A COMPREHENSIVE PANEL OF NEAR-FULL-LENGTH CLONES AND REFERENCE SEQUENCES FOR NON-SUBTYPE-B ISOLATES OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1

Non-subtype B viruses cause the vast majority of new human immunodefic iency virus type 1 (HIV-I) infections worldwide and are thus the major focus of international vaccine efforts. Although their geographic dis semination is carefully monitored, their immunogenic and biological pr operties remain largely unknown, in part because well-characterized vi rological reference reagents are lacking. In particular, full-length c lones and sequences are rare, since subtype classification is frequent ly based on small PCR-derived viral fragments. There are only five pro viral clones available for viruses other than subtype B, and these rep resent only 3 of the 10 proposed (group M) sequence subtypes, This lac k of reference sequences also confounds the identification and analysi s of mosaic (recombinant) genomes, which appear to be arising with inc reasing frequency in areas where multiple sequence subtypes cocirculat e, To generate a more representative panel of non-subtype B reference reagents, we have cloned (by long PCR or lambda phage techniques) and sequenced 10 near-full-length HIV-1 genomes (lacking less than 80 bp o f long terminal repeat sequences) from primary isolates collected at m ajor epicenters of the global AIDS pandemic, Detailed phylogenetic ana lyses identified six that represented nonrecombinant members of HIV-1 subtypes A (92UG037.1), C (92BR025.8), D (84ZR85.1 and 93UG114.1), F ( 93BR020.1), and H (90CF056.1), the last two comprising the first full- length examples of these subtypes, Four others were found to be comple x mosaics of subtypes A and C (92RW009.6), A and G (92NG083.2 and 92NG 003.1), and B and F (93BR029.4), again emphasizing the impact of inter subtype recombination on global HIV-1 diversification. Although a numb er of clones had frameshift mutations or translational stop codons in major open reading frames, all the genomes contained a complete set of genes and three had intact genomic organizations without inactivating mutations, Reconstruction of one of these (94UG114.1) yielded replica tion-competent virus that grew to high titers in normal donor peripher al blood mononuclear cell cultures, This panel of non-subtype B refere nce genomes should prove valuable for structure-function studies of ge netically diverse viral gene products, the generation of subtype-speci fic immunological reagents, and the production of DNA-and protein-base d subunit vaccines directed against a broader spectrum of viruses.