Reverse Transcriptase Assay Research Articles

A solid phase reverse transcriptase micro-assay for the detection of human immunodeficiency virus and other retroviruses in cell culture supernatants

A simple and rapid solid-phase reverse transcriptase assay was developed based on the use of poly(rA):oligo(dT) 12–18 as template primer immobilized on DEAE cellulose paper squares to detect human immunodeficiency virus (HIV) and/or Other retroviruses in cell culture supernatants. It was found that PEG (per se) — up to 4% concentrations (w/v) — did not inhibit reverse transcriptase activity. Optimal conditions of the assay were determined. This solid-phase technique is much faster and more convenient than the methods described previously.

Preparation of a Detailed Restriction Map of the Avian Leukosis Virus MAV-2(O)

Unintegrated MAV-2(O) DNA was isolated from infected chicken embryo fibroblasts and inserted into the lambda bacteriophage vector lambda gtWES lambda B. Three x 10(6) bacteriophage plaques were screened, yielding a total of seven clones, six of which contained DNA representing the complete MAV-2(O) genome. Viral DNA was isolated from four of the clones and was used to transfect chicken embryo fibroblasts. All four clones produced virus as monitored by reverse transcriptase assay. When the four cloned viruses were inoculated into 10-day-old embryos, all hatched chickens developed osteopetrosis. One clone, lambda 9, induced osteopetrosis at a rate of onset and severity identical to that induced by the MAV-2(O) parental stock. This clone was selected for further study. To facilitate restriction mapping, the viral DNA from lambda 9 was subcloned into plasmid vector pUC 12 to construct a plasmid called p9. Cleavage of p9 DNA with single and multiple restriction endonucleases and hybridization with gene-specific probes identified the restriction fragments obtained. A comprehensive restriction map of cloned MAV-2(O) was generated and is compared with published maps and sequences of other avian retroviruses.

Use of monoclonal antibodies for the detection and quantification of HIV1 core protein p25: Comparative evaluation of in vitro HIV1 infection by immunofluorescence, antigen capture ELISA and reverse transcriptase assays

A two-site enzyme linked immunosorbent assay (ELISA) was developed to detect and quantify the HIV1 core protein p25 in the cell-free supernatant from virus-infected CEM cell culture, and compared with other assays. The assay, based on a sandwich method, employs two monoclonal antibodies (mAb) directed against different epitopes on the p25 core protein of HIV1, one used for p25 antigen capture and the other as a biotinylated probe. This immunoassay is sensitive enough to detect as little as 30 pg/ml of recombinant p25, the range of sensitivity of commercial kits, and therefore compares favourably with the conventional reverse transcriptase assay. Moreover, several hundred assays can be monitored quite conveniently by this simple ELISA procedure, which represents a useful tool for detection of HIV1 replication in microculture systems and rapid screening of antiretroviral agents using the reference strain HIV1-BRU as a model system.

Spectrum of biological properties of human immunodeficiency virus (HIV-1) isolates

In vitro assessment of biological properties of 14 independent isolates of human immunodeficiency virus type 1 (HIV-1) was performed in order to gain insight into the spectrum of behavioral diversity of HIV-1s and to attempt to identify phenotypic traits that may be eventually correlated with in vivo pathogenesis. All of these biologically cloned isolates were found to spread very slowly in most cell cultures, requiring 8–10 weeks for virus to spread from a few infected cells to around 10 5 cells. If viral synergistic activity was also present, as in HTLV-1-infected cells, HIV-1 spread was greatly accelerated. The isolates varied in their cellular tropisms, having as much as 100,000-fold difference in their tropisms for various human CD4-positive cell lines. Several HIV isolates were dual-tropic for both T and promonocytic cells, but some of these isolates did not readily infect U937 promonocytes while readily infecting THP-1 promonocytes. Both the slow spread and extreme tropisms of HIV-1 isolates have practical implications for titering HIVs and for initiating any studies examining the interaction between a given isolate and any given cell. Some isolates did not score readily by reverse transcriptase assays while others did and this did not reflect the amount of infectious virus produced. These findings raise questions about the reliability of HIV quantitation by RT assay. The HIV isolates further varied in their ability to kill and/or fuse cells, whereas some induced cytopathology more efficiently in a given cell line than others, even though the latter appeared to replicate as well. Finally, most isolates killed cells without syncytia formation, demonstrating that cell-to-cell fusion is a minor mechanism of cytopathology. The properties observed for each HIV isolate appeared to be stable phenotypes for that virus and the diversity of biological behavior raises the possibility that independent HIV isolates may differ in their virulence properties in vivo as well.

