitro Insertion Mutagenesis of Gram-negative Bacteria,” Gene 52:147-15 (1987), which is hereby incorporated by reference) such that truncated forms of the RSP virus polypeptide or protein, that lack various amounts of the C-terminus, can be produced or (ii) delete various internal portions of the protein. Alternatively, the sequence can be used to amplify any portion of the coding region, such that it can be cloned into a vector supplying both transcription and translation start signals.
  • Suitable DNA molecules are those that hybridize to a DNA molecule comprising a nucleotide sequence of at least 15 continuous bases of SEQ. ID. No. 1 under stringent conditions characterized by a hybridization buffer comprising 0.9M sodium citrate (“SSC”) buffer at a temperature of 37° C. and remaining bound when subject to washing with SSC buffer at 37° C.; and preferably in a hybridization buffer comprising 20% formamide in 0.9M saline/0.9M SSC buffer at a temperature of 42° C. and remaining bound when subject to washing at 42° C. with 0.2×SSC buffer at 42° C.
  • Variants may also (or alternatively) be modified by, for example, the deletion or addition of nucleotides that have minimal influence on the properties, secondary structure and hydropathic nature of the encoded protein or polypeptide. For example, the nucleotides encoding a protein or polypeptide may be conjugated to a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or post-translationally directs transfer of the protein. The nucleotide sequence may also be altered so that the encoded protein or polypeptide is conjugated to a linker or other sequence for ease of synthesis, purification, or identification of the polypeptide.
  • The protein or polypeptide of the present invention is preferably produced in purified form (preferably, at least about 80%, more preferably 90%, pure) by conventional techniques. Typically, the protein or polypeptide of the present invention is isolated by lysing and sonication. After washing, the lysate pellet is re-suspended in buffer containing Tris-HCl. During dialysis, a precipitate forms from this protein solution. The solution is centrifuged, and the pellet is washed and re-suspended in the buffer containing Tris-HCl. Proteins are resolved by electrophoresis through an SDS 12% polyacrylamide gel.
  • The DNA molecule encoding the RSP virus protein or polypeptide of the present invention can be incorporated in cells using conventional recombinant DNA technology. Generally, this involves inserting the DNA molecule into an expression system to which the DNA molecule is heterologous (i.e., not normally present). The heterologous DNA molecule is inserted into the expression system or vector in proper sense orientation and correct reading frame. The vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences.
  • U.S. Pat. No. 4,237,224 to Cohen and Boyer, which is hereby incorporated by reference, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including procaryotic organisms and eukaryotic cells grown in tissue culture.
  • Recombinant genes may also be introduced into viruses, such as vaccinia virus. Recombinant viruses can be generated by transfection of plasmids into cells infected with virus.
  • Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gt11, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKCO101, SV 40, pBluescript II SK +/− or KS+/− (see “Stratagene Cloning Systems” Catalog (1993) from Stratagene, La Jolla, Calif., which is hereby incorporated by reference), pQE, pIH821, pGEX, pET series (see Studier et. al., “Use of T7 RNA Polymerase to Direct Expression of Cloned Genes,” Gene Expression Technology, vol. 185 (1990), which is hereby incorporated by reference), and any derivatives thereof. Suitable vectors are continually being developed and identified. Recombinant molecules can be introduced into cells via transformation, transduction, conjugation, mobilization, or electroporation. The DNA sequences are cloned into the vector using standard cloning procedures in the art, as described by Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1982), which is hereby incorporated by reference.
  • A variety of host-vector systems may be utilized to express the protein-encoding sequence(s). Primarily, the vector system must be compatible with the host cell used. Host-vector systems include but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); and plant cells infected by bacteria or transformed via particle bombardment (i.e. biolistics). The expression elements of these vectors vary in their strength and specificities. Depending upon the host-vector system utilized, any one of a number of suitable transcription and translation elements can be used.
  • Different genetic signals and processing events control many levels of gene expression (e.g., DNA transcription and messenger RNA (“mRNA”) translation).
  • Transcription of DNA is dependent upon the presence of a promoter which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis. The DNA sequences of eukaryotic promoters differ from those of procaryotic promoters. Furthermore, eukaryotic promoters and accompanying genetic signals may not be recognized in or may not function in a procaryotic system, and, further, procaryotic promoters are not recognized and do not function in eukaryotic cells.
  • Similarly, translation of mRNA in procaryotes depends upon the presence of the proper procaryotic signals which differ from those of eukaryotes; Efficient translation of mRNA in procaryotes requires a ribosome binding site called the Shine-Dalgarno (“SD”) sequence on the mRNA. This sequence is a short nucleotide sequence of mRNA that is located before the start codon, usually AUG, which encodes the amino-terminal methionine of the protein. The SD sequences are complementary to the 3′-end of the 16S rRNA (ribosomal RNA) and probably promote binding of mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome. For a review on maximizing gene expression, see Roberts and Lauer, Methods in Enzymology, 68:473 (1979), which is hereby incorporated by reference.
  • Promoters vary in their “strength” (i.e., their ability to promote transcription). For the purposes of expressing a cloned gene, it is desirable to use strong promoters in order to obtain a high level of transcription and, hence, expression of the gene. Depending upon the host cell system utilized, any one of a number of suitable promoters may be used. For instance, when cloning in E. coli, its bacteriophages, or plasmids, promoters such as the T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the PR and PL promoters of coliphage lambda and others, including but not limited, to lacUV5, ompF, bla, Ipp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacV5 (tac) promoter or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.
  • Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promoter unless specifically induced. In certain operons, the addition of specific inducers is necessary for efficient transcription of the inserted DNA. For example, the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside). A variety of other operons, such as trp, pro, etc., are under different controls.
  • Specific initiation signals are also required for efficient gene transcription and translation in procaryotic cells. These transcription and translation initiation signals may vary in “strength” as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively. The DNA expression vector, which contains a promoter, may also contain any combination of various “strong” transcription and/or translation initiation signals. For instance, efficient translation in E. coli requires a Shine-Dalgarno (“SD”) sequence about 7-9 bases 5′ to the initiation codon (“ATG”) to provide a ribosome binding site. Thus, any SD-ATG combination that can be utilized by host cell ribosomes may be employed. Such combinations include but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DNA or other techniques involving incorporation of synthetic nucleotides may be used.
  • Once the isolated DNA molecules encoding the various Rupestris stem pitting associated virus proteins or polypeptides, as described above, have been cloned into an expression system, they are ready to be incorporated into a host cell. Such incorporation can be carried out by the various forms of transformation noted above, depending upon the vector/host cell system. Suitable host cells include, but are not limited to, bacteria, virus, yeast, mammalian cells, insect, plant, and the like.
  • The present invention also relates to RNA molecules which encode the various RSP virus proteins or polypeptides described above. The transcripts can be synthesized using the host cells of the present invention by any of the conventional techniques. The mRNA can be translated either in vitro or in vivo. Cell-free systems typically include wheat-germ or reticulocyte extracts. In vivo translation can be effected, for example, by microinjection into frog oocytes.
  • One aspect of the present invention involves using one or more of the above DNA molecules encoding the various proteins or polypeptides of a RSP virus to transform grape plants in order to impart RSP resistance to the plants. The mechanism by which resistance is imparted in not known. In one hypothetical mechanism, the transformed plant can express the coat protein or polypeptide, and, when the transformed plant is inoculated by a RSP virus, such as RSPaV-1, the expressed coat protein or polypeptide surrounds the virus, thereby preventing translation of the viral DNA.
  • In this aspect of the present invention, the subject DNA molecule incorporated in the plant can be constitutively expressed. Alternatively, expression can be regulated by a promoter which is activated by the presence of RSP virus. Suitable promoters for these purposes include those from genes expressed in response to RSP virus infiltration.
  • The isolated DNA molecules of the present invention can be utilized to impart RSP virus resistance for a wide variety of grapevine plants. The DNA molecules are particularly well suited to imparting resistance to Vitis scion or rootstock cultivars. Scion cultivars which can be protected include those commonly referred to as Table or Raisin Grapes, such as Alden, Almeria, Anab-E-Shahi, Autumn Black, Beauty Seedless, Black Corinth, Black Damascus, Black Malvoisie, Black Prince, Blackrose, Bronx Seedless, Burgrave, Calmeria, Campbell Early, Canner, Cardinal, Catawba, Christmas, Concord, Dattier, Delight, Diamond, Dizmar, Duchess, Early Muscat, Emerald Seedless, Emperor, Exotic, Ferdinand de Lesseps, Fiesta, Flame seedless, Flame Tokay, Gasconade, Gold, Himrod, Hunisa, Hussiene, Isabella, Italia, July Muscat, Khandahar, Katta, Kourgane, Kishmishi, Loose Perlette, Malaga, Monukka, Muscat of Alexandria, Muscat Flame, Muscat Hamburg, New York Muscat, Niabell, Niagara, Olivette blanche, Ontario, Pierce, Queen, Red Malaga, Ribier, Rish Baba, Romulus, Ruby Seedless, Schuyler, Seneca, Suavis (IP 365), Thompson seedless, and Thomuscat. They also include those used in wine production, such as Aleatico, Alicante Bouschet, Aligote, Alvarelhao, Aramon, Baco blanc (22A), Burger, Cabernet franc, Cabernet, Sauvignon, Calzin, Carignane, Charbono, Chardonnay, Chasselas dore, Chenin blanc, Clairette blanche, Early Burgundy, Emerald Riesling, Feher Szagos, Fernao Pires, Flora, French Colombard, Fresia, Furmint, Gamay, Gewurztraminer, Grand noir, Gray Riesling, Green Hungarian, Green Veltliner, Grenache, Grillo, Helena, Inzolia, Lagrein, Lambrusco de Salamino, Malbec, Malvasia bianca, Mataro, Melon, Merlot, Meunier, Mission, Montua de Pilas, Muscadelle du Bordelais, Muscat blanc, Muscat Ottonel, Muscat Saint-Vallier, Nebbiolo, Nebbiolo fino, Nebbiolo Larnpia, Orange Muscat, Palomino, Pedro Ximenes, Petit Bouschet, Petite Sirah, Peverella, Pinot noir, Pinot Saint-George, Primitivo di Gioa, Red Veltliner, Refosco, Rkatsiteli, Royalty, Rubired, Ruby abernet, Saint-Emilion, Saint Macaire, Salvador, Sangiovese, Sauvignon blanc, Sauvignon gris, Sauvignon vert, Scarlet, Seibel 5279, Seibel 9110, Seibel 13053, Semillon, Servant, Shiraz, Souzao, Sultana Crimson, Sylvaner, Tannat, Teroldico, Tinta Madeira, Tinto cao, Touriga, Traminer, Trebbiano Toscano, Trousseau, Valdepenas, Viognier, Walschriesling, White Riesling, and Zinfandel. Rootstock cultivars which can be protected include Couderc 1202, Couderc 1613, Couderc 1616, Couderc 3309, Dog Ridge, Foex 33 EM, Freedom, Ganzin 1 (A×R #1), Harmony, Kober 5BB, LN33, Millardet & de Grasset 41B, Millardet & de Grasset 420A, Millardet & de Grasset 101-14, Oppenheim 4 (SO4), Paulsen 775, Paulsen 1045, Paulsen 1103, Richter 99, Richter 110, Riparia Gloire, Ruggeri 225, Saint-George, Salt Creek, Teleki 5A, Vitis rupestris Constantia, Vitis California, and Vitis girdiana.
  • Plant tissue suitable for transformation include leaf tissue, root tissue, meristems, zygotic and somatic embryos, and anthers. It is particularly preferred to utilize embryos obtained from anther cultures.
  • The expression system of the present invention can be used to transform virtually any plant tissue under suitable conditions. Tissue cells transformed in accordance with the present invention can be grown in vitro in a suitable medium to impart RSPaV resistance. Transformed cells can be regenerated into whole plants such that the protein or polypeptide imparts resistance to RSPaV in the intact transgenic plants. In either case, the plant cells transformed with the recombinant DNA expression system of the present invention are grown and caused to express that DNA molecule to produce one of the above-described RSPaV proteins or polypeptides and, thus, to impart RSPaV resistance.
  • In producing transgenic plants, the DNA construct in a vector described above can be microinjected directly into plant cells by use of micropipettes to transfer mechanically the recombinant DNA. Crossway, Mol. Gen. Genetics, 202:179-85 (1985), which is hereby incorporated by reference. The genetic material may also be transferred into the plant cell using polyethylene glycol. Krens, et al., Nature, 296:72-74 (1982), which is hereby incorporated by reference.
  • One technique of transforming plants with the DNA molecules in accordance with the present invention is by contacting the tissue of such plants with an inoculum of a bacteria transformed with a vector comprising a gene in accordance with the present invention which imparts RSPaV resistance. Generally, this procedure involves inoculating the plant tissue with a suspension of bacteria and incubating the tissue for 48 to 72 hours on regeneration medium without antibiotics at 25-28° C.
  • Bacteria from the genus Agrobacterium can be utilized to transform plant cells. Suitable species of such bacterium include Agrobacterium tumefaciens and Agrobacterium rhizogenes. Agrobacterium tumefaciens (e.g., strains C58, LBA4404, or EHA105) is particularly useful due to its well-known ability to transform plants.
  • Heterologous genetic sequences can be introduced into appropriate plant cells, by means of the Ti plasmid of A. tumefaciens or the R1 plasmid of A. rhizogenes. The Ti or R1 plasmid is transmitted to plant cells on infection by Agrobacterium and is stably integrated into the plant genome. J. Schell, Science, 237:1176-83 (1987), which is hereby incorporated by reference.
  • After transformation, the transformed plant cells must be regenerated.
  • Plant regeneration from cultured protoplasts is described in Evans et al., Handbook of Plant Cell Cultures, Vol. 1: (MacMillan Publishing Co., New York, 1983); and Vasil I. R. (ed.), Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I, 1984, and Vol. III (1986), which are hereby incorporated by reference.
  • It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to, all major species of sugarcane, sugar beets, cotton, fruit trees, and legumes.
  • Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts or a petri plate containing explants is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced in the callus tissue. These embryos germinate as natural embryos to form plants. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is usually reproducible and repeatable.
  • After the expression cassette is stably incorporated in transgenic plants, it can be transferred to other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
  • Once transgenic plants of this type are produced, the plants themselves can be cultivated in accordance with conventional procedure so that the DNA construct is present in the resulting plants. Alternatively, transgenic seeds are recovered from the transgenic plants. These seeds can then be planted in the soil and cultivated using conventional procedures to produce transgenic plants.
  • Another approach to transforming plant cells with a gene which imparts resistance to pathogens is particle bombardment (also known as biolistic transformation) of the host cell. This can be accomplished in one of several ways. The first involves propelling inert or biologically active particles at cells. This technique is disclosed in U.S. Pat. Nos. 4,945,050, 5,036,006, and 5,100,792, all to Sanford et al., and in Emerschad et al., “Somatic Embryogenesis and Plant Development from Immature Zygotic Embryos of Seedless Grapes (Vitis vinifera),” Plant Cell Reports, 14:6-12 (1995) (“Emerschad (1995)”), which are hereby incorporated by reference. Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and to be incorporated within the interior thereof. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the heterologous DNA. Alternatively, the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle. Biologically active particles (e.g., dried bacterial cells containing the vector and heterologous DNA) can also be propelled into plant cells.
