Reverse Transcriptase Activity Assay Research Articles

Small molecule ONC201 inhibits HIV-1 replication in macrophages via FOXO3a and TRAIL

Despite the success of antiretroviral therapy (ART), eradication of HIV-1 from brain reservoirs remains elusive. HIV-1 brain reservoirs include perivascular macrophages that are behind the blood-brain barrier and difficult to access by ART. Macrophages express transcription factor FOXO3a and the TNF superfamily cytokine TRAIL, which are known to target HIV-1-infected macrophages for viral inhibition. ONC201 is a novel and potent FOXO3a activator capable of inducing TRAIL. It can cross the blood-brain barrier, and has shown antitumor effects in clinical trials. We hypothesized that activation of FOXO3a/TRAIL by ONC201 will inhibit HIV-1 replication in macrophages. Using primary human monocyte-derived macrophages, we demonstrated that ONC201 dose-dependently decreased replication levels of both HIV-1 laboratory strain and primary strains as determined by HIV-1 reverse transcriptase activity assay. Consistent with data on HIV-1 replication, ONC201 also reduced intracellular and extracellular p24, viral RNA, and integrated HIV-1 DNA in infected macrophages. Blocking TRAIL or knockdown of FOXO3a with siRNA reversed ONC201-mediated HIV-1 suppression, suggesting that ONC201 inhibits HIV-1 through FOXO3a and TRAIL. The anti-HIV-1 effect of ONC201 was further validated in vivo in NOD/scid-IL-2Rgcnull mice. After intracranial injection of HIV-1-infected macrophages into the basal ganglia, we treated the mice daily with ONC201 through intraperitoneal injection for six days. ONC201 significantly decreased p24 levels in both the macrophages and the brain tissues, suggesting that ONC201 suppresses HIV-1 in vivo. Therefore, ONC201 can be a promising drug candidate to combat persistent HIV-1 infection in the brain.

Can HIV reverse transcriptase activity assay be a low-cost alternative for viral load monitoring in resource-limited settings?

ObjectiveTo evaluate the performance and cost of an HIV reverse transcriptase-enzyme activity (HIV-RT) assay in comparison to an HIV-1 RNA assay for routine viral load monitoring in resource limited settings.DesignA...

Reverse Transcriptase in Action: FRET-Based Assay for Monitoring Flipping and Polymerase Activity in Real Time.

Reverse transcriptase (RT) of human immunodeficiency virus-1 (HIV-1) is a multifunctional enzyme that catalyzes the conversion of the single stranded viral RNA genome into double-stranded DNA, competent for host-cell integration. RT is endowed with RNA- and DNA-dependent DNA polymerase activity and DNA-directed RNA hydrolysis (RNase H activity). As a key enzyme of reverse transcription, RT is a key target of currently used highly active antiretroviral therapy (HAART), though RT inhibitors offer generally a poor resistance profile, urging new RT inhibitors to be developed. Using single molecule fluorescence approaches, it has been recently shown that RT binding orientation and dynamics on its substrate play a critical role in its activity. Currently, most in vitro RT activity assays, inherently end-point measurements, are based on the detection of reaction products by using radio-labeled or chemically modified nucleotides. Here, we propose a simple and continuous real-time Förster resonance energy transfer (FRET) based-assay for the direct measurement of RT's binding orientation and polymerase activity, with the use of conventional steady-state fluorescence spectroscopy. Under our working conditions, the change in binding orientation and the primer elongation step can be visualized separately on the basis of their opposite fluorescence changes and their different kinetics. The assay presented can easily discriminate non-nucleoside RT inhibitors from nucleoside RT inhibitors and determine reliably their potency. This one-step and one-pot assay constitutes an improved alternative to the currently used screening assays to disclose new anti-RT drugs and identify at the same time the class to which they belong.

Natural Plant Alkaloid (Emetine) Inhibits HIV-1 Replication by Interfering with Reverse Transcriptase Activity.

