Sex Differences in Human Immunodeficiency Virus Persistence and Reservoir Size During Aging.

BACKGROUND: Sex differences in human immunodeficiency virus (HIV) reservoir dynamics remain underexplored. METHODS: Longitudinal samples from virally suppressed midlife women (n = 59, median age 45 years) and age-matched men (n = 31) were analyzed retrospectively. At each time point, we measured sex hormones (by means of enzyme-linked immunosorbent assay) and cellular HIV DNA and RNA (by means of digital droplet polymerase chain reaction). Number of inducible HIV RNA+ cells, which provides an upper estimate of the replication-competent reservoir, was quantified longitudinally in a different subset of 14 women, across well-defined reproductive stages. Mixed-effects models included normalized reservoir outcomes and sex, time since antiretroviral therapy (ART) initiation, and the sex-by-time interaction as predictors. RESULTS: At ART initiation, women and men had median (interquartile range [IQR]) CD4+ T-cell counts of 204/µL (83-306/µL) versus 238/µL (120-284/µL), respectively; median ages of 45 (42-48) versus 47 (43-51) years; and median follow-up times of 79.2/µL (60.5-121.1/µL) versus 66.2/µL (43.2-80.6/µL) months. We observed a significant decline of total HIV DNA over time in both men and women (P < .01). However, the rates of change differed significantly between the sexes (P < .01), with women having a significantly slower rate of decline than men, more pronounced with age. By contrast, the levels of inducible HIV RNA increased incrementally over time in women during reproductive aging (P < .01). CONCLUSIONS: In contrast to men, in whom the HIV reservoir steadily declines with aging, the HIV reservoir in women is more dynamic. Total HIV DNA (including intact and defective genomes) declines more slowly in women than in men, while the inducible HIV RNA+ reservoir, which is highly enriched in replication-competent virus, increases in women after menopause.

Subclinical Cytomegalovirus and Epstein-Barr Virus Shedding Is Associated with Increasing HIV DNA Molecular Diversity in Peripheral Blood during Suppressive Antiretroviral Therapy.

Cytomegalovirus (CMV) almost universally infects persons with HIV (PWH), and it is a driver of persistent inflammation and HIV persistence. The mechanisms underlying the association between CMV (and possibly other herpesviruses) and HIV persistence are unclear. Serially collected blood samples were obtained from men who have sex with men (MSM) who started antiretroviral therapy (ART) within 1 year of their estimated date of HIV infection (EDI). Total CMV and Epstein-Barr virus (EBV) DNA were quantified in peripheral blood mononuclear cells by droplet digital PCR (ddPCR). Deep sequencing of the HIV DNA partial env gene was performed, and the dynamics of viral diversity over time were analyzed in relation to CMV and EBV shedding status. In total, 37 MSM PWH were included and followed for a median of 23 months (IQR, 22 to 28). Participants started ART within a median of 3.1 months (IQR, 1.5 to 6.5) after EDI and remained virally suppressed thereafter. A total of 18 participants (48.6%) were classified as high EBV shedders, while 19 (51.4%) were classified as CMV shedders. In longitudinal analyses, normalized molecular diversity levels tended to increase over time among participants with detectable CMV and high EBV DNA (0.03 ± 0.02, P = 0.08), while they significantly declined among participants with no/low viral shedding (-0.04 ± 0.02, P = 0.047, interaction P < 0.01). Subclinical CMV and EBV shedding could contribute to the dynamics of the HIV DNA reservoir during suppressive ART. Whether persistent CMV/EBV replication could be targeted as a strategy to reduce the size of the latent HIV reservoir is an avenue that should be explored.IMPORTANCE As part of this study, we evaluated the molecular characteristics of the HIV DNA reservoir over time during antiretroviral treatment (ART) in relation to those of other chronic viral infections (i.e., cytomegalovirus [CMV] and Epstein-Barr virus [EBV]). We demonstrated that the presence of CMV and high-level EBV DNA in peripheral blood cells was associated with changes in HIV DNA molecular diversity. Specifically, HIV DNA molecular diversity increased over time among participants with detectable CMV and high-level EBV DNA, while it significantly declined among participants with no/low viral shedding. Although the current study design does not allow causality to be inferred, it does support the theory that persistent CMV and EBV shedding could contribute to the dynamics of the HIV DNA reservoir during suppressive ART, even when ART is initiated during the earliest phases of HIV infection.

