Purification and Partial Characterization of Isometric Virus-like Particles in Kalanchoe Species

Isometric virus-like particles (IVLP) were detected in crude sap from Kalanchoe pinnata, K. daigremontiana and K. tubiflora plants showing a mild mosaic on the leaves. These particles of 35 nm in diameter were transmitted mechanically to several test plants but not to healthy Kalanchoe. Air temperatures above 30 °C hindered the infection process. The IVLP were purified from systematically infected Nicotiana benthamiana using Triton X-100 as clarifying agent followed byP, EG precipitation. IVLP were degraded by organic solvents and formed aggregates in the presence of 2 mmol/1 CaCl₂. The particles occurred in relatively low concentration in plant sap and lost infectivity in leaves frozen at -70 °C for one week and in purified preparations kept at 4 °C. In buffer crude sap of N. benthamiana IVLP had a thermal inactivation point between 45 and 50 °C on a longevity in vitro of 20 h at 25 °C. Particles contained one nucleoprotein component witha molecular weight of 46,000 daltons and a ssRNA species which, when denatured, had a molecular weight of 1.2 × 10⁶. IVLP purified preparations exhibited a typical nucleoprotein absorption spectrum with a maximum at 254–260 nm and a minimum at 240,–243 nm and a A 260/280 ratio of 1.56. The buoyant density of the IVLP was 1.32 g/ml calculated by isopycnic centrifugation on CsCl. Ultrastructural studies in infected leaves of K. pinnata indicated that IVLP caused an increase in chloroplast volume, distortion of the grana and reduced the number of thylakiods per grana. IVLP infection also impared the diurnal pattern of synthesis and hydrolysis of starch, characteristic of CAM plants. The non-serological reaction of the IVLP with antisera specific to members of 7 different groups of spherical viruses as well as the combination of physicochemical properties and host range, exhibited by these particles impeded their taxonomic location. In nature, young Kalanchoe plantlets acquire the IVLP through their physical connections with the infected mature leaves.     

Assessment of yield losses as a result of co-infection by maize streak virus and maize stripe virus in Mauritius

The effect of co-infection by maize streak virus (MSV) and maize stripe virus (MStV) on plant growth and grain yield was investigated in a susceptible variety of maize (Zea mays), ZS 5206, in Mauritius. Under natural conditions MSV, transmitted by the leafhopper Cicadulina mbila, was normally established before MStV, which is vectored by the planthopper Peregrinus maidis; as a result, MStV symptoms were often partially or completely masked by those of MSV, making MStV detection by symptomatology very unreliable. MSV and MStV were diagnosed by ELISA and MStV by a novel method of detecting the MStV-coded non-capsid protein. The maize hybrid ZS 5206 was inoculated with either MSV, MStV or both, at two stages in the growth cycle (3–5 or 7–10 leaf stage). A greater reduction in plant growth was observed in plants inoculated singly with MStV (80% and 29% for first and second stage, respectively) than with MSV (50% and 23%, respectively). No cobs were produced by plants singly infected with MStV at the first stage, or co-infected with MSV and MStV at both stages; however, marginal grain production was recorded in plants singly infected with MSV at the first stage (91% reduction), or infected either with MSV or MStV, at the second stage (65% and 80% reduction, respectively). In maize hybrid ZS 5206, MStV is more virulent than MSV; co-infection by both viruses causes greater reductions in plant growth and grain yield than single infection by either virus at a given stage of plant development. In the event of co-infection by MSV and MStV, yield losses can be erroneously attributed to MSV only if the symptoms of MStV are masked by those of the former and if adequate methods for MStV detection are not used.     

Biological activity of invitro synthesised protein: Binding of Semliki Forest virus capsid protein to the large ribosomal subunit

Cell-free translation of the Semliki Forest virus-specific 26S RNA yielded primarily capsid protein. After treatment of the protein synthesising reaction with 25 mM EDTA, the capsid protein cosedimented with the large ribosomal subunit in sucrose gradients, and banded with the subunit at a density of 1.54 gm/cm³ in CsCl. Exposure to 0.5 M KCl released the protein from the subunit. Similar binding of the virus capsid protein to the large ribosomal subunit has been observed in infected HeLa cells, although its function is not clear. The nonstructural proteins, which are the major products translated from the virion 42S RNA, did not associate with sedimenting structures.     

