La transcriptase inverse (RT) est un hétéro-dimère p66/p51 avec des activités ADN polymérase et ribonucléase H qui jouent un rôle critique dans le cycle viral du VIH-1. La RT convertit l’ARN génomique viral simple brin en ADN proviral double brin dans le cytoplasme de la cellule infectée. L’efficacité de la RT est augmentée par la protéine de la nucléocapside (NC) grâce à son activité chaperonne vis-à-vis des acides nucléiques et/ou une coopération entre les deux protéines. Dans ce travail, nous avons étudié par la technique de smFRET (single molecule Fluorescence Resonance Energy Transfer) les effets de la NC sur l’interaction entre la RT et un substrat d’ADN au niveau de deux sites de pause de la synthèse de l’ADN(+). Nous avons d’abord réalisé et validé le montage de smFRET. Lors de la validation du montage avec des fluorophores Cy3 encapsulés dans des vésicules lipidiques, nous avons mis en évidence deux mécanismes différents entrainant le photoblanchiment du Cy3. Ensuite, après avoir déterminé les propriétés de liaison à l’équilibre de la RT et la NC sur différents substrats amorce/matrice à l’aide de mesures d’ensemble en solution, nous avons confirmé par smFRET que la RT adopte plusieurs conformations sur son substrat d’ADN, incluant celle qui conduit à la polymérisation de l’ADN. En présence de la NC, nous n’avons observé qu’une réorganisation modérée des différentes conformations du complexe RT/substrat. Par contre, une réorganisation beaucoup plus importante est induite par la NC en présence du dNTP, avec une très forte exaltation des conformations compétentes pour la polymérisation. Nous avons également montré que la NC augmente l’efficacité de synthèse de l’ADN au niveau de sites de pause en diminuant ou en augmentant le temps de dissociation du complexe RT/substrat/dNTP selon le type de site de pause. L’ensemble de ces données permet de mieux comprendre les mécanismes polyvalents par lesquels NC facilite l’activité de la RT. / The reverse transcriptase (RT) is a p66/p51 hetero-dimer with DNA polymerase and ribonuclease H activities which plays a critical role in the viral cycle of HIV-1. RT converts the viral genomic RNA to proviral DNA in the cytoplasm of infected cells. The efficiency of RT is increased by the nucleocapsid protein (NC) through its nucleic acids chaperone properties and/or via direct interaction with RT. In the present work, we investigated the effects of NC on the interaction between RT and its DNA substrate attwo pause sites during the synthesis of (+)DNA by using the smFRET (single molecule Fluorescence Resonance Energy Transfer) technique. In a first step, we implemented and validated the smFRET set-up. Within the validation step, using Cy3 fluorophores encapsulated, in lipid vesicles, we monitored the photobleaching of Cy3 dyes and found out that it was governed by two parallel mechanisms. In a second step, we determined the affinity of RT and NC to different primer/template substrates by using steady-state fluorescence. Then, we confirmed by smFRET that RT adopts different conformations on its DNA substrate, including the one that leads to DNA polymerization. In the presence of NC, we observed only a moderate reorganization of the different conformations of RT/substrate complex. However, NC was found to induce a more important reorganization in the presence of dNTP, with a very strong promotion of the polymerization-competent conformations. We also showed that NC increases the efficiency of DNA synthesis at pause sites by either decreasing or increasing the dissociation time of the RT/substrate/dNTP complex, depending on the type of pause site. Together, these data allow us to further elucidate the mechanisms by which NC facilitates the RT.