HIV RNA Rebound in Seminal Plasma after Antiretroviral Treatment Interruption.

If strategies currently in development succeed in eradicating HIV reservoirs in peripheral blood and lymphoid tissues, residual sources of virus may remain in anatomic compartments. Paired blood and semen samples were collected from 12 individuals enrolled in a randomized, double-blind, placebo-controlled therapeutic vaccine clinical trial in people with HIV (PWH) who began antiretroviral therapy (ART) during acute or early infection (ClinicalTrials registration no. NCT01859325). After the week 56 visit (postintervention), all participants interrupted ART. At the first available time points after viral rebound, we sequenced HIV-1 env (C2-V3), gag (p24), and pol (reverse transcriptase) regions amplified from cell-free HIV RNA in blood and seminal plasma using the MiSeq Illumina platform. Comprehensive sequence and phylogenetic analyses were performed to evaluate viral population structure, compartmentalization, and viral diversity in blood and seminal plasma. Compared to that in blood, HIV RNA rebound in semen occurred significantly later (median of 66 versus 42 days post-ART interruption, P < 0.01) and reached lower levels (median 164 versus 16,090 copies/ml, P < 0.01). Three of five participants with available sequencing data presented compartmentalized viral rebound between blood and semen in one HIV coding region. Despite early ART initiation, HIV RNA molecular diversity was higher in semen than in blood in all three coding regions for most participants. Higher HIV RNA molecular diversity in the genital tract (compared to that in blood plasma) and evidence of compartmentalization illustrate the distinct evolutionary dynamics between these two compartments after ART interruption. Future research should evaluate whether the genital compartment might contribute to viral rebound in some PWH interrupting ART.IMPORTANCE To cure HIV, we likely need to target the reservoirs in all anatomic compartments. Here, we used sophisticated statistical and phylogenetic methods to analyze blood and semen samples collected from 12 persons with HIV who began antiretroviral therapy (ART) during very early HIV infection and who interrupted their ART as part of a clinical trial. First, we found that HIV RNA rebound in semen occurred significantly later and reached lower levels than in blood. Second, we found that the virus in semen was genetically different in some participants compared to that in blood. Finally, we found increased HIV RNA molecular diversity in semen compared to that in blood in almost all study participants. These data suggest that the HIV RNA populations emerging from the genital compartment after ART interruption might not be the same as those emerging from blood plasma. Future research should evaluate whether the genital compartment might contribute to viral rebound in some people with HIV (PWH) interrupting ART.

Factors predictive of optimal peristomal skin status in patients with an ostomy: a secondary analysis.

Peristomal skin problems are common in ostomy patients and are connected to decreased quality of life and patient independence, as well as increased treatment costs. This study analysed the factors related to peristomal skin changes in order to better define the clinical profile of patients with optimal peristomal skin status. Secondary analysis of data from a nationwide, cross-sectional, retrospective, multicentre study performed in Spain (Uses and Attitudes in Ostomy) (Bueno Cruz et al, 2021) evaluated clinical data, including demographics, preoperative stoma site marking, type of ostomy, device used, frequency of leakage and scores from the Ostomy Skin Tool (OST) and quality of life (QoL) questionnaires. Risk factors for peristomal skin changes were analysed using multivariate analysis, and a predictive nomogram to anticipate optimal peristomal skin status (defined here as discolouration (D), erosion (E), and tissue overgrowth (T) (DET) score of 0) was developed. Some 871 patients with an ostomy using different commercial devices in Spain were evaluated. Multivariate analysis to predict optimal peristomal skin status revealed leakage frequency, patient age, type of ostomy, preoperative siting and type of baseplate used were independent predictors of peristomal skin status. Optimal peristomal skin care should be a treatment goal for nurse specialists in stomal therapy, and its individual influencing variables should be taken into account by nurses specialising in ostomy care.

