Analysis of the origin and evolutionary history of HIV-1 CRF28_BF and CRF29_BF reveals a decreasing prevalence in the AIDS epidemic of Brazil

Background

HIV-1 subtype B and subtype F are prevalent in the AIDS epidemic of Brazil. Recombinations between these subtypes have generated at least four BF circulating recombinant forms (CRFs). CRF28_BF and CRF29_BF are among the first two BF recombinants being identified in Brazil and they contributed significantly to the epidemic. However, the evolution and demographic histories of the CRFs are unclear.

Methodology/Principal Findings

A collection of gag and pol sequences sampled within Brazil was screened for CRF28_BF-like and CRF29_BF-like recombination patterns. A Bayesian coalescent framework was employed to delineate the phylogenetic, divergence time and population dynamics of the virus having CRF28_BF-like and CRF29_BF-like genotype. These recombinants were phylogenetically related to each other and formed a well-supported monophyletic clade dated to 1988–1989. The effective number of infections by these recombinants grew exponentially over a five-year period after their emergence, but then decreased toward the present following a logistic model of population growth. The demographic pattern of both recombinants closely resembles those previously reported for CRF31_BC.

Conclusions

We revealed that HIV-1 recombinants of the CRF28_BF/CRF29_BF clade are still circulating in the Brazilian population. These recombinants did not exhibit a strong founder effect and showed a decreasing prevalence in the AIDS epidemic of Brazil. Our data suggested that multiple URFs may also play a role in shaping the epidemic of recombinant BF HIV-1 in the region.     

Ultra-Deep Sequencing of HIV-1 near Full-Length and Partial Proviral Genomes Reveals High Genetic Diversity among Brazilian Blood Donors

Background

Here, we aimed to gain a comprehensive picture of the HIV-1 diversity in the northeast and southeast part of Brazil. To this end, a high-throughput sequencing-by-synthesis protocol and instrument were used to characterize the near full length (NFLG) and partial HIV-1 proviral genome in 259 HIV-1 infected blood donors at four major blood centers in Brazil: Pro-Sangue foundation (São Paulo state (SP), n 51), Hemominas foundation (Minas Gerais state (MG), n 41), Hemope foundation (Recife state (PE), n 96) and Hemorio blood bank (Rio de Janeiro (RJ), n 70).

Materials and Methods

A total of 259 blood samples were obtained from 195 donors with long-standing infections and 64 donors with a lack of stage information. DNA was extracted from the peripheral blood mononuclear cells (PBMCs) to amplify the HIV-1 NFLGs from five overlapping fragments. The amplicons were molecularly bar-coded, pooled, and sequenced by Illumina paired-end protocol.

Results

Of the 259 samples studied, 208 (80%) NFLGs and 49 (18.8%) partial fragments were de novo assembled into contiguous sequences and successfully subtyped. Of these 257 samples, 183 (71.2%) were pure subtypes consisting of clade B (n = 167, 65%), C (n = 10, 3.9%), F1 (n = 4, 1.5%), and D (n = 2, 0.7%). Recombinant viruses were detected in 74 (28.8%) samples and consist of unique BF1 (n = 41, 15.9%), BC (n = 7, 2.7%), BCF1 (n = 4, 1.5%), CF1 and CDK (n = 1, 0.4%, each), CRF70_BF1 (n = 4, 1.5%), CRF71_BF1 (n = 12, 4.7%), and CRF72_BF1 (n = 4, 1.5%). Evidence of dual infection was detected in four patients coinfected with the same subtype (n = 3) and distinct subtype (n = 1).

Conclusion

Based on this work, subtype B appears to be the prevalent subtype followed by a high proportion of intersubtype recombinants that appeared to be arising continually in this country. Our study represents the largest analysis of the viral NFLG ever undertaken worldwide and provides insights into the understanding the genesis of the HIV-1 epidemic in this particular area of South America and informs vaccine design and clinical trials.     

Deep Sequencing of HIV-1 near Full-Length Proviral Genomes Identifies High Rates of BF1 Recombinants Including Two Novel Circulating Recombinant Forms (CRF) 70_BF1 and a Disseminating 71_BF1 among Blood Donors in Pernambuco,Brazil

Background

The findings of frequent circulation of HIV-1 subclade F1 viruses and the scarcity of BF1 recombinant viruses based on pol subgenomic fragment sequencing among blood donors in Pernambuco (PE), Northeast of Brazil, were reported recently. Here, we aimed to determine whether the classification of these strains (n = 26) extends to the whole genome sequences.

Methods

Five overlapping amplicons spanning the HIV near full-length genomes (NFLGs) were PCR amplified from peripheral blood mononuclear cells (PBMCs) of 26 blood donors. The amplicons were molecularly bar-coded, pooled, and sequenced by Illumina paired-end protocol. The prevalence of viral variants containing drug resistant mutations (DRMs) was compared between plasma and PBMCs.

