RANTES and MIP-1beta mRNA expression in human peripheral blood mononuclear cells: transcript quantification using NASBA technology.

The importance of chemokines in the immune response, as well as in a range of specific disease states, is becoming increasingly apparent. The role of CC- (or beta-) chemokines and their receptors in the pathology and mechanisms of HIV-1 infection has served to intensify interest in these factors. Although the functionality of these factors resides in their protein forms, assays for the detection and quantification of these protein factors in clinical samples are not readily available. Consequently, we designed NASBA-based assays for the quantification of the mRNA encoding two members of the CC-chemokine family: RANTES and MIP-1beta. The NASBA-based assays are extremely sensitive, accurate, and reproducible across a dynamic range of at least four orders of magnitude. Inter-assay performance is comparable to intra-assay performance. We applied these methods to the analysis of normal human PBMC and PBMC from HIV-1 infected individuals. Although MIP-1beta mRNA levels are higher than RANTES levels in both populations, RANTES levels in HIV-1+ patients are higher than in normal individuals. The utility of these assays in longitudinal studies of specific subpopulations of cells, as well as their potential use in clinical diagnostics, is discussed.

Detection of dengue viral RNA using a nucleic acid sequence-based amplification assay.

Faster techniques are needed for the early diagnosis of dengue fever and dengue hemorrhagic fever during the acute viremic phase of infection. An isothermal nucleic acid sequence-based amplification (NASBA) assay was optimized to amplify viral RNA of all four dengue virus serotypes by a set of universal primers and to type the amplified products by serotype-specific capture probes. The NASBA assay involved the use of silica to extract viral nucleic acid, which was amplified without thermocycling. The amplified product was detected by a probe-hybridization method that utilized electrochemiluminescence. Using normal human plasma spiked with dengue viruses, the NASBA assay had a detection threshold of 1 to 10 PFU/ml. The sensitivity and specificity of the assay were determined by testing 67 dengue virus-positive and 21 dengue virus-negative human serum or plasma samples. The "gold standard" used for comparison and evaluation was the mosquito C6/36 cell culture assay followed by an immunofluorescent assay. Viral infectivity titers in test samples were also determined by a direct plaque assay in Vero cells. The NASBA assay was able to detect dengue viral RNA in the clinical samples at plaque titers below 25 PFU/ml (the detection limit of the plaque assay). Of the 67 samples found positive by the C6/36 assay, 66 were found positive by the NASBA assay, for a sensitivity of 98.5%. The NASBA assay had a specificity of 100% based on the negative test results for the 21 normal human serum or plasma samples. These results indicate that the NASBA assay is a promising assay for the early diagnosis of dengue infections.

Macrophage-derived chemokine gene expression in human and macaque cells: mRNA quantification using NASBA technology.

Macrophage-derived chemokine (MDC) is a CC-chemokine that inhibits infection by both macrophage- and T cell-tropic strains of HIV-1. This suppressor activity has led to great interest in fully characterizing the role of MDC in the pathogenesis of HIV-1 infection. Methods for the quantitation of constitutive levels of MDC protein in vivo are lacking. In this report, we describe the development and performance of a NASBA-based assay for the quantification of MDC mRNA expression in human and macaque cells. Although the constitutive in vivo levels of MDC mRNA in macaque and human T lymphocytes were low, in vitro activation of these cells greatly increased MDC transcription. Levels in the human and macaque cells were comparable under all conditions tested. Positive correlations between MDC transcription and protein expression were observed. The results indicate that this assay is extremely sensitive and reproducible over a five log dynamic range, and effectively quantifies MDC mRNA in resting and activated T cells. This assay may therefore permit characterization of the role of MDC in HIV-1/SIV pathogenesis, and in vaccine-induced immune responses.

Quantitative evaluation of simian immunodeficiency virus infection using NASBA technology.

