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5 cells per 60-mm dish) were incubated for 24 hours after seeding and then transfected with 12 μg of DNA (including 0.5 μg of infectious KOS DNA, 5 μg of mutant plasmid DNA, and 6.5 μg of carrier DNA). Five days later, when generalized cytopathic effects were apparent, cultures were harvested and then frozen and thawed three times. The cell suspension was sonicated briefly, diluted 1:10 in medium, passed through a 0.22-μm filter, plated on monolayers of G33 cells-BHK21 cells expressing the UL6 protein [A. H. Patel et al., Virology 217, 111 (1996)] - and overlaid with methylcellulose. After incubation at 37°C for 4 days, plates were stained for 24 hours with medium containing neutral red, individual plaques were picked, and isolates were screened for their ability to produce cytopathic effects in G33 cells but not in Vero cells. Isolates with this phenotype were plaque-purified three times.
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5 cells per 60-mm dish) were incubated for 24 hours after seeding and then transfected with 12 μg of DNA (including 0.5 μg of infectious KOS DNA, 5 μg of mutant plasmid DNA, and 6.5 μg of carrier DNA). Five days later, when generalized cytopathic effects were apparent, cultures were harvested and then frozen and thawed three times. The cell suspension was sonicated briefly, diluted 1:10 in medium, passed through a 0.22-μm filter, plated on monolayers of G33 cells-BHK21 cells expressing the UL6 protein [A. H. Patel et al., Virology 217, 111 (1996)] - and overlaid with methylcellulose. After incubation at 37°C for 4 days, plates were stained for 24 hours with medium containing neutral red, individual plaques were picked, and isolates were screened for their ability to produce cytopathic effects in G33 cells but not in Vero cells. Isolates with this phenotype were plaque-purified three times.
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m mutant viruses at a multiplicity of infection of 10 PFU per cell. Uninfected and infected cells were harvested 18 to 20 hours after infection by scraping into the medium and were centrifugea at 1500 revolutions per minute and 4°C for 10 min. The cell pellet was resuspended in 5 ml of phosphate-buffered saline (PBS), repelleted, and resuspended again in 1 ml of PBS. The cell suspension was stored at -70°C overnight, thawed at 37°C, and sonicated for 1 min. Samples were pelleted at 4°C for 10 min, and the supernatant fluid was divided into aliquots and stored at -70°C. Protein concentrations were determined by measuring absorbance at 280 nm and using bovine serum albumin as a standard.
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The antigen used for in vitro stimulation was prepared from extracts of Vero cells infected with HSV-1 (KOS) (0.01 PFU per cell) and harvested 3 days after infection [W. Cai and P. A. Scharfer, J. Virol. 66, 2904 (1992)]. The virus suspension was inactivated by UV light (254 nm) for 20 min at a distance of 5 cm [L. Morrison and D. Knipe, J. Virol. 68, 689 (1994)]. Control antigen was prepared in the same way from extracts of uninfected cells.
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The antigen used for in vitro stimulation was prepared from extracts of Vero cells infected with HSV-1 (KOS) (0.01 PFU per cell) and harvested 3 days after infection [W. Cai and P. A. Scharfer, J. Virol. 66, 2904 (1992)]. The virus suspension was inactivated by UV light (254 nm) for 20 min at a distance of 5 cm [L. Morrison and D. Knipe, J. Virol. 68, 689 (1994)]. Control antigen was prepared in the same way from extracts of uninfected cells.
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We would like to thank D. Knipe for providing pSG10, A. H. Patel for G33 cells, J. Glorioso for KO82 mutant virus, and A. Angel for assistance in preparation of the manuscript. Supported by NIH grant AI 37562 to H.C.
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