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Biomedical subjects

R I Francki

Publications and source records attributed to R I Francki.

At least 19 recordsLinked to original sources
PubMed1995

Mixed-subunit capsids can be assembled in vitro with coat protein subunits from two cucumoviruses.

Virus particles were reassembled in vitro from tomato aspermy virus strain V (V-TAV) RNA and a mixture of subunits prepared from V-TAV and 35S-labelled cucumber mosaic virus strain T (T-CMV). Immunodiffusion tests showed that the reassembled particles reacted with polyclonal antisera raised against both V-TAV and T-CMV. Radioactivity was found in the precipitin line formed between the reassembled particles and antiserum raised against T-CMV as well as in the precipitin line formed between the reassembled particles and antiserum raised against V-TAV. This shows that 35S-labelled T-CMV protein subunits were incorporated with V-TAV protein subunits into the same particles. Thus, coat proteins of V-TAV and T-CMV can co-assemble and form mixed-subunit capsids in vitro.

Capsid↗
PubMed1994

Alleged reaction in gel-immunodiffusion of an IgM monoclonal antibody with alfalfa mosaic virus and cucumber mosaic virus is an artefact.

A previously reported spurious serological cross-reaction between alfalfa mosaic virus (AMV) and cucumber mosaic virus (CMV), which had been defined by the reaction in gel-immunodiffusion tests of a single IgM monoclonal antibody (MAb), MAb 8, was no longer detected in the presence of 0.1 M-NaCl. The non-specific reactivity of this IgM was also confirmed in Western blotting assays. When skimmed milk was used as a blocking agent and as a diluent of antibodies, MAb 8 failed to recognize AMV and CMV coat proteins. Hence, it is concluded that the alleged cross-reaction between AMV and CMV is due to non-specific binding of MAb 8 and that there is no evidence for a serological relationship between these two viruses.

Alfalfa mosaic virus↗
PubMed1992

Improved ELISA for the detection of barley yellow dwarf virus in grasses.

BYDV, a phloem restricted luteovirus, occurs in very low concentration in grasses. Several modifications to the virus extraction procedure were tested for their effect on ELISA detection of BYDV from grasses. The most efficient grinding method was to dip plastic bags containing samples in liquid nitrogen prior to crushing with a hand roller. Overnight incubation of samples at 25 degrees C in 0.1 M citrate buffer, pH 6.0, or in 0.1 M phosphate buffer, pH 7.0, prior to testing increased the optical density (OD) obtained without increasing the background. However, the highest OD was obtained when celluclast was added to the incubation buffer. The end point for detection was in general one to two steps higher with incubation and one further step higher after adding enzyme to the incubation buffer.

Enzyme-Linked Immunosorbent Assay↗
PubMed1992

Insect-mediated transmission of mixed and reassorted cucumovirus genomic RNAs.

Transmissions of virus using the aphid Myzus persicae were performed using plants co-infected with two cucumoviruses, tomato aspermy virus (V-TAV) and cucumber mosaic virus (M-CMV). Five of the aphid-transmitted progeny viruses (3.7%) induced symptoms distinct from those induced by either parental virus. Northern blot hybridization analysis of encapsidated RNAs from these novel progeny demonstrated that all of the RNA profiles were characteristic of pseudo-recombinants, i.e. viruses with reassorted genomic RNAs. The two larger RNAs, 1 and 2, originated from V-TAV, whereas RNA 3 was derived from M-CMV. A more sensitive RNase protection assay analysis of both unencapsidated and encapsidated RNAs revealed the presence of minor populations of V-TAV-derived RNA 3 in all of these novel progeny, and of M-CMV-derived RNA 1 (and presumably RNA 2) in one of the progeny. A bias against the encapsidation of the minor populations of RNAs by the M-CMV coat protein was observed, suggesting that there is specificity or competition with regard to the encapsidation of cucumoviral RNAs in vivo. This study demonstrates that insect vectors can mediate the establishment of pseudorecombinants with mixed populations of RNA 3.

Animals↗
PubMed1991

Some observations on the binding properties of alfalfa mosaic virus to polystyrene and its significance to indirect ELISA.

The adsorption and retention properties of native (unfixed) and glutaraldehyde-fixed alfalfa mosaic virus (AMV) antigens to the polystyrene of ELISA plates were studied using [35S]-labelled virus preparations. It was shown that adsorption was a temperature-dependent, relatively slow process which varied between different AMV isolates. The amount of virus antigen adsorbed was dependent on the type and pH of the suspending buffer. Although native virus antigen adsorbed very efficiently at high pH when the particles had dissociated, significant amounts also adsorbed at pH 7.0, or lower. However, glutaraldehyde-fixed virus particles which retained their integrity even at pH as high as 9.6, adsorbed much more efficiently than native virus antigen above pH 9.0, but hardly at all around pH 7.0. The wide variation in adsorption of AMV antigen to microtitre plates under even slightly different conditions had significant influence on ELISA readings, which calls for extreme caution in interpreting serological results from indirect ELISA when antigen is used to coat the microtitre plates.

