This project will test the hypothesis that the perivascular macrophage is a critical cellular target in SIV infection. In the first specific aim, the Principal Investigator and his associates will determine the in vivo tropism of SIV for discrete subpopulations of brain macrophages during the progression of SIV encephalitis (SIVE). This will be performed in both retrospective and prospective studies. In the retrospective studies, the researchers will analyze animals previously infected with uncloned SIVmac251, a strain that leads to a higher percentage of animals developing SIVE than molecularly cloned strains. These studies will consist primarily of immunohistochemical analyses of banked tissue using monoclonal antibodies against a number of macrophage markers, and of differentiating perivascular brain macrophage from microglia by the differential reactivity with antibodies against CD45 and CD14. Expression of macrophage markers will be correlated with expression of SIV DNA and RNA using in situ hybridization. For the prospective studies, the researchers will isolate relatively pure populations of perivascular and parenchymal macrophages/microglia from SIVmac251-infected macaques. Infectivity will be quantified by the recovery of virus using a permissive cell line, and by quantitative PCR. For these experiments, the researchers will use animals sacrificed at two weeks after infection--a time when the Investigator believes that the predominantly infected cell is perivascular--and compare the results with animals sacrificed at later time points. In the second specific aim, the researchers will identify unique viral genotypes within the swarm of viruses used for inoculation. These studies are based on the observation that specific SIV and HIV sequences are associated with infection of target organs. To accomplish this aim, the researchers will isolate subpopulations of brain macrophages using CD14 and FACS. Viral sequences from the sorted cells will be amplified using PCR, and the genotypes for env and nef will be analyzed. The choice of genes for these experiments is based on the observation that these two genes are of particular relevance in the determination of neurotropism, as indicated in previous work from the NERPRC. The CNS macrophage tropic sequences will be inserted into an SIVmac239 backbone, and the replication in an in vitro system will then be analyzed. Ultimately, these recombinant viruses will be tested in monkeys.
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