This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Primary SIVsm isolates are significantly less pathogenic in Rh than the serially passaged SIVmac251/239. Our hypothesis is that the use of SIVsm strains representing different clades will generate a better model for AIDS pathogenesis. Our objective is to construct and characterize in vitro and in vivo infectious molecular clones of SIVsm strains belonging to different, well-defined phylogenetic lineages (clades). Total RNA from the sera of SIV infected animals M926, M946 (lineage 2), D215 (Lineage 6), FTQ (Lineage 5) and M923 (Lineage 1, recombinant strain) was isolated. An 800 bp RT-PCR fragment spanning the 3' end of the LTR sequence was generated, as well as an approximately 1.3 kb RT-PCR fragment encompassing the nef gene and extending into the 5' end of the LTR. Using an overlap-extension technique, an approximately 2 kb DNA fragment derived from all these viruses was generated and five clones from each strain were sequenced directly to produce consensus virus sequences. cDNA fragments were also cloned into a PCR II TOPO vector and 4 clones of each derived virus were sequenced. An approximately 600 bp RT-PCR product specific for SIVsmm isolates viruses was generated. These DNA sequences, in combination with the described above 2 kb DNA fragments, allowed us to design specific primers to produce the 9 kb of the virus. An attempt to generate an RT-PCR product of approximately 9 kb long was not successful. The entire nucleotide sequences representing the M926, D215 and FTQ were generated as overlapping RT-PCR fragments of approximately 2 kb, 5 kb and 4 kb. We are in the process assembling entire viruses. An array of infectious molecular clones, better controlled by the immune system will provide enhanced and very much needed tools to the research community for AIDS immunopathogenesis and vaccine development investigating heterotypic protection.
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