opcration, and SO, emissions were substantially

rcduccd by 1910 [K. J. Seigworth, A m . For. 49,

521 (1943)l.
16. National Occanic and Atmos heric Administration, Cli~naticAt1a.v of' the 8ttited Stcrtes (National Climatic Ccntcr, Ashcville, N.C., 1977);
F. C. Kornegay, pcrsonal communlcatlon.
17. Therc are five coal-fircd power plants in the Oak
Ridgc area. Kingston burns 4.0 x 10' kg of coal
per year. Bull Run, 2.3 x 10' k ~ l y c a r ;Y-12,
I .I x lod kglyear; K-25, 3.2 x 10 kglyear; and
thc OKNL power plant, 2.5 x 107 kglyear; the
total in thc Oak Ridge area is approximately
6.5 x lo!' kglyear.
18. Iron is most available to plants in thc ferrous
form: uptakc into bladcs of Macrocy.sti.s and
Pha.veolus roots and shoots has been shown to
be dependent on thc prcreduction of Fe(ll1) to
Fe(I1); S. L. Manlcy, Plant Phy.vio1. 68, 914
(1981). Iron uptakc is also cnhanccd by rcduction of rhi~osphcrcpH [A. N. Sarkar and R. G.
Wyn Jones, Plrrnt Soil 6 6 , 361 (1982)l.
19. T. A. Hagan, unpublished work. Data included
in S. B. McLaughlin, paper prcsentcd at thc

U.S.-Conarlictn Confirr>ncr>on firest Hr~sponsrJs to Acirlic Deoosition. Orono, Mainc. 2 to 4

August 1983.
C. P. Baes 111, thesis, Emory Univcrsity, Atlanta, Ga. (1977).
We thank S. E. Lindberg, R. R. Turner, R. S.
Turncr, T. J. Blasing, D. N. Duvick, 0 . U.
Brakcr, T. A. Hagan, D. R. Hcine, and H. G.
Davis for their hclpful discussions and technical
assistancc: thc Laboratorv of Trcc-Rine Rcscarch, Univcrsity of ~ r c z o n a , for proGiding
ring-width nicasurements for thc Norris, Tenn.,
short-leaf pincs; and the National Park Service
for allowing us to collect samplcs in the Great
Smoky Mountains National park. Research
sponsored by thc U.S. Environmental Protection Agcncy under interagency agrccmcnt EPA
82-D-X0533 and with the U.S. Dcpartmcnt of
Energy undcr contract W-7405-cng-26 with
Union Carbidc Corporation. Publication 2299,
Environmental Scicnces Division, Oak Ridge
National Laboratory.

family are T-cell tropic (12, 19); (iii)

