989 resultados para Dasheen mosaic virus
Resumo:
Dasheen mosaic potyvirus (DsMV) is an important virus affecting taro. The virus has been found wherever taro is grown and infects both the edible and ornamental aroids, causing yield losses of up to 60%. The presence of DsMV, and other viruses,prevents the international movement of taro germplasm between countries. This has a significant negative impact on taro production in many countries due to the inability to access improved taro lines produced in breeding programs. To overcome this problem, sensitive and reliable virus diagnostic tests need to be developed to enable the indexing of taro germplasm. The aim of this study was to generate an antiserum against a recombinant DsMV coat protein (CP) and to develop a serological-based diagnostic test that would detect Pacific Island isolates of the virus. The CP-coding region of 16 DsMV isolates from Papua New Guinea, Samoa, Solomon Islands, French Polynesia, New Caledonia and Vietnam were amplified,cloned and sequenced. The size of the CP-coding region ranged from 939 to 1038 nucleotides and encoded putative proteins ranged from 313 to 346 amino acids, with the molecular mass ranging from 34 to 38 kDa. Analysis ofthe amino acid sequences revealed the presence of several amino acid motifs typically found in potyviruses,including DAG, WCIE/DN, RQ and AFDF. When the amino acid sequences were compared with each other and the DsMV sequences on the database, the maximum variability was21.9%. When the core region ofthe CP was analysed, the maximum variability dropped to 6% indicating most variability was present in the N terminus. Within seven PNG isolates ofDsMV, the maximum variability was 16.9% and 3.9% over the entire CP-coding region and core region, respectively. The sequence ofPNG isolate P1 was most similar to all other sequences. Phylogenetic analysis indicated that almost all isolates grouped according to their provenance. Further, the seven PNG isolates were grouped according to the region within PNG from which they were obtained. Due to the extensive variability over the entire CP-coding region, the core region ofthe CP ofPNG isolate Pl was cloned into a protein expression vector and expressed as a recombinant protein. The protein was purified by chromatography and SDS-PAGE and used as an antigen to generate antiserum in a rabbit. In western blots, the antiserum reacted with bands of approximately 45-47 kDa in extracts from purified DsMV and from known DsMV -infected plants from PNG; no bands were observed using healthy plant extracts. The antiserum was subsequently incorporated into an indirect ELISA. This procedure was found to be very sensitive and detected DsMV in sap diluted at least 1:1,000. Using both western blot and ELISA formats,the antiserum was able to detect a wide range ofDsMV isolates including those from Australia, New Zealand, Fiji, French Polynesia, New Caledonia, Papua New Guinea, Samoa, Solomon Islands and Vanuatu. These plants were verified to be infected with DsMV by RT-PCR. In specificity tests, the antiserum was also found to react with sap from plants infected with SCMV, PRSV-P, PRSV-W, but not with PVY or CMV -infected plants.
Resumo:
Abacá mosaic virus (AbaMV) is related to members of the sugarcane mosaic virus subgroup of the genus Potyvirus. The ~2 kb 3′ terminal region of the viral genome was sequenced and, in all areas analysed, found to be most similar to Sugarcane mosaic virus (SCMV) and distinct from Johnsongrass mosaic virus (JGMV), Maize dwarf mosaic virus (MDMV) and Sorghum mosaic virus (SrMV). Cladograms of the 3′ terminal region of the NIb protein, the coat protein core and the 3′ untranslated region showed that AbaMV clustered with SCMV, which was a distinct clade and separate from JGMV, MDMV and SrMV. The N-terminal region of the AbaMV coat protein had a unique amino acid repeat motif different from those previously published for other strains of SCMV. The first experimental transmission of AbaMV from abacá (Musa textilis) to banana (Musa sp.), using the aphid vectors Rhopalosiphum maidis and Aphis gossypii, is reported. Polyclonal antisera for the detection of AbaMV in western blot assays and ELISA were prepared from recombinant coat protein expressed in E. coli. A reverse transcriptase PCR diagnostic assay, with microtitre plate colourimetric detection, was developed to discriminate between AbaMV and Banana bract mosaic virus, another Musa-infecting potyvirus. Sequence data, host reactions and serological relationships indicate that AbaMV should be considered a distinct strain of SCMV, and the strain designation SCMV-Ab is suggested.
