Evolutionary History of HIV-1 Subtype B and CRF01_AE Transmission Clusters among Men Who Have Sex with Men (MSM) in Kuala Lumpur,Malaysia

HIV-1 epidemics among men who have sex with men (MSM) continue to expand in developed and developing countries. Although HIV infection in MSM is amongst the highest of the key affected populations in many countries in Southeast Asia, comprehensive molecular epidemiological study of HIV-1 among MSM remains inadequate in the region including in Malaysia. Here, we reported the phylodynamic profiles of HIV-1 genotypes circulating among MSM population in Kuala Lumpur, Malaysia. A total of n = 459 newly-diagnosed treatment-naïve consenting subjects were recruited between March 2006 and August 2012, of whom 87 (18.9%) were self-reported MSM. Transmitted drug resistance mutations were absent in these isolates. Cumulatively, phylogenetic reconstructions of the pro-rt gene (HXB2∶2253–3275) showed that HIV-1 subtype B and CRF01_AE were predominant and contributed to approximately 80% of the total HIV-1 infection among MSM. In addition to numerous unique transmission lineages within these genotypes, twelve monophyletic transmission clusters of different sizes (2–7 MSM sequences, supported by posterior probability value of 1) were identified in Malaysia. Bayesian coalescent analysis estimated that the divergence times for these clusters were mainly dated between 1995 and 2005 with four major transmission clusters radiating at least 12 years ago suggesting that active spread of multiple sub-epidemic clusters occurred during this period. The changes in effective population size of subtype B showed an exponential growth within 5 years between 1988 and 1993, while CRF01_AE lineage exhibited similar expansion between 1993 and 2003. Our study provides the first insight of the phylodynamic profile of HIV-1 subtype B and CRF01_AE circulating among MSM population in Kuala Lumpur, Malaysia, unravelling the importance of understanding transmission behaviours as well as evolutionary history of HIV-1 in assessing the risk of outbreak or epidemic expansion.     

HIV-1 Diversity,Drug-Resistant Mutations,and Viral Evolution among High-Risk Individuals in Phase II HIV Vaccine Trial Sites in Southern China

HIV-1 prevalence in Guangxi, China, has been growing since 1996, when the first case was reported. Over half of HIV-1 positive patients in Guangxi Province were injecting drug users (IDUs), possibly because of the province’s location near drug-trafficking routes. Since a phase II HIV vaccine trial is ongoing there, a current characterization of the subtypes of HIV-1 among IDUs in Guangxi would provide critical information for future HIV vaccine trials, as well as further control and prevention of HIV-1 transmission. Thus, we conducted a molecular epidemiological investigation of HIV-1 samples from 2008–2010 among IDUs in multiple cities in Guangxi Province. Our results, based on the gag/pol fragment, indicated a very high proportion (78.47%) of HIV-1 CRF08_BC recombinants, some CRF01_AE (15.38%) recombinants, and a low proportion of CRF07_BC (6.15%) recombinants among the IDUs. The high proportion of CRF08 HIV-1 strains among recent IDUs matches the vaccine candidate constructs. However, future vaccine development should also incorporate CRF01-targeted vaccine candidates. Distinct Env sequence evolution patterns were observed for CRF08_BC and CRF01_AE, indicating that different local selection pressures have been exerted on these two HIV-1 subtypes. Unique drug-resistant mutations were also detected, and our data indicate that HIV treatment programs should consider pre-existing drug-resistant mutations.     

Characteristics of HIV-1 Molecular Epidemiology in Suzhou,China, from 2009 to 2014

正Dear Editor,Human immunodeficiency virus(HIV)and acquired immunodeficiency syndrome(AIDS)remain major public health problems in China,where approximately 780,000people currently live with HIV,according to the 2012 China AIDS Response Progress Report.Suzhou was an early epidemic region in Jiangsu province and the current situation of     

Multiple Independent Introductions of HIV-1 CRF01_AE Identified in China: What Are the Implications for Prevention?

Background

HIV-1 CRF01_AE accounts for an important fraction of HIV infections in Asia including China, but little is known about the phylogenetic and evolutionary history of this CRF (circulating recombinant form). In the current study, we collected a large number of 1,957 CRF01_AE gag p17 sequences with known sampling year (1990-2010) from 5 global regions representing 15 countries to better understand the phylogenetic relationships and epidemic history of CRF01_AE strains in China.

