Molecular characterization and population structure of blackberry vein banding associated virus, new Ampelovirus associated with yellow vein disease.

Blackberry yellow vein disease is the most important viral disease of blackberry in the United States. Experiments were conducted to characterize a new virus identified in symptomatic plants. Molecular analysis revealed a genome organization resembling Grapevine leafroll-associated virus 3, the type species of the genus Ampelovirus in the family Closteroviridae. The genome of the virus, provisionally named blackberry vein banding associated virus (BVBaV), consists of 18,643 nucleotides and contains 10 open reading frames (ORFs). These ORFs encode closterovirid signature replication-associated and quintuple gene block proteins, as well as four additional proteins of unknown function. Phylogenetic analyses of taxonomically relevant products consistently placed BVBaV in the same cluster with GLRaV-3 and other members of the subgroup I of the genus Ampelovirus. The virus population structure in the U.S. was studied using the replication associated polyprotein 1a, heat shock 70 homolog and minor coat proteins of 25 isolates. This study revealed significant intra-species variation without any clustering among isolates based on their geographic origin. Further analyses indicated that these proteins are under stringent purifying selections. High genetic variability and incongruent clustering of isolates suggested the possible involvement of recombination in the evolution of BVBaV.

Experimental and bioinformatic evidence that raspberry leaf blotch emaravirus P4 is a movement protein of the 30K superfamily.

Emaravirus is a recently described genus of negative-strand RNA plant viruses. Emaravirus P4 protein localizes to plasmodesmata, suggesting that it could be a viral movement protein (MP). In the current study, we showed that the P4 protein of raspberry leaf blotch emaravirus (RLBV) rescued the cell-to-cell movement of a defective potato virus X (PVX) that had a deletion mutation in the triple gene block 1 movement-associated protein. This demonstrated that RLBV P4 is a functional MP. Sequence analyses revealed that P4 is a distant member of the 30K superfamily of MPs. All MPs of this family contain two highly conserved regions predicted to form ß-strands, namely ß1 and ß2. We explored by alanine mutagenesis the role of two residues of P4 (Ile106 and Asp127) located in each of these strands. We also made the equivalent substitutions in the 29K MP of tobacco rattle virus, another member of the 30K superfamily. All substitutions abolished the ability to complement PVX movement, except for the I106A substitution in the ß1 region of P4. This region has been shown to mediate membrane association of 30K MPs; our results show that it is possible to make non-conservative substitutions of a well-conserved aliphatic residue within ß1 without preventing the membrane association or movement function of P4.

Population structure of blackberry chlorotic ringspot virus in the United States.

Blackberry chlorotic ringspot virus is a subgroup 1 ilarvirus, detected in several rosaceous hosts exhibiting disease symptoms in Europe and the United States. The population structure of the virus was studied using isolates collected from wild and cultivated plants from six states in the United States. The results suggest a homogeneous virus population in the United States, similar to what observed within single orchards for other ilarviruses. Given the lack of evidence for host or geography-driven adaptation, it is hypothesized that the virus was recently introduced into the New World.

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.

The complete sequence of a UK strain of black raspberry necrosis virus.

The complete nucleotide sequence of a UK strain of the sadwavirus Black raspberry necrosis virus (BRNV) was obtained by amplification and sequencing of virus RNA from infected plants grown in a raspberry plantation in Aylth, Scotland. The RNA1 was 7,572 nucleotides (nt) in size and RNA2 was 6,350 nt in size, each excluding the 3' poly-A tail. The RNA1- and RNA2-encoded polyproteins are predicted to be processed into (RNA1) a protease cofactor, an RNA helicase, the VpG, a 3C-like protease, an RNA-dependent RNA polymerase and an AlkB protein, and (RNA2) a movement protein and two capsid proteins.

Genomic sequences of blackberry chlorotic ringspot virus and strawberry necrotic shock virus and the phylogeny of viruses in subgroup 1 of the genus Ilarvirus.

Three members of subgroup 1 of the genus Ilarvirus: blackberry chlorotic ringspot (BCRV), strawberry necrotic shock (SNSV), and tobacco streak viruses (TSV), may infect Rubus and Fragaria species. All cause symptoms similar to those previously attributed to infection by TSV alone. Although similarities exist among the genomic sequences of the three, phylogenetic analysis shows them to be distinct viruses. These viruses and Parietaria mottle virus, the other currently accepted member of subgroup 1, appear to have evolved from a common ancestral virus, share conserved motifs in the products of the genomic RNAs, and constitute a distinct subgroup within the genus.

Identification and molecular characterization of a marafivirus in Rubus spp.

