A New Overlay for Plaquing Animal Viruses

Mirchamsy, H.; Rapp, Fred

doi:10.3181/00379727-129-33237

Cited by 36 publications

(17 citation statements)

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“…Type A12 strain 119 FMDV was grown in rolling-bottle cultures of a baby hamster kidney (BHK) cell line passage 21, clone 13 in a Tris-buffered modified Eagle medium containing 0.5% lactalbumin hydrolysate, 10% tryptose phosphate, and no serum (9). Virus was concentrated 100-fold by two cycles of precipitation with polyethylene glycol (13), and infectivity was determined by plaque assay in BK cell cultures by using a 0.6% gum tragacanth overlay and crystal violet staining at 48 h (6 (ii) Low-multiplicity infection. Cells were pretreated with agent and drained as in (i).…”

Section: Methodsmentioning

confidence: 99%

Effect of Zinc and Other Chemical Agents on Foot-and-Mouth Disease Virus Replication

Polatnick

Bachrach

1978

Antimicrob Agents Chemother

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Chemical agents reported to inhibit the growth of various ribonucleic acid and deoxyribonucleic acid viruses were tested against foot-and-mouth disease virus in cell culture. These included Zn2+, aurintricarboxylic acid, polyribocytidylic acid, polyriboinosinic acid, phosphonoacetic acid, and the viral contact inactivator Nmethyl isatin,-thiosemicarbazone alone and with CUSO4. The most effective agent, Zn2+, inhibited foot-and-mouth disease virus production in primary calf kidney cells by 1 log unit at 0.05 mM Zn2+ and completely at 0.50 mM. Zinc was inhibitory even when added late in infection and was nontoxic to uninfected cells as measured by protein and nucleic acid syntheses. Polyacrylamide gel patterns of [IS]methionine-labeled, virus-specific proteins showed increasing amounts of higher-molecular-weight material, in accord with reports that Zn2+ inhibits posttranslational cleavages of other picornavirus precursor polypeptides.Certain antiviral agents (6) have been shown to inhibit the production of foot-and-mouth disease virus (FMDV) in cell cultures (1,7,8). These agents are primarily amino acid, purine, and pyrimidine analogs, as well as substituted thiosemicarbazones. The present report examines the effectiveness of Zn2+, aurintricarboxylic acid (ATA), polyribocytidylic acid, polyriboinosinic acid, phosphonoacetic acid, and N-methyl isatin,8-thiosemicarbazone (methisazone) alone and in conjunction with Cu2+. The most inhibitory of these, Zn2+, is shown to interfere with the cleavage of high-molecular-weight precursors of FMDV-specific proteins, as has been reported for post-translational cleavages of other picornavirus precursor polypeptides (2). MATERIALS AND METHODSCells and media. Primary bovine kidney (BK) monolayer cultures were grown in 4-ounce (120-ml) prescription bottles containing Hanks balanced salts solution (HS), 0.5% lactalbuini hydrolysate, and 6% bovine serum. The chemical agents, unless otherwise noted, were dissolved in HS in 1.0% tris(hydroxymethyl)aminomethane (Tris) buffer (pH 7.5) containing 0.2% glucose, 4 mM glutamine (HS-TB), and basal Eagle amino acids (HS-TB-AA).Viru8. Type A12 strain 119 FMDV was grown in rolling-bottle cultures of a baby hamster kidney (BHK) cell line passage 21, clone 13 in a Tris-buffered modified Eagle medium containing 0.5% lactalbumin hydrolysate, 10% tryptose phosphate, and no serum (9). Virus was concentrated 100-fold by two cycles of precipitation with polyethylene glycol (13), and infectivity was determined by plaque assay in BK cell cultures by using a 0.6% gum tragacanth overlay and crystal violet staining at 48 h (6).Chemicals. Copper sulfate, zinc acetate, and zinc chloride were from standard commercial sources. ATA was from the Aldrich Chemical Co., Milwaukee, Wis., methisazone from Nutritional Biochemicals Corp., Cleveland, Ohio, and polyribocytidylic and polyriboinosinic acids were purchased from Miles Laboratories, Inc., Elkhart, Ind. Phosphonoacetic acid was a gift of Abbot Laboratories, North Chicago, Ill.Inhibition test. The action of th...