A genotipagem do vírus da hepatite C (VHC) é a principal ferramenta para prognóstico e tempo de tratamento. Dependendo do genótipo infectado existem diferentes esquemas e tempo de tratamento. O objetivo deste trabalho foi desenvolver padronizar e validar um método de genotipagem por PCR em tempo real com base na análise da região NS5B. Esta região apresenta um grau de polimorfismo que permite identificar de modo mais acurado tanto os tipos como os subtipos do VHC. Para isto foram desenhados dois conjuntos de primers e sondas. Neste trabalho desenvolvemos um método one-step modificado em uma reação triplex em que ocorre a identificação dos genótipos (1a, 1b, 3a) e em outro set a identificação dos genótipos (2a, 2b, 2c). Os resultados obtidos pelo método de genotipagem em tempo real concordaram em 100% com os resultados de seqüênciamento da região NS5B quando excluímos amostras que foram identificados como mistura de genótipos no método desenvolvido e classificados somente como um único genótipo no seqüênciamento. Houve uma boa concordância entre o método desenvolvido e o seqüênciamento da região NS5B pelo coeficiente de Kappa (k= 0,6222; p=0,0020). O método de desenvolvido conseguiu detectar 97,93% (190/194) do genótipo 1, 86,11% (31/36) do genótipo 2 e 100% (80/80) do genótipo 3. A média da sensibilidade foi de 97%. Quando comparamos a genotipagem por PCR em tempo real e LiPA nas 310 amostras analisadas não houve resultados discordantes em relação ao genótipo. Entretanto, 26,24% (79/301) das amostras analisadas apresentaram resultados discordantes em relação ao subtipo quando comparados os dois métodos. Foi determinada a sensibilidade analítica do método através de um ponto do painel da OptiQuant que foi diluído de modo seriado e a sensibilidade relativa foi realizada através de amostras de plasma de pacientes com carga viral determinada pelo Cobas Amplicor. O limite mínimo de detecção determinado foi de 125UI/ml para o genótipo 3a, 250 UI/ml para o genótipo 1b e 2b e 500 UI/ml para o genótipo 1a. Somados a isso, o método desenvolvido tem um custo de R$ 58,00, um valor nove vezes menor que o método comercial utilizado em nosso laboratório. Além disso, no método desenvolvido, o tempo trabalhado cai para menos de 2 horas sem a necessidade de manipulação constante em comparação com LiPA que necessita em torno de 16 horas, devido ao vários passos de hibridizações e lavagens. No presente trabalho o método de genotipagem por PCR em tempo real para o VHC mostrou-se eficiente e capaz de identificar de um modo acurado os diferentes genótipos e seus subtipos e contribuir para um entendimento melhor do verdadeiro papel dos genótipos e seus subtipos e da variabilidade genética na história natural da infecção do VHC. / Hepatitis C virus (HCV) genotyping is the most significant predictor of response to antiviral therapy. Depending on the infecting HCV genotyping different antiviral regimens have been proposed as well as the length of different treatment. The aim of this study was to develop and evaluate a new real time PCR of HCV genotyping based in NS5B region. This region has sequencing heterogeneity and can accurately identify both type and subtype of HCV. Furthermore, we compared the real time PCR with LiPA and sequencing of NS5B region. We developed a new one-step modified method in triplex reaction where we identified in two sets genotypes (1a, 1b, 3a) and (2a, 2b, 2c). Results obtained by real time PCR agreed 100% with those obtained by NS5B sequencing when excluded samples with mixed of HCV genotypes identified by real time PCR genotyping and in NS5B sequencing all samples were classified only as only one genotype. We found a good concordance for the analysis of genotype concordance between genotyping by real time and sequencing of NS5B region through the coefficient kappa (k= 0,6222; p=0,0020). The method developed detected 97,93% (190/194) of genotype 1, 86,11% (31/36) of genotype 2 and 100% (80/80) of genotype 3, with the overall sensitivity of this new method being 97%. Among 310 samples only two samples had discordant results at type level when comparing real time PCR and LiPA. However, 26,24% (79/301) had discordant results at subtype level when comparing LiPA and real time PCR genotyping of HCV. In order to measure the analytical sensitivity of the real time assay, one member of the panel OptiQuant HCV RNA was diluted. The relative sensitivity was determined by analysis the clinical specimens based upon the initial HCV RNA concentration determined by Cobas Amplicor. The lower limit of detection was estimated to be 125 IU/ml for genotype 3a, 250 IU/ml for genotype 1b and 2b, and 500 IU/ml for genotype 1a. Finally, the cost of each reaction are about R$ 58,00 nine fold lower than the commercial method available in Brazil. Manipulation time of real time PCR genotyping is about 2 hours, in comparison to LiPA that requires about 16 hours due to various hybridization steps and washing. This study was demonstrated an efficient method of identification in a accurate way. HCV genotyping which is important to understand the role of genotypes and subtypes, as well as of genomic variability in the natural history of HCV infection.

