Purification of carrot red leaf virus and evidence from four serological tests for its relationship to luteoviruses

Carrot red leaf virus (CRLV) was purified from infected chervil by centrifuging whole plant extracts at low speed and incubating the resuspended pellets with Driselase; the digest was then treated with 1% (v/v) Triton X-100 and the virus concentrated by centrifugation twice at high speed through a layer of 20% sucrose. The preparations (about 1 μg virus/g tissue) contained isometric particles c. 25 nm in diameter which formed a single u.v.-absorbing component in sucrose density gradients. Chervil seedlings exposed to aphids (Cavariella aegopodii) that had been injected with or had fed on fractions from the u.v.-absorbing zone developed typical symptoms of infection with CRLV. CRLV particles had a sedimentation coefficient (s_20,w) of 104 S, buoyant density in CsCl of 1.403 g/cm³ and A₂₆₀/A₂₈₀ of 1.62. Antiserum with a gel-diffusion titre of 1/512 was obtained from a rabbit injected intradermally with 100 μg purified virus. CRLV was detected by immunosorbent electron microscopy and enzyme-linked immunosorbent assay in extracts of the petioles and leaf midribs of infected chervil and in groups of five to 20 viruliferous C. aegopodii. Analysis of antiserum/virus reactions by density gradient centrifugation showed that CRLV is distantly related to all luteoviruses tested; its relationships were closest to barley yellow dwarf virus (RPV strain), and perhaps also to beet western yellows virus, more distant to tobacco necrotic dwarf, potato leafroll and bean leafroll viruses, and very distant to barley yellow dwarf (MAV strain) and soybean dwarf viruses. Some of these relationships were detected by double diffusion in agarose gels and by electron microscopy of antiserum/virus mixtures. Immunosorbent electron microscopy detected all these relationships but suggested that CRLV was more closely related to tobacco necrotic dwarf and potato leafroll viruses than to barley yellow dwarf virus (RPV strain). The results show that CRLV should be considered a definitive member of the luteovirus group, and provide confirmation of recent evidence that potato leafroll virus is a luteovirus.     

Changes in the activity of phosphogluconate dehydrogenase and its regulation in tobacco infected with PVY

The content and changes in the activity of phosphogluconate dehydrogenase were followed in leaf tissues of tobacco plants infected with the potato virus Y (PVY) in the acute phase of infection. The activity of the enzyme was higher in virus-infected tissues during the entire experimental period compared with the values found in healthy control plants in both crude homogenate and partially purified enzyme preparation. The courses of the activity curves of both the crude extract and partially purified enzyme preparations were consistent and correlated with the reproduction curve of the virus. These results suggest that increased activity of the enzyme was a result of its coarse regulation and indicate its immediate involvement in“de novo” biosynthesis of the virus via the oxidative pentose phosphate pathway.     

Preparation of mycoplasma immunogens from plants and a comparison of polyclonal and monoclonal antibodies made against primula yellows MLO-associated antigens

A method is described for obtaining from plants partially purified preparations of mycoplasma-like organisms (MLO) which are suitable for use as immunogens for polyclonal or monoclonal antibody production, and as antigens for directly coating ELISA plates. Using this method a mouse monoclonal antibody to primula yellows MLO was prepared, and its characteristics compared with those of primula yellows polyclonal antibodies from rabbits and also against polyclonal antibodies made to similar preparations of European aster yellows MLO. No serological distinction was obtained between any of the homologous or heterologous combinations of antibody and MLO preparation using ELISA, fluorescence microscopy with FITC-labelled antibodies, or immunoprobes of western blots of partially purified MLO preparations. By contrast, there were no cross-reactions between the primula or aster yellows antibodies or MLO preparations and preparations of clover phyllody or tomato big bud MLOs or their respective polyclonal antibodies. The primula yellows MLO monoclonal and polyclonal antibodies, and also the European aster yellows MLO polyclonal antibodies, all appeared to recognize only a single major antigen of approximate M, = 22 400 daltons. Some possible explanations for the apparent specificity of the polyclinic antisera for a single antigen, and the relevance to MLO preparation procedures are discussed.     

Some characteristics of monoclonal antibodies to a British M A V-like isolate of barley yellow dwarf virus

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.     

