Syphilis and HIV-1 co-infection: influence on CD4 T cell count, HIV-1 viral load and treatment response

DEFF Research Database (Denmark)

Kofoed, Kristian; Gerstoft, Jan; Mathiesen, Lars Reinhardt

2006-01-01

OBJECTIVES: To assess the effect of human immunodeficiency virus (HIV)-1 and syphilis coinfection on HIV-ribonucleic acid (RNA) viral load, CD4 cell count, and the response in rapid plasmin reagin (RPR) to treatment of the syphilis infection. STUDY DESIGN: Cases of syphilis diagnosed during 1 year...... in HIV-infected patients in Copenhagen were included. HIV-RNA, CD4 cell counts, and RPR-serology were measured before, during, and after syphilis. RESULTS: Forty-one patients were included. CD4 cell count decreased significantly during infection in patients with primary and secondary stages of syphilis...... (mean 106 cells/mm, P = 0.03). Treatment of syphilis was associated with an increase in the CD4 cell count and a decrease in HIV-RNA in the overall group (mean 66 cells/mm and -0.261 RNA log10 copies/ml, P = 0.02 and 0.04). The serological response rates for 15 patients treated with penicillin and 25...

Associations between HIV-RNA-based indicators and virological and clinical outcomes

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Laut, Kamilla G; Shepherd, Leah C; Pedersen, Court

2016-01-01

OBJECTIVES: To evaluate and compare the performance of six HIV-RNA-based quality of care indicators for predicting short-term and long-term outcomes. DESIGN: Multinational cohort study. METHODS: We included EuroSIDA patients on antiretroviral therapy (ART) with at least three viral load measureme......OBJECTIVES: To evaluate and compare the performance of six HIV-RNA-based quality of care indicators for predicting short-term and long-term outcomes. DESIGN: Multinational cohort study. METHODS: We included EuroSIDA patients on antiretroviral therapy (ART) with at least three viral load...... measurements after baseline (the latest of 01/01/2001 or entry into EuroSIDA). Using multivariate Poisson regression, we modelled the association between short-term (resistance, triple-class failure) and long-term (all-cause mortality, any AIDS/non-AIDS clinical event) outcomes and the indicators: viraemia...

HIV Viral Load

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... PF4 Antibody Hepatitis A Testing Hepatitis B Testing Hepatitis C Testing HER2/neu Herpes Testing High-sensitivity C-reactive Protein (hs-CRP) Histamine Histone Antibody HIV Antibody and HIV Antigen (p24) HIV Antiretroviral Drug Resistance Testing, Genotypic HIV Viral Load HLA Testing HLA- ...

MicroRNA profile changes in human immunodeficiency virus type 1 (HIV-1 seropositive individuals

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Smith Stephen M

2008-12-01

Full Text Available Abstract MicroRNAs (miRNAs play diverse roles in regulating cellular and developmental functions. We have profiled the miRNA expression in peripheral blood mononuclear cells from 36 HIV-1 seropositive individuals and 12 normal controls. The HIV-1-positive individuals were categorized operationally into four classes based on their CD4+ T-cell counts and their viral loads. We report that specific miRNA signatures can be observed for each of the four classes.

Meticulous plasma isolation is essential to avoid false low-level viraemia in Roche Cobas HIV-1 viral load assays.

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Mortier, Virginie; Vancoillie, Leen; Dauwe, Kenny; Staelens, Delfien; Demecheleer, Els; Schauvliege, Marlies; Dinakis, Sylvie; Van Maerken, Tom; Dessilly, Géraldine; Ruelle, Jean; Verhofstede, Chris

2017-10-24

Pre-analytical sample processing is often overlooked as a potential cause of inaccurate assay results. Here we demonstrate how plasma, extracted from standard EDTA-containing blood collection tubes, may contain traces of blood cells consequently resulting in a false low-level HIV-1 viral load when using Roche Cobas HIV-1 assays. The presence of human DNA in Roche Cobas 4800 RNA extracts and in RNA extracts from the Abbott HIV-1 RealTime assay was assessed by quantifying the human albumin gene by means of quantitative PCR. RNA was extracted from plasma samples before and after an additional centrifugation and tested for viral load and DNA contamination. The relation between total DNA content and viral load was defined. Elevated concentrations of genomic DNA were detected in 28 out of 100 Cobas 4800 extracts and were significantly more frequent in samples processed outside of the AIDS Reference Laboratory. An association between genomic DNA presence and spurious low-level viraemia results was demonstrated. Supplementary centrifugation of plasma before RNA extraction eliminated the contamination and the false viraemia. Plasma isolated from standard EDTA-containing blood collection tubes may contain traces of HIV DNA leading to false viral load results above the clinical cutoff. Supplementary centrifugation of plasma before viral load analysis may eliminate the occurrence of this spurious low-level viraemia.

The semen microbiome and its relationship with local immunology and viral load in HIV infection.

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Cindy M Liu

2014-07-01

Full Text Available Semen is a major vector for HIV transmission, but the semen HIV RNA viral load (VL only correlates moderately with the blood VL. Viral shedding can be enhanced by genital infections and associated inflammation, but it can also occur in the absence of classical pathogens. Thus, we hypothesized that a dysregulated semen microbiome correlates with local HIV shedding. We analyzed semen samples from 49 men who have sex with men (MSM, including 22 HIV-uninfected and 27 HIV-infected men, at baseline and after starting antiretroviral therapy (ART using 16S rRNA gene-based pyrosequencing and quantitative PCR. We studied the relationship of semen bacteria with HIV infection, semen cytokine levels, and semen VL by linear regression, non-metric multidimensional scaling, and goodness-of-fit test. Streptococcus, Corynebacterium, and Staphylococcus were common semen bacteria, irrespective of HIV status. While Ureaplasma was the more abundant Mollicutes in HIV-uninfected men, Mycoplasma dominated after HIV infection. HIV infection was associated with decreased semen microbiome diversity and richness, which were restored after six months of ART. In HIV-infected men, semen bacterial load correlated with seven pro-inflammatory semen cytokines, including IL-6 (p = 0.024, TNF-α (p = 0.009, and IL-1b (p = 0.002. IL-1b in particular was associated with semen VL (r(2  = 0.18, p = 0.02. Semen bacterial load was also directly linked to the semen HIV VL (r(2 = 0.15, p = 0.02. HIV infection reshapes the relationship between semen bacteria and pro-inflammatory cytokines, and both are linked to semen VL, which supports a role of the semen microbiome in HIV sexual transmission.

Discordant CSF/plasma HIV-1 RNA in individuals on virologically suppressive antiretroviral therapy in Western India.

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Dravid, Ameet N; Natrajan, Kartik; Kulkarni, Milind M; Saraf, Chinmay K; Mahajan, Uma S; Kore, Sachin D; Rathod, Niranjan M; Mahajan, Umakant S; Wadia, Rustom S

2018-02-01

Aim of this study was to estimate the prevalence of cerebrospinal fluid (CSF)/Plasma HIV-1 RNA discordance in virologically suppressed individuals presenting with incident neurologic symptoms.In this retrospective cohort study conducted between March 1, 2009, and March 1, 2017, HIV-1 infected adults exposed to atleast 12 months of antiretroviral therapy (ART) and having plasma viral load (VL) CSF/Plasma HIV-1 RNA discordance by measuring HIV-1 RNA in collected plasma and CSF samples. CSF/plasma HIV-1 RNA discordance was defined as either detectable CSF HIV-1 RNA (VL > 20 copies/mL) with an undetectable plasma RNA (complete viral suppression, VL ≤20 copies/mL) or CSF HIV-1 RNA ≥ 0.5 log10 higher than plasma RNA when plasma VL was between 20 and 1000 copies/mL (low-level viremia, LLV).Out of 1584 virologically suppressed patients, 71 (4.4%) presented with incident neurologic symptoms. Twenty out of 71 (28.2%) patients were diagnosed with CSF/Plasma HIV-1 discordance. Median plasma and CSF VL in patients with discordance was 120 [interquartile range (IQR): CSF HIV-1 genotypic resistance testing was done showed mutations that would compromise efficacy of prescribed ART regimen. Prevalence of CSF/plasma HIV-1 RNA discordance was higher among neurologically symptomatic patients with plasma LLV as compared with those with complete viral suppression (70% vs 11.8%, P CSF/plasma HIV-1 RNA discordance indicates replication of HIV-1 that has adapted to the CNS or has developed antiretroviral drug resistance. Larger studies should be performed to study incidence of discordance in India. This will help in managing patients presenting with neurologic symptoms on suppressive ART with appropriate neuroeffective therapy.

Decrease in Seminal HIV-1 RNA Load After Praziquantel Treatment of Urogenital Schistosomiasis Coinfection in HIV-Positive Men—An Observational Study

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Midzi, Nicholas; Mduluza, Takafira; Mudenge, Boniface

2017-01-01

BACKGROUND: Urogenital schistosomiasis due to Schistosoma hematobium infection is hypothesized to cause increased HIV-1 RNA shedding in semen in HIV co-infected men as result of chronic egg-induced inflammation in the prostate and the seminal vesicles. The effect of treatment with the antihelmint...... targeting praziquantel as a supplementary preventive measure of sexual transmission of HIV-1 in S. haematobium endemic areas in sub-Saharan Africa....

Physician experience and rates of plasma HIV-1 RNA suppression among illicit drug users: an observational study

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Sangsari Sassan

2012-01-01

Full Text Available Abstract Background Despite the availability of antiretroviral therapy (ART, suboptimal treatment outcomes have been observed among HIV-seropositive illicit drug users. As there is an urgent need to improve responses to antiretroviral therapy among this population, we undertook this study to evaluate the role of physician experience on rates of plasma HIV-1 RNA suppression following initiation of ART. Methods Using data from a community-recruited cohort of HIV-positive illicit drug users, we used Cox proportional hazards regression to model the time to plasma viral HIV RNA Results Between May 1996 and December 2008, 267 individuals initiated ART among whom 227 (85% achieved a plasma HIV RNA Conclusions In this setting of universal HIV/AIDS care, illicit drug users with more experienced physicians exhibited faster rates of plasma viral load suppression. These findings argue for specialized services to help optimize HIV treatment outcomes among this population.

The Semen Microbiome and Its Relationship with Local Immunology and Viral Load in HIV Infection

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Liu, Cindy M.; Osborne, Brendan J. W.; Hungate, Bruce A.; Shahabi, Kamnoosh; Huibner, Sanja; Lester, Richard; Dwan, Michael G.; Kovacs, Colin; Contente-Cuomo, Tania L.; Benko, Erika; Aziz, Maliha

2014-01-01

Semen is a major vector for HIV transmission, but the semen HIV RNA viral load (VL) only correlates moderately with the blood VL. Viral shedding can be enhanced by genital infections and associated inflammation, but it can also occur in the absence of classical pathogens. Thus, we hypothesized that a dysregulated semen microbiome correlates with local HIV shedding. We analyzed semen samples from 49 men who have sex with men (MSM), including 22 HIV-uninfected and 27 HIV-infected men, at baseline and after starting antiretroviral therapy (ART) using 16S rRNA gene-based pyrosequencing and quantitative PCR. We studied the relationship of semen bacteria with HIV infection, semen cytokine levels, and semen VL by linear regression, non-metric multidimensional scaling, and goodness-of-fit test. Streptococcus, Corynebacterium, and Staphylococcus were common semen bacteria, irrespective of HIV status. While Ureaplasma was the more abundant Mollicutes in HIV-uninfected men, Mycoplasma dominated after HIV infection. HIV infection was associated with decreased semen microbiome diversity and richness, which were restored after six months of ART. In HIV-infected men, semen bacterial load correlated with seven pro-inflammatory semen cytokines, including IL-6 (p = 0.024), TNF-α (p = 0.009), and IL-1b (p = 0.002). IL-1b in particular was associated with semen VL (r2 = 0.18, p = 0.02). Semen bacterial load was also directly linked to the semen HIV VL (r2 = 0.15, p = 0.02). HIV infection reshapes the relationship between semen bacteria and pro-inflammatory cytokines, and both are linked to semen VL, which supports a role of the semen microbiome in HIV sexual transmission. PMID:25058515

Micro RNA in Exosomes from HIV-Infected Macrophages

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William W. Roth

2015-12-01

Full Text Available Exosomes are small membrane-bound vesicles secreted by cells that function to shuttle RNA and proteins between cells. To examine the role of exosomal micro RNA (miRNA during the early stage of HIV-1 infection we characterized miRNA in exosomes from HIV-infected macrophages, compared with exosomes from non-infected macrophages. Primary human monocytes from uninfected donors were differentiated to macrophages (MDM which were either mock-infected or infected with the macrophage-tropic HIV-1 BaL strain. Exosomes were recovered from culture media and separated from virus particles by centrifugation on iodixanol density gradients. The low molecular weight RNA fraction was prepared from purified exosomes. After pre-amplification, RNA was hybridized to microarrays containing probes for 1200 miRNA species of known and unknown function. We observed 48 miRNA species in both infected and uninfected MDM exosomes. Additionally, 38 miRNAs were present in infected-cell exosomes but not uninfected-cell exosomes. Of these, 13 miRNAs were upregulated in exosomes from HIV-infected cells, including 4 miRNA species that were increased by more than 10-fold. Though numerous miRNA species have been identified in HIV-infected cells, relatively little is known about miRNA content in exosomes from these cells. In the future, we plan to investigate whether the upregulated miRNA species we identified are increased in exosomes from HIV-1-positive patients.

Comparison of sampling methods to measure HIV RNA viral load in female genital tract secretions.

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Jaumdally, Shameem Z; Jones, Heidi E; Hoover, Donald R; Gamieldien, Hoyam; Kriek, Jean-Mari; Langwenya, Nontokozo; Myer, Landon; Passmore, Jo-Ann S; Todd, Catherine S

2017-03-01

How does menstrual cup (MC) compare to other genital sampling methods for HIV RNA recovery? We compared HIV RNA levels between MC, endocervical swab (ECS), and ECS-enriched cervicovaginal lavage (eCVL) specimens in 51 HIV-positive, antiretroviral therapy-naive women at enrollment, 3 and 6 months, with order rotated by visit. Paired comparisons were analyzed with McNemar's exact tests, signed-rank tests, and an extension of Somer's D for pooled analyses across visits. MC specimens had the highest proportion of quantifiable HIV VL at enrollment and month 3, but more MC specimens (n=12.8%) were insufficient for testing, compared with ECS (2%, P=0.006) and eCVL (0%, P<0.001). Among sufficient specimens, median VL was significantly higher for MC (2.62 log 10 copies/mL) compared to ECS (1.30 log 10 copies/mL, P<0.001) and eCVL (1.60 log 10 copies/mL, P<0.001) across visits. MC may be more sensitive than eCVL and CVS, provided insufficient specimens are reduced. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Factors influencing cerebrospinal fluid and plasma HIV-1 RNA detection rate in patients with and without opportunistic neurological disease during the HAART era

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Aleixo Agdemir W

2007-12-01

Full Text Available Abstract Background In the central nervous system, HIV replication can occur relatively independent of systemic infection, and intrathecal replication of HIV-1 has been observed in patients with HIV-related and opportunistic neurological diseases. The clinical usefulness of HIV-1 RNA detection in the cerebrospinal fluid (CSF of patients with opportunistic neurological diseases, or the effect of opportunistic diseases on CSF HIV levels in patients under HAART has not been well defined. We quantified CSF and plasma viral load in HIV-infected patients with and without different active opportunistic neurological diseases, determined the characteristics that led to a higher detection rate of HIV RNA in CSF, and compared these two compartments. Methods A prospective study was conducted on 90 HIV-infected patients submitted to lumbar puncture as part of a work-up for suspected neurological disease. Seventy-one patients had active neurological diseases while the remaining 19 did not. Results HIV-1 RNA was quantified in 90 CSF and 70 plasma samples. The HIV-1 RNA detection rate in CSF was higher in patients with neurological diseases, in those with a CD4 count lower than 200 cells/mm3, and in those not receiving antiretroviral therapy, as well as in patients with detectable plasma HIV-1 RNA. Median viral load was lower in CSF than in plasma in the total population, in patients without neurological diseases, and in patients with toxoplasmic encephalitis, while no significant difference between the two compartments was observed for patients with cryptococcal meningitis and HIV-associated dementia. CSF viral load was lower in patients with cryptococcal meningitis and neurotoxoplasmosis under HAART than in those not receiving HAART. Conclusion Detection of HIV-1 RNA in CSF was more frequent in patients with neurological disease, a CD4 count lower than 200 cells/mm3 and detectable plasma HIV-1. Median HIV-1 RNA levels were generally lower in CSF than in

Interactions between the HIV-1 Unspliced mRNA and Host mRNA Decay Machineries

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Daniela Toro-Ascuy

2016-11-01

Full Text Available The human immunodeficiency virus type-1 (HIV-1 unspliced transcript is used both as mRNA for the synthesis of structural proteins and as the packaged genome. Given the presence of retained introns and instability AU-rich sequences, this viral transcript is normally retained and degraded in the nucleus of host cells unless the viral protein REV is present. As such, the stability of the HIV-1 unspliced mRNA must be particularly controlled in the nucleus and the cytoplasm in order to ensure proper levels of this viral mRNA for translation and viral particle formation. During its journey, the HIV-1 unspliced mRNA assembles into highly specific messenger ribonucleoproteins (mRNPs containing many different host proteins, amongst which are well-known regulators of cytoplasmic mRNA decay pathways such as up-frameshift suppressor 1 homolog (UPF1, Staufen double-stranded RNA binding protein 1/2 (STAU1/2, or components of miRNA-induced silencing complex (miRISC and processing bodies (PBs. More recently, the HIV-1 unspliced mRNA was shown to contain N6-methyladenosine (m6A, allowing the recruitment of YTH N6-methyladenosine RNA binding protein 2 (YTHDF2, an m6A reader host protein involved in mRNA decay. Interestingly, these host proteins involved in mRNA decay were shown to play positive roles in viral gene expression and viral particle assembly, suggesting that HIV-1 interacts with mRNA decay components to successfully accomplish viral replication. This review summarizes the state of the art in terms of the interactions between HIV-1 unspliced mRNA and components of different host mRNA decay machineries.

HIV Viral Load: MedlinePlus Lab Test Information

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... this page: https://medlineplus.gov/labtests/hivviralload.html HIV Viral Load To use the sharing features on this page, please enable JavaScript. What is an HIV Viral Load? An HIV viral load is a ...

Co-occurrence of Trichomonas vaginalis and bacterial vaginosis and vaginal shedding of HIV-1 RNA.

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Fastring, Danielle R; Amedee, Angela; Gatski, Megan; Clark, Rebecca A; Mena, Leandro A; Levison, Judy; Schmidt, Norine; Rice, Janet; Gustat, Jeanette; Kissinger, Patricia

2014-03-01

Trichomonas vaginalis (TV) and bacterial vaginosis (BV) are independently associated with increased risk of vaginal shedding in HIV-positive women. Because these 2 conditions commonly co-occur, this study was undertaken to examine the association between TV/BV co-occurrence and vaginal shedding of HIV-1 RNA. HIV-positive women attending outpatient HIV clinics in 3 urban US cities underwent a clinical examination; were screened for TV, BV, Neisseria gonorrhoeae, Chlamydia trachomatis, and vulvovaginal candidiasis; and completed a behavioral survey. Women shedding HIV-1 RNA vaginally (≥50 copies/mL) were compared with women who had an undetectable (women who were TV positive and BV positive or had co-occurrence of TV/BV had higher odds of shedding vaginally when compared with women who did not have these conditions. In this sample of 373 HIV-positive women, 43.1% (n = 161) had co-occurrence of TV/BV and 33.2% (n = 124) were shedding HIV-1 RNA vaginally. The odds of shedding HIV vaginally in the presence of TV alone or BV alone and when TV/BV co-occurred were 4.07 (95% confidence interval [CI], 1.78-9.37), 5.65 (95% CI, 2.64-12.01), and 18.63 (95% CI, 6.71-51.72), respectively, when compared with women with no diagnosis of TV or BV, and after adjusting for age, antiretroviral therapy status, and plasma viral load. T. vaginalis and BV were independently and synergistically related to vaginal shedding of HIV-1 RNA. Screening and prompt treatment of these 2 conditions among HIV-positive women are important not only clinically but for HIV prevention, as well.

Semen Bacterial Concentrations and HIV-1 RNA Shedding Among HIV-1-Seropositive Kenyan Men.

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Korhonen, Christine J; Srinivasan, Sujatha; Huang, Dandi; Ko, Daisy L; Sanders, Eduard J; Peshu, Norbert M; Krieger, John N; Muller, Charles H; Coombs, Robert W; Fredricks, David N; Graham, Susan M

2017-03-01

HIV-1 is transmitted through semen from men to their sexual partners. Genital infections can increase HIV-1 RNA shedding in semen, but shedding also occurs in the absence of typical pathogens. We hypothesized that higher bacterial concentrations in semen would be associated with higher HIV-1 RNA levels. We analyzed semen samples from 42 HIV-1-seropositive Kenyan men using quantitative polymerase chain reaction (PCR) to assess bacterial concentrations and real-time PCR to measure HIV-1 RNA levels. Generalized estimation equations were used to evaluate associations between these 2 measures. Broad-range 16S rRNA gene PCR with pyrosequencing was performed on a subset of 13 samples to assess bacterial community composition. Bacteria were detected in 96.6% of 88 samples by quantitative PCR. Semen bacterial concentration and HIV-1 RNA levels were correlated 0.30 (P = 0.01). The association between bacterial concentration and HIV-1 RNA detection was not significant after adjustment for antiretroviral therapy (ART) (adjusted odds ratio: 1.27, 95% CI: 0.84 to 1.91). Factors associated with semen bacterial concentration included insertive anal sex (adjusted beta 0.92, 95% CI: 0.12 to 1.73) and ART use (adjusted beta: -0.77, 95% CI: -1.50 to 0.04). Among 13 samples with pyrosequencing data, Corynebacterium spp., Staphylococcus spp., and Streptococcus spp. were most frequently detected. Most of these HIV-1-infected men had bacteria in their semen. ART use was associated with undetectable semen HIV-1 RNA and lower semen bacterial concentrations, whereas insertive anal sex was associated with higher bacterial concentrations. Additional studies evaluating the relationship between semen bacteria, inflammation, mucosal immunity, and HIV-1 shedding are needed to understand implications for HIV-1 transmission.

Association between feline immunodeficiency virus (FIV) plasma viral RNA load, concentration of acute phase proteins and disease severity.

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Kann, Rebecca K C; Seddon, Jennifer M; Kyaw-Tanner, Myat T; Henning, Joerg; Meers, Joanne

2014-08-01

Veterinarians have few tools to predict the rate of disease progression in FIV-infected cats. In contrast, in HIV infection, plasma viral RNA load and acute phase protein concentrations are commonly used as predictors of disease progression. This study evaluated these predictors in cats naturally infected with FIV. In older cats (>5 years), log10 FIV RNA load was higher in the terminal stages of disease compared to the asymptomatic stage. There was a significant association between log10 FIV RNA load and both log10 serum amyloid A concentration and age in unwell FIV-infected cats. This study suggests that viral RNA load and serum amyloid A warrant further investigation as predictors of disease status and prognosis in FIV-infected cats. Copyright © 2014 Elsevier Ltd. All rights reserved.

Screening Yield of HIV Antigen/Antibody Combination and Pooled HIV RNA Testing for Acute HIV Infection in a High-Prevalence Population.

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Peters, Philip J; Westheimer, Emily; Cohen, Stephanie; Hightow-Weidman, Lisa B; Moss, Nicholas; Tsoi, Benjamin; Hall, Laura; Fann, Charles; Daskalakis, Demetre C; Beagle, Steve; Patel, Pragna; Radix, Asa; Foust, Evelyn; Kohn, Robert P; Marmorino, Jenni; Pandori, Mark; Fu, Jie; Samandari, Taraz; Gay, Cynthia L

2016-02-16

Although acute HIV infection contributes disproportionately to onward HIV transmission, HIV testing has not routinely included screening for acute HIV infection. To evaluate the performance of an HIV antigen/antibody (Ag/Ab) combination assay to detect acute HIV infection compared with pooled HIV RNA testing. Multisite, prospective, within-individual comparison study conducted between September 2011 and October 2013 in 7 sexually transmitted infection clinics and 5 community-based programs in New York, California, and North Carolina. Participants were 12 years or older and seeking HIV testing, without known HIV infection. All participants with a negative rapid HIV test result were screened for acute HIV infection with an HIV Ag/Ab combination assay (index test) and pooled human immunodeficiency virus 1 (HIV-1) RNA testing. HIV RNA testing was the reference standard, with positive reference standard result defined as detectable HIV-1 RNA on an individual RNA test. Number and proportion with acute HIV infections detected. Among 86,836 participants with complete test results (median age, 29 years; 75.0% men; 51.8% men who have sex with men), established HIV infection was diagnosed in 1158 participants (1.33%) and acute HIV infection was diagnosed in 168 participants (0.19%). Acute HIV infection was detected in 134 participants with HIV Ag/Ab combination testing (0.15% [95% CI, 0.13%-0.18%]; sensitivity, 79.8% [95% CI, 72.9%-85.6%]; specificity, 99.9% [95% CI, 99.9%-99.9%]; positive predictive value, 59.0% [95% CI, 52.3%-65.5%]) and in 164 participants with pooled HIV RNA testing (0.19% [95% CI, 0.16%-0.22%]; sensitivity, 97.6% [95% CI, 94.0%-99.4%]; specificity, 100% [95% CI, 100%-100%]; positive predictive value, 96.5% [95% CI, 92.5%-98.7%]; sensitivity comparison, P testing detected 82% of acute HIV infections detectable by pooled HIV RNA testing. Compared with rapid HIV testing alone, HIV Ag/Ab combination testing increased the relative HIV diagnostic yield (both

DBR1 siRNA inhibition of HIV-1 replication

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Naidu Yathi

2005-10-01

Full Text Available Abstract Background HIV-1 and all retroviruses are related to retroelements of simpler organisms such as the yeast Ty elements. Recent work has suggested that the yeast retroelement Ty1 replicates via an unexpected RNA lariat intermediate in cDNA synthesis. The putative genomic RNA lariat intermediate is formed by a 2'-5' phosphodiester bond, like that found in pre-mRNA intron lariats and it facilitates the minus-strand template switch during cDNA synthesis. We hypothesized that HIV-1 might also form a genomic RNA lariat and therefore that siRNA-mediated inhibition of expression of the human RNA lariat de-branching enzyme (DBR1 expression would specifically inhibit HIV-1 replication. Results We designed three short interfering RNA (siRNA molecules targeting DBR1, which were capable of reducing DBR1 mRNA expression by 80% and did not significantly affect cell viability. We assessed HIV-1 replication in the presence of DBR1 siRNA and found that DBR1 knockdown led to decreases in viral cDNA and protein production. These effects could be reversed by cotransfection of a DBR1 cDNA indicating that the inhibition of HIV-1 replication was a specific effect of DBR1 underexpression. Conclusion These data suggest that DBR1 function may be needed to debranch a putative HIV-1 genomic RNA lariat prior to completion of reverse transcription.

HIV-1 nef suppression by virally encoded microRNA

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Brisibe Ebiamadon

2004-12-01

Full Text Available Abstract Background MicroRNAs (miRNAs are 21~25-nucleotides (nt long and interact with mRNAs to trigger either translational repression or RNA cleavage through RNA interference (RNAi, depending on the degree of complementarity with the target mRNAs. Our recent study has shown that HIV-1 nef dsRNA from AIDS patients who are long-term non-progressors (LTNPs inhibited the transcription of HIV-1. Results Here, we show the possibility that nef-derived miRNAs are produced in HIV-1 persistently infected cells. Furthermore, nef short hairpin RNA (shRNA that corresponded to a predicted nef miRNA (~25 nt, miR-N367 can block HIV-1 Nef expression in vitro and the suppression by shRNA/miR-N367 would be related with low viremia in an LTNP (15-2-2. In the 15-2-2 model mice, the weight loss, which may be rendered by nef was also inhibited by shRNA/miR-N367 corresponding to suppression of nef expression in vivo. Conclusions These data suggest that nef/U3 miRNAs produced in HIV-1-infected cells may suppress both Nef function and HIV-1 virulence through the RNAi pathway.

Semen Bacterial Concentrations and HIV-1 RNA Shedding Among HIV-1–Seropositive Kenyan Men

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Srinivasan, Sujatha; Huang, Dandi; Ko, Daisy L.; Sanders, Eduard J.; Peshu, Norbert M.; Krieger, John N.; Muller, Charles H.; Coombs, Robert W.; Fredricks, David N.; Graham, Susan M.

2017-01-01

Introduction: HIV-1 is transmitted through semen from men to their sexual partners. Genital infections can increase HIV-1 RNA shedding in semen, but shedding also occurs in the absence of typical pathogens. We hypothesized that higher bacterial concentrations in semen would be associated with higher HIV-1 RNA levels. Methods: We analyzed semen samples from 42 HIV-1–seropositive Kenyan men using quantitative polymerase chain reaction (PCR) to assess bacterial concentrations and real-time PCR to measure HIV-1 RNA levels. Generalized estimation equations were used to evaluate associations between these 2 measures. Broad-range 16S rRNA gene PCR with pyrosequencing was performed on a subset of 13 samples to assess bacterial community composition. Results: Bacteria were detected in 96.6% of 88 samples by quantitative PCR. Semen bacterial concentration and HIV-1 RNA levels were correlated 0.30 (P = 0.01). The association between bacterial concentration and HIV-1 RNA detection was not significant after adjustment for antiretroviral therapy (ART) (adjusted odds ratio: 1.27, 95% CI: 0.84 to 1.91). Factors associated with semen bacterial concentration included insertive anal sex (adjusted beta 0.92, 95% CI: 0.12 to 1.73) and ART use (adjusted beta: −0.77, 95% CI: −1.50 to 0.04). Among 13 samples with pyrosequencing data, Corynebacterium spp., Staphylococcus spp., and Streptococcus spp. were most frequently detected. Conclusion: Most of these HIV-1–infected men had bacteria in their semen. ART use was associated with undetectable semen HIV-1 RNA and lower semen bacterial concentrations, whereas insertive anal sex was associated with higher bacterial concentrations. Additional studies evaluating the relationship between semen bacteria, inflammation, mucosal immunity, and HIV-1 shedding are needed to understand implications for HIV-1 transmission. PMID:27861240

Distribution of HIV RNA in CSF and Blood is linked to CD4/CD8 Ratio During Acute HIV.

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Chan, Phillip; Patel, Payal; Hellmuth, Joanna; Colby, Donn J; Kroon, Eugène; Sacdalan, Carlo; Pinyakorn, Suteeraporn; Jagodzinski, Linda; Krebs, Shelly; Ananworanich, Jintanat; Valcour, Victor; Spudich, Serena

2018-05-07

HIV RNA levels in the plasma and cerebrospinal fluid (CSF) are correlated in chronic HIV infection but their dynamics have not been characterized during acute infection. This study analyzed predictors of CSF HIV RNA and relative degree of CNS viral transmigration expressed as plasma minus CSF HIV log10 RNA (PCratio) during untreated acute HIV infection. CSF immune markers were compared between groups with different PCratio. 117 mostly male (97%) participants in the RV254 cohort in Bangkok, Thailand, had median age 28 years and an estimated median 18 days duration of infection; forty-three (37%) were Fiebig stages I/II. Twenty-seven (23%) had CSF HIV RNA CSF HIV RNA and PCratio of 3.76 and 2.36 Log10 copies/mL, respectively. HIV RNA peaked at Fiebig III in plasma and Fiebig IV in CSF. In multivariable analyses, plasma HIV RNA and CD4/CD8 ratio independently correlated with CSF HIV RNA (pCSF neopterin, sCD163, IL-6 and sCD14 levels (all pCSF HIV RNA and PCratio, suggesting that immune responses modulate CNS viral entry at early infection.

A universal real-time PCR assay for the quantification of group-M HIV-1 proviral load.

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Malnati, Mauro S; Scarlatti, Gabriella; Gatto, Francesca; Salvatori, Francesca; Cassina, Giulia; Rutigliano, Teresa; Volpi, Rosy; Lusso, Paolo

2008-01-01

Quantification of human immunodeficiency virus type-1 (HIV-1) proviral DNA is increasingly used to measure the HIV-1 cellular reservoirs, a helpful marker to evaluate the efficacy of antiretroviral therapeutic regimens in HIV-1-infected individuals. Furthermore, the proviral DNA load represents a specific marker for the early diagnosis of perinatal HIV-1 infection and might be predictive of HIV-1 disease progression independently of plasma HIV-1 RNA levels and CD4(+) T-cell counts. The high degree of genetic variability of HIV-1 poses a serious challenge for the design of a universal quantitative assay capable of detecting all the genetic subtypes within the main (M) HIV-1 group with similar efficiency. Here, we describe a highly sensitive real-time PCR protocol that allows for the correct quantification of virtually all group-M HIV-1 strains with a higher degree of accuracy compared with other methods. The protocol involves three stages, namely DNA extraction/lysis, cellular DNA quantification and HIV-1 proviral load assessment. Owing to the robustness of the PCR design, this assay can be performed on crude cellular extracts, and therefore it may be suitable for the routine analysis of clinical samples even in developing countries. An accurate quantification of the HIV-1 proviral load can be achieved within 1 d from blood withdrawal.

The Effects of Viral Load Burden on Pregnancy Loss among HIV-Infected Women in the United States

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Jordan E. Cates

2015-01-01

Full Text Available Background. To evaluate the effects of HIV viral load, measured cross-sectionally and cumulatively, on the risk of miscarriage or stillbirth (pregnancy loss among HIV-infected women enrolled in the Women’s Interagency HIV Study between 1994 and 2013. Methods. We assessed three exposures: most recent viral load measure before the pregnancy ended, log10 copy-years viremia from initiation of antiretroviral therapy (ART to conception, and log10 copy-years viremia in the two years before conception. Results. The risk of pregnancy loss for those with log10 viral load >4.00 before pregnancy ended was 1.59 (95% confidence interval (CI: 0.99, 2.56 times as high as the risk for women whose log10 viral load was ≤1.60. There was not a meaningful impact of log10 copy-years viremia since ART or log10 copy-years viremia in the two years before conception on pregnancy loss (adjusted risk ratios (aRRs: 0.80 (95% CI: 0.69, 0.92 and 1.00 (95% CI: 0.90, 1.11, resp.. Conclusions. Cumulative viral load burden does not appear to be an informative measure for pregnancy loss risk, but the extent of HIV replication during pregnancy, as represented by plasma HIV RNA viral load, predicted loss versus live birth in this ethnically diverse cohort of HIV-infected US women.

The Effects of Viral Load Burden on Pregnancy Loss among HIV-Infected Women in the United States.

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Cates, Jordan E; Westreich, Daniel; Edmonds, Andrew; Wright, Rodney L; Minkoff, Howard; Colie, Christine; Greenblatt, Ruth M; Cejtin, Helen E; Karim, Roksana; Haddad, Lisa B; Kempf, Mirjam-Colette; Golub, Elizabeth T; Adimora, Adaora A

2015-01-01

To evaluate the effects of HIV viral load, measured cross-sectionally and cumulatively, on the risk of miscarriage or stillbirth (pregnancy loss) among HIV-infected women enrolled in the Women's Interagency HIV Study between 1994 and 2013. We assessed three exposures: most recent viral load measure before the pregnancy ended, log10 copy-years viremia from initiation of antiretroviral therapy (ART) to conception, and log10 copy-years viremia in the two years before conception. The risk of pregnancy loss for those with log10 viral load >4.00 before pregnancy ended was 1.59 (95% confidence interval (CI): 0.99, 2.56) times as high as the risk for women whose log10 viral load was ≤1.60. There was not a meaningful impact of log10 copy-years viremia since ART or log10 copy-years viremia in the two years before conception on pregnancy loss (adjusted risk ratios (aRRs): 0.80 (95% CI: 0.69, 0.92) and 1.00 (95% CI: 0.90, 1.11), resp.). Cumulative viral load burden does not appear to be an informative measure for pregnancy loss risk, but the extent of HIV replication during pregnancy, as represented by plasma HIV RNA viral load, predicted loss versus live birth in this ethnically diverse cohort of HIV-infected US women.

Repeat testing of low-level HIV-1 RNA: assay performance and implementation in clinical trials.

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White, Kirsten; Garner, Will; Wei, Lilian; Eron, Joseph J; Zhong, Lijie; Miller, Michael D; Martin, Hal; Plummer, Andrew; Tran-Muchowski, Cecilia; Lindstrom, Kim; Porter, James; Piontkowsky, David; Light, Angela; Reiske, Heinz; Quirk, Erin

2018-05-15

Assess the performance of HIV-1 RNA repeat testing of stored samples in cases of low-level viremia during clinical trials. Prospective and retrospective analysis of randomized clinical trial samples and reference standards. To evaluate assay variability of the Cobas AmpliPrep/Cobas TaqMan HIV-1 Test, v2.0, three separate sources of samples were utilized: the World Health Organization (WHO) HIV reference standard (assayed using 50 independent measurements at six viral loads <200 copies/ml), retrospective analysis of four to six aliquots of plasma samples from four clinical trial participants, and prospective repeat testing of 120 samples from participants in randomized trials with low-level viremia. The TaqMan assay on the WHO HIV-1 RNA standards at viral loads <200 copies/ml performed within the expected variability according to assay specifications. However, standards with low viral loads of 36 and 18 copies/ml reported values of ≥ 50 copies/ml in 66 and 18% of tests, respectively. In participants treated with antiretrovirals who had unexpected viremia of 50-200 copies/ml after achieving <50 copies/ml, retesting of multiple aliquots of stored plasma found <50 copies/ml in nearly all cases upon retesting (14/15; 93%). Repeat testing was prospectively implemented in four clinical trials for all samples with virologic rebound of 50-200 copies/ml (n = 120 samples from 92 participants) from which 42% (50/120) had a retest result of less than 50 copies/ml and 58% (70/120) retested ≥ 50 copies/ml. The TaqMan HIV-1 RNA assay shows variability around 50 copies/ml that affects clinical trial results and may impact clinical practice. In participants with a history of viral load suppression, unexpected low-level viremia may be because of assay variability rather than low drug adherence or true virologic failure. Retesting a stored aliquot of the same sample may differentiate between assay variability and virologic failure as the source of viremia

Undetectable plasma viral load predicts normal survival in HIV-2-infected people in a West African village

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Ricard Dominique

2010-05-01

Full Text Available Abstract Background There have been no previous studies of the long-term survival and temporal changes in plasma viral load among HIV-2 infected subjects. Methods 133 HIV-2 infected and 158 HIV-uninfected subjects from a rural area in North-west Guinea-Bissau, West Africa were enrolled into a prospective cohort study in 1991 and followed-up to mid-2009. Data were collected on four occasions during that period on HIV antibodies, CD4% and HIV-2 plasma viral load. Results Median age (interquartile range [IQR] of HIV-2 infected subjects at time of enrollment was 47 (36, 60 years, similar to that of HIV-uninfected control subjects, 49 (38, 62 (p = 0.4. Median (IQR plasma viral load and CD4 percentage were 347 (50, 4,300 copies/ml and 29 (22, 35 respectively. Overall loss to follow-up to assess vital status was small, at 6.7% and 6.3% for HIV-2 infected and uninfected subjects respectively. An additional 17 (12.8% and 16 (10.1% of HIV-2 infected and uninfected subjects respectively were censored during follow-up due to infection with HIV-1. The mortality rate per 100 person-years (95% CI was 4.5 (3.6, 5.8 among HIV-2 infected subjects compared to 2.1 (1.6, 2.9 among HIV-uninfected (age-sex adjusted rate ratio 1.9 (1.3, 2.8, p Viral load measurements were available for 98%, 78%, 77% and 61% HIV-2 infected subjects who were alive and had not become super-infected with HIV-1, in 1991, 1996, 2003 and 2006 respectively. Median plasma viral load (RNA copies per ml (IQR did not change significantly over time, being 150 (50, 1,554; n = 77 in 1996, 203 (50, 2,837; n = 47 in 2003 and 171 (50, 497; n = 31 in 2006. Thirty seven percent of HIV-2 subjects had undetectable viraemia ( Conclusions A substantial proportion of HIV-2 infected subjects in this cohort have stable plasma viral load, and those with an undetectable viral load (37% at study entry had a normal survival rate. However, the sequential laboratory findings need to be interpreted with caution given

Dynamics of HIV-1 RNA Near the Plasma Membrane during Virus Assembly.

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Sardo, Luca; Hatch, Steven C; Chen, Jianbo; Nikolaitchik, Olga; Burdick, Ryan C; Chen, De; Westlake, Christopher J; Lockett, Stephen; Pathak, Vinay K; Hu, Wei-Shau

2015-11-01

To increase our understanding of the events that lead to HIV-1 genome packaging, we examined the dynamics of viral RNA and Gag-RNA interactions near the plasma membrane by using total internal reflection fluorescence microscopy. We labeled HIV-1 RNA with a photoconvertible Eos protein via an RNA-binding protein that recognizes stem-loop sequences engineered into the viral genome. Near-UV light exposure causes an irreversible structural change in Eos and alters its emitted fluorescence from green to red. We studied the dynamics of HIV-1 RNA by photoconverting Eos near the plasma membrane, and we monitored the population of photoconverted red-Eos-labeled RNA signals over time. We found that in the absence of Gag, most of the HIV-1 RNAs stayed near the plasma membrane transiently, for a few minutes. The presence of Gag significantly increased the time that RNAs stayed near the plasma membrane: most of the RNAs were still detected after 30 min. We then quantified the proportion of HIV-1 RNAs near the plasma membrane that were packaged into assembling viral complexes. By tagging Gag with blue fluorescent protein, we observed that only a portion, ∼13 to 34%, of the HIV-1 RNAs that reached the membrane were recruited into assembling particles in an hour, and the frequency of HIV-1 RNA packaging varied with the Gag expression level. Our studies reveal the HIV-1 RNA dynamics on the plasma membrane and the efficiency of RNA recruitment and provide insights into the events leading to the generation of infectious HIV-1 virions. Nascent HIV-1 particles assemble on plasma membranes. During the assembly process, HIV-1 RNA genomes must be encapsidated into viral complexes to generate infectious particles. To gain insights into the RNA packaging and virus assembly mechanisms, we labeled and monitored the HIV-1 RNA signals near the plasma membrane. Our results showed that most of the HIV-1 RNAs stayed near the plasma membrane for only a few minutes in the absence of Gag, whereas

An RNA-binding compound that stabilizes the HIV-1 gRNA packaging signal structure and specifically blocks HIV-1 RNA encapsidation.

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Ingemarsdotter, Carin K; Zeng, Jingwei; Long, Ziqi; Lever, Andrew M L; Kenyon, Julia C

2018-03-14

NSC260594, a quinolinium derivative from the NCI diversity set II compound library, was previously identified in a target-based assay as an inhibitor of the interaction between the HIV-1 (ψ) stem-loop 3 (SL3) RNA and Gag. This compound was shown to exhibit potent antiviral activity. Here, the effects of this compound on individual stages of the viral lifecycle were examined by qRT-PCR, ELISA and Western blot, to see if its actions were specific to the viral packaging stage. The structural effects of NSC260594 binding to the HIV-1 gRNA were also examined by SHAPE and dimerization assays. Treatment of cells with NSC260594 did not reduce the number of integration events of incoming virus, and treatment of virus producing cells did not affect the level of intracellular Gag protein or viral particle release as determined by immunoblot. However, NSC260594 reduced the incorporation of gRNA into virions by up to 82%, without affecting levels of gRNA inside the cell. This reduction in packaging correlated closely with the reduction in infectivity of the released viral particles. To establish the structural effects of NSC260594 on the HIV-1 gRNA, we performed SHAPE analyses to pinpoint RNA structural changes. NSC260594 had a stabilizing effect on the wild type RNA that was not confined to SL3, but that was propagated across the structure. A packaging mutant lacking SL3 did not show this effect. NSC260594 acts as a specific inhibitor of HIV-1 RNA packaging. No other viral functions are affected. Its action involves preventing the interaction of Gag with SL3 by stabilizing this small RNA stem-loop which then leads to stabilization of the global packaging signal region (psi or ψ). This confirms data, previously only shown in analyses of isolated SL3 oligonucleotides, that SL3 is structurally labile in the presence of Gag and that this is critical for the complete psi region to be able to adopt different conformations. Since replication is otherwise unaffected by NSC260594

Comparison of HIV DNA and RNA in gut-associated lymphoid tissue of HIV-infected controllers and noncontrollers.

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Hatano, Hiroyu; Somsouk, Ma; Sinclair, Elizabeth; Harvill, Kara; Gilman, Lee; Cohen, Michelle; Hoh, Rebecca; Hunt, Peter W; Martin, Jeffrey N; Wong, Joseph K; Deeks, Steven G; Yukl, Steven A

2013-09-10

HIV-infected controllers have provided novel insights into mechanisms of viral control. We investigated the degree to which HIV DNA and RNA are present in gut-associated lymphoid tissue (GALT) of controllers. Cross-sectional cohort study. Colorectal biopsy pieces were obtained from five untreated noncontrollers, five ART-suppressed patients, and nine untreated controllers. Rectal HIV DNA was lower in controllers (median 496 copies/10(6) CD4 T cells) than in untreated noncontrollers (117483 copies/10(6) CD4+ T cells, P = 0.001) and ART-suppressed patients (6116 copies/10(6) CD4 T cells, P = 0.004). Similarly, rectal HIV RNA was lower in controllers (19 copies/10(6) CD4 T cells) than in noncontrollers (15210 copies/10(6) CD4+ T cells, P = 0.001) and ART-suppressed patients (1625 copies/10(6) CD4+ T cells, P = 0.0599). Rectal HIV RNA/DNA ratios were not statistically different between the three groups. Despite being able to maintain very low plasma HIV RNA levels in the absence of antiretroviral therapy (ART), HIV-infected controllers have readily measurable levels of HIV DNA and RNA in GALT. As expected, controllers had lower rectal HIV DNA and RNA compared with untreated noncontrollers and ART-suppressed individuals. Compared with the mechanisms of 'natural' viral control of controllers, long-term ART does not reduce the total HIV reservoir to the level of controllers.

Longer duration of homelessness is associated with a lower likelihood of non-detectable plasma HIV-1 RNA viral load among people who use illicit drugs in a Canadian setting.

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Loh, Jane; Kennedy, Mary Clare; Wood, Evan; Kerr, Thomas; Marshall, Brandon; Parashar, Surita; Montaner, Julio; Milloy, M-J

2016-11-01

Homelessness is common among people who use drugs (PWUD) and, for those living with HIV/AIDS, an important contributor to sub-optimal HIV treatment outcomes. This study aims to investigate the relationship between the duration of homelessness and the likelihood of plasma HIV-1 RNA viral load (VL) non-detectability among a cohort of HIV-positive PWUD. We used data from the ACCESS study, a long-running prospective cohort study of HIV-positive PWUD linked to comprehensive HIV clinical records including systematic plasma HIV-1 RNA VL monitoring. We estimated the longitudinal relationship between the duration of homelessness and the likelihood of exhibiting a non-detectable VL (i.e., effects modelling. Between May 1996 and June 2014, 922 highly active antiretroviral therapy-exposed participants were recruited and contributed 8188 observations. Of these, 4800 (59%) were characterized by non-detectable VL. Participants reported they were homeless in 910 (11%) interviews (median: six months, interquartile range: 6-12 months). A longer duration of homelessness was associated with lower odds of VL non-detectability (adjusted odds ratio = 0.71 per six-month period of homelessness, 95% confidence interval: 0.60-0.83) after adjustment for age, ancestry, drug use patterns, engagement in addiction treatment, and other potential confounders. Longer durations of episodes of homelessness in this cohort of HIV-positive illicit drug users were associated with a lower likelihood of plasma VL non-detectability. Our findings suggest that interventions that seek to promptly house homeless individuals, such as Housing First approaches, might assist in maximizing the clinical and public health benefits of antiretroviral therapy among people living with HIV/AIDS.

Dynamics of breast milk HIV-1 RNA with unilateral mastitis or abscess.

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Semrau, Katherine; Kuhn, Louise; Brooks, Daniel R; Cabral, Howard; Sinkala, Moses; Kankasa, Chipepo; Thea, Donald M; Aldrovandi, Grace M

2013-03-01

Mastitis and abscess in HIV-infected women increase the risk of breastfeeding transmission of HIV. Guidelines encourage women to stop breastfeeding on the affected breast and feed on the contralateral breast. However, impact of breast pathology on breast milk HIV dynamics is unknown. HIV RNA was quantified in 211 breast milk samples collected before, during, and after a clinical mastitis or an abscess diagnosis from 38 HIV-infected women participating in a Zambian breastfeeding study. HIV RNA quantity was compared between affected and unaffected breasts over time using generalized estimating equation models. A sample of 115 women without breast pathology was selected as a control group. In the affected breast, breast milk HIV RNA quantity increased from the pre- to during-pathology period by log(10) 0.45 copies per milliliter [95% confidence interval (CI): 0.16 to 0.74], and after symptom resolution, HIV RNA levels were no different from prepathology levels (log10 -0.04 copies per milliliter 95% CI: -0.33 to 0.25). In the contralateral, unaffected breast, HIV RNA quantity did not significantly increase (log(10) 0.15 copies per milliliter, 95% CI: -0.41 to 0.10). Increase was more marked in women with abscess or with a greater number of mastitis symptoms. HIV RNA was not significantly different between affected and unaffected women, except at the time of diagnosis. Breast milk HIV RNA increased modestly in the affected breast with unilateral mastitis or abscess and returned to prepathology levels with symptom resolution. Contralateral HIV RNA was not affected. Results support guidelines encouraging feeding from the contralateral breast to minimize the risk of HIV transmission associated with unilateral breast pathology.

HIV-1 viral load measurement in venous blood and fingerprick blood using Abbott RealTime HIV-1 DBS assay.

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Tang, Ning; Pahalawatta, Vihanga; Frank, Andrea; Bagley, Zowie; Viana, Raquel; Lampinen, John; Leckie, Gregor; Huang, Shihai; Abravaya, Klara; Wallis, Carole L

2017-07-01

HIV RNA suppression is a key indicator for monitoring success of antiretroviral therapy. From a logistical perspective, viral load (VL) testing using Dried Blood Spots (DBS) is a promising alternative to plasma based VL testing in resource-limited settings. To evaluate the analytical and clinical performance of the Abbott RealTime HIV-1 assay using a fully automated one-spot DBS sample protocol. Limit of detection (LOD), linearity, lower limit of quantitation (LLQ), upper limit of quantitation (ULQ), and precision were determined using serial dilutions of HIV-1 Virology Quality Assurance stock (VQA Rush University), or HIV-1-containing armored RNA, made in venous blood. To evaluate correlation, bias, and agreement, 497 HIV-1 positive adult clinical samples were collected from Ivory Coast, Uganda and South Africa. For each HIV-1 participant, DBS-fingerprick, DBS-venous and plasma sample results were compared. Correlation and bias values were obtained. The sensitivity and specificity were analyzed at a threshold of 1000 HIV-1 copies/mL generated using the standard plasma protocol. The Abbott HIV-1 DBS protocol had an LOD of 839 copies/mL, a linear range from 500 to 1×10 7 copies/mL, an LLQ of 839 copies/mL, a ULQ of 1×10 7 copies/mL, and an inter-assay SD of ≤0.30 log copies/mL for all tested levels within this range. With clinical samples, the correlation coefficient (r value) was 0.896 between DBS-fingerprick and plasma and 0.901 between DBS-venous and plasma, and the bias was -0.07 log copies/mL between DBS-fingerprick and plasma and -0.02 log copies/mL between DBS-venous and plasma. The sensitivity of DBS-fingerprick and DBS-venous was 93%, while the specificity of both DBS methods was 95%. The results demonstrated that the Abbott RealTime HIV-1 assay with DBS sample protocol is highly sensitive, specific and precise across a wide dynamic range and correlates well with plasma values. The Abbott RealTime HIV-1 assay with DBS sample protocol provides an

Full Viral Suppression, Low-Level Viremia, and Quantifiable Plasma HIV-RNA at the End of Pregnancy in HIV-Infected Women on Antiretroviral Treatment.

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Baroncelli, Silvia; Pirillo, Maria F; Tamburrini, Enrica; Guaraldi, Giovanni; Pinnetti, Carmela; Degli Antoni, Anna; Galluzzo, Clementina M; Stentarelli, Chiara; Amici, Roberta; Floridia, Marco

2015-07-01

There is limited information on full viral suppression and low-level HIV-RNA viremia in HIV-infected women at the end of pregnancy. We investigated HIV-RNA levels close to delivery in women on antiretroviral treatment in order to define rates of complete suppression, low-level viremia, and quantifiable HIV-RNA, exploring as potential determinants some clinical and viroimmunological variables. Plasma samples from a national study in Italy, collected between 2003 and 2012, were used. According to plasma HIV-RNA levels, three groups were defined: full suppression (target not detected), low-level viremia (target detected but HIV-RNA (≥37 copies/ml). Multivariable logistic regression was used to define determinants of full viral suppression and of quantifiable HIV-RNA. Among 107 women evaluated at a median gestational age of 35 weeks, 90 (84.1%) had HIV-RNA HIV-RNA was 109 copies/ml (IQR 46-251), with only one case showing resistance (mutation M184V; rate: 9.1%). In multivariable analyses, women with higher baseline HIV-RNA levels and with hepatitis C virus (HCV) coinfection were significantly more likely to have quantifiable HIV-RNA in late pregnancy. Full viral suppression was significantly more likely with nonnucleoside reverse transcriptase inhibitor (NNRTI)-based regimens and significantly less likely with higher HIV-RNA in early pregnancy. No cases of HIV transmission occurred. In conclusion, HIV-infected pregnant women showed a high rate of viral suppression and a low resistance rate before delivery. In most cases no target HIV-RNA was detected in plasma, suggesting a low risk of subsequent virological rebound and development of resistance. Women with high levels of HIV-RNA in early pregnancy and those who have concomitant HCV infection should be considered at higher risk of having quantifiable HIV-RNA at the end of pregnancy.

Integrase inhibitors in late pregnancy and rapid HIV viral load reduction.

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Rahangdale, Lisa; Cates, Jordan; Potter, JoNell; Badell, Martina L; Seidman, Dominika; Miller, Emilly S; Coleman, Jenell S; Lazenby, Gweneth B; Levison, Judy; Short, William R; Yawetz, Sigal; Ciaranello, Andrea; Livingston, Elizabeth; Duthely, Lunthita; Rimawi, Bassam H; Anderson, Jean R; Stringer, Elizabeth M

2016-03-01

Minimizing time to HIV viral suppression is critical in pregnancy. Integrase strand transfer inhibitors (INSTIs), like raltegravir, are known to rapidly suppress plasma HIV RNA in nonpregnant adults. There are limited data in pregnant women. We describe time to clinically relevant reduction in HIV RNA in pregnant women using INSTI-containing and non-INSTI-containing antiretroviral therapy (ART) options. We conducted a retrospective cohort study of pregnant HIV-infected women in the United States from 2009 through 2015. We included women who initiated ART, intensified their regimen, or switched to a new regimen due to detectable viremia (HIV RNA >40 copies/mL) at ≥20 weeks gestation. Among women with a baseline HIV RNA permitting 1-log reduction, we estimated time to 1-log RNA reduction using the Kaplan-Meier estimator comparing women starting/adding an INSTI in their regimen vs other ART. To compare groups with similar follow-up time, we also conducted a subgroup analysis limited to women with ≤14 days between baseline and follow-up RNA data. This study describes 101 HIV-infected pregnant women from 11 US clinics. In all, 75% (76/101) of women were not taking ART at baseline; 24 were taking non-INSTI containing ART, and 1 received zidovudine monotherapy. In all, 39% (39/101) of women started an INSTI-containing regimen or added an INSTI to their ART regimen. Among 90 women with a baseline HIV RNA permitting 1-log reduction, the median time to 1-log RNA reduction was 8 days (interquartile range [IQR], 7-14) in the INSTI group vs 35 days (IQR, 20-53) in the non-INSTI ART group (P pregnancy. Inclusion of an INSTI may play a role in optimal reduction of HIV RNA for HIV-infected pregnant women presenting late to care or failing initial therapy. Larger studies are urgently needed to assess the safety and effectiveness of this approach. Copyright © 2016 Elsevier Inc. All rights reserved.

Correlates of HIV-1 genital shedding in Tanzanian women.

