The human immunodeficiency virus type 1 envelope confers higher rates of replicative fitness to perinatally transmitted viruses than to nontransmitted viruses

Selection of a minor viral genotype during perinatal transmission of human Immunodeficiency virus type 1 (HIV-1) has been observed, but there is a lack of information on the correlation of the restrictive transmission with biological properties of the virus, such as replicative fitness. Recombinant viruses expressing the enhanced green fluorescent protein or the Discosoma sp. red fluorescent (DsRed2) protein carrying the V1 to V5 regions of env from seven mother-infant pairs (MIPs) infected by subtype C HIV-1 were constructed, and competition assays were carried out to compare the fitness between the transmitted and nontransmitted viruses. Flow cytometry was used to quantify the frequency of infected cells, and the replicative fitness was determined based on a calculation that takes into account replication of competing viruses in a single infection versus dual infections. Transmitted viruses from five MIPs with the mothers chronically infected showed a restrictive env genotype, and all the recombinant viruses carrying the infants' Env had higher replicative fitness than those carrying the Env from the mothers. This growth fitness is lineage specific and can be observed only within the same MIP. In contrast, in two MIPs where the mothers had undergone recent acute infection, the viral Env sequences were similar between the mothers and infants and showed no further restriction in quasispecies during perinatal transmission. The recombinant viruses carrying the Env from the infants' viruses also showed replication fitness similar to those carrying the mothers' Env proteins. Our results suggest that newly transmitted viruses from chronically infected mothers have been selected to have higher replicative fitness to favor transmission, and this advantage is conferred by the V1 to V5 region of Env of the transmitted viruses. This finding has important implications for vaccine design or development of strategies to prevent HIV-1 transmission.     

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.     

Restriction of HIV-1 Genotypes in Breast Milk Does Not Account for the Population Transmission Genetic Bottleneck That Occurs following Transmission

Background

Breast milk transmission of HIV-1 remains a major route of pediatric infection. Defining the characteristics of viral variants to which breastfeeding infants are exposed is important for understanding the genetic bottleneck that occurs in the majority of mother-to-child transmissions. The blood-milk epithelial barrier markedly restricts the quantity of HIV-1 in breast milk, even in the absence of antiretroviral drugs. The basis of this restriction and the genetic relationship between breast milk and blood variants are not well established.

Methodology/Principal Findings

We compared 356 HIV-1 subtype C gp160 envelope (env) gene sequences from the plasma and breast milk of 13 breastfeeding women. A trend towards lower viral population diversity and divergence in breast milk was observed, potentially indicative of clonal expansion within the breast. No differences in potential N-linked glycosylation site numbers or in gp160 variable loop amino acid lengths were identified. Genetic compartmentalization was evident in only one out of six subjects in whom contemporaneously obtained samples were studied. However, in samples that were collected 10 or more days apart, six of seven subjects were classified as having compartmentalized viral populations, highlighting the necessity of contemporaneous sampling for genetic compartmentalization studies. We found evidence of CXCR4 co-receptor using viruses in breast milk and blood in nine out of the thirteen subjects, but no evidence of preferential localization of these variants in either tissue.

Conclusions/Significance

Despite marked restriction of HIV-1 quantities in milk, our data indicate intermixing of virus between blood and breast milk. Thus, we found no evidence that a restriction in viral genotype diversity in breast milk accounts for the genetic bottleneck observed following transmission. In addition, our results highlight the rapidity of HIV-1 env evolution and the importance of sample timing in analyses of gene flow.     

Significantly longer envelope V2 loops are characteristic of heterosexually transmitted subtype B HIV-1 in Trinidad

Background

In Trinidad and the wider Caribbean, subtype B Human Immunodeficiency Virus-type 1 (HIV-1B) overwhelmingly accounts for HIV infection among heterosexuals; this contrasts with the association of HIV-1B with homosexual transmission and injecting drug use globally. The HIV envelope contains genetic determinants of cell tropism and evasion from immune attack. In this study we investigate the genetic properties of the env V1-C4 of HIV-1B soon after transmission to Trinidadian heterosexuals. This will reveal distinctive genetic features of the strains that cause the HIV-1B epidemic in Trinidad and generate insights to better understand their properties.