Involvement of Volatile Substances in Systemic Resistance of Barley Against Erysiphe graminis f. sp. hordei Induced by Pruning of Leaves

Horsegram yellow mosaic disease was shown to be caused by a geminivirus; horsegram yellow mosaic virus (HYMV). The virus could not be transmitted by mechanical sap inoculation. Leaf dip and purified virus preparations showed geminate virus particles, measuring 15-18 * 30 nm. An antiserum for HYMV was produced and in enzyme-linked immunosorbent assay (ELISA) and immunosorbent electron microscopy (ISEM) tests HYMV was detected in leaf extracts of fieldinfected bambara groundnut, french bean, groundnut, limabean, mungbean, pigeonpea and soybean showing yellow mosaic symptoms. Bemisia tabaci fed on purified HYMV through a parafilm membrane transmitted the virus to all the hosts listed above but not to Ageratum conyzoides, okra, cassava, cowpea, Croton bonplandianus, Lab-lab purpureus, Malvastrum coromandalianum and tomato. No reaction was obtained in ELISA and ISEM tests between HYMV antibodies and extracts of plants diseased by whitefly-transmitted agents in India such as A. conyzoides yellow mosaic, okra yellow vein mosaic, C. bonplandianus, yellow vein mosaic, M. coromandalianum yellow vein mosaic, tomato leaf curl and cassava mosaic. HYMV was also not found to be related serologically to bean golden mosaic, virus.     

Further evidence on the nature of the dependence of carrot mottle virus on carrot red leaf virus for transmission by aphids

Preparations were made from chervil plants doubly infected with carrot mottle virus (CMotV) and its helper virus, carrot red leaf (CRLV), on which it depends for transmission by the aphid Cavariella aegopodii, by the procedure developed previously for CRLV. The preparations contained 25 nm isometric particles which were indistinguishable from those of CRLV but possessed aphid-transmissible infectivity of both viruses and manually transmissible infectivity of CMotV. Only one sedimenting and buoyant density component was detected. The manually transmissible CMotV infectivity was resistant to freezing and to organic solvents, treatments that destroyed the CMotV infectivity in extracts from singly infected plants. The aphid-transmissible CMotV infectivity in preparations from CRLV/ CMotV-infected plants, and that in extracts from CRLV/CMotV-carrying C. aegopodii, was abolished by treatment with CRLV antiserum but not with normal serum. These results show that transmission of CMotV by C. aegopodii is dependent on the packaging of its RNA in coats composed partially or entirely of CRLV particle protein. The aphid Myzus persicae does not transmit CRLV or CMotV from plants mixedly or singly infected with these viruses but it is a vector of beet western yellows virus (BWYV) and potato leafroll virus (PLRV) and it transmitted CMotV from plants that also contained either of these viruses. This suggests that the coat proteins of BWYV and PLRV can substitute for that of CRLV in packaging CMotV nucleic acid and thereby confer on it their own vector specificities.     

Detection of a Luteovirus in Groundnut Rosette Diseased Groundnuts (Arachis hypogaea) by Enzyme-linked Immunosorbent Assay and Immunoelectron Microscopy

In groundnut rosette diseased groundnut plants collected near Zaria, Nigeria, a luteovirus was detected by ELISA and ISEM. In ELISA only beet western yellows virus antiserum reacted, while in ISEM luteovirus particles were trapped by antisera beet western yellows virus, potato leafroll virus, pea leafroll virus and barley yellow dwarf virus. The data are in agreement with the interpretation that the assistor of groundnut rosette virus is possibly a member of the luteovirus group.     

Studies on beet western yellows virus in oilseed rape (Brassica napus ssp. oleifera) and sugar beet (Beta vulgaris)

Oilseed rape (Brassica napus L. ssp. oleifera) was studied as a potential overwintering host for the sugar-beet yellowing viruses, beet yellows virus (BYV) and beet mild yellowing virus (BMYV), and their principal vector, Myzus persicae. In spring 1982, plants infected with a virus which reacted positively in enzyme-linked immunosorbent assay (ELISA) with BMYV antibody globulin were found in oilseed-rape crops; none of the plants contained virus which reacted with BYV antibody globulin. This virus was subsequently identified as beet western yellows virus (BWYV). No leaf symptoms could be consistently associated with infection of oilseed rape, but the virus was reliably detected by sampling any leaf on an infected oilseed-rape plant. Some isolates from oilseed rape did infect sugar beet in glasshouse tests, but the proportions of inoculated plants which became infected were low. Apparently there is therefore little danger of much direct transmission of BWYV by M. persicae from oilseed rape to sugar beet in spring. BWYV was introduced to and spread within oilseed-rape crops in autumn by M. persicae, and autumn-sown oilseed rape proved to be a potentially important overwintering host for M. persicae. In a survey of 80 autumn-sown crops of oilseed rape in East Anglia, northern England and Scotland in spring 1983, 78 were shown to be extensively infected with BWYV. Experimental plots of oilseed rape with 100% BWYV-infection yielded approximately 13.4% less oil than plots with 18% virus infection, the result of a decrease in both seed yield and oil content.     

