HIV gene expression from intact proviruses positioned in bacterial artificial chromosomes at integration sites previously identified in latently infected T cells

AbstractHIV integration predominantly occurs in introns of transcriptionally active genes. To study the impact of the integration site on HIV gene expression, a complete HIV-1 provirus (with GFP as a fusion with Nef) was inserted into bacterial artificial chromosomes (BACs) at three sites previously identified in latent T cells of patients: topoisomerase II (Top2A), DNA methyltransferase 1 (DNMT1), or basic leucine transcription factor 2 (BACH2). Transfection of BAC-HIV into 293T cells resulted in a fourfold difference in production of infectious HIV-1. Cell lines were established that contained BAC–Top2A, BAC–DNMT1, or BAC–BACH2, but only BAC–DNMT1 spontaneously produced virus, albeit at a low level. Stimulation with TNF-α resulted in virus production from four of five BAC–Top2A and all BAC–DNMT1 cell lines, but not from the BAC–BACH2 lines. The results of these studies highlight differences between integration sites identified in latent T cells to support virus production and reactivation from latency

Accuracy of Fitbit Activity Trackers During Walking in a Controlled Setting

Activity trackers are widely used to measure daily physical activity. Many devices have been shown to measure steps more accurately at higher intensities, however, it is also important to determine the accuracy of these new devices at measuring steps while walking at a pace similar to that used during most daily activities. PURPOSE: To assess the accuracy of 6 popular activity trackers at measuring steps while walking on a treadmill. METHODS: Twenty-six college students (Mean±SD; 22.1±3.7yrs; 25.1±4.0kg/m2; 13 male) walked 500 steps at 3mph on a treadmill while wearing 6 different activity trackers (Pedometer, Fitbit Blaze, Charge HR, Alta, Flex, Zip, One). The Charge HR was placed two fingers above the right wrist while the Flex was next to the wrist bone. The Blaze was placed two fingers above the left wrist while the Alta was next to the wrist bone. The Fitbit Zip and the One were aligned with the hipbone on the left and right waistband respectively. Steps were counted by a trained researcher using a hand tally counter. Missing values were replaced with the mean value for that device. Step counts were correlated between Fitbit devices and the pedometer and tally counter using Pearson correlations. Significance was set at p\u3c0.05. Mean bias scores were calculated between the step counts for each device and the tally counter. Mean Absolute Percent Error (MAPE) values were also calculated for each device relative to the tally counter. RESULTS: Fitbit Zip and One were significantly correlated with the tally counter (r=0.50, p\u3c0.05; r=0.68, p\u3c0.01, respectively) while the other devices were not significantly correlated. Mean bias and MAPE values were as follows: Device (Mean Bias/MAPE) Pedometer (-0.2±39.2/3.8±6.8), Blaze (34.5±67.1/9.9±11.3), Charge HR (-12.6±61.5/7.0±10.3), Alta (-85.0±70.8/17.1±14.1), Flex (49.5±242.4/19.7±45.3), Zip (1.8±3.4/0.4±0.6), One (0.2±2.1/0.3±0.3). Fitbit Zip and One were within one half percent of actual steps while wrist-worn Fitbits ranged from 7.0-19.7% from actual step counts. CONCLUSION: Consistent with previous research, activity trackers worn at the waist provide the most accurate step counts compared to wrist-worn models. Differences found in wrist-worn models may result in significant over- or underestimation of activity levels when worn for long periods of time

Comparison of Smartphone Pedometer Apps on a Treadmill versus Outdoors

Previous research has focused on the accuracy of smartphone pedometer apps in laboratory settings, however less information is available in outdoor (free living) environments. PURPOSE: Determine the accuracy of 5 smartphone apps at recording steps at a walking speed in a laboratory versus an outdoor setting. METHODS: Twenty-three healthy college students consented (Mean±SD; 22±3.8yrs; BMI 24.9±4.13kg/m2) to participate in 2 separate visits. During the first visit participants walked 500 steps at 3mph on a treadmill while wearing a pedometer and a smartphone placed in the pocket using 5 pedometer apps concurrently (Moves, Google Fit (G-Fit), Runtastic, Accupedo, S-Health). During the second visit, participants walked 400 meters at 3mph on a sidewalk outside. Actual steps for each visit were recorded using a hand tally counter device. Zero and negative values were replaced with the mean value for that trial. Statistical analyses were performed using IBM SPSS 23.0. Mean bias scores were calculated between the step count for each app and the respective tally count for each trial. Mean bias scores were correlated between trials for each app using Pearson correlations and significance was set at p\u3c0.05. Mean Absolute Percent Error (MAPE) values were also calculated for each app for both trials. RESULTS: G-Fit recorded 2 zero values and 2 negative values and Moves recorded 1 zero value. Mean bias scores were significantly correlated between the indoor and outdoor protocols for the pedometer (r=0.67, p\u3c0.01) and S-Health (r=0.46, p\u3c0.5). The remaining apps were not correlated between protocols. The outdoor protocol producing a greater mean bias for the outdoor protocol for G-Fit, Runtastic, and Accupedo (mean bias ± SD indoor, outdoor; -4.3±53.1, -19.3±120.0; -10.7±63.3, -33.4±118.7; 16.0±143.6, 79.0±75.0; respectively) and a greater mean bias for the indoor protocol for the pedometer, Moves, and S-Health (mean bias indoor, outdoor; -1.4±41.5, 0.0±34.1; -117.4±196.7, -42.2±209.6; 11.3±28.4, 0.0±58.7; respectively). MAPE was below 5% for the pedometer and S-Health for both trials. CONCLUSION: Apps with the lowest error in a controlled setting may be less affected when used in other settings, while apps with greater variation in a controlled setting may be affected when used in a different environment