Virus-like particles in the take-all fungus, Gaeumannomyces graminis

Isometric virus-like particles (VLP) measuring 35 nm and 27 nm occurred in cultured mycelium of Gaeumannomyces graminis var. tritici and G. graminis var. avenae. These VLP had, respectively, sedimentation coefficients (s°_{20, W}) 148S and 110S and ultraviolet absorption (maximum 260 nm, minimum 240 nm) typical of nucleoprotein (A260:280 = 1.6, A260:240 = 1.2). Preparations of the 35 nm particles had two major and one minor component in caesium chloride, and 27 nm particles had two components (buoyant densities 1.37, 1.36, 1.30, 1.35, and 1.29 g/cm³ respectively). Preparations of the 35 nm particles or 35 nm plus 27 nm particles had one major protein species with estimated molecular weight 70000 daltons. The 35 nm VLP were absent from 11 isolates of G. graminis var. tritici from first cereal crops after fallow or non-susceptible break crops; two of these contained the 27 nm particles. More than half of 145 isolates, from cereals after 2–12 consecutive susceptible crops, contained either 35 nm or 27 nm VLP. VLP were not confined to G. graminis isolates from soils exhibiting ‘take-all decline’ nor consistently associated with weak pathogenicity or with isolates of unusual growth, morphology, pigmentation, lysis or readiness to form perithecia. Isolates with one kind of particle were mostly more pathogenic and those with both kinds less pathogenic than isolates without VLP. The proportion of isolates with 27 nm and 35 nm particles increased progressively in samples from different consecutive crops during the first 9 years of cropping, then decreased. Isolates did not gain or lose VLP during infection and re-isolation from wheat seedlings grown in sand. Four ‘infected’ isolates were freed from VLP either by culturing ascospores or by growing hyphal tips excised from colonies kept near their thermal death point. Both VLP appeared in cultures which had undergone anastomosis with infected isolates.     

Semliki Forest virus capsid protein associates with the 60S ribosomal subunit in infected cells.

Semlike forest virus capsid protein cosedimented with the large ribosomal subunit at 60S in sucrose gradients after treatment of cytoplasm from infected cells with Triton X-100 and EDTA. In CsCl gradients the capsid protein banded with the subunit at a density of 1.56 to 1.57 g/cm3. Most of the capsid protein could be detached from the 60S structure by treatment with 0.8 M KCl. The ribonucleoprotein of the 26S RNA had a sedimentation value of 53S and a density of 1.50 g/cm3 and could thus be separated from the 60S structure. The data suggest that the capsid protein binds to the large ribosomal subunit, but not to the viral 26S RNA.     

Purification and Properties of the Geminivirus Euphorbia Mosaic Virus

Euphorbia mosaic virus was purified from infected plants of Nicotiana benthamiana. Highest concentrations of virus particles were found in infected plant tissue between 10–12 days after inoculation. The enzyme driselase assisted in purification of the virus particles from the infected tissue yielding about 600 μg/kg of plant material. Purified preparations showed a maximum absorption at 260–263 nm and the ratio of absorption at 260 and 280 nm was 1.4. The viral nucleic acid was digestedby DNase I and S₁ Nuclease but not RNase A. A single coat protein with a MW of 32,000 d and two DNA bands with a MW 0.96 × 10⁶ d (2870 nucleotides) and 0.90 × 10⁶ d (2700 nucleotides) were associated with the purified virus particles. Virus specific DNA was isolated from infected tissue between 7 and 15 days after inoculations.     

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.     

Characterization of Maize Iranian Mosaic Virus and Comparison with Hawaiian and Other Isolates of Maize Mosaic Virus (Rhabdoviridae)