  • Once a grape plant tissue is transformed in accordance with the present invention, it is regenerated to form a transgenic grape plant. Generally, regeneration is accomplished by culturing transformed tissue on medium containing the appropriate growth regulators and nutrients to allow for the initiation of shoot meristems. Appropriate antibiotics are added to the regeneration medium to inhibit the growth of Agrobacterium and to select for the development of transformed cells. Following shoot initiation, shoots are allowed to develop tissue culture and are screened for marker gene activity.
  • The DNA molecules of the present invention can be made capable of transcription to a messenger RNA that does not translate to the protein. This is known as RNA-mediated resistance. When a Vitis scion or rootstock cultivar is transformed with such a DNA molecule, the DNA molecule can be transcribed under conditions effective to maintain the messenger RNA in the plant cell at low level density readings. Density readings of between 15 and 50 using a Hewlet ScanJet and Image Analysis Program are preferred.
  • A portion of one or more DNA molecules of the present invention as well as other DNA molecules can be used in a transgenic grape plant in accordance with U.S. patent application Ser. No. 09/025,635, which is hereby incorporated herein by reference.
  • The RSPaV protein or polypeptide can also be used to raise antibodies or binding portions thereof or probes. The antibodies can be monoclonal or polyclonal.
  • Monoclonal antibody production may be effected by techniques which are well-known in the art. Basically, the process involves first obtaining immune cells (lymphocytes) from the spleen of a manunal (e.g., mouse) which has been previously immunized with the antigen of interest either in vivo or in vitro. The antibody-secreting lymphocytes are then fused with (mouse) myeloma cells or transformed cells, which are capable of replicating indefinitely in cell culture, thereby producing an immortal, immunoglobulin-secreting cell line. The resulting fused cells, or hybridomas, are cultured, and the resulting colonies screened for the production of the desired monoclonal antibodies. Colonies producing such antibodies are cloned, and grown either in vivo or in vitro to produce large quantities of antibody. A description of the theoretical basis and practical methodology of fusing such cells is set forth in Kohler and Milstein, Nature, 256:495 (1975), which is hereby incorporated by reference.
  • Mammalian lymphocytes are immunized by in vivo immunization of the animal (e.g., a mouse) with the protein or polypeptide of the present invention. Such immunizations are repeated as necessary at intervals of up to several weeks to obtain a sufficient titer of antibodies. Following the last antigen boost, the animals are sacrificed and spleen cells removed.
  • Fusion with mammalian myeloma cells or other fusion partners capable of replicating indefinitely in cell culture is effected by standard and well-known techniques, for example, by using polyethylene glycol (“PEG”) or other fusing agents. (See Milstein and Kohler, Eur. J. Immunol., 6:511 (1976), which is hereby incorporated by reference.) This immortal cell line, which is preferably murine, but may also be derived from cells of other mammalian species, including but not limited to rats and humans, is selected to be deficient in enzymes necessary for the utilization of certain nutrients, to be capable of rapid growth, and to have good fusion capability. Many such cell lines are known to those skilled in the art, and others are regularly described.
  • Procedures for raising polyclonal antibodies are also well known. Typically, such antibodies can be raised by administering the protein or polypeptide of the present invention subcutaneously to New Zealand white rabbits which have first been bled to obtain pre-immune serum. The antigens can be injected at a total volume of 100 μl per site at six different sites. Each injected material will contain synthetic surfactant adjuvant pluronic polyols, or pulverized acrylamide gel containing the protein or polypeptide after SDS-polyacrylamide gel electrophoresis. The rabbits are then bled two weeks after the first injection and periodically boosted with the same antigen three times every six weeks. A sample of serum is then collected 10 days after each boost. Polyclonal antibodies are then recovered from the serum by affinity chromatography using the corresponding antigen to capture the antibody. Ultimately, the rabbits are euthanized with pentobarbital 150 mg/Kg IV. This and other procedures for raising polyclonal antibodies are disclosed in Harlow et. al., editors, Antibodies: A Laboratory Manual (1988), which is hereby incorporated by reference.
  • In addition to utilizing whole antibodies, binding portions of such antibodies can be used. Such binding portions include Fab fragments, F(ab′)2 fragments, and Fv fragments. These antibody fragments can be made by conventional procedures, such as proteolytic fragmentation procedures, as described in Goding, Monoclonal Antibodies: Principles and Practice, New York: Academic Press, pp. 98-118 (1983), which is hereby incorporated by reference.
  • The present invention also relates to probes found either in nature or prepared synthetically by recombinant DNA procedures or other biological procedures. Suitable probes are molecules that bind to RSP viral antigens identified by the polyclonal antibodies of the present invention or bind to the nucleic acid of RSPaV. Such probes can be, for example, proteins, peptides, lectins, or nucleic acids.
  • The antibodies or binding portions thereof or probes can be administered to RSPaV infected scion cultivars or rootstock cultivars. Alternatively, at least the binding portions of these antibodies can be sequenced, and the encoding DNA synthesized. The encoding DNA molecule can be used to transform plants together with a promoter which causes expression of the encoded antibody when the plant is infected by an RSPaV. In either case, the antibody or binding portion thereof or probe will bind to the virus and help prevent the usual stem pitting response.
  • Antibodies raised against the proteins or polypeptides of the present invention or binding portions of these antibodies can be utilized in a method for detection of RSPaV in a sample of tissue, such as tissue from a grape scion or rootstock. Antibodies or binding portions thereof suitable for use in the detection method include those raised against a replicase, proteins or polypeptides of the triple gene block, or a coat protein or polypeptide in accordance with the present invention. Any reaction of the sample with the antibody is detected using an assay system which indicates the presence of RSPaV in the sample. A variety of assay systems can be employed, such as enzyme-linked immunosorbent assays, radioimmunoassays, gel diffusion precipitin reaction assays, immunodiffusion assays, agglutination assays, fluorescent immunoassays, protein A immunoassays, or immunoelectrophoresis assays.
  • Alternatively, the RSPaV can be detected in such a sample using the DNA molecules of the present, RNA molecules of the present invention, or DNA or RNA fragments thereof, as probes in nucleic acid hybridization assays for detecting the presence of complementary virus DNA or RNA in the various tissue samples described above. The nucleotide sequence is provided as a probe in a nucleic acid hybridization assay or a gene amplification detection procedure (e.g., using a polymerase chain reaction procedure). The nucleic acid probes of the present invention may be used in any nucleic acid hybridization assay system known in the art, including, but not limited to, Southern blots (Southern, E. M., “Detection of Specific Sequences Among DNA Fragments Separated by Gel Electrophoresis,” J. Mol. Biol. 98:503-17 (1975), which is hereby incorporated by reference), Northern blots (Thomas, P. S., “Hybridization of Denatured RNA and Small DNA Fragrnents Transferred to Nitrocellulose,” Proc. Nat'l Acad. Sci. USA, 77:5201-05 (1980), which is hereby incorporated by reference), and Colony blots (Grunstein, M., et al., “Colony Hybridization: A Method for the Isolation of Cloned cDNAs that Contain a Specific Gene,” Proc. Nat'l Acad. Sci. USA, 72:3961-65 (1975), which is hereby incorporated by reference). Alternatively, the isolated DNA molecules of the present invention or RNA transcripts thereof can be used in a gene amplification detection procedure (e.g., a polymerase chain reaction). Erlich, H. A., et. al., “Recent Advances in the Polymerase Chain Reaction,” Science 252:1643-51 (1991), which is hereby incorporated by reference. Any reaction with the probe is detected so that the presence of RSP virus in the sample is indicated. Such detection is facilitated by providing the DNA molecule of the present invention with a label. Suitable labels include a radioactive compound, a fluorescent compound, a chemiluminescent compound, an enzymatic compound, or other equivalent nucleic acid labels.
  • Depending upon the desired scope of detection, it is possible to utilize probes having nucleotide sequences that correspond with conserved or variable regions of the ORF or UTR. For example, to distinguish RSPaV from other related viruses (as described herein), it is desirable to use probes which contain nucleotide sequences that correspond to sequences more highly conserved among all RSPaV strains. Also, to distinguish between different RSPaV strains (e.g., RSPaV-1, RSP47-4, RSP158), it is desirable to utilize probes containing nucleotide sequences that correspond to sequences less highly conserved among the RSP virus strains.
  • Nucleic acid (DNA or RNA) probes of the present invention will hybridize to complementary RSPaV-1 nucleic acid under stringent conditions. Less stringent conditions may also be selected. Generally, stringent conditions are selected to be about 50° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. The Tm is dependent upon the solution conditions and the base composition of the probe, and may be calculated using the following equation:
    T m=79.8° C.+(18.5×Log[Na+])+
    +(58.4° C.×%[G+C])
    −(820/#bp in duplex)
    −(0.5×% formamide)
    Nonspecific binding may also be controlled using any one of a number of known techniques such as, for example, blocking the membrane with protein-containing solutions, addition of heterologous RNA, DNA, and SDS to the hybridization buffer, and treatment with RNase. Generally, suitable stringent conditions for nucleic acid hybridization assays or gene amplification detection procedures are as set forth above. More or less stringent conditions may also be selected.
  • The development of a rapid detection method for RSP is a major breakthrough, because the only detection method now available is through inoculation of St. George grape indicators, which takes two to three years to develop symptoms. A serological or nucleic acid based detection tests developed for RSP will take only 1 to 2 days and it is less expensive. The woody indicator test on St. George costs $250 per sample, while a serological or nucleic acid based test would cost $30-50 per sample. Moreover, the rapid tests will speed up the introduction of grape imports into the US from the current three years to about six months. These applications will be valuable wherever grapes are grown. Since RSP is part of the rugose wood complex, development of rapid detection methods will be invaluable in determining the significance of RSP in the rugose wood complex. This will allow an investigator to determine whether RSP alone can cause the rugose wood complex or if other components are needed. In addition, these rapid detection methods are very useful to evaluate the resistance of transgenic plants to Rupestris stem pitting associated virus.
    • EXAMPLES
  • The following examples are provided to illustrate embodiments of the present invention but are by no means intended to limit its scope.
    • Example 1 Grapevine Materials for dsRNA Analysis
  • Samples from 15 accessions that induced pitting on graft-inoculated St. George were collected from the National Grapevine Germplasm Repository of the USDA Plant Genetic Resources Unit (PGRU) at Geneva and used for dsRNA analysis. Positive controls used included Thompson Seedless (RSP105) (Golino, “The Davis Grapevine Virus Collection,” Am. J. Enology Viticulture, 43:200-05 (1992), which is hereby incorporated by reference) from the FPMS, University of California (Davis) and Pinot Noir (SVP1186-09A2), which was kindly provided by Dr. R. Johnson of Center for Plant Health, Agriculture Canada, Sidney, British Columbia. Negative controls as judged by indexing on St. George included Freedom from the PGRU at Geneva, N.Y., and Verduzzo 233A. The latter was kindly provided by Dr. P. Silvano of the Sezione di Fitovirologia, ERSA Servizio Chimico-Agrario e della Certificazione, Pozzuolo del Friuh (UD), Italy.
    • Example 2 Grapevine Materials for RT-PCR
  • Dormant cuttings of 138 grapevine selections were collected from USA, Canada, Italy, and Portugal over three years. Samples included Vitis vinifera cultivars, hybrids, V. riparia, and rootstocks. 117 grapevine selections were indexed on St. George for RSP and other RW diseases. Pinot noir (1186-9A2) from Agriculture Canada, Center for Plant Health (Sidney, Canada) and Thompson seedless (RSP105) from University of California (Davis) were included as positive controls. Sauvignon blanc, generated from shoot tip tissue culture and tested free of viruses and viroids was provided by Dr. J. Semancik (University of California at Riverside) and used as a healthy control. In addition, six seedlings of five Vitis species were also included as negative controls.
    • Example 3 dsRNA Isolation and Analysis
  • Methods for isolating dsRNA were described by Hu et al., “Characterization of Closterovirus-like Particles Associated with Grapevine Leafroll Disease,” J. Phytopathology, 128:1-14 (1990), which is hereby incorporated by reference, except that 1×STE with 15% ethanol (instead of 16.5%) was used to wash CF-11 cellulose columns prior to elution of dsRNAs. The dsRNAs were isolated from leaves, petioles, and the phloem tissue of dormant canes, electrophoresed on 1% agarose or low melting temperature agarose gels, and analyzed by staining with ethidium bromide (EtBr). Hind EII digested lambda DNA was used as markers to estimate the sizes of the dsRNA molecules.
    • Example 4 cDNA Synthesis and Cloning
  • The extremely low yield of dsRNA and the limited quantity of RSP-infected grape materials precluded the use of a single RSP-infected grapevine accession as the source of dsRNA for cloning purpose. Therefore, dsRNA preparations from Colobel 257, Ravat 34, Couderc 28-112, and Seyval were pooled and used as templates for cDNA synthesis. In order to get pure templates for cloning, dsRNA bands were excised from low melting temperature agarose gels after electrophoresis and recovered by extraction with phenol and chloroform (Sambrook et al., Molecular Cloning: A LaboratorEy Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), which is hereby incorporated by reference). The same recovery procedure was repeated once more. The purified dsRNA was denatured with 20 mM methyl mercuric hydroxide and cDNAs were synthesized using slightly modified methods of Jelkmann et al., “Cloning of Four Viruses from Small Quantities of Double-Stranded RNA,” Phytopathology, 79:1250-53 (1989), which is incorporated herein be reference. The cDNA fragments were first blunt-ended with T4 DNA polymerase at 12° C. T4 DNA ligase was used to add EcoR I adapters to both ends of the cDNAs. Subsequently, the cDNA molecules with cohesive ends were ligated to EcoR I-prepared arms of lambda ZAP II. Finally, the resulting recombinant phages were packed into Gigapack II packaging extract following manufacturer's instructions (Stratagene, La Jolla, Calif.).
    • Example 5 Identification of cDNA Clones Specific to the dsRNA
  • Plaque hybridization was used to screen cDNA clones by transferring recombinant cDNA plaques to nylon membranes and hybridizing to 32P-labeled first-strand cDNA probes generated from the dsRNA according to manufacturer's recommendations (Du Pont, 1987). Clones with strong hybridization signals were converted into pBluescript SK through in vivo excision (Stratagene, 1991). After digestion of the resulting plasmids with EcoR I, 20 clones were selected and further analyzed in Southern hybridization with radio labeled first strand cDNA probes synthesized from the dsRNA. The specificity of two selected clones to the dsRNA was confirmed by Northern analysis using 32P labeled inserts of the two clones.
    • Example 6 Bridging Gaps Between Clones
  • To bridge the gap between clones RSP3 and RSP94, a pair of specific primers were used in RT-PCR to generate cDNA fragments from the dsRNA. RSP3-RSP94 primer 1 (sense, nt 3629-3648) has a nucleotide sequence corresponding to SEQ. ID. No. 41 as follows:
    GCTTCAGCAC TTGGAAGGCG 20
  • RSP3-RSP94 primer 2 (antisense, nt 4350-4366) has a nucleotide sequence corresponding to SEQ. ID. No. 42 as follows:
    CACACAGTGG CCAGCCT 17