Methodology/Principal Findings

CRF01_AE gag p17 sequences were retrieved from public databases to explore phylogenetic relationships and phylogeographic dynamics of CRF01_AE in Asia by using maximum-likelihood phylogenetics and Bayesian coalescent-based analyses. We found close phylogenetic relationships between sequences from Thailand, Vietnam and China. Moreover, at least 5 independent introductions and 5 independent autochthonous clades of CRF01_AE, which descended from Thailand or Vietnam were identified in China from 1991 through 2003.

Conclusion/Significance

The current study not only defines the migration of CRF01_AE clades to/in Asia, but also demonstrates the criticalness of identifying the circulating strains in the population for the development of vaccine and microbicides.     

柳州和南宁HIV-1亚型分布及耐药情况调查

为了解柳州和南宁两市HIV-1亚型分布和耐药情况,在柳州和南宁招募HIV感染者和AIDS患者共304名,采集外周静脉血,从血浆中提取HIVRNA,扩增HIVpol基因并测序。将获得的序列进行系统进化树分析,结果表明柳州的HIV-1毒株中存在CRF01_AE和CRF07_BC两种亚型,其中CRF01_AE毒株占75.2%,CRF07_BC毒株占24.8%;南宁的HIV-1毒株中存在CRF01_AE、CRF08_BC、B亚型和C亚型共4种亚型,其中CRF01_AE和CRF08_BC仍是南宁最主要的亚型,CRF01_AE占85.8%,CRF08_BC占11.5%。根据所得的序列资料进行HIV-1耐药性分析,计算耐药率。计算结果表明,柳州未治疗和治疗研究对象的耐药率分别为3.3%和8.7%,南宁未治疗和治疗研究对象的耐药率分别为1.4%和27.5%。     

Molecular Evolution of the HIV-1 Thai Epidemic between the Time of RV144 Immunogen Selection to the Execution of the Vaccine Efficacy Trial

The RV144 HIV-1 vaccine trial (Thailand, 2003 to 2009), using immunogens genetically matched to the regional epidemic, demonstrated the first evidence of efficacy for an HIV-1 vaccine. Here we studied the molecular evolution of the HIV-1 epidemic from the time of immunogen selection to the execution of the efficacy trial. We studied HIV-1 genetic diversity among 390 volunteers who were deferred from enrollment in RV144 due to preexisting HIV-1 infection using a multiregion hybridization assay, full-genome sequencing, and phylogenetic analyses. The subtype distribution was 91.7% CRF01_AE, 3.5% subtype B, 4.3% B/CRF01_AE recombinants, and 0.5% dual infections. CRF01_AE strains were 31% more diverse than the ones from the 1990s Thai epidemic. Sixty-nine percent of subtype B strains clustered with the cosmopolitan Western B strains. Ninety-three percent of B/CRF01_AE recombinants were unique; recombination breakpoint analysis showed that these strains were highly embedded within the larger network that integrates recombinants from East/Southeast Asia. Compared to Thai sequences from the early 1990s, the distance to the RV144 immunogens increased 52% to 68% for CRF01_AE Env immunogens and 12% to 29% for subtype B immunogens. Forty-three percent to 48% of CRF01_AE sequences differed from the sequence of the vaccine insert in Env variable region 2 positions 169 and 181, which were implicated in vaccine sieve effects in RV144. In conclusion, compared to the molecular picture at the early stages of vaccine development, our results show an overall increase in the genetic complexity of viruses in the Thai epidemic and in the distance to vaccine immunogens, which should be considered at the time of the analysis of the trial results.     

Molecular Epidemiology of HIV-1 in Jilin Province,Northeastern China: Emergence of a New CRF07_BC Transmission Cluster and Intersubtype Recombinants

Objective

To investigate the HIV-1 molecular epidemiology among newly diagnosed HIV-1 infected persons living in the Jilin province of northeastern China.

Methods

Plasma samples from 189 newly diagnosed HIV-1 infected patients were collected between June 2010 and August 2011 from all nine cities of Jilin province. HIV-1 nucleotide sequences of gag P17–P24 and env C2–C4 gene regions were amplified using a multiplex RT-PCR method and sequenced. Phylogenetic and recombination analyses were used to determine the HIV-1 genotypes.

