Identification and characterisation of tomato torrado virus, a new plant picorna-like virus from tomato

Abstract

A new virus was isolated from tomato plants from the Murcia region in Spain which showed symptoms of 'torrado disease'; very distinct necrotic, almost burn-like symptoms on leaves of infected plants. The virus particles are isometric with a diameter of approximately 28 nm. The viral genome consists of two (+)ssRNA molecules of 7793 (RNA1) and 5389 nts (RNA2). RNA1 contains one open reading frame (ORF) encoding a predicted polyprotein of 241 kDa that shows conserved regions with motifs typical for a protease-cofactor, a helicase, a protease and an RNA-dependent RNA polymerase. RNA2 contains two, partially overlapping ORFs potentially encoding proteins of 20 and 134 kDa. These viral RNAs are encapsidated by three proteins with estimated sizes of 35, 26 and 23 kDa. Direct protein sequencing mapped these coat proteins to ORF2 on RNA2. Phylogenetic analyses of nucleotide and derived amino acid sequences showed that the virus is related to but distinct from viruses belonging to the genera Sequivirus, Sadwavirus and Cheravirus. This new virus, for which the name tomato torrado virus is proposed, most likely represents a member of a new plant virus genus.

Fig. 1

Typical initial symptoms of tomato torrado virus on tomato leaves: necrotic spots, surrounded by a light green or yellow area, beginning at the base of a leaflet

Fig. 2

Denaturating agarose gel electrophoresis of RNAs extracted from ToTV virions and stained with orthotoluidine blue. 1 (M): molecular size standard (Invitrogen 0.24–9.5-kb RNA Ladder); 2 (T): RNA purified from ToTV top component; 3 (B): RNA purified from ToTV bottom component. Arrows indicate positions of the ∼5.5-kb and ∼8-kb RNA bands

Fig. 3

A: Silver-stained capsid proteins after denaturing polyacrylamide gelelectrophoresis (SDS-PAGE). 1 and 4 (M): molecular weight markers (Bio-Rad silver stain markers, low range), 2: ToTV-T (top band of purified virions in Cs₂SO₄ buoyant density gradient centrifugation) and 3: ToTV-B (bottom band of purified virions after Cs₂SO₄ buoyant density gradient centrifugation). B: Amino acid sequence of RNA2-ORF2. Positions of the three coat proteins (Vp35, Vp26 and Vp23) are indicated by boxes. Putative cleavage site areas are indicated by dotted lines. Peptides found in MS=MS analysis are shaded

Fig. 4

Genome organization of the tomato torrado virus isolate PRI-ToTV0301. Relative positions of regions containing motifs of protease-cofactor (PRO-co), helicase (HEL), protease (PRO), and RNA-dependent RNA polymerase (RdRp) on RNA1 and of the three coat proteins (Vp35, Vp26 and Vp23) and putative movement protein (MP) on RNA2 are indicated

Fig. 5

Phylogenetic analysis of ToTV and related viruses based on the alignment of A) the region between the protease CG motif and GDD RdRp motif (aa 1041-1498 of RNA1-ORF1) and B) the helicase region between motifs A and C (aa 382–495 of RNA1-ORF1). Sequences included in the analysis are those of (with virus acronyms, genus and accession numbers in parentheses): acute bee paralysis virus (ABPV; unassigned species in the family Dicistroviridae; NP_066241), apple latent spherical virus (ALSV; Cheravirus NP_620568), avian encephalomyelitis virus (AEV; Hepatovirus; NP_653151), beet ringspot virus (BRSV; Nepovirus; NP_620112), blackcurrant reversion virus (BRV; Nepovirus; NP_612604), broad bean wilt virus-2 (BBWV-2; Fabavirus; AAK27841), cherry rasp leaf virus (CRLV; Cheravirus; YP_081444), cowpea mosaic virus (CPMV; {urComovirus}; NP_613283), grapevine fanleaf virus (GFLV; Nepovirus; NP_619689), hepatitis A virus (HAV; Hepatovirus; NP_041008), maize chlorotic dwarf virus (MCDV; Waikavirus; NP_619716), navel orange infectious mottling virus (NIMV; Sadwavirus; BAA74537), parsnip yellow fleck virus (PYFV; Sequivirus; NP_619734), rice tungro spherical virus (RTSV; Waikavirus; NP_042507), satsuma dwarf virus (SDV; Sadwavirus; NP_620566), Solenopsis invicta virus (SinV; unassigned species in the family Dicistroviridae; YP_164440), strawberry mottle virus (SMoV; Sadwavirus; NP_599086). strawberry latent ringspot virus (SLRSV; Sadwavirus; NC_006764), stocky prune virus (StPV; Cheravirus; DQ143874). Potato virus Y (PVY; Potyvirus; ABA28320) was used as an outgroup sequence in the analyses. The numbers at each node are the bootstrap values for 1000 replicates. The scale bar represents the number of residue substitutions per site