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.     

A distinct strain of lucerne Australian latent virus in white clover in New Zealand

A strain of lucerne Australian latent virus (LALV) was widespread in Trifolium repens (white clover) in Northland, New Zealand, occurring in 20–100% of plants tested on 23 of 26 farms. Infected plants showed chlorotic line patterns or were symptomless. In agar gel double diffusion serological tests, white clover isolates of LALV differed from lucerne isolates by two or three twofold dilution steps of antisera. In mechanical inoculation tests, white clover isolates did not infect lucerne and lucerne isolates did not systemically infect white clover. White clover and lucerne isolates also differed in their reactions in some other hosts. Six LALV isolates from white clover were indistinguishable in double diffusion serological tests and by enzyme-linked immunosorbent assay, although the isolates could be differentiated by some reactions in test plants. Purified LALV particles of lucerne and white clover isolates contained two nucleoprotein components and two RNA species of molecular weight 2.82 × 10⁶ (8150 nucleotides) and 2.46 × 10⁶ (7100 nucleotides) (estimated under denaturing conditions).     

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/*.     

Host range, purification and some properties of rubus Chinese seed-borne virus

A previously undescribed virus, for which the name rubus Chinese seed-borne virus (RCSV) is proposed, was isolated from a single, symptomless plant of an unidentified Rubus species grown from seed collected in the wild in the People's Republic of China, Experimentally RCSV infected 23 out of 39 spp. in six out of eight families. The virus was seed-transmitted in Chenopodium quinoa (100%) and Nicotiana bigelowii (27%). RCSV was not transmitted by the nematodes Xiphinema diversicaudatum or X. index. The particles of RCSV were isometric, c. 30 nm in diameter with some penetrated by negative stains. In thin sections particles were found in double walled tubular structures with an outer membrane enclosing one or more tubules. In crude extracts some particles were found within single-walled tubules. Two virus-associated bands were seen in sucrose density gradients of purified preparations. The upper band was not infective and consisted of penetrated particles apparently devoid of nucleic acid. The lower, infective band was resolved into two components, of density 1.452 and 1.461 g/ml, in caesium chloride isopycnic gradients. There were two polypeptides (mol. wts c. 47 000 and 25 200 daltons) and two nucleic acid species (one of mol. wt c. 1.4 × 10⁶ daltons; the second was poorly defined by the methods used but was of higher molecular weight). RCSV was distantly related serologically (6–7 SDI) to the type isolate of strawberry latent ringspot virus (SLRV) and also reacted with antisera to serologicaly distinct grape and olive isolates of SLRV. It did not react with antisera to 10 other isometric viruses.     

Narcissus latent, a virus with filamentous particles and a novel combination of properties

As previously reported, narcissus latent virus (NLV) has flexuous filamentous particles measuring c. 650 nm × 13 nm, is manually transmissible to Nicotiana clevelandii and Tetragonia expansa, and is transmitted by the aphid Myzus persicae following brief acquisition access periods. In contrast to previous reports the virus particle protein has an apparent mol. wt of c. 45 kD. Moreover, infected cells in N. clevelandii leaves contain cytoplasmic inclusion bodies resembling those of potyviruses. In vitro translation of NLV RNA produced only one major product (mol. wt c. 25 kD) which was not precipitated by antisera to virus particle protein or to cytoplasmic inclusion protein. Antisera to 12 potyviruses and nine carlaviruses failed to react with sap containing NLV particles. Similarly antiserum to NLV particles did not react with particles of seven potyviruses or four carlaviruses. A weak reaction was detected between NLV particles and antiserum to particles of maclura mosaic virus (MMV), a virus which resembles NLV in particle morphology and particle-protein size, and in inducing pinwheel inclusions. The cytoplasmic inclusion proteins (CIPs) of NLV, MMV and from narcissus plants with yellow stripe symptoms were serologically inter-related. These proteins were also serologically related to, and had mol. wt similar to, the CIP of members of the potyvirus group. Particles with the size and antigenic specificity of those of NLV were found consistently in narcissus plants with yellow stripe disease. Narcissus latent and narcissus yellow stripe viruses therefore seem to be synonymous and, together with MMV, have properties distinct from those of any previously described virus group.     