Resumo:
We have characterised six Australian Cucumber mosaic virus (CMV) strains belonging to different subgroups, determined by the sequence of their complete RNA 3 and by their host range and the symptoms they cause on species in the Solanaceae, Cucurbitaceae and on sweet corn. These data allowed classification of strains into the known three CMV subgroups and identification of plant species able to differentiate the Australian strains by symptoms and host range. Western Australian strains 237 and Twa and Queensland strains 207 and 242 are closely related members of CMV subgroup IA, which cause similar severe symptoms on Nicotiana species. Strains 207 and 237 (subgroup IA) were the only strains tested which systemically infected sweet corn. Strain 243 caused the most severe symptoms of all strains on Nicotiana species, tomato and capsicum and appears to be the first confirmed subgroup IB strain reported in Australia. Based on pair-wise distance analysis and phylogeny of RNA 3, as well as mild disease symptoms on Nicotiana species, CMV 241 was assigned to subgroup II, as the previously described Q-CMV and LY-CMV.
Resumo:
A panel of 19 monoclonal antibodies (mAbs) was used to study the immunological variability of Lettuce mosaic virus (LMV), a member of the genus Potyvirus, and to perform a first epitope characterization of this virus. Based on their specificity of recognition against a panel of 15 LMV isolates, the mAbs could be clustered in seven reactivity groups. Surface plasmon resonance analysis indicated the presence, on the LMV particles, of at least five independent recognition/binding regions, correlating with the seven mAbs reactivity groups. The results demonstrate that LMV shows significant serological variability and shed light on the LMV epitope structure. The various mAbs should prove a new and efficient tool for LMV diagnostic and field epidemiology studies.
Resumo:
Cucumber mosaic virus (CMV) was found by reverse transcription polymerase chain reaction (RT-PCR) to be not fully systemic in naturally infected kava (Piper methysticum) plants in Fiji. Twenty-six of 48 samples (54%) from various tissues of three recently infected plants were CMV-positive compared with 7/51 samples (14%) from three long-term infections (plants affected by dieback for more than 1 year). The virus was also found to have a limited ability to move into newly formed stems. CMV was detected in only 2/23 samples taken from re-growth stems arising from known CMV infected/dieback affected plants. Mechanical inoculation experiments conducted in Fiji indicate that the known kava intercrop plants banana (Musa spp.), pineapple (Ananas comosus), peanut (Arachis hypogaea) and the common weed Mikania micrantha are potential hosts for a dieback-causing strain of CMV It was not possible to transmit the virus mechanically to the common kava intercrop plants taro (Colocasia esculenta), Xanthosoma sp., sweet potato (Ipomoea batatas), yam (Dioscorea alata), papaya (Carica papaya) or the weed Momordica charantia. Implications of the results of this research on a possible integrated disease management strategy are discussed.
Resumo:
Carrot was confirmed as a new natural and experimental host of Watermelon mosaic virus by serology, host reactions and sequence comparisons of the coat protein.
Resumo:
Although monocotyledonous-plant-infecting mastreviruses (in the family Geminiviridae) are known to cause economically significant crop losses in certain areas of the world, in Australia, they pose no obvious threat to agriculture. Consequently, only a few Australian monocot-infecting mastreviruses have been described, and only two have had their genomes fully sequenced. Here, we present the third full-genome sequence of an Australian monocot-infecting mastrevirus from Bromus catharticus belonging to a distinct species, which we have tentatively named Bromus catharticus striate mosaic virus (BCSMV). Although the genome of this new virus shares only 57.7% sequence similarity with that of its nearest known relative, Digitaria didactyla striate mosaic virus (DDSMV; also from Australia), it has features typical of all other known mastrevirus genomes. Phylogenetic analysis showed that both the full genome and each of its probable expressed proteins group with the two other characterised Australian monocot-infecting mastreviruses. Besides the BCSMV genome sequence revealing that Australian monocot-infecting mastrevirus diversity rivals that seen in Africa, it has enabled us, for the first, to time detect evidence of recombination amongst the Australian viruses. Specifically, it appears that DDSMV possesses a short intergenic region sequence that has been recombinationally derived from either BCSMV or a close relative that has not yet been identified.
