Species: Carnation ringspot virus
|This is a revised version of DPV 308|
Steven A. Lommel
Department of Plant Pathology, North Carolina State University, Box 7616, Raleigh, NC 27695-7616, USA.
Host Range and Symptomatology
Transmission by Vectors
Transmission through Seed
Transmission by Grafting
Transmission by Dodder
Nucleic Acid Hybridization
Stability in Sap
Properties of Particles
Properties of Infective Nucleic Acid
Relations with Cells and Tissues
Ecology and Control
Synonym Anjermozaiek virus (Noordam et al., 1951).
A virus with icosahedral isometric particles approximately 34 nm in diameter composed of 180 copies of a Mr 38,000 capsid protein and two genomic ssRNA species of 3.8 and 1.4 kb. The virus was once cosmopolitan in carnations, but is now only infrequently detected due to rigorous detection and sanitation. It also infects grapevine and a number of orchard crops in Central and Eastern Europe. It is readily mechanically transmitted and is also transmitted through the soil without the aid of a biological vector.
Transmitted experimentally to over 133 species in 25 families (Hollings & Stone, 1970; Kleinhempel et al., 1980). In nature, infects carnations (Dianthus caryophyllus) and a wide range of orchard trees (including plum, pear, apple, sour cherry, and sweet cherry), and grapevines (Richter et al., 1978; Fritzsche et al., 1979; Kleinhempel et al., 1980; Kegler et al., 1983). Has also been identified in several weed species, particularly Stellaria media growing within infected fruit orchards (Rudel et al., 1977; Fritzsche et al., 1979; Kegler et al., 1983). The experimental host range is much broader than that found in nature (Hollings & Stone, 1970). Easily mechanically transmissible to a wide range of herbaceous species, and can systemically infect a number of members of the Solanaceae, Leguminosae, Cucurbitaceae and Compositae. Infects an even larger number of plants non-systemically (Hollings & Stone, 1970). On experimental systemic hosts, causes concentric ringspots with necrotic centres on the inoculated leaves and mosaics, necrotic flecks and often veinal necrosis on the systemically infected leaves (Hollings & Stone, 1970).
Chenopodium amaranticolor and C. quinoa. Local necrotic lesions in 2-4 days (Fig.4); usually not systemic.
Dianthus barbatus (Sweet William). Inoculated leaves exhibit flecks, rings and ringspots after 4-7 days (Fig.5) followed by systemic chlorotic and semi-necrotic rings and flecks. Not all clones exhibit obvious symptoms or support large concentrations of the virus.
Gomphrena globosa. Local necrotic rings develop in 2-4 days after inoculation followed by systemic flecking, mottle and distortion.
Phaseolus vulgaris (French bean). Local chlorotic dots in 4-5 days, becoming white and necrotic; irregular systemic spotting and necrotic veinal flecks, later growth symptomless though infected.
Tetragonia expansa. Local white necrotic dots in 2-3 days, sometimes followed by systemic necrotic flecks.
Dianthus barbatus is an acceptable host for maintaining cultures. Nicotiana clevelandii, and to a lesser extent Phaseolus vulgaris and Vigna unguiculata, are good hosts from which to purify the virus.
Chenopodium amaranticolor, C. quinoa and Vigna unguiculata ssp. sinensis are useful local lesion hosts.
Easily purified from infected Nicotiana clevelandii or cowpea plants. Fresh tissue can yield 150 mg virus/kg; frozen tissue yields significantly less virus.
Method 1 (Tremaine et al., 1976). Extract 100 g infected tissue 3 weeks after inoculation in 200 ml 0.2 M sodium acetate buffer (pH 5.0) containing 20 mM sodium diethyl dithiocarbamate and 0.1% 2-mercaptoethanol. Adjust the extract to pH 5.0 with 10% acetic acid, and leave at 4°C for 4 h. Clarify by low speed centrifugation and precipitate the virus particles by adding polyethylene glycol (mol. wt 6000) to 8.0% (w/v). Suspend the pellet from low speed centrifugation in 0.1 M sodium acetate buffer (pH 5.0) and give the preparation one cycle of differential centrifugation (20 min at 12,000 g; 90 min at 95,000 g). Further purify by repeated cycles of differential centrifugation and rate zonal sucrose density gradient centrifugation.
Method 2 (Hollings & Stone, 1965). Must be used for strains that precipitate at pH 5.0. Extract each 100 g tissue in 125 ml 50 mM phosphate buffer (pH 7.6) containing 0.1% thioglycollic acid. Clarify by stirring with 8.5% butan-1-ol overnight at 2°C. The virus particles are then concentrated and purified by differential centrifugation.
