Reverse Transcriptase Assay Research Articles

Simian retrovirus D serogroup 1 has a broad cellular tropism for lymphoid and nonlymphoid cells.

Simian acquired immunodeficiency syndrome is a fatal immunosuppressive disease caused by type D retroviruses such as simian acquired immunodeficiency syndrome retrovirus type 1 (SRV-1). The disease is characterized by generalized lymphadenopathy, opportunistic infections, and lymphoid depletion with defects in both humoral and cell-mediated immunity. To understand how SRV-1 infection relates to the immune defect, we studied in vivo-infected lymphocytes from SRV-1-positive macaques with and without clinical signs of immunosuppressive disease. B and T helper/inducer and T suppressor/cytotoxic lymphocytes were purified by panning or by flow cytometry. Neutrophils were purified by dextran sedimentation, and platelets were purified by low-speed centrifugation. In vitro infection studies were also done with HUT78, H9, K562, rhesus lung fibroblast, rhesus monkey kidney, and bat lung cells. SRV-1 in lymphocytes or culture supernatants was detected by the induction of syncytia in cocultivated Raji cells and was confirmed by immunofluorescence, electron microscopy, or reverse transcriptase assay. We found that B and T helper/inducer lymphocytes were infected in all animals tested. The number of infected T suppressor/cytotoxic cells was generally lower than that of the other cell subsets, and not all animals in this subset had SRV-1 infections. All other cells exposed in vitro to SRV-1, except bat lung cells, were able to be infected. These findings show that SRV-1 has a broad cell tropism for lymphoid and nonlymphoid cell types.

Enzyme immunoassay for detection of human immunodeficiency virus antigens in cell cultures

A sensitive and specific avidin-biotin enzyme immunoassay that uses immunoreagents from naturally infected individuals was formulated for the detection of human immunodeficiency virus (HIV) antigens. A total of 500 cell culture samples from 111 cultures were tested in this assay and in a reverse transcriptase (RT) assay. Of 353 samples that were positive in the immunoassay, 174 were positive and 179 were negative in the RT assay. The specificity of the immunoassay results was supported by the failure of samples to react with nonimmune serum, by the ability of an anti-HIV type 1 monoclonal antibody to block the reactivity of selected samples, and by the appearance of RT activity in samples drawn from some cultures after a longer period of cultivation. HIV antigens were detected in 174 of 176 RT-positive samples (sensitivity, 98.9%). A comparison of the kinetics of antigen production and RT activity revealed that detectable antigen levels frequently preceded the appearance of RT activity. Thus, 50% of virus-containing cultures were identified within 9 days by immunoassay compared with 14 days by RT assay. In addition, RT activity was often detected intermittently in cultures sampled on several days, whereas antigen levels did not decline after initial appearance. Enzyme immunoassays for HIV antigen detection that use easily obtained reagents and simple technologies could facilitate laboratory and clinical research which requires cultivation of viruses.

Expression of soluble, enzymatically active, human immunodeficiency virus reverse transcriptase in Escherichia coli and analysis of mutants.

We have constructed a plasmid that, when introduced into Escherichia coli, induces the synthesis of large quantities of a protein with an apparent molecular mass of 66 kDa that differs from human immunodeficiency virus (HIV) RNA-dependent DNA polymerase (deoxynucleoside-triphosphate:DNA deoxynucleotidyltransferase or reverse transcriptase, EC 2.7.7.49) only in that it has two additional amino-terminal amino acids. This protein is soluble in E. coli extracts, is active in reverse transcriptase assays, and shows inhibition profiles with dideoxy-TTP and dideoxy-GTP that are indistinguishable from the viral enzyme. The deletion of 23 amino-terminal or carboxyl-terminal amino acids or the insertion of 5 amino acids at position 143 substantially decreases the polymerizing activity of the HIV reverse transcriptase made in E. coli. The properties of a 51-kDa reverse transcriptase-related protein made in E. coli suggests that the p51 found in the virion probably does not have substantial polymerizing activity. The full-length HIV reverse transcriptase and the various mutant proteins produced in E. coli should be quite useful for structural and biochemical analyses as well as for the production of antibodies.

