Vol. 64, No.

11

JOURNAL OF VIROLOGY, Nov. 1990, p. 5290-5294

0022-538X/90/115290-05$02.00/0
Copyright C 1990, American Society for Microbiology

Detection of Human T-Cell Leukemia Virus Type I (HTLV-I)

Provirus in an Infected Cell Line and in Peripheral Mononuclear
Cells of Blood Donors by the Nested Double Polymerase Chain
Reaction Method: Comparison with HTLV-I Antibody Tests
CHIEKO MATSUMOTO,* SHIGEKI MITSUNAGA, TAKASHI OGUCHI, YOSHITADA MITOMI,
TOORU SHIMADA, AKIKO ICHIKAWA, JUNNOSUKE WATANABE, AND KUSUYA NISHIOKA
The Japanese Red Cross Central Blood Center, Hiroo 4-1-31, Shibuya-ku, Tokyo 150, Japan
Received 30 May 1990/Accepted 27 July 1990

Human T-cell leukemia virus type I (HTLV-I) provirus DNA from the cultured cell line HUT 102 and from
peripheral mononuclear cells (PBMC) of anti-HTLV-I antibody-positive Japanese blood donors was detected
by the nested double polymerase chain reaction (PCR) method. This procedure consists of a first amplification
and a second amplification with the products of the first amplification and primers interior to the first primers.
Using this method, we demonstrated that it is possible to detect single-template DNA. Polyacrylamide gel
electrophoresis of the nested double PCR products, with our primers, revealed three bands with excess
amounts of template DNA, two bands with moderate amounts, and a single band with limited amounts. The
amount of provirus in PBMC was roughly estimated from the results of the nested double PCR. Particle
agglutination (PA) assays and indirect immunofluorescence testing (IF) with mixed MT-2 cells and Molt-4 cells
as targets to detect anti-HTLV-I antibody were performed, and the results were compared with those of the
nested double PCR of the pX region. None of the 101 PA-negative samples were positive in either the IF or PCR
test. Of the 155 samples that were antibody positive by the PA assay, 57 were positive by both PCR and IF.
Furthermore, the results of the IF and PCR tests coincided completely. It was therefore concluded that the IF
method is most appropriate for confirmation of the PA assay currently used in most diagnostic laboratories and
blood centers.

The results of PCR detection of the HTLV-I provirus in

peripheral mononuclear cells (PBMC) obtained from donated blood are compared with the results of antibody
testing of sera by the PA and IF methods in this report.

To prevent posttransfusion infection by human T-cell

leukemia virus type I (HTLV-I), anti-HTLV-I antibody has
been used since 1986 to screen donated blood in all Japanese
Red Cross blood centers in a particle agglutination (PA)
assay. However, some samples were judged to be antibody
negative by indirect immunofluorescence testing (IF) in spite
of a positive PA assay (11, 29), particularly low-titer sera.
Therefore, it was necessary to find some means of determining the presence of HTLV-I in such sera. Accordingly, we
attempted to detect HTLV-I provirus DNA by the polymerase chain reaction (PCR) directly.
PCR was first reported by Mullis et al. (8) and Saiki et al.
(23), and since then many modifications and applications
have been described (2, 6, 15, 25). This method has been
used for detection of viral genomes of human immunodeficiency virus (20), cytomegalovirus (8), hepatitis B virus (13),
and HTLV-I (1, 4, 22).
We adopted the nested double PCR method, in which two
amplifications are used under nonradioactive conditions (18,
19). We began by amplifying a DNA sample with Thermus
aquaticus polymerase and one pair of primers. Next, a
portion of the products was amplified again with another pair
of primers that were located inside the first pair. After the
second amplification, almost all nonspecific background
observed at the first amplification disappeared, and the
desired product was confirmed by using the inner primers.
Finally, the amplified products, even if they had originated
from a very small number of templates, were visualized by
polyacrylamide gel electrophoresis (PAGE) stained with
ethidium bromide.