Purification and some particle properties of anthriscus yellows virus, a phloem-limited semi-persistent aphid-borne virus

Anthriscus yellows virus (AYV), a phloem-limited virus transmitted in the semi-persistent manner by the aphid Cavariella aegopodii, was purified by treatment of leaf extracts with cellulasc, followed by differential and sucrose density gradient centrifugation. ‘The preparations contained isometric particles c. 29 nm in diameter which were unstable unless stored in buffer at pH 8.0 containing 1 mM CaCl₂,. The particles sedimented as two components, ’full‘ nucleoprotein particles with A₂₆₀/A₂₈₀= 1.83 containing about 42% nucleic acid, and ’empty‘ protein shells with A₂₆₀,/A₂₈₀= 0.73; their buoyant densities in CsCl solutions were 1.52 and 1.27 g/cm³. Respectively. Yields of ihe nircleoprotein particles were c. 1.75 mg/kg leaf tissue. The particles contained a single species of RNA, of mol. wt 3.6 × 10 “(10 000 nucleotides). Particle protein preparations contained four electrophoretic species, of mol. wt (× 10³) 35.0, 28.3, 23.3 and 22.3.C. aegopodii did not transmit AYV from purified preparations. A rabbit injected with AYV preparations produced antibodies that coated AYV particles in electron microscope tests, but gave variable reactions in immunosorbent electron microscopy (ISEM), depending on the composition of the medium. No reactions were obtained in enzyme-linked inimunosorbent asjay (ELISA). No serological relationship was detected in ISEM between AYV and any of 10 viruses that resembled it in one or more properties.     

Use of monoclonal antibody to potato leafroll virus for detecting groundnut rosette assistor virus by ELISA*

Three of 10 monoclonal antibodies (MAbs) produced to potato leafroll luteovirus (PLRV) were found to react in triple antibody sandwich ELISA (TAS-ELISA) with groundnut rosette assistor luteovirus (GRAV), though none reacted with four other luteoviruses (barley yellow dwarf, bean leaf roll, beet western yellows or carrot red leaf)- The most effective PLRV MAb, SCR 6, was used in TAS-ELISA to detect isolates of GRAV from groundnut plants with chlorotic, green and mosaic forms of rosette from Nigeria and Malawi. The test also detected GRAV in extracts of single Aphis craccivora.     

The use of monoclonal antibodies to detect beet mild yellowing virus and beet western yellows virus in aphids

Information on infectivity of the aphids which invade sugar beet root crops each Spring is required for forecasting incidence and providing advice on control of virus yellows. Monoclonal antibodies, produced in the USA to barley yellow dwarf virus (BYDV) and in Canada to beet western yellows virus (BWYV), were used to distinguish between sugar-beet-infecting strains of the luteovirus beet mild yellowing virus (BMYV), and the non-beet-infecting strains of the closely-related BWYV in plant and aphid tissue. Totals of 773 immigrant winged Myzuspersicae and 124 Macrosiphum euphorbiae were caught in water traps in a crop of sugar beet between 25 April and 5 August 1990. Using the monoclonal antibodies and an amplified ELISA, 67%M. persicae and 19%M. euphorbiae were shown to contain BWYV; 8%M. persicae and 7%M. euphorbiae contained BMYV. In studies with live winged aphids collected from the same sugar beet field during May, 25 of 60 M. persicae and two of 13 M. euphorbiae transmitted BWYV to the indicator host plant Montia perfoliata; two M. persicae and two M. euphorbiae transmitted BMYV. In another study three of 65 M. persicae and one of three M. euphorbiae in which only BWYV was detected, transmitted this virus to sugar beet.     

