Properties and partial purification of infective material from plants containing groundnut rosette virus*

Groundnut plants with chlorotic rosette disease contain a manually transmissible virus, groundnut rosette (GRV), which is also transmitted in the persistent (circulative) manner by aphids (Aphis craccivora), but only from plants that are co-infected with a manually non-transmissible luteovirus, groundnut rosette assistor virus (GRAV). Strains of GRV from plants with chlorotic or green forms of rosette are called GRV(C) and GRV(G) respectively. An isolate of GRV(C) from Nigeria remained infective in Nicotiana clevelandii leaf extracts for 1 day at room temperature and for 15 days at 4d?C, but lost infectivity after 1 day at -20d?C or after dilution to 10^--⁴. Its infectivity and longevity in vitro were not altered by addition of 1 mg/litre bentonite to the extraction buffer. Infectivity in leaf extracts was abolished by treatment with 50% (v/v) ether, 10% (v/v) chloroform or 8% (v/v) n-butanol, but not by treatment for 30 min with RNase A at up to 100 ng/ml. In attempts to purify GRV(C), nearly all the infectivity from N. clevelandii extracts was found in the pellets from centrifugation at 65 000 g for 1. 5 h; infectivity also occurred in a cell membrane fraction that collected at the top of a 30% sucrose ‘cushion’ containing 4% polyethylene glycol and 0.2 M NaCI. However, no virus-like particles were found in either type of preparation by electron microscopy. Nucleic acid preparations made directly from GRV(C)-infected N. clevelandii leaves were very infective; this infectivity was totally inactivated by treatment for 30 min with RNase A at 10 ng/ml in buffers of both low and high ionic strength and was therefore attributed to ssRNA. When nucleic acid preparations were electrophoresed in gels no virus-specific bands were visible but the position of the infectivity indicated that the infective ssRNA has an apparent mol. wt of c. 1.55 × 10⁶. A similar mol. wt was indicated by the rate of sedimentation of the infective ssRNA in sucrose gradients. Preparations of dsRNA made from GRV(C)-infected N. clevelandii leaves contained a species of mol. wt c. 3.0 × 10⁶; in addition some dsRNA preparations contained an abundant component of mol. wt c. 0.6 × 106 together with several other components of intermediate mol. wt. Similar patterns of bands were observed in dsRNA preparations made from Nigerian-grown groundnut material infected with GRV(C) alone, or with GRV(C) + GRAV, or with GRV(G) + GRAV. The properties of GRV closely resemble those of two other viruses that depend on luteoviruses for transmission by aphids, carrot mottle virus and lettuce speckles mottle virus.     

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.     

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.     

Viruses associated with chlorotic rosette and green rosette diseases of groundnut in Nigeria*

Groundnut (Arachis hypogaea) plants from Nigeria with chlorotic rosette disease contained a manually transmissible virus, considered to be a strain of groundnut rosette virus (GRV(C)). GRV(C) infected nine out of 32 species in three out of nine families. It caused local lesions without systemic infection in Chenopodium amaranticolor, C. murale and C. quinoa, and systemic symptoms in Glycine max, Nicotiana benthamiana, N. clevelandii and Phaseolus vulgaris as well as in groundnut. Some ‘rosette-resistant’ groundnut lines were also infected. GRV(C) was transmitted by Aphis craccivora, but only from groundnut plants that were also infected with an aphid-transmissible second virus, which was not manually transmissible and was considered to be groundnut rosette assistor virus (GRAV). Plants infected with GRAV contained isometric particles c. 25 nm in diameter which were detectable by immunosorbent electron microscopy on grids coated with antisera to several luteoviruses, especially with antisera to bean leaf roll, potato leafroll and beet western yellows viruses. No virus-like particles were observed in extracts from plants infected with GRV(C) alone. A single groundnut plant obtained from Nigeria with symptoms of green rosette contained luteovirus particles, presumed to be of GRAV, and yielded a manually transmissible virus that induced symptoms similar to those of GRV(C) in C. amaranticolor but gave only mild or symptomless infection of N. benthamiana and N. clevelandii. It was considered to be a strain of GRV and designated GRV(G).     

