IDENTIFICATION OF SINGLE AND DUAL INFECTIONS WITH DISTINCT SUBTYPES OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 BY USING RESTRICTION-FRAGMENT-LENGTH-POLYMORPHISM ANALYSIS

The simultaneous presence of multiple HIV-1 subtypes has become common in communities with the growth of the pandemic, As a consequence, the potentiality for an increased frequency of HIV-1 mixed infections cau sed by viruses of distinct subtypes could be expected. Thus, there is a need to estimate the prevalence and geographic distribution of infec tions caused by viruses of a singular subtype as well as coinfections caused by two or more HIV-1 strains of distinct subtypes, To address t his need, we have developed a genetic method based on restriction frag ment length polymorphism (RFLP) to screen for these two types of infec tions within infected populations. In this assay, restriction enzymes may be used to predict the phylogroup of HIV-1 infected samples, A 297 bp pol fragment spanning the entire viral protease gene and a 311 bp fragment of the p24 gag region are used for this analysis, The viral r egions are amplified by nested PCR using DNA templates from uncultured peripheral blood mononuclear cells (PBMC) or virus culture. Classific ation of HIV-I strains to well defined subtypes B, D, F, and A/C is do ne by sequential endonuclease restriction analysis of a PCR amplified- protease gene followed by analysis of the p24 gag region. The electrop horetic migration patterns visualized by ethidium bromide staining or by radiolabeled probes are then determined on a 10% polyacrylamide gel , In infections caused by viruses of a singular subtype, a single rest riction pattern is detected, whereas in multiple infections caused by two or more viral strains of different subtypes, the combination of di fferent digestion patterns are observed in infected individuals, Using this methodology we have screened for genetic variations in HIV-1 pro viral DNA from thirty-three Brazilian samples, Our RFLP procedure clas sified thirty-two samples as single infections caused by viruses of su btypes B (31) and F (1), and one sample as dual infection caused by di stinct viral strains, Subsequent sequence and phylogenetic analysis of the viral protease gene in lymphocytes of all these patients confirme d our RFLP findings in single infections, and demonstrated the existen ce of two distinct HIV-1 strains of subtypes F and D in a patient whic h lymphocytes showed the simultaneous presence of two different digest ion patterns. As up to now, single infections caused by subtype D vari ants were not identified in Brazil, our data provide the first evidenc e of subtype D HIV-1 in this country. Because sequencing of HIV provir al DNA is not particularly practical for large-scale molecular epidemi ological studies, the protease/gag-based RFLP screening method will be useful to predict the phylogroup of HIV-1, and to identify multiple i nfections caused by HIV-1 strains of distinct subtypes. We believe tha t this information is crucial for both evaluation of the HIV-1/AIDS pa ndemic and intervention strategies.