_Archives
Vi rology
Arch Virol (t992) 126:107-117
© Springer-Verlag 1992 Printed in Austria
Properties of a previously undeseribed grapevine nepovirus from Tunisia R. Ouertani 1, V. Savino 2, A. Minafra 2, D. Boscia 2, M. A. Castellano 2, G. P. Martelli 2, and N. Greco 2
1Istituto Agronomico Mediterraneo, Valenzano 2Dipartimento di Protezione delle Piante dalle Malattie, Universit/t degli Studi and Centro di Studio del CNR sui Virus e le Virosi delle Colture Mediterranee, Bari, Italy Accepted February 7, 1992
Summary. A virus with isometric particles c. 30 nm in diameter and angular contour was isolated by inoculation of sap from a Tunisian grapevine with mild mottling and leaf deformation. The virus sedimented in sucrose density gradients as three components: T (empty shells), M (particles containing a molecule of ssRNA with an apparent size of 5,800 nucleotides, constituting 35% of the particle weight) and B (particles containing a molecule of ssRNA with apparent size of 6,800 nucleotides, constituting 41% of the particle weight). Virus particles had buoyant densities of 1.31 (T), 1.45 (M), and 1.49 g/cm 3 (B) in cesium chloride equilibrium gradients. The coat protein subunits consisted of a single polypeptide with mol. wt. of c. 59,000 daltons. An antiserum was produced with a titer of 1 : 256, which did not react with healthy plant antigens. Cells of artificially infected herbaceous hosts showed cytoplasmic vesiculate-vacuolate inclusion bodies, virus-containing tubules, mostly associated with plasmodesmata and/ or cell wall protrusions, and crystalline aggregates of virus particles and empty capsids. The physicochemical and ultrastructural properties of this virus resemble very much those of nepoviruses. However, it was serologically unrelated to t9 different members of the group, including all those reported to infect grapevines. Therefore, the virus is possibly a hitherto unreported nepovirus for which the name of grapevine Tunisian ringspot virus (GTRV) is proposed.
Introduction The virus described in the present paper is a seemingly new member of the Nepovirus group [5-], isolated by mechanical inoculation from Tunisian grapevines in the course of a survey for virus and virus diseases of Vitis in the Mediterranean area [9]. The source plant was a vine of unknown variety which
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did n o t s h o w perceptible signs o f disease except for an exceedingly mild mottling and d e f o r m a t i o n o f the leaves. This vine did n o t c o n t a i n mechanically transmissible viruses o t h e r t h a n the one described hereafter, for which the n a m e o f grapevine Tunisian ringspot virus ( G T R V ) is p r o p o s e d .
Materials and methods Transmission and culture of the virus isolate GTRV was isolated from grapevine leaves crushed in a mortar in the presence of a 1 : 1 mixture of 0.1 M phosphate buffer pH 7.2 and 2.5% aqueous nicotine solution. The slurry was rubbed onto celite-dusted leaves of herbaceous hosts and subsequently those plants showing symptoms were kept as virus cultures and used for subinoculations for host range determination. Grapevine seedlings of cv. Mission at the coltyledonary stage were kept in the dark for 2 weeks prior to mechanical inoculation with concentrated, partially purified virus preparations. Latent infections were ascertained by back inoculations to Chenopodium
quinoa. Purification and fractionation of virus particles Systemically infected plants of Cucumis sativus were harvested about 12 days after inoculation and utilized immediately or after storage for a few days in a refrigerator. Infected tissues were processed in either of two ways: (a) Extraction by homogenizing in a blender with 1 to 2 vol. of 0,1 M phosphate buffer p H 7 containing 0.1% thioglycohc acid, straining through muslin, addition of 5% Mgactivated bentonite [3], centfifugation at 10,000g for 15min, and addition of 10% polyethylene glycol (PEG, mol. wt. 6,000) plus 1% NaC1. After stirring in the cold for about 1 h, the precipitate was collected by low-speed centrifugation (t0,000 g for 10rain), resuspended in 0.02 M phosphate buffer pH 7.2, and centrifuged at 68,000 g for 2 h or 86,000 g for 90 min. (b) Extraction as above in 0.07 M phosphate buffer pH 7 containing 1% mercaptoacetate and 0.01 M EDTA, addition to filtrate of an equal volume of a 1 : 1 mixture of chloroformbutanol [18], centrifugation at 3,000 g for 20 min, addition to the supernatant of 10% PEG and 1% NaC1, and application of two cycles of differential centrifugation. Final pellets were resuspended in 0.02 M phosphate buffer pH 7. Fractionation of concentrated partially purified virus preparations was in 10-40% linear sucrose desity gradient columns centrifuged at 24,000 rpm in a Beckman SW.27 rotor. The gradients were scanned at 254 nm with a ISCO ultraviolet absorbance monitor and the peaks corresponding to virus fractions were collected separately, dialyzed against 0.02 M phosphate buffer pH 7.2 and concentrated by high-speed centrifugafion. Centrifugafion at equilibrium was in caesium chloride gradients. Purified virus was mixed with a CsC1 solution in 0.02 M phosphate buffer pH 7.2 (initial density 1.33 g/cm 3) and centrifuged for 18h at 36,000rpm in a Beckman SW.41 rotor. The virus bands were collected by piercing with a microsyringe the side of the tubes and then dialyzed against 0.02 M phosphate buffer pH 7.2.
