As the pandemic caused by HIV-1 spreads, analysis of lentiviral pathobiology and drug development need to proceed rapidly, but progress is slowed by the lack of a practical small animal model. The use of laboratory mice would offer well-studied genetics, immunology and metabolism, and the availability of many immunological reagents, but no murine lentivirus is known yet. The overall goal of this proposal is to establish a murine model for lentivirus-induced immunodeficiency. Thus, we shall: 1. Trap cricetid an murid rodents worldwide, identify the animals, collect sera, freeze splenocytes to preserve viability and extract DNA directly from some organs. 2. Screen feral rodent sera by Western blot with known lentiviral antigens, using strips from type B and C murine retroviruses as controls, to priortize regions or animal species for further collection. 3. Directly isolate virus from viably frozen splenocytes, which will be stimulated with concanavalin A and recombinant human IL-2, cocultivated with cells derived from Mus musculus and screened for cytopathic effects (CPE). Supernatants will be tested for Mg2+ preferring reverse transcriptase activity, a hallmark for all known lentiviruses. Virus will be biologically cloned by end point dilution. 4. Directly clone lentiviral sequences from genome DNA via polymerase chain reaction (PCR). A mixture of primers from well conserved gag-pol regions shared by the known lentiviruses but not by Type B or C murine retroviruses will be used to generate probes for subsequent cloning. Computer analysis of rodent virus DNA sequences will be used to construct phylogenetic relationships with known viruses. 5. Molecularly clone Hirt DNA from biologically cloned viruses or proviral sequences yielding positive PCR signals, and characterize candidate murine lentiviruses in vitro. After transfecting full length proviral DNA into a panel of cell lines, progeny virus will be tested for cross-reactivity with antisera against known viruses, electron microscopy, CPE, cell surface receptor dependency, and host range. Rodent lentiviral gene functions will be compared to those of HIV. 6. Analyze molecularly cloned viruses for pathogenicity in vivo, initially using neonates of inbred laboratory mice (Mus musculus) or clean colonies of other species of Mus of Peromyscus. If no laboratory stains can be infected, pathogen-free stains of wild rodents will be developed. 7. Test known pharmacological inhibitors of HIV replication for their potential to inhibit propagation of the newly discovered murine lentivirus(es) in vitro and in vivo. The significance of this work lies in the rational approach to isolate murine lentiviruses. Our multidisciplinary team of experienced investigators using tissue culture and direct cloning techniques promises success.
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