An Evaluation of Methods for the Concentration and Purification of Influenza Virus

Abstract

The concentration and purification of influenza virus by means of differential centrifugation in a vacuum type centrifuge, by adsorption on and elution from adult chicken red cells, by elution of the precipitate formed on freezing and thawing of allantoic fluid, by adsorption on and elution from embryonic chick red cells, and by combinations of the first method with each of the three succeeding methods, have been studied. Over-all yields of virus of about 50 to 70 per cent were obtained by these methods and combinations of methods except for somewhat lower yields when adsorption on and elution from adult chicken red cells was employed. However, the purified products obtained by methods involving only the use of red cells or the freezing and thawing technique were found to contain about 80 per cent of non-virus protein. The purified products obtained when differential centrifugation was used either alone or in combination with any one of the other methods were found to be indistinguishable and to consist of a fairly homogeneous component having a sedimentation constant of about 600 S. Such preparations possessed about 22,000 chicken red cell agglutinating units per mg. of protein nitrogen and solutions containing only about 10–14 gm. of the materials gave 50 per cent infectivity end points in chick embryos. The Sharples centrifuge was found to be almost as efficient as the vacuum type centrifuge for the concentration and purification of influenza virus and, because of its larger capacity, is recommended for the preparation of purified virus on a large scale.

Amino Acid Composition of Highly Purified Viral Particles of Influenza a and B

“…Preparation of Virus for Assay.--Higldy purified preparations of the PR8 and Lee strains of influenza virus were obtained from allantoie fluids of infected chick embryos by a combination of the methods of differential centrifugation and adsorption on and elution from chicken red cells (8)(9)(10)(11)(12). Such preparations were found to consist of particles which were highly active biologically and which were uniform in size, in electrochemical behavior, and in serological reactions (11).…”

“…In view of these findings, it was naturally of interest to determine whether or not similar chemical differences exist between strains or types of an animal virus. Highly purified preparations of influenza viruses (8)(9)(10)(11)(12) were available for this purpose and the PR8 strain of influenza A and the Lee strain of influenza B were chosen for comparison. These viruses produce clinically indistinguishable diseases (13) and appear t 9 be very similar in gross chemical properties (9).…”

The Preparation and Properties of Influenza Virus Vaccines Concentrated and Purified by Differential Centrifugation

“…The recent passage history of the PR8 strain has been described (48). When received in this laboratory the Lee strain had been isolated from ferret B75 following 8 passages in ferrets and then passed 137 times in mice and 21 times in chick embryos.…”

The Nucleic Acid and Carbohydrate of Influenza Virus

“…However, the Sharples concentrate was found to contain much less glucosamine than the other fractions. This apparent anomaly led to the discovery that all of the allantoic fluid was not being removed from the Sharples bowl by the final wash with phosphate buffer (2). As a result, the Shalples concentrate contained, in addition to the comparatively large virus particles and those of the well characterized "sedimentable protein of normal allantoic fluid" (4), components of allantoic fluid which were much too small to have been included in the virus concentrate by virtue of their sedimentation characteristics.…”

“…before use. Lot 2 was obtained from infectious aliantoic fluid by a combination of the methods of differential centrifugation and adsorption on and elution from chicken red cells as follows: The virus was concentrated and partially purified by means of the Sharples centrifuge (2). This concentrate was diluted with 0.1 ~ phosphate buffer at pH 7 until it contained about 400 to 500 standard CCA units per ml.…”