Previous work indicated that herpes simplex virus type 1 (HSV-1) is a mitogen for mouse spleen cultures, as monitored by uptake of H-thymidine. We observed variable responses of mouse spleen cultures to different viral preparations. The variable responses, which did not follow normal dose—response relationships, were not due to HSV-1 strain differences, altered response kinetics or the presence or absence of defective viral particles, but to mycoplasma contamination of viral stocks. Mycoplasma-free (MF) HSV-1 stocks were prepared by transfection of MF NHF cells with HSV-1 DNA. MF HSV-1 infection of spleen cultures resulted in a five- to sixfold stimulation of DNA synthesis and stimulation of a polyclonal antibody response. Heat treatment (56 °C for 1 h) and antibiotics were used to distinguish mycoplasma and HSV-1-induced spleen culture mitogenic responses. The mycoplasma-induced mitogenic activity was found to be heat labile and sensitive to gentamicin and chloramphenicol. In contrast, the HSV-1-induced response was not affected by gentamicin or chloramphenicol and heat treatment resulted in only a 50% loss of activity.


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