Ipecac alkaloids are secondary metabolites produced in the medicinal plant Psychotria ipecacuanha. Emetine is the main alkaloid of ipecac and one of the active compounds in syrup of Ipecac with emetic property. Here we evaluated emetine’s potential as an antiviral agent against Human Immunodeficiency Virus. We performed in vitro Reverse Transcriptase (RT) Assay and Natural Endogenous Reverse Transcriptase Activity Assay (NERT) to evaluate HIV RT inhibition. Emetine molecular docking on HIV-1 RT was also analyzed. Phenotypic assays were performed in non-lymphocytic and in Peripheral Blood Mononuclear Cells (PBMC) with HIV-1 wild-type and HIV-harboring RT-resistant mutation to Nucleoside Reverse Transcriptase Inhibitors (M184V). Our results showed that HIV-1 RT was blocked in the presence of emetine in both models: in vitro reactions with isolated HIV-1 RT and intravirion, measured by NERT. Emetine revealed a strong potential of inhibiting HIV-1 replication in both cellular models, reaching 80% of reduction in HIV-1 infection, with low cytotoxic effect. Emetine also blocked HIV-1 infection of RT M184V mutant. These results suggest that emetine is able to penetrate in intact HIV particles, and bind and block reverse transcription reaction, suggesting that it can be used as anti-HIV microbicide. Taken together, our findings provide additional pharmacological information on the potential therapeutic effects of emetine.

673. Establishment of a National Reference Standard for Reverse Transcriptase Activity Assay and Its Applicability in Quality Control of Gene therapy Products

The reverse transcriptase (RT) activity assay is widely used in the quality control of biological products including gene therapy products for it can detect all infectious retroviruses in biological starting materials, cell banks and final products. But the lack of reference standard for RT activity results in significant systemic error and incomparability between laboratories. To standardize RT activity assay, this study established a sensitive real time PCR and developed the first national reference standard, which was prepared from a raw material of recombinant moloney murine leukemia virus reverse transcriptase. Our tests indicated that the candidate reference standard met relevant requirements of the national reference standard. The activity value of candidate RT standard was determined as 26.4 U per ampoule by a collaborative calibration among four laboratories, with the within-laboratory geometric coefficients of variation (GCVs) ranging from 2.7% to 9.6% and the between-laboratory GCV values being 9.8%. The thermal accelerated stability test showed that no loss of activity was observed when the candidate RT standard was stored at 4°C, 25°C and 37 °C compared with -20°C even after 9 months. The applicability study showed that the cell bank and viral bank of an adenovirus gene therapy product were positive and the final products were negative. As a conclusion, the candidate preparation was suitable to serve as a Chinese national standard for RT activity assay and can be used in the quality control of gene therapy products.

Prevalence and characterization of murine leukemia virus contamination in human cell lines.

Contaminations of cell cultures with microbiological organisms are well documented and can be managed in cell culture laboratories applying reliable detection, elimination and prevention strategies. However, the presence of viral contaminations in cell cultures is still a matter of debate and cannot be determined with general detection methods. In the present study we screened 577 human cell lines for the presence of murine leukemia viruses (MLV). Nineteen cell lines were found to be contaminated with MLV, including 22RV1 which is contaminated with the xenotropic murine leukemia virus-related virus variant of MLV. Of these, 17 cell lines were shown to produce active retroviruses determined by product enhanced reverse transcriptase PCR assay for reverse transcriptase activity. The contaminated cell lines derive from various solid tumor types as well as from leukemia and lymphoma types. A contamination of primary human cells from healthy volunteers could not be substantiated. Sequence analyses of 17 MLV PCR products and five complete MLV genomes of different infected cell lines revealed at least three groups of related MLV genotypes. The viruses harvested from the supernatants of infected cell cultures were infectious to uninfected cell cultures. In the course of the study we found that contamination of human genomic DNA preparations with murine DNA can lead to false-positive results. Presumably, xenotransplantations of the human tumor cells into immune-deficient mice to determine the tumorigenicity of the cells are mainly responsible for the MLV contaminations. Furthermore, the use of murine feeder layer cells during the establishment of human cell lines and a cross-contamination with MLV from infected cultures might be sources of infection. A screening of cell cultures for MLV contamination is recommended given a contamination rate of 3.3%.

