Alfalfa Mosaic Virus Particles Research Articles

Effects of the Number of Genome Segments on Primary and Systemic Infections with a Multipartite Plant RNA Virus

Multipartite plant viruses were discovered because of discrepancies between the observed dose response and predictions of the independent-action hypothesis (IAH) model. Theory suggests that the number of genome segments predicts the shape of the dose-response curve, but a rigorous test of this hypothesis has not been reported. Here, Alfalfa mosaic virus (AMV), a tripartite Alfamovirus, and transgenic Nicotianatabacum plants expressing no (wild type), one (P2), or two (P12) viral genome segments were used to test whether the number of genome segments necessary for infection predicts the dose response. The dose-response curve of wild-type plants was steep and congruent with the predicted kinetics of a multipartite virus, confirming previous results. Moreover, for P12 plants, the data support the IAH model, showing that the expression of virus genome segments by the host plant can modulate the infection kinetics of a tripartite virus to those of a monopartite virus. However, the different types of virus particles occurred at different frequencies, with a ratio of 116:45:1 (RNA1 to RNA2 to RNA3), which will affect infection kinetics and required analysis with a more comprehensive infection model. This analysis showed that each type of virus particle has a different probability of invading the host plant, at both the primary- and systemic-infection levels. While the number of genome segments affects the dose response, taking into consideration differences in the infection kinetics of the three types of AMV particles results in a better understanding of the infection process.

First Report of Alfalfa mosaic virus Associated with Severe Mosaic and Mottling of Pepper (Capsicum annuum) and White Clover (Trifolium repens) in Oklahoma.

Alfalfa mosaic virus (AMV), a member of the genus Alfamovirus, family Bromoviridae (1), has been reported in 44 states in the United States excluding Oklahoma. During a cucurbit survey in the summer of 2010, severe mosaic and mottling symptoms were observed on many peppers (Capsicum annuum) and white clover (Trifolium repens) plants in Tulsa, Oklahoma. Symptomatic leaf samples from 15 pepper and two white clover plants were collected in the Bixby area and analyzed serologically by dot-immunobinding assay (DIBA) using specific polyclonal antibodies against AMV (Agdia, Inc). Seven out of 15 pepper samples and both white clover samples were tested positive by DIBA to AMV. The remaining symptomatic samples were positive to Cucumber mosaic virus (CMV). Total RNA was extracted from DIBA positive AMV samples by Tri-reagent method. A small aliquot of total RNA was tested by reverse transcription (RT)-PCR using specific primers: AMV-F 5' GTCCGCGATCTCTTAAAT 3' and AMV-R 5' GAAGTTTGGGTCGAGAGA 3' that were designed to amplify 900 bp of the AMV-RNA 3. Analysis of the PCR products on agarose gel electrophoreses showed that all tested samples showed a band of the expected size while DIBA negative AMV samples did not produce any band. The amplified PCR product (900 bp) obtained from pepper and white clover were cleaned with PCR purification kit (Qiagen, Germantown, MD) and directly sequenced bi-directionally using the above primers. Sequence analysis confirmed that this virus shared 97% identity at nucleotide sequence with RNA 3 of AMV isolate from Madison-USA (GenBank Accession No. K02703). For biological and morphological characterization of the virus, eight pepper plants were mechanically inoculated using 0.1 M K2HPO4 buffer (pH 7.2) with total RNA extracted from AMV positive pepper or white clover plant samples. One to two weeks post-inoculation, all inoculated plants produced severe mosaic, mottling, and stunting. Virus-like particles preparations were obtained from these symptomatic plants according to our previously described method (2) and electron microcopy examination showed typical AMV particles. These biological and morphological data further confirmed the presence of AMV infecting pepper and clover in Oklahoma. AMV is a significant pathogen worldwide and infects more than 600 species in 70 families, especially alfalfa, pepper, soybean, and tobacco (3). AMV has a worldwide distribution, including the United States, and particularly the Midwestern U.S. where the incidence of the virus is on the rise recently because of the presence of its vector (Aphis glycines) (4). To our knowledge, this is the first report of AMV infecting crops in Oklahoma, which could pose a threat to other economic crops grown in Oklahoma, especially soybean.

