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.     

Detection of BMYV and BWYV isolates using monoclonal antibodies and radioactive RNA probes, and relationships among luteoviruses

In order to discriminate between sugar beet infecting beet mild yellowing virus (BMYV) and other isolates of beet western yellows virus (BWYV), monoclonal antibodies (MAbs) and radioactive riboprobes were used. With MAbs prepared against BMYV or potato leafroll virus (PLRV) no distinction could be established between BMYV and BWYV. Seven probes were synthesised from a lettuce infecting BWYV isolate; their localisation in the genome is known and they cover almost its entire length. Probes from the '3 part of the genome hybridised with all BMYV and BWYV isolates whereas those from the '5 part did not recognise BMYV isolates, showing that a divergent '5 region exists in the genomes of BMYV and BWYV. Probes also readily detected the virus in single aphids. The relevance of this finding for epidemiological studies is discussed.
MAbs and riboprobes were also tested against other luteoviruses (PLRV; barley yellow dwarf virus (BYDV) MAV, PAV and RPV strains). The serological relationship between BMYV and PLRV was confirmed and an epitope common to PLRV and BYDV-RPV was found. Using probes, PLRV and BYDV-RPV were found to share domains of homology with BWYV. BYDV-PAV showed weak homology with BWYV, while BYDV-MAV showed none.     

Nucleotide sequence of beet western yellows virus RNA.

The nucleotide sequence of the genomic RNA (5641 nt) of beet western yellow virus (BWYV) isolated from lettuce has been determined and its genetic organization deduced. The sequence of the 3'terminal 2208 nt of RNA of a second BWYV isolate, obtained from sugarbeet, was also determined and was found to be very similar but not identical to that of the lettuce isolate. The complete sequence of BWYV RNA contains six long open reading frames (ORFs). A cluster of three of these ORFs, including the coat protein cistron, display extensive amino acid sequence homology with corresponding ORFs of a second luteovirus, the PAV isolate of barley yellow dwarf virus (BYDV) (1,2). The ORF corresponding to the putative viral RNA-dependant RNA polymerase, on the other hand, resembles that of southern bean mosaic virus. There is circumstantial evidence that expression of the BWYV RNA polymerase ORF may involve a translational frameshift mechanism. The ORF immediately following the coat protein cistron may be translated by in-frame readthrough of the coat protein cistron amber termination codon. Similar mechanisms have been proposed for expression of the corresponding ORFs of BYDV(PAV) (1).     

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.     

Beet luteovirus coat protein sequence variation

The sequences of cDNA clones covering the coat protein genes of 29 isolates of beet mild yellowing virus from sugar beet and beet western yellows virus mainly from oilseed rape were compared. The sequences could be partitioned into seven distinct clusters falling into three main groups. Group 1 isolates were found both in oilseed rape and sugar beet mainly from north Europe; group 2 isolates were from hosts other than sugar beet in England and France; group 3 isolates were beet-specific and found from northern Italy and Iran. The factors affecting this variation and its significance in relation to coat protein-mediated protection are discussed.     

The N-terminal region of the luteovirus readthrough domain determines virus binding to Buchnera GroEL and is essential for virus persistence in the aphid.

Luteoviruses and the luteovirus-like pea enation mosaic virus (PEMV; genus Enamovirus) are transmitted by aphids in a circulative, nonreplicative manner. Acquired virus particles persist for several weeks in the aphid hemolymph, in which a GroEL homolog, produced by the primary endosymbiont of the aphid, is abundantly present. Six subgroup II luteoviruses and PEMV displayed a specific but differential affinity for Escherichia coli GroEL and GroEL homologs isolated from the endosymbiotic bacteria of both vector and nonvector aphid species. These observations suggest that the basic virus-binding capacity resides in a conserved region of the GroEL molecule, although other GroEL domains may influence the efficiency of binding. Purified luteovirus and enamovirus particles contain a major 22-kDa coat protein (CP) and lesser amounts of an approximately 54-kDa readthrough protein, expressed by translational readthrough of the CP into the adjacent open reading frame. Beet western yellows luteovirus (BWYV) mutants devoid of the readthrough domain (RTD) did not bind to Buchnera GroEL, demonstrating that the RTD (and not the highly conserved CP) contains the determinants for GroEL binding. In vivo studies showed that virions of these BWYV mutants were significantly less persistent in the aphid hemolymph than were virions containing the readthrough protein. These data suggest that the Buchnera GroEL-RTD interaction protects the virus from rapid degradation in the aphid. Sequence comparison analysis of the RTDs of different luteoviruses and PEMV identified conserved residues potentially important in the interaction with Buchnera GroEL.     