    After gel electrophoresis, PCR amplified cDNA bands were excised from gels and recovered with the phenol/chloroform method (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), which is hereby incorporated by reference).
  • The same strategy was employed to bridge the gap between clones RSP94 and RSP95. RSP94—RSP95 primer 1 (sense, nt 5272-5291) has a nucleotide sequence corresponding to SEQ. ID. No. 43 as follows:
    GGAGGTGCGT TGTGGTTATG 20
  • RSP94-RSP95 primer 2 (antisense, nt 6791-6808) has a nucleotide sequence corresponding to SEQ. ID. No. 44 as follows:
    CCGTGGCACT GCACACCC 17
    • Example 7 Obtaining Nucleotide Sequences on Both Termini of RSPaV-1 Genome
  • To obtain the terminal 3′ end sequences, a primer (sense, nt 8193-8210) having a nucleotide sequence corresponding to SEQ. ID. No. 45 as follows:
    GGAGGTGACC ACATTACG 18
  • and a (dT) 18 primer were used in RT-PCR to amplify cDNA from the dsRNA. Resulting PCR products were cloned into TA vector pCRII (Invitrogen) and sequenced. This approach was based on the assumption that the RSP associated dsRNA contained a poly (A) tail. For the terminal 5′ end, the dsRNA was first tagged with poly (A) using yeast Poly (A) polymerase (USB) (Pappu et al., “Nucleotide Sequence and Organization of Eight 3′ Open Reading Frames of the Citrus tristeza Closterovirus Genome,” Virology 199:35-46 (1994), which is hereby incorporated by reference) and then used as templates to generate cDNA fragments by RT-PCR using (dT) 18 primer and primer (antisense, nt 429-449) having a nucleotide sequence corresponding to SEQ. ID. NO. 46 as follows:
    CATCACGACT TGTCACAAAC C 21
    • Example 8 Nucleotide Sequencing
  • CsCl or alkaline/PEG (polyethylene glycol) purified plasmids (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), which is hereby incorporated by reference; Applied Biosystems, Inc.) and RT-PCR amplified cDNA fragments were sequenced for completion on both strands. Nucleotide sequencing was done manually with Sequenase version 2.0 kit (USB) or automatically on ABI 373 automated sequencer with Taq DyeDeoxy™ terminator cycle sequencing kit (Applied Biosystems, Inc.). Vector primers (T3, T7, M13 Forward, and M13 Reverse) were used in initial sequencing and sequences were completed by primer walking strategy.
    • Example 9 Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
  • Two pairs of primers were designed for RT-PCR: (1) RSP95F1 and RSP95R1; and (2) RSP149F1 and RSP149R1. Primer RSP95F1, an antisense strand primer, has a nucleotide sequence corresponding to SEQ. ID. NO. 47 as follows:
    TGGGCCTCCA CTTCTTC 17
  • Primer RSP95R1, a sense strand primer, has a nucleotide sequence corresponding to SEQ. ID. No. 48 as follows:
    GGGGTTGCCT GAAGAT 16
  • Primer RSP149F1, an antisense strand primer, has a nucleotide sequence corresponding to SEQ. ID. No. 49 as follows:
    ACACCTGCTG TGAAAGC 17
  • Primer RSP149R1, a sense strand primer, has a nucleotide sequence corresponding to SEQ. ID. No. 50 as follows:
    GGCCAAGGTT CAGTTTG 17

