[Potato leafroll virus: purification, serologic detection and quantitative analysis in plants].
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The Potato Leafroll Virus has been purified from Potato. An antiserum obtained allows the use of the Enzyme Linked-Immunosorbent Assay, a direct means to detect the PLRV in the plant and to assess its real concentration.Keywords:
Potato leafroll virus
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A Japanese isolate of barley yellow dwarf virus (BYDV) was purified from infected plants by an improved procedure. The procedure included extraction of the virus by grinding the frozen plant tissues in a meat grinder followed by regrinding in phosphate buffer with motar and pestle for extended periods (more than 2hr) in room temperature, clarification of the sap with chloroform, concentration of the virus by polyethylene glycol, two cycles of differential centrifugation and a sucrose density gradient centrifugation. The average yield of the virus was 44μg per 100g tissues. The Japanese isolate had particle diameter of 27.3nm (2% PTA, pH 5.0) and UV absorbance spectrum with an average A260/A280 ratio of 1.71. In ELISA using the double antibody sandwich method, the virus reacted only with homologous antiserum but not with antiserum to potato leafroll virus (PLRV), and vice versa. The virus, however, reacted slightly with heterologous antisera to other luteoviruses such as PLRV, soybean dwarf and beet western yellows viruses in agar gel double diffusion tests.
Barley yellow dwarf
Potato leafroll virus
Differential centrifugation
Luteovirus
Mottle
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SUMMARY In immunosorbent electron microscopy tests, large numbers of particles of a Scottish isolate of potato leafroll virus (PLRV) became attached to grids coated with antisera prepared to Canadian, Japanese or Swiss isolates of PLRV. Moderate numbers of particles became attached using antiserum to tobacco necrotic dwarf virus or to bean leafroll virus, smaller numbers using antiserum to soybean dwarf virus or to barley yellow dwarf virus (RPV strain) and a few particles became attached using antiserum to barley yellow dwarf virus (MAV strain) or to beet western yellows virus. A similar pattern of antigenic relationships was deduced from tests in which the binding of antibody molecules to PLRV particles exposed to different concentrations of the antisera was assessed by electron microscopy. It is concluded that these viruses should all be included in the luteovirus group and that their apparent degree of relationship to PLRV is: tobacco necrotic dwarf virus > bean leafroll virus > soybean dwarf virus > barley yellow dwarf virus (RPV strain) > barley yellow dwarf virus (MAV strain).
Luteovirus
Barley yellow dwarf
Potato leafroll virus
Strain (injury)
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SUMMARY Rat monoclonal antibodies (MAbs) specific for a British F (MAV‐like) isolate of barley yellow dwarf virus (BYDV) were produced and studied. In indirect ELISA using an antiserum to BYDV‐F to trap virus from infected sap, the MAbs were shown to be specific for MAV‐like isolates of BYDV from Britain, USA and Sweden but, in this test, they did not detect PAV‐, RPV‐, SGV‐ or RMV‐ like isolates of BYDV. In similar tests using homologous antisera to trap the viruses, the MAbs did not detect BYDV‐PAV or ‐RPV or two other luteoviruses (potato leafroll and beet western yellows). One of the MAbs (MAFF 2) was partially purified from ascitic fluid, and used successfully in ELISA as a coating antibody and when conjugated to the enzyme alkaline phosphatase. Also, MAFF 2 successfully trapped BYDV‐F particles when used to coat electron microscope grids. In indirect ELISA using three MAbs (MAFF 2, MAC 91 and MAC 92) it was possible to type the three major strain groups of BYDV, viz. MAV, PAV and RPV‐like strains from Britain, USA and Europe.
Barley yellow dwarf
Luteovirus
Potato leafroll virus
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Like typical luteoviruses, Potato leafroll virus (PLRV) cannot be transmitted mechanically by rubbing plants with solutions containing virus particles. However, PLRV was found to be mechanically transmissible from extracts of plants that had been inoculated by viruliferous aphids and then post-inoculated with Pea enation mosaic virus-2 (PEMV-2). Unlike the asymptomatic infections induced by either virus alone, double infections in Nicotiana benthamiana induced necrotic symptoms with some line patterning and vein yellowing. Infective PLRV was recovered from a purified virus preparation by inoculating plants mechanically with purified virus particles mixed with PEMV-2. Similarly, Beet mild yellowing virus was readily transmitted mechanically from mixtures containing PEMV-2. PLRV was also transmissible from mixtures made with extracts of plants infected with Groundnut rosette virus, although less efficiently than from mixtures containing PEMV-2. This novel means of transmitting PLRV, and perhaps other poleroviruses, should prove very useful in a number of fields of luteovirus research.
