APPENDIX B 
I) Intravenous exposure to Amphotropic Retrovirus. 
Three normal rhesus monkey and one animal immunosuppressed with prednisone 
and cyclosporine A received intravenous injection of amphotropic retrovirus (mean 
dose - 7.2x10^ ffu/animal) . Monitoring the animals serum for replication 
competent virus demonstrated a rapid disappearance of virus, with no detectable 
virus present 30 min after exposure. Tne animals have been followed for a mean of 
10.8 months with no detectable retroviremia, both by the S+/L- assay and 3T3 
rescue. The rapid clearance may, in part, be related to the ability of primate 
complement to inactive murine retroviruses. 
Clinically the animals have had no temperature elevation, abnormal behavior 
or change in complete blood count or blood chemistries. Physical exam has been 
unremarkable except for 2 animals that developed asymptomatic lymphadenopathy 7-8 
days after virus infusion. Lymphadenopathy resolved spontaneously within 7 days. 
Enlarged lymph nodes were surgically removed and attempts to document viral 
infection and replication were performed on the tissue directly and after 
culturing. We were unable to demonstrate evidence of retroviral sequences or 
recover infection virus. Since the animals received viral supernate, which 
contains 10% fetal calf serum it is possible that the adenopathy represents an 
immune response to injected antigens rather than retroviral replication. 
Western blot analysis has demonstrated the development of antibodies to the 
p30 antigen of murine leukemia virus. Of the 4 animals, 3 have developed 
detectable antibody 30 to 120 days after exposure. 
Summary ; Murine amphotropic retrovirus is cleared rapidly from the blood stream 
and replicating virus could not be detected in lymphocytes from animals with 
lymphadenopathy. Animals have shown no clinical consequences with a mean follow 
up of 10.8 months (range: 7.5 to 13.4). Antibodies appear between 30 and 110 days 
and are detectable at one year of follow-up. 
II) Exposure to retrovirus by implantation of infected virus producing autologous 
fibroblasts and intraperitoneal injection of viral supernate. 
To increase the intensity of retroviral exposure we chose to circumvent 
complement inactivation by introducing the virus on collagen pads containing 
autologous skin fibroblasts infected in vitro and shown to be producing helper 
virus. In addition we injected these fibroblasts, with viral supernate, 
intraperitoneally . The one animal studied was immunosuppressed with cyclosporine 
prior to treatment. Using 3T3 amplification we were able to detect helper virus 
up to 2 days post infusion. Collagen pads were removed at various intervals and 
were shown to be producing virus at the time of removal. Virus was recovered from 
peritoneal lavage fluid one week after inoculation. Lymphadenopathy developed 
systemically but most significantly in the area draining the collagen pads. Lymph 
node and peripheral blood lymphocytes were shown to be producing retrovirus. 
Virus could not be recovered from stool and urine samples. Immunosuppression was 
discontinued and the animal immune system allowed to recover. Lymph node tissue, 
peripheral blood lymphocytes, peritoneal lavage fluid and serum samples evaluated 
84 days after exposure had no detectable retrovirus. Antibodies to p30 antigen 
were detectable at this time. 
Recombinant DNA Research, Volume 13 
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