Adsorption↗
PubMed1991

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.

Antibodies, Viral↗
PubMed1991

Change in phenotype and encapsidated RNA segments of an isolate of alfalfa mosaic virus: an influence of host passage.

A local lesion isolate of alfalfa mosaic virus (AMV-N20) from lucerne was found to encapsidate two extra RNAs in addition to the four major RNAs (RNA1, -2, -3 and -4). These were resolved by gel electrophoresis both under native conditions and after glyoxal denaturation. The RNA with an electrophoretic mobility between that of RNAs 2 and 3 was designated RNA31, that between RNAs 3 and 4 was designated RNA3s. Sucrose density gradient centrifugation analysis of AMV-N20 showed six instead of the normal four nucleoprotein components, the additional two presumably representing encapsidated RNAs 31 and 3s. RNAs 31 and 3s were both shown by Northern blot hybridization to be unrelated to host plant RNA and to contain the AMV coat protein gene sequence, which resides in RNA3. Primer extension of the RNAs 31 and 3s using a primer complementary to the 3' common terminus of all genomic AMV RNAs provided further evidence that they contained AMV sequences. RNA31 represents an addition of about 255 nucleotides, compared with RNA3, and RNA3s represents a loss of about 308 nucleotides. The coat proteins of variants encapsidating either RNA31, -3 or -3s had the same Mr indicating that the addition or deletion of the nucleotides was outside the coat protein gene. Serial mechanical passage of AMV-N20 over 5 years in four host species led both to changes in the composition of the RNA3 mixture, and to changes in symptom severity. For example, following passage in Nicotiana clevelandii, RNA3 was lost whereas passage in either N. glutinosa, Chenopodium quinoa or C. amaranticolor resulted in the loss of RNA31. No association was found between the changes in RNA3 and phenotypic changes that resulted from continuous passage for 5 years. Phenotypic changes with passage are thus presumably determined by mutations elsewhere in the virus genome.

Blotting, Northern↗
PubMed1990

Reducing agents interfere with the detection of lettuce necrotic yellows virus in infected plants by immunoblotting with monoclonal antibodies.

A procedure is described for the detection of lettuce necrotic yellows virus (LNYV) nucleocapsid protein (N) or envelope glycoprotein (G) by immuno-blotting with their respective monoclonal antibodies. The antigens can be detected in 1-10 mg of fresh tissue from systemically infected Nicotiana glutinosa leaves showing prominent symptoms. The composition of the extraction buffer played a crucial role in the recovery of antigenically active proteins. Procedures involving tissue extraction in the presence of the reducing agents 2-mercaptoethanol (2-ME) or dithiothreitol (DTT) failed to detect either the N or G antigen. For optimum detection of the N or G antigens, leaf tissue was ground with 10 mM phosphate buffer, pH 7.6 (PB), containing 3% (w/v) sodium dodecyl sulphate (SDS) and 1 mM of the protease inhibitor N-p-tosyl-L-lysine chloromethyl ketone (TLCK). The extract was then mixed with an equal volume of dissociation buffer containing 2-ME before electrophoresis and immunoblotting.

Animals↗
PubMed1990

Indirect double antibody sandwich ELISA for detecting alfalfa mosaic virus in aphids after short probes on infected plants.

An indirect double antibody sandwich enzyme-linked immunosorbent assay (IDAS-ELISA) system using antibodies elicited in rabbits and chickens is described for the detection of alfalfa mosaic virus (AMV). The method is capable of detecting 40 pg of homologous purified AMV and was shown to be suitable for detecting the virus in aphids. AMV in the order of 150 pg was detected in single aphids. It was shown that a significant proportion of insects could acquire this or higher amounts of virus during 1 min probes on the leaves of infected plants. The IDAS-ELISA should find applicability in research on the acquisition of viruses by aphids and in epidemiological studies for detecting viruses in insects from traps.

Animals↗
PubMed1990

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.

Animals↗
PubMed1990

Isolation of a subterranean clover mottle virus-like satellite RNA from lucerne infected with lucerne transient streak virus.