Development of an enzyme-linked immunosorbent assay based on the murine leukemia virus p30 capsid protein

Retroviral vectors derived from the murine leukemia virus (MuLV) are widely used as the starting material in the development of vectors for gene therapy and critical in answering questions relating to viral pathogenesis. The p30 capsid (CA) is the major viral core protein and an internal group antigen in MuLV. In this study, an enzyme-linked immunosorbent assay (ELISA) was developed for quantitation of MuLV infectious particles with p30 CA core antigen protein. The ELISA was developed using several goat-polyclonal serum against MuLV p30 generated by the NCI as primary antibody and a rat-monoclonal antibody to CA available from ATCC. The MuLV p30 CA antigen was standardized against recombinant MuLV p30 CA expressed from bacteria. The assay is sensitive, accurate and linear within a defined concentration range of CA. Comparison with different MuLV quantitative methods including reporter gene transfer, reverse transcriptase activity assay, and viral RNA quantitative PCR, showed this ELISA protocol to be highly quantifiable within defined ranges, which can be correlated with infectious viral titer.

Increased Virus Replication and Cytotoxicity of Non-pathogenic Simian Human Immuno Deficiency Viruses-NM-3rN After Serial Passage in a Monkey-Derived Cell Line

Background:Infection and disease induction of variants of HIV type 1 (HIV-1) in vivo, especially their persistence, replication and rate of disease progression, have been found to depend on phenotypic characteristics. However, the mechanism (s) underlying these diverse phenotypic characteristics remain poorly understood.Aim:It was aimed at determining whether a SHIV that had been adapted to a monkey-derived cell line could be used to explain the mechanism that underlies adaptive evolution of a virus to its host cell environment.Materials and Methods:Standard procedures in virology such as cell culturing, FACS analysis and ELISA were employed to measure virus replication and growth kinetics, cell viability, reverse transcriptase (RT) activity assay and CD4 cells down-regulation.Results:After about 20 passages, LT efficiently adapted to the monkey-derived cell line and replicated much better than the parent virus. LT accumulated a number of mutations in its entire genome with a majority of them being monkey cell-specific.Conclusion:Thus we think we have obtained a virus that may enable studies to determine which of these mutations are specifically related to in vitro viral replication and which are specifically related to cytotoxicity so as to explain the mechanism associated with viral cytotoxicity and host cell specificity.

Point of care testing of HIV in children younger than 18 months with three different HIV virological assays. Experience from a district hospital in a resource-limited setting

Diagnosis of HIV in children younger than 18 months can be challenging in developing countries because requires the use of HIV virological tests. In this study we describe the experience with three commercial assays, HIV- 1 DNA polymerase chain reaction (PCR) (Roche Amplicor 1.5) with dried blood spot, HIV-1 RNA PCR (Roche COBAS TaqMan) with plasma and reverse transcriptase activity assay (Cavidi Exavir Load 3) with plasma in a rural setting of India. Sensitivity and specificity were 98.1% (95% confidence interval [CI] 90- 100) and 99.3% (95% CI 97.9-99.9) for the HIV- 1 RNA PCR assay, 66.7% (95% CI 29.9-92.5) and 100% (95% CI 96.8-100) for the HIV-1 DNA PCR assay, and 100% (95% CI 48-100) and 98.7% (95% CI 92.8-100) for the reverse transcriptase activity assay respectively. The low sensitivity of the HIV-1 DNA PCR assay in this setting is worrisome and warrants further investigations.

In Vitro Anti-HIV-1 Activity of the Aqueous Extract of Asterina Pectinifera

An aqueous extract of starfish, Asterina pectinifera, was investigated for its anti-HIV-1 efficiency in vitro on human T-cell lines. A. pectinifera significantly maintained the viability of HIV-infected cells as much as 86% of the untreated infected control cells at the non-toxic concentrations (0.05~4 mg/mL) in CEM-SS cells. Anti-HIV-1 activity of A. pectinifera extract was further supported by quantification of syncytia formation, reverse transcriptase activity and Western blot analysis in C8166, CEM-SS and H9 cells, respectively. Current results demonstrated a notable inhibition of HIV-1 induced syncytia formation and HIV-1 reverse transcriptase activity assay with EC50 of 0.71 mg/mL and 0.65 mg/mL, respectively. Moreover, A. pectinifera extract treatment decreased the production of p24 protein and gene expression of HIV-1 viral infection factor in a dose-dependent manner according to immunoblot and reverse transcription polymerase chain reaction analysis. In the light of current study, it can be concluded that A. pectinifera contains highly potential anti-HIV-1 components and a further investigation for active compound isolation is urged.

