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
Chloroquine, a compound widely used clinically for the prophylaxis and therapy of malaria, was recently found to inhibit the replication of herpes simplex virus (HSV) in HeLa cell cultures (1). This observation and the current lack of clinically effective drugs of low toxicity for the treatment of severe systemic human herpesvirus infections prompted the present study. We have investigated the therapeutic efficacy of chloroquine on lethal systemic infection produced by HSV-1 and HSV-2 in the newborn mouse. The lethal endpoint of this assay system (2) is unequivocal and highly reproducible. Additionally, manifestations suggestive of central nervous system involvement which resemble those observed in human herpesvirus encephalitis result from the intraperitoneal inoculation of the virus (2). These features and the observation that chloroquine inhibited HSV replication in mouse embryo (ME) cell cultures made assay of the antiviral activity of chloroquine in HSV-infected newborn mice seem appropriate and advantageous.Materials and Methods. Viruses. Stock strains of herpesvirus type 1 (68-157) and herpesvirus type 2 (68-1 5 8 ) , originally isolated in human embryo kidney cell cultures and passaged twice in rabbit kidney cell cultures were kindly provided by Dr. Fred Rapp. Virus pools for use in these experiments were prepared in cultures of BHK-21 cells ( 3 ) . Cytopathic effects were complete 48 hr after inoculation, at which time the entire cultures were frozen (-70") and thawed three times, centrifuged (15 min, 2000 rpm, 4', International Centrifuge Model PR2), and the clarified supernates stored in screw-capped vials at -70' until use. Cells and Media. HeLa cells and secondary mouse embryo (ME) cells were propagated in Eagle's medium, enriched with 10% tryptose phosphate broth and 5 % fetal calf serum (FCS) (Hyland Laboratories), (ETC) , containing penicillin 100 units/ml, streptomycin 100 pg/ml and mycostatin, 50 units/ml. Chloroquine. Chloroquine was added to tissue culture media as chloroquine hydrochloride (Aralen R, Winthrop Laboratories, New York, New York). It was added to media at a final concentration of 36 pg of chloroquine base/ml, the amount present in 60 pg of chloroquine diphosphate ( 1 ) . In vitro HSV growth curves and virus assays. Confluent monolayers of HeLa or secondary ME cells in 30 ml plastic tissue culture flasks (Falcon Plastics No. 3012) were drained and infected with 0.2 ml of virus suspended in 0.02 5 M Tris (hydroxymethyl)aminomethane buffer (TBS) (pH 7.4), containing 3% FCS. The number of cells present at the time of infection was determined by cell counts performed on replicate cultures. When virus adsorption was complete (37O, 2 hr, intermittent manual rotation), the inoculum was removed with three washes of phosphate-buffered saline (PBS) , and the cultures fed with 4 ml of ETC containing 5% FCS. At appropriate intervals after washing, replicate whole cultures were harvested by freezing and stored at -70" until thawed for virus plaque assays. They were then frozen and thawed twi...
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