A Plaque Assay for Respiratory Syncytial Virus

Abstract

The plaque assay first described for animal viruses by Dulbecco ( 1 ) , and since applied to a large number of viral agents (2), has the advantage of greater accuracy than is obtainable by endpoint dilution methods. I t also provides a highly reproducible and sensitive method for quantitation of specific neutralizing antibody. The development of such a method for respiratory syncytial virus seemed of importance because of recent interest in the problem of differentiating the consequences of reinfection in humans from those of primary infection with this agent (3-6). The present communication describes a plaque assay for respiratory syncytial (RS) virus in a continuous human epithelial cell line, HEp-2.Materials and methods. Virus. The virus used was the Long strain of respiratory syncytial virus, first isolated from the throat of a patient with bronchopneumonia( 7 ) . It had been passed 6 times in tissue culture of KB cells, twice in Chang liver epithelium and once in HEp-2 cells. Virus pools used in all experiments were obtained after either one or two further passages in HEp-2 cells.Tissue culture. HEp-2 cells derived from a strain initially obtained from Microbiological Associates, Bethesda, Md., were grown as monolayers in 32 oz medicine bottles. The cells were grown for 6 consecutive passages in Eagle's minimum essential medium (MEM) (8) in Hanks' salt solution containing 10% unheated calf serum, 0.011 mg/ml Achromy-sin@ and 0.1 mgJml KanamycinQ. Thereafter they were subcultured without antibiotics. Tests for presence of pleuropneumoriia-like organisms(9) in the cells were repeatedly negative when carried out both aerobically and in an atmosphere of 5% C02 and 95% nitrogen.The cells were removed from the glass by

Activity and Regulation of Alpha Interferon in Respiratory Syncytial Virus and Human Metapneumovirus Experimental Infections

“…RSV A2 was grown in HEp-2 cells (American Type Culture Collection, Manassas, VA) and purified by polyethylene glycol precipitation, followed by centrifugation on 35 to 65% discontinuous sucrose gradients as described elsewhere (42,55). The virus titer was determined by a methylcellulose plaque assay (30). The hMPV strain CAN97-83 was obtained from the Centers for Disease Control, Atlanta, GA, with permission from Guy Boivin at the Research Center in Infectious Diseases, Regional Virology Laboratory, Laval University, Quebec, Canada.…”

“…Homogenized samples were centrifuged twice at 10,000 ϫ g for 1 min. Serial twofold dilutions of the supernatant were tested by a methylcellulose plaque assay (30). For hMPV titration, serial twofold serial dilutions of lung homogenates were titrated in LLC-MK2 cell monolayers in 96-well flat-bottomed plates (as described for virus stock preparation).…”

IκB Kinase Is a Critical Regulator of Chemokine Expression and Lung Inflammation in Respiratory Syncytial Virus Infection

“…The virus was divided into aliquots, quick-frozen, and stored at Ϫ70°C until use. Virus titers were determined by a methylcellulose plaque assay (21). As a control, noninfected HEp-2 cells were accordingly prepared and diluted before application.…”

Alveolar Macrophages Contribute to the Pathogenesis of Human Metapneumovirus Infection while Protecting against Respiratory Syncytial Virus Infection

“…RSV (Long strain) was grown in HEp-2 cells (American Type Culture Collection, Manassas, VA) and purified as described elsewhere (22,23). The virus titer was determined by a methylcellulose plaque assay (24).…”