Short Communication: Molecular Epidemiology of HIV Type 1 in the Republic of Dagestan,Russian Federation: Virtually Uniform Circulation of Subtype A,Former Soviet Union Variant,with Predominance of the V77I PR Subvariant

R ussia is the European country with the greatest number of HIV-1 infections, with 940,000 cases estimated at the end of 2007. ¹ The main transmission mode is through needle sharing among injecting drug users (IDUs), although secondary transmission via heterosexual contact has experienced an important increase in recent years. ² The HIV-1 epidemic in Russia is dominated by a subtype A variant ^{3 –5} of Central African origin, ⁶ which, after an initial limited transmission by heterosexual contact, ⁶ began to spread among IDUs in the southern Ukrainian city of Odessa in early 1995, ^7,8 with subsequent propagation into Russia since 1996. ⁹ This variant, which dominates the epidemics of most former Soviet Union (FSU) countries, is often referred to as A_FSU. The A_FSU variant has split into diverse subvariants, the most prevalent of which is the one containing the characteristic V77I substitution in protease ^4,10,11 (henceforth designated V77I_PR), which circulates widely in diverse areas of Russia and other FSU countries. Other subvariants were recently identified in St. Petersburg (A_SP1, A_SP2, and A_SP3), one of which (A_SP1) comprised around 60% of the infections in the city. ¹¹ Subtype B circulates in Russia mostly among homosexual men. ^3,4,11 Other genetic forms circulating at lower prevalences in Russia are CRF03_AB, ^5,12,13 subtype C, ^4,11 and subtype F. ¹⁴

In recent years there have been reports on a rapid increase of HIV-1 infections in the North Caucasus area of Russia, including Chechnya and Dagestan ¹⁵ (Fig. 1). However, no data on the distribution of HIV-1 variants or on the prevalence of drug resistance mutations in this area of the Russian Federation have been published. In the Caucasus area, there are reports only from the neighboring South Caucasus countries of Georgia and Azerbaijan, in which A_FSU is predominant, representing 70% and 90.2% of the infections, respectively, with subtype B representing 26% and 7.3% of the infections, respectively. ^16,17

OPEN IN VIEWER

FIG. 1.

Chronological evolution of the HIV-1 epidemic in Dagestan. ¹⁵

In this study, we analyzed 41 samples from HIV-1-infected individuals collected in Dagestan in November 2006. Subjects were from the cities of Derbent (n = 21), Mahachkala (n = 18), Hasaburt (n = 1), and Buinaks (n = 1). Samples from Derbent were collected at the local prison and those from residents in Mahachkala, Hasaburt, and Buinaks were collected at the Republic's Center for Infectious Diseases, AIDS and Prevention in Mahachkala. Thirty six were injecting drug users and five were infected by heterosexual contact. Ages ranged from 20 to 52 years old (mean 28.1). None was on antiretroviral drug treatment. RNA extraction from plasma and reverse transcriptase polymerase chain reaction (RT-PCR) amplification and sequencing of protease and a segment of reverse transcriptase (PR-RT) were done as previously described. ¹⁸ Briefly, RNA was extracted from plasma using the reagents provided in the Nuclisens kit (Biomerieux, Marcy l'Etoile, France); RT-PCR was performed with a mixture of SuperScript III (Invitrogen, Carlsbad, CA) and Biotaq DNA polymerase (Bioline, London, UK), followed by nested PCR performed with Biotaq DNA polymerase, enzymatic purification of the PCR product using exonuclease I and shrimp alkaline phosphatase, and sequencing with the ABI Prism BigDye Terminator kit, with analysis in ABI PRISM 3700 automated sequencer (Applied Biosystems, Foster City, CA). The sequenced segment used for phylogenetic analysis is 1.2 kb long, comprising nucleotides 2253–3452 in the HXB2 proviral genome. Phylogenetic trees were constructed via maximum likelihood (ML) using Treefinder. ¹⁹ For tree construction, we included A_FSU references, and within this variant, sequences of viruses representative of the V77I_PR subvariant, and the recently identified St. Petersburg clusters (A_SP1, A_SP2, and A_SP3). The best-fit model of sequence evolution was selected under the Akaike information criterion using jModeltest v.0.1.1. ²⁰ The reliability of tree topologies was assessed using 100 bootstrap replicates. The presence of antiretroviral drug resistance-associated mutations was analyzed with the Stanford University HIVdb Program. ²¹

The ML tree obtained with the Dagestan and reference sequences is shown in Fig. 2. Forty sequences clustered with the A_FSU variant, and only one sequence was of subtype B. Of the 40 A_FSU sequences, 27 (67.5%) formed a cluster corresponding to the V77I_PR subvariant, and 4 (10%) formed another cluster nested within the A_SP2 subcluster, a minor A_FSU variant of St. Petersburg. ¹¹ Within the V77I_PR clade, 13 (48.1%) viruses formed a local subcluster, 10 of which were from Derbent (Figs. 2 and 3). To examine the relationship of the Dagestan viruses with viruses from neighboring areas, sequences of all subtype A sequences from Georgia ¹⁶ and Azerbaijan ¹⁷ available at the Los Alamos HIV Sequence Database ²² were included in the alignment, which was analyzed with Treefinder. In the resulting tree, a virus from Dagestan (Dag42) clustered with a virus from Azerbaijan (02AZ098) and another two viruses of Dagestan (Dag03 and Dag35) clustered with a virus from Georgia (03GEMZ008), with 91% and 84% bootstrap values, respectively (results not shown). No antiretroviral drug resistance mutations were detected in any of the samples.