The most commonly used animal model for the study of HIV-1 infection in humans is the infection of non-human primates by simian immunodeficiency virus (SIV). The animal hosts used most frequently are different species of macaques, which are readily infected with SIV, and can therefore be used to study natural infection, pathogenesis, therapy, and vaccine efficacy. The study of HIV-1 infection in humans relies heavily on the quantification of HIV-1 load (i.e. viral RNA) in patient plasma. Given the importance of HIV-1 RNA levels in humans, it follows that SIV RNA levels in animals are also relevant to the study of infection in this model system. This report describes the development of the isothermal amplification-based NASBA technology for the quantification of SIV RNA load in macaque plasma. Evaluation of the assay using model systems demonstrated that the assay is accurate and reproducible over nearly four orders of magnitude. Viral RNA load data were compared to other infection measurements in the macaque system. Further, the assay was used to provide copy number levels of SIV RNA in macaque plasma samples, permitting characterization of viral load during the course of SIV infection.

Genotyping of the CCR5 chemokine receptor by isothermal NASBA amplification and differential probe hybridization.

The human CCR5 chemokine receptor functions as a coreceptor with CD4 for infection by macrophage-tropic isolates of human immunodeficiency virus type 1 (HIV-1). A mutated CCR5 allele which encodes a protein that does not function as a coreceptor for HIV-1 has been identified. Thus, expression of the wild-type and/or mutation allele is relevant to determining the infectability of patient peripheral blood mononuclear cells (PBMC) and affects disease progression in vivo. We developed a qualitative CCR5 genotyping assay using NASBA, an isothermal nucleic acid amplification technology. The method involves three enzymes and two oligonucleotides and targets the CCR5 mRNA, which is expressed in PBMC at a copy number higher than 2, the number of copies of DNA present encoding the gene. The single oligonucleotide set amplifies both alleles, and genotyping is achieved by separate hybridizations of wild-type- and mutation-specific probes directly to the single-stranded RNA amplification product. Assay sensitivity and specificity were demonstrated with RNAs produced in vitro from plasmid clones bearing the DNA encoding each allele. No detectable cross-reactivity between wild-type and mutation probes was found, and 50 copies of each allele were readily detectable. Analysis of patient samples found that 20% were heterozygous and 1% were homozygous for the CCR5 mutation. Thus, NASBA is a sensitive and specific means of rapidly determining CCR5 genotype and provides several technical advantages over alternative assay systems.

NASBA technology: isothermal RNA amplification in qualitative and quantitative diagnostics.

Nucleic acid amplification technologies allow for the development of highly sensitive and specific diagnostic assays. The capacity to amplify and detect analyte targets, which may be present in a clinical sample as a single copy; is characteristic of many of these amplification technologies. NASBA is an isothermal method of nucleic acid amplification with such capability, and is particularly well suited for the amplification of RNA analytes. NASBA utilizes the coordinated activities of three enzymes (AMV-RT, RNase H, T7 RNA polymerase), and two oligonucleotide primers which are specific for the analyte target. The amplification process is part of a total system which includes a versatile nucleic acid isolation procedure, and powerful detection methodology. In this report, the development of NASBA technology for the detection of human Retrovirus RNA will be discussed. Specifically, a qualitative NASBA assay for the RNA of HTLV I, and a quantitative NASBA assay for HIV-1 will be described.

Detection of HIV-1 infection in vitro using NASBA: an isothermal RNA amplification technique.

Establishment of a sensitive infection assay for HIV-1 is essential for successful screening of antiviral agents and neutralizing antibodies. In this report, an infection assay is described which measures the expression of viral genomic RNA and spliced mRNA intermediates in infected cells by an amplification-based technique called NASBA. The extreme sensitivity of this method permits the detection of viral RNA in peripheral blood mononuclear cells (PBMC) within 48 h of infection by a low dose of virus. Similarly, spliced HIV-1 mRNA could be detected within 24 h of infection of CEM cells by HIV-1IIIB. This NASBA-based infection assay was shown to titer the neutralization of the HIV-1IIIB isolate by serum from an infected human and by a monoclonal antibody to gp120. Furthermore, the inhibitory effects of azidothymidine (AZT) and soluble CD4 on HIV-1IIIB infection were quantitated by this assay. The early detection of virus by NASBA minimizes the contribution of secondary infection, thereby permitting more accurate evaluation of antiviral agents and neutralizing antibodies. This assay may be useful for the study of infection of phenotypically distinct HIV-1 isolates, which differ in terms of their replication kinetics.