Les ARN sont des molécules aux propriétés diverses interagissant les uns avec les autres dans le but de médier des fonctions spécifiques. L’évaluation de l’abondance relative des ARN est une étape cruciale à la compréhension de la stoechiométrie nécessaire à ces fonctions. Cependant, plusieurs biais et limitations s’imposent avec l’utilisation de différentes techniques d’évaluation, dont le séquençage d’ARN (RNA-Seq), qui sont problématiques dans l’estimation de l’abondance de différents types d’ARN. De récentes études ont exposé les avantages d’un protocole novateur de RNA-Seq qui utilise une rétrotranscriptase thermostable d’intron de groupe II (TGIRT) d’origine bactérienne afin de réduire ces biais. Ce mémoire fait la comparaison entre différentes techniques de RNA-Seq afin d’élucider si l’utilisation de TGIRT en RNA-Seq offre une représentation plus juste du transcriptome. Les comparaisons avec les valeurs d’abondance de différents types d’ARN décrites dans la littérature ainsi qu’obtenues expérimentalement par notre groupe pointent au fait que TGIRT donne une meilleure estimation de l’abondance relative des ARN, et plus particulièrement des ARN hautement structurés. Cette meilleure estimation de l’ensemble de la composition du transcriptome permet de faire des observations sur les rapports d’abondance entre des ARN codants et non-codants fonctionnellement apparentés. Notamment, des ratios d’expression constants entre les ARN non-codants associés à des RNP et les ARNm codants pour leur facteurs protéiques ont été observés. Ceci suggère la présence d’une régulation transcriptionnelle commune nécessaire à la stoechiométrie de ces complexes. Une forte disparité dans l’expression des snoRNA et de leurs gènes hôtes, dépendant du type de snoRNA et de gène hôte a par ailleurs été constatée, corroborant une régulation distincte de la stabilité de ces transcrits. Dans l’ensemble, nos données suggèrent que la méthode TGIRT-Seq est la plus appropriée dans l’évaluation du transcriptome entier et ouvre donc la voie à des analyses plus holistiques par RNA-Seq en donnant une estimation plus juste de l’abondance relative des transcrits d’ARN. / Abstract : RNA are molecules with a wide range of properties that can interact with one another to mediate specific function. The evaluation of RNA abundance is a crucial step in understanding the stoichiometry needed for these functions. However, many limitations and biases come with the use of different techniques, including RNA sequencing (RNA-Seq), which affects the estimation of the abundance of different RNA types. Recent studies have exposed the advantages of a new RNA-Seq protocol using a thermostable group II intron reverse transcriptase (TGIRT) of bacterial origin to reduce these biases. This thesis makes the comparison between different RNA-Seq techniques to elucidate if the use of TGIRT in RNA-Seq offers a more representative depiction of the transcriptome. The comparisons with the abundance values given in literature and obtained experimentally by our group agree with the fact that TGIRT gives a better estimation of the relative abundance of RNA, especially highly structured RNA. This better estimation of the transcriptomic landscape allows many observations on the abundance relations between coding and non-coding RNA that are functionally related. Namely, constant expression ratios between RNP associated noncoding RNA and the mRNA that codes for their associated proteins have been observed. This suggests the presence of a common transcriptional regulation which is necessary for the stoichiometry of these complexes. A strong disparity in the expression of snoRNA and their host genes depending on snoRNA and host gene types has also been observed and corroborate a distinct regulation of these transcripts’ stability. In summary, our data suggest that the TGIRT-Seq method is the most appropriate to evaluate the transcriptome and thus opens the way to more holistic RNA-Seq analyses by giving a better estimation of RNA transcripts relative abundance.