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Clare Tanton

2011-03-01

Full Text Available Understanding the correlates of HIV shedding is important to inform strategies to reduce HIV infectiousness. We examined correlates of genital HIV-1 RNA in women who were seropositive for both herpes simplex virus (HSV-2 and HIV-1 and who were enrolled in a randomised controlled trial of HSV suppressive therapy (aciclovir 400 mg b.i.d vs. placebo in Tanzania.Samples, including a cervico-vaginal lavage, were collected and tested for genital HIV-1 and HSV and reproductive tract infections (RTIs at randomisation and 6, 12 and 24 months follow-up. Data from all women at randomisation and women in the placebo arm during follow-up were analysed using generalised estimating equations to determine the correlates of cervico-vaginal HIV-1 RNA detection and load.Cervico-vaginal HIV-1 RNA was detected at 52.0% of 971 visits among 482 women, and was independently associated with plasma viral load, presence of genital ulcers, pregnancy, bloody cervical or vaginal discharge, abnormal vaginal discharge, cervical ectopy, Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis, an intermediate bacterial vaginosis score and HSV DNA detection. Similar factors were associated with genital HIV-1 RNA load.RTIs were associated with increased presence and quantity of genital HIV-1 RNA in this population. These results highlight the importance of integrating effective RTI treatment into HIV care services.

Interactions Between HIV-1 Gag and Viral RNA Genome Enhance Virion Assembly

DEFF Research Database (Denmark)

Dilley, Kari A; Nikolaitchik, Olga A; Galli, Andrea

2017-01-01

between Gag and viral RNA are required for the enhancement of particle production. Taken together, these studies are consistent with our previous hypothesis that specific dimeric viral RNA:Gag interactions are the nucleation event of infectious virion assembly, ensuring that one RNA dimer is packaged......Most HIV-1 virions contain two copies of full-length viral RNA, indicating that genome packaging is efficient and tightly regulated. However, the structural protein Gag is the only component required for the assembly of noninfectious virus-like particles and the viral RNA is dispensable...... in this process. The mechanism that allows HIV-1 to achieve such high efficiency of genome packaging when a packageable viral RNA is not required for virus assembly is currently unknown. In this report, we examined the role of HIV-1 RNA in virus assembly and found that packageable HIV-1 RNA enhances particle...

Analysis of correlation between cerebrospinal fluid and plasma HIV-1 RNA levels in patients with neurological opportunistic diseases

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Paulo Pereira Christo

2011-08-01

Full Text Available The question of whether HIV-1 RNA in cerebrospinal fluid (CSF is derived from viral replication in the central nervous system or simply reflects the transit of infected lymphocytes from the blood compartment has long been a matter of debate. Some studies found no correlation between CSF and plasma viral load, whereas others did. The lack of a correlation between the two compartments suggests that the presence of HIV-1 RNA is not simply due to the passive passage of the virus from blood to CSF but rather due to intrathecal replication. To evaluate the correlation between plasma and CSF HIV-1 RNA levels and to identify situations in which there is no correlation between the two compartments, seventy patients were prospectively studied. The association between CSF and plasma viral load was evaluated in the total population and in subgroups of patients with similar characteristics. A correlation between the CSF and plasma compartments was observed for patients undergoing highly active antiretroviral therapy (HAART, those with a CD4 T lymphocyte count lower than 200 cells/mm³, and those with increased CSF protein content. On the other hand, no correlation was observed for patients without adequate virological control, who had a CD4 count higher than 200 cells/mm³ and who did not use HAART. The correlation between the two compartments observed in some patients suggests that CSF HIV-1 RNA levels may reflect plasma levels in these subjects. In contrast, the lack of a correlation between the two compartments in patients who were not on HAART and who had normal CSF proteins and a poor virological control possibly indicates compartmentalization of the virus in CSF and, consequently, plasma-independent intrathecal viral replication.

RNA interference-based therapeutics for human immunodeficiency virus HIV-1 treatment: synthetic siRNA or vector-based shRNA?

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Subramanya, Sandesh; Kim, Sang-Soo; Manjunath, N; Shankar, Premlata

2010-02-01

Despite the clinical benefits of highly active antiretroviral therapy (HAART), the prospect of life-long antiretroviral treatment poses significant problems, which has spurred interest in developing new drugs and strategies to treat HIV infection and eliminate persistent viral reservoirs. RNAi has emerged as a therapeutic possibility for HIV. We discuss progress in overcoming hurdles to translating transient and stable RNAi enabling technologies to clinical application for HIV; covering the past 2 - 3 years. HIV inhibition can be achieved by transfection of chemically or enzymatically synthesized siRNAs or by DNA-based vector systems expressing short hairpin RNAs (shRNAs) that are processed intracellularly into siRNA. We compare these approaches, focusing on technical and safety issues that will guide the choice of strategy for clinical use. Introduction of synthetic siRNA into cells or its stable endogenous production using vector-driven shRNA have been shown to suppress HIV replication in vitro and, in some instances, in vivo. Each method has advantages and limitations in terms of ease of delivery, duration of silencing, emergence of escape mutants and potential toxicity. Both appear to have potential as future therapeutics for HIV, once the technical and safety issues of each approach are overcome.

Different patterns of HIV-1 DNA after therapy discontinuation

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Ghinelli Florio

2005-09-01

Full Text Available Abstract Background By persisting in infected cells for a long period of time, proviral HIV-1 DNA can represent an alternative viral marker to RNA viral load during the follow-up of HIV-1 infected individuals. In the present study sequential blood samples of 10 patients under antiretroviral treatment from 1997 with two NRTIs, who refused to continue any antiviral regimen, were analyzed for 16 – 24 weeks to study the possible relationship between DNA and RNA viral load. Methods The amount of proviral DNA was quantified by SYBR green real-time PCR in peripheral blood mononuclear cells from a selected group of ten patients with different levels of plasmatic viremia (RNA viral load. Results Variable levels of proviral DNA were found without any significant correlation between proviral load and plasma HIV-1 RNA levels. Results obtained showed an increase or a rebound in viral DNA in most patients, suggesting that the absence of therapy reflects an increase and/or a persistence of cells containing viral DNA. Conclusion Even though plasma HIV RNA levels remain the basic parameter to monitor the intensity of viral replication, the results obtained seem to indicate that DNA levels could represent an adjunct prognostic marker in monitoring HIV-1 infected subjects.

Differences in HIV Type 1 RNA Plasma Load Profile of Closely Related Cocirculating Ethiopian Subtype C Strains: C and C '

NARCIS (Netherlands)

Ayele, Workenesh; Mekonnen, Yared; Messele, Tsehaynesh; Mengistu, Yohannes; Tsegaye, Aster; Bakker, Margreet; Berkhout, Ben; Dorigo-Zetsma, Wendelien; Wolday, Dawit; Goudsmit, Jaap; Coutinho, Roel; de Baar, Michel; Paxton, William A.; Pollakis, Georgios

2010-01-01

Two HIV-1 subtype C subclusters have been identified in Ethiopia (C and C') with little knowledge regarding their biological or clinical differences. We longitudinally monitored HIV-1 viral loads and CD4(+) T cell counts for 130 subtype C-infected individuals from Ethiopia over 5 years. The genetic

The thermodynamics of Pr55Gag-RNA interaction regulate the assembly of HIV.

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Hanumant S Tanwar

2017-02-01

Full Text Available The interactions that occur during HIV Pr55Gag oligomerization and genomic RNA packaging are essential elements that facilitate HIV assembly. However, mechanistic details of these interactions are not clearly defined. Here, we overcome previous limitations in producing large quantities of full-length recombinant Pr55Gag that is required for isothermal titration calorimetry (ITC studies, and we have revealed the thermodynamic properties of HIV assembly for the first time. Thermodynamic analysis showed that the binding between RNA and HIV Pr55Gag is an energetically favourable reaction (ΔG<0 that is further enhanced by the oligomerization of Pr55Gag. The change in enthalpy (ΔH widens sequentially from: (1 Pr55Gag-Psi RNA binding during HIV genome selection; to (2 Pr55Gag-Guanosine Uridine (GU-containing RNA binding in cytoplasm/plasma membrane; and then to (3 Pr55Gag-Adenosine(A-containing RNA binding in immature HIV. These data imply the stepwise increments of heat being released during HIV biogenesis may help to facilitate the process of viral assembly. By mimicking the interactions between A-containing RNA and oligomeric Pr55Gag in immature HIV, it was noted that a p6 domain truncated Pr50Gag Δp6 is less efficient than full-length Pr55Gag in this thermodynamic process. These data suggest a potential unknown role of p6 in Pr55Gag-Pr55Gag oligomerization and/or Pr55Gag-RNA interaction during HIV assembly. Our data provide direct evidence on how nucleic acid sequences and the oligomeric state of Pr55Gag regulate HIV assembly.

Changes in HIV-1 subtypes B and C genital tract RNA in women and men after initiation of antiretroviral therapy.

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Fiscus, Susan A; Cu-Uvin, Susan; Eshete, Abel Tilahun; Hughes, Michael D; Bao, Yajing; Hosseinipour, Mina; Grinsztejn, Beatriz; Badal-Faesen, Sharlaa; Dragavon, Joan; Coombs, Robert W; Braun, Ken; Moran, Laura; Hakim, James; Flanigan, Timothy; Kumarasamy, N; Campbell, Thomas B

2013-07-01

Combination antiretroviral therapy (cART) reduces genital tract human immunodeficiency virus type 1 (HIV-1) load and reduces the risk of sexual transmission, but little is known about the efficacy of cART for decreasing genital tract viral load (GTVL) and differences in sex or HIV-1 subtype. HIV-1 RNA from blood plasma, seminal plasma, or cervical wicks was quantified at baseline and at weeks 48 and 96 after entry in a randomized clinical trial of 3 cART regimens. One hundred fifty-eight men and 170 women from 7 countries were studied (men: 55% subtype B and 45% subtype C; women: 24% subtype B and 76% subtype C). Despite similar baseline CD4(+) cell counts and blood plasma viral loads, women with subtype C had the highest GTVL (median, 5.1 log10 copies/mL) compared to women with subtype B and men with subtype C or B (4.0, 4.0, and 3.8 log10 copies/mL, respectively; P female genital tract may serve as a reservoir of persistent HIV-1 replication during cART and affect the use of cART to prevent sexual and perinatal transmission of HIV-1.

Molecular mimicry of human tRNALys anti-codon domain by HIV-1 RNA genome facilitates tRNA primer annealing.

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Jones, Christopher P; Saadatmand, Jenan; Kleiman, Lawrence; Musier-Forsyth, Karin

2013-02-01

The primer for initiating reverse transcription in human immunodeficiency virus type 1 (HIV-1) is tRNA(Lys3). Host cell tRNA(Lys) is selectively packaged into HIV-1 through a specific interaction between the major tRNA(Lys)-binding protein, human lysyl-tRNA synthetase (hLysRS), and the viral proteins Gag and GagPol. Annealing of the tRNA primer onto the complementary primer-binding site (PBS) in viral RNA is mediated by the nucleocapsid domain of Gag. The mechanism by which tRNA(Lys3) is targeted to the PBS and released from hLysRS prior to annealing is unknown. Here, we show that hLysRS specifically binds to a tRNA anti-codon-like element (TLE) in the HIV-1 genome, which mimics the anti-codon loop of tRNA(Lys) and is located proximal to the PBS. Mutation of the U-rich sequence within the TLE attenuates binding of hLysRS in vitro and reduces the amount of annealed tRNA(Lys3) in virions. Thus, LysRS binds specifically to the TLE, which is part of a larger LysRS binding domain in the viral RNA that includes elements of the Psi packaging signal. Our results suggest that HIV-1 uses molecular mimicry of the anti-codon of tRNA(Lys) to increase the efficiency of tRNA(Lys3) annealing to viral RNA.

Comparación de los métodos de cuantificación de carga viral de VIH: COBAS® AmpliPrep/COBAS® TaqMan HIV-1 test, v 2.0, y VERSANT HIV-1 RNA 1.0 Assay (kPCR Comparison of COBAS® AmpliPrep/COBAS® TaqMan HIV-1 test, v 2.0 and VERSANT HIV-1 RNA 1.0 (kPCR assays for HIV-1 plasma viral load

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María Isabel Múnera-Jaramillo

2012-03-01

Full Text Available Objetivo. El propósito del estudio fue evaluar el desempeño del método VERSANTHIV-1RNA 1.0 Assay® (kPCR (Siemens, para la cuantificación de la carga viral en pacientes con VIH-1, en comparación con el método COBAS® AmpliPrep/COBAS TaqMan HIV-1 test®, v2.0 (Roche Diagnostics (CAP/CTM. Métodos. Las muestras fueron tomadas en dos tubos con EDTA, de 60 pacientes remitidos por el médico tratante para pruebas de carga viral como parte de su control de rutina de VIH/sida, y fueron procesadas para la cuantificación del ARN del VIH-1 por ambas técnicas. Se hizo análisis de regresión y se calcularon los coeficientes de correlación de Pearson, y los de correlación y concordancia de Lin. Se evalúo la concordancia entre las dos técnicas mediante el método de Bland-Altman. Resultados. El promedio de la carga viral por el método CAP/CTM fue 3,2±1,4 long10 copias/ml y, por el método kPCR, 3,0±1,3 long10 copias/ml. El 86,7 % de muestras presentó diferencias entre los dos métodos, menores de 0,5 long10 copias/ml, y el 13,3 % presentó diferencias mayores. El coeficiente de correlación de Pearson entre los dos métodos fue de 0,97 (IC95% 0,95-0,99 y el índice kappa ponderado entre los dos métodos en diferentes rangos de concentración, fue de 0,91 (IC95% 0,87-0,96. El promedio de las diferencias entre las mediciones fue 0,22 long10 copias/ml (IC95% -0,45 a 0,89. Conclusión. Las dos técnicas evaluadas fueron comparables, con el método kPCR se observaron resultados más bajos.Objective: The purpose of this study was to evaluate the performance of the kPCR VERSANT (™ 440 HIV-1RNA 3.0 Assay® (Siemens method for the quantification of viral load in HIV-1 patients, compared to the COBAS AmpliPrep/COBASTaqMan HIV-1 test®, v. 2.0 (Roche Diagnostics (CAP/CTM. Methods: Samples were taken in 2 tubes with EDTA, in 60 patients referred by the attending physician for viral load tests as part of their routine control of HIV/AIDS, and were

Receptor-targeted aptamer-siRNA conjugate-directed transcriptional regulation of HIV-1

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Zhou, Jiehua; Lazar, Daniel; Li, Haitang; Xia, Xin; Satheesan, Sangeetha; Charlins, Paige; O'Mealy, Denis; Akkina, Ramesh; Saayman, Sheena; Weinberg, Marc S.; Rossi, John J.; Morris, Kevin V.

2018-01-01

Gene-based therapies represent a promising therapeutic paradigm for the treatment of HIV-1, as they have the potential to maintain sustained viral inhibition with reduced treatment interventions. Such an option may represent a long-term treatment alternative to highly active antiretroviral therapy. Methods: We previously described a therapeutic approach, referred to as transcriptional gene silencing (TGS), whereby small noncoding RNAs directly inhibit the transcriptional activity of HIV-1 by targeting sites within the viral promoter, specifically the 5' long terminal repeat (LTR). TGS differs from traditional RNA interference (RNAi) in that it is characterized by concomitant silent-state epigenetic marks on histones and DNA. To deliver TGS-inducing RNAs, we developed functional RNA conjugates based on the previously reported dual function of the gp120 (A-1) aptamer conjugated to 27-mer Dicer-substrate anti-HIV-1 siRNA (dsiRNA), LTR-362. Results: We demonstrate here that high levels of processed guide RNAs localize to the nucleus in infected T lymphoblastoid CEM cell line and primary human CD4+ T-cells. Treatment of the aptamer-siRNA conjugates induced TGS with an ~10-fold suppression of viral p24 levels as measured at day 12 post infection. To explore the silencing efficacy of aptamer-siRNA conjugates in vivo, HIV-1-infected humanized NOD/SCID/IL2 rγnull mice (hu-NSG) were treated with the aptamer-siRNA conjugates. Systemic delivery of the A-1-stick-LTR-362 27-mer siRNA conjugates suppressed HIV-1 infection and protected CD4+ T cell levels in viremia hu-NSG mice. Principle conclusions: Collectively these data suggest that the gp120 aptamer-dsiRNA conjugate design is suitable for systemic delivery of small RNAs that can be used to suppress HIV-1. PMID:29556342

Impact of tuberculosis treatment on CD4 cell count, HIV RNA, and p24 antigen in patients with HIV and tuberculosis

DEFF Research Database (Denmark)

Wejse, Christian; Furtado, A.; Camara, C.

2013-01-01

To describe HIV RNA levels during tuberculosis (TB) infection in patients co-infected with TB and HIV. Moreover, to examine the p24 antigen profile during TB treatment.......To describe HIV RNA levels during tuberculosis (TB) infection in patients co-infected with TB and HIV. Moreover, to examine the p24 antigen profile during TB treatment....

What do we measure when we measure cell-associated HIV RNA.

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Pasternak, Alexander O; Berkhout, Ben

2018-01-29

Cell-associated (CA) HIV RNA has received much attention in recent years as a surrogate measure of the efficiency of HIV latency reversion and because it may provide an estimate of the viral reservoir size. This review provides an update on some recent insights in the biology and clinical utility of this biomarker. We discuss a number of important considerations to be taken into account when interpreting CA HIV RNA measurements, as well as different methods to measure this biomarker.

The HIV-1 leader RNA conformational switch regulates RNA dimerization but does not regulate mRNA translation

NARCIS (Netherlands)

Abbink, Truus E. M.; Ooms, Marcel; Haasnoot, P. C. Joost; Berkhout, Ben

2005-01-01

The untranslated leader RNA is the most conserved part of the human immunodeficiency virus type I (HIV-1) genome. It contains many regulatory motifs that mediate a variety of steps in the viral life cycle. Previous work showed that the full-length leader RNA can adopt two alternative structures: a

Editing of HIV-1 RNA by the double-stranded RNA deaminase ADAR1 stimulates viral infection

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Doria, Margherita; Neri, Francesca; Gallo, Angela; Farace, Maria Giulia; Michienzi, Alessandro

2009-01-01

Adenosine deaminases that act on dsRNA (ADARs) are enzymes that target double-stranded regions of RNA converting adenosines into inosines (A-to-I editing) thus contributing to genome complexity and fine regulation of gene expression. It has been described that a member of the ADAR family, ADAR1, can target viruses and affect their replication process. Here we report evidence showing that ADAR1 stimulates human immuno deficiency virus type 1 (HIV-1) replication by using both editing-dependent and editing-independent mechanisms. We show that over-expression of ADAR1 in HIV-1 producer cells increases viral protein accumulation in an editing-independent manner. Moreover, HIV-1 virions generated in the presence of over-expressed ADAR1 but not an editing-inactive ADAR1 mutant are released more efficiently and display enhanced infectivity, as demonstrated by challenge assays performed with T cell lines and primary CD4+ T lymphocytes. Finally, we report that ADAR1 associates with HIV-1 RNAs and edits adenosines in the 5′ untranslated region (UTR) and the Rev and Tat coding sequence. Overall these results suggest that HIV-1 has evolved mechanisms to take advantage of specific RNA editing activity of the host cell and disclose a stimulatory function of ADAR1 in the spread of HIV-1. PMID:19651874

Effect of mild-to-moderate smoking on viral load, cytokines, oxidative stress, and cytochrome P450 enzymes in HIV-infected individuals.

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Anusha Ande

Full Text Available Mild-to-moderate tobacco smoking is highly prevalent in HIV-infected individuals, and is known to exacerbate HIV pathogenesis. The objective of this study was to determine the specific effects of mild-to-moderate smoking on viral load, cytokine production, and oxidative stress and cytochrome P450 (CYP pathways in HIV-infected individuals who have not yet received antiretroviral therapy (ART. Thirty-two human subjects were recruited and assigned to four different cohorts as follows: a HIV negative non-smokers, b HIV positive non-smokers, c HIV negative mild-to-moderate smokers, and d HIV positive mild-to-moderate smokers. Patients were recruited in Cameroon, Africa using strict selection criteria to exclude patients not yet eligible for ART and not receiving conventional or traditional medications. Those with active tuberculosis, hepatitis B or with a history of substance abuse were also excluded. Our results showed an increase in the viral load in the plasma of HIV positive patients who were mild-to-moderate smokers compared to individuals who did not smoke. Furthermore, although we did not observe significant changes in the levels of most pro-inflammatory cytokines, the cytokine IL-8 and MCP-1 showed a significant decrease in the plasma of HIV-infected patients and smokers compared with HIV negative non-smokers. Importantly, HIV-infected individuals and smokers showed a significant increase in oxidative stress compared with HIV negative non-smoker subjects in both plasma and monocytes. To examine the possible pathways involved in increased oxidative stress and viral load, we determined the mRNA levels of several antioxidant and cytochrome P450 enzymes in monocytes. The results showed that the levels of most antioxidants are unaltered, suggesting their inability to counter oxidative stress. While CYP2A6 was induced in smokers, CYP3A4 was induced in HIV and HIV positive smokers compared with HIV negative non-smokers. Overall, the findings suggest

Effectiveness of etravirine-based therapy for treatment-experienced HIV-infected patients.

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Huerta García, Gloria; Mata-Marín, José Antonio; Domínguez-Hermosillo, Juan Carlos; Chavez-García, Marcelino; Banda-Lara, Marco Issac; Nuñez-Rodríguez, Nohemi; Cruz-Herrera, Javier Enrique; Sandoval-Ramírez, Jorge Luis; Villagómez-Ruiz, Alfredo; Manjarrez-Tellez, Bulmaro; Gaytan-Martínez, Jesús Enrique

2016-06-30

Treatment options are limited for HIV-1-infected individuals who have received extensive previous antiretroviral therapy. ETV has shown significant clinical benefits in treatment-experienced HIV-1+ patients with antiretroviral resistance. The aim of this study was to evaluate the effectiveness of ETV plus optimized background regimen in real-life conditions in a cohort of highly HIV-1 antiretroviral-experienced patients. Retrospective cohort of treatment-experienced HIV-1-infected adults with virological failure who started therapy with an ETV-containing regimen. The effectiveness was evaluated using HIV-1 RNA viral load and changes in CD4+ cell count after 48 weeks of treatment. Forty-two patients ≥ 16 years of age were included; 74% were men, and the median age was 45 years (IQR 41-53). All participants had prior non-nucleoside reverse transcriptase inhibitor use (55% nevirapine, 83%, efavirenz, and 28% both). Baseline median HIV-1 RNA viral load was 15,598 copies/mL (IQR 2651-84,175) and CD4+ cell count was 276 cells/mL (IQR 155-436). After 48 weeks of treatment, 90.5% (95% CI 78-96) of patients had HIV-1 RNA viral load treatment to a median of 407 cells/mL (IQR 242-579); p HIV-1 RNA viral load ≥ 100,000 copies/mL (OR 7.6; 95% CI 1.2-44.80; p = 0.025). Our study provides clinically important evidence of the effectiveness and safety of ETV in highly antiretroviral-experienced HIV-1-infected patients.

Unnecessary antiretroviral treatment switches and accumulation of HIV resistance mutations; two arguments for viral load monitoring in Africa.

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Sigaloff, Kim C E; Hamers, Raph L; Wallis, Carole L; Kityo, Cissy; Siwale, Margaret; Ive, Prudence; Botes, Mariette E; Mandaliya, Kishor; Wellington, Maureen; Osibogun, Akin; Stevens, Wendy S; van Vugt, Michèle; de Wit, Tobias F Rinke

2011-09-01

This study aimed to investigate the consequences of using clinicoimmunological criteria to detect antiretroviral treatment (ART) failure and guide regimen switches in HIV-infected adults in sub-Saharan Africa. Frequencies of unnecessary switches, patterns of HIV drug resistance, and risk factors for the accumulation of nucleoside reverse transcriptase inhibitor (NRTI)-associated mutations were evaluated. Cross-sectional analysis of adults switching ART regimens at 13 clinical sites in 6 African countries was performed. Two types of failure identification were compared: diagnosis of clinicoimmunological failure without viral load testing (CIF only) or CIF with local targeted viral load testing (targeted VL). After study enrollment, reference HIV RNA and genotype were determined retrospectively. Logistic regression assessed factors associated with multiple thymidine analogue mutations (TAMs) and NRTI cross-resistance (≥2 TAMs or Q151M or K65R/K70E). Of 250 patients with CIF switching to second-line ART, targeted VL was performed in 186. Unnecessary switch at reference HIV RNA <1000 copies per milliliter occurred in 46.9% of CIF only patients versus 12.4% of patients with targeted VL (P < 0.001). NRTI cross-resistance was observed in 48.0% of 183 specimens available for genotypic analysis, comprising ≥2 TAMs (37.7%), K65R (7.1%), K70E (3.3%), or Q151M (3.3%). The presence of NRTI cross-resistance was associated with the duration of ART exposure and zidovudine use. Clinicoimmunological monitoring without viral load testing resulted in frequent unnecessary regimen switches. Prolonged treatment failure was indicated by extensive NRTI cross-resistance. Access to virological monitoring should be expanded to prevent inappropriate switches, enable early failure detection and preserve second-line treatment options in Africa.

Herpes simplex virus type 2 (HSV-2) genital shedding in HSV-2-/HIV-1-co-infected women receiving effective combination antiretroviral therapy.

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Péré, Héléne; Rascanu, Aida; LeGoff, Jérome; Matta, Mathieu; Bois, Frédéric; Lortholary, Olivier; Leroy, Valériane; Launay, Odile; Bélec, Laurent

2016-03-01

The dynamics of genital shedding of HSV-2 DNA was assessed in HIV-1-infected women taking combination antiretroviral therapy (cART). HIV-1 RNA, HIV-1 DNA and HSV DNA loads were measured during 12-18 months using frozen plasma, PBMC and cervicovaginal lavage samples from 22 HIV-1-infected women, including 17 women naive for antiretroviral therapy initiating cART and 5 women with virological failure switching to a new regimen. Nineteen (86%) women were HSV-2-seropositive. Among HSV-2-/HIV-1-co-infected women, HIV-1 RNA loads showed a rapid fall from baseline after one month of cART, in parallel in paired plasma and cervicovaginal secretions. In contrast, HIV-1 DNA loads did not show significant variations from baseline up to 18 months of treatment in both systemic and genital compartments. HSV DNA was detected at least once in 12 (63%) of 19 women during follow up: HSV-2 shedding in the genital compartment was observed in 11% of cervicovaginal samples at baseline and in 16% after initiating or switching cART. Cervicovaginal HIV-1 RNA loads were strongly associated with plasma HIV-1 RNA loads over time, but not with cervicovaginal HSV DNA loads. Reactivation of genital HSV-2 replication frequently occurred despite effective cART in HSV-2-/HIV-1-co-infected women. Genital HSV-2 replication under cART does not influence cervicovaginal HIV-1 RNA or DNA shedding. © The Author(s) 2015.

Increasing cerebrospinal fluid chemokine concentrations despite undetectable cerebrospinal fluid HIV RNA in HIV-1-infected patients receiving antiretroviral therapy

NARCIS (Netherlands)

Gisolf, E. H.; van Praag, R. M.; Jurriaans, S.; Portegies, P.; Goudsmit, J.; Danner, S. A.; Lange, J. M.; Prins, J. M.

2000-01-01

Only limited data on cerebrospinal fluid (CSF) HIV-1 RNA responses and markers of local inflammation in CSF during antiretroviral therapy are available. HIV-RNA, soluble tumor necrosis factor (TNF)-receptor (sTNFr)-II, monocyte chemoattractant protein (MCP)-1, and interferon-gamma-inducible protein

RNA interactions in the 5' region of the HIV-1 genome

DEFF Research Database (Denmark)

Damgaard, Christian Kroun; Andersen, Ebbe Sloth; Knudsen, Bjarne

2004-01-01

The untranslated leader of the dimeric HIV-1 RNA genome is folded into a complex structure that plays multiple and essential roles in the viral replication cycle. Here, we have investigated secondary and tertiary structural elements within the 5' 744 nucleotides of the HIV-1 genome using...... a combination of bioinformatics, enzymatic probing, native gel electrophoresis, and UV-crosslinking experiments. We used a recently developed RNA folding algorithm (Pfold) to predict the common secondary structure of an alignment of 20 divergent HIV-1 sequences. Combining this analysis with biochemical data, we...

Full Viral Suppression, Low-Level Viremia, and Quantifiable Plasma HIV-RNA at the End of Pregnancy in HIV-Infected Women on Antiretroviral Treatment

OpenAIRE

Baroncelli, Silvia; Pirillo, Maria F.; Tamburrini, Enrica; Guaraldi, Giovanni; Pinnetti, Carmela; Antoni, Anna Degli; Galluzzo, Clementina M.; Stentarelli, Chiara; Amici, Roberta; Floridia, Marco

2015-01-01

There is limited information on full viral suppression and low-level HIV-RNA viremia in HIV-infected women at the end of pregnancy. We investigated HIV-RNA levels close to delivery in women on antiretroviral treatment in order to define rates of complete suppression, low-level viremia, and quantifiable HIV-RNA, exploring as potential determinants some clinical and viroimmunological variables. Plasma samples from a national study in Italy, collected between 2003 and 2012, were used. According ...

Relação entre diagnóstico citopatológico de neoplasia intra-epitelial cervical e índices de células CD4+ e de carga viral em pacientes HIV-soropositivas Association of cervical intraepithelial neoplasia with CD4 T cell counts and viral load in HIV-infected women

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Raquel Autran Coelho

2004-03-01

viral load in HIV-positive patients. METHODS: one hundred and fifteen HIV patients were evaluated retrospectively in the present study, during the period from January 2002 to April 2003, at a university hospital. Eighty-three patients presented cervical intraepithelial neoplasia (CIN in Pap smear, in comparison with thirty-two with no lesions. Patients were divided into three groups, according to CD4 counts: CD4 more than 500 cells/mm³, between 200 and 500 cells/mm³, and less than 200 cells/mm³, and other three groups, according to HIV viral load: less than 10,000 HIV-RNA copies/mL, between 10,000 and 100,000 HIV-RNA copies/mL, or more than 100,000 HIV-RNA copies/mL. Correlation was investigated by the Fisher test. RESULTS: of the eighty-three patients with CIN, 73% presented CD4 counts less than 500 cells/mm³. In all CD4 groups, more than 50% of the patients presented CIN. According to the viral load, 71.7% of the patients with less than 10,000 HIV-RNA copies/mL presented CIN I, compared with 11.3% that showed CIN III. In the group with higher viral load (>100.000 HIV-RNA copies/mL, 61.5% showed CIN I and 30.8% presented CIN III. CONCLUSION: association between viral load and CIN was established (p=0.013, which was not observed with CD4 cell counts and CIN. Concomitant cervicovaginal infection was considered a potential confounding factor.

Performance of the BioPlex 2200 HIV Ag-Ab assay for identifying acute HIV infection.

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Eshleman, Susan H; Piwowar-Manning, Estelle; Sivay, Mariya V; Debevec, Barbara; Veater, Stephanie; McKinstry, Laura; Bekker, Linda-Gail; Mannheimer, Sharon; Grant, Robert M; Chesney, Margaret A; Coates, Thomas J; Koblin, Beryl A; Fogel, Jessica M

Assays that detect HIV antigen (Ag) and antibody (Ab) can be used to screen for HIV infection. To compare the performance of the BioPlex 2200 HIV Ag-Ab assay and two other Ag/Ab combination assays for detection of acute HIV infection. Samples were obtained from 24 individuals (18 from the US, 6 from South Africa); these individuals were classified as having acute infection based on the following criteria: positive qualitative RNA assay; two negative rapid tests; negative discriminatory test. The samples were tested with the BioPlex assay, the ARCHITECT HIV Ag/Ab Combo test, the Bio-Rad GS HIV Combo Ag-Ab EIA test, and a viral load assay. Twelve (50.0%) of 24 samples had RNA detected only ( > 40 to 13,476 copies/mL). Ten (43.5%) samples had reactive results with all three Ag/Ab assays, one sample was reactive with the ARCHITECT and Bio-Rad assays, and one sample was reactive with the Bio-Rad and BioPlex assays. The 11 samples that were reactive with the BioPlex assay had viral loads from 83,010 to >750,000 copies/mL; 9/11 samples were classified as Ag positive/Ab negative by the BioPlex assay. Detection of acute HIV infection was similar for the BioPlex assay and two other Ag/Ab assays. All three tests were less sensitive than a qualitative RNA assay and only detected HIV Ag when the viral load was high. The BioPlex assay detected acute infection in about half of the cases, and identified most of those infections as Ag positive/Ab negative. Copyright © 2018 Elsevier B.V. All rights reserved.

HIV-1 RNAs are Not Part of the Argonaute 2 Associated RNA Interference Pathway in Macrophages.

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Valentina Vongrad

Full Text Available MiRNAs and other small noncoding RNAs (sncRNAs are key players in post-transcriptional gene regulation. HIV-1 derived small noncoding RNAs (sncRNAs have been described in HIV-1 infected cells, but their biological functions still remain to be elucidated. Here, we approached the question whether viral sncRNAs may play a role in the RNA interference (RNAi pathway or whether viral mRNAs are targeted by cellular miRNAs in human monocyte derived macrophages (MDM.The incorporation of viral sncRNAs and/or their target RNAs into RNA-induced silencing complex was investigated using photoactivatable ribonucleoside-induced cross-linking and immunoprecipitation (PAR-CLIP as well as high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP, which capture Argonaute2-bound miRNAs and their target RNAs. HIV-1 infected monocyte-derived macrophages (MDM were chosen as target cells, as they have previously been shown to express HIV-1 sncRNAs. In addition, we applied small RNA deep sequencing to study differential cellular miRNA expression in HIV-1 infected versus non-infected MDMs.PAR-CLIP and HITS-CLIP data demonstrated the absence of HIV-1 RNAs in Ago2-RISC, although the presence of a multitude of HIV-1 sncRNAs in HIV-1 infected MDMs was confirmed by small RNA sequencing. Small RNA sequencing revealed that 1.4% of all sncRNAs were of HIV-1 origin. However, neither HIV-1 derived sncRNAs nor putative HIV-1 target sequences incorporated into Ago2-RISC were identified suggesting that HIV-1 sncRNAs are not involved in the canonical RNAi pathway nor is HIV-1 targeted by this pathway in HIV-1 infected macrophages.

Antiretroviral treatment, viral load of mothers & perinatal HIV transmission in Mumbai, India

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Swati P Ahir

2013-01-01

Full Text Available Background & objectives: Mother-to-child transmission (MTCT is the most significant route of HIV transmission in children below the age of 15 yr. In India, perinatal HIV transmission, even after treatment, accounts for 5.4 per cent of HIV cases. The present study was conducted to evaluate the efficacy of anti-retro viral therapy (ART or prophylactic treatment (PT to control maternal viral load in HIV positive women, and its effect on vertical HIV transmission to their infants. Methods: A total of 58 HIV positive women were enrolled at the time of delivery and their plasma samples were obtained within 24 h of delivery for estimation of viral load. Viral load analysis was completed in 38 women. Infants received single dose nevirapine within 2 h of birth and zidovudine for 6 wk. At the end of 18 month follow up, HIV positive or negative status was available in 28 infants. Results: Results revealed undetectable levels of viral load in 58.3 per cent of women with ART compared to 30.7 per cent of women with PT. No women on ART had viral load more than 10,000 copies/ml, whereas seven (26.9%, P=0.07 women receiving PT had this viral load. Median CD4 count of women on PT (483 cells/μl was high compared to the women on ART (289 cells/ μl. At the end of 18 months follow up, only two children were HIV positive, whose mothers were on PT. One had in utero transmission; infection detected within 48 h of delivery, while the other child was infected post partum as HIV was detected at six months follow up. Interpretation & conclusions: Women who received a single dose of nevirapine during delivery had higher levels of viral load than women on ART. Combination drug therapy for pregnant women is now a standard of care in most of the western countries; use of nevirapine monotherapy at the time of delivery in our settings is not effective in controlling viral load. This highlights initiation of ART in pregnant women to control their viral load and thus to inhibit

Design and implementation of an external quality assessment program for HIV viral load measurements using dried blood spots.

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Prach, Lisa M; Puren, Adrian; Lippman, Sheri A; Carmona, Sergio; Stephenson, Sophie; Cutler, Ewalde; Barnhart, Scott; Liegler, Teri

2015-03-01

An external quality assurance program was developed for HIV-1 RNA viral load measurements taken from dried blood spots using a reference panel and field-collected specimens. The program demonstrated that accurate and reproducible quantitation can be obtained from field-collected specimens. Residual proviral DNA may confound interpretation in virologically suppressed subjects. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Viral protein Nef is detected in plasma of half of HIV-infected adults with undetectable plasma HIV RNA.

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Jana Ferdin

Full Text Available To address the role of translationally active HIV reservoir in chronic inflammation and non-AIDS related disorders, we first need a simple and accurate assay to evaluate viral protein expression in virally suppressed subjects.We optimized an HIV Nef enzyme-linked immunosorbent assay (ELISA and used it to quantify plasma Nef levels as an indicator of the leaky HIV reservoir in an HIV-infected cohort.This study accessed 134 plasma samples from a well-characterized cohort study of HIV-infected and uninfected adults in San Francisco (the SCOPE cohort. We optimized an ELISA for detection of plasma Nef in HIV-negative subjects and HIV-infected non-controllers, and evaluated its utility to quantify plasma Nef levels in a cross-sectional study of ART-suppressed and elite controller HIV-infected subjects.Here, we describe the performance of an optimized HIV Nef ELISA. When we applied this assay to the study cohort we found that plasma Nef levels were correlated with plasma HIV RNA levels in untreated disease. However, we were able to detect Nef in plasma of approximately half of subjects on ART or with elite control, despite the lack of detectable plasma HIV RNA levels using standard assays. Plasma Nef levels were not consistently associated with CD4+ T-cell count, CD8+ T-cell count, self-reported nadir CD4+ T-cell count or the CD4+/CD8+ T-cell ratio in HIV-infected subjects.Since plasma HIV RNA levels are undetectable in virally suppressed subjects, it is reasonable to assume that viral protein expression in leaky reservoir, and not plasma virions, is the source of Nef accumulating in plasma. To examine this further, improvements of the assay sensitivity, by lowering the background through improvements in the quality of Nef antibodies, and detailed characterization of the HIV reservoirs are needed.

Chimeric peptide-mediated siRNA transduction to inhibit HIV-1 infection.

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Bivalkar-Mehla, Shalmali; Mehla, Rajeev; Chauhan, Ashok

2017-04-01

Persistent human immunodeficiency virus 1 (HIV-1) infection provokes immune activation and depletes CD4 +  lymphocytes, leading to acquired immunodeficiency syndrome. Uninterrupted administration of combination antiretroviral therapy (cART) in HIV-infected patients suppresses viral replication to below the detectable level and partially restores the immune system. However, cART-unresponsive residual HIV-1 infection and elusive transcriptionally silent but reactivatable viral reservoirs maintain a permanent viral DNA blue print. The virus rebounds within a few weeks after interruption of suppressive therapy. Adjunct gene therapy to control viral replication by ribonucleic acid interference (RNAi) is a post-transcriptional gene silencing strategy that could suppress residual HIV-1 burden and overcome viral resistance. Small interfering ribonucleic acids (siRNAs) are efficient transcriptional inhibitors, but need delivery systems to reach inside target cells. We investigated the potential of chimeric peptide (FP-PTD) to deliver specific siRNAs to HIV-1-susceptible and permissive cells. Chimeric FP-PTD peptide was designed with an RNA binding domain (PTD) to bind siRNA and a cell fusion peptide domain (FP) to enter cells. FP-PTD-siRNA complex entered and inhibited HIV-1 replication in susceptible cells, and could be a candidate for in vivo testing.

Acute HIV Infection in Pregnancy: The Case for Third Trimester Rescreening

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Jocelyn Wertz

2011-01-01

Full Text Available Combination testing with anti-HIV Elisa and Western blot is both sensitive and specific for diagnosis of established HIV-1 infection but could not detect acute HIV infection (AHI. AHI is a time of extremely high viral load, which may correlate to increased risk of horizontal or vertical transmission. Thus, early identification of AHI could allow for interventions to decrease transmission. However, recognition of AHI can be challenging as symptoms could be absent or nonspecific, therefore, AHI is often not detected, particularly in pregnancy. We present a case report of AHI in a pregnant woman who presented with headache and fever. She tested negative for HIV in the first trimester and at time of AHI at 26 3/7 weeks by anti-HIV Elisa, but was diagnosed with AHI based on an HIV RNA viral load of 434,000 copies/mL. This report presents a case for improved awareness of AHI in pregnancy, and the need for repeat HIV testing in late pregnancy, and highlighted that early detection of AHI might be possible with adding HIV RNA testing at time of standard anti-HIV Elisa screening test in pregnancy. Novel laboratory approaches including pooling of sera for HIV RNA could reduce the cost of HIV RNA testing.

HIV Infection Status as a Predictor of Hepatitis C Virus RNA Testing in Primary Care

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Yartel, Anthony K.; Morgan, Rebecca L.; Rein, David B.; Brown, Kimberly Ann; Kil, Natalie B.; Massoud, Omar I.; Fallon, Michael B.; Smith, Bryce D.

2015-01-01

Introduction Receipt of hepatitis C virus (HCV) RNA testing following a positive HCV antibody (anti-HCV+) test result to establish current infection is a quality indicator for HCV-related care. This study examines HIV infection status as a predictor of HCV RNA test receipt after an anti-HCV+ result in the primary care setting. Methods Electronic medical records of anti-HCV+ patients from a multisite retrospective study of patients aged ≥18 years who utilized one or more primary care outpatient services during 2005–2010 were analyzed in 2014. A multivariable logistic regression model examined the independent relationships between patient characteristics and receipt of HCV RNA testing. Results Among 1,115 anti-HCV+ patients, 133 (11.9%) were also HIV-positive. Of these, 77.4% (n=103) underwent HCV RNA testing to determine current infection status. By contrast, 66.7% (n=654/980) of anti-HCV+ patients who were HIV-negative received HCV RNA testing. Following multivariable adjustment, the odds of receiving HCV RNA testing were higher among anti-HCV+ patients who were also HIV-positive (AOR=1.9, 95% CI=1.2, 3.0), compared with their HIV-negative counterparts. Elevated alanine aminotransferase level was also associated with receipt of HCV RNA testing (AOR=1.9, 95% CI=1.4, 2.4). Black race was associated with decreased odds of receiving HCV RNA testing (AOR=0.7, 95% CI=0.5, 1.0). Conclusions HIV infection status is independently associated with the likelihood of receiving HCV RNA testing following an anti-HCV+ result. One quarter of anti-HCV+ patients who were also HIV-positive and one third of their HIV-negative counterparts, respectively, did not receive testing to establish active HCV infection, which is imperative for appropriate care and treatment. PMID:25896194

The HIV-1 Rev/RRE system is required for HIV-1 5' UTR cis elements to augment encapsidation of heterologous RNA into HIV-1 viral particles

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Ma Hong

2011-06-01

Full Text Available Abstract Background The process of HIV-1 genomic RNA (gRNA encapsidation is governed by a number of viral encoded components, most notably the Gag protein and gRNA cis elements in the canonical packaging signal (ψ. Also implicated in encapsidation are cis determinants in the R, U5, and PBS (primer binding site from the 5' untranslated region (UTR. Although conventionally associated with nuclear export of HIV-1 RNA, there is a burgeoning role for the Rev/RRE in the encapsidation process. Pleiotropic effects exhibited by these cis and trans viral components may confound the ability to examine their independent, and combined, impact on encapsidation of RNA into HIV-1 viral particles in their innate viral context. We systematically reconstructed the HIV-1 packaging system in the context of a heterologous murine leukemia virus (MLV vector RNA to elucidate a mechanism in which the Rev/RRE system is central to achieving efficient and specific encapsidation into HIV-1 viral particles. Results We show for the first time that the Rev/RRE system can augment RNA encapsidation independent of all cis elements from the 5' UTR (R, U5, PBS, and ψ. Incorporation of all the 5' UTR cis elements did not enhance RNA encapsidation in the absence of the Rev/RRE system. In fact, we demonstrate that the Rev/RRE system is required for specific and efficient encapsidation commonly associated with the canonical packaging signal. The mechanism of Rev/RRE-mediated encapsidation is not a general phenomenon, since the combination of the Rev/RRE system and 5' UTR cis elements did not enhance encapsidation into MLV-derived viral particles. Lastly, we show that heterologous MLV RNAs conform to transduction properties commonly associated with HIV-1 viral particles, including in vivo transduction of non-dividing cells (i.e. mouse neurons; however, the cDNA forms are episomes predominantly in the 1-LTR circle form. Conclusions Premised on encapsidation of a heterologous RNA into

Marcadores virológicos no convencionales en pacientes infectados con el virus de la inmunodeficiencia humana: ADN HIV-T, ADN HIV- 2LTR y ARN de HIV Non conventional virological markers in HIV-infected patients: T-HIV DNA, 2LTR-HIV DNA and HIV RNA

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Rosana Gariglio

2004-10-01

Full Text Available La terapia antirretroviral de alta eficacia (TAAE induce una reducción marcada y persistente de la viremia plasmática, contribuyendo a disminuir la mortalidad y morbilidad de los pacientes HIV-positivos. Así, la carga viral (CV es el método de referencia para evaluar la eficacia terapéutica. Sin embargo, aun en presencia de una TAAE eficiente no se ha logrado la erradicación viral. En este estudio analizamos la presencia del ADN total de HIV (ADN HIV-T, del ADN no integrado con 2LTR (ADN HIV-2LTR y del ARN de HIV, en un grupo de 55 pacientes HIV-positivos en distintos estadios clínicos, con y sin TAAE, mediante ensayos de PCR con revelado colorimétrico en microplaca, optimizados en nuestro laboratorio. La sensibilidad clínica del ARN del HIV fue evaluada con el bDNA, resultando del 74% y del 64%, respectivamente, con una concordancia del 85%. Este ensayo podría ser utilizado en el seguimiento de pacientes bajo TAAE. El ADN HIV-2LTR resultó positivo en el 54% aunque estuvo ausente en pacientes con elevada CV. Este marcador se consideraba un producto lábil y su presencia se asociaba a infección reciente. Sin embargo, actuales evidencias ponen en discusión su estabilidad por lo que su significado clínico debe ser reconsiderado. La ausencia del ADN HIV-2LTR en pacientes con CV detectable puede relacionarse con la heterogeneidad de la secuencia utilizada para su detección. El ADN HIV-T estuvo presente en el 100% de las muestras y resultaría relevante como marcador de remisión cuando se dispongan de terapias que efectivamente erradiquen la infección.Highly active antiretroviral therapy (HAART induces a persistent reduction of the plasmatic viremia, contributing to decrease mortality and morbidity of infected people with human immunodeficiency virus (HIV. Thus, viral load (VL is the reference method to evaluate therapy effectiveness. However, even in the presence of efficient HAART viral eradication was yet not achieved. In this

HIV RNA Suppression during and after Pregnancy among Women in the HIV Outpatient Study, 1996 to 2015.

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Patel, Monita; Tedaldi, Ellen; Armon, Carl; Nesheim, Steven; Lampe, Margaret; Palella, Frank; Novak, Richard; Sutton, Madeline; Buchacz, Kate

2018-01-01

To examine HIV viral suppression during/after pregnancy. Prospective observational cohort. We identified pregnancies from 1996 to 2015. We examined HIV RNA viral load (VL), VL suppression (≤500 copies/mL), and antiretroviral therapy (ART) status at pregnancy start, end, and 6 months postpartum. We estimated risk ratios (RRs) and 95% confidence intervals (CIs) for VL nonsuppression. Among 253 pregnancies analyzed, 34.8% of women exhibited VL suppression at pregnancy start, 60.1% at pregnancy end, and 42.7% at 6 months postpartum. Median VL (log 10 copies/mL) was 2.80 (interquartile range [IQR]: 1.40-3.85) at pregnancy start, 1.70 (IQR: 1.40-2.82) at pregnancy end, and 2.30 (IQR: 1.40-3.86) at postpartum. Risk of postpartum VL nonsuppression was also lower among women on ART and with VL suppression at pregnancy end (versus those not; adjusted RR = 0.30, 95% CI: 0.17-0.53). Maintaining VL suppression among US women remains a challenge, particularly during postpartum. Achieving VL suppression earlier during pregnancy benefits women subsequently.

Hepatitis C virus (HCV) RNA profiles among chronic HIV/HCV-coinfected individuals in ESPRIT; spontaneous HCV RNA clearance observed in nine individuals.

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Grint, D; Tedaldi, E; Peters, L; Mocroft, A; Edlin, B; Gallien, S; Klinker, H; Boesecke, C; Kokordelis, P; Rockstroh, J K

2017-07-01

Studies have shown that hepatitis C virus (HCV) RNA levels remain stable over time in HIV/HCV-coinfected individuals taking combination antiretroviral therapy (cART), while spontaneous clearance of HCV RNA during the persistent infection phase has been documented only rarely among those with the CC interleukin (IL)-28B genotype. This study describes HCV RNA profiles and factors associated with changes over time in HCV RNA levels in the ESPRIT study. HIV/HCV-coinfected individuals positive for HCV RNA were included in the study. Follow-up was counted from the first HCV RNA positive test and censored at the initiation of interferon-based treatment. HCV RNA and IL-28B measurements were performed in the same reference laboratory. Random effects mixed models were used to analyse changes over time in HCV RNA. A total of 312 ESPRIT patients were included in the study (151 in the arm receiving subcutaneous recombinant IL-2 and 161 in the control arm). Most of the patients were white (89%) and male (76%), and they had a median of 5 HCV RNA measurements per person [interquartile range (IQR) 3-6; range 1-9]. Median follow-up was 5 years (IQR: 2-6 years). At baseline, 96% of patients were taking cART and 93% had undetectable HIV RNA. Mean HCV RNA levels decreased by 13% per year over the study period [95% confidence interval (CI) 8-18%; P < 0.0001]. Baseline HCV RNA levels and the change over time in HCV RNA did not differ by randomization arm (P = 0.16 and P = 0.56, respectively). Nine individuals spontaneously cleared HCV RNA during follow-up [IL-28B genotypes: CC, five patients (56%); CT, four patients (44%)]. HCV RNA levels decreased over time in this population with well-controlled HIV infection. Spontaneous clearance of HCV RNA was documented in five individuals with IL-28B genotype CC and four with the CT genotype. © 2016 British HIV Association.

RRE-dependent HIV-1 Env RNA effects on Gag protein expression, assembly and release

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López, Claudia S.; Sloan, Rachel; Cylinder, Isabel; Kozak, Susan L.; Kabat, David; Barklis, Eric

2014-01-01

The HIV-1 Gag proteins are translated from the full-length HIV-1 viral RNA (vRNA), whereas the envelope (Env) protein is translated from incompletely spliced Env mRNAs. Nuclear export of vRNAs and Env mRNAs is mediated by the Rev accessory protein which binds to the rev-responsive element (RRE) present on these RNAs. Evidence has shown there is a direct or indirect interaction between the Gag protein, and the cytoplasmic tail (CT) of the Env protein. Our current work shows that env gene expression impacts HIV-1 Gag expression and function in two ways. At the protein level, full-length Env expression altered Gag protein expression, while Env CT-deletion proteins did not. At the RNA level, RRE-containing Env mRNA expression reduced Gag expression, processing, and virus particle release from cells. Our results support models in which Gag is influenced by the Env CT, and Env mRNAs compete with vRNAs for nuclear export. - Highlights: • At the protein level, full-length HIV-1 Env alters Gag protein expression. • HIV-1 Env RNA expression reduces Gag levels and virus release. • Env RNA effects on Gag are dependent on the RRE. • RRE-containing Env RNAs compete with vRNAs for nuclear export

Detection of HIV and HCV RNA in semen from Brazilian coinfected men using multiplex PCR before and after semen washing Detecção do RNA do HIV e HCV em sêmen de homens brasileiros, usando PCR multiplex antes e depois do "semen washing"

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Cynthia Liliane Motta do Canto

2006-08-01

Full Text Available INTRODUCTION: Prolonged survival of patients under HAART has resulted in new demands for assisted reproductive technologies. HIV serodiscordant couples wish to make use of assisted reproduction techniques in order to avoid viral transmission to the partner or to the newborn. It is therefore essential to test the effectiveness of techniques aimed at reducing HIV and HCV loads in infected semen using molecular biology tests. METHODS: After seminal analysis, semen samples from 20 coinfected patients were submitted to cell fractioning and isolation of motile spermatozoa by density gradient centrifugation and swim-up. HIV and HCV RNA detection tests were performed with RNA obtained from sperm, seminal plasma and total semen. RESULTS: In pre-washing semen, HIV RNA was detected in 100% of total semen samples, whereas HCV RNA was concomitantly amplified in only one specimen. Neither HIV nor HCV were detected either in the swim-up or in the post-washing semen fractions. CONCLUSIONS: Reduction of HIV and/or HCV shedding in semen by density gradient centrifugation followed by swim-up is an efficient method. These findings lead us to believe that, although semen is rarely found to contain HCV, semen processing is highly beneficial for HIV/HCV coinfected individuals.O aumento da sobrevida dos pacientes que utilizam terapêutica antiretroviral altamente eficaz (HAART- Highly Active Antiretroviral Therapy trouxe uma nova demanda de casais sorodiscordantes que desejam filhos. Como esses casais não podem abandonar o uso de preservativos, torna-se indispensável tratar o sêmen infectado com técnicas laboratoriais eficazes que além de isolar os melhores espermatozóides, reduzam a carga viral do HIV e HCV a níveis indetectáveis. Para isso, são utilizadas técnicas de semen washing, associadas a testes ultra sensíveis de biologia molecular. Após análise seminal, sêmen de 20 pacientes co-infectados HIV-HCV foram submetidos a fracionamento celular e

Promiscuous RNA binding ensures effective encapsidation of APOBEC3 proteins by HIV-1.

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Luis Apolonia

2015-01-01

Full Text Available The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3 proteins are cell-encoded cytidine deaminases, some of which, such as APOBEC3G (A3G and APOBEC3F (A3F, act as potent human immunodeficiency virus type-1 (HIV-1 restriction factors. These proteins require packaging into HIV-1 particles to exert their antiviral activities, but the molecular mechanism by which this occurs is incompletely understood. The nucleocapsid (NC region of HIV-1 Gag is required for efficient incorporation of A3G and A3F, and the interaction between A3G and NC has previously been shown to be RNA-dependent. Here, we address this issue in detail by first determining which RNAs are able to bind to A3G and A3F in HV-1 infected cells, as well as in cell-free virions, using the unbiased individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP method. We show that A3G and A3F bind many different types of RNA, including HIV-1 RNA, cellular mRNAs and small non-coding RNAs such as the Y or 7SL RNAs. Interestingly, A3G/F incorporation is unaffected when the levels of packaged HIV-1 genomic RNA (gRNA and 7SL RNA are reduced, implying that these RNAs are not essential for efficient A3G/F packaging. Confirming earlier work, HIV-1 particles formed with Gag lacking the NC domain (Gag ΔNC fail to encapsidate A3G/F. Here, we exploit this system by demonstrating that the addition of an assortment of heterologous RNA-binding proteins and domains to Gag ΔNC efficiently restored A3G/F packaging, indicating that A3G and A3F have the ability to engage multiple RNAs to ensure viral encapsidation. We propose that the rather indiscriminate RNA binding characteristics of A3G and A3F promote functionality by enabling recruitment into a wide range of retroviral particles whose packaged RNA genomes comprise divergent sequences.

Cis elements and trans-acting factors involved in the RNA dimerization of the human immunodeficiency virus HIV-1.

