Molecular changes in Nef with progression to AIDS. We have compared nef gene sequences isolated by polymerase chain reaction from peripheral blood lymphocyte DNA of macaques which had been inoculated with either biologically (E11S) or molecularly cloned SIV/Mne (clone 8). Two samples from each animal obtained early (wk 2-8) or late (wk 21-137) after infection were analyzed. Four substitutions in the predicted Nef amino acid sequence were seen in all animals at the late time point, and a fifth in all but one. Animals inoculated with virus already containing the """"""""late version"""""""" nef gene developed a more aggressive disease. Recombinant virus containing a macaque adapted nef (MA-nef) on the clone 8 backbone was threefold more infectious on SMAGI cells than the original virus. A lymphocyte line infected with SIV-clone8MA-nef contained a large proportion of cells carrying proviruses with defective nef genes. These findings suggest that the nef gene of the cloned SIV/Mne had undergone attenuating mutations during propagation in tissue culture which were """"""""corrected"""""""" in vivo. Chimeric viruses containing the late nef gene sequence associated with AIDS will be tested for pathogenicity in macaques in vivo. Screening macaques for the presence of type D retroviruses. The SIV macaque model is the preferred nonhuman primate model for AIDS related vaccine, antiviral, and pathogenesis research. A necessary component of this animal model is the availability of macaques that are free of retrovirus infections that might confound pathogenesis and vaccine studies with SIV. Macaques are known to be infected with various retroviruses, including type D viruses (SRV), STLV-I, and foamy (spumaretroviruses). SRV infection is prevalent in wild-caught macaques as well as in colony born animals. Five neutralization types have been identified (SRV1 - SRV5). These infectious type D retroviruses cause an often fatal immunosuppressive disease in macaques that is manifested by progressive weight loss, persistent diarrhea, anemia, opportunistic infections, and unusual neoplasms. Since the experimental infection of macaques with SIV results in similar clinical outcomes, animals used in AIDS experiments must be free of other retrovirus infections. Three species of macaques, rhesus, cynomolgus, and pigtailed, have been screened for the presence of type D retroviruses (SRV). A combination of serological, virus isolation and DNA polymerase chain reaction (PCR) assays were used to identify SRV infected macaques. Serological screening for SRV alone is not sufficient because not all infected animals seroconvert. Virus isolation by coculture of peripheral blood mononuclear cells (PBMCs) with various indicator cell lines is time consuming (4 to 8 weeks) and variable due to the presence of antibodies and differing virus titers in infected animals. Several PCR primers were compared for their sensitivity and ability to detect SRV subtypes 1-5 present in PBMC DNA. One primer set has been identified as being the most sensitive for the detection of SRV-1, SRV-2, and Mason Pfizer monkey virus (MPMV, SRV-3). This primer set detected one DNA proviral copy per 50,000 cells and identified the greatest number of SRV positive animals. We have previously reported on a recombinant vaccine which is 100% effective in preventing SRV-2 infection in vaccinated animals. Additional studies are planned to test this vaccine in a colony setting (group-housed macaques) for efficacy in preventing infection from a variety of SRV isolates.