Potato leafroll virus
Luteovirus
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SUMMARY Attachment of virus particles to antiserum‐coated electron microscope grids (immunosorbent electron microscopy) provided a test that was at least a thousand times more sensitive than conventional electron microscopy for detecting potato leafroll (PLRV) and potato mop‐top (PMTV) viruses. The identity of the attached virus particles was confirmed by exposing them to additional virus antibody, which coated the particles. PLRV particles (up to 50/μm 2 of grid area) were detected in extracts of infected potato leaves and tubers, infected Physalis floridana leaves, and single virus‐carrying aphids. On average, Myzus persicae yielded 10–30 times more PLRV particles than did Macrosiphum euphorbiae . PMTV particles (up to 10/μm 2 of grid area) were detected in extracts of inoculated tobacco leaves, and of infected Arran Pilot potato tubers with symptoms of primary infection. Particles from tobacco leaves were of two predominant lengths, about 125 nm or about 290 nm, and fewer particles of other lengths were found than in previous work, in which partially purified or purified preparations of virus particles were examined, using grids not coated with antiserum.
Potato leafroll virus
Myzus persicae
Luteovirus
Physalis
Macrosiphum euphorbiae
Potato virus Y
Immunoelectron microscopy
Rose bengal
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Barley yellow dwarf virus (BYDV) is one of the most devastating plant viruses and belongs to a ubiquitous plant virus group. In China, four BYDV strains (GPV, GAV, PAV and RMV) have been identified based on their specific aphid vectors and serological properties. Among the four identified strains, the GAV is the most common BYDV strain in China. To diagnose, forecast of BYDV GAV, two reliable serological assays for BYDV GAV detection were established. We purified virion from a confirmed BYDV GAV source and used it as the immunogen to produce monoclonal antibodies against the virus. Using the hybridoma technology, three highly specific murine monoclonal antibodies were produced and two serological assays [antigen-coated-plate enzyme-linked immunosorbent assay (ACP-ELISA) and dot enzyme-linked immunosorbent assay (dot-ELISA)] were established for the BYDV GAV detection. All three monoclonal antibodies reacted strongly and specifically with the BYDV GAV strain in crude leaf extracts. Titers of the monoclonal antibodies in ascitic fluids were up to 10−7 by indirect-ELISA. These three monoclonal antibodies (18A1, 18A9 and 12A11) all belonged to the isotype IgG1, kappa light chain. The highest dilution points for the three antibodies during the ACP-ELISA using infected crude leaf extracts were 1:163,840, 1:81,920 and 1:81,920 (w/v, g · mL−1), respectively. Result of dot-ELISA showed a successful detection of BYDV GAV strain in 1:5,120 (w/v, g · mL−1) diluted wheat leaf crude extracts. Analysis of 22 field wheat leaf samples and 33 aphid samples from the Shaanxi Province in China, using the two newly developed assays confirmed the presence of BYDV GAV in about 80 % of the wheat samples and 18 % of the aphid samples. All three monoclonal antibodies are highly sensitive and specific to the BYDV GAV. The two newly developed serological assays are simple and effective. These two assays, particularly the dot-ELISA, are useful for high throughput detection of BYDV GAV in host plants and aphid vectors.
Luteovirus
Barley yellow dwarf
Immunogen
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Mouse line RBF/Dn immunized with beet western yellows virus (BWYV) resulted in two hybridoma clones secreting BWYV-specific antibodies. Another mouse line (BALB/c) immunized with a combination of BWYV and potato leafroll virus (PLRV) resulted in 17 hybridomas that tested positive for either BWYV or PLRV and one that reacted with an epitope common to both viruses. Selected monoclonal antibodies specific to either BWYV or PLRV were used in ELISA tests to detect luteovirus strains and isolates (...)
Luteovirus
Potato leafroll virus
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Studying the transmission of potato leafroll virus (PLRV) by Myzus persicae from infected Physalis floridana plants, revealed that the ability of aphids to transmit the virus differed widely among individuals and strongly depended on the biotype of the vector aphid and the age of the virus source. Virus transmission was more frequent by M. persicae from top leaves that hardly showed any disease symptom than from bottom leaves with pronounced symptoms. The percentage of successful PLRV transmission decreased with the infection age. Furthermore, the median latency period (LP 50 ) of the virus was significantly shorter in aphids that fed on top leaves than on bottom leaves. The differences in virus transmission from bottom and top leaves could not be explained by the amount of viral antigen in the sources, since a higher concentration of viral antigen was detected in bottom leaves than in top leaves. The feeding behaviour of aphids on the virus sources also did not account for the observed differences; the honeydew excretion rate of M.persicae nymphs feeding on bottom leaves was higher than on top leaves of infected plants (Chapter 1).To investigate to what extent the amount of virus acquired by M. persicae contributed to differences in virus transmission, the sensitivity of the double antibody sandwich (DAS) variant of the enzyme-linked immunosorbent assay (ELISA) had to be increased. To this end, the procedure was modified by incubating sample and conjugate simultaneously (cocktail-ELISA), and by amplifying the enzyme reaction in which a dephosphorylated substrate catalytically triggers an enzyme-mediated redox cycle. Cocktail-ELISA preceding enzyme amplification was 10- to 15-fold more sensitive than DAS-ELISA and could easily be applied to detect viral antigen in individual nymphs which had been feeding for only a short period of time on infected P. floridana plants (Chapter 2).Nymphs that had fed on bottom leaves with pronounced symptoms of P. floridana plants infected with PLRV acquired considerably less virus in the same