Comparable sensitivities for detection of human immunodeficiency virus by sensitive reverse transcriptase and antigen capture enzyme-linked immunosorbent assays

The sensitive overnight reverse transcriptase and the antigen capture enzyme-linked immunosorbent assays were tested for their ability to detect human immunodeficiency virus. The two assays were able to quantitate as few as 75 to 130 virus particles. Thus, both tests offer similar sensitivities for determining the presence of the virus.

HIV p24 ELISA for quantitation of HIV infection and replication in vitro

The presented p24 ELISA performs better than other methods to follow HIV infection in cell culture: it is more sensitive, more specific and less laborious than the reverse transcriptase assay or advanced histochemical procedures. It is especially well suited for the study of glycosylation inhibitors on HIV replication since p24 itself is not glycosylated.

Practical diagnostic testing for human immunodeficiency virus.

Since the discovery of human immunodeficiency virus (HIV) as the causative agent of acquired immunodeficiency syndrome in 1983, there has been a proliferation of diagnostic tests. These assays can be used to detect the presence of HIV antibody, HIV antigen, HIV ribonucleic and deoxyribonucleic acids, and HIV reverse transcriptase. Enzyme-linked immunosorbent assays, Western blot, radioimmunoprecipitation assays, indirect immunofluorescence assays, reverse transcriptase assays, and several molecular hybridization techniques are currently available. Enzyme-linked immunosorbent, Western blot, and indirect immunofluorescence assays for HIV antibody are very sensitive, specific, and adaptable to most laboratories. An enzyme-linked immunosorbent assay for HIV antigen is also readily adaptable to most laboratories and will be commercially available soon. While the other assays are more tedious, they are valuable confirmatory tests and are suitable for reference laboratories. The biohazards of performing HIV testing can be minimized with proper biosafety measures.

Antiviral Effects of 3′-Azido-3′-Deoxythymidine, 2′,3′-Dideoxycytidine, and 2′,3′-Dideoxyadenosine Against Simian Acquired Immunodeficiency Syndrome-Associated Type D Retrovirus In Vitro

The simian acquired immunodeficiency syndrome (SAIDS) in macaques at the Washington Regional Primate Research Center is associated with a type D retrovirus known as SAIDS-D/WA. We tested the ability of 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxycytidine (ddC), and 2',3'-dideoxyadenosine (ddA) to inhibit the in vitro cytopathic effect (syncytium formation) and infectivity of the SAIDS-D/WA virus. Raji cell cultures were infected with virus and treated with various concentrations of AZT, ddC, and ddA. The ability of these drugs to inhibit replication of the SAIDS-D/WA virus in Raji cells was monitored by syncytium formation, expression of viral antigen, and reverse transcriptase assay. At concentrations of 4, 40, and 400 microM, AZT completely blocked the viral infectivity and inhibited the cytopathic effect of SAIDS-D/WA. Likewise, ddC was inhibitory at concentrations of 5 and 50 microM and ddA was inhibitory at 100 and 200 microM. AZT, ddC, and ddA became cytostatic to Raji cells with increasing drug concentrations. AZT also partially inhibited SAIDS-D/WA replication in previously infected Raji cell cultures, and viral inhibition increased in response to the concentration of AZT. These data indicate that AZT, ddC, and ddA are effective antiretroviral agents that merit further evaluation, including clinical trials, in animal models with AIDS-like diseases.

Characterization of Vero cells

A cell line used in the production of biologicals should be free of infectious agents, and ‘described with respect to cytogenetic characteristics and tumorigenicity’. Vero, a continuous cell line derived from a normal African green monkey kidney, was examined for the presence of retroviruses and for tumorigenic potential. We were unable to detect the presence of retroviruses by reverse transcriptase assay, electron microscopy or hybridization of cellular genomic DNA with Mason-Pfizer monkey virus DNA probes. In addition, passage 156 Vero cells did not form progressively growing tumors in nude mice or grow with high efficiency in soft agarose.