This study has involved the design, synthesis and evaluation of novel HIV-1 enzyme inhibitors accessed by synthetic elaboration of Baylis-Hillman adducts. Several series of complex coumarin-AZT and cinnamate ester-AZT conjugates have been prepared, in high yields, by exploiting the click reaction between appropriate Baylis-Hillman derived precursors and azidothymidine (AZT), all of which have been fully characterised using spectroscopic techniques. These conjugates, designed as potential dual-action HIV-1 inhibitors, were tested against the appropriate HIV-1 enzymes, i.e. HIV-1 reverse transcriptase and protease or HIV-1 reverse transcriptase and integrase. A number of the ligands have exhibited % inhibition levels and IC50 values comparable to drugs in clinical use, permitting their identification as lead compounds for the development of novel dual-action inhibitors. In silico docking of selected ligands into the active sites of the respective enzymes has provided useful insight into binding conformations and potential hydrogen-bonding interactions with active-site amino acid residues. A series of furocoumarin carboxamide derivatives have been synthesised in four steps starting from resorcinol and these compounds have also been tested for HIV-1 integrase inhibition activity. The structures of unexpected products isolated from Aza-Baylis-Hillman reactions of N-tosylaldimines have been elucidated by spectroscopic analysis, and confirmed by single crystal X-ray analysis. A mechanism for what appears to be an unprecedented transformation has been proposed. Microwave-assisted SeO₂ oxidation of Baylis-Hillman-derived 3-methylcoumarins has provided convenient and efficient access to coumarin-3-carbaldehydes, and a pilot study has revealed the potential of these coumarin-3-carbaldehydes as scaffolds for the construction of tricyclic compounds. The HCl-catalysed reaction of tert-butyl acrylate derived Baylis-Hillman adducts has been shown to afford 3-(chloromethyl)coumarins and α-(chloromethyl)cinnamic acids, the Zstereochemistry of the latter being established by X-ray crystallography. ¹H NMR-based experimental kinetic and DFT-level theoretical studies have been undertaken to establish the reaction sequence and other mechanistic details. Base-catalysed cyclisation on the other hand, has been shown to afford 2H-chromene rather than coumarin derivatives.

Polycyclic aromatic hydrocarbons, produced by anthropological and natural activities, are hazardous through formation of oxidative radicals and DNA adducts. Growth of Mycobacterium sp. strain KMS, isolated from a contaminated soil, on the model hydrocarbon pyrene induced specific proteins. My work extends the study of isolate KMS to the gene level to understand the pathways and regulation of pyrene utilization. Genes encoding pyrene-induced proteins were clustered on a 72 kb section on the KMS chromosome but some also were duplicated on plasmids. Skewed GC content and presence of integrase and transposase genes suggested horizontal transfer of pyrene-degrading gene islands that also were found with high conservation in five other pyrene-degrading Mycobacterium isolates. Transcript analysis found both plasmid and chromosomal genes were induced by pyrene. These processes may enhance the survival of KMS in hydrocarbon-contaminated soils when other carbon sources are limited. KMS also grew on benzoate, confirming the functionality of an operon containing genes distinct from those in other benzoate-degrading bacteria. Growth on benzoate but not on pyrene induced a gene, benA, encoding a benzoate dioxygenase α-subunit, but not the pyrene-induced nidA encoding a pyrene dioxygenase α-subunit; the differential induction correlated with differences in promoter sequences. Diauxic growth occurred when pyrene cultures were amended with benzoate or acetate, succinate, or fructose, and paralleled delayed expression of nidA. Single phase growth and normal expression of benA was observed for benzoate single and mixed cultures. The nidA promoters had potential cAMP-CRP binding sites, suggesting that cAMP could be involved in carbon repression of pyrene metabolism. Growth on benzoate and pyrene requires gluconeogenesis. Intermediary metabolism in isolate KMS involves expression from genes encoding a novel malate:quinone oxidoreductase and glyoxylate shunt enzymes. Generation of C3 structures involves transcription of genes encoding malic enzyme, phosphoenolpyruvate carboxykinase, and phosphoenolpyruvate synthase. Carbon source modified the transcription patterns for these genes. My findings are the first to show duplication of pyrene-degrading genes on the chromosome and plasmids in Mycobacterium isolates and expression from a unique benzoate-degrading operon. I clarified the routes for intermediary metabolism leading to gluconeogenesis and established a potential role for cAMP-mediated catabolite repression of pyrene utilization.

It has been found in previous studies that there is a correlation between the placenta regulatory genes and the weight gain of the mother, Body Mass Index (BMI) as well as the birthweight of the fetus. When the mother gains weight / is overweight, this will affect the gene expression in the placenta, and in turn this triggers the weight gain of the fetus. The aim of the study was to investigate the correlation between the lipoprotein lipase gene and the mother's BMI, weight gain and the child's birth weight by extracting RNA from the placentas and analysing its quality and concentration. cDNA was generated from RNA using reverse transcription and gene expression was amplified using real-time PCR. The data from real-time PCR was used in the comparative Ct-method to calculate a 2˄(-ΔΔCt)-value which represents the RNA-level of the LPL-gene. Lastly this value was analysed by using the two-statistic methods, Pearson's rank correlation and Spearman's correlation, which showed that the value of the correlation coefficient for all the variables was close to the value of zero. The closer the value is to zero, the weaker the association becomes between the different variables. The correlation was 0.045, 0.112 and 0.044 for the child's birth weight, mother's BMI respective weight gain. The results from this study shows that there is no correlation between LPL and the mother's weight gain, BMI, or the child's birth weight.