Results

Of the 26 samples studied, 20 NFLGs and 4 partial fragments were de novo assembled into contiguous sequences and successfully subtyped. Two distinct BF1 recombinant profiles designated CRF70_BF1 and CRF71_BF1, with 4 samples in profile I and 11 in profile II were detected and thus constitute two novel recombinant forms circulating in PE. Evidence of dual infections was detected in four patients co-infected with distinct HIV-1 subtypes. According to our estimate, the new CRF71_BF1 accounts for 10% of the HIV-1 circulating strains among blood donors in PE. Discordant data between the plasma and PBMCs-virus were found in 15 of 24 donors. Six of these strains displayed major DRMs only in PBMCs and four of which had detectable DRMs changes at prevalence between 1-20% of the sequenced population.

Conclusions

The high percentage of the new RF71_BF1 and other BF1 recombinants found among blood donors in Pernambuco, coupled with high rates of transmitted DRMs and dual infections confirm the need for effective surveillance to monitor the prevalence and distribution of HIV variants in a variety of settings in Brazil.     

Generation of a Recombinant Gag Virus-Like-Particle Panel for the Evaluation of p24 Antigen Detection by Diagnostic HIV Tests

Background

Detection of HIV-1 p24 antigen permits early identification of primary HIV infection and timely intervention to limit further spread of the infection. Principally, HIV screening should equally detect all viral variants, but reagents for a standardised test evaluation are limited. Therefore, we aimed to create an inexhaustible panel of diverse HIV-1 p24 antigens.

Methods

We generated a panel of 43 recombinantly expressed virus-like particles (VLPs), containing the structural Gag proteins of HIV-1 subtypes A-H and circulating recombinant forms (CRF) CRF01_AE, CRF02_AG, CRF12_BF, CRF20_BG and group O. Eleven 4^th generation antigen/antibody tests and five antigen-only tests were evaluated for their ability to detect VLPs diluted in human plasma to p24 concentrations equivalent to 50, 10 and 2 IU/ml of the WHO p24 standard. Three tests were also evaluated for their ability to detect p24 after heat-denaturation for immune-complex disruption, a pre-requisite for ultrasensitive p24 detection.

Results

Our VLP panel exhibited an average intra-clade p24 diversity of 6.7%. Among the 4^th generation tests, the Abbott Architect and Siemens Enzygnost Integral 4 had the highest sensitivity of 97.7% and 93%, respectively. Alere Determine Combo and BioRad Access were least sensitive with 10.1% and 40.3%, respectively. Antigen-only tests were slightly more sensitive than combination tests. Almost all tests detected the WHO HIV-1 p24 standard at a concentration of 2 IU/ml, but their ability to detect this input for different subtypes varied greatly. Heat-treatment lowered overall detectability of HIV-1 p24 in two of the three tests, but only few VLPs had a more than 3-fold loss in p24 detection.

Conclusions

The HIV-1 Gag subtype panel has a broad diversity and proved useful for a standardised evaluation of the detection limit and breadth of subtype detection of p24 antigen-detecting tests. Several tests exhibited problems, particularly with non-B subtypes.     

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.     

Evolving Molecular Epidemiological Profile of Human Immunodeficiency Virus 1 in the Southwest Border of China

Background

We have previously reported in Xishuangbanna (Banna) Dai Autonomous Prefecture, a well-developed tourist destination in the southwest border of China, that HIV-1 transmitted dominantly through heterosexual contact with less divergent genotypes and few drug resistant mutations [1]. Due to the rapid increase of newly diagnosed HIV-1 cases per year in Banna in recent years, it’s important to evaluate the evolution of HIV-1 molecular epidemiology for the better understanding of ongoing HIV-1 outbreak in this region.

Methodology/Principal Findings

By sequencing of HIV-1 pol genes and phylogenetic analysis, we conducted a molecular epidemiologic study in 352 HIV-1-seropositive highly active antiretroviral treatment (HAART)-naïve individuals newly diagnosed at the Banna Center for Disease Control and Prevention between 2009 and 2011. Of 283 samples (84.1% taken from heterosexually acquired adults, 10.6% from needle-sharing drug users, 2.8% from men who have sex with men, 0.4% from children born from HIV-1-infected mothers, and 2.1% remained unknown) with successful sequencing for pol gene, we identified 108 (38.2%) HIV-1 subtype CRF08_BC, 101 (35.7%) CRF01_AE, 49 (17.3%) CRF07_BC, 5 (1.8%) C/CRF57_BC, 3 (1.1%) B’, 1 (0.4%) B/CRF51_01B, and 16 (5.7%) unique recombinants forms. Among these infected individuals, 104 (36.7%) cases showed drug resistant or resistance-relevant mutations, and 4 of them conferring high-level resistance to 3TC/FTC, EFV/NVP or NFV. Phylogenetic analysis revealed 21 clusters (2–7 sequences) with only 21.2% (60/283) sequences involved.