MATERIALS AND METHODS

Synthesis of oligonucleotides. Oligonucleotides were synthesized on an Applied Biosystems 381A DNA synthesizer
by the phosphoramidate method and purified with oligonucleotide purification cartridges (Applied Biosystems Inc.).
Source and isolation of DNA. DNA from two human T-cell
lines or PBMC was isolated by the phenol-chloroform
method. One of the cell lines, CEM, was HTLV-I noninfected, and the other, HUT 102, was HTLV-I infected (21).
PBMC were obtained from voluntary blood donors in our
blood center.
Nested double PCR. The pX region of the HTLV-I genome
was amplified as shown in Fig. 1 by using DNA sequence
information from Seiki et al. (24). The first amplification was
carried out with primers 1 (5'-AGGGTTTGGACAG
AGTCTT-3') and 2 (5'-AAGGACCTTGAGGGTCTTAG3'). The second amplification was carried out with primers 3
(5'-CTTTTCGGATACCCAGTCTAC-3') and 4 (5'-GGTTC
TCTGGGTGGGGAAGGAG-3') and 10 ,ul of the first-amplification products as template DNA. All reactions were
performed in a volume of 100 ,ul containing 50 pmol of each
primer, 50 mM Tris hydrochloride (pH 8.8), 10 mM MgCl2,
10 mM (NH4)2SO4, template DNA, 2.5 U of T. aquaticus
polymerase (Perkin-Elmer Cetus), and 1.5 mM each dATP,
dCTP, dGTP, and dTTP. Reactions were carried out for 30
cycles at an annealing temperature of 60C for 1 min, a
polymerization temperature of 72C for 2 min, and a heat

* Corresponding author.
5290

VOL. 64, 1990

NESTED DOUBLE PCR TO DETECT HTLV-I PROVIRUS

73312

7567
*a

a a -

primer 1

-job,
I.
primer 2
(731 2-7330)
ner 3 :3 ~qwl L
(7548-7567)
prirrner 3= -hprimer 4
(733 1-7 '351);
(7525-7546)

216
235
237

I_

"

256

FIG. 1. Amplification of the HTLV-I pX region by the nested

double PCR method. The first amplification was carried out with
primers 1 and 2. A portion of the amplified product was amplified
again with primers 3 and 4. The distance between primers is shown
(in bases).

denaturation temperature of 94C for 1 min on a PerkinElmer Cetus DNA thermal cycler.
Analysis of the PCR products. A portion (4 IL) from each of
the completed PCR reactions was mixed with 4 ,ul of loading
buffer and subjected to PAGE on 5% polyacrylamide gels.
PAGE was performed in Tris-borate buffer (pH 8.0). After
completion of PAGE, the gel was stained with ethidium
bromide and photographed under UV transillumination.
Detection of anti-HTLV-I antibody. (i) PA assay. A PA
assay was used for mass screening for anti-HTLV-I antibodies in sera from donors from all Red Cross blood centers in
Japan. A Serodia-ATLA kit (Fujirebio Inc., Tokyo, Japan)
was used. A final serum dilution of 1:16 or higher that caused
agglutination of the antigen-coated particle was considered
positive.
(ii) IF test. IF was performed by the method of Hinuma et

256

al. (7) with some modifications, with mixed targets of

HTLV-I-infected and noninfected cells. Mixtures of carefully maintained MT-2 cells, an HTLV-I-infected T-cell line
derived from cord blood cells which were cocultured with
T-cells bearing adult T-cell leukemia (17), and Molt-4 cells,
used as a noninfected human T-cell line, were fixed with cold
acetone and used as antibody target cells. The ratio of MT-2
cells to Molt-4 cells was 1:3. Fixed cells were incubated with
serum samples for 30 min at 37C, and after the cells were
washed with phosphate-buffered saline, pH 7.2, fluorescein
isothiocyanate-conjugated rabbit anti-human immunoglobulin G was added. Incubation was continued for 30 min at
37C, followed by washing with phosphate-buffered saline
and water. Stained cells were observed under a fluorescence
microscope. When only MT-2 cells, i.e., one-fourth of the
cell population, were stained, this result was judged to be IF
positive. When both MT-2 cells and Molt-4 cells were
stained, this was judged to be nonspecific.
RESULTS
Detection of HTLV-I genome in HUT 102 cell DNA. PAGE
patterns of the product of the first 30 cycles of PCR with
primers 1 and 2, expected to be 256 bp in length, are shown
in Fig. 2a. A band corresponding to 256 bp was obtained with
130 pg of HUT 102 cell DNA. Nonspecific background was
observed in the upper region. The products of repeated PCR
with the same primers, 1 and 2, in the second amplification
are shown in Fig. 2b. Only 256-bp bands with several
nonspecific background bands were observed with an endpoint of 15 pg of HUT 102 cell DNA, which was nine times
more sensitive than that obtained with single PCR. The
b o(Repeated fPCHj