Análisis econométrico sobre el consumo de agua subterránea por el sector agropecuario en Guanajuato, México

Los factores que afectan el consumo de agua subterránea en los sectores agrícola de riego y pecuario en el estado de Guanajuato fueron estimados con un modelo de ecuaciones simultáneas, para lo cual se utilizó información estadística anual de 1980 a 2007. Los resultados encontrados muestran que la cantidad consumida de agua responde de manera inelástica a cambios en el precio, con elasticidades de -0.0527 para la agricultura de riego y de -0.00486 para la ganadería. Estas elasticidades indican que en el sector pecuario el consumo de agua es más insensible a cambios en el precio, en comparación a la agricultura de riego y, por ende, políticas de administración del consumo deberán considerar aumentos en el precio del agua en este sector.Los factores que afectan el consumo de agua subterránea en los sectores agrícola de riego y pecuario en el estado de Guanajuato fueron estimados con un modelo de ecuaciones simultáneas, para lo cual se utilizó información estadística anual de 1980 a 2007. Los resultados encontrados muestran que la cantidad consumida de agua responde de manera inelástica a cambios en el precio, con elasticidades de -0.0527 para la agricultura de riego y de -0.00486 para la ganadería. Estas elasticidades indican que en el sector pecuario el consumo de agua es más insensible a cambios en el precio, en comparación a la agricultura de riego y, por ende, políticas de administración del consumo deberán considerar aumentos en el precio del agua en este sector

Use of Dried Blood Spots to Elucidate Full-Length Transmitted/Founder HIV-1 Genomes.

BACKGROUND: Identification of HIV-1 genomes responsible for establishing clinical infection in newly infected individuals is fundamental to prevention and pathogenesis research. Processing, storage, and transportation of the clinical samples required to perform these virologic assays in resource-limited settings requires challenging venipuncture and cold chain logistics. Here, we validate the use of dried-blood spots (DBS) as a simple and convenient alternative to collecting and storing frozen plasma. METHODS: We performed parallel nucleic acid extraction, single genome amplification (SGA), next generation sequencing (NGS), and phylogenetic analyses on plasma and DBS. RESULTS: We demonstrated the capacity to extract viral RNA from DBS and perform SGA to infer the complete nucleotide sequence of the transmitted/founder (TF) HIV-1 envelope gene and full-length genome in two acutely infected individuals. Using both SGA and NGS methodologies, we showed that sequences generated from DBS and plasma display comparable phylogenetic patterns in both acute and chronic infection. SGA was successful on samples with a range of plasma viremia, including samples as low as 1,700 copies/ml and an estimated ∼50 viral copies per blood spot. Further, we demonstrated reproducible efficiency in gp160 env sequencing in DBS stored at ambient temperature for up to three weeks or at -20°C for up to five months. CONCLUSIONS: These findings support the use of DBS as a practical and cost-effective alternative to frozen plasma for clinical trials and translational research conducted in resource-limited settings

Prevalence of viral load suppression, predictors of virological failure and patterns of HIV drug resistance after 12 and 48 months on first-line antiretroviral therapy: a national cross-sectional survey in Uganda.

OBJECTIVES: We implemented the WHO cross-sectional survey protocol to determine rates of HIV viral load (VL) suppression (VLS), and weighted prevalence, predictors and patterns of acquired drug resistance (ADR) in individuals with virological failure (VF) defined as VL ≥1000 copies/mL. METHODS: We enrolled 547 and 1064 adult participants on first-line ART for 12 (±3) months (ADR12) and ≥48 months (ADR48), respectively. Dried blood spots and plasma specimens were collected for VL testing and genotyping among the VFs. RESULTS: VLS was 95.0% (95% CI 93.4%-96.5%) in the ADR12 group and 87.9% (95% CI 85.0%-90.9%) in the ADR48 group. The weighted prevalence of ADR was 96.1% (95% CI 72.9%-99.6%) in the ADR12 and 90.4% (95% CI 73.6-96.8%) in the ADR48 group, out of the 30 and 95 successful genotypes in the respective groups. Initiation on a zidovudine-based regimen compared with a tenofovir-based regimen was significantly associated with VF in the ADR48 group; adjusted OR (AOR) 1.96 (95% CI 1.13-3.39). Independent predictors of ADR in the ADR48 group were initiation on a zidovudine-based regimen compared with tenofovir-based regimens, AOR 3.16 (95% CI 1.34-7.46) and ART duration of ≥82 months compared with <82 months, AOR 1.92 (95% CI 1.03-3.59). CONCLUSIONS: While good VLS was observed, the high prevalence of ADR among the VFs before they underwent the recommended three intensive adherence counselling (IAC) sessions followed by repeat VL testing implies that IAC prior to treatment switching may be of limited benefit in improving VLS