Conclusion/Significance

In contrast to our previous findings, CRF08_BC, replaced CRF01_AE, became the dominant genotype of HIV-1 in Banna prefecture. The viral strains with drug resistance mutations were detected frequently in newly diagnosed HIV-1-infected individuals in this region.     

Heterogeneous Evolution of HIV-1 CRF01_AE in Men Who Have Sex with Men (MSM) and Other Populations in China

Introduction

The HIV epidemic in men who have sex with men (MSM) continues to grow in most countries. However, the phylodynamic and virological differences among HIV-1 strains circulating in MSM and other populations are not well characterized.

Methods

Nearly full-length genomes (NFLGs) of the HIV-1 CRF01_AE were obtained from the Los Alamos HIV database. Phylogenetic analyses were conducted using the NFLG, gag, pol and env genes, using the maximum likelihood method. Selection pressure analyses at the codon level were performed for each gene in the phylogenetic clusters using PAML.

Results

Sequences isolated from MSM in China clustered in Clusters 1 (92.5%) and 2 (85.71%). The major risk factor for Cluster 3 was heterosexual transmission (62.16%). The ratio of non-synonymous to synonymous substitutions in the env gene (0.7–0.75) was higher than the gag (0.26–0.34) or pol (0.21–0.26) genes. In env gene, Cluster 1 (4.56×10^-3subs/site/year) and 2 (6.01×10^-3subs/site/year) had higher evolutionary rates than Cluster 3 (1.14×10^-3subs/site/year). Positive selection affected 4.2–6.58% of the amino acid sites in the env gene. Two sites (HXB2:136 and 316) evolved similarly in Clusters 1 and 2, but not Cluster 3.

Conclusion

The HIV-1 CRF01_AE in MSM is evolving differently than in other populations.     

Physical Activity and Bone Health in Schoolchildren: The Mediating Role of Fitness and Body Fat

Background

The relationship between physical activity (PA) and bone health is well known, although the role of percent body fat (%BF) and fitness as confounders or mediators in this relationship remains uncertain.

Objective

To examine whether the association between PA and bone mineral content (BMC) is mediated by %BF and cardiorespiratory fitness (CRF).

Methods

In this cross sectional study, BMC, total %BF (by DXA), vigorous PA (VPA), CRF, age and height were measured in 132 schoolchildren (62 boys, aged 8–11 years). ANCOVA was used to test differences in BMC by %BF, CRF and VPA, controlling for different sets of confounders. Simple mediation analyses and serial multiple mediation analyses were fitted to examine whether the relationship between PA and BMC is mediated by %BF and fitness.

Results

Children with high %BF had higher total body BMC than their peers after controlling for all sets of confounders. Children with good CRF or VPA had significantly less total body BMC after controlling for age and sex but in children with good CRF this inverse relation disappeared after adjusting by %BF. %BF and CRF both act as a full mediator in the association between VPA and BMC, after inclusion of the potential confounders in the models.

Conclusion

Fitness and %BF seem to have a mediator role on the relationship between physical activity and bone mass.     

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.     

A Scoring Tool to Identify East African HIV-1 Serodiscordant Partnerships with a High Likelihood of Pregnancy

Introduction

HIV-1 prevention programs targeting HIV-1 serodiscordant couples need to identify couples that are likely to become pregnant to facilitate discussions about methods to minimize HIV-1 risk during pregnancy attempts (i.e. safer conception) or effective contraception when pregnancy is unintended. A clinical prediction tool could be used to identify HIV-1 serodiscordant couples with a high likelihood of pregnancy within one year.

Methods

Using standardized clinical prediction methods, we developed and validated a tool to identify heterosexual East African HIV-1 serodiscordant couples with an increased likelihood of becoming pregnant in the next year. Datasets were from three prospectively followed cohorts, including nearly 7,000 couples from Kenya and Uganda participating in HIV-1 prevention trials and delivery projects.

Results

The final score encompassed the age of the woman, woman’s number of children living, partnership duration, having had condomless sex in the past month, and non-use of an effective contraceptive. The area under the curve (AUC) for the probability of the score to correctly predict pregnancy was 0.74 (95% CI 0.72–0.76). Scores ≥7 predicted a pregnancy incidence of >17% per year and captured 78% of the pregnancies. Internal and external validation confirmed the predictive ability of the score.

Discussion

A pregnancy likelihood score encompassing basic demographic, clinical and behavioral factors defined African HIV-1 serodiscordant couples with high one-year pregnancy incidence rates. This tool could be used to engage African HIV-1 serodiscordant couples in counseling discussions about fertility intentions in order to offer services for safer conception or contraception that align with their reproductive goals.     

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.     

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.     

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.