HIV-1 Clade B and C Isolates Exhibit Differential Replication: Relevance to Macrophage-Mediated Neurotoxicity

HIV-associated neurocognitive disorders (HAND) continue to be a consequence of HIV-1 infection among clade B-infected individuals. In contrast, the incidence of severe neurological impairment is lower among clade C-infected patients in regions of Sub-Saharan Africa and India. Biological aspects such as replication, cytopathicity, inflammatory response, and neurotoxicity unique to each clade influence neuropathogenicity and ultimately affect the clinical outcome of the disease. We hypothesize that productive infection by clade C isolates leads to macrophage-mediated neurotoxicity, although to a lesser extent than clade B isolates. Using a panel of primary isolates of clades B and C we demonstrated that clade B has higher replication efficiency in monocyte-derived macrophages (MDM) through reverse transcriptase activity assay and HIV-1 p24 antigen ELISA. To test the neurotoxicity of clades B and C, we used an in vitro neurotoxicity model. Conditioned medium from clade B-infected MDM was neurotoxic to rat and human neuron cultures. In contrast, clade C isolates mediated neurotoxicity when a higher initial viral titer was used for MDM infection. Furthermore, neurotoxicity mediated by isolates of both clades correlated with virus replication in MDM. Together, these results suggest that in comparison to clade B, primary isolates of clade C have slower replication kinetics in primary MDM, leading to lower levels of macrophage-mediated neurotoxicity. Elucidating the differences in replication and macrophage-mediated neurotoxicity between isolates of HIV-1 clades B and C will provide important insights needed to clarify the disparity seen in HAND incidence.

Influence of chitosan nanofiber scaffold on porcine endogenous retroviral expression and infectivity in pig hepatocytes

To investigate the influence of chitosan nanofiber scaffold on the production and infectivity of porcine endogenous retrovirus (PERV) expressed by porcine hepatocytes. Freshly isolated porcine hepatocytes were cultured with or without chitosan nanofiber scaffold (defined as Nano group and Hep group) for 7 d. The daily collection of culture medium was used to detect reverse transcriptase (RT) activity with RT activity assay kits and PERV RNA by reverse transcription-polymerase chain reaction (PCR) and real time PCR with the PERV specific primers. And Western blotting was performed with the lysates of daily retrieved cells to determine the PERV protein gag p30. Besides, the in-vitro infectivity of the supernatant was tested by incubating the human embryo kidney 293 (HEK293) cells. The similar changing trends between two groups were observed in real time PCR, RT activity assay and Western blotting. Two peaks of PERV expression at 10H and Day 2 were found and followed by a regular decline. No significant difference was found between two groups except the significantly high level of PERV RNA at Day 6 and PERV protein at Day 5 in Nano group than that in Hep group. And in the in-vitro infection experiment, no HEK293 cell was infected by the supernatant. Chitosan nanofiber scaffold might prolong the PERV secreting time in pig hepatocytes but would not obviously influence its productive amount and infectivity, so it could be applied in the bioartificial liver without the increased risk of the virus transmission.

Nano-NRTIs: Efficient Inhibitors of HIV Type-1 in Macrophages with a Reduced Mitochondrial Toxicity

Macrophages serve as a depot for HIV type-1 (HIV-1) in the central nervous system. To efficiently target macrophages, we developed nanocarriers for potential brain delivery of activated nucleoside reverse transcriptase inhibitors (NRTIs) called nano-NRTIs. Nanogel carriers consisting of poly(ethylene glycol) (PEG)- or Pluronic-polyethylenimine (PEI) biodegradable networks, star PEG-PEI or poly(amidoamine) dendrimer-PEI-PEG dendritic networks, as well as nanogels decorated with brain-targeting peptide molecules, specifically binding to the apolipoprotein E receptor, were synthesized and evaluated. Nano-NRTIs were obtained by mixing aqueous solutions of zidovudine 5'-triphosphate or didanosine 5'-triphosphate and nanocarriers, followed by freeze-drying. Intracellular accumulation, cytotoxicity and antiviral activity of nano-NRTIs were monitored in monocyte-derived macrophages (MDMs). HIV-1 viral activity in infected MDMs was measured by a reverse transcriptase activity assay following treatment with nano-NRTIs. Mitochondrial DNA depletion in MDMs and human HepG2 cells was assessed by quantitative PCR. Nanogels were efficiently captured by MDMs and demonstrated low cytotoxicity, and no antiviral activity without drugs. All nano-NRTIs demonstrated high efficacy of HIV-1 inhibition at drug levels as low as 1 μmol/l, representing a 4.9- to 14-fold decrease in 90% effective drug concentrations as compared with NRTIs, whereas 50% cytotoxicity effects started at 200× higher concentrations. Nano-NRTIs with a core-shell structure and decorated with brain-targeting peptides displayed the highest antiviral efficacy. Mitochondrial DNA depletion, a major cause of NRTI neurotoxicity, was reduced threefold compared with NRTIs at application of selected nano-NRTIs. Nano-NRTIs demonstrated a promising antiviral efficacy against HIV-1 in MDMs and showed strong potential as nanocarriers for delivery of antiviral drugs to macrophages harbouring in the brain.