Resumo:
The properties of the S-strain of cucumber mosaic virus (S-CMV) and the B-strain of tomato aspermy virus (B-TAV) have been studied with respect to their (i) size and sedimentation behavior, (ii) requirement of divalent metal ions for stability, (iii) sensitivity towards chloride salts and the anionic detergent sodium dodecyl sulfate, (iv) solubility in ammonium sulfate-containing buffers, and (v) pH-dependent structural transitions. The results indicate that the coat protein of B-TAV is more hydrophobic than the other well-studied strains of TAV and CMV. Circular dichroism and uv absorption studies reveal pH-dependent structural transitions, although these do not result in particle swelling. These transitions appear to alter the strength of protein-nucleic acid interactions in these viruses.
Resumo:
Protein-protein interactions play a Crucial role in Virus assembly and stability. With the view of disrupting capsid assembly and capturing smaller oligomers, interfacial residue mutations were carried Out in the coat protein gene of Sesbania Mosaic Virus, a T=3 ss (+) RNA plant virus. A single point mutation of a Trp 170 present at the five-fold interface of the virus to a charged residue (Glu or Lys) arrested assembly of virus like particles and resulted in stable Soluble dimers of the capsid Protein. The X-ray crystal structure of one of the isolated dimer mutants - rCP Delta N65W170K was determined to a resolution of 2.65 angstrom. Detailed analysis of the dimeric mutant protein structure revealed that a number of Structural changes take place, especially in the loop and interfacial regions during the course of assembly. The isolated chiller was ``more relaxed'' than the dimer found in the T=3 or T=1 capsids. The isolated dimer does not bind Ca2+ ion and consequently four C-terminal residues are disordered. The FG loop, which interacts with RNA in the Virus, has different conformations in the isolated dimer and the intact Virus Suggesting its flexible nature and the conformational changes that accompany assembly. The isolated choler mutant was much less stable when compared to the assembled capsids, suggesting the importance of inter-subunit interactions and Ca2+ mediated interactions in the stability of the capsids. With this study, SeMV becomes the first icosahedral virus for which X-ray crystal Structures of T=3, T=1 capsids as well as a smaller oligomer of the capsid protein have been determined.
Resumo:
Sesbania mosaic virus (SMV) is an isometric, ss-RNA plant virus found infecting Sesbania grandiflora plants in fields near Tirupathi, South India. The virus particles, which sediment at 116 S at pH 5.5, swell upon treatment with EDTA at pH 7.5 resulting in the reduction of the sedimentation coefficient to 108 S. SMV coat protein amino acid sequence was determined and found to have approximately 60% amino acid sequence identity with that of southern bean mosaic virus (SBMV). The amino terminal 60 residue segment, which contains a number of positively charged residues, is less well conserved between SMV and SBMV when compared to the rest of the sequence. The 3D structure of SMV was determined at 3.0 Å resolution by molecular replacement techniques using SBMV structure as the initial phasing model. The icosahedral asymmetric unit was found to contain four calcium ions occurring in inter subunit interfaces and three protein subunits, designated A, B and C. The conformation of the C subunit appears to be different from those of A and B in several segments of the polypeptide. These observations coupled with structural studies on SMV partially depleted of calcium suggest a plausible mechanisms for the initiation of the disassembly of the virus capsid.
Resumo:
The complete genome of an Australian isolate of zantedeschia mild mosaic virus (ZaMMV) causing mosaic symptoms on Alocasia sp. (designated ZaMMVAU) was cloned and sequenced. The genome comprises 9942 nucleotides (excluding the poly-A tail) and encodes a polyprotein of 3167 amino acids. The sequence is most closely related to a previously reported ZaMMV isolate from Taiwan (ZaMMV-TW), with 82 and 86 % identity at the nucleotide and amino acid level, respectively. Unlike the amino acid sequence of ZaMMV-TW, however, ZaMMV-AU does not contain a polyglutamine stretch at the N-terminus of the coat-protein-coding region upstream of the DAG motif. This is the first report of ZaMMV from Australia and from Alocasia sp.