Arabidopsis thaliana is an asymptomatic host of Alfalfa mosaic virus

The susceptibility of Arabidopsis thaliana ecotypes to infection by Alfalfa mosaic virus (AMV) was evaluated. Thirty-nine ecotypes supported both local and systemic infection, 26 ecotypes supported only local infection, and three ecotypes could not be infected. No obvious symptoms characteristic of virus infection developed on the susceptible ecotypes under standard conditions of culture. Parameters of AMV infection were characterized in ecotype Col-0, which supported systemic infection and accumulated higher levels of AMV than the symptomatic host Nicotiana tabacum. The formation of infectious AMV particles in infected Col-0 was confirmed by infectivity assays on a hypersensitive host and by electron microscopy of purified virions. Replication and transcription of AMV was confirmed by de novo synthesis of AMV subgenomic RNA in Col-0 protoplasts transfected with AMV RNA or plasmids harboring AMV cDNAs.

A damaging outbreak of arabis mosaic nepovirus in blackcurrant, the occurrence of other nepoviruses in Ribes species, and the demonstration that alfalfa mosaic virus is the cause of interveinal white mosaic in blackcurrant

Summary.In a crop of blackcurrant (Ribes nigrum), cv. Baldwin in Eire, chlorotic mottling and ringspot symptoms in leaves on plants and severe crop loss was associated with infection with arabis mosaic nepovirus (ArMV) and the presence in the soil of its nematode vector, Xiphinema diversicaudatum. This is only the second report of ArMV damaging a crop of blackcurrant. Tomato black ring (TBRV) and raspberry ringspot nepoviruses were detected in single plants of redcurrant (R. rubrum) in England and flowering currant (R. sanguineum) in Scotland respectively; each of these infected plants showed foliar chlorotic line‐pattern symptoms. This is the first record of TBRV in redcurrant. A single blackcurrant plant in New Zealand showing symptoms typical of those described for interveinal white mosaic disease, contained alfalfa mosaic virus (AMV). When AMV particles were purified and concentrated from herbaceous test plants and mechanically inoculated to young blackcurrant plants, several became infected with AMV and most infected plants developed systemic symptoms typical of the original disease. This provides the strongest evidence to date that AMV is the causal agent of interveinal white mosaic disease.

Effect of glutaraldehyde-fixation on the immunogenicity, particle stability and antigenic reactivity of alfalfa mosaic virus, and the specificity of elicited antibodies

Glutaraldehyde-fixation was shown to stabilize the structural integrity of alfalfa mosaic virus (AMV) particles as well as to increase their immunogenicity and antigenic reactivity. The antigenic reactivity of the particles was substantially increased irrespective of whether the antibodies were from animals immunized with native or fixed AMV, or preparations of coat protein subunits isolated from the virus. No significant changes in the antigenic specificity of AMV particles were detected following glutaraldehyde-fixation. Thus it is possible to raise antisera to AMV with higher titres by using fixed virus as immunogen.

Serological Differentiation of Some Strains of Alfalfa Mosaic Virus with Polyclonal Antibodies

Five biologically distinct but antigenically similar alfalfa mosaic virus (AMV) strains were tested for minor antigenic differences by immunodiffusion tests and indirect enzyme-linked immunosorbent assay (ELISA) by using antisera elicited to native and glutaraldehyde-fixed AMV particles of each of the virus strains. In addition, antisera to coat protein preparations of two of the strains were also used. In immunodiffusion tests with antisera to fixed virus particles and either fixed or native virus preparations as test antigens, no antigenic differences were detected among any of the strains (...)

Differentiation and Antigenic Characterization of Closely Related Alfalfa Mosaic Virus Strains with Monoclonal Antibodies

A panel of 15 mouse monoclonal antibodies (MAbs) was raised against five strains of alfalfa mosaic virus (AMV) which were closely related antigenically but biologically distinct. A wide diversity of MAb specificity was revealed by screening them in three formats of indirect ELISA, using native and glutaraldehyde-fixed AMV particles as well as isolated coat protein preparations. Of these MAbs, seven reacted specifically with only one AMV strain in at least one ELISA format and at least one MAb was capable of identifying each of the strains. One of the MAbs reacted with a cryptotope, whereas the other recognized different subtypes of either metatopes or neotopes, indicating that the AMV particle has a complex antigenic structure. Only two of the MAbs precipitated AMV in agarose gels. Another two, which recognized epitopes on coat protein subunits, also reacted well in immunoblots. One of the precipitating MAbs recognized an epitope which appears to be common to AMV and cucumber mosaic virus.

