The Purification and Concentration of Influenza Virus by Means of Alcohol Precipitation

Hr, Cox; J, Van der Scheer

doi:10.2307/4585914

Cited by 16 publications

(12 citation statements)

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“…Based on the CsCI equilibrium density gradient sedimentation technique of Meselson, Stahl, and Vinograd (8), the following method was devised for purification of adenoviruses. Crude type 5 virus in PBS was precipitated by the addition of chilled methanol to a final concentration of 30 per cent (v/v) (9). The mixture was held at -25°C for 1 hour and the precipitate sedimented by a single cycle of eentrifugation at 1800 g for 30 minutes.…”

Section: Purification Of Type 5 a Denovirus--a Preparation Of Highlymentioning

confidence: 99%

Structure of Type 5 Adenovirus

Wilcox

Ginsberg

1963

The Journal of Experimental Medicine

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Infection of mamm~.lian cells with agents of the adenovirus group elicits production of at least three major soluble antigens. These proteins have been separated on the basis of their immunological, biological, and physiochemical properties into: (a) a group-specific antigen common to all adenovirus types; (b) a type-specific antigen unique for each type; and (c) a toxin-like material which also possesses group specificity (1, 2). The exact role of the soluble antigens in the virus synthetic process has been under investigation. Indirect evidence suggests that the soluble antigens represent virus-structural protein subunits produced in large excess of the amount utilized for synthesis of infectious virus (3, 4). A rigorous confirmation of this hypothesis requires evidence that the virus particle contains antigens which are identical with the soluble antigens. I t is the purpose of this communication to present data which indicate that the antigens of type 5 adenovirus particles are immunologically and physically identical with the soluble antigens produced in mammalian cells infected by this agent. Materials and MethodsTissue Culture.--Epithellal-like cells of the HeLa (Gey) or KB llne were cultured as monolayers in bottles using Eagle's basal medium supplemented with 10 per cent human serum or in screw-capped test tubes employing 40 per cent human serum in Hanks' balanced salt solution (BSS) by methods previously described (5).Virus.--The prototype strain of type 5 adenovirus utilized in previous studies was employed (2, 3). Virus pools were prepared by infecting monolayer cultures of KB cells maintained in a medium consisting of 67.5 per cent Scherer's amino acid-vitamin mixture, 25 per cent tryptose phosphate broth and 7.5 per cent chicken serum (termed maintenance mixture). Infected Cells were incubated at 36°C until cytopathic effects were complete, at which time the cells were recovered by centrifugation from the harvested cell suspension, and resuspended in phosphate-buffered saline (PBS). Resuspended cells were disrupted by freezing and thawing 6 times and the cell debris removed by centrifugation at 6600 g for 15 minutes in a Spinco

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Section: Purification Of Type 5 a Denovirus--a Preparation Of Highlymentioning

confidence: 99%

Structure of Type 5 Adenovirus

Wilcox

Ginsberg

1963

The Journal of Experimental Medicine

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“…Precipitation methods for the concentration and purification of influenza virus were among the first to be described (Cox et al, 1947). In 1966, the use of gradient density centrifugation with zonal ultracentrifuges was introduced for large-scale preparations (Reimer et al, 1966(Reimer et al, , 1967.…”

Section: Introductionmentioning

confidence: 99%

Purification of cell culture‐derived human influenza A virus by size‐exclusion and anion‐exchange chromatography