Immunopharmacological study of sulfaded schizophyllan (SPG) I. — Its action as a mitogen and anti-HIV agent

Sulfated schizophyllans of differing sulfur content were prepared and their mitogenic activities and effects on human immunodeficiency virus (HIV) were investigated in vitro. The sulfated schizophyllans were found to posses mitogenic activity. Anti-viral activity was evaluated in terms of cytopathmic effect, and by the fluorescence antibody technique, reverse transcriptase assay and cell proliferation assay. Schizophyllans with a higher sulfur content showed potent anti-HIV effects on Molt-4 clone No. 8 or MT-4 cells infected with HIV as well as on lymphocytes from AIDS patients. It is suggested that the sulfur content of schizophyllans is the most important factor for inhibiting growth of HIV, rather than molecular weight or kinds of sugar component. Thus, sulfated schizophyllans appear applicable as anti-HIV drugs.

Analysis of Nonproductive Human Immunodeficiency Virus Type 1 Infection of Human Fetal Dorsal Root Ganglia Glial Cells

Direct infection of glia by human immunodeficiency virus type 1 (HIV-1) has been suggested as one of several mechanisms responsible for the severe neurologic complications observed in both neonates and adults with the acquired immunodeficiency syndrome. We have demonstrated by protein immunoblotting analysis that HIV-1 infection of human fetal glial cells isolated from the dorsal root ganglia (DRG) of the developing human peripheral nervous system results in viral gag antigen expression with little, if any, detectable env gene products. No cytopathogenicity was evident in the infected cell population. Blot hybridization analyses indicate transient expression of the HIV-1 genome with maximum levels of virus-specific RNA being observed between 2 and 3 days postinfection and decreasing below the limits of detection by 16 days postinfection. To determine whether infection of the human fetal DRG glial cell population culminates in the production and release of infectious HIV-1, cocultivation and reverse transcriptase assays were performed. Direct assay of HIV-1-infected neural cell supernatants as well as exposure of permissive SupT1 cells to these HIV-1-infected neural cell supernatants resulted in no demonstrable reverse transcriptase activity in either the HIV-1-infected DRG glial cell supernatants or the SupT1 cell supernatants. Although transmission electron microscopy analyses have suggested the absence of intracellular viral particles, highly electron-dense inclusions in the cytoplasm of HIV-1-infected DRG glial cells were observed. The nature of the intracellular cytoplasmic inclusions is under current investigation. Cumulatively, these data suggest that the interaction of HIV-1 with human fetal DRG neural cells results in transient expression of the HIV genome culminating in a nonproductive infection.

高感度逆転写酵素活性測定法のヒト免疫不全ウイルス (HIV) 2型への応用

Sensitive reverse transcriptase assay was applied to human immunodeficiency virus type 2. The kinetics of this assay, stability of the enzyme and the effect of BSA to this assay indicated that the condition of this assay should be 37 degrees C in reaction temperature. The sensitivity of this assay increased by adding more than 10 micrograms/ml of BSA. The sensitivity of this assay is at least four times more than that of CPE assay using Molt-4 cell. Other HIV-2 isolate, LAV-2 in culture medium was also detectable in this condition. Moreover reverse transcriptase inhibiting antibody that specifically inhibits HIV-2 reverse transcriptase was found by this assay.

Transient expression of human immunodeficiency virus type 1 genome results in a nonproductive infection in human fetal dorsal root ganglia glial cells

Human immunodeficiency virus type 1 (HIV-1), the etiologic agent of acquired immunodeficiency syndrome (AIDS), has been implicated in the generation of AIDS-associated neurologic dysfunction. We are currently examining the replicative processes involved in HIV-1 infection of selected human fetal neural cell populations in vitro. To determine whether infection of the human fetal dorsal root ganglia (DRG) glial cell population culminates in the production and release of infectious HIV-1, cocultivation and reverse transcriptase (RT) assays were performed. Direct assay of HIV-1 infected neural cell supernatants as well as exposure of permissive SupTi cells to these HIV-1-infected neural cell supernatants detected no RT activity in eitherthe HIV-1-infected DRG glial cell supernatants orthe SupTi cell supernatants. When SupTl cells were cocultivated with the HIV-1-infected neural cells for 24-hr intervals, RT activity was detected in the SupTi supernatants from cocultures initiated less than 2 days after infection (most likely resulting from infectious input virus) but not from cocultures initiated on 3, 5, 10, and 30 days after infection. Hybridization analysis demonstrated transient expression of HIV-1 cytoplasmic mRNA with accumulation reaching a maximum level by 2 to 3 days postinfection, declining thereafter with low, but detectable, levels at 16 days postinfection. In addition, polymerase chain reaction amplification in conjunction with DNA blot hybridization detected HIV-1-specific proviral DNA at 3 days postinfection. Cumulatively, these data suggest that HIV-1 infection of human fetal DRG glial cells culminates in a nonproductive infection with expression of at least a fraction of the virus genome but no detectable infectious virus production.