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Darlix, J L; Gabus, C; Nugeyre, M T; Clavel, F; Barré-Sinoussi, F

1990-12-05

The retroviral genome consists of two identical RNA molecules joined at their 5' ends by the Dimer Linkage Structure (DLS). To study the mechanism of dimerization and the DLS of HIV-1 RNA, large amounts of bona fide HIV-1 RNA and of mutants have been synthesized in vitro. We report that HIV-1 RNA forms dimeric molecules and that viral nucleocapsid (NC) protein NCp15 greatly activates dimerization. Deletion mutagenesis in the RNA 5' 1333 nucleotides indicated that a small domain of 100 nucleotides, located between positions 311 to 415 from the 5' end, is necessary and sufficient to promote HIV-1 RNA dimerization. This dimerization domain encompasses an encapsidation element located between the 5' splice donor site and initiator AUG of gag and shows little sequence variations in different strains of HIV-1. Furthermore, cross-linking analysis of the interactions between NC and HIV-1 RNA (311 to 415) locates a major contact site in the encapsidation element of HIV-1 RNA. The genomic RNA dimer is tightly associated with nucleocapsid protein molecules in avian and murine retroviruses, and this ribonucleoprotein structure is believed to be the template for reverse transcription. Genomic RNA-protein interactions have been analyzed in human immunodeficiency virus (HIV) virions and results showed that NC protein molecules are tightly bound to the genomic RNA dimer. Since retroviral RNA dimerization and packaging appear to be under the control of the same cis element, the encapsidation sequences, and trans-acting factor, the NC protein, they are probably related events in the course of virion assembly.

Expression of Herpes Simplex Virus Thymidine Kinase/Ganciclovir by RNA Trans-Splicing Induces Selective Killing of HIV-Producing Cells

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Carin K. Ingemarsdotter

2017-06-01

Full Text Available Antiviral strategies targeting hijacked cellular processes are less easily evaded by the virus than viral targets. If selective for viral functions, they can have a high therapeutic index. We used RNA trans-splicing to deliver the herpes simplex virus thymidine kinase-ganciclovir (HSV-tk/GCV cell suicide system into HIV-producing cells. Using an extensive in silico bioinformatics and RNA structural analysis approach, ten HIV RNA trans-splicing constructs were designed targeting eight different HIV splice donor or acceptor sites and were tested in cells expressing HIV. Trans-spliced mRNAs were identified in HIV-expressing cells using qRT-PCR with successful detection of fusion RNA transcripts between HIV RNA and the HSV-tk RNA transcripts from six of ten candidate RNA trans-splicing constructs. Conventional PCR and Sanger sequencing confirmed RNA trans-splicing junctions. Measuring cell viability in the presence or absence of GCV expression of HSV-tk by RNA trans-splicing led to selective killing of HIV-producing cells using either 3′ exon replacement or 5′ exon replacement in the presence of GCV. Five constructs targeting four HIV splice donor and acceptor sites, D4, A5, A7, and A8, involved in regulating the generation of multiple HIV RNA transcripts proved to be effective for trans-splicing mediated selective killing of HIV-infected cells, within which individual constructs targeting D4 and A8 were the most efficient.

A Pilot Study to Increase the Efficiency of HIV Outreach Testing Through the Use of Timely and Geolocated HIV Viral Load Surveillance Data

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Jennings, Jacky M.; Schumacher, Christina; Perin, Jamie; Myers, Tanya; Fields, Nathan; Greiner Safi, Amelia; Chaulk, Patrick

2018-01-01

Background Eliminating HIV transmission in a population necessitates identifying population reservoirs of HIV infection and subgroups most likely to transmit. HIV viral load is the single most important predictor of HIV transmission. The objective of this analysis was to evaluate whether a public health practice pilot project based on community viral load resulted in increases in the proportion of time spent testing in high viral load areas (process measure) and 3 outcome measures—the number and percent of overall HIV diagnoses, new diagnoses, and high viral load positives—in one mid-Atlantic US city with a severe HIV epidemic. Methods The evaluation was conducted during three, 3-month periods for 3 years and included the use of community viral load, global positioning system tracking data, and statistical testing to evaluate the effectiveness of the pilot project. Results The proportion of time spent outreach testing in high viral load areas (69%–84%, P the overall number and percent of HIV positives ((60 (3%) to 127 (6%), P The number and percent of new diagnoses (3 (0.1%) to 6 (0.2%)) and high viral load positives (5 (0.2%) to 9 (0.4%)) increased, but the numbers were too small for statistical testing. Discussion These results suggest that using community viral load to increase the efficiency of HIV outreach testing is feasible and may be effective in identifying more HIV positives. The pilot project provides a model for other public health practice demonstration projects. PMID:29420450

HIV-1 matrix dependent membrane targeting is regulated by Gag mRNA trafficking.

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Jing Jin

Full Text Available Retroviral Gag polyproteins are necessary and sufficient for virus budding. Productive HIV-1 Gag assembly takes place at the plasma membrane. However, little is known about the mechanisms by which thousands of Gag molecules are targeted to the plasma membrane. Using a bimolecular fluorescence complementation (BiFC assay, we recently reported that the cellular sites and efficiency of HIV-1 Gag assembly depend on the precise pathway of Gag mRNA export from the nucleus, known to be mediated by Rev. Here we describe an assembly deficiency in human cells for HIV Gag whose expression depends on hepatitis B virus (HBV post-transcriptional regulatory element (PRE mediated-mRNA nuclear export. PRE-dependent HIV Gag expressed well in human cells, but assembled with slower kinetics, accumulated intracellularly, and failed to associate with a lipid raft compartment where the wild-type Rev-dependent HIV-1 Gag efficiently assembles. Surprisingly, assembly and budding of PRE-dependent HIV Gag in human cells could be rescued in trans by co-expression of Rev-dependent Gag that provides correct membrane targeting signals, or in cis by replacing HIV matrix (MA with other membrane targeting domains. Taken together, our results demonstrate deficient membrane targeting of PRE-dependent HIV-1 Gag and suggest that HIV MA function is regulated by the trafficking pathway of the encoding mRNA.

Relationship between hunger, adherence to antiretroviral therapy and plasma HIV RNA suppression among HIV-positive illicit drug users in a Canadian setting.

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Anema, Aranka; Kerr, Thomas; Milloy, M-J; Feng, Cindy; Montaner, Julio S G; Wood, Evan

2014-04-01

Food insecurity may be a barrier to achieving optimal HIV treatment-related outcomes among illicit drug users. This study therefore, aimed to assess the impact of severe food insecurity, or hunger, on plasma HIV RNA suppression among illicit drug users receiving antiretroviral therapy (ART). A cross-sectional Multivariate logistic regression model was used to assess the potential relationship between hunger and plasma HIV RNA suppression. A sample of n = 406 adults was derived from a community-recruited open prospective cohort of HIV-positive illicit drug users, in Vancouver, British Columbia (BC), Canada. A total of 235 (63.7%) reported "being hungry and unable to afford enough food," and 241 (59.4%) had plasma HIV RNA hunger was associated with lower odds of plasma HIV RNA suppression (Odds Ratio = 0.59, 95% confidence interval [CI]: 0.39-0.90, p = 0.015). In multivariate analyses, this association was no longer significant after controlling for socio-demographic, behavioral, and clinical characteristics, including 95% adherence (Adjusted Odds Ratio [AOR] = 0.65, 95% CI: 0.37-1.10, p = 0.105). Multivariate models stratified by 95% adherence found that the direction and magnitude of this association was not significantly altered by the adherence level. Hunger was common among illicit drug users in this setting. Although, there was an association between hunger and lower likelihood of plasma HIV RNA suppression, this did not persist in adjusted analyses. Further research is warranted to understand the social-structural, policy, and physical factors shaping the HIV outcomes of illicit drug users.

Deciphering the role of the Gag-Pol ribosomal frameshift signal in HIV-1 RNA genome packaging.

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Nikolaitchik, Olga A; Hu, Wei-Shau

2014-04-01

A key step of retroviral replication is packaging of the viral RNA genome during virus assembly. Specific packaging is mediated by interactions between the viral protein Gag and elements in the viral RNA genome. In HIV-1, similar to most retroviruses, the packaging signal is located within the 5' untranslated region and extends into the gag-coding region. A recent study reported that a region including the Gag-Pol ribosomal frameshift signal plays an important role in HIV-1 RNA packaging; deletions or mutations that affect the RNA structure of this signal lead to drastic decreases (10- to 50-fold) in viral RNA packaging and virus titer. We examined here the role of the ribosomal frameshift signal in HIV-1 RNA packaging by studying the RNA packaging and virus titer in the context of proviruses. Three mutants with altered ribosomal frameshift signal, either through direct deletion of the signal, mutation of the 6U slippery sequence, or alterations of the secondary structure were examined. We found that RNAs from all three mutants were packaged efficiently, and they generate titers similar to that of a virus containing the wild-type ribosomal frameshift signal. We conclude that although the ribosomal frameshift signal plays an important role in regulating the replication cycle, this RNA element is not directly involved in regulating RNA encapsidation. To generate infectious viruses, HIV-1 must package viral RNA genome during virus assembly. The specific HIV-1 genome packaging is mediated by interactions between the structural protein Gag and elements near the 5' end of the viral RNA known as packaging signal. In this study, we examined whether the Gag-Pol ribosomal frameshift signal is important for HIV-1 RNA packaging as recently reported. Our results demonstrated that when Gag/Gag-Pol is supplied in trans, none of the tested ribosomal frameshift signal mutants has defects in RNA packaging or virus titer. These studies provide important information on how HIV-1

A predominance of R5-like HIV genotypes in vaginal secretions is associated with elevated plasma HIV-1 RNA levels and the absence of anti-retroviral therapy

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Lacour Nedra

2008-07-01

Full Text Available Abstract HIV expressed in genital secretions provides the inoculum from which transmitting variants are selected, both in sexual transmission and mother-to-infant transmission during partuition. Characterization of HIV levels and genotypes found in vaginal secretions and the impact of anti-retroviral therapy (ART on this virus can provide valuable insight for the prevention of HIV transmission. Vaginal HIV was evaluated in a cohort of 43 women attending a New Orleans HIV outpatient clinic. Predominant vaginal genotypes were characterized as R5- or X4-like by heteroduplex tracking analyses of the envelope V3 region. Most women (67.4% shed R5-like genotypes in vaginal secretions which was associated with elevated plasma HIV levels (≥ 10,000 copies HIV-RNA/mL and absence of ART. Because R5-like genotypes are more frequently associated with transmission, these observations suggest that the majority of women shedding HIV in genital secretions present a transmission risk. The levels of vaginal virus were similar between both groups, but shedding of X4-like genotypes was associated with lower plasma viral loads and the use of ART, suggesting that ART use may impact the genotypes of virus found in the female genital compartment.

Validation of Performance of the Gen-Probe Human Immunodeficiency Virus Type 1 Viral Load Assay with Genital Swabs and Breast Milk Samples

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DeVange Panteleeff, Dana; Emery, Sandra; Richardson, Barbra A.; Rousseau, Christine; Benki, Sarah; Bodrug, Sharon; Kreiss, Joan K.; Overbaugh, Julie

2002-01-01

Human immunodeficiency type 1 (HIV-1) continues to spread at an alarming rate. The virus may be transmitted through blood, genital secretions, and breast milk, and higher levels of systemic virus in the index case, as measured by plasma RNA viral load, have been shown to correlate with increased risk of transmitting HIV-1 both vertically and sexually. Less is known about the correlation between transmission and HIV-1 levels in breast milk or genital secretions, in part because reliable quantitative assays to detect HIV-1 in these fluids are not available. Here we show that the Gen-Probe HIV-1 viral load assay can be used to accurately quantify viral load in expressed breast milk and in cervical and vaginal samples collected on swabs. Virus could be quantified from breast milk and swab samples spiked with known amounts of virus, including HIV-1 subtypes A, C, and D. As few as 10 copies of HIV-1 RNA could be detected above background threshold levels in ≥77% of assays performed with spiked breast milk supernatants and mock swabs. In genital swab samples from HIV-1-infected women, similar levels of HIV-1 RNA were consistently detected in duplicate swabs taken from the same woman on the same clinic visit, suggesting that the RNA values from a single swab sample can be used to measure genital viral load. PMID:12409354

Seminal Plasma HIV-1 RNA Concentration Is Strongly Associated with Altered Levels of Seminal Plasma Interferon-γ, Interleukin-17, and Interleukin-5

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Hoffman, Jennifer C.; Anton, Peter A.; Baldwin, Gayle Cocita; Elliott, Julie; Anisman-Posner, Deborah; Tanner, Karen; Grogan, Tristan; Elashoff, David; Sugar, Catherine; Yang, Otto O.

2014-01-01

Abstract Seminal plasma HIV-1 RNA level is an important determinant of the risk of HIV-1 sexual transmission. We investigated potential associations between seminal plasma cytokine levels and viral concentration in the seminal plasma of HIV-1-infected men. This was a prospective, observational study of paired blood and semen samples from 18 HIV-1 chronically infected men off antiretroviral therapy. HIV-1 RNA levels and cytokine levels in seminal plasma and blood plasma were measured and analyzed using simple linear regressions to screen for associations between cytokines and seminal plasma HIV-1 levels. Forward stepwise regression was performed to construct the final multivariate model. The median HIV-1 RNA concentrations were 4.42 log10 copies/ml (IQR 2.98, 4.70) and 2.96 log10 copies/ml (IQR 2, 4.18) in blood and seminal plasma, respectively. In stepwise multivariate linear regression analysis, blood HIV-1 RNA level (pplasma HIV-1 RNA level. After controlling for blood HIV-1 RNA level, seminal plasma HIV-1 RNA level was positively associated with interferon (IFN)-γ (p=0.03) and interleukin (IL)-17 (p=0.03) and negatively associated with IL-5 (p=0.0007) in seminal plasma. In addition to blood HIV-1 RNA level, cytokine profiles in the male genital tract are associated with HIV-1 RNA levels in semen. The Th1 and Th17 cytokines IFN-γ and IL-17 are associated with increased seminal plasma HIV-1 RNA, while the Th2 cytokine IL-5 is associated with decreased seminal plasma HIV-1 RNA. These results support the importance of genital tract immunomodulation in HIV-1 transmission. PMID:25209674

A Conserved Target Site in HIV-1 Gag RNA is Accessible to Inhibition by Both an HDV Ribozyme and a Short Hairpin RNA

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Robert J Scarborough

2014-01-01

Full Text Available Antisense-based molecules targeting HIV-1 RNA have the potential to be used as part of gene or drug therapy to treat HIV-1 infection. In this study, HIV-1 RNA was screened to identify more conserved and accessible target sites for ribozymes based on the hepatitis delta virus motif. Using a quantitative screen for effects on HIV-1 production, we identified a ribozyme targeting a highly conserved site in the Gag coding sequence with improved inhibitory potential compared to our previously described candidates targeting the overlapping Tat/Rev coding sequence. We also demonstrate that this target site is highly accessible to short hairpin directed RNA interference, suggesting that it may be available for the binding of antisense RNAs with different modes of action. We provide evidence that this target site is structurally conserved in diverse viral strains and that it is sufficiently different from the human transcriptome to limit off-target effects from antisense therapies. We also show that the modified hepatitis delta virus ribozyme is more sensitive to a mismatch in its target site compared to the short hairpin RNA. Overall, our results validate the potential of a new target site in HIV-1 RNA to be used for the development of antisense therapies.

Interleukin-27 is differentially associated with HIV viral load and CD4+ T cell counts in therapy-naive HIV-mono-infected and HIV/HCV-co-infected Chinese.

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Lai He

Full Text Available Human Immunodeficiency Virus (HIV infection and the resultant Acquired Immunodeficiency Syndrome (AIDS epidemic are major global health challenges; hepatitis C virus (HCV co-infection has made the HIV/AIDS epidemic even worse. Interleukin-27 (IL-27, a cytokine which inhibits HIV and HCV replication in vitro, associates with HIV infection and HIV/HCV co-infection in clinical settings. However, the impact of HIV and HCV viral loads on plasma IL-27 expression levels has not been well characterized. In this study, 155 antiretroviral therapy-naïve Chinese were recruited. Among them 80 were HIV- and HCV-negative healthy controls, 45 were HIV-mono-infected and 30 were HIV/HCV-co-infected. Plasma level HIV, HCV, IL-27 and CD4+ number were counted and their correlation, regression relationships were explored. We show that: plasma IL-27 level was significantly upregulated in HIV-mono-infected and HIV/HCV-co-infected Chinese; HIV viral load was negatively correlated with IL-27 titer in HIV-mono-infected subjects whereas the relationship was opposite in HIV/HCV-co-infected subjects; and the relationships between HIV viral loads, IL-27 titers and CD4+ T cell counts in the HIV mono-infection and HIV/HCV co-infection groups were dramatically different. Overall, our results suggest that IL-27 differs in treatment-naïve groups with HIV mono-infections and HIV/HCV co-infections, thereby providing critical information to be considered when caring and treating those with HIV mono-infection and HIV/HCV co-infection.

Development of antibody-modified chitosan nanoparticles for the targeted delivery of siRNA across the blood-brain barrier as a strategy for inhibiting HIV replication in astrocytes.

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Gu, Jijin; Al-Bayati, Karam; Ho, Emmanuel A

2017-08-01

RNA interference (RNAi)-mediated gene silencing offers a novel treatment and prevention strategy for human immunodeficiency virus (HIV) infection. HIV was found to infect and replicate in human brain cells and can cause neuroinfections and neurological deterioration. We designed dual-antibody-modified chitosan/small interfering RNA (siRNA) nanoparticles to deliver siRNA across the blood-brain barrier (BBB) targeting HIV-infected brain astrocytes as a strategy for inhibiting HIV replication. We hypothesized that transferrin antibody and bradykinin B2 antibody could specifically bind to the transferrin receptor (TfR) and bradykinin B2 receptor (B2R), respectively, and deliver siRNA across the BBB into astrocytes as potential targeting ligands. In this study, chitosan nanoparticles (CS-NPs) were prepared by a complex coacervation method in the presence of siRNA, and antibody was chemically conjugated to the nanoparticles. The antibody-modified chitosan nanoparticles (Ab-CS-NPs) were spherical in shape, with an average particle size of 235.7 ± 10.2 nm and a zeta potential of 22.88 ± 1.78 mV. The therapeutic potential of the nanoparticles was evaluated based on their cellular uptake and gene silencing efficiency. Cellular accumulation and gene silencing efficiency of Ab-CS-NPs in astrocytes were significantly improved compared to non-modified CS-NPs and single-antibody-modified CS-NPs. These results suggest that the combination of anti-Tf antibody and anti-B2 antibody significantly increased the knockdown effect of siRNA-loaded nanoparticles. Thus, antibody-mediated dual-targeting nanoparticles are an efficient and promising delivery strategy for inhibiting HIV replication in astrocytes. Graphical abstract Graphic representation of dual-antibody-conjugated chitosan nanoparticles for the targeted delivery of siRNA across the blood-brain barrier (BBB) for inhibiting HIV replication in astrocytes. a Nanoparticle delivery to the BBB and penetration. b Tf

Antiretroviral-treated HIV-1 patients can harbour resistant viruses in CSF despite an undetectable viral load in plasma.

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Soulie, Cathia; Grudé, Maxime; Descamps, Diane; Amiel, Corinne; Morand-Joubert, Laurence; Raymond, Stéphanie; Pallier, Coralie; Bellecave, Pantxika; Reigadas, Sandrine; Trabaud, Mary-Anne; Delaugerre, Constance; Montes, Brigitte; Barin, Francis; Ferré, Virginie; Jeulin, Hélène; Alloui, Chakib; Yerly, Sabine; Signori-Schmuck, Anne; Guigon, Aurélie; Fafi-Kremer, Samira; Haïm-Boukobza, Stéphanie; Mirand, Audrey; Maillard, Anne; Vallet, Sophie; Roussel, Catherine; Assoumou, Lambert; Calvez, Vincent; Flandre, Philippe; Marcelin, Anne-Geneviève

2017-08-01

HIV therapy reduces the CSF HIV RNA viral load (VL) and prevents disorders related to HIV encephalitis. However, these brain disorders may persist in some cases. A large population of antiretroviral-treated patients who had a VL > 1.7 log 10 copies/mL in CSF with detectable or undetectable VL in plasma associated with cognitive impairment was studied, in order to characterize discriminatory factors of these two patient populations. Blood and CSF samples were collected at the time of neurological disorders for 227 patients in 22 centres in France and 1 centre in Switzerland. Genotypic HIV resistance tests were performed on CSF. The genotypic susceptibility score was calculated according to the last Agence Nationale de Recherche sur le Sida et les hépatites virales Action Coordonnée 11 (ANRS AC11) genotype interpretation algorithm. Among the 227 studied patients with VL > 1.7 log 10 copies/mL in CSF, 195 had VL detectable in plasma [median (IQR) HIV RNA was 3.7 (2.7-4.7) log 10 copies/mL] and 32 had discordant VL in plasma (VL plasma compared with patients with plasma VL > 1.7 log 10 copies/mL. Resistance to antiretrovirals was observed in CSF for the two groups of patients. Fourteen percent of this population of patients with cognitive impairment and detectable VL in CSF had well controlled VL in plasma. Thus, it is important to explore CSF HIV (VL and genotype) even if the HIV VL is controlled in plasma because HIV resistance may be observed. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Cervical Shedding of HIV-1 RNA Among Women With Low Levels of Viremia While Receiving Highly Active Antiretroviral Therapy

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Neely, Michael N.; Benning, Lorie; Xu, Jiaao; Strickler, Howard D.; Greenblatt, Ruth M.; Minkoff, Howard; Young, Mary; Bremer, James; Levine, Alexandra M.; Kovacs, Andrea

2011-01-01

Background Among women with low o r undetectable quantities of HIV-1 RNA in plasma, factors associated with genital HIV-1 RNA shedding, including choice of treatment regimen, are poorly characterized. Methods We measured HIV-1 RNA in cervical swab specimens obtained from participants in the Women’s Interagency HIV Study who had concurrent plasma viral RNA levels <500 copies/mL, and we assessed factors associated with genital HIV shedding. The study was powered to determine the relative effects of antiretroviral protease inhibitors (PIs) versus nonnucleoside reverse transcriptase inhibitors (NNRTIs) on viral RNA shedding. Results Overall, 44 (15%) of 290 women had detectable HIV-1 RNA in cervical specimens. In the final multivariate model, shedding was independently associated with NNRTI (vs. PI) use (odds ratio [OR], 95% confidence interval [CI]: 2.24, 1.13 to 4.45) and illicit drug use (OR, 95% CI: 2.41, 0.96 to 5.69). Conclusions This is the largest study to define risks for genital HIV-1 RNA shedding in women with low/undetectable plasma virus. Shedding in this population was common, and NNRTI-based highly active antiretroviral therapy (HAART) (vs. PI-based HAART) was associated with genital HIV shedding. Further study is required to determine the impact of these findings on transmission of HIV from mother to child or to sexual partners. PMID:17106279

Mechanistic evaluation of the pros and cons of digital RT-LAMP for HIV-1 viral load quantification on a microfluidic device and improved efficiency via a two-step digital protocol.

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Sun, Bing; Shen, Feng; McCalla, Stephanie E; Kreutz, Jason E; Karymov, Mikhail A; Ismagilov, Rustem F

2013-02-05

Here we used a SlipChip microfluidic device to evaluate the performance of digital reverse transcription-loop-mediated isothermal amplification (dRT-LAMP) for quantification of HIV viral RNA. Tests are needed for monitoring HIV viral load to control the emergence of drug resistance and to diagnose acute HIV infections. In resource-limited settings, in vitro measurement of HIV viral load in a simple format is especially needed, and single-molecule counting using a digital format could provide a potential solution. We showed here that when one-step dRT-LAMP is used for quantification of HIV RNA, the digital count is lower than expected and is limited by the yield of desired cDNA. We were able to overcome the limitations by developing a microfluidic protocol to manipulate many single molecules in parallel through a two-step digital process. In the first step we compartmentalize the individual RNA molecules (based on Poisson statistics) and perform reverse transcription on each RNA molecule independently to produce DNA. In the second step, we perform the LAMP amplification on all individual DNA molecules in parallel. Using this new protocol, we increased the absolute efficiency (the ratio between the concentration calculated from the actual count and the expected concentration) of dRT-LAMP 10-fold, from ∼2% to ∼23%, by (i) using a more efficient reverse transcriptase, (ii) introducing RNase H to break up the DNA:RNA hybrid, and (iii) adding only the BIP primer during the RT step. We also used this two-step method to quantify HIV RNA purified from four patient samples and found that in some cases, the quantification results were highly sensitive to the sequence of the patient's HIV RNA. We learned the following three lessons from this work: (i) digital amplification technologies, including dLAMP and dPCR, may give adequate dilution curves and yet have low efficiency, thereby providing quantification values that underestimate the true concentration. Careful

Antiretroviral treatment is associated with increased attentional load-dependent brain activation in HIV patients.

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Chang, L; Yakupov, R; Nakama, H; Stokes, B; Ernst, T

2008-06-01

The purpose of this paper was to determine whether antiretroviral medications, especially the nucleoside analogue reverse transcriptase inhibitors, lead to altered brain activation due to their potential neurotoxic effects in patients with human immunodeficiency virus (HIV) infection. Forty-two right-handed men were enrolled in three groups: seronegative controls (SN, n = 18), HIV subjects treated with antiretroviral medications (HIV+ARV, n = 12), or not treated with antiretroviral medications (HIV+NARV, n = 12). Each subject performed a set of visual attention tasks with increasing difficulty or load (tracking two, three or four balls) during functional magnetic resonance imaging. HIV subjects, both groups combined, showed greater load-dependent increases in brain activation in the right frontal regions compared to SN (p-corrected = 0.006). HIV+ARV additionally showed greater load-dependent increases in activation compared to SN in bilateral superior frontal regions (p-corrected = 0.032) and a lower percent accuracy on the performance of the most difficult task (tracking four balls). Region of interest analyses further demonstrated that SN showed load-dependent decreases (with repeated trials despite increasing difficulty), while HIV subjects showed load-dependent increases in activation with the more difficult tasks, especially those on ARVs. These findings suggest that chronic ARV treatments may lead to greater requirement of the attentional network reserve and hence less efficient usage of the network and less practice effects in these HIV patients. As the brain has a limited reserve capacity, exhausting the reserve capacity in HIV+ARV would lead to declined performance with more difficult tasks that require more attention.

Nuclear retention of multiply spliced HIV-1 RNA in resting CD4+ T cells.

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Kara G Lassen

2006-07-01

Full Text Available HIV-1 latency in resting CD4+ T cells represents a major barrier to virus eradication in patients on highly active antiretroviral therapy (HAART. We describe here a novel post-transcriptional block in HIV-1 gene expression in resting CD4+ T cells from patients on HAART. This block involves the aberrant localization of multiply spliced (MS HIV-1 RNAs encoding the critical positive regulators Tat and Rev. Although these RNAs had no previously described export defect, we show that they exhibit strict nuclear localization in resting CD4+ T cells from patients on HAART. Overexpression of the transcriptional activator Tat from non-HIV vectors allowed virus production in these cells. Thus, the nuclear retention of MS HIV-1 RNA interrupts a positive feedback loop and contributes to the non-productive nature of infection of resting CD4+ T cells. To define the mechanism of nuclear retention, proteomic analysis was used to identify proteins that bind MS HIV-1 RNA. Polypyrimidine tract binding protein (PTB was identified as an HIV-1 RNA-binding protein differentially expressed in resting and activated CD4+ T cells. Overexpression of PTB in resting CD4+ T cells from patients on HAART allowed cytoplasmic accumulation of HIV-1 RNAs. PTB overexpression also induced virus production by resting CD4+ T cells. Virus culture experiments showed that overexpression of PTB in resting CD4+ T cells from patients on HAART allowed release of replication-competent virus, while preserving a resting cellular phenotype. Whether through effects on RNA export or another mechanism, the ability of PTB to reverse latency without inducing cellular activation is a result with therapeutic implications.

Bacterial vaginosis, alterations in vaginal flora and HIV genital shedding among HIV-1-infected women in Mozambique

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Robert D Kirkcaldy

2011-05-01

Full Text Available Objectives. We investigated whether abnormal vaginal flora, including bacterial vaginosis (BV, are associated with detection of cervical HIV-1 RNA among HIV-infected women in Mozambique. Methods. We obtained clinical data and vaginal specimens from HIV-infected women registering for their first visit at one of two HIV care clinics in Mozambique. We compared women with detectable cervical HIV viral load (≥40 copies/ml with women with undetectable cervical HIV. Results. We enrolled 106 women. Women with abnormal vaginal flora (intermediate Nugent scores, 4 - 6 were more likely to have detectable cervical HIV RNA then women with normal vaginal flora (adjusted odds ratio 7.2 (95% confidence interval 1.8 - 29.1, adjusted for CD4 count. Women with BV had a non-significantly higher likelihood of detectable cervical HIV than women with normal flora. Conclusions. Abnormal vaginal flora were significantly associated with cervical HIV expression. Further research is needed to confirm this relationship.

Maternal characteristics during pregnancy and risk factors for positive HIV RNA at delivery: a single-cohort observational study (Brescia, Northern Italy

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Magoni Michele

2011-02-01

Full Text Available Abstract Background Detectable HIV RNA in mothers at delivery is an important risk factor for HIV transmission to newborns. Our hypothesis was that, in migrant women, the risk of detectable HIV RNA at delivery is greater owing to late HIV diagnosis. Therefore, we examined pregnant women by regional provenance and measured variables that could be associated with detectable HIV RNA at delivery. Methods A observational retrospective study was conducted from January 1999 to May 2008. Univariate and multivariable regression analyses (generalized linear models were used, with detectable HIV RNA at delivery as dependent variable. Results The overall population comprised 154 women (46.8% migrants. Presentation was later in migrant women than Italians, as assessed by CD4-T-cell count at first contact (mean 417/mm3 versus 545/mm3, respectively; p = 0.003. Likewise, HIV diagnosis was made before pregnancy and HAART was already prescribed at the time of pregnancy in more Italians (91% and 75%, respectively than migrants (61% and 42.8%, respectively. A subgroup of women with available HIV RNA close to term (i.e., ≤30 days before labour was studied for risk factors of detectable HIV RNA (≥50 copies/ml at delivery. Among 93 women, 25 (26.9% had detectable HIV RNA. A trend toward an association between non-Italian nationality and detectable HIV RNA at delivery was demonstrated by univariate analysis (relative risk, RR = 1.86; p = 0.099. However, by multivariable regression analysis, the following factors appeared to be more important: lack of stable (i.e., ≥14 days antiretroviral therapy at the time of HIV RNA testing (RR = 4.3; p 3, RR = 0.94; p = 0.038. Conclusions These results reinforce the importance of extensive screening for HIV infection, earlier initiation of antiretroviral therapy and stricter monitoring of pregnant women to reduce the risk of detectable HIV RNA at delivery. Public health interventions should be particularly targeted to migrant

Is the virulence of HIV changing? A meta-analysis of trends in prognostic markers of HIV disease progression and transmission

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Herbeck, Joshua T.; Müller, Viktor; Maust, Brandon S.; Ledergerber, Bruno; Torti, Carlo; Di Giambenedetto, Simona; Gras, Luuk; Günthard, Huldrych F.; Jacobson, Lisa P.; Mullins, James I.; Gottlieb, Geoffrey S.

2013-01-01

Objective The potential for changing HIV-1 virulence has significant implications for the AIDS epidemic, including changing HIV transmission rates, rapidity of disease progression, and timing of ART. Published data to date have provided conflicting results. Design We conducted a meta-analysis of changes in baseline CD4+ T-cell counts and set point plasma viral RNA load over time in order to establish whether summary trends are consistent with changing HIV-1 virulence. Methods We searched PubMed for studies of trends in HIV-1 prognostic markers of disease progression and supplemented findings with publications referenced in epidemiological or virulence studies. We identified 12 studies of trends in baseline CD4+ T-cell counts (21 052 total individuals), and eight studies of trends in set point viral loads (10 785 total individuals), spanning the years 1984–2010. Using random-effects meta-analysis, we estimated summary effect sizes for trends in HIV-1 plasma viral loads and CD4+ T-cell counts. Results Baseline CD4+ T-cell counts showed a summary trend of decreasing cell counts [effect=−4.93 cells/µl per year, 95% confidence interval (CI) −6.53 to −3.3]. Set point viral loads showed a summary trend of increasing plasma viral RNA loads (effect=0.013 log10 copies/ml per year, 95% CI −0.001 to 0.03). The trend rates decelerated in recent years for both prognostic markers. Conclusion Our results are consistent with increased virulence of HIV-1 over the course of the epidemic. Extrapolating over the 30 years since the first description of AIDS, this represents a CD4+ T cells loss of approximately 148 cells/µl and a gain of 0.39 log10 copies/ml of viral RNA measured during early infection. These effect sizes would predict increasing rates of disease progression, and need for ART as well as increasing transmission risk. PMID:22089381

Cytoplasmic HIV-1 RNA is mainly transported by diffusion in the presence or absence of Gag protein

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Chen, Jianbo; Grunwald, David; Sardo, Luca

2014-01-01

. In this report, we visualized HIV-1 RNA and monitored its movement in the cytoplasm by using single-molecule tracking. We observed that most of the HIV-1 RNA molecules move in a nondirectional, random-walk manner, which does not require an intact cytoskeletal structure, and that the mean-squared distance...... traveled by the RNA increases linearly with time, indicative of diffusive movement. We also observed that a single HIV-1 RNA molecule can move at various speeds when traveling through the cytoplasm, indicating that its movement is strongly affected by the immediate environment. To examine the effect of Gag...

RNA glycosidase and other agents target Tat to inhibit HIV-1 transcription.

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Harrich, David; Jin, Hongping

2018-03-20

The HIV-1 tat gene encodes a small 86-104 amino acid protein depending on the HIV-1 strain. Tat is essential for HIV-1 replication through interactions with numerous cellular transcription factors. The interaction between Tat and P-TEFb, which is a cellular protein complex composed of cyclin T1 and CDK9, delivers P-TEFb to the newly transcribed viral mRNAs where phosphorylation of RNA polymerase II by CDK9 leads to highly efficient mRNA transcription. It has long been recognized that Tat is a potential anti-HIV-1 target and possibly a viral Achilles' heel. However, specifically targeting Tat without affecting normal host cell functions has been challenging. Means to inactivate Tat have been reported that includes small compounds, transdominant negative Tat proteins, and by plant-derived antivirals. Investigations of these agents have reported encouraging outcomes that inform and may hopefully affect strategies for a functional HIV-1 cure. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

HIV shedding in cervico-vaginal secretions in pregnant women.

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Gardella, Barbara; Roccio, Marianna; Maccabruni, Anna; Mariani, Bianca; Panzeri, Lucia; Zara, Francesca; Spinillo, Arsenio

2011-07-01

The purpose of this study was to evaluate the presence of HIV-1 in cervico-vaginal secretions of pregnant as compared to non-pregnant HIV-seropositive women. We compared 43 known HIV seropositive pregnant patients versus 241 age-matched (± 2 years) control non-pregnant HIV-seropositive subjects. In pregnant patients blood and cervico-vaginal samples were obtained during each trimester of pregnancy. In control subjects the same samples were obtained at enrolment. HIV-1 RNA was measured in plasma; proviral HIV-1 DNA, cell-associated and cell-free HIV-1 RNA in cervico-vaginal secretion by competitive polymerase chain reaction (cRT-PCR) and reverse transcriptase PCR. The genital shedding of HIV-DNA (22/43 as compared to 79/241, p = 0.02), and cell-free HIV-RNA detection (26/43 as compared to 72/241, p pregnant than in non pregnant women. Pregnancy correlated with a significant positive trend in the cervico-vaginal load of HIV-DNA (Spearman Rho= 0.149, p= 0.012), and cell-free HIV-RNA (Spearman Rho= 0.253, p HIV-RNA transcripts (Spearman Rho = 0.06, p= 0.31). After correction for potential confounders, first trimester pregnant women had increased rates of genital HIV- DNA (odds ratio = 1.94, 95% confidence interval = 1.01 3.78) and cell-free HIV-RNA (odds ratio = 4.07, 95% confidence interval = 1.97 8.41) detection compared to nonpregnant controls. The shedding of genital HIV was increased in pregnant compared to non pregnant subjects, even in patients with undetectable viremia. In this low-risk HIV-positive population the risks of vertical or horizontal transmissions should not be underestimated.

Comparison of droplet digital PCR and seminested real-time PCR for quantification of cell-associated HIV-1 RNA

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Kiselinova, Maja; Pasternak, Alexander O.; de Spiegelaere, Ward; Vogelaers, Dirk; Berkhout, Ben; Vandekerckhove, Linos

2014-01-01

Cell-associated (CA) HIV-1 RNA is considered a potential marker for assessment of viral reservoir dynamics and antiretroviral therapy (ART) response in HIV-infected patients. Recent studies employed sensitive seminested real-time quantitative (q)PCR to quantify CA HIV-1 RNA. Digital PCR has been

HIV-1 transmitting couples have similar viral load set-points in Rakai, Uganda.

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T Déirdre Hollingsworth

2010-05-01

Full Text Available It has been hypothesized that HIV-1 viral load set-point is a surrogate measure of HIV-1 viral virulence, and that it may be subject to natural selection in the human host population. A key test of this hypothesis is whether viral load set-points are correlated between transmitting individuals and those acquiring infection. We retrospectively identified 112 heterosexual HIV-discordant couples enrolled in a cohort in Rakai, Uganda, in which HIV transmission was suspected and viral load set-point was established. In addition, sequence data was available to establish transmission by genetic linkage for 57 of these couples. Sex, age, viral subtype, index partner, and self-reported genital ulcer disease status (GUD were known. Using ANOVA, we estimated the proportion of variance in viral load set-points which was explained by the similarity within couples (the 'couple effect'. Individuals with suspected intra-couple transmission (97 couples had similar viral load set-points (p = 0.054 single factor model, p = 0.0057 adjusted and the couple effect explained 16% of variance in viral loads (23% adjusted. The analysis was repeated for a subset of 29 couples with strong genetic support for transmission. The couple effect was the major determinant of viral load set-point (p = 0.067 single factor, and p = 0.036 adjusted and the size of the effect was 27% (37% adjusted. Individuals within epidemiologically linked couples with genetic support for transmission had similar viral load set-points. The most parsimonious explanation is that this is due to shared characteristics of the transmitted virus, a finding which sheds light on both the role of viral factors in HIV-1 pathogenesis and on the evolution of the virus.

Sexual Activity Without Condoms and Risk of HIV Transmission in Serodifferent Couples When the HIV-Positive Partner Is Using Suppressive Antiretroviral Therapy

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Rodger, Alison J; Cambiano, Valentina; Bruun, Tina

2016-01-01

IMPORTANCE: A key factor in assessing the effectiveness and cost-effectiveness of antiretroviral therapy (ART) as a prevention strategy is the absolute risk of HIV transmission through condomless sex with suppressed HIV-1 RNA viral load for both anal and vaginal sex. OBJECTIVE: To evaluate the ra...

Quantification of Human T-lymphotropic virus type I (HTLV-I) provirus load in a rural West African population: no enhancement of human immunodeficiency virus type 2 pathogenesis, but HTLV-I provirus load relates to mortality

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Ariyoshi, Koya; Berry, Neil; Cham, Fatim; Jaffar, Shabbar; Schim van der Loeff, Maarten; Jobe, Ousman; N'Gom, Pa Tamba; Larsen, Olav; Andersson, Sören; Aaby, Peter; Whittle, Hilton

2003-01-01

Human T-lymphotropic virus type I (HTLV-I) provirus load was examined in a cohort of a population in Guinea-Bissau among whom human immunodeficiency virus (HIV) type 2 is endemic. Geometric mean of HIV-2 RNA load among HTLV-I-coinfected subjects was significantly lower than that in subjects infected

The diagnosis of symptomatic acute antiretroviral syndrome during the window period with antigen/antibody testing and HIV viral load

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Daniel O. Griffin

Full Text Available Despite much focus on moving toward a cure to end the epidemic human immunodeficiency virus (HIV epidemic there are still thousands of new infections occurring every year in the United States. Although there is ongoing transmission of HIV in the United States and a growing population of people living with HIV, the acute presentation of HIV infection can be challenging to diagnose and is often not considered when patients present to healthcare providers. Although in certain states there are HIV testing laws that require that all persons between the ages of 13 and 64 be offered HIV testing in an opt-out approach, many patient presenting with an acute illness, that would warrant diagnostic testing for HIV, leave without having an HIV test performed for either diagnostic or screening purposes.We describe the case of a woman who presented to medical attention with symptoms later confirmed to be due to acute HIV infection. She was initially discharged from the hospital and only underwent HIV testing with confirmation of her diagnosis after readmission. We describe the algorithm where fourth generation testing combined with HIV viral load testing allowed for the diagnosis of acute HIV prior to the development of a specific immunoglobulin response. Consideration of this diagnosis, improved HIV screening, and understanding of the use of antigen/antibody screening tests, combined with Multispot and HIV viral RNA detection, when appropriate, can allow for early diagnosis of HIV before progression of disease and before undiagnosed patient spread the infection to new contacts.

A prospective study of the effect of pregnancy on CD4 counts and plasma HIV-1 RNA concentrations of antiretroviral-naive HIV-1-infected women.

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Heffron, Renee; Donnell, Deborah; Kiarie, James; Rees, Helen; Ngure, Kenneth; Mugo, Nelly; Were, Edwin; Celum, Connie; Baeten, Jared M

2014-02-01

In HIV-1-infected women, CD4 count declines occur during pregnancy, which has been attributed to hemodilution. However, for women who have not initiated antiretroviral therapy, it is unclear if CD4 declines are sustained beyond pregnancy and accompanied by increased viral levels, which could indicate an effect of pregnancy on accelerating HIV-1 disease progression. In a prospective study among 2269 HIV-1-infected antiretroviral therapy-naive women from 7 African countries, we examined the effect of pregnancy on HIV-1 disease progression. We used linear mixed models to compare CD4 counts and plasma HIV-1 RNA concentrations between pregnant, postpartum, and nonpregnant periods. Women contributed 3270 person-years of follow-up, during which time 476 women became pregnant. In adjusted analysis, CD4 counts were an average of 56 (95% confidence interval: 39 to 73) cells/mm lower during pregnant compared with nonpregnant periods and 70 (95% confidence interval: 53 to 88) cells/mm lower during pregnant compared with postpartum periods; these results were consistent when restricted to the subgroup of women who became pregnant. Plasma HIV-1 RNA concentrations were not different between pregnant and nonpregnant periods (P = 0.9) or pregnant and postpartum periods (P = 0.3). Neither CD4 counts nor plasma HIV-1 RNA levels were significantly different in postpartum compared with nonpregnant periods. CD4 count declines among HIV-1-infected women during pregnancy are temporary and not sustained in postpartum periods. Pregnancy does not have a short-term impact on plasma HIV-1 RNA concentrations.

Engineering and Validation of a Vector for Concomitant Expression of Rare Transfer RNA (tRNA and HIV-1 nef Genes in Escherichia coli.

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Siti Aisyah Mualif

Full Text Available Relative ease in handling and manipulation of Escherichia coli strains make them primary candidate to express proteins heterologously. Overexpression of heterologous genes that contain codons infrequently used by E. coli is related with difficulties such as mRNA instability, early termination of transcription and/or translation, deletions and/or misincorporation, and cell growth inhibition. These codon bias -associated problems are addressed by co-expressing ColE1-compatible, rare tRNA expressing helper plasmids. However, this approach has inadequacies, which we have addressed by engineering an expression vector that concomitantly expresses the heterologous protein of interest, and rare tRNA genes in E. coli. The expression vector contains three (argU, ileY, leuW rare tRNA genes and a useful multiple cloning site for easy in-frame cloning. To maintain the overall size of the parental plasmid vector, the rare tRNA genes replaced the non-essential DNA segments in the vector. The cloned gene is expressed under the control of T7 promoter and resulting recombinant protein has a C-terminal 6His tag for IMAC-mediated purification. We have evaluated the usefulness of this expression vector by expressing three HIV-1 genes namely HIV-1 p27 (nef, HIV-1 p24 (ca, and HIV-1 vif in NiCo21(DE3 E.coli and demonstrated the advantages of using expression vector that concomitantly expresses rare tRNA and heterologous genes.

Single-Cell RNA-Seq Reveals Transcriptional Heterogeneity in Latent and Reactivated HIV-Infected Cells.

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Golumbeanu, Monica; Cristinelli, Sara; Rato, Sylvie; Munoz, Miguel; Cavassini, Matthias; Beerenwinkel, Niko; Ciuffi, Angela

2018-04-24

Despite effective treatment, HIV can persist in latent reservoirs, which represent a major obstacle toward HIV eradication. Targeting and reactivating latent cells is challenging due to the heterogeneous nature of HIV-infected cells. Here, we used a primary model of HIV latency and single-cell RNA sequencing to characterize transcriptional heterogeneity during HIV latency and reactivation. Our analysis identified transcriptional programs leading to successful reactivation of HIV expression. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Sexual Activity Without Condoms and Risk of HIV Transmission in Serodifferent Couples When the HIV-Positive Partner Is Using Suppressive Antiretroviral Therapy

NARCIS (Netherlands)

Rodger, Alison J.; Cambiano, Valentina; Bruun, Tina; Vernazza, Pietro; Collins, Simon; van Lunzen, Jan; Corbelli, Giulio Maria; Estrada, Vicente; Geretti, Anna Maria; Beloukas, Apostolos; Asboe, David; Viciana, Pompeyo; Gutiérrez, Félix; Clotet, Bonaventura; Pradier, Christian; Gerstoft, Jan; Weber, Rainer; Westling, Katarina; Wandeler, Gilles; Prins, Jan M.; Rieger, Armin; Stoeckle, Marcel; Kümmerle, Tim; Bini, Teresa; Ammassari, Adriana; Gilson, Richard; Krznaric, Ivanka; Ristola, Matti; Zangerle, Robert; Handberg, Pia; Antela, Antonio; Allan, Sris; Phillips, Andrew N.; Lundgren, Jens; Pompeyo, V.; Trastoy, M.; Palacio, R.; Gutiérrez, F.; Masiá, M.; Padilla, S.; Robledano, C.; Clotet, B.; Coll, P.; Peña, J.; Estrada, V.; Rodrigo, M.; Santiago, E.; Rivero, A.; Antela, A.; Losada, E.

2016-01-01

IMPORTANCE A key factor in assessing the effectiveness and cost-effectiveness of antiretroviral therapy (ART) as a prevention strategy is the absolute risk of HIV transmission through condomless sex with suppressed HIV-1 RNA viral load for both anal and vaginal sex. OBJECTIVE To evaluate the rate of

HIV-1 Reservoir Association with Immune Activation

Directory of Open Access Journals (Sweden)

Alejandro Vallejo

2015-09-01

Full Text Available In this issue of EBioMedicine, Ruggiero and colleagues describe immune activation biomarkers associated with the size of the HIV reservoir in a carefully designed cross-sectional study. The cohort consists of a homogeneous sample of HIV-1-infected patients with long-term plasma HIV-1 RNA suppression under antiretroviral treatment (ART. It is crucial to explore the potential utility of biomarkers that are easier (less labor intensive, less expensive to measure than integrated HIV DNA load, in order to quickly and accurately quantify cellular reservoirs of HIV.

Structural insights into RNA processing by the human RISC-loading complex.

Science.gov (United States)

Wang, Hong-Wei; Noland, Cameron; Siridechadilok, Bunpote; Taylor, David W; Ma, Enbo; Felderer, Karin; Doudna, Jennifer A; Nogales, Eva

2009-11-01

Targeted gene silencing by RNA interference (RNAi) requires loading of a short guide RNA (small interfering RNA (siRNA) or microRNA (miRNA)) onto an Argonaute protein to form the functional center of an RNA-induced silencing complex (RISC). In humans, Argonaute2 (AGO2) assembles with the guide RNA-generating enzyme Dicer and the RNA-binding protein TRBP to form a RISC-loading complex (RLC), which is necessary for efficient transfer of nascent siRNAs and miRNAs from Dicer to AGO2. Here, using single-particle EM analysis, we show that human Dicer has an L-shaped structure. The RLC Dicer's N-terminal DExH/D domain, located in a short 'base branch', interacts with TRBP, whereas its C-terminal catalytic domains in the main body are proximal to AGO2. A model generated by docking the available atomic structures of Dicer and Argonaute homologs into the RLC reconstruction suggests a mechanism for siRNA transfer from Dicer to AGO2.

Quantification of Human T-lymphotropic virus type I (HTLV-I) provirus load in a rural West African population: no enhancement of human immunodeficiency virus type 2 pathogenesis, but HTLV-I provirus load relates to mortality

DEFF Research Database (Denmark)

Ariyoshi, K; Berry, N; Cham, F

2003-01-01

Human T-lymphotropic virus type I (HTLV-I) provirus load was examined in a cohort of a population in Guinea-Bissau among whom human immunodeficiency virus (HIV) type 2 is endemic. Geometric mean of HIV-2 RNA load among HTLV-I-coinfected subjects was significantly lower than that in subjects...... infected with HIV-2 alone (212 vs. 724 copies/mL; P=.02). Adjusted for age, sex, and HIV status, the risk of death increased with HTLV-I provirus load; mortality hazard ratio was 1.59 for each log10 increase in HTLV-I provirus copies (P=.038). There is no enhancing effect of HTLV-I coinfection on HIV-2...... disease, but high HTLV-I provirus loads may contribute to mortality....

Long-term effectiveness of highly active antiretroviral therapy (HAART) in perinatally HIV-infected children in Denmark

DEFF Research Database (Denmark)

Bracher, Linda; Valerius, Niels Henrik; Rosenfeldt, Vibeke

2007-01-01

children treated with HAART. Initial HAART included 2 nucleoside reverse-transcriptase inhibitors in combination with either a protease inhibitor (n =38) or a non-nucleoside reverse-transcriptase inhibitor (n =12). 19 (39%) patients were previously treated with mono- or dual therapy. Baseline......The long-term impact of highly active antiretroviral therapy (HAART) on HIV-1 infected children is not well known. The Danish Paediatric HIV Cohort Study includes all patients ... characteristics were median CD4 percentage 14% and HIV-RNA viral load 4.9 log(10). Within the first 12 weeks of therapy approximately 60% achieved HIV-RNA viral load children changed the components of HAART. The proportion of children with CD4...

Improving laboratory efficiencies to scale-up HIV viral load testing.

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Alemnji, George; Onyebujoh, Philip; Nkengasong, John N

2017-03-01

Viral load measurement is a key indicator that determines patients' response to treatment and risk for disease progression. Efforts are ongoing in different countries to scale-up access to viral load testing to meet the Joint United Nations Programme on HIV and AIDS target of achieving 90% viral suppression among HIV-infected patients receiving antiretroviral therapy. However, the impact of these initiatives may be challenged by increased inefficiencies along the viral load testing spectrum. This will translate to increased costs and ineffectiveness of scale-up approaches. This review describes different parameters that could be addressed across the viral load testing spectrum aimed at improving efficiencies and utilizing test results for patient management. Though progress is being made in some countries to scale-up viral load, many others still face numerous challenges that may affect scale-up efficiencies: weak demand creation, ineffective supply chain management systems; poor specimen referral systems; inadequate data and quality management systems; and weak laboratory-clinical interface leading to diminished uptake of test results. In scaling up access to viral load testing, there should be a renewed focus to address efficiencies across the entire spectrum, including factors related to access, uptake, and impact of test results.

A prospective study of the effect of pregnancy on CD4 counts and plasma HIV-1 RNA concentrations of antiretroviral-naive HIV-1 infected women

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Heffron, Renee; Donnell, Deborah; Kiarie, James; Rees, Helen; Ngure, Kenneth; Mugo, Nelly; Were, Edwin; Celum, Connie; Baeten, Jared M.

2014-01-01

Background In HIV-1 infected women, CD4 count declines occur during pregnancy, which has been attributed to hemodilution. However, for women who have not initiated antiretroviral therapy (ART), it is unclear if CD4 declines are sustained beyond pregnancy and accompanied by increased viral levels, which could indicate an effect of pregnancy on accelerating HIV-1 disease progression. Methods In a prospective study among 2269 HIV-1 infected ART-naïve women from 7 African countries, we examined the effect of pregnancy on HIV-1 disease progression. We used linear mixed models to compare CD4 counts and plasma HIV-1 RNA concentrations between pregnant, postpartum and non-pregnant periods. Results Women contributed 3270 person-years of follow-up, during which time 476 women became pregnant. In adjusted analysis, CD4 counts were an average of 56 (95% CI 39-73) cells/mm3 lower during pregnant compared to non-pregnant periods and 70 (95% CI 53-88) cells/mm3 lower during pregnant compared to postpartum periods; these results were consistent when restricted to the subgroup of women who became pregnant. Plasma HIV-1 RNA concentrations were not different between pregnant and non-pregnant periods (p=0.9) or pregnant and postpartum periods (p=0.3). Neither CD4 counts nor plasma HIV-1 RNA levels were significantly different in postpartum compared to non-pregnant periods. Conclusion CD4 count declines among HIV-1 infected women during pregnancy are temporary and not sustained in postpartum periods. Pregnancy does not have a short term impact on plasma HIV-1 RNA concentrations. PMID:24442226

HIV-1 and herpes simplex virus type-2 genital shedding among co-infected women using self-collected swabs in Chiang Rai, Thailand.

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Forhan, S E; Dunne, E F; Sternberg, M R; Whitehead, S J; Leelawiwat, W; Thepamnuay, S; Chen, C; Evans-Strickfaden, Tt; McNicholl, J M; Markowitz, L E

2012-08-01

We analysed 528 genital self-collected swabs (SCS) from 67 HIV-1 and herpes simplex virus type-2 (HSV-2) co-infected women collected during the placebo month of a randomized crossover clinical trial of suppressive acyclovir in Chiang Rai, Thailand. In this first longitudinal study of HIV-1 and HSV-2 co-infected women using genital SCS specimens, we found frequent mucosal HIV-1 shedding. Overall, 372 (70%) swabs had detectable HIV-1 RNA with median HIV-1 viral load of 2.61 log(10) copies/swab. We found no statistically significant association between detectable HIV-1 RNA and HSV-2 DNA in the same SCS specimen (adjusted odds ratio [aOR] 1.40; 95% confidence intervals [CI], 0.78-2.60, P = 0.25). Only baseline HIV-1 plasma viral load was independently associated with genital HIV-1 RNA shedding (aOR, 7.6; 95% CI, 3.3-17.2, P genital sampling, and inclusion of genital sites other than the cervix.

Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: Implications for viral genomic RNA packaging

OpenAIRE

Webb, Joseph A.; Jones, Christopher P.; Parent, Leslie J.; Rouzina, Ioulia; Musier-Forsyth, Karin

2013-01-01

The mechanism underlying the selective packaging of genomic RNA into HIV-1 virions is not known. This paper provides important new biophysical insights into the nature of protein–RNA interactions responsible for HIV-1 genome packaging by quantifying the electrostatic and hydrophobic contributions to specific and nonspecific RNA.

Correlation of immune activation with HIV-1 RNA levels assayed by real-time RT-PCR in HIV-1 Subtype C infected patients in Northern India

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Agarwal, Atima; Sankaran, Sumathi; Vajpayee, Madhu; Sreenivas, V; Seth, Pradeep; Dandekar, Satya

2014-01-01

Background Assays with specificity and cost effectiveness are needed for the measurement of HIV-1 burden to monitor disease progression or response to anti-retroviral therapy (ART) in HIV-1 subtype C infected patients. Objectives The objective of this study was to develop and validate an affordable; one step Real-Time RT-PCR assay with high specificity and sensitivity to measure plasma HIV-1 loads in HIV-1 subtype C infected patients. Results We developed an RT-PCR assay to detect and quantitate plasma HIV-1 levels in HIV-1 subtype C infected patients. An inverse correlation between plasma viral loads (PVL) and CD4+ T-cell numbers was detected at all CDC stages. Significant correlations were found between CD8+ T-cell activation and PVL, as well as with the clinical and immunological status of the patients. Conclusions The RT-PCR assay provides a sensitive method to measure PVL in HIV-1 subtype C infected patients. Viral loads correlated with immune activation and can be used to monitor HIV care in India. PMID:17962068

High levels of T lymphocyte activation in Leishmania-HIV-1 co-infected individuals despite low HIV viral load

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Grinsztejn Beatriz

2010-12-01

Full Text Available Abstract Background Concomitant infections may influence HIV progression by causing chronic activation leading to decline in T-cell function. In the Americas, visceral (AVL and tegumentary leishmaniasis (ATL have emerged as important opportunistic infections in HIV-AIDS patients and both of those diseases have been implicated as potentially important co-factors in disease progression. We investigated whether leishmaniasis increases lymphocyte activation in HIV-1 co-infected patients. This might contribute to impaired cellular immune function. Methods To address this issue we analyzed CD4+ T absolute counts and the proportion of CD8+ T cells expressing CD38 in Leishmania/HIV co-infected patients that recovered after anti-leishmanial therapy. Results We found that, despite clinical remission of leishmaniasis, AVL co-infected patients presented a more severe immunossupression as suggested by CD4+ T cell counts under 200 cells/mm3, differing from ATL/HIV-AIDS cases that tends to show higher lymphocytes levels (over 350 cells/mm3. Furthermore, five out of nine, AVL/HIV-AIDS presented low CD4+ T cell counts in spite of low or undetectable viral load. Expression of CD38 on CD8+ T lymphocytes was significantly higher in AVL or ATL/HIV-AIDS cases compared to HIV/AIDS patients without leishmaniasis or healthy subjects. Conclusions Leishmania infection can increase the degree of immune system activation in individuals concomitantly infected with HIV. In addition, AVL/HIV-AIDS patients can present low CD4+ T cell counts and higher proportion of activated T lymphocytes even when HIV viral load is suppressed under HAART. This fact can cause a misinterpretation of these laboratorial markers in co-infected patients.