acquisition access period than nymphs feeding on top leaves. The observed dissimilarity between the viral antigen content in the virus sources and in the aphids feeding on them suggests that the availability of the virus for acquisition by aphids is considerably lower in bottom than in top leaves (Chapter 3). Changes in availability of virus for acquisition were also noticed on eight potato genotypes with different levels of field resistance to PLRV. Early in the growing season, a lower virus titer in the secondarily-infected potato plants resulted in a lower capacity of the plants to act as a virus source. As plants aged, and symptoms became apparent, the virus was still readily detectable in the plants but virus acquisition by M. persicae was impaired and did not correlate anymore with the amount of viral antigen in the source (Chapter 4). PLRV acquisition and transmission by M. persicae from artificial diets containing purified PLRV demonstrated that the amount of viral antigen in the nymphs, and the percentage of viruliferous nymphs were linearly related to the log 10 transformed virus concentration in the diet. Therefore, the amount of viral antigen present in aphids is a more reliable parameter in deducing the potential of a plant to act as a virus source, than the viral antigen concentration of the plant itself.Artificial diet studies furthermore demonstrated that the LP 50 of the virus in M. persicae was not influenced by the virus concentration in the diet, but by interactions between the virus and its vector. Four-day old M. persicae nymphs displayed a longer LP 50 than one-day old nymphs. PLRV purified from top leaves of infected P.floridana was transmitted with a significantly shorter LP 50 than virus purified from bottom leaves. This finding shows that the previously observed differences in virus transmission from intact top and bottom leaves of P.floridana can not be solely explained by differences in the virus concentration available for acquisition. As similar amounts of purified virus from top and bottom leaves in artificial diet were fed to the aphids, it is likely that intrinsic properties of the virus also determine the transmissibility of the virus occurring in top and bottom leaves. This may concern changes at the surface of the viral capsid (Chapter 3).To study possible relationships between the transmissibility of PLRV and protein structures at the surface of the viral capsid, monoclonal antibodies (MAbs) were generated to PLRV. After two fusion experiments, nine different MAbs to PLRV were selected, and the topological relationships and the nature of the epitopes, to which they were directed, determined (Chapter 5). PLRV isolates which differed in their transmissibility by M. persicae, were tested in a triple antibody sandwich ELISA with this panel of MAbs. It was shown that four MAbs reacted significantly stronger with isolates which were readily transmitted than with the poorly transmitted isolates. Moreover, when mixtures of PLRV and MAbs suspensions were fed to M.persicae, these four MAbs reduced the probability of virus transmission and significantly increased the latency period of the virus in its vector. Hence, two lines of evidence indicate that the epitopes to which these MAbs were directed might be functionally involved in virus transmission by M.persicae (Chapter 6). The four MAbs reacted with conformation-dependent epitopes which are supposedly dependent on the tertiary protein structures. Competitive binding assays indicated that these epitopes strongly overlap which each other (Chapter 5). Anti-idiotypic antibodies (AiAbs) were raised to the four MAbs in rabbits. They may be considered as blueprints of the epitopes to which the MAbs are directed. When suspensions containing these AiAbs were fed to M.persicae prior to virus acquisition, PLRV transmission was reduced by up to 71% (Chapter 6). AiAbs may have blocked specific sites in the aphid which have a function in transcellular transport of virus particles.
Myzus persicae
Potato leafroll virus
Luteovirus
Honeydew
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An antiserum was raised against a fusion protein containing part of the 56K polypeptide (P5) encoded by the open reading frame (ORF) at the 3' end of the genome of potato leafroll virus (PLRV). This antiserum reacted specifically with 80K and 90K polypeptides in PLRV-infected protoplasts, with a 90K polypeptide in infected potato tissue and with a 53K polypeptide in protein extracted from purified particles of PLRV. Monoclonal antibodies raised against purified PLRV particles also reacted with these polypeptides, as well as with the 23K coat protein. Virus particles partially purified from infected protoplasts contained some 90K polypeptide as well as the major 23K coat protein. The ORFs of the 23K coat protein and P5 are contiguous and in frame. The results suggest that the P5 polypeptide of PLRV occurs in infected cells as part of a readthrough protein comprising the 23K coat protein joined to the P5 amino acid sequence. Moreover the readthrough protein can be assembled into virus particles as a minor component together with the main 23K component. The P5 protein may thus contribute to properties of PLRV determined by its virus particle surface.
Stop codon
Coat
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The aphid Myzus persicae (Sulz.) was shown to transmit potato spindle tuber viroid (PSTVd) to potato clone DTO-33 from source plants doubly infected with potato leafroll virus (PLRV) and PSTVd. Transmission was of the persistent type and did not occur when the insects were allowed to feed on singly infected plants. Only low levels of PSTVd were associated with purified PLRV virions, but its resistance to digestion with micrococcal nuclease indicates that the viroid RNA is encapsidated within the PLRV particles. Epidemiological surveys carried out at three locations in China revealed a strong correlation between PSTVd infection and the presence of PLRV, suggesting that PLRV can facilitate PSTVd spread under field conditions.
Potato leafroll virus
Myzus persicae
Luteovirus
Micrococcal nuclease
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