Nicotiana velutina mosaic virus: purification, properties and affinities with other rod-shaped viruses

Nicotiana velutina mosaic virus (NVMV), found in Australia, was transmitted by inoculation of sap to twenty species in the Solanaceae and Chenopodiaceae, and to Gomphrena globosa; its host range closely resembles that of potato mop-top virus (PMTV). Infectivity was abolished when sap was kept at room temperature between 1 and 4 days, or when heated for 10 min between 60 and 70 °C. NVMV was frequently transmitted through the seed of four Nicotiana spp. NVMV and PMTV were purified by a method that involved redissolving virus particles sedimented by low speed centrifugation of leaf extracts, followed by sedimentation through sucrose cushions. NVMV preparations contain rod-shaped particles about 18 nm wide and with a large range of lengths, the commonest being 125–150 nm. The particles have a helical structure with a pitch of 2–9 nm, break easily, and contain a single protein of apparent mol. wt. 21|400, slightly larger than that of PMTV (19 800). In serological tests assessed by electron microscopy, no relationship was detected between NVMV and PMTV, or barley stripe mosaic, beet necrotic yellow vein, soil-borne wheat mosaic, tobacco mosaic or tobacco rattle viruses. However, antiserum to soil-borne wheat mosaic virus reacted quite strongly with PMTV and weakly with tobacco mosaic virus. NVMV is considered to be a distinct member of the tobamovirus group; its frequent transmission through seed may be an adaptation to the arid environment where it was found. Its cryptogram is */*:*/*:E/E:S/*.     

Distribution, host range, properties and purification of cassava latent virus, a geminivirus

Cassava latent virus (CLV) is almost entirely confined in East Africa to upland cassava-growing areas west of the Rift Valley, where it is often associated with cassava mosaic disease (it was isolated from 27 of 38 cassava plants with mosaic, but not from 24 without mosaic). However, it is not the causal agent, because it was not recovered from any of 31 mosaic-diseased plants in coastal districts. All attempts to return CLV to cassava failed. The host range of CLV appears to be limited to Euphorbiaceae (Manihot) and Solanaceae (Nicotiana, Datura, Nicandra, Solanum). N. clevelandii proved the most useful assay and propagation host. The dilution end-point of CLV was about 10^-3, thermal inactivation point about 55°C, and longevity in vitro about 3 days. CLV was purified by clarification of leaf extracts with butanol/chloroform mixtures. Purified preparations (A 260/A 280 ratio c. 16) contained numerous 30 20 nm paired particles with a sedimentation coefficient (s₂₀w) of 76 S. Treatment with RNase and DNase showed that the viral nucleic acid is DNA; CLV closely resembles maize streak virus but is not related to it serologically. The cryptogram for CLV is D/1: 0.8/*: S/S: S/*, geminivirus group.     

Host range, purification and serological properties of heracleum latent virus

Heracleum latent virus (HLV occurs commonly in wild plants of Heracleum sphondylium (hogweed) in Scotland without causing symptoms. It was transmitted manually or by aphids (Cavariella aegopodii, C. pastinacae or C. theobaldi) to 37 of 105 species in 11 of 18 families (especially to members of the Amaranthaceae, Chenopodiaceae, Solanaceae and Umbelliferae), but was not transmitted through seed of four species tested. It has very flexuous filamentous particles c. 730 × 12 nm in phosphotungstate, with obvious cross-banding of pitch 3–8 nm. Leaf extracts lost infectivity after 1–2 days at 22°C, 10 min at 40–50°C and after dilution 10^-4-10^-5. Infectivity in leaf extracts was not stabilised by addition of Mg²⁺, Ca²⁺ or Ni²⁺, but was abolished by EDTA. HLV was purified by bentonite clarification, precipitation with polyethylene glycol (mol. wt 6000), and differential centrifugation. Its properties resemble those of the tentative closterovirus, apple chlorotic leaf spot (ACLSV), but no serological relationship was detected to this or to any of 18 other filamentous viruses, including six definitive closteroviruses. No cross-protection was observed between HLV, ACLSV and apple stem grooving virus.     