Detection of human immunodeficiency virus-infected lymphoid cells at low frequency by flow cytometry

Flow cytometric detection of human immunodeficiency virus (HIV)-infected lymphoid cells at low frequencies is described. Infected cells from human T lymphoid cell lines H9 and A3.01 were detected at frequencies as low as 10 −4 following indirect immunofluorescence labeling. For labeling, cells were treated with an HIV-inactivating, permeabilizing fixative followed by binding of a monoclonal antibody specific for the HIV major core protein p24, and then by binding of fluorescein isothiocyanate-conjugated F(ab′) 2 fragments of goat anti-mouse immunoglobulin antibody. We compared two fixation procedures, one using a mixture of methanol and acetone, the other a three-step fixation using methanol, paraformaldehyde and Triton X-100. The latter fixation protocol was found to be superior in its ability to resolve mixtures of infected and uninfected cells. The method allowed determination of the percentage of the cell population that was infected and the relative amount of p24 antigen per cell. At analysis rates of several thousand cells/s, detection of HIV-infected cells as rare events was possible. Excellent agreement was obtained between flow cytometric evaluation and reverse transcriptase (RT) assay of infected H9 cells cocultured with uninfected H9 cells in various proportions for 7 days. In time course of infection experiments, cultures infected by small numbers of viral particles were positive by flow cytometry up to 3 days earlier than by RT assay.

Comparison of antigen assay and reverse transcriptase assay for detecting human immunodeficiency virus in culture

We compared an antigen capture assay (Abbott Laboratories, North Chicago, Ill.) with a reverse transcriptase assay to identify and quantify human immunodeficiency virus (HIV) in culture. In direct comparisons of serial dilutions of lymphadenopathy-associated virus type 1, the antigen assay was 100-fold more sensitive than the reverse transcriptase assay in detecting the virus. The antigen assay reacted strongly with 60 different HIV isolates but did not cross-react with human T-cell lymphotropic virus type I, human T-cell lymphotropic virus type II, cytomegalovirus, varicella-zoster virus, herpes simplex virus type 1, Epstein-Barr virus, adenovirus type 5, or poliovirus type 1 or with extracts from four different control human cell lines and eight different phytohemagglutinin-stimulated normal human lymphocytes. Peripheral blood lymphocyte samples from 50 individuals were evaluated by both the antigen assay and the reverse transcriptase assay. The cells from the 34 seropositive individuals were all positive by the antigen assay (range, 3 to 9 days; average time, 5.9 days) and the reverse transcriptase assay (range, 7 to 16 days; average time, 9.6 days). Cells from the 16 seronegative individuals were negative by both assays. These results indicate that the antigen assay is an important addition to the monitoring of HIV production in the lymphocytes of infected patients.

Resumption of virus production after human immunodeficiency virus infection of T lymphocytes in the presence of azidothymidine

The new antiviral agent, azidothymidine (AZT; BW A509U), is currently the only successful drug in use for patients with acquired immunodeficiency syndrome. The effect of this thymidine analog, 3'-azido-3'-deoxythymidine, on the replication of the lymphadenopathy-associated virus strain of the human immunodeficiency virus was evaluated by using susceptible H9 and Jurkat cells. Cells were pretreated with concentrations of drug ranging from 0.5 to 100 microM, infected, and maintained in medium containing drug. Virus production was assayed by reverse transcriptase assays, and virus-specific DNA was analyzed by Southern blots probed with cloned human immunodeficiency virus sequences. At 4 to 8 days postinfection, infected cells without drug reached a peak of reverse transcriptase activity that was sustained. Increasing concentrations of AZT caused increasing delays in virus production; however, replicate cultures at nontoxic levels of the drug (up to 25 microM) eventually produced as much virus as did non-drug-treated infected cells, despite the continued presence of the drug. Levels of intracellular, unintegrated, virus-specific DNA paralleled reverse transcriptase levels. Virus-caused cytopathic effect was likewise delayed in drug-treated cultures. Virus recovered from H9 cultures after 25 microM AZT treatment did not appear resistant to AZT.

In situ hybridization and immunocytochemistry for improved assessment of human immunodeficiency virus cultures.