Phylogeography of HIV-1 suggests that Ugandan fishing communities are a sink for, not a source of, virus from general populations

Although fishing communities (FCs) in Uganda are disproportionately affected by HIV-1 relative to the general population (GP), the transmission dynamics are not completely understood. We earlier found most HIV-1 transmissions to occur within FCs of Lake Victoria. Here, we test the hypothesis that HIV-1 transmission in FCs is isolated from networks in the GP. We used phylogeography to reconstruct the geospatial viral migration patterns in 8 FCs and 2 GP cohorts and a Bayesian phylogenetic inference in BEAST v1.8.4 to analyse the temporal dynamics of HIV-1 transmission. Subtype A1 (pol region) was most prevalent in the FCs (115, 45.1%) and GP (177, 50.4%). More recent HIV transmission pairs from FCs were found at a genetic distance (GD) <1.5% than in the GP (Fisher's exact test, p = 0.001). The mean time depth for pairs was shorter in FCs (5 months) than in the GP (4 years). Phylogeographic analysis showed strong support for viral migration from the GP to FCs without evidence of substantial viral dissemination to the GP. This suggests that FCs are a sink for, not a source of, virus strains from the GP. Targeted interventions in FCs should be extended to include the neighbouring GP for effective epidemic control

High Multiplicity Infection by HIV-1 in Men Who Have Sex with Men

Elucidating virus-host interactions responsible for HIV-1 transmission is important for advancing HIV-1 prevention strategies. To this end, single genome amplification (SGA) and sequencing of HIV-1 within the context of a model of random virus evolution has made possible for the first time an unambiguous identification of transmitted/founder viruses and a precise estimation of their numbers. Here, we applied this approach to HIV-1 env analyses in a cohort of acutely infected men who have sex with men (MSM) and found that a high proportion (10 of 28; 36%) had been productively infected by more than one virus. In subjects with multivariant transmission, the minimum number of transmitted viruses ranged from 2 to 10 with viral recombination leading to rapid and extensive genetic shuffling among virus lineages. A combined analysis of these results, together with recently published findings based on identical SGA methods in largely heterosexual (HSX) cohorts, revealed a significantly higher frequency of multivariant transmission in MSM than in HSX [19 of 50 subjects (38%) versus 34 of 175 subjects (19%); Fisher's exact p = 0.008]. To further evaluate the SGA strategy for identifying transmitted/founder viruses, we analyzed 239 overlapping 5′ and 3′ half genome or env-only sequences from plasma viral RNA (vRNA) and blood mononuclear cell DNA in an MSM subject who had a particularly well-documented virus exposure history 3–6 days before symptom onset and 14–17 days before peak plasma viremia (47,600,000 vRNA molecules/ml). All 239 sequences coalesced to a single transmitted/founder virus genome in a time frame consistent with the clinical history, and a molecular clone of this genome encoded replication competent virus in accord with model predictions. Higher multiplicity of HIV-1 infection in MSM compared with HSX is consistent with the demonstrably higher epidemiological risk of virus acquisition in MSM and could indicate a greater challenge for HIV-1 vaccines than previously recognized

HIV-1 Gag-Pol Sequences from Ugandan Early Infections Reveal Sequence Variants Associated with Elevated Replication Capacity.

The ability to efficiently establish a new infection is a critical property for human immunodeficiency virus type 1 (HIV-1). Although the envelope protein of the virus plays an essential role in receptor binding and internalization of the infecting virus, the structural proteins, the polymerase and the assembly of new virions may also play a role in establishing and spreading viral infection in a new host. We examined Ugandan viruses from newly infected patients and focused on the contribution of the Gag-Pol genes to replication capacity. A panel of Gag-Pol sequences generated using single genome amplification from incident HIV-1 infections were cloned into a common HIV-1 NL4.3 pol/env backbone and the influence of Gag-Pol changes on replication capacity was monitored. Using a novel protein domain approach, we then documented diversity in the functional protein domains across the Gag-Pol region and identified differences in the Gag-p6 domain that were frequently associated with higher in vitro replication