An improved procedure for the purification of apple chlorotic leaf spot virus

1992 ◽  
Vol 72 (3) ◽  
pp. 937-941
Author(s):  
Delano James ◽  
Paul L. Monette
Keyword(s):  
Density Gradient ◽  
Leaf Spot ◽  
Gradient Centrifugation ◽  
Chenopodium Quinoa ◽  
Sucrose Density Gradient ◽  
High Yield ◽  
Spot Virus ◽  
Virus Particles ◽  
Intact Virus ◽  
Chlorotic Leaf

A reproducible procedure is described for the purification of apple chlorotic leaf spot virus (CLSV) from Chenopodium quinoa. Two cycles of sucrose density-gradient centrifugation in a magnesium-containing buffer were used. A relatively high yield of virus particles was obtained with little degradation observed. The average yield was 2.3 mg 100 g−1 fresh leaf tissue. The modal lengths of purified virus particles and particles from leaf sap were 692 nm and 704 nm, respectively.Key words: Chenopodium quinoa, sucrose density gradient, magnesium-containing buffer, intact virus

Two-step centrifugation method. A simplification of the density-gradient procedure for the purification of influenza virus

1973 ◽  
Vol 19 (5) ◽  
pp. 633-638 ◽  
Author(s):  
D. J. S. Arora ◽  
V. Pavilanis ◽  
P. Robert
Keyword(s):  
Influenza Virus ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Sucrose Concentration ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Virus Preparation ◽  
Virus Particles ◽  
Centrifugation Method ◽  
Degree Of Purification

In the present investigation, centrifugation of an influenza virus preparation by the sucrose density-gradient method was carried out to determine the "upper" and "lower" concentrations of sucrose at which viral activity resided. Virus was suspended in an "upper" concentration of sucrose and was centrifuged, thus resulting in a suspension containing virus free from impurities. The sucrose concentration in the suspension containing virus was then reduced to the "lower" concentration of sucrose and the suspension was recentrifuged. Virus particles sedimented, leaving impurities in the suspension. As well as achieving the same degree of purification of the virus sample as obtained by density-gradient centrifugation, this method also succeeded in concentrating influenza virus.

scholarly journals Characterization of Apricot pseudo-chlorotic leaf spot virus, A Novel Trichovirus Isolated from Stone Fruit Trees

2005 ◽  
Vol 95 (4) ◽  
pp. 420-426 ◽  
Author(s):  
D. Liberti ◽  
A. Marais ◽  
L. Svanella-Dumas ◽  
M. J. Dulucq ◽  
D. Alioto ◽  
...  
Keyword(s):  
Leaf Spot ◽  
Chenopodium Quinoa ◽  
Fruit Trees ◽  
Open Reading Frames ◽  
Spot Virus ◽  
Phylogenetic Reconstructions ◽  
Wide Geographical Distribution ◽  
Chlorotic Leaf ◽  
Reading Frames

A trichovirus closely related to Apple chlorotic leaf spot virus (ACLSV) was detected in symptomatic apricot and Japanese plum from Italy. The Sus2 isolate of this agent cross-reacted with anti-ACLSV polyclonal reagents but was not detected by broad-specificity anti- ACLSV monoclonal antibodies. It had particles with typical trichovirus morphology but, contrary to ACLSV, was unable to infect Chenopodium quinoa and C. amaranticolor. The sequence of its genome (7,494 nucleotides [nt], missing only ≈30 to 40 nt of the 5′ terminal sequence) and the partial sequence of another isolate were determined. The new virus has a genomic organization similar to that of ACLSV, with three open reading frames coding for a replication-associated protein (RNA-dependent RNA polymerase), a movement protein, and a capsid protein, respectively. However, it had only ≈65 to 67% nucleotide identity with sequenced isolates of ACLSV. The differences in serology, host range, genome sequence, and phylogenetic reconstructions for all viral proteins support the idea that this agent should be considered a new virus, for which the name Apricot pseudo-chlorotic leaf spot virus (APCLSV) is proposed. APCLSV shows substantial sequence variability and has been recovered from various Prunus sources coming from seven countries, an indication that it is likely to have a wide geographical distribution.