Intrasubtype B HIV-1 Superinfection Correlates with Delayed Neutralizing Antibody Response.

Understanding whether the neutralizing antibody (NAb) response impacts HIV-1 superinfection and how superinfection subsequently modulates the NAb response can help clarify correlates of protection from HIV exposures and better delineate pathways of NAb development. We examined associations between the development of NAb and the occurrence of superinfection in a well-characterized, antiretroviral therapy (ART)-naive, primary infection cohort of men who have sex with men. Deep sequencing was applied to blood plasma samples from the cohort to detect cases of superinfection. We compared the NAb activity against autologous and heterologous viruses between 10 participants with intrasubtype B superinfection and 19 monoinfected controls, matched to duration of infection and risk behavior. Three to 6 months after primary infection, individuals who would later become superinfected had significantly weaker NAb activity against tier 1 subtype B viruses (P = 0.003 for SF-162 and P = 0.017 for NL4-3) and marginally against autologous virus (P = 0.054). Lower presuperinfection NAb responses correlated with weaker gp120 binding and lower plasma total IgG titers. Soon after superinfection, the NAb response remained lower, but between 2 and 3 years after primary infection, NAb levels strengthened and reached those of controls. Superinfecting viruses were typically not susceptible to neutralization by presuperinfection plasma. These observations suggest that recently infected individuals with a delayed NAb response against primary infecting and tier 1 subtype B viruses are more susceptible to superinfection.IMPORTANCE Our findings suggest that within the first year after HIV infection, a relatively weak neutralizing antibody response against primary and subtype-specific neutralization-sensitive viruses increases susceptibility to superinfection in the face of repeated exposures. As natural infection progresses, the immune response strengthens significantly in some superinfected individuals. These findings will inform HIV vaccine design by providing testable correlates of protection from initial HIV infection.

Incidence and prevalence of intrasubtype HIV-1 dual infection in at-risk men in the United States.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) dual infection (DI) has been associated with decreased CD4 T-cell counts and increased viral loads; however, the frequency of intrasubtype DI is poorly understood. We used ultradeep sequencing (UDS) to estimate the frequency of DI in a primary infection cohort of predominantly men who have sex with men (MSM). METHODS: HIV-1 genomes from longitudinal blood samples of recently infected, therapy-naive participants were interrogated with UDS. DI was confirmed when maximum sequence divergence was excessive and supported by phylogenetic analysis. Coinfection was defined as DI at baseline; superinfection was monoinfection at baseline and DI at a later time point. RESULTS: Of 118 participants, 7 were coinfected and 10 acquired superinfection. Superinfection incidence rate was 4.96 per 100 person-years (95% confidence interval [CI], 2.67-9.22); 6 occurred in the first year and 4 in the second. Overall cumulative prevalence of intrasubtype B DI was 14.4% (95% CI, 8.6%-22.1%). Primary HIV-1 incidence was 4.37 per 100 person-years (95% CI, 3.56-5.36). CONCLUSIONS: Intrasubtype DI was frequent and comparable to primary infection rates among MSM in San Diego; however, superinfection rates declined over time. DI is likely an important component of the HIV epidemic dynamics, and development of stronger immune responses to the initial infection may protect from superinfection.

Dynamics of viral evolution and neutralizing antibody response after HIV-1 superinfection.

