Detailed Molecular Epidemiologic Characterization of HIV-1 Infection in Bulgaria Reveals Broad Diversity and Evolving Phylodynamics

<div><p>Limited information is available to describe the molecular epidemiology of HIV-1 in Bulgaria. To better understand the genetic diversity and the epidemiologic dynamics of HIV-1 we analyzed 125 new polymerase (<i>pol</i>) sequences from Bulgarians diagnosed through 2009 and 77 <i>pol</i> sequences available from our previous study from persons infected prior to 2007. Epidemiologic and demographic information was obtained from each participant and phylogenetic analysis was used to infer HIV-1 evolutionary histories. 120 (59.5%) persons were infected with one of five different HIV-1 subtypes (A1, B, C, F1 and H) and 63 (31.2%) persons were infected with one of six different circulating recombinant forms (CRFs; 01_AE, 02_AG, 04_cpx, 05_DF, 14_BG, and 36_cpx). We also for the first time identified infection with two different clusters of unique A-like and F-like sub-subtype variants in 12 persons (5.9%) and seven unique recombinant forms (3.5%), including a novel J/C recombinant. While subtype B was the major genotype identified and was more prevalent in MSM and increased between 2000–2005, most non-B subtypes were present in persons ≥45 years old. CRF01_AE was the most common non-B subtype and was higher in women and IDUs relative to other risk groups combined. Our results show that HIV-1 infection in Bulgaria reflects the shifting distribution of genotypes coincident with the changing epidemiology of the HIV-1 epidemic among different risk groups. Our data support increased public health interventions targeting IDUs and MSM. Furthermore, the substantial and increasing HIV-1 genetic heterogeneity, combined with fluctuating infection dynamics, highlights the importance of sustained and expanded surveillance to prevent and control HIV-1 infection in Bulgaria.</p> </div

Phylogenetic relationship of Bulgarian HIV-1 subtype B polymerase sequences.

<p>The 691-bp alignment was composed of 104 Bulgarian HIV-1 B strains and 45 subtype B reference sequences from the Los Alamos HIV database. Antiretroviral resistance-associated mutations were stripped from the alignment. The tree was rooted by using an HIV-1 subtype A1 strain as the outgroup. Tree topology was inferred using maximum likelihood analysis implemented in MEGA5. Support for each node was determined using 1,000 bootstrap replications with only values ≥70 shown. Scale bar indicates the number of nucleotide substitutions per site. Nearly identical tree topologies were also obtained with Bayesian analysis. Bulgarian sequences are shown using green branches and taxon names.</p

Inferred phylogenetic relationships of Bulgarian HIV-1 subtypes.

<p>Tree structure was inferred using maximum likelihood analysis of polymerase sequences implemented in MEGA5. Support for each node was determined using 1,000 bootstrap replications with only values ≥70 shown. Scale bar indicates the number of nucleotide substitutions per site. Antiretroviral resistance-associated mutations were stripped from the alignments. Nearly identical tree topologies were also obtained with Bayesian analysis. (<b>A</b>) <b>Subtype 02_AG.</b> The 777-bp alignment was composed of 15 HIV-1 02_AG strains from Bulgaria and 71 02_AG reference sequences from the Los Alamos HIV database. The tree was rooted by using HIV-1 01_AE strain as the outgroup. Bulgarian sequences are shown using green branches and taxon names. Taxon names in red represent Bulgarian IDUs. (<b>B</b>) <b>Circulating recombinant form (CRF) 01_AE.</b> The 689-bp alignment was composed of 40 HIV-1 CRF 01_AE strains from Bulgaria and 60 subtype CRF 01_AE reference sequences from the Los Alamos HIV database. The tree was rooted by using HIV-1 subtype J as the outgroup. Bulgarian sequences are shown using green branches and taxon names. Taxon names in red represent Bulgarian IDUs.</p

Trends in Bulgarian HIV-1 subtype (A) prevalence and (B) transmission route.

<p>HIV-1 genotypes were stratified into four time periods of the epidemic consisting of early (1986–1995), late 1990s (1996–1999), and the first (2000–2005) and second halves (2006–2009) of the last decade.</p

Geographic distribution of HIV-1 subtypes within Bulgaria.

<p>Pie charts show genotype distribution within the most populous cities of Sofia, Plovdiv, Burgas, and Varna. Numbers of each subtype are in parentheses for the smaller towns.</p

Inferred phylogenetic relationships of Bulgarian HIV-1 subtypes C and H.

