Sedimentation Behaviour and Electron Microscopic Examination of Purified Influenza Virus

Abstract

. Melt)ourne, AustraliaPurified preparations of influenza virus were examined in the ultracentrifuge and electron microscope principally to assess the degree of purification achieved. As indicated in the main paper (Ada & Perry, 1956) the same purification procedure was used foir each batch of virus. METHODS Ultracentrifugation.The virus was examined in 0.85 yo NaCl solution (w/v) a t a concentration varying between 0.2 and 0.4 yo (w/v). The ultracentrifuge was air-driven of the Beams-Pickels type. The speed of the rotor was 185 r.p.s. and the temperature c. 20". The sedimentation was observed with a schlieren optical system having an inclined second slit. The light was of 5461 A. from a mercury arc. Viscosity measurements were carried out in an Ostwald viscometer at 25".Electron microscopy. A Siemens microscope, type UM 100, was used. The specimens to be examined were dialysed at 0" against 0.8 yo (w/v) ammonium carbonate solution, sprayed on to nitrocellulose films at a pressure of 10 lh./sq.in., air dried and shadowed with gold manganin. RESULTSSedirne?&tatkm behaviour. Most samples of standard virus showed a single peak of S,, = 620 -800 xIn some cases, a small amount ( < 20 yo) of a slower component (S2* = 350-500 x 1O-l3) was present. The sedimentation constant was independent of concentration (within experimental error) over the range studied (0-2-0-4 yo). Incomplete virus, P R 8 strain, whether made by two or three passages of undiluted inoculum, consistently showed decreased sedimentation constants, varying between 400 and 550 x 10-13. Incomplete virus was usually more heterogeneous than standard virus. Relative viscosities of the virus solutions were of a similar order to those found by Lauffer & Stanley (1944), who investigated material purified by extensive difl'erential centrifugation. In P1.1, fig. 1, are shown sedimentation diagrams of purified preparations of several A and B strains and OF incomplete influenza virus. Electron microscopy. P1. 1, figs. 2-4, show typical fields of purified preparations of standard PR8, standard LEE and incomplete P R 8 respectively.

The multiplication of influenza virus

“…Well studied strains such as PR8 and LEE were found to consist mainly of irregularly round objects, approximately 75 mji, in diameter (Williams and Wyckoff, 1945), Occasionally elongated forms appeared which were quite distinct in size and appearance (Mosley and Wyckoff, 1946;Heiraoats, 1948). These observations were confirmed by Chu, Dawson and Elford (1949), who found that these filaments were extremely rare in old laboratory strains such as PR8 and LEE, The physical nature of ' 'incomplete virus" (von Magnus, 1947(von Magnus, , 1951b has also been intensively investigated, Gard and von Magnus (1947) and Gard et al, (1952) Schlesinger, 1954;Toss and Wengel, 1955;Pye et al, 1956;Hollos, 1957) Once the identity of the virus particle was established Williams (1950). No attempt was made in either of theee studies to relate particle counts to biological activity.…”

Structure and Development of Viruses as Observed in the Electron Microscope

“…It is of interest in this regard that morphologic evidence supporting the similarity between the cytoplasmic fragments and incomplete virus is provided by electron microscopic examination of purified preparations. Dried-down suspensions of incomplete virus contain particles which are pleomorphic, generally larger than infectious virus and flattened in appearance, often resembling empty sacs (18,(23)(24)(25)(26)(27). Moreover, many of the forms observed in sectioned pellets (28) closely resemble the particles illustrated in this paper.…”

“…1 and noted in a previous publication, reference 8). Filaments, however, do not appear to constitute the incomplete forms associated with undiluted inocula for they were not especially numerous in the thin sections examined, nor do they appear to make up a significant proportion of the particles seen in purified preparations (18,(23)(24)(25)(26)(27)(28).…”