Particle properties of parsnip yellow fleck virus

Particle preparations of parsnip yellow fleck virus (PYFV) isolates A-421 and P-121, representing the two major serotypes, were made by clarifying leal extracts with ether or butan-1-ol and concentrating the virus particles by precipitation with polyethylene glycol and differential centrifugation. The preparations contained c. 31 nm-diameter particles comprising two sedimenting components. Top component (T) consisted of stain-penetrable protein shells with A₂₆₀/A₂₈₀= 0.8–0.9, sedimentation coefficient (S₂₀) = 56 S (A-421) or 60 S (P-121), and buoyant density = 1.297 g/cm³. Bottom component (B) consisted of nucleoprotein particles, not penetrable by negative stain, with A₂₆₀/A₂₈₀= 1.9, sedimentation coefficient (S⁰_20.w) = 148 S (A-421) or 153 S (P-121), and buoyant density = 1.520 g/cm³ (A-421) or 1.490 g/cm³ (P-121). Yields of B component particles were up to c. 1 mg/100 g leaf tissue (both isolates); yields of T component particles were up to c. 0.6 mg (A-421) or 5.5 mg (P-121) per 100 g leaf tissue. PYFV particles were found to contain a single RNA species (mol. wt c. 3.4 × 10⁶, c. 9800 nucleotides), constituting 40% of the particle weight, and three polypeptide species, of mol. wt (× 10 ³) 30 , 26 and 24 (A-421) or 31 , 26 and 23 (P-121).     

Further properties of black raspberry latent virus, and evidence for its relationship to tobacco streak virus

Purified preparations of an isolate of black raspberry latent virus (BRLV) contained quasispherical particles with a mean diameter of 28·5 nm; these particles were resolved into three sedimenting components (s_{20, w}= 82S, 95S and 104S), but when centrifuged to equilibrium in caesium chloride solution they formed a single infective band (σ= 1·35 g/cm³). During electrophoresis in polyacrylamide gels, virus particles separated into three classes, and virus RNA was resolved into three major (mol. wt 1·35, 1·10 and 0·85 × 10⁶) and one minor (mol. wt 0·4 × 10⁶) component. The protein from virus particles had an estimated mol. wt of 28000. Isolates of BRLV were found to be serologically related but not identical to some strains of tobacco streak virus. No symptoms developed in black raspberry seedlings infected with BRLV by mechanical inoculation, nor in eight red raspberry cultivars infected by graft inoculation. However, graft inoculation of BRLV to Rubus henryi, R. phoenicolasius and Himalaya blackberry induced symptoms typical of necrotic shock disease.     

Biological and biochemical properties of an isolate of cherry rasp leaf virus from red raspberry

A mechanically transmissible virus obtained from symptomless plants of a red raspberry selection imported into Scotland from Quebec, Canada was indistinguishable serologically from a cherry isolate of cherry rasp leaf virus (CRLV). The raspberry isolate, CRLV-R, was graft transmitted to several virus indicator species and cultivars of Rubus without inducing noticeable symptoms. In Chenopodium quinoa sap, CRLV-R lost infectivity after dilution to 10^-5 or heating for 10 min at 60°C but was infective after 16 days (the longest period tested) at 18°, 4° or - 15°C. The virus particles are isometric, c. 28 nm in diameter, and were purified with difficulty from infected C. murale and C. quinoa plants. The particles comprise two nucleoprotein components with sedimentation coefficients of 89 and 115 S and are prone to aggregate during purification. When centrifuged to equilibrium in CS₂SO₄ solution, purified virus preparations formed two major components with p= 1·28 and 1·36 g/cm³. Virus particles contained two RNA species which, when denatured in glyoxal and electrophoresed in agarose gels, had estimated mol. wt of 2·56 × 10⁶ (RNA-1) and 1·26 × 10⁶ (RNA–2). Infectivity of CRLV-R RNA was abolished by treatment with proteinase K, suggesting that the RNA is linked to protein necessary for infectivity; RNA molecules contained polyadenylate. In reticulocyte lysates, CRLV-R RNA stimulated the incorporation of ³H-leucine, mainly into two polypeptides of estimated mol. wt 200 000 and 102 000. When electrophoresed in polyacrylamide gels, protein obtained from CRLV-R particles purified by centrifugation to equilibrium in Cs₂SO₄ separated into three bands with estimated mol. wt 26 000 , 23 000 and 21 000.     