No description available.

The use of a surface plasmon biosensor fills a missing link in kinetic studies of enzymes, since it measures directly the interaction between biomolecules and allows determination of parameters that are determined only indirectly in activity assays. The present thesis deals with kinetic and dynamic aspects of ligand binding to two viral enzymes: the human cytomegalovirus (HCMV) protease and the human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). The improved description of interactions presented herein will contribute to the discovery and development of antiviral drugs. The biosensor method provided new insights into the interaction between serine proteases and a peptide substrate, as well as substrate-induced conformational changes of the enzymes. The direct binding assay served as a tool for characterising the binding mechanism of HCMV protease inhibitors. Kinetic details of the interaction between HIV-1 RT and non-nucleoside reverse transcriptase inhibitors (NNRTIs) were unravelled. The recorded sensorgrams revealed several forms of complexity. A general binding model for the analysis was derived from the data, describing a two-state mechanism for the enzyme and a high- and a low-affinity interaction with the inhibitor. Interaction kinetic constants were determined for the clinically used NNRTIs and several investigational inhibitors. The established method was applied to investigate the mechanism of resistance against NNRTIs. Amino acid substitutions in the NNRTI-binding site resulted in both decreased association rates and increased dissociation rates for the inhibitors. The K103N and the L100I substitution also interfered with the formation of the binding site, thereby facilitating inhibitor binding and unbinding. Finally, thermodynamic analysis revealed that, despite the hydrophobic character of the interaction, NNRTI binding was mainly enthalpy-driven at equilibrium. Large entropy contributions in the association and dissociation indicated that binding is associated with a dynamic effect in the enzyme.

O transporte de iodo para tireóide é mediado pelo simportador sódio-iodo (NIS), glicoproteína de 643 aminoácidos localizada na região basolateral da célula folicular que acopla a entrada de sódio e iodo para o interior da célula. A clonagem do gene NIS em 1996 foi o primeiro passo na investigação dos mecanismos moleculares responsáveis pela diminuição da captação do radioiodo nos tumores benignos e malignos da tireóide. Estudos prévios sobre a expressão do gene NIS baseados em quantificação do transcrito e/ou análise imuno-histoquímica da proteína, mostraram resultados bastante divergentes. A maioria dos estudos com RT-PCR mostrou redução ou até ausência do transcrito do NIS. Os estudos mais recentes de imunohistoquímica, no entanto, mostraram aumento da expressão da proteína NIS, ao invés de diminuição. Poucos foram os estudos que fizeram análise do transcrito e da proteína concomitantemente nas mesmas amostras. Este estudo teve como objetivo quantificar o RNAm do gene NIS e avaliar a expressão e localização celular da proteína NIS, através das técnicas de RT-PCR em tempo real e exame imunohistoquímico, respectivamente. Foram estudadas 30 amostras de nódulos de tireóide, sendo 14 nódulos benignos e 16 nódulos malignos, sempre pareados com o tecido não-tumoral do mesmo paciente. Através de exame cintilográfico, verificouse que 100% dos nódulos malignos e 85,7% dos benignos eram hipocaptantes. Houve diminuição da expressão gênica do NIS em 78,6% dos nódulos benignos, em 81,2% dos carcinomas utilizando o gene PSMC6 como controle interno e em 100% dos carcinomas com o gene GAPDH como controle interno. O exame imunohistoquímico da proteína NIS revelou positividade da proteína NIS no citoplasma em 100% dos tecidos não-tumorais, 100% dos nódulos benignos e 93,75% dos nódulos malignos, não sendo estatisticamente diferentes entre si. A positividade na membrana basolateral ocorreu em 23,3% das amostras não-tumorais, em 14,3% dos nódulos benignos e em 12,5% dos nódulos malignos, não se detectando diferença significativa entre os grupos. A comparação da intensidade da imunoexpressão do NIS no citoplasma entre os nódulos versus os tecidos não-nodulares mostrou que esta foi maior em 64,3% dos nódulos benignos e em 87,5% dos malignos. Houve associação entre diminuição da quantificação gênica do NIS tumoral e aumento da imuno-expressão da proteína NIS no citoplasma tumoral em 50% dos nódulos benignos, 87,5% dos nódulos malignos, quando se utilizou o GAPDH como controle interno e, em 68,75% dos nódulos malignos quando se utilizou o PSMC6. A diminuição da expressão gênica do NIS