Replication-competent murine retroviruses (RCRs) can arise unexpectedly and spontaneously from retrovirus-based packaging cell systems used in human gene therapy products. RCRs can infect a wide variety of primate cells including human. Since the risk associated with RCRs in man is unknown, rigorous testing for RCRs is recommended to exclude possible contamination of product. Although previous studies have shown that RCRs were cleared when inoculated into normal and moderately immunosuppressed monkeys (Cornetta et al, 1991), a subsequent study reported RCR to be associated with development of lymphomas in severely immunosuppressed rhesus monkeys (Donahue et al, 1992). To evaluate the potential of RCR infection in humans, 4 normal, juvenile rhesus monkeys were inoculated with a vector virus preparation which contained RCR (provided by Genetic Therapy Inc., Gaithersburg, MD). The monkeys were monitored regularly for virus infection by 1) isolation of infectious MuLV from the PBMCs; 2) Western blot analysis for antibody response; and 3) DNA PCR for persistence of viral sequences in the host. Clinical changes were monitored by serum chemistry, hematology and physical examination. The results of a 3-year analysis indicated that all 4 animals were infected with RCR. There was early, transient virus isolation; persistence of viral DNA sequences in the PBMCs and long-term antibody response. Each animal responded uniquely with respect to the kinetics of infection and antibody response. In two of the animals (AG6 and A4W), where high virus infection was initially established, a gradual decrease in the number of WBC and lymphocytes was seen over the 3 year period; however, no abnormalities were noted on preliminary bone marrow examination. In the beginning of the 6th year post-inoculation A4W was euthanized due to diarrhea and significant weight loss. Clinical and histological evaluation indicated retroviral-mediated immune suppression. To investigate the role of RCR in the disease, the absence of SRV was confirmed by PCR and antibody testing by Dr. N. Lerche (Calif. Primate Center). In situ hybridization analysis of various tissues are currently underway for the detection of RCR as well as for simian foamy virus (SFV) which was also found (retrospectively) to be present in the animal at the time of inoculation. The results will be important in evaluating the potential risk of long-term RCR infection in man.