A comparative study of red clover vein mosaic virus and some other plant viruses.
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The role of recombination in the evolution of Cucumber mosaic virus (CMV) was analyzed in a collection of Spanish isolates from 1989 to 2002. Isolates were characterized by ribonuclease protection assay using six RNA probes, two for each of the three genomic RNAs, which allowed the identification of the analyzed regions as belonging to CMV isolates in subgroups IA, IB, and II. Most isolates belonged to subgroups IA (64%) and IB (12%), 5% were reassortants among subgroups IA, IB, or II, and 17% were recombinants between these groups. Recombinants at RNA3 were significantly more frequent than recombinants at RNAs 1 and 2. One IB-IA recombinant RNA3 was as frequent in central Spain as the IA RNA3. The genetic structure of the virus population suggested that reassortants and most recombinant genotypes were selected against and was consistent with a higher biological cost of reassortment than recombination. Data also suggest that recombinants that encode hybrid proteins are at a higher disadvantage than recombinants that exchange whole ORFs.
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Plant viruses pose a significant threat to global agriculture and require efficient tools for their timely detection. We present AutoPVPrimer, an innovative pipeline that integrates artificial intelligence (AI) and machine learning to accelerate the development of plant virus primers. The pipeline uses Biopython to automatically retrieve different genomic sequences from the NCBI database to increase the robustness of the subsequent primer design. The design_primers_with_tuning module uses a random forest classifier that optimizes parameters and provides flexibility for different experimental conditions. Quality control measures, including the evaluation of poly-X content and melting temperature, increase primer reliability. Unique to AutoPVPrimer is the visualize_primer_dimer module, which supports the visual evaluation of primer dimers-a feature missing in other tools. Primer specificity is validated via primer BLAST, which contributes to the overall efficiency of the pipeline. AutoPVPrimer has been successfully applied to the tomato mosaic virus, proving its adaptability and efficiency. The modular design allows customization by the user and extends the applicability to different plant viruses and experimental scenarios. The pipeline represents a significant advance in primer design and provides researchers with an effective tool to accelerate molecular biology experiments. Future developments aim to extend compatibility and incorporate user feedback to consolidate AutoPVPrimer as an innovative contribution to the bioinformatics toolbox and a promising resource for the advancement of plant virology research.
Plant virus cell-to-cell movement and subsequent systemic transport are governed by a series of mechanisms involving various virus and plant factors. Specialized virus encoded movement proteins (MPs) control the cell-to-cell transport of viral nucleoprotein complexes through plasmodesmata. MPs of different viruses have diverse properties and each interacts with specific host factors that also have a range of functions. Most viruses are then transported via the phloem as either nucleoprotein complexes or virions, with contributions from host and virus proteins. Some virus proteins contribute to the establishment and maintenance of systemic infection by inhibiting RNA silencing-mediated degradation of viral RNA. In spite of all the different movement strategies and the viral and host components, there are possible functional commonalities in virus-host interactions that govern viral spread through plants.
The filamentous particles of poplar mosaic virus (PopMV) were detected in palisade, mesophyll and parenchyma cells of systemically infected leaves of Nicotiana clevelandii and N. megalosiphon. The particles were sometimes found scattered throughout the cytoplasm, but more often they occurred loosely associated within ovoid aggregates (up to 3.5 x 1.5 mum) adjacent to chloroplasts and/or mitochondria. PopMV did not induce the formation of 'pinwheel' inclusions, nor other conspicuous ultrastructural or cytopathic effects in infected cells. These observations provide additional evidence that PopMV is a Carlavirus and not a Potyvirus nor a mite-borne virus. PopMV particles were also detected occasionally within plasmodesmata.
Over the last 15 years, interest in plant virus evolution has re-emerged, as shown by the increasing number of papers published on this subject. In recent times, research in plant virus evolution has been viewed from a molecular, rather than populational, standpoint, and there is a need for work aimed at understanding the processes involved in plant virus evolution. However, accumulated data from analyses of experimental and natural populations of plant viruses are beginning to delineate some trends that often run contrary to accepted opinion: (1) high mutation rates are not necessarily adaptive, as a large fraction of the mutations are deleterious or lethal; (2) in spite of high potential for genetic variation, populations of plant viruses are not highly variable, and genetic stability is the rule rather than the exception; (3) the degree of constriction of genetic variation in virus-encoded proteins is similar to that in their eukaryotic hosts and vectors; and (4) in spite of huge census sizes of plant virus populations, selection is not the sole factor that shapes their evolution, and genetic drift may be important. Here, we review recent advances in understanding plant virus evolution, and describe the experimental and analytical methods most suited to this purpose.