associada a maior imuno-positividade intracitoplasmática da proteína NIS se deve a provável incapacidade de migração da proteína para a membrana plasmática. / The iodide uptake by epithelial thyroid cells requires the expression of sodium iodide symporter (NIS), a transmembrane glycoprotein of 643 amino acids. NIS is located at basolateral plasma membrane of the thyroid follicular cells and couples the transport of iodide and sodium to this cells. NIS gene was cloned in 1996, being the first step of investigation of the mechanisms responsible for the lower uptake of radioiodide by benign and malignant thyroid tumors. Previous studies about expression of NIS gene based in quantification by RT-PCR and/or immunohistochemistry analysis showed divergent data. The majority of RT-PCR studies showed reduction or even absence of transcript of NIS in malignant tumors. Recent studies of immunostaining showed that many tumors overexpress rather than under express NIS. Only a few studies have made the analysis of the transcript and the protein at the same time in the same samples. The objective of this study was to investigate NIS transcript levels and the presence and location of NIS protein, using real time RT-PCR and immunohistochemistry, respectively. It was included 30 samples of thyroid tumors, 14 benign and 16 malignant ones, always compared to adjacent non-tumoral samples. Scintigraphic findings showed that 100% of malignant tumors and 87.5% of benign ones were ?cold?. RT-PCR data revealed lower gene expression in 78.6% of the benign tumors, 81.2% of the carcinomas using PSMC6 as a housekeeping gene and 100% of the carcinomas using GHPDH as a housekeeping gene, when paired to the normal tissue samples. Immunohistochemical staining revealed presence of NIS protein in 100% of the nontumoral samples, 100% of the benign tumors and 93.75% of the malignant tumors. No statistical differences were detect in this data. NIS protein was identified at basolateral membrane in 23.3% of non-tumoral samples, 14.3% of benign tumors and 12.5% of malignant tumors. No statistical differences were detect in this data. Higher quantities of intracytoplasmatic NIS protein were detect in 64.3% of benign tumors and in 87.5% of malignant tumors when compared to non-tumoral samples. Association between lower gene expression and higher presence of intracytoplasmatic NIS protein was found in 50% of benign tumors, 87.5% of malignant tumors using GHPDH as a housekeeping gene and 68.75% of malignant tumors using PSMC6 as a housekeeping gene. The reduced gene expression of NIS in thyroid tumors with strong intracytoplasmatic staining may be due to his incapacity in migrating to cellular membrane.

<p>Reverse transcriptase (RT) is a crucial enzyme for retrovirus replication, and its presence in the virion is indispensable for infectivity. This thesis illustrates the use of RT activity assays as tools for quantitation and characterization of different retroviruses, particularly HIV. </p><p>A non radioactive assay, using microtiter plates, for the RT of Moloney murine leukemia virus (MMuLV) was developed. Assay conditions for MMuLV and HIV-1 RT, together with isozyme specific RT activity blocking antibodies, were shown useful for discrimination between RTs from different retrovirus genera. RT activity assay for HIV-1 was found to quantitate different subtypes more equally efficient than p24 antigen assays did.</p><p>Viral load (VL), the amount of HIV particles in the blood, is an important marker of the clinical status of an infected person. A method for VL determination based on RT activity (ExaVir Load) was developed. After plasma pretreatment, to inactivate cellular DNA polymerases, virions in patient plasma were immobilized on a gel, which was washed to remove disturbing factors. The virions were lysed with a detergent containing buffer and the lysate eluted. Finally, the RT activity in the lysate was determined and found to correlate strongly to VL by RNA according to a PCR based standard method (Roche Amplicor 1.5). The second version of the method was able to measure VL down to approximately 400 HIV-1 RNA copies/ml. The usefulness of RT from the VL procedure for determination of susceptibility towards anti-HIV drugs was demonstrated, and the results were in agreement with genotypic data. </p><p>Due to its technical simplicity, and ability to detect a broad range of HIV-1 subtypes, ExaVir Load and the drug susceptibility application are interesting for clinical use, particularly but not only in resource limited settings. The concept is also potentially useful for research purposes, e.g. in combination with specific RT assay conditions.</p>