An undescribed virus with isometric particles and a diameter of ca. 30 nm was identified in diseased samples of wild and cultivated Rubus species and molecularly characterized. Its genome was 6,463 nt, excluding the 3'-terminal poly(A) tail, and contained a single open reading frame coding for a 2,035-amino-acid-long precursor polypeptide (p223). The amino terminal portion of p223, identified as a replication-associated polyprotein, contained conserved motifs of methyltransferase, endopeptidase/protease, helicase and RNA-dependent RNA polymerase. The carboxy terminus of the large polypeptide is involved in the formation of two viral coat protein subunits with deduced molecular masses of 23 and 21 kDa. Pairwise comparisons and phylogenetic analyses showed closest relationships of this virus with oat blue dwarf virus and citrus sudden death-associated virus, sharing levels of genome sequence conservation far below the species demarcation level established for tymovirids. Our data indicate that this virus, for which the name blackberry virus S (BlVS) is proposed, is a hitherto undescribed species of the genus Marafivirus, family Tymoviridae. A survey conducted in Mississippi, USA, has shown that BlVS is also present in cultivated Rubus germplasm. This work represents the first report of a marafivirus infecting small fruits.

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.

Strawberry necrotic shock virus is a distinct virus and not a strain of Tobacco streak virus.

Fragaria (strawberry) and Rubus species (blackberry, wild blackberry, red raspberry and black raspberry) were thought to be infected with distinct isolates of Tobacco streak virus (TSV). Employing serology and nucleic acid hybridization it has been shown that these isolates form a cluster distinct from other strains of TSV. In this study we have cloned and sequenced the complete RNA 3 of an isolate of TSV from strawberry (Fragaria) as well as the coat protein (CP) gene of 14 additional isolates of TSV originating from Fragaria and Rubus species. Our data suggest that the isolates of TSV that infect Fragaria and Rubus belong to a distinct virus for which we propose the name Strawberry necrotic shock virus (SNSV). The RNA 3 of SNSV contains 2248 nucleotides, 43 more than the type isolate of TSV from white clover (TSV-WC), with a CP gene that is 669 nucleotides long, in contrast to the 714-7 nucleotides of the TSV CP sequences found in the database. The movement protein gene of SNSV is 897 nucleotides in length, 27 more than that of the TSV-WC isolate of TSV. The CP genes of the 15 Fragaria and Rubus isolates that we studied form two distinct phylogenetic clusters that share about 95% amino acid sequence identity, while they only share 60-65% amino acid sequence identity with TSV-WC.

Characterization and complete nucleotide sequence of Strawberry mottle virus: a tentative member of a new family of bipartite plant picorna-like viruses.

An isolate of Strawberry mottle virus (SMoV) was transferred from Fragaria vesca to Nicotiana occidentalis and Chenopodium quinoa by mechanical inoculation. Electron micrographs of infected tissues showed the presence of isometric particles of approximately 28 nm in diameter. SMoV-associated tubular structures were also conspicuous, particularly in the plasmodesmata of C. quinoa. DsRNA extraction of SMoV-infected N. occidentalis yielded two bands of 6.3 and 7.8 kbp which were cloned and sequenced. Gaps in the sequence, including the 5' and 3' ends, were filled using RT-PCR and RACE. The genome of SMoV was found to consist of RNA1 and RNA2 of 7036 and 5619 nt, respectively, excluding a poly(A) tail. Each RNA encodes one polyprotein and has a 3' non-coding region of approximately 1150 nt. The polyprotein of RNA1 contains regions with identities to helicase, viral genome-linked protein, protease and polymerase (RdRp), and shares its closest similarity with RNA1 of the tentative nepovirus Satsuma dwarf virus (SDV). The polyprotein of RNA2 displayed some similarity to the large coat protein domain of SDV and related viruses. Phylogenetic analysis of the RdRp region showed that SMoV falls into a separate group containing SDV, Apple latent spherical virus, Naval orange infectious mottling virus and Rice tungro spherical virus. Given the size of RNA2 and the presence of a long 3' non-coding region, SMoV is more typical of a nepovirus, although atypically for a nepovirus it is aphid transmissible. We propose that SMoV is a tentative member of an SDV-like lineage of picorna-like viruses.

Aphid pests of strawberry crops and their parasitoids in France.

The pest status of aphids on strawberry crops has changed with time. They were mainly considered with respect to virus transmission. Now, they are rather considered as sap-feeders and honeydew producers. As such, low populations regulated by natural enemies can be tolerated and systematic use of insecticides avoided. A survey of the species present in the different areas of production has been undertaken during 1999 and 2000 in France. During this survey, well known species of the strawberry crops have been found, like Chaetosiphon fragaefolii, Aphis forbesi or Acyrthosiphon rogersii. Widely polyphagous species like Aphis gossypii, Aulacorthum solani, Myzus ascalonicus or Macrosiphum euphorbiae were also present quite often. Eventually, aphids of the Rosaceae, not considered before as important pests of the strawberries, like Rhodobium porosum, have also been found in many different areas. The biology of the different species and their natural enemies have to be taken into account with respect to integrated control of strawberry pests.