OPEN IN VIEWER

FIG. 2.

Maximum likelihood phylogenetic tree of PR-RT sequences from Dagestan. The analyzed segment comprises nucleotide positions 2253–3452 in the HXB2 proviral genome. Names of viruses from Dagestan start with Dag. A_FSU references are underlined. Sequences preceded by A_SP1, A_SP2, and A_SP3 represent recently described clusters of St. Petersburg. The tree was constructed with Treefinder ¹⁹ using the TVM + Γ + I substitution model selected by jModeltest ²⁰ under the Akaike information criterion. A subcluster of viruses comprising viruses mainly from Derbent is bracketed on the right. Only bootstrap values of 70% or greater of relevant clusters are shown.

OPEN IN VIEWER

FIG. 3.

Distribution of HIV-1 clades in Dagestan.

The results, therefore, indicate an overwhelming predominance (97.6%) of the A_FSU variant in the Dagestan epidemic. This is similar to the prevalence reported in Azerbaijan (90.2%), ¹⁷ and higher than that reported in Georgia (70%). ¹⁶ Among the A_FSU viruses, the V77I_PR subvariant is predominant, representing approximately two-thirds of the viruses. This subvariant has also been detected in Georgia, where it represents 36.4% of A_FSU viruses. ¹⁶ Subcluster analysis has not been reported in Azerbaijan, ¹⁷ but our own analysis with sequences retrieved from databases shows that 44.8% A_FSU viruses from this country are of the V77I_PR subvariant (results not shown). Further phylogenetic structure was detected in the V77I_PR subvariant, with 10 viruses from Derbent and 3 from Mahachkala forming a subcluster nested within it.

The results reported here and elsewhere ^4,6,10,11 show that the A_FSU variant has undergone multiple successive splits along its expansion in FSU countries, representing successive introductions at different geographic areas and at different times. Some subvariants have remained largely confined to a geographic area, such as those recently identified in St. Petersburg, although sporadic introductions have occurred into distant areas, ¹¹ including, as here reported, Dagestan. On the other hand, one of the A_FSU subvariants, V77I_PR is widely circulating at different geographic areas of Russia and other A_FSU countries, representing a substantial proportion of viruses in some of them. ^{4,10,11,16,17,23} We recently reported its presence in 17.4% of A_FSU samples from St Petersburg, ¹¹ and in a previous study on samples from diverse FSU countries, the great majority of them from Russia, we found V77I_PR in 49.4% subtype A viruses. Outside of Russia, it is also circulating in Georgia (36.4%), ¹⁶ Azerbaijan (44.8%), ¹⁷ and Kazakhstan (58.8%). ²³ The current study now reports its presence in Dagestan, where it represents 67.5% of A_FSU viruses in this Republic of the Russian Federation. This subvariant has undergone further splits, as shown here in the cluster identified in Derbent. The molecular epidemiological study of the HIV-1 epidemic in FSU countries, therefore, reveals the progressively increasing complexity of the HIV-1 evolution along its expansion, with successive splits of HIV-1 clades into different subclades and subsequent formation of intraclade recombinant forms (unpublished results). The increasing complexity of the HIV-1 evolution along its expansion, as evidenced in the A_FSU epidemic, should be taken into consideration in a future revision of the nomenclature system of HIV-1 genetic forms, which is not properly addressed by the current classification. ²⁴

The identification of large proportions of viruses grouping into phylogenetic clusters within the A_FSU variant is in contrast to findings in most countries where subtype B is predominant, in which, with some exceptions, ^25,26,27 no such structured phylogenies are found, except for relatively minor clusters of recent origin. ^28,29 One of the reasons for this difference may be that in longstanding epidemics, such as those of subtype B in Western countries, the distinction among clusters may be blurred by extensive intrasubtype recombination, which accumulates progressively over time and interferes with the reconstruction of the original phylogenetic structure of the viruses by using the usual phylogeny inference methods. In the case of the subtype A_FSU epidemic, being relatively young, the original phylogenetic structure may still remain relatively intact, allowing reconstruction of phylogenetic clusters with commonly used programs.

In conclusion, the HIV-1 epidemic in Dagestan is largely dominated by the A_FSU variant, with a predominance of the V77I_PR subvariant, within which approximately half of the viruses belong to a local subcluster, most of them from the city of Derbent. Sporadic relationships were found with viruses from St. Petersburg, Georgia, and Azerbaijan. The phylogenetic characterization and the knowledge on the distribution of HIV-1 variants circulating in Russia and other FSU countries will contribute to tracking the expansion of the epidemic in this geographic area and may be important in examining correlations of HIV-1 clades with immune responses relevant for vaccine development and with biological features of the virus.