A modification of the polyethylene glycol technique for the purification of small quantities of tobacco mosaic virus.

An adaptation of the polyethylene glycol method for the purification of tobacco mosaic virus (sunnhemp strain) from small (2 g) samples of French bean lamina is described. The virus can be precipitated from 1-2 ml of clarified sap by the addition of an equal volume of 8% polyethylene glycol 6000 (w/v) (PEG), and pelleted by centrifugation for 10 min at 1,8000 x g. Details are given of tests which establish that the purity and quality of the virus obtained by the small-scale method are as good as those achieved by the established large-scale method. Even with very low virus titres, the small-scale method effectively precipitates total tobacco mosaic virus nucleoprotein without substantial loss of infectivity. The results are reproducible and in a relatively short time it is possible to purify the virus from large numbers of replicate samples.     

Lymphocytic Choriomeningitis virus. I. Concentration and purification of the infectious virus.

Two procedures for the purification of infectious lymphocytic choriomeningitis virus from cell culture fluid have been developed. If large quantities of very pure virus are to be prepared, infected L cells are maintained with a medium supplemented with calf serum, the proteins of which have been largely removed by pretreatment with polyethylene glycol. Two days after infection of the cultures, the media are collected and the virus is concentrated by treatment with polyethylene glycol 40,000. Purification with a 10,000-fold increase of specific infectivity is achieved with steric chromatography on controlled-pore glass beads with pore sizes of 42 to 44 nm and centrifugation in density gradients prepared with amido trizoate. An alternative method begins with precipitation of the virus from infected cell cuture medium with zinc acetate, followed by controlled-pore glass chromatography and density centrifugation in a discontinuous sucrose gradient. Purification thus obtained is 200-fold in terms of specific infectivity.     

Polyethylene glycol precipitation for recovery of pathogenic viruses, including hepatitis A virus and human rotavirus, from oyster, water, and sediment samples

Polyethylene glycol 6000 precipitation was found to be an effective concentration method that enhanced the chances for detecting human virus pathogens in environmental samples. Percent recoveries from eluates of fresh and estuarine waters with 8% polyethylene glycol 6000 averaged 86 for hepatitis A virus, 77 for human rotavirus Wa, 87 for simian rotavirus SA11, and 68 for poliovirus. Percent recoveries of 97, 40, 97 and 105, respectively, for the same viruses were obtained from oyster eluates by the same procedure. Percent recoveries of 97 for hepatitis A virus and 78 for human rotavirus Wa were obtained from sediment eluates containing 2 M NaNO3 with a final concentration of 15% polyethylene glycol 6000. The polyethylene glycol method was shown to be more effective than the organic flocculation method for recovery of hepatitis A virus and rotaviruses Wa and SA11, but not of poliovirus 1 in laboratory studies. In field trials, hepatitis A virus or rotavirus or both were recovered from 12 of 18 eluates by polyethylene glycol, compared with recovery from 9 of 18 eluates by organic flocculation from fresh and estuarine waters subject to pollution.     

Polyethylene glycol precipitation for recovery of pathogenic viruses, including hepatitis A virus and human rotavirus, from oyster, water, and sediment samples.

Polyethylene glycol 6000 precipitation was found to be an effective concentration method that enhanced the chances for detecting human virus pathogens in environmental samples. Percent recoveries from eluates of fresh and estuarine waters with 8% polyethylene glycol 6000 averaged 86 for hepatitis A virus, 77 for human rotavirus Wa, 87 for simian rotavirus SA11, and 68 for poliovirus. Percent recoveries of 97, 40, 97 and 105, respectively, for the same viruses were obtained from oyster eluates by the same procedure. Percent recoveries of 97 for hepatitis A virus and 78 for human rotavirus Wa were obtained from sediment eluates containing 2 M NaNO3 with a final concentration of 15% polyethylene glycol 6000. The polyethylene glycol method was shown to be more effective than the organic flocculation method for recovery of hepatitis A virus and rotaviruses Wa and SA11, but not of poliovirus 1 in laboratory studies. In field trials, hepatitis A virus or rotavirus or both were recovered from 12 of 18 eluates by polyethylene glycol, compared with recovery from 9 of 18 eluates by organic flocculation from fresh and estuarine waters subject to pollution.     

Concentration of Rous Sarcoma Virus from Tissue Culture Fluids with Polyethylene Glycol

Concentration of Rous sarcoma virus from tissue culture fluids with polyethylene glycol, with and without NaCl or dextran sulfate, resulted in significant and highly variable losses caused by entrapment of virus particles in proteinaceous debris. Treatment of concentrated preparations with Pronase greatly enhanced the recovery of virions. Maximum recovery of virus particles was obtained by the addition of 8% polyethylene glycol and 0.4 M NaCl to tissue culture fluids, followed by Pronase treatment of the concentrated virus preparations.     

Large-scale purification of Japanese encephalitis virus from infected mouse brain for preparation of vaccine.

