Reverse Transcriptase Activity Assay Research Articles

A new method for measuring reverse transcriptase activity by ELISA

A new and sensitive assay of reverse transcriptase (RT) activity of retroviruses measures the incorporation of digoxigenin-labelled dUTP in newly synthesized DNA instead of radioactively labelled ( 3H- or 32P-)dTTP. To avoid difficulties associated with separation of non-incorporated nucleotides from the newly synthesized DNA, biotin-labelled dUTP is added to the reaction mixture in very low concentrations. After reverse transcription, the newly synthesized, doubly labelled DNA is immobilized on streptavidin-coated ELISA wells and evaluated photometrically by binding of peroxidase-conjugated anti-digoxigenin-antibodies (sheep) and subsequent colour development with 2,2'-azino-di[3-ethylbenzthiazolin-sulfonate(6)] (ABTS R) as substrate. For better standardization, it is suggested that RT activity is given in units (one unit of RT is the amount of enzyme incorporating one nanomole of labelled dNTP in 10 min at 37°C into an acid precipitable DNA) rather than in cpm (counts per minute). The method is specific and easy to perform.

Porcine retrovirus: optimal conditions for its biochemical detection.

The assay of reverse transcriptase (RT) activity was used to detect the presence of retrovirus in porcine cells. A set of optimal assay conditions was determined to design a sensitive, quantitative and reproducible RT assay for porcine systems. The template-primer poly(rA).oligo(dT) was an absolute requirement. The presence of Mn++ was indispensable, with an optimal concentration of 0.25 mM. Monocations (K+, Na+) at 50 mM greatly enhanced, but their high doses inhibited the reaction. The pH of the medium influenced very much the reaction, especially with non-purified virus samples, with which the RT activity was inhibited at pHs above 8.2. Non-ionic detergents at 1% enhanced several-fold the RT activity. It was also shown that porcine retrovirus could be spontaneously reactivated in porcine cell lines by in vitro long-term propagation and transmitted to pigs by inoculation with virus-producing cells.

HIV reverse transcriptase inhibiting antibodies detected by a new technique: Relation to p24 and gp41 antibodies, HIV antigenemia and clinical variables

A new assay for HIV reverse transcriptase activity inhibiting antibodies (RTI-ab) was used for the analysis of a large collection of sera sampled before and after confirmation of HIV infection. In this assay HIV-RT was preincubated with diluted serum, after which residual RT activity was determined by a technique using a template coupled to macrobeads and 125I-lodo-deoxyuridine-triphosphate as the tracer-substrate. Of the 936 sera analysed, 818 were found positive for RTI-ab, and 824 were positive in Western blot (Wb). The prevalence of RTI-ab compared to Wb was therefore 99.3%. The corresponding figure for 930 sera analysed for envelope-ab, i.e., gp41-ab, was 823 positive, and of these 930 sera 815 were Wb positive, giving a comparative prevalence of 101%. In contrast, only 678 samples of 993 analyzed for core ab, i.e., p24, were positive, giving a prevalence of 77.0% as 880 of these samples were Wb positive. Thus, RTI-ab was as prevalent as gp41-ab, and although the analyses of RTI-ab amounts in different stages showed decreasing levels in stage IV compared to stages II or III, all of the sera except 1 were found positive in stages III and IV. Further, it was found that both the few RTI-ab negative samples in stage II and the few RTI-ab positive samples among Wb negative sera were sampled in connection with seroconversion. The specificity of the RTI-ab assay was 100% in a test of 200 serum samples from HIV negative blood donors. It was concluded that RTI-ab analyses can be made highly sensitive and specific and useful for studies of HIV infection.

Histocompatible miniature boar model: Selection of transformed cell lines of b and t lineages producing retrovirus

A lymphoblastoid cell line (B1) was isolated in culture following a brief exposure to 5-azacytidine from peripheral-blood mononuclear cells of a boar previously injected with cells (Shimozuma) producing porcine retrovirus (Tsukuba-1) and suffering a severe non-neoplastic syndrome at autopsy. B1 cell line and 5 of its sublines were propagated for more than 100 generations, retaining doubling times comprised between 16.8 and 27.5 hr and growing readily in agarose or agar (plating efficiency: 5 to 50%). Karyotype analyses showed that 4 sublines were nearly diploid, except for cells of L14, which displayed a monosomy affecting chromosome 18 pair. Two sublines (L35 and L45) were considered as being of T-cell lineage, since MSA, antigen was observed on the surface of approximately 30% of cells. Three sublines (L23, L14 and L52) were considered of B-cell lineage, since membrane immunoglobulins were observed on the cell surface. In addition, sublines L23 and L52 were actively secreting immunoglobulin of mu isotype. Retrovirus particles were evidenced in gradient-purified preparation of 200-fold-concentrated cell culture supernatants of the B1 cell line, L14, L35 and L52 sublines, using both a reverse transcriptase activity assay and electron microscopic observation. These cell lines can be used to select for porcine retrovirus variants with transforming potential for lymphocytes of B and T lineages.