Sinqle PCR)

bp

4;

5291

ii
1 1I0

;_

;r 1

256 bp

c (Nested Double
1 2 3 4 5

PCOR
;-

56I6

25-237-

.Pll.l

2 1 6 /-

bp

FIG. 2. PAGE of PCR products. (a) HUT 102 cell and CEM cell DNAs were amplified for 30 cycles with primers 1 and 2 (single PCR).
Portions of these were amplified for a further 30 cycles with the same primers 1 and 2 (b) or with the inner-position primers 3 and 4 (c). In
each amplification, 1 ,ug of CEM cell DNA plus 0, 3.3 x 104 pg, 1.1 x 104 pg, 3.7 x 103 pg, 1.2 x 103 pg, 4 x 102 pg, 1.3 x 102 pg, 4.5 x 101
pg, 1.5 x 101 pg, 5 pg, or 1.6 pg of HUT 102 cell DNA (lanes 1 to 11, respectively) were used as template DNA.

5292

J. VIROL.

MATSUMOTO ET AL.
1

9 10

products of nested double PCR with the inner-position

primers 3 and 4 (216 bp in length) are shown in Fig. 2c. A
distinct 216-bp band without background was observed with
1.6 pg of HUT 102 cell DNA, showing a sensitivity more
than 80 times higher than single PCR. With 15 to 130 pg of
HUT 102 cell DNA, two bands (216 and 235 to 237 bp in
length) were observed, and with more than 400 pg of cell
DNA, three bands (216, 235 to 237, and 256 bp in length)
were observed.
The results of nested double PCR with 0.5 pg of HUT 102
cell DNA as a starting template are shown in Fig. 3. Four of
10 PCR tests carried out with 0.5 pg of HUT 102 cell DNA
showed distinct single bands of 216 bp without nonspecific
background. One HUT 102 cell contains 8 to 10 copies of the
pX region of HTLV-I (26), and 150 human diploid cells
contain 1 ng of genomic DNA (assuming a haploid genome
size of 3 x 109 bp). It was calculated that 0.5 pg of HUT 102
cell DNA contains one or no molecules of the pX region
DNA, and it was thereby determined that one template DNA
can be detected by the nested double PCR.
Comparison of nested double PCR for detection of the
HTLV-I genome and anti-HTLV-I antibody testing in donated
blood. Detection in blood donor sera of HTLV-I provirus
DNA in PBMC by nested double PCR and anti-HTLV-I
antibody by the PA and IF methods was carried out in a
blind test, and the results were compared. DNA (1 ,ug) was
extracted from PBMC (1.5 x 105 cells) from each blood
donor and used for the nested double PCR test (some of
them are shown in Fig. 4). Of the 256 blood donors, 101 were
negative for anti-HTLV-I antibody by both PA and IF, and
no HTLV-I provirus DNA was detected.
The results of PCR and IF for the other 155 samples which
were positive by PA testing are summarized in Table 1 and
are classified by PA titers. Fifty-seven samples were antibody positive by IF, and provirus was detected in the same
57 samples without exception. Almost all serum samples
with antibody titers lower than 64 in the PA assay were
negative for antibody by IF, and provirus was not detected
in the PBMC of these individuals. Two subjects in this group
were positive for antibody by IF and for provirus by the
nested double PCR. Four of eight serum samples with a titer
of 128 by PA were both IF and provirus positive. Fifty-one
cases with a titer of 256 and higher were positive in both IF
for serum antibody detection and the nested double PCR test
for HTLV-I provirus detection. The results of IF for
HTLV-I antibody coincided completely with the results of

0o

2 16-

216 bp

FIG. 3. PAGE of the nested double PCR products. HUT 102 cell
and CEM cell DNAs were first amplified for 30 cycles with primers
1 and 2. In the second step, a pair of interior primers, 3 and 4, were
used and amplified 30 times. HUT 102 cell DNA (0.5 pg) and 1 jig of
CEM cell DNA were used as template DNA in every lane.

