Identification of putative viroplasms within banana cells infected by banana streak MY virus.

The badnavirus replication cycle is poorly understood and most knowledge is based on extrapolations from model viruses such as Cauliflower mosaic virus (CaMV). However, in contrast to CaMV, badnaviruses are thought not to produce viroplasms and therefore it has been a mystery as to where virion assembly occurs. In this study, ultrathin sections of a banana leaf infected with a badnavirus, banana streak MY virus (BSMYV), were examined by transmission electron microscopy. Electron-dense inclusion bodies (EDIBs) were sporadically distributed in parenchymatous tissues of the leaf, most commonly in the palisade and spongy mesophyll cells. These EDIBs had a characteristic structure, comprising an electron-dense core, a single, encircling lacuna and an outer ring of electron-dense material. However, much less frequently, EDIBs with two or three lacunae were observed. In the outer ring, densely packed virions were visible with a shape and size consistent with that expected for badnaviruses. Immunogold labelling was done with primary antibodies that detected the N-terminus of the capsid protein and strong labelling of the outer ring but not the central core or lacuna was observed. It is concluded that the EDIBs that were observed are equivalent in function to the viroplasms of CaMV, although obviously different in composition as there is not a paralogue of the transactivation/viroplasm protein in the badnavirus genome. It is postulated that production of a viroplasm could be a conserved characteristic of all members of the Caulimoviridae.

Infection of non-host model plant species with the narrow-host-range Cacao swollen shoot virus.

Cacao swollen shoot virus (CSSV) is a major pathogen of cacao (Theobroma cacao) in Africa, and long-standing efforts to limit its spread by the culling of infected trees have had very limited success. CSSV is a particularly difficult virus to study, as it has a very narrow host range, limited to several tropical tree species. Furthermore, the virus is not mechanically transmissible, and its insect vector can only be used with difficulty. Thus, the only efficient means to infect cacao plants that have been experimentally described so far are by particle bombardment or the agroinoculation of cacao plants with an infectious clone. We have genetically transformed three non-host species with an infectious form of the CSSV genome: two experimental hosts widely used in plant virology (Nicotiana tabacum and N. benthamiana) and the model species Arabidopsis thaliana. In transformed plants of all three species, the CSSV genome was able to replicate, and, in tobacco, CSSV particles could be observed by immunosorbent electron microscopy, demonstrating that the complete virus cycle could be completed in a non-host plant. These results will greatly facilitate the preliminary testing of CSSV control strategies using plants that are easy to raise and to transform genetically.

Sequence analysis of shorter than genome length episomal Banana streak OL virus like sequences isolated from banana in India.

Electron microscopy and sequencing of reverse transcriptase and ribonuclease H (RT/RNase H) region of Badnavirus genome from two banana cultivars: Poovan (triploid: AAB) and Safed velchi (diploid: AB), exhibiting leaf streak symptoms, confirmed the association of Banana streak OL virus (BSOLV). As per ICTV species demarcation threshold of 80 % identity in RT/RNase H region, both the isolates were identified as BSOLV. Rolling circle and end-to-end amplification showed the association of two short episomal BSOLV variants: BSOLV-IN1 and BSOLV-IN2 from Poovan and Safed velchi banana, respectively. The genome sizes of both isolates were 6,950 nucleotides long, but shorter than the typical BSOLV genome of 7,389 bp. Open reading frames (ORFs) 1 and 2 of shorter BSOLV isolates shared almost complete nucleotide identity (>99 %) to that of BSOLV. However, the ORF 3 (5,130 bp) and intergenic region (IGR), 886 bp, showed deletions compared with ORF 3 (5,499 bp) and IGR (956 bp) of BSOLV. In phylogenetic analysis for ORF 3 polyprotein, both the isolates clustered with BSOLV, Banana streak CA virus (BSCAV), and Sugarcane bacilliform GA virus (SCBGAV). Identical ORF 1, ORF 2, and the presence of all the conserved domains in short ORF 3 and promoter elements in IGR indicated that these isolates represent replicationally competent shorter variants of BSOLV. These two shorter-than-BSOLV genome sequences and two other identical banana streak virus sequences in GenBank (BSV-TRY; DQ859899 and BSV-GD; DQ451009) might have evolved due to error-prone reverse transcription and splicing or excision from the integrated sequences by homologous recombination in natural banana hybrids under field conditions.

In situ localization of cacao swollen shoot virus in agroinfected Theobroma cacao.

Cacao swollen shoot virus (CSSV) is a small non-enveloped bacilliform virus with a double-stranded DNA genome. A very restricted host range and difficulties in transmitting the virus, either mechanically or via its natural vector, have hindered the study of cacao swollen shoot disease. As an alternative to the particle-bombardment method previously reported, we investigated another approach to infect Theobroma cacao. A greater-than-unit length copy (1.2) of the CSSV DNA genome was cloned into the Agrobacterium binary vector pBin 19 and was transferred into young plants via Agrobacterium tumefaciens. Typical leaf symptoms and stem swelling were observed seven and eleven weeks post inoculation, respectively. Viral DNA, CSSV coat protein and virions were detected in leaves with symptoms. Agroinfected plants were used to study the in situ localization of CSSV and its histopathologic effects in planta. In both leaves and petioles, virions were only seen in the cytoplasm of phloem companion cells and of a few xylem parenchyma cells. Light microscopy showed that stem swelling results from a proliferation of the xylem, phloem and cortex cells.

Tubules containing virions are present in plant tissues infected with Commelina yellow mottle badnavirus.

Tubular structures containing bacilliform virions were observed in cell-free extracts of Commelina diffusa infected with Commelina yellow mottle badnavirus (CoYMV). The exterior of the tubule reacted with antibodies to CoYMV movement protein, but not with antibodies to virus coat protein. Similar tubular structures containing bacilliform particles were also observed in ultrathin sections of CoYMV-infected C. diffusa. These tubular structures traversed the cell wall at points where this was thickened or protruded. No similar structures were observed in healthy C. diffusa. These observations support the hypothesis that the virion-containing tubular structures observed in cell-free extracts are the same as those observed in situ, that these structures are composed, at least in part, of virus movement protein, and that they play a role in the cell-to-cell trafficking of virions of CoYMV.

Detection of sugarcane bacilliform virus in sugarcane germplasm.

Sugarcane bacilliform virus (SCBV), a badnavirus was found in sugarcane genotypes of Saccharum officinarum L., S. barberi Jesw., S. sinense Roxb., S. robustum Brand and Jesw., and Saccharum hybrids. In most of the suspected genotypes the virus was found associated with clear foliar symptoms. However, certain symptom-free clones carried the virus too. The virus was detected by immuno-electron microscopy (IEM) and enzyme-linked immunosorbent assay (ELISA) in suspected clones. The virions measured about 108-118 x 20-21 nm in size. The virus was serologically closely related to another badnavirus, banana streak virus (BSV). Virus titer was low in most of the genotypes. However, a close correlation between symptoms expression and virus titer existed in some genotypes.