The particles swell at pH >7.0. Swelling is prevented or reversed by divalent cations. The particles dissociate in 0.1 M tris-HCl buffer, pH 7.5, containing 10 mM EDTA and 1 M NaCl. Stable particles can be reconstituted by dialysis in 0.1 M tris-HCl , pH 7.0 with divalent cations (Tremaine & Dodds, 1985). The particles are stabilized by pH-dependent protein-protein interactions and by RNA- protein interactions. Strain A particles can form aggregates of six virus particles and linked aggregates (Fig.7). R and N strain particles aggregate in a temperature-reversible manner but the transition occurs at lower temperatures with particles of the R strain than with those of the N strain. N strain particles are readily dissociated at room temperature in low concentrations of SDS at pH 7.0. A and R strain particles are more stable than N strain particles (Tremaine & Ronald, 1976; Ronald & Tremaine, 1976; Tremaine et al., 1976, 1983, 1984).
Sedimentation coefficient (s20,w): N strain 133 S at pH 5.0, 125 S at pH 7.5 (Tremaine & Dodds, 1985).
Particle weight: 8.526 x 106 daltons based on calculations from the complete nucleotide sequence and assuming that the shell contains 180 copies of the capsid protein (Kendall & Lommel, 1992; Ryabov et al., 1994).
Electrophoretic mobility: in free boundary electrophoresis the virus particles are isoelectric at pH 4.5; at pH 6.0 to 8.0 the virus boundary moves to the anode as an increasingly broad Schlieren peak which continues to broaden when the polarity is reversed (Tremaine & Dodds, 1985).
Diffusion coefficient (D20,w x 10-7 cm2/sec): 1.48 (Kalmakoff & Tremaine, 1967).
Absorbance at 260 nm (1 mg/ml, 1 cm light path): 6.46 (Kalmakoff & Tremaine, 1967).
A260/A280: 1.67 corrected for light-scattering (Tremaine & Dodds, 1985).
Buoyant density in CsCl: 1.366 g/cm3. When limited proteolysis of virus occurs, additional species with buoyant densities of 1.369 and 1.374 are present.
Nucleic acid. ssRNA, constituting 20% of the particle weight. The genome is composed of two species of RNA termed RNA-1 and RNA-2 (Fig.9). It is not known how the RNAs are packaged into virions (Hamilton & Tremaine, 1996). Particle preparations yield 2-3 times as many molecules of RNA-2 as of RNA-1, so that some particles must contain RNA-2 alone. Some evidence suggests that each particle contains one copy of each RNA, whereas other evidence suggests that there are two particle classes, one containing one molecule of RNA-1 and the other containing three molecules of RNA-2. RNA-1, 3840 nt (accession no. L18870; Ryabov et al., 1994) with a mol. wt of 1.246 x 106, is composed of 1037 A, 903 C, 972 G and 928 U residues for molar percentages of 27.01%, 23.52%, 25.31%, and 24.17%, respectively. RNA-2, 1403 nt (accession no. M88589; Kendall & Lommel, 1992) with a mol. wt of 0.455 x 106, is composed of 380 A, 364 U, 318 C, and 340 G residues for molar percentages of 27.1%, 25.96%, 22.68%, and 24.25%, respectively.
Protein. Capsid protein can be isolated by first swelling the virus at 5-10 mg/ml in 0.1 M tris-HCl, 10 mM EDTA, pH 7.5 to 8.0, for at least 1 h at 0°C, and then adding an equal volume of 2.0 M NaCl and subjecting the mixture to rate zonal density gradient centrifugation in a 5-35% sucrose density gradient in 0.1 M tris-HCl buffer, pH 7.0, containing 1.0 M NaCl (Tremaine & Dodds, 1985). Virus particles contain a single polypeptide species comprising 80% of the particle weight. The polypeptide has a Mr of 37,900 but limited proteolysis may occur in stored preparations to yield polypeptides of Mr 36,000 and 34,000. The polypeptide is composed of 345 amino acid residues.
RNA-1 encodes two ORFs. The first ORF (p88) initiates at the first methionine codon, 58 nt from the 5' terminus (Fig.10). The p88 ORF is capable of encoding a 769 amino acid Mr 87,776 polypeptide. This ORF is interrupted by a retrovirus-like -1 ribosomal frameshifting element (Kim & Lommel, 1998) yielding a pre-readthrough 236 amino acid Mr 27,158 polypeptide (p27) that is identical to the first 236 amino acids of the 88 kd protein (Kim & Lommel, 1994). The 3'-most ORF encodes the 345 amino acid Mr 37, 921 capsid protein (p38). This ORF is followed by a 436 nt 3'-terminal non-coding sequence. RNA-2 contains a single ORF encoding a 303 amino acid Mr 33,730 movement protein (p34) (Kendall & Lommel, 1992). The ORF initiates at the first start codon at nt 47 and is followed by a 3'-terminal 441 nt non-coding region (Fig.10). RNA-1 directs the synthesis of 27, 57, 88 and 37 kd polypeptides in vitro. The 57 kd polypeptide has not been observed in vivo and the independent production of this protein may well be an in vitro translation artifact (Kim & Lommel, 1994). The RNA-1-encoded 88 kd polypeptide contains the conserved Glycine-Aspartate-Aspartate ("GDD") motif present in all RNA-dependent RNA polymerases (Ryabov et al., 1994). In addition, extensive amino acid sequence similarity exists between the 27 kd and 88 kd polypeptides and the replicases encoded by all of the species within the Tombusviridae. The capsid protein is not necessary for cell-to-cell movement, but is required for rapid systemic infection through the vascular tissue. The RNA-2 encoded 34 kd movement protein is essential for cell-to-cell and long distance movement.