Methodology/Principal Findings

Quasispecies sampling was performed on the env V1-C4 of HIV-1B strains soon after transmission to heterosexual Trinidadians in a cohort of seroconverters. Phylogenetic relationships were determined for these quasispecies and the length and number of asparagine (N) linked glycosylation sites (NLGS) in their variable loops compared to that for HIV-1B globally. Signature amino acids within the constant domains of the env V1-C4 were identified for heterosexually transmitted HIV-1B from Trinidad relative to HIV-1B globally. HIV-1B obtained from Trinidadian heterosexuals soon after seroconversion had significantly longer V2 loops with one more glycosylation site, shorter V3 loops and no significant difference in V1 or V4 when compared to HIV-1B obtained soon after seroconversion from infected individuals in the rest of the world. HIV-1B soon after seroconversion and during chronic infection of Trinidadians was not significantly different, suggesting that distinctly long V2 loops are characteristic of HIV-1B in Trinidad. A threonine deletion at position 319 (T319-) along with the substitutions R315K and S440R were found to be distinctly associated with HIV-1B from Trinidad compared to HIV-1B globally.

Conclusions

This finding of distinctive genetic features that are characteristic of HIV-1B strains from Trinidad is consistent with the Trinidad epidemic being established by a founder strain or closely related founder strains of HIV-1B.     

Evaluation of cervical mucosa in transmission bottleneck during acute HIV-1 infection using a cervical tissue-based organ culture

Background

Although there are different strains of HIV-1 in a chronically infected individual, only one or limited virus strains are successfully transmitted to a new individual. The reason for this “transmission bottleneck” is as yet unknown.

Methodology/Principal Findings

A human cervical explant model was used to measure HIV-1 transmission efficiency of viral strains from chronic infections, and transmitter/founder variants. We also evaluated the genetic characteristics of HIV-1 variants in the inoculums compared to those transmitted across the cervical mucosa. Eight different HIV-1 isolates were used in this study, six chronic isolates and two transmitter/founder viruses. The transmission efficiency of the chronic and transmitter/founder virus isolates and the viral diversity of chronic isolates before and after viral transmission were assessed. The results indicate that transmitter/founder viruses did not display higher transmission efficiency than chronic HIV-1 isolates. Furthermore, no evidence for a difference in diversity was found between the inoculums and transmitted virus strains. Phylogenetic analysis indicated that the sequences of variants in the inoculums and those present in transmitted virus intermingled irrespective of co-receptor usage. In addition, the inoculum and transmitted variants had a similar pairwise distance distribution.

Conclusion

There was no selection of a single or limited number of viral variants during HIV-1 transmission across the cervical mucosa in the organ culture model, indicating that the cervical mucosa alone may not produce the transmission bottleneck of HIV-1 infection observed in vivo.     

Sexually-Transmitted/Founder HIV-1 Cannot Be Directly Predicted from Plasma or PBMC-Derived Viral Quasispecies in the Transmitting Partner

Objective

Characterization of HIV-1 sequences in newly infected individuals is important for elucidating the mechanisms of viral sexual transmission. We report the identification of transmitted/founder viruses in eight pairs of HIV-1 sexually-infected patients enrolled at the time of primary infection (“recipients”) and their transmitting partners (“donors”).

Methods

Using a single genome-amplification approach, we compared quasispecies in donors and recipients on the basis of 316 and 376 C2V5 env sequences amplified from plasma viral RNA and PBMC-associated DNA, respectively.

