The HIV type 1 epidemic in Bulgaria involves multiple subtypes and is sustained by continuous viral inflow from West and East European countries

Abstract

Little is known about the HIV-1 epidemic in Balkan countries. To fill the gap, we investigated the viral genetic diversity in Bulgaria, by sequencing and phylogenetic characterization of 86 plasma samples collected between 2002 and 2006 from seropositive individuals diagnosed within 1986-2006. Analysis of pol gene sequences assigned 51% of the samples to HIV-1 subtype B and 27% to subtype A1. HIV-1 subtype C, F, G, H, and a few putative recombinant forms were also found. Phylogenetic and molecular clock analysis showed a continuous exchange of subtype A and B between Bulgaria and Western as well as other Eastern European countries. At least three separate introductions of HIV-1 subtype A and four of HIV-1 subtype B have occurred within the past 25 years in Bulgaria. The central geographic location of Bulgaria, the substantial genetic heterogeneity of the epidemic with multiple subtypes, and the significant viral flow observed to and from the Balkan countries have the potential to modify the current HIV-1 epidemiological structure in Europe and highlight the importance of more extensive and continuous monitoring of the epidemic in the Balkans.

FIG. 1.

Maximum likelihood phylogenetic analysis of HIV-1B pol sequences. The data set included 41 HIV-1B strains from Bulgaria and 91 subtype B reference sequences downloaded from the Los Alamos HIV database. The tree was rooted by using two HIV-1A strains as the outgroup. Branch lengths were estimated with the best fitting nucleotide substitution model according to a hierarchical likelihood ratio test, and were drawn in scale with the bar at the bottom indicating 0.02 nucleotide substitutions per site. One asterisk (*) along a branch represents significant statistical support for the clade subtending that branch (p < 0.001 in the zero-branch-length test, Bayesian posterior probability >90%, and bootstrap support >75%). The color of each external branch represents the country of origin of the sequence corresponding to that branch, according to the legend in the figure. FSU (Former Soviet Union) strains include sequences from Russia, Bielorussia, Ukraine, and Slovenia (the specific country of origin is indicated by the arrows). Broken boxes highlight statistically supported monophyletic clades including Bulgarian strains. The inferred time of the most recent common ancestor of each supported Bulgarian clade is also indicated on the right of the highlighted clade. Dates and 95% high posterior density interval (within square brackets) were estimated using a Bayesian relaxed molecular clock (see Materials and Methods).

FIG. 2.

Maximum likelihood phylogenetic analysis of HIV-1A1 pol sequences. The data set included 18 HIV-1A strains from Bulgaria and 137 subtype A1 reference sequences downloaded from the Los Alamos HIV database. The tree was rooted by using two HIV-1B strains as the outgroup. Branch lengths were estimated with the best fitting nucleotide substitution model according to a hierarchical likelihood ratio test, and were drawn to scale with the bar at the bottom indicating 0.01 nucleotide substitutions per site. One asterisk (*) along a branch represents significant statistical support for the clade subtending that branch (p < 0.001 in the zero-branch-length test, Bayesian posterior probability >90%, and bootstrap support >75%). The color of each external branch represents the country of origin of the sequence corresponding to that branch, according to the legend in the figure. The Blood Transfusion clade and Bulgaria West Europe clade are the two major monophyletic clades containing the Bulgarian isolates. The FSU (Former Soviet Union) clade includes strains from Russia, Bielorussia, Ukraine, Slovenia, Czech Republic, and two strains from Bulgaria. Dates and 95% high posterior density intervals (within square brackets) were estimated using a Bayesian relaxed molecular clock (see Materials and Methods).

FIG. 3.

Maximum parsimony migration patterns of Bulgarian sequences to/from different geographic areas. The bubblegram shows the frequency of gene flow (migrations) to/from different geographic areas, as the percentage of the total observed migrations estimated from the maximum likelihood trees for different subtypes with a modified version of the Slatkin and Maddison test (see Materials and Methods). The surface of each circle is proportional to the percentage of observed migrations given within the circle. The highlighted row indicates inferred HIV-1 outflow from Bulgaria to different geographic areas. The highlighted column indicates inferred HIV-1 inflow to Bulgaria from different geographic areas. (A) HIV-1A subtype migration from/to Africa, Bulgaria, East Europe, and West Europe estimated from the tree given in Fig. 1.(B) HIV-1B subtype migration from/to the United States, Bulgaria, East Europe, and West Europe estimated from the tree given in Fig. 2.