Journal of Clinical Microbiology, June 1999, p. 1704-1708, Vol. 37, No. 6
0095-1137/99/$04.00+0
Improved Mg2+-Based Reverse
Transcriptase Assay for Detection of Primate Retroviruses
Johnna F.
Sears,1
Roy
Repaske,2,
and
Arifa S.
Khan1,*
Laboratory of Retrovirus Research, Division
of Viral Products, Center for Biologics Evaluation and Research,
U.S. Food and Drug Administration,1 and
Laboratory of Molecular Microbiology, National Institute of
Allergy and Infectious Diseases,2 Bethesda,
Maryland 20892
Received 30 December 1998/Accepted 22 February 1999
 |
ABSTRACT |
The reverse transcriptase (RT) assay is a simple, relatively
inexpensive, widely used assay that can detect all retroviruses (known
and novel retroviruses as well as infectious and defective retroviruses) on the basis of the divalent cation requirement of their
RT enzyme, i.e., Mg2+ or Mn2+. Descriptions of
various RT assays have been published; however, they cannot be directly
applied to the analysis of biological products or clinical samples
without further standardization to determine the lower limit of virus
detection (sensitivity), assay variability (reproducibility), or
ability to detect different retroviruses (specificity). We describe the
detection of type E and type D primate retroviruses, which may be
pathogenic for humans, by a new 32P-based,
Mg2+-containing RT assay. The results show that the
sensitivity of detection is <3.2 50% tissue culture infective doses
(TCID50s) for human immunodeficiency virus type 1 (HIV-1)
and <1 TCID50 for simian immunodeficiency virus isolated
from a rhesus macaque (SIVmac). Analysis of recombinant
HIV-1 RT enzyme indicated that 10
5 U, which is equivalent
to 4.25 × 104 virions, could be detected.
Additionally, genetically distinct type D retroviruses such as simian
AIDS retrovirus and squirrel monkey retrovirus were also detected in
the assay with similar sensitivities. Thus, the improved RT assay can
be used to detect genetically divergent Mg2+-dependent
retroviruses of human and simian origin that can infect human cells and
that therefore pose a potential health risk to humans.
| |
INTRODUCTION |
All retroviruses can be divided into
two categories on the basis of the presence of an Mg2+- or
Mn2+-requiring RNA-dependent DNA polymerase that is termed
reverse transcriptase (RT) (1, 22) and that is critical in
the retroviral life cycle (23, 24). Each group includes
retroviruses of diverse origins that are structurally distinct and
genetically divergent but that share similar cation requirements for
their RT activity. For example, different retrovirus types
(21) such as avian type C retroviruses, primate type D
retroviruses, and primate type E lentiviruses, which includes human
immunodeficiency virus (HIV) type 1 (HIV-1), can be grouped together on
the basis of the presence of an Mg2+-requiring RT in these
viruses. Thus, the detection of RT activity can generally indicate the
presence of a retrovirus in the absence of specific information
regarding its genome or protein content. Although RT assays generally
detect about 105 to 106 virus particles and are
not as sensitive as infectivity or PCR assays, they are broadly
reactive and have been used for the detection and isolation of
different types of novel retroviruses including HIV-1 (2,
6). In addition, RT assays are routinely used in infectivity
studies for the rapid and easy monitoring of retrovirus infection and
replication. The detection of small amounts of retrovirus by the RT
assay may be made possible by virus amplification in a susceptible cell
line or by increasing the virus concentration in a sample so that it is
above the detection limit of the assay, e.g., by centrifugation. The RT
assay is also widely used for analysis of potential retroviral
contaminants in biological products, which may be introduced during
passage through animals, during propagation in cell substrates, or from
biological raw materials used in production (12).
Several Mg2+-based RT assays have been developed; however,
the viruses used in most of the studies have been avian myeloblastosis virus or HIV-1. Furthermore, the previous studies describe details regarding assay development; however, there is little information about
assay standardization, including sensitivity of virus detection, ability to detect different retroviruses, or assay variability. Such
information is especially important when an RT assay is used to
demonstrate the absence of retroviral contaminants in biological products and in analyses of clinical samples from potentially infected
individuals. Current HIV-1 RT assays have been used in infectivity
studies, neutralization assays, and assessments of antiviral effects.
The original assays used [3H]deoxynucleoside
triphosphates to extend the oligonucleotide primer to produce the cDNA
copy of the homopolymer template (2, 6, 10). Modifications
of the RT assays have been made to increase the sensitivity of virus
detection which include the use of 32P- and
125I-radiolabeled nucleotide substrates (7, 25).