Occurrence, purification and properties of narcissus tip necrosis virus

Narcissus tip necrosis virus (NTNV), a previously undescribed virus, was detected in the Netherlands and the United Kingdom in plants of twenty-one cultivars of trumpet, large-cupped, small-cupped, double, tazetta and poeticus narcissus. In some cultivars distinct leaf symptoms were sometimes associated with infection but in others infected plants remained symptomless and detection was dependent on serological tests. The virus was readily transmitted by manual inoculation to narcissus, but it failed to infect any of forty-six other plant species from fourteen families. Up to 50 mg of virus/kg of tissue were obtained by differential centrifugation of narcissus leaf extracts previously clarified with either diethyl ether, n-butanol or a mixture of n-butanol and chloroform. The virus particles are isometric, c. 30 nm in diameter, have a sedimentation coefficient (^s°₂₀ w) Of 123 S a buoyant density of 1·356 g/cm³, migrate as a single component in polyacrylamide gel electrophoresis, and contain a single RNA species of mol. wt 1·6×10⁶ and two major polypeptides of mol. wt 42000 and 39000. Although NTNV resembles tombusviruses it showed no serological relationship to the type member or six putative members of this group or to thirty-four other viruses with isometric particles. Its present cryptogram is R/*:1.6/(18):S/S:S/*.     

Peanut green mosaic virus — a member of the potato virus Y group infecting groundnut (Arachis hypogaea) in India

A virus, now named peanut green mosaic virus (PGMV), was isolated from groundnut (Arachis hypogaea) in India and identified as a member of the potato virus Y group by electron microscopy, aphid transmission, and its chemical properties. It was sap transmissible to 16 species of the Leguminosae, Solanaceae, Chenopodiaceae, Aizoaceae and Pedaliaceae; Phaseolus vulgaris was a good local lesion host. PGMV remained infective in buffered groundnut leaf sap at dilutions of 10^-3 after 3 to 4 days at 25 °C, or heating for 10 min to 55 °C but not 60 °C. PGMV was transmitted in the non-persistent manner by Aphis gossypii and Myzus persicae but was not seed-borne. Purified virus preparations contained flexuous filamentous particles c. 750 nm long which sedimented as a single component with a sedimentation coefficient (S°_20w) of 171S, and contained a single polypeptide (mol. wt 34 500 daltons) and one nucleic acid species (mol. wt 3.25 × 10⁶ daltons). PGMV is serologically unrelated to peanut mottle virus (PMV) and other viruses infecting leguminous crops. Infected leaves contained cylindrical, cytoplasmic inclusions.     

Garlic yellow streak virus, a potyvirus infecting garlic in New Zealand

In New Zealand, all garlic (Allium sativum) plants tested were infected by a virus with flexuous filamentous particles 700–800 nm long. This virus, called garlic yellow streak virus (GYSV), infected only two of 12 species tested and was transmitted to garlic by the aphid Myzus persicae in a non-persistent manner. In garlic sap, GYSV was infective at a dilution of 10^-4 but not 10^-3, after heating for 10 min at 60°C but not 65°C, and after 2 days but not 3 days at 25°C. The yield of virus, purified from naturally infected garlic, was 3–4 mg/kg fresh leaf. Preparations had A₂₆₀/A₂₈₀= 1.28 and A_man/A_min= 1.08. The virus particles had a sedimentation coefficient of 149S and a buoyant density in CsCl of 1.334 g/cm³. Mol. wt estimates for the virus nucleic acid were 2.95 × 10⁶ by electrophoresis in polyacrylamide gels and 3.46 × 10⁶ from the sedimentation coefficient (41.4S) in linear-log sucrose density gradients. Two polypeptides were detected in virus preparations; one (mol. wt 30 500) was possibly a breakdown product of the other (mol. wt 33 000). GYSV was serologically distantly related to onion yellow dwarf and leek yellow stripe viruses but was considered to be a separate virus because it differed from them in host range.     