    RSP95F1/R1 were used in RT-PCR to test samples collected in 1994. RSP149R1/F1, alone or together with RSP95F1I/R1, were used to test samples collected in 1995 and 1996. To avoid bias in the judgment of RT-PCR results, blind tests were conducted for samples from Canada in 1995 and 1996. The indexing results of these samples were kept untold until the RT-PCR tests were complete.
  • dsRNAs were denatured with methylmercuric hydroxide (CH4HgOH) and reverse transcribed into cDNAs with Moloney murine leukemia virus (MMLV) or Avian Myeloblastosis Virus (AMV) reverse transcriptases (Promega) at 42° C. for 1 to 3 h. Five of 20 μl of the RT reactions were added to PCR mix and amplified in thermal cycler (HYBAID OmniGene, National Labnet Company) with Taq DNA polymerase (buffer B, Promega) using the following parameters: initial denaturation at 94° C. for 5 min, 40 cycles of amplification at 94° C. for 45 s, 52° C. for 1 min, and 72° C. for 1 min, and a final extension at 72° C. for 10 min. PCR products were analyzed by electrophoresis on 1% agarose gels containing ethidium bromide. Hae III digested Phix 174 fragments were used as molecular weight markers.
    • Example 10 Southern Blot
  • DNA fragments amplified by PCR from cDNA clone RSP149 with primers RSP149F1/R1 were labeled with 32P by random priming and used as probes. Products of RT-PCR of randomly selected grapevines including 26 positives and 6 negatives by RT-PCR were electrophoresed on an 0.8% agarose gel, transferred to nylon membranes, and hybridized to the probes following manufacturer's instructions (Du Pont).
    • Example 11 Computer Assisted Analysis of Sequences and Genome Structure of RSPaV-1
  • Sequences were assembled with SeqMan program and potential open reading frames were generated with MapDraw program (DNASTAR, Madison, Wis.). BLAST program of the NCBI (the National Center for Biotechnology Information) was used to search for homologies in DNA and protein databases. Clustal analysis (with identity weight table) of MegAlign (DNASTAR) was employed to reveal sequence similarities between the putative proteins of RSPaV-1 and the analogous proteins of ASPV (Jelkmann, “Nucleotide Sequences of Apple Stem Pitting Virus and of the Coat Protein of a Similar Virus from Pear Associated with Vein Yellows Disease and Their Relationship with Potex- and Carlaviruses,” J. General Virology, 75:1535-42 (1994), which is hereby incorporated by reference) and PVM (Zavriev et al., “Complete Nucleotide Sequence of Genomic RNA of the Potato M-Virus,” Molecular Biology (Mosk.) 25:761-69 (1991), which is hereby incorporated by reference). In addition, nucleotide sequences of the untranslated regions (UTR) of these three viruses were also compared using MagAlign, as shown in FIGS. 6A and 6B.
    • Example 12 Consistent Association of a High Molecular Weight dsRNA with RSP
  • The 15 grapevine accessions used in this study were previously indexed on St. George where 12 accessions induced typical RSP symptoms (i.e., a narrow strip of small pits below the inoculum bud). FIG. 1A illustrates these typical RSP symptoms. A good correlation was found between the presence of the specific dsRNA and the indexing results on St. George. As shown in FIG. 2A and recorded in Table 1 below, twelve grapevine accessions with typical RSP symptoms revealed a dsRNA of ca. 8.7 kb with gel electrophoresis. In addition, a smaller dsRNA of about 6.6 kb was observed in Colobel 257 and Seyval. In contrast, although Aminia and Canandaigua elicited deep pits and grooves around the woody cylinder of St. George, they did not reveal visible dsRNA of expected size in repeated experiments. Freedom, which indexed negative for RSP on St.
  • George, did not reveal visible dsRNA. Although two dsRNA bands were observed in Verduzzo 233A (which was indexed free of RSP on St. George), they were not specific to RSP based on the fact that they were larger or smaller than the 8.7 kb dsRNA associated with RSP (FIG. 2A) and that they did not hybridize to the RSP-specific probe in Northern analysis (FIG. 2B). In addition, the two dsRNA species isolated from Verduzzo 233A were not observed in other healthy grapevines such as Cabernet Franc and LN 33.
    TABLE 1
    St. George
    Accessions and Parentage Indicator dsRNA Northern
    Aminia (Carter X Black Hamburg) + − −
    Bertille Seyve 3408 (BS 872 X + + +
    Seibel 5410)
    Bertille Seyve 5563 (Seibel 6905 + + +
    X BS 3445)
    Canandaigua (V. labrusca X + − −
    V. vinifera)
    Colobel 257 (Seibel 6150 X + + +
    Seibel 5455)
    Couderc 28-112 (Emily X V. rupestris) + + +
    Freedom (Couderc 1613 X Dog Ridge) − − −
    Grande Glabre (V. riparia) + + +
    Ill 344-1 (BS 2667 X Seibel 6905) + +† −†
    Joffre (V. vinifera X V. riparia + + +
    X V. rupestris)
    Ravat 34 (Berlandieri X Chardonnay) + + +
    Seyval (Seibel 4995 X Seibel 4986) + + +
    Seyve Villard 14-287 (V. labrusca + + +
    X V. rupestris X V. aestiv
    X V. cinerea X V. vinifera)
    Seyve Villard 3160 (Seibel 5163 + + +
    X Seibel 2049)
    Verdelet (Seibel 5455 X Seibel 4938) + + +
    Controls
    Pinot Noir (V. vinifera) + + +
    Thompson seedless (V. vinifera) + NT +
    Verduzzo 233A − −‡ −