Resumo:
Sesbania mosaic virus (SeMV) is a ss-RNA (4149 nt) plant sobemovirus isolated from farmer's field around Tirupathi, Andhra Pradesh. The viral capsid (30 nm diameter) consists of 180 copies of protein subunits (MW 29 kDa) organized with icosahedral symmetry. In order to understand the mechanism of assembly of SeMV, a large number of deletion and substitution mutants of the coat protein (CP) were constructed. Recombinant SeMV CP (rCP) as well as the N-terminal rCP deletion mutant Delta N22 were found to assemble in E. coli into virus-like particles (VLPs). Delta N36 and Delta N65 mostly formed smaller particles consisting of 60 protein subunits. Although particlem assembly was not affected due to the substitution of aspartates (D14 and D149) that coordinate calcium ions by asparagines, the stability of the resulting capsids was drastically reduced. Deletion of residues forming a characteristic beta-annulus at the icosahedral 3-folds did not affect the assembly of VLPs. Mutation of a single tryptophan, which occurs near the icosahedral fivefold axis to glutamate or lysine, resulted in the disruption of the capsid leading to soluble dimers that resembled the quasi-dimer structure of the native virus. Replacement of positively charged residues in the amino terminal segment of CP resulted in the formation of empty shells. Based on these observations, a plausible mechanism of assembly is proposed.
Resumo:
Sesbania mosaic virus (SeMV) is a single-stranded positive-sense RNA plant virus belonging to the genus Sobemovirus. The movement protein (MP) encoded by SeMV ORF1 showed no significant sequence similarity with MPs of other genera, but showed 32% identity with the MP of Southern bean mosaic virus within the Sobemovirus genus. With a view to understanding the mechanism of cell-to-cell movement in sobemoviruses, the SeMV MP gene was cloned, over-expressed in Escherichia coli and purified. Interaction of the recombinant MP with the native virus (NV) was investigated by ELISA and pull-down assays. It was observed that SeMV MP interacted with NV in a concentration- and pH-dependent manner. Analysis of N- and C-terminal deletion mutants of the MP showed that SeMV MP interacts with the NV through the N- terminal 49 amino acid segment. Yeast two-hybrid assays confirmed the in vitro observations, and suggested that SeMV might belong to the class of viruses that require MP and NV/coat protein for cell-to-cell movement.
Resumo:
Sesbania mosaic virus (SMV) is a plant virus infecting Sesbania grandiflora plants in Andhra Pradesh, India. Amino acid sequence of the tryptic peptides of SMV coat protein were determined using a gas phase sequenator. These sequences showed identical amino acids at 69% of the positions when aligned with the corresponding residues of southern bean mosaic virus (SBMV).Crystals diffracting to better than 3 Å resolution were obtained by precipitating the virus with ammonium sulphate. The crystals belonged to rhombohedral space group R3 with α = 291·4 Å and α = 61·9°. Three-dimensional X-ray diffraction data on these crystals were collected to a resolution of 4·7 Å, using a Siemens-Nicolet area detector system. Self-rotation function studies revealed the icosahedral symmetry of the virus particles, as well as their precise orientation in the unit cell. Cross-rotation function and modelling studies with SBMV showed that it is a valid starting model for SMV structure determination. Low resolution phases computed using a polyalanine model of SBMV were subjected to refinement and extension by real-space electron density averaging and solvent flattening. The final electron density map revealed a polypeptide fold similar to SBMV. The single disulphide bridge of SBMV coat protein is retained in SMV. Four icosahedrally independent cation binding sites have been tentatively identified. Three of these sites, related by a quasi threefold axis, are also found in SBMV. The fourth site is situated on the quasi threefold axis. Aspartic acid residues, which replace Ile218 of SBMV from the quasi threefold-related subunits are suitable ligands to the cation at this site