An established and persistent disease problem in commercial carnations. The virus has diminished as a major problem in recent years as a result of vigilant and effective control measures (Lommel et al., 1983a). Infected nuclear stock plants, from which production cuttings are derived, serve as sources of virus inoculum in commercial glasshouses. Roguing, selection, indexing, and meristem tip culture are all effectively employed to ensure that these stock plants are free of the virus (Rybalko & Kharuta, 1978; Koev et al., 1983). Spread in carnations occurs as a result of careless propagation and leaf and root contact. In orchard crops, it has been observed that nematode infestation increases incidence (Kegler & Kegler, 1981). Cultural practices and chemicals that control nematode populations reduce the spread of the virus in orchard crops.
The most effective control is by sanitation during plant propagation. In vegetatively propagated hosts such as carnation and fruit trees, eradication of virus in plant shoots or meristems by chemo- and thermotherapy is routinely accomplished to produce certified virus free nuclear stocks which are then commercially propagated under a strict sanitation regime (Rybalko & Kharuta, 1978). More recently, antiviral agents, usually based on nucleotide analogs such as 5-fluoro- uracil, are added to the media during meristem tip culture. Production from virus-free nuclear stocks coupled with constant monitoring for CRSV infection by ELISA has ensured the production of CRSV-free commercial carnations (van Ruiten, 1987). The nearly world-wide establishment of a virus-free certified carnation propagation program has ensured that virus has not returned in epidemic forms (Ebbels, 1979).
Carnation var. Joker, (left) healthy plant, (right) infected plant showing typical symptoms of infection with CRSV (stunting and shortened internodes).
Carnation var. Joker, (left) healthy flower, (right) flower infected with CRSV.
Systemic symptoms on Dianthus caryophyllus leaves. The leaf at the top is healthy and the other three leaves are exhibiting typical symptoms including tip necrosis.
Local lesions on Chenopodium amaranticolor.
Symptoms on inoculated leaf of Dianthus barbatus.
Local lesions on Vigna unguiculata ssp. sinensis (cowpea).
Electron micrographs of particles of strain A negatively stained with 1% uranyl acetate. Bar represent 100 nm. (a) cluster of 12 virus particles seen from the three-fold axis; (b) similar cluster seen from the five-fold axis; (c) dimer cluster of 23 virus particles; (d) linear trimer cluster; (e) angled trimer cluster; (f) linear tetramer cluster.
(a) Electron micrograph of negatively stained CRSV virions, (b) A presumed structural model of the virion based on the structure at 3 Å resolution of tomato bushy stunt and turnip crinkle viruses, which share significant amino acid and structural homology with all members of the Dianthovirus genus, including CRSV (Giesman-Cookmeyer et al., 1995). The model is a T=3 structure with 5:3:2 symmetry. Depending on their position in the virion, identical capsid protein subunits adopt slightly different conformational states A, B, and C (termed quasi-equivalence) to allow formation of the virion. The protruding domains (P) of the subunits are thought to account for the granular appearance of the virions in the electron microscope.
(Left) Photograph of agarose gel electrophoretic patterns of genomic ssRNA species isolated from virus particles, (right) polyacrylamide gel electrophoretic patterns of dsRNA species isolated from virus infected tissue. (A) CRSV, (B) RCNMV.
Genome organization and replication strategy. Lines represent non-coding regions and boxes represent ORFs with the sizes of the respective proteins (or readthrough products) indicated within. The nt numbers for start and stop codons defining the ORFs are indicated above and below, respectively. The -1 ribosomal frameshifting signal is identified by -1 FS. The line under RNA-1 depicts the subgenomic RNA for the 1.5 kb capsid protein.
Ultrathin section of cytoplasmic crystal composed of virus particles from inoculated cowpea leaf. Bar represents 0.5 µm.
Section of infected Dianthus barbatus leaf showing nucleus containing: v, virus particles; t, tubules; and cr, protein crystal. Bar represents 0.5 µm.