Analysis of viral nucleic acid Nucleic acid was obtained from unfractionated purified virus preparations suspended in 0.02 M phosphate buffer, by incubating at room temperature for 10min in presence of 1 mM EDTA and 1% SDS, then extracting twice with I vol. of water-saturated phenol [ 1]. The aqueous phase was washed twice with ether and the nucleic acid was collected by ethanol precipitation overnight at - 20 °C. Extracted nucleic acid was electrophoresed in
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1.2% agarose gels in TBE buffer (22.5 mM Tris, 22.5 mM boric acid, 0.5 mM EDTA pH 8.3) after denaturation with 50% formamide at 65 °C for 5 rain. Reference markers were E. coli ribosomal RNAs (2,904 nucleotides and 1,541 nt, respectively) and grapevine fanleaf nepovirus (GFLV) genomic RNAs 1 and 2 (6,800 nt and 3,900 nt, respectively). The gels were stained with ethidium bromide. The type and strandedness of nucleic acid was ascertained by enzymic digestion with DNase-free pancreatic RNase (2 pg/ml) in high (0.3 M SSC buffer) or low-salt (0.003 M SSC buffer) (SSC = 0.15 M sodium chloride and 0.015 M sodium citrate), or with RNasefree DNase (2.5 gg/ml) in 10 mM magnesium chloride. For RNA infectivity assays, each electrophoretic band was cut from agarose gels, soaked in an Eppendorf tube with 400 pl of TE buffer (0.01 M Tris and 0.001 M EDTA, pH 7.8), incubated at 65 °C for 10 min, extracted twice with phenol (400 pl) and chloroform (400 gl), and precipitated with ethanol at - 20 °C. Nucleic acid was resuspended in an autoclavesterilized inoculation buffer (0.05 M glycine, 0.03 M potassium phosphate, 1% bentonite, 1% celite) and rubbed onto C. sativus cotyledons.
Analysis of coat protein Purified virus preparations were disrupted by boiling for 5 rain in the presence of 10% SDS and 2% 2-mercaptoethanol in 0.4 M Tris-HC1 buffer pH 6.8. Protein samples were electrophoresed with a Protean II apparatus (Bio Rad) in 12.5% polyacrylamide slab gels using Laemmli's [7] discontinuous buffer system and stained with Coomassie brilliant blue. The molecular weight of the protein subunit was determined by comparison with the following reference markers (mol. wt. in parentheses): a-lactoalbumin (14,400), soybean trypsin inhibitor (20,100), carbonic anhydrase (30,000), ovalbumin (43,000), bovine serum albumin (67,000), phosphorylase B (94,000). Western blotting was done using a Trans-blot cell (Bio Rad). Electrophoretic bands of dissociated coat protein of GTRV and GFLV were transferred (20 V constant voltage for 1 h) to polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Millipore). Membrane "blocking", incubation with polyclonal immunoglobulins, rinsing and staining was as described by Hu et al. [6].
Serology An antiserum to GTRV was raised using purified preparations from CsC1 gradients. A rabbit was injected intramuscularly with antigen (0.8rag nucleoproteins) emulsified in Freund's incomplete adjuvant followed by two intravenous injections (0.5 rag/injection) one week apart. Antiserum collection began 8 days after the last injection. The titre of different serum samples was determined by immunodiffusion in agar plates (Oxoid Agar no. 1 0.7%, NaC1 0.85%, sodium azide 0.02%).
Electron microscopy Purified virus preparations were negatively stained with 2% aqueous uranyl acetate. Thin sections of local lesions and systemically infected leaf tissues of C. sativus and Nicotiana clevelandii were prepared according to standard procedures [ 10]. Tissue samples were fixed in 4% glutaraldehyde in 0.05 M phosphate buffer pH 7.2, postfixed at 4°C in 1% osmium tetroxide, dehydrated in graded ethanol dilutions and embedded in Spurr's resin. Thin sections were double stained with uranyl acetate and lead citrate before observation with a Philips 201 C electron microscope. Controls consisted of tissue samples from healthy seedlings.
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Host range and symptomatology GTRV had a moderately wide experimental host range infecting either with visible symptoms or symptomlessly 14 of 19 plant species, belonging to 6 different botanical families. Nicotianan tabacum cv. White Burley and Samsun, N. glutinosa, Ocymum basilicum, Phaseolus vulgaris cv. La Victoire, and P. aureus did not develop symptoms and proved not to be infected when back-inoculated to C. quinoa. Gomphrena globosa and N. benthamiana were latently infected, whereas N. tabacum cv. Xanthi, N. rustica and N. cavicola reacted only with chlorotic or necrotic local lesions. Systemic symptoms consisting of vein clearing, mottling or chlorotic ringspotting were shown by N. clevelandii, N. megalosiphon, N. rotundifolia, N. occidentalis, Petunia hybrida, and Cucurbita pepo cv. Striata d'Italia. Symptoms in other diagnostic species were as follows. Chenopodium amaranticolor: minute necrotic local lesions in 5-6 days, not followed by systemic infection; C. quinoa: chlorotic lesions in 5-6 days, systemic mottling, deformation of the leaves and apical necrosis; and C. sativus cv. Delicatezza: chlorotic local lesions in inoculated cotyledons in 4-6 days, severe mottling and necrotic flecks of systemically infected leaves. Of 50 mechanically inoculated grapevine seedlings, 18 survived. Of these, three were infected by GTRV, which could readily be recovered by sap inoculation. Eight months after inoculation, infected seedlings grown under glasshouse conditions did not show visible symptoms.
Stability in leaf extracts Extracts of C. sativus leaves in distilled water were no longer infective after a dilution of 10 -3, heating for 10min at 65°C, and/or a week storage at room temperature.
Virus purification and fractionation Clarification of sap extracts with either magnesium-bentonite or chloroformbutanol removed most of the host components. The results were consistent with both methods but virus yield was low, with a seasonal variation ranging from 1.5 to 6mg/kg of leaf tissue. Partially purified, unfractionated preparations contained two types of isometric particles c. 30 nm in diameter, i.e., empty shells penetrated by the stain and apparently intact particles (Fig. 1 D). These preparations were infectious when inoculated to assay hosts (C. sativusand C. quinoa) and exhibited u.v. absorption spectrum of nucleoproteins with E m a × = 260 nm, Emin = 240 nm, and E 260/E 280 = 1.8.
Properties of fractionated virus Sucrose density gradient centrifugation separated virus preparations into three components (Fig. 1 A). When seen in the electron microscope, the slowest-
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Fig.1. A U.V.-absorption trace of a GTRV preparation after centrifugation through 1040% sucrose density gradients. Three centrifugal components denoted T, M, and B are clearly discernible. Sedimentation is from left to right (arrow). B Particles from T component. Most of the particels are empty capsids penetrated by uranyl acetate. C Particles from components M and B. All particles are apparently intact. D Purified lmfractionated GTRV preparation showing both "empty" and "full" virus particles. Bars: 100 nm
sedimenting fraction (T) mostly contained e m p t y shells (Fig. 1 B) whereas the two faster sedimenting c o m p o n e n t s (M and B) consisted of apparently intact particles (Fig. 1 C).