bP

FIG. 4. Detection of HTLV-I provirus in HUT 102 cells and

PBMC from blood donors by the nested double PCR method.
Products of nested double PCR were observed by PAGE (5%
polyacrylamide) CEM cell DNA (1 ,ug) plus HUT 102 cell DNA at 4,
40, 400, 2,000, or 0 pg (lanes 1 to 5, respectively) were used as
control templates, and 1 ,ug of cellular DNA obtained from PBMC of
five blood donors (lanes 6 to 10) was subjected to amplification.

the nested double PCR for detection of provirus DNA in

PBMC.
Estimation of the number of HTLV-I proviruses. The
nested double PCR DNA samples were titrated on the basis
of the number of bands which appeared on PAGE gels (Table
2). As shown in Fig. 2c, a single band (216 bp) was obtained
with 1.6 to 5 pg of HUT 102 cell DNA, two bands (216 and
235 to 237 bp) were obtained with 15 to 45 pg, and three
bands (216, 235 to 237, and 256 bp) were obtained with more
than 130 pg. It was calculated that 1,000 pg of HUT 102 cell
DNA contains 1,200 to 1,500 molecules of pX region DNA.
Therefore, it was estimated that one, two, and three bands
came from 2 to 7, 22 to 70, and more than 200 proviruses,
respectively.
The nested double PCR for detection of provirus was
carried out with 1 Fg each of DNA prepared from approximately 1.5 x 105 PBMC from 57 individuals who were
anti-HTLV-I antibody positive by IF. Of these, 2 showed a
single band (216 bp), 7 showed two bands (216 bp and 235 to
237 bp), and 48 showed three bands (216, 235 to 237, and 256
bp). Therefore, the majority (about 84%) of HTLV-I-antibody-positive samples contained more than 200 HTLV-I
proviruses in 1.5 x 105 PBMC and only a minor proportion
TABLE 1. Comparison of HTLV-I antibody and provirus
detection by PCR and anti-HTLV-I antibody testing by
PA and IF'
PA titer
(final serum dilution)

---8
16
32
64
128
256
512

No. of samples positive by:

No. of samples
(n = 256)

IF (antibody)

PCR (provirus)

101
55
29
8
8
14
10
11
12
2
6

0
1
1
0
4
12
10
10
11
2
6

0
1
1
0
4
12
10
10
11
2
6

1,024
2,048
4,096
-8,192
"The HTLV-I pX region was amplified by nested double PCR with T.
aquanticus polymerase. Samples tested by IF and PCR were identical.

VOL. 64, 1990

NESTED DOUBLE PCR TO DETECT HTLV-1 PROVIRUS

5293

TABLE 2. Number of bands in PAGE gels of nested double PCR products and antibody titer of HTLV-I in adult carriers
No. of
bands
bands
32
64
16

1
2
3

No. of samples with PA

128

256

512

titer":

1,024

2,048

10

1
10

4,096

-8,192

1
5

1
1

1
3

3
7

10

HUT 102

Estimated copies

1.6-5
15-45
-130

2-7
22-70
.200

DNA
of
~~~~~~~~
~~~~~~~~~~cell
~ pX regionb
(pg)

a The PA titer represents the final serum dilution.

b Estimated from data in Fig. 2.