    Symbols:

    *Probe used was insert from cDNA clone RSP149.

    †A faint dsRNA band could be observed on the gel after electrophoresis but no hybridization signal could be seen in Northern analysis.

    ‡Although two dsRNA bands were observed in Verduzzo 233A, they were not specific to RSP, because they were either larger or smaller than the RSP-associated 8.7 kbp dsRNA and they did not hybridize to the probe in Northern analysis.
  • The yield of dsRNA was low and varied significantly among different accessions. When a comparable amount of phloem tissue (14 g for Bertille Seyve 5563 and Couderc 28-112; 18.5 g for the others) was used to isolate dsRNA, Colobel 257, Seyval, Ravat 34, Grande Glabre, and Seyve Villard 14-287 displayed strong dsRNA bands, while Bertille Seyve 5563, Couderc 28-112, Joffre, and Verdelet showed weak bands after staining with EtBr, as shown in FIG. 2A. Bertille Seyve 3408 and Seyve Villard 3160 were analyzed in separate experiments and dsRNA bands of the same size were observed.
    • Example 13 Selection and Specificity of cDNA Clones
  • A total of 182 clones were selected after plaque hybridization. Eighty clones with strong hybridization signals were subcloned into pBluescript SK through in vivo excision. Resulting plasmids were shown to have inserts ranging from 0.3 to 3.0 kb. A total of 20 clones with inserts of ca. 0.8 kb or larger were selected. Southern analysis of these 20 clones to radio labeled first strand cDNA probes derived from the dsRNA resulted in 15 clones with strong hybridization signals. Several of these clones were used to determine the genome sequence of the dsRNA: RSP3, RSP28, RSP94, RSP140, RSP95, and TA5. Another clone (RSP149), which was 97% similar in nucleotide sequence to RSP95, was used as one of the two probes in Northern hybridization.
  • Northern hybridization was employed to confirm the specific relationship of clones RSP95 and RSP149 to the isolated dsRNA. These two clones gave the strongest reaction in Southern analysis described above. Initial experiments showed that RSP95 insert hybridized with the dsRNA isolated from three accessions (Colobel 257, Seyval, and Ravat 34), from which the template dsRNAs used in cDNA synthesis were isolated. As shown in FIG. 2B and indicated in Table 1, use of RSP 149 insert as the probe showed that this clone hybridized with the dsRNA of ca. 8.7 kb isolated from RSP infected grapevines. Furthermore, the intensity of hybridization signals corresponded to that of the dsRNA bands observed on agarose gels stained with EtBr. Colobel 257, Seyval, Ravat 34, Grande Glabre, and Serve Villard 14-287 reacted strongly; Bertille Seyve 5563, Couderc 28-112, Joffre, and Verdelet had weak hybridization signals. The result for Ill 344-1 was not conclusive. Aminia and Canandaigua did not show visible dsRNAs or hybridization in Northern analysis. Bertille Seyve 3408, which was tested in a separate experiment, did show a ca. 8.7 kb dsRNA which hybridized to the probe from RSP149. Freedom and Verduzzo 233A, which had indexed negative for RSP on St. George, were also negative in Northern blot.
    • Example 14 Nucleotide Sequence and Genome Structure of RSPaV-1
  • Six cDNA clones and three RT-PCR amplified cDNA fragments (identified as RSPA, RSPB, and RSPC) were sequenced on both strands and used to obtain the complete nucleotide sequence of a viral agent, which is shown in FIG. 3A. The genome of RSPaV-1 consisted of 8726 nts excluding a poly (A) tail on the 3′ end. The sequence of RSPA indicated that the 5′ first base of the RSPaV-1 genome appeared to be a cytosine (C). Clone TA5, which represented the 3′ end of the RSPaV-1 genome, contained a stretch of adenines (A) preceded by a cytosine.
  • MapDraw analysis, shown at FIG. 3B, indicated that the genome of RSPaV-1 had five potential ORFs on its positive strand, while no ORFs were observed on the negative strand (data not shown). ORF1 (nt 62 to 6547 of SEQ. ID. No. 1) has a nucleotide sequence corresponding to SEQ. ID. NO. 2. ORF1 believed to encode a protein or polypeptide having a molecular weight of about 244 kDa and an amino acid sequence corresponding to SEQ. ID. No. 3. According to Lutcke et al., “Selection of AUG Initiation Codons Differs in Plants and Animals,” Eur. Mol. Biol. J., 6:43-48 (1987), which is hereby incorporated by reference, the start codon of ORF1 was in a favorable context: GCAAUGGC, where the “GC” after the start codon is important for initiating translation in a plant system. ORF2 (nt 6578 to 7243 of SEQ. ID. No. 1) has a nucleotide sequence corresponding to SEQ. ID. No. 4. ORF2 is believed to encode a protein or polypeptide having a molecular weight of about 24.4 kDa and an amino acid sequence corresponding to SEQ. ID. NO. 5. The first two ORFs were separated by an intergenic region of 30 nts. ORF3 (nt 7245 to 7598 of SEQ. ID. NO. 1) has a nucleotide sequence corresponding to SEQ. ID. No. 6. ORF3 is believed to encode a protein or polypeptide having a molecular weight of about 12.8 kDa and an amino acid sequence corresponding to SEQ. ID. NO. 7. ORF4 (nt 7519 to 7761 of SEQ. ID. NO. 1), which overlapped with ORF3 by 80 nts, has a nucleotide sequence corresponding to SEQ. ID. No. 8. ORF3 is believed to encode a protein or polypeptide having a molecular weight of about 8.4 kDa and an amino acid sequence corresponding to SEQ. ID. No. 9. Nine nucleotides downstream of ORF4 was the start of ORF5 (nt 7771 to 8550 of SEQ. ID. No. 1), which has a nucleotide sequence corresponding to SEQ. ID. No. 10. ORF5 is believed to encode a protein or polypeptide having a molecular weight of about 28 kDa and an amino acid sequence corresponding to SEQ. ID. No. 11. Downstream of ORF5 was the 3′ end LJTR of 176 nts. Although computer assisted analysis indicated that two shorter ORFs may exist as alternatives to ORF1 and ORF5, neither of them were in good contexts for translation initiation.
    • Example 15 Comparison of the RSPaV-1 Genome with ASPV and PVM Carlavirus Genomes
  • The arrangement of the ORFs and the amino acid sequences of RSPaV-1 showed similarities to those of PVX (Skryabin et al., “The Nucleotide Sequence of Potato Virus X RNA,” Nucleic Acids Res. 16: 10929-30 (1988), which is hereby incorporated by reference), PVM (Zavriev et al., “Complete Nucleotide Sequence of Genomic RNA of the Potato M-Virus,” Molecular Biology (Mosk.) 25:761-69 (1991), which is hereby incorporated by reference), and ASPV (Jelkmann, “Nucleotide Sequences of Apple Stem Pitting Virus and of the Coat Protein of a Similar Virus from Pear Associated with Vein Yellows Disease and Their Relationship with Potex- and Carlaviruses,” J. General Virology 75:1535-42 (1994), which is hereby incorporated by reference), with the latter two being the most similar to RSPaV-1. A representation of the sequence comparison is shown in FIG. 3B and the percent identities in amino acid sequences of the ORF of RSPaV-1 and the corresponding ORF of ASPV, PVM, and PVX are shown in Table 2 below. These analyses suggest that the ORFs of RSPaV-1 are compared with those of PVM and ASPV.
    TABLE 2
    Replicase Coat
    ORF1 Protein
    Region I Region II Triple Gene Block ORF5
    aa 1-372 aa 1354-2161 Total ORF2 ORF3 ORF4 Total aa 142-245
    ASPV 49.2 57.5 39.6 38.0 39.3 27.1 31.3 49.5
    PVM 47.2 53.2 37.6 34.8 31.2 19.0 21.2 33.3
    PVX 18.9 20.4 15.7 23.5 31.3 22.9 27.4 42.9
  • When the total amino acid sequence of RSPaV-1 ORF 1 was used for comparison, it showed 39.6% and 37.6% identities with the replicases of ASPV and PVM respectively (Table 2). These homologies were mainly found in regions I (aa 1 to 372) and II (aa 1354-2161), which are at the N and C terminal portions of the putative replicase, respectively, shown at FIGS. 4A and 4B. Within region I, the identities of RSPaV-1 with ASPV and PVM were 49.2% and 47.2%, respectively (Table 2). The methyltransferase domain, which is conserved in Sindbis-like superfamily of plant viruses (Rozanov et al., “Conservation of the Putative Methyltransferase Domain: A Hallmark of the “Sindbis-like” Supergroup of Positive-Strand RNA Viruses,” J. General Virology 73:2129-34 (1992), which is hereby incorporated by reference), was found in this region (FIG. 4A). Region II, on the other hand, showed even higher identities: 57.5% with ASPV and 53.2% with PVM (Table 2). A NTP binding motif “GXXXXGKS/T” (aa 1356 to 1363) (“X” stands for any amino acid residue), which is conserved in helicase proteins and helicase domains of eukaryotic positive strand RNA viruses (Gorbalenya et al., “A Novel Superfamily of Nucleotide Triphosphate-Binding Motif Containing Proteins which are Probably Involved in Duplex Unwinding in DNA and RNA Replication and Recombination,” FEBS Letters, 235:16-24 (1988), which is hereby incorporated by reference), was found in the beginning of region II (FIG. 4B). The amino acid sequences of this motif in ASPV and PVM were identical to that of RSPaV-1 except for one position. Furthermore, amino acid sequence surrounding the GDD motif, which is conserved in all RNA dependent RNA polymerases of positive strand RNA viruses (Koonin, “The Phylogeny of RNA-Dependent RNA Polymerases of Positive-Strand RNA Viruses,” J. Gen. Virology 72:2197-2206 (1991), which is hereby incorporated by reference), was located near the C terminus of the RSPaV-1 replicase protein and showed high identities to those of ASPV and PVM (FIG. 4B). Other conserved residues of positive strand RNA viruses as described by Koonin, “The Phylogeny of RNA-Dependent RNA Polymerases of Positive-Strand RNA Viruses,” J. Gen. Virology 72:2197-2206 (1991), which is hereby incorporated by reference, were also found in this region. Based on these information, it was concluded that ORF1 of RSPaV-1 codes for the putative replicase protein.
  • The triple gene block is a common feature of several groups of plant viruses including carlaviruses, potexviruses, and ASPV. Comparison of RSPaV-1 ORF2 with those of PVM and ASPV showed evenly distributed homologies in amino acid sequence: 38.0% identity to ASPV and 34.8% to PVM (Table 2). The N terminal region of the 24.4K protein (ORF2) contained the consensus sequence “GXGKS S/T” (aa 31 to 36) (FIG. 5A), which is observed in its counterparts in carlaviruses (Zavriev et al., “Complete Nucleotide Sequence of Genomic RNA of the Potato M-Virus,” Molecular Biology (Mosk.) 25:761-69 (1991), which is hereby incorporated by reference) and a number of ATP and GTP binding proteins (Zimmem, “Evolution of RNA Viruses,” in RNA Genetics, Holland et al., eds., CRC Press, Boca Raton, Fla., USA (1987), which is hereby incorporated by reference). The 12.8K protein of RSPaV-1 encoded by ORF3 had 39.3% and 31.2% identities with its counterparts in ASPV and PVM respectively (Table 2). However, most of the matching occurred in a region from aa 29 to 62, among which 18 aa were fully conserved in all three viruses (FIG. 5B). These 12-13K proteins may function in membrane binding (Morozov et al., “Nucleotide Sequence of the Open Reading Frames Adjacent to the Coat Protein in Potato Virus X Genome,” FEBS Letters 213:438-42 (1987), which is hereby incorporated by reference). The 8.4K protein encoded by RSPaV-1 ORF4, in contrast, showed much lower identities: 27.1% with that of ASPV and 19.0% with that of PVM (Table 2). However, four residues “TGES” (aa 38 to 41) were conserved in all three viruses (FIG. 5C). In vitro studies indicated that the analogous 7K protein of PVM may bind to single or double stranded nucleic acids (Gramstat et al., “The 12 kDa Protein of Potato Virus M Displays Properties of a Nucleic Acid-Binding Regulatory Protein,” FEBS Letters, 276:34-38 (1990), which is hereby incorporated by reference) and to plasma membrane (Morozov et al., “In vitro Membrane Binding of the Translation Products of the Carlavirus 7-kDa Protein Genes,” Virology 183:782-85 (1991), which is hereby incorporated by reference).
  • A sequence similarity search in a DNA database revealed identities between the putative protein encoded for by RSPaV-1 ORF5 to the coat proteins (CPs) of several groups of plant viruses, indicating that RSPaV-1 ORF5 may code for the coat protein. MegAlign analysis revealed that RSPaV-1 ORF5 had 31.3% and 21.2% identities with the CPs of ASPV and PVM, respectively (Table 2). Most of the identities were found in the C terminal portion of the coat proteins (aa 142 to 245 for RSPaV-1), while the N terminal portions were quite variable in the numbers and sequences of amino acid residues. When the C terminal portion of RSPaV-1 CP was compared to the corresponding regions of ASPV and PVM, it showed 49.5% and 33.3% identities with ASPV and PVM, respectively (Table 2). In addition, the “RR/QX-XFDF” motif was found in the central region of RSPaV-1 CP (FIG. 5D). This motif is conserved in the CPs of positive strand RNA viruses with filamentous morphology and were reported to be involved in salt bridge formation (Dolja et al., “Phylogeny of Capsid Proteins of Rod-Shaped and Filamentous RNA Plant Virus: Two Families with Distinct Patterns of Sequence and Probably Structure Conservation,” Virology, 184:79-86 (1991), which is hereby incorporated by reference). Therefore, it is believed that ORF5 encodes a putative coat protein.
  • MegAlign analysis, shown in FIGS. 6A and 6B, revealed that the 3′ UTR of RSPaV-1 is more similar to that of PVM than to that of ASPV. For example, in a 75 nts stretch, RSPaV-1 had 68% identity with PVM. Within this region, 21 consecutive nucleotides were identical between these two viruses. The significance of this conservation in nucleotide sequence remains to be explored. In contrast, the 5′ UTR of RSPaV-1 did not reveal significant similarities with those of PVM and ASPV.
  • It has been have shown that an 8.7 kbp dsRNA is consistently associated with grapevines that indexed positively on St. George for RSP. Sequence analyses of the dsRNA provide evidence that a virus is involved in RSP, which has now been named RSPaV-1. The complete nucleotide sequence of RSPaV-1 was determined from overlapping cDNA clones and RT-PCR-amplified cDNA fragments generated from the dsRNA. The RSPaV-1 genome has five ORFs coding for the putative replicase (ORF1), the triple gene block (ORF24), and the CP(ORF5). The existence of these ORFs and their potential to code for structural and non-structural viral proteins were further supported by the identification of conserved motifs which are the signatures of various viral proteins.
  • This work confirms and extends the findings of Walter and Cameron (“Double-stranded RNA Isolated from Grapevines Affected by Rupestris Stem Pitting Disease,” Am. J. Enology and Viticulture 42:175-79 (1991), which is hereby incorporated by reference), and Azzam and Gonsalves (“Detection of dsRNA in Grapevines Showing Symptoms of Rupestris Stem Pitting Disease and the Variabilities Encountered,” Plant Disease 75:960-64 (1991), which is hereby incorporated by reference), who observed a major dsRNA species of about 8.0-8.3 kbp in RSP-infected grapevines. In addition, such work also observed a smaller dsRNA of ca. 6.6 kbp. A dsRNA of similar size was also observed here, but it was consistently detected in only Colobel 257 and Seyval. The relationship, if any, of this smaller dsRNA to RSP remains to be determined. The small dsRNA of ca. 0.359 kbp, which Monette et al. (“Double-stranded RNA from Rupestris Stem Pitting-Affected Grapevines,” Vitis 28:13744 (1989), which is hereby incorporated by reference) isolated from RSP-infected grapevines growing in tissue culture, was not observed.
  • Electron microscopy evidence also suggests that RSP is caused by filamentous virus(es). Tzeng et al. (“Anatomical and Tissue Culture Studies of Rupestris Stem Pitting-Affected Grapevines,” Botan. Bulletin of Acad. Sinica (Taipei) 34:73-82 (1993), which is hereby incorporated by reference) observed flexuous filamentous virus aggregates in the phloem parenchyma cells of young shoots of Sylvner grapevines that had indexed positively for RSP. Monette and Godkin (“Detection of Capillovirus-like Particles in a Grapevine Affected with Rugose Wood,” Vitis 34:24142 (1995), which is hereby incorporated by reference) observed a filamentous virus in Sauvignon blanc infected by RSP and LNSG. The relationship of these virus particles to RSP disease remains to be studied.
  • Evidence suggests that the cDNA library generated from the isolated dsRNA templates is not homogeneous for only RSPaV-1. During the process of sequencing cDNA clones, several clones (e.g., RSP474 and RSP158) were identified with high, but not identical, sequence similarities to RSPaV-1.
  • RSPaV-1 has the most similarities to ASPV, which has not yet been grouped into a virus genus. Both viruses have the same genome organization and their ORFs code for putative proteins of similar sizes, except that the coat protein of ASPV is significantly larger (44 kDa) than that of RSPaV-1 (28 kDa). Comparisons of RSPaV-1 with PVM carlavirus show some similarities in genome organization except that RSPaV-1 lacks ORF6 which is located at the 3′ end of PVM genome. Although the genome organization of RSPaV-1 is similar to PVX potexvirus, the latter has a much smaller putative replicase. RSPaV-1 has no relation to grape viruses whose genomes have been sequenced so far. The closest possibilities, GVA (Minafra et al., “Grapevine virus A: Nucleotide Sequence, Genome Organization, and Relationship in the Trichovirus Genus,” Arch. Virology 142:417-23 (1997), which is hereby incorporated by reference) and GVB (Saldarelli et al., “The Nucleotide Sequence and Genomic Organization of Grapevine Virus B,” J. General Virology 77:2645-52 (1996), which is hereby incorporated by reference), have different genome structures than RSPaV-1.
    • Example 16 Specific and Universal Primers and the Detection of Different Strains of RSPaV by Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
  • Among the 138 grapevine entries collected, 25 indexed negatively and 93 indexed positively for RSP on St. George, while the others were not indexed (see Tables 3-7 below). Symptoms induced by RSP on the woody cylinder of St. George after graft inoculation with chip-buds can be divided into two types. The first type is called “specific”, that is, pits and/or grooves being restricted to the area on the woody cylinder below the inoculation sites. The other is called “nonspecific”, that is, pits and/or grooves being present above, around, and below the inoculation sites.
    TABLE 3
    Cultivar/Accession ID Index St.G RT-PCR Source
    Almeria K3 P 661 1483-13D1 − − C
    Auxerrois CL 56 658-1A2 − −a C
    Auxerrois CL 56 658-1A1-1A2 − − C
    GM 32458 604-8A2-2A2 − − C
    GM 7117-10 1347-16A1 − −a C
    Italia 1186-5B1 − − C
    Pslanka (H) 23-10A2-2A2 − − C
    Ventura (V. 51061) 1166-2A1 − − C
    (H)
    Verdelet (H) 1170-3C2-2S1 − − C
    Verduzzo (V) 233A − − I
    Vivant (V. 63331) (H) 1166-3A1 − − C
    Control
    Sauvignon Blanc (V) AV-4 #2 − −a U