Investigating the incidence and prevalence of HIV-1 superinfection is challenging due to the complex dynamics of two infecting strains. The superinfecting strain can replace the initial strain, be transiently expressed, or persist along with the initial strain in distinct or in recombined forms. Various selective pressures influence these alternative scenarios in different HIV-1 coding regions. We hypothesized that the potency of the neutralizing antibody (NAb) response to autologous viruses would modulate viral dynamics in env following superinfection in a limited set of superinfection cases. HIV-1 env pyrosequencing data were generated from blood plasma collected from 7 individuals with evidence of superinfection. Viral variants within each patient were screened for recombination, and viral dynamics were evaluated using nucleotide diversity. NAb responses to autologous viruses were evaluated before and after superinfection. In 4 individuals, the superinfecting strain replaced the original strain. In 2 individuals, both initial and superinfecting strains continued to cocirculate. In the final individual, the surviving lineage was the product of interstrain recombination. NAb responses to autologous viruses that were detected within the first 2 years of HIV-1 infection were weak or absent for 6 of the 7 recently infected individuals at the time of and shortly following superinfection. These 6 individuals had detectable on-going viral replication of distinct superinfecting virus in the env coding region. In the remaining case, there was an early and strong autologous NAb response, which was associated with extensive recombination in env between initial and superinfecting strains. This extensive recombination made superinfection more difficult to identify and may explain why the detection of superinfection has typically been associated with low autologous NAb titers.

Perfluoroalkylated substances in the global tropical and subtropical surface oceans.

In this study, perfluoroalkylated substances (PFASs) were analyzed in 92 surface seawater samples taken during the Malaspina 2010 expedition which covered all the tropical and subtropical Atlantic, Pacific and Indian oceans. Nine ionic PFASs including C6-C10 perfluoroalkyl carboxylic acids (PFCAs), C4 and C6-C8 perfluoroalkyl sulfonic acids (PFSAs) and two neutral precursors perfluoroalkyl sulfonamides (PFASAs), were identified and quantified. The Atlantic Ocean presented the broader range in concentrations of total PFASs (131-10900 pg/L, median 645 pg/L, n = 45) compared to the other oceanic basins, probably due to a better spatial coverage. Total concentrations in the Pacific ranged from 344 to 2500 pg/L (median = 527 pg/L, n = 27) and in the Indian Ocean from 176 to 1976 pg/L (median = 329, n = 18). Perfluorooctanesulfonic acid (PFOS) was the most abundant compound, accounting for 33% of the total PFASs globally, followed by perfluorodecanoic acid (PFDA, 22%) and perfluorohexanoic acid (PFHxA, 12%), being the rest of the individual congeners under 10% of total PFASs, even for perfluorooctane carboxylic acid (PFOA, 6%). PFASAs accounted for less than 1% of the total PFASs concentration. This study reports the ubiquitous occurrence of PFCAs, PFSAs, and PFASAs in the global ocean, being the first attempt, to our knowledge, to show a comprehensive assessment in surface water samples collected in a single oceanic expedition covering tropical and subtropical oceans. The potential factors affecting their distribution patterns were assessed including the distance to coastal regions, oceanic subtropical gyres, currents and biogeochemical processes. Field evidence of biogeochemical controls on the occurrence of PFASs was tentatively assessed considering environmental variables (solar radiation, temperature, chlorophyll a concentrations among others), and these showed significant correlations with some PFASs, but explaining small to moderate percentages of variability. This suggests that a number of physical and biogeochemical processes collectively drive the oceanic occurrence and fate of PFASs in a complex manner.

Using ultradeep pyrosequencing to study HIV-1 coreceptor usage in primary and dual infection.

HIV-1 dual infection (DI) and CXCR4 (X4) coreceptor usage are associated with accelerated disease progression but frequency and dynamics of coreceptor usage during DI is unknown. Ultradeep sequencing was used to interrogate for DI and infer coreceptor usage in longitudinal blood samples of 102 subjects. At baseline, X4 usage was high (23 subjects harbored X4 variants) and was not associated with infection duration or DI. Coreceptor usage changed over time in 12 of 47 participants, and X4 usage emerged in 4 of 41 monoinfections vs 2 of 5 superinfections (P = .12), suggesting a weak statistical trend toward occurrence of superinfection and acquiring X4 usage.

Impact of influenza and pneumococcal vaccines on HIV persistence and immune dynamics during suppressive antiretroviral therapy.

