Diversity of human immunodeficiency virus type 1 subtype A and CRF03_AB protease in Eastern Europe: selection of the V77I variant and its rapid spread in injecting drug user populations

Abstract

To characterize polymorphisms of the subtype A protease in the former Soviet Union, proviral DNA samples were obtained, with informed consent, from 119 human immunodeficiency virus type 1 (HIV-1)-positive untreated injecting drug users (IDUs) from 16 regions. All individuals studied have never been treated with antiretroviral drugs. The isolates were defined as IDU-A (n = 115) and CRF03_AB (n = 4) by using gag/env HMA/sequencing. The pro region was analyzed by using sequencing and original HIV-ProteaseChip hybridization technology. The mean of pairwise nucleotide distance between 27 pro sequences (23 IDU-A and 4 CRF03_AB) was low (1.38 +/- 0.79; range, 0.00 to 3.23). All sequences contained no primary resistance mutations. However, 13 of 23 (56.5%) subtype A isolates bore the V77I substitution known as the secondary protease mutation. V77I was associated with two synonymous substitutions in triplets 31 and 78, suggesting that all V77I-bearing viruses evolved from a single source in 1997. Hybridization analysis showed that 55 of 115 (47.8%) HIV-1 isolates contained V77I, but this variant was not found in any of 31 DNA samples taken from regions, where the HIV-1 epidemic among IDUs started earlier 1997, as well as in any of four CRF03_AB isolates. The results of analysis of 12 additional samples derived from epidemiologically linked subjects showed that in all four epidemiological clusters the genotype of the donor and the recipients was the same irrespective of the route of transmission. This finding demonstrates the transmission of the V77I mutant variant, which is spreading rapidly within the circulating viral pool in Russia and Kazakhstan. The continued molecular epidemiological and virological monitoring of HIV-1 worldwide thus remains of great importance.

FIG. 1.

Phylogenetic tree of Eastern European subtype A nucleotide sequences encoding protease. Horizontal lines indicate nucleotide difference between sequences; vertical spacing is for clarity only. The bootstrap value (of 100 replicates) supporting a particular cluster is placed next to the nodes. The Kaliningrad CRF03_AB recombinant sequences are marked with an “r.” Nucleotide sequences bearing the V77I substitution are marked with asterisks. Phylogenetic relationships were determined as described in Materials and Methods. The Eastern European nucleotide sequences from the Ukraine (98UA0116), Belarus (97BL006 and BY10443), Russia (RU98001 and KAL153), Moldova (97MD10 to -19), and Spain (LP27) published previously (GenBank accession numbers AF413987, AF193275, AF414006, AF193277, AF193276, AF400675 to AF400680, and AF455622, respectively) (18, 25, 26, 28, 34), as well as the representative sequences of genetic subtypes A1, A2, B, and C from the Los Alamos database, were also included in the analysis.

FIG. 2.

Alignment of amino acid sequences deduced from the HIV-1 subtype A protease gene. The protease sequences from IDUs are aligned with the subtype A consensus sequence derived from IDUs previously. The subtype A1 consensus and subtype B consensus from the Los Alamos database, as well as the subtype A IDU protease sequences published previously, were also included in the alignment.

FIG. 3.

Scheme of HIV-ProteaseChip. The darkly shaded pads contain oligonucleotides corresponding to the wild-type sequence. The lightly shaded pads contain the mutant oligonucleotide probes.

FIG. 4.

Geographic distribution of the V77I subtype A HIV-1 IDU variant, as well as a wild-type subtype A IDU strain, in the former Soviet Union from 1997 to 2003. Assignments are based upon analysis of several independent cases from each region (Tables 1 and 3).