preferentially infect helper T cells
(OKT4') (12, 19); (iv) have cytopathic
effects on various human and mammalian cells, as demonstrated by their induction of cell syncytia formation (20); (v)
can alter some T-cell functions (21); (vi)
can in some cases selectively kill T cells
(22); and (viii) may be transmitted by
intimate contact and blood products (9).
Also consistent w ~ t han HTLV etiology
were the results of Essex and Lee and
their colleagues showing the presence of
antibod~esto cell membrane antigens of
HTLV-infected cells in serum samples
27 Octobcr 1983; accepted 6 March 1984
from more than 40 percent of patients
with AIDS (23). This antigen has since
been defined as part of the envelope of
HTLV (24). The more frequent detection
Detection, Isolation, and Continuous Production of Cytopathic
in AIDS patients of antibodies to a membrane protein rather than to HTLV-I
Retroviruses (HTLV-III) from Patients with AIDS and Pre-AIDS
internal structural core proteins (25), toAbstract. A cell system was developedfor the reproducible detection of human T- gether with the low incidence of isolalymphotropic retroviruses (HTLVfumily)from patients with the acquired immunode- tions of HTLV-I or HTLV-I1 from AIDS
patients, also suggested that a new varificiency syndrome (AILIS) or with signs or symptoms that frequently precede AIDS
(pre-AIDS). The cells are speciJic clones,from a permissive human neoplastic T-cell ant of HTLV might be present.
The original detection and isolation of
line. Some q f t h e clones permanently grow and contin~rouslyproduce large amounts
of virus after infection with cytopathic (HTLV-III) variants of these viruses. One HTLV-I were made possible by the discytopathic effect ogHTLV-III in this system is the arrangement of multiple nuclei in a covery of T-cell growth factor (TCGF)
characteristic ring formation in giant cells of the infected T-cell population. These (26), also called interleukin 2 (1L-2),
structures can be used as an indicator to detect HTLV-III in clinical specimens. This which stimulates the growth of different
system opens the way to the routine detection of HTLV-III and related cytopathic subsets of normal and neoplastic mature
variants o f HTLV in patients with AIDS or pre-AIDS and in healthy carriers, and it T cells (29, and by the development of
provides large amounts of virus for detailed molec~rlgrand immunological analyses. sensitive assays for reverse transcriptase
(R?'), an enzyme characteristic of retroEpidemiologic data suggest that the cell lymphotropic retroviruses (HTLV) viruses (28). ?'he procedures used previacquired immunodeficiency syndrome (Y) that includes two major, well-charac- ously for the transmission and continu(AIDS) is caused by an infectious agent terized subgroups of human retrovir- ous production of HTLV-I and -11 were
that is horizontally transmitted by inti- uses, called human T-cell leukemia-lym- first worked out in mammalian cells
phoma viruses, HTLV-I (9-12) and transformed by avian sarcoma virus (29).
mate contact or blood products (1-3).
Though the disease is manifested by op- HTLV-I1 (9, 11, 13). The most common These methods involved cocultivation of
portunistic infections, predominantly isolate, HTLV-I, is obtained mainly the transformed cells with cells permisPneumocystis carinii pneumonia ( 4 ) ,and from patients with mature T-cell mal~g- sive for the particular virus strain. Norby Kaposi's sarcoma (S), the underlying nancies (9, 12). Seroepidemiological mal human T cells in cocultivation exdisorder affects the patient's cell-mediat- studies, the biological effects of the virus periments preferentially yielded HTLV
ed immunity (6), resulting in absolute In vltro, and nucleic acid hybridization of both subgroups. Some of these viruses
lymphopenia and reduced subpopula- data indicate that HTLV-I is etiological- showed an immortalizing (transforming)
tions of helper ?' lymphocytes (OK1'4 I). ly associated with the T-cell malignancy capability for certaln target T cells (9,
Moreover, before a complete clinical of adults that is endemic in certain areas 12). We thought that HTLV variants that
manifestation of the disease occurs, its of the south of Japan (14), the Caribbean. have cytopathic effects on their target
prodrome, pre-AIDS, is frequently char- (IS), and Africa (16). H1'LV-I1 was first cells but do not immortalize them might
acterized by unexplained chronic lymph- isolated from a patient with a T-cell be more important in the cause of AIDS.
adenopathy or leukopenia involving variant of hairy cell leukemia (13). To In fact, such variants were frequently
helper 1' lymphocytes (5, 6). This leads date, this is the only reported isolate of but only transiently detected when norto the severe immune deficiency of the HTLV-I1 from a patient with a neoplas- mal T cells were used as targets in coculpatient and suggests that a specific sub- tic disease. Virus isolation and seroepi- tivation or cell-free transmission experiset of 1' cells could be a primary target demiological data show that both HTLV- ments. This transience was our main
for an infectious agent. Although pa- 1 and HTLV-I1 can sometimes be found obstacle to the isolation of these cytopathic variants of HTLV from patients
tients with AIDS or pre-AIDS are often in patients with AIDS (17).
That a retrovirus of the HTLV family with AIDS or pre-AIDS. We subsechronically infected with cytomegalovirus (7) or hepatitis B virus (a), for vari- mlght be an etiological agent of AIDS quently found a cell line that is highly
ous reasons these appear to be opportu- was suggested by the findings (i) that susceptible to and permissive for cytonistic or coincidental infections. We another retrovirus, feline leukemia virus, pathic variants of HTLV. This cell line
have proposed that AIDS may be caused causes immgne deficiency in cats (18); can grow permanently after infection
by a virus from the family of human T- and that (ii) retroviruses of the HTLV with the virus. We report here the estab-

lishment and characterization of this new

immortalized T-cell population and its
use in the isolation and continuous highlevel production of HTLV variants from
patients with AIDS and pre-AIDS.
Several neoplastic human cell lines

established in vitro were assayed for

susceptibility to infection with HTLV-I
and -11 and with many of the more cytopathic retroviruses isolated from AIDS
patients (30). One neoplastic aneuploid
T-cell line, derived from an adult with

Fig. 1. Light and electron microscopic examination of clone H41HTLV-111. (a) H41HTLV-111
cells were characterized by the presence of large multinucleated cells that showed, with
Giemsa-Wright staining, a characteristic arrangement of their nuclei (x350). (b) Electron
micrograph of the cells showing the presence of extracellular viral particles (~60,000).

