SUMMARY: Particle counts of influenza virus preparations were made by two independent techniques ; there was good agreement in the counts found by the two methods. Counts made on four strains of influenza virus and one strain of 'incomplete' virus showed that at the agglutination end-point there was about one virus particle per red cell. Parallel infectivity measurements of these strains made under optimal conditions showed that about ten virus particles corresponded to one ID 50.

A strain of influenza virus A which produced abundant filaments was more efficient in agglutinating red cells than non-filamentous strains. This was shown by counts of the number of particles (i.e. filaments and spheres)/agglutinating dose ; the ratios found were 106*6 for the filamentous, and for the non-filamentous strains. The increased efficiency of agglutination was shown by filtration experiments to be due to the filaments. There was no corresponding increase in the efficiency of initiating infection. Treatment of a filamentous preparation with ultrasonic vibrations caused breakdown of the filaments, an increase in agglutination titre but no change in the infectivity titre.In a recent study of counts of influenza virus particles by electron microscopy it was found that for four strains of virus examined there was an average of virus particles/agglutinating dose, as defined below (Donald & Isaacs, 1954). One strain gave a figure of in two different experiments, however, and it was noticed that these two preparations contained a number of filamentous forms of virus. The possibility was considered, therefore, that virus filaments might show different agglutinating or infective behaviour from spherical forms. This possibility was investigated with a strain of influenza virus, A/Persian Gulf/2/52, known to produce many filamentous forms. This paper reports the results of our investigation. MATERIALS AND METHODSThe techniques for counting and biological assay of influenza virus described in an earlier paper were used with only a few modifications.Technique of counting. Previously two techniques were employed-a spray technique and counts of the number of particles adsorbed on fowl red cell ghosts; these two techniques gave essentially the same results. The spray technique was unsuitable for the present experiments, however, since virus filaments were not found in sprayed materials. The red cell technique was therefore used alone. For purposes of counting each filament was counted as a single particle. Agglutinating dose. An agglutinating dose (A.D.) is defined as the least amount of virus which causes partial (50 yo ) agglutination of 0.25 ml. of a 1 yo suspension of chick red cells (approximately 107.15 cells) in a pattern test. Infectivity titrations. Titrations were carried out in the allantoic cavity of the chick embryo as described previously and with 4 eggs/half-log dilution. Ultrasonic treatment. For the 350 kc. treatment a quartz crystal oscillator 21-2

A form of " hot wire " apparatus is described in which the hot wire is replaced by a thin-walled nickel tube � in. in diameter mounted in a similar nickel tube in, in diameter. The gas under investigation occupies the narrow annular space between the two tubes. It is shown that the apparatus is completely free from convection up to pressures somewhat greater than 1 atm, and that the temperature discontinuity effect is inappreciable down to pressures of a few cm. of mercury. The apparatus is used to test the pressure dependence of the thermal conductivity of several polyatomic gases. The thermal conductivity (at 0 �C.) of the gases carbon dioxide and methane is found to increase slowly with the pressure over the range 10-80 cm. of mercury. It is suggested that this effect may be taken as evidence of the incomplete participation of the energy of vibration of the molecules in the conduction process.

SUMMARY: The electron microscope was used to make particle counts of the viruses of Newcastle disease of fowls, fowl plague, mumps and influenza C. Two counting techniques previously described were also used, and correlations with simultaneous measurements of haemagglutination and infectivity were made.In a previous study (Donald & Isaacs, 1954a) counts were made in the electron microscope of influenza virus particles, and related to measurements of haemagglutination and infectivity of the preparations carried out under optimal conditions. It was found that at the agglutination end-point there was about one virus particle/red cell. It was also found that about ten virus particles of the standard influenza strains corresponded to the minimal infective dose. The particle count methods developed for that study are extended here to the related haemagglutinating viruses of Newcastle disease, fowl plague, mumps and influenza C. It was not always possible to use both counting techniques for each of these viruses ; however, the modifications developed for each particular case and the results obtained are presented in this paper. ' Herts.' strain; mumps-Enders EMA 41 strain; influenza C-Taylor's 1233 strain (1949). MATERIALS AND METHODS VirusBoth particle-counting techniques and the methods of biological assay were described in an earlier paper (Donald & Isaacs, 1954a). Certain modifications were introduced for each of the above viruses.Preparation of virus material. With influenza C lo-day eggs were inoculated amniotically since this virus does not grow well in the allantoic cavity, and the allantoic fluid was harvested after 2 days. Dilutions of of the seed virus were used for inoculation.Newcastle disease and fowl plague viruses were passaged in the allantoic cavity of Wday eggs and harvested after 18 hr. of incubation a t 3 5 ' . A dilution of of the seed virus was used for Newcastle disease virus, for fowl plague.Mumps virus was passaged in the allantois of 8-day eggs and harvested after 5 days of incubation at 35O. A dilution of of the seed virus was used for inoculation.Mod@cations to haemagglutination titration. Titrations with influenza C were carried out in the cold room (+Z0) with pre-chilled reagents, as this virus elutes spontaneously from red cells at room temperature. Titrations with

. 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.