Maize Iranian mosaic virus (MIMV) was characterized and compared with isolates of Maize mosaic virus (MMV, genus Nucleorhabdovirus, family Rhabdoviridae) in insect transmission, cytopathology and ultrastructure of infected maize cells, virion proteins and serologically. MIMV is naturally transmitted by Ribautodelphax notabilis, a delphacid planthopper, in Iran. In this study, another planthopper, Peregrinus maidis, vector of MMV, transmitted MIMV with an estimated efficiency of 0.4–1.6% following feeding on MIMV‐infected maize plants and 64% following injection of MIMV into the hemolymph, suggesting that P. maidis gut tissues largely blocked MIMV transmission. MIMV and MMV‐HI (Hawaii) induced similar cytopathologies in cells of infected maize leaves, with virions budding through inner nuclear and endoplasmic reticulum membranes. In thin sections, virions of MIMV were significantly shorter than those of MMV‐HI. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis of virions of MIMV, MMV‐HI, MMV‐CR (Costa Rica) and MMV‐FL (Florida) yielded six proteins of which four were identified as the putative G, N, P and M proteins of plant rhabdoviruses. The N, P and M proteins of MIMV migrated faster in gels than those of the MMV isolates indicating a lower molecular weight, whereas the bands corresponding to the G proteins migrated similarly for both viruses. Polyclonal antibodies to MMV‐HI failed to react with virions of MIMV in enzyme‐linked immunosorbent assay (ELISA) and with MIMV proteins in Western blots. In contrast, these antibodies reacted strongly with MMV‐HI and MMV‐FL virions in ELISA and with MMV‐HI, MMV‐CR and MMV‐FL proteins in Western blots. Further, in ELISA, polyclonal antibodies to MMV‐MR (Mauritius) reacted weakly with MIMV virions but strongly with MMV‐HI and MMV‐FL virions. Thus, it is concluded that MIMV is a new virus of the Nucleorhabdovirus genus that may be distantly related to MMV.     

Staphylococcus,paramyxovirus-like,rickettsia-like and other structures in Peregrinus maidis (Homoptera,delphacidae)

In an ultrastructural study of the delphacid planthopper, Peregrinus maidis, vector of maize mosaic virus (MMV) and maize stripe virus (MStpV), the following structures were found in several organs of MMV-inoculative, MStpV-inoculative, and noninoculative insects: (a) paramyxovirus-like particles (PLP), (b) filamentous rhabdo-like structures (FRS), (c) spiked double-membrane structures (SDS) that were always associated, and sometimes contiguous, with FRS, and (d) rickettsia-like structures (RLS). In salivary gland acini, the PLP, FRS, and SDS were usually found in extracellular spaces between basal infoldings of plasma membranes, whereas the RLS were intracytoplasmic or intranuclear. The PLP and FRS appeared to bud through plasma membranes. The colony of P. maidis in which these structures were found suffered from retarded development and premature death. Limited trials to isolate a paramyxovirus from this colony were unsuccessful. However, further trials led to the isolation of Staphylococcus sciuri, which proved pathogenic when injected into P. maidis and two cicadellid leafhoppers; Dalbulus maidis and Graminella nigrifrons.     

Electrical Penetration Graphs from Peregrinus maidis on a susceptible maize hybrid

The feeding behavior of Peregrinus maidis (Ashmead) (Homoptera: Delphacidae), vector of maize mosaic virus (MMV) and maize stripe virus (MStpV) in maize (Zea mays L.), has been studied by Electrical Penetration Graph (EPG). The different recordings collected have allowed the temporal distinction of three EPG signal classes. These class 1, class 2 and class 3 signals are correlated through histological sections to the feeding activities of probing, xylem ingestion and phloem ingestion, respectively. Although these signals are described by various statistical parameters, only the median allows significant differentiation between class 2 and class 3 signals, the others varying from one insect to the next. On the other hand, spectral analysis is used to describe the signal classes by associating a characteristic frequency spectrum to each. This study treats the importance of such analysis in characterizing and comparing the signals of various piercing and sucking insects.     

Multiple molecular forms of murine thymocyte-stimulating factor

Murine thymocyte stimulating factor (TSF) was found to sediment in sucrose density gradients on a broad band with peaks at about 2.60 S and 2.0 S. Two main peaks of TSF activity (with buoyant densities of 1.34 and 1.28 g/ml) were found in CsCl density gradients. Gel chromatography on Sephadex G-100 columns of the material sedimented in sucrose or CsCl density gradients originated multiple peaks of TSF activity with various molecular weights. Heterogeneity of molecular forms of TSF was also found upon dilution of the factor. The lowest molecular weights found were 4000 and 4700 daltons. When Sephadex fractions containing the low molecular weight material were pooled and rerun on Sephadex columns, molecular species with a wide range of molecular weights were found. Temperature also affects the appearance of the low molecular weight forms of TSF. Most of the experiments presented in this work were carried out with Sephadex-purified TSF. Multiple molecular forms, however, and, in particular, the forms with molecular weights of 4000 and 4700 daltons were found also with TSF-Fraction IIIa, a highly purified preparation of this factor.     

