Centrifugation and Ultrafiltration Studies on Allantoic Fluid Preparations of Influenza Virus

Abstract

The property of influenza virus suspensions to agglutinate red blood ceils appears to be closely associated with the infective activity of the virus (1). The complement-fixing antigen, on the other hand, has been shown to consist of two distinct fractions: one is intimately associated with the hemagglutinin and infective virus particle, while the other has a smaller size and is readily separable from the virus particle by centrifugation or adsorption with red blood ceils (2). A preliminary report (3) showed that the infectivity and hemagglutinin of the PR8 and Lee strains of influenza virus were associated with particles which were at least 60 m/z in diameter. Subsequent papers by other workers (4, 5) have confirmed this latter finding and have set the size of the PR8 virus particle at 80 m# and that of the Lee virus particle at 100 m#.The present paper presents a further study of the various properties of influenza virus and their interrelationships, by means of experiments in the angle centrifuge and the optical ultracentrifuge, and by filtration with graded collodion membranes. It will be shown that the application of a synthetic density gradient, already described (6), to influenza virus suspensions minimizes convective disturbances in the angle centrifuge and permits the demonstration of sedimenting virus boundaries which can be identified by infectivity, agglutination, and complement fixation tests. MethodsPreparation of Virus Suspension.--The PR8 strain of influenza A virus (7) and the Lee strain of influenza B virus (8) were used in these experiments. Mlantoic fluid suspensions of each virus strain were prepared by a technique already described (9). Briefly, it consisted of inoculating the allantoic sac of 11 day old white Leghorn embryos with 0.2 cc. of a 10 -s dilution of allantoic fluid known to contain virus in high titer. After incubation at 37°C. for 48 hours, the eggs were chilled overnight at about 4°C. The blood-free allantoic fluids were then removed, pooled, and cleared by centrifugation at about 1800 R.1,.~r. for 5 minutes. Without further treatment the fresh allantoie fluids were subjected to the experimental procedures to be described, and all tests were made on the same day.Infectivity Tests.--The virus suspensions were diluted in steps of 10 -°'5, and 0.05 cc. samples were instilled intranasally into albino Swiss mice under light ether anesthesia. 301

A Sedimentable Component of Allantoic Fluid and Its Relationship to Influenza Viruses

“…The sedimentation constants obtained are listed in Table I and can be compared with the values of 600 to 800 reported for PR8 virus particles (6,9,12). A single boundary, which was usually quite sharp, was observed with preparations containing 2 to 3 rag.…”

“…However, the presence of a 30 S component in centrifugally purified preparations of F12 influenza virus has been well established (10,18) and it has recently been shown that this component, contrary to earlier conclusions, is not the virus (10). In addition, a non-viral small component has been observed in purified preparations of Lee virus (29) and components having sedimentation constants ranging from 75 S to 450 S have been reported for preparations of PR8 virus (12,30). The small components in purified preparations of influenza virus are demonstrable by means of the ultracentrifuge mainly in concentrated solutions; i.e., 10 to 30 rag.…”

The Production of Fever by Influenzal Viruses

“…virus has shown that the infective and hemagglutinating properties of the virus are removed with the 600-S component (14). The toxic factor described by Henle and Henle (9) has not been dissociated from the infective particle.…”

Studies on Host-Virus Interactions in the Chick Embryo-Influenza Virus System

“…If these calculations were restricted to the seed virus actually adsorbed onto the cells, the increase would be nearly 100-fold. Since agglutination by intact virus can be detected when the number of virus particles nearly equals that of the red cells (5,6,8,9,23,24) its efficiency per se cannot be improved. The seed virus would have to disintegrate, therefore, into 30 or 100 smaller units, respectively, if the NIHA obtained in the above experiments represented breakdown products.…”