Additionally, RT assays with increased sensitivities have been
developed with nonisotopically labeled nucleotides; however, this was
achieved after a prolonged incubation, i.e., 15 to 24 h (3,
5, 20). In this paper we describe the standardization of a new
32P-based RT cocktail, with a 2-h incubation period, for
the general detection of retroviruses that contain
Mg2+-requiring RT, including type E lentiviruses (e.g.,
HIV-1 and simian immunodeficiency virus [SIV]) as well as two
distinct type D retroviruses, i.e., simian AIDS retrovirus (SRV)
(4, 16) and squirrel monkey retrovirus (SMRV)
(8).
| |
MATERIALS AND METHODS |
RT assays.
The new RT cocktail contains the following: 6 mM
MgCl2, 3 µg of poly(A) (P-L Biochemicals Inc. Milwaukee,
Wis.) per ml, 0.021 µg of p(dT)12-18 (Pharmacia Biotech,
Piscataway, N.J.) per ml, 0.12% Nonidet P-40, 24 mM triethanolamine
(TEA; U.S. Biochemical Corp., Cleveland, Ohio), and 28.8 mM EGTA (U.S.
Biochemical Corp.). TEA-EGTA was formulated separately by mixing 3.93 ml of TEA and 13.146 g of EGTA in a final volume of 300 ml (pH 8.0).
This cocktail was aliquoted and stored at 
20°C such that it would
undergo only two freeze-thaws prior to its use. Additionally, the
cocktail was thawed on ice or quickly at 37°C and was immediately put
on ice. Under these handling conditions the cocktail was found to remain stable for at least a year, as determined by the RT activity of
a standard control virus. Just prior to use of the cocktail, the
following were added per milliliter of chilled cocktail: 4 µl of 1 M
dithiothreitol, 3 µl of [
-32P]dTTP (1.5 µCi; >400
Ci/mmol; Amersham Corp., Arlington Heights, Ill.), and 1 µl of
104 M dTTP. The dithiothreitol and nonradioactive dTTP
solutions were also aliquoted individually and stored at 20°C to
avoid additional freezing and thawing. Ten microliters of test sample was incubated with 50 µl of the RT cocktail in a screw-cap, 1.5-ml conical polypropylene tube (Starstedt, Arlington, Tex.) for 2 h in
a 37°C water bath. Five microliters of the reaction mixture was
spotted, in duplicate, onto DE81 paper (Whatman, Maidstone, United
Kingdom), air dried, and washed at room temperature on a rocker in 2×
SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) four times
each for 5 min and two times each for 1 min with 95% ethanol. The
paper was air dried and exposed overnight at 80°C to X-OMAT AR film
(Kodak, Rochester, N.Y.). The next day the spots were cut out and
counted in a scintillation counter.
In all the experiments described in this paper, the radioisotope was
used prior to or on the manufacturer's reference date. Dilutions of
viruses and enzyme were prepared as described below and were
immediately used for RT analyses. The negative control was complete
medium (described below).
Viruses.
HIV-1 was prepared in human peripheral blood
mononuclear cells (PBMCs) by using strain LAI, which had been grown in
CEM clone 12D7 cells (obtained as strain LAV.04 from
M. A. Martin, National Institute of Allergy and Infectious
Diseases). For virus titration, human PBMCs (106 cells/ml)
were stimulated with phytohemagglutinin (PHA; Murex Diagnostics,
Dartford, United Kingdom), which was used at a final concentration of
250 ng/ml in complete RPMI 1640 medium containing 10% fetal bovine
serum (Hyclone, Logan, Utah), 10 mM HEPES (Biofluids, Rockville Md.),
250 U of penicillin per ml, 250 µg of streptomycin per ml, and 2 mM
L-glutamine (Life Technologies, Gibco BRL, Grand Island,
N.Y.). After 3 days, the PHA-containing medium was replaced with medium
containing 10% interleukin-2 (Hemagen Diagnostics, Columbia, Md.), and
the cells were resuspended at a final concentration of 106
cells/0.1 ml. Virus infection was set up in a 24-well plate: 0.1 ml of
101 to 106 serial dilutions of virus were
initially incubated for 1 h at 37°C with 0.1 ml of
106 PBMCs. After 1 h, 1.8 ml of medium was added and
the cells were cultured in a 5% CO2 atmosphere at 37°C.