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Unfractionated virus preparations centrifuged at equilibrium in CsC1, also yielded three components banding at densities of 1.31 (T), 1.45 (M), and 1.49 g/ cm 3 (B). From these values, nucleic acid contents of about 35 and 41% were calculated for M and B fractions, respectively [17]. When purified preparations of GTRV and GFLV were mixed and centrifuged at equilibrium in CsC1, T and B components of both viruses sedimented together, each couple forming a single band at different density levels. By contrast, M components were clearly separated banding at densities of 1.45 g/cm 3 (GTRV) and 1.41 g/cm 3 (GFLV), respectively. When inoculated to assay hosts, T fractions did not induce symptoms, contrary to mixtures of M and B components which were highly infectious. After separation by two successive cycles of density gradient centrifugation in sucrose columns, M component gave rise to a single non-infective sedimenting band. However, despite of several attempts, no complete separation of B from M component could be achieved, so that B fractions always retained a residual infectivity.
Identification and properties of nucleic acid Infectious nucleic acid was extracted successfully from unfractionated virus preparations or from pooled M and B particles. No nucleic acid was detected in T particles. Electrophoresis of nucleic acid extracts from M and B components showed two main species migrating very closely to one another (Fig. 2 A). These were resitant to DNase but were degraded by pancreatic RNase in both low and high salt (not shown) and were therefore identified as single-stranded RNA. The slowest nucleic acid species of GTRV (RNA-1) migrated at the same rate as RNA-1 of GFLV (tool. wt. 2.4 x 10 6 daltons, c. 6,800 nt) (Fig. 2 A), whereas mol. wt. of the faster migrating RNA-2 was estimated to be c. 2 x 106 daltons (c. 5,800 nt). Satisfactory separation of the two RNAs could be obtained by two successive electrophoretic runs (Fig. 2 B) but after elution and pooling no infectivity was recovered.
Virus coat protein A single protein with an estimated tool. wt. of c. 59,000 daltons was observed in gels loaded with preparations from unfractionated virus (Fig. 2 C, lanes 1 and 2). GTRV coat protein subunits had a slower migration rate than those of GFLV (mol. wt. 57,000) from which they were clearly separated, even when mixed preparations were etectrophoresed (Fig. 2 C, lane 2). In Western blots, the protein bands of GTRV and GFLV were specifically and selectively recognized only by the homologous antisera (not shown).
Serology GTRV was moderately immunogenic. The antiserum obtained had a titer of 1 : 256 in gel double diffusion plates and did not react visibly with healthy plant
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Fig. 2. A Electropherogram of a GTRV nucleic acid extract showing two closely migrating bands (2). Reference markers in 1 (genomie GFLV RNAs) and 3 (E. coli ribosomal RNAs). Note that RNAs- 1 of GFLV and GTRV migrate at the same rate. B Separation of GTRV genomic RNAs (1) after two electrophoretic runs. RNA-1 and RNA-2 in 2 and 3, respectively. C Electropherogram of dissociated capsid proteins of GTRV (1) and GFLV (3) alone or in mixture (2). Reference markers in 4 (numbers are the corresponding tool. wt.) extracts nor with GFLV preparations. Single precipitin lines merging at the point of junction were formed when the antiserum was allowed to react with different GTRV fractions. No reaction was detected when partially purified GTRV was tested with antisera to the following nepoviruses, which include all those known to infect grapevines (asterisk): *arabis mosaic (ArMV), *artichoke Italian latent (AILV), cherry leafroll (CLRV), cherry rasp leaf (CRLV), *grapevine Bulgarian latent (GBLV), *grapevine chrome mosaic (GCMV), *raspberry ringspot (RRV), *strawberry latent ringspot (SLRV), *tomato black ring (TBRV), *tomato ringspot (ToRSV), *tobacco ringspot (TRSV), artichoke yellow ringspot (AYRSV), chicory yellow mottle (CYMV), *peach rosette mosaic
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(PRMV), cocoa necrosis (CNV), *blueberry leaf mottle (BBLMV), and myrobalan latent ringspot (MyLRV).
Cytopathology Regardless of the type of infection (local or systemic) or the host (C. sativus, N. clevelandii) the cytopathology was the same. The general cytology of many cells was relatively well preserved and their major organelles had an appearance comparable to that of healthy controls. Other cells, however, showed a cytopathic condition due to fragmentation of the ground cytoplasm deriving from abundant secondary vacuolation and to the abnormal development of membranes in the form of a proliferated endoplasmic reticulum and excessive vesiculation. These membranous materials accumulated in certain cytoplasmic areas giving rise to conspicuous vesiculate-vacuolate inclusion bodies (Fig. 3 A). Structural modification of the cell wall-plasmalemma interface consisting of multivesicular structures (paramural bodies) were rather frequent, together with cell wall outgrowths developing at the level of plasmodesmata and protruding into the cytoplasm (Fig. 3 D). Virus particles appeared as electron opaque rounded bodies with a regular and smooth outline and a diameter of c. 22 nm. Virions were either scattered at random in the cytoplasm or gathered in single rows inside tubular structures c. 42 nm in width, or grouped to form rather large aggregates. Virus-containing tubules were rarely seen free in the cytoplasm. Sometimes they gathered in the proximity of paramural bodies or, more often, were connected with plasmodesmata and/or contained in the cell wall outgrowths (Fig. 3 D). Ribbon-shaped paracrystalline aggregates of virus particles were seen in the vacuoles (Fig. 3 B). These could also contain clusters of virus particles embedded in a densely staining amorphous matrix, resembling those associated with olive latent ringspot nepovirus infections [2], or relatively large viral aggregates in which the particles were more or less arranged in a crystalline lattice. Several of these crystals were made up of both heavily stained solid particles, interpreted as profiles of intact virions, and doughnut-shaped particles with an electron lucent center, likely to represent empty virus capsids (Fig. 3 C). Virions could also be clustered inside plasmodesmatal dilations (Fig. 3 E).
