Sex extreme Bulgaria

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Try out PMC Labs and tell us what you think. Learn More. 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 and from seropositive individuals diagnosed within — 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.

The central geographic location of Bulgaria, the substantial genetic heterogeneity of the epidemic with multiple subtypes, and the ificant 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.

C urrent epidemiological data indicate that the impact of the HIV-1 epidemic on Balkan countries, which are part of the central European region, is still limited. The annual of HIV-1 diagnoses increased from 15—20 in the early s to 40—60 within the past 5 years, with a peak of 91 diagnoses infollowing implementation of second generation surveillance systems. According to recent serological surveys conducted in Bulgaria's largest cities and towns, the prevalence of HIV-1 infection is only 0.

Following the end of the cold war a decade ago, most Balkan countries have been affected by dramatic political and socioeconomic changes, which could contribute to the spread of HIV infection. Given the central geographic location of Bulgaria at the crossing point between Western Europe, Eastern Europe, and the Middle East, defining the origin and evolution of HIV in Bulgaria has obvious epidemiological relevance. However, although basic information on the characteristics of HIVinfected individuals in Bulgaria is available, little is known about the HIV origin and distribution of different circulating subtypes.

The small amount of information from other Balkan countries indicates that there is high genetic diversity, with subtype B being predominant in Yugoslavia and subtype A in Albania. The objective of the present study was to investigate the molecular diversity and epidemiology of HIV-1 subtypes circulating in Bulgaria. To this end, we sequenced a fragment of the HIV-1 pol gene amplified from serum samples collected between and within a cohort of infected subjects under monitoring in different Bulgarian hospitals.

By employing high-resolution phylogenetic and phylogeographic analysis, as well as molecular clock estimates, we obtained, for the first time, detailed information about the origin and the epidemiological history of the two most prevalent viral subtypes HIV-1 A and B in this Balkan country. Eighty-six serum samples were obtained from persons who were diagnosed with HIV infection [58 treated with highly active antiretroviral therapy HAART23 naive, and 5 no treatment data available] between and in Bulgaria. The samples were linked to demographic and clinical data through an anonymous numerical code, in accordance with the ethical standards of the National HIV Confirmatory Laboratory of Sofia.

The PCR products 1. For each aligned data set, the best fitting nucleotide substitution model was tested with a hierarchical likelihood ratio test, using a neighbor-ing NJ tree with LogDet corrected distances as base tree.

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The heuristic search for the best tree was performed using an NJ tree as the starting tree and the TBR branch-swapping algorithm. NJ trees were also obtained with ML estimated pairwise distances using the best fitting nucleotide substitution model.

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Statistical support for specific clades in each phylogeny was obtained with the ML-based zero branch length test for the ML tree, 12 by bootstrapping replicates for the NJ trees, and by calculating the posterior probability of each monophyletic clade for the Bayesian tree.

Trees were rooted by ML rooting by selecting the rooted tree with the best likelihood under the molecular clock constraint or by outgroup rooting. Multiple sequence alignments of Bulgarian and reference HIV strains are available from the authors upon request. Ultrametric trees were obtained by enforcing a molecular clock on the inferred genealogy and reestimating the branch lengths and substitution parameters with ML using the ly selected evolutionary model.

The clock hypothesis was tested with the likelihood ratio test.

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Each chain was run for 20, generations, with sampling every generations. A one-character data matrix is obtained from the original data set by asing to each taxon in the tree a one-letter code indicating its country or geographic region of origin. Then, the putative origin of each ancestral sequence i. The final tree length, i. Observed genealogies ificantly shorter than random trees indicate the presence of subdivided populations with restricted gene flow. Specific migrations among different countries character states were traced with the State changes and stasis tool MacClade softwarewhich counts the of changes in a tree for each pairwise character state.

When multiple MPRs were present as in our data setsthe algorithm calculated the average migration count over all possible MPRs for each pair. The resulting pairwise migration matrix was then normalized, and a randomization test with 10, matrices obtained from 10, random trees by random ing-splitting of the original tree was performed to assess the statistical ificance of the observed migration counts. NJ and Bayesian trees gave similar topologies data not shown. The Bulgarian subtype B strains were highly interspersed within the tree.

Four monophyletic clades of Bulgarian sequences, highlighted by the boxes in Fig. All four clades clustered subtype B sequences from individuals infected through sexual transmission. Sequences from individuals infected through other transmission routes were intermixed in the tree and failed to cluster within any supported monophyletic clade. Clade IV in particular was closely related to strains from Ukraine. The high degree of intermixing between Bulgarian and foreign sequences and the phylogenetic position of the four statistically supported clades are evidence of several independent introductions of HIV-1 subtype B into Bulgaria from both East and West Europe.