OBJECTIVE: We sought to determine if standard influenza and pneumococcal vaccines can be used to stimulate HIV reservoirs during antiretroviral therapy (ART). DESIGN: A prospective, randomized, double-blinded, placebo-controlled, crossover trial of two clinically recommended vaccines (influenza and pneumococcal). METHODS: Persons with HIV on ART ( N  = 54) were enrolled in the clinical trial. Blood was collected at baseline and days 2,4,7,14, and 30 postimmunizations. Levels of cellular HIV RNA and HIV DNA were measured by ddPCR. Expression of immunological markers on T cell subsets was measured by flow cytometry. Changes in unspliced cellular HIV RNA from baseline to day 7 postinjection between each vaccine and placebo was the primary outcome. RESULTS: Forty-seven participants completed at least one cycle and there were no serious adverse events related to the intervention. We observed no significant differences in the change in cellular HIV RNA after either vaccine compared with placebo at any timepoint. In secondary analyses, we observed a transient increase in total HIV DNA levels after influenza vaccine, as well as increased T cell activation and exhaustion on CD4 + T cells after pneumococcal vaccine. CONCLUSION: Clinically recommended vaccines were well tolerated but did not appear to stimulate the immune system strongly enough to elicit significantly noticeable HIV RNA transcription during ART.Clinicaltrials.gov identifier: NCT02707692.

Identification of system-level features in HIV migration within a host.

OBJECTIVE: Identify system-level features in HIV migration within a host across body tissues. Evaluate heterogeneity in the presence and magnitude of these features across hosts. METHOD: Using HIV DNA deep sequencing data generated across multiple tissues from 8 people with HIV, we represent the complex dependencies of HIV migration among tissues as a network and model these networks using the family of exponential random graph models (ERGMs). ERGMs allow for the statistical assessment of whether network features occur more (or less) frequently in viral migration than might be expected by chance. The analysis investigates five potential features of the viral migration network: (1) bi-directional flow between tissues; (2) preferential migration among tissues in the same biological system; (3) heterogeneity in the level of viral migration related to HIV reservoir size; (4) hierarchical structure of migration; and (5) cyclical migration among several tissues. We calculate the Cohran's Q statistic to assess heterogeneity in the magnitude of the presence of these features across hosts. The analysis adjusts for missing data on body tissues. RESULTS: We observe strong evidence for bi-directional flow between tissues; migration among tissues in the same biological system; and hierarchical structure of the viral migration network. This analysis shows no evidence for differential level of viral migration with respect to the HIV reservoir size of a tissue. There is evidence that cyclical migration among three tissues occurs less frequent than expected given the amount of viral migration. The analysis also provides evidence for heterogeneity in the magnitude that these features are present across hosts. Adjusting for missing tissue data identifies system-level features within a host as well as heterogeneity in the presence of these features across hosts that are not detected when the analysis only considers the observed data. DISCUSSION: Identification of common features in viral migration may increase the efficiency of HIV cure efforts as it enables targeting specific processes.

Occurrence of aerosol-bound fullerenes in the Mediterranean Sea atmosphere.

This work describes the assessment of a selection of fullerenes including C(60) and C(70) fullerene, N-methylfulleropyrrolidine, C(60) pyrrolidine tris-acid ethyl ester, [6,6]-Phenyl-C(61) butyric acid butyl ester and [6,6]-Thienyl C(61) butyric acid methyl ester, in airborne particulate from the Mediterranean Sea collected during two sampling campaigns from Barcelona to Istanbul and Alexandria, respectively. The analysis of the samples was carried out using a new method based on liquid chromatography coupled to mass spectrometry (LC-MS) presenting sensitivities between 5.4 and 20.9 pg/m(3). A total number of 43 samples covering the different basins of Mediterranean Sea were analyzed. Fullerenes were detected in all analyzed samples and quantifiable concentrations were found in 28 of the analyzed samples. The median of C(60) and C(70) fullerenes aerosol phase concentrations were 0.06 ng/m(3) and 0.48 ng/m(3) respectively for the Mediterranean Sea atmosphere. C(70) fullerene was the most frequently detected compound and also it was found in the higher concentrations for most samples, reaching 233.8 ng/m(3). The modeled back-trajectories disclose that those samples with higher concentrations of fullerenes were related to air masses which had been circulating over regions with an intense industrial activity, but the variability of the C(70)/C(60) ratio suggests multiple different sources. These results are related to the incidental emissions from urban and industrial development, underpinning the need of studying the possible risks associated to carbon nanoparticles in the environment and the need of evaluating the possible consequences of their ubiquitous occurrence.