<p>Tree structure was inferred using maximum likelihood analysis of polymerase sequences implemented in MEGA5. Support for each node was determined using 1,000 bootstrap replications with only values ≥70 shown. Scale bar indicates the number of nucleotide substitutions per site. Antiretroviral resistance-associated mutations were stripped from the alignments. Nearly identical tree topologies were also obtained with Bayesian analysis. The 690-bp alignment consisted of 9 HIV-1 C and H strains from Bulgaria and 29 Group M reference sequences from the Los Alamos HIV database. The tree was rooted by using an HIV-1 subtype J strain as the outgroup. Bulgarian sequences are shown using green branches and taxon names.</p

Identification of novel HIV-1 in Bulgaria.

<p>Phylogenetic relationships were inferred using Bayesian methods implemented in the program BEAST using a relaxed molecular clock and constant population size tree prior. Antiretroviral resistance-associated mutations were stripped from the alignment. Posterior probabilities >0.6 are provided at the nodes. The scale bar indicates relative units of time. Nearly identical tree topologies were also obtained with maximum likelihood analysis. (<b>A</b>) <b>F-like sub-subtypes.</b> The 758-bp alignment was composed of 9 subtype F and F-like sub-subtype polymerase sequences from Bulgaria and 43 subtype F reference sequences from the Los Alamos HIV database and highly related sequences identified using BLAST. The tree was rooted by using three HIV-1 subtype J strains as the outgroup. (<b>B</b>) <b>A-like sub-subtypes.</b> The 754-bp alignment consisted of 10 subtype A and A-like sub-subtype polymerase sequences from Bulgaria and 121 subtype A reference sequences from the Los Alamos HIV database and highly related sequences identified using BLAST. Antiretroviral resistance-associated mutations were stripped from the alignment. The tree was rooted by using three HIV-1 J strains as the outgroup.</p

Inferred phylogenetic relationships of Bulgarian HIV-1 subtypes.

<p>Tree structure was inferred using maximum likelihood analysis of polymerase sequences implemented in MEGA5. Support for each node was determined using 1,000 bootstrap replications with only values ≥70 shown. Scale bar indicates the number of nucleotide substitutions per site. Antiretroviral resistance-associated mutations were stripped from the alignments. Nearly identical tree topologies were also obtained with Bayesian analysis. (<b>A</b>) <b>Multiple Circulating and unique recombinant forms (CRF and URF, respectively).</b> The 696-bp alignment was composed of one CRF04_cpx, four CRF05_DF, two CRF14_BG, and one CRF36_cpx strains and one CRF43_02G-like strain from Bulgaria and 71 related reference sequences from the Los Alamos HIV database. The tree was rooted by using an HIV-1 subtype J strain as the outgroup. Bulgarian sequences are shown using green branches and taxon names. (<b>B</b>) <b>CRF04_cpx.</b> The 1043-bp alignment was composed of one HIV-1 CRF04_cpx1 strain from Bulgaria and 69 reference sequences from the Los Alamos HIV database and related sequences identified by BLAST. The tree was rooted by using an HIV-1 subtype J sequence as the outgroup. Bulgarian sequence is depicted using a green branch and taxon name. <b>(C) Identification of novel HIV-1 URFs in Bulgaria.</b> The 696-bp alignment was composed of six HIV-1 URF strains from Bulgaria and 68 reference sequences from the Los Alamos HIV database. The tree was rooted by using an HIV-1 subtype J strain as the outgroup. Bulgarian sequences are shown using green branches and taxon names. Bulgarian sequence #297 was shown by SimPlot analysis to be a novel recombinant of subtype J and C sequences.</p

Risk factors associated with major HIV-1 subtypes in Bulgaria, results from multivariable logistic regression model.

<p>1. Odds ratios (OR) are the estimated odds of a subtype occurring in the study population for a given risk factor relative to the referent group (Ref) as compared to occurrence of all other subtypes.</p><p>2. CL, confidence limit.</p><p>3. <i>p</i>, is the p-value for differences in subtype prevalence compared to the referent group.</p

HIV-1 subtype diversity in samples from 202 Bulgarian residents.

<p>1. Self-reported travel outside of Bulgaria which may not accurately reflect geographic origin of infection.</p