Nucleotide diversity in three different genomic regions of Venezuelan HIV type 1 isolates: a subtyping update.

Abstract

73 HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 (HIV-1) infection continues its rapid global spread. A short time ago a few HIV-1 subtypes based on env sequences were identified, and outside the African continent most HIV-1 isolates belonged to subtype B. However, the rapid genetic diversification of HIV1 is reflected in the current separation of isolates into two groups (M and O), and an increasing number of subtypes within each group. New subtypes are finding their way outside the geographical areas where they were first isolated.1,2 Continuous reports of additional a non-subtype Bo HIV-1 infections around the world underscore the need for surveillance to track HIV-1 variants and to further define the molecular basis of their genetic variation. In the American continent and the Caribbean region, HIV-1 subtype B is dominant. However, the detection of HIV-1 of different subtypes in this area (i.e., subtypes A, C, D, E, and F, the group O) exemplifies the spread of HIV1 clades well beyond their original geographical locations.4±6 In addition, with more than 1.5 million estimated HIV infections, Latin America and the Caribbean follow Africa and southern Asia as the macroregion with the highest number of estimated HIV infections. We have undertaken the first molecular characterization of HIV-1 isolates from Venezuela.3 These initial viruses (VE1 to VE8) were isolated in 1991, and an analysis of pol and env genomic segments identified them as belonging to subtype B. In the present report 20 additional isolates of HIV-1 from Venezuela have been molecularly characterized on the basis of three distinct, noncontiguous genomic regions: the long terminal repeat (LTR), the first exon of the tat gene, and the env region encoding the C2V3 domain of gp120, with the aim of determining the extent of their genetic variation, and to characterize sequence relationships and evolutionary pattern in recent years. Twenty-eight blood samples were obtained in two periods: a first group of 8 samples (VE1 to VE8) was collected in 1991,3 and the second group includes samples from 1995. Epidemiological and clinical data are provided in Table 1. Proviral DNA was extracted as previously described. Three genomic regions were amplified for each DNA sample: 560 nucleotides of the HIV-1 LTR (positions 2 285 to 1 275 from the transcription start site), the first exon of the tat gene (216 bp), and an internal fragment of 1.2 kb spanning the gp120 V1±V5-coding region of env. Nested polymerase chain reaction (PCR) was used for all amplifications. PCR conditions and primers used to amplify the LTR region and the first exon of tat have been previously described.7 To amplify the V1±V5-coding region of env, the primers described in Delwart et al.8 were used. The PCR products of env were compared with those of 14 previously characterized HIV-1 strains1 by a heteroduplex mobility assay (HMA) as previously described. Nucleotide sequencing was performed using the fmol method (Promega, Madison, WI), followed by treatment with terminal deoxynucleotidyl transferase, as described.9 LTR (nucleotides 170 to 730 of the CAM1 genome1) and tat (nucleotides 5832 to 6047 of the CAM1 genome1) were sequenced using the inner primers LTR3/LTR4, and TAT3/TAT4, respectively. The C2V3-coding region (nucleotides 6817 to 7218 of the CAM1 genome) was sequenced as described previously.9 Initial subtyping experiments involved characterization of the new isolates by heteroduplex mobility assay,8 using reference plasmid DNA corresponding to HIV-1 subtypes A to H (heteroduplex mobility analysis HIV-1 env subtyping kit protocol version 3; NIH AIDS Research and Reference Reagent Program). Comparison of 28 Venezuelan samples with standard probes of several subtypes showed unequivocal electrophoretic patterns, corresponding to subtype B (data not shown). The epidemiological relationships of HIV-1 isolates from Venezuela with other HIV-1 isolates were further analyzed by phylogenetic analyses based on nucleotide sequences of the LTR region, the first exon of tat, and the C2V3 domain of the gp120coding region. Venezuelan HIV-1 isolates clustered with subtype B viruses in all cases (Fig. 1).10±14 Nucleotide diversities among Venezuelan samples were similar for the three genomic segments analyzed. Pairwise comparisons showed an average divergence of 6.6% (range of 1.4 to 11.0%), 8.0% (1.2 to 14.9%), and 9.4% (1.5 to 13.8%) for LTR, tat, and env, respectively. Average genetic distances be-