Table 1. Response of cloned T-cell populations to infection with HTLV-111. Single-cell clones
were isolated as described (34, 35) from a long-term cultured aneuploid HT cell line exhibiting
mature T-cell phenotype [OKT3+ (62 percent), OKT4+ (39 percent), and OKT8 ] as determined by cytofluorometry w ~ t ha fluo~escence-activatedcell sorter. The cultures are routinely
maintained in RPMl 1640 medium containing 20 percent fetal calf serum (FCS) and antibiotics.
The terminal cell density of the parental cell culture, seeded at a concentration of 2 x lo5 cells
per milliliter of culture media, was in the range of 106.to 1.5 x 10" cells per milliliter after 5 days
of culture.
Characteristics
after infection
Total cel~l~number
( x lo6)
At 6 days
At 14 days
Multinucleated cells (%)*
At 6 days
At 14 days
Immunofluorescence uositive cells (%)I'
At 6 days
Rabbit antiserum to HTLV-111
Patient serum (E.T.)
At 14 days
Rabbit a n t i s e ~ mto HTLV-111Patient serum
Reverse transcriptase activity (x lo4 cpm/ml)S
At 6 days
At 14 days

Clones*
H3

H4

H6

H9

1
2.2

1.5
7.3

1.5 0.3
7.5 10

HI7 H31 H35 H38

0.4
4.7

0.3
5.0
14
45

0.5
4.5
30
60

1.8
3.2
45
60

24
45

42
48

32
45

7
30

55
56

56
29

32
21

39 21
10
87
32
ND ND ND ND 73

50
45

74
47

60
56

97
78

13
22

71
61

40
43

20
22

80
89

2.4 1.8 2.1 4.1 2.6 1.4 1.7 2.5

16.2 18.1 16.1 20.2 17.1 13.4 15.1 18.2

*Cell smears were prepared from cultures 6 and 14 days after infection and stained with Wright-Giemsa. Cells
with more than five nuclei were considered to be multinucleated. Cloned cells from uninfected cultures also
contained some multinucleated giant cells; however, the arrangement of the multiple nuclei in a characteristic
ring formation (see Fig. la) was lacking and the number of these cells was much less (0.7 to 10
percent). . +Cells wefe washed with phosphate-buffered saline (PBS) and resuspended in the same buffer at
concentration 1 9 cells per milliliter. Approximately 50 yl of cell suspension was spotted on a slide, air dried,
and fixed in acetone for 10 minutes at room temperature. Slides were stored at -20C until use. Twenty
microliters of either rabbit antiserum to HTLV-111 (diluted 1: 2000 in PBS) or serum from the patient (E.T.)
diluted 1:8 in P.BS was applied to cells and incubated for 50 minutes at 37C. The fluorescein.conjugated
antiserum to rabbit or human immunoglobulin G was diluted and applied to the fixed cells for 30 minutes at
room temperature. Slid,es were then washed extensively before microscopic examinations, The uninfected
parental cell line as wekt as the clones were cofisistently negative in these assays. ND, not done.
$Virus
particles were precipitated from cell-free supernatant as follows: 0.3 ml of 4M NaCl and 3.6 ml of 30 percent
(weight to volume) polyethylene glycol (Carbowax 6000) were added to 8 ml of harvested culture fluids and
the suspension was placed on ice overnight. The suspension was centrifuged in a Sorvall RC-3 centrifuge at
2000 revlmin at. 4C for 30 minutes. The precipitate was. resuspended in 300 pl of 50 percent (by volume)
glycerol (25 mhf tris-HCI, pH 7;5, 5 mM dithiothreitol, 150 mM KCI, and 0.025 percent Triton X-100)..Virus
particles were disrupted by add~tionof 100 pI d 0 . 9 ercent Triton X-100 to 1.5M KCI. Reverse transcrlptase
assays were performed as previously described (lo,.&) (see comments to Fig. 2b) and expressed In counts per
minute per milliliter of culture medium.
498