Moreover, the existence of independent monophyletic clades implies the presence of separate transmission networks for subtype B within the Balkan countries. Maximum likelihood phylogenetic analysis of HIV-1B pol sequences. 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, 11 and were drawn in scale with the bar at the bottom indicating 0.

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. 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.

Strains 21A and A, which clustered together at the top of the tree, were isolated from two subjects infected through blood transfusion, whereas the strains in the remaining two clades indicated in the figure as Bulgaria West Europe clade and FSU clade, respectively were isolated from persons infected through sexual contact.

The Bulgarian blood transfusion subtype A1 clade appears to be closely related to a monophyletic clade that includes both West European and African sequences.

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A second statistically supported monophyletic clade included most of the Bulgarian subtype A1 strains Although a monophyletic group ing a strain from the former Yugoslavia and one from Greece appeared at the origin of the clade see Fig. The third statistically supported clade included two Bulgarian subtype A strains, in addition to sequences originating from different Eastern European countries. Overall, each cluster is likely to represent a separate introduction into Bulgaria of HIV-1A1 from at least two different geographic areas West and East Europe by different transmission routes sexual transmission and blood transfusion.

Maximum likelihood phylogenetic analysis of HIV-1A1 pol sequences. 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, 11 and were drawn to scale with the bar at the bottom indicating 0. The Blood Transfusion clade and Bulgaria West Europe clade are the two major monophyletic clades containing the Bulgarian isolates.

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To estimate the time of the most recent common ancestor tMRCA of the supported Bulgarian clades within subtype A and B, we implemented a strict molecular clock model, assuming a constant evolutionary rate along each subtype tree. The other two subtype A Bulgarian clades Fig. The trees given in Figs. The null hypothesis of panmixia i. A substantial gene outflow of HIV-1A1 strains from Africa to the other regions, including Bulgaria, was also evident The gene flow from Bulgaria to West Europe was 5.

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. The present study is the first detailed investigation of the HIV-1 molecular epidemiology in Bulgaria.

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For example, The molecular clock analysis estimated that HIV-1B was present in Bulgaria since the early s, with a lower limit estimate for the first introduction in that is within the same time frame as the introduction of HIV-1 subtype B in North America. Both molecular clock and viral gene flow analyses indicated a dynamic epidemic over the past 25 years, with multiple introductions from foreign countries and ongoing gene outflow from Bulgaria to West and East Europe.

Although fewer than cumulative AIDS cases have been identified in Bulgaria to date, our data suggest that the actual prevalence of HIV-1 in Bulgaria may be underestimated. The dynamic patterns of the HIV-1 epidemic within the country point out an enormous potential for spreading within the next few years, both within the country and to other neighboring countries in Europe. In this regard, three factors seem to be highly relevant: 1 the central geographic location of Bulgaria at the crossing between Western Europe, Eastern Europe, and the Middle East, 2 the high genetic heterogeneity of the HIV-1 epidemic in the Balkan countries revealed by the molecular characterization of our samples, and 3 the ongoing sociopolitical changes that have been affecting Bulgaria, as well as other countries in Eastern and Central Europe, since the end of the cold war.

In fact, phylogenetic analysis indicated that at least three independent introductions of the HIV-1A1 subtype have occurred in Bulgaria, and that the majority of the HIV-1A Bulgarian strains in our sample were more closely related to West European subtype A sequences. Phylogeographic migration analysis showed that the largest HIV-1A1 outflow occurred from the African continent, a finding consistent with the African origin and the high seroprevalence of subtype A1 in Africa.

The findings are consistent with the central geographic location of Bulgaria and point out that continuous surveillance of the epidemic in this Balkan country may be of great importance for the monitoring and prediction of future HIV-1 epidemiological trends in the rest of Europe. A recent study explored the history of the HIV epidemic in the United Kingdom within defined risk groups. Most of the Bulgarian clades included viral strains from individuals infected exclusively through sexual contact. However, the presence of distinct and well-supported monophyletic clades within both HIV-1A1 and HIV-1B phylogeny suggests that after each initial introduction of the virus in a specific population, separate transmission clusters have been evolving along independent phylogenetic lineages.

Better characterization and continuous monitoring of such groups are going to be crucial to understand in detail the epidemiology of HIV-1 in Bulgaria and to assess the efficacy of prevention and therapy in controlling the epidemic. Some possible limits and biases of the present study should be mentioned. First, we cannot exclude the possibility that the study population is not truly representative of all HIV cases in Bulgaria.

In particular, most persons included in our analysis had treatment failure; thus it is possible that persons in an advanced stage of disease i. The possible consequences could be 1 underestimation of the increase in the circulation of different clades in recent infections or 2 overrepresentation of transmission clusters due to the elevated virulence of the circulating virus.

Sex extreme Bulgaria

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The HIV Type 1 Epidemic in Bulgaria Involves Multiple Subtypes and Is Sustained by Continuous Viral Inflow from West and East European Countries