Partial purification and some properties of wineberry latent, a virus obtained from Rubus phoenicolasius

Wineberry latent virus (WLV) was obtained from a single symptomless plant of American wineberry (Rubus phoenicolasius) originally imported from the United States of America. On graft inoculation, WLV infected but induced no distinctive symptoms in several Rubus species including those used as indicators for known Rubus viruses. It was not seed-borne in wineberry. WLV was mechanically transmitted to several herbaceous species but induced local lesions in only a few; it was weakly systemic in some Chenopodium species. Infective C. quinoa sap lost infectivity after diluting to 10^-4, heating for 10 min at 70°C, and storage either for 8 days at 18°C or for 32 days at 4°C. Sap from infected plants contained flexuous filamentous particles c. 510°12 nm. WLV was partially purified by extracting infected C. quinoa leaves in 0·05 M tris-HCl buffer (pH ₇) containing 0·2% thio-glycerol and 10% (v/v) chloroform and concentrating virus by precipitation with 7% (w/v) polyethylene glycol (PEG, mol. wt 6000) and 0·1 NaCl. The virus was then pelleted through a 30% (w/v) sucrose pad containing 7% PEG+0·1 M NaCl and finally sedimented through a sucrose density-gradient. These preparations had A_260/280 ratios of 1·26, contained end to end aggregates of WLV particles and formed a partly polydispersed peak in the analytical ultracentrifuge. WLV did not react with antisera to four potex-viruses, or to apple chlorotic leaf spot or apple stem grooving viruses.     

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.     

Properties of broad bean yellow band virus, a possible new tobravirus

A virus was transmitted from broad bean plants in Apulia (Southern Italy) with leaves showing yellow rings, line patterns or yellow vein banding and malformations and necrosis of pods. Symptoms in some, but not all, test plants were similar to those induced by tobraviruses. Purified virus preparations contained two classes of rod-shaped particles containing c. 5% nucleic acid with sedimentation coefficients of 186S and 276S. After centrifugation to equilibrium in CsCl gradients, two components were resolved, with buoyant densities of 1·298 and 1·316 g/cm³. Unfractionated virus preparations contained two species of single-stranded RNA with mol. wts of c. 1·06 × 10⁶ and 2·48 × 10⁶ and one species of coat protein with mol. wt of c. 21 300. The modal lengths of the two classes of particles, both in plant sap and in purified preparations, were 77 nm (S particles) and 202 nm (L particles). L particles accumulated in infected cells in paracrystalline aggregates, whereas S particles were randomly distributed in the cytoplasm of cells. The virus was serologically unrelated to two isolates of tobacco rattle virus and two isolates of pea early-browning virus. The virus, named broad bean yellow band, is considered a distinct tobravirus.     

Pathology and properties of tulip virus X, a new potexvirus

Tulip virus X (TVX), a previously undescribed mechanically transmissible virus, causes chlorotic and necrotic lesions in leaves and streaks of intensified pigmentation in tepals of tulip plants. The virus infected 22 of 42 other plant species in 10 of 14 families, but most host species were infected only erratically. TVX is best propagated in Chenopodium quinoa and assayed in C. amaranticolor. Spindleshaped inclusions were observed in epidermal cells of C. amaranticolor leaves. Leaf extracts from C. quinoa contained flexuous filamentous particles measuring c. 495 ×13 nm. The extracts were infective after dilution to 10^-9, after heating for 10 min at 60 °C but not at 65 °C, and after storage at c. 20 °C for 30 days or at -20 °C for 6 months. TVX particles were purified (500 μg/g C. quinoa leaf) from tissue extracts in 0.067 M phosphate buffer containing 10 mM EDTA at pH 7, by twice precipitating the virus with 8% polyethylene glycol in 0.2 M NaCl followed by differential centrifugation. The virus particles have a sedimentation coefficient (s_{20, w}) of 102 S. They contain a protein of mol. wt c. 22 500 and a nucleic acid that, when glyoxalated, migrates in agarose gel like single-stranded RNA of mol. wt 2.05 × 10⁶. TVX particles tend to aggregate, and evidence was obtained that a 118 S component which was consistently observed in purified preparations and in infective sap is an end-to-end dimer. A distant serological relationship was found between particles of TVX and those of viola mottle and hydrangea ringspot viruses, but no serological relationship was detected to nine other potexviruses. TVX is considered to be a distinct and definitive member of the potexvirus group.     