Pregnant and breastfeeding women: A priority population for HIV viral load monitoring.

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Myer, Landon; Essajee, Shaffiq; Broyles, Laura N; Watts, D Heather; Lesosky, Maia; El-Sadr, Wafaa M; Abrams, Elaine J

2017-08-01

Landon Myer and colleagues discuss viral load monitoring for pregnant HIV-positive women and those breastfeeding; ART treatments can suppress viral load and are key to preventing transmission to the child.

Suppression of HIV-1 viral load after multiple changes in high active ...

African Journals Online (AJOL)

1-infected patients. However, the virus persists ... chronological changes in HIV viral load and CD4+ T-cell count, and treatment outcomes of multiple combinations of .... Lewin SR, Rouzioux C. HIV cure and eradication: how will we get from the ...

Nuclear Factor 90, a cellular dsRNA binding protein inhibits the HIV Rev-export function

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St-Laurent Georges

2006-11-01

Full Text Available Abstract Background The HIV Rev protein is known to facilitate export of incompletely spliced and unspliced viral transcripts to the cytoplasm, a necessary step in virus life cycle. The Rev-mediated nucleo-cytoplasmic transport of nascent viral transcripts, dependents on interaction of Rev with the RRE RNA structural element present in the target RNAs. The C-terminal variant of dsRNA-binding nuclear protein 90 (NF90ctv has been shown to markedly attenuate viral replication in stably transduced HIV-1 target cell line. Here we examined a mechanism of interference of viral life cycle involving Rev-NF90ctv interaction. Results Since Rev:RRE complex formations depend on protein:RNA and protein:protein interactions, we investigated whether the expression of NF90ctv might interfere with Rev-mediated export of RRE-containing transcripts. When HeLa cells expressed both NF90ctv and Rev protein, we observed that NF90ctv inhibited the Rev-mediated RNA transport. In particular, three regions of NF90ctv protein are involved in blocking Rev function. Moreover, interaction of NF90ctv with the RRE RNA resulted in the expression of a reporter protein coding sequences linked to the RRE structure. Moreover, Rev influenced the subcellular localization of NF90ctv, and this process is leptomycin B sensitive. Conclusion The dsRNA binding protein, NF90ctv competes with HIV Rev function at two levels, by competitive protein:protein interaction involving Rev binding to specific domains of NF90ctv, as well as by its binding to the RRE-RNA structure. Our results are consistent with a model of Rev-mediated HIV-1 RNA export that envisions Rev-multimerization, a process interrupted by NF90ctv.

HIV DNA Is Frequently Present within Pathologic Tissues Evaluated at Autopsy from Combined Antiretroviral Therapy-Treated Patients with Undetectable Viral Loads.

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Lamers, Susanna L; Rose, Rebecca; Maidji, Ekaterina; Agsalda-Garcia, Melissa; Nolan, David J; Fogel, Gary B; Salemi, Marco; Garcia, Debra L; Bracci, Paige; Yong, William; Commins, Deborah; Said, Jonathan; Khanlou, Negar; Hinkin, Charles H; Sueiras, Miguel Valdes; Mathisen, Glenn; Donovan, Suzanne; Shiramizu, Bruce; Stoddart, Cheryl A; McGrath, Michael S; Singer, Elyse J

2016-10-15

HIV infection treatment strategies have historically defined effectiveness through measuring patient plasma HIV RNA. While combined antiretroviral therapy (cART) can reduce plasma viral load (pVL) to undetectable levels, the degree that HIV is eliminated from other anatomical sites remains unclear. We investigated the HIV DNA levels in 229 varied autopsy tissues from 20 HIV-positive (HIV(+)) cART-treated study participants with low or undetectable plasma VL and cerebrospinal fluid (CSF) VL prior to death who were enrolled in the National Neurological AIDS Bank (NNAB) longitudinal study and autopsy cohort. Extensive medical histories were obtained for each participant. Autopsy specimens, including at least six brain and nonbrain tissues per participant, were reviewed by study pathologists. HIV DNA, measured in tissues by quantitative and droplet digital PCR, was identified in 48/87 brain tissues and 82/142 nonbrain tissues at levels >200 HIV copies/million cell equivalents. No participant was found to be completely free of tissue HIV. Parallel sequencing studies from some tissues recovered intact HIV DNA and RNA. Abnormal histological findings were identified in all participants, especially in brain, spleen, lung, lymph node, liver, aorta, and kidney. All brain tissues demonstrated some degree of pathology. Ninety-five percent of participants had some degree of atherosclerosis, and 75% of participants died with cancer. This study assists in characterizing the anatomical locations of HIV, in particular, macrophage-rich tissues, such as the central nervous system (CNS) and testis. Additional studies are needed to determine if the HIV recovered from tissues promotes the pathogenesis of inflammatory diseases, such as HIV-associated neurocognitive disorders, cancer, and atherosclerosis. It is well-known that combined antiretroviral therapy (cART) can reduce plasma HIV to undetectable levels; however, cART cannot completely clear HIV infection. An ongoing question is

Combinatorial RNA-based gene therapy for the treatment of HIV/AIDS.

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Chung, Janet; DiGiusto, David L; Rossi, John J

2013-03-01

HIV/AIDS continues to be a worldwide health problem and viral eradication has been an elusive goal. HIV+ patients are currently treated with combination antiretroviral therapy (cART) which is not curative. For many patients, cART is inaccessible, intolerable or unaffordable. Therefore, a new class of therapeutics for HIV is required to overcome these limitations. Cell and gene therapy for HIV has been proposed as a way to provide a functional cure for HIV in the form of a virus/infection resistant immune system. In this review, the authors describe the standard therapy for HIV/AIDS, its limitations, current areas of investigation and the potential of hematopoietic stem cells modified with anti-HIV RNAs as a means to affect a functional cure for HIV. Cell and gene therapy for HIV/AIDS is a promising alternative to antiviral drug therapy and may provide a functional cure. In order to show clinical benefit, multiple mechanisms of inhibition of HIV entry and lifecycle are likely to be required. Among the most promising antiviral strategies is the use of transgenic RNA molecules that provide protection from HIV infection. When these molecules are delivered as gene-modified hematopoietic stem and progenitor cells, long-term repopulation of the patient's immune system with gene-modified progeny has been observed.

Probing the HIV-1 genomic RNA trafficking pathway and dimerization by genetic recombination and single virion analyses.

Directory of Open Access Journals (Sweden)

Michael D Moore

2009-10-01

Full Text Available Once transcribed, the nascent full-length RNA of HIV-1 must travel to the appropriate host cell sites to be translated or to find a partner RNA for copackaging to form newly generated viruses. In this report, we sought to delineate the location where HIV-1 RNA initiates dimerization and the influence of the RNA transport pathway used by the virus on downstream events essential to viral replication. Using a cell-fusion-dependent recombination assay, we demonstrate that the two RNAs destined for copackaging into the same virion select each other mostly within the cytoplasm. Moreover, by manipulating the RNA export element in the viral genome, we show that the export pathway taken is important for the ability of RNA molecules derived from two viruses to interact and be copackaged. These results further illustrate that at the point of dimerization the two main cellular export pathways are partially distinct. Lastly, by providing Gag in trans, we have demonstrated that Gag is able to package RNA from either export pathway, irrespective of the transport pathway used by the gag mRNA. These findings provide unique insights into the process of RNA export in general, and more specifically, of HIV-1 genomic RNA trafficking.

Self-Perceived Viral Load and Sexual Risk Behavior Among Known HIV-Positive MSM in San Francisco, 2014.

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Guigayoma, John; Chen, Yea-Hung; Snowden, Jonathan M; Santos, Glenn-Milo; Hecht, Jennifer; Raymond, H Fisher

2017-07-01

Self-perceived viral suppression status among men who have sex with men (MSM) may impact HIV risk transmission behaviors. We conducted a 2014 cross-sectional survey of MSM in San Francisco and assessed differences in sexual risk behavior among known HIV-positive MSM based on viral suppression of HIV. We collected demographics, self-perceived viral load status, and sexual risk behavior and tested for viral load levels through laboratory assays. Men were categorized in a hierarchical schema of sexual risk behavior categories based on responses to questions regarding recent partners' HIV status, condom use, and sexual positioning. We used Fisher exact tests to assess for differences based on self-perceived viral load status. Out of a sample of 96 known HIV-positive men, 59 men self-reported an undetectable HIV viral load and 9 men self-reported a detectable viral load consented to confirmatory laboratory testing. The sample of self-reported undetectable men had gradually larger proportions of higher-risk sexual practices, whereas the sample of detectable men was evenly distributed across sexual practices. This association was not statistically significant (P = 0.91). Self-perceived viral suppression may influence sexual practices of known HIV-positive MSM, but small sample size, especially within the detectable category, hinders our ability to determine statistical significance. More research is necessary to assess how HIV-positive men account for viral load in sexual decision-making practices, and this research may inform resource allocation and clinical recommendations to maintain the health of MSM populations.

Virologic response to tipranavir-ritonavir or darunavir-ritonavir based regimens in antiretroviral therapy experienced HIV-1 patients: a meta-analysis and meta-regression of randomized controlled clinical trials.

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Asres Berhan

Full Text Available The development of tipranavir and darunavir, second generation non-peptidic HIV protease inhibitors, with marked improved resistance profiles, has opened a new perspective on the treatment of antiretroviral therapy (ART experienced HIV patients with poor viral load control. The aim of this study was to determine the virologic response in ART experienced patients to tipranavir-ritonavir and darunavir-ritonavir based regimens.A computer based literature search was conducted in the databases of HINARI (Health InterNetwork Access to Research Initiative, Medline and Cochrane library. Meta-analysis was performed by including randomized controlled studies that were conducted in ART experienced patients with plasma viral load above 1,000 copies HIV RNA/ml. The odds ratios and 95% confidence intervals (CI for viral loads of <50 copies and <400 copies HIV RNA/ml at the end of the intervention were determined by the random effects model. Meta-regression, sensitivity analysis and funnel plots were done. The number of HIV-1 patients who were on either a tipranavir-ritonavir or darunavir-ritonavir based regimen and achieved viral load less than 50 copies HIV RNA/ml was significantly higher (overall OR = 3.4; 95% CI, 2.61-4.52 than the number of HIV-1 patients who were on investigator selected boosted comparator HIV-1 protease inhibitors (CPIs-ritonavir. Similarly, the number of patients with viral load less than 400 copies HIV RNA/ml was significantly higher in either the tipranavir-ritonavir or darunavir-ritonavir based regimen treated group (overall OR = 3.0; 95% CI, 2.15-4.11. Meta-regression showed that the viral load reduction was independent of baseline viral load, baseline CD4 count and duration of tipranavir-ritonavir or darunavir-ritonavir based regimen.Tipranavir and darunavir based regimens were more effective in patients who were ART experienced and had poor viral load control. Further studies are required to determine their consistent

The impact of nevirapine- versus protease inhibitor-based regimens on virological markers of HIV-1 persistence during seemingly suppressive ART.

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Kiselinova, Maja; Anna, Maria; Malatinkova, Eva; Vervish, Karen; Beloukas, Apostolos; Messiaen, Peter; Bonczkowski, Pawel; Trypsteen, Wim; Callens, Steven; Verhofstede, Chris; De Spiegelaere, Ward; Vandekerckhove, Linos

2014-01-01

The source and significance of residual plasma HIV-1 RNA detection during suppressive ART remain controversial. It has been proposed that nevirapine (NVP)-based regimens achieve a greater HIV-1 RNA suppression than regimens containing a protease inhibitor (PI). The aim of this study was to compare the effect of receiving NVP- vs PI-based ART on the virological markers of HIV persistence in peripheral blood. The study population comprised 161 HIV-1 infected patients receiving either NVP-based (n=81) or PI-based (n=80) ART and showing a HIV-1 RNA load stably suppressed ART, with median (IQR) levels of 5 (3-6) and 5 (3-8) copies/mL, respectively. HIV-1 RNA detection was associated with shorter duration of suppressive ART regardless of treatment arm (p=0.007), and lower CD4 nadir (p=0.015). HIV-1 DNA levels were median 282 (120-484) and 213 (87-494) copies/106 PBMCs in the two groups respectively, and were lowest (ART HIV-1 RNA load (p=0.0001). In this comprehensive characterization of patients on long-term suppressive ART, we did not observe evidence for a greater suppressive activity of NVP-based over PI-based therapy on plasma and intracellular markers of virus persistence. Overall excellent correlation was observed between the markers, allowing the identification of a subset of treated patients with low HIV-1 expression as an important cohort for future HIV cure studies.

High-throughput SHAPE analysis reveals structures in HIV-1 genomic RNA strongly conserved across distinct biological states.

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Kevin A Wilkinson

2008-04-01

Full Text Available Replication and pathogenesis of the human immunodeficiency virus (HIV is tightly linked to the structure of its RNA genome, but genome structure in infectious virions is poorly understood. We invent high-throughput SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension technology, which uses many of the same tools as DNA sequencing, to quantify RNA backbone flexibility at single-nucleotide resolution and from which robust structural information can be immediately derived. We analyze the structure of HIV-1 genomic RNA in four biologically instructive states, including the authentic viral genome inside native particles. Remarkably, given the large number of plausible local structures, the first 10% of the HIV-1 genome exists in a single, predominant conformation in all four states. We also discover that noncoding regions functioning in a regulatory role have significantly lower (p-value < 0.0001 SHAPE reactivities, and hence more structure, than do viral coding regions that function as the template for protein synthesis. By directly monitoring protein binding inside virions, we identify the RNA recognition motif for the viral nucleocapsid protein. Seven structurally homologous binding sites occur in a well-defined domain in the genome, consistent with a role in directing specific packaging of genomic RNA into nascent virions. In addition, we identify two distinct motifs that are targets for the duplex destabilizing activity of this same protein. The nucleocapsid protein destabilizes local HIV-1 RNA structure in ways likely to facilitate initial movement both of the retroviral reverse transcriptase from its tRNA primer and of the ribosome in coding regions. Each of the three nucleocapsid interaction motifs falls in a specific genome domain, indicating that local protein interactions can be organized by the long-range architecture of an RNA. High-throughput SHAPE reveals a comprehensive view of HIV-1 RNA genome structure, and further

Plasma HIV-1 RNA viral load rebound among people who inject drugs receiving antiretroviral therapy (ART) in a Canadian setting: an ethno-epidemiological study.

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Small, Will; Milloy, M J; McNeil, Ryan; Maher, Lisa; Kerr, Thomas

2016-01-01

People who inject drugs (PWID) living with HIV often experience sub-optimal antiretroviral therapy (ART) treatment outcomes, including HIV plasma viral load (PVL) rebound. While previous studies have identified risk factors for PVL rebound among PWID, no study has examined the perspectives of PWID who have experienced PVL rebound episodes. We conducted an ethno-epidemiological study to investigate the circumstances surrounding the emergence of rebound episodes among PWID in Vancouver, BC, Canada. Comprehensive clinical records linked to a community-based prospective observational cohort of HIV-positive drug users were used to identify PWID who had recently experienced viral rebound. In-depth qualitative interviews with 16 male and 11 female participants explored participant perspectives regarding the emergence of viral rebound. A timeline depicting each participant's HIV viral load and adherence to ART was used to elicit discussion of circumstances surrounding viral rebound. Viral rebound episodes were shaped by interplay between various individual, social, and environmental factors that disrupted routines facilitating adherence. Structural-environmental influences resulting in non-adherence included housing transitions, changes in drug use patterns and intense drug scene involvement, and inadequate care for co-morbid health conditions. Social-environmental influences on ART adherence included poor interactions between care providers and patients producing non-adherence, and understandings of HIV treatment that fostered intentional treatment discontinuation. This study describes key pathways which led to rebound episodes among PWID receiving ART and illustrates how environmental forces may increase vulnerability for non-adherence leading to treatment failure. Our findings have potential to help inform interventions and supports that address social-structural forces that foster non-adherence among PWID.

A European multicientre study on the comparison of HIV-1 viral loads between VERIS HIV-1 Assay and Roche COBAS® TAQMAN® HIV-1 test, Abbott RealTime HIV-1 Assay, and Siemens VERSANT HIV-1 Assay.

Science.gov (United States)

Braun, Patrick; Delgado, Rafael; Drago, Monica; Fanti, Diana; Fleury, Hervé; Hofmann, Jörg; Izopet, Jacques; Kühn, Sebastian; Lombardi, Alessandra; Mancon, Alessandro; Marcos, Mª Angeles; Mileto, Davide; Sauné, Karine; O'Shea, Siobhan; Pérez-Rivilla, Alfredo; Ramble, John; Trimoulet, Pascale; Vila, Jordi; Whittaker, Duncan; Artus, Alain; Rhodes, Daniel

2017-07-01

Viral load monitoring is essential for patients under treatment for HIV. Beckman Coulter has developed the VERIS HIV-1 Assay for use on the novel, automated DxN VERIS Molecular Diagnostics System. ¥ OBJECTIVES: Evaluation of the clinical performance of the new quantitative VERIS HIV-1 Assay at multiple EU laboratories. Method comparison with the VERIS HIV-1 Assay was performed with 415 specimens at 5 sites tested with COBAS ® AmpliPrep/COBAS ® TaqMan ® HIV-1 Test, v2.0, 169 specimens at 3 sites tested with RealTime HIV-1 Assay, and 202 specimens from 2 sites tested with VERSANT HIV-1 Assay. Patient monitoring sample results from 4 sites were also compared. Bland-Altman analysis showed the average bias between VERIS HIV-1 Assay and COBAS HIV-1 Test, RealTime HIV-1 Assay, and VERSANT HIV-1 Assay to be 0.28, 0.39, and 0.61 log 10 cp/mL, respectively. Bias at low end levels below 1000cp/mL showed predicted bias to be <0.3 log 10 cp/mL for VERIS HIV-1 Assay versus COBAS HIV-1 Test and RealTime HIV-1 Assay, and <0.5 log 10 cp/mL versus VERSANT HIV-1 Assay. Analysis on 174 specimens tested with the 0.175mL volume VERIS HIV-1 Assay and COBAS HIV-1 Test showed average bias of 0.39 log 10 cp/mL. Patient monitoring results using VERIS HIV-1 Assay demonstrated similar viral load trends over time to all comparators. The VERIS HIV-1 Assay for use on the DxN VERIS System demonstrated comparable clinical performance to COBAS ® HIV-1 Test, RealTime HIV-1 Assay, and VERSANT HIV-1 Assay. Copyright © 2017 Elsevier B.V. All rights reserved.

Opening of the TAR hairpin in the HIV-1 genome causes aberrant RNA dimerization and packaging

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Das Atze T

2012-07-01

Full Text Available Abstract Background The TAR hairpin is present at both the 5′ and 3′ end of the HIV-1 RNA genome. The 5′ element binds the viral Tat protein and is essential for Tat-mediated activation of transcription. We recently observed that complete TAR deletion is allowed in the context of an HIV-1 variant that does not depend on this Tat-TAR axis for transcription. Mutations that open the 5′ stem-loop structure did however affect the leader RNA conformation and resulted in a severe replication defect. In this study, we set out to analyze which step of the HIV-1 replication cycle is affected by this conformational change of the leader RNA. Results We demonstrate that opening the 5′ TAR structure through a deletion in either side of the stem region caused aberrant dimerization and reduced packaging of the unspliced viral RNA genome. In contrast, truncation of the TAR hairpin through deletions in both sides of the stem did not affect RNA dimer formation and packaging. Conclusions These results demonstrate that, although the TAR hairpin is not essential for RNA dimerization and packaging, mutations in TAR can significantly affect these processes through misfolding of the relevant RNA signals.

Polymorphisms of large effect explain the majority of the host genetic contribution to variation of HIV-1 virus load

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Coulonges, Cedric; Bartha, István; Lenz, Tobias L.; Deutsch, Aaron J.; Bashirova, Arman; Buchbinder, Susan; Carrington, Mary N.; Cossarizza, Andrea; Dalmau, Judith; De Luca, Andrea; Goedert, James J.; Gurdasani, Deepti; Haas, David W.; Herbeck, Joshua T.; Johnson, Eric O.; Kirk, Gregory D.; Lambotte, Olivier; Luo, Ma; Mallal, Simon; van Manen, Daniëlle; Martinez-Picado, Javier; Meyer, Laurence; Miro, José M.; Mullins, James I.; Obel, Niels; Poli, Guido; Sandhu, Manjinder S.; Schuitemaker, Hanneke; Shea, Patrick R.; Theodorou, Ioannis; Walker, Bruce D.; Weintrob, Amy C.; Winkler, Cheryl A.; Wolinsky, Steven M.; Raychaudhuri, Soumya; Goldstein, David B.; Telenti, Amalio; de Bakker, Paul I. W.; Zagury, Jean-François; Fellay, Jacques

2015-01-01

Previous genome-wide association studies (GWAS) of HIV-1–infected populations have been underpowered to detect common variants with moderate impact on disease outcome and have not assessed the phenotypic variance explained by genome-wide additive effects. By combining the majority of available genome-wide genotyping data in HIV-infected populations, we tested for association between ∼8 million variants and viral load (HIV RNA copies per milliliter of plasma) in 6,315 individuals of European ancestry. The strongest signal of association was observed in the HLA class I region that was fully explained by independent effects mapping to five variable amino acid positions in the peptide binding grooves of the HLA-B and HLA-A proteins. We observed a second genome-wide significant association signal in the chemokine (C-C motif) receptor (CCR) gene cluster on chromosome 3. Conditional analysis showed that this signal could not be fully attributed to the known protective CCR5Δ32 allele and the risk P1 haplotype, suggesting further causal variants in this region. Heritability analysis demonstrated that common human genetic variation—mostly in the HLA and CCR5 regions—explains 25% of the variability in viral load. This study suggests that analyses in non-European populations and of variant classes not assessed by GWAS should be priorities for the field going forward. PMID:26553974

Undetectable hepatitis C virus RNA during syphilis infection in two HIV/HCV-co-infected patients

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Salado-Rasmussen, Kirsten; Knudsen, Andreas; Krarup, Henrik Bygum

2014-01-01

BACKGROUND: Treponema pallidum, the causative agent of syphilis, elicits a vigorous immune response in the infected host. This study sought to describe the impact of syphilis infection on hepatitis C virus (HCV) RNA levels in patients with HIV and chronic HCV infection. METHODS: Patients......-α), interferon gamma (IFN-γ), and IFN-γ-inducible protein 10 kDa (IP-10). RESULTS: Undetectable HCV RNA at the time of early latent syphilis infection was observed in 2 patients with HIV and chronic HCV infection. After treatment of the syphilis infection, HCV RNA levels increased again in patient 1, whereas...... patient 2 initiated HCV therapy and remained HCV RNA-negative. Available plasma samples obtained before and after the episode with undetectable HCV RNA were phylogenetically identical, making the possibility of spontaneous clearance and HCV reinfection less likely. The IL-10, TNF-α, and IP-10 levels...

HIV-Infected Ugandan Women on Antiretroviral Therapy Maintain HIV-1 RNA Suppression Across Periconception, Pregnancy, and Postpartum Periods.

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Matthews, Lynn T; Ribaudo, Heather B; Kaida, Angela; Bennett, Kara; Musinguzi, Nicholas; Siedner, Mark J; Kabakyenga, Jerome; Hunt, Peter W; Martin, Jeffrey N; Boum, Yap; Haberer, Jessica E; Bangsberg, David R

2016-04-01

HIV-infected women risk sexual and perinatal HIV transmission during conception, pregnancy, childbirth, and breastfeeding. We compared HIV-1 RNA suppression and medication adherence across periconception, pregnancy, and postpartum periods, among women on antiretroviral therapy (ART) in Uganda. We analyzed data from women in a prospective cohort study, aged 18-49 years, enrolled at ART initiation and with ≥1 pregnancy between 2005 and 2011. Participants were seen quarterly. The primary exposure of interest was pregnancy period, including periconception (3 quarters before pregnancy), pregnancy, postpartum (6 months after pregnancy outcome), or nonpregnancy related. Regression models using generalized estimating equations compared the likelihood of HIV-1 RNA ≤400 copies per milliliter, pregnancy, and 89% of postpartum visits, and was more likely during periconception (adjusted odds ratio, 2.15) compared with nonpregnant periods. Average ART adherence was 90% [interquartile range (IQR), 70%-98%], 93% (IQR, 82%-98%), 92% (IQR, 72%-98%), and 88% (IQR, 63%-97%) during nonpregnant, periconception, pregnant, and postpartum periods, respectively. Average adherence pregnancy were virologically suppressed at most visits, with an increased likelihood of suppression and high adherence during periconception follow-up. Increased frequency of 72-hour gaps suggests a need for increased adherence support during postpartum periods.

Options to Expand HIV Viral Load Testing in South Africa: Evaluation of the GeneXpert® HIV-1 Viral Load Assay.

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Natasha Gous

Full Text Available Expansion of HIV viral load (VL testing services are required to meet increased targets for monitoring patients on antiretroviral treatment. South Africa currently tests >4million VLs per annum in 16 highly centralised, automated high-throughput laboratories. The Xpert HIV-1 VL assay (Cepheid was evaluated against in-country predicates, the Roche Cobas Taqmanv2 and Abbott HIV-1RT, to investigate options for expanding VL testing using GeneXpert's random access, polyvalent capabilities and already established footprint in South Africa with the Xpert MTB/RIF assay (207 sites. Additionally, the performance of Xpert HIV-1VL on alternative, off-label specimen types, Dried Blood Spots (DBS and whole blood, was investigated.Precision, accuracy (agreement and clinical misclassification (1000cp/ml of Xpert HIV-1VL plasma was compared to Taqmanv2 (n = 155 and Abbott HIV-1 RT (n = 145. Misclassification of Xpert HIV-1VL was further tested on DBS (n = 145 and whole blood (n = 147.Xpert HIV-1VL demonstrated 100% concordance with predicate platforms on a standardised frozen, plasma panel (n = 42 and low overall percentage similarity CV of 1.5% and 0.9% compared to Taqmanv2 and Abbott HIV-1 RT, respectively. On paired plasma clinical specimens, Xpert HIV-1VL had low bias (SD 0.32-0.37logcp/ml and 3% misclassification at the 1000cp/ml threshold compared to Taqmanv2 (fresh and Abbott HIV-1 RT (frozen, respectively. Xpert HIV-1VL on whole blood and DBS increased misclassification (upward by up to 14% with increased invalid rate. All specimen testing was easy to perform and compatible with concurrent Xpert MTB/RIF Tuberculosis testing on the same instrument.The Xpert HIV-1VL on plasma can be used interchangeably with existing predicate platforms in South Africa. Whole blood and DBS testing requires further investigation, but polyvalency of the GeneXpert offers a solution to extending VL testing services.

Dissection of specific binding of HIV-1 Gag to the 'packaging signal' in viral RNA.

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Comas-Garcia, Mauricio; Datta, Siddhartha Ak; Baker, Laura; Varma, Rajat; Gudla, Prabhakar R; Rein, Alan

2017-07-20

Selective packaging of HIV-1 genomic RNA (gRNA) requires the presence of a cis -acting RNA element called the 'packaging signal' (Ψ). However, the mechanism by which Ψ promotes selective packaging of the gRNA is not well understood. We used fluorescence correlation spectroscopy and quenching data to monitor the binding of recombinant HIV-1 Gag protein to Cy5-tagged 190-base RNAs. At physiological ionic strength, Gag binds with very similar, nanomolar affinities to both Ψ-containing and control RNAs. We challenged these interactions by adding excess competing tRNA; introducing mutations in Gag; or raising the ionic strength. These modifications all revealed high specificity for Ψ. This specificity is evidently obscured in physiological salt by non-specific, predominantly electrostatic interactions. This nonspecific activity was attenuated by mutations in the MA, CA, and NC domains, including CA mutations disrupting Gag-Gag interaction. We propose that gRNA is selectively packaged because binding to Ψ nucleates virion assembly with particular efficiency.

Cardiac Gene Expression Knockdown Using Small Inhibitory RNA-Loaded Microbubbles and Ultrasound.

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Jonathan A Kopechek

Full Text Available RNA interference has potential therapeutic value for cardiac disease, but targeted delivery of interfering RNA is a challenge. Custom designed microbubbles, in conjunction with ultrasound, can deliver small inhibitory RNA to target tissues in vivo. The efficacy of cardiac RNA interference using a microbubble-ultrasound theranostic platform has not been demonstrated in vivo. Therefore, our objective was to test the hypothesis that custom designed microbubbles and ultrasound can mediate effective delivery of small inhibitory RNA to the heart. Microbubble and ultrasound mediated cardiac RNA interference was tested in transgenic mice displaying cardiac-restricted luciferase expression. Luciferase expression was assayed in select tissues of untreated mice (n = 14. Mice received intravenous infusion of cationic microbubbles bearing small inhibitory RNA directed against luciferase (n = 9 or control RNA (n = 8 during intermittent cardiac-directed ultrasound at mechanical index of 1.6. Simultaneous echocardiography in a separate group of mice (n = 3 confirmed microbubble destruction and replenishment during treatment. Three days post treatment, cardiac luciferase messenger RNA and protein levels were significantly lower in ultrasound-treated mice receiving microbubbles loaded with small inhibitory RNA directed against luciferase compared to mice receiving microbubbles bearing control RNA (23±7% and 33±7% of control mice, p<0.01 and p = 0.03, respectively. Passive cavitation detection focused on the heart confirmed that insonification resulted in inertial cavitation. In conclusion, small inhibitory RNA-loaded microbubbles and ultrasound directed at the heart significantly reduced the expression of a reporter gene. Ultrasound-targeted destruction of RNA-loaded microbubbles may be an effective image-guided strategy for therapeutic RNA interference in cardiac disease.

Selective translational repression of HIV-1 RNA by Sam68DeltaC occurs by altering PABP1 binding to unspliced viral RNA

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Soros Vanessa

2008-10-01

Full Text Available Abstract HIV-1 structural proteins are translated from incompletely spliced 9 kb and 4 kb mRNAs, which are transported to the cytoplasm by Crm1. It has been assumed that once in the cytoplasm, translation of incompletely spliced HIV-1 mRNAs occurs in the same manner as host mRNAs. Previous analyses have demonstrated that Sam68 and a mutant thereof, Sam68ΔC, have dramatic effects on HIV gene expression, strongly enhancing and inhibiting viral structural protein synthesis, respectively. While investigating the inhibition of incompletely spliced HIV-1 mRNAs by Sam68ΔC, we determined that the effect was independent of the perinuclear bundling of the viral RNA. Inhibition was dependent upon the nuclear export pathway used, as translation of viral RNA exported via the Tap/CTE export pathway was not blocked by Sam68ΔC. We demonstrate that inhibition of HIV expression by Sam68ΔC is correlated with a loss of PABP1 binding with no attendant change in polyadenosine tail length of the affected RNAs. The capacity of Sam68ΔC to selectively inhibit translation of HIV-1 RNAs exported by Crm1 suggests that it is able to recognize unique characteristics of these viral RNPs, a property that could lead to new therapeutic approaches to controlling HIV-1 replication.

CD4+ T cell count, HIV-1 viral loads and demographic variables of newly identified patients with HIV infection in Wuhan, China.

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Liu, Man-Qing; Tang, Li; Kong, Wen-Hua; Zhu, Ze-Rong; Peng, Jin-Song; Wang, Xia; Yao, Zhong-Zhao; Schilling, Robert; Zhou, Wang

2013-10-01

In China, the rate of human immunodeficiency virus (HIV) testing is increasing among men who have sex with men. The purpose of the present study was to describe HIV-related biomarkers and selected demographic variables of persons with newly diagnosed HIV/AIDS, among men who have sex with men in particular, in Wuhan China. Demographic indicators, and CD4+ T cell counts and HIV-1 viral load were collected from individuals newly identified as HIV-1 antibody positive during 2011. Of 176 enrolled patients, 132 (75.0%) were men who have sex with men. This group was significantly younger and had higher CD4+ T cell counts than patients who were likely infected through heterosexual contact. Most men who have sex with men (56.6%) were discovered by initiative investigation. Among heterosexual patients CD4+ T cell counts and HIV-1 viral load were significantly correlated; among the group of men who have sex with men, no such association was found. Copyright © 2013 Wiley Periodicals, Inc.

Low levels of HIV-1 RNA detected in the cerebrospinal fluid after up to 10 years of suppressive therapy are associated with local immune activation.

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Dahl, Viktor; Peterson, Julia; Fuchs, Dietmar; Gisslen, Magnus; Palmer, Sarah; Price, Richard W

2014-09-24

Though combination antiretroviral therapy reduces the concentration of HIV-1 RNA in both plasma and cerebrospinal fluid (CSF) below the detection limit of clinical assays, low levels of HIV-1 RNA are frequently detectable in plasma using more sensitive assays. We examined the frequency and magnitude of persistent low-level HIV-1 RNA in CSF and its relation to the central nervous system (CNS) immune activation. CSF and plasma HIV-1 RNA were measured using the single-copy assay with a detection limit of 0.3 copies/ml in 70 CSF and 68 plasma samples from 45 treated HIV-1-infected patients with less than 40 copies/ml of HIV-1 RNA in both fluids by standard clinical assays. We also measured CSF neopterin to assess intrathecal immune activation. Theoretical drug exposure was estimated using the CNS penetration-efficacy score of treatment regimens. CSF HIV-1 RNA was detected in 12 of the 70 CSF samples (17%) taken after up to 10 years of suppressive therapy, compared to 39 of the 68 plasma samples (57%) with a median concentration of less than 0.3 copies/ml in CSF compared to 0.3 copies/ml in plasma (P < 0.0001). CSF samples with detectable HIV-1 RNA had higher CSF neopterin levels (mean 8.2 compared to 5.7 nmol/l; P = 0.0085). Patients with detectable HIV-1 RNA in CSF did not differ in pretreatment plasma HIV-1 RNA levels, nadir CD4 cell count or CNS penetration-efficacy score. Low-level CSF HIV-1 RNA and its association with elevated CSF neopterin highlight the potential for the CNS to serve as a viral reservoir and for persistent infection to cause subclinical CNS injury.

Neurocognitive and neuroinflammatory correlates of PDYN and OPRK1 mRNA expression in the anterior cingulate in postmortem brain of HIV-infected subjects.

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Yuferov, Vadim; Butelman, Eduardo R; Ho, Ann; Morgello, Susan; Kreek, Mary Jeanne

2014-01-09

Chronic inflammation may contribute to neuropsychological impairments in individuals with HIV, and modulation of this inflammatory response by opiate receptor ligands is important in light of the prevalence of drug use in HIV populations. Exogenous MOR and KOR agonists have differential effects on central nervous system (CNS) immunity and, while some data suggest KOR agonists are immunosuppressive, the KOR agonist dynorphin has been shown to stimulate human monocyte chemotaxis. In this study, we examined mRNA levels of endogenous opioid receptors OPRK1 and OPRM1, prodynorphin (PDYN), macrophage scavenger receptor CD163, and microglia/macrophage marker CD68 in the caudate and anterior cingulate of postmortem brains from HIV-positive and HIV-negative subjects. Brain tissues of HIV-infected (n = 24) and control subjects (n = 15) were obtained from the Manhattan HIV Brain Bank. Quantification of the gene mRNA was performed using SYBR Green RT-PCR. CD68 and CD163 were increased in HIV-positive (HIV+) compared to HIV-negative (HIV-) individuals in both brain regions. There were higher OPRK1 (P <0.005), and lower PDYN mRNA (P <0.005) levels in the anterior cingulate of HIV+ compared to HIV- subjects. This difference between the clinical groups was not found in the caudate. There was no difference in the levels of OPRM1 mRNA between HIV+ and HIV- subjects. Using linear regression analysis, we examined the relationship of OPRK1 and PDYN mRNA levels in the HIV+ subjects with seven cognitive domain T scores of a neuropsychological test battery. Within the HIV+ subjects, there was a positive correlation between anterior cingulate PDYN mRNA levels and better T-scores in the motor domain. Within the HIV+ subjects there were also positive correlations of both OPRK1 and PDYN mRNA levels with the anti-inflammatory marker CD163, but not with proinflammatory CD68 levels. In this setting, decreased PDYN mRNA may reflect a homeostatic mechanism to reduce monocyte

Hepatitis C virus (HCV) RNA profiles among chronic HIV/HCV-coinfected individuals in ESPRIT; spontaneous HCV RNA clearance observed in nine individuals

DEFF Research Database (Denmark)

Grint, D; Tedaldi, Ellen; Peters, L

2017-01-01

OBJECTIVES: Studies have shown that hepatitis C virus (HCV) RNA levels remain stable over time in HIV/HCV-coinfected individuals taking combination antiretroviral therapy (cART), while spontaneous clearance of HCV RNA during the persistent infection phase has been documented only rarely among tho...

The relative prognostic value of plasma HIV RNA levels and CD4 lymphocyte counts in advanced HIV infection

DEFF Research Database (Denmark)

Cozzi-Lepri, A; Katzenstein, T L; Ullum, H

1998-01-01

. RESULTS: The plasma HIV RNA (median 101410 copies/ml; range (range 200-7200000) and the CD4 lymphocyte count (median 250 cells x 10(6)/l; range 1-1247) were negatively correlated (Pearson r = -0.53; P

Human papillomavirus load in benign HPV-associated oral lesions from HIV/AIDS individuals.

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Camacho-Aguilar, S; Ramírez-Amador, V; Rosendo-Chalma, P; Guido-Jiménez, M; García-Carrancá, A; Anaya-Saavedra, G

2018-03-01

Although HPV emerged as a crucial carcinogenic and prognostic biomarker in head and neck cancer, and considering the increase in HPV-associated oral lesions (HPV-OLs) in HIV individuals, molecular information about HPV-OLs is scarce; thus, our aim was to determine viral loads in HPV-OLs from HIV/AIDS individuals. HIV/AIDS subjects with HPV-OL were included in this cross-sectional study. Following informed consent, biopsies were obtained. HPV detection and typing were carried out by PCR and sequencing (MY09/11, GP5+/6+). HPV-13 and HPV-32 loads were determined by a high-resolution melting assay. For statistical analysis, X 2 , Fisher's exact, and Mann-Whitney U tests were applied, using SPSS software (v.23). Twenty-nine HIV subjects (median age 38 years, 93% males) were included. Most were AIDS individuals (72.4%) under HAART (89.7%). Twenty-two (75.9%) participants had more than one HPV-OL (four with florid presentations), mostly multifocal epithelial hyperplasia (62%), being HPV-13 (26%) and HPV-32 (31%) the most frequent types. HPV load was higher in individuals with multiple HPV-OLs than in solitary lesions (4.9 vs. 3.2 Log 10 copies/ml, p = .090) and in HPV-32 + than in HPV-13 + (8.3 vs. 6.4 Log 10 copies/ml, p = .014). Multiple HPV-OLs showed high HPV loads, possibly indicating transcriptional activity of the virus; however, in the HIV setting, the individual and local immunological response could be the key process. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. All rights reserved.

Inhibition of HIV transmission in human cervicovaginal explants and humanized mice using CD4 aptamer-siRNA chimeras

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Wheeler, Lee Adam; Trifonova, Radiana; Vrbanac, Vladimir; Basar, Emre; McKernan, Shannon; Xu, Zhan; Seung, Edward; Deruaz, Maud; Dudek, Tim; Einarsson, Jon Ivar; Yang, Linda; Allen, Todd M.; Luster, Andrew D.; Tager, Andrew M.; Dykxhoorn, Derek M.; Lieberman, Judy

2011-01-01

The continued spread of the HIV epidemic underscores the need to interrupt transmission. One attractive strategy is a topical vaginal microbicide. Sexual transmission of herpes simplex virus type 2 (HSV-2) in mice can be inhibited by intravaginal siRNA application. To overcome the challenges of knocking down gene expression in immune cells susceptible to HIV infection, we used chimeric RNAs composed of an aptamer fused to an siRNA for targeted gene knockdown in cells bearing an aptamer-binding receptor. Here, we showed that CD4 aptamer-siRNA chimeras (CD4-AsiCs) specifically suppress gene expression in CD4+ T cells and macrophages in vitro, in polarized cervicovaginal tissue explants, and in the female genital tract of humanized mice. CD4-AsiCs do not activate lymphocytes or stimulate innate immunity. CD4-AsiCs that knock down HIV genes and/or CCR5 inhibited HIV infection in vitro and in tissue explants. When applied intravaginally to humanized mice, CD4-AsiCs protected against HIV vaginal transmission. Thus, CD4-AsiCs could be used as the active ingredient of a microbicide to prevent HIV sexual transmission. PMID:21576818

Endogenous MCM7 microRNA cluster as a novel platform to multiplex small interfering and nucleolar RNAs for combinational HIV-1 gene therapy.

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Chung, Janet; Zhang, Jane; Li, Haitang; Ouellet, Dominique L; DiGiusto, David L; Rossi, John J

2012-11-01

Combinational therapy with small RNA inhibitory agents against multiple viral targets allows efficient inhibition of viral production by controlling gene expression at critical time points. Here we explore combinations of different classes of therapeutic anti-HIV-1 RNAs expressed from within the context of an intronic MCM7 (minichromosome maintenance complex component-7) platform that naturally harbors 3 microRNAs (miRNAs). We replaced the endogenous miRNAs with anti-HIV small RNAs, including small interfering RNAs (siRNAs) targeting HIV-1 tat and rev messages that function to induce post-transcriptional gene silencing by the RNA interference pathway, a nucleolar-localizing RNA ribozyme that targets the conserved U5 region of HIV-1 transcripts for degradation, and finally nucleolar trans-activation response (TAR) and Rev-binding element (RBE) RNA decoys designed to sequester HIV-1 Tat and Rev proteins inside the nucleolus. We demonstrate the versatility of the MCM7 platform in expressing and efficiently processing the siRNAs as miRNA mimics along with nucleolar small RNAs. Furthermore, three of the combinatorial constructs tested potently suppressed viral replication during a 1-month HIV challenge, with greater than 5-log inhibition compared with untransduced, HIV-1-infected CEM T lymphocytes. One of the most effective constructs contains an anti-HIV siRNA combined with a nucleolar-localizing U5 ribozyme and TAR decoy. This represents the first efficacious example of combining Drosha-processed siRNAs with small nucleolar ribonucleoprotein (snoRNP)-processed nucleolar RNA chimeras from a single intron platform for effective inhibition of viral replication. Moreover, we demonstrated enrichment/selection for cells expressing levels of the antiviral RNAs that provide optimal inhibition under the selective pressure of HIV. The combinations of si/snoRNAs represent a new paradigm for combinatorial RNA-based gene therapy applications.

Dataset of longitudinal analysis of tear cytokine levels, CD4, CD8 counts and HIV viral load in dry eye patients with HIV infection

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Praveen Kumar Balne

2017-04-01

Full Text Available The data presented in this article shows the longitudinal analysis of tear fluid cytokine profiles, blood CD4 and CD8 counts and HIV viral load in 34 dry eye patients with HIV infection during the HAART therapy. Clinical samples were collected from HIV patients with dry eye disease at the time of presentation to the clinic (visit 1, three months (visit 2 and 6 months (visit 3 after the presentation. At each time point tear samples were evaluated for 41 cytokines using Luminex bead based multiplex assay and blood samples were tested for HIV viral load and CD4 and CD8 counts.

Novel RNA Duplex Locks HIV-1 in a Latent State via Chromatin-mediated Transcriptional Silencing

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Chantelle Ahlenstiel

2015-01-01

Full Text Available Transcriptional gene silencing (TGS of mammalian genes can be induced by short interfering RNA (siRNA targeting promoter regions. We previously reported potent TGS of HIV-1 by siRNA (PromA, which targets tandem NF-κB motifs within the viral 5′LTR. In this study, we screened a siRNA panel with the aim of identifying novel 5′LTR targets, to provide multiplexing potential with enhanced viral silencing and application toward developing alternate therapeutic strategies. Systematic examination identified a novel siRNA target, si143, confirmed to induce TGS as the silencing mechanism. TGS was prolonged with virus suppression >12 days, despite a limited ability to induce post- TGS. Epigenetic changes associated with silencing were suggested by partial reversal by histone deacetylase inhibitors and confirmed by chromatin immunoprecipitation analyses, which showed induction of H3K27me3 and H3K9me3, reduction in H3K9Ac, and recruitment of argonaute-1, all characteristic marks of heterochromatin and TGS. Together, these epigenetic changes mimic those associated with HIV-1 latency. Further, robust resistance to reactivation was observed in the J-Lat 9.2 cell latency model, when transduced with shPromA and/or sh143. These data support si/shRNA-mediated TGS approaches to HIV-1 and provide alternate targets to pursue a functional cure, whereby the viral reservoir is locked in latency following antiretroviral therapy cessation.

Rational design of micro-RNA-like bifunctional siRNAs targeting HIV and the HIV coreceptor CCR5.

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Ehsani, Ali; Saetrom, Pål; Zhang, Jane; Alluin, Jessica; Li, Haitang; Snøve, Ola; Aagaard, Lars; Rossi, John J

2010-04-01

Small-interfering RNAs (siRNAs) and micro-RNAs (miRNAs) are distinguished by their modes of action. SiRNAs serve as guides for sequence-specific cleavage of complementary mRNAs and the targets can be in coding or noncoding regions of the target transcripts. MiRNAs inhibit translation via partially complementary base-pairing to 3' untranslated regions (UTRs) and are generally ineffective when targeting coding regions of a transcript. In this study, we deliberately designed siRNAs that simultaneously direct cleavage and translational suppression of HIV RNAs, or cleavage of the mRNA encoding the HIV coreceptor CCR5 and suppression of translation of HIV. These bifunctional siRNAs trigger inhibition of HIV infection and replication in cell culture. The design principles have wide applications throughout the genome, as about 90% of genes harbor sites that make the design of bifunctional siRNAs possible.

The prion protein has RNA binding and chaperoning properties characteristic of nucleocapsid protein NCP7 of HIV-1.

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Gabus, C; Derrington, E; Leblanc, P; Chnaiderman, J; Dormont, D; Swietnicki, W; Morillas, M; Surewicz, W K; Marc, D; Nandi, P; Darlix, J L

2001-06-01

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases associated with the accumulation of a protease-resistant form of the prion protein (PrP). Although PrP is conserved in vertebrates, its function remains to be identified. In vitro PrP binds large nucleic acids causing the formation of nucleoprotein complexes resembling human immunodeficiency virus type 1 (HIV-1) nucleocapsid-RNA complexes and in vivo MuLV replication accelerates the scrapie infectious process, suggesting possible interactions between retroviruses and PrP. Retroviruses, including HIV-1 encode a major nucleic acid binding protein (NC protein) found within the virus where 2000 NC protein molecules coat the dimeric genome. NC is required in virus assembly and infection to chaperone RNA dimerization and packaging and in proviral DNA synthesis by reverse transcriptase (RT). In HIV-1, 5'-leader RNA/NC interactions appear to control these viral processes. This prompted us to compare and contrast the interactions of human and ovine PrP and HIV-1 NCp7 with HIV-1 5'-leader RNA. Results show that PrP has properties characteristic of NCp7 with respect to viral RNA dimerization and proviral DNA synthesis by RT. The NC-like properties of huPrP map to the N-terminal region of huPrP. Interestingly, PrP localizes in the membrane and cytoplasm of PrP-expressing cells. These findings suggest that PrP is a multifunctional protein possibly participating in nucleic acid metabolism.

Perceived viral load, but not actual HIV-1-RNA load, is associated with sexual risk behaviour among HIV-infected homosexual men

NARCIS (Netherlands)

Stolte, Ineke G.; de Wit, John B. F.; van Eeden, Arne; Coutinho, Roel A.; Dukers, Nicole H. T. M.

2004-01-01

BACKGROUND: Increases in sexual risk behaviour and sexually transmitted infections among HIV-infected homosexual men after the introduction of highly active antiretroviral therapy (HAART) confirm the need for innovative prevention activities. The present study focused on time trends in sexual risk

HIV-1 Tat C-mediated regulation of tumor necrosis factor receptor-associated factor-3 by microRNA 32 in human microglia

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Mishra Ritu

2012-06-01

Full Text Available Abstract Background HIV-1 Tat protein is known to be associated with neuroinflammation, a condition that develops in almost half of patients infected with HIV-1. HIV-1 Tat can alter glial neuroprotective functions, leading to neurotoxicity within the CNS. HIV-1 Tat is known to be secreted from productively infected cells and can affect neighboring uninfected cells by modulating cellular gene expression in a bystander fashion. Methods We were interested to study whether exogenous exposure to HIV-1 Tat-C protein perturbs the microRNA (miRNA expression profile of human microglial cells, leading to altered protein expression. We used protein expression and purification, miRNA overexpression, miRNA knockdown, transfection, site-directed mutagenesis, real-time PCR, luciferase assay and western blotting techniques to perform our study. Results HIV-1 Tat-C treatment of human microglial cells resulted in a dose-dependent increase in miR-32 expression. We found that tumor necrosis factor-receptor–associated factor 3 TRAF3 is a direct target for miR-32, and overexpression of miR-32 in CHME3 cells decreased TRAF3 both at the mRNA and the protein level. Recovery of TRAF3 protein expression after transfection of anti-miR-32 and the results of the luciferase reporter assay provided direct evidence of TRAF3 regulation by miR-32. We found that the regulation of interferon regulatory factor 3 (IRF3 and IRF7 is controlled by cellular levels of TRAF3 protein in microglial cells, as after overexpression of miR-32 and application of anti-miR-32, expression levels of IRF3 and IRF7 were inversely regulated by expression levels of TRAF3. Thus, our results suggest a novel miRNA mediated mechanism for regulation of TRAF3 in human microglial cells exposed to HIV-1 Tat C protein. These results may help to elucidate the detrimental neuroinflammatory consequences of HIV-1 Tat C protein in bystander fashion. Conclusion HIV-1 Tat protein can modulate TRAF3 expression through

Nanoparticle orientation to control RNA loading and ligand display on extracellular vesicles for cancer regression

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Pi, Fengmei; Binzel, Daniel W.; Lee, Tae Jin; Li, Zhefeng; Sun, Meiyan; Rychahou, Piotr; Li, Hui; Haque, Farzin; Wang, Shaoying; Croce, Carlo M.; Guo, Bin; Evers, B. Mark; Guo, Peixuan

2018-01-01

Nanotechnology offers many benefits, and here we report an advantage of applying RNA nanotechnology for directional control. The orientation of arrow-shaped RNA was altered to control ligand display on extracellular vesicle membranes for specific cell targeting, or to regulate intracellular trafficking of small interfering RNA (siRNA) or microRNA (miRNA). Placing membrane-anchoring cholesterol at the tail of the arrow results in display of RNA aptamer or folate on the outer surface of the extracellular vesicle. In contrast, placing the cholesterol at the arrowhead results in partial loading of RNA nanoparticles into the extracellular vesicles. Taking advantage of the RNA ligand for specific targeting and extracellular vesicles for efficient membrane fusion, the resulting ligand-displaying extracellular vesicles were capable of specific delivery of siRNA to cells, and efficiently blocked tumour growth in three cancer models. Extracellular vesicles displaying an aptamer that binds to prostate-specific membrane antigen, and loaded with survivin siRNA, inhibited prostate cancer xenograft. The same extracellular vesicle instead displaying epidermal growth-factor receptor aptamer inhibited orthotopic breast cancer models. Likewise, survivin siRNA-loaded and folate-displaying extracellular vesicles inhibited patient-derived colorectal cancer xenograft.

Quality control assessment of human immunodeficiency virus type 2 (HIV-2) viral load quantification assays: results from an international collaboration on HIV-2 infection in 2006

NARCIS (Netherlands)

Damond, Florence; Benard, Antoine; Ruelle, Jean; Alabi, Abraham; Kupfer, Bernd; Gomes, Perpetua; Rodes, Berta; Albert, Jan; Böni, Jürg; Garson, Jeremy; Ferns, Bridget; Matheron, Sophie; Chene, Geneviève; Brun-Vezinet, Françoise; Goubau, Patrick; Campa, Pauline; Descamps, Diane; Simon, François; Taieb, Audrey; Autran, Brigitte; Cotten, Matt; Jaye, Assan; Peterson, Kevin; Rowland-Jones, Sarah; Rockstroh, Jürgen; Schwarze-Zander, Carolynne; de Wolf, Frank; van Sighem, Ard; Reiss, Peter; van der Loeff, Maarten Schim; Schutten, Martin; Camacho, Ricardo; Mansinho, Kamal; Antunes, Francisco; Luis, Franca; Valadas, Emilia; Toro, Carlos; Soriano, Vicente; Gyllensten, Katarina; Sonnerborg, Anders; Yilmaz, Aylin; Gisslén, Magnus; Calmy, Alexandra; Rickenbach, Martin; Pillay, Deenan; Tosswill, Jennifer; Anderson, Jane; Chadwick, David

2008-01-01

Human immunodeficiency virus type 2 (HIV-2) RNA quantification assays used in nine laboratories of the ACHI(E)V(2E) (A Collaboration on HIV-2 Infection) study group were evaluated. In a blinded experimental design, laboratories quantified three series of aliquots of an HIV-2 subtype A strain, each

Prolonged elevation of viral loads in HIV-1-infected children

African Journals Online (AJOL)

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2010-11-09

Nov 9, 2010 ... treatment i.e. mean difference (signed-rank test) in viral load “before” and .... We used Abbott Determine and Unigold as the rapid HIV test kits. The ..... N: Number of patients, *: Wilcoxon signed ranks test for Median difference ...

A Multiple siRNA-Based Anti-HIV/SHIV Microbicide Shows Protection in Both In Vitro and In Vivo Models.

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Sandhya Boyapalle

Full Text Available Human immunodeficiency virus (HIV types 1 and 2 (HIV-1 and HIV-2 are the etiologic agents of AIDS. Most HIV-1 infected individuals worldwide are women, who acquire HIV infections during sexual contact. Blocking HIV mucosal transmission and local spread in the female lower genital tract is important in preventing infection and ultimately eliminating the pandemic. Microbicides work by destroying the microbes or preventing them from establishing an infection. Thus, a number of different types of microbicides are under investigation, however, the lack of their solubility and bioavailability, and toxicity has been major hurdles. Herein, we report the development of multifunctional chitosan-lipid nanocomplexes that can effectively deliver plasmids encoding siRNA(s as microbicides without adverse effects and provide significant protection against HIV in both in vitro and in vivo models. Chitosan or chitosan-lipid (chlipid was complexed with a cocktail of plasmids encoding HIV-1-specific siRNAs (psiRNAs and evaluated for their efficacy in HEK-293 cells, PBMCs derived from nonhuman primates, 3-dimensional human vaginal ectocervical tissue (3D-VEC model and also in non-human primate model. Moreover, prophylactic administration of the chlipid to deliver a psiRNA cocktail intravaginally with a cream formulation in a non-human primate model showed substantial reduction of SHIV (simian/human immunodeficiency virus SF162 viral titers. Taken together, these studies demonstrate the potential of chlipid-siRNA nanocomplexes as a potential genetic microbicide against HIV infections.

HIV Maintains an Evolving and Dispersed Population in Multiple Tissues during Suppressive Combined Antiretroviral Therapy in Individuals with Cancer.

