SUMMARYMass determinations on highly purified influenza virus preparations were performed using the technique of scanning transmission electron microscopy. The masses of the three strains, X49, B/Singapore/222/79 and B/Hong Kong/8/73 were determined. The average value was 174 × 106 daltons with only small differences between the three strains. The mass of virus particles after removal of the protruding spike proteins, haemagglutinin and neuraminidase by bromelain treatment was determined to be 86 × 106 daltons. From the mass difference and the known molecular weight of the spike proteins the number of spikes was estimated to lie in the range 400 to 500.Influenza virus is a complex virus belonging to the Orthomyxoviridae. The virus is composed of protein, lipid, carbohydrate and RNA; there are four major proteins involved in the virus architecture. The outer virus envelope consists of a lipid bilayer from which the so-called spikes project externally. These spikes consist of trimers and tetramers of haemagglutinin (HA) and neuraminidase (NA) respectively. The inner core of the virus consists of M protein, nucleoprotein and RNA. The molecular biology of the virus was reviewed recently (Webster et al., 1982).There is no general agreement on the size, shape and exact composition of the virus. Most of the information on its structure was obtained from biochemical studies (Schulze, 1973;Laver, 1973), electron microscopic results (Wrigley, 1979;Nermut & Frank, 1971), X-ray crystallographic work (Wilson et al., 1981 ;Varghese et al., 1983) and solution scattering data (Mellema et al., 1981). Here we describe the determination of particle weight and spike content by scanning transmission electron microscopy (STEM). This method consists of scanning a focused electron beam across the specimen and obtaining a digital signal from the scattered electrons that can be computer-processed. Under certain well defined conditions the scattering intensity is proportional to the scattering mass (see Freeman & Leonard, 1981).As proposed by Freeman & Leonard, we determined particle masses by using an internal standard. Influenza particle masses were determined by comparison with tobacco mosaic virus which has a very well known mass per unit length. A linear relationship was assumed between the scattering mass and the scattering intensity; this assumption holds under our specific measurement conditions (acceleration voltage, carbon film thickness, etc.) up to a thickness of approximately 60 nm. From shadowing experiments the thickness of the unstained, flattened, influenza particles was found to be 48 + 4 nm, which is in the linear range. The validity of the mass determination method was indicated by the very good reproduction of published data on masses of a number of plant viruses with well known molecular weights. A full description of our implementation of the STEM mass determination method will be published elsewhere (R. W. H. Ruigrok et al., unpublished results).Influenza virus preparations were obtained from DUPHAR (Weesp, The Net...
