Purification and properties of Australian lucerne latent virus, a seed-borne virus having affinities with nepoviruses

An isolate of Australian lucerne latent virus (ALLV) from lucerne in New Zealand was mechanically transmitted to a few herbaceous hosts. It induced diagnostic symptoms in several species of the Chenopodiaceae, but was symptomless in most other hosts including lucerne and Trifolium subterraneum. It was seed transmitted in lucerne. When assayed to Chenopodium quinoa, infective C. quinoa sap lost infectivity after diluting to 10^-4, heating for 10 min at 55°C and storage for 4 days at 4°C. ALLV was purified from infected C. quinoa or pea plants by extracting sap in 0.1 m borate buffer (pH 7) containing 0.2% 2-mercaptoethanol and clarifying with 15% bentonite suspension, high and low speed centrifugation and sucrose density gradient centrifugation. Purified virus preparations contained isometric particles about 25 nm in diameter and sedimented as three virus components with sedimentation coefficients (s_20-w⁰) of 56 S, 128 S and 133 S. The 56 S component appeared to consist of nucleic acid-free protein shells. Polyacrylamide gel electrophoresis of virus preparations showed that ALLV contained a single protein species of mol. wt 55 000 and two RNA species of mol. wt 2.1 × 10⁶ and 2.4 × 10⁶. An antiserum to ALLV had an homologous titre of 1/256 to purified virus but failed to detect ALLV in infective sap of C. quinoa, pea or lucerne. Purified ALLV failed to react to antisera to 28 distinct isometric plant viruses including those to 10 nepoviruses.     

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.     

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.     

Purification and properties of elm mottle virus

A virus obtained commonly from Wych elm (Ulmus glabra) in Scotland showing ringspot and line-pattern leaf symptoms was serologically related to elm mottle virus (EMotV) from East Germany. The virus was seed-borne in elm and was transmitted by inoculation of sap to elm and twenty-one herbaceous species. No symptoms developed in infected elm seedlings kept in the glasshouse. In Chenopodium quinoa sap, EMotV lost infectivity after diluting to 10^-4, after 10 min at 60 ^oC, or 9 days at 18 ^oC. When purified from C. quinoa sap by clarification with n-butanol (8-5 %, v/v) and differential centrifugation, preparations contained quasi-spherical particles mostly 26–29 nm m diameter (mean = 28 nm) which sedimented as three nucleo-protein components with sedimentation coefficients (s^o₂o, w) of 83, 88 and 1 or S; most infectivity was associated with the 101 S component but infectivity was enhanced by adding the slower sedimenting components. When centrifuged to equilibrium in caesium chloride solution at 4 ^oC, purified virus preparations were largely degraded and contained many non-infective particles c. 15–22 nm in diameter, and intact infective particles which formed a band of density c. 1–34 g/cm³. Polyacrylamide gel electrophoresis indicated that EMotV contained a single major protein species of estimated mol. wt. 25000 and five RNA species of estimated mol. wt. 1–30, 1.15, 0–82, 0 39 and 0–30 times10⁶. Gel electrophoresis of RNA extracted from the separated components indicated that the 101 S component contained 1–30 x io⁶ mol. wt. RNA and the 83 S component 0–82 times 10⁶ mol. wt. RNA. In these and other properties, EMotV resembles the serologically unrelated tobacco streak virus.     

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.     

Host range, properties and purification of raspberry bushy dwarf virus

Raspberry bushy dwarf virus (RBDV) was found in all plants of Lloyd George raspberry with bushy dwarf disease and occurred occasionally in plants of some other cultivars. It was transmitted by inoculation of sap to fifty-five other species in twelve families of flowering plants and infected most of them symptomlessly. It caused systemic symptoms in some species of Amaranthaceae, Chenopodiaceae and Cucurbitaceae, and necrotic local lesions in some Leguminosae. It did not induce bushy dwarf disease when returned to Lloyd George raspberry. Chenopodium quinoa was used for propagating the virus and Vigna cylindrica for local lesion assay. In C. quinoa sap, RBDV lost infectivity when diluted 10^-4, heated for 10 min at 65 °C or stored for 4 days at 22 °C. Preparations made by twice precipitating the virus at pH 4·8 and resuspending it at pH 7·0, followed by ultracentrifugation and exclusion chromatography in columns of 2 % agarose beads, contained isometric particles about 33 nm in diameter, which sedimented as two components, with sedimentation coefficients of 111 and 116S. Only a few particles, all of them disrupted, were seen in preparations mounted in phosphotungstate, but the particles were well preserved in uranyl formate provided that they were first dispersed in a saxlt such as MgCl₂ instead of distilled water. Many particles were oval in outline as though distorted during drying. No serological relationship was detected between RBDV and twenty-four other isometric viruses nor between RBDV and the filamentous virus apple chlorotic leafspot, to which it was previously thought to be related. An isolate of loganberry degeneration virus was serologically indistinguishable from RBDV.     