Results

Based on all sequences generated, the subtype/CFR distribution was as follows: CRF01_AE (58.1%), CRF07_BC (13.2%), subtype B’ (13.2%), recombinant viruses (8.1%), subtype B (3.7%), CRF02_AG (2.9%), subtype C (0.7%). In addition to finding CRF01_AE strains from previously reported transmission clusters 1, 4 and 5, a new transmission cluster was described within the CRF07_BC radiation. Among 11 different recombinants identified, 10 contained portions of gene regions from the CRF01_AE lineage. CRF02_AG was found to form a transmission cluster of 4 in local Jilin residents.

Conclusions

Our study presents a molecular epidemiologic investigation describing the complex structure of HIV-1 strains co-circulating in Jilin province. The results highlight the critical importance of continuous monitoring of HIV-infections, along with detailed socio-demographic data, in order to design appropriate prevention measures to limit the spread of new HIV infections.     

Phylodynamic Profile of HIV-1 Subtype B,CRF01_AE and the Recently Emerging CRF51_01B among Men Who Have Sex with Men (MSM) in Singapore

HIV-1 subtype B and CRF01_AE are the predominant infecting subtypes among men who have sex with men (MSM) in Singapore. The genetic history, population dynamics and pattern of transmission networks of these genotypes remain largely unknown. We delineated the phylodynamic profiles of HIV-1 subtype B, CRF01_AE and the recently characterized CRF51_01B strains circulating among the MSM population in Singapore. A total of 105 (49.5%) newly-diagnosed treatment-naïve MSM were recruited between February 2008 and August 2009. Phylogenetic reconstructions of the protease gene (HXB2: 2239 – 2629), gp120 (HXB2: 6942 – 7577) and gp41 (HXB2: 7803 – 8276) of the env gene uncovered five monophyletic transmission networks (two each within subtype B and CRF01_AE and one within CRF51_01B lineages) of different sizes (involving 3 – 23 MSM subjects, supported by posterior probability measure of 1.0). Bayesian coalescent analysis estimated that the emergence and dissemination of multiple sub-epidemic networks occurred between 1995 and 2005, driven largely by subtype B and later followed by CRF01_AE. Exponential increase in effective population size for both subtype B and CRF01_AE occurred between 2002 to 2007 and 2005 to 2007, respectively. Genealogical estimates suggested that the novel CRF51_01B lineages were probably generated through series of recombination events involving CRF01_AE and multiple subtype B ancestors. Our study provides the first insight on the phylodynamic profiles of HIV-1 subtype B, CRF01_AE and CRF51_01B viral strains circulating among MSM in Singapore.     

A Comprehensive Mapping of HIV-1 Genotypes in Various Risk Groups and Regions across China Based on a Nationwide Molecular Epidemiologic Survey

Background

China is experiencing a dynamic HIV/AIDS epidemic. While serology based surveillance systems have reported the spread of HIV/AIDS, detailed tracking of its transmission in populations and regions is not possible without mapping it at the molecular level. We therefore conducted a nationwide molecular epidemiology survey across the country.

Methods

HIV-1 genotypes were determined from 1,408 HIV-positive persons newly diagnosed in 2006. The prevalence of each genotype was estimated by weighting the genotype’s prevalence from each province- and risk-specific subpopulation with the number of reported cases in the corresponding subgroups in that year.

Results

CRF07_BC (35.5%), CRF01_AE (27.6%), CRF08_BC (20.1%), and subtype B'' (9.6%) were the four main HIV-1 strains in China. CRF07_BC and CRF08_BC were the primary drivers of infection among injecting drug users in northeastern and southeastern China, respectively, and subtype B'' remained dominant among former plasma donors in central China. In contrast, all four strains occurred in significant proportions among heterosexuals nationwide, pointing to an expansion of the HIV-1 epidemic from high-risk populations into the general population. CRF01_AE also replaced subtype B as the principal driver of infection among men-who-have-sex-with-men.

Conclusions

Our study provides the first comprehensive baseline data on the diversity and characteristics of HIV/AIDS epidemic in China, reflecting unique region- and risk group-specific transmission dynamics. The results provide information critical for designing effective prevention measures against HIV transmission.     

