Activation of Latent Murine Cytomegalovirus in Cardiac Explant and Cell Cultures

Cytomegalovirus (CMV) produces a latent infection in its host. The actively or latently infected transplanted kidney and heart are sources of debilitating CMV infection in transplant recipients [1], but the organs and cellular sites of viral latency and the mechanisms of CMV activation have not been fully defined. Latent murine cytomegalovirus (MCMV) has been activated in vitro by cocultivating spleen cells or peritoneal macrophages with allogeneic, syngeneic, or both mouse embryo cultures (MECs) [2-5]. MCMV has also been activated from explant cultures of spleens and prostate glands, cultured with or without MECs [6, 7]. Cultures of salivary glands, liver, kidney, and brain from latently infected mice, however, have failed to yield MCMV in vitro [8]. Latently infected, heterotopic cardiac isografts are the source of lethal MCMV infection in the immunosuppressed recipient [9]. Results presented below demonstrate the activation of latent MCMV from cardiac explants and dissociated cardiac cell cultures when cultured alone or with allogeneic MECs. The Smith strain of MCMV was maintained in this laboratory by serial passage of CD-1 Swiss mice (Charles River Breeding Laboratory, Wilmington, Mass) followed by salivary gland harvest as described elsewhere [9]. The stock virus had a titer of 2 x 10' pfu/ml. Noninfected salivary gland homogenates (NSG) were prepared in a similar manner after inoculation of weanling mice with saline. Virus stocks and NSG were stored at 70 C until use. Latent infection was established by ip inoculation of five-weekold female BALB/c mice (Cumberland View Farms, Clinton, Tenn) with 104 pfu of MCMV (0.1 ml of infected salivary gland homogenate). These mice were then maintained in our animal facility for four to six months, at which time their hearts were used for tissue culture. Control animals had been injected with an equal volume of NSG and maintained for four to six months in the same way. These animals showed no evidence of virus by salivary gland culture or by antibody testing at the time of death. Explants of cardiac tissue were prepared by aseptically removing hearts from latently infected or NSG-inoculated mice and placing them in cold, lactated Ringers solution with 507o dextrose. Hearts were transferred into petri dishes containing fresh Ringers solution and cut into 1x 2-mm pieces that were kept on ice. Secondary MECs (prepared as previously described [9] from pathogen-free CD-1 Swiss mice) seeded the previous day in six-well plates (Falcon Plastics, Oxnard, Calif) containing RPMI medium with