Ubiquitous net volatilization of polycyclic aromatic hydrocarbons from soils and parameters influencing their soil-air partitioning.

Soils are a major reservoir of organic pollutants, and soil-air partitioning and exchange are key processes controlling the regional fate of pollutants. Here, we report and discuss the soil concentrations of polycyclic aromatic hydrocarbons (PAHs), their soil fugacities, the soil-air partition coefficients (K(SA)) and soil-air gradients for rural and semirural soils, in background areas of N-NE Spain and N-NW England. Different sampling campaigns were carried out to assess seasonal variability and differences between sampling sites. K(SA) values were dependent on soil temperature and soil organic quantity and type. Soil fugacities of phenanthrene and its alkyl homologues were 1-2 orders of magnitude higher than their ambient air fugacities for all sampling sites and periods. The soil to air fugacity ratio was correlated with soil temperature and soil redox potential. Similar trends for other PAHs were found but with lower fugacity ratios. The ubiquitous source of PAHs from background soils to the atmosphere found in all temperate regions in different seasons provides an indirect evidence of potential in situ generation of two to four ring PAHs and their alkyl homologues in the surface soil. We discuss this hypothetical biogenic source and other potential processes that could drive the high soil to air fugacity ratios of some PAHs.

Factors influencing the soil-air partitioning and the strength of soils as a secondary source of polychlorinated biphenyls to the atmosphere.

Soils are a major reservoir of persistent organic pollutants, and soil-air partitioning and exchange are key processes controlling the atmospheric concentrations and regional fate of pollutants. Here, we report and discuss the concentrations of polychlorinated biphenyls (PCBs) in soils, their measured fugacities in soil, the soil-air partition coefficients (K(SA)) and soil-air fugacity gradients in rural background areas of N-NE Spain and N-NW England. Four sampling campaigns were carried out to assess seasonal and daily variability and differences between sampling sites. K(SA) values were significantly dependent on soil temperature and soil organic matter quantity, and to a minor extent organic matter type. All the PCB congeners in the soil are close to equilibrium with the atmosphere at rural Ebro sites, but soil fugacities tend to be higher than ambient air fugacities in early and late summer, consistent with the influence of temperature on soil-air partitioning. Therefore, during warm periods, soils increment their strength as secondary sources to the atmosphere. The mixture of PCBs found in the atmosphere is clearly strongly influenced by the mixture of PCBs which escape from soil, with significant correlations between them (R(2) ranging between 0.35 and 0.74 and p-level <0.001 for the Ebro sampling sites). Conversely, the close-to-equilibrium to net sink status of rural UK sites, suggest a close coupling of air and soil concentrations, but it is not possible to elucidate the importance of these soils as secondary sources yet, and presumably there are still significant primary sources to the regional/global environment.

Presence of asymptomatic cytomegalovirus and Epstein--Barr virus DNA in blood of persons with HIV starting antiretroviral therapy is associated with non-AIDS clinical events.