Quantification of feline immunodeficiency virus in a newly established feline T-lymphoblastoid cell line (MYA-1 cells)

A quantitative method for analysis of feline immunodeficiency virus (FIV) was established by the indirect immunofluorescence (IF) test using MYA-1 cells infected with the serially diluted viruses. When we compared this method with the reverse transcriptase (RT) activity assay, the quantitative analysis by the indirect IF test was more sensitive than that by RT activity assay. The relationship between TCID50 measured by the indirect IF test and RT activity was linear. However, the RT activity assay was not suitable for measurement of low amounts of FIV in the supernatants of FIV-infected cultures.

Immunoassay for detection of feline immunodeficiency virus core antigen

The feline immunodeficiency virus (FIV) is a recently identified feline lentivirus that has been found at significant levels in domestic cat populations worldwide. A microdilution plate format, monoclonal antibody-based enzyme-linked immunosorbent assay was developed for the detection of the FIV group-associated antigen (gag) designated p24. Assays of serially diluted samples containing disrupted virus showed that the assay had a sensitivity limit of approximately 0.2 ng/ml for FIV p24. The assay was approximately eightfold more sensitive than the assay for viral reverse transcriptase activity when it was tested with diluted tissue culture samples. A qualitative confirmation assay by standard antibody inhibition techniques was coupled to the screening test methodology. The test was used to detect and confirm the presence of virus in cultured feline lymphocytes from infected animals.

Non-productive chronic infection of human cells by a xenotropic murine type-C retrovirus

Summary We report the establishment and characterization of a non-productive (NP) chronic infection of a human cell line (FL) by a xenotropic murine type-C retrovirus from Mus molossinus mouse (Mol-MuLV-X). The NP chronically infected cells (FL/Mol) do not show any virus particles detectable by electron microscopy, nor do they release any virus detectable either by reverse transcriptase activity assay or by inoculation of culture supernatants onto permissive cells. Most of the FL cells are shown to be infected, as demonstrated by specific immunofluorescence. The totality of provirus DNA genetic information is present, as demonstrated by specific molecular hybridization, and infectious Mol-MuLV-X virions can be rescued from co-cultures with permissive cells after fusion with polyethylene glycol. The integrated provirus DNA is transcribed, and both main species (24S and 35S) of viral RNA are synthesized. Antigenic determinants of both virus p30gag and gp70env proteins are easily detected. Polyacrylamide gel electrophoresis analysis of immunoprecipitated cell lysates of NP FL/Mol cells did not show any anomaly in the synthesis of the virus p30gag protein and its precursors. Anti-gp70 immune serum precipitated the 85 kd gp70 precursor and a gp70 which migrated slightly faster than that found in productively infected human cell lines.

Isolation and properties of Moloney murine leukemia virus mutants: use of a rapid assay for release of virion reverse transcriptase.

A rapid assay for retroviral reverse transcriptase activity released into the culture medium by infected cells was developed. With the assay, 4,000 clonally infected cell lines could be tested in a few hours. We have adapted the assay for use as a screen for the detection of spontaneous viral mutants. Mutants of Moloney murine leukemia virus have been isolated which (i) produce a thermolabile reverse transcriptase, (ii) are temperature sensitive for release of enzyme activity, or (iii) can only productively infect cells already producing gag-related polypeptides. The assay has also been useful for the isolation of nonproducer cells infected with various replication-defective transforming viruses.

Transfection of XC plaques with DNA from murine leukemia virus producer cells

Transfection of leukemia virus resulting in virus production has been accomplished in the murine system using cellular DNA containing a vertically established leukemia virus. Virus production was detected in a bioassay by XC plaque formation and in a biochemical assay for reverse transcriptase activity. Infection by DNA was successful using the Calcium method but not with the DEAE dextran method. XC plaques were observed as early as the first subculture of transfected cells.