Search for a retrovirus in long-term cultured cerebrospinal fluid cells and peripheral blood mononuclear cells from patients with multiple sclerosis.

Long-term peripheral blood mononuclear cell (MNC) cultures stimulated with interleukin 2 (IL-2) or IL-2 + phytohemagglutinin were established from 33 multiple sclerosis (MS) patients, 9 with other neurological diseases (OND), and 24 normal controls (C). Cultures were analysed for growth characteristics, reverse transcriptase (RT) in the culture medium, 2'-5' oligoadenylate synthetase in the cells, and cell morphology. None of these parameters differed in the MS group compared with the OND and C groups. Furthermore, 11 cerebrospinal fluid cell cultures were established without feeder cells. Morphology studies of the cells and RT assays of the supernatants from these cultures were normal. Induction studies by dexamethasone and 2-bromo-5'-deoxyuridine in 2 of these cultures did not reveal any signs of a virus. The significance of these results for the retrovirus hypothesis is discussed.

Susceptibility of EBV-carrying B cell lines to infection by HIV-1: variability of production of progeny virus and expression of viral antigens

We have examined 3 different EBV-carrying B cell lines, in terms of ability to be super-infected by the human immunodeficiency virus (HIV-1), and have followed these lines, after infection by HIV-1, over a period of 3 months. We found significant variation among different HIV-1 strains in terms of the multiplicity of infection required to initiate infection in these EBV-positive cell lines. Persistent infection by HIV-1 in the absence of detectable cytopathic effects could be demonstrated, as evaluated by a variety of techniques, including reverse transcriptase assay and immunofluorescence. The results indicate also that all of these cell lines produced progeny HIV-1 intermittently, with large amounts of virus production on some days but not others. In contrast, they were all able to continuously express p24.

Detection of human immunodeficiency virus (HIV) in cultures of lymphocytes from HIV-seropositive persons with special reference to an enzyme immunoassay

Three different assay systems for monitoring the production of human immunodeficiency virus (HIV) in infected cells were compared. These were (1) a reverse transcriptase assay (RTA), (2) an indirect immunofluorescence assay (IFA), and (3) an enzyme immunoassay (EIA) for detecting HIV antigen in vitro. MOLT-4 subclone 1 cells were infected with serial dilutions of the HTLV-IIIB strain of HIV. Six days after infection, production of HIV was examined by the three methods. EIA was most sensitive showing 64-fold higher sensitivity than RTA, which in turn was fourfold more sensitive than IFA. We applied EIA for detecting HIV in the lymphocyte cultures derived from 18 HIV-seropositive persons. By means of the three systems HIV was isolated from lymphocyte cultures derived from one of nine (11%) asymptomatic persons and from five of nine (56%) symptomatic patients. Interestingly, it was noticed that four samples of lymphocytes did not allow the isolation of HIV but gave positive results by EIA only. Thus, the detection rate of HIV was increased by the application of EIA, to three of nine (33%) persons in the asymptomatic group and to seven of nine (78%) patients in the symptomatic group. Possible use of this assay system for following the development of acquired immunodeficiency syndrome is discussed.

Recovery of Human Immunodeficiency Virus and Detection of p24 Antigen in Bronchoalveolar Lavage Fluid from Adult Patients with AIDS

Published reports indicate that HIV is recovered from BAL fluid of patients with AIDS who have LIP but not with other AIDS-related pulmonary disease. Our experience has been different. Ten BAL specimens from nine patients with AIDS were cultured directly in peripheral blood mononuclear cells, and all ten cultures were positive for HIV as indicated by examination of the culture supernatant by reverse transcriptase assay and enzyme immunoassay for HIV antigen. Five of the specimens were also positive for Pneumocystis carinii, and other pulmonary diagnoses included histoplasmosis, lymphoma, Kaposi's sarcoma, and aspiration pneumonia. Five additional BAL specimens were cultured after freezing at -70 degrees C, but only two were culture-positive for HIV (p = 0.022; FET). This study indicates that HIV can be recovered from the BAL fluid in most patients with AIDS, unrelated to the type of pulmonary disease. In contrast to cultures, HIV antigen was detected in the BAL fluid of only one patient, and that patient had LIP with noncaseating granulomas. Therefore, HIV culture is not useful in the diagnosis of LIP, but HIV antigen detection should be studied further. All BAL fluids should be considered potentially infectious.