    Symbols:

    V., Vitis vinefera;

    R., Vitis riparia;

    H., hybrid;

    C., Canada;

    I., Italy;

    U., USA;

    P., Portugal;

    a, tested by RSP149F1/R1 and 95F1/R1 and results agree to each other;

    b, tested by 95F1/R1 only
  • TABLE 4
    Index
    Cultivar/Accession ID St.G RT-PCR Source
    Aragonez (Temperanillo) 238 − + P
    Albalonga 1058-4A2-2A1 − + C
    Cabernet Franc (V) 147A − + I
    Chardonnay (V) 80A − + I
    Ehrenfelser PM 1 (V) 1169-1A1 − + C
    Freedom (H) PI 588370 − +a U
    Harslevellu P 679 1483-2B1 − + C
    Heroldrebe 1318-2A1 − + C
    Malvasia Fina 340 − + P
    Perle of Zala 1407-5A1 − + C
    Refosco (V) 181A − + I
    San Giovese 1497-2A1 − + C
    Brunello CL BBS 11
    Touriga Francesa 313 − + P

    Symbols:

    V., Vitis vinefera;

    R., Vitis riparia;

    H., hybrid;

    C., Canada;

    I., Italy;

    U., USA;

    P., Portugal;

    a, tested by RSP149F1/R1 and 95F1/R1 and results agree to each other;

    b, tested by 95F1/R1 only
  • TABLE 5
    Index RT-
    Cultivar/Accession ID St.G PCR Source
    Albalonga 1058-4A2-1A2 + + C
    Aminia (H) PI 588306 + + U
    Antao Vaz CL 245 + + P
    Aragonez (Temperanillo) 350 + + P
    Auxerrois CL 56 658-1A1 + + C
    Badacsony-10 1407-1A1 + + C
    Bertille Seyve 3408 (H) GVIT 348 + +b U
    Bertille Seyve 5563 (H) PI 181647 + +a U
    Blauer Spatburgunder Q1378-1 + +b C
    Blauer Zwiegelt/5BB 1240-1A1 + +a C
    Bonbino B 9 1586-17P3 + + C
    Brant (H) 1078-1A1 + + C
    Cabernet Franc (V) 151A + + I
    Cabernet Sauvignon (V) 124A + + I
    Cardinal Q390-13 + +b C
    Chardonnay (V) Q661-4 + +b C
    Chardonnay CL 116 (V) 1021-13A2 + +a C
    Chardonnay (V) 128B + +b I
    Chardonnay (V) 72A + +b I
    Chardonnay (V) 73A + +b I
    Chardonnay (V) 83A + + I
    Chazan CL 538 1346-6A1 + +a C
    Chenin Blanc CL 220 1555-6A1 + + C
    Colobel 257 (Seibel 8357) (H) PI 588062 + +a U
    Couderc 28-112 (H) PI 588248 + +a U
    De Chaunac S9549 (H) Q659-1 + +b C
    Durella 3 1586-13P1 + + C
    Esgana cao 276 + + P
    Egri Csillagok-30 1407-3A1 + + C
    Gamay Precoce 1500-2A1 + + C
    GM 31875 782-18A1 + +a C
    GM 32458 604-8A1 + + C
    GM 32458 782-21B1 + + C
    GM 6417-7 1347-7A1 + + C
    GM 6497-4 1347-14A1 + + C
    GM 7116-10 1362-4A1 + + C
    GM 7117-13 1347-17A2 + + C
    Grande Glabre (R) 279897 + +a U
    Gyongyriziling 1407-4A1 + + C
    ILL 344-1 (H) GVIT 658 + +a U
    Joffre (Kuhlmann 187-1) (H) GVIT 381 + +a U
    Koret (H) Q1179-7 + +b C
    Malvasia (V) 153A + + I
    Malvasia (V) 161A + + I
    Merlot CL 447 (V) 1236-17A1 + + C
    Moureto 87 + + P
    Moureto 96 + + P
    Muscat De Hambourg CL 202 1346-5A1 + + C
    Perle of Csaba Q806-1 + +b C
    Pinot Chardonnay CL 76 (V) 949-3A2 + +a C
    Pinot Chardonny CL 277 (V) 949-8B1 + + C
    Pinot Grigio (V) 104A + +b I
    Pinot Grigio (V) 108A + +b I
    Pinot Grigio (V) 114A + + I
    Pollux B6-18 1357-4A1 + + C
    Pslanka (H) 23-10A2 + + C
    Ravat 34 PI 588247 + +a U
    Refosco (V) 190A + +? I
    Refosco (V) 195A + + I
    Riesling CL 49 (V) 1555-2A1 + +a C
    San Giovese Brunello 1497-3B1 + + C
    CL E BS 4
    Schew-Rebe 778-6A1 + +a C
    Semillon CL 299 (V) 1555-7A1 + +a C
    Seyval Blanc PI 588309 + +a U
    (Seyve Villard 5-276) (H)
    Seyve Villard 14-287 (H) PI 588246 + +a U
    Seyve Villard 3160 (H) PI 181630 + +a U
    Titan Q1235-1 + +b C
    Verdelet (H) PI 186260 + +a U
    Verdelho 274 + + P
    Verduzzo (V) 222A + +b I
    Verduzzo (V) 226A + +b I
    Verduzzo (V) 239A + + I
    Vidal Blanc 1200-5A1 + +a C
    Weiser Burgunder Q782-40 + +b C
    3309 C 330-4A1 + + C
    420 A 1483-4A1 + + C
    7542 Q1386-1 + +b C
    Pinot Noir (V) 1186-9A2 + +a C
    Thompson Seedless (V) RSP105 + +a U