Further studies on double-stranded RNA species from raspberry plants infected with aphid-borne viruses

Analysis by electrophoresis in polyacrylamide gels, followed by silver staining, of dsRNA extracted from many samples of raspberry leaves infected with raspberry leaf mottle virus (RLMV) and/or raspberry leaf spot virus (RLSV) failed to detect reliably any significant quantities of dsRNA species in excess of 1·0 × 10⁶mol. wt. This contrasts with results reported from Canada where three dsRNA species of estimated mol. wt 2·6 × 10⁶1·6 × 10⁶and 1·1 × 10⁶were consistently associated with infection with RLSV but none were associated with RLMV. However, in Scotland, four dsRNA species of estimated mol. wt 2·4 × 10⁶1·6 × 10⁶0·7 × 10⁶and 0·3 × 10⁶were detected in raspberry infected with apple mosaic ilarvirus. These results suggest that the dsRNA species reported from Canada are not those of RLSV but are probably those of a second virus, possibly an ilarvirus, which occurs together with RLSV and/or induces similar symptoms. A few samples from plants infected with RLMV and RLSV contained very small amounts of two dsRNA species of estimated mol. wt 4·7 × 10⁶and 4·5 × 10⁶. It is not known whether these species are those of RLMV and RLSV.     

Some properties of an isolate of tobacco rattle virus from Northern Ireland

A virus obtained from soil in which potato plants had shown severe spraing symptoms induced symptoms on indicator plants typical of tobacco rattle virus (TRY). Purified virus preparations of a local-lesion isolate contained particles of two modal lengths, 192 nm and 94 nm containing RNA molecules of mol. wt 2.4 × 10⁶ and 1.23 × 10⁶. Virus coat protein had a mol. wt of c. 21 500. The virus was serologically distantly related to TRY (SYM) and pea early browning virus (PEBV) SP5, but did not react with TRY (CAM) or TRY (PRN) antisera. However, cDNA hybridisation indicated that the virus was more closely related to TRY (PRN) than either TRY (SYM) or PEBV (SP5). The virus isolate has been designated TRY (NI).     

Purification and properties of ginger chlorotic fleck virus

A previously undescribed isometric virus, named ginger chlorotic fleck virus (GCFV), was detected in ginger (Zingiber officinale) imported into Australia from a number of countries. The geographical distribution of the virus is uncertain, but is thought to include India, Malaysia and Mauritius. The virus apparently does not occur in Australian commercial ginger plantings. The virus has isometric particles c. 30 nm in diameter, with a sedimentation coefficient of 111 S, and was readily purified from infected ginger with yields of 50–90 mg/kg leaf tissue. Purified preparations contained a major species of single-stranded RNA of mol. wt 1.50 × 10⁶ and a major coat protein species of mol. wt 29.0 × 10³. At pH 7, the particles formed a single zone in both caesium chloride and caesium sulphate gradients, with buoyant densities of 1.355 g cm^-3 (fixed virus) and 1. 297 g cm^-3 (unfixed virus), respectively. The virus particles migrated as two electrophoretic components and were labile when treated with 10 mM EDTA, 1 M NaCI, 10 mM tris pH 8.25 or when negatively stained with potassium phosphotungstate. GCFV was mechanically transmitted only to ginger, and was not transmitted by the aphids Myzus persicae. Pentalonia nigronervosa, Rhopalosiphum maidis or R. padi. Possible affinities of GCFV with the sobemo-virus group are discussed. The present cryptogram of GCFV is ^R/_l: ^1.5/₂₀: ^S/_S: ^S/_*.     

