Isolation and Characterization of Influenza Virus B (Lee Strain)

Summary A particulate component possessing the biological properties of the influenza virus B (Lee strain), as determined by chicken red blood cell agglutination, precipitation, complement fixation and infectivity for chick embryos, has been isolated in purified preparations from the chorio-allantoic fluid of chick embryos. Concentration and purification were accomplished by adsorption on and elution from chicken red blood cells followed by ultracentrifugation of the eluate at 27,000 g for 1 hour. Electron micrographs of the concentrates revealed circular or ovoid images, measurements of which indicated an average particle diameter of 97.3 mμ. In diagrams of sedimentation-velocity there was present a single slightly diffuse boundary corresponding to a sedimentation-constant of S20° = 832 × 10−13. The probable relation of diffuseness of the boundary to variation in particle size in electron micrographs has been discussed in connection with similar findings with influenza virus A. The yield of virus from 6,600 ml of chorio-allantoic fluid was 137 mg or 2.1 mg per 100 ml of the fluid. On the basis of nitrogen, the degree of concentration was about 20 times and on volume, practicable for working conditions, 150 times. Preliminary analysis indicated that the virus is a complex containing 21 per cent fat-solvent extractive of which 5.7 per cent are neutral fat, 10.2 per cent phospholipid and 5.1 per cent cholesterol. The non-lipid fraction was 66 per cent of the whole virus and gave the qualitative tests for protein and for desoxy-pentose. It contained 0.44 per cent phosphorus, which, if all was associated with nucleic acid, indicated the possible presence of 3.5 per cent nucleic acid of the desoxypentose type in the whole virus. The total phosphorus of the whole virus was 0.7 per cent. The total carbohydrate of the whole virus was 9 per cent, an amount greatly in excess of that accounted for in nucleic acid. The specific volume determined by pyknometer was 0.865. A comparison of the characters of the influenza virus B with those found for influenza virus A reveals certain differences between the two agents. The influenza virus A appeared to consist principally of bean- or kidney-shaped particles whereas the particles of influenza virus B were more nearly spherical bodies. An average diameter of 77.6 mμ was obtained from electron micrographs of the influenza virus A, and the sedimentation-constant was S20° = 724 × 10−13. The diameter of the influenza virus A particles calculated from the density and sedimentation-constant, assuming a spherical shape, was 80 mμ. A similar calculation for influenza virus B gave a value of 99.8 mμ, for the particle diameter. The differences in the respective sedimentation-constants and the diameters of influenza viruses A and B, determined from sedimentation-velocity and electron micrographs, were of a magnitude which indicated a significant difference in the sizes of the two agents.