Results

Both DNA and RNA sequences indicated very homogeneous viral populations in all recipients, suggesting transmission of a single variant, even in cases of recent sexually transmitted infections (STIs) in donors (n = 2) or recipients (n = 3). In all pairs, the transmitted/founder virus was derived from an infrequent variant population within the blood of the donor. The donor variant sequences most closely related to the recipient sequences were found in plasma samples in 3/8 cases and/or in PBMC samples in 6/8 cases. Although donors were exclusively (n = 4) or predominantly (n = 4) infected by CCR5-tropic (R5) strains, two recipients were infected with highly homogeneous CXCR4/dual-mixed-tropic (X4/DM) viral populations, identified in both DNA and RNA. The proportion of X4/DM quasispecies in donors was higher in cases of X4/DM than R5 HIV transmission (16.7–22.0% versus 0–2.6%), suggesting that X4/DM transmission may be associated with a threshold population of X4/DM circulating quasispecies in donors.

Conclusions

These suggest that a severe genetic bottleneck occurs during subtype B HIV-1 heterosexual and homosexual transmission. Sexually-transmitted/founder virus cannot be directly predicted by analysis of the donor’s quasispecies in plasma and/or PBMC. Additional studies are required to fully understand the traits that confer the capacity to transmit and establish infection, and determine the role of concomitant STIs in mitigating the genetic bottleneck in mucosal HIV transmission.     

Origin and dynamics of HIV-1 subtype C infection in India

Objective

To investigate the geographical origin and evolution dynamics of HIV-1 subtype C infection in India.

Design

Ninety HIV-1 subtype C env gp120 subtype C sequences from India were compared with 312 env gp120 reference subtype C sequences from 27 different countries obtained from Los Alamos HIV database. All the HIV-1 subtype C env gp120 sequences from India were used for the geographical origin analysis and 61 subtype C env gp120 sequences with known sampling year (from 1991 to 2008) were employed to determine the origin of HIV infection in India.

Methods

Phylogenetic analysis of HIV-1 env sequences was used to investigate the geographical origin and tMRCA of Indian HIV-1 subtype C. Evolutionary parameters including origin date and demographic growth patterns of Indian subtype C were estimated using a Bayesian coalescent-based approach under relaxed molecular clock models.

Findings

The majority of the analyzed Indian and South African HIV-1 subtype C sequences formed a single monophyletic cluster. The most recent common ancestor date was calculated to be 1975.56 (95% HPD, 1968.78–1981.52). Reconstruction of the effective population size revealed three phases of epidemic growth: an initial slow growth, followed by exponential growth, and then a plateau phase approaching present time. Stabilization of the epidemic growth phase correlated with the foundation of National AIDS Control Organization in India.

Interpretation

Indian subtype C originated from a single South African lineage in the middle of 1970s. The current study emphasizes not only the utility of HIV-1 sequence data for epidemiological studies but more notably highlights the effectiveness of community or government intervention strategies in controlling the trend of the epidemic.     

Uterine epithelial cell regulation of DC-SIGN expression inhibits transmitted/founder HIV-1 trans infection by immature dendritic cells

Background

Sexual transmission accounts for the majority of HIV-1 infections. In over 75% of cases, infection is initiated by a single variant (transmitted/founder virus). However, the determinants of virus selection during transmission are unknown. Host cell-cell interactions in the mucosa may be critical in regulating susceptibility to infection. We hypothesized in this study that specific immune modulators secreted by uterine epithelial cells modulate susceptibility of dendritic cells (DC) to infection with HIV-1.

Methodology/Principal Findings

Here we report that uterine epithelial cell secretions (i.e. conditioned medium, CM) decreased DC-SIGN expression on immature dendritic cells via a transforming growth factor beta (TGF-β) mechanism. Further, CM inhibited dendritic cell-mediated trans infection of HIV-1 expressing envelope proteins of prototypic reference. Similarly, CM inhibited trans infection of HIV-1 constructs expressing envelopes of transmitted/founder viruses, variants that are selected during sexual transmission. In contrast, whereas recombinant TGF- β1 inhibited trans infection of prototypic reference HIV-1 by dendritic cells, TGF-β1 had a minimal effect on trans infection of transmitted/founder variants irrespective of the reporter system used to measure trans infection.