Kalbfuss

Wolff

Morenweiser³

et al. 2006

Biotech & Bioengineering

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A process comprising of size-exclusion chromatography (SEC) and anion-exchange chromatography (AEC) was investigated for downstream processing of cell culture-derived influenza A virus. Human influenza virus A/PR/8/34 (H1N1) was propagated in serum-free medium using MDCK cells as a host. Concentrates of the virus were prepared from clarified and inactivated cell culture supernatants by cross-flow ultrafiltration as described before. SEC on Sepharose 4 FF resulted in average product yields of 85% based on hemagglutination (HA) activity. Productivity was maximized to 0.15 column volumes (cv) of concentrate per hour yielding a reduction in total protein and host cell DNA (hcDNA) to 35 and 34%, respectively. AEC on Sepharose Q XL was used to separate hcDNA from virus at a salt concentration of 0.65 M sodium chloride. Product yields >80% were achieved for loads >160 kHAU/mL of resin. The reduction in hcDNA was 67-fold. Split peak elution and bimodal particle volume distributions suggested aggregation of virions. Co-elution with hcDNA and constant amounts of hcDNA per dose indiciated association of virions to hcDNA. An overall product yield of 52% was achieved. Total protein was reduced more than 19-fold; hcDNA more than 500-fold by the process. Estimation of the dose volume from HA activity predicted a protein content at the limit for human vaccines. Reduction of hcDNA was found insufficient (about 500 ng per dose) requiring further optimization of AEC or additional purification steps. All operations were selected to be scalable and independent of the virus strain rendering the process suitable for vaccine production. Biotechnol. Bioeng. 2007;96: 932-944.

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“…Attempts at Purification.--Because differential centrifugation is a relatively inefficient fracfionation procedure producing losses of up to 85 per cent, 4 chemical methods of virus purification were applied to mumps-infected amniotic fluid. The methods of Cox (11) and Pollard (12), precipitating virus from 30 per cent methanol at -10°C., completely destroyed the mumps hemolysin. Utilizing the selective elution of virus from cellulose anion exchange columns (DEAE-SF and ECTEOLA-SF, Brown and Co.) by the method of Hoyer et al (13), only 4 per cent virus recovery was achieved.…”

Section: Studies With Sheep Red Blood Cells--mentioning

confidence: 99%

Studies of the Hemolysis of Red Blood Cells by Mumps Virus

Soule

Marinetti

Morgan

1959

The Journal of Experimental Medicine

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The hemolysis of chicken red blood cells by mumps virus (1) has been shown to exhibit many of the properties of an enzymatic reaction (2, 3). Although subsequent studies showed that the hemolysin possessed certain properties in common with lecithinase A (2-5), the last study in this series, by Moberly, Marinetti, and Morgan (6), demonstrated conclusively that the mechanism of hemolysis was not the conversion of red cell lecithin by mumps virus to the hemolytic compound lysolecithin. Evidence was offered however that another specific change was taking place in the cell stroma with hemolysis. This was manifest as a decrease in the size of the sphingomyelin spot on phosphatide chromatograms prepared from lipide extracts of hemolyzed chicken red blood cells, when compared to unhemolyzed controls.The present study is an attempt to clarify the role of sphingomyelin in which the phosphatides and non-phosphorous-containing lipides of chicken and human red blood cells were examined quantitatively to detect possible alterations following hemolysis. In addition the lipides of the virus itself were examined, and the virus was reacted with isolated phosphatide substrates in an attempt to define a specific enzyme-substrate system. Materials and MethodsVirus.--The Enders strain of mumps virus was inoculated into the ananiotic sac of 8-day embryonated eggs which were then incubated for 96 hours. Surviving eggs were refrigerated for 12 hours and the amniotic fluid harvested. Pooled fluids with a hemagghitination titer of at least 1:512 were concentrated by differential centrifugation at 0°C. The supematant fluid obtained after centrifugation at 3,000 R.p.~r. for 20 minutes was spun in the Spinco ultracentrifuge (model L) with No. 40 rotor at 30,000 R.P.~. for 20 minutes. The white pellet obtained was resuspended in 10 per cent of the original volume of 0.16 molar buffer with a

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The Purification and Concentration of Influenza Virus by Means of Alcohol Precipitation

Cited by 16 publications

References 1 publication

Structure of Type 5 Adenovirus

Structure of Type 5 Adenovirus

Purification of cell culture‐derived human influenza A virus by size‐exclusion and anion‐exchange chromatography

Studies of the Hemolysis of Red Blood Cells by Mumps Virus

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