lymphoid leukemia, was found to be

susceptible to infection with the new
cytopathic virus isolates. This cell line,
termed HT, has produced HTLV-variants in sufficient quantities to permit the
development of specific immunologic reagents and nucleic acid probes that can
be used to characterize new isolates and
compare them with HTLV-I and HTLVI1 (30). These cytopathic variants differ
from HTLV-I and -11 not only in their
biological effects but also in several immunological assays and in their morphology (31). They nevertheless have many
properties similar to HTLV-I and -11.
For example, they are T4 lymphotropic,
they have a similar RT (30), they crossreact with several structural proteins in
heterologous radioimmune assays with
serum from AIDS patients and with antisera to the virus raised in animals (31),
and they induce syncytia. These new
HTLV isolates are collectively designated HTLV-111, although it is not yet
proved that they are identical.
The cell line HT was tested for HTLV
before being infected in vitro and was
negative by all criteria including lack of
proviral sequences (32). Continuous production of HTLV-111 was obtained after
repeated exposure of parental HT cells
(3 x 10' cells pretreated with polybrene)
to concentrated culture fluids harvested
from short-term cultures of T cells
(grown with TCGF) obtained from patients with AIDS or pre-AIDS. The concentrated fluids were first shown to contain particle-associated RT. When cell
proliferation declined, usually 10 to 20
days after exposure to the culture fluids,
the fresh (uninfected) HT cells were added to the cultures. Culture fluids from
the infected parental cell line were positive for particulate RT activity, and
about 20 percent of the infected cell
population was positive in an indirect
immune fluorescence assay in which we
used serum fi-om a hemophilia patient
with pre-AIDS (patient E.T.). Serum
from E.T. also contained antibodies to
proteins of disrupted HTLV-111 (33) but
did not react with cells infected with
HTLV-I or HTLV-11.
The parental T-cell population was extensively cloned in order to select the
most permissive clones that would preserve high rates of growth and virus
production (for example, see clones 4
and 9 in Table 1). A total of 51 single-cell
clones were obtained by both capillary
(34) and limited dilution (35) techniques
using irradiated mononuclear cells from
peripheral blood of a healthy donor as a
feeder. The clones were infected with
HTLV-111 by exposure to concentrated
SCIENCE, VOL. 224

cells of each clone and 0. I

virus (2 x
ml of virus). Then cell growth and morphology, expression of cellular viral antigens, and RT activity in culture fluids
were assessed 6 and 14 days after infection. Results for eight of these clones are
shown in Table 1. Although all of these
clones were susceptible to and permissive for the virus, there were considerable differences in their ability to proliferate after infection. For example, the
cell number decreased by 10 to 90 percent from the initial cell count within 6
days after infection. The percentage of
T cells positive for viral antigens ranged
from 10 to 80 percent, as determined by
immune fluorescence assays with serum
from patient E.T. and with antiserum
from rabbits infected repeatedly with
disrupted HTLV-111. At 14 days after
infection, the total cell number and the
proportion of HTLV-I11 positive cells
had increased in all eight clones. The
virus positive cultures consistently
showed a high proportion of round giant
cells containing numerous nuclei (Fig.
la). These cells resemble those induced
by H'TLV-I and -11 (9) except that the
nuclei exhibit a characteristic ring formation. Electron microscopic examinations
showed that the cells released considerable amounts of virus (Fig. Ib).
Both virus production and cell viability of the infected clone H4 (H4IHTLV111) were monitored for several months.
Although virus production fluctuated
(Fig. 2a), culture fluids harvested and
assayed at approximately 14-day intervals consistently showed particulate RT
activity which has been followed for
over 5 months. The viability of the cells
ranged from 65 to 85 percent and the
doubling time of the cell population was
approximately 30 to 40 hours (data not
shown). Thus the data show that this
permanently growing T-cell population
can continuously produce HTLV-111.
The yield of virus from H4IHTLV-I11
cells was assessed by purification of concentrated culture fluids through a sucrose density gradient and assays of particulate KT activity in each fraction collected from the gradient. As shown in
Fig. 2b, the highest RT activity was
found at a density of 1.16 glml, which IS
similar to other retroviruses. The highest
RT activity was found in the fractions
with the largest amount of virus, as determined by electron microscopy. The
actual number of viral particles determined by this method was estimated (36)
to be about 10" per liter of culture fluid.
We have used clones H4 and H9 for
the long-term propagation of HTLV-111
from patients with AIDS and pre-AIDS.
4 MAY 1984

12

16

Fractcon number

Tlme (months)

Fig. 2. (a) Continuous HTLV-111 production from H4lHTLV.-111 in long-term crilture was
characterized by fluctuation in the amount of releasedvirus as assessed by RT activity in the
culture fluid (for details, see Table 1 and Fig. 2b). Viability of the infected cells was in the range
of 60 to 90 percent. (b) Sucrose, density gradient banding of HTLV-111 show;ed the highest
particulate RT activity at a density of 1.16 glml. A cell-free virus concentrate from. a culture of
H4lHTLV-111 was layered on a 20 to 60 percent (by weight) sucrose gradient in 10 mM tris-HCI
(pH 7.4) containing 0. IM NaCl and 1 mM EDTA and centrifuged overnight a%35,000 revlmin in
a Spinco SW47 rotor. Fractions of 0.7 ml were collected from the bottom of the gradient and
portions were assayed for RT ( 0 )with (dT),5 . (A*), being used asthe prilner template and Mg2+
as the divalent cation according to the methods described earlier (10,28). Depsity of sucrose (X)
was determined by refractive index measurements.

