Species: Rice tungro spherical virus
|This is a revised version of DPV 67|
Department of Disease and Stress Biology, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK
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
Rice tungro disease was first described by Anon. (1964) and Rivera & Ou (1965). The first description of rice tungro spherical virus (RTSV) was by Galvez (1968) and the role of RTSV as a member of the virus complex that causes tungro disease was recognized by Hibino et al. (1978). The cause of waika disease of rice, first described by Saito (1977), was reported by Anjaneyulu et al. (1995).
A virus with isometric particles about 30 nm in diameter, which contain a single molecule of positive-sense ssRNA of over 12 kb, and three proteins, CP1, CP2 and CP3, of Mr 22,900, 22,300 and 33,000, respectively. The virus is transmitted in a semi-persistent manner by leafhoppers; it is not mechanically transmissible. It occurs in Oryza species in South and South-East Asia. It acts as a helper for leafhopper transmission of Rice tungro bacilliform virus (RTBV). In most cultivars of rice, RTSV causes no symptoms but in some cultivars it causes a mild stunting disease (waika); the RTSV/RTBV complex causes a severe disease (tungro).
Early host range studies on RTSV are unreliable as its association with RTBV was not then recognized. Host range studies are also constrained by the ability of the vector species to transmit the virus to the plant species tested. RTSV appears to have a restricted host range limited to members of the Poaceae and Cyperaceae (Echinochloa crus-galli, E. glabescens, E. colona, Leptochloa chinensis, Leersia hexandra, Oryza sativa, Panicum repens, Cyperus rotundus) (Khan et al., 1991). RTSV causes few, if any, symptoms in these hosts.
Because of lack of symptoms, the most reliable diagnosis is by serological or nucleic acid-based techniques. The virus can be propagated in rice cultivar TN1.
1. Method of Cabauatan & Hibino (1988):
Harvest rice plants 50 days after infection and homogenize each 500 g material in 1 L 0.01 M sodium ethylene-diamine-tetra-acetate (EDTA), pH 8.0. Incubate the extract with 3 g Driselase (Kyowa Hakko Kogyo Co. Ltd., Tokyo) for 1 h at room temperature,1 h at 40 °C, and then centrifuge for 10 min at 15,000g. Add polyethylene glycol (mol. wt 8000), NaCl and Triton X-100 to 7%, 0.2 M and 1% respectively and stir the mixture for 1 h at room temperature, centrifuge at 30,000g for 30 min and resuspend the pellets in 20 ml cold 0.01 M EDTA, pH 8.0. Centrifuge at 11,000g for 10 min, retain the supernatant fluid and centrifuge it at 100,000g for 60 min. Resuspend the pellet in 2 ml cold 0.01 M phosphate buffer (PB), pH 7.4, layer on 10-50% sucrose density gradients (in PB) and centrifuge in a Beckman SW27 rotor at 25,000 rev./min for 2.5 h at 4 °C. Recover the virus-containing band, dilute in 0.01 M PB and centrifuge at 130,000g for 1 h. Resuspend the resulting pellet in 1 ml PB and centrifuge at 11,000g for 10 min. The virus is found in the supernatant fluid.
2. Method of Jones et al. (1991):
Freeze infected rice leaves in liquid N2, grind and thaw each 100 g tissue into 400 ml 0.1 M sodium citrate, pH 5.9. Add Celluclast (Novo Enzymes) to 5% (v/v), incubate the mixture at 30 °C for 2 h and centrifuge at 10,000 rev./min for 10 min. Resuspend the pellet in 200 ml citrate buffer and 5% Celluclast, incubate for 1 h at 30 °C and centrifuge as above. Pool the supernatant fractions from both centrifugations and add polyethylene glycol (mol. wt 6000), NaCl and Triton X-100 to give 7%, 0.2 M and 1% respectively. Incubate the mixture for 2 h at room temperature, then centrifuge at 12,000 rev./min for 10 min. Resuspend the pellet in 20 ml citrate buffer and centrifuge through a 5% sucrose cushion at 36,000 rev./min for 2.5 h in a Beckman Ti40 rotor at 4 °C. Resuspend the pellet in 4 ml citrate buffer. Further purification and, if necessary, separation from RTBV can be effected by rate zonal or isopycnic gradient centrifugation.