    Symbols:

    V., Vitis vinefera;

    R., Vitis riparia;

    H., hybrid;

    C., Canada;

    I., Italy;

    U., USA;

    P., Portugal;

    a, tested by RSP149F1/R1 and 95F1/R1 and results agree to each other;

    b, tested by 95F1/R1 only.
  • TABLE 6
    Index
    Cultivar/Accession ID St.G RT-PCR Source
    Aligote Q637-2B2 + −b C
    Aragonez (Temperanillo) 232 + − P
    Canandaigua (H) GVIT 566 + −a U
    Challenger (H) Q1338-1 + −b C
    Fercal CL 242 1551-4A1 + −a C
    GM 7746-6 1362-6A1 + − C
    Gravesac CL 264 1551-3A1 + −a C
    Honey Red 1339-6A1 + − C
    Kee-Wah-Din (H) 1278-1A1 + − C
    Periquita 72 + − P
    Tajoznyt Izumrud (H) Q2-2 + −b C
    Thurling 1047-4A2-1A2 + − C
    Verdelet 1170-3D2-2A1 + − C
    5BB CL 114 1236-2A1 + − C
    Alphonse Lavalle NI + I
    Ancellotta NI + I
    Chardonnay (V) 127 NI + I
    Kober 5BB? 100 NI + I
    Moscato d'Adda
    7 NI + I
    Periquita 624 NI + P
    Periquita 633 NI + P
    Riesling (V) 3 NI + I
    Seyval (H) Peterson NI + U
    Terrano
    1/1/3/K NI + I
    Thurling 1047-4A2-2A1 NI − C
    Tocai Rosso 19 1586-21P4 NI + C
    Trebbiano Toscano 67 NI − I
    Vidal Peterson NI + U

    Symbols:

    V., Vitis vinefera;

    R., Vitis riparia;

    H., hybrid;

    NI, not indexed;

    C., Canada;

    I., Italy;

    U., USA;

    P., Portugal;

    a, tested by RSP149F1/R1 and 95F1/R1 and results agree to each other;

    b, tested by 95F1/R1 only
  • TABLE 7
    Cultivar/Accession ID Index St.G RT-PCR Source
    V. acerifolia PI 588448 NI − U
    V. acerifolia PI 588449 NI − U
    V. cinerea PI 588446 NI − U
    V. monticola PI 588454 NI − U
    V. riparia PI 495622 NI − U
    V. sp. yenshanesis PI 588421 NI − U

    Symbols:

    V., Vitis vinefera;

    R., Vitis riparia;

    H., hybrid;

    NI, not indexed;

    C., Canada;

    I., Italy;

    U., USA;

    P., Portugal;

    a, tested by RSP149F1/R1 and 95F1/R1 and results agree to each other;

    b, tested by 95F1/R1 only
  • Among the 93 RSP-infected grapevines, 79 (85%) produced cDNA fragments of expected sizes in repeated RT-PCR using RSP149F1/R1 primers (SEQ. ID. Nos. 49 and 50) and/or RSP95F1/R1 primers (SEQ. ID. Nos. 47 and 48), while the other 14 were negative (see Tables 5 and 6). Interestingly, 12 of 14 (85.7%) grapevine accessions which were not indexed for RSP also produced cDNA fragments of expected size in RT-PCR (see Table 6). Sauvignon blanc healthy control) was negative in repeated RT-PCR (see Table 3).
  • Results of RT-PCR for grapevines indexed negatively for RSP were surprising (see Tables 3 and 4). While 11 were negative in RT-PCR tests (excluding Sauvignon blanc healthy control), the other 13 produced cDNA fragments of expected sizes.
  • Since RSPaV-1 was detected not only from grapevines which indexed positively for RSP but also from some of the grapevines indexed negatively for RSP, a search for more healthy materials for RT-PCR tests became necessary. As the majority of plant viruses do not pass on through seeds, grapevine seedlings are probably free of RSPaV-1. Based on this assumption, six seedlings from five Vitis species were included in RT-PCR (see Table 7). None of them produce cDNA of expected size in RT-PCR using RSP149R1/F1 primers (SEQ. ID. Nos. 49 and 50).
  • The data described above (and shown in Tables 3-7) indicate that RSPaV-1 is closely associated with RSP and that it is likely the causal agent of RSP. RT-PCR detected RSPaV-1 specific sequences from most of the RSP-infected grapevines collected from a wide range of viticultural regions of the world. Among the 93 grapevine accessions indexed positively for RSP on St. George, 85% were positive in RT-PCR (see Table 5). The data also suggests that RT-PCR has the potential to be used as a standard method for diagnosing RSP. This method is advantageous over the biological indexing on indicator St. George, because it is simpler, quicker, and more sensitive.
  • RT-PCR did not detect RSPaV-1 sequences from 14 of the grapevine accessions indexed positively for RSP (see Table 6). The discrepancy between RT-PCR and indicator indexing can be attributed to the existence in grapevines of different viruses or strains of the same virus which may all induce similar pitting and/or grooving symptoms on St. George upon graft-inoculation. It is believed these agents are only slightly different from RSPaV-1 at the level of their nucleotide sequences, but significant enough to hinder them from being detected by RT-PCR using RSPaV-1 specific primers.
  • It is likely that many RSPaV strains have genomes with nucleotide sequences that are highly similar to the nucleotide sequence of the RSPaV-1 genome. Evidence that supports this hypothesis includes the finding of a highly conserved region of ca. 600 bps among the nucleotide sequences of RSPaV-1 (type strain) and seven other cDNA clones, as shown in FIG. 9. The nucleotide sequence identities of these strains to RSPaV-1 (type strain) range from 83.6% to 98.4%. If oligonucleotides are chosen which are conserved among all these strains (i.e., with one or only a few mismatches), then the oligonucleotides should function as universal primers, allowing all of the strains to be detected by RT-PCR. Based on this theory, a primer pair (BM98-3F/BM98-3R) can be designed to amplify a DNA fragment of 320 bps from all these clones. BM98-3F has a nucleotide sequence corresponding to SEQ. ID. No. 51 as follows:
    GATGAGGTCCAGTTGTTTCC 20
  • BM98-3R has a nucleotide sequence corresponding to SEQ. ID. No. 52 as follows:
    ATCCAAAGGACCTTTTGACC 20

    Primers BM98-3F/BM98-3R can be used in RT-PCR to test further some of the grapevine samples which were negative for RSPaV in RT-PCR using RSP95F1/RSP95R1 primers (SEQ. ID. Nos. 47 and 48, respectively) or RSP149F1/RSP149R1 primers (SEQ. ID. Nos. 49 and 50, respectively). Results show that 6 of the 9 samples included were positive for RSPaV in RT-PCR using BM98-3F/BM98-3R primers. This indicates that these universal primers can be used to achieve even higher detection rates.
  • Another pair of primers (BM98-1F/BM98-1R) can be designed in a way that they can amplify DNA of 760 bps from RSPaV-1, RSP47-4, and RSP158. BM98-1F has a nucleotide sequence corresponding to SEQ. ID. No. 53 as follows:
    CTTGATGAGTACTTGTC 17
  • BM98-1R has a nucleotide sequence corresponding to SEQ. ID. No. 54 as follows:
    GCAAGGATTTGGATGGC 17