HIV reverse transcriptase activity assay: a feasible surrogate for HIV viral load measurement in China

The quantitation of HIV viral load using an assay that measures the activity of reverse transcriptase (RT) may provide an alternative strategy for the monitoring of HIV viral load within resource-limited areas in China. Plasma viral load analyses of 215 samples from 87 patients infected with HIV were detected using the RT activity assay (ExaVir Load versions 2 and 3; Cavidi, Uppsala, Sweden) and RT polymerase chain reaction (PCR) (COBAS TaqMan 48, Amplink version 3.2; Roche Molecular Systems, Branchburg, NJ). The RT activity assay versions 3 (RT3) and 2 (RT2) could detect 95.3% and 86.9% of samples with measurable RNA by RT-PCR, respectively. A stronger correlation was observed between viral loads detected by RT3 and RT-PCR than between RT2 and RT-PCR ( r = 0.95, P < 0.001, and r = 0.92, P < 0.001, respectively). The correlation between serial samples collected from 6 patients at 1, 3, 6, 12, 18, and 24 months after beginning triple combination antiretroviral therapy, using the 2 different methodologies, was also strong ( r = 0.99, P < 0.001, for RT3 and RT-PCR, r = 0.98, P < 0.001, for RT2 and RT-PCR). The viral loads detected by RT activity assay were inversely correlated with CD4 + T-cell counts. Reproducibility of the RT activity assay was assessed by testing 3 samples in triplicate by 3 different operators. Viral load testing using assays that measure HIV RT activity is an affordable, feasible, simple, and reliable alternative for HIV RNA viral load determination in laboratories in China and other developing countries.

Antiviral Efficacy of the Novel Compound BIT225 against HIV-1 Release from Human Macrophages

Building on previous findings that amiloride analogues inhibit HIV-1 replication in monocyte-derived macrophages (MDM), Biotron Limited has generated a library of over 300 small-molecule compounds with significant improvements in anti-HIV-1 activity. Our lead compound, BIT225, blocks Vpu ion channel activity and also shows anti-HIV-1 activity, with a 50% effective concentration of 2.25+/-0.23 microM (mean+/-the standard error) and minimal in vitro toxicity (50% toxic concentration, 284 microM) in infected MDM, resulting in a selectivity index of 126. In this study, we define the antiretroviral efficacy of BIT225 activity in macrophages, which are important drug targets because cells of the monocyte lineage are key reservoirs of HIV-1, disseminating virus to the peripheral tissues as they differentiate into macrophages. In assays with acutely and chronically HIV-1Ba-L-infected MDM, BIT225 resulted in significant reductions in viral integration and virus release as measured by real-time PCR and a reverse transcriptase (RT) activity assay at various stages of monocyte-to-macrophage differentiation. Further, the TZM-bl assay showed that the de novo virus produced at low levels in the presence of BIT225 was less infectious than virus produced in the absence of the compound. No antiviral activity was observed in MDM chronically infected with HIV-2, which lacks Vpu, confirming our initial targeting of and screening against this viral protein. The activity of BIT225 is post-virus integration, with no direct effects on the HIV-1 enzymes RT and protease. The findings of this study suggest that BIT225 is a late-phase inhibitor of the viral life cycle, targeting Vpu, and is a drug capable of significantly inhibiting HIV-1 release from both acute and chronically infected macrophages.