BACKGROUND: Even with antiretroviral therapy (ART), persons with HIV (PWH) experience increased morbidity and mortality. Cytomegalovirus (CMV) and Epstein--Barr virus (EBV) co-infections likely exacerbate inflammatory-related diseases. OBJECTIVE: To determine if presence of detectable CMV or EBV DNA in peripheral blood mononuclear cells (PBMC) is associated with non-AIDS events among PWH receiving modern ART. DESIGN: We performed a case--control study of PWH starting ART and HIV-suppressed at year 1 and thereafter, 140 cases who experienced non-AIDS events and 305 matched controls. Events included myocardial infarction, stroke, malignancy, serious bacterial infection or death. METHODS: Blood samples were studied pre-ART, 1-year post-ART and pre-event. Controls had an event-free follow-up equal or greater than cases. CMV and EBV DNA levels were measured in PBMC. Conditional logistic regression analysis assessed associations and adjusted for relevant covariates; Spearman's correlations compared CMV and EBV DNA levels with other biomarkers. RESULTS: CMV DNA was detected in PBMC of 25% of participants, EBV DNA was detected in more than 90%. Higher EBV DNA levels were associated with increased risk of events at all time points (odds ratio (OR) per one IQR = 1.5-1.7, all P < 0.009). At year 1, detectable CMV DNA was associated with increased risk of events in most adjusted models (OR = 1.4-1.8, P values ranging 0.03-0.17). Higher levels of CMV and EBV DNA correlated with multiple inflammatory markers and lower CD4/CD8 ratio. CONCLUSION: In PWH starting ART, detection of CMV and EBV DNA in PBMC was associated with development of non-AIDS events. Clinical trials will be needed to understand causal mechanisms and ways to interrupt them.

HIV Trafficking Between Blood and Semen During Early Untreated HIV Infection.

BACKGROUND: Understanding the dynamics of HIV across anatomic compartments is important to design effective eradication strategies. In this study, we evaluated viral trafficking between blood and semen during primary HIV infection in 6 antiretroviral-naive men who have sex with men. METHODS: Deep sequencing data of HIV env were generated from longitudinal blood plasma, peripheral blood mononuclear cells, and seminal plasma samples. The presence or absence of viral compartmentalization was assessed using tree-based Slatkin-Maddison and distance-based Fst methods. Phylogeographic analyses were performed using a discrete Bayesian asymmetric approach of diffusion with Markov jump count estimation to evaluate the gene flow between blood and semen during primary HIV infection. Levels of DNA from human herpesviruses and selected inflammatory cytokines were also measured on genital secretions collected at baseline to evaluate potential correlates of increased viral migration between anatomic compartments. RESULTS: We detected varying degrees of compartmentalization in all 6 individuals evaluated. None of them maintained viral compartmentalization between blood and seminal plasma throughout the analyzed time points. Phylogeographic analyses revealed that the HIV population circulating in blood plasma populated the seminal compartment during the earliest stages of infection. In our limited data set, we found no association between local inflammation or herpesvirus shedding at baseline and viral trafficking between semen and blood. CONCLUSIONS: The early spread of virus from blood plasma to genital tract and the complex viral interplay between these compartments suggest that viral eradication efforts will require monitoring viral subpopulations in anatomic sites and viral trafficking during the course of infection.

Characterizing the multiplicity of HIV founder variants during sexual transmission among MSM.

Transmission of multiple founder variants has been associated with faster HIV disease progression. Many studies have attempted to determine the number of founder variants, mainly by analysis of sequence diversity and/or tree topology from acutely HIV-infected individuals. We hypothesized that adding sequence data collected from source partners might improve resolution and characterization of transmission events. Blood plasma samples were collected from both the source and recipient in thirty epidemiologically- and phylogenetically linked transmission pairs. All were men who have sex with men, sampled on average 70 days (range 11-170) after the recipient's estimated date of infection. Next generation sequencing (454 FLX, Roche) of HIV-1 env (C2-V3) was performed for all samples. Inspection of sequence alignments, highlighter plots, phylogenetic tree topologies and sequence diversity were used to determine the multiplicity of founder viruses with and without the inclusion of source data. Using only recipient sequence data, we were able to resolve multiplicity in twenty-six of the thirty transmission pairs (87 percent). Among them, five presented with a high viral diversity at baseline (>0.10 subst/site), consistent with multiple founders. By incorporating sequence data collected from the source partner, we were able to characterize all thirty transmission pairs. Overall, sixteen transmission events (53.3 percent) involved multiple founders. Results obtained by combining sequence data from recipient and source were congruent for nineteen of the twenty-six (73 percent) cases where conclusions were made using only recipient sequence data. The multiplicity of founders was associated with significantly higher HIV RNA levels (P = 0.04). To further evaluate the transmission bottleneck, we focused on single founder transmissions (fourteen of the thirty), and identified four recipients (28.6 percent) that had founder variants that were inferred to arise from minority viral populations in the source. These source clades ranged from 1.0 to 5.4 percent of the sampled population. Incorporating sequence data from the source increased of the ability to determine the multiplicity of founder variants, reduced misclassification, and allowed us to infer the transmission of minority variants.