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.     

Purification and properties of a small latent matrix metalloproteinase of the rat uterus

A small metalloproteinase that digests Azocoll was found in the uterus of the rat. Its activity increased to high levels during the postpartum period in parallel with the breakdown of the extracellular matrix exclusive of collagen (Sellers, A., and Woessner, J.F., Jr. (1980) Biochem. J. 189, 521-531). This enzyme has now been purified almost 7,000-fold to homogeneity from 12 g of tissue using molecular sieve chromatography, blue sepharose chromatography, and zinc-chelate chromatography. Gel electrophoresis with sodium dodecyl sulfate and dithiothreitol gives Mr = 28,000 for the latent form of the enzyme and Mr = 19,000 for the active form that arises spontaneously or by treatment with aminophenylmercuric acetate. The enzyme digests components of the extracellular matrix including gelatins of types I, III, IV, and V, fibronectin, and proteoglycan. It digests the alpha 2(I) chain of gelatin in preference to the alpha 1(I) chain and cleaves dinitrophenyl-Pro-Leu-Gly-Ile-Ala-Gly-Pro-D-Arg. It cleaves the B chain of insulin at two points: Ala14-Leu15 and Tyr16-Leu17. It has no action on collagens of types I, III, IV, or V at 26 degrees C and no action on elastin or phenylazo-Pro-Leu-Gly-Pro-D-Arg. The pH optimum is at pH 7 and the pI at 5.9. The enzyme requires zinc and calcium ions for activity; cobalt and strontium can partially replace these metal ions. The enzyme is not inhibited by low levels of phosphoramidon or Zincov. Its properties clearly distinguish it from collagenase, gelatinase (matrix metalloproteinase 2), and stromelysin (matrix metalloproteinase 3); it therefore constitutes a further member of the family of extracellular matrix metalloendopeptidases. The name matrix metalloproteinase 7 is proposed.     

Some properties of grapevine Bulgarian latent virus

Grapevine Bulgarian latent virus (GBLV), obtained from symptomless vines grown in Bulgaria, was readily transmitted by inoculation of sap to a restricted range of hosts. The virus was re-inoculated into virus-free rooted cuttings and seedlings of several grapevine cultivars without inducing symptoms. Purified preparations of GBLV contained isometric particles c. 30 nm in diameter which sedimented as three components at 52 , c. 120 and 1275, respectively. The slow-sedimenting component (T) contained non-infective protein shells, whereas the two heavier classes of particles (J5_X, B₂) each contained one molecule of single-stranded ribonucleic acid (RNA) with mol. wts of 2.1 and 2.2 times 10⁶ daltons. The RNA content of B₁ and B₂ components were 39 and 41 % by particle weight respectively. The capsid was composed of one type of protein with a mol. wt of 54000. GBLV did not react with antisera to any of twenty-eight viruses with isometric particles. Its present cryptogram is R/1: 2-2/41 +2-1/39:S/S:S/*.     

Purification, properties and transmission of parsnip yellow fleck, a semi-persistent, aphid-borne virus

Parsnip yellow fleck virus (PYFV) is the commonest cause of virus-like symptoms in parsnip plants in Britain: it is sap-transmissible but systemically infects few species outside the Umbelliferae. It has isometric particles 29–31 mμ in diameter, a sedimentation coefficient of 167s, and loses infectivity in sap after dilution to 10^-3-10^-4, heating for 10 min at 57·5–65°C, or storage at room temperature for 4–7 days. Two isolates, from parsnip and Anthriscus sylvestris respectively, are only distantly serologically related. The aphid Cavariella aegopodii transmits PYFV in a semi-persistent manner from A. sylvestris but not from parsnip. Transmission by aphids apparently depends on the presence in A. sylvestris or other source plants of a second virus, anthriscus yellows (AYV), which is persistent in the vector and not manually transmissible. PYFV was therefore not transmitted by aphids from manually inoculated plants or from parsnip or other plants immune to AYV. In controlled experiments, C. aegopodii transmitted PYFV (both A. sylvestris and parsnip isolates) from chervil plants inoculated separately with PYFV and AYV, but not from plants inoculated only with PYFV.     