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Rose, Rebecca; Lamers, Susanna L; Nolan, David J; Maidji, Ekaterina; Faria, N R; Pybus, Oliver G; Dollar, James J; Maruniak, Samuel A; McAvoy, Andrew C; Salemi, Marco; Stoddart, Cheryl A; Singer, Elyse J; McGrath, Michael S

2016-10-15

While combined antiretroviral therapy (cART) can result in undetectable plasma viral loads, it does not eradicate HIV infection. Furthermore, HIV-infected individuals while on cART remain at an increased risk of developing serious comorbidities, such as cancer, neurological disease, and atherosclerosis, suggesting that during cART, tissue-based HIV may contribute to such pathologies. We obtained DNA and RNA env, nef, and pol sequences using single-genome sequencing from postmortem tissues of three HIV(+) cART-treated (cART(+)) individuals with undetectable viral load and metastatic cancer at death and performed time-scaled Bayesian evolutionary analyses. We used a sensitive in situ hybridization technique to visualize HIV gag-pol mRNA transcripts in cerebellum and lymph node tissues from one patient. Tissue-associated virus evolved at similar rates in cART(+) and cART-naive (cART(-)) patients. Phylogenetic trees were characterized by two distinct features: (i) branching patterns consistent with constant viral evolution and dispersal among tissues and (ii) very recently derived clades containing both DNA and RNA sequences from multiple tissues. Rapid expansion of virus near death corresponded to wide-spread metastasis. HIV RNA(+) cells clustered in cerebellum tissue but were dispersed in lymph node tissue, mirroring the evolutionary patterns observed for that patient. Activated, infiltrating macrophages were associated with HIV RNA. Our data provide evidence that tissues serve as a sanctuary for wild-type HIV during cART and suggest the importance of macrophages as an alternative reservoir and mechanism of virus spread. Combined antiretroviral therapy (cART) reduces plasma HIV to undetectable levels; however, removal of cART results in plasma HIV rebound, thus highlighting its inability to entirely rid the body of infection. Additionally, HIV-infected individuals on cART remain at high risk of serious diseases, which suggests a contribution from residual HIV. In

The RNA helicase DDX1 is involved in restricted HIV-1 Rev function in human astrocytes

International Nuclear Information System (INIS)

Fang Jianhua; Acheampong, Edward; Dave, Rajnish; Wang Fengxiang; Mukhtar, Muhammad; Pomerantz, Roger J.

2005-01-01

Productive infection by human immunodeficiency virus type I (HIV-1) in the central nervous system (CNS) involves mainly macrophages and microglial cells. A frequency of less than 10% of human astrocytes is estimated to be infectable with HIV-1. Nonetheless, this relatively low percentage of infected astrocytes, but associated with a large total number of astrocytic cells in the CNS, makes human astrocytes a critical part in the analyses of potential HIV-1 reservoirs in vivo. Investigations in astrocytic cell lines and primary human fetal astrocytes revealed that limited HIV-1 replication in these cells resulted from low-level viral entry, transcription, viral protein processing, and virion maturation. Of note, a low ratio of unspliced versus spliced HIV-1-specific RNA was also investigated, as Rev appeared to act aberrantly in astrocytes, via loss of nuclear and/or nucleolar localization and diminished Rev-mediated function. Host cellular machinery enabling Rev function has become critical for elucidation of diminished Rev activity, especially for those factors leading to RNA metabolism. We have recently identified a DEAD-box protein, DDX1, as a Rev cellular co-factor and now have explored its potential importance in astrocytes. Cells were infected with HIV-1 pseudotyped with envelope glycoproteins of amphotropic murine leukemia viruses (MLV). Semi-quantitative reverse transcriptase-polymerase chain reactions (RT-PCR) for unspliced, singly-spliced, and multiply-spliced RNA clearly showed a lower ratio of unspliced/singly-spliced over multiply-spliced HIV-1-specific RNA in human astrocytes as compared to Rev-permissive, non-glial control cells. As well, the cellular localization of Rev in astrocytes was cytoplasmically dominant as compared to that of Rev-permissive, non-glial controls. This endogenous level of DDX1 expression in astrocytes was demonstrated directly to lead to a shift of Rev sub-cellular distribution dominance from nuclear and/or nucleolar to

Alcohol Types and HIV Disease Progression Among HIV-Infected Drinkers Not Yet on Antiretroviral Therapy in Russia and Uganda.

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Asiimwe, Stephen B; Fatch, Robin; Patts, Gregory; Winter, Michael; Lloyd-Travaglini, Christine; Emenyonu, Nneka; Muyindike, Winnie; Kekibiina, Allen; Blokhina, Elena; Gnatienko, Natalia; Kruptisky, Evgeny; Cheng, Debbie M; Samet, Jeffrey H; Hahn, Judith A

2017-11-01

In HIV-infected drinkers, alcohol types more likely to cause inflammation could plausibly increase the risk of HIV disease progression. We therefore assessed the association between alcohol type and plasma HIV RNA level (HIV viral load) among HIV-infected drinkers not on antiretroviral therapy (ART) in Russia and Uganda. We analyzed the data of participants from cohorts in Russia and Uganda and assessed their HIV viral load at enrollment by the alcohol type predominantly consumed. We defined predominant alcohol type as the alcohol type contributing >50% of total alcohol consumption in the 1 month (Russia) or 3 months (Uganda) prior to enrollment. Using multiple linear regression, we compared log 10 HIV viral load by predominant alcohol type, controlling for age, gender, socioeconomic status, total number of standard drinks, frequency of drinking ≥6 drinks/occasion, and in Russia, history of injection drug use. Most participants (99.2% of 261 in Russia and 98.9% of 352 in Uganda) predominantly drank one alcohol type. In Russia, we did not find evidence for differences in viral load levels between drinkers of fortified wine (n = 5) or hard liquor (n = 49), compared to drinkers of beer/low-ethanol-content cocktails (n = 163); however, wine/high-ethanol-content cocktail drinkers (n = 42) had higher mean log 10 viral load than beer/low-ethanol-content cocktail drinkers (β = 0.38, 95% CI 0.07-0.69; p = 0.02). In Uganda, we did not find evidence for differences in viral load levels between drinkers of locally-brewed beer (n = 41), commercially-distilled spirits (n = 38), or locally-distilled spirits (n = 43), compared to drinkers of commercially-made beer (n = 218); however, wine drinkers (n = 8) had lower mean log 10 HIV viral load (β = -0.65, 95% CI -1.36 to 0.07, p = 0.08), although this did not reach statistical significance. Among HIV-infected drinkers not yet on ART in Russia and Uganda, we observed an association between the

Effect of genital herpes on cervicovaginal HIV shedding in women co-infected with HIV AND HSV-2 in Tanzania.

Science.gov (United States)

Todd, Jim; Riedner, Gabriele; Maboko, Leonard; Hoelscher, Michael; Weiss, Helen A; Lyamuya, Eligius; Mabey, David; Rusizoka, Mary; Belec, Laurent; Hayes, Richard

2013-01-01

To compare the presence and quantity of cervicovaginal HIV among HIV seropositive women with clinical herpes, subclinical HSV-2 infection and without HSV-2 infection respectively; to evaluate the association between cervicovaginal HIV and HSV shedding; and identify factors associated with quantity of cervicovaginal HIV. Four groups of HIV seropositive adult female barworkers were identified and examined at three-monthly intervals between October 2000 and March 2003 in Mbeya, Tanzania: (1) 57 women at 70 clinic visits with clinical genital herpes; (2) 39 of the same women at 46 clinic visits when asymptomatic; (3) 55 HSV-2 seropositive women at 60 clinic visits who were never observed with herpetic lesions; (4) 18 HSV-2 seronegative women at 45 clinic visits. Associations of genital HIV shedding with HIV plasma viral load (PVL), herpetic lesions, HSV shedding and other factors were examined. Prevalence of detectable genital HIV RNA varied from 73% in HSV-2 seronegative women to 94% in women with herpetic lesions (geometric means 1634 vs 3339 copies/ml, p = 0.03). In paired specimens from HSV-2 positive women, genital HIV viral shedding was similar during symptomatic and asymptomatic visits. On multivariate regression, genital HIV RNA (log10 copies/mL) was closely associated with HIV PVL (β = 0.51 per log10 copies/ml increase, 95%CI:0.41-0.60, pgenital HIV than the presence of herpetic lesions. These data support a role of HSV-2 infection in enhancing HIV transmissibility.

Methamphetamine inhibits HIV-1 replication in CD4+ T cells by modulating anti-HIV-1 miRNA expression.

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Mantri, Chinmay K; Mantri, Jyoti V; Pandhare, Jui; Dash, Chandravanu

2014-01-01

Methamphetamine is the second most frequently used illicit drug in the United States. Methamphetamine abuse is associated with increased risk of HIV-1 acquisition, higher viral loads, and enhanced HIV-1 pathogenesis. Although a direct link between methamphetamine abuse and HIV-1 pathogenesis remains to be established in patients, methamphetamine has been shown to increase HIV-1 replication in macrophages, dendritic cells, and cells of HIV transgenic mice. Intriguingly, the effects of methamphetamine on HIV-1 replication in human CD4(+) T cells that serve as the primary targets of infection in vivo are not clearly understood. Therefore, we examined HIV-1 replication in primary CD4(+) T cells in the presence of methamphetamine in a dose-dependent manner. Our results demonstrate that methamphetamine had a minimal effect on HIV-1 replication at concentrations of 1 to 50 μmol/L. However, at concentrations >100 μmol/L, it inhibited HIV-1 replication in a dose-dependent manner. We also discovered that methamphetamine up-regulated the cellular anti-HIV-1 microRNAs (miR-125b, miR-150, and miR-28-5p) in CD4(+) T cells. Knockdown experiments illustrated that up-regulation of the anti-HIV miRNAs inhibited HIV-1 replication. These results are contrary to the paradigm that methamphetamine accentuates HIV-1 pathogenesis by increasing HIV-1 replication. Therefore, our findings underline the complex interaction between drug use and HIV-1 and necessitate comprehensive understanding of the effects of methamphetamine on HIV-1 pathogenesis. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Gastrointestinal viral load and enteroendocrine cell number are associated with altered survival in HIV-1 infected individuals.

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Guido van Marle

Full Text Available Human immunodeficiency virus type 1 (HIV-1 infects and destroys cells of the immune system leading to an overt immune deficiency known as HIV acquired immunodeficiency syndrome (HIV/AIDS. The gut associated lymphoid tissue is one of the major lymphoid tissues targeted by HIV-1, and is considered a reservoir for HIV-1 replication and of major importance in CD4+ T-cell depletion. In addition to immunodeficiency, HIV-1 infection also directly causes gastrointestinal (GI dysfunction, also known as HIV enteropathy. This enteropathy can manifest itself as many pathological changes in the GI tract. The objective of this study was to determine the association of gut HIV-1 infection markers with long-term survival in a cohort of men who have sex with men (MSM enrolled pre-HAART (Highly Active Antiretroviral Therapy. We examined survival over 15-years in a cohort of 42 HIV-infected cases: In addition to CD4+ T cell counts and HIV-1 plasma viral load, multiple gut compartment (duodenum and colon biopsies were taken by endoscopy every 6 months during the initial 3-year period. HIV-1 was cultured from tissues and phenotyped and viral loads in the gut tissues were determined. Moreover, the tissues were subjected to an extensive assessment of enteroendocrine cell distribution and pathology. The collected data was used for survival analyses, which showed that patients with higher gut tissue viral load levels had a significantly worse survival prognosis. Moreover, lower numbers of serotonin (duodenum and somatostatin (duodenum and colon immunoreactive cell counts in the gut tissues of patients was associated with significant lower survival prognosis. Our study, suggested that HIV-1 pathogenesis and survival prognosis is associated with altered enteroendocrine cell numbers, which could point to a potential role for enteroendocrine function in HIV infection and pathogenesis.

Systemic Immune Activation and HIV Shedding in the Female Genital Tract.

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Spencer, LaShonda Y; Christiansen, Shawna; Wang, Chia-Hao H; Mack, Wendy J; Young, Mary; Strickler, Howard D; Anastos, Kathryn; Minkoff, Howard; Cohen, Mardge; Geenblatt, Ruth M; Karim, Roksana; Operskalski, Eva; Frederick, Toni; Homans, James D; Landay, Alan; Kovacs, Andrea

2016-02-01

Plasma HIV RNA is the most significant determinant of cervical HIV shedding. However, shedding is also associated with sexually transmitted infections (STIs) and cervical inflammation. The mechanism by which this occurs is poorly understood. There is evidence that systemic immune activation promotes viral entry, replication, and HIV disease progression. We hypothesized that systemic immune activation would be associated with an increase in HIV genital shedding. Clinical assessments, HIV RNA in plasma and genital secretions, and markers of immune activation (CD38(+)DR(+) and CD38(-)DR(-)) on CD4(+) and CD8(+) T cells in blood were evaluated in 226 HIV+ women enrolled in the Women's Interagency HIV Study. There were 569 genital evaluations of which 159 (28%) exhibited HIV RNA shedding, defined as HIV viral load >80 copies per milliliter. We tested associations between immune activation and shedding using generalized estimating equations with logit link function. In the univariate model, higher levels of CD4(+) and CD8(+) T-cell activation in blood were significantly associated with genital tract shedding. However, in the multivariate model adjusting for plasma HIV RNA, STIs, and genital tract infections, only higher levels of resting CD8(+) T cells (CD38(-)DR(-)) were significantly inversely associated with HIV shedding in the genital tract (odds ratios = 0.44, 95% confidence interval: 0.21 to 0.9, P = 0.02). The association of systemic immune activation with genital HIV shedding is multifactorial. Systemic T-cell activation is associated with genital tract shedding in univariate analysis but not when adjusting for plasma HIV RNA, STIs, and genital tract infections. In addition, women with high percentage of resting T cells are less likely to have HIV shedding compared with those with lower percentages. These findings suggest that a higher percentage of resting cells, as a result of maximal viral suppression with treatment, may decrease local genital activation, HIV

The role of Vif oligomerization and RNA chaperone activity in HIV-1 replication.

Science.gov (United States)

Batisse, Julien; Guerrero, Santiago; Bernacchi, Serena; Sleiman, Dona; Gabus, Caroline; Darlix, Jean-Luc; Marquet, Roland; Tisné, Carine; Paillart, Jean-Christophe

2012-11-01

The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells that involve most natural HIV-1 target cells. Vif counteracts the packaging of two cellular cytidine deaminases named APOBEC3G (A3G) and A3F by diverse mechanisms including the recruitment of an E3 ubiquitin ligase complex and the proteasomal degradation of A3G/A3F, the inhibition of A3G mRNA translation or by a direct competition mechanism. In addition, Vif appears to be an active partner of the late steps of viral replication by participating in virus assembly and Gag processing, thus regulating the final stage of virion formation notably genomic RNA dimerization and by inhibiting the initiation of reverse transcription. Vif is a small pleiotropic protein with multiple domains, and recent studies highlighted the importance of Vif conformation and flexibility in counteracting A3G and in binding RNA. In this review, we will focus on the oligomerization and RNA chaperone properties of Vif and show that the intrinsic disordered nature of some Vif domains could play an important role in virus assembly and replication. Experimental evidence demonstrating the RNA chaperone activity of Vif will be presented. Copyright © 2012 Elsevier B.V. All rights reserved.

APOBEC3G inhibits HIV-1 RNA elongation by inactivating the viral trans-activation response element.

Science.gov (United States)

Nowarski, Roni; Prabhu, Ponnandy; Kenig, Edan; Smith, Yoav; Britan-Rosich, Elena; Kotler, Moshe

2014-07-29

Deamination of cytidine residues in viral DNA is a major mechanism by which APOBEC3G (A3G) inhibits vif-deficient human immunodeficiency virus type 1 (HIV-1) replication. dC-to-dU transition following RNase-H activity leads to viral cDNA degradation, production of non-functional proteins, formation of undesired stop codons and decreased viral protein synthesis. Here, we demonstrate that A3G provides an additional layer of defense against HIV-1 infection dependent on inhibition of proviral transcription. HIV-1 transcription elongation is regulated by the trans-activation response (TAR) element, a short stem-loop RNA structure required for elongation factors binding. Vif-deficient HIV-1-infected cells accumulate short viral transcripts and produce lower amounts of full-length HIV-1 transcripts due to A3G deamination of the TAR apical loop cytidine, highlighting the requirement for TAR loop integrity in HIV-1 transcription. We further show that free single-stranded DNA (ssDNA) termini are not essential for A3G activity and a gap of CCC motif blocked with juxtaposed DNA or RNA on either or 3'+5' ends is sufficient for A3G deamination. These results identify A3G as an efficient mutator and that deamination of (-)SSDNA results in an early block of HIV-1 transcription. Copyright © 2014 Elsevier Ltd. All rights reserved.

Nanoparticles containing siRNA to silence CD4 and CCR5 reduce expression of these receptors and inhibit HIV-1 infection in human female reproductive tract tissue explants

Directory of Open Access Journals (Sweden)

Susan K. Eszterhas

2011-09-01

Full Text Available Human Immunodeficiency Virus-type 1 (HIV- 1 binds to CD4 and CCR5 receptors on target cells in the human female reproductive tract. We sought to determine whether reducing levels of messenger RNA (mRNA transcripts that encode these receptors in female reproductive tract cells could protect mucosal tissue explants from HIV- 1 infection. Explants prepared from the endometrium, endocervix, and ectocervix of hysterectomy tissues from HIV-1 sero-negative women were exposed to nanoparticles containing CD4- and CCR5-specific short-interfering RNA (siRNA sequences. Explants were then exposed two days later to HIV-1, and HIV-1 reverse transcripts were measured five days post-infection. Explants treated with nanoparticles containing CD4- and CCR5-specific siRNA showed reduced levels of CD4 and CCR5 transcripts, and significantly lower levels of HIV-1 reverse transcripts compared to those treated with an irrelevant siRNA. In female reproductive tract explants and in peripheral blood cell cultures, siRNA transfection induced the secretion of IFN-alpha (IFN-α, a potent antiviral cytokine. In female mice, murine-specific Cd4-siRNA nanoparticles instilled within the uterus significantly reduced murine Cd4 transcripts by day 3. Our findings demonstrate that siRNA nanoparticles reduce expression of HIV-1 infectivity receptors in human female reproductive tract tissues and also inhibit HIV-1 infection. Murine studies demonstrate that nanoparticles can penetrate the reproductive tract tissues in vivo and silence gene expression. The induction of IFN-α after siRNA transfection can potentially contribute to the antiviral effect. These findings support the therapeutic development of nanoparticles to deliver siRNA molecules to silence host cell receptors in the female reproductive tract as a novel microbicide to inhibit mucosal HIV-1 transmission.

Association of High-Risk Human Papillomavirus with Genital Tract Mucosal Immune Factors In HIV-Infected Women

Science.gov (United States)

Buckley, Niall; Huber, Ashley; Lo, Yungtai; Castle, Philip E.; Kemal, Kimdar; Burk, Robert D.; Strickler, Howard D.; Einstein, Mark H.; Young, Mary; Anastos, Kathryn; Herold, Betsy C.

2015-01-01

Problem High-risk human papillomavirus (HR-HPV) is prevalent in HIV-infected women and may be associated with mucosal changes that promote HIV replication. Method of Study Innate immune molecules, antimicrobial activity, HIV RNA, and HPV DNA genotypes were measured in a cross-sectional study of 128 HIV-infected women categorized into HPV-16 (n=8), other HR-HPV (n=41), and non-HR-HPV controls (n=79). Results Compared to controls, HR-HPV groups had higher plasma viral loads (p=0.004), lower CD4 cells (p=0.02), more genital tract HIV RNA (p=0.03), greater number of different HPV types (p<0.001), higher cervicovaginal lavage (CVL) IL-1α (p=0.03) and human beta defensin 2 (HBD2) (p=0.049), and less anti-HIVBal activity (p=0.03). HPV-16 remained significantly associated with higher HBD2 (p=0.03), higher IL-1α (p=0.009), and lower anti-HIVBaL activity (p=0.03) compared to controls after adjusting for plasma viral load and CD4 T cell count. Conclusion HR-HPV is associated with mucosal changes in HIV-infected women that could adversely impact genital tract health. PMID:26685115

Time to viral load suppression in antiretroviral-naive and -experienced HIV-infected pregnant women on highly active antiretroviral therapy: implications for pregnant women presenting late in gestation.

Science.gov (United States)

Aziz, N; Sokoloff, A; Kornak, J; Leva, N V; Mendiola, M L; Levison, J; Feakins, C; Shannon, M; Cohan, D

2013-11-01

To compare time to achieve viral load HIV-infected antiretroviral (ARV) -naive versus ARV-experienced pregnant women on highly active antiretroviral therapy (HAART). Retrospective cohort study. Three university medical centers, USA. HIV-infected pregnant women initiated or restarted on HAART during pregnancy. We calculated time to viral load HIV-infected pregnant women on HAART who reported at least 50% adherence, stratifying based on previous ARV exposure history. Time to HIV viral load HIV-infected pregnant women, comprising 76 ARV-naive and 62 ARV-experienced. Ninety-three percent of ARV-naive women achieved a viral load HIV log10 viral load was associated with a later time of achieving viral load HIV log10 viral load was associated with a longer time of achieving viral load Pregnant women with ≥50% adherence, whether ARV-naive or ARV-experienced, on average achieve a viral load HIV log10 viral load were all statistically significant predictors of earlier time to achieve viral load <400 copies/ml and <1000 copies/ml. Increased CD4 count was statistically significant as a predictor of earlier time to achieve viral load <1000 copies/ml. © 2013 The Authors BJOG An International Journal of Obstetrics and Gynaecology © 2013 RCOG.

HIV-1 resistance conferred by siRNA cosuppression of CXCR4 and CCR5 coreceptors by a bispecific lentiviral vector

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Akkina Ramesh

2005-01-01

Full Text Available Abstract Background RNA interference (RNAi mediated by small interfering RNAs (siRNAs has proved to be a highly effective gene silencing mechanism with great potential for HIV/AIDS gene therapy. Previous work with siRNAs against cellular coreceptors CXCR4 and CCR5 had shown that down regulation of these surface molecules could prevent HIV-1 entry and confer viral resistance. Since monospecific siRNAs targeting individual coreceptors are inadequate in protecting against both T cell tropic (X4 and monocyte tropic (R5 viral strains simultaneously, bispecific constructs with dual specificity are required. For effective long range therapy, the bispecific constructs need to be stably transduced into HIV-1 target cells via integrating viral vectors. Results To achieve this goal, lentiviral vectors incorporating both CXCR4 and CCR5 siRNAs of short hairpin design were constructed. The CXCR4 siRNA was driven by a U6 promoter whereas the CCR5 siRNA was driven by an H1 promoter. A CMV promoter driven EGFP reporter gene is also incorporated in the bispecific construct. High efficiency transduction into coreceptor expressing Magi and Ghost cell lines with a concomitant down regulation of respective coreceptors was achieved with lentiviral vectors. When the siRNA expressing transduced cells were challenged with X4 and R5 tropic HIV-1, they demonstrated marked viral resistance. HIV-1 resistance was also observed in bispecific lentiviral vector transduced primary PBMCs. Conclusions Both CXCR4 and CCR5 coreceptors could be simultaneously targeted for down regulation by a single combinatorial lentiviral vector incorporating respective anti-coreceptor siRNAs. Stable down regulation of both the coreceptors protects cells against infection by both X4 and R5 tropic HIV-1. Stable down regulation of cellular molecules that aid in HIV-1 infection will be an effective strategy for long range HIV gene therapy.

Engineering HIV-1-resistant T-cells from short-hairpin RNA-expressing hematopoietic stem/progenitor cells in humanized BLT mice.

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Gene-Errol E Ringpis

Full Text Available Down-regulation of the HIV-1 coreceptor CCR5 holds significant potential for long-term protection against HIV-1 in patients. Using the humanized bone marrow/liver/thymus (hu-BLT mouse model which allows investigation of human hematopoietic stem/progenitor cell (HSPC transplant and immune system reconstitution as well as HIV-1 infection, we previously demonstrated stable inhibition of CCR5 expression in systemic lymphoid tissues via transplantation of HSPCs genetically modified by lentiviral vector transduction to express short hairpin RNA (shRNA. However, CCR5 down-regulation will not be effective against existing CXCR4-tropic HIV-1 and emergence of resistant viral strains. As such, combination approaches targeting additional steps in the virus lifecycle are required. We screened a panel of previously published shRNAs targeting highly conserved regions and identified a potent shRNA targeting the R-region of the HIV-1 long terminal repeat (LTR. Here, we report that human CD4(+ T-cells derived from transplanted HSPC engineered to co-express shRNAs targeting CCR5 and HIV-1 LTR are resistant to CCR5- and CXCR4- tropic HIV-1-mediated depletion in vivo. Transduction with the combination vector suppressed CXCR4- and CCR5- tropic viral replication in cell lines and peripheral blood mononuclear cells in vitro. No obvious cytotoxicity or interferon response was observed. Transplantation of combination vector-transduced HSPC into hu-BLT mice resulted in efficient engraftment and subsequent stable gene marking and CCR5 down-regulation in human CD4(+ T-cells within peripheral blood and systemic lymphoid tissues, including gut-associated lymphoid tissue, a major site of robust viral replication, for over twelve weeks. CXCR4- and CCR5- tropic HIV-1 infection was effectively inhibited in hu-BLT mouse spleen-derived human CD4(+ T-cells ex vivo. Furthermore, levels of gene-marked CD4(+ T-cells in peripheral blood increased despite systemic infection with either

Human Papillomavirus prevalence, viral load and cervical intraepithelial neoplasia in HIV-infected women

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José E. Levi

Full Text Available HIV-infected women from São Paulo city were enrolled in a cross-sectional study on Human Papillomavirus (HPV and cervical intraepithelial neoplasia (CIN prevalence and their association with laboratory markers of AIDS, namely HIV viral load and CD4+ cell counts. A cervical specimen was collected and submitted to Hybrid Capture, a test for HPV viral load determination. HPV-DNA was detected in 173 of 265 women (64.5%. Twenty (7.5% women were infected by one or more low-risk viruses, 89 (33% by one or more high-risk viruses, and 64 (24% harbored at least one HPV type from each risk group. Abnormal smears were observed in 19% of the patients, though there were no invasive carcinomas. Severely immunosuppressed patients (CD4/µL <100 were at the greatest risk of having a cytological abnormality and a high high-risk HPV viral load.

Human Papillomavirus prevalence, viral load and cervical intraepithelial neoplasia in HIV-infected women

Directory of Open Access Journals (Sweden)

Levi José E.

2002-01-01

Full Text Available HIV-infected women from São Paulo city were enrolled in a cross-sectional study on Human Papillomavirus (HPV and cervical intraepithelial neoplasia (CIN prevalence and their association with laboratory markers of AIDS, namely HIV viral load and CD4+ cell counts. A cervical specimen was collected and submitted to Hybrid Capture, a test for HPV viral load determination. HPV-DNA was detected in 173 of 265 women (64.5%. Twenty (7.5% women were infected by one or more low-risk viruses, 89 (33% by one or more high-risk viruses, and 64 (24% harbored at least one HPV type from each risk group. Abnormal smears were observed in 19% of the patients, though there were no invasive carcinomas. Severely immunosuppressed patients (CD4/µL <100 were at the greatest risk of having a cytological abnormality and a high high-risk HPV viral load.

Liver stiffness is not associated with short- and long-term plasma HIV RNA replication in immunocompetent patients with HIV infection and with HIV/HCV coinfection

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Parisi, Saverio Giuseppe; Basso, Monica; Mengoli, Carlo; Scaggiante, Renzo; Andreis, Samantha; Franzetti, Marzia Maria; Cattelan, Anna Maria; Zago, Daniela; Cruciani, Mario; Andreoni, Massimo; Piovesan, Sara; Palù, Giorgio; Alberti, Alfredo

2017-01-01

Background Human immunodeficiency virus (HIV) may be directly responsible for liver damage but there are contrasting data regarding the influence of detectable plasma viremia. We analyzed the influence of plasma HIV RNA (pHIV) detectability and of other clinical and viro-immunological variables on liver stiffness (LS) measurement in adult immunocompetent HIV-monoinfected patients and in patients coinfected with hepatitis C virus (HCV). Methods Logistic regression analysis was performed using the value of LS>7.1 kPa as the dependent variable. A linear regression model was applied using LS measurement after log10 transformation (lkpa) as the dependent variable and we analyzed the predicted values versus the observed lkpa values; pHIV was classified as detectable or undetectable in the 12- and 36-month study periods before LS measurement. Results We studied 251 patients (178 with HIV monoinfection), most of whom were on antiviral treatment; 36-month study time was available for 154 subjects. The mean CD4+ cell count was 634 cells/mm3 in HIV-monoinfected patients and 606 cells/mm3 in coinfected patients. No difference in LS was found between patients with detectable or undetectable pHIV in either the 12- or the 36-month study period before transient elastography. The mean LS was higher in HIV/HCV coinfected patients (P<0.0001) than in the HIV-monoinfected subjects; lkpa was positively correlated with HCV coinfection (P<0.0001) and aspartate aminotransferase levels (P<0.0001). Detectable pHIV failed to reach significance. Eight HIV-monoinfected patients had a predicted LS measurement lower than the observed one, while eight patients had the opposite result. Conclusion LS was not correlated with ongoing HIV replication during the 12- and 36-month study periods in immunocompetent HIV-monoinfected and HIV/HCV-coinfected patients. PMID:28845109

Stability of hepatitis C virus (HCV) RNA levels among interferon-naïve HIV/HCV-coinfected individuals treated with combination antiretroviral therapy

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Grint, D; Peters, L; Reekie, J

2013-01-01

Infection with hepatitis C virus (HCV) is a major cause of chronic liver disease. High HCV RNA levels have been associated with poor treatment response. This study aimed to examine the natural history of HCV RNA in chronically HCV/HIV-coinfected individuals.......Infection with hepatitis C virus (HCV) is a major cause of chronic liver disease. High HCV RNA levels have been associated with poor treatment response. This study aimed to examine the natural history of HCV RNA in chronically HCV/HIV-coinfected individuals....

Ebola Virus RNA in Semen from an HIV-Positive Survivor of Ebola.

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Purpura, Lawrence J; Rogers, Emerson; Baller, April; White, Stephen; Soka, Moses; Choi, Mary J; Mahmoud, Nuha; Wasunna, Christine; Massaquoi, Moses; Kollie, Jomah; Dweh, Straker; Bemah, Philip; Ladele, Victor; Kpaka, Jonathan; Jawara, Mary; Mugisha, Margaret; Subah, Onyekachi; Faikai, Mylene; Bailey, Jeff A; Rollin, Pierre; Marston, Barbara; Nyenswah, Tolbert; Gasasira, Alex; Knust, Barbara; Nichol, Stuart; Williams, Desmond

2017-04-01

Ebola virus is known to persist in semen of male survivors of Ebola virus disease (EVD). However, maximum duration of, or risk factors for, virus persistence are unknown. We report an EVD survivor with preexisting HIV infection, whose semen was positive for Ebola virus RNA 565 days after recovery from EVD.

Effect of menstrual cycle on HIV-1 levels in the peripheral blood and genital tract. WHS 001 Study Team.

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Reichelderfer, P S; Coombs, R W; Wright, D J; Cohn, J; Burns, D N; Cu-Uvin, S; Baron, P A; Coheng, M H; Landay, A L; Beckner, S K; Lewis, S R; Kovacs, A A

2000-09-29

To assess the variation in HIV-1 over the menstrual cycle, including RNA levels in the female genital tract, plasma HIV-1-RNA levels, CD4 cell counts, and culturable virus. A prospective analysis of 55 HIV-1-infected women. Blood and genital tract specimens were collected weekly over 8 weeks, spanning two complete menstrual cycles. Applying repeated-measures models that used menses as the reference level, the variation in viral RNA levels was compared in endocervical canal fluid and cells (collected by Sno-strips and cytobrush, respectively) and ectocervicovaginal lavage (CVL) fluid. Repeated-measures models were also used to assess the variation in plasma CD4 cell counts and viral load. Shedding patterns differed among the three sampling methods, independent of genital tract co-infections. Genital tract HIV-1-RNA levels from CVL fluid and endocervical canal cytobrush specimens were highest during menses and lowest immediately thereafter (P = 0.001 and P = 0.04). The HIV-1-RNA level in endocervical canal fluid was highest in the week preceding menses (P = 0.003). The menstrual cycle had no effect on blood levels of RNA (P = 0.62), culturable virus (P = 0.34), or CD4 cell counts (P = 0.55). HIV-1-RNA levels were higher in endocervical canal fluid than in peripheral blood plasma during the late luteal phase (P = 0.03). HIV-1-RNA levels vary with the menstrual cycle in the female genital tract but not the blood compartment. HIV-1-RNA levels are higher in endocervical canal fluid than in blood plasma. These findings may have important implications for sex-specific pathogenesis, heterosexual transmission, and contraceptive hormone interventions in HIV-1-infected women.

Virological and immunological profiles among patients with undetectable viral load followed prospectively for 24 months

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Katzenstein, T.; Ullum, H.; Røge, B.T.

2003-01-01

OBJECTIVE: To quantify HIV-RNA in plasma, in lymphoid tissue and proviral DNA in peripheral blood mononuclear cells and to relate these to immunological markers among patients with plasma viral load counts of

Residual viraemia in HIV-1-infected patients with plasma viral load

DEFF Research Database (Denmark)

Ostrowski, S R; Katzenstein, T L; Pedersen, B K

2008-01-01

Despite undetectable viral load in conventional assays, probably all human immunodeficiency virus (HIV)-1 infected patients have residual viraemia (RV) detectable by ultra-sensitive assays. To study this issue, this study investigated virologic and immunologic consequences of RV in highly active......-count, CD4+HLA-DR+, CD8+HLA-DR+CD38+, CD4+CD45RA-CD45RO+, CD8+CD45RA-CD45RO+, CD4+CD45RA+CD62L+, CD8+CD45RA+CD62L+ T cells, IgG or IgM. In conclusion, RV was associated with increased blood levels of soluble immune activation markers in HAART-treated HIV-1-infected patients. The finding that RV...... antiretroviral therapy (HAART)-treated HIV-1-infected patients with plasma HIV-1 RNA or=1 episode with TMA-RV whereas 9 patients had undetectable TMA-RV throughout the study-period. Time-points with TMA-RV and PCR-RV were associated with higher circulating sTNFrII (+0.234 ng/ml, P = 0.030) and beta(2...

Comparison of the Abbott m2000 HIV-1 Real-Time and Roche AMPLICOR Monitor v1.5 HIV-1 assays on plasma specimens from Rakai, Uganda.

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Ssebugenyi, I; Kizza, A; Mpoza, B; Aluma, G; Boaz, I; Newell, K; Laeyendecker, O; Shott, J P; Serwadda, D; Reynolds, S J

2011-07-01

The need for viral load (VL) monitoring of HIV patients receiving antiretroviral therapy (ART) in resource-limited settings (RLS) has become apparent with studies showing the limitations of immunological monitoring. We compared the Abbott m2000 Real-Time (Abbott) HIV-1 assay with the Roche AMPLICOR Monitor v1.5 (Roche) HIV-1 assay over a range of VL concentrations. Three hundred and eleven plasma samples were tested, including 164 samples from patients on ART ≥ six months and 147 from ART-naïve patients. The Roche assay detected ≥400 copies/mL in 158 (50.8%) samples. Of these, Abbott produced 145 (91.8%) detectable results ≥400 copies/mL; 13 (8.2%) samples produced discrepant results. Concordance between the assays for detecting HIV-1 RNA ≥400 copies/mL was 95.8% (298/311). The sensitivity, specificity, positive predictive value and negative predictive value of Abbott to detect HIV-1 RNA ≥400 copies/mL were 91.8%, 100%, 100% and 92.2%, respectively. For the 151 samples with HIV-1 RNA ≥400 copies/mL for both assays, a good linear correlation was found (r = 0.81, P Abbott assay performed well in our setting, offering an alternative methodology for HIV-1 VL for laboratories with realtime polymerase chain reaction (PCR) capacity.

Growth patterns reflect response to antiretroviral therapy in HIV-positive infants: potential utility in resource-poor settings.

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Benjamin, D K; Miller, W C; Ryder, R W; Weber, D J; Walter, E; McKinney, R E

2004-01-01

Laboratory monitoring of HIV-infected children is the current standard of care in the United States to guide the appropriate use of antiretroviral therapy (ART). Although ART is becoming a reality in some developing countries, laboratory monitoring of ART is costly, necessitating creative approaches to monitoring. As an initial step to guide monitoring of HIV progression in low resource settings, we assessed the utility of the physical examination to predict clinical progression of HIV. We conducted a retrospective cohort study of HIV-infected children using data from Pediatric AIDS Clinical Trials Group Protocol 300. We developed a clinical predictive model, and compared the utility of the clinical model to the change in HIV RNA viral load as diagnostic tests of ART failure. The clinical model incorporated treatment regimen, age, and height velocity: a three-level clinical predictive model provided likelihood ratios of 0.3, 3.9, and 14. For decline in RNA the likelihood ratios were 0.2 (> 1 log decline), 1.4, and 3.5 (> log increase). We developed a simple clinical predictive model that was able to predict clinical progression of HIV after initiation of new ART. The clinical model performed similarly to using changes in HIV RNA viral load. These data should be validated internationally and prospectively, because the test subjects were from a resource rich environment and growth patterns in undernourished children may be impacted differently by HIV and its treatment. The model was most pertinent to children 36 months of age or younger, and was conducted in children receiving monotherapy and dual therapy.

A miRNA Signature for Cognitive Deficits and Alcohol Use Disorder in Persons Living with HIV/AIDS

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Dorota Wyczechowska

2017-11-01

Full Text Available HIV-associated neurocognitive disorders (HAND affects more than half of persons living with HIV-1/AIDS (PLWHA. Identification of biomarkers representing the cognitive status of PLWHA is a critical step for implementation of successful cognitive, behavioral and pharmacological strategies to prevent onset and progression of HAND. However, the presence of co-morbidity factors in PLWHA, the most common being substance abuse, can prevent the identification of such biomarkers. We have optimized a protocol to profile plasma miRNAs using quantitative RT-qPCR and found a miRNA signature with very good discriminatory ability to distinguish PLWHA with cognitive impairment from those without cognitive impairment. Here, we have evaluated this miRNA signature in PLWHA with alcohol use disorder (AUD at LSU Health Sciences Center (LSUHSC. The results show that AUD is a potential confounding factor for the miRNAs associated with cognitive impairment in PLWHA. Furthermore, we have investigated the miRNA signature associated with cognitive impairment in an independent cohort of PLWHA using plasma samples from the CNS HIV Antiretroviral Therapy Effects Research (CHARTER program. Despite differences between the two cohorts in socioeconomic status, AUD, and likely misuse of illicit or prescription drugs, we validated a miRNA signature for cognitive deficits found at LSUHSC in the CHARTER samples.

Persistent CSF but not Plasma HIV RNA, is Associated with Increased Risk of New-onset Moderate-to-Severe Depressive Symptoms; A Prospective Cohort Study

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Hammond, Edward R.; Crum, Rosa M.; Treisman, Glenn J.; Mehta, Shruti H.; Clifford, David B.; Ellis, Ronald J.; Gelman, Benjamin B.; Grant, Igor; Letendre, Scott L.; Marra, Christina M; Morgello, Susan; Simpson, David M.; McArthur, Justin C.

2016-01-01

Major depressive disorder is the most common neuropsychiatric complication in human immunodeficiency virus (HIV) infections and is associated with worse clinical outcomes. We determined if detectable cerebrospinal fluid (CSF) HIV ribonucleic acid (RNA) at threshold ≥50 copies/ml is associated with increased risk of depression. The CNS HIV Anti-Retroviral Therapy Effects Research (CHARTER) cohort is a six-center US-based prospective cohort with bi-annual follow-up 674 participants. We fit linear mixed models (N=233) and discrete-time survival models (N=154; 832 observations), to evaluate trajectories of Beck Depression Inventory (BDI) II scores, and the incidence of new-onset moderate-to-severe depressive symptoms (BDI≥17) among participants, on combination antiretroviral therapy (cART), who were free of depression at study entry, and received a minimum of three CSF examinations over 2,496 person-months follow-up. Detectable CSF HIV RNA (threshold ≥50 copies/ml) at any visit was associated with a 4.7-fold increase in new-onset depression at subsequent visits adjusted for plasma HIV RNA and treatment adherence; hazard ratio (HR)=4.76, (95% CI: 1.58–14.3); P=0.006. Depression (BDI) scores were 2.53 points higher (95% CI: 0.47–4.60; P=0.02) over 6 months if CSF HIV RNA was detectable at a prior study visit in fully adjusted models including age, sex, race, education, plasma HIV RNA, duration and adherence of cART, and lifetime depression diagnosis by DSM-IV criteria. Persistent CSF but not plasma HIV RNA, is associated with an increased risk for new-onset depression. Further research evaluating the role of immune activation and inflammatory markers may improve our understanding of this association. PMID:26727907

Forced evolution of a regulatory RNA helix in the HIV-1 genome

NARCIS (Netherlands)

Berkhout, B.; Klaver, B.; Das, A. T.

1997-01-01

The 5'and 3'end of the HIV-1 RNA genome forms a repeat (R) element that encodes a double stem-loop structure (the TAR and polyA hairpins). Phylogenetic analysis of the polyA hairpin in different human and simian immunodeficiency viruses suggests that the thermodynamic stability of the helix is

Respiratory Syncytial Virus (RSV RNA loads in peripheral blood correlates with disease severity in mice

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Torres Juan

2010-09-01

Full Text Available Abstract Background Respiratory Syncytial Virus (RSV infection is usually restricted to the respiratory epithelium. Few studies have documented the presence of RSV in the systemic circulation, however there is no consistent information whether virus detection in the blood correlates with disease severity. Methods Balb/c mice were inoculated with live RSV, heat-inactivated RSV or medium. A subset of RSV-infected mice was treated with anti-RSV antibody 72 h post-inoculation. RSV RNA loads were measured by PCR in peripheral blood from day 1-21 post-inoculation and were correlated with upper and lower respiratory tract viral loads, the systemic cytokine response, lung inflammation and pulmonary function. Immunohistochemical staining was used to define the localization of RSV antigens in the respiratory tract and peripheral blood. Results RSV RNA loads were detected in peripheral blood from day 1 to 14 post-inoculation, peaked on day 5 and significantly correlated with nasal and lung RSV loads, airway obstruction, and blood CCL2 and CXCL1 expression. Treatment with anti-RSV antibody reduced blood RSV RNA loads and improved airway obstruction. Immunostaining identified RSV antigens in alveolar macrophages and peripheral blood monocytes. Conclusions RSV RNA was detected in peripheral blood upon infection with live RSV, followed a time-course parallel to viral loads assessed in the respiratory tract and was significantly correlated with RSV-induced airway disease.

High Maternal HIV-1 Viral Load During Pregnancy Is Associated With Reduced Placental Transfer of Measles IgG Antibody

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Farquhar, Carey; Nduati, Ruth; Haigwood, Nancy; Sutton, William; Mbori-Ngacha, Dorothy; Richardson, Barbra; John-Stewart, Grace

2012-01-01

Background Studies among HIV-1–infected women have demonstrated reduced placental transfer of IgG antibodies against measles and other pathogens. As a result, infants born to women with HIV-1 infection may not acquire adequate passive immunity in utero and this could contribute to high infant morbidity and mortality in this vulnerable population. Methods To determine factors associated with decreased placental transfer of measles IgG, 55 HIV-1–infected pregnant women who were enrolled in a Nairobi perinatal HIV-1 transmission study were followed. Maternal CD4 count, HIV-1 viral load, and HIV-1–specific gp41 antibody concentrations were measured antenatally and at delivery. Measles IgG concentrations were assayed in maternal blood and infant cord blood obtained during delivery to calculate placental antibody transfer. Results Among 40 women (73%) with positive measles titers, 30 (75%) were found to have abnormally low levels of maternofetal IgG transfer (<95%). High maternal HIV-1 viral load at 32 weeks’ gestation and at delivery was associated with reductions in placental transfer (P < 0.0001 and P = 0.0056, respectively) and infant measles IgG concentrations in cord blood (P < 0.0001 and P = 0.0073, respectively). High maternal HIV-1–specific gp41 antibody titer was also highly correlated with both decreased placental transfer (P = 0.0080) and decreased infant IgG (P < 0.0001). Conclusions This is the first study to evaluate the relationship between maternal HIV-1 viremia, maternal HIV-1 antibody concentrations, and passive immunity among HIV-1–exposed infants. These data support the hypothesis that high HIV-1 viral load during the last trimester may impair maternofetal transfer of IgG and increases risk of measles and other serious infections among HIV-1–exposed infants. PMID:16280707

Hepatitis C virus infection in HIV-infected patients.

Science.gov (United States)

Sulkowski, Mark S

2007-10-01

The hepatitis C virus (HCV) is a spherical enveloped RNA virus of the Flaviviridae family, classified within the Hepacivirus genus. Since its discovery in 1989, HCV has been recognized as a major cause of chronic hepatitis and hepatic fibrosis that progresses in some patients to cirrhosis and hepatocellular carcinoma. In the United States, approximately 4 million people have been infected with HCV, and 10,000 HCVrelated deaths occur each year. Due to shared routes of transmission, HCV and HIV co-infection are common, affecting approximately one third of all HIV-infected persons in the United States. In addition, HIV co-infection is associated with higher HCV RNA viral load and a more rapid progression of HCV-related liver disease, leading to an increased risk of cirrhosis. HCV infection may also impact the course and management of HIV disease, particularly by increasing the risk of antiretroviral drug-induced hepatotoxicity. Thus, chronic HCV infection acts as an opportunistic disease in HIV-infected persons because the incidence of infection is increased and the natural history of HCV infection is accelerated in co-infected persons. Strategies to prevent primary HCV infection and to modify the progression of HCV-related liver disease are urgently needed among HIV/HCV co-infected individuals.

Safety and immunogenicity of therapeutic DNA vaccination in individuals treated with antiretroviral therapy during acute/early HIV-1 infection.

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Eric S Rosenberg

2010-05-01

Full Text Available An effective therapeutic vaccine that could augment immune control of HIV-1 replication may abrogate or delay the need for antiretroviral therapy. AIDS Clinical Trials Group (ACTG A5187 was a phase I/II, randomized, placebo-controlled, double-blinded trial to evaluate the safety and immunogenicity of an HIV-1 DNA vaccine (VRC-HVDNA 009-00-VP in subjects treated with antiretroviral therapy during acute/early HIV-1 infection. (clinicaltrials.gov NCT00125099Twenty healthy HIV-1 infected subjects who were treated with antiretroviral therapy during acute/early HIV-1 infection and had HIV-1 RNA<50 copies/mL were randomized to receive either vaccine or placebo. The objectives of this study were to evaluate the safety and immunogenicity of the vaccine. Following vaccination, subjects interrupted antiretroviral treatment, and set-point HIV-1 viral loads and CD4 T cell counts were determined 17-23 weeks after treatment discontinuation.Twenty subjects received all scheduled vaccinations and discontinued antiretroviral therapy at week 30. No subject met a primary safety endpoint. No evidence of differences in immunogenicity were detected in subjects receiving vaccine versus placebo. There were also no significant differences in set-point HIV-1 viral loads or CD4 T cell counts following treatment discontinuation. Median set-point HIV-1 viral loads after treatment discontinuation in vaccine and placebo recipients were 3.5 and 3.7 log(10 HIV-1 RNA copies/mL, respectively.The HIV-1 DNA vaccine (VRC-HIVDNA 009-00-VP was safe but poorly immunogenic in subjects treated with antiretroviral therapy during acute/early HIV-1 infection. Viral set-points were similar between vaccine and placebo recipients following treatment interruption. However, median viral load set-points in both groups were lower than in historical controls, suggesting a possible role for antiretroviral therapy in persons with acute or early HIV-1 infection and supporting the safety of

Inhibition of both HIV-1 reverse transcription and gene expression by a cyclic peptide that binds the Tat-transactivating response element (TAR RNA.

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Matthew S Lalonde

2011-05-01

Full Text Available The RNA response element TAR plays a critical role in HIV replication by providing a binding site for the recruitment of the viral transactivator protein Tat. Using a structure-guided approach, we have developed a series of conformationally-constrained cyclic peptides that act as structural mimics of the Tat RNA binding region and block Tat-TAR interactions at nanomolar concentrations in vitro. Here we show that these compounds block Tat-dependent transcription in cell-free systems and in cell-based reporter assays. The compounds are also cell permeable, have low toxicity, and inhibit replication of diverse HIV-1 strains, including both CXCR4-tropic and CCR5-tropic primary HIV-1 isolates of the divergent subtypes A, B, C, D and CRF01_AE. In human peripheral blood mononuclear cells, the cyclic peptidomimetic L50 exhibited an IC(50 ∼250 nM. Surprisingly, inhibition of LTR-driven HIV-1 transcription could not account for the full antiviral activity. Timed drug-addition experiments revealed that L-50 has a bi-phasic inhibition curve with the first phase occurring after HIV-1 entry into the host cell and during the initiation of HIV-1 reverse transcription. The second phase coincides with inhibition of HIV-1 transcription. Reconstituted reverse transcription assays confirm that HIV-1 (- strand strong stop DNA synthesis is blocked by L50-TAR RNA interactions in-vitro. These findings are consistent with genetic evidence that TAR plays critical roles both during reverse transcription and during HIV gene expression. Our results suggest that antiviral drugs targeting TAR RNA might be highly effective due to a dual inhibitory mechanism.

CD32 is expressed on cells with transcriptionally active HIV but does not enrich for HIV DNA in resting T cells.

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Abdel-Mohsen, Mohamed; Kuri-Cervantes, Leticia; Grau-Exposito, Judith; Spivak, Adam M; Nell, Racheal A; Tomescu, Costin; Vadrevu, Surya Kumari; Giron, Leila B; Serra-Peinado, Carla; Genescà, Meritxell; Castellví, Josep; Wu, Guoxin; Del Rio Estrada, Perla M; González-Navarro, Mauricio; Lynn, Kenneth; King, Colin T; Vemula, Sai; Cox, Kara; Wan, Yanmin; Li, Qingsheng; Mounzer, Karam; Kostman, Jay; Frank, Ian; Paiardini, Mirko; Hazuda, Daria; Reyes-Terán, Gustavo; Richman, Douglas; Howell, Bonnie; Tebas, Pablo; Martinez-Picado, Javier; Planelles, Vicente; Buzon, Maria J; Betts, Michael R; Montaner, Luis J

2018-04-18

The persistence of HIV reservoirs, including latently infected, resting CD4 + T cells, is the major obstacle to cure HIV infection. CD32a expression was recently reported to mark CD4 + T cells harboring a replication-competent HIV reservoir during antiretroviral therapy (ART) suppression. We aimed to determine whether CD32 expression marks HIV latently or transcriptionally active infected CD4 + T cells. Using peripheral blood and lymphoid tissue of ART-treated HIV + or SIV + subjects, we found that most of the circulating memory CD32 + CD4 + T cells expressed markers of activation, including CD69, HLA-DR, CD25, CD38, and Ki67, and bore a T H 2 phenotype as defined by CXCR3, CCR4, and CCR6. CD32 expression did not selectively enrich for HIV- or SIV-infected CD4 + T cells in peripheral blood or lymphoid tissue; isolated CD32 + resting CD4 + T cells accounted for less than 3% of the total HIV DNA in CD4 + T cells. Cell-associated HIV DNA and RNA loads in CD4 + T cells positively correlated with the frequency of CD32 + CD69 + CD4 + T cells but not with CD32 expression on resting CD4 + T cells. Using RNA fluorescence in situ hybridization, CD32 coexpression with HIV RNA or p24 was detected after in vitro HIV infection (peripheral blood mononuclear cell and tissue) and in vivo within lymph node tissue from HIV-infected individuals. Together, these results indicate that CD32 is not a marker of resting CD4 + T cells or of enriched HIV DNA-positive cells after ART; rather, CD32 is predominately expressed on a subset of activated CD4 + T cells enriched for transcriptionally active HIV after long-term ART. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Anti-HIV Antibody Responses and the HIV Reservoir Size during Antiretroviral Therapy.

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Sulggi A Lee

Full Text Available A major challenge to HIV eradication strategies is the lack of an accurate measurement of the total burden of replication-competent HIV (the "reservoir". We assessed the association of anti-HIV antibody responses and the estimated size of the reservoir during antiretroviral therapy (ART.We evaluated anti-HIV antibody profiles using luciferase immunoprecipitation systems (LIPS assay in relation to several blood-based HIV reservoir measures: total and 2-LTR DNA (rtPCR or droplet digital PCR; integrated DNA (Alu PCR; unspliced RNA (rtPCR, multiply-spliced RNA (TILDA, residual plasma HIV RNA (single copy PCR, and replication-competent virus (outgrowth assay. We also assessed total HIV DNA and RNA in gut-associated lymphoid tissue (rtPCR. Spearman correlations and linear regressions were performed using log-transformed blood- or tissue-based reservoir measurements as predictors and log-transformed antibody levels as outcome variables.Among 51 chronically HIV-infected ART-suppressed participants (median age = 57, nadir CD4+ count = 196 cells/mm3, ART duration = 9 years, the most statistically significant associations were between antibody responses to integrase and HIV RNA in gut-associated lymphoid tissue (1.17 fold-increase per two-fold RNA increase, P = 0.004 and between antibody responses to matrix and integrated HIV DNA in resting CD4+ T cells (0.35 fold-decrease per two-fold DNA increase, P = 0.003. However, these associations were not statistically significant after a stringent Bonferroni-adjustment of P<0.00045. Multivariate models including age and duration of ART did not markedly alter results.Our findings suggest that anti-HIV antibody responses may reflect the size of the HIV reservoir during chronic treated HIV disease, possibly via antigen recognition in reservoir sites. Larger, prospective studies are needed to validate the utility of antibody levels as a measure of the total body burden of HIV during treatment.

Trofile HIV co-receptor usage assay.

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Low, Andrew J; McGovern, Rachel A; Harrigan, P Richard

2009-03-01

The introduction of CCR5 antagonists increases the options available for constructing therapeutic drug regimens for HIV-positive patients. However, as these drugs do not inhibit HIV variants that use the CXCR4 co-receptor, a pretreatment test is required to determine accurately HIV co-receptor usage (tropism) before initiating CCR5 antagonist-based therapy. To discuss the Monogram Trofile assay as a diagnostic tool for determining HIV tropism by critically reviewing reported literature and available data. Monogram Trofile has become, largely by default, the de facto standard for HIV tropism assay. However, there is significant room for improvement in the speed, cost and availability of the test. Furthermore, the test is not quantitative, requires high-input HIV RNA viral loads, and produces results that are less biologically stable than expected. These technical considerations may limit the use of CCR5 antagonists in therapy. Nevertheless, this test is likely to remain the most widely used tropism diagnostic for the short term. We expect that a more practical and possibly more accurate method for measuring HIV tropism can be developed.

Genital herpes simplex virus type 2 infection in humanized HIV-transgenic mice triggers HIV shedding and is associated with greater neurological disease.

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Nixon, Briana; Fakioglu, Esra; Stefanidou, Martha; Wang, Yanhua; Dutta, Monica; Goldstein, Harris; Herold, Betsy C

2014-02-15

Epidemiological studies consistently demonstrate synergy between herpes simplex virus type 2 (HSV-2) and human immunodeficiency virus type 1 (HIV-1). Higher HIV-1 loads are observed in coinfected individuals, and conversely, HIV-1 is associated with more-severe herpetic disease. A small animal model of coinfection would facilitate identification of the biological mechanisms underlying this synergy and provide the opportunity to evaluate interventions. Mice transgenic for HIV-1 provirus and human cyclin T1 under the control of a CD4 promoter (JR-CSF/hu-cycT1) were intravaginally infected with HSV-2 and evaluated for disease progression, HIV shedding, and mucosal immune responses. HSV-2 infection resulted in higher vaginal HIV loads and genital tissue expression of HIV RNA, compared with HSV-uninfected JR-CSF/hu-cycT1 mice. There was an increase in genital tract inflammatory cells, cytokines, chemokines, and interferons in response to HSV-2, although the kinetics of the response were delayed in HIV-transgenic, compared with control mice. Moreover, the JR-CSF/hu-cycT1 mice exhibited earlier and more-severe neurological disease. The latter was associated with downregulation of secretory leukocyte protease inhibitor expression in neuronal tissue, a molecule with antiinflammatory, antiviral, and neuroprotective properties. JR-CSF/hu-cycT1 mice provide a valuable model to study HIV/HSV-2 coinfection and identify potential mechanisms by which HSV-2 facilitates HIV-1 transmission and HIV modulates HSV-2-mediated disease.