Discussion Many of the features shown by GTRV are typical of nepoviruses [13]: (a) transmissibility by inoculation of sap; (b) size and outward appearance of virus
Fig. 3. A A vesiculate-vacuolate inclusion body (IB) in the cytoplasm of a systemically infected cell of N. clevelandii. B Rows of virus particles in a ribbon-like paracrystalline aggregate in a systemicallyinfected C. sativus cell. C A large crystalline structure containing many empty viral capsids. Dark-stained spots are intact virus particles. D Virus-containing tubules with plasmodesmata and developing cell wall (CW) outgrowths (arrows). E Virus particles clustering within plasmodesmatal dilations. Bars: 200 nm
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particles; (c) presence of three centrifugal components (T, M, and B) all consisting of a single type of coat protein subunit with mol. wt. of c. 59,000, but deprived of nucleic acid (T) or containing one molecule each of ssRNA with an apparent size of c. 5,800 nt (M) or 6,800 nt (B), respectively, both of which are essential for infectivity; (d) cytological modifications, i.e., presence of large vesiculate-vacuolate cytoplasmic inclusion bodies, virus-containing tubules and cell wall outgrowths [4, 10]. Cells infected by GTRV, however, displayed some unusual cytopathological features. For instance, groups of free virus particles were seen in plasmodesmatal dilations similar to those reported for cucumoand luteoviruses, but contrary to what is known for nepoviruses, whose association with plasmodesmata seems to be almost invariably mediated by tubular structures [8]. Furthermore, viral crystalline aggregates were much larger than those usually induced by other members of the group and consisted of a mixture of complete virions and empty capsids. With other nepoviruses (e.g., GFLV, ArMV, and AYRSV) when aggregates of empty capsids are formed, they are rather small, have a loose paracrystaUine structure, do not contain full particles, and are primarily located in the nucleus [15, 16]. Based on physicochemical properties, the nepovirus group has been divided into subgroups [12, 14]. Because of its properties, GTRV appears to belong to the tomato ringspot virus subgroup characterized by a homogeneous B component (containing one molecule of RNA-1 only) and an RNA-2 with a tool. wt. equal or above 2 x 10 6 daltons. Of the 12 viruses included in this subgroup [11], eight (AYRSV, BBLMV, CLRV, CYMV, GBLV, MyLRSV, PRMV, and ToRSV) were tested serologically and found to be unrelated to GTRV. The tested viruses comprised the four known to infect grapevines (BBLMV, GBLV, PRMV, ToRSV). GTRV therefore seems to be a previously undescribed member of the nepovirus group. GTRV does not constitute an economic threat for grapevines. Besides its rare occurrence, the virus does not appear to be pathogenic, as demonstrated by the virtual absence of symptoms in naturally infected vines and artificially inoculated seedlings. It has, however, a scientific importance for it represents the first new nepovirus recorded in a woody host in the southern bank of the Mediterranean.
Acknowledgements Grateful thanks are expressed to Dr. D. C. RamsdeU for the gift of antisera to BBLMV and PRMV.
References 1. Diener TO, Schneider IR (1968) Virus degradation and nucleic acid release in singlephase phenol systems. Arch Biochem Biophys 124:401-412 2. Di Franco A, Martelli GP, Russo M (1983) An ultrastructural study of olive latent ringspot virus in Gomphrenaglobosa. J Submicroscop Cytol 15:539-548 3. Dunn DB, Hitchborn JH (1965) The use of bentonite in the purification of plant viruses. Virology 25:171-192
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4. Francki RIB, Milne RG, Hatta T (1985) Atlas of plant viruses, vol 2. CRC Press, Boca Raton 5. Harrison BD, Murant AF (1978) Nepovirus group. CMI/AAB Description of Plant Viruses, no 185 6. Hu JS, Gonsalves D, Teliz D (1990) Characterization of closterovirus-like particles associated with grapevine leafroll disease. J Phytopathol 128:1-14 7. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacterial phage T 4. Nature 227:680-685 8. Martelli GP (1980) Ultrastructural aspects of possible defence reactions in virus-infected plants. Microbiologica 3:639-691 9, Martelli GP (1986) Virus and virus-like diseases of the grapevine in the Mediterranean area. FAO Plant Protect Bull 34:25-42 10., Martelli GP, Russo M (1984) Use of thin sectioning for the visualization and identification of plant viruses. In: Maramorosch K, Koprowski H (eds) Methods in virology, vol 8. Academic Press, New York, pp 143-224 11. Martelli GP, Taylor CE (1990) Distribution of viruses and their nematode vectors. In: Harris KF (ed) Advances in disease vector research, vol 6. Springer, Berlin Heidelberg New York Tokyo, pp 151-189 12. Martelli GP, Quacquarelli A, GaUitelli D, Savino V, Piazzolla P (1978) A tentative grouping of nepoviruses. Phytopathol Medit 17:147 13. Mutant AF (1981) Nepoviruses. In: Kurstak E (ed) Handbook of plant virus infections and comparative diagnosis. Elsevier/North-Holland, Amsterdam, pp 19'7-238 14. Mutant AF, Taylor M (1978) Estimates of molecular weight ofnepovirus RNA species by polyacrylamide gel etectrophoresis under denaturing conditions. J Gen Virol 41: 53-61 15. Russo M (1985) Electron microscopy of grapevine virus infections. Phytopathol Medit 24:144-147 16. Russo M, Martelli GP, Rana GL, Kyriakopoulou PE (1978) The ultrastructure of artichoke yellow ringspot virus infections. Microbiologica 1:81-99 17. Seghal OP, Jean JL, Bhalla RB, Soong MM, Krause GF (1970) Correlation between buoyant densities and ribonucleic acid content in viruses. Phytopathology 60: 17781784 18. Steere RL (1956) Purification and properties of tobacco ringspot virus. Phytopathology 46:60-69 Authors' address: G. P. Martelli, Dipartimento di Protezione delle Piante dalle Malattie, Universifft degli Studi di Bari, Via Amendola 165 A, 1-70126 Bari, Italy. Received November 19, 1991