The replication of positive-strand RNA plant viruses, which involves both virus-encoded and plant-encoded proteins, takes place in two stages: synthesis of a negative-strand RNA using the genomic positive-strand RNA as a template and synthesis of progeny positive-strand RNA using the negative-strand RNA as a template. Using gel mobility shift and photochemical crosslinking assays, we have identified three proteins of M(r) 32K, 50K and 100K in extracts of tobacco and spinach leaves that bind to the 3'-terminal sequences of the negative-strand RNA of three diverse positive-strand RNA plant viruses. The 32K protein was purified to near homogeneity by chromatography on columns of Macro-prep high Q, heparin-sepharose, single-stranded DNA cellulose and poly(U)-sepharose. No binding of any of the three proteins to the 3'-termini of the positive-strand RNA or the 5'-termini of the positive-strand RNA or negative-strand RNA of any of the three viruses, or the 3'-termini of the mRNAs of two chloroplast genes, psbA or petD, could be detected. We propose that 3'-terminal negative-strand RNA binding proteins, which may be widespread in the plant kingdom, could be utilised by at least three different positive-strand RNA plant viruses for the initiation of positive-strand RNA synthesis.
Seedlings of Kennedya rubicunda were manually inoculated with Kennedya yellow mosaic virus and planted along with comparable virus-free seedlings in a coastal bushland site where the plant and virus were common. Virus infection decreased the growth rate of the plants by about one-third; however, virus-free plants disappeared more than twice as quickly as infected plants as a result of grazing by herbivores. In palatability trials using grated carrot bait, caged young rabbits preferred bait mixed with powdered virus-free K.rubicunda leaves to that mixed with virus-infected leaf powder. However, 'polyamine extracts' of the leaves did not have a comparable effect.
Certain plant viruses require a 'helper component' in order to be transmitted by aphids. The helper component for potyviruses is a virus-encoded protein with a subunit molecular weight of between 50,000 and 60,000. Aphid transmission of caulimoviruses is associated with the presence of an 18,000 molecular weight polypeptide. The mode of action of the helper component is the subject of speculation.
The DNAs of several plant viruses were analyzed for the presence of alternating purine-pyrimidine sequences that can potentially undergo B to Z transition. The DNA of the caulimoviruses (plant retroviruses) was compared with that of the geminiviruses, with the cDNA of an RNA plant virus, and with several computer-generated random sequences. Our analysis indicates that potential Z-DNA sites tend to be restricted in the DNA of the caulimoviruses, whereas the same does not occur significantly in the other viral DNAs examined. This result is discussed in relation to the mode of replication of the caulimoviral DNA and offers additional evidence of the existence of selection processes regulating the frequency and distribution of the Z-DNA sites in the different genomes.
Nonspecific binding of immunoglobulins to coat protein of cucumber mosaic virus and several other plant viruses was observed in Western immunoblots, and to a much lesser extent in enzyme-linked immunosorbent assays. The binding appears to occur between immunoglobulin molecules and the basic domains of viral coat proteins which bind to RNA during encapsidation. In all cases tested, the nonspecific reactions could be prevented by addition of 5 U/ml of heparin to the incubation buffers.
Capsid proteins of a number of plant viruses are permissive to genetic modifications in which foreign polypeptides are inserted in exposed loops or at their C termini. Plant viruses with these genetic alterations often grow at wild-type levels, providing gram quantities of modified viruses. Presented polypeptides studied most extensively correspond to antigenic epitopes of animal viruses and in some cases appropriately altered plant viruses generate neutralizing antibodies to the cognate animal virus when the plant virus is used as a vaccine. Structure-based analyses of these animal-plant virus chimeras have led to rational alterations to the presentation in efforts to increase the efficacy of the presented peptide.
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A new chromatographic medium, Convective Interaction Media(CIM) disk monolithic columns, was applied to plant virus concentration. The ability of the columns to concentrate highly diluted plant viruses was tested on a model plant virus, rod-shaped tomato mosaic virus (ToMV). Enzyme-linked immunosorbent assay (ELISA) was used for the quantitative analysis. The virus was concentrated using a strong anion exchanger, CIM quaternary amine (QA) disk monolithic column. A high salt concentration was used to elute the concentrated virus from the columns. It has been demonstrated that ToMV, which had been diluted considerably below the sensitivity of ELISA, was concentrated by several orders of magnitude in the one-step procedure. Concentrated virus preparations could be used directly for ELISA testing. In comparison with methods described for concentrating plant viruses from irrigation water, the above procedure may provide a much faster and more efficient way to concentrate highly diluted plant viruses. The procedure could be applied to the testing of other highly diluted plant viruses, and to concentrating viruses for antiserum production.
A yeast homologous recombination system was used to generate mutants and chimeras in the genome of Potato leafroll virus (PLRV). A yeast-bacteria shuttle vector was developed that allows mutants and chimeras generated in yeast to be transformed into Escherichia coli for confirmation of the mutations and transformed into Agrobacterium tumefaciens to facilitate agroinfection of plants by the mutant PLRV genomes. The advantages of the system include the high frequency of recovered mutants generated by yeast homologous recombination, the ability to generate over 20 mutants and chimeras using only two restriction endonuclease sites, the ability to introduce multiple additional sequences using three and four DNA fragments, and the mobilization of the same plasmid from yeast to E. coli, A. tumefaciens, and plants. The wild-type PLRV genome showed no loss of virulence after sequential propagation in yeast, E. coli, and A. tumefaciens. Moreover, many PLRV clones with mutations generated in the capsid protein and readthrough domain of the capsid protein replicated and moved throughout plants. This approach will facilitate the analysis of plant-virus interactions of in vivo-generated mutants for many plant viruses, especially those not transmissible mechanically to plants.