Conclusions/Significance

Our results provide the first direct evidence for uterine epithelial cell regulation of dendritic cell transmission of infection with reference and transmitted/founder HIV-1 variants. These findings have immediate implications for designing strategies to prevent sexual transmission of HIV-1.     

Phylogenetic Relatedness of Circulating HIV-1C Variants in Mochudi,Botswana

Background

Determining patterns of HIV transmission is increasingly important for the most efficient use of modern prevention interventions. HIV phylogeny can provide a better understanding of the mechanisms underlying HIV transmission networks in communities.

Methods

To reconstruct the structure and dynamics of a local HIV/AIDS epidemic, the phylogenetic relatedness of HIV-1 subtype C env sequences obtained from 785 HIV-infected community residents in the northeastern sector of Mochudi, Botswana, during 2010–2013 was estimated. The genotyping coverage was estimated at 44%. Clusters were defined based on relatedness of HIV-1C env sequences and bootstrap support of splits.

Results

The overall proportion of clustered HIV-1C env sequences was 19.1% (95% CI 17.5% to 20.8%). The proportion of clustered sequences from Mochudi was significantly higher than the proportion of non-Mochudi sequences that clustered, 27.0% vs. 14.7% (p = 5.8E-12; Fisher exact test). The majority of clustered Mochudi sequences (90.1%; 95% CI 85.1% to 93.6%) were found in the Mochudi-unique clusters. None of the sequences from Mochudi clustered with any of the 1,244 non-Botswana HIV-1C sequences. At least 83 distinct HIV-1C variants, or chains of HIV transmission, in Mochudi were enumerated, and their sequence signatures were reconstructed. Seven of 20 genotyped seroconverters were found in 7 distinct clusters.

Conclusions

The study provides essential characteristics of the HIV transmission network in a community in Botswana, suggests the importance of high sampling coverage, and highlights the need for broad HIV genotyping to determine the spread of community-unique and community-mixed viral variants circulating in local epidemics. The proposed methodology of cluster analysis enumerates circulating HIV variants and can work well for surveillance of HIV transmission networks. HIV genotyping at the community level can help to optimize and balance HIV prevention strategies in trials and combined intervention packages.     

Viral linkage in HIV-1 seroconverters and their partners in an HIV-1 prevention clinical trial

Background

Characterization of viruses in HIV-1 transmission pairs will help identify biological determinants of infectiousness and evaluate candidate interventions to reduce transmission. Although HIV-1 sequencing is frequently used to substantiate linkage between newly HIV-1 infected individuals and their sexual partners in epidemiologic and forensic studies, viral sequencing is seldom applied in HIV-1 prevention trials. The Partners in Prevention HSV/HIV Transmission Study (ClinicalTrials.gov #{"type":"clinical-trial","attrs":{"text":"NCT00194519","term_id":"NCT00194519"}}NCT00194519) was a prospective randomized placebo-controlled trial that enrolled serodiscordant heterosexual couples to determine the efficacy of genital herpes suppression in reducing HIV-1 transmission; as part of the study analysis, HIV-1 sequences were examined for genetic linkage between seroconverters and their enrolled partners.

Methodology/Principal Findings

We obtained partial consensus HIV-1 env and gag sequences from blood plasma for 151 transmission pairs and performed deep sequencing of env in some cases. We analyzed sequences with phylogenetic techniques and developed a Bayesian algorithm to evaluate the probability of linkage. For linkage, we required monophyletic clustering between enrolled partners'' sequences and a Bayesian posterior probability of ≥50%. Adjudicators classified each seroconversion, finding 108 (71.5%) linked, 40 (26.5%) unlinked, and 3 (2.0%) indeterminate transmissions, with linkage determined by consensus env sequencing in 91 (84%). Male seroconverters had a higher frequency of unlinked transmissions than female seroconverters. The likelihood of transmission from the enrolled partner was related to time on study, with increasing numbers of unlinked transmissions occurring after longer observation periods. Finally, baseline viral load was found to be significantly higher among linked transmitters.