Chenopodium necrosis: a distinctive strain of tobacco necrosis virus isolated from river water

A distinctive strain of tobacco necrosis virus (TNV) of unknown source was repeatedly isolated from water of the River Avon (Warwickshire) and two of its tributaries (R. Swift and R. Alne) using a technique developed for the concentration and isolation of water-borne bacteriophages. The same strain was isolated from the rivers Cam and Thames and from Lake Esthwaite (Cumbria) together with tomato bushy stunt virus. The TNV strain, designated Chenopodium necrosis (TNV-CN) was mechanically transmissible to C. amaranticolor and C. quinoa in both of which it caused local lesions and systemic infection. TNV-CN caused no infection when inoculated to tobacco (Nicotiana tabacum cv. White Burley) plants. The virus was not adsorbed to soil, could be isolated from leachate of soil in which systemically infected C. quinoa were grown and C. quinoa plants became infected when grown in soil watered with suspensions of the virus. The virus was not transmitted by Myzus persicae but was vectored by the zoospores of a lettuce isolate of Olpidium brassicae. TNV-CN was infective after 10 min at 85 °C., 3 wk at 20 °C and when diluted to 10^-8 but not 10^-9. Purified virus preparations contained c. 26 nm isometric virus particles. TNV-CN contained single-stranded RNA (mol. wt 1·5 × 10⁶) and one protein (mol. wt c. 26·4 × 10³) which co-electrophoresed in polyacrylamide gels with the protein of the D strain of TNV (TNV-D). Analytical centrifugation of TNV-CN indicated a single component virus with the same sedimentation coefficient (s₂₀, w= 115S) and buoyant density (1·385) in a CsCl gradient as those of TNV-D. TNV-CN and TNV-D were indistinguishable serologically.     

Crimson clover latent virus - a newly recognised seed-borne virus infecting crimson clover (Trigolium incarnatum)

Crimson clover latent virus (CCLV) was detected in five seed lots of crimson clover (Trifolium incarnatum) from Europe and in one from the United States of America. Ninety-seven per cent of all crimson clover plants examined were found to be infected but were without symptoms. Keeping crimson clover plants at 32–38°C for 34 days failed to free them from CCLV. The virus was not transmitted by Myzus persicae, but was transmitted by inoculation of sap to Chenopodium album, C. amaranticolor and C. quinoa. Twenty-four other plant species from seven families were not infected. CCLV was best propagated in C. quinoa in which it caused stunting and systemic chlorosis. Sap from infected C. quinoa was infective after dilution to 10^-2 but not 10^-3, after 10 min at 60°C but not 65°C, and after 20 days at 20°C. In neutral phosphotungstate, CCLV had isometric particles c. 26 nm in diameter with a hexagonal profile. About 20 to 80 A_1cm,260 units of purified virus were obtained from 1 kg of infected C. quinoa or C. amaranticolor leaves by extraction in 0.5 M phosphate buffer, pH 7.5, containing 0.01 M ethylene diamine tetra-acetate and 0.4% 2–mercaptoethanol and clarification with chloroform-butanol followed by two precipitations with polyethylene glycol (mol. wt 6000) and several cycles of differential centrifugation. Purified virus sedimented as three components with sedimentation coefficients (s°_{20, w}) of 52S, 101S and 122S. The 101S and 122S components had buoyant densities in CsCl of 1.438 and 1.495 g/cm³ respectively. From these values the nucleic acid content of the 101S and 122S components was estimated to be 32–35% and 40–41% respectively. The virus contained a single protein with an estimated mol. wt of 52 000 and two single-stranded RNA species of estimated mol. wt 1.6 × 10⁶ and 2.2 × 10⁶. CCLV was serologically unrelated to 31 other morphologically similar viruses. Although its vector is unknown, CCLV seems to have affinities with nepoviruses. The cryptogram of CCLV is R/1:2.2/40–41 + 1.6132–35:S/S:S/*.     