HIV-associated neurocognitive disorder is associated with HIV-1 dual infection.

OBJECTIVE: Compared with HIV monoinfection, HIV dual infection has been associated with decreased CD4 T-cell counts and increased viral loads. The same markers are also associated with the development of HIV-associated neurocognitive disorder (HAND), which continues to be a prevalent problem in the era of combination antiretroviral therapy (ART). We sought to determine the relationship between dual infection and HAND. METHODS: Participants on ART (N = 38) underwent deep sequencing of four PCR-amplified HIV coding regions derived from peripheral blood mononuclear cell DNA samples. Phylogenetic analyses were performed to evaluate whether two distinct viral lineages, that is, dual infection, were present in the same individual. All study participants underwent neurocognitive, substance use, and neuromedical assessments at each study visit. RESULTS: Of 38 participants, nine (23.7%) had evidence of dual infection. Using clinical ratings, global neurocognitive impairment was identified in 21 (55%) participants, and multivariate analysis demonstrated a significant association between dual infection and impairment; odds ratio (95% confidence interval) = 18.3 (1.9, 414.2), P = 0.028. Neurocognitive impairment was also associated with lower current (P = 0.028) and nadir (P = 0.043) CD4 T-cell counts. CONCLUSIONS: Deep sequencing of HIV DNA populations in blood mononuclear cell identified dual infection in nearly a quarter of HIV-infected adults receiving ART, and dual infection was associated with HAND. Dual infection may contribute to the development of HAND, perhaps because of increased viral diversity. Further investigation is needed to determine how dual infection results in worse neurocognitive performance.

Rapid Sequencing of Complete env Genes from Primary HIV-1 Samples.

The ability to study rapidly evolving viral populations has been constrained by the read length of next-generation sequencing approaches and the sampling depth of single-genome amplification methods. Here, we develop and characterize a method using Pacific Biosciences' Single Molecule, Real-Time (SMRT®) sequencing technology to sequence multiple, intact full-length human immunodeficiency virus-1 env genes amplified from viral RNA populations circulating in blood, and provide computational tools for analyzing and visualizing these data.

Clade-Specific Quantitative Analysis of Photosynthetic Gene Expression in Prochlorococcus.

Newly designed primers targeting rbcL (CO2 fixation), psbA (photosystem II) and rnpB (reference) genes were used in qRT-PCR assays to assess the photosynthetic capability of natural communities of Prochlorococcus, the most abundant photosynthetic organism on Earth and a major contributor to primary production in oligotrophic oceans. After optimizing sample collection methodology, we analyzed a total of 62 stations from the Malaspina 2010 circumnavigation (including Atlantic, Pacific and Indian Oceans) at three different depths. Sequence and quantitative analyses of the corresponding amplicons showed the presence of high-light (HL) and low-light (LL) Prochlorococcus clades in essentially all 182 samples, with a largely uniform stratification of LL and HL sequences. Synechococcus cross-amplifications were detected by the taxon-specific melting temperatures of the amplicons. Laboratory exposure of Prochlorococcus MED4 (HL) and MIT9313 (LL) strains to organic pollutants (PAHs and organochlorine compounds) showed a decrease of rbcL transcript abundances, and of the rbcL to psbA ratios for both strains. We propose this technique as a convenient assay to evaluate effects of environmental stressors, including pollution, on the oceanic Prochlorococcus photosynthetic function.