The authors characterized the early intracellular events involved in human immunodeficiency virus (HIV) replication after in vitro inoculation into cultures of susceptible human T-cell lines and phytohemagglutinin-stimulated peripheral-blood mononuclear cells (PMCs). Within 24 hours of infection, in situ hybridization with HIV DNA probe detected cytoplasmic viral RNA. Viral core antigen was detected in infected cells over the subsequent two to ten days by means of an immunocytochemical assay employing monoclonal antibodies. Several days later, cell-free virus was detected by both reverse transcriptase assay and a p25gag antigen-capture assay. When these methods were applied to monitor cultures of ten sero-positive persons' PMCs, a similar progression of virus replication was apparent: cytoplasmic viral RNA was detected in infected PMCs by day 3, with the subsequent appearance of intracellular viral proteins (days 6-9) and cell-free virus (days 12-21). In situ hybridization and immunocytochemistry offer complementary, sensitive, and specific approaches for monitoring the early stages of acquired immune deficiency syndrome virus replication in vitro.

ISOLATION OF HIV FROM AUSTRALIAN PATIENTS WITH AIDS, AIDS RELATED CONDITIONS AND HEALTHY ANTIBODY POSITIVE INDIVIDUALS*

The Fairfield Hospital experience with isolation of HIV from peripheral blood leucocytes, cerebrospinal fluid and semen is described. To date HIV has been isolated from single specimens of blood from 45% of patients with AIDS, 35% of patients with lymphadenopathy syndrome AIDS-related complex or ARC and 14% of asymptomatic antibody positive individuals. HIV was recovered from peripheral blood leucocytes in the presence of phytohemagglutinin and interleukin-2. The presence of virus in the supernatant fluid was detected using reverse transcriptase and immunofluorescence assays. Supernatants with borderline activity were confirmed by infection of a continuous cell line.

Inactivation of human immunodeficiency virus by Betadine.

Human immunodeficiency virus (HIV), the etiological agent of the acquired immunodeficiency syndrome (AIDS), was treated with either Betadine (povidone-iodine) Solution or Betadine Surgical Scrub. HIV inactivation was analyzed using the viral reverse transcriptase assay or by observing the cytopathic effect produced in HIV-infected, H-9, T-cell cultures. The minimum effective Betadine dose was 0.25% for complete inactivation of HIV that was treated for various time intervals (immediate vortex to ten minutes). The titer of HIV stocks used in these experiments (10(5) TCID50 per mL) was greater than amounts generally detected in clinical specimens. Our results provide a rationale for the use of povidone-iodine as a topical antiseptic against HIV in the clinic or laboratory.

Comparison of enzyme immunoassay and reverse transcriptase assay for detection of HIV in culture supernates

An enzyme immunoassay (EIA) was developed for detection of human immunodeficiency virus antigen (HIV Ag) in tissue culture supernatants. The assay was found to be specific for HIV and cheaper, easier to perform and more sensitive than the generally used reverse transcriptase (RT) assay. Cultures of peripheral blood leucocytes (PBL) from 106 patients with acquired immune deficiency syndrome (AIDS), AIDS related complex (ARC), healthy anti-HIV positive subjects and healthy anti-HIV negative subjects were held for 35 days and the supernatant fluid tested at regular intervals by EIA and RT. Of these 106 cultures, the presence of HIV was detected by EIA in 27 and by RT in 21. While six cultures were positive by EIA alone, none were positive by RT alone; the specificity of the results in the six EIA positive RT negative cultures was confirmed by subculture. In the 21 cultures in which HIV was detected by both techniques, the EIA became positive first on 10 occasions; in the remaining cultures both tests became positive at the same time. The HIV Ag assay reduces the time taken to process specimens and thus increases the efficiency and reduces the cost of isolation procedures.

Liquid competition radioimmunoassay for the detection and quantitation of the HIV p24

Productive infection of permissive cell cultures by HIV has been detected by different assays of which the measurement of reverse transcriptase (RT) activity has been considered highly specific and sensitive. Here we describe the production and characterization of a mouse hybridoma cell line, MB12, secreting monoclonal antibodies to HIV p24, the major core protein, and the use of this monoclonal antibody to develop a type specific indirect liquid competitive radioimmunoassay (RIA) capable of providing earlier detection of the replicating virus than the RT assay. This assay also provides a quantitative analysis of HIV p24, which can be used to study the viral replication in permissive cell cultures. The ease of methodology and the adaptability of the competitive RIA to various assay conditions make this immunoassay suitable for the study of HIV expression in infected cell cultures.