Narcissus late season yellows potyvirus: symptoms, properties and serological detection

Narcissus late season yellows virus (NLSYV) was obtained, apparently unmixed with other viruses, in selected naturally infected narcissus plants. The virus was not transmitted by manual inoculation to any of the 12 herbaceous species tested but was transmitted by Myzus persicae to virus-tested narcissus plants, which then developed symptoms characteristic of late season yellows. Typically, symptoms develop late in the growing season but their expression is unreliable. NLSYV particles, which measured c. 750 ± 12 nm, were purified from fresh leaf extracts of narcissus in 0·3 m sodium citrate containing 10 mm sodium EDTA and 10 mm sodium DIECA by differential centrifugation followed by clarification with diethyl ether, gel filtration and isopycnic banding in caesium sulphate. Using antibodies from antisera prepared to virus particles, NLSYV was detected by ELISA in 14 of the 18 cultivars examined and nine seemed totally infected. Detection of the virus by immunosorbent electron microscopy was as sensitive as by ELISA but neither test detected all infections. The virus was detected by ELISA only in samples collected after flowering and capsid antigen concentration was greatest in the distal region of leaves. The reliability of detection depended on the cultivar. Immuno-electron microscopy tests confirmed the occurrence of a distinct potyvirus, presumably narcissus degeneration, in narcissus cv. Grand Soleil d'Or, failed to detect any virus other than NLSYV in narcissus plants affected by white streak disease, and showed that NLSYV is related to three of five other potyviruses tested.     

Failure of British isolates of beet western yellows virus to infect potato

Potato cultivars were tested for susceptibility to two British isolates of beet western yellows virus originally obtained from sugar beet and oil seed rape. Neither isolate was transmitted by Myzus persicae to virus-free potato plants, either by itself or in association with potato leafroll virus.     

Specificity of the effect of sap-transmissible viruses in increasing the accumulation of luteoviruses in co-infected plants

The concentration of potato leafroll luteovirus (PLRV) (c. 1300 ng/g leaf) in singly infected Nicotiana clevelandii plants was increased up to 10-fold in plants co-infected with each of several potyviruses, or with narcissus mosaic potexvirus, carrot mottle virus or each of three tobravirus isolates. With the tobraviruses, PLRV concentration was increased equally by co-infection with either NM-type isolates (coat protein-free cultures containing RNA-1) or M-type isolates (particle-producing cultures containing RNA-1 and RNA-2). In contrast, the accumulation of PLRV was not substantially affected by co-infection with either of two nepoviruses, cucumber mosaic cucumovirus, broad bean mottle bromovirus, alfalfa mosaic virus, pea enation mosaic virus or parsnip yellow fleck virus. The specificity of these interactions between PLRV and sap-transmissible viruses was retained in tests made in Nicotiana benthamiana and when beet western yellows luteovirus was used instead of PLRV.     

TYROSINE HYDROXYLASE IN BOVINE CAUDATE NUCLEUS

Approximately 80 per cent of tyrosine hydroxylase activity in bovine caudate nucleus was particle-bound. The rest of the activity was found in the soluble fraction. The enzyme activity in crude tissue preparations was inhibited, probably by the presence of endogenous inhibitors. Dilution of crude tissue preparations such as the crude mitochondrial fraction caused an increase in the specific activity. The particle-bound enzyme was solubilized by incubation with trypsin. The presence of deoxycholate increased the degree of solubilization. The activity of the solubilized enzyme from the washed particles was also inhibited, but the subsequent purification by ammonium sulphate could eliminate the inhibition. The solubilized enzyme was partially purified by ammonium sulphate fractionation and Sephadex G-150 chromatography. A tetrahydropteridine and ferrous ion were required as cofactors for the partially purified enzyme. Among various divalent cations, only ferrous ion could activate the partially purified enzyme. The enzyme was inhibited by L-α-methyl-p-tyrosine and catecholamines such as dopamine. The optimum pH was found between 5.5 and 6.0. K_m values toward tyrosine, 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine and Fe²⁺, were approximately 5 × 10^?5 M, 1 × 10^?4 M and 4 × 10^?4 M, respectively.     