Conclusions/Significance

In this first use of HIV-1 sequencing to establish endpoints in a large clinical trial, more than one-fourth of transmissions were unlinked to the enrolled partner, illustrating the relevance of these methods in the design of future HIV-1 prevention trials in serodiscordant couples. A hierarchy of sequencing techniques, analysis methods, and expert adjudication contributed to the linkage determination process.     

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.     

Genetically Related Human Immunodeficiency Virus Type 1 in Three Adults of a Family with No Identified Risk Factor for Intrafamilial Transmission

A small number of cases of human immunodeficiency virus (HIV) infection have been reported in individuals with no identified risk factors for transmission. We report on the seroconversion of the 61-year-old mother and the subsequent finding of HIV seropositivity in the 66-year-old father of a 31-year-old AIDS patient. Extensive investigation failed to identify any risk factor for intrafamilial transmission. We conducted a genetic analysis and determined the amino acid signature patterns of the V3, V4, and V5 hypervariable domains and flanking regions in the HIV-1 gp120 env gene of 26 clones derived from proviral DNA in peripheral blood mononuclear cells of the members of the family. env sequences of the viruses isolated from the patients were compared with sequences of HIV-1 subtype B viruses from Europe and local field isolates. Phylogenetic analysis revealed that the sequences of the viruses isolated from the patients were genetically related and formed an intrafamilial cluster of HIV-1 distinct from other subtype B viruses. Interindividual nucleotide variability in the C2-V3 and V4-C4-V5 domains ranged between 1.2 and 5.0% and between 2.2 and 7.5%, respectively, whereas divergence between HIV strains from the patients and control viral strains ranged from 6.6 to 29.3%. The amino acid signature patterns of viral clones from the three patients were closely related. In the C2-V3 region, two minor clones derived from the son’s virus showed less nucleotide divergence (mean, 3.5 and 3.9%) than did the clones derived from the viruses of both parents or the seven other predominant clones derived from the virus from the son (mean, 5.4%). The top of the V3 loop of the last two clones and of all viral clones from the parents exhibited an unusual GPGG sequence. This is the first report of genotypic relatedness of HIV-1 in three adults of the same family in the absence of identified risk factor for transmission between the members of the family. Our findings suggest that atypical transmission of HIV may occur.     

The HIV-1 Subtype C Epidemic in South America Is Linked to the United Kingdom

Background

The global spread of HIV-1 has been accompanied by the emergence of genetically distinct viral strains. Over the past two decades subtype C viruses, which predominate in Southern and Eastern Africa, have spread rapidly throughout parts of South America. Phylogenetic studies indicate that subtype C viruses were introduced to South America through a single founder event that occurred in Southern Brazil. However, the external route via which subtype C viruses spread to the South American continent has remained unclear.

Methodology/Principal Findings

We used automated genotyping to screen 8,309 HIV-1 subtype C pol gene sequences sampled within the UK for isolates genetically linked to the subtype C epidemic in South America. Maximum likelihood and Bayesian approaches were used to explore the phylogenetic relationships between 54 sequences identified in this screen, and a set of globally sampled subtype C reference sequences. Phylogenetic trees disclosed a robustly supported relationship between sequences from Brazil, the UK and East Africa. A monophyletic cluster comprised exclusively of sequences from the UK and Brazil was identified and dated to approximately the early 1980s using a Bayesian coalescent-based method. A sub-cluster of 27 sequences isolated from homosexual men of UK origin was also identified and dated to the early 1990s.

Conclusions

Phylogenetic, demographic and temporal data support the conclusion that the UK was a crucial staging post in the spread of subtype C from East Africa to South America. This unexpected finding demonstrates the role of diffuse international networks in the global spread of HIV-1 infection, and the utility of globally sampled viral sequence data in revealing these networks. Additionally, we show that subtype C viruses are spreading within the UK amongst men who have sex with men.     