Vi rology
Arch Virol (t992) 126:107-117
© Springer-Verlag 1992 Printed in Austria
Properties of a previously undeseribed grapevine nepovirus from Tunisia R. Ouertani 1, V. Savino 2, A. Minafra 2, D. Boscia 2, M. A. Castellano 2, G. P. Martelli 2, and N. Greco 2
1Istituto Agronomico Mediterraneo, Valenzano 2Dipartimento di Protezione delle Piante dalle Malattie, Universit/t degli Studi and Centro di Studio del CNR sui Virus e le Virosi delle Colture Mediterranee, Bari, Italy Accepted February 7, 1992
Summary. A virus with isometric particles c. 30 nm in diameter and angular contour was isolated by inoculation of sap from a Tunisian grapevine with mild mottling and leaf deformation. The virus sedimented in sucrose density gradients as three components: T (empty shells), M (particles containing a molecule of ssRNA with an apparent size of 5,800 nucleotides, constituting 35% of the particle weight) and B (particles containing a molecule of ssRNA with apparent size of 6,800 nucleotides, constituting 41% of the particle weight). Virus particles had buoyant densities of 1.31 (T), 1.45 (M), and 1.49 g/cm 3 (B) in cesium chloride equilibrium gradients. The coat protein subunits consisted of a single polypeptide with mol. wt. of c. 59,000 daltons. An antiserum was produced with a titer of 1 : 256, which did not react with healthy plant antigens. Cells of artificially infected herbaceous hosts showed cytoplasmic vesiculate-vacuolate inclusion bodies, virus-containing tubules, mostly associated with plasmodesmata and/ or cell wall protrusions, and crystalline aggregates of virus particles and empty capsids. The physicochemical and ultrastructural properties of this virus resemble very much those of nepoviruses. However, it was serologically unrelated to t9 different members of the group, including all those reported to infect grapevines. Therefore, the virus is possibly a hitherto unreported nepovirus for which the name of grapevine Tunisian ringspot virus (GTRV) is proposed.
Introduction The virus described in the present paper is a seemingly new member of the Nepovirus group [5-], isolated by mechanical inoculation from Tunisian grapevines in the course of a survey for virus and virus diseases of Vitis in the Mediterranean area [9]. The source plant was a vine of unknown variety which
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did n o t s h o w perceptible signs o f disease except for an exceedingly mild mottling and d e f o r m a t i o n o f the leaves. This vine did n o t c o n t a i n mechanically transmissible viruses o t h e r t h a n the one described hereafter, for which the n a m e o f grapevine Tunisian ringspot virus ( G T R V ) is p r o p o s e d .
Materials and methods Transmission and culture of the virus isolate GTRV was isolated from grapevine leaves crushed in a mortar in the presence of a 1 : 1 mixture of 0.1 M phosphate buffer pH 7.2 and 2.5% aqueous nicotine solution. The slurry was rubbed onto celite-dusted leaves of herbaceous hosts and subsequently those plants showing symptoms were kept as virus cultures and used for subinoculations for host range determination. Grapevine seedlings of cv. Mission at the coltyledonary stage were kept in the dark for 2 weeks prior to mechanical inoculation with concentrated, partially purified virus preparations. Latent infections were ascertained by back inoculations to Chenopodium
quinoa. Purification and fractionation of virus particles Systemically infected plants of Cucumis sativus were harvested about 12 days after inoculation and utilized immediately or after storage for a few days in a refrigerator. Infected tissues were processed in either of two ways: (a) Extraction by homogenizing in a blender with 1 to 2 vol. of 0,1 M phosphate buffer p H 7 containing 0.1% thioglycohc acid, straining through muslin, addition of 5% Mgactivated bentonite [3], centfifugation at 10,000g for 15min, and addition of 10% polyethylene glycol (PEG, mol. wt. 6,000) plus 1% NaC1. After stirring in the cold for about 1 h, the precipitate was collected by low-speed centrifugation (t0,000 g for 10rain), resuspended in 0.02 M phosphate buffer pH 7.2, and centrifuged at 68,000 g for 2 h or 86,000 g for 90 min. (b) Extraction as above in 0.07 M phosphate buffer pH 7 containing 1% mercaptoacetate and 0.01 M EDTA, addition to filtrate of an equal volume of a 1 : 1 mixture of chloroformbutanol [18], centrifugation at 3,000 g for 20 min, addition to the supernatant of 10% PEG and 1% NaC1, and application of two cycles of differential centrifugation. Final pellets were resuspended in 0.02 M phosphate buffer pH 7. Fractionation of concentrated partially purified virus preparations was in 10-40% linear sucrose desity gradient columns centrifuged at 24,000 rpm in a Beckman SW.27 rotor. The gradients were scanned at 254 nm with a ISCO ultraviolet absorbance monitor and the peaks corresponding to virus fractions were collected separately, dialyzed against 0.02 M phosphate buffer pH 7.2 and concentrated by high-speed centrifugafion. Centrifugafion at equilibrium was in caesium chloride gradients. Purified virus was mixed with a CsC1 solution in 0.02 M phosphate buffer pH 7.2 (initial density 1.33 g/cm 3) and centrifuged for 18h at 36,000rpm in a Beckman SW.41 rotor. The virus bands were collected by piercing with a microsyringe the side of the tubes and then dialyzed against 0.02 M phosphate buffer pH 7.2.
