Wendy J. McGavin

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.

A new badnavirus in Ribes species, its detection by PCR, and its close association with Gooseberry vein banding disease

Gooseberry vein banding disease (GVBD) affects Ribes species and cultivars worldwide. It is the second most important virus-like disease in these crops after black currant reversion disease. In this paper, we describe a bacilliform virus, Gooseberry vein banding associated virus (GVBAV), which is associated closely with GVBD, and provide evidence that GVBAV is a distinct species within the genus Badnavirus. Purified GVBAV particles were ca. 120 × 30 nm in size and contained dsDNA. The sequence of a 1.5-kb DNA fragment amplified from viral genomic DNA was similar to those of a wide range of badnaviruses and contained motifs characteristic of the RNase H domain of the badnavirus open reading frame (ORF) III polyprotein. Phylogenetic analyses suggest that GVBAV is most closely related to Spiraea yellow leaf spot virus. Using sequence derived from the polymerase chain reaction (PCR)-amplified DNA fragment, virus-specific primers were designed. These primers were used in PCR to assay for GVBAV in a range of Ribes germplasm affected with GVBD, with other unrelated virus-like diseases and viruses found in Ribes, and in healthy plants. GVBAV was detected in all of 58 GVBD-affected plants from diverse sources, but not from healthy Ribes plants nor from plants infected with other viruses.

Some coat protein constituents from strawberry latent ringspot virus expressed in transgenic tobacco protect plants against systematic invasion following root inoculation by nematode vectors.

The coding sequences in RNA2 for the coat proteins (CP) of strawberry latent ringspot virus (SLRSV) were modified and amplified using polymerase chain amplification reactions (PCR) to facilitate their expression inAgrobacterium tumefaciens-transformedNicotiana tabacum Xanthi-nc. The coding sequences for the smaller capsid protein (S, 29kDa) and that for the theoretical precursor of L and S (P, 73kDa) had ATG ‘initiation’ codon sequences added at the 5′-proximal Ser/Gly (S/G) cleavage site in the unmodified sequence. The sequence coding for the larger of the two proteins of mature SLRSV capsids (L, 44kDa) had an ATG codon added at its 5′ S/G site and a TAG ‘stop’ codon sequence added at the 3′-proximal S/G site. The P, L and S proteins were expressedin planta to a maximum concentration of 0.01 % of total extractable proteins but did not assemble into virus-like particles. When challenged by mechanical inoculation with virus particles or viral RNA, and compared with control plants, tobacco plants (primary transgenic clones or S1 and S2, kanamycin-resistant seedlings) expressing the virus capsid subunits separately, or their precursor, decreased the accumulation of SLRSV particles in inoculated leaves and fewer plants became invaded systemically. In experiments in which the roots of seedlings were exposed to SLRSV-carrying vector nematodes (Xiphinema diversicaudatum), SLRSV was detected in the roots of non-transformed control tobacco plants (6/20) and in transgenic tobacco expressing the L protein (7/40), but not in any of 25 tobacco plants expressing the S protein or in 35 expressing the P protein. This is the second example of CP-mediated resistance to virus inoculation by nematode vectors.

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.

Newly identified RNAs of raspberry leaf blotch virus encoding a related group of proteins.

Members of the genus Emaravirus, including Raspberry leaf blotch virus (RLBV), are enveloped plant viruses with segmented genomes of negative-strand RNA, although the complete genome complement for any of these viruses is not yet clear. Currently, wheat mosaic virus has the largest emaravirus genome comprising eight RNAs. Previously, we identified five genomic RNAs for RLBV; here, we identify a further three RNAs (RNA6-8). RNA6-8 encode proteins that have clear homologies to one another, but not to any other emaravirus proteins. The proteins self-interacted in yeast two-hybrid and bimolecular fluorescence complementation (BiFC) experiments, and the P8 protein interacted with the virus nucleocapsid protein (P3) using BiFC. Expression of two of the proteins (P6 and P7) using potato virus X led to an increase in virus titre and symptom severity, suggesting that these proteins may play a role in RLBV pathogenicity; however, using two different tests, RNA silencing suppression activity was not detected for any of the RLBV proteins encoded by RNA2-8.

Comparisons of some properties of two laboratory variants of Raspberry bushy dwarf virus (RBDV) with those of three previously characterised RBDV isolates

The properties of two laboratory variants of Raspberry bushy dwarf virus (RBDV), genus Idaeovirus, were compared with those of their parental sources and with two naturally occurring variants. Isolate RB is a natural variant able to overcome the resistance to RBDV present in some red raspberry cultivars. Isolate M is a serological variant from black raspberry. Laboratory variant D1, was derived from the Scottish type isolate (D200) by continuous sub-culture in Chenopodium quinoa. Laboratory variant Can-S was derived from an isolate infecting Canby red raspberry in Canada (Can) after passage through Nicotiana benthamiana. All isolates reacted with a polyclonal antiserum to isolate D200 in agarose gel double-diffusion tests but, whereas isolates D200, RB, Can and Can-S were serologically indistinguishable, the precipitin lines formed by these isolates each spurred over those formed by isolates D1 and M. All six isolates reacted strongly with the polyclonal antiserum in double antibody sandwich and plate-trapped antigen (PTA) forms of ELISA and in Western blotting (WB) and when each of four monoclonal antibodies (Mabs) to an unnamed red raspberry isolate from Canada was used to detect antigen trapped by the polyclonal antiserum. However, the virus isolates differed in their reactions to these four Mabs in PTA-ELISA and in WB. Isolates RB, Can and Can-S behaved similarly in these tests as did isolates D200 and D1, but isolate M was distinct. In herbaceous test plants, variants D1 and Can-S were readily distinguished from their parental sources and from the other two isolates by producing either no symptoms (D1) or very severe symptoms (Can-S) in hosts. Unlike all other isolates studied world-wide, Can-S failed to infect C. quinoa systemically but induced severe necrotic local lesions in this and other hosts.Reverse transcription-polymerase chain reaction was used to amplify the gene encoding the coat protein (CP) in RNA-2, and a region of the gene encoding the polymerase in RNA-1. The nucleotide sequences of the CP genes of the six isolates were > 96% identical but isolate Can-S was the most distinctive. However, the similarity between Can-S and its parent isolate (Can) was no greater than the similarity between Can-S and the other isolates, suggesting that Can-S may not have arisen as the result of a mutation from isolate Can. Sequence comparisons of parts of the polymerase gene of isolates R15, D1, D200 and Can-S showed that they were 95–98% identical.

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.

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).

Blackcurrant reversion virus: Validation of an improved diagnostic test, accelerating testing in breeding and certification of blackcurrants

A study was commenced in 2005 to provide a validated diagnostic test for the detection of Blackcurrant reversion virus (BRV) to be used as an alternative to the conventional test recommended for use in the UK certification scheme. A range of cultivars previously virus indexed and held as nuclear stock (Baldwin, Ben Lomond and Ben Tirran) were grafted with known BRV-positive scions and assessed over the following four years. Data was collected through visual observations and buds sampled and tested with a single round RT-PCR using two new primer sets. For the method to be accepted for use in Scotland the detection rate must be equal or better than that of the existing grafting method. Statistical analysis of our results for the detection of both the European (E) and Russian (R) forms of Reversion disease in the three cultivars provides a validation for this test. We suggest that this method provides a faster throughput test for BRV even when no symptoms are visible on the plants, and so is suitable for adoption into the guidelines followed by both UK and European authorities for detection of BRV in blackcurrant propagation material.

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.