After 18 months of antiretroviral therapy,total HIV DNA decreases more pronouncedly in patients infected by CRF01_AE than in those infected by subtype B and CRF07_BC

Whether the amount of HIV DNA is associated with the subtype of HIV‐1 after antiretroviral therapy (ART) has not been reported. In the present study, the amount of HIV DNA and RNA and CD4+T counts in blood and semen prior to and after 18 months of ART were compared in 48 patients infected by CRF01_AE, subtype B or CRF07_BC of HIV‐1. Viral RNA was suppressed and CD4 cell count recovery achieved in all patients. The level of HIV DNA were similar before ART; however, patients with CRF01_AE had less HIV DNA after ART than those with subtype B and CRF07_BC infection. According to prediction of co‐receptor usage by Geno2Pheno and PSSM in combination, more than 35.6% of clones for CRF01_AE were predicted as CXCR4‐using before ART, whereas less than 6% of those for subtype B and CRF07_BC were predicted as CXCR4‐using. After 18 months of ART, no CXCR4‐using clones were predicted in any of the subtypes. Despite more HIV RNA and fewer CD4 + T cells in patients with CRF01_AE before therapy, no significant differences (P > 0.05) in viral RNA or CD4 cell counts were observed between the subtypes after 18 months of ART. Thus, 18 months of antiretroviral therapy was more efficient in patients with CRF01_AE. Considering that successful ART dramatically reduces the viral load in both blood and semen, risks of sexual transmission of HIV were reduced, contributing to prevention of rapid spread of HIV among men who have sex with men in the region.

     

HIV-1 Genetic Diversity and Its Impact on Baseline CD4+T Cells and Viral Loads among Recently Infected Men Who Have Sex with Men in Shanghai,China

The HIV-1 epidemic among men who have sex with men (MSM) has been spreading throughout China. Shanghai, a central gathering place for MSM, is facing a continuously increasing incidence of HIV-1 infection. In order to better understand the dynamics of HIV-1 diversity and its influence on patient’s immune status at baseline on diagnosis, 1265 newly HIV-1-infected MSM collected from January 2009 to December 2013 in Shanghai were retrospectively analyzed for genetic subtyping, CD4+T cell counts, and viral loads. HIV-1 phylogenetic analysis revealed a broad viral diversity including CRF01_AE (62.13%), CRF07_BC (24.51%), subtype B (8.06%), CRF55_01B (3.24%), CER67_01B (0.95%), CRF68_01B (0.4%), CRF08_BC (0.08%) and CRF59_01B (0.08%). Twenty-four unique recombination forms (URFs) (1.98%) were identified as well. Bayesian inference analysis indicated that the introduction of CRF01_AE strain (1997) was earlier than CRF07_BC strain (2001) into MSM population in Shanghai based on the time of the most recent common ancestor (tMRCA). Three epidemic clusters and five sub-clusters were found in CRF01_AE. Significantly lower CD4+T cell count was found in individuals infected with CRF01_AE than in those infected with CRF07_BC infection (P<0.01), whereas viral load was significantly higher those infected with CRF01_AE than with CRF07_BC (P<0.01). In addition, the patients with >45 years of age were found to have lower CD4+T cell counts and higher viral loads than the patients with <25 years of age (P<0.05). This study reveals the presence of HIV-1 subtype diversity in Shanghai and its remarkable influence on clinical outcome. A real-time surveillance of HIV-1 viral diversity and phylodynamics of epidemic cluster, patient’s baseline CD4+T cell count and viral load would be of great value to monitoring of disease progression, intervention for transmission, improvement of antiretroviral therapy strategy and design of vaccines.     

HIV-1 Genetic Characteristics and Transmitted Drug Resistance among Men Who Have Sex with Men in Kunming,China

Background

Yunnan has been severely affected by HIV/AIDS in China. Recently, the reported prevalence of HIV-1 among men who have sex with men (MSM) in Yunnan was high in China. To monitor dynamic HIV-1 epidemic among Yunnan MSM, HIV-1 genetic characteristics and transmitted drug resistance (TDR) were investigated.

