The complete nucleotide sequence of pelargonium leaf curl virus.

Investigation of a tombusvirus isolated from tulip plants in Scotland revealed that it was pelargonium leaf curl virus (PLCV) rather than the originally suggested tomato bushy stunt virus. The complete sequence of the PLCV genome was determined for the first time, revealing it to be 4789 nucleotides in size and to have an organization similar to that of the other, previously described tombusviruses. Primers derived from the sequence were used to construct a full-length infectious clone of PLCV that recapitulates the disease symptoms of leaf curling in systemically infected pelargonium plants.

Cassava Ivorian bacilliform virus is a member of the genus Anulavirus.

The complete genomic sequence of Cassava Ivorian bacilliform virus (CIBV) is described. The virus has a genomic organization similar to that of pelargonium zonate spot virus (PZSV), the type member of the genus Anulavirus, but it is most closely related to a second, recently described, anulavirus, Amazon lily mild mottle virus (ALiMMV).

Raspberry leaf blotch virus, a putative new member of the genus Emaravirus, encodes a novel genomic RNA.

A new, segmented, negative-strand RNA virus with morphological and sequence similarities to other viruses in the genus Emaravirus was discovered in raspberry plants exhibiting symptoms of leaf blotch disorder, a disease previously attributed to the eriophyid raspberry leaf and bud mite (Phyllocoptes gracilis). The virus, tentatively named raspberry leaf blotch virus (RLBV), has five RNAs that each potentially encode a single protein on the complementary strand. RNAs 1, 2 and 3 encode, respectively, a putative RNA-dependent RNA polymerase, a glycoprotein precursor and the nucleocapsid. RNA4 encodes a protein with sequence similarity to proteins of unknown function that are encoded by the genomes of other emaraviruses. When expressed transiently in plants fused to green or red fluorescent protein, the RLBV P4 protein localized to the peripheral cell membrane and to punctate spots in the cell wall. These spots co-localized with GFP-tagged tobacco mosaic virus 30K cell-to-cell movement protein, which is itself known to associate with plasmodesmata. These results suggest that the P4 protein may be a movement protein for RLBV. The fifth RLBV RNA, encoding the P5 protein, is unique among the sequenced emaraviruses. The amino acid sequence of the P5 protein does not suggest any potential function; however, when expressed as a GFP fusion, it localized as small aggregates in the cytoplasm near to the periphery of the cell.

Genome activation by raspberry bushy dwarf virus coat protein.

Two sets of infectious cDNA clones of raspberry bushy dwarf virus (RBDV) have been constructed, enabling either the synthesis of infectious RNA transcripts or the delivery of infectious binary plasmid DNA by infiltration of Agrobacterium tumefaciens. In whole plants and in protoplasts, inoculation of RBDV RNA1 and RNA2 transcripts led to a low level of infection, which was greatly increased by the addition of RNA3, a subgenomic RNA coding for the RBDV coat protein (CP). Agroinfiltration of RNA1 and RNA2 constructs did not produce a detectable infection but, again, inclusion of a construct encoding the CP led to high levels of infection. Thus, RBDV replication is greatly stimulated by the presence of the CP, a mechanism that also operates with ilarviruses and alfalfa mosaic virus, where it is referred to as genome activation. Mutation to remove amino acids from the N terminus of the CP showed that the first 15 RBDV CP residues are not required for genome activation. Other experiments, in which overlapping regions at the CP N terminus were fused to the monomeric red fluorescent protein, showed that sequences downstream of the first 48 aa are not absolutely required for genome activation.

Improved PCR Detection of Blackcurrant reversion virus in Ribes and Further Evidence that It Is the Causal Agent of Reversion Disease.

Within 5 years of mechanically inoculating blackcurrant cultivars with partially purified preparations of particles of Blackcurrant reversion virus (BRV), infected plants developed leaf and flower bud symptoms typical of reversion disease, demonstrating that BRV is the causal agent of this disease. To improve the erratic immunocapture reverse transcriptase-polymerase chain reaction (RT-PCR) detection of BRV in Ribes plants, various stepwise changes were made to the original protocol. Significant improvement in the reliability and sensitivity of BRV detection was made by extracting RNA from trapped BRV particles using Triton-X 100, the design of new primers with higher annealing temperatures, and the use of 'Ready-to-go' RT-PCR beads. These features, combined with other minor changes to the protocol, improved BRV detection in reverted blackcurrant plants from <50% to >90% but the reliability of BRV detection in red currant was always very much less and was possible only using nested PCR that was developed for this purpose.

Incidence and Distribution of Raspberry bushy dwarf virus in Commercial Red Raspberry (Rubus idaeus) Crops in Scotland.

A survey was done in 1998 to determine whether Raspberry bushy dwarf virus (RBDV) was established in raspberry fruiting plantations in Scotland. Raspberry-producing holdings were selected according to geographical area and size. Samples (201), each comprising 60 shoots per stock, were obtained from 77 holdings and tested by enzyme-linked immunosorbent assay (ELISA). ELISA-positive shoots from each infected stock were grafted onto cultivar Glen Clova, which is resistant to the Scottish-type isolate of RBDV (RBDV-S), to establish whether the virus is a resistance-breaking (RB) isolate. RBDV was detected in 22% of the stocks sampled, with 2 to 80% incidence of infection. No RBDV was in any of the 40 plantations containing cultivars resistant to RBDV-S or in Glen Clova plants, which were grafted successfully with samples from 15 infected plantations, indicating that no RB isolates were detected. The percentage of infected plantations increased with time from the planting date. In order to investigate possible sources of infection, ELISA for RBDV was made in 1999 on samples of stocks of raspberry cultivars entered for the lowest certified grade (Standard Grade) in Scotland and, in 1994 to 1997, on certified stocks planted with material originating from outside Scotland. No RBDV was detected in any of the samples. RBDV was found only rarely in samples of wild raspberry in Angus and Perthshire.