Large volumes of Japanese encephalitis (JE) virus propagated in mouse brain can be easily purified by polyethylene glycol 6,000. By using the polyethylene glycol precipitation method, mouse hemoglobin was almost all separated from the viral suspension, and consequently the total amount of nonviral protein in the viral suspension decreased. The recovery of infectivity was about 100%. The removal of residual polyethylene glycol in the viral suspension was possible without difficulty by means of ethanol precipitation. This method is recommended as an initial step in large-scale purification of Japanese encephalitis virus propagated in mouse brain because it is simple, rapid, and inexpensive.     

Purification of Large Amounts of Murine Ribonucleic Acid Tumor Viruses Produced in Roller Bottle Cultures

Murine ribonucleic acid tumor viruses and C-type virus particles are produced in relatively large quantities in roller bottle cultures. The viruses present in large volumes of culture fluids can be purified by a simple two-step procedure involving polyethylene glycol precipitation and equilibrium centrifugation in sucrose density gradients.     

水体病毒浓缩方法的建立和优化研究

采用氯化钙(CaC l2)、聚乙二醇(PEG,pH7.0)、聚乙二醇(PEG,pH11.5)、三氯化铝(A lC l3)沉淀、Am icon Utcra超滤离心装置和硝酸纤维素吸附膜6种浓缩方法,浓缩人工添加于水体的1型脊髓灰质炎疫苗病毒(PV1),并对浓缩实验条件进行选择和优化。结果表明,CaC l2和聚乙二醇(pH7.0)沉淀法适用于浓缩大容量水体中的病毒,而超滤离心管浓缩法适用于小容量水体,这3种浓缩方法的病毒回收率均达到100%。     

Purification and Aggregation of Influenza Virus by Precipitation with Polyethylene Glycol

Influenza virus may be purified and rendered free of extraneous proteins by precipitation and aggregation with polyethylene glycol at polymer concentrations of 1 to 4%. The precipitated virus is superior antigenically to the virus in monomeric and in the ether dissociated forms. When the virus is precipitated at polyethylene glycol concentrations of 5% and higher the virus is not aggregated and is associated with extraneous protein which co-precipitates with the infectious agent.     

Eliciting antigen-specific egg-yolk IgY with naked DNA

Immunization with naked DNA was used to elicit chicken egg yolk antibodies (IgY). Layer hens were inoculated with plasmid DNA encoding the enhanced green fluorescent protein, the fusion protein of Newcastle disease virus, and VP2 of African horse sickness virus. IgY was extracted from egg yolks by polyethylene glycol precipitation. Specific antibodies were present in the yolks of eggs from hens immunized with each of the three different plasmids. This approach to raising polyclonal antibodies obviates the need to produce and purify large quantities of proteins for immunization and can potentially yield large amounts of diagnostically or therapeutically useful reagents.     

17. Purification and Aggregation of Influenza Virus by Precipitation with Polyethylene Glycol

ABSTRACT

Influenza virus may be purified and rendered free of extraneous proteins by precipitation and aggregation with polyethylene glycol at polymer concentrations of 1 to 4%. The precipitated virus is superior antigenically to the virus in monomeric and in the ether dissociated forms. When the virus is precipitated at polyethylene glycol concentrations of 5% and higher the virus is not aggregated and is associated with extraneous protein which co-precipitates with the infectious agent.     

The use of polyethylene glycol in concentration and purification of several bovine viruses.

The polyethylene glycol (PEG) precipitation method was used for the concentration and purification of eight bovine viruses. Good results were obtained from four viruses, parainfluenza--3 virus, bovine enterovirus, bovine adenovirus, and bovine parvovirus. No satisfactory results of concentration were obtained from bovine reovirus, bovine viral diarrhea virus, and infectious bovine rhinotracheitis virus. The failure of concentration of the four viruses seems to be ascribed rather to the resuspending of virus from the virus--PEG precipitate than to the precipitation of virus from infective culture fluid. This method can be applied as the initial step to the concentration of parainfluenza--3 virus, bovine enterovirus, bovine adenovirus, and bovine parvovirus from a large volume of material, since it is simple, rapid, and inexpensive.     

Comparison between inactivated Sendai virus, polyethylene glycol and Tween 80 as permeabilizing agents for introduction of neurospora endonuclease into X-irradiated cells

Inactivated Sendai virus, polyethylene glycol and Tween 80 were employed as agents to make X-irradiated CHO cells permeable for Neurospora endonuclease, in studies designed to evaluate the influence of this enzyme on the frequencies of X-ray-induced chromosome aberrations. Polyethylene glycol and Tween 80 were found not to be very efficient in making cells permeable. Besides, polyethylene glycol was found to increase the frequencies of X-ray-induced chromosomal aberrations.     

Purification of hepatitis A virus from chimpanzee stools.

Hepatitis A virus antigen was purified from early acute-phase chimpanzee stools by a rapid three-step procedure using 7% polyethylene glycol precipitation, CsCl banding, and Sepharose 2B column chromatography. Electron microscopic examination of the hepatitis A virus entigen preparation revealed highly purified hepatitis A virus particles.     

Virus detection on grapes.