Purification and properties of cassava green mottle, a previously undescribed virus from the Solomon Islands

A sap-transmissible virus obtained from cassava with a green mottle disease occurring at Choiseul, Solomon Islands, was transmitted to 30 species in 12 plant families and was readily seed-borne in Nicotiana clevelandii. In cassava plants infected by inoculation with sap, the first leaves to be infected systemically developed a mottle with some necrosis whereas leaves produced subsequently were symptomless but contained the virus. Most other species developed chlorotic or necrotic local lesions and systemic mottle or necrosis. This was followed, in several species, by production of small symptomless virus-containing leaves. The virus was cultured in N. clevelandii; Chenopodium quinoa was used for local-lesion assays. Leaf extracts from infected N. clevelandii were infective after dilution to 10–⁵ but usually not at 10–⁶, after heating for 10 min at 60°C but not at 65°C, and after storage at 20°C for at least 12 days. The virus has isometric particles of 26 nm diameter which sediment as three components, all containing a protein of mol. wt c. 53000. The two fastest sedimenting components respectively contain single-stranded RNA of mol. wt, estimated after glyoxylation, c. 2.9 × 10⁶ and 2.3 × 10⁶. Both RNA species are needed for infection of plants. In tests with antiserum prepared to purified virus particles, the virus was detected in cassava and N. clevelandii by gel-diffusion precipitin tests, by immunosorbent electron microscopy and by ELISA. Despite its similarity to nepoviruses, the virus did not react with antisera to 18 members of the group. It was named cassava green mottle virus and is considered to be a previously undescribed nepovirus.     

Identification and characterisation of a newly described potyvirus in West Africa: Asystasia mottle virus

Asystasia mottle virus (AsMV) was detected serologically in samples of Asystasia gangetica with mottle symptoms, from several areas of tropical West Africa. It infected 12 species systemically and induced local lesions in a further four. The virus lost infectivity after dilution to 10^-4, after 10 min at 75 °C and after 3 days at 27°C. Purified virus had an A₂₆₀/A₂₈₀ ratio of c. 1·2 and a protein subunit mol. wt of c. 33 000. Particles were c. 750 nm long and cytoplasmic inclusions typical of potyviruses were seen in ultrathin sections of infected leaves. The antiserum prepared had a titre of 1/1024 in microprecipitin tests but purified virus failed to react with 31 antisera to known potyviruses. The virus was transmitted in the non-persistent manner by Aphis spiraecola but only very infrequently. On the basis of these properties, AsMV is considered to be a new member of the potyvirus group.     

Further properties of wineberry latent virus and evidence for its possible involvement in calico disease

The flexuous filamentous particles of wineberry latent virus (WLV) were found to measure 620. 12 nm and not 510. 12 nm as previously reported. Analysis of dsRNA from infected plants detected a major species of c. 5.7. 10⁶ mol. wt and minor species of lower mol. wt. Purified virus particles formed a major and a minor buoyant density component in solutions of caesium salts with densities of 1.26 and 1.25 g cm^-3 in Cs₂SO₄ and 1.30 and 1.29 g cm^-3 in CsCl. The particles contained a single nucleic acid species, presumably single stranded RNA, and a single polypeptide of estimated mol. wt 2.78. 10⁶ and 31 000 respectively. In indirect ELISA, purified particles of WLV and particles in plant sap failed to react specifically with antiserum to nine carlaviruses, 12 potexviruses, three capilloviruses or apple chlorotic leafspot closterovirus, nor was WLV found to react with several of these antisera in immunosorbent electron microscopy or immunoblots. In Marion and Olallie blackberry, WLV in mixture with raspberry bushy dwarf virus (RBDV), but not RBDV alone, induced veinal line-pattern symptoms resembling those of calico disease reported from the USA.     