(3.5%) contained fewer than 10 proviruses in the same

number of PBMC.
DISCUSSION
The method described here as nested double PCR was
used for detection of HTLV-I provirus in HUT 102 cells and
found to be extremely sensitive. It can be used to detect a
single H'rLV-I genome in cellular DNA with almost no
nonspecific background.
Wit, moderate amounts (22 to 70 genomes) of template
provirus DNA, two bands, 216 and 235 to 237 bp in length,
were observed. The 216-bp band corresponds to the region
between primers 3 and 4, which were added in the nested
second amplification (Fig. 1). In this amplification, moderate
aniounts of 256-base products (corresponding to the distance
between primers 1 and 2) might be produced in the first
amplification, and these were annealed with only primer 3 or
4 in the second amplification. Annealing of primer 3 to
256-base products of the first amplification produced 237base plus-strand products, and annealing of primer 4 to
256-base products produced 235-base minus-strand products. The amounts of both the 237-base plus-strand and
235-base minus-strand products were expected to be 30-fold
greater than the amount of template 256-base products used
in the second 30-cycle amplification. Under these conditions, in addition to the 216-base plus- and minus-strand
products, 237-base plus-strand products coupled with 216base minus-strand products and 235-base minus-strand products coupled with 216-base plus-strand products might be
produced. Distinguishing between the 235-base product and
237-base product was difficult, and they appeared as a single
band on PAGE. Therefore, two bands (216 bp and partially
single-stranded 235 and 237 bases) were observed on PAGE.
With a limited amount (one to seven genomes) of template
provirus DNA, only one band, 216 bp in length, was observed. We assume that the first amplification with a small
amount of template DNA produced only a limited amount of
256-bp-length products. In addition, in the second amplification, 237-base plus-strand and 235-base minus-strand
products might have been produced in amounts 30-fold
greater than the template 256-base products, but their quantities were not high enough to be detected by PAGE.
Accordingly, only the 216-bp plus- and minus-strand products in the second amplification were observed by PAGE.
With excess DNA from 200 provirus genomes or more,
large amounts of 256-bp DNA were produced in the first
amplification, and they annealed with only primer 3 or 4. A
large number of 237-base plus-strand/216-base minus-strand
and 235-base minus-strand/216-base plus-strand products
might have been produced, as postulated above. If so,
237-base plus strands would be easily coupled with 235-base
minus strands and partially single-stranded 256-base products composed of 237-base plus and 235-base minus strands
might be formed. Therefore, in addition to 216-bp products

and 235- and 237-base products, a third band of 256 bases

should appear, and three bands would be detected.
We amplified the portion of the HTLV-I provirus pX
region at positions 7312 to 7567. It overlaps the coding region
of p40far and p27rex, which regulate replication of HTLV-I at
the transcriptional and posttranscriptional levels, respectively (3, 5, 10). Therefore, the region is considered indispensable for HTLV-I provirus multiplication in vivo. It is
believed that the region we amplified is adequate for detecting HTLV-I provirus in PBMC of HTLV-I-infected individuals.
Although the PA test has been widely used in blood
screening as one of the simplest, speediest, and most specific
tests with high sensitivity (8, 12), false-positive results can
be a problem, especially when using samples with low
antibody titers. Therefore, tests for detecting the presence of
HTLV-I in blood donors are urgently required to rule out the
possibility of transfusing infected blood. In addition, when
the presence of this virus can be determined with certainty,
blood donors can be notified about their status with respect
to HTLV-I infection. For these purposes, cocultivation with
HTLV-I-noninfected T cells (16, 27) and detection of the
HTLV-I genome (14, 28) have been considered. Our nested
double PCR test has now been demonstrated to be the most
sensitive and specific test for detection of the HTLV-I
genome in PBMC of donors. However, at present, both
cocultivation and the PCR test are time-consuming, complicated, and costly as routine confirmation tests.
As described in this report, the results of our IF method
with carefully maintained MT-2 cells and Molt-4 cells as
positive and negative targets, respectively, for HTLV-I
antibody testing coincided completely with those of the
nested double PCR test regardless of the antibody titer
determined by PA. As preliminary tests, IF tests with cold
acetone-fixed Molt-4 cells as the target were carried out with
100 serum samples which were proven to be positive for
HTLV-I antibody by both PA and IF against HTLV-Iinfected MT-2 cells. The results were all negative against
Molt-4 cells. Therefore, when Molt-4 cells were stained, this
was judged to be nonspecific. Although Molt-4 cells are
smaller than MT-2 cells, sometimes it is difficult to distinguish them only by size. Therefore, mixtures of MT-2 cells
and Molt-4 cells at a 1:3 ratio were prepared and used as
antibody target cells for the routine confirmation test. When
one-fourth of the larger cells were stained, this was judged to
be IF positive as described above. The end titer of the PA
test and IF test for five anti-HTLV-I antibody-positive
serum samples did not show any difference between the two
methods, showing that the PA assay is no more sensitive
than IF tests. Therefore, serum samples giving low antibody
titers on the PA assay and negative results for IF and PCR
are considered false-positive samples. Therefore, at present,
the IF method described here is now considered the most

5294

MATSUMOTO ET AL.