Production of HIV-1 vif mRNA Is Modulated by Natural Nucleotide Variations and SLSA1 RNA Structure in SA1D2prox Genomic Region

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Masako Nomaguchi

2017-12-01

Full Text Available Genomic RNA of HIV-1 contains localized structures critical for viral replication. Its structural analysis has demonstrated a stem-loop structure, SLSA1, in a nearby region of HIV-1 genomic splicing acceptor 1 (SA1. We have previously shown that the expression level of vif mRNA is considerably altered by some natural single-nucleotide variations (nSNVs clustering in SLSA1 structure. In this study, besides eleven nSNVs previously identified by us, we totally found nine new nSNVs in the SLSA1-containing sequence from SA1, splicing donor 2, and through to the start codon of Vif that significantly affect the vif mRNA level, and designated the sequence SA1D2prox (142 nucleotides for HIV-1 NL4-3. We then examined by extensive variant and mutagenesis analyses how SA1D2prox sequence and SLSA1 secondary structure are related to vif mRNA level. While the secondary structure and stability of SLSA1 was largely changed by nSNVs and artificial mutations introduced to restore the original NL4-3 form from altered ones by nSNVs, no clear association of the two SLSA1 properties with vif mRNA level was observed. In contrast, when naturally occurring SA1D2prox sequences that contain multiple nSNVs were examined, we attained significant inverse correlation between the vif level and SLSA1 stability. These results may suggest that SA1D2prox sequence adapts over time, and also that the altered SA1D2prox sequence, SLSA1 stability, and vif level are mutually related. In total, we show here that the entire SA1D2prox sequence and SLSA1 stability critically contribute to the modulation of vif mRNA level.

Hsc70/Hsp90 chaperone machinery mediates ATP-dependent RISC loading of small RNA duplexes.

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Iwasaki, Shintaro; Kobayashi, Maki; Yoda, Mayuko; Sakaguchi, Yuriko; Katsuma, Susumu; Suzuki, Tsutomu; Tomari, Yukihide

2010-07-30

Small silencing RNAs--small interfering RNAs (siRNAs) or microRNAs (miRNAs)--direct posttranscriptional gene silencing of their mRNA targets as guides for the RNA-induced silencing complex (RISC). Both siRNAs and miRNAs are born double stranded. Surprisingly, loading these small RNA duplexes into Argonaute proteins, the core components of RISC, requires ATP, whereas separating the two small RNA strands within Argonaute does not. Here we show that the Hsc70/Hsp90 chaperone machinery is required to load small RNA duplexes into Argonaute proteins, but not for subsequent strand separation or target cleavage. We envision that the chaperone machinery uses ATP and mediates a conformational opening of Ago proteins so that they can receive bulky small RNA duplexes. Our data suggest that the chaperone machinery may serve as the driving force for the RISC assembly pathway. Copyright 2010 Elsevier Inc. All rights reserved.

Effect of genital herpes on cervicovaginal HIV shedding in women co-infected with HIV AND HSV-2 in Tanzania.

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Jim Todd

Full Text Available To compare the presence and quantity of cervicovaginal HIV among HIV seropositive women with clinical herpes, subclinical HSV-2 infection and without HSV-2 infection respectively; to evaluate the association between cervicovaginal HIV and HSV shedding; and identify factors associated with quantity of cervicovaginal HIV.Four groups of HIV seropositive adult female barworkers were identified and examined at three-monthly intervals between October 2000 and March 2003 in Mbeya, Tanzania: (1 57 women at 70 clinic visits with clinical genital herpes; (2 39 of the same women at 46 clinic visits when asymptomatic; (3 55 HSV-2 seropositive women at 60 clinic visits who were never observed with herpetic lesions; (4 18 HSV-2 seronegative women at 45 clinic visits. Associations of genital HIV shedding with HIV plasma viral load (PVL, herpetic lesions, HSV shedding and other factors were examined.Prevalence of detectable genital HIV RNA varied from 73% in HSV-2 seronegative women to 94% in women with herpetic lesions (geometric means 1634 vs 3339 copies/ml, p = 0.03. In paired specimens from HSV-2 positive women, genital HIV viral shedding was similar during symptomatic and asymptomatic visits. On multivariate regression, genital HIV RNA (log10 copies/mL was closely associated with HIV PVL (β = 0.51 per log10 copies/ml increase, 95%CI:0.41-0.60, p<0.001 and HSV shedding (β = 0.24 per log10 copies/ml increase, 95% CI:0.16-0.32, p<0.001 but not the presence of herpetic lesions (β = -0.10, 95%CI:-0.28-0.08, p = 0.27.HIV PVL and HSV shedding were more important determinants of genital HIV than the presence of herpetic lesions. These data support a role of HSV-2 infection in enhancing HIV transmissibility.

Intracellular interactions between APOBEC3G, RNA, and HIV-1 Gag: APOBEC3G multimerization is dependent on its association with RNA

OpenAIRE

Friew, Yeshitila N; Boyko, Vitaly; Hu, Wei-Shau; Pathak, Vinay K

2009-01-01

Abstract Background Host restriction factor APOBEC3G (A3G) blocks human immunodeficiency virus type 1 (HIV-1) replication by G-to-A hypermutation, and by inhibiting DNA synthesis and provirus formation. Previous reports have suggested that A3G is a dimer and its virion incorporation is mediated through interactions with viral or nonviral RNAs and/or HIV-1 Gag. We have now employed a bimolecular fluorescence complementation assay (BiFC) to analyze the intracellular A3G-A3G, A3G-RNA, and A3G-Ga...

Integrated and Total HIV-1 DNA Predict Ex Vivo Viral Outgrowth.

Directory of Open Access Journals (Sweden)

Maja Kiselinova

2016-03-01

Full Text Available The persistence of a reservoir of latently infected CD4 T cells remains one of the major obstacles to cure HIV. Numerous strategies are being explored to eliminate this reservoir. To translate these efforts into clinical trials, there is a strong need for validated biomarkers that can monitor the reservoir over time in vivo. A comprehensive study was designed to evaluate and compare potential HIV-1 reservoir biomarkers. A cohort of 25 patients, treated with suppressive antiretroviral therapy was sampled at three time points, with median of 2.5 years (IQR: 2.4-2.6 between time point 1 and 2; and median of 31 days (IQR: 28-36 between time point 2 and 3. Patients were median of 6 years (IQR: 3-12 on ART, and plasma viral load (<50 copies/ml was suppressed for median of 4 years (IQR: 2-8. Total HIV-1 DNA, unspliced (us and multiply spliced HIV-1 RNA, and 2LTR circles were quantified by digital PCR in peripheral blood, at 3 time points. At the second time point, a viral outgrowth assay (VOA was performed, and integrated HIV-1 DNA and relative mRNA expression levels of HIV-1 restriction factors were quantified. No significant change was found for long- and short-term dynamics of all HIV-1 markers tested in peripheral blood. Integrated HIV-1 DNA was associated with total HIV-1 DNA (p<0.001, R² = 0.85, us HIV-1 RNA (p = 0.029, R² = 0.40, and VOA (p = 0.041, R2 = 0.44. Replication-competent virus was detected in 80% of patients by the VOA and it correlated with total HIV-1 DNA (p = 0.039, R² = 0.54. The mean quantification difference between Alu-PCR and VOA was 2.88 log10, and 2.23 log10 between total HIV-1 DNA and VOA. The levels of usHIV-1 RNA were inversely correlated with mRNA levels of several HIV-1 restriction factors (TRIM5α, SAMHD1, MX2, SLFN11, pSIP1. Our study reveals important correlations between the viral outgrowth and total and integrated HIV-1 DNA measures, suggesting that the total pool of HIV-1 DNA may predict the size of the

The connection domain in reverse transcriptase facilitates the in vivo annealing of tRNALys3 to HIV-1 genomic RNA

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Niu Meijuan

2004-10-01

Full Text Available Abstract The primer tRNA for reverse transcription in HIV-1, tRNALys3, is selectively packaged into the virus during its assembly, and annealed to the viral genomic RNA. The ribonucleoprotein complex that is involved in the packaging and annealing of tRNALys into HIV-1 consists of Gag, GagPol, tRNALys, lysyl-tRNA synthetase (LysRS, and viral genomic RNA. Gag targets tRNALys for viral packaging through Gag's interaction with LysRS, a tRNALys-binding protein, while reverse transcriptase (RT sequences within GagPol (the thumb domain bind to tRNALys. The further annealing of tRNALys3 to viral RNA requires nucleocapsid (NC sequences in Gag, but not the NC sequences GagPol. In this report, we further show that while the RT connection domain in GagPol is not required for tRNALys3 packaging into the virus, it is required for tRNALys3 annealing to the viral RNA genome.

Clinical and Emotional Factors Related to Erectile Dysfunction in HIV-Infected Men.

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Fumaz, Carmina R; Ayestaran, Aintzane; Perez-Alvarez, Nuria; Muñoz-Moreno, Jose A; Ferrer, Maria Jose; Negredo, Eugenia; Clotet, Bonaventura

2017-05-01

The prevalence and associated factors of erectile dysfunction (ED) in Human Immunodeficiency Virus (HIV)-infected men remain controversial. The authors evaluated ED, clinical, and emotional variables in a group of 501 HIV-infected men in a cross-sectional 4-month observational study. ED was assessed using the International Index of Erectile Function-5 and emotional status using the Hospital Anxiety and Depression (HAD) questionnaire. Median age (interquartile range) was 42 (35, 48) years. Time since HIV diagnosis was 6.3 (2.6, 17.1) years, 92% were taking antiretroviral treatment and 81.8% had an HIV-RNA viral load emotional status seemed to play a fundamental role in its presence.

Early HIV Diagnosis Leads to Significantly Decreased Costs in the First 2 Years of HIV Care in an Urban Charity Hospital in New Orleans.

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Halperin, Jason; Katz, Morgan; Pathmanathan, Ishani; Myers, Leann; Van Sickels, Nicholas; Seal, Paula Sereebutra; Richey, Lauren E

We undertook a retrospective cohort study of patients with a positive HIV test in the emergency department who were then linked to care. Inpatient, outpatient, and emergency costs were collected for the first 2 years after HIV diagnosis. Fifty-six patients met the inclusion criteria; they were predominantly uninsured (73%) and African American (89%). The median total cost for a newly diagnosed patient over the first 2 years was US$36 808, driven predominantly by outpatient costs of US$17 512. Median inpatient and total costs were significantly different between the lowest (499 cells/mm 3 ) CD4 count categories (US$21 878 vs US$6607, P <.05; US$61 378 vs US$18 837, P <.05, respectively). Total costs were significantly different between viral load categories <100 000 HIV-RNA copies/mL and ≥100 000 HIV-RNA copies/mL (US$28 219 vs US$49 482, P <.05). Costs were significantly lower among patients diagnosed earlier in their disease. Decreased cost is another factor supporting early diagnosis and linkage to care for patients with HIV.

Effectiveness of tipranavir versus darunavir as a salvage therapy in HIV-1 treatment-experienced patients.

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Domínguez-Hermosillo, Juan Carlos; Mata-Marin, José Antonio; Herrera-González, Norma Estela; Chávez-García, Marcelino; Huerta-García, Gloria; Nuñez-Rodríguez, Nohemí; García-Gámez, José Gerardo; Jiménez-Romero, Anai; Gaytán-Martínez, Jesús Enrique

2016-09-30

Although both tipranavir (TPV) and darunavir (DRV) represent important options for the management of patients with multi-protease inhibitor (PI)-resistant human immunodeficiency virus (HIV), currently there are no studies comparing the effectiveness and safety of these two drugs in the Mexican population. The aim of this study was to compare the effectiveness of TPV versus DRV as a salvage therapy in HIV-1 treatment-experienced patients. This was a comparative, prospective, cohort study. Patients with HIV and triple-class drug resistance evaluated at the Hospital de Infectología "La Raza", National Medical Center, were included. All patients had the protease and retrotranscriptase genotype; resistance mutation interpretation was done using the Stanford database. A total of 35 HIV-1 triple-class drug-resistant patients were analyzed. All of them received tenofovir and raltegravir, 22 received darunavir/ritonavir (DRV/r), and 13 received tipranavir/ritonavir (TPV/r) therapies. The median baseline RNA HIV-1 viral load and CD4+ cell count were 4.34 log (interquartile range [IQR], 4.15-4.72) and 267 cells/mm3 (IQR, 177-320) for the DRV/r group, and 4.14 log (IQR, 3.51-4.85) and 445 cells/mm3 (IQR, 252-558) for the TPV/r group. At week 24 of treatment, 91% of patients receiving DRV/r and 100% of patients receiving TPV/r had an RNA HIV-1 viral load HIV-1 patients who were highly experienced in antiretroviral therapy.

Disseminated HIV-Associated Kaposi’s Sarcoma With High CD4 Cell Count And Low Viral Load

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Diana Pereira Anjos

2017-12-01

Full Text Available Kaposi’s sarcoma is considered an acquired immunodeficiency syndrome-defining illness and is caused by human herpesvirus 8. It has been associated with patients infected with human immunodeficiency virus (HIV who have CD4 T lymphocytes <200 cells/uL and high viral loads. We report a case of a 23-year old woman infected with HIV-1 and receiving antiretroviral treatment since diagnosis, with high CD4 cell count and low viral load that presented with disseminated Kaposi’s sarcoma. Clinicians should be aware of the occurrence of Kaposi’s sarcoma despite robust CD4 cell counts.

Factors associated with mother-to-child transmission of HIV-1 despite a maternal viral load EPF-ANRS CO1).

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Tubiana, Roland; Le Chenadec, Jerome; Rouzioux, Christine; Mandelbrot, Laurent; Hamrene, Karima; Dollfus, Catherine; Faye, Albert; Delaugerre, Constance; Blanche, Stephane; Warszawski, Josiane

2010-02-15

The rate of mother-to-child transmission (MTCT) of human immunodeficiency virus (HIV) type 1 is as low as 0.5% in non-breast-feeding mothers who delivered at term while receiving antiretroviral therapy with a plasma viral load <500 copies/mL. This situation accounted for 20% of the infected children born during the period 1997-2006 in the French Perinatal Cohort. We aimed to identify factors associated with such residual transmission risk. We performed a case-control study nested in the aforementioned subpopulation of the French Perinatal Cohort. Nineteen case patients (transmitters) and 60 control subjects (nontransmitters) were included. Case patients and control subjects did not differ by geographical origin, gestational age at HIV diagnosis, type of antiretroviral therapy received, or elective Cesarean delivery. Case patients were less often receiving treatment at the time that they conceived pregnancy than control subjects (16% vs 45%; P=.017). A lower proportion of case patients had a viral load <500 copies/mL, compared with control subjects, at 14 weeks (0% vs 38.1%; P=.02), 28 weeks (7.7% vs 62.1%; P=.005), and 32 weeks: (21.4% vs 71.1%; P=.004). The difference remained significant when we restricted analysis to the 10 of 16 intrapartum transmission cases. In a multivariate analysis at 30+/-4 weeks adjusted for viral load, CD4(+) T cell count, and time at antiretroviral therapy initiation, viral load was the only factor independently associated with MTCT of HIV (adjusted odds ratio, 23.2; 95% confidence interval, 3.5-553; P<.001). Early and sustained control of viral load is associated with a decreasing residual risk of MTCT of HIV-1. Guidelines should take into account not only CD4(+) T cell count and risk of preterm delivery, but also baseline HIV-1 load for deciding when to start antiretroviral therapy during pregnancy.

Viral load, CD4+ T-lymphocyte counts and antibody titres in HIV-1 ...

African Journals Online (AJOL)

Viral load, CD4+ T-lymphocyte counts and antibody titres in HIV-1 infected untreated children in Kenya; implication for immunodeficiency and AIDS progression. Washingtone Ochieng, Dorington Ogoyi, Francis J Mulaa, Simon Ogola, Rachel Musoke, Moses G Otsyula ...

Select neurocognitive impairment in HIV-infected women: associations with HIV viral load, hepatitis C virus, and depression, but not leukocyte telomere length.

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Chantelle J Giesbrecht

Full Text Available Through implementation of combination antiretroviral therapy (cART remarkable gains have been achieved in the management of HIV infection; nonetheless, the neurocognitive consequences of infection remain a pivotal concern in the cART era. Research has often employed norm-referenced neuropsychological scores, derived from healthy populations (excluding many seronegative individuals at high risk for HIV infection, to characterize impairments in predominately male HIV-infected populations.Using matched-group methodology, we assessed 81 HIV-seropositive (HIV+ women with established neuropsychological measures validated for detection of HIV-related impairments, as well as additional detailed tests of executive function and decision-making from the Cambridge Neuropsychological Test Automated Battery (CANTAB.On validated tests, the HIV+ women exhibited impairments that were limited to significantly slower information processing speed when compared with 45 HIV-seronegative (HIV- women with very similar demographic backgrounds and illness comorbidities. Additionally, select executive impairments in shifting attention (i.e., reversal learning and in decision-making quality were revealed in HIV+ participants. Modifiers of neurocognition in HIV-infected women included detectable HIV plasma viral load, active hepatitis C virus co-infection, and self-reported depression symptoms. In contrast, leukocyte telomere length (LTL, a marker of cellular aging, did not significantly differ between HIV+ and HIV- women, nor was LTL associated with overall neurocognition in the HIV+ group.The findings suggest that well-managed HIV infection may entail a more circumscribed neurocognitive deficit pattern than that reported in many norm-referenced studies, and that common comorbidities make a secondary contribution to HIV-related neurocognitive impairments.

Low-cost HIV-1 diagnosis and quantification in dried blood spots by real time PCR.

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Mehta, Nishaki; Trzmielina, Sonia; Nonyane, Bareng A S; Eliot, Melissa N; Lin, Rongheng; Foulkes, Andrea S; McNeal, Kristina; Ammann, Arthur; Eulalievyolo, Vindu; Sullivan, John L; Luzuriaga, Katherine; Somasundaran, Mohan

2009-06-05

Rapid and cost-effective methods for HIV-1 diagnosis and viral load monitoring would greatly enhance the clinical management of HIV-1 infected adults and children in limited-resource settings. Recent recommendations to treat perinatally infected infants within the first year of life are feasible only if early diagnosis is routinely available. Dried blood spots (DBS) on filter paper are an easy and convenient way to collect and transport blood samples. A rapid and cost effective method to diagnose and quantify HIV-1 from DBS is urgently needed to facilitate early diagnosis of HIV-1 infection and monitoring of antiretroviral therapy. We have developed a real-time LightCycler (rtLC) PCR assay to detect and quantify HIV-1 from DBS. HIV-1 RNA extracted from DBS was amplified in a one-step, single-tube system using primers specific for long-terminal repeat sequences that are conserved across all HIV-1 clades. SYBR Green dye was used to quantify PCR amplicons and HIV-1 RNA copy numbers were determined from a standard curve generated using serially diluted known copies of HIV-1 RNA. This assay detected samples across clades, has a dynamic range of 5 log(10), and %CV real-time systems demonstrated similar performance. The accuracy, reliability, genotype inclusivity and affordability, along with the small volumes of blood required for the assay suggest that the rtLC DBS assay will be useful for early diagnosis and monitoring of pediatric HIV-1 infection in resource-limited settings.

Inhibition of HIV Replication by Cyclic and Hairpin PNAs Targeting the HIV-1 TAR RNA Loop

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Upert, Gregory; Di Giorgio, Audrey; Upadhyay, Alok; Manvar, Dinesh; Pandey, Nootan; Pandey, Virendra N.; Patino, Nadia

2012-01-01

Human immunodeficiency virus-1 (HIV-1) replication and gene expression entails specific interaction of the viral protein Tat with its transactivation responsive element (TAR), to form a highly stable stem-bulge-loop structure. Previously, we described triphenylphosphonium (TPP) cation-based vectors that efficiently deliver nucleotide analogs (PNAs) into the cytoplasm of cells. In particular, we showed that the TPP conjugate of a linear 16-mer PNA targeting the apical stem-loop region of TAR impedes Tat-mediated transactivation of the HIV-1 LTR in vitro and also in cell culture systems. In this communication, we conjugated TPP to cyclic and hairpin PNAs targeting the loop region of HIV-1 TAR and evaluated their antiviral efficacy in a cell culture system. We found that TPP-cyclic PNAs containing only 8 residues, showed higher antiviral potency compared to hairpin PNAs of 12 or 16 residues. We further noted that the TPP-conjugates of the 8-mer cyclic PNA as well as the 16-mer linear PNA displayed similar antiviral efficacy. However, cyclic PNAs were shown to be highly specific to their target sequences. This communication emphasizes on the importance of small constrained cyclic PNAs over both linear and hairpin structures for targeting biologically relevant RNA hairpins. PMID:23029603

Inhibition of HIV Replication by Cyclic and Hairpin PNAs Targeting the HIV-1 TAR RNA Loop

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Gregory Upert

2012-01-01

Full Text Available Human immunodeficiency virus-1 (HIV-1 replication and gene expression entails specific interaction of the viral protein Tat with its transactivation responsive element (TAR, to form a highly stable stem-bulge-loop structure. Previously, we described triphenylphosphonium (TPP cation-based vectors that efficiently deliver nucleotide analogs (PNAs into the cytoplasm of cells. In particular, we showed that the TPP conjugate of a linear 16-mer PNA targeting the apical stem-loop region of TAR impedes Tat-mediated transactivation of the HIV-1 LTR in vitro and also in cell culture systems. In this communication, we conjugated TPP to cyclic and hairpin PNAs targeting the loop region of HIV-1 TAR and evaluated their antiviral efficacy in a cell culture system. We found that TPP-cyclic PNAs containing only 8 residues, showed higher antiviral potency compared to hairpin PNAs of 12 or 16 residues. We further noted that the TPP-conjugates of the 8-mer cyclic PNA as well as the 16-mer linear PNA displayed similar antiviral efficacy. However, cyclic PNAs were shown to be highly specific to their target sequences. This communication emphasizes on the importance of small constrained cyclic PNAs over both linear and hairpin structures for targeting biologically relevant RNA hairpins.

Mesoporous silica nanorods toward efficient loading and intracellular delivery of siRNA

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Chen, Lijue; She, Xiaodong; Wang, Tao; Shigdar, Sarah; Duan, Wei; Kong, Lingxue

2018-02-01

The technology of RNA interference (RNAi) that uses small interfering RNA (siRNA) to silence the gene expression with complementary messenger RNA (mRNA) sequence has great potential for the treatment of cancer in which certain genes were usually found overexpressed. However, the carry and delivery of siRNA to the target site in the human body can be challenging for this technology to be used clinically to silence the cancer-related gene expression. In this work, rod shaped mesoporous silica nanoparticles (MSNs) were developed as siRNA delivery system for specific intracellular delivery. The rod MSNs with an aspect ratio of 1.5 had a high surface area of 934.28 m2/g and achieved a siRNA loading of more than 80 mg/g. With the epidermal growth factor (EGF) grafted on the surface of the MSNs, siRNA can be delivered to the epidermal growth factor receptor (EGFR) overexpressed colorectal cancer cells with high intracellular concentration compared to MSNs without EGF and lead to survivin gene knocking down to less than 30%.

Improved detection of CXCR4-using HIV by V3 genotyping: application of population-based and "deep" sequencing to plasma RNA and proviral DNA.

Science.gov (United States)

Swenson, Luke C; Moores, Andrew; Low, Andrew J; Thielen, Alexander; Dong, Winnie; Woods, Conan; Jensen, Mark A; Wynhoven, Brian; Chan, Dennison; Glascock, Christopher; Harrigan, P Richard

2010-08-01

Tropism testing should rule out CXCR4-using HIV before treatment with CCR5 antagonists. Currently, the recombinant phenotypic Trofile assay (Monogram) is most widely utilized; however, genotypic tests may represent alternative methods. Independent triplicate amplifications of the HIV gp120 V3 region were made from either plasma HIV RNA or proviral DNA. These underwent standard, population-based sequencing with an ABI3730 (RNA n = 63; DNA n = 40), or "deep" sequencing with a Roche/454 Genome Sequencer-FLX (RNA n = 12; DNA n = 12). Position-specific scoring matrices (PSSMX4/R5) (-6.96 cutoff) and geno2pheno[coreceptor] (5% false-positive rate) inferred tropism from V3 sequence. These methods were then independently validated with a separate, blinded dataset (n = 278) of screening samples from the maraviroc MOTIVATE trials. Standard sequencing of HIV RNA with PSSM yielded 69% sensitivity and 91% specificity, relative to Trofile. The validation dataset gave 75% sensitivity and 83% specificity. Proviral DNA plus PSSM gave 77% sensitivity and 71% specificity. "Deep" sequencing of HIV RNA detected >2% inferred-CXCR4-using virus in 8/8 samples called non-R5 by Trofile, and <2% in 4/4 samples called R5. Triplicate analyses of V3 standard sequence data detect greater proportions of CXCR4-using samples than previously achieved. Sequencing proviral DNA and "deep" V3 sequencing may also be useful tools for assessing tropism.

Effectiveness and Risk Factors for Virological Outcome of Raltegravir-Based Therapy for Treatment-Experienced HIV-Infected Patients.

Science.gov (United States)

Mata-Marín, José Antonio; Smeke, Ariane Estrella Weiser; Rodriguez, Mariana Rotzinger; Chávez-García, Marcelino; Banda-Lara, Marco Isaac; Rios, Alma Minerva Pérez; Nuñez-Rodríguez, Nohemí; Domínguez-Hermosillo, Juan Carlos; Sánchez, Alberto Chaparro; Juarez-Kasusky, Irene; Herrera, Javier Enrique Cruz; Ramírez, Jorge Luis Sandoval; Gaytán-Martínez, Jesús

2017-03-01

We evaluated the effectiveness of a raltegravir (RAL)-containing regimen plus an optimized background regimen in HIV-1 highly treatment-experienced patients. A retrospective cohort, multicentre study was conducted. Adult (>16 years old) HIV treatment-experience patients starting therapy with a RAL-containing regimen were included. Effectiveness was evaluated as the percentage of patients with an undetectable HIV-1 RNA viral load (treatment failure. Of the 107 patients in the cohort, 86% were men, the median age was 45 years [interquartile range (IQR) 40-52] and the median number of previous regimens was six (IQR 4-7). After 48 weeks of treatment, 73% (IQR 63-80%) of patients (n = 78) had a viral load of HIV-1 RNA of 40 years [odds ratio (OR) 5.61; 95% confidence interval (CI) 1.61-18.84; P = 0.006] and use of tenofovir in the regimen (OR 0.16; 95% CI 0.03-0.80; P = 0.026). In this Mexican cohort, RAL achieved high rates of virological suppression and an increase in CD4+ cell count in highly treatment-experienced patients infected with HIV-1. Age >40 years was associated with a good virological outcome, contrary to tenofovir use, which was associated with a poor virological outcome.

Real-Time PCR in HIV/Trypanosoma cruzi Coinfection with and without Chagas Disease Reactivation: Association with HIV Viral Load and CD4+ Level

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de Freitas, Vera Lúcia Teixeira; da Silva, Sheila Cristina Vicente; Sartori, Ana Marli; Bezerra, Rita Cristina; Westphalen, Elizabeth Visone Nunes; Molina, Tatiane Decaris; Teixeira, Antonio R. L.; Ibrahim, Karim Yaqub; Shikanai-Yasuda, Maria Aparecida

2011-01-01

Background Reactivation of chronic Chagas disease, which occurs in approximately 20% of patients coinfected with HIV/Trypanosoma cruzi (T. cruzi), is commonly characterized by severe meningoencephalitis and myocarditis. The use of quantitative molecular tests to monitor Chagas disease reactivation was analyzed. Methodology Polymerase chain reaction (PCR) of kDNA sequences, competitive (C-) PCR and real-time quantitative (q) PCR were compared with blood cultures and xenodiagnosis in samples from 91 patients (57 patients with chronic Chagas disease and 34 with HIV/T. cruzi coinfection), of whom 5 had reactivation of Chagas disease and 29 did not. Principal Findings qRT-PCR showed significant differences between groups; the highest parasitemia was observed in patients infected with HIV/T. cruzi with Chagas disease reactivation (median 1428.90 T. cruzi/mL), followed by patients with HIV/T. cruzi infection without reactivation (median 1.57 T. cruzi/mL) and patients with Chagas disease without HIV (median 0.00 T. cruzi/mL). Spearman's correlation coefficient showed that xenodiagnosis was correlated with blood culture, C-PCR and qRT-PCR. A stronger Spearman correlation index was found between C-PCR and qRT-PCR, the number of parasites and the HIV viral load, expressed as the number of CD4+ cells or the CD4+/CD8+ ratio. Conclusions qRT-PCR distinguished the groups of HIV/T. cruzi coinfected patients with and without reactivation. Therefore, this new method of qRT-PCR is proposed as a tool for prospective studies to analyze the importance of parasitemia (persistent and/or increased) as a criterion for recommending pre-emptive therapy in patients with chronic Chagas disease with HIV infection or immunosuppression. As seen in this study, an increase in HIV viral load and decreases in the number of CD4+ cells/mm3 and the CD4+/CD8+ ratio were identified as cofactors for increased parasitemia that can be used to target the introduction of early, pre-emptive therapy. PMID

Comparison of the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 test v1.0 with v2.0 in HIV-1 viral load quantification

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Yi-Ching Tung

2015-04-01

Full Text Available Roche modified the COBAS AmpliPrep/COBAS TaqMan human immunodeficiency virus type 1 (HIV-1 test version 1.0 (CAP/CTM v1.0, resulting in the COBAS AmpliPrep/COBAS TaqMan HIV-1 test version 2.0 (CAP/CTM v2.0. The aim of this study was to evaluate the performance of the CAP/CTM v2.0 and to compare this performance with that of the CAP/CTM v1.0. The study was conducted in a small local study group in Kaohsiung Medical University Hospital, Kaohsiung, Taiwan. A total of 86 plasma samples from HIV-1-seropositive patients were tested using the two assays. The correlation and concordance of results between the two assays were calculated. The CAP/CTM v2.0 generated higher values than did the CAP/CTM v1.0, and five samples (5.8% yielded a difference of > 1 log10 copies/mL. In addition, our data show that CAP/CTM v1.0 and CAP/CTM v2.0 yielded relatively consistent values for 23 samples with low viral loads (< 200 copies/mL. Furthermore, when viral loads were in a medium range (2–5 log10 copies/mL, the results of the two assays were more compatible. This study shows a good correlation between CAP/CTM v1.0 and v2.0 in HIV-1 viral load measurement. Further attention must be paid to those cases in which measured viral loads present larger differences between the two assays.

HIV-1 pre-mRNA commitment to Rev mediated export through PSF and Matrin 3

International Nuclear Information System (INIS)

Kula, Anna; Gharu, Lavina; Marcello, Alessandro

2013-01-01

Human immunodeficiency virus gene expression and replication are regulated at several levels. Incompletely spliced viral RNAs and full-length genomic RNA contain the RRE element and are bound by the viral trans-acting protein Rev to be transported out of the nucleus. Previously we found that the nuclear matrix protein MATR3 was a cofactor of Rev-mediated RNA export. Here we show that the pleiotropic protein PSF binds viral RNA and is associated with MATR3. PSF is involved in the maintenance of a pool of RNA available for Rev activity. However, while Rev and PSF bind the viral pre-mRNA at the site of viral transcription, MATR3 interacts at a subsequent step. We propose that PSF and MATR3 define a novel pathway for RRE-containing HIV-1 RNAs that is hijacked by the viral Rev protein.

Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: implications for viral genomic RNA packaging.

Science.gov (United States)

Webb, Joseph A; Jones, Christopher P; Parent, Leslie J; Rouzina, Ioulia; Musier-Forsyth, Karin

2013-08-01

Despite the vast excess of cellular RNAs, precisely two copies of viral genomic RNA (gRNA) are selectively packaged into new human immunodeficiency type 1 (HIV-1) particles via specific interactions between the HIV-1 Gag and the gRNA psi (ψ) packaging signal. Gag consists of the matrix (MA), capsid, nucleocapsid (NC), and p6 domains. Binding of the Gag NC domain to ψ is necessary for gRNA packaging, but the mechanism by which Gag selectively interacts with ψ is unclear. Here, we investigate the binding of NC and Gag variants to an RNA derived from ψ (Psi RNA), as well as to a non-ψ region (TARPolyA). Binding was measured as a function of salt to obtain the effective charge (Zeff) and nonelectrostatic (i.e., specific) component of binding, Kd(1M). Gag binds to Psi RNA with a dramatically reduced Kd(1M) and lower Zeff relative to TARPolyA. NC, GagΔMA, and a dimerization mutant of Gag bind TARPolyA with reduced Zeff relative to WT Gag. Mutations involving the NC zinc finger motifs of Gag or changes to the G-rich NC-binding regions of Psi RNA significantly reduce the nonelectrostatic component of binding, leading to an increase in Zeff. These results show that Gag interacts with gRNA using different binding modes; both the NC and MA domains are bound to RNA in the case of TARPolyA, whereas binding to Psi RNA involves only the NC domain. Taken together, these results suggest a novel mechanism for selective gRNA encapsidation.

Full-length RNA structure prediction of the HIV-1 genome reveals a conserved core domain

DEFF Research Database (Denmark)

Sükösd, Zsuzsanna; Andersen, Ebbe Sloth; Seemann, Ernst Stefan

2015-01-01

of the HIV-1 genome is highly variable in most regions, with a limited number of stable and conserved RNA secondary structures. Most interesting, a set of long distance interactions form a core organizing structure (COS) that organize the genome into three major structural domains. Despite overlapping...

Co-financing for viral load monitoring during the course of antiretroviral therapy among patients with HIV/AIDS in Vietnam: A contingent valuation survey.

Science.gov (United States)

Nguyen, Quyen Le Thi; Nguyen, Long Hoang; Tran, Bach Xuan; Phan, Huong Thi Thu; Le, Huong Thi; Nguyen, Hinh Duc; Tran, Tho Dinh; Do, Cuong Duy; Nguyen, Cuong Manh; Thuc, Vu Thi Minh; Latkin, Carl; Zhang, Melvyn W B; Ho, Roger C M

2017-01-01

Viral load testing is considered the gold standard for monitoring HIV treatment; however, given its high cost, some patients cannot afford viral load testing if this testing is not subsidized. Since foreign aid for HIV/AIDS in Vietnam is rapidly decreasing, we sought to assess willingness to pay (WTP) for viral load and CD4 cell count tests among HIV-positive patients, and identified factors that might inform future co-payment schemes. A multi-site cross-sectional survey was conducted with 1133 HIV-positive patients on antiretroviral therapy (ART) in Hanoi and Nam Dinh. Patients' health insurance coverage, quality of life, and history of illicit drug use were assessed. A contingent valuation approach was employed to measure patients' WTP for CD4 cell count and viral load testing. HIV-positive patients receiving ART at provincial sites reported more difficulty obtaining health insurance (HI) and had the overall the poorest quality of life. Most patients (90.9%) were willing to pay for CD4 cell count testing; here, the mean WTP was valued at US$8.2 (95%CI = 7.6-8.8 US$) per test. Most patients (87.3%) were also willing to pay for viral load testing; here, mean WTP was valued at US$18.6 (95%CI = 16.3-20.9 US$) per test. High income, high education level, and hospitalization were positively associated with WTP, while co-morbidity with psychiatric symptoms and trouble paying for health insurance were both negatively related to WTP. These findings raise concerns that HIV-positive patients in Vietnam might have low WTP for CD4 cell count and viral load testing. This means that without foreign financial subsidies, many of these patients would likely go without these important tests. Treating psychiatric co-morbidities, promoting healthcare services utilization, and removing barriers to accessing health insurance may increase WTP for monitoring of HIV/AIDS treatment among HIV+-positive Vietnamese patients.

The allosteric HIV-1 integrase inhibitor BI-D affects virion maturation but does not influence packaging of a functional RNA genome.

Directory of Open Access Journals (Sweden)

Nikki van Bel

Full Text Available The viral integrase (IN is an essential protein for HIV-1 replication. IN inserts the viral dsDNA into the host chromosome, thereby aided by the cellular co-factor LEDGF/p75. Recently a new class of integrase inhibitors was described: allosteric IN inhibitors (ALLINIs. Although designed to interfere with the IN-LEDGF/p75 interaction to block HIV DNA integration during the early phase of HIV-1 replication, the major impact was surprisingly found on the process of virus maturation during the late phase, causing a reverse transcription defect upon infection of target cells. Virus particles produced in the presence of an ALLINI are misformed with the ribonucleoprotein located outside the virus core. Virus assembly and maturation are highly orchestrated and regulated processes in which several viral proteins and RNA molecules closely interact. It is therefore of interest to study whether ALLINIs have unpredicted pleiotropic effects on these RNA-related processes. We confirm that the ALLINI BI-D inhibits virus replication and that the produced virus is non-infectious. Furthermore, we show that the wild-type level of HIV-1 genomic RNA is packaged in virions and these genomes are in a dimeric state. The tRNAlys3 primer for reverse transcription was properly placed on this genomic RNA and could be extended ex vivo. In addition, the packaged reverse transcriptase enzyme was fully active when extracted from virions. As the RNA and enzyme components for reverse transcription are properly present in virions produced in the presence of BI-D, the inhibition of reverse transcription is likely to reflect the mislocalization of the components in the aberrant virus particle.

The allosteric HIV-1 integrase inhibitor BI-D affects virion maturation but does not influence packaging of a functional RNA genome.

Science.gov (United States)

van Bel, Nikki; van der Velden, Yme; Bonnard, Damien; Le Rouzic, Erwann; Das, Atze T; Benarous, Richard; Berkhout, Ben

2014-01-01

The viral integrase (IN) is an essential protein for HIV-1 replication. IN inserts the viral dsDNA into the host chromosome, thereby aided by the cellular co-factor LEDGF/p75. Recently a new class of integrase inhibitors was described: allosteric IN inhibitors (ALLINIs). Although designed to interfere with the IN-LEDGF/p75 interaction to block HIV DNA integration during the early phase of HIV-1 replication, the major impact was surprisingly found on the process of virus maturation during the late phase, causing a reverse transcription defect upon infection of target cells. Virus particles produced in the presence of an ALLINI are misformed with the ribonucleoprotein located outside the virus core. Virus assembly and maturation are highly orchestrated and regulated processes in which several viral proteins and RNA molecules closely interact. It is therefore of interest to study whether ALLINIs have unpredicted pleiotropic effects on these RNA-related processes. We confirm that the ALLINI BI-D inhibits virus replication and that the produced virus is non-infectious. Furthermore, we show that the wild-type level of HIV-1 genomic RNA is packaged in virions and these genomes are in a dimeric state. The tRNAlys3 primer for reverse transcription was properly placed on this genomic RNA and could be extended ex vivo. In addition, the packaged reverse transcriptase enzyme was fully active when extracted from virions. As the RNA and enzyme components for reverse transcription are properly present in virions produced in the presence of BI-D, the inhibition of reverse transcription is likely to reflect the mislocalization of the components in the aberrant virus particle.

Effect of antiretroviral drugs on maternal CD4 lymphocyte counts, HIV-1 RNA levels, and anthropometric parameters of their neonates Efeito das drogas anti-retrovirais sobre os valores dos linfócitos TCD4, RNA do HIV-1 e parâmetros antropométricos de neonatos de gestantes portadoras do HIV-1

Directory of Open Access Journals (Sweden)

Patrícia El Beitune

2005-06-01

Full Text Available PURPOSE: To study the effect of antiretroviral drugs administered during pregnancy on CD4 lymphocyte counts and HIV-1 RNA levels of pregnant women and on the anthropometric parameters of their neonates. METHODS: A prospective study was conducted on 57 pregnant women and their neonates divided into 3 groups: ZDV Group, HIV-infected mothers taking zidovudine (n = 20; triple therapy (TT Group, mothers taking zidovudine + lamivudine + nelfinavir (n = 25, and Control Group, normal women (n = 12. CD4 lymphocyte counts and HIV-1 RNA levels of pregnant women were analyzed during two periods of pregnancy. The perinatal prognosis took into account preterm rates, birth weight, intrauterine growth restriction, perinatal death, and vertical transmission of HIV-1. Data were analyzed statistically using the nonparametric chi-square, Mann-Whitney, Friedman, Kruskal-Wallis, and Wilcoxon matched pairs tests, with the level of significance set at P OBJETIVOS: Estudar o efeito das drogas anti-retrovirais sobre a quantificação dos linfócitos TCD4 e RNA do HIV-1 de gestantes portadoras do HIV-1 e parâmetros antropométricos de seus neonatos. MÉTODOS: Estudo prospectivo avaliando 57 gestantes e seus neonatos em três grupos: Grupo AZT, gestantes portadoras do HIV utilizando zidovudina (n=20; Grupo TT, mães utilizando zidovudina+lamivudina+nelfinavir (n=25, e Grupo Controle, mulheres saudáveis (n=12. A quantificação dos linfócitos TCD4 e RNA do HIV-1 de gestantes portadoras do HIV foi analisada em dois períodos durante a gestação. O prognóstico perinatal levou em consideração as taxas de pré-termos, restrição de crescimento intra-útero, mortalidade perinatal e transmissão vertical do HIV-1. Os dados foram analisados utilizando-se testes não paramétricos de qui-quadrado, Mann-Whitney, Friedman, Kruskal-Wallys e Wilcoxon para amostras pareadas, considerando-se significativos valores associados a p<0,05. RESULTADOS: Observou-se homogeneidade entre

Correlation of virus load in plasma and lymph node tissue in human immunodeficiency virus infection. INCAS Study Group. Italy, Netherlands, Canada, Australia, and (United) States.

Science.gov (United States)

Harris, M; Patenaude, P; Cooperberg, P; Filipenko, D; Thorne, A; Raboud, J; Rae, S; Dailey, P; Chernoff, D; Todd, J; Conway, B; Montaner, J S

1997-11-01

The impact of long-term changes in plasma viremia, produced by effective combination antiretroviral therapy, on human immunodeficiency virus (HIV) burden within tissue reservoirs is unknown. Fifteen patients who had received at least 1 year of therapy with two or three drug combinations of zidovudine, didanosine, and nevirapine had suitable samples of lymph node tissue obtained by ultrasound-guided core needle biopsy. HIV RNA was extracted from homogenized tissue samples and quantitated using a modified branched DNA assay. Results were correlated with antiretroviral treatment effect on the basis of plasma virus load measurements over the preceding 12-18 months. A statistically significant negative correlation was observed between magnitude of treatment effect on plasma viremia and lymph node virus load. These data suggest that combinations of antiretroviral drugs that produce sustained suppression of plasma HIV RNA may also be able to reduce the virus burden in lymphoid tissues.

Hepatitis B and A vaccination in HIV-infected adults: A review.

Science.gov (United States)

Mena, G; García-Basteiro, A L; Bayas, J M

2015-01-01

Hepatitis B and A account for considerable morbidity and mortality worldwide. Immunization is the most effective means of preventing hepatitis B and A. However, the immune response to both hepatitis vaccines seems to be reduced in HIV-infected subjects. The aim of this review was to analyze the immunogenicity, safety, long-term protection and current recommendations of hepatitis B and A vaccination among HIV-infected adults. The factors most frequently associated with a deficient level of anti-HBs or IgG anti-HAV after vaccination are those related to immunosuppression (CD4 level and HIV RNA viral load) and to the frequency of administration and/or the amount of antigenic load per dose. The duration of the response to both HBV and HAV vaccines is associated with suppression of the viral load at vaccination and, in the case of HBV vaccination, with a higher level of antibodies after vaccination. In terms of safety, there is no evidence of more, or different, adverse effects compared with HIV-free individuals. Despite literature-based advice on the administration of alternative schedules, revaccination after the failure of primary vaccination, and the need for periodic re-evaluation of antibody levels, few firm recommendations are found in the leading guidelines.

Hepatitis B and A vaccination in HIV-infected adults: A review

Science.gov (United States)

Mena, G; García-Basteiro, AL; Bayas, JM

2015-01-01

Hepatitis B and A account for considerable morbidity and mortality worldwide. Immunization is the most effective means of preventing hepatitis B and A. However, the immune response to both hepatitis vaccines seems to be reduced in HIV-infected subjects. The aim of this review was to analyze the immunogenicity, safety, long-term protection and current recommendations of hepatitis B and A vaccination among HIV-infected adults. The factors most frequently associated with a deficient level of anti-HBs or IgG anti-HAV after vaccination are those related to immunosuppression (CD4 level and HIV RNA viral load) and to the frequency of administration and/or the amount of antigenic load per dose. The duration of the response to both HBV and HAV vaccines is associated with suppression of the viral load at vaccination and, in the case of HBV vaccination, with a higher level of antibodies after vaccination. In terms of safety, there is no evidence of more, or different, adverse effects compared with HIV-free individuals. Despite literature-based advice on the administration of alternative schedules, revaccination after the failure of primary vaccination, and the need for periodic re-evaluation of antibody levels, few firm recommendations are found in the leading guidelines. PMID:26208678

Isolation and characterization of 20'-F-RNA aptamers against whole HIV-1 subtype C envelope pseudovirus

CSIR Research Space (South Africa)

London, GM

2015-01-01

Full Text Available Aptamers, which are artificial nucleic acid ligands akin to antibodies in function, represent a new class of molecules that can prevent HIV infection. In this study, we isolated RNA aptamers against whole HV-1CAP45 enveloped pseudotyped virus...

New Highly Sensitive Real-Time PCR Assay for HIV-2 Group A and Group B DNA Quantification.

Science.gov (United States)

Bertine, Mélanie; Gueudin, Marie; Mélard, Adeline; Damond, Florence; Descamps, Diane; Matheron, Sophie; Collin, Fidéline; Rouzioux, Christine; Plantier, Jean-Christophe; Avettand-Fenoel, Véronique

2017-09-01

HIV-2 infection is characterized by a very low replication rate in most cases and low progression. This necessitates an approach to patient monitoring that differs from that for HIV-1 infection. Here, a new highly specific and sensitive method for HIV-2 DNA quantification was developed. The new test is based on quantitative real-time PCR targeting the long terminal repeat (LTR) and gag regions and using an internal control. Analytical performance was determined in three laboratories, and clinical performance was determined on blood samples from 63 patients infected with HIV-2 group A ( n = 35) or group B ( n = 28). The specificity was 100%. The 95% limit of detection was three copies/PCR and the limit of quantification was six copies/PCR. The within-run coefficients of variation were between 1.03% at 3.78 log 10 copies/PCR and 27.02% at 0.78 log 10 copies/PCR. The between-run coefficient of variation was 5.10%. Both manual and automated nucleic acid extraction methods were validated. HIV-2 DNA loads were detectable in blood cells from all 63 patients. When HIV-2 DNA was quantifiable, median loads were significantly higher in antiretroviral-treated than in naive patients and were similar for groups A and B. HIV-2 DNA load was correlated with HIV-2 RNA load ( r = 0.68; 95% confidence interval [CI], 0.4 to 0.8; P < 0.0001). Our data show that this new assay is highly sensitive and quantifies the two main HIV-2 groups, making it useful for the diagnosis of HIV-2 infection and for pathogenesis studies on HIV-2 reservoirs. Copyright © 2017 American Society for Microbiology.

Comparison of the RealTime HIV-1, COBAS TaqMan 48 v1.0, Easy Q v1.2, and Versant v3.0 assays for Determination of HIV-1 Viral Loads in a Cohort of Canadian Patients with Diverse HIV Subtype Infections▿

OpenAIRE

Church, Deirdre; Gregson, Daniel; Lloyd, Tracie; Klein, Marina; Beckthold, Brenda; Laupland, Kevin; Gill, M. John

2010-01-01

HIV clinics in Canada provide care to an increasing number of patients born outside of Canada with HIV-1 non-B subtype infections. Because the Easy Q HIV-1 v1.2 assay (EQ; bioMérieux) failed to detect some non-B subtype infections, a multiassay HIV-1 viral load (VL) study was conducted with patients with diverse HIV subtype infections. Patients were enrolled from the Southern Alberta HIV Clinic (SAC), Calgary, Alberta, Canada (n = 349) and the McGill HIV Clinic (MHC), Montreal, Quebec, Canada...

HIV-1 persistence following extremely early initiation of antiretroviral therapy (ART during acute HIV-1 infection: An observational study.

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Timothy J Henrich

2017-11-01

Full Text Available It is unknown if extremely early initiation of antiretroviral therapy (ART may lead to long-term ART-free HIV remission or cure. As a result, we studied 2 individuals recruited from a pre-exposure prophylaxis (PrEP program who started prophylactic ART an estimated 10 days (Participant A; 54-year-old male and 12 days (Participant B; 31-year-old male after infection with peak plasma HIV RNA of 220 copies/mL and 3,343 copies/mL, respectively. Extensive testing of blood and tissue for HIV persistence was performed, and PrEP Participant A underwent analytical treatment interruption (ATI following 32 weeks of continuous ART.Colorectal and lymph node tissues, bone marrow, cerebral spinal fluid (CSF, plasma, and very large numbers of peripheral blood mononuclear cells (PBMCs were obtained longitudinally from both participants and were studied for HIV persistence in several laboratories using molecular and culture-based detection methods, including a murine viral outgrowth assay (mVOA. Both participants initiated PrEP with tenofovir/emtricitabine during very early Fiebig stage I (detectable plasma HIV-1 RNA, antibody negative followed by 4-drug ART intensification. Following peak viral loads, both participants experienced full suppression of HIV-1 plasma viremia. Over the following 2 years, no further HIV could be detected in blood or tissue from PrEP Participant A despite extensive sampling from ileum, rectum, lymph nodes, bone marrow, CSF, circulating CD4+ T cell subsets, and plasma. No HIV was detected from tissues obtained from PrEP Participant B, but low-level HIV RNA or DNA was intermittently detected from various CD4+ T cell subsets. Over 500 million CD4+ T cells were assayed from both participants in a humanized mouse outgrowth assay. Three of 8 mice infused with CD4+ T cells from PrEP Participant B developed viremia (50 million input cells/surviving mouse, but only 1 of 10 mice infused with CD4+ T cells from PrEP Participant A (53 million input

Mechanisms of human immunodeficiency virus type 2 RNA packaging

DEFF Research Database (Denmark)

Ni, Na; Nikolaitchik, Olga A; Dilley, Kari A

2011-01-01

do not support the cis-packaging hypothesis but instead indicate that trans packaging is the major mechanism of HIV-2 RNA packaging. To further characterize the mechanisms of HIV-2 RNA packaging, we visualized HIV-2 RNA in individual particles by using fluorescent protein-tagged RNA-binding proteins......Human immunodeficiency virus type 2 (HIV-2) has been reported to have a distinct RNA packaging mechanism, referred to as cis packaging, in which Gag proteins package the RNA from which they were translated. We examined the progeny generated from dually infected cell lines that contain two HIV-2...... proviruses, one with a wild-type gag/gag-pol and the other with a mutant gag that cannot express functional Gag/Gag-Pol. Viral titers and RNA analyses revealed that mutant viral RNAs can be packaged at efficiencies comparable to that of viral RNA from which wild-type Gag/Gag-Pol is translated. These results...

Dual R3R5 tropism characterizes cerebrospinal fluid HIV-1 isolates from individuals with high cerebrospinal fluid viral load.

Science.gov (United States)

Karlsson, Ulf; Antonsson, Liselotte; Ljungberg, Bengt; Medstrand, Patrik; Esbjörnsson, Joakim; Jansson, Marianne; Gisslen, Magnus

2012-09-10

To study the use of major and alternative coreceptors by HIV-1 isolates obtained from paired plasma and cerebrospinal fluid (CSF) samples. Paired plasma and CSF isolates from HIV-1-infected individuals with varying clinical, virologic, and immunologic parameters were assessed for the ability to infect indicator cells expressing a panel of coreceptors with documented expression in the central nervous system (CNS). HIV-1 isolates obtained from plasma and CSF in 28 individuals with varying viral load, CD4 T-cell counts, and with or without AIDS-defining disease were analyzed for the ability to infect NP2.CD4 cells stably expressing a panel of HIV coreceptors (CCR5, CXCR4, CCR3, CXCR6, GPR1, APJ, ChemR23, RDC-1 or BLT1). All isolates from both plasma and CSF utilized CCR5 and/or CXCR4. However, the ability to use both CCR3 and CCR5 (R3R5) was more pronounced in CSF isolates and correlated with high CSF viral load and low CD4 T-cell count. Notably, four out of five CSF isolates of subtype C origin exhibited CXCR6 use, which coincided with high CSF viral load despite preserved CD4 T-cell counts. The use of other alternative coreceptors was less pronounced. Dual-tropic R3R5 HIV-1 isolates in CSF coincide with high CSF viral load and low CD4 T-cell counts. Frequent CXCR6 use by CSF-derived subtype C isolates indicates that subtype-specific differences in coreceptor use may exist that will not be acknowledged when assessing plasma virus isolates. The findings may also bare relevance for HIV-1 replication within the CNS, and consequently, for the neuropathogenesis of AIDS.

The RNA binding protein HuR does not interact directly with HIV-1 reverse transcriptase and does not affect reverse transcription in vitro

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Gronenborn Angela M

2010-05-01

Full Text Available Abstract Background Lemay et al recently reported that the RNA binding protein HuR directly interacts with the ribonuclease H (RNase H domain of HIV-1 reverse transcriptase (RT and influences the efficiency of viral reverse transcription (Lemay et al., 2008, Retrovirology 5:47. HuR is a member of the embryonic lethal abnormal vision protein family and contains 3 RNA recognition motifs (RRMs that bind AU-rich elements (AREs. To define the structural determinants of the HuR-RT interaction and to elucidate the mechanism(s by which HuR influences HIV-1 reverse transcription activity in vitro, we cloned and purified full-length HuR as well as three additional protein constructs that contained the N-terminal and internal RRMs, the internal and C-terminal RRMs, or the C-terminal RRM only. Results All four HuR proteins were purified and characterized by biophysical methods. They are well structured and exist as monomers in solution. No direct protein-protein interaction between HuR and HIV-1 RT was detected using NMR titrations with 15N labeled HuR variants or the 15N labeled RNase H domain of HIV-1 RT. Furthermore, HuR did not significantly affect the kinetics of HIV-1 reverse transcription in vitro, even on RNA templates that contain AREs. Conclusions Our results suggest that HuR does not impact HIV-1 replication through a direct protein-protein interaction with the viral RT.

HIV-DNA in the genital tract of women on long-term effective therapy is associated to residual viremia and previous AIDS-defining illnesses.

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Thierry Prazuck

Full Text Available OBJECTIVES: To assess the impact of long-term combined antiretroviral therapy (cART on HIV-RNA and HIV-DNA levels in cervicovaginal secretions of HIV-1-infected women with sustained undetectable plasma RNA viral load (PVL; to explore factors predictive of residual viral shedding; and to evaluate the risk of heterosexual transmission. METHODS: Women with undetectable PVL (6 months were included in this cross-sectional study. HIV-RNA and HIV-DNA were measured in blood and cervicovaginal lavage fluid (CVL. Women were systematically tested for genital infections. The risk of transmission to male partners during unprotected intercourse was estimated. RESULTS: Eighty-one women composed the study population: all had HIV-RNA <40 copies/mL in CVL. HIV-DNA was detectable in CVL of 29/78 patients (37%. There was a weak positive correlation between HIV-DNA levels in PBMCs and CVL (r = 0.20; p = 0.08. In multivariate analysis, two factors were associated with HIV-DNA detection in CVL: previous AIDS-defining illnesses (OR = 11; 95%CI = 2-61 and current residual viremia (20HIV-DNA detection in CVL. Twenty-eight percent of the women had unprotected intercourse with their regular HIV-seronegative male partner, for between 8 and 158 months. None of their male partners became infected, after a total of 14 000 exposures. CONCLUSION: In our experience, HIV-RNA was undetectable in the genital tract of women with sustained control of PVL on cART. HIV-DNA shedding persisted in about one third of cases, with no substantial evidence of residual infectiousness.