Activation of Prunus necrotic ringspot virus and rose mosaic virus by RNa 4 components of some llarviruses

RNA extracted from rose mosaic virus (RMV) and from necrotic ringspot virus-G(NRSV) separated into four sedimenting components in sucrose density gradients. The components were designated RNAs 1, 2, 3, and 4 in order of decreasing sedimentation velocities. Sodium dodecyl sulfate (SDS)-denatured NRSV and RMV preparations showed a prominent protein species of about 25,000 daltons when electrophoresed in polyacrylamide gels. In addition, two closely migrating protein species of about 19,000 daltons were also observed in SDS-denatured RMV preparations. Preparations containing RNA 1+2+3 and preparations containing RNA 4 were obtained by two successive cycles of sucrose density gradient centrifugation. Neither preparation was infectious alone. Mixtures of RNA 1+2+3 from each virus became infectious, however, with the addition of homologous RNA 4. RMV protein also activated RMV-RNA 1+2+3 preparations. NRSV-RNA 4 efficiently activated RMV-RNA 1+2+3, but RMV-RNA 4 had very little ability to activate NRSV-RNA 1+2+3 even though it efficiently activated its homologous RNA 1+2+3 preparations. RNA 4 preparations from alfalfa mosaic virus (AMV) and citrus leaf rugose virus (CLRV) activated RMV-RNA 1+2+3. Comparable concentrations of RMV-RNA 4, however, failed to activate RNA 1+2+3 from AMV and from CLRV. RNA from CLRV, RMV, and citrus variegation virus (CVV) efficiently uncoated AMV particles. However, AMV-RNA and CLRV-RNA 4 uncoated CVV but not CLRV or RMV. The evidence presented indicated that viruses in the new Ilarvirus group [Shepherd, R. J., et al. (1976). Intervirology 6, 181–1841 have similar infectivity requirements; that is, mixtures of RNA 1+2+3 are not infectious but can be activated by either RNA 4 or coat protein.

The uncoating of alfalfa mosaic virus by its own RNA

Upon addition of alfalfa mosaic virus (AMV)-RNA to AMV particles the latter are rapidly changed into highly RNase-sensitive, slow-sedimenting structures. These structures still contain intact RNA but they band at a higher density in CsCl gradients than virus particles and lack the typical bacilliform virus structure. The disintegration of the particle structure is thought to be due to the free RNA combining with some of the protein subunits. The presence of protein subunits on RNA molecules was demonstrated by means of biological assay. The reaction is dependent on the concentrations of virus and RNA, and on the time of incubation. When particles of two other viruses were brought in contact with their own RNA or with AMV-RNA no reaction was observed. Addition of other RNAs to AMV particles also had no effect. Because of the specificity of the reaction, we postulate the existence of sites with a high affinity for AMV coat protein on the AMV-RNAs. These sites may be related to the biological role of the coat protein. The fact that the AMV structure becomes disturbed upon addition of its own RNA confirms the idea that in this virus particle the protein-protein interactions are of minor importance.

The in vivo behaviour of twenty-four strains of alfalfa mosaic virus

The in vivo behaviour of 24 strains of alfalfa mosaic virus (AMV) in cells of tobacco leaves was investigated by electron microscopy of thin sections. In the cytoplasm the particles of 13 of the strains packed in four distinct aggregation forms which are described in detail. These aggregation forms appear to be strain specific. The particles of the other 11 strains were not found to aggregate in the cytoplasm of tobacco. In an attempt to find how two of the types of cytoplasmic aggregates of AMV particles were formed, samples were taken at regular intervals from plants infected with Caldy and 15 64 strains. Aggregates were found in Caldy infected plants from the earliest stage (2 days) at which virus particles were detected. Particles were found in 15 64 infected plants 2 days after inoculation and aggregates, first detected in the third day sample, became common by the fifth day; but the cause of the aggregates was not detected. Possible mechanisms of aggregation formation are discussed. Aggregates which resembled those formed by virus particles were found in nuclei of tobacco plants infected with 15 64 , AMV 425, and VRU strains. AMV-like particles were extracted from nuclei from plants infected with 15 64 strain. Aggregates of long rod-shaped particles were found in the nuclei of plants infected with three strains. Those associated with 15 64 and ATCC 91 strains were in loose aggregates whereas those associated with Caldy strain were in large crystalline aggregates. Both types of aggregate of long tubular particles were usually found near the nucleoli. The long rod-shaped particles extracted from nuclei from 15 64 and Caldy infected tobacco were indistinguishable in the electron microscope.

Electron microscopy on in Vivo aggregation forms of a strain of alfalfa mosaic virus

The particle length distribution of alfalfa mosaic virus (AMV) particles from crude preparations from tobacco leaves did not differ significantly from the length distribution of particles in preparations of AMV purified from tobaccos at the same stage of infection. In older infections components with particles longer than bottom component were found. Two aggregation forms of AMV particles were found in sections and in negatively stained crude preparations. One form was found only in the vacuole, and the other in the cytoplasm. The structures of the aggregates are described, and the differences between them are discussed.