Quantitation of human immunodeficiency virus type 1 viral load in plasma using reverse transcriptase activity assay at a district hospital laboratory in Botswana: A decentralization pilot study

Roche COBAS Amplicor monitor version 1.5 assay is considered gold standard for viral load monitoring in Botswana. Due to its demand for elaborate infrastructure, viral load testing has been confined to the national HIV reference laboratories. Cavidi ExaVir Load version 2 assay was considered as a potential alternative to decentralize viral load testing to the rural/remote hospital laboratories and thus increase access to therapy. This study compared the performance of ExaVir Load v2 assay at a district hospital laboratory in Serowe and COBAS Amplicor monitor v1.5 assay at the Botswana Harvard HIV Reference Laboratory using quality assessment samples and plasma from HIV-positive individuals. ExaVir Load v2 and COBAS Amplicor monitor v1.5 assays had very good agreement; Kappa statistic 0.951. The COBAS Amplicor monitor v1.5 and ExaVir Load v2 assays detected HIV-1 RNA in 84 and 86 samples but did not detect HIV-1 RNA in 221 and 219 samples, respectively. The two assays detected HIV-1 RNA concordantly in 82 samples and were strongly correlated ( r = 0.8554, P < 0.0001). ExaVir Load v2 assay provided a simple and reliable alternative viral load system that is adaptable to district hospital laboratories. The cost per test is less than RT-PCR. The ExaVir Load v2 systems have since been placed in four more district and primary hospital laboratories.

Evaluation of HIV-1 viral load and genotypic resistance assays for resource-limited settings

There is an urgent need for low-cost assays for HIV-1 quantitation to ensure adequate follow-up of HIV-infected patients on antiretroviral therapy (ART) in resource-limited countries. Two low-cost viral load assays are evaluated, a reverse transcriptase activity assay (ExavirLoad v2, Cavidi) and a real-time reverse transcriptase PCR assay (Generic HIV viral load, Biocentric). Both tests were compared with the ultrasensitive HIV Amplicor Monitor assay. Samples were collected in Mombasa, Kenya, from 20 HIV-1 seronegative and 150 HIV-1 seropositive individuals of whom 50 received antiretroviral treatment (ART). The ExavirLoad and the Generic HIV viral load assay were performed in a local laboratory in Mombasa, the Amplicor Monitor assay (version 1.5, Roche Diagnostics) was performed in Ghent, Belgium. ExavirLoad and Generic HIV viral load reached a sensitivity of 98.3% and 100% and a specificity of 80.0% and 90.0%, respectively. Linear regression analyses revealed good correlations between the Amplicor Monitor and the Generic HIV viral load (r= 0.935, p< 0.001) with high accuracy (100.1%), good precision (5.5%) and a low percent similarity coefficient of variation (5.4%). Bland–Altman analysis found 95% of the samples within clinically acceptable limits of agreement (−1.19 to 0.87 log copies/ml). Although, the ExavirLoad also showed a good linear correlation with the Amplicor Monitor (r= 0.901, p< 0.001), a problem with false positive results was more significant. The cost per test remains relatively high (US$ 30 for ExavirLoad and US$ 20 for the Generic HIV viral load). Hence, false positive results and the need for an expensive PCR instrument for the Generic HIV viral load assays still limit the implementation of these tests in less equipped, less experienced laboratories. © 2007 Elsevier B.V. All rights reserved.

Evaluation of two commercially available alternatives for HIV-1 viral load testing in resource-limited settings

There is an urgent need for low-cost assays for HIV-1 quantitation to ensure adequate follow-up of HIV-infected patients on antiretroviral therapy (ART) in resource-limited countries. Two low-cost viral load assays are evaluated, a reverse transcriptase activity assay (ExavirLoad v2, Cavidi) and a real-time reverse transcriptase PCR assay (Generic HIV viral load, Biocentric). Both tests were compared with the ultrasensitive HIV Amplicor Monitor assay. Samples were collected in Mombasa, Kenya, from 20 HIV-1 seronegative and 150 HIV-1 seropositive individuals of whom 50 received antiretroviral treatment (ART). The ExavirLoad and the Generic HIV viral load assay were performed in a local laboratory in Mombasa, the Amplicor Monitor assay (version 1.5, Roche Diagnostics) was performed in Ghent, Belgium. ExavirLoad and Generic HIV viral load reached a sensitivity of 98.3% and 100% and a specificity of 80.0% and 90.0%, respectively. Linear regression analyses revealed good correlations between the Amplicor Monitor and the Generic HIV viral load ( r = 0.935, p < 0.001) with high accuracy (100.1%), good precision (5.5%) and a low percent similarity coefficient of variation (5.4%). Bland–Altman analysis found 95% of the samples within clinically acceptable limits of agreement (−1.19 to 0.87 log copies/ml). Although, the ExavirLoad also showed a good linear correlation with the Amplicor Monitor ( r = 0.901, p < 0.001), a problem with false positive results was more significant. The cost per test remains relatively high (US$ 30 for ExavirLoad and US$ 20 for the Generic HIV viral load). Hence, false positive results and the need for an expensive PCR instrument for the Generic HIV viral load assays still limit the implementation of these tests in less equipped, less experienced laboratories.

