dengue-2 virus infection of human mononuclear cell lines

7/28/2019 Dengue-2 Virus Infection of Human Mononuclear Cell Lines

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Arch Virol (1990) 110:91-101 ArchivesVfrology by Springer-Verlag 1990

Dengue-2 virus infection of human mononuclear cell lines andestablishment of persistent infections

I. Kurane, U. Kontny, J. Janus, and F. A. EnnisDivision of Infectious Diseases, Department of Medicine, University of MassachusettsMe dical Center, Worcester, Massachusetts, U.S .A.

Acc epted September 19, 1989

Summary. Twenty three human mononuclear cel l l ines including ten myelo-mo noc ytic cell l ines, eight B cell lines and f ive T cell l ines, were exam ined todeterm ine wheth er they co uld be infected with dengue-2 virus. All the cell l ineswere infected with dengue-2 virus as determined by imm unof luorescent s tainingand by virus t i t rat ion of cul ture su pernata nt f luids. K562, J iyoye and J urkat ,respectively, showed the highest percentage of infected cells of these myelo-monocytic, B and T cell l ines. Antibody to dengue-2 virus at subneutralizingconcen trat ions augme nted dengue-2 virus infect ion ofm yelo mo noc yticcell lines,but not of B cell l ines or of T cell lines.

Pers is tent dengue-2 virus infect ion was es tablished using a mye lomon ocyticcell line (K562), a B cell line (Raji), and a T cell line (HSB-2). These cell linesmaintained a high percentage (more than 70%) of dengue-2 virus ant igen-positive cells for at least 25 weeks. Very low titers o f infectious dengue-2 viruswere detected in the cu lture su pern atan t f luids of the persistently infected cells.Dengue -2 virus a ntigen-positive Raji cell clones were established from persist-ently-infected Raji cells using limiting dilutions and all of the cells in theseclones were dengue-2 virus antigen-positive. These f indings demo nstrate t ha t avar iety of hum an mo nonu clea r cel l lines can be infected with dengue-2 virusand may be useful as models for the analysis of dengue virus-human cel l in-teractions in dengue virus infections.

IntroductionDengue virus infect ions are ser ious heal th problems in m any areas of the w or ld;Southeast Asia, Central and South Am erica [14] . Dengue viruses belong to thefamily Flaviviridae, genus Flavivirus and are t ransmit ted to humans by mos-quitoes. Dengue virus infections cause two forms o f syndro mes; den gue fever(DF) and dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS)


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92 I. Ku ran e et al.[ 1 2 ] . D F i s a s e l f- l im i t e d f e br il e d is e a se a n d a m o s t c o m m o n t y p e o f d e n g u e .I n s o m e s i t u a t i o n s p a t i e n t s i n f e c t e d w i t h d e n g u e v i r u s d e v e l o p l i f e - th r e a t e n i n gc o m p l i c a t i o n s s u c h a s h e m o r r h a g i c m a n i f e s t a t i o n s a n d s h o c k , w h i c h a r e c a l le dD H F / D S S . T h e p a th o g e n es i s o f D F a n d D H F / D S S h a s n o t b e en el u ci da te d .I t i s s p e c u l a t e d t h a t f o l l o w i n g f e e d i n g b y m o s q u i t o e s , t h e v i r u s e n t e r b l o o ds t r e a m v i a l y m p h a t i c s 1 -30] a n d p e r i p h e r a l b l o o d m o n o n u c l e a r ce ll s ( P B M C ) ,e s p e c ia l ly m o n o c y t e s , s u p p o r t v i r u s i n f e c ti o n s [ 1 6 ] . T h u s , s t u d y o f d e n g u e v i r u s-P B M C i n t e r a ct i o n is i m p o r t a n t t o u n d e r s t a n d t h e p a th o g e n e s i s o f d e n g u e v ir u si n f e c t i o n s . M a n y h u m a n n a o n o n u c l e a r c e l l l i n e s a r e a v a i l a b l e ; t h e r e f o r e , w ed e c i d e d t o e x a m i n e a v a r ie t y o f h u m a n m o n o n u c l e a r c el l l in e s t o d e t e r m i n ew h e t h e r t h e y c o u l d b e i n f e c t e d w i t h d e n g u e v i r u s a n d b e u s e f u l f o r f u t u r e s t u d i es .i n t h i s p a p e r w e a t t e m p t t o i n f e c t 2 3 h u m a n m o n o n u c l e a r ce ll l in e s, in c l u d i n gt e n m y e l o m o n o c y t i c c e ll li n e s, e i g h t B c e l l l i ne s a n d f i ve T c e l l l i n es w i t h d e n g u e -2 v ir u s . A l l t h e c e ll li n e s c a n b e i n f e c t e d w i t h d e n g u e - 2 v i r u s . Pe r s i s t e n t d e n g u e -2 v ir u s i n f e c t i o n is e s ta b l i s h e d u s i n g K 5 6 2 ( m y e l o m o n o c y t i c ) , R a j i ( B ), a n dHSB-2 (T) ce l l l ines .