Analysis of viral nucleic acid Nucleic acid was obtained from unfractionated purified virus preparations suspended in 0.02 M phosphate buffer, by incubating at room temperature for 10min in presence of 1 mM EDTA and 1% SDS, then extracting twice with I vol. of water-saturated phenol [ 1]. The aqueous phase was washed twice with ether and the nucleic acid was collected by ethanol precipitation overnight at - 20 °C. Extracted nucleic acid was electrophoresed in
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1.2% agarose gels in TBE buffer (22.5 mM Tris, 22.5 mM boric acid, 0.5 mM EDTA pH 8.3) after denaturation with 50% formamide at 65 °C for 5 rain. Reference markers were E. coli ribosomal RNAs (2,904 nucleotides and 1,541 nt, respectively) and grapevine fanleaf nepovirus (GFLV) genomic RNAs 1 and 2 (6,800 nt and 3,900 nt, respectively). The gels were stained with ethidium bromide. The type and strandedness of nucleic acid was ascertained by enzymic digestion with DNase-free pancreatic RNase (2 pg/ml) in high (0.3 M SSC buffer) or low-salt (0.003 M SSC buffer) (SSC = 0.15 M sodium chloride and 0.015 M sodium citrate), or with RNasefree DNase (2.5 gg/ml) in 10 mM magnesium chloride. For RNA infectivity assays, each electrophoretic band was cut from agarose gels, soaked in an Eppendorf tube with 400 pl of TE buffer (0.01 M Tris and 0.001 M EDTA, pH 7.8), incubated at 65 °C for 10 min, extracted twice with phenol (400 pl) and chloroform (400 gl), and precipitated with ethanol at - 20 °C. Nucleic acid was resuspended in an autoclavesterilized inoculation buffer (0.05 M glycine, 0.03 M potassium phosphate, 1% bentonite, 1% celite) and rubbed onto C. sativus cotyledons.
Analysis of coat protein Purified virus preparations were disrupted by boiling for 5 rain in the presence of 10% SDS and 2% 2-mercaptoethanol in 0.4 M Tris-HC1 buffer pH 6.8. Protein samples were electrophoresed with a Protean II apparatus (Bio Rad) in 12.5% polyacrylamide slab gels using Laemmli's [7] discontinuous buffer system and stained with Coomassie brilliant blue. The molecular weight of the protein subunit was determined by comparison with the following reference markers (mol. wt. in parentheses): a-lactoalbumin (14,400), soybean trypsin inhibitor (20,100), carbonic anhydrase (30,000), ovalbumin (43,000), bovine serum albumin (67,000), phosphorylase B (94,000). Western blotting was done using a Trans-blot cell (Bio Rad). Electrophoretic bands of dissociated coat protein of GTRV and GFLV were transferred (20 V constant voltage for 1 h) to polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Millipore). Membrane "blocking", incubation with polyclonal immunoglobulins, rinsing and staining was as described by Hu et al. [6].
Serology An antiserum to GTRV was raised using purified preparations from CsC1 gradients. A rabbit was injected intramuscularly with antigen (0.8rag nucleoproteins) emulsified in Freund's incomplete adjuvant followed by two intravenous injections (0.5 rag/injection) one week apart. Antiserum collection began 8 days after the last injection. The titre of different serum samples was determined by immunodiffusion in agar plates (Oxoid Agar no. 1 0.7%, NaC1 0.85%, sodium azide 0.02%).
Electron microscopy Purified virus preparations were negatively stained with 2% aqueous uranyl acetate. Thin sections of local lesions and systemically infected leaf tissues of C. sativus and Nicotiana clevelandii were prepared according to standard procedures [ 10]. Tissue samples were fixed in 4% glutaraldehyde in 0.05 M phosphate buffer pH 7.2, postfixed at 4°C in 1% osmium tetroxide, dehydrated in graded ethanol dilutions and embedded in Spurr's resin. Thin sections were double stained with uranyl acetate and lead citrate before observation with a Philips 201 C electron microscope. Controls consisted of tissue samples from healthy seedlings.
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Host range and symptomatology GTRV had a moderately wide experimental host range infecting either with visible symptoms or symptomlessly 14 of 19 plant species, belonging to 6 different botanical families. Nicotianan tabacum cv. White Burley and Samsun, N. glutinosa, Ocymum basilicum, Phaseolus vulgaris cv. La Victoire, and P. aureus did not develop symptoms and proved not to be infected when back-inoculated to C. quinoa. Gomphrena globosa and N. benthamiana were latently infected, whereas N. tabacum cv. Xanthi, N. rustica and N. cavicola reacted only with chlorotic or necrotic local lesions. Systemic symptoms consisting of vein clearing, mottling or chlorotic ringspotting were shown by N. clevelandii, N. megalosiphon, N. rotundifolia, N. occidentalis, Petunia hybrida, and Cucurbita pepo cv. Striata d'Italia. Symptoms in other diagnostic species were as follows. Chenopodium amaranticolor: minute necrotic local lesions in 5-6 days, not followed by systemic infection; C. quinoa: chlorotic lesions in 5-6 days, systemic mottling, deformation of the leaves and apical necrosis; and C. sativus cv. Delicatezza: chlorotic local lesions in inoculated cotyledons in 4-6 days, severe mottling and necrotic flecks of systemically infected leaves. Of 50 mechanically inoculated grapevine seedlings, 18 survived. Of these, three were infected by GTRV, which could readily be recovered by sap inoculation. Eight months after inoculation, infected seedlings grown under glasshouse conditions did not show visible symptoms.
Stability in leaf extracts Extracts of C. sativus leaves in distilled water were no longer infective after a dilution of 10 -3, heating for 10min at 65°C, and/or a week storage at room temperature.
Virus purification and fractionation Clarification of sap extracts with either magnesium-bentonite or chloroformbutanol removed most of the host components. The results were consistent with both methods but virus yield was low, with a seasonal variation ranging from 1.5 to 6mg/kg of leaf tissue. Partially purified, unfractionated preparations contained two types of isometric particles c. 30 nm in diameter, i.e., empty shells penetrated by the stain and apparently intact particles (Fig. 1 D). These preparations were infectious when inoculated to assay hosts (C. sativusand C. quinoa) and exhibited u.v. absorption spectrum of nucleoproteins with E m a × = 260 nm, Emin = 240 nm, and E 260/E 280 = 1.8.
Properties of fractionated virus Sucrose density gradient centrifugation separated virus preparations into three components (Fig. 1 A). When seen in the electron microscope, the slowest-
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Fig.1. A U.V.-absorption trace of a GTRV preparation after centrifugation through 1040% sucrose density gradients. Three centrifugal components denoted T, M, and B are clearly discernible. Sedimentation is from left to right (arrow). B Particles from T component. Most of the particels are empty capsids penetrated by uranyl acetate. C Particles from components M and B. All particles are apparently intact. D Purified lmfractionated GTRV preparation showing both "empty" and "full" virus particles. Bars: 100 nm
sedimenting fraction (T) mostly contained e m p t y shells (Fig. 1 B) whereas the two faster sedimenting c o m p o n e n t s (M and B) consisted of apparently intact particles (Fig. 1 C).