Alcohol suppresses IL-2-induced CC chemokine production by natural killer cells.

Natural killer (NK) cells are a critical component of the host innate immune system. We investigated whether alcohol impairs NK cell function, particularly production of CC chemokines induced by interleukin (IL)-2, the natural ligands for CCR5 receptor. Primary NK cells and NK cell line (YTS) were cultured with or without alcohol (10 to 80 mM) for three hours. The culture supernatants were then harvested and used to treat human peripheral blood monocyte-derived macrophages and a HeLa cell line, which expresses CD4, CCR5, and CXCR4 receptors (MAGI cells). CC chemokine expression by YTS and primary NK cells treated with or without alcohol was analyzed with the real-time RT-PCR and ELISA. [Ca(2)(+)]i and Western blot assays were used to determine calcium-mediated intracellular signaling pathway and NF-kappaB p65 expression. HIV strains (Bal and UG024) were used to infect macrophages and MAGI cells. In addition, ADA (macrophage-tropic strain) and murine leukemia virus (MLV) envelope-pseudotyped HIV infection was carried out in macrophages. HIV infectivity was determined by HIV reverse transcriptase (RT) and beta-galactosidase activity assays. Alcohol inhibited IL-2-induced CC chemokine (CCL3 and CCL4) expression by NK cells. Functional tests demonstrated that this reduced expression of CC chemokines was associated with diminished anti-HIV ability of NK cells. Alcohol also reduced the ability of NK cells to response to CCL3-mediated chemotaxis. Alcohol inhibited IL-2-induced NF-kappaB p65 protein expression and calcium mobilization by NK cells. Alcohol, through the inhibition of IL-2-induced NF-kappaB p65 protein expression and intracellular calcium mobilization, suppressed NK cell production of CC chemokines. This suppression of CC chemokine production was associated with diminished anti-HIV activity of NK cells. Thus, by inhibiting NK cell-mediated innate immunity against HIV, alcohol consumption may have a cofactor role in the immunopathogenesis of HIV disease.

Plasma virion reverse transcriptase activity and heat dissociation-boosted p24 assay for HIV load in Burkina Faso, West Africa

In resource-limited settings, the requirement for inexpensive, easy-to-perform viral load monitoring has increased with greater antiretroviral drug availability. To evaluate feasibility, in Burkina Faso, of a simple assay for plasma HIV reverse transcriptase (RT) activity quantification compared to heat dissociation-boosted (HDB) p24 antigen and RNA-based quantifications in plasma samples from HIV-infected patients. : Plasma viraemia was quantified by RT activity, HDB-p24 and RNA copies in 84 samples from 70 HIV-1 group M-infected patients (82% non-B subtype, 93% treatment naive), including serial samples from nine patients. RT activity detected 86% of plasma samples containing measurable RNA copies; corresponding to 0, 93 and 100% of samples with 1.7-4.0 log(10), 4.1-4.8 log(10) and 4.9-6.7 log(10) RNA copies/ml, respectively. HDB-p24 detected 77% of plasma samples containing measurable RNA copies; corresponding to 27, 80 and 86% of samples with 1.7-4.0 log(10), 4.1-4.8 log(10) and 4.9-6.7 log(10) RNA copies/ml, respectively. Measurement error based on one-way analysis of variance between RT activity and HDB-p24 values with RNA copies showed good agreement with RT activity (ME, <10%), however poorer agreement was obtained with HDB-p24 values (ME, >10%). Patient follow up showed a similar pattern of viraemia with RNA and RT activity assays. Field trials in Burkina Faso support the practical use of plasma RT activity assay as an affordable alternative for HIV viral load determination in regions where RNA detection remains difficult to perform. HDB-p24 use requires further evaluation before being considered as an alternative method in African HIV-infected patient follow up.