Salt-stable association of simian virus 40 capsid with simian virus 40 DNA

In 8 M CsCl, a fraction of the wild-type previrions and tsB228 nucleoprotein complexes lose their core histones but retain their capsid. These histone-depleted complexes appear in the electron microscope as a protein shell attached to supercoiled DNA. Consistent with this result, we find that in 1 M NaCl, the wild-type previrions dissociate into two populations of nucleoprotein complexes. One population sediments between 50 and 140 S and morphologically resembles the shell-DNA complexes isolated in CsCl gradients. The other population is comprised primarily of nucleoproteins which sediment at 40 S.     

Arracacha virus A, a newly recognised virus infecting arracacha (Arracacia xanthorrhiza; Umbelliferae) in the Peruvian Andes

Arracacha virus A (AVA), a previously undescribed virus, is common in arracacha (Arracacia xanthorrhiza; Umbelliferae) in the Huanuco region of the Peruvian Andes. AVA was not transmitted by Myzus persicae, but was transmitted by inoculation of sap to 38 species from 10 families out of 63 species from 12 families tested. AVA was best propagated and assayed in Chenopodium quinoa and Nicotiana clevelandii in which it caused severe diseases. Sap from infected C. quinoa was occasionally infective after dilution to 10^-4 but not 10^-5, after 10 min at 65 °C but not 70 °C, and after 15 days at 20 °C. In neutral phosphotungstate, AVA has isometric particles c. 26 nm in diameter with a hexagonal profile, some of which were either fully or partially penetrated by the negative stain. Up to 50–200 E₂₆₀^1cm units of purified virus was obtained from 1 kg of infected N. clevelandii leaf by extraction in 0.05 M phosphate buffer at pH 7.5 containing 0.05 M ethylene diaminetetra-acetate, and clarification with chloroform, followed by differential precipitation with ammonium sulphate and three cycles of differential centrifugation. Purified virus sedimented as three components with sedimentation coefficients (S_20w^°) of 50 S, 92 S and 125 S and E₂₆₀/E₂₈₀ ratios of 0.65, 1.50 and 1.85 respectively. At equilibrium in CsCl gradients, buoyant densities of the 50, 92 and 125 S components were 1.32, 1.45 and 1.52 g/cm³ respectively. From the sedimentation coefficients and buoyant densities, the nucleic acid contents of the 92 S and 125 S components were estimated at 30–35% and 43–44% respectively. Only the 125 S component seemed to be infective but its infectivity was greater when mixed with the 92 S component. All three components contained a single protein with a molecular weight of 53 000. AVA was not serologically related to any of 33 other morphologically similar viruses. Although the vector is unknown, its properties suggest that it is a member of the nepovirus group. The cryptogram of AVA is */*: */43–44 +*/30–35: S/S:S/*.     

Some Properties of Cucumber Fruit Streak Virus

An isometric virus c. 30 nm in diameter with a single RNA species (mol.wt 1.45 × 10⁶) isolated from cucumber plants from the island of Crete (Greece) is described under the name of cucumber fruit streak virus (CFSV). The most evident symptom on naturally infected plants consisted of longitudinal chlorotic streak of the fruits. In glasshouse, the virus was soil-transmitted to C. sativus, and, mechanically, to a wide range of herbaceous hosts, most of which were infected only locally. Purified virus preparations sedimented as a single component with sedimentation coefficient of 132S. At equilibrium these preparations were homogeneous in CsCl gradients but formed two bands in Cs₂SO₄ gradients. Virus particles were stabilized by forces involving divalent cations, pH-dependent bonds and salt links between protein and RNA. Although some of the properties of CFSV are similar to those of other small spherical viruses with single RNA species there are differences which do not allow for the assignment of the virus to any of established taxonomic group of plant viruses.     

Intermediate in adenovirus type 2 replication.

Replicating chromosomes, called intermediate DNA, have been extracted from the adenovirus replication complex. Compared to mature molecules, intermediate DNA had a greater buoyant density in CsCl gradients and ethidium bromide-cesium chloride gradients. Digestion of intermediate DNA with S1 endonuclease, but not with RNase, abolished the difference in densities. These properties suggest that replicating molecules contain extensive regions of parental single strands. Although intermediate DNA sedimented faster than marker viral DNA in neutral sucrose gradients, single strands longer than unit length could not be detected after alkaline denaturation. Integral size classes of nascent chains in intermediate DNA suggest a relationship between units of replication and the nucleoprotein structure of the virus chromosome. Adenovirus DNA was replicated at a rate of 0.7 x 10-6 daltons/min. Although newly synthesized molecules had the same sedimentation coefficient and buoyant density as mature chromosomes, they still contained single-strand interruptions. Complete joining of daughter strands required an additional 15 to 20 min.     

