Establishment of herpes simplex virus latency in vitro with cycloheximide

Human embryonic lung cells were infected with herpes simplex virus (HSV), treated with 10 micrograms/ml or more of cycloheximide for 24 h, incubated at 37 degrees C, and then shifted to 40.5 degrees C for various periods of time (0 to 40 days) without cycloheximide treatment. No infectious virus was detected after freezing and thawing of the cultures; however, infectious virus was recovered after temperature shift-down to 37 degrees C or superinfection with human cytomegalovirus (HCMV). The time course for formation of infectious centres after temperature shift-down was examined with and without HCMV superinfection during incubation at 40.5 degrees C. Two patterns of latently infected cells were identified: one pattern showed spontaneous reactivation of virus after temperature shift-down, and the second showed reactivation of HSV after superinfection with HCMV. The first pattern showed a rapid decrease in the number of infectious centres with time, whereas the second maintained a steady reactivation rate up to 40 days at 40.5 degrees C. The same tendency was observed for infectious centre formation at 37 degrees C with and without HCMV superinfection in the HSV latency system established with (E)-5-(2-bromovinyl)-2'-deoxyuridine and interferon treatment.