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Section: Methodsmentioning

confidence: 99%

Effect of Zinc and Other Chemical Agents on Foot-and-Mouth Disease Virus Replication

Polatnick

Bachrach

1978

Antimicrob Agents Chemother

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“…Tenfold dilutions of virus were made, and each of two monolayers was inoculated with 0.1 ml of inoculum. After a 2 h adsorption period at 37 °C, monolayers were overlaid with modified MEM (Schloer & Hanson, 1968) containing 5% bovine serum and 0.8% gum tragacanth (Mirchamsy & Rapp, 1968). Monolayers were stained at 6 days post-inoculation with 0-2 % crystal violet containing 2 % formaldehyde.…”

Section: Methodsmentioning

confidence: 99%

Purification of African Malignant Catarrhal Fever Virus using a Two-phase Aqueous Polymer System

Schloer

Breese

1982

Journal of General Virology

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SUMMARYThe WCll isolate of malignant catarrhal fever virus (MCFV) was purified by a method employing phase separation of the virus in an aqueous polymer system. Virus was grown in primary bovine thyroid cells. The virus released into the extracellular medium was purified. Initial concentration and partial purification of MCFV occurred after separation of the virus into the dextran phase following the addition of 10 % (w/v) polyethylene glycol and 8 % (w/v) dextran T10 to the extracellular virus. Further purification was achieved by centrifugation into a 20 % (w/v) Ficoll cushion followed by centrifugation to equilibrium by flotation in a 15 to 36 % (w/w) CsC1 or 30 to 60 % (w/w) sucrose gradient. Virus recovery was monitored using a plaque assay on bovine thyroid cells and ranged from 3 to 23 % of the input extracellular virus.

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“…4-oz bottles were seeded and after 2-3 days, when plaques were macroscopically observable, the bottles were decanted and the monolayers were stained with crystal violet (0.2% in ethanol). The syncytial plaques also appeared under fluid tragacanth [18] which was added 1 day after the start of cocultivation. For staining, the overlay was removed on the 3rd day and plaques were stained as mentioned above.…”

Section: Methodsmentioning

confidence: 99%

Isolation and Characterization of a Defective Measles Virus from Brain Biopsies of Three Patients in Iran with Subacute Sclerosing Panencephalitis

Mirchamsy¹,

Bahrami²,

Shafyi³

et al. 1978

Intervirology

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Three cytopathic strains of subacute sclerosing panencephalitis (SSPE) virus were isolated from brain biopsies of three patients. These strains were isolated and maintained by cocultivation of infected brain cells with fresh Vero cells. The biological characteristics of two strains were studied. It was found that these strains remain cell-associated after repeated cocultivations with Vero cells and produce plaques under fluid medium or traga-canth overlay. The correlation with measles virus was demonstrated by the plaque reduction test as well as by the immunofluorescence test. Large numbers of nucleocapsids were observed in the cytoplasm of infected cells but none in nuclei. Intracerebral inoculation of monkeys, adult guinea pigs, newborn and adult hamsters or mice was followed by acute encephalitis and death.

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A New Overlay for Plaquing Animal Viruses

Cited by 36 publications

References 1 publication

Effect of Zinc and Other Chemical Agents on Foot-and-Mouth Disease Virus Replication

Effect of Zinc and Other Chemical Agents on Foot-and-Mouth Disease Virus Replication

Purification of African Malignant Catarrhal Fever Virus using a Two-phase Aqueous Polymer System

Isolation and Characterization of a Defective Measles Virus from Brain Biopsies of Three Patients in Iran with Subacute Sclerosing Panencephalitis

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