Ullucus virus C, a newly recognised comovirus infecting Ullucus tuberosus (Basellaceae)

Ullucus virus C (UVC) is a comovirus prevalent in Ullucus tuberosus grown at high altitudes in the Bolivian and Peruvian Andes. It was transmitted mechanically to U. tuberosus (Basellaceae) and to five of 26 species from three of eight other families, infecting U. tuberosus symptomlessly but inducing conspicuous systemic infection in Chenopodium amaranticolor and C. quinoa. Sap from infected C. quinoa was usually infective after 10 min at 70 but not 75 °C, after dilution to 10^-7 but not 10^-8, and after 8 but not 16 wk at 20 °C. UVC was not transmitted by either of two aphid species (Aphis gossypii and Myzus persicae) or through seed of C. quinoa, but it was transmitted by leaf contact between infected and healthy plants. UVC has isometric particles which, in neutral phosphotungstate, are c. 28 nm in diameter. The particles sediment as three components (T, M and B) with sedimentation coefficients (s^?_{20, w}) of 51 S (T), 95 S (M) and 116 S (B). M component particles have a buoyant density (g cm^-3) in caesium chloride of 1.404, and B component particles separated into minor and major sub-components with densities of 1.409 and 1.463, respectively. T, M and B particles were serologically indistinguishable, and each contained similar relative amounts of two polypeptides of mol. wts 20 700 and 45 100. T particles contained only protein, but M particles also contained c. 30% ss-RNA of mol. wt 1–45 ×10⁶ and B particles c. 38% ss-RNA of mol. wt 2·2 × 10⁶. The virus is serologically distantly related to cowpea mosaic virus but, as it showed no relationship to any of 11 other similar viruses, it is probably a distinct member of the comovirus group.     

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.     

Properties of a carlavirus causing a latent infection of pepino (Solanum muricatum)

Pepino (Solanum muricatum) cuttings imported from Chile contained a latent virus which was transmitted by inoculation of sap to Chenopodium quinoa but not to 21 other species. The virus was transmitted by the aphid, Myzus persicae. In C. quinoa sap, the virus lost infectivity when diluted between 10^-3 and 10^-4, heated for 10 min between 65 and 70 °C, or stored at room temperature for 4 to 6 days. The virus particles were straight or slightly flexuous filaments 660 to 680 nm long. Up to 15 mg virus per 100 g C. quinoa leaves was obtained by clarification with a mixture of chloroform and carbon tetrachloride. Purified preparations had A_max/A_min= 1.11, A₂₆₀/A₂₈₀= 1–30, A^0.₂₆₀^1%= 2.8, and contained a single sedimenting component with a sedimentation coeficient of 149s and a buoyant density in CsCl of 1–318. The virus particles contained 5.5% of single-stranded RNA of mol. wt 2.4×10⁶ (estimated by gel electrophoresis of undenatured RNA) and sedimentation coefficient 38.5S, and a single polypeptide of mol. wt 33 000. The virus is distantly serologically related to potato S and carnation latent viruses and is considered a new member of the carlavirus group. The name pepino latent virus is proposed. The cryptogram for this virus is R/1: 2.4/5–5: E/E: S/Ve/Ap.     

Purification and properties of lucerne Australian symptomless virus, a new virus infecting lucerne in Australia

A virus with isometric particles c. 26–28 nm in diameter isolated from naturally infected lucerne (Medicago sativa) in Australia and reported there to be a strain of lucerne Australian latent virus (LALV), is shown to be a distinct virus. The virus, called lucerne Australian symptomless (LASV), was mechanically transmitted to 10 of 22 plant species inoculated, but only induced symptoms in three Chenopodium species and Gomphrena globosa. Virus particles occurred in relatively low concentrations in plant sap, and the virus could not be reliably maintained in culture by serial transmission to plants during winter (October-April). During the summer, sap of infected C. quinoa remained infective after diluting 10^-2 but not 10^-3, after heating for 10 min at 50 but not 55 ^oC and after storage for 24 days (the longest period tested) at 20, 4 and -15 ^oC. LASV was seed-borne to 6% of C. quinoa seedlings. Partially purified preparations of virus particles contained one nucleoprotein component with a sedimentation coefficient of c. BOS. Particles contained two polypeptide species of estimated mol. wts 26 000 and 40 000, and two ssRNA species which, when denatured in glyoxal, had apparent mol. wts of 2–5 times 10⁶ and 1–4 times 10⁶. The infectivity of virus RNA was abolished by incubation with proteinase K. Purified particles of LASV reacted with homologous antiserum (gel diffusion titre 1:256) but not with antiserum to LALV or to 13 other plant viruses with isometric particles including arracacha B (AVB), broad bean wilt, rubus Chinese seed-borne (RCSV) and strawberry latent ringspot (SLRV) viruses, and five comoviruses. These properties distinguish LASV from LALV and from all recognised nepoviruses and comoviruses. Its closest affinities are with SLRV, RCSV and possibly AVB; these viruses may comprise a distinct virus group or nepovirus subgroup.     