    Other “universal primers” can be designed manually or with computer programs (such as PrimerSelect) in the same way so that they contain conserved regions of nucleotide sequences for different strains of RSPaV-1.
  • RT-PCR detected RSPaV-1 sequences from 54% of grapevines negative for RSP as judged by indexing on St. George (see Tables 3 and 4). Several possibilities may account for this discrepancy. First, RT-PCR is much more sensitive than indicator indexing. Virus(es) of extremely low concentration may not induce visible symptoms on St. George within the standard indexing period, while they can be detected by RT-PCR. Second, judging indexing results can, in some cases, be very subjective. For example, it is very difficult to reach a conclusion on whether a grapevine is infected with RSP when only one or a few small pits are present on the woody cylinder of St. George. Third, uneven distribution of virus(es) within grapevines and the relatively limited number of replicates of St. George indicators may result in the failure to detect RSP-infection.
  • RSP seems to be widespread in different types of grapevines including V. vinifera, hybrids, V. riparia, and rootstocks. It occurs in a wide range of geographic regions including North America, Europe, Australia, and possibly many other countries as well. Testing grapevines from other areas of the world using RSPaV-1 specific primers will provide definitive information on the exact distribution of RSP throughout the world. It is also interesting to investigate whether RSP is transmitted by any vectors in nature.
  • RSP is a disease under quarantine in Washington and New York of the USA. Since this work and the work of others (Golino and Butler, “A Preliminary Analysis of Grapevine Indexing Records at Davis, California,” in Proceedings of the 10th Meeting of the ICVG, pp. 369-72, Rumbos et al., eds., Volos, Greece (1990); Azzam and Gonsalves, “Detection of dsRNA in Grapevines Showing Symptoms of Rupestris Stem Pitting Disease and the Variabilities Encountered,” Plant Disease, 75:96-964 (1991); Garau, “Kober Stem Grooving and Grapevine Virus A: A Possible Relationship,” in Extended Abstracts of the 11th Meeting of the International Council for the Study of Viruses and Virus Diseases of the Grapevine, p. 54, Montreux, Switzerland (1993); Credi, “Characterization of Grapevine Rugose Wood Sources from Italy,” Plant Disease, 82:1288-92 (1997), all of which are hereby incorporated by reference) showed that RSP is so wide-spread, it is questionable whether or not RSP should be kept under plant quarantine any longer. The devlopment and advance of rapid diagnostic methods will also allow us to investigate on the economic damage caused by RSP.
  • According to Goheen (“Rupestris Stem Pitting,” in Compendium of Grape Diseases, p. 53, Pearson and Goheen, eds., American Phytopathological Society Press, St. Paul, Minn., USA (1988), which is hereby incorporated by reference), RSP is a disease which induces, after graft-inoculation with a chip bud from an infected grapevine, a row of small pits on the woody cylinder of St. George below the point of inoculation. This definition may not be comprehensive. Indexing record indicated that two types of stem pitting (specific vs. nonspecific) were often observed on the woody cylinder of St. George upon graft inoculation with chip buds. For example, among 16 RSP-positive grapevines collected from Canada in 1995, eight developed specific type symptoms, while the others produced nonspecific symptoms. Credi (“Characterization of Grapevine Rugose Wood Sources from Italy,” Plant Disease, 82:1288-92 (1997), which is hereby incorporated by reference) also observed these two types of stem pitting in his indexing work. However, from the primers used in RT-PCR, as described above, RSPaV-1 was detected in grapevines showing both types of symptoms on St. George.
  • Thus, RT-PCR detected RSPaV-1 sequences from a wide range of grapevines collected from a number of major grapevine growing countries. The data clearly suggest that RSPaV-1 is closely associated with Rupestris stem pitting of grapevines and that it is likely the causal virus of RSP. Use of “universal” primers which can detect multiple agents which are highly similar to RSPaV-1 in nucleotide sequences would improve the detection rate by RT-PCR In addition, antibodies produced against bacteria-expressed coat proteins of RSPaV-1 will help in finding the viral particles from RSP infected grapevines and in rapid detection of RSP.
    • Example 17 Southern Hybridization
  • To confirm the specificity of the RT-PCR products to RSPaV-1, Southern blot hybridization was conducted using 32P labeled probe specific to RSPaV-1. As shown in FIG. 7, the Southern blot hybridization confirmed the results of the RT-PCR in each of the tested samples. Specifically, cDNA fragments amplified by RT-PCR from 16 selected RT-PCR positive samples hybridized with the probe.
    • Example 18 Constructing Expression Systems, Expression of a Fusion Protein Containing the RSPaV-1 Coat Protein, Production of Antibodies Against the Fusion Protein and Their Use in Detecting RSPaV-1 from Grapevines
  • The coat protein gene (SEQ. ID. No. 10) of RSPaV-1 was cloned into the EcoRI and HindIII sites of the polylinker region of a protein expression vector pMAL-c2 which, upon induction by inducer IPTG, produces a fusion protein containing maltose binding protein (MBP) and the coat protein of RSPaV-1. The fusion protein of expected size (ca. 71 KDa) was produced in E. coli bacteria after induction with IPTG. This fusion protein was purified through affinity chromatography using an amylose column. Purified fusion protein was used as an antigen to immunize a rabbit (by subcutaneous injection along the back) with the following scheme:
      • first injection, 400 μg fusion protein in 0.5 ml column buffer with Freund's complete adjuvant;
      • second injection, 100 μg of protein in 0.5 ml column buffer with Freund's incomplete adjuvant; and
      • third injection, 100 μg of protein in 0.5 ml buffer with Freund's incomplete adjuvant.
        Blood containing the antibodies was collected 70 days after the first injection. The antibodies were recovered and successfully used in an enzyme linked immunoabsorbent assay to detect the presence of virus particles (i.e., coat protein) of RSPaV-1 from a variety of tissue types of grapevines infected with RSP.
  • The antibodies produced against the expressed RSPaV-1 coat protein, therefore, are useful in the identification of the particles associated with RSP disease of grapevines, in the purification of the particles of RSPaV-1, and in the development of a serological diagnosis for RSP in grapevine. The use of the antibodies is suitable for detecting different strains of RSPaV-1. Because the coat proteins for strains RSP47-4 and RSP158 have high amino acid identities with the coat protein of RSPaV-1, it is very likely that the antibodies raised against RSPaV-1 coat protein will also detect other strains. Antibodies can be used in an ELISA to assay rapidly a large number of samples, thus making commercial development and utilization of diagnostic kits possible.
    • Example—19 Transformation of Grapevines with a Vector Containing RSPaV-1 Coat Protein Gene and Analysis of Transgenic Grapevines for Resistance to RSP
  • The DNA molecule coding for the RSPaV-1 coat protein (e.g., SEQ. ID. No. 10) was cloned into a pEPT8 plant expression vector that contains the double 35S enhancer at restriction sites SalI and BamHI. The resulting recombinant plasmid, designated pEPT8/RSPaV-1 coat protein, was then cloned into the plant transformation vector pGA482G, which has resistance genes to gentamycin and tetracycline as selection markers. The resultant pGA482G containing pEPT8/RSPaV-1 CP was used to transform grapevines using the Agrobacterium method.
  • The rootstock Vitis rupestris Scheele St. George was used in genetic transformation. Anthers were excised aseptically from flower buds. The pollen was crushed on a microscope slide with acetocarmine to observe the cytological stage (Bouquet et al., “Influence du Gentype sur la Production de cals: Dembryoides et Plantes Entieres par Culture Danthers in vitro dans le Genre Vitis,” C. R. Acad. Sci. Paris III 295:560-74 (1982), which is hereby incorporated by reference). This was done to determine which stage was most favorable for callus induction.
  • Anthers were plated under aseptic condition at a density of 40 to 50 per 9 cm diameter Petri dish containing MSE. Plates were cultured at 28° C. in the dark. After 60 days, embryos were induced and transferred to hormone-free medium (HMG) for differentiation. Torpedo stage embryos were transferred to MGC medium yo promote embryo germination. Cultures were maintained in the dark at 26-28° C. and transferred to fresh medium at 3-4 week intervals. Elongated embryos were transferred to rooting medium (5-8 embryos per jar). The embryos were grown in a tissue culture room at 25° C. with a daily 16 h photoperiod (76 μmol. s) to induce shoot and root formation. After plants developed roots, they were transplanted to soil in the greenhouse.
  • The protocols used for transformation were modified from those described by Scorza et al., “Transformation of Grape (Vitis vinifera L.) Zygotic-Derived Somatic Embryos and Regeneration of Transgenic Plants,” Plant Cell Rpt. 14:589-92 (1995), which is hereby incorporated by reference. Overnight cultures of Agrobacterium strain C58Z707 or LBA4404 were grown in LB medium at 28° C. in a shaking incubator. Bacteria were centrifuged for 5 minutes at 3000-5000 rpm and re-suspended in MS liquid medium (OD 1.0 at A600 nm). Calli with embryos were immersed in the bacterial suspension for 15-30 minutes, blotted dry, and transferred to HMG medium with or without acetosyringone (100 μM). Embryogenic calli were co-cultivated with the bacteria for 48 h in the dark at 28° C. The plant material was then washed in MS liquid plus cefotaxime (300 mg/ml) and carbenicillin (200 mg/ml) 2-3 times. To select transgenic embryos, the material was transferred to HMG medium containing either 20 or 40 mg/L kanamycin, 300 mg/L cefotaxime, and 200 mg/L carbenicillin. Alternatively, after co-cultivation, embryogenic calli were transferred to initiation MSE medium containing 25 mg/l kanamycin plus the same antibiotics listed above. All plant materials were incubated in continuous darkness at 28° C. After growth on selection medium for 3 months, embryos were transferred to HMG or MGC without kanamycin to promote elongation of embryos. They were then transferred to rooting medium without antibiotics. Non-transformed calli were grown on the same media with and without kanamycin to verify the efficiency of the kanamycin selection process.
  • The X-gluc (5-bromo-4-chloro-3-indoyl-β-glucuronidase) histochemical assay was used to detect GUS (β-glucuronidase) activity in embryos and plants that were transformed with constructs containing the GUS gene that survived kanamycin selection. All propagated plants were screened using an enzyme linked immunoabsorbent assay (ELISA) system (5 Prime-3 Prime, Boulder, Co.) to detect the NPTII (neomycin phosphotransferase II) protein in leaf extracts. ELISA tests with respective coat protein (CP)-specific antibodies were used to assay for CP. ELISA results were read on an SLT Spectra ELISA reader (Tecan U.S. Inc., Research Triangle Park, N.C.) 15-60 minutes after the substrate was added.
  • PCR analysis was carried out to detect the presence of transgene sequences in grape plants. Genomic DNA was isolated from transformed and non-transformed grape plants according to the method of Lodhi et al., “A Simple and Efficient Method for DNA Extraction from Grapevine Cultivars and Vitis Species,” Plant Mol. Biol. Rpt. 12:6-13 (1994), which is hereby incorporated by reference. Primer sets included those of specific primers to the transgene. DNA was initially denatured at 94° C. for 3 minutes, then amplified by 35 cycles of 1 minute at 94° C. (denaturing), 1 minute at 52° C. (annealing), and 2 minutes at 72° C. (polymerizing). Reaction samples were directly loaded and electrophoresed in 1.5% agarose gels.
  • Southern analysis of transformants was accomplished by extracting genomic DNA from young leaves of transformed and non-transformed plants (3309C) as described above. DNA (10 μg) was digested with the restriction enzyme Bgl II, electrophoresed on a 0.8% agarose gel in TAE buffer and transferred to a Genescreen Plus membrane by capillary in 10×SSC. A probe was prepared by random primer labeling of a PCR amplified gene coding sequence with radioisotope 32P-dATP (Dupont, NEN). Pre-hybridization and hybridization steps were carried out at 65° C. following the manufacturer's instruction. The autoradiograph was developed after overnight exposure.
  • Although the invention has been described in detail for the purposes of illustration, it is understood that such detail is solely for that purpose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.

Claims (11)

1. An isolated protein or polypeptide corresponding to a protein or polypeptide of a Rupestris stem pitting associated virus.
2-17. (cancelled)
18. An isolated DNA molecule encoding a protein or polypeptide according to claim 1.
19-34. (cancelled)
35. A host cell transformed with a heterologous DNA molecule according to claim 18.
36. The host cell according to claim 35, wherein the host cell is selected from a group consisting of Agrobacterium vitis and Agrobacterium tumefaciens.
37. The host cell according to claim 35, wherein the host cell is a grape cell.
38. The host cell according to claim 35, wherein the protein or polypeptide is selected from a group consisting of a replicase, a coat protein, and a protein of a triple gene block.
39. A transgenic Vitis scion cultivar or rootstock cultivar comprising the DNA molecule according to claim 18.
40. A transgenic Vitis scion cultivar or rootstock cultivar according to claim 39, wherein the protein or polypeptide is selected from a group consisting of a replicase, a coat protein, and a protein of a triple gene block.
41-63. (cancelled).
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