Evidence for Limited Genetic Compartmentalization of HIV-1 between Lung and Blood

Background

HIV-1 is frequently detected in the lungs of infected individuals and is likely important in the development of pulmonary opportunistic infections. The unique environment of the lung, rich in alveolar macrophages and with specialized local immune responses, may contribute to differential evolution or selection of HIV-1.

Methodology and Findings

We characterized HIV-1 in the lung in relation to contemporaneous viral populations in the blood. The C2-V5 region of HIV-1 env was sequenced from paired lung (induced sputum or bronchoalveolar lavage) and blood (plasma RNA and proviral DNA from sorted or unsorted PBMC) from 18 subjects. Compartmentalization between tissue pairs was assessed using 5 established tree or distance-based methods, including permutation tests to determine statistical significance. We found statistical evidence of compartmentalization between lung and blood in 10/18 subjects, although lung and blood sequences were intermingled on phylogenetic trees in all subjects. The subject showing the greatest compartmentalization contained many nearly identical sequences in BAL sample, suggesting clonal expansion may contribute to reduced viral diversity in the lung in some cases. However, HIV-1 sequences in lung were not more homogeneous overall, nor were we able to find a lung-specific genotype associated with macrophage tropism in V3. In all four subjects in whom predicted X4 genotypes were found in blood, predicted X4 genotypes were also found in lung.

Conclusions

Our results support a picture of continuous migration of HIV-1 between circulating blood and lung tissue, with perhaps a very limited degree of localized evolution or clonal replication.     

Effect of HIV-1 Subtypes on Disease Progression in Rural Uganda: A Prospective Clinical Cohort Study

Objective

We examined the association of HIV-1 subtypes with disease progression based on three viral gene regions.

Design

A prospective HIV-1 clinical cohort study in rural Uganda.

Methods

Partial gag, env and pol genes were sequenced. Cox proportional hazard regression modelling was used to estimate adjusted hazard ratios (aHRs) of progression to: CD4≤250, AIDS onset and death, adjusted for sex, age and CD4 count at enrolment.

Results

Between 1990 and 2010, 292 incident cases were subtyped: 25% had subtype A, 45% had D, 26% had A/D recombinants, 1% had C and 4% were other recombinant forms. Of the 278 incident cases included in the disease progression analysis, 62% progressed to CD4≤250, 32% to AIDS, and 34% died with a higher proportion being among subtype D cases. The proportions of individuals progressing to the three endpoints were significantly higher among individuals infected with subtype D. Throughout the study period, individuals infected with subtype D progressed faster to CD4≤250, adjusted HR (aHR), (95% CI) = 1.72 (1.16–2.54), but this was mainly due to events in the period before antiretroviral therapy (ART) introduction, when individuals infected with subtype D significantly progressed faster to CD4≤250 than subtype A cases; aHR (95% CI) = 1.78 (1.01–3.14).

Conclusions

In this population, HIV-1 subtype D was the most prevalent and was associated with faster HIV-1 disease progression than subtype A. Further studies are needed to examine the effect of HIV-1 subtypes on disease progression in the ART period and their effect on the virological and immunological ART outcomes.     

Evidence of at least two introductions of HIV-1 in the Amerindian Warao population from Venezuela

Background

The Venezuelan Amerindians were, until recently, free of human immunodeficiency virus (HIV) infection. However, in 2007, HIV-1 infection was detected for the first time in the Warao Amerindian population living in the Eastern part of Venezuela, in the delta of the Orinoco river. The aim of this study was to analyze the genetic diversity of the HIV-1 circulating in this population.

Methodology/Principal Findings

The pol genomic region was sequenced for 16 HIV-1 isolates and for some of them, sequences from env, vif and nef genomic regions were obtained. All HIV-1 isolates were classified as subtype B, with exception of one that was classified as subtype C. The 15 subtype B isolates exhibited a high degree of genetic similarity and formed a highly supported monophyletic cluster in each genomic region analyzed. Evolutionary analyses of the pol genomic region indicated that the date of the most recent common ancestor of the Waraos subtype B clade dates back to the late 1990s.