Uranyl Acetate and Rotary Shadowing to Increase the Contrast of Small Aggregated Virus Particles

1969 ◽  
Vol 27 ◽  
pp. 424-425
Author(s):  
Lee F. Ellis ◽  
Richard M. Van Frank ◽  
Walter J. Kleinschmidt
Keyword(s):  
Electron Microscopy ◽  
Tissue Culture ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Uranyl Acetate ◽  
Interferon Inducer ◽  
Virus Particles ◽  
Staining Methods ◽  
Rotary Shadowing

The extract from Penicillum stoliniferum, known as statolon, has been purified by density gradient centrifugation. These centrifuge fractions contained virus particles that are an interferon inducer in mice or in tissue culture. Highly purified preparations of these particles are difficult to enumerate by electron microscopy because of aggregation. Therefore a study of staining methods was undertaken.

The Combining Ability of Glycoproteins IIb, IIIa and Ca2+ in EDTA-Treated Nonaggregable Platelets

1983 ◽  
Vol 50 (04) ◽  
pp. 848-851 ◽  
Author(s):  
Marjorie B Zucker ◽  
David Varon ◽  
Nicholas C Masiello ◽  
Simon Karpatkin
Keyword(s):  
Density Gradient ◽  
Combining Ability ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Electrophoretic Pattern ◽  
Sucrose Density Gradient ◽  
Irreversible Loss ◽  
Sucrose Density Gradient Centrifugation ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

SummaryPlatelets deprived of calcium and incubated at 37° C for 10 min lose their ability to bind fibrinogen or aggregate with ADP when adequate concentrations of calcium are restored. Since the calcium complex of glycoproteins (GP) IIb and IIIa is the presumed receptor for fibrinogen, it seemed appropriate to examine the behavior of these glycoproteins in incubated non-aggregable platelets. No differences were noted in the electrophoretic pattern of nonaggregable EDTA-treated and aggregable control CaEDTA-treated platelets when SDS gels of Triton X- 114 fractions were stained with silver. GP IIb and IIIa were extracted from either nonaggregable EDTA-treated platelets or aggregable control platelets with calcium-Tris-Triton buffer and subjected to sucrose density gradient centrifugation or crossed immunoelectrophoresis. With both types of platelets, these glycoproteins formed a complex in the presence of calcium. If the glycoproteins were extracted with EDTA-Tris-Triton buffer, or if Triton-solubilized platelet membranes were incubated with EGTA at 37° C for 30 min, GP IIb and IIIa were unable to form a complex in the presence of calcium. We conclude that inability of extracted GP IIb and IIIa to combine in the presence of calcium is not responsible for the irreversible loss of aggregability that occurs when whole platelets are incubated with EDTA at 37° C.

scholarly journals Mapping the RNA-binding domain on the Apple chlorotic leaf spot virus movement protein

2005 ◽  
Vol 86 (1) ◽  
pp. 225-229 ◽  
Author(s):  
Masamichi Isogai ◽  
Nobuyuki Yoshikawa
Keyword(s):  
Leaf Spot ◽  
Rna Binding ◽  
Movement Protein ◽  
Binding Domain ◽  
Sequence Specificity ◽  
Nucleic Acid Binding ◽  
Spot Virus ◽  
Uv Crosslinking ◽  
Chlorotic Leaf ◽  
Rna Binding Domain

The RNA-binding properties of the cell-to-cell movement protein (MP) of Apple chlorotic leaf spot virus were analysed. MP was expressed in Escherichia coli and was used in UV-crosslinking analysis, using a digoxigenin–UTP-labelled RNA probe and gel-retardation analysis. The analyses demonstrated that MP bound cooperatively to single-stranded RNA (ssRNA). When analysed for NaCl dependence of the RNA-binding activity, the majority of the MP could bind ssRNA even in binding buffer with 1 M NaCl. Furthermore, competition binding experiments showed that the MP bound preferentially to ssRNA and single-stranded DNA without sequence specificity. MP deletion mutants were used to identify the RNA-binding domain by UV-crosslinking analysis. Amino acid residues 82–126 and 127–287 potentially contain two independently active, single-stranded nucleic acid-binding domains.

Molecular Characterization and Distribution of Apple Chlorotic Leaf Spot Virus on Apple in China

2013 ◽  
Vol 162 (5) ◽  
pp. 284-290
Author(s):  
Hao Duan ◽  
Zhirui Ji ◽  
Shutong Wang ◽  
Tongle Hu ◽  
Yanan Wang ◽  
...  
Keyword(s):  
Molecular Characterization ◽  
Leaf Spot ◽  
Spot Virus ◽  
Chlorotic Leaf
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