Particle purification and properties of cassava Ivorian bacilliform virus

A virus found in cassava from the north-west of the Ivory Coast was transmitted by inoculation with sap extracts to herbaceous species in six plant families. Chenopodium quinoa was used as a propagation host and C. murale was used for local lesion assays. The virus particles are bacilliform, c. 18 nm in diameter, with predominant lengths of 42,49 and 76 nm and a structure apparently similar to that found in alfalfa mosaic virus. Purified preparations of virus particles had A₂₆₀/A₂₈₀ of 1.7 ±0.05, contained one protein of M_rc. 22 000, and yielded three species of RNA with M_r (× 10^-6) of c. 0.7, 0.8 and 1.2. Although the virus particles were poorly immunogenic, an antiserum was produced and the virus was detected by enzyme-linked immunosorbent assay (DAS-ELISA) in leaf extracts at concentrations down to c. 6 ng/ml. Four other field isolates were also detected, including a strain which caused only mild systemic symptoms in C. quinoa instead of necrosis. The naturally infected cassava source plants were also infected with African cassava mosaic virus (ACMV) but when the new virus was cultured in Nicotiana benthamiana, either separately or together with ACMV, its concentration was the same. The new virus did not react with antisera to several plant viruses with small bacilliform or quasi-bacilliform particles, and alfalfa mosaic virus reacted only weakly and inconsistently with antiserum to the cassava virus. The new virus, for which the name cassava Ivorian bacilliform virus is proposed, is tentatively classified as the second member of the alfalfa mosaic virus group.     

Host range, purification and some properties of two carlaviruses from hop (Humulus lupulus): hop latent and American hop latent

Hop latent virus (HLV) occurs in virtually all commercial hop plants in England, without causing apparent symptoms. It was transmitted between hop plants in a non-persistent manner by the aphid Phorodon humuli, but was not seed-borne in hop. The virus infected six species in four families out of 40 in 13 families which were inoculated, but infection was systemic only in Dianthus deltoides and hop. Only Phaseolus vulgaris and Chenopodium murale developed symptoms. Purification of HLV from hop extracts was hampered by aggregation of virus particles but this was minimised either by resuspending pellets in phosphate-buffered saline containing Tween 20 or by avoiding ultra-centrifugation. Virus was purified from extracts treated with Triton X-100 by precipitation with polyethylene glycol (PEG) followed either by centrifugation through sucrose density gradients or by exclusion chromatography through columns of Sephadex G-25 and Sepharose 4B. Purified preparations contained filamentous particles c. 675 × 14 nm composed of c. 6% single stranded RNA of mol. wt c. 2.9 × 10⁶ and a single protein species of mol. wt c 33 000. Immunosorbent electron microscopy (IEM) decoration tests suggested that HLV was serologically related to carnation latent, Helenium virus S, lily symptomless and Nerine latent viruses. American hop latent virus (AHLV) was found in two introductions to England from Corvallis, USA in 1975 and 1976. It was transmitted between hop plants in the non-persistent manner by P. humuli. The virus infected 17 species in seven families out of 41 species in 13 families which were mechanically inoculated and was systemic in nine species. It did not cause symptoms in any of five English hop cultivars. C. quinoa was a convenient propagation host and countable local necrotic lesions and ringspots occurred in leaves of Datura stramonium. AHLV was purified by PEG precipitation and centrifugation in sucrose density gradients. Preparations contained filamentous particles c. 680 × 15 nm composed of c. 6% single-stranded RNA of mol. wt c. 3.0 × 10⁶ and a single protein species of mol. wt c. 33 000. In IEM decoration tests AHLV was serologically related to Nerine latent virus but did not react with antisera to 14 other carlaviruses.     

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.