J. VIROL.

appropriate confirmatory test for most diagnostic laboratories and blood centers.
ACKNOWLEDGMENTS
We thank T. Juji, K. Tokunaga, S. Kuwata, and E. Tokunaga for
useful suggestions on PCR and K. Nakamura for technical collaboration.
LITERATURE CITED
1. Bhagavati, S., G. Ehrlich, R. W. Kula, S. Kwok, J. Sninsky, V.
Udani, and B. J. Poiesz. 1988. Detection of human T-cell
lymphoma/leukemia virus type I DNA and antigen in spinal fluid
and blood of patients with chronic progressive myelopathy. N.
Engl. J. Med. 318:1141-1147.
2. Chehab, F. F., and Y. W. Kan. 1989. Detection of specific DNA
sequences by fluorescence amplification: a color complementation assay. Proc. Natl. Acad. Sci. USA 86:9178-9182.
3. Chen, I. S. Y., D. J. Slamon, J. D. Rosenblatt, N. P. Shah, S. G.
Quan, and W. Wachsman. 1985. The x gene is essential for
HTLV replication. Science 229:54-58.
4. Duggan, D. B., G. D. Ehrlich, F. P. Davey, S. Kwok, J. Sninsky,
J. Goldberg, L. Baltrucki, and B. J. Poiesz. 1988. HTLV-Iinduced lymphoma mimicking Hodgkin's disease. Diagnosis by
polymerase chain reaction amplification of specific HTLV-I
sequences in tumor DNA. Blood 71:1027-1032.
5. Fujisawa, J., M. Seiki, T. Kiyokawa, and M. Yoshida. 1985.
Functional activation of the long terminal repeat of human
T-cell leukemia virus type I by a trans-acting factor. Proc. Natl.
Acad. Sci. USA 82:2277-2281.
6. Gyllensten, U. B., and H. A. Erlich. 1988. Generation of singlestranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus. Proc. Natl.
Acad. Sci. USA 85:7652-7656.
7. Hinuma, Y., K. Nagata, M. Hanaoka, M. Nakai, T. Matsumoto,
K. Kinoshita, S. Shirakawa, and I. Miyoshi. 1981. Adult T-cell
leukemia: antigen in an ATL cell line and detection of antibodies
to the antigen in human sera. Proc. Natl. Acad. Sci. USA
78:6476-6480.
8. Hsia, K., D. H. Spector, J. Lawrie, and S. A. Spector. 1989.
Enzymatic amplification of human cytomegalovirus sequences
by polymerase chain reaction. J. Clin. Microbiol. 27:1802-1809.
9. Ikeda, M., R. Fujino, T. Matsui, T. Yoshida, H. Komoda, and J.
Imai. 1984. A new agglutination test for serum antibodies to
adult T-cell leukemia virus. Gann 75:845-848.
10. Inoue, J., M. Yoshida, and M. Seiki. 1987. Transcriptional (p40y)
and post-transcriptional (p27x`I1) regulators are required for the
expression and replication of human T-cell leukemia virus type
I genes. Proc. Natl. Acad. Sci. USA 84:3653-3657.
11. Kamihira, S., S. Nakashima, M. Ichimaru, Y. Moriuchi, Y.
Oyakawa, H. Okuda, M. Kanamura, and T. Oota. 1987. The
problems concerning assay of anti-ATLA by immunofluorescence, enzymeimmuno assays, gelatin particle agglutination and
Western blot methods. Nippon Yuketsu Gakkai-shi 33:7-11.
12. Kamihira, S., S. Nakasima, Y. Oyakawa, Y. Moriuti, M. Ichimaru, H. Okuda, M. Kanamura, and T. Oota. 1987. Transmission of human T cell lymphotropic virus type I by blood
transfusion before and after mass screening of sera from sero-

positive donors. Vox Sang. 52:43-44.

13. Kaneko, S., R. H. Miller, S. M. Feinstone, M. Unoura, K.
Kobayashi, N. Hattori, and R. H. Purcell. 1989. Detection of
serum hepatitis B virus DNA in patients with chronic hepatitis
using the polymerase chain reaction assay. Proc. Natl. Acad.
Sci. USA 86:312-316.
14. Kinoshita, K., T. Amagasaki, S. Ikeda, J. Suzuyama, K. Toriya,
K. Nishino, M. Tagawa, M. Ichimaru, S. Kamihira, Y. Yamada,
S. Momita, M. Kusano, T. Morikawa, S. Fujita, Y. Ueda, N. Ito,

15.
16.

17.

18.

19.
20.

21.

22.

23.

24.

25.

26.
27.

28.