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.     

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.     

Detection of the luteoviruses, beet mild yellowing virus and beet western yellows virus, in aphids caught in sugar-beet and oilseed rape crops, 1990–1993

The incidence of beet mild yellowing luteovirus (BMYV) and non-beet-infecting strains of beet western yellows luteovirus (BWYV) in individual winged aphids, caught in yellow water-traps, in sugar beet during the spring and early summer, and in oilseed rape plots in the autumn, was monitored using monoclonal antibodies in ELISA tests from 1990 to 1993. Between 0% and 8% of the Myzus persicae trapped in sugar beet each year carried BMYV, whereas 0% to 4% caught in oilseed rape in the autumn contained this virus. In 1990, 6.5% of Macrosiphum euphorbiae trapped in sugar beet contained BMYV, but in subsequent years less than 1% were carrying virus. Much higher proportions (26–67%) of the M. persicae tested from sugar beet contained BWYV, and similar proportions tested from oilseed rape (24–45%) also carried this virus in the autumn. In contrast only 3–19% of the M. euphorbiae caught in sugar beet contained BWYV, and none in oilseed rape. In 1991 and 1992 large numbers of Breuicoryne brassicae were caught in the plot of oilseed rape, of which over 50% contained BWYV; none were carrying BMYV. In transmission studies between 1990 and 1992, 1% and 27% of M. persicae transmitted BMYV and BWYV respectively to indicator plants; subsequent ELISA tests on the same aphids showed that 3% and 33% respectively contained the two viruses. One percent of M. euphorbiae transmitted BMYV, but none were found to contain BMYV using ELISA; 15% transmitted BWYV whilst only 5% were found to carry the virus. In 1992 and 1993 the incidence of BMYV-infection in the sugar-beet fields in which aphids had been trapped ranged from 1.2%, in a field which had received granular pesticide (aldicarb) at drilling plus three foliar aphicidal sprays, to 39.5% in a field which had received only one foliar spray. In 1992 in a sugar-beet crop which had received no aphicidal treatments, and where 2.8% of immigrant M. persicae and 2.5% of M. euphorbiae contained BMYV, 11.6% of plants developed BMYV infection. Lowest levels of infection were associated with the use of granular pesticides at drilling. In 1990, 80% of oilseed rape plants in a field plot were infested with a mean of seven wingless M. persicae per plant by mid-December; 37% of these plants were infected with BWYV. The studies show that M. persicae is the principal vector of BWYV, and large proportions of winged M. persicae carry the virus, in contrast to BMYV, which is consistent with the common occurrence of BWYV in brassica crops such as oilseed rape.     

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     

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.     

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.     

Structural requirements for efficient translational frameshifting in the synthesis of the putative viral RNA-dependent RNA polymerase of potato leafroll virus.

The putative RNA-dependent RNA polymerase of potato leafroll luteovirus (PLRV) is expressed by -1 ribosomal frameshifting in the region where the open reading frames (ORF) of proteins 2a and 2b overlap. The signal responsible for efficient frameshift is composed of the slippery site UUUAAAU followed by a sequence that has the potential to adopt two alternative folding patterns, either a structure involving a pseudoknot, or a simple stem-loop structure. To investigate the structure requirements for efficient frameshifting, mutants in the stem-loop or in the potential pseudoknot regions of a Polish isolate of PLRV (PLRV-P) have been analyzed. Mutations that are located in the second stem (S2) of the potential pseudoknot structure, but are located in unpaired regions of the alternative stem-loop structure, reduce frameshift efficiency. Deletion of the 3' end sequence of the alternative stem-loop structure does not reduce frameshift efficiency. Our results confirm that -1 frameshift in the overlap region depends on the slippery site and on the downstream positioned sequence, and propose that in PLRV-P a pseudoknot is required for efficient frameshifting. These results are in agreement with those recently published for the closely related beet western yellows luteovirus (BWYV).     