HTLV-I11 was isolated from four patients by the cocultivation method- and
from one patient by cell-free infection of
these T-cell clones (Table 2). The transmission was monitored by RT activity,
electron microscopic examinations, and
expression of viral protein. When the H4
cells thus infected were fixed with acetone and tested with rabbit antiserum to
HTLV-I11 and with serum from patients
E.T., the percentage of positive cells
was between 5 and 80 percent. HTLV111 has,also been isolated in our labora-

tory from a total of 48 patlents by the

more convent~onalmethods for lsolat~on
of HTLV (30). Some of these ~rolates
have now successfully *been transmitted
to the HT clones for production and
detalled analyses.
A few T-lymphocyte retroviruses that
dlffered from HTLV-I and -11 but were
associated wlth lymphadenopathy syndrome were detected e a r l l e ~(37, 361).
One such virus, called LAV, was reported to be unrelated to HTLV-I or -11 (38).
from 37.5 per, Moreover, serum sam,@les

Table 2. Isolation of HTLV-111 from patients with AIDS and pre-AIDS.

Patient*

R.F.
S.N.

B.K.
L.S.
W.T.

Or~g~n

Diagnosis

AIDS (heterosexual)
Hemophiliac
(lymphadenopathy)
AIDS (homosexual)
AIDS (homosexual)
Hemophiliac
(lymphadenopathy)
~

Percent positlve
cells In
lmmune
" fluorescence
RT
Aet~vity
assay
( X lo4
cpm)
~ a b b ~ tSerum
antlfrom

serum " E.T.

Haiti
United States

0.2%
6.3

80
10

United States
United States
United States

0.24
0.13
3.2

44
64
69

.. - -

-~ - - - - - - - -

33
ND

5
19.
ND

Electron
micros-

'OPY

ND

+
+
+

ND

---~

*Cocultivation with H4 recipient T-cell clone was performed with fresh mononuclear cells from periphel-al
blood of patients R.F. and S.N., respectivel). For patients B . K . and L.S. tocultivation was performed wlth T
cells grown in the presence of' exogenous TCGF (I0 pel-cent by volume) fol- 1.0 days. The ratio of' recipient to
donor (patients') cells was 1 :5. The mixed cultures were maintained in RPMI I640 medium (containing 20
percent FCS and antibiotics) in the absence of'exogenou.~TCCiF. H9 cells were also infected by exposing the
cells to concentrated culture fluids harvested from~T-cellcultures of' patient W.T.The cultures were gl-own in
the presence of exogenous TCGF for 2 week, before the culture fluids were harvested and concentrated.
Cells of H9 clones were treated with polybrene (2 pglml) for 20 minutes and 2 x 106cells were exposed for 1
hour to 0.5 ml of 100-fold concentrated culture fluids positive for part~culatcKT activity.
1-HT1.V-I11
virus expression in cells infected by the coculture and cell-free methods was assayed approximately I month
after cultivation in vitro. Note a con5iderable fluctuation in HTLV-111 e x p ~ a s i o n .For detail, of the KT and
indirect Immune fluore,cence as,ays see Table 1.