Preliminary characterization of a reovirus isolated from golden ide Leuciscus idus melanotus

Some characteristics of a reovirus recently isolated from golden ide Leuciscus idus melanotus and tentatively designated as golden ide reovirus (GIRV) were determined. Spherical non-enveloped particles with an outer capsid of about 70 nm and an inner capsid of about 50 nm were observed by electron microscopy. The density of the virus determined in CsCl gradients was 1.36 g ml-1. The genome contained 11 segments of dsRNA. GIRV differed from other aquareoviruses by a slight reduction of infectivity after treatment with chloroform and by the absence of forming syncytia in cell monolayers.     

Intracytoplasmic A Particles: Mouse Mammary Tumor Virus Nucleoprotein Cores?

A rapid method for the isolation of intracytoplasmic A particles, the putative intracellular nucleoprotein cores of mouse mammary tumor virus (MTV), is presented. Spontaneous C3H/He mouse mammary tumors and transplantable mouse Leydig cell tumor were used as source material. Large aggregations of intracytoplasmic A particles were separated from cellular contaminants on discontinuous sucrose gradients and subsequently further purified by isopycnic banding in linear sucrose gradients. The purified particles were solubilized in sodium dodecyl sulfate, and the structural proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified mouse mammary tumor virions were solubilized, and their proteins were analyzed in the same way. Comparison of co-electrophoretic gels indicated a lack of correlation in the molecular size of the major structural proteins in intracytoplasmic A particles and MTV. The three major proteins of the A particles were calculated to be 80,000; 35,000; and 20,000 daltons. Five major polyacrylamide gel electrophoresis bands were obtained with purified MTV; these were 90,000; 69,000; 55,000; 37,000; and 27,500 daltons. These figures showed good correlation with those published for MTV by Nowinski et al. These results suggest the need for the reexamination of the current tenet that intracytoplasmic particles represent intracellular MTV nucleoprotein cores.     

Physical properties of phage 299

Summary Phage 299, on equilibrium sedimentation in CsCl, gives 3 major bands whose relative proportions depend on growth conditions. One band is whole heads without tails, the other two are infectious phage of differing degrees of disarray and stability. Electron micrographs show that the infectious phage has a head about 60 nm across, probably icosahedral in shape, and a straight tail of approximately 140 nm in length. The tail assemblies appear defective and incomplete. Sedimentation in a sucrose gradient of the DNA extracted from phage 299 is monodisperse with a molecular weight of 20.6±1.5×10⁶ daltons based on comparison with λ and 186 phage DNA’s. The DNA has a base composition of 51.7% guanine and cytosine as determined by bouyant density in CsCl. A comparison of its denaturation behavior by analysis of the hyperchromic shift at 260 nm with that of phage P2 suggests a considerable number of common characteristics, and an absence of a low guanine and cytosine portion on the part of 299 which amounts to approximately 10% of the total DNA.     

Characterisation of watermelon curly mottle virus, a geminivirus distinct from squash leaf curl virus

Purified preparations of watermelon curly mottle virus (WCMoV), a whitefly-transmitted geminivirus, contained dimeric or geminate particles of 20 times 30 nm and the virus was transmissible by mechanical means. Virus yields ranged from 100–150 μg/100 g leaf tissue. Purified preparations exhibited a typical nucleoprotein absorbance profile with a maximum absorbance at 258 nm, and A280 / A260 ratio of 0.61–0.64. Infectivity was associated with two light-scattering, virus-containing bands following sucrose density gradient centrifugation. The viral capsid protein was resolved as a doublet by SDS-PAGE. The estimated mol. wts of the two bands within the doublet were 29 100 (±1550) and 27 733 (±1550), respectively. DNA isolated from virus particles was resolved by gel electrophoresis into two circular single-stranded DNA bands of approximately 2.6 to 2.7 kb. The two bands are believed to represent the individual components of a bipartite genome, characteristic of previously described whitefly-transmitted geminiviruses.