Conclusions/Significance

At least two independent introductions of HIV-1 have occurred in the Warao Amerindians from Venezuela. The HIV-1 subtype B was successfully established and got disseminated in the community, while no evidence of local dissemination of the HIV-1 subtype C was detected in this study. These results warrant further surveys to evaluate the burden of this disease, which can be particularly devastating in this Amerindian population, with a high prevalence of tuberculosis, hepatitis B, among other infectious diseases, and with limited access to primary health care.     

In vivo emergence of HIV-1 highly sensitive to neutralizing antibodies

Background

The rapid and continual viral escape from neutralizing antibodies is well documented in HIV-1 infection. Here we report in vivo emergence of viruses with heightened sensitivity to neutralizing antibodies, sometimes paralleling the development of neutralization escape.

Methodology/Principal Findings

Sequential viral envs were amplified from seven HIV-1 infected men monitored from seroconversion up to 5 years after infection. Env-recombinant infectious molecular clones were generated and tested for coreceptor use, macrophage tropism and neutralization sensitivity to homologous and heterologous serum, soluble CD4 and monoclonal antibodies IgG1b12, 2G12 and 17b. We found that HIV-1 evolves sensitivity to contemporaneous neutralizing antibodies during infection. Neutralization sensitive viruses grow out even when potent autologous neutralizing antibodies are present in patient serum. Increased sensitivity to neutralization was associated with susceptibility of the CD4 binding site or epitopes induced after CD4 binding, and mediated by complex envelope determinants including V3 and V4 residues. The development of neutralization sensitive viruses occurred without clinical progression, coreceptor switch or change in tropism for primary macrophages.

Conclusions

We propose that an interplay of selective forces for greater virus replication efficiency without the need to resist neutralizing antibodies in a compartment protected from immune surveillance may explain the temporal course described here for the in vivo emergence of HIV-1 isolates with high sensitivity to neutralizing antibodies.     

Identification of Human MicroRNA-Like Sequences Embedded within the Protein-Encoding Genes of the Human Immunodeficiency Virus

Background

MicroRNAs (miRNAs) are highly conserved, short (18–22 nts), non-coding RNA molecules that regulate gene expression by binding to the 3′ untranslated regions (3′UTRs) of mRNAs. While numerous cellular microRNAs have been associated with the progression of various diseases including cancer, miRNAs associated with retroviruses have not been well characterized. Herein we report identification of microRNA-like sequences in coding regions of several HIV-1 genomes.

Results

Based on our earlier proteomics and bioinformatics studies, we have identified 8 cellular miRNAs that are predicted to bind to the mRNAs of multiple proteins that are dysregulated during HIV-infection of CD4+ T-cells in vitro. In silico analysis of the full length and mature sequences of these 8 miRNAs and comparisons with all the genomic and subgenomic sequences of HIV-1 strains in global databases revealed that the first 18/18 sequences of the mature hsa-miR-195 sequence (including the short seed sequence), matched perfectly (100%), or with one nucleotide mismatch, within the envelope (env) genes of five HIV-1 genomes from Africa. In addition, we have identified 4 other miRNA-like sequences (hsa-miR-30d, hsa-miR-30e, hsa-miR-374a and hsa-miR-424) within the env and the gag-pol encoding regions of several HIV-1 strains, albeit with reduced homology. Mapping of the miRNA-homologues of env within HIV-1 genomes localized these sequence to the functionally significant variable regions of the env glycoprotein gp120 designated V1, V2, V4 and V5.

Conclusions

We conclude that microRNA-like sequences are embedded within the protein-encoding regions of several HIV-1 genomes. Given that the V1 to V5 regions of HIV-1 envelopes contain specific, well-characterized domains that are critical for immune responses, virus neutralization and disease progression, we propose that the newly discovered miRNA-like sequences within the HIV-1 genomes may have evolved to self-regulate survival of the virus in the host by evading innate immune responses and therefore influencing persistence, replication and/or pathogenicity.