14. Y. Hinuma et rrl., Proc. Nutl. Acad. Sci. 1J.S.A. 28. R. C. Gitllo, F. W. Kuscetti, K. E. Gallagher, in
cent of patients with AIDS were found to
78,6476 (1981); M. Robert-Guroffel ul., Scirncc7
Nemrrlopoic~lrcM e c h a n i ~ m s ,B. Clarkson, P. A.
react with it (38). In contrast, H'I'LV-111
215, 975 (1982); V. S. Kalyanaraman 1.1 ul.,
Marks, J . Till, Eds. (Cold Spring Harbor 13re,s,
79,
I653
(1982).
P
n
~
c
Natl.
.
Acad.
Sci.
U
.
S
.
A
.
Cold Spring Harbor, N.Y., 1978), vol. 5, p. 671.
is
HTLV-l arid -I1 (31~39) and'
15. W. A. Blattner 1.1 a/., Int. J . Cancrr 30, 257
29. J. Svoboda, Nutl. Canerr Inst. M o n o ~ r .17, 277
by all criteria, this new virus belongs to
(1964); 5. Svohoda and K. Dourmashki~i,J. G r n .
(1982).
Virol. 4, 523 (1969); M. I'opovic, J. Svoboda, J.
W. C. Saxingee' et 01.. in Humun T-cell Leukethe HTLV family of retroviruses.
ad- 16. miu
Suni, A. Vaheri, 1.. Ponten, Int. J . Cancer 19,
Viru.re.r, K. C. Gallo, M. Essex, L. Gro,,,
834 (1977); M. I'opovic, J. Svoboda, F. I..
Eds. (Cold Spring Harhor Press, Cold Spring
dition, more than 85 percent of serum
Kissclyov, K. Polakova, Folio Uiol. 26, 244
Harbor, N.Y., in press).
samples from AIDS patients are reactive
Scirncr 220, 865 (1983); E ,
( 1980).
17, R, C. ciallo
Gelmann 1.1 a/., ibid., p. 862; M. I'opovic rl al.,
30. K. C. Gallo rl a/., Scic71zc.e 224, 500 (1984).
with proteins of HTLV-111 (33). These
31. J. Schiipbach, M. I'opovic, K. V. Gilden, M. A.
in preparation.
findings suggest that HTLV-111 and LAV
Gonda, M. G. Sarngadharan, R. C. Gallo, ihid.
18. M. Essex, W. D. Hardy, Jr., S. M. Cotter, R.
224, 503 (1984).
M.
Jakowski,
A.
Sliski,
Infect.
Immloz.
11,
470
may be different, However, it is possible
32. G. Shaw and F. Wong-Staal, unpublished data.
(1975); W. D. Hardy, Jr., 1.1 ul., C'an~.rrh ' r ~ .36,
that this is due to insufficient character33. M. G. Sarngadharan, M. Popovic, L. Bruch, J.
582 (1976); I,. J. Anderwn, 0. Jarret, H. M
I.aird, J. Nrrtl. Cancer 1n.rt. 47, 807 (1971).
Schiipbach, R. C. Gallo, Science 224, 506
''ation of
because the virus has not 1% R , C. Gallo (,I., Cnncrr N P J ,43, 3892 (1983);
(1984).
F. Wong-Staal 1.1 a / . , Nutarr (London) 302, 626
34. M. Popovlc, M. Grofova, N. Valentova, D.
yet been transmitted to a permanently
Simkov~c,Neoplrr.rmrr 18, 257 (1971).
line for true
growing
and 20. (1983).
35. H. F. Bach, B. J. Alter, B. M. Widmer, M. S.
K. Nagy, 1'. Clapham, K. Cheinsong-Popov, K.
SegaU, 1). Dunlap, ~ m m u n o l ~. r v 54,
111t.
J
.
C'rrncer
32,
3.21
(1983).
. 5 (1981).
A.
Weiss,
therefore has been difficult to obtain in
21. M. Popovic rl ul., ~n preparat~on.
36. J. H. Monroe and P. M. Brandt, Appl. Microhiquantity.
22. H Mitsuya, H. Ci. Guo, M. Megson, C. D.
01. 20 259 (1970).
Trainor, M. S. Keitz, S. Broder, Sciencc7 223,
37. F. Ba;ll-?ilnouss~ et ul., Science 220,868 (1983).
The transient expression of cytopathic
38. I.. Montagnier el ul., in Human T-Cell 1.rukc~1293 (1984).
variants of HTLV in cells from AIDS 23. M. E s e x el a/., ihid. 220, 859 (1983).
rniu Vir~rse.\,K. C. Gallo, M. E,sex, L. Gro,,,
. Schupbpbach, M. G . Sarngadharan, K. C. C;allo,
Eds. (Cold Spring Harbor Press, Cold Spring
patients and the previous lack of a cell 24. Jihid.,
in pre,s; T . H. I.ee 1.1 a/., Proc. Nrrtl.
Harbor, N.Y., in press).
Acrrd. Sci. U.S.A., in press.
39. S. Arya rt al., in preparation.
system that could maintain growth and
25. M. Robert-Guron' ct a/., in preparation.
40. We thank B. Kramarsky for help in electron
still be susceptible to and permissive for 26 L). A , M
examination
~ F. W.~ ~
~ ~R. ~C. ~~ ;~ ~ l l ~
, ~ , microscopic
~
~
~ of HTLV-111
i
,infected
cells, E. Kichardwn and K. Zicht for techn~cal
Science
193,
1007
(1976).
the virus represented a major obstacle in
27. F. W. Ruscetti, D. A. Morgan, K. C. Gallo, .I.
help, and A. M a ~ r u c afor her editorial assistdetection, isolation, and elucidation of
~
~ 119, 131~ (1977); B.~ J . ~ ' o ~~ e s rF., W.~
ance.
~
r
,
Kuscetti, J. W. Mier, A. M. Woods, R. C. Gallo,
agent of AIDS. l-he
the precise
30 March 1984; accepted 19 April 1984
P n ~ cN
. atl. Acad. Sci. U . S . A . 77, 6134 (1980).
establishment of T-cell populations that
continuously grow and produce virus
after infection opens the way to the
routine detection of cytopathic variants
Frequent Detection and Isolation of Cytopathic Retroviruses
of HTLV in AIDS patients and provides
the first opportunity for detailed immu- (HTLV-111) from Patients with AIDS and at Risk for AIDS
nological (31,33) and molecular analyses
pcitic.rzts with the' acquired immunoAbstract. Peripheral blood lymphoc~ytc~s,from
of these viruses.
.syrzdrom~(AIDS) or with .signs or symptoms t/i~t,fr.cyuentlyprc~c~c~de
AIDS
MIKULASPOPOVIC. dclJic.i~nc.y
(prc-AIDS)were grown in vitro with c1ddc.d T-cell growth factor and us.sciyed,for the.
Labordtoty q f Turnor Cell Biology,
expression and relc.cise of hutnurz 7-lymphotropic retrovir~rsc's(HZ'L,V).Retroviruses
Nationcil Cancer Institute,
belonging to the. NTLV jilmily citz(1 cwllcctively dcsignatcd HTLV-Ill wc.re isoluted
Bethesdu, Muryland 20205
fiom a total of48 subjects irzclr~ding18 o f 2 1 pciticnts with prc-AIDS, threc. offour
M. G. SARNGADHARAN
rliniccilly normal mothers o f juveniles with AILIS, 26 of 72 cidr~ltand juvc>nile pciDepartment c$ Cell Biology,
tic.nts with AIDS, and j>om one. o f 22 normcil mcilc izomo.sc~xuc11subjc~cts. No
Litton Bionetirs, Itzr.,
c1etc.ctc.d in or isolated Ji.orrz 115 nortnal Izctcro.sc~xuu1subjcrts. The.
HXV-kll
Kensington, Maryland 20895
ofthc.
tzumb~r
o
JHTLV-Ill
i.solutc~.src~porfc'd17c'r.c' urzdere.stimutes the true prc~~~cilencc~
EI,IZABETH
READ
K o e ~ C.
~ rGALLO virus since mciny spcvimens rvcrc7 rc.ccivcd in un.sati,rfirctory c.ondition. Othc'r dater
.show thclt sc~rums a ~ p l c ~ . ~ , f ici. ohigh
m proportion qfA1L)S patients rontciin antibodies
L,aboratory q f Tumor Cc.11 Biology,
to HZ'LV-Ill. That these nc'w i.so1ute.s circ' mc.mber.s of'the NZ'L,VJamily but difc7r
National Cnnrer Institute
from the prc~~~ious
isol~tclsknowtz as IITLV-I citzd HTLV-I1 is indic.citcvi by their.
References and Notes
mor~~holorical,
bioloriccil, and immunologic~cilrhurcic~teristic.~.
Thc7sc. rc.sult.s and
1. Centers for Di,case Control Task ,Force On
issue.
.sr~ggc..st
that
f1TI.V-111
may
be the. primary
thosc
rcportcd
c.lscwlzcrc
in
this
Kaposi', Sarcoma and Opportunistic Infections,
ruu.sc7 of AIDS.
N. Engl. J . Med. 306, 248 (1982).
~ j c i s

