The ability of the influenza viruses to elute from erythrocytes appears to be dependent upon the presence of active neuraminidase in the viral particle(1,2,3,4). The parainfluenza viruses are also able to agglutinate and elute from erythrocytes (5), but direct evidence for neuraminidase has been lacking. This paper presents the results of investigation into the enzymatic properties of parainfluenza virus (type 2) in relation to infectivity, hemagglutinin and receptor specificity.
Materials and methods. A line of stable human amnion cellst was grown at 37°C in Eagle's basal medium with Hanks' balanced salt solution and 10-15% calf serum. Type 2 parainfluenza virus (CA virus) was obtained from The American Type Culture Collection. Washed monolayers in 32 ounce bottles were infected with 10-3105 TCID-30 of virus, and the virus harvested 3-4 days later. Virus was concentrated from culture fluid by centrifugation at 50,000 to 100,000 g for 60 to 90 rutes, and resuspended to 1/100 the original volume in 0.9% NaCl. Cells remaining in the culture flasks were scraped from the glass into saline, washed once, resuspended in a volume of 5-10 ml and subjected to ultrasonic vibration for 10 minutes in a Raytheon Sonic Oscillator, model DF 101. The sonicate, combined with virus concentrated from culture fluid, was clarified by centrifugation at 3,000 rpm. The final preparation gave a hemagglutinin titer of 1/512 to 1/2560 and contained 105 to 106 TCID50 per ml. Hemagglutination tests were performed at 4°C using 0.4 ml of virus dilution and 0.1 ml of 2% washed chicken ery throcy tes in 0.9% NaCl buffered to pH 7.2 with M/15 phosphate. Hemagglutination inhibition tests were performed at 4°C in buffered saline using 5 hemagglutinating units of virus for 0.1 ml of 2% chicken ery throcy tes in a final volume of 0.7 ml. End points were read by pattern after 30 minutes. For infectivity titrations, washed monolayers of stable human amnion cells in screw cap tubes were inoculated in duplicate with 0.2 ml of serial tenfold dilutions of virus, and fresh growth medium was added after 45 minutes' incubation at 37°C. Three days later the cultures were examined for hemadsorption with guinea pig ery throcy tes (6). The virus titer was taken as the highest dilution that produced 2 or 3 areas of hemadsorption in one or both duplicate tubes. The neuraminidase activity in concentrated virus preparations was estimated by measuring the rate at which N-acetylneuraminic acid was liberated from a standard substrate, Collocalia mucoid(7), and expressed as a percentage of the total N-acetylneuraminic acid obtainable by hydrolysis at 80°C for 20 minutes in 0.1 n H2SO4. In various experiments, Collocalia mucoid and virus sonicate preparations were mixed in the proportion of 2 to 20 mg of mucoid per mg of viral sonicate nitrogen measured by the microKjeldahl procedure (8). 0.2 ml aliquots of the mixture were withdrawn at zero time, and subsequently at appropriate intervals, heated to 75°C for 10 minutes to destroy enzyme activity, and stored at 4°C until analyzed for free N-acetylneuraminic acid by the thiobarbituric acid method of Warren(9). A sample of the standard preparation of mucoid was hydrolyzed with each set of determinations.