After 3 to 4 days, half of the medium was removed and was replaced with
fresh medium. On day 7 postinfection, the supernatant was filtered
through a 0.45-µm-pore-size filter unit (Spin-X Centrifuge Tube
Filters; Costar, Cambridge, Mass.) and collected for the RT assay. The virus infections were set up in quadruplicate. The virus titer was
calculated as described by Reed and Muench (18) and was expressed as the 50% tissue culture infectious dose
(TCID50) per milliliter. The titer of the HIV-1 stock was
104.5 TCID50s/ml in human PBMCs.
SIVmac was obtained by ligation of cloned 5' and 3' DNA
fragments of SIVmac-mm239 (11) and transfection
in 174× CEM cells. The virus obtained was propagated in rhesus monkey
PBMCs to produce a large-scale virus stock (6a). The titer
of the monkey-grown SIVmac stock was determined as
described above for HIV-1, except that the monkey PBMCs were stimulated
with 500 ng of PHA per ml. The titer of SIVmac239 at
passage 1 in autologous monkey PBMCs was determined as
104.0 TCID50s/ml.
For RT assay analysis of HIV-1 and SIV, each virus was serially diluted
in complete RPMI 1640 medium. At least two independently prepared
dilutions were assayed in at least two separate RT assays.
SRV type 1 (SRV-1) and SMRV were purchased from Advanced
Biotechnologies Inc. (Columbia, Md.) and were at 8.26 × 107 and 1.92 × 1011 virus particles/ml,
respectively, on the basis of electron microscopy. Serial dilutions of
the viruses were made in complete RPMI 1640 medium. For each virus, one
dilution series was used and the RT assay was done in duplicate.
RT enzyme.
Recombinant HIV-1 RT was purchased from
Worthington Biochemical Corporation (Freehold, N.J.; supplied as 48.7 U/µl). Serial dilutions of the enzyme were made in complete medium
and were used immediately in RT assays.
| |
RESULTS |
The linear range of the RT reaction was determined by
assaying the RT activity at various times ranging from 30 to 180 min (Fig. 1). Independent RT reactions with
10 µl of undiluted HIV-1 and SIVmac were set up for each
time point as described in Materials and Methods. Each reaction was
terminated at the times indicated in Fig. 1 by spotting in duplicate 5 µl of the reaction mixture onto DE81 filter paper. All the reaction
mixtures were spotted on a single filter paper until the reaction
mixture from last time point was spotted. The paper was then washed as
described in Materials and Methods. The results indicated that the RT
activities for HIV and SIV were directly proportional to the reaction
times up to 180 min. The rate of dTTP incorporation was found to be directly proportional to the virus concentration as well as to the
amount of [32P]dTTP used in the assay (data not shown).
The RT activity increased with the amount of [32P]dTTP
with 1 to 10 µl (0.5 to 5 µCi) of the radiolabeled deoxynucleoside triphosphate.
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FIG. 1.
Linear range of the RT assay. The linearity of the RT
assay was determined by HIV-1LAI and
SIVmac-mm239. Ten microliters of undiluted virus was
assayed at the indicated time intervals. The RT activity is indicated.
The mean ± standard deviation from one experiment is indicated.
 , HIV-1;  , SIV;  , medium.
|
|
The sensitivity of detection of type E lentiviruses in the RT assay was
determined with HIV-1 and SIV. The results are presented in Table
1. Ten microliters of undiluted virus
(first sample) and serial dilutions were assayed for RT activity. The
sensitivity of detection of HIV-1 was <3.2 TCID50s. A
similar sensitivity of detection was seen with a different,
independently prepared HIV-1 stock (13). The sensitivity of
detection of SIVmac was <1 TCID50.
Furthermore, in repeated, parallel experiments, the sensitivity of
HIV-1 and SIV detection was 10-fold greater by the new RT assay than by
a previously published assay (25). In a comparison of the
new RT assay with another HIV-1 RT assay published by Hoffman et al.
(10), sevenfold greater RT activity was detected when two
HIV-1 concentrations were tested with the new cocktail
(18a). To further assess assay sensitivity, RT analysis was
done with serial dilutions of recombinant HIV-1 RT. Similar results
were obtained with two independently prepared dilutions. The results of
RT analysis of one dilution series, which was assayed in duplicate, are
presented in Fig. 2. The sensitivity of detection was 105 U of HIV-1 RT
enzyme.
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FIG. 2.
Detection of recombinant HIV-1 RT enzyme. Serial
dilutions of HIV-1 RT enzyme were analyzed by the RT assay. The RT
activity is indicated. The mean ± standard deviation was
calculated for four spots obtained from two RT assays which were done
in duplicate with one dilution series.
|
|
The ability of the new RT assay to detect other retroviruses
containing an Mg2+-requiring RT enzyme, such as type D
retroviruses, was assessed with SRV and SMRV. In this case the
sensitivity of detection was based upon the use of virus stocks with
known numbers of particles, as determined by electron microscopy, and
therefore may be less accurate than when the HIV-1 and SIV stocks were
used, in which case the number of infectious particles was determined
on the basis of an infectivity assay. The results presented in Table 2 indicate that about 2 × 105 virions of SMRV were detected and that about 8 × 104 virions of SRV were detected.