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 and properties of blackberry chlorotic ringspot, a new virus species in subgroup 1 of the genus Ilarvirus found naturally infecting blackberry in the UK

A mechanically transmissible virus was isolated from Bedford Giant blackberry plants showing chlorotic mottling and ringspot symptoms growing in Scotland. It infected several herbaceous test plants, many of them symptomlessly. This virus was also transmitted to several Rubus species and cultivars by graft inoculation with scions from the field‐infected Bedford Giant plant. Most grafted plants were infected symptomlessly, but Himalaya Giant blackberry and the hybrid berry Tayberry developed symptoms similar to those in the infected Bedford Giant plant. In the sap of infected Chenopodium quinoa, the virus lost infectivity when diluted 10^?4 but not 10^?3, after 6 h and 48 h when kept at 20°C and 4°C, respectively, but was infective for more than 8 days when kept at ?15°C. Preparations of purified virus from infected C. quinoa or spinach sedimented as three major nucleoprotein components and consisted of quasi‐isometric particles that varied in size from 24 to 32 nm in diameter and that were not penetrated by negative stain. Such virus particle preparations contained a major polypeptide of ca 28 kDa and three single‐stranded RNA species of estimated size 3.2, 2.8 and 2.1 kb. The complete sequence of the largest RNA (RNA 1, 3478 nt) and the partial sequence of the other RNAs (1863 and 2102 nt long, respectively) were determined and compared with sequences in databases. These findings, together with the biological and biochemical properties of this virus, indicate that it should be regarded as a distinct species in subgroup 1 of the genus Ilarvirus even though it was serologically unrelated to existing members of this subgroup. The virus showed a very distant serological relationship with prune dwarf virus (PDV) but differed significantly from it in the amino acid sequence of its coat protein, experimental host range and symptomatology and was unrelated to PDV at the molecular level. The virus, tentatively named blackberry chlorotic ringspot virus, is therefore a newly described virus and the first ilarvirus found naturally infecting Rubus in the UK.     

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.     

Attempts at Multiplication,Purification, Electron Microscopy,and Characterisation of Three Isolates of the Strawberry Mottle Agent

Three isolates of strawberry mottle agent (SMA) from strawberry plants were regularly maintained and multiplied by mechanical inoculation onChenopodium quinoa plants showing mosaic and mottle symptoms. The use of 5 mM borate buffer pH 8.6 or tap water pH 6.6-7.9 with 4 % (m/v) charcoal for homogenization resulted usually in 100 % infection. The total of 2090 plants were infected from 2264 inoculated ones under the same conditions. The infectivity of SMA isolates in crude sap ofC. quinoa was retained from 48 h to 72 h at 20 °C. The dilution end points of SMA isolates were 10^-3 while the inactivation temperatures were between 50 and 55 °C. The infectivity of SMA isolates in frozen leaves ofC. quinoa was detected still after six months. Purification procedure of SMA is based on using low molar 25 mM borate buffer pH 8.3 with cysteine hydrochloride, DIECA and Tween 20 for homogenization of infectedC. quinoa leaves, polyethyleneglycol precipitation, clarification with octanol, low and high speed centrifugation and sucrose density-gradient centrifugation. Partially purified preparations are highly infectious, causing mosaic, mottling and tip necrosis ofC. quinoa plants. The agent could not be completely separated from host proteins and it could not be concentrated to a high extent. Isometric virus-like particles 14-16 nm were observed in partially purified preparations.     

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.