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Unfractionated virus preparations centrifuged at equilibrium in CsC1, also yielded three components banding at densities of 1.31 (T), 1.45 (M), and 1.49 g/ cm 3 (B). From these values, nucleic acid contents of about 35 and 41% were calculated for M and B fractions, respectively [17]. When purified preparations of GTRV and GFLV were mixed and centrifuged at equilibrium in CsC1, T and B components of both viruses sedimented together, each couple forming a single band at different density levels. By contrast, M components were clearly separated banding at densities of 1.45 g/cm 3 (GTRV) and 1.41 g/cm 3 (GFLV), respectively. When inoculated to assay hosts, T fractions did not induce symptoms, contrary to mixtures of M and B components which were highly infectious. After separation by two successive cycles of density gradient centrifugation in sucrose columns, M component gave rise to a single non-infective sedimenting band. However, despite of several attempts, no complete separation of B from M component could be achieved, so that B fractions always retained a residual infectivity.
Identification and properties of nucleic acid Infectious nucleic acid was extracted successfully from unfractionated virus preparations or from pooled M and B particles. No nucleic acid was detected in T particles. Electrophoresis of nucleic acid extracts from M and B components showed two main species migrating very closely to one another (Fig. 2 A). These were resitant to DNase but were degraded by pancreatic RNase in both low and high salt (not shown) and were therefore identified as single-stranded RNA. The slowest nucleic acid species of GTRV (RNA-1) migrated at the same rate as RNA-1 of GFLV (tool. wt. 2.4 x 10 6 daltons, c. 6,800 nt) (Fig. 2 A), whereas mol. wt. of the faster migrating RNA-2 was estimated to be c. 2 x 106 daltons (c. 5,800 nt). Satisfactory separation of the two RNAs could be obtained by two successive electrophoretic runs (Fig. 2 B) but after elution and pooling no infectivity was recovered.
Virus coat protein A single protein with an estimated tool. wt. of c. 59,000 daltons was observed in gels loaded with preparations from unfractionated virus (Fig. 2 C, lanes 1 and 2). GTRV coat protein subunits had a slower migration rate than those of GFLV (mol. wt. 57,000) from which they were clearly separated, even when mixed preparations were etectrophoresed (Fig. 2 C, lane 2). In Western blots, the protein bands of GTRV and GFLV were specifically and selectively recognized only by the homologous antisera (not shown).
Serology GTRV was moderately immunogenic. The antiserum obtained had a titer of 1 : 256 in gel double diffusion plates and did not react visibly with healthy plant
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Fig. 2. A Electropherogram of a GTRV nucleic acid extract showing two closely migrating bands (2). Reference markers in 1 (genomie GFLV RNAs) and 3 (E. coli ribosomal RNAs). Note that RNAs- 1 of GFLV and GTRV migrate at the same rate. B Separation of GTRV genomic RNAs (1) after two electrophoretic runs. RNA-1 and RNA-2 in 2 and 3, respectively. C Electropherogram of dissociated capsid proteins of GTRV (1) and GFLV (3) alone or in mixture (2). Reference markers in 4 (numbers are the corresponding tool. wt.) extracts nor with GFLV preparations. Single precipitin lines merging at the point of junction were formed when the antiserum was allowed to react with different GTRV fractions. No reaction was detected when partially purified GTRV was tested with antisera to the following nepoviruses, which include all those known to infect grapevines (asterisk): *arabis mosaic (ArMV), *artichoke Italian latent (AILV), cherry leafroll (CLRV), cherry rasp leaf (CRLV), *grapevine Bulgarian latent (GBLV), *grapevine chrome mosaic (GCMV), *raspberry ringspot (RRV), *strawberry latent ringspot (SLRV), *tomato black ring (TBRV), *tomato ringspot (ToRSV), *tobacco ringspot (TRSV), artichoke yellow ringspot (AYRSV), chicory yellow mottle (CYMV), *peach rosette mosaic
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(PRMV), cocoa necrosis (CNV), *blueberry leaf mottle (BBLMV), and myrobalan latent ringspot (MyLRV).
Cytopathology Regardless of the type of infection (local or systemic) or the host (C. sativus, N. clevelandii) the cytopathology was the same. The general cytology of many cells was relatively well preserved and their major organelles had an appearance comparable to that of healthy controls. Other cells, however, showed a cytopathic condition due to fragmentation of the ground cytoplasm deriving from abundant secondary vacuolation and to the abnormal development of membranes in the form of a proliferated endoplasmic reticulum and excessive vesiculation. These membranous materials accumulated in certain cytoplasmic areas giving rise to conspicuous vesiculate-vacuolate inclusion bodies (Fig. 3 A). Structural modification of the cell wall-plasmalemma interface consisting of multivesicular structures (paramural bodies) were rather frequent, together with cell wall outgrowths developing at the level of plasmodesmata and protruding into the cytoplasm (Fig. 3 D). Virus particles appeared as electron opaque rounded bodies with a regular and smooth outline and a diameter of c. 22 nm. Virions were either scattered at random in the cytoplasm or gathered in single rows inside tubular structures c. 42 nm in width, or grouped to form rather large aggregates. Virus-containing tubules were rarely seen free in the cytoplasm. Sometimes they gathered in the proximity of paramural bodies or, more often, were connected with plasmodesmata and/or contained in the cell wall outgrowths (Fig. 3 D). Ribbon-shaped paracrystalline aggregates of virus particles were seen in the vacuoles (Fig. 3 B). These could also contain clusters of virus particles embedded in a densely staining amorphous matrix, resembling those associated with olive latent ringspot nepovirus infections [2], or relatively large viral aggregates in which the particles were more or less arranged in a crystalline lattice. Several of these crystals were made up of both heavily stained solid particles, interpreted as profiles of intact virions, and doughnut-shaped particles with an electron lucent center, likely to represent empty virus capsids (Fig. 3 C). Virions could also be clustered inside plasmodesmatal dilations (Fig. 3 E).