Natural controlled HIV infection: Preserved HIV-specific immunity despite undetectable replication competent virus

International Nuclear Information System (INIS)

Kloosterboer, Nico; Groeneveld, Paul H.P.; Jansen, Christine A.; Vorst, Teun J.K. van der; Koning, Fransje; Winkel, Carel N.; Duits, Ashley J.; Miedema, Frank; Baarle, Debbie van; Rij, Ronald P. van; Brinkman, Kees; Schuitemaker, Hanneke

2005-01-01

Long-term non-progressive HIV infection, characterized by low but detectable viral load and stable CD4 counts in the absence of antiviral therapy, is observed in about 5% of HIV-infected patients. Here we identified four therapy naive individuals who are strongly seropositive for HIV-1 but who lack evidence of detectable HIV p24 antigen, plasma RNA, and proviral DNA in routine diagnostic testing. With an ultrasensitive PCR, we established that frequencies of pol proviral DNA sequences were as low as 0.2-0.5 copies/10 6 PBMC. HIV could not be isolated using up to 30 x 10 6 patient PBMC. One individual was heterozygous for CCR5 Δ32, but CCR5 expression on CD4 + T cells was normal to high in all four individuals. In vitro R5 and X4 HIV-1 susceptibility of CD8-depleted PBMC of all study subjects was significantly lower than the susceptibility of CD8-depleted PBMC of healthy blood donors. All individuals expressed protective HLA-B*58s alleles and showed evidence of HIV-specific cellular immunity either by staining with HLA-B*57 tetramers folded with an HIV RT or gag peptide or after stimulation with HIV-1 p24 gag, RT, or nef peptides in ELIspot analysis. HIV-specific CD4 + T helper cells were demonstrated by proliferation of CD4 + T cells and intracellular staining for IL-2 and IFNγ after stimulation with an HIV-gag peptide pool. Sera of all individuals showed antibody-mediated neutralization of both R5 and X4 HIV-1 variants. These data implicate that very low-level antigen exposure is sufficient for sustained HIV-specific immunity and suggest the possibility of a multi-factorial control of HIV infection

Hepatitis C viral load, genotype 3 and interleukin-28B CC genotype predict mortality in HIV and hepatitis C-coinfected individuals

DEFF Research Database (Denmark)

Clausen, Louise Nygaard; Astvad, Karen; Ladelund, Steen

2012-01-01

OBJECTIVE: We hypothesized that hepatitis C virus (HCV) load and genotype may influence all-cause mortality in HIV-HCV-coinfected individuals. DESIGN AND METHODS: Observational prospective cohort study. Mortality rates were compared in a time-updated multivariate Poisson regression analysis....... RESULTS: We included 264 consecutive HIV-HCV-coinfected individuals. During 1143 person years at risk (PYR) 118 individuals died [overall mortality rate 10 (95% confidence interval; 8, 12)/100 PYR]. In multivariate analysis, a 1 log increase in HCV viral load was associated with a 30% higher mortality......) CC genotype was associated with 54% higher mortality risk [aMRR: 1.54 (0.89, 3.82] compared to TT genotype. CONCLUSION: High-HCV viral load, HCV genotype 3 and IL28B genotype CC had a significant influence on the risk of all-cause mortality among individuals coinfected with HIV-1. This may have...

Relationship of HIV Reservoir Characteristics with Immune Status and Viral Rebound Kinetics in an HIV Therapeutic Vaccine Study

Science.gov (United States)

Li, Jonathan Z.; Heisey, Andrea; Ahmed, Hayat; Wang, Hongying; Zheng, Lu; Carrington, Mary; Wrin, Terri; Schooley, Robert T.; Lederman, Michael M.; Kuritzkes, Daniel R.

2014-01-01

Objectives To evaluate the impact of therapeutic HIV vaccination on the HIV reservoir, and assess the relationship of the viral reservoir with HIV-specific immune status and viral rebound kinetics. Design Retrospective analysis of ACTG A5197, a randomized, placebo-controlled trial of a therapeutic rAd5 HIV-1 gag vaccine. Methods Participants received vaccine/placebo at weeks 0, 4, and 26 prior to a 16-week analytic treatment interruption (ATI) at week 38. Cell-associated HIV-1 RNA and DNA (CA-RNA and CA-DNA) and HIV-1 residual viremia (RV) were quantified at weeks 0, 8, and 38. HIV-specific CD4+/CD8+ activity were assessed by an intracellular cytokine staining assay. Results At study entry, CA-RNA and CA-DNA levels were correlated inversely with the numbers of HIV-specific CD4+ interferon-γ-producing cells (CA-RNA: r = −0.23, P=0.03 and CA-DNA: r = −0.28, P<0.01, N=93). Therapeutic HIV vaccination induced HIV-specific CD4+ activity, but did not significantly affect levels of CA-RNA or CA-DNA. Vaccine recipients with undetectable RV at week 8 had higher frequencies of HIV-specific CD4+ and CD8+ interferon-γ-producing cells (undetectable versus detectable RV: 277 versus 161 CD4+ cells/106 lymphocytes, P=0.03 and 1326 versus 669 CD8+ cells/106 lymphocytes, P=0.04). Pre-ATI CA-RNA and CA-DNA were associated with post-ATI plasma HIV set point (CA-RNA: r = 0.51, P<0.01 and CA-DNA: r = 0.47, P<0.01). Conclusions Vaccine-induced T-cell responses were associated with a modest transient effect on RV, but more potent immune responses and/or combination treatment with latency-reversing agents are needed to reduce the HIV reservoir. HIV reservoir measures may act as biomarkers of post-ATI viral rebound kinetics. PMID:25254301

A pilot evaluation of whole blood finger-prick sampling for point-of-care HIV viral load measurement: the UNICORN study.

Science.gov (United States)

Fidler, Sarah; Lewis, Heather; Meyerowitz, Jodi; Kuldanek, Kristin; Thornhill, John; Muir, David; Bonnissent, Alice; Timson, Georgina; Frater, John

2017-10-20

There is a global need for HIV viral load point-of-care (PoC) assays to monitor patients receiving antiretroviral therapy. UNICORN was the first study of an off-label protocol using whole blood finger-prick samples tested with and without a simple three minute spin using a clinic-room microcentrifuge. Two PoC assays were evaluated in 40 HIV-positive participants, 20 with detectable and 20 with undetectable plasma viral load (pVL) (<20 copies/ml). Using 100 µl finger-prick blood samples, the Cepheid Xpert HIV-1 Viral Load and HIV-1 Qual cartridges were compared with laboratory pVL assessment (TaqMan, Roche). For participants with undetectable viraemia by TaqMan, there was poor concordance without centrifugation with the TaqMan platform with only 40% 'undetectable' using Xpert VL and 25% 'not detected' using the Qual assay. After a 3 minute spin, 100% of samples were undetectable using either assay, showing full concordance with the TaqMan assay. Defining a lower limit of detection of 1000 copies/ml when including a spin, there was 100% concordance with the TaqMan platform with strong correlation (rho 0.95 and 0.94; p < 0.0001 for both assays). When including a simple microcentrifugation step, finger-prick PoC testing was a quick and accurate approach for assessing HIV viraemia, with excellent concordance with validated laboratory approaches.

Concerted motions in HIV-1 TAR RNA may allow access to bound state conformations: RNA dynamics from NMR residual dipolar couplings.

Science.gov (United States)

Al-Hashimi, Hashim M; Gosser, Yuying; Gorin, Andrey; Hu, Weidong; Majumdar, Ananya; Patel, Dinshaw J

2002-01-11

Ground-state dynamics in RNA is a critical precursor for structural adaptation observed ubiquitously in protein-RNA recognition. A tertiary conformational analysis of the stem-loop structural element in the transactivation response element (TAR) from human immunodeficiency virus type 1 (HIV-I) RNA is presented using recently introduced NMR methods that rely on the measurement of residual dipolar couplings (RDC) in partially oriented systems. Order matrix analysis of RDC data provides evidence for inter-helical motions that are of amplitude 46(+/-4) degrees, of random directional character, and that are executed about an average conformation with an inter-helical angle between 44 degrees and 54 degrees. The generated ensemble of TAR conformations have different organizations of functional groups responsible for interaction with the trans-activator protein Tat, including conformations similar to the previously characterized bound-state conformation. These results demonstrate the utility of RDC-NMR for simultaneously characterizing RNA tertiary dynamics and average conformation, and indicate an avenue for TAR complex formation involving tertiary structure capture. Copyright 2001 Academic Press.

Male circumcision significantly reduces prevalence and load of genital anaerobic bacteria.

Science.gov (United States)

Liu, Cindy M; Hungate, Bruce A; Tobian, Aaron A R; Serwadda, David; Ravel, Jacques; Lester, Richard; Kigozi, Godfrey; Aziz, Maliha; Galiwango, Ronald M; Nalugoda, Fred; Contente-Cuomo, Tania L; Wawer, Maria J; Keim, Paul; Gray, Ronald H; Price, Lance B

2013-04-16

Male circumcision reduces female-to-male HIV transmission. Hypothesized mechanisms for this protective effect include decreased HIV target cell recruitment and activation due to changes in the penis microbiome. We compared the coronal sulcus microbiota of men from a group of uncircumcised controls (n = 77) and from a circumcised intervention group (n = 79) at enrollment and year 1 follow-up in a randomized circumcision trial in Rakai, Uganda. We characterized microbiota using16S rRNA gene-based quantitative PCR (qPCR) and pyrosequencing, log response ratio (LRR), Bayesian classification, nonmetric multidimensional scaling (nMDS), and permutational multivariate analysis of variance (PerMANOVA). At baseline, men in both study arms had comparable coronal sulcus microbiota; however, by year 1, circumcision decreased the total bacterial load and reduced microbiota biodiversity. Specifically, the prevalence and absolute abundance of 12 anaerobic bacterial taxa decreased significantly in the circumcised men. While aerobic bacterial taxa also increased postcircumcision, these gains were minor. The reduction in anaerobes may partly account for the effects of circumcision on reduced HIV acquisition. The bacterial changes identified in this study may play an important role in the HIV risk reduction conferred by male circumcision. Decreasing the load of specific anaerobes could reduce HIV target cell recruitment to the foreskin. Understanding the mechanisms that underlie the benefits of male circumcision could help to identify new intervention strategies for decreasing HIV transmission, applicable to populations with high HIV prevalence where male circumcision is culturally less acceptable.

Dissecting HIV Virulence: Heritability of Setpoint Viral Load, CD4+ T-Cell Decline, and Per-Parasite Pathogenicity.

Science.gov (United States)

Bertels, Frederic; Marzel, Alex; Leventhal, Gabriel; Mitov, Venelin; Fellay, Jacques; Günthard, Huldrych F; Böni, Jürg; Yerly, Sabine; Klimkait, Thomas; Aubert, Vincent; Battegay, Manuel; Rauch, Andri; Cavassini, Matthias; Calmy, Alexandra; Bernasconi, Enos; Schmid, Patrick; Scherrer, Alexandra U; Müller, Viktor; Bonhoeffer, Sebastian; Kouyos, Roger; Regoes, Roland R

2018-01-01

Pathogen strains may differ in virulence because they attain different loads in their hosts, or because they induce different disease-causing mechanisms independent of their load. In evolutionary ecology, the latter is referred to as "per-parasite pathogenicity". Using viral load and CD4+ T-cell measures from 2014 HIV-1 subtype B-infected individuals enrolled in the Swiss HIV Cohort Study, we investigated if virulence-measured as the rate of decline of CD4+ T cells-and per-parasite pathogenicity are heritable from donor to recipient. We estimated heritability by donor-recipient regressions applied to 196 previously identified transmission pairs, and by phylogenetic mixed models applied to a phylogenetic tree inferred from HIV pol sequences. Regressing the CD4+ T-cell declines and per-parasite pathogenicities of the transmission pairs did not yield heritability estimates significantly different from zero. With the phylogenetic mixed model, however, our best estimate for the heritability of the CD4+ T-cell decline is 17% (5-30%), and that of the per-parasite pathogenicity is 17% (4-29%). Further, we confirm that the set-point viral load is heritable, and estimate a heritability of 29% (12-46%). Interestingly, the pattern of evolution of all these traits differs significantly from neutrality, and is most consistent with stabilizing selection for the set-point viral load, and with directional selection for the CD4+ T-cell decline and the per-parasite pathogenicity. Our analysis shows that the viral genotype affects virulence mainly by modulating the per-parasite pathogenicity, while the indirect effect via the set-point viral load is minor. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

A Method to Predict the Structure and Stability of RNA/RNA Complexes.

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Xu, Xiaojun; Chen, Shi-Jie

2016-01-01

RNA/RNA interactions are essential for genomic RNA dimerization and regulation of gene expression. Intermolecular loop-loop base pairing is a widespread and functionally important tertiary structure motif in RNA machinery. However, computational prediction of intermolecular loop-loop base pairing is challenged by the entropy and free energy calculation due to the conformational constraint and the intermolecular interactions. In this chapter, we describe a recently developed statistical mechanics-based method for the prediction of RNA/RNA complex structures and stabilities. The method is based on the virtual bond RNA folding model (Vfold). The main emphasis in the method is placed on the evaluation of the entropy and free energy for the loops, especially tertiary kissing loops. The method also uses recursive partition function calculations and two-step screening algorithm for large, complicated structures of RNA/RNA complexes. As case studies, we use the HIV-1 Mal dimer and the siRNA/HIV-1 mutant (T4) to illustrate the method.

Immune restoration does not invariably occur following long-term HIV-1 suppression during antiretroviral therapy. INCAS Study Group.

Science.gov (United States)

Pakker, N G; Kroon, E D; Roos, M T; Otto, S A; Hall, D; Wit, F W; Hamann, D; van der Ende, M E; Claessen, F A; Kauffmann, R H; Koopmans, P P; Kroon, F P; ten Napel, C H; Sprenger, H G; Weigel, H M; Montaner, J S; Lange, J M; Reiss, P; Schellekens, P T; Miedema, F

1999-02-04

Current antiretroviral treatment can induce significant and sustained virological and immunological responses in HIV-1-infected persons over at least the short- to mid-term. In this study, long-term immune reconstitution was investigated during highly active antiretroviral therapy. Patients enrolled in the INCAS study in The Netherlands were treated for 102 weeks (range 52-144 weeks) with nevirapine (NVP) + zidovudine (ZDV) (n = 9), didanosine (ddl) + ZDV (n = 10), or NVP + ddl + ZDV (n = 10). Memory and naïve CD4+ and CD8+ T cells were measured using CD45RA and CD27 monoclonal antibodies (mAb), T-cell function was assayed by CD3 + CD28 mAb stimulation, and plasma HIV-1 RNA load was measured by ultra-direct assay (cut-off < 20 copies/ml). Compared to both double combination regimens the triple combination regimen resulted in the most sustained increase in CD4+ T cells (change in CD4+, + 253 x 10(6) cells/l; standard error, 79 x 10(6) cells/l) and reduction of plasma HIV-1 RNA. In nine patients (31%) (ddl + ZDV, n = 2; NVP + ddl + ZDV, n = 7) plasma HIV-1 RNA levels remained below cut-off for at least 2 years. On average, these long-term virological responders demonstrated a significantly higher increase of naïve and memory CD4+ T cells (P = 0.01 and 0.02, respectively) as compared with patients with a virological failure, and showed improved T-cell function and normalization of the naïve; memory CD8+ T-cell ratio. However, individual virological success or failure did not predict the degree of immunological response. T-cell patterns were independent of baseline CD4+ T-cell count, T-cell function, HIV-1 RNA load or age. Low numbers of naïve CD4+ T cells at baseline resulted in modest long-term naïve T-cell recovery. Patients with prolonged undetectable plasma HIV-1 RNA levels during antiretroviral therapy do not invariably show immune restoration. Naïve T-cell recovery in the setting of complete viral suppression is a gradual process, similar to that reported

Activation of HIV Transcription with Short-Course Vorinostat in HIV-Infected Patients on Suppressive Antiretroviral Therapy

Science.gov (United States)

Solomon, Ajantha; Ghneim, Khader; Ahlers, Jeffrey; Cameron, Mark J.; Smith, Miranda Z.; Spelman, Tim; McMahon, James; Velayudham, Pushparaj; Brown, Gregor; Roney, Janine; Watson, Jo; Prince, Miles H.; Hoy, Jennifer F.; Chomont, Nicolas; Fromentin, Rémi; Procopio, Francesco A.; Zeidan, Joumana; Palmer, Sarah; Odevall, Lina; Johnstone, Ricky W.; Martin, Ben P.; Sinclair, Elizabeth; Deeks, Steven G.; Hazuda, Daria J.; Cameron, Paul U.; Sékaly, Rafick-Pierre; Lewin, Sharon R.

2014-01-01

Human immunodeficiency virus (HIV) persistence in latently infected resting memory CD4+ T-cells is the major barrier to HIV cure. Cellular histone deacetylases (HDACs) are important in maintaining HIV latency and histone deacetylase inhibitors (HDACi) may reverse latency by activating HIV transcription from latently infected CD4+ T-cells. We performed a single arm, open label, proof-of-concept study in which vorinostat, a pan-HDACi, was administered 400 mg orally once daily for 14 days to 20 HIV-infected individuals on suppressive antiretroviral therapy (ART). The primary endpoint was change in cell associated unspliced (CA-US) HIV RNA in total CD4+ T-cells from blood at day 14. The study is registered at ClinicalTrials.gov (NCT01365065). Vorinostat was safe and well tolerated and there were no dose modifications or study drug discontinuations. CA-US HIV RNA in blood increased significantly in 18/20 patients (90%) with a median fold change from baseline to peak value of 7.4 (IQR 3.4, 9.1). CA-US RNA was significantly elevated 8 hours post drug and remained elevated 70 days after last dose. Significant early changes in expression of genes associated with chromatin remodeling and activation of HIV transcription correlated with the magnitude of increased CA-US HIV RNA. There were no statistically significant changes in plasma HIV RNA, concentration of HIV DNA, integrated DNA, inducible virus in CD4+ T-cells or markers of T-cell activation. Vorinostat induced a significant and sustained increase in HIV transcription from latency in the majority of HIV-infected patients. However, additional interventions will be needed to efficiently induce virus production and ultimately eliminate latently infected cells. Trial Registration ClinicalTrials.gov NCT01365065 PMID:25393648

Activation of HIV transcription with short-course vorinostat in HIV-infected patients on suppressive antiretroviral therapy.

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Julian H Elliott

2014-10-01

Full Text Available Human immunodeficiency virus (HIV persistence in latently infected resting memory CD4+ T-cells is the major barrier to HIV cure. Cellular histone deacetylases (HDACs are important in maintaining HIV latency and histone deacetylase inhibitors (HDACi may reverse latency by activating HIV transcription from latently infected CD4+ T-cells. We performed a single arm, open label, proof-of-concept study in which vorinostat, a pan-HDACi, was administered 400 mg orally once daily for 14 days to 20 HIV-infected individuals on suppressive antiretroviral therapy (ART. The primary endpoint was change in cell associated unspliced (CA-US HIV RNA in total CD4+ T-cells from blood at day 14. The study is registered at ClinicalTrials.gov (NCT01365065. Vorinostat was safe and well tolerated and there were no dose modifications or study drug discontinuations. CA-US HIV RNA in blood increased significantly in 18/20 patients (90% with a median fold change from baseline to peak value of 7.4 (IQR 3.4, 9.1. CA-US RNA was significantly elevated 8 hours post drug and remained elevated 70 days after last dose. Significant early changes in expression of genes associated with chromatin remodeling and activation of HIV transcription correlated with the magnitude of increased CA-US HIV RNA. There were no statistically significant changes in plasma HIV RNA, concentration of HIV DNA, integrated DNA, inducible virus in CD4+ T-cells or markers of T-cell activation. Vorinostat induced a significant and sustained increase in HIV transcription from latency in the majority of HIV-infected patients. However, additional interventions will be needed to efficiently induce virus production and ultimately eliminate latently infected cells.ClinicalTrials.gov NCT01365065.

Novel targeted siRNA-loaded hybrid nanoparticles: preparation, characterization and in vitro evaluation.

Science.gov (United States)

Dim, Nneka; Perepelyuk, Maryna; Gomes, Olukayode; Thangavel, Chellappagounder; Liu, Yi; Den, Robert; Lakshmikuttyamma, Ashakumary; Shoyele, Sunday A

2015-09-26

siRNAs have a high potential for silencing critical molecular pathways that are pathogenic. Nevertheless, their clinical application has been limited by a lack of effective and safe nanotechnology-based delivery system that allows a controlled and safe transfection to cytosol of targeted cells without the associated adverse effects. Our group recently reported a very effective and safe hybrid nanoparticle delivery system composing human IgG and poloxamer-188 for siRNA delivery to cancer cells. However, these nanoparticles need to be optimized in terms of particle size, loading capacity and encapsulation efficiency. In the present study, we explored the effects of certain production parameters on particle size, loading capacity and encapsulation efficiency. Further, to make these nanoparticles more specific in their delivery of siRNA, we conjugated anti-NTSR1-mAb to the surface of these nanoparticles to target NTSR1-overexpressing cancer cells. The mechanism of siRNA release from these antiNTSR1-mAb functionalized nanoparticles was also elucidated. It was demonstrated that the concentration of human IgG in the starting nanoprecipitation medium and the rotation speed of the magnetic stirrer influenced the encapsulation efficiency, loading capacity and the size of the nanoparticles produced. We also successfully transformed these nanoparticles into actively targeted nanoparticles by functionalizing with anti-NTSR1-mAb to specifically target NTSR1-overexpressing cancer cells, hence able to avoid undesired accumulation in normal cells. The mechanism of siRNA release from these nanoparticles was elucidated to be by Fickian diffusion. Using flow cytometry and fluorescence microscopy, we were able to confirm the active involvement of NTSR1 in the uptake of these anti-NTSR1-mAb functionalized hybrid nanoparticles by lung adenocarcinoma cells. This hybrid nanoparticle delivery system can be used as a platform technology for intracellular delivery of siRNAs to NTSR1

Episodes of Guillain-Barré syndrome associated with the acute phase of HIV-1 infection and with recurrence of viremia

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Castro Gleusa de

2006-01-01

Full Text Available We report a severe case of Guillain-Barré syndrome (GBS characterized by flaccid areflexive tetraplegia and signs of autonomic instability related to acute HIV-1 infection, and the occurrence of relapse episodes coinciding with the detection of HIV-1 RNA in blood during the phase of irregular treatment with antiretroviral agents. The patient has been asymptomatic for 3 years and has an HIV-1 load below the limit of detection. The recurrence of GBS in this case may be related to alterations of the immunologic response caused by disequilibrium in the host-HIV relationship due to the increase in HIV-1 viremia.

Association of a 3' untranslated region polymorphism in proprotein convertase subtilisin/kexin type 9 with HIV viral load and CD4+ levels in HIV/hepatitis C virus coinfected women.

Science.gov (United States)

Kuniholm, Mark H; Liang, Hua; Anastos, Kathryn; Gustafson, Deborah; Kassaye, Seble; Nowicki, Marek; Sha, Beverly E; Pawlowski, Emilia J; Gange, Stephen J; Aouizerat, Bradley E; Pushkarsky, Tatiana; Bukrinsky, Michael I; Prasad, Vinayaka R

2017-11-28

To assess variation in genes that regulate cholesterol metabolism in relation to the natural history of HIV infection. Cross-sectional and longitudinal analysis of the Women's Interagency HIV Study. We examined 2050 single nucleotide polymorphisms (SNPs) in 19 genes known to regulate cholesterol metabolism in relation to HIV viral load and CD4 T-cell levels in a multiracial cohort of 1066 antiretroviral therapy-naive women. Six SNPs were associated with both HIV viral load and CD4 T-cell levels at a false discovery rate of 0.01. Bioinformatics tools did not predict functional activity for five SNPs, located in introns of nuclear receptor corepressor 2, retinoid X receptor alpha (RXRA), and tetratricopeptide repeat domain 39B. Rs17111557 located in the 3' untranslated region of proprotein convertase subtilisin/kexin type 9 (PCSK9) putatively affects binding of hsa-miR-548t-5p and hsa-miR-4796-3p, which could regulate PCSK9 expression levels. Interrogation of rs17111557 revealed stronger associations in the subset of women with HIV/hepatitis C virus (HCV) coinfection (n = 408, 38% of women). Rs17111557 was also associated with low-density lipoprotein cholesterol levels in HIV/HCV coinfected (β: -10.4; 95% confidence interval: -17.9, -2.9; P = 0.007), but not in HIV monoinfected (β:1.2; 95% confidence interval: -6.3, 8.6; P = 0.76) women in adjusted analysis. PCSK9 polymorphism may affect HIV pathogenesis, particularly in HIV/HCV coinfected women. A likely mechanism for this effect is PCSK9-mediated regulation of cholesterol metabolism. Replication in independent cohorts is needed to clarify the generalizability of the observed associations.

Performance Evaluation of the Bioneer AccuPower® HIV-1 Quantitative RT-PCR kit: Comparison with the Roche COBAS® AmpliPrep/COBAS TaqMan® HIV-1 Test Ver.2.0 for Quantification of HIV-1 Viral Load in Indonesia.

Science.gov (United States)

Kosasih, Agus Susanto; Sugiarto, Christine; Hayuanta, Hubertus Hosti; Juhaendi, Runingsih; Setiawan, Lyana

2017-08-08

Measurement of viral load in human immunodeficiency virus type 1 (HIV-1) infected patients is essential for the establishment of a therapeutic strategy. Several assays based on qPCR are available for the measurement of viral load; they differ in sample volume, technology applied, target gene, sensitivity and dynamic range. The Bioneer AccuPower® HIV-1 Quantitative RT-PCR is a novel commercial kit that has not been evaluated for its performance. This study aimed to evaluate the performance of the Bioneer AccuPower® HIV-1 Quantitative RT-PCR kit. In total, 288 EDTA plasma samples from the Dharmais Cancer Hospital were analyzed with the Bioneer AccuPower® HIV-1 Quantitative RT-PCR kit and the Roche COBAS? AmpliPrep/COBAS® TaqMan® HIV-1 version 2.0 (CAP/CTM v2.0). The performance of the Bioneer assay was then evaluated against the Roche CAP/CTM v2.0. Overall, there was good agreement between the two assays. The Bioneer assay showed significant linear correlation with CAP/CTM v2.0 (R2=0.963, plaboratories.

High rate of hepatitis C virus (HCV) recurrence in HIV-infected individuals with spontaneous HCV RNA clearance

DEFF Research Database (Denmark)

Peters, L; Mocroft, A; Soriano, V

2014-01-01

OBJECTIVES: Following resolution of hepatitis C virus (HCV) infection, recurrence has been shown to occur in some persons with repeated exposure to HCV. We aimed to investigate the rate and factors associated with HCV RNA recurrence among HIV-1-infected patients with prior spontaneous HCV RNA cle......-up. Our findings underline the importance of maintaining focus on preventive measures to reduce IDU and sharing of contaminated needles. Clinicians should maintain a high degree of vigilance to identify patients with new HCV infection early....

HIV-1 splicing is controlled by local RNA structure and binding of splicing regulatory proteins at the major 5' splice site

NARCIS (Netherlands)

Mueller, Nancy; Berkhout, Ben; Das, Atze T.

2015-01-01

The 5' leader region of the human immunodeficiency virus 1 (HIV-1) RNA genome contains the major 5' splice site (ss) that is used in the production of the many spliced viral RNAs. This splice-donor (SD) region can fold into a stable stem-loop structure and the thermodynamic stability of this RNA

Hepatitis C prevalence among HIV-infected patients in Guinea-Bissau

DEFF Research Database (Denmark)

Hønge, Bo Langhoff; Jespersen, Sanne; Medina, Candida

2014-01-01

Objectives: To estimate the prevalence and determine the clinical presentation of risk factors of hepatitis C virus (HCV) among HIV-infected patients in Bissau, Guinea-Bissau. Methods: In this cross-sectional study, we included individuals who had a routine blood analysis performed during...... the period April 28 to September 30, 2011. Patient samples were tested for HCV antibodies (anti-HCV) with a chemiluminescence test (Architect, Abbott, USA) and INNO-LIA HCV Score (Innogenetics, Belgium). HCV viral load and genotype were analyzed using an in-house real-time PCR method. Results: In total, 576...... patients were included (417 HIV-1, 104 HIV-2, and 55 HIV-1/2). Ten (1.7%) patients were anti-HCV-positive and eight (1.4%) patients had detectable HCV RNA; all were genotype 2. In a multivariable logistic regression analysis, age >50 years was associated with anti-HCV reactivity (p

Stavudine loaded gelatin liposomes for HIV therapy: Preparation, characterization and in vitro cytotoxic evaluation

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Nayak, Debasis; Boxi, Ankita; Ashe, Sarbani; Thathapudi, Neethi Chandra; Nayak, Bismita, E-mail: nayakb@nitrkl.ac.in

2017-04-01

Despite continuous research and availability of 25 different active compounds for treating chronic HIV-1 infection, there is no absolute cure for this deadly disease. Primarily, the residual viremia remains hidden in latently infected reservoir sites and persistently release the viral RNA into the blood stream. The study proposes the dual utilization of the prepared stavudine-containing nanoformulations to control the residual viremia as well as target the reservoir sites. Gelatin nanoformulations containing very low dosage of stavudine were prepared through classical desolvation process and were later loaded in soya lecithin-liposomes. The nanoformulations were characterized through dynamic light scattering (DLS), Transmission electron microscopy (TEM), X-ray diffraction (XRD) and ATR-FTIR. All the formulations were in nano regime with high hemocompatibility and exhibited dose-dependent cytotoxicity towards Raw 264.7 macrophages. Among the various formulations, SG-3 (Stavudine-Gelatin Nanoformulation sample 3) and SG-LP-3 (Stavudine-Gelatin Nano-Liposome formulation sample 3) showed the best results in terms of yield, size, charge, encapsulation efficiency, hemocompatibility and % cell viability. For the first time, liposomal delivery of antiretroviral drugs using nanocarriers has been demonstrated using very low dosage (lower than the recommended WHO dosage) showing the prominent linear release of stavudine for up to 12 h which would reduce the circulatory viremia as well as reach the sanctuary reservoir sites due to their nanosize. This method of liposomal delivery of antiretroviral drugs in very low concentrations using nanocarriers could provide a novel therapeutic alternative to target HIV reservoir sites. - Highlights: • Stavudine entrapped gelatin nanocarriers prepared with two step desolvation process • Linear and release of stavudine from liposomal formulations up to 12 h • All the SG nanoparticles and SG-LP formulations showed negligible

Stavudine loaded gelatin liposomes for HIV therapy: Preparation, characterization and in vitro cytotoxic evaluation

International Nuclear Information System (INIS)

Nayak, Debasis; Boxi, Ankita; Ashe, Sarbani; Thathapudi, Neethi Chandra; Nayak, Bismita

2017-01-01

Despite continuous research and availability of 25 different active compounds for treating chronic HIV-1 infection, there is no absolute cure for this deadly disease. Primarily, the residual viremia remains hidden in latently infected reservoir sites and persistently release the viral RNA into the blood stream. The study proposes the dual utilization of the prepared stavudine-containing nanoformulations to control the residual viremia as well as target the reservoir sites. Gelatin nanoformulations containing very low dosage of stavudine were prepared through classical desolvation process and were later loaded in soya lecithin-liposomes. The nanoformulations were characterized through dynamic light scattering (DLS), Transmission electron microscopy (TEM), X-ray diffraction (XRD) and ATR-FTIR. All the formulations were in nano regime with high hemocompatibility and exhibited dose-dependent cytotoxicity towards Raw 264.7 macrophages. Among the various formulations, SG-3 (Stavudine-Gelatin Nanoformulation sample 3) and SG-LP-3 (Stavudine-Gelatin Nano-Liposome formulation sample 3) showed the best results in terms of yield, size, charge, encapsulation efficiency, hemocompatibility and % cell viability. For the first time, liposomal delivery of antiretroviral drugs using nanocarriers has been demonstrated using very low dosage (lower than the recommended WHO dosage) showing the prominent linear release of stavudine for up to 12 h which would reduce the circulatory viremia as well as reach the sanctuary reservoir sites due to their nanosize. This method of liposomal delivery of antiretroviral drugs in very low concentrations using nanocarriers could provide a novel therapeutic alternative to target HIV reservoir sites. - Highlights: • Stavudine entrapped gelatin nanocarriers prepared with two step desolvation process • Linear and release of stavudine from liposomal formulations up to 12 h • All the SG nanoparticles and SG-LP formulations showed negligible

Prevalence of Oral Manifestations and Their Association with CD4/CD8 Ratio and HIV Viral Load in South India

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Sharma Gaurav

2011-01-01

Full Text Available The objective of the present research was to determine the prevalence of oral manifestations in an HIV infected population from south India and evaluate their association with HIV viral load and CD4/CD8 ratio. Intraoral examination of 103 patients, whose CD4/CD8 ratio was available, were conducted. HIV viral loads were available for thirty patients only. The prevalence of oral manifestations was 80.6% (83/103. The most common oromucosal lesion was erythematous candidiasis (EC (38.8% followed by melanotic hyperpigmentation (35.9%. Patients having any oral manifestation had a mean CD4/CD8 ratio of 0.24. EC had positive predictive value of 85.0% for CD4/CD8 ratio 20,000 copies/mL (20,000 copies/mL.

Use of human papillomavirus DNA, E6/E7 mRNA, and p16 immunocytochemistry to detect and predict anal high-grade squamous intraepithelial lesions in HIV-positive and HIV-negative men who have sex with men.

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Nittaya Phanuphak

Full Text Available Men who have sex with men (MSM are at high risk of having anal cancer. Anal high-grade squamous intraepithelial lesion (HSIL is the precursor of anal cancer. We explored the use of different biomarkers associated with human papillomavirus (HPV infection and HPV-mediated cell transformation to detect and predict HSIL among HIV-positive and HIV-negative MSM.A total of 123 HIV-positive and 123 HIV-negative MSM were enrolled and followed for 12 months. High-resolution anoscopy (HRA with biopsies were performed at every visit along with anal sample collection for cytology, high-risk HPV DNA genotyping, HPV E6/E7 mRNA, and p16 immunocytochemistry. Performance characteristics and area under the receiver operator characteristics curve were calculated for these biomarkers at baseline, and Cox regression compared the usefulness of these biomarkers in predicting incident HSIL. High-risk HPV DNA, E6/E7 mRNA, and p16 immunocytochemistry each identified 43-46% of MSM whose baseline test positivity would trigger HRA referral. E6/E7 mRNA had the highest sensitivity (64.7% and correctly classified the highest number of prevalent HSIL cases. With the exception of p16 immunochemistry, most tests showed significant increases in sensitivity but decreases specificity versus anal cytology, while the overall number of correctly classified cases was not significantly different. Baseline or persistent type 16 and/or 18 HPV DNA was the only test significantly predicting incident histologic HSIL within 12 months in models adjusted for HIV status and low-grade squamous intraepithelial lesions at baseline.Countries with a high HIV prevalence among MSM and limited HRA resources may consider using biomarkers to identify individuals at high risk of HSIL. E6/E7 mRNA had the highest sensitivity for prevalent HSIL detection regardless of HIV status, whereas type 16 and/or 18 HPV DNA performed best in predicting development of incident HSIL within 12 months.

Novel HIV-1 knockdown targets identified by an enriched kinases/phosphatases shRNA library using a long-term iterative screen in Jurkat T-cells.

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Sylvie Rato

2010-02-01

Full Text Available HIV-1 is a complex retrovirus that uses host machinery to promote its replication. Understanding cellular proteins involved in the multistep process of HIV-1 infection may result in the discovery of more adapted and effective therapeutic targets. Kinases and phosphatases are a druggable class of proteins critically involved in regulation of signal pathways of eukaryotic cells. Here, we focused on the discovery of kinases and phosphatases that are essential for HIV-1 replication but dispensable for cell viability. We performed an iterative screen in Jurkat T-cells with a short-hairpin-RNA (shRNA library highly enriched for human kinases and phosphatases. We identified 14 new proteins essential for HIV-1 replication that do not affect cell viability. These proteins are described to be involved in MAPK, JNK and ERK pathways, vesicular traffic and DNA repair. Moreover, we show that the proteins under study are important in an early step of HIV-1 infection before viral integration, whereas some of them affect viral transcription/translation. This study brings new insights for the complex interplay of HIV-1/host cell and opens new possibilities for antiviral strategies.

EBV AND HHV-6 CIRCULATING SUBTYPES IN PEOPLE LIVING WITH HIV IN BURKINA FASO, IMPACT ON CD4 T CELL COUNT AND HIV VIRAL LOAD

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Lassina TRAORE

2017-09-01

Full Text Available Epstein Barr Virus (EBV and Human Herpes Virus 6 (HHV-6 are responsible for severe diseases, particularly in immunocompromised persons. There are poor data on the infection with these opportunistic viruses in Burkina Faso. The purpose of this study is to characterize EBV and HHV-6 subtypes and to assess their impact on CD4 T cell count, HIV-1 viral load and antiretroviral treatment in people living with HIV-1. The study population consisted of 238 HIV-positive patients with information on CD4 count, HIV-1 viral load and HAART. Venous blood samples collected on EDTA tubes were used for EBV and HHV-6 Real Time PCR subtyping. An infection rate of 6.7% (16/238 and 7.1% (17/238 were found respectively for EBV and HHV-6 in the present study. Among EBV infections, similar prevalences were noted for both subtypes (3.9% [9/238] for EBV-1 vs 4.6% [11/238] for EBV-2 with 2.1% (5/238 of co-infection. HHV-6A infection represented 6.3% (15/238 of the study population against 5.0% (12/238 for HHV-6B. . EBV-2 infection was significantly higher in patients with CD4 count ≥ 500 compared to those with CD4 count less than 500 cells (1.65% vs 8.56%, p = 0,011. The prevalence of EBV and HHV-6 infections were almost similar in HAART-naive and HAART-experienced patients. The present study provides information on the prevalence of EBV and HHV-6 subtypes in people living with HIV-1 in Burkina Faso. The study also suggests that HAART treatment has no effect on infection with these opportunistic viruses in people living with HIV-1.

Magnetic bead/capture DNA/glucose-loaded nanoliposomes for amplifying the glucometer signal in the rapid screening of hepatitis C virus RNA.

Science.gov (United States)

Tu, Haijian; Lin, Kun; Lun, Yongzhi; Yu, Liuming

2018-06-01

A digital detection strategy based on a portable personal glucometer (PGM) was developed for the simple, rapid, and sensitive detection of hepatitis C virus (HCV) RNA, involving the release of glucose-loaded nanoliposomes due to coupling-site-specific cleavage by the endonuclease BamHI. The glucose-loaded nanoliposomes were synthesized using a reversed-phase evaporation method and provided an amplified signal at the PGM in the presence of HCV RNA. Initially, a 21-mer oligonucleotide complementary to HCV RNA was covalently conjugated to a magnetic bead through the amino group at the 5' end of the oligonucleotide, and then bound to a glucose-loaded liposome by typical carbodiimide coupling at its 3' end. In the presence of the target HCV RNA, the target hybridized with the oligonucleotide to form double-stranded DNA. The symmetrical duplex sequence 5'-GGATCC-3' between guanines was then catalytically cleaved by BamHI, which detached the glucose-loaded liposome from the magnetic bead. Following magnetic separation of the bead, the detached glucose-loaded liposome was lysed using Triton X-100 to release the glucose molecules within it, which were then detected as an amplified signal at the digital PGM. Under optimal conditions, the PGM signal increased with increasing HCV RNA, and displayed a strongly linear dependence on the level of HCV RNA for concentrations ranging from 10 pM to 1.0 μM. The detection limit (LOD) of the system was 1.9 pM. Good reproducibility and favorable specificity were achieved in the analysis of the target HCV RNA. Human serum samples containing HCV RNA were analyzed using this strategy, and the developed sensing platform was observed to yield satisfactory results based on a comparison with the corresponding results from a Cobas ® Amplicor HCV Test Analyzer. Graphical abstract A digital detection strategy utilizing a personal glucometer was developed for the detection of hepatitis C virus RNA. The strategy involved the use of the

The export receptor Crm1 forms a dimer to promote nuclear export of HIV RNA.

Science.gov (United States)

Booth, David S; Cheng, Yifan; Frankel, Alan D

2014-12-08

The HIV Rev protein routes viral RNAs containing the Rev Response Element (RRE) through the Crm1 nuclear export pathway to the cytoplasm where viral proteins are expressed and genomic RNA is delivered to assembling virions. The RRE assembles a Rev oligomer that displays nuclear export sequences (NESs) for recognition by the Crm1-Ran(GTP) nuclear receptor complex. Here we provide the first view of an assembled HIV-host nuclear export complex using single-particle electron microscopy. Unexpectedly, Crm1 forms a dimer with an extensive interface that enhances association with Rev-RRE and poises NES binding sites to interact with a Rev oligomer. The interface between Crm1 monomers explains differences between Crm1 orthologs that alter nuclear export and determine cellular tropism for viral replication. The arrangement of the export complex identifies a novel binding surface to possibly target an HIV inhibitor and may point to a broader role for Crm1 dimerization in regulating host gene expression.

Continued indinavir versus switching to indinavir/ritonavir in HIV-infected patients with suppressed viral load.

NARCIS (Netherlands)

Arnaiz, J.A.; Mallolas, J.; Podzamczer, D.; Gerstoft, J.; Lundgren, J.D.; Cahn, P.; Fatkenheuer, G.; D'Arminio-Monforte, A.; Casiro, A.; Reiss, P.; Burger, D.M.; Stek Jr, M.; Gatell, J.M.

2003-01-01

OBJECTIVE: To compare continued indinavir (IDV) 8-hourly (q8h) with switching to indinavir/ritonavir (IDV/RTV) 12-hourly (q12h) in HIV-positive patients having suppressed viral load with IDV q8h plus two nucleoside reverse transcriptase inhibitors (NRTI). DESIGN: Multicentre, international,

Continued indinavir versus switching to indinavir/ritonavir in HIV-infected patients with suppressed viral load

NARCIS (Netherlands)

Arnaiz, Juan A.; Mallolas, Josep; Podzamczer, Daniel; Gerstoft, Jan; Lundgren, Jens D.; Cahn, Pedro; Fätkenheuer, Gerd; D'Arminio-Monforte, Antonella; Casiró, Arnaldo; Reiss, Peter; Burger, David M.; Stek, Michael; Gatell, José M.

2003-01-01

Objective: To compare continued indinavir (IDV) 8-hourly (q8h) with switching to indinavir/ritonavir (IDV/RTV) 12-hourly (q12h) in HIV-positive patients having suppressed viral load with IDV q8h plus two nucleoside reverse transcriptase inhibitors (NRTI). Design: Multicentre, international,

Evaluation of a cost effective in-house method for HIV-1 drug resistance genotyping using plasma samples.

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Devidas N Chaturbhuj

Full Text Available OBJECTIVES: Validation of a cost effective in-house method for HIV-1 drug resistance genotyping using plasma samples. DESIGN: The validation includes the establishment of analytical performance characteristics such as accuracy, reproducibility, precision and sensitivity. METHODS: The accuracy was assessed by comparing 26 paired Virological Quality Assessment (VQA proficiency testing panel sequences generated by in-house and ViroSeq Genotyping System 2.0 (Celera Diagnostics, US as a gold standard. The reproducibility and precision were carried out on five samples with five replicates representing multiple HIV-1 subtypes (A, B, C and resistance patterns. The amplification sensitivity was evaluated on HIV-1 positive plasma samples (n = 88 with known viral loads ranges from 1000-1.8 million RNA copies/ml. RESULTS: Comparison of the nucleotide sequences generated by ViroSeq and in-house method showed 99.41±0.46 and 99.68±0.35% mean nucleotide and amino acid identity respectively. Out of 135 Stanford HIVdb listed HIV-1 drug resistance mutations, partial discordance was observed at 15 positions and complete discordance was absent. The reproducibility and precision study showed high nucleotide sequence identities i.e. 99.88±0.10 and 99.82±0.20 respectively. The in-house method showed 100% analytical sensitivity on the samples with HIV-1 viral load >1000 RNA copies/ml. The cost of running the in-house method is only 50% of that for ViroSeq method (112$ vs 300$, thus making it cost effective. CONCLUSIONS: The validated cost effective in-house method may be used to collect surveillance data on the emergence and transmission of HIV-1 drug resistance in resource limited countries. Moreover, the wide applications of a cost effective and validated in-house method for HIV-1 drug resistance testing will facilitate the decision making for the appropriate management of HIV infected patients.

GB Virus C (GBV-C Infection in Hepatitis C Virus (HCV Seropositive Women with or at Risk for HIV Infection.

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Jason T Blackard

Full Text Available GB virus C (GBV-C may have a beneficial impact on HIV disease progression; however, the epidemiologic characteristics of this virus are not well characterized. Behavioral factors and gender may lead to differential rates of GBV-C infection; yet, studies have rarely addressed GBV-C infections in women or racial/ethnic minorities. Therefore, we evaluated GBV-C RNA prevalence and genotype distribution in a large prospective study of high-risk women in the US.438 hepatitis C virus (HCV seropositive women, including 306 HIV-infected and 132 HIV-uninfected women, from the HIV Epidemiologic Research Study were evaluated for GBV-C RNA. 347 (79.2% women were GBV-C RNA negative, while 91 (20.8% were GBV-C RNA positive. GBV-C positive women were younger than GBV-C negative women. Among 306 HIV-infected women, 70 (22.9% women were HIV/GBV-C co-infected. Among HIV-infected women, the only significant difference between GBV-negative and GBV-positive women was age (mean 38.4 vs. 35.1 years; p<0.001. Median baseline CD4 cell counts and plasma HIV RNA levels were similar. The GBV-C genotypes were 1 (n = 31; 44.3%, 2 (n = 36; 51.4%, and 3 (n = 3; 4.3%. The distribution of GBV-C genotypes in co-infected women differed significantly by race/ethnicity. However, median CD4 cell counts and log10 HIV RNA levels did not differ by GBV-C genotype. GBV-C incidence was 2.7% over a median follow-up of 2.9 (IQR: 1.5, 4.9 years, while GBV-C clearance was 35.7% over a median follow-up of 2.44 (1.4, 3.5 years. 4 women switched genotypes.Age, injection drug use, a history of sex for money or drugs, and number of recent male sex partners were associated with GBV-C infection among all women in this analysis. However, CD4 cell count and HIV viral load of HIV/HCV/GBV-C co-infected women were not different although race was associated with GBV-C genotype.

Viral dynamics in primary HIV-1 infection. Karolinska Institutet Primary HIV Infection Study Group.

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Lindbäck, S; Karlsson, A C; Mittler, J; Blaxhult, A; Carlsson, M; Briheim, G; Sönnerborg, A; Gaines, H

2000-10-20

To study the natural course of viremia during primary HIV infection (PHI). Eight patients were followed from a median of 5 days from the onset of PHI illness. Plasma HIV-1 RNA levels were measured frequently and the results were fitted to mathematical models. HIV-1 RNA levels were also monitored in nine patients given two reverse transcriptase inhibitors and a protease inhibitor after a median of 7 days from the onset of PHI illness. HIV-1 RNA appeared in the blood during the week preceding onset of PHI illness and increased rapidly during the first viremic phase, reaching a peak at a mean of 7 days after onset of illness. This was followed by a phase of rapidly decreasing levels of HIV-1 RNA to an average of 21 days after onset. Viral density continued to decline thereafter but at a 5- to 50-fold lower rate; a steady-state level was reached at a median of 2 months after onset of PHI. Peak viral density levels correlated significantly with levels measured between days 50 and 600. Initiation of antiretroviral treatment during PHI resulted in rapidly declining levels to below 50 copies/mL. This study demonstrates the kinetic phases of viremia during PHI and indicates two new contributions to the natural history of HIV-1 infection: PHI peak levels correlate with steady-state levels and HIV-1 RNA declines biphasically; an initial rapid decay is usually followed by a slow decay, which is similar to the initial changes seen with antiviral treatment.

HIVsirDB: a database of HIV inhibiting siRNAs.

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Atul Tyagi

Full Text Available Human immunodeficiency virus (HIV is responsible for millions of deaths every year. The current treatment involves the use of multiple antiretroviral agents that may harm patients due to their toxic nature. RNA interference (RNAi is a potent candidate for the future treatment of HIV, uses short interfering RNA (siRNA/shRNA for silencing HIV genes. In this study, attempts have been made to create a database HIVsirDB of siRNAs responsible for silencing HIV genes.HIVsirDB is a manually curated database of HIV inhibiting siRNAs that provides comprehensive information about each siRNA or shRNA. Information was collected and compiled from literature and public resources. This database contains around 750 siRNAs that includes 75 partially complementary siRNAs differing by one or more bases with the target sites and over 100 escape mutant sequences. HIVsirDB structure contains sixteen fields including siRNA sequence, HIV strain, targeted genome region, efficacy and conservation of target sequences. In order to facilitate user, many tools have been integrated in this database that includes; i siRNAmap for mapping siRNAs on target sequence, ii HIVsirblast for BLAST search against database, iii siRNAalign for aligning siRNAs.HIVsirDB is a freely accessible database of siRNAs which can silence or degrade HIV genes. It covers 26 types of HIV strains and 28 cell types. This database will be very useful for developing models for predicting efficacy of HIV inhibiting siRNAs. In summary this is a useful resource for researchers working in the field of siRNA based HIV therapy. HIVsirDB database is accessible at http://crdd.osdd.net/raghava/hivsir/.

Toxoplasmic encephalitis in an HIV infected pregnant woman: successful outcome for both mother and child

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Susie Andries Nogueira

Full Text Available This report describes a case of Toxoplasma encephalitis during pregnancy of an HIV infected woman who was severely immunosuppressed (CD4: 17 cells/mm3, had a high viral load (RNA PCR:230,000 copies/ml, was treated with sulfadiazine, pyrimethamine and folinic acid for toxoplasmosis and was being treated with highly potent antiretroviral drugs (AZT, 3TC and nelfinavir for HIV infection. The newborn was born through an elective C-section, received six weeks of AZT according to the 076 protocol and was clinically normal at birth. Subsequently he had two RNA PCR negatives for HIV, seroreverted and had no clinical or laboratory evidence of congenital toxoplasmosis. Despite the concerns of the use of these combined therapies on the foetus during pregnancy, their efficacy illustrates that keeping the mother alive and in good health is an important strategy to protect the unborn child from acquiring these two infections.

Sensitivity and specificity of dried blood spots for HIV-1 viral load quantification

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Pannus, Pieter; Claus, Maarten; Gonzalez, Maria Mercedes Perez; Ford, Nathan; Fransen, Katrien

2016-01-01

Abstract The use of dried blood spots (DBS) instead of plasma as a specimen type for HIV-1 viral load (VL) testing facilitates the decentralization of specimen collection and can increase access to VL testing in resource-limited settings. The performance of DBS for VL testing is lower, however, when compared to the gold standard sample type plasma. In this diagnostic accuracy study, we evaluated 3 VL assays with DBS. Participants were recruited between August 2012 and April 2015. Both plasma and DBS specimens were prepared and tested for HIV-1 VL with the Roche CAP/CTM HIV-1 test v2.0, the Abbott RealTime HIV-1, and the bioMérieux NucliSENS EasyQ HIV-1 v2.0. Sensitivity and specificity to detect treatment failure at a threshold of 1000 cps/mL with DBS were determined. A total of 272 HIV-positive patients and 51 HIV-negative people were recruited in the study. The mean difference or bias between plasma and DBS VL was 25% of the specimens differed by >0.5 log cps/mL. All 3 assays had comparable sensitivities around 80% and specificities around 90%. Upward misclassification rates were around 10%, but downward misclassification rates ranged from 20.3% to 23.6%. Differences in between assays were not statistically significant (P > 0.1). The 3 VL assays evaluated had suboptimal performance with DBS but still performed better than immunological or clinical monitoring. Even after the introduction of the much-anticipated point-of-care VL devices, it is expected that DBS will remain important as a complementary option for supporting access to VL monitoring, particularly in rural, resource-limited settings. Manufacturers should accelerate efforts to develop more reliable, sensitive and specific methods to test VL on DBS specimens. PMID:27902602

Fab’-bearing siRNA TNFα-loaded nanoparticles targeted to colonic macrophages offer an effective therapy for experimental colitis

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Hamed, Laroui; Emilie, Viennois; Xiao, Bo; Canup, Brandon S.; Duke, Geem; Denning, Timothy L.; Didier, Merlin

2014-01-01

Patients suffering from Inflammatory Bowel Disease (IBD) are currently treated by systemic drugs that can have significant side effects. Thus, it would be highly desirable to target TNFα siRNA (a therapeutic molecule) to the inflamed tissue. Here, we demonstrate that TNFα siRNA can be efficiently loaded into nanoparticles (NPs) made of poly (lactic acid) poly (ethylene glycol) block copolymer (PLA-PEG), and that grafting of the Fab’ portion of the F4/80 Ab (Fab’-bearing) onto the NP surface via maleimide/thiol group-mediated covalent bonding improves the macrophage (MP)-targeting kinetics of the NPs to RAW264.7 cells in vitro. Direct binding was shown between MPs and the Fab’-bearing NPs. Next, we orally administered hydrogel (chitosan/alginate)-encapsulated Fab’-bearing TNFα-siRNA-loaded NPs to 3% dextran sodium sulfate (DSS)-treated mice and investigated the therapeutic effect on colitis. In vivo, the release of TNFα-siRNA-loaded NPs into the mouse colon attenuated colitis more efficiently when the NPs were covered with Fab’-bearing, compared to uncovered NPs. All DSS-induced parameters of colonic inflammation (e.g., weight loss, myeloperoxidase activity, and Iκbα accumulation) were more attenuated Fab’-bearing NPs loaded with TNFα siRNA than without the Fab’-bearing. Grafting the Fab’-bearing onto the NPs improved the kinetics of endocytosis as well as the MP-targeting ability, as indicated by flow cytometry. Collectively, our results show that Fab’-bearing PLA-PEG NPs are powerful and efficient nanosized tools for delivering siRNAs into colonic macrophages. PMID:24810114

Determination of HIV Status in African Adults With Discordant HIV Rapid Tests.

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Fogel, Jessica M; Piwowar-Manning, Estelle; Donohue, Kelsey; Cummings, Vanessa; Marzinke, Mark A; Clarke, William; Breaud, Autumn; Fiamma, Agnès; Donnell, Deborah; Kulich, Michal; Mbwambo, Jessie K K; Richter, Linda; Gray, Glenda; Sweat, Michael; Coates, Thomas J; Eshleman, Susan H

2015-08-01

In resource-limited settings, HIV infection is often diagnosed using 2 rapid tests. If the results are discordant, a third tie-breaker test is often used to determine HIV status. This study characterized samples with discordant rapid tests and compared different testing strategies for determining HIV status in these cases. Samples were previously collected from 173 African adults in a population-based survey who had discordant rapid test results. Samples were classified as HIV positive or HIV negative using a rigorous testing algorithm that included two fourth-generation tests, a discriminatory test, and 2 HIV RNA tests. Tie-breaker tests were evaluated, including rapid tests (1 performed in-country), a third-generation enzyme immunoassay, and two fourth-generation tests. Selected samples were further characterized using additional assays. Twenty-nine samples (16.8%) were classified as HIV positive and 24 of those samples (82.8%) had undetectable HIV RNA. Antiretroviral drugs were detected in 1 sample. Sensitivity was 8.3%-43% for the rapid tests; 24.1% for the third-generation enzyme immunoassay; 95.8% and 96.6% for the fourth-generation tests. Specificity was lower for the fourth-generation tests than the other tests. Accuracy ranged from 79.5% to 91.3%. In this population-based survey, most HIV-infected adults with discordant rapid tests were virally suppressed without antiretroviral drugs. Use of individual assays as tie-breaker tests was not a reliable method for determining HIV status in these individuals. More extensive testing algorithms that use a fourth-generation screening test with a discriminatory test and HIV RNA test are preferable for determining HIV status in these cases.

Penetration of polymeric nanoparticles loaded with an HIV-1 inhibitor peptide derived from GB virus C in a vaginal mucosa model.

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Ariza-Sáenz, Martha; Espina, Marta; Bolaños, Nuria; Calpena, Ana Cristina; Gomara, María José; Haro, Isabel; García, María Luisa

2017-11-01

Despite the great effort to decrease the HIV infectivity rate, current antiretroviral therapy has several weaknesses; poor bioavailability, development of drug resistance and poor ability to access tissues. However, molecules such as peptides have emerged asa new expectative to HIV eradication. The vaginal mucosa is the main spreading point of HIV. There are natural barriers such as the vaginal fluid which protects the vaginal epithelium from any foreign agents reaching it. This work has developed and characterized Nanoparticles (NPs) coated with glycol chitosan (GC), loaded with an HIV-1 inhibitor peptide (E2). In vitro release and ex vivo studies were carried out using the vaginal mucosa of swine and the peptide was determined by HPLC MS/MS validated method. Moreover, the peptide was labeled with 5(6)-carboxyfluoresceine and entrapped into the NPs to carried out in vivo studies and to evaluate the NPs penetration and toxicity in the vaginal mucosa of the swine. The mean size of the NPs, ξ and the loading percentage were fundamental features for to reach the vaginal tissue and to release the peptide within intercellular space. The obtained results suggesting that the fusion inhibitor peptides loaded into the NPs coated with GC might be a new way to fight the HIV-1, due to the formulation might reach the human epithelial mucosa and release peptide without any side effects. Copyright © 2017 Elsevier B.V. All rights reserved.