Studies on broccoli necrotic yellows virus

Electron microscopy of infected D. stramonium cells showed that the virus particles occurred in the cytoplasm. Particles were mostly bacilliform and measured 297 ± 18 times 64 ± 4 nm. In negatively stained leaf homogenates, particles were mostly disrupted; intact particles measured 267 ± 20 times 69 ± 6 nm. In brussels-sprout cells containing BNYV and CIMV, BNYV particles were rarely found compared with those of CIMV, and they occurred within and near CIMV inclusion bodies. BNYV particles were also found in extracts of virus-carrying Brevicoryne brassicae. Broccoli necrotic yellows (BNYV) and cauliflower mosaic (CIMV) viruses occurred together in naturally infected Brussels sprout plants, which showed conspicuous symptoms, and in cauliflower. BNYV was transmitted to and maintained in Datura stramonium and Hyoscyamus niger. It was partially purified from D. stramonium. Using these preparations, from which inhibitor had been removed, BNYV was manually transmitted to cauliflower, causing mild symptoms, and to Brussels sprout, causing a symptomless infection. BNYV also infected Sinapis alba but not cabbage, lettuce, Sonchus oleraceus or Plantago major. BNYV was transmitted by Brevicoryne brassicae but not by Myzus persicae, Hyperomyzus lactucae or Aleyrodes proletella.     

Purification and particle properties of groundnut rosette assistor virus and production of a specific antiserum*

Purified preparations of the luteovirus, groundnut rosette assistor virus (GRAV), were made by treatment of groundnut leaf extracts with cellulase, followed by sucrose density gradient centrifugation. Yields of virus particles were about 0·5-1·0 mg/kg leaf material. The preparations contained isometric particles c. 28 nm in diameter with a sedimentation coefficient (s_{20, w}) of 115 S, a buoyant density in Cs₂SO₄ of 1·34 g/cm³, and A₂₆₀/A₂₈₀ of 1·86. The particles contained a single species of nucleic acid (presumably RNA), of mol. wt 2·09 × 10⁶and with no detectable polyadenylate sequence, and a single protein species, of mol. wt 24 × 10³. An antiserum produced in a rabbit had a titre of 1/256 in gel diffusion tests and detected GRAV in leaf extracts by ELISA. GRAV particles reacted in F(ab')₂-ELISA and immunosorbent electron microscopy (ISEM) tests with antisera to bean leaf roll, potato leafroll and tobacco necrotic dwarf luteoviruses, but did not react with antisera to carrot red leaf luteovirus.     

Serological Detection of Beet Western Yellows Luteovirus in the Non-vector,Brevicoryne brassicae (Homoptera: Aphididae)

The interaction between beet western yellows luteovirus (BWYV) and the aphid species Brevicoryne brassicae was investigated using virus transmission and serological detection experiments. This species failed to transmit a BWYV isolate from infected to healthy oilseed rape plants, although virus was readily detected by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) in single B. brassicae adults. When virus-carrying adults were tested by ELISA after different inoculation access periods, the number of virus-positive individuals decreased after 5 days, whereas with the efficient vector Myzus persicae, virus-positive individuals were found even after 10 days. This confirms the inability of B. brassicae to transmit BWYV, even though it may acquire the virus. It is suggested that B. brassicae, as compared with the efficient vector M. persicae, may serve as an experimental model for studying the mechanisms of the luteovirus-vector specificity     

The use of enzyme-linked immunosorbent assay to detect, and discriminate between, small iridescent viruses

The enzyme-linked immunosorbent assay (ELISA) double antibody method provided an efficient method for detecting iridescent virus (type 22) in purified preparations and extracts of Galleria mellonella larvae; 10 ng of purified virus/ml were detected with confidence. The ELISA method discriminated between the five iridescent viruses tested.     

Development and application of ELISA assays for the detection of two members of the family Luteoviridae infecting legumes: Pea enation mosaic virus (genus Enamovirus) and Bean leafroll virus (genus Luteovirus)

An antigen‐coated plate enzyme‐linked immunosorbent assay (ACP‐ELISA) method was developed and validated for the detection of Bean leafroll virus (BLRV) and Pea enation mosaic virus (PEMV), two of the important viral pathogens of several legume crops. The coat protein (CP) gene of each of the viruses was bacterially expressed as a fusion protein containing an N‐terminal hexa‐histidine tag and used as an antigen to produce antisera in rabbits. The antiserum to BLRV could detect the virus in leaf samples in up to 1:1000 dilution, and the PEMV antiserum detected the homologous virus in leaf samples of dilutions up to 1:6400. No serological cross‐reactivity was observed between anti‐BLRV and anti‐PEMV sera. The ACP‐ELISA assays were then used for estimating the prevalence of these two viruses in alfalfa, pea and vetch over a three‐state area in the US Pacific Northwest over a 2‐year period and virus incidence was mapped. Availability of rapid and sensitive ELISA assays facilitate virus disease mapping efforts and screening germplasm for virus resistance.