2. J. P. Hanranhan, G. 1'. W o r m e r , C. P. Maquire, I.. J. DeLorenzo, G . Davi.;, ihid. 307,498

(1982).
3. J. W. Curran et a/., ihid. 310, 69 (1984).
4, ~~Pneumocystl, pneunlonia-Los
Angele,,fl
Morbid. Mortrrl. Wc7rkly Rep. 30, 250 (19x1).
5. "Kaposi', sarcoma and pneumocystis pneumonia among homosexual n l e n - ~ e w york c i t y
and California-" ihid.9 P. 305; A. E. FrkdmanKlein rt ul.. Ann. Int. Med. 96, 693 (1982).
N. ~
~J . ~~ ~1305,d. 1425
.
6. M. Gottlieb at
(1981); J. Masur r t ctl., ihid., p. 1431.
7, C, Urmacher, P, Myskowski, M. Ochoa, M.
. 569 (1982).
Kris, B. Saki. A m . J . M P ~72,
8. D. R. Francls and J . E. Maynard, Epr(lrmio1.
N , Enh,I,
Rrv, 17 (1979); N , Clunleck at
Med. 310, 492 (1984).
9. R. C . Gallo, in Cuncrr Srrrvrys. L. M. Franks.
L. M. Wyke, R, A , Weiss, ~ d (oxford
~ . univ,
Press, Oxford, in press).
10. B . J . Poiesz rt ( I / . , Proc. Null. Acad. Sci.
U . S . A .77,7415 (1980); M . Yoshida, 1. Miyoshl,
Y. Hinuma, ihid. 79, 2031 (1982).
11. M. S . Reitz, M. Popovic. B. F. Haynes, S . C.
Clark, R. C. Gallo, Virolr,,yy 26, 688 (1983).
12. M. Popovic rt a/., Scirncr 219. 856 (1983).
ihid, 218. 571 (1982,,
13,
S. Ka,yanaraman at

,,.