It should be noted that because 32P was used in the assay,
the results could be monitored by both scintillation counting and autoradiography. A parallel analysis was done in all cases; it was
found that a weakly positive signal was easier to visualize from the
autoradiogram. On the basis of autoradiography, a result was positive
if the counts were 50% above the background RT activity.
| |
DISCUSSION |
The RT assay is widely used for the general detection of
known and novel retroviruses. This is primarily because it is easy, quick, and relatively inexpensive to perform. Recently, highly sensitive PCR-based RT assays which can detect 3 to 100 virions have
been developed (9, 17, 19); however, their use at this time
is limited because the assay is technically demanding and expensive.
Thus, parallel efforts have continued to increase the sensitivity of
retrovirus detection by the conventional RT assays.
In this paper, we describe the standardization of an improved
Mg2+-based RT assay which can detect different types of
primate retroviruses. The sensitivity of the assay was determined to be
<1 TCID50 of SIVmac and <3.2
TCID50s of HIV-1. Analysis of the recombinant HIV-1 RT
enzyme indicated the detection of 105 U, which, on the
basis of the molecular weight (117,000), was equivalent to 3.4 × 106 molecules of RT. Since it is reported that 80 molecules
of RT are present per HIV-1 particle (15), the sensitivity
of detection was calculated to be <4.25 × 104
virions. These results thus indicate a possible 1:10,000 ratio of
infectious virus to total particles, which is consistent with previously reported results (14). In addition to primate
lentiviruses, type D retroviruses of genetically diverse origins were
detected at about 8 × 104 virions of SRV, which was
isolated from an Old World monkey, and about 2 × 105
virions of SMRV, which was isolated from a New World monkey.
The sensitivity of detection was achieved with a relatively short
incubation time (2 h), in contrast to some other RT assays that require
prolonged incubation times (i.e., 15 to 24 h) for increased
detection (5, 20). The sensitivity of the new RT assay for
HIV and SIV detection can be further improved by increasing the
incubation time to up to 3 h and/or by increasing the amount of
[32P]dTTP. Conversely, with samples containing adequate
amounts of virus, a minimum amount of radiolabeled dTTP and/or a
reduced reaction time can be used, since the counts obtained are
directly proportional to the incubation time and to the
[32P]dTTP concentration.
The results obtained with the new RT assay were highly reproducible;
similar results were obtained with regard to the sensitivity of
detection of HIV and SIV when independently prepared virus stocks or
dilutions were used. However, some variability in the counts per minute
incorporated can occur between different assays, especially due to
handling, e.g., when the sample or the radioisotope is pipetted or when
the final reaction mixture is spotted onto the filter paper. We have
reduced interassay variability by using designated and accurately
calibrated pipetting devices for the different handling procedures. One
caveat of the new RT assay is that if the reaction is done in a
CO2 incubator, screw-cap tubes must be used to avoid
lowering of the pH in the reaction, which suppresses the RT activity
(18b).
The RT assay has been used for the detection of known and novel
retroviruses from infected cells both in assessing the replication of
viruses and for evaluating antiretroviral treatments (e.g., in the case
of treatments for HIV-1 infection). We have used the RT assay described
in this paper as a general detection strategy in a multicombinational
analysis with specific detection strategies such as DNA and RNA PCR
assays to demonstrate the absence of detectable HIV or SIV in several
monovalent lots of oral, poliovirus vaccine (13). The new RT
assay was especially useful because of its increased sensitivity for
the detection of retroviral RT compared to those of other, similar
assays and because of its low background signal.
| |
ACKNOWLEDGMENTS |
The following reagents were obtained through the AIDS
Research and Reference Reagent Program, Division of AIDS, National
Institute of Allergy and Infectious Diseases: p239SpE3' and p239SpSp5'
cloned DNAs (from Ronald Desrosiers). We thank Teresa A. Galvin for
preparing the titered SIVmac-mm239 stock, S. Tabriz Ali for
preparing the titered HIV-1 stock, and Theodore Bryan for technical
assistance. We also acknowledge Malcolm A. Martin for support in
formulation of the RT cocktail and Keith Peden, Hana Golding, and
Muhammad Shahabuddin for review of the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratory of
Retrovirus Research, Division of Viral Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, 1401 Rockville Pike, HFM-454, Rockville, MD 20852-1448. Phone: (301) 827-0791. Fax: (301) 496-1810. E-mail: khan{at}cber.fda.gov.
Retired.
| |
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Journal of Clinical Microbiology, June 1999, p. 1704-1708, Vol. 37, No. 6
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Abstract
Introduction