A circular, viroid-like satellite RNA (sat RNA) was detected in lucerne transient streak virus (LTSV), from lucerne in South Australia. It was larger than the previously reported sat RNA of LTSV, being similar in size and sequence homology to the 388 nucleotide sat RNA previously shown to be encapsidated by subterranean clover mottle virus (SCMoV) isolated from subterranean clover in Western Australia. This indicates that under field conditions, very similar sat RNAs can be associated with two distantly related sobemoviruses, LTSV and SCMoV. The natural hosts of these viruses are lucerne and subterranean clover, respectively.

Australia↗
PubMed1989

Kinetics of velvet tobacco mottle virus satellite RNA synthesis and encapsidation.

Synthesis of circular (RNA 2) and linear (RNA 3) molecules of velvet tobacco mottle virus (VTMoV) satellite RNA (sat RNA) has been studied by incubating strips of tissues excised from systemically infected Nicotiana clevelandii in solutions of [14C]uridine. After a short lag, RNA and virus synthesis proceeded at a constant rate for at least 24 hr, during which time most of the synthesis was directed to the production of RNAs 2 and 3. The kinetics of [14C]uridine incorporation into the sat RNA molecules after increasing times of incubation and during pulses of [14C]uridine followed by chase incubation with excess [12C]uridine suggest that RNA 3 is a percursor of RNA 2. However, not all the RNA 3 synthesized was shown to end up as RNA 2, even after 72 hr of incubation. Several lines of evidence are presented supporting the conclusion that VTMoV-infected cells contain large pools of unencapsidated sat RNA. It is suggested that the sat RNA may have a greater affinity for the VTMoV replicase than the helper viral RNA which results in copious production of the sat RNA.

In Vitro Techniques↗
PubMed1989

Preparation of soluble, biologically active alfalfa mosaic virus coat protein and its CaCl2-induced degradation.

A method for the preparation of soluble protein from five biologically distinct alfalfa mosaic virus (AMV) isolates is described. Highly purified AMV was dissociated with 1 M CaCl2 in 10 mM sodium acetate, pH 6.0, and the precipitated RNA was removed by centrifugation. The protein was dialysed against 10 mM sodium acetate, pH 6.0, containing 0.1 M CaCl2. If the salt concentration was reduced further, proteins from some AMV isolates precipitated. Proteins prepared by this method were shown to be immunoreactive and to activate the infectivity of the AMV genome. However, during prolonged exposure of the protein to buffers containing 0.1 M CaCl2, it undergoes slow proteolysis thereby losing its ability to activate the AMV genome but not its immunoreactivity.

Animals↗
PubMed1988

Analysis of lettuce necrotic yellows virus structural proteins with monoclonal antibodies and concanavalin A.

Three major structural proteins of lettuce necrotic yellow virus (LNYV) were identified by discontinuous polyacrylamide gel electrophoresis (PAGE) to have Mr approximately 78,000 (G), 57,000 (N), and 19,000 (M). Unreduced G and M proteins had faster mobilities in PAGE indicating the presence of disulfide bonds. The G protein was shown to be glycosylated with a complex network of oligosaccharides containing beta-N-acetylchitobiose N-linked to asparagine residues of the protein. Up to 17 additional minor bands were also detected in silver-stained electrophoretograms. In Western immunoblots, 9 of these (Mr approximately 27,000-220,000) were recognized by a monoclonal antibody to the N protein and another 6 (Mr approximately 58,000-180,000) with a monoclonal antibody to the G protein, indicating that they were degradation products or aggregates of these two viral proteins. Two minor silver-stained bands failed to react with either of the monoclonal antibodies, but were recognized by polyclonal anti-LNYV serum and are probably the L (Mr approximately 190,000) and NS (Mr approximately 38,000) viral proteins.

Antibodies, Monoclonal↗
PubMed1987

Nonspecific binding of immunoglobulins to coat proteins of certain plant viruses in immunoblots and indirect ELISA.

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.

Antibody Specificity↗
PubMed1986

Taxonomy of cucurbit-infecting tobamoviruses as determined by serological and molecular hybridization analyses.

A number of cucurbit-infecting tobamoviruses have been reported in the past but there is confusion about their identity and relationships. Cucumber viruses 3 (CV3) and 4 (CV4) were originally described in the United Kingdom whereas the watermelon (W) and cucumber (C) 'isolates' of cucumber green mottle mosaic virus (CGMMV) came from Japan. The results of serological studies and RNA-cDNA analyses have shown that CV3, CV4 and CGMMV-W are very closely related, whereas CGMMV-C is quite different. It is concluded that there are two distinct tobamoviruses that infect cucurbits; they are only very remotely related to each other and to a number of other tobamoviruses. It is suggested that the name CGMMV be retained to include the isolates or strains CV3, CV4 and CGMMV-W, and that a new name, kyuri green mottle mosaic virus (KGMMV), be given to CGMMV-C.

Antigens, Viral↗