500

The acquired immunodeficiency syndrome known as AIDS was initially recognized as a separate disease entity in
1981 ( I ) . Groups rcported to be at risk
for AIDS include homosexual or bisexua1 males (about 70 percent of reported
cases), intravenous drug users (about 17
percent of cases), and Haitian immigrants to the United States (about 5
percent of cases). Also at risk are heterosexual contacts of members of the highest risk group, hemophiliacs treated with
blood products pooled from donors, recipients of multiple blood transfusions,
and infants born of parents belonging to
the high-risk groups (2). AIDS is diag-

nosed as a severe, unexplained, immune

deficiency that usually involves a reduction in the number of helper T lymphocytes and is accompanied by multiple
opportunistic infections or malignancies.
A number of other clinical manifestations, when occurring in members of a
group at risk for AIDS, are identified as
its prodrome (pre-AIDS). These include
unexplained chronic lymphadenopathy or
leukopenia involving a reduction in the
number of helper T lymphocytes ( 1 , 2).
The increasing incidence of this disease,
the types of patients atfected, and other
epidemiological data suggest the existence of an infectious etiologic agent that
SCIENCE, VO1.. 224

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Detection, Isolation, and Continuous Production of Cytopathic Retroviruses (HTLV-III)
from Patients with AIDS and Pre-AIDS
Mikulas Popovic; M. G. Sarngadharan; Elizabeth Read; Robert C. Gallo
Science, New Series, Vol. 224, No. 4648. (May 4, 1984), pp. 497-500.
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References and Notes

10

Detection and Isolation of Type C Retrovirus Particles from Fresh and Cultured
Lymphocytes of a Patient with Cutaneous T-Cell Lymphoma
Bernard J. Poiesz; Francis W. Ruscetti; Adi F. Gazdar; Paul A. Bunn; John D. Minna; Robert C.
Gallo
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Isolation and Transmission of Human Retrovirus (Human T-Cell Leukemia Virus)

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14

Adult T-Cell Leukemia: Antigen in an ATL Cell Line and Detection of Antibodies to the
Antigen in Human Sera
Yorio Hinuma; Kinya Nagata; Masao Hanaoka; Masuyo Nakai; Tadashi Matsumoto; Ken-Ichiro
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Natural Antibodies to Human Retrovirus HTLV in a Cluster of Japanese Patients with

Adult T Cell Leukemia
Marjorie Robert-Guroff; Yoshinobu Nakao; Kunihiro Notake; Yohei Ito; Ann Sliski; Robert C.
Gallo
Science, New Series, Vol. 215, No. 4535. (Feb. 19, 1982), pp. 975-978.
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14

Natural Antibodies to the Structural Core Protein (p24) of the Human T-Cell Leukemia
(Lymphoma) Retrovirus Found in Sera of Leukemia Patients in Japan
V. S. Kalyanaraman; M. G. Sarngadharan; Y. Nakao; Y. Ito; T. Aoki; R. C. Gallo
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17

Isolation of Human T-Cell Leukemia Virus in Acquired Immune Deficiency Syndrome

(AIDS)
Robert C. Gallo; Prem S. Sarin; E. P. Gelmann; Marjorie Robert-Guroff; Ersell Richardson; V. S.
Kalyanaraman; Dean Mann; Gurdip D. Sidhu; Rosalyn E. Stahl; Susan Zolla-Pazner; Jacque
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Transformation and Cytopathogenic Effect in an Immune Human T-Cell Clone Infected by

HTLV-I
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Selective in vitro Growth of T Lymphocytes from Normal Human Bone Marrows

Doris Anne Morgan; Francis W. Ruscetti; Robert Gallo
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30

Frequent Detection and Isolation of Cytopathic Retroviruses (HTLV-III) from Patients with
AIDS and at Risk for AIDS
Robert C. Gallo; Syed Z. Salahuddin; Mikulas Popovic; Gene M. Shearer; Mark Kaplan; Barton F.
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Antibodies Reactive with Human T-Lymphotropic Retroviruses (HTLV-III) in the Serum of

Patients with AIDS
M. G. Sarngadharan; Mikulas Popovic; Lilian Bruch; Jrg Schpbach; Robert C. Gallo
Science, New Series, Vol. 224, No. 4648. (May 4, 1984), pp. 506-508.
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37

Isolation of a T-Lymphotropic Retrovirus from a Patient at Risk for Acquired Immune

Deficiency Syndrome (AIDS)
F. Barr-Sinoussi; J. C. Chermann; F. Rey; M. T. Nugeyre; S. Chamaret; J. Gruest; C. Dauguet; C.
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