Methods

Blood samples from 131 newly HIV-1 diagnosed MSM were continuously collected at fixed sites from January 2010 to December 2012 in Kunming City, Yunnan Province. Partial gag, pol and env genes were sequenced. Phylogenetic, evolutionary and genotypic drug resistance analyses were performed.

Results

Multiple genotypes were identified among MSM in Kunming, including CRF01_AE (64.9%), CRF07_BC (25.2%), unique recombinant forms (URFs, 5.3%), subtype B (3.1%) and CRF08_BC (1.5%). CRF01_AE and CRF07_BC were the predominant strains. The mean of genetic distance within CRF01_AE were larger than that within CRF07_BC. The estimated introducing time of CRF01_AE in Yunnan MSM (1996.9) is earlier than that of CRF07_BC (2002.8). In this study, subtype B was first identified in Yunnan MSM. CRF08_BC seems to be the distinctive strain in Yunnan MSM, which was seldom found among MSM outside Yunnan. The proportion of URFs increased, which further contributed to genetic diversity among MSM. Strikingly, genetic relatedness was found among these strains with MSM isolates from multiple provinces, which suggested that a nationwide transmission network may exist. TDR-associated mutations were identified in 4.6% individuals. The multivariate analysis revealed that non-native MSM and divorced/widowed MSM were independently associated with a higher TDR rate.

Conclusion

This work revealed diverse HIV-1 genetics, national transmission networks and a baseline level of TDR in MSM. These findings enhance our understanding of the distribution and evolution of HIV-1 in MSM, and are valuable for developing HIV prevention strategies for MSM.     

Genome Sequence of a Novel HIV-1 Circulating Recombinant Form 54_01B from Malaysia

We report here the first novel HIV-1 circulating recombinant form (CRF) 54_01B (CRF54_01B) isolated from three epidemiologically unlinked subjects of different risk groups in Malaysia. These recently sampled recombinants showed a complex genome organization composed of parental subtype B′ and CRF01_AE, with identical recombination breakpoints observed in the gag, pol, and vif genes. Such a discovery highlights the ongoing active generation and spread of intersubtype recombinants involving the subtype B′ and CRF01_AE lineages and indicates the potential of the new CRF in bridging HIV-1 transmission among different risk groups in Southeast Asia.     

Emerging Variability in HIV-1 Genetics among Recently Infected Individuals in Yunnan,China

Background

Yunnan has the longest endured Human Immunodeficiency Virus-1 (HIV-1) epidemic in China, and the genetic diversity of HIV-1 constitutes an essential characteristic of molecular epidemiology in this region. To obtain a more comprehensive picture of the dynamic changes in Yunnan’s HIV-1 epidemic, a cross-sectional molecular epidemiological investigation was carried out among recently infected individuals.

Methodology/Principal Findings

We sequenced partial gag (HXB2∶781–1861) and env (HXB2∶7002–7541) genes from 308 plasma samples of recently infected patients. With phylogenetic analysis, 130 specimens generated interpretable genotyping data. We found that the circulating genotypes included: CRF08_BC (40.8%), unique recombinant forms (URFs, 27.7%), CRF01_AE (18.5%), CRF07_BC (9.2%), subtype B (2.3%) and C (1.5%). CRF08_BC was the most common genotype, and was predominant in both intravenous drug users (IDUs) and heterosexually transmitted populations. CRF08_BC and CRF07_BC still predominated in eastern Yunnan, but CRF08_BC showed increasing prevalence in western Yunnan. Strikingly, the URFs raised dramatically in most regions of Yunnan. Seven different types of URFs were detected from 12 prefectures, suggesting that complicated and frequent recombination is a salient feature of Yunnan’s HIV-1 epidemic. Among URFs, two BC clusters with distinctive recombination patterns might be potential new CRF_BCs. CRF01_AE was no longer confined to the prefectures bordering Myanmar, and had spread to the eastern part of Yunnan, especially the capital city of Kunming, with a large number of infections in the transient population. The ratios of the main genotypes showed no statistical differences between infected IDUs and heterosexually transmitted infections.

Conclusions/Significance

The changing patterns of the dominant HIV-1 genotypes in Yunnan indicate the complex evolving dynamic nature of the epidemic. Understanding new trends in molecular epidemiology of HIV-1 infection is critical for adjusting current prevention strategies and vaccine development in Yunnan.     