Virus particles contain one species of positive-sense ssRNA. The complete nucleotide sequence has been determined for the type isolate (12,226 nucleotides; accession number NC001632) (Shen et al., 1993; Thole & Hull, 1996) and for the resistance-breaking strain, Vt6 (12,171 nucleotides; accession number AB064963) (Isogal et al., 2000).
Polyacrylamide gel electrophoresis of viral coat protein reveals three species, CP1, CP2 and CP3, of 21.5, 24 and 31 kDa (Hibino et al., 1991) or 25, 26 and 35 kDa (Jones et al., 1991), respectively. Identification of cleavage sites by N-terminal sequencing shows that CP1 is 22.9 kDa and CP2 is 22.3 kDa (Shen et al., 1993; Zhang et al., 1993); the C-terminus of CP3 has not been identified. In western blots of crude extracts from infected plants, an antiserum to CP3 identifies a band at 33 kDa and several in the range 40-42 kDa; the nature of these larger bands is unknown (Druka et al., 1996).
The genome encodes a polyprotein of 393 kDa (Fig.3) and has two possible small open reading frames (ORFs) at the 3' end (Shen et al., 1993); however, the nature of these 3' ORFs has been questioned (Thole & Hull, 1996).
The cleavage sites of two of the three coat protein species (CP1 and CP2) and of the C-terminal cysteine protease and polymerase products have been determined (Shen et al., 1993; Zhang et al., 1993; Thole & Hull, 1998). Little is known of the products N-terminal to the coat proteins or in the region downstream of CP3. An antiserum to E. coli-expressed CP1 detected a product of 32 kDa, much smaller than the expected 70 kDa (Hull, 1996); the region downstream of CP3 contains the sequence motif for a nucleotide triphosphate binding protein (Shen et al., 1993).
RTSV on its own causes no significant damage in most rice cultivars. However, it provides the transmission capability to RTBV in the economically important tungro complex and thus has considerable importance in relation to that disease. It spreads from old to seedling plants in overlapping cropping systems and is likely to spread from latent infections in weeds harbouring the leafhopper, Nephotettix virescens, surrounding rice paddies. Tungro infection occurs in periodic epidemics (Savary et al., 1993; Anjaneyulu et al., 1995).
Control of RTSV is either by use of insecticides (see Anjaneyulu et al., 1995) or by resistance breeding. Rice breeding programmes have attempted to introduce resistance to RTSV into elite cultivars. Resistance to the vector is not very durable (Dahal et al., 1990). Resistance to the virus occurs in varieties TKM6 (Hibino et al., 1988) and TW5 (Azzam et al., 2001), that of TKM6 co-segregating with leafhopper resistance (Sebastian et al., 1996); however, RTSV strain Vt6 breaks this resistance (Cabauatan et al., 1995). A single recessive gene controls the TKM6 resistance, whereas in TW5 two recessive genes are involved (Azzam et al., 2001). Transformation of rice with coat protein or replicase genes affords protection against the virus (Sivamani et al., 1999; Huet et al., 1999).
The rice green leafhopper, Nephotettix virescens.
Particles of RTSV negatively stained in uranyl acetate. Bar represents 100 nm.
Genome organization of RTSV. The horizontal line represents the genomic positive-sense ssRNA with the 3' polyadenylation (An) indicated. The box shows the polyprotein with below the positions of the processed coat proteins (CP1, CP2 and CP3), protease (Pro) and polymerase (Pol) indicated together with the motif for the NTP binding site.
Thin sections of rice plant, cultivar TN1, jointly infected with RTSV and RTBV showing both viruses in the cytoplasm. Bar represents 400 nm. (From Sta. Cruz et al., 1993).
Thin sections of rice plant, cultivar TN1, jointly infected with RTSV and RTBV showing RTSV embedded in an inclusion body in the cytoplasm. Bar represents 400 nm. (From Sta. Cruz et al., 1993).
Thin sections of rice plant, cultivar TN1, jointly infected with RTSV and RTBV showing RTSV as crystalline aggregates. Bar represents 400 nm. (From Sta. Cruz et al., 1993).
Thin sections of rice plant, cultivar TN1, jointly infected with RTSV and RTBV showing vesicles (VE) containing fibres (F) and RTSV particles. Bar represents 400 nm. (From Sta. Cruz et al., 1993).