Discussion Many of the features shown by GTRV are typical of nepoviruses [13]: (a) transmissibility by inoculation of sap; (b) size and outward appearance of virus
Fig. 3. A A vesiculate-vacuolate inclusion body (IB) in the cytoplasm of a systemically infected cell of N. clevelandii. B Rows of virus particles in a ribbon-like paracrystalline aggregate in a systemicallyinfected C. sativus cell. C A large crystalline structure containing many empty viral capsids. Dark-stained spots are intact virus particles. D Virus-containing tubules with plasmodesmata and developing cell wall (CW) outgrowths (arrows). E Virus particles clustering within plasmodesmatal dilations. Bars: 200 nm
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particles; (c) presence of three centrifugal components (T, M, and B) all consisting of a single type of coat protein subunit with mol. wt. of c. 59,000, but deprived of nucleic acid (T) or containing one molecule each of ssRNA with an apparent size of c. 5,800 nt (M) or 6,800 nt (B), respectively, both of which are essential for infectivity; (d) cytological modifications, i.e., presence of large vesiculate-vacuolate cytoplasmic inclusion bodies, virus-containing tubules and cell wall outgrowths [4, 10]. Cells infected by GTRV, however, displayed some unusual cytopathological features. For instance, groups of free virus particles were seen in plasmodesmatal dilations similar to those reported for cucumoand luteoviruses, but contrary to what is known for nepoviruses, whose association with plasmodesmata seems to be almost invariably mediated by tubular structures [8]. Furthermore, viral crystalline aggregates were much larger than those usually induced by other members of the group and consisted of a mixture of complete virions and empty capsids. With other nepoviruses (e.g., GFLV, ArMV, and AYRSV) when aggregates of empty capsids are formed, they are rather small, have a loose paracrystaUine structure, do not contain full particles, and are primarily located in the nucleus [15, 16]. Based on physicochemical properties, the nepovirus group has been divided into subgroups [12, 14]. Because of its properties, GTRV appears to belong to the tomato ringspot virus subgroup characterized by a homogeneous B component (containing one molecule of RNA-1 only) and an RNA-2 with a tool. wt. equal or above 2 x 10 6 daltons. Of the 12 viruses included in this subgroup [11], eight (AYRSV, BBLMV, CLRV, CYMV, GBLV, MyLRSV, PRMV, and ToRSV) were tested serologically and found to be unrelated to GTRV. The tested viruses comprised the four known to infect grapevines (BBLMV, GBLV, PRMV, ToRSV). GTRV therefore seems to be a previously undescribed member of the nepovirus group. GTRV does not constitute an economic threat for grapevines. Besides its rare occurrence, the virus does not appear to be pathogenic, as demonstrated by the virtual absence of symptoms in naturally infected vines and artificially inoculated seedlings. It has, however, a scientific importance for it represents the first new nepovirus recorded in a woody host in the southern bank of the Mediterranean.
Acknowledgements Grateful thanks are expressed to Dr. D. C. RamsdeU for the gift of antisera to BBLMV and PRMV.
References 1. Diener TO, Schneider IR (1968) Virus degradation and nucleic acid release in singlephase phenol systems. Arch Biochem Biophys 124:401-412 2. Di Franco A, Martelli GP, Russo M (1983) An ultrastructural study of olive latent ringspot virus in Gomphrenaglobosa. J Submicroscop Cytol 15:539-548 3. Dunn DB, Hitchborn JH (1965) The use of bentonite in the purification of plant viruses. Virology 25:171-192
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4. Francki RIB, Milne RG, Hatta T (1985) Atlas of plant viruses, vol 2. CRC Press, Boca Raton 5. Harrison BD, Murant AF (1978) Nepovirus group. CMI/AAB Description of Plant Viruses, no 185 6. Hu JS, Gonsalves D, Teliz D (1990) Characterization of closterovirus-like particles associated with grapevine leafroll disease. J Phytopathol 128:1-14 7. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacterial phage T 4. Nature 227:680-685 8. Martelli GP (1980) Ultrastructural aspects of possible defence reactions in virus-infected plants. Microbiologica 3:639-691 9, Martelli GP (1986) Virus and virus-like diseases of the grapevine in the Mediterranean area. FAO Plant Protect Bull 34:25-42 10., Martelli GP, Russo M (1984) Use of thin sectioning for the visualization and identification of plant viruses. In: Maramorosch K, Koprowski H (eds) Methods in virology, vol 8. Academic Press, New York, pp 143-224 11. Martelli GP, Taylor CE (1990) Distribution of viruses and their nematode vectors. In: Harris KF (ed) Advances in disease vector research, vol 6. Springer, Berlin Heidelberg New York Tokyo, pp 151-189 12. Martelli GP, Quacquarelli A, GaUitelli D, Savino V, Piazzolla P (1978) A tentative grouping of nepoviruses. Phytopathol Medit 17:147 13. Mutant AF (1981) Nepoviruses. In: Kurstak E (ed) Handbook of plant virus infections and comparative diagnosis. Elsevier/North-Holland, Amsterdam, pp 19'7-238 14. Mutant AF, Taylor M (1978) Estimates of molecular weight ofnepovirus RNA species by polyacrylamide gel etectrophoresis under denaturing conditions. J Gen Virol 41: 53-61 15. Russo M (1985) Electron microscopy of grapevine virus infections. Phytopathol Medit 24:144-147 16. Russo M, Martelli GP, Rana GL, Kyriakopoulou PE (1978) The ultrastructure of artichoke yellow ringspot virus infections. Microbiologica 1:81-99 17. Seghal OP, Jean JL, Bhalla RB, Soong MM, Krause GF (1970) Correlation between buoyant densities and ribonucleic acid content in viruses. Phytopathology 60: 17781784 18. Steere RL (1956) Purification and properties of tobacco ringspot virus. Phytopathology 46:60-69 Authors' address: G. P. Martelli, Dipartimento di Protezione delle Piante dalle Malattie, Universifft degli Studi di Bari, Via Amendola 165 A, 1-70126 Bari, Italy. Received November 19, 1991