Phylodynamics of HIV-1 from a phase III AIDS vaccine trial in Bangkok, Thailand

Background

In 2003, a phase III placebo-controlled trial (VAX003) was completed in Bangkok, Thailand. Of the 2,546 individuals enrolled in the trial based on high risk for infection through injection drug use (IDU), we obtained clinical samples and HIV-1 sequence data (envelope glycoprotein gene gp120) from 215 individuals who became infected during the trial. Here, we used these data in combination with other publicly available gp120 sequences to perform a molecular surveillance and phylodynamic analysis of HIV-1 in Thailand.

Methodology and Findings

Phylogenetic and population genetic estimators were used to assess HIV-1 gp120 diversity as a function of vaccination treatment, viral load (VL) and CD4⁺ counts, to indentify transmission clusters and to investigate the timescale and demographics of HIV-1 in Thailand. Three HIV-1 subtypes were identified: CRF01_AE (85% of the infections), subtype B (13%) and CRF15_AE (2%). The Bangkok IDU cohort showed more gp120 diversity than other Asian IDU cohorts and similar diversity to that observed in sexually infected individuals. Moreover, significant differences (P<0.02) in genetic diversity were observed in CRF01_AE IDU with different VL and CD4⁺ counts. No phylogenetic structure was detected regarding any of the epidemiological and clinical factors tested, although high proportions (35% to 50%) of early infections fell into clusters, which suggests that transmission chains associated with acute infection play a key role on HIV-1 spread among IDU. CRF01_AE was estimated to have emerged in Thailand in 1984.5 (1983–1986), 3–6 years before the first recognition of symptomatic patients (1989). The relative genetic diversity of the HIV-1 population has remained high despite decreasing prevalence rates since the mid 1990s.

Conclusions

Our study and recent epidemiological reports indicate that HIV-1 is still a major threat in Thailand and suggest that HIV awareness and prevention needs to be strengthened to avoid AIDS resurgence.     

Higher frequency of premature stop codon mutations at vpu gene of human immunodeficiency virus type 1 CRF01_AE compared with those of other subtypes

Our previous study demonstrated the anti-apoptosis function of the human immunodeficiency virus type 1 (HIV-1) vpu gene product in normal CD4+ T lymphocytes. In this study, using sequences obtained from the HIV sequence database, we compared vpu sequences from 184 preparations of various subtypes of HIV-1 from diverse geographical regions. Our analysis revealed that CRF01_AE isolates had premature stop codon mutations at the vpu gene at a much higher rate (36%) than other subtypes (0-9%). The premature stop codon mutations in vpu existed mostly at two amino acid residues: the methionine initiation codon and the boundary between the transmembrane (TM) and cytoplasmic domains. The mutations at the latter site were more often detected in CRF01_AE. The higher mutation rates at vpu in CRF01_AE were confirmed by sequence comparison of polymerase chain reaction products newly obtained directly from the DNA extracted from peripheral blood mononuclear cells (PBMCs), but not from the RNA from the plasma, in CRF01_AE- and subtype B-infected individuals. This finding may indicate the possibility that the more abundant population of HIV-1 CRF01_AE is able to induce apoptosis in CD4+ T lymphocytes than the populations of other subtypes.     

HIV-1 genetic diversity in Western Brittany,France

We describe human immunodeficiency virus type 1 (HIV-1) diversity in Western Brittany, France, and trace the dissemination of HIV-1 non-B subtype infection. The strategy for HIV-1 subtyping used involved subtype specific enzyme immunoassays, heteroduplex mobility assays and phylogenetic analysis of the sequences of env encoding the V3 loop region. Samples were obtained from 567 patients: 465 (82%) were of subtype B and 66 (11.6%) were not (20 were subtype A, 11 subtype C, four subtype D, seven subtype F, five subtype G and 19 others with circulating recombinant forms: 4CRF01_AE, 11CRF02_AG, 1H, 3CRF11_cpx). These findings are consistent with other studies of French populations. There is an epidemiological correlation between subtype B and homosexual or heterosexual contamination in France and between non-B subtype and heterosexual contamination in Africa.     

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

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 (pol) sequences from Bulgarians diagnosed through 2009 and 77 pol 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.