Infection of cultured human thymic epithelial cells by human immunodeficiency virus

We have succeeded in growing the HTLV-III B strain of human immunodeficiency virus type 1 (HIV-1) in cultured human thymic epithelial (TE) cells. Expression of the HIV-1 proteins p17 and p24 was detected by immunofluorescence and reached a peak at 3 days after infection. Antigen capture and reverse transcriptase assays were used to detect HIV-1 in culture fluids, with positive results also being realized. The infection was cytolytic; cellular disarrangement, increased numbers of Hassall's corpuscles, and giant cells first appeared in monolayers of TE cells at 2 days after inoculation. By 4 days these changes were increased, and by 7 days, retraction and involution of TE cells were evident. The infection of TE cells by HIV-1 was blocked by preincubation with monoclonal OKT4A antibodies directed against CD4 target molecules.

Combinations of isoprinosine and 3'-azido-3'-deoxythymidine in lymphocytes infected with human immunodeficiency virus type 1

Since clinical trials are being planned with the immunomodulating drug isoprinosine combined with the antiviral drug 3'-azido-3'-deoxythymidine (AZT) in patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex, it is important to determine the type of antiviral interaction produced by these drugs in vitro. Such a combined modality may not only produce enhanced antiviral effects but also may have a valuable immunorestorative action. The interaction of several ratios of AZT and isoprinosine on the replication of human immunodeficiency virus type 1 in human peripheral blood mononuclear cells was determined by reverse transcriptase assay of disrupted virus obtained from supernatants of cells that were exposed to virus and the drugs separately and in combination and by a human immunodeficiency virus type 1 p24 enzyme immunoassay of the same supernatants. The correlation between the reverse transcriptase and enzyme immunoassay data was high. The antiviral activity of AZT alone was neither diminished nor augmented when AZT was used in combination with isoprinosine. Isoprinosine did not enhance virus yield when used alone or in combination with AZT in peripheral blood mononuclear cells, nor did it affect the growth of uninfected cells. The in vitro results indicate that this combination did not decrease the efficacy of AZT or exacerbate virus replication.

Comparison of antigen immunoassay and reverse transcriptase assay for monitoring human immunodeficiency virus infection in an antiviral trial

We compared the Abbott enzyme immunoassay for human immunodeficiency virus (HIV) antigen with the reverse transcriptase assay (RTA) as a means of monitoring HIV infection during an antiviral trial. The Abbott enzyme immunoassay detected HIV earlier than RTA whether or not the patients were antigenemic and appears to be superior to RTA for detecting HIV in cultures used for monitoring clinical trials.

Addition of substitution of simian virus 40 enhancer sequences into the Moloney murine leukemia virus (M-MuLV) long terminal repeat yields infectious M-MuLV with altered biological properties

Moloney murine leukemia virus (M-MuLV) is a replication-competent retrovirus which induces T-cell lymphoma in mice. The enhancer sequences present within the M-MuLV long terminal repeat (LTR) region of the proviral genome have been shown to influence the disease specificity of the virus strongly. We examined the contribution of the M-MuLV enhancers to the transcriptional activity and pathogenesis of M-MuLV by constructing LTRs containing heterologous enhancer elements. The simian virus 40 enhancer region (72- and 21-base-pair repeats) was inserted into the U3 region (at -150 base pairs) of the M-MuLV LTR (Mo + SV) and also into a deleted form of the LTR which lacks the M-MuLV enhancer sequences (delta Mo + SV). These chimeric LTRs were used to generate infectious M-MuLVs by transfection of corresponding proviral plasmids into mouse fibroblasts. The relative infectivities of Mo + SV and delta Mo + SV recombinant viruses as determined by rat XC cell plaque assay and reverse transcriptase assay were 60 to 70% of wild-type M-MuLV levels. To study the pathogenicity of these two recombinant viruses, we inoculated newborn NIH Swiss mice with either Mo + SV or delta Mo + SV M-MuLV. Both viruses induced disease more slowly than M-MuLV, which induces disease 2 to 4 months postinoculation. Mo + SV M-MuLV-inoculated animals became moribund at 3 to 13 months postinoculation, whereas delta Mo + SV M-MuLV-inoculated animals became moribund at 6 to 24 months postinoculation. The tumors induced by the two viruses were characterized histologically and molecularly. Mo + SV M-MuLV-induced tumors were primarily T-cell-derived lymphoblastic lymphomas containing extensive rearrangements of the T-cell receptor beta gene. In contrast, delta Mo + SV M-MuLV induced pre-B- and B-cell lymphoblastic lymphomas, B-cell-derived follicular-center cell lymphomas, and acute myeloid leukemia. The delta Mo + SV tumor DNAs from B-lineage tumors were typically rearranged at the immunoglobulin gene loci and contained germ line configurations of the T-cell receptor beta gene. Southern blot hybridization confirmed that the tumor DNAs contained the predicted Mo + SV M-MuLV or delta Mo + SV M-MuLV provirus.