Sensitive reverse transcriptase assay to detect and quantitate human immunodeficiency virus

A sensitive biochemical assay of viral reverse transcriptase (RT) was developed that is useful for both the detection and quantitation of human immunodeficiency virus (HIV), the agent responsible for acquired immune deficiency syndrome in humans. This assay gave a 20- to 40-fold increase in enzyme activity over the current method used for RT detection of HIV. The test is based on a previous biochemical study showing the unusual stability of avian oncornavirus RNA-dependent DNA polymerases at 30 degrees C for at least 2 days. Our study shows that the HIV polymerase is stable at 30 to 37 degrees C for up to 3 days. By using this sensitive RT assay, as few as 250 HIV virions can be quantitated directly in tissue culture medium. This assay should prove useful in studies in which the detection of HIV or the quantitation of the number of virions is required.

Detection of Human Immunodeficiency Virus Core Protein in Plasma by Enzyme Immunoassay

A sensitive enzyme immunoassay was developed for detecting human immunodeficiency virus (HIV) core antigen. Assay sensitivity was 3.67 pmol/L of purified HIV core protein, and 1 or 100 in-vitro infectious units/mL of HIV in purified virus preparations or cell culture supernatants, respectively. Enzyme immunoassay sensitivity exceeded that of reverse transcriptase assay by 1000-fold. Core antigen was detected in whole plasma from 41% of symptomatic subjects and 13% of asymptomatic subjects seropositive for HIV. After plasma fractionation, antigenemia was found in 60% of symptomatic subjects and in 33% of asymptomatic subjects seropositive for HIV. Fifty-seven percent of samples from which HIV could be isolated in lymphocyte culture had detectable quantities of core antigen in plasma. However, at least 87% of samples with measurable antigen in plasma had HIV isolated from lymphocyte cultures. Antigenemia was associated with reduced T-cell number and symptomatic disease, and may be a useful marker for disease progression.

Comparison of reverse transcriptase assay with the Retro-Tek viral capture assay for detection of human immunodeficiency virus

The reverse transcriptase (RT) assay for human immunodeficiency virus (HIV) is tedious, expensive, and nonspecific for HIV activity. Because the Retro-Tek viral capture assay (VCA) (Cellular Products, Inc., Buffalo, NY) is relatively quick, inexpensive, and specific for HIV antigens, we compared the sensitivity and specificity of the two assays in a blinded fashion to see if the VCA could replace the RT assay. Specifically, peripheral mononuclear cells were cocultured from 11 Western blot-positive acquired immunodeficiency syndrome (AIDS) patients. The culture supernatant fluids were tested every 4-7 days for at least 28 days by VCA for HIV antigens, and by RT assay for retrovirus activity. Ten seronegative healthy donors with no risk factors were also cocultured and tested. Seven (64%) of 11 AIDS patients were positive by VCA and confirmed by RT assay in each instance. Concordance between VCA and RT assay was 100% in each patient. Time-to-positive detection was 7-28 days for both assays. Once a culture became positive by either assay, subsequent culture supernatant fluids remained positive by both assays until the culture was discarded, usually at day 28. The ten healthy seronegative donors were negative by both assays. These results suggest that the VCA is preferable to the RT assay for detection of HIV.

Detection of human immunodeficiency virus by reverse transcriptase assay, antigen capture assay, and radioimmunoassay

The reverse transcriptase assay, antigen capture assay, and radioimmunoassay were compared for the detection of human immunodeficiency virus (HIV) in culture fluids of virus-infected lymphocytes. The reverse transcriptase assay and the antigen capture assay were compared for 962 samples, and the two tests displayed comparable sensitivities (98.5% agreement) in detecting HIV antigen. In addition, these two tests displayed similar sensitivities when examined for the kinetics of HIV appearance following in vitro infection of normal lymphocytes. We also found the antigen capture assay to be as sensitive as the radioimmunoassay in detecting HIV antigen in culture fluids. Furthermore, all three tests were found to be reasonably concordant when applied simultaneously to the detection of HIV antigen in cultures. The antigen capture assay, however, is relatively fast, can handle a large number of samples, does not require radioactive material, and is less expensive than the other two tests. Therefore, the antigen capture assay appears to be the method of choice for the detection of HIV in routine virus culturing.