Grapes inoculated with poliovirus 1 and coxsackievirus B5 were washed with water, 0.5% polyehtylene glycol, or phosphate-buffered saline with 1% serum. These washes were equally efficient at removing virus but much of the virus in the water was noninfectious until treated with 0.5% polyethylene glycol.     

Formation of heterosymplasts in human reticular cell cultures]

The cell fusion has been studied in human reticular cell cultures J-96 and J-41 treated with the Sendai virus or with polyethylene glycol 1000 and 6000. The J-96 cells have a high alkaline phosphatase activity, in J-41 cells the enzyme is not detectable. No heterogenous alkaline phosphatase activity was seen in the protoplasm of symplasts 18 hours after virus cell fusion. It has been shown with polyethylene glycol treatment that during the fusion of cells J-96 and J-41 the enzyme activity was spreading over the symplast protoplasm.     

Concentration and detection of hepatitis A virus and rotavirus from shellfish by hybridization tests.

A modified polyethylene glycol precipitation method for concentration of virus followed by a new method to recover nucleic acid was used to detect hepatitis A virus (HAV) and rotavirus (SA11) in shellfish (oysters and hard-shell clams) by hybridization tests. Infectious virus, seeded into relatively large quantities of shellfish, was recovered consistently, with greater than 90% efficiency as measured by either in situ hybridization (HAV) or plaque assay (rotavirus SA11). Viral nucleic acid for dot blot hybridization assays was extracted and purified from virus-containing polyethylene glycol concentrates. Separation of shellfish polysaccharides from nucleic acid was necessary before viral RNA could be detected by dot blot hybridization. Removal of shellfish polysaccharides was accomplished by using the cationic detergent cetyltrimethylammonium bromide (CTAB). Use of CTAB reduced background interference with hybridization signals, which resulted in increased hybridization test sensitivity. After polysaccharide removal, dot blot hybridization assays could detect approximately 10(6) physical particles (corresponding to approximately 10(3) infectious particles) of HAV and 10(4) PFU of SA11 rotavirus present in 20-g samples of oyster and clam meats. These studies show continuing promise for the development of uniform methods to directly detect human viral pathogens in different types of shellfish. However, practical applications of such methods to detect noncultivatable human viral pathogens of public health interest will require additional improvements in test sensitivity.     

Concentration and detection of hepatitis A virus and rotavirus from shellfish by hybridization tests.

A modified polyethylene glycol precipitation method for concentration of virus followed by a new method to recover nucleic acid was used to detect hepatitis A virus (HAV) and rotavirus (SA11) in shellfish (oysters and hard-shell clams) by hybridization tests. Infectious virus, seeded into relatively large quantities of shellfish, was recovered consistently, with greater than 90% efficiency as measured by either in situ hybridization (HAV) or plaque assay (rotavirus SA11). Viral nucleic acid for dot blot hybridization assays was extracted and purified from virus-containing polyethylene glycol concentrates. Separation of shellfish polysaccharides from nucleic acid was necessary before viral RNA could be detected by dot blot hybridization. Removal of shellfish polysaccharides was accomplished by using the cationic detergent cetyltrimethylammonium bromide (CTAB). Use of CTAB reduced background interference with hybridization signals, which resulted in increased hybridization test sensitivity. After polysaccharide removal, dot blot hybridization assays could detect approximately 10(6) physical particles (corresponding to approximately 10(3) infectious particles) of HAV and 10(4) PFU of SA11 rotavirus present in 20-g samples of oyster and clam meats. These studies show continuing promise for the development of uniform methods to directly detect human viral pathogens in different types of shellfish. However, practical applications of such methods to detect noncultivatable human viral pathogens of public health interest will require additional improvements in test sensitivity.     

Somatic hybridization between male sterile Nicotiana tabacum and N. glutinosa through protoplast fusion

Summary Protoplasts derived from suspension cultured cells of cytoplasmic male sterile Nicotiana tabacum (N. debneyi cytoplasm) and of fertile N. glutinosa were fused with the aid of polyethylene glycol (PEG). Out of 1,089 colonies developed from PEG-treated protoplasts, 29 restored whole plants.A somatic hybrid plant was selected on the basis of isoelectrofocusing analysis of Fraction I protein in leaves of regenerated plants. A newly created hybrid contained small subunits of both parents but only a N. glutinosa type large subunit.Male sterile character was conserved in a hybrid plant while leaf morphology was intermediate between the parents. By tobacco mosaic virus infection tests, the hybrid's leaves showed resistant symptoms, hypersensitive local lesions, which were due to N. glutinosa nuclear genome expression.Abbreviations PEG Polyethylene glycol - TMV Tobacco mosaic virus     

Simplified procedure for transient transformation of plant protoplasts using polyethylene glycol treatment

A modification of the polyethylene glycol-mediated transformation procedure which eliminates the manual polyethylene glycol dilution step is presented. A transformation mixture of protoplasts, DNA and polyethylene glycol was plated directly onto agarose blocks after incubation. The procedure was simple and fast, thereby suitable for screening the gene activity of large numbers of plasmid constructions. It has been tested for both maize and rice protoplasts.