Detection of human immunodeficiency virus by antigen capture ELISA in culture fluids and blood specimens

A newly developed HIV p24 antigen capture ELISA was compared with the reverse transcriptase assay (RT) for the detection of human immunodeficiency virus (HIV) in culture fluids of virus-infected lymphocytes. The comparison was made by testing 350 culture fluids derived from lymphocyte cultures of 88 patients and collected 7, 14, 21 and 28 days after co-cultivation with lymphocytes from healthy donors. For 87 out of 88 patients there was agreement in the results of the two methods (98·9%) but the antigen capture assay gave positive results 7–14 days earlier than the RT assay with culture fluid specimens from five patients; specimens from only one patient gave discrepant results with a constantly negative RT activity but positive HIV p24 antigen detection at the second and third passage in culture. We also investigated the ability of the antigen capture assay to detect the antigen directly in 246 clinical specimens (including serum, plasma, lymphocytes and cerebrospinal fluid) from 100 seropositive patients, and found that 16% of the patients were positive. These and the former results compared well with those obtained using commercial antigen capture assays. A similar percentage of positivity (16·2%) was observed in a panel of 37 serum specimens from seropositive patients whose antibody response pattern was evaluated by Western blot analysis. Besides, a higher percentage of positivity (66·6%) was observed in a panel of 12 plasma specimens from hemophiliac children for whom virus isolation was also performed.

Expression in Escherichia coli of a Moloney murine leukemia virus reverse transcriptase whose structure closely resembles the viral enzyme

We have constructed an expression plasmid containing the portion of the Moloney murine leukemia virus genome encoding the reverse transcriptase (RT). When introduced into Escherichia coli this plasmid induces the synthesis of a 70-kDa protein. The RT made in E. coli differs from the viral protein only in that there are two new amino acids, methionine and glycine, substituted for the threonine found at the N terminus of the viral enzyme. Approximately half of the E. coli synthesized RT enzyme is soluble in cell extracts. This protein is active in an RT assay, and like the enzyme purified from virions, is more active in the presence of Mn 2+ than Mg 2+. We have also constructed a plasmid that induces the synthesis of an RT-integration protein fusion.

A comparison of reverse transcriptase and antigen capture assays for the detection of HIV

HIV antigen capture assay (ELISA) and reverse transcriptase assay were compared for the detection of HIV. Purified virus and the supernatant of infected cells were used for this study. Antigen capture assay was found to be at least one hundred times more sensitive than the RT assay, and for a large number of samples easier to perform. This assay would be useful for clinical and laboratory diagnosis.

Interaction between rifabutin and human immunodeficiency virus type 1: inhibition of replication, cytopathic effect, and reverse transcriptase in vitro

Rifabutin (ansamycin; LM427), a semisynthetic rifamycin derivative, inhibits the replication of human immunodeficiency virus type 1, the virus etiologically linked to acquired immunodeficiency syndrome. The inhibition of virus production was observed in human immunodeficiency virus type 1-infected peripheral blood mononuclear cells both by reverse transcriptase assay and virus-antigen assay. In addition, rifabutin effectively reduced human immunodeficiency virus type 1-associated cytopathic effect to OKT4-positive cells. Virion-associated reverse transcriptase was also markedly inhibited by rifabutin. The inhibitory dose of rifabutin was nontoxic to lymphoid cells, which further suggests that it might serve as a useful agent in the therapeutic modalities of acquired immunodeficiency syndrome.