Thermodynamic studies of a series of homologous HIV-1 TAR RNA ligands reveal that loose binders are stronger Tat competitors than tight ones.

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Pascale, Lise; Azoulay, Stéphane; Di Giorgio, Audrey; Zenacker, Laura; Gaysinski, Marc; Clayette, Pascal; Patino, Nadia

2013-06-01

RNA is a major drug target, but the design of small molecules that modulate RNA function remains a great challenge. In this context, a series of structurally homologous 'polyamide amino acids' (PAA) was studied as HIV-1 trans-activating response (TAR) RNA ligands. An extensive thermodynamic study revealed the occurence of an enthalpy-entropy compensation phenomenon resulting in very close TAR affinities for all PAA. However, their binding modes and their ability to compete with the Tat fragment strongly differ according to their structure. Surprisingly, PAA that form loose complexes with TAR were shown to be stronger Tat competitors than those forming tight ones, and thermal denaturation studies demonstrated that loose complexes are more stable than tight ones. This could be correlated to the fact that loose and tight ligands induce distinct RNA conformational changes as revealed by circular dichroism experiments, although nuclear magnetic resonance (NMR) experiments showed that the TAR binding site is the same in all cases. Finally, some loose PAA also display promising inhibitory activities on HIV-infected cells. Altogether, these results lead to a better understanding of RNA interaction modes that could be very useful for devising new ligands of relevant RNA targets.

Nef exosomes isolated from the plasma of individuals with HIV-associated dementia (HAD) can induce Aβ(1-42) secretion in SH-SY5Y neural cells.

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Khan, Mahfuz B; Lang, Michelle J; Huang, Ming-Bo; Raymond, Andrea; Bond, Vincent C; Shiramizu, Bruce; Powell, Michael D

2016-04-01

In the era of combined antiretroviral therapy (CART), many of the complications due to HIV-1 infection have diminished. One exception is HIV-associated neurocognitive disorder (HAND). HAND is a spectrum of disorders in cognitive function that ranges from asymptomatic disease to severe dementia (HAD). The milder form of HAND has actually remained the same or slightly increased in prevalence in the CART era. Even in individuals who have maintained undetectable HIV RNA loads, viral proteins such as Nef and Tat can continue to be expressed. In this report, we show that Nef protein and nef messenger RNA (mRNA) are packaged into exosomes that remain in circulation in patients with HAD. Plasma-derived Nef exosomes from patients with HAD have the ability to interact with the neuroblastoma cell line SH-SY5Y and deliver nef mRNA. The mRNA can induce expression of Nef in target cells and subsequently increase expression and secretion of beta-amyloid (Aβ) and Aβ peptides. Increase secretion of amyloid peptide could contribute to cognitive impairment seen in HAND.

The National NeuroAIDS Tissue Consortium brain gene array: two types of HIV-associated neurocognitive impairment.

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Benjamin B Gelman

Full Text Available The National NeuroAIDS Tissue Consortium (NNTC performed a brain gene expression array to elucidate pathophysiologies of Human Immunodeficiency Virus type 1 (HIV-1-associated neurocognitive disorders.Twenty-four human subjects in four groups were examined A Uninfected controls; B HIV-1 infected subjects with no substantial neurocognitive impairment (NCI; C Infected with substantial NCI without HIV encephalitis (HIVE; D Infected with substantial NCI and HIVE. RNA from neocortex, white matter, and neostriatum was processed with the Affymetrix® array platform.With HIVE the HIV-1 RNA load in brain tissue was three log(10 units higher than other groups and over 1,900 gene probes were regulated. Interferon response genes (IFRGs, antigen presentation, complement components and CD163 antigen were strongly upregulated. In frontal neocortex downregulated neuronal pathways strongly dominated in HIVE, including GABA receptors, glutamate signaling, synaptic potentiation, axon guidance, clathrin-mediated endocytosis and 14-3-3 protein. Expression was completely different in neuropsychologically impaired subjects without HIVE. They had low brain HIV-1 loads, weak brain immune responses, lacked neuronally expressed changes in neocortex and exhibited upregulation of endothelial cell type transcripts. HIV-1-infected subjects with normal neuropsychological test results had upregulation of neuronal transcripts involved in synaptic transmission of neostriatal circuits.Two patterns of brain gene expression suggest that more than one pathophysiological process occurs in HIV-1-associated neurocognitive impairment. Expression in HIVE suggests that lowering brain HIV-1 replication might improve NCI, whereas NCI without HIVE may not respond in kind; array results suggest that modulation of transvascular signaling is a potentially promising approach. Striking brain regional differences highlighted the likely importance of circuit level disturbances in HIV/AIDS. In

Single Tablet Regimen Usage and Efficacy in the Treatment of HIV Infection in Australia

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B. Armstrong

2015-01-01

Full Text Available Single tablet regimens (STRs for HIV infection improve patient satisfaction, quality of life, medication adherence, and virological suppression compared to multitablet regimens (MTRs. This is the first study assessing STR uptake and durability in Australia. This retrospective audit of all patients receiving an STR (n=299 at a large Sydney HIV clinic (January 2012–December 2013 assessed patient demographics, treatment prior to STR, HIV RNA load and CD4 during MTR and STR dosing, and reasons for STR switch. 206 patients switched from previous antiretroviral treatment to an STR, of which 88% switched from an MTR. Reasons for switching included desire to simplify treatment (57%, reduced side effects or toxicity (18%, and cost-saving for the patient. There was no switching for virological failure. Compared to when on an MTR, patients switching to an STR had significantly lower HIV RNA counts (p<0.001 and significantly higher CD4 counts (p<0.001. The discontinuation rate from STR was very low and all patients who switched to an STR maintained virological suppression throughout the study duration, although the study is limited by the absence of a control group.

Performance of the Xpert HIV-1 Viral Load Assay: a Systematic Review and Meta-analysis.

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Nash, Madlen; Huddart, Sophie; Badar, Sayema; Baliga, Shrikala; Saravu, Kavitha; Pai, Madhukar

2018-04-01

Viral load (VL) is the preferred treatment-monitoring approach for HIV-positive patients. However, more rapid, near-patient, and low-complexity assays are needed to scale up VL testing. The Xpert HIV-1 VL assay (Cepheid, Sunnyvale, CA) is a new, automated molecular test, and it can leverage the GeneXpert systems that are being used widely for tuberculosis diagnosis. We systematically reviewed the evidence on the performance of this new tool in comparison to established reference standards. A total of 12 articles (13 studies) in which HIV patient VLs were compared between Xpert HIV VL assay and a reference standard VL assay were identified. Study quality was generally high, but substantial variability was observed in the number and type of agreement measures reported. Correlation coefficients between Xpert and reference assays were high, with a pooled Pearson correlation ( n = 8) of 0.94 (95% confidence interval [CI], 0.89, 0.97) and Spearman correlation ( n = 3) of 0.96 (95% CI, 0.86, 0.99). Bland-Altman metrics ( n = 11) all were within 0.35 log copies/ml of perfect agreement. Overall, Xpert HIV-1 VL performed well compared to current reference tests. The minimal training and infrastructure requirements for the Xpert HIV-1 VL assay make it attractive for use in resource-constrained settings, where point-of-care VL testing is most needed. Copyright © 2018 Nash et al.

The effect of HIV infection and HCV viremia on inflammatory mediators and hepatic injury-The Women's Interagency HIV Study.

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Sheila M Keating

Full Text Available Hepatitis C virus infection induces inflammation and while it is believed that HIV co-infection enhances this response, HIV control may reduce inflammation and liver fibrosis in resolved or viremic HCV infection. Measurement of systemic biomarkers in co-infection could help define the mechanism of inflammation on fibrosis and determine if HIV control reduces liver pathology. A nested case-control study was performed to explore the relationship of systemic biomarkers of inflammation with liver fibrosis in HCV viremic and/or seropositive women with and without HIV infection. Serum cytokines, chemokines, growth factors and cell adhesion molecules were measured in HIV uninfected (HIV-, n = 18, ART-treated HIV-controlled (ARTc, n = 20, uncontrolled on anti-retroviral therapy (ARTuc, n = 21 and elite HIV controllers (Elite, n = 20. All were HCV seroreactive and had either resolved (HCV RNA-; <50IU/mL or had chronic HCV infection (HCV RNA+. In HCV and HIV groups, aspartate aminotransferase to platelet ratio (APRI was measured and compared to serum cytokines, chemokines, growth factors and cell adhesion molecules. APRI correlated with sVCAM, sICAM, IL-10, and IP-10 levels and inversely correlated with EGF, IL-17, TGF-α and MMP-9 levels. Collectively, all HCV RNA+ subjects had higher sVCAM, sICAM and IP-10 compared to HCV RNA-. In the ART-treated HCV RNA+ groups, TNF-α, GRO, IP-10, MCP-1 and MDC were higher than HIV-, Elite or both. In ARTuc, FGF-2, MPO, soluble E-selectin, MMP-9, IL-17, GM-CSF and TGF-α are lower than HIV-, Elite or both. Differential expression of soluble markers may reveal mechanisms of pathogenesis or possibly reduction of fibrosis in HCV/HIV co-infection.

Preclinical safety and efficacy of an anti–HIV-1 lentiviral vector containing a short hairpin RNA to CCR5 and the C46 fusion inhibitor

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Orit Wolstein

2014-01-01

Full Text Available Gene transfer has therapeutic potential for treating HIV-1 infection by generating cells that are resistant to the virus. We have engineered a novel self-inactivating lentiviral vector, LVsh5/C46, using two viral-entry inhibitors to block early steps of HIV-1 cycle. The LVsh5/C46 vector encodes a short hairpin RNA (shRNA for downregulation of CCR5, in combination with the HIV-1 fusion inhibitor, C46. We demonstrate here the effective delivery of LVsh5/C46 to human T cell lines, peripheral blood mononuclear cells, primary CD4+ T lymphocytes, and CD34+ hematopoietic stem/progenitor cells (HSPC. CCR5-targeted shRNA (sh5 and C46 peptide were stably expressed in the target cells and were able to effectively protect gene-modified cells against infection with CCR5- and CXCR4-tropic strains of HIV-1. LVsh5/C46 treatment was nontoxic as assessed by cell growth and viability, was noninflammatory, and had no adverse effect on HSPC differentiation. LVsh5/C46 could be produced at a scale sufficient for clinical development and resulted in active viral particles with very low mutagenic potential and the absence of replication-competent lentivirus. Based on these in vitro results, plus additional in vivo safety and efficacy data, LVsh5/C46 is now being tested in a phase 1/2 clinical trial for the treatment of HIV-1 disease.

Associations Between Medicare Part D and Out-of-Pocket Spending, HIV Viral Load, Adherence, and ADAP Use in Dual Eligibles With HIV.

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Belenky, Nadya; Pence, Brian W; Cole, Stephen R; Dusetzina, Stacie B; Edmonds, Andrew; Oberlander, Jonathan; Plankey, Michael W; Adedimeji, Adebola; Wilson, Tracey E; Cohen, Jennifer; Cohen, Mardge H; Milam, Joel E; Golub, Elizabeth T; Adimora, Adaora A

2018-01-01

The implementation of Medicare part D on January 1, 2006 required all adults who were dually enrolled in Medicaid and Medicare (dual eligibles) to transition prescription drug coverage from Medicaid to Medicare part D. Changes in payment systems and utilization management along with the loss of Medicaid protections had the potential to disrupt medication access, with uncertain consequences for dual eligibles with human immunodeficiency virus (HIV) who rely on consistent prescription coverage to suppress their HIV viral load (VL). To estimate the effect of Medicare part D on self-reported out-of-pocket prescription drug spending, AIDS Drug Assistance Program (ADAP) use, antiretroviral adherence, and HIV VL suppression among dual eligibles with HIV. Using 2003-2008 data from the Women's Interagency HIV Study, we created a propensity score-matched cohort and used a difference-in-differences approach to compare dual eligibles' outcomes pre-Medicare and post-Medicare part D to those enrolled in Medicaid alone. Transition to Medicare part D was associated with a sharp increase in the proportion of dual eligibles with self-reported out-of-pocket prescription drug costs, followed by an increase in ADAP use. Despite the increase in out-of-pocket costs, both adherence and HIV VL suppression remained stable. Medicare part D was associated with increased out-of-pocket spending, although the increased spending did not seem to compromise antiretroviral therapy adherence or HIV VL suppression. It is possible that increased ADAP use mitigated the increase in out-of-pocket spending, suggesting successful coordination between Medicare part D and ADAP as well as the vital role of ADAP during insurance transitions.

Enrichment of intersubtype HIV-1 recombinants in a dual infection system using HIV-1 strain-specific siRNAs

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2011-01-01

Background Intersubtype HIV-1 recombinants in the form of unique or stable circulating recombinants forms (CRFs) are responsible for over 20% of infections in the worldwide epidemic. Mechanisms controlling the generation, selection, and transmission of these intersubtype HIV-1 recombinants still require further investigation. All intersubtype HIV-1 recombinants are generated and evolve from initial dual infections, but are difficult to identify in the human population. In vitro studies provide the most practical system to study mechanisms, but the recombination rates are usually very low in dual infections with primary HIV-1 isolates. This study describes the use of HIV-1 isolate-specific siRNAs to enrich intersubtype HIV-1 recombinants and inhibit the parental HIV-1 isolates from a dual infection. Results Following a dual infection with subtype A and D primary HIV-1 isolates and two rounds of siRNA treatment, nearly 100% of replicative virus was resistant to a siRNA specific for an upstream target sequence in the subtype A envelope (env) gene as well as a siRNA specific for a downstream target sequence in the subtype D env gene. Only 20% (10/50) of the replicating virus had nucleotide substitutions in the siRNA-target sequence whereas the remaining 78% (39/50) harbored a recombination breakpoint that removed both siRNA target sequences, and rendered the intersubtype D/A recombinant virus resistant to the dual siRNA treatment. Since siRNAs target the newly transcribed HIV-1 mRNA, the siRNAs only enrich intersubtype env recombinants and do not influence the recombination process during reverse transcription. Using this system, a strong bias is selected for recombination breakpoints in the C2 region, whereas other HIV-1 env regions, most notably the hypervariable regions, were nearly devoid of intersubtype recombination breakpoints. Sequence conservation plays an important role in selecting for recombination breakpoints, but the lack of breakpoints in many conserved

Blood neutrophil counts in HIV-infected patients with pulmonary tuberculosis: association with sputum mycobacterial load.

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Andrew D Kerkhoff

Full Text Available Increasing evidence suggests that neutrophils play a role in the host response to Mycobacterium tuberculosis. We determined whether neutrophil counts in peripheral blood are associated with tuberculosis (TB and with mycobacterial load in sputum in HIV-infected patients.Adults enrolling in an antiretroviral treatment (ART clinic in a Cape Town township were screened for TB regardless of symptoms. Paired sputum samples were examined using liquid culture, fluorescence microscopy, and the Xpert MTB/RIF assay. Absolute neutrophil counts (ANC were measured in blood samples. Of 602 HIV-infected patients screened, 523 produced one or more sputum samples and had complete results available for analysis. Among these 523 patients, the median CD4 count was 169×10(9/L (IQR, 96-232 and median ANC was 2.6×10(9/L (IQR, 1.9-3.6. Culture-positive pulmonary tuberculosis was diagnosed in 89 patients. Patients with TB had a median ANC of 3.4×10(9/L (IQR, 2.4-5.1 compared to 2.5×10(9/L (IQR, 1.8-3.4 among those who were culture negative (p7.5×10(9/L; p = 0.0005. Patients were then classified into four mutually exclusive groups with increasing sputum mycobacterial load as defined by the results of culture, Xpert MTB/RIF and sputum smear microscopy. Multivariable analyses demonstrated that increasing sputum mycobacterial load was positively associated with blood ANC ≥2.6×10(9/L and with neutrophilia.Increased blood neutrophil counts were independently associated with pulmonary TB and sputum mycobacterial burden in this HIV-infected patient group. This observation supports the growing body of literature regarding the potential role for neutrophils in the host response to TB.

Viral load and genomic integration of HPV 16 in cervical samples from HIV-1-infected and uninfected women in Burkina Faso.

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Rousseau, Marie-Noelle Didelot; Costes, Valérie; Konate, Issouf; Nagot, Nicolas; Foulongne, Vincent; Ouedraogo, Abdoulaye; Van de Perre, Philippe; Mayaud, Philippe; Segondy, Michel

2007-06-01

The relationships between human papillomavirus type 16 (HPV 16) viral load, HPV 16 integration status, human immunodeficiency virus type 1 (HIV-1) status, and cervical cytology were studied among women enrolled in a cohort of female sex workers in Burkina Faso. The study focused on 24 HPV 16-infected women. The HPV 16 viral load in cervical samples was determined by real-time PCR. Integration ratio was estimated as the ratio between E2 and E6 genes DNA copy numbers. Integrated HPV16 viral load was defined as the product of HPV 16 viral load by the integration ratio. High HPV 16 viral load and high integration ratio were more frequent among women with squamous intraepithelial lesions compared with women with normal cytology (33% vs. 11%, and 33% vs. 0%, respectively), and among women with high-grade squamous intraepithelial lesions compared with women without high-grade squamous intraepithelial lesions (50% vs. 17%, and 50% vs. 11%, respectively). High HPV 16 DNA load, but not high integration ratio, was also more frequent among HIV-1-positive women (39% vs. 9%; and 23% vs. 18%, respectively). The absence of statistical significance of these differences might be explained by the small study sample size. High-integrated HPV 16 DNA load was significantly associated with the presence of high-grade squamous intraepithelial lesions (50% vs. 5%, P = 0.03) in univariate and multivariate analysis (adjusted odds-ratio: 19.05; 95% confidence interval (CI), 1.11-328.3, P = 0.03), but not with HIV-1 or other high-risk HPV types (HR-HPV). Integrated HPV 16 DNA load may be considered as a useful marker of high-grade cervical lesions in HPV 16-infected women. (c) 2007 Wiley-Liss, Inc.

Dimerization of human immunodeficiency virus (type 1) RNA: stimulation by cations and possible mechanism.

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Marquet, R; Baudin, F; Gabus, C; Darlix, J L; Mougel, M; Ehresmann, C; Ehresmann, B

1991-05-11

The retroviral genome consists of two identical RNA molecules joined close to their 5' ends by the dimer linkage structure. Recent findings indicated that retroviral RNA dimerization and encapsidation are probably related events during virion assembly. We studied the cation-induced dimerization of HIV-1 RNA and results indicate that all in vitro generated HIV-1 RNAs containing a 100 nucleotide domain downstream from the 5' splice site are able to dimerize. RNA dimerization depends on the concentration of RNA, mono- and multivalent cations, the size of the monovalent cation, temperature, and pH. Up to 75% of HIV-1 RNA is dimeric in the presence of spermidine. HIV-1 RNA dimer is fairly resistant to denaturing agents and unaffected by intercalating drugs. Antisense HIV-1 RNA does not dimerize but heterodimers can be formed between HIV-1 RNA and either MoMuLV or RSV RNA. Therefore retroviral RNA dimerization probably does not simply proceed through mechanisms involving Watson-Crick base-pairing. Neither adenine and cytosine protonation, nor quartets containing only guanines appear to determine the stability of the HIV-1 RNA dimer, while quartets involving both adenine(s) and guanine(s) could account for our results. A consensus sequence PuGGAPuA found in the putative dimerization-encapsidation region of all retroviral genomes examined may participate in the dimerization process.

2012 HIV Diagnostics Conference: the molecular diagnostics perspective.

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Branson, Bernard M; Pandori, Mark

2013-04-01

2012 HIV Diagnostic Conference Atlanta, GA, USA, 12-14 December 2012. This report highlights the presentations and discussions from the 2012 National HIV Diagnostic Conference held in Atlanta (GA, USA), on 12-14 December 2012. Reflecting changes in the evolving field of HIV diagnostics, the conference provided a forum for evaluating developments in molecular diagnostics and their role in HIV diagnosis. In 2010, the HIV Diagnostics Conference concluded with the proposal of a new diagnostic algorithm which included nucleic acid testing to resolve discordant screening and supplemental antibody test results. The 2012 meeting, picking up where the 2010 meeting left off, focused on scientific presentations that assessed this new algorithm and the role played by RNA testing and new developments in molecular diagnostics, including detection of total and integrated HIV-1 DNA, detection and quantification of HIV-2 RNA, and rapid formats for detection of HIV-1 RNA.

Translation initiation in bacterial polysomes through ribosome loading on a standby site on a highly translated mRNA

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Andreeva, Irena

2018-01-01

During translation, consecutive ribosomes load on an mRNA and form a polysome. The first ribosome binds to a single-stranded mRNA region and moves toward the start codon, unwinding potential mRNA structures on the way. In contrast, the following ribosomes can dock at the start codon only when the first ribosome has vacated the initiation site. Here we show that loading of the second ribosome on a natural 38-nt-long 5′ untranslated region of lpp mRNA, which codes for the outer membrane lipoprotein from Escherichia coli, takes place before the leading ribosome has moved away from the start codon. The rapid formation of this standby complex depends on the presence of ribosomal proteins S1/S2 in the leading ribosome. The early recruitment of the second ribosome to the standby site before translation by the leading ribosome and the tight coupling between translation elongation by the first ribosome and the accommodation of the second ribosome can contribute to high translational efficiency of the lpp mRNA. PMID:29632209

Field evaluation of an open and polyvalent universal HIV-1/SIVcpz/SIVgor quantitative RT-PCR assay for HIV-1 viral load monitoring in comparison to Abbott RealTime HIV-1 in Cameroon.

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Guichet, Emilande; Aghokeng, Avelin; Eymard-Duvernay, Sabrina; Vidal, Nicole; Ayouba, Ahidjo; Mpoudi Ngole, Eitel; Delaporte, Eric; Ciaffi, Laura; Peeters, Martine

2016-11-01

With the increasing demand of HIV viral load (VL) tests in resource-limited countries (RLCs) there is a need for assays at affordable cost and able to quantify all known HIV-1 variants. VLs obtained with a recently developed open and polyvalent universal HIV-1/SIVcpz/SIVgor RT-qPCR were compared to Abbott RealTime HIV-1 assay in Cameroon. On 474 plasma samples, characterized by a wide range of VLs and a broad HIV-1 group M genetic diversity, 97.5% concordance was observed when using the lower detection limit of each assay. When using the threshold of 3.00 log 10 copies/mL, according to WHO guidelines to define virological failure (VF) in RLCs, the concordance was 94.7%, 360/474 versus 339/474 patients were identified with VF with the new assay and Abbott RealTime HIV-1, respectively. Higher VLs were measured with the new assay, +0.47 log 10 copies/mL (95% CI; 0.42-0.52) as shown with Bland-Altman analysis. Eleven samples from patients on VF with drug resistance were not detected by Abbott RealTime HIV-1 versus two only with the new assay. Overall, our study showed that the new assay can be easily implemented in a laboratory in RLCs with VL experience and showed good performance on a wide diversity of HIV-1 group M variants. Copyright © 2016 Elsevier B.V. All rights reserved.

Computerized counseling reduces HIV-1 viral load and sexual transmission risk: findings from a randomized controlled trial.

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Kurth, Ann E; Spielberg, Freya; Cleland, Charles M; Lambdin, Barrot; Bangsberg, David R; Frick, Pamela A; Severynen, Anneleen O; Clausen, Marc; Norman, Robert G; Lockhart, David; Simoni, Jane M; Holmes, King K

2014-04-15

Evaluate a computerized intervention supporting antiretroviral therapy (ART) adherence and HIV transmission prevention. Longitudinal randomized controlled trial. An academic HIV clinic and a community-based organization in Seattle. In a total of 240 HIV-positive adults on ART, 209 completed 9-month follow-up (87% retention). Randomization to computerized counseling or assessment only, 4 sessions over 9 months. HIV-1 viral suppression, and self-reported ART adherence and transmission risks, compared using generalized estimating equations. Overall, intervention participants had reduced viral load: mean 0.17 log10 decline, versus 0.13 increase in controls, P = 0.053, and significant difference in ART adherence baseline to 9 months (P = 0.046). Their sexual transmission risk behaviors decreased (odds ratio = 0.55, P = 0.020), a reduction not seen among controls (odds ratio = 1.1, P = 0.664), and a significant difference in change (P = 0.040). Intervention effect was driven by those most in need; among those with detectable virus at baseline (>30 copies/mL, n = 89), intervention effect was mean 0.60 log10 viral load decline versus 0.15 increase in controls, P = 0.034. ART adherence at the final follow-up was 13 points higher among intervention participants versus controls, P = 0.038. Computerized counseling is promising for integrated ART adherence and safer sex, especially for individuals with problems in these areas. This is the first intervention to report improved ART adherence, viral suppression, and reduced secondary sexual transmission risk behavior.

High level of viral suppression and low switch rate to second-line antiretroviral therapy among HIV-infected adult patients followed over five years: retrospective analysis of the DART trial.

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Cissy Kityo

Full Text Available In contrast to resource-rich countries, most HIV-infected patients in resource-limited countries receive treatment without virological monitoring. There are few long-term data, in this setting, on rates of viral suppression or switch to second-line antiretroviral therapy. The DART trial compared clinically driven monitoring (CDM versus routine laboratory (CD4/haematology/biochemistry and clinical monitoring (LCM in HIV-infected adults initiating therapy. There was no virological monitoring in either study group during follow-up, but viral load was measured in Ugandan participants at trial closure. Two thousand three hundred and seventeen (2317 participants from this country initiated antiretroviral therapy with zidovudine/lamivudine plus tenofovir (n = 1717, abacavir (n = 300, or nevirapine (n = 300. Of 1896 (81.8% participants who were alive and in follow-up at trial closure (median 5.1 years after therapy initiation, 1507 (79.5% were on first-line and 389 (20.5% on second-line antiretroviral therapy. The overall switch rate after the first year was 5.6 per 100 person-years; the rate was substantially higher in participants with low baseline CD4 counts (<50 cells/mm3. Among 1207 (80.1% first-line participants with viral load measured, HIV RNA was <400 copies/ml in 963 (79.8%, 400-999 copies/ml in 37 (3.1%, 1,000-9,999 copies/ml in 110 (9.1%, and ≥10,000 copies/ml in 97 (8.0%. The proportion with HIV RNA <400 copies/ml was slightly lower (difference 7.1%, 95% CI 2.5 to 11.5% in CDM (76.3% than in LCM (83.4%. Among 252 (64.8% second-line participants with viral load measured (median 2.3 years after switch, HIV RNA was <400 copies/ml in 226 (89.7%, with no difference between monitoring strategies. Low switch rates and high, sustained levels of viral suppression are achievable without viral load or CD4 count monitoring in the context of high-quality clinical care.ISRCTN13968779.

Preanalytic process linked to spuriously elevated HIV viral loads: improvement on an FDA-approved process.

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Procop, Gary W; Taege, Alan J; Starkey, Colleen; Tungsiripat, Marisa; Warner, Diane; Schold, Jesse D; Yen-Lieberman, Belinda

2017-09-01

The processing of specimens often occurs in a central processing area within laboratories. We demonstrated that plasma centrifuged in the central laboratory but allowed to remain within the primary tube following centrifugation was associated with spuriously elevated HIV viral loads compared with recentrifugation of the plasma just prior to testing. Copyright © 2016 Elsevier Inc. All rights reserved.

A randomized, double-blind trial comparing combinations of nevirapine, didanosine, and zidovudine for HIV-infected patients: the INCAS Trial. Italy, The Netherlands, Canada and Australia Study.

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Montaner, J S; Reiss, P; Cooper, D; Vella, S; Harris, M; Conway, B; Wainberg, M A; Smith, D; Robinson, P; Hall, D; Myers, M; Lange, J M

1998-03-25

were 23% (11/47), 25% (13/53), and 12% (6/51) for the zidovudine plus nevirapine, zidovudine plus didanosine, and triple drug therapy groups, respectively (P=.08). Triple drug therapy with zidovudine, didanosine, and nevirapine led to a substantially greater and sustained decrease in plasma viral load than the 2-drug regimens studied. Our results also suggest that suppression of viral replication, as demonstrated by a decrease in the plasma HIV-1 RNA load below the level of quantitation of the most sensitive test available, may at least forestall the development of resistance.

RNA helicase MOV10 functions as a co-factor of HIV-1 Rev to facilitate Rev/RRE-dependent nuclear export of viral mRNAs

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Huang, Feng; Zhang, Junsong; Zhang, Yijun; Geng, Guannan; Liang, Juanran; Li, Yingniang; Chen, Jingliang; Liu, Chao; Zhang, Hui

2015-01-01

Human immunodeficiency virus type 1 (HIV-1) exploits multiple host factors during its replication. The REV/RRE-dependent nuclear export of unspliced/partially spliced viral transcripts needs the assistance of host proteins. Recent studies have shown that MOV10 overexpression inhibited HIV-1 replication at various steps. However, the endogenous MOV10 was required in certain step(s) of HIV-1 replication. In this report, we found that MOV10 potently enhances the nuclear export of viral mRNAs and subsequently increases the expression of Gag protein and other late products through affecting the Rev/RRE axis. The co-immunoprecipitation analysis indicated that MOV10 interacts with Rev in an RNA-independent manner. The DEAG-box of MOV10 was required for the enhancement of Rev/RRE-dependent nuclear export and the DEAG-box mutant showed a dominant-negative activity. Our data propose that HIV-1 utilizes the anti-viral factor MOV10 to function as a co-factor of Rev and demonstrate the complicated effects of MOV10 on HIV-1 life cycle. - Highlights: • MOV10 can function as a co-factor of HIV-1 Rev. • MOV10 facilitates Rev/RRE-dependent transport of viral mRNAs. • MOV10 interacts with Rev in an RNA-independent manner. • The DEAG-box of MOV10 is required for the enhancement of Rev/RRE-dependent export.

RNA helicase MOV10 functions as a co-factor of HIV-1 Rev to facilitate Rev/RRE-dependent nuclear export of viral mRNAs

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Huang, Feng; Zhang, Junsong; Zhang, Yijun; Geng, Guannan; Liang, Juanran; Li, Yingniang; Chen, Jingliang [Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Liu, Chao, E-mail: liuchao9@mail.sysu.edu.cn [Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Zhang, Hui [Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China)

2015-12-15

Human immunodeficiency virus type 1 (HIV-1) exploits multiple host factors during its replication. The REV/RRE-dependent nuclear export of unspliced/partially spliced viral transcripts needs the assistance of host proteins. Recent studies have shown that MOV10 overexpression inhibited HIV-1 replication at various steps. However, the endogenous MOV10 was required in certain step(s) of HIV-1 replication. In this report, we found that MOV10 potently enhances the nuclear export of viral mRNAs and subsequently increases the expression of Gag protein and other late products through affecting the Rev/RRE axis. The co-immunoprecipitation analysis indicated that MOV10 interacts with Rev in an RNA-independent manner. The DEAG-box of MOV10 was required for the enhancement of Rev/RRE-dependent nuclear export and the DEAG-box mutant showed a dominant-negative activity. Our data propose that HIV-1 utilizes the anti-viral factor MOV10 to function as a co-factor of Rev and demonstrate the complicated effects of MOV10 on HIV-1 life cycle. - Highlights: • MOV10 can function as a co-factor of HIV-1 Rev. • MOV10 facilitates Rev/RRE-dependent transport of viral mRNAs. • MOV10 interacts with Rev in an RNA-independent manner. • The DEAG-box of MOV10 is required for the enhancement of Rev/RRE-dependent export.

The Human Nuclear Exosome Targeting Complex Is Loaded onto Newly Synthesized RNA to Direct Early Ribonucleolysis

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Michal Lubas

2015-01-01

Full Text Available The RNA exosome complex constitutes the major nuclear eukaryotic 3′-5′ exonuclease. Outside of nucleoli, the human nucleoplasmic exosome is directed to some of its substrates by the nuclear exosome targeting (NEXT complex. How NEXT targets RNA has remained elusive. Using an in vivo crosslinking approach, we report global RNA binding sites of RBM7, a key component of NEXT. RBM7 associates broadly with RNA polymerase II-derived RNA, including pre-mRNA and short-lived exosome substrates such as promoter upstream transcripts (PROMPTs, enhancer RNAs (eRNAs, and 3′-extended products from snRNA and replication-dependent histone genes. Within pre-mRNA, RBM7 accumulates at the 3′ ends of introns, and pulse-labeling experiments demonstrate that RBM7/NEXT defines an early exosome-targeting pathway for 3′-extended snoRNAs derived from such introns. We propose that RBM7 is generally loaded onto newly synthesized RNA to accommodate exosome action in case of available unprotected RNA 3′ ends.

Hepatitis B and C Co-Infection in HIV Patients from the TREAT Asia HIV Observational Database: Analysis of Risk Factors and Survival

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Chen, Marcelo; Wong, Wing-Wai; Law, Matthew G.; Kiertiburanakul, Sasisopin; Yunihastuti, Evy; Merati, Tuti Parwati; Lim, Poh Lian; Chaiwarith, Romanee; Phanuphak, Praphan; Lee, Man Po; Kumarasamy, Nagalingeswaran; Saphonn, Vonthanak; Ditangco, Rossana; Sim, Benedict L. H.; Nguyen, Kinh Van; Pujari, Sanjay; Kamarulzaman, Adeeba; Zhang, Fujie; Pham, Thuy Thanh; Choi, Jun Yong; Oka, Shinichi; Kantipong, Pacharee; Mustafa, Mahiran; Ratanasuwan, Winai; Durier, Nicolas; Chen, Yi-Ming Arthur

2016-01-01

Background We assessed the effects of hepatitis B (HBV) or hepatitis C (HCV) co-infection on outcomes of antiretroviral therapy (ART) in HIV-infected patients enrolled in the TREAT Asia HIV Observational Database (TAHOD), a multi-center cohort of HIV-infected patients in the Asia-Pacific region. Methods Patients testing HBs antigen (Ag) or HCV antibody (Ab) positive within enrollment into TAHOD were considered HBV or HCV co-infected. Factors associated with HBV and/or HCV co-infection were assessed by logistic regression models. Factors associated with post-ART HIV immunological response (CD4 change after six months) and virological response (HIV RNA <400 copies/ml after 12 months) were also determined. Survival was assessed by the Kaplan-Meier method and log rank test. Results A total of 7,455 subjects were recruited by December 2012. Of patients tested, 591/5656 (10.4%) were HBsAg positive, 794/5215 (15.2%) were HCVAb positive, and 88/4966 (1.8%) were positive for both markers. In multivariate analysis, HCV co-infection, age, route of HIV infection, baseline CD4 count, baseline HIV RNA, and HIV-1 subtype were associated with immunological recovery. Age, route of HIV infection, baseline CD4 count, baseline HIV RNA, ART regimen, prior ART and HIV-1 subtype, but not HBV or HCV co-infection, affected HIV RNA suppression. Risk factors affecting mortality included HCV co-infection, age, CDC stage, baseline CD4 count, baseline HIV RNA and prior mono/dual ART. Shortest survival was seen in subjects who were both HBV- and HCV-positive. Conclusion In this Asian cohort of HIV-infected patients, HCV co-infection, but not HBV co-infection, was associated with lower CD4 cell recovery after ART and increased mortality. PMID:26933963

Management of HIV Infection during Pregnancy in the United States: Updated Evidence-Based Recommendations and Future Potential Practices

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Bassam H. Rimawi

2016-01-01

Full Text Available All HIV-infected women contemplating pregnancy should initiate combination antiretroviral therapy (cART, with a goal to achieve a maternal serum HIV RNA viral load beneath the laboratory level of detection prior to conceiving, as well as throughout their pregnancy. Successfully identifying HIV infection during pregnancy through screening tests is essential in order to prevent in utero and intrapartum transmission of HIV. Perinatal HIV transmission can be less than 1% when effective cART, associated with virologic suppression of HIV, is given during the ante-, intra-, and postpartum periods. Perinatal HIV guidelines, developed by organizations such as the World Health Organization, American College of Obstetricians and Gynecologists, and the US Department of Health and Human Services, are constantly evolving, and hence the aim of our review is to provide a useful concise review for medical providers caring for HIV-infected pregnant women, summarizing the latest and current recommendations in the United States.

Pregnancy loss and role of infant HIV status on perinatal mortality among HIV-infected women

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Kim Hae-Young

2012-08-01

Full Text Available Abstract Background HIV-infected women, particularly those with advanced disease, may have higher rates of pregnancy loss (miscarriage and stillbirth and neonatal mortality than uninfected women. Here we examine risk factors for these adverse pregnancy outcomes in a cohort of HIV-infected women in Zambia considering the impact of infant HIV status. Methods A total of 1229 HIV-infected pregnant women were enrolled (2001–2004 in Lusaka, Zambia and followed to pregnancy outcome. Live-born infants were tested for HIV by PCR at birth, 1 week and 5 weeks. Obstetric and neonatal data were collected after delivery and the rates of neonatal ( Results The ratio of miscarriage and stillbirth per 100 live-births were 3.1 and 2.6, respectively. Higher maternal plasma viral load (adjusted odds ratio [AOR] for each log10 increase in HIV RNA copies/ml = 1.90; 95% confidence interval [CI] 1.10–3.27 and being symptomatic were associated with an increased risk of stillbirth (AOR = 3.19; 95% CI 1.46–6.97, and decreasing maternal CD4 count by 100 cells/mm3 with an increased risk of miscarriage (OR = 1.25; 95% CI 1.02–1.54. The neonatal mortality rate was 4.3 per 100 increasing to 6.3 by 70 days. Intrauterine HIV infection was not associated with neonatal morality but became associated with mortality through 70 days (adjusted hazard ratio = 2.76; 95% CI 1.25–6.08. Low birth weight and cessation of breastfeeding were significant risk factors for both neonatal and early mortality independent of infant HIV infection. Conclusions More advanced maternal HIV disease was associated with adverse pregnancy outcomes. Excess neonatal mortality in HIV-infected women was not primarily explained by infant HIV infection but was strongly associated with low birth weight and prematurity. Intrauterine HIV infection contributed to mortality as early as 70 days of infant age. Interventions to improve pregnancy outcomes for HIV-infected women are needed to

Markovian negentropies in bioinformatics. 1. A picture of footprints after the interaction of the HIV-1 Psi-RNA packaging region with drugs.

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Díaz, Humberto González; de Armas, Ronal Ramos; Molina, Reinaldo

2003-11-01

Many experts worldwide have highlighted the potential of RNA molecules as drug targets for the chemotherapeutic treatment of a range of diseases. In particular, the molecular pockets of RNA in the HIV-1 packaging region have been postulated as promising sites for antiviral action. The discovery of simpler methods to accurately represent drug-RNA interactions could therefore become an interesting and rapid way to generate models that are complementary to docking-based systems. The entropies of a vibrational Markov chain have been introduced here as physically meaningful descriptors for the local drug-nucleic acid complexes. A study of the interaction of the antibiotic Paromomycin with the packaging region of the RNA present in type-1 HIV has been carried out as an illustrative example of this approach. A linear discriminant function gave rise to excellent discrimination among 80.13% of interacting/non-interacting sites. More specifically, the model classified 36/45 nucleotides (80.0%) that interacted with paromomycin and, in addition, 85/106 (80.2%) footprinted (non-interacting) sites from the RNA viral sequence were recognized. The model showed a high Matthews' regression coefficient (C = 0.64). The Jackknife method was also used to assess the stability and predictability of the model by leaving out adenines, C, G, or U. Matthews' coefficients and overall accuracies for these approaches were between 0.55 and 0.68 and 75.8 and 82.7, respectively. On the other hand, a linear regression model predicted the local binding affinity constants between a specific nucleotide and the aforementioned antibiotic (R2 = 0.83,Q2 = 0.825). These kinds of models may play an important role either in the discovery of new anti-HIV compounds or in the elucidation of their mode of action. On request from the corresponding author (humbertogd@cbq.uclv.edu.cu or humbertogd@navegalia.com).

Correlates of HIV-1 viral suppression in a cohort of HIV-positive drug users receiving antiretroviral therapy in Hanoi, Vietnam

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Jordan, Michael R; La, Hanh; Nguyen, Hien Duc; Sheehan, Heidi; Lien, Trinh Thi Minh; Van Dang, Duong; Hellinger, James; Wanke, Christine; Tang, Alice M

2009-01-01

Summary Injection drug users bear the burden of HIV in Vietnam and are a focus of national treatment programs. To date, determinants of successful therapy in this population are unknown. Substance use and clinical correlates of viral suppression were studied in 100 HIV-1 infected drug users receiving antiretroviral therapy (ART) for at least 6 months in Hanoi, Vietnam. Mean age of the cohort was 29.9 + 4.9 years; all were men. A majority of patients (73%) achieved viral suppression (HIV-RNA 95% adherence (p<0.01) and current use of trimethoprim/sulfamethoxazole (p<0.01); current or ever diagnosed with tuberculosis was associated with viral non-suppression (p=0.006). Tobacco use was prevalent (84%), and surprisingly 48% of patients reported active drug use; neither was associated with viral non-suppression. This is the first study to document successful ART treatment in a population of Vietnamese drug users; rates of viral suppression are comparable to other international populations. The 28% of patients without HIV-1 suppression highlights the need for adherence promotion, risk reduction programs, and population based surveillance strategies for assessing the emergence of HIV drug resistance in settings where access to viral load and drug resistance testing is limited. PMID:19451329

HIV-1 virological remission lasting more than 12 years after interruption of early antiretroviral therapy in a perinatally infected teenager enrolled in the French ANRS EPF-CO10 paediatric cohort: a case report.

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Frange, Pierre; Faye, Albert; Avettand-Fenoël, Véronique; Bellaton, Erianna; Descamps, Diane; Angin, Mathieu; David, Annie; Caillat-Zucman, Sophie; Peytavin, Gilles; Dollfus, Catherine; Le Chenadec, Jerome; Warszawski, Josiane; Rouzioux, Christine; Sáez-Cirión, Asier

2016-01-01

Durable HIV-1 remission after interruption of combined antiretroviral therapy (ART) has been reported in some adults who started treatment during primary infection; however, whether long-term remission in vertically infected children is possible was unknown. We report a case of a young adult perinatally infected with HIV-1 with viral remission despite long-term treatment interruption. The patient was identified in the ANRS EPF-CO10 paediatric cohort among 100 children infected with HIV perinatally who started ART before 6 months of age. HIV RNA viral load and CD4 cell counts were monitored from birth. Ultrasensitive HIV RNA, peripheral blood mononuclear cell (PBMC)-associated HIV DNA, HIV-specific T-cell responses (ie, production of cytokines and capacity to suppress HIV infection), reactivation of the CD4 cell reservoir (measured by p24 ELISA and HIV RNA in supernatants upon phytohaemagglutinin activation of purified CD4 cells), and plasma concentrations of antiretroviral drugs were assessed after 10 years of documented control off therapy. The infant was born in 1996 to a woman with uncontrolled HIV-1 viraemia and received zidovudine-based prophylaxis for 6 weeks. HIV RNA and DNA were not detected 3 days and 14 days after birth. HIV DNA was detected at 4 weeks of age. HIV RNA reached 2·17× 10(6) copies per mL at 3 months of age and ART was started. HIV RNA was undetectable 1 month later. ART was discontinued by the family at some point between 5·8 and 6·8 years of age. HIV RNA was undetectable at 6·8 years of age and ART was not resumed. HIV RNA has remained below 50 copies per mL and CD4 cell counts stable through to 18·6 years of age. After 11·5 years of control off treatment, HIV RNA was below 4 copies per mL and HIV DNA was 2·2 log10 copies per 10(6) PBMCs. The HLA genotype showed homozygosity at several loci (A*2301-, B*1503/4101, C*0210/0802, DRB1*1101-, and DQB1*0602-). HIV-specific CD8 T-cell responses and T-cell activation were weak. Findings

Dolutegravir as maintenance monotherapy for HIV (DOMONO): a phase 2, randomised non-inferiority trial.

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Wijting, Ingeborg; Rokx, Casper; Boucher, Charles; van Kampen, Jeroen; Pas, Suzan; de Vries-Sluijs, Theodora; Schurink, Carolina; Bax, Hannelore; Derksen, Maarten; Andrinopoulou, Eleni-Rosalina; van der Ende, Marchina; van Gorp, Eric; Nouwen, Jan; Verbon, Annelies; Bierman, Wouter; Rijnders, Bart

2017-12-01

The high genetic barrier to resistance of dolutegravir might allow for its use as maintenance monotherapy in patients with HIV. We investigated whether dolutegravir monotherapy was non-inferior to combination antiretroviral therapy (ART) for maintaining virological suppression in patients with HIV-1 infection successfully treated with combination ART. We did this open-label, phase 2, randomised non-inferiority trial at two medical centres in the Netherlands. Eligible patients (aged ≥18 years) were on combination ART, had been virologically suppressed (HIV RNA <50 copies per mL) for at least 6 months, and had CD4 nadirs of 200 cells per μL or higher, HIV RNA zeniths of 100 000 copies per mL or less, and no history of virological failure. Patients were randomly assigned (1:1), via a web-based block randomisation method (variable block sizes of 4 and 6), to switch to dolutegravir monotherapy (50 mg once a day) either immediately or after a delay of 24 weeks of continued combination ART. Randomisation was stratified by HIV RNA zenith (<50 000 copies per mL vs 50 000-99 999 copies per mL). Investigators and patients were not masked to group allocation. The primary endpoint was the proportion of patients with plasma HIV RNA viral loads of less than 200 copies per mL at week 24, with a non-inferiority margin of 12%. We did analyses in the on-treatment and intention-to-treat populations. This trial is registered with ClinicalTrials.gov, NCT02401828. Between March 10, 2015, and Feb 4, 2016, we randomly assigned 51 patients to the immediate switch group and 53 patients to the delayed switch group. One patient who received immediate monotherapy discontinued treatment at week 12 because of disturbed sleep. At week 24, dolutegravir monotherapy was non-inferior to combination ART, with plasma HIV RNA loads of 200 copies per mL or higher observed in 2% (1/50) of patients in the immediate switch group and in no patients in the delayed switch group (difference 2%, 95% CI

Influenza vaccination of HIV-1-positive and HIV-1-negative former intravenous drug users.

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Amendola, A; Boschini, A; Colzani, D; Anselmi, G; Oltolina, A; Zucconi, R; Begnini, M; Besana, S; Tanzi, E; Zanetti, A R

2001-12-01

The immunogenicity of an anti-influenza vaccine was assessed in 409 former intravenous drug user volunteers and its effect on the levels of HIV-1 RNA, proviral DNA and on CD4+ lymphocyte counts in a subset HIV-1-positive subjects was measured. HIV-1-positive individuals (n = 72) were divided into three groups on the basis of their CD4+ lymphocyte counts, while the 337 HIV-1-negative participants were allocated into group four. Haemagglutination inhibiting (HI) responses varied from 45.8 to 70% in the HIV-1-positive subjects and were significantly higher in group four (80.7% responses to the H1N1 strain, 81.6% to the H3N2 strain, and 83% to the B strain). The percentage of subjects with HI protective antibody titres (> or = 1:40) increased significantly after vaccination, especially in HIV-1 uninfected subjects. Immunization caused no significant changes in CD4+ counts and in neither plasma HIV-1 RNA nor proviral DNA levels. Therefore, vaccination against influenza may benefit persons infected by HIV-1. Copyright 2001 Wiley-Liss, Inc.

Identifying HIV-1 dual infections

Directory of Open Access Journals (Sweden)

Cornelissen Marion

2007-09-01

Full Text Available Abstract Transmission of human immunodeficiency virus (HIV is no exception to the phenomenon that a second, productive infection with another strain of the same virus is feasible. Experiments with RNA viruses have suggested that both coinfections (simultaneous infection with two strains of a virus and superinfections (second infection after a specific immune response to the first infecting strain has developed can result in increased fitness of the viral population. Concerns about dual infections with HIV are increasing. First, the frequent detection of superinfections seems to indicate that it will be difficult to develop a prophylactic vaccine. Second, HIV-1 superinfections have been associated with accelerated disease progression, although this is not true for all persons. In fact, superinfections have even been detected in persons controlling their HIV infections without antiretroviral therapy. Third, dual infections can give rise to recombinant viruses, which are increasingly found in the HIV-1 epidemic. Recombinants could have increased fitness over the parental strains, as in vitro models suggest, and could exhibit increased pathogenicity. Multiple drug resistant (MDR strains could recombine to produce a pan-resistant, transmittable virus. We will describe in this review what is presently known about super- and re-infection among ambient viral infections, as well as the first cases of HIV-1 superinfection, including HIV-1 triple infections. The clinical implications, the impact of the immune system, and the effect of anti-retroviral therapy will be covered, as will as the timing of HIV superinfection. The methods used to detect HIV-1 dual infections will be discussed in detail. To increase the likelihood of detecting a dual HIV-1 infection, pre-selection of patients can be done by serotyping, heteroduplex mobility assays (HMA, counting the degenerate base codes in the HIV-1 genotyping sequence, or surveying unexpected increases in the

Trans-activation of the 5' to 3' viral DNA strand transfer by nucleocapsid protein during reverse transcription of HIV1 RNA.

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Darlix, J L; Vincent, A; Gabus, C; de Rocquigny, H; Roques, B

1993-08-01

Two DNA strand transfer reactions take place during reverse transcription of the retroviral genome. The first transfer, that of the minus-strand strong stop DNA from the 5' end of the viral RNA to the 3' end, has been studied in vitro with two RNAs mimicking the 5' and 3' regions of the HIV1 genome and with nucleocapsid protein, NCp7, and reverse transcriptase. The results show that NCp7 strongly activates the 5' to 3' DNA strand transfer during reverse transcription while a basic peptide resembling NCp7 is inactive. Activation of the first transfer by several NCp7 derived peptides and the influence of the terminal redundancies (R) present at the 5' and 3' ends of HIV1 RNA were also examined. The first transfer is optimal in the presence of intact NCp7 and necessitates R on both the 5' and 3' RNAs. Sequencing of full length viral DNA products reveals approximately 40% misincorporations at the first nucleotide beyond the transfer point. If such base misincorporations occur during proviral DNA synthesis with possible homologous recombinations it may well contribute to the high level of genetic variability of HIV.

Elucidating the Burden of HIV in Tissues Using Multiplexed Immunofluorescence and In Situ Hybridization: Methods for the Single-Cell Phenotypic Characterization of Cells Harboring HIV In Situ.

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Vasquez, Joshua J; Hussien, Rajaa; Aguilar-Rodriguez, Brandon; Junger, Henrik; Dobi, Dejan; Henrich, Timothy J; Thanh, Cassandra; Gibson, Erica; Hogan, Louise E; McCune, Joseph; Hunt, Peter W; Stoddart, Cheryl A; Laszik, Zoltan G

2018-02-01

Persistent tissue reservoirs of HIV present a major barrier to cure. Defining subsets of infected cells in tissues is a major focus of HIV cure research. Herein, we describe a novel multiplexed in situ hybridization (ISH) (RNAscope) protocol to detect HIV-DNA (vDNA) and HIV-RNA (vRNA) in formalin-fixed paraffin-embedded (FFPE) human tissues in combination with immunofluorescence (IF) phenotyping of the infected cells. We show that multiplexed IF and ISH (mIFISH) is suitable for quantitative assessment of HIV vRNA and vDNA and that multiparameter IF phenotyping allows precise identification of the cellular source of the ISH signal. We also provide semi-quantitative data on the impact of various tissue fixatives on the detectability of vDNA and vRNA with RNAscope technology. Finally, we describe methods to quantitate the ISH signal on whole-slide digital images and validation of the quantitative ISH data with quantitative real-time PCR for vRNA. It is our hope that this approach will provide insight into the biology of HIV tissue reservoirs and to inform strategies aimed at curing HIV.

GeneXpert HIV-1 quant assay, a new tool for scale up of viral load monitoring in the success of ART programme in India.

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Kulkarni, Smita; Jadhav, Sushama; Khopkar, Priyanka; Sane, Suvarna; Londhe, Rajkumar; Chimanpure, Vaishali; Dhilpe, Veronica; Ghate, Manisha; Yelagate, Rajendra; Panchal, Narayan; Rahane, Girish; Kadam, Dilip; Gaikwad, Nitin; Rewari, Bharat; Gangakhedkar, Raman

2017-07-21

Recent WHO guidelines identify virologic monitoring for diagnosing and confirming ART failure. In view of this, validation and scale up of point of care viral load technologies is essential in resource limited settings. A systematic validation of the GeneXpert® HIV-1 Quant assay (a point-of-care technology) in view of scaling up HIV-1 viral load in India to monitor the success of national ART programme was carried out. Two hundred nineteen plasma specimens falling in nine viral load ranges (5 L copies/ml) were tested by the Abbott m2000rt Real Time and GeneXpert HIV-1 Quant assays. Additionally, 20 seronegative; 16 stored specimens and 10 spiked controls were also tested. Statistical analysis was done using Stata/IC and sensitivity, specificity, PPV, NPV and %misclassification rates were calculated as per DHSs/AISs, WHO, NACO cut-offs for virological failure. The GeneXpert assay compared well with the Abbott assay with a higher sensitivity (97%), specificity (97-100%) and concordance (91.32%). The correlation between two assays (r = 0.886) was statistically significant (p performance and rapidity will aid in timely diagnosis of ART failures, integrated HIV-TB management and will facilitate the UNAIDS 90-90-90 target.

Prevalence of human immunodeficiency virus RNA and antibody in first-time, lapsed, and repeat blood donations across five international regions and relative efficacy of alternative screening scenarios.

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Bruhn, Roberta; Lelie, Nico; Custer, Brian; Busch, Michael; Kleinman, Steven

2013-10-01

Twenty-one blood organizations from five geographical regions provided HIV individual donation (ID)-NAT and serology data on 11,787,610 donations. Infections were classified as anti-HIV-/RNA+ window period (WP), anti-HIV+/RNA+ concordant positive (CP) or anti-HIV+/RNA- elite controller (EC). Residual risk and efficacy of several screening scenarios were estimated for first time, lapsed and repeat donations. WP residual risk estimates assumed a 50% infectious dose of 3.16 virions and a 50% detection limit of 2.7 HIV RNA copies/mL for ID-NAT and 10,000 copies/mL for p24Ag. Infectivity for CP (100%) and EC (2.2%) donations was estimated based on viral load distributions and 100-fold reduced infectivity by antibody neutralization as reported elsewhere. Efficacy was calculated as proportion of transmission risk removed from baseline (i.e. in absence of any screening). There was no significant difference in transmission risk between lapsed and repeat donations in any region. Risk was 3.8-fold higher in first time than combined lapsed/repeat donations in South Africa but not in other regions. Screening strategies were most efficacious at interdicting infectious transfusions in first time (98.7-99.8%) followed by lapsed (97.6-99.7%) and repeat (86.8-97.7%) donations in all regions combined. In each donor category the efficacy of ID-NAT alone (97.7-99.8%) was superior to that of minipool (MP)-NAT/anti-HIV (95.0-99.6%) and p24 Ag/anti-HIV (89.8-99.1%). Efficacy patterns were similar by donor/donation status in each region despite large differences in HIV prevalence and transmission risk. As similar data become available for HBV and HCV, this modeling may be useful in cost effectiveness analyses of alternative testing scenarios. © 2013 American Association of Blood Banks.

Expansion of Viral Load Testing and the Potential Impact on HIV Drug Resistance.

Science.gov (United States)

Raizes, Elliot; Hader, Shannon; Birx, Deborah

2017-12-01

The US President's Emergency Plan for AIDS Relief (PEPFAR) supports aggressive scale-up of antiretroviral therapy (ART) in high-burden countries and across all genders and populations at risk toward global human immunodeficiency virus (HIV) epidemic control. PEPFAR recognizes the risk of HIV drug resistance (HIVDR) as a consequence of aggressive ART scale-up and is actively promoting 3 key steps to mitigate the impact of HIVDR: (1) routine access to routine viral load monitoring in all settings; (2) optimization of ART regimens; and (3) routine collection and analysis of HIVDR data to monitor the success of mitigation strategies. The transition to dolutegravir-based regimens in PEPFAR-supported countries and the continuous evolution of HIVDR surveillance strategies are essential elements of PEPFAR implementation. Published by Oxford University Press for the Infectious Diseases Society of America 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US.