Characterisation of an Australian isolate of tomato yellow top virus

An Australian isolate of tomato yellow top virus (TYTV-A) was transmitted in the persistent manner by the aphid Myzus persicae. Its host range was mainly restricted to the Solanaceae, though Capsella bursa-pastoris and Gomphrena globosa were symptomlessly infected. TYTV-A was purified from Physalis Joridana, using an enzyme-assisted method in which the initial tissue homogenate was incubated with cellulase. Yields of purified virus were 100–900 μg/kg tissue and depended on the age of the infected plants. Maximum yields were obtained 4–5 wk after inoculation. The particles of TYTV-A were c. 24 nm in diameter, had a buoyant density of 1.34 in caesium sulphate and a coat protein mol. wt of c. 25.7 × 10³. TYTV-A was shown to be closely related serologically to potato leafroll virus (PLRV), TYTV from New Zealand and more distantly related to several other luteoviruses. An antiserum to TYTV-A was used in enzyme-linked immunosorbent assay tests to detect TYTV in field-infected tomato plants and also luteoviruses from potato plants with leafroll symptoms. It is clear that TYTV-A is a luteovirus closely related to PLRV.     

Purification, Properties and Serology of Strawberry Mild Yellow-Edge Virus

Oregon isolate My-18 of strawberry mild yellow-edge virus (SMYEV) was purified by comminution in liquid nitrogen, extraction in 0.1 M phosphate, 0.01 M DIECA, 1 % thioglycollic acid (pH 7.0) and differential and rate-zonal density gradient (dg) centrifugation. The resulting ultraviolet-absorbing dg band (A_{254 nm}), not seen in healthy control preparations, contained isometric, 23 mm-diameter, virus-like particles. The partially purified MY-18 virus was not transmitted to Fragaria vesca by means of membrane-fed or injected Chaetosiphon fragaefolii. MY-18 has an in vivo thermal inactivation point between 45 and 50 °C as determined by feeding C. fragaefolii on detached leaves that had been immersed in water for 10 min at various temperatures. In ELISA, rabbit antisera against MY-18 differentiated between partially purified preparations from root and leaf tissue and between crude root but not crude leaf extracts from healthy and MY-18-infected Fragaria. Our data support the generally held hypothesis that SMYEV is a luteovirus. However, comparative ISEM and ELISA tests failed to reveal any serological releationship between MY-18 and potato leafroll, beet western yellows, legume yellows, pea leafroll, or tobacco necrotic dwarf viruses.     

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.     

Aphid transmission of beet western yellows luteovirus requires the minor capsid read-through protein P74.

Beet western yellows luteovirus is obligately transmitted by the aphid Myzus persicae in a circulative, non-propagative fashion. Virus movement across the epithelial cells of the digestive tube into the hemocoel and from the hemocoel into the accessory salivary glands is believed to occur by receptor-mediated endocytosis and exocytosis. Virions contain two types of protein; the major 22 kDa capsid protein and the minor read-through protein, P74, which is composed of the major capsid protein fused by translational read-through to a long C-terminal extension called the read-through domain. Beet western yellows virus carrying various mutations in the read-through domain was tested for its ability to be transmitted to test plants by aphids fed on agro-infected plants and semi-purified or purified virus preparations. The results establish that the read-through domain carries determinants that are essential for aphid transmission. The findings also reveal that the read-through domain is important for accumulation of the virus in agro-infected plants.     

The influence of primary infection date and establishment of vector populations on the spread of yellowing viruses in sugar beet

In three field experiments in 1985 and 1986, we studied the effect of the date of primary infection on the spread of beet yellows closterovirus (BYV) and beet mild yellowing luteovirus (BMW) from artificially inoculated sugar beet plants. Laboratory-reared vector aphids, Myzus persicae, were placed on these sources of virus. There was no substantial natural immigration of vectors or viruses. In two experiments, one with BMYV in 1985 and the other in BYV in 1986, populations of vector aphids remained low and there was little virus spread, i.e. c. 50 infected plants from one primarily infected source. The cause of this small amount of spread was the low number of vector aphids. In the third experiment, with BYV in 1986, large populations of M. persicae developed and there was substantial virus spread: c. 2000 infected plants in the plots which were inoculated before canopy closure. In later-inoculated plots in the same experiment, there was much less spread: c. 100 infected plants per virus source plant. Differences between fields in predator impact are implicated as the most probable factor causing differences in vector establishment and virus spread between these three experiments. Virus spread decreased with later inoculation in all three experiments. A mathematical model of virus spread incorporating results from our work has been used to calculate how the initial proportion of infected plants in a crop affects the final virus incidence. This model takes into account the effect of predation on the development of the aphid populations. The processes underlying the spread and its timing are discussed.