29.

and M. Yoshida. 1985. Preleukemic state of adult T cell leukemia: abnormal T lymphocytosis induced by human adult T cell
leukemia-lymphoma virus. Blood 66:120-127.
Loh, E. Y., J. F. Elliott, S. Cwirla, L. L. Lanier, and M. M.
Davis. 1984. Polymerase chain reaction with single-sided specificity: analysis of T cell receptor 8 chain. Science 243:217-220.
Miyamoto, K., N. Tomita, A. Ishii, T. Nishizaki, K. Kitajima, T.
Tanaka,T. Nakamura, S. Watanabe, and T. Oda. 1984. Transformation of ATLA-negative leukocytes by blood components
from anti-ATLA-positive donors in vitro. Int. J. Cancer 33:721725.
Miyoshi, I., I. Kubonishi, S. Yoshimoto, T. Akagi, Y. Ohtsuki, Y.
Shiraishi, K. Nagata, and Y. Hinuma. 1981. Type C virus
particles in a cord T-cell line derived by co-cultivating normal
human cord leukocytes and human leukaemic T cells. Nature
(London) 294:770-771.
Mullis, K., F. Faloona, S. Scharf, R. Saiki, G. Horn, and H.
Erlich. 1986. Specific enzymatic amplification of DNA in vitro:
the polymerase chain reaction. Cold Spring Harbor Symp.
Quant. Biol. 51:263-273.
Mullis, K. B., and F. A. Faloona. 1987. Specific synthesis of
DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 155:335-350.
Ou, C., S. Kwok, S. W. Mitchell, D. H. Mack, J. J. Sninsky,
J. W. Krebs, P. Feorino, D. Warfield, and G. Schochetman.
1988. DNA amplification for direct detection of HIV-I in DNA
of peripheral blood mononuclear cells. Science 239:295-297.
Poiesz, B. J., F. W. Ruscetti, A. F. Gazdar, P. A. Bunn, J. D.
Minna, and R. C. Gallo. 1980. Detection and isolation of type C
retrovirus particles from fresh and cultured lymphocytes of a
patient with cutaneous T-cell lymphoma. Proc. Nati. Acad. Sci.
USA 77:7415-7419.
Reddy, E. P., M. Sandberg-Wollheim, R. V. Mettus, P. E. Ray,
E. DeFreitas, and H. Koprowsky. 1989. Amplification and molecular cloning of HTLV-I sequences from DNA of multiple
sclerosis patients. Science 243:529-533.
Saiki, R. K., T. L. Bugawan, G. T. Horn, K. B. Mullis, and
H. A. Erlich. 1986. Analysis of enzymatically amplified P-globin
and HLA-DQa DNA with allele-specific oligonucleotide
probes. Nature (London) 324:163-166.
Seiki, M., S. Hattori, Y. Hirayama, and M. Yoshida. 1983.
Human adult T-cell leukemia virus: complete nucleotide sequence of the provirus genome integrated in leukemia cell DNA.
Proc. Natl. Acad. Sci. USA 80:3618-3622.
Sheffield, V. C., D. R. Cox, L. S. Lerman, and R. M. Myers.
1989. Attachment of a 40-base-pair G+C-rich sequence (GCclamp) to genomic DNA fragments by the polymerase chain
reaction results in improved detection of single-base changes.
Proc. Natl. Acad. Sci. USA 86:232-236.
Watanabe, T., M. Seiki, and M. Yoshida. 1984. HTLV type I
(U.S. isolate) and ATLV (Japanese isolate) are the same species
of human retrovirus. Virology 133:238-241.
Yamamoto, N., M. Okada, Y. Koyanagi, M. Kannagi, and Y.
Hinuma. 1982. Transformation of human leukocytes by cocultivation with an adult T cell leukemia virus producer cell line.
Science 217:737-739.
Yoshida, M., M. Seiki, K. Yamaguchi, and K. Takatsuki. 1984.
Monoclonal integration of human T-cell leukemia provirus in all
primary tumors of adult T-cell leukemia suggests causative role
of human T-cell leukemia virus in the disease. Proc. Natl. Acad.
Sci. USA 81:2534-2537.
Yoshida, T., S. Kobayashi, and N. Yamamoto. 1986. Discrepancy between gelatin particle agglutination assay and indirect
immunofluorescence assay for antibodies to adult T cell leukemia associated antigen (ATLA). J. Clin. Exp. Med. (Igaku-noayumi) 136:387-388.