Some hosts, properties and possible affinities of a labile virus from Hypochoeris radicata (Compositae)

Hypochoeris mosaic virus (HMV) is common in Hypochoeris radicata (‘cat's ear’) in western Canada. It infected 10 of 53 mechanically inoculated species in five of twelve families, but was not transmitted by aphids or through seed or soil. Sap from infected Nicotiana clevelandii was sometimes infective after dilution to 10^-1 and occasionally 10², after 10 min at 45 but not 50°C, and after 1 but not 2 days at 20°C. Infectivity of crude nucleic acid extracts from infected leaves was rapidly abolished by RNase but not by DNase. Host sap contained very few rod-shaped particles or particle fragments mostly 21.0–22.5 nm in diameter, and up to 420 nm long but with predominant lengths of 120–140 and 240–260 nm. Many rods in purified virus preparations were less than 240 nm long, and the majority were c. 140 nm or shorter. The particles had a helical substructure with a pitch of 2.58 nm and contained a single type of protein of estimated mol. wt 24.5 × 10³. HMV showed no serological relationship to eight morphologically similar viruses (beet necrotic yellow vein, broad bean necrosis, barley stripe mosaic, peanut clump, potato mop-top, Nicotiana velutina mosaic, wheat soil-borne mosaic and defective strains of tobacco mosaic). It is probably a hitherto undescribed tobamovirus.     

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.     

Serological detection of ryegrass mosaic virus and ryegrass seed-borne virus

Ryegrass mosaic virus (RMV) was reliably detected in both perennial (S24) and Italian (S22) ryegrass, by enzyme-linked immunosorbent assay (ELISA) when plants had been infected for 8 wk. ELISA detected more infections in field-grown perennial ryegrass cv. Premo than either visual assessment or electron microscopy. However, with plants of Italian ryegrass cultivars only recently infected with RMV, positive reactions were more difficult to separate from the reactions of RMV-free plants, which varied considerably with cultivar, some giving high absorbance values. Immunosorbent electron microscopy showed that the RMV antiserum also contained antibodies to ryegrass seed-borne virus (RGSV), suggesting that these high values were caused by RGSV infection in the material tested.     

Purification and properties of elderberry latent virus, one of four sap-transmissible viruses obtained from American elder (Sambucus canadensis L.)

Cherry leaf roll, tomato black ring and two previously unrecorded viruses were transmitted to Chenopodium quinoa from Sambucus canadensis plants imported from the U.S.A. Of the two newly recognized viruses one, code-named elderberry virus A, has filamentous particles about 650 times 15 nm; the other, named elderberry latent virus (ELV), was transmitted to several herbaceous species but remained symptomless in elder and most other hosts. In C. quinoa sap ELV lost infectivity after dilution to 10^--⁵ to 10^-- ⁶ , 10 min at 85–90°C, and 7 days at 18°C. Infectivity of nucleic acid extracts was abolished by ribonuclease in 0.2 m sodium chloride. ELV was purified from C. quinoa leaf extracts that were clarified with chloroform, by precipitation at pH 5 and differential centrifugation. Purified preparations contained numerous isometric particles c. 30 nm in diameter and a few particles c. 17 nm in diameter. In 0.06 M phosphate buffer ELV sedimented as a major 112 S (calculated for infinite dilution) component and a 48 S minor component. ELV showed no serological relationship to twenty-seven other isometric plant viruses. Its present cryptogram is R/I: *I*:S/S:S/*.     

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.