Distinct variation pattern in the env of macrophage-tropic simian immunodeficiency virus in vivo demonstrated by denaturing gradient gel electrophoresis.

Genetic variations, occurring during the infection in the AIDS animal model using a molecular clone virus, often consist of single nucleotide substitutions distributed sparsely in the viral genome. A highly sensitive method was developed to detect such mutations, consisting of denaturing gradient gel electrophoresis (DGGE) and direct sequencing. The genetic variation in the env of a macrophage tropic simian immunodeficiency virus (SIV) in a macaque infected chronically was examined by this technique and compared with that of a T cell tropic SIV in an animal infected chronically. A whole env sequence was amplified by 11 sets of PCR for DNA ranging from 300 to 420 bp. The amplified DNA was subjected to mutational screening by DGGE and to subsequent direct sequencing. Imaging analysis of separated DNA bands by DGGE provided information on the proportion of each variant DNA. The validity of this technique was confirmed by sequencing of variant DNAs cloned by limiting dilutions. Thus, this technique is suitable for analysis of genetic variations in the AIDS animal model using a molecular clone virus.