Materials and methodsCell lines

We used hu ma n myelo mono cytic celt lines K562 [26], HEL 92-1-7 [28], JO SK-I,-K,-M,and -S [31], U937 [34], THP-1 [37], KG-1 [20], and H L-60 [8], h um an B cell lines Jiyoye[32], ARH-77 [6], IM-9 [10], Raji [9], HS-Sultan [17], CA 46 [-27], Daud i [18], andRamos [19], and human T cell lines Jurkat, CEM [11], HSB-2 [1], Molt3 and Molt4[29]. K562, HEL92-1-7, U937, THP-1, KG-1, HL-60, Jiyoye, ARH-77, IM-9, Rail, HS-Sultan, CA46, Daudi, Ramos, CEM, HSB-2, Molt3, and Molt4 were purchased fromAmerican Tissue Culture Collection (ATCC). JOSK-I, JOSK-K, JOSK-M, and JOSK-Swere provid ed by Dr. Mas atsugu Oh ta of Jichi Medical School, Japan. CA46, D audi, andJurk at were provided by D r. John Sullivan of University of Massachusetts Medical Center,Worcester, MA, U.S.A. All cell lines were mainta ined in R PM I 1640 me dium (FlowLaboratories, McL ean, VA, U.S.A.) supplemen ted with 10% fetal bovine serum (FBS)(GIBCO Laboratories, G rand Island, NY, U.S.A.), penicillin (100 U/m l) and strep tomycin(100 pg/ml).

Virus and antibodyDen gue-2 virus, New Gu inea C strain, was used for infection. Th e virus was received fromDr. Walter E. Brandt of the Walter Reed Army Institute of Research, Washington, DC.The virus has been passed in mouse brain and was then propagated in Aedes albopictus(mosquito) cells (C6/36) as previously described [22]. The titer of the virus pool used inthese experiments was 1 x 10 7 plaque formin g unit (p.f.u.)/mt in Vero cells using previouslydescribed meth ods [22]. Ascitic fluid from mice hyperimm unized with dengue-2 virus wasused as a source of anti-dengue-2 virus antibody. This an tibody was also supplied by Dr.Brandt. T he titer o f this antibody was 1,024 as determined by a plaque neutralization test[23]. Hy perim mu ne ascitic fluid was heated at 56 C for 30rain to d estroy comp lemen tactivity before use.

Infection of cells with dengue virusCells were washed once in RP M I 1640 containing 1% FBS and suspended at a concentrationof 2 x 105 cells in 0.1 ml. Th ey were the n incu bated with deng ue-2 virus or with d engue-2


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Dengue-2 virus infection of hu ma n cell l ines 93virus antibo dy complexes at 37 C for 2 h. These v irus-antibody complexes were preparedby ad ding 10 gl of anti-dengue-2 virus antibody at a dilution of 1 : 200 to 5 x 10 6 p.f .u, o fdengue-2 virus in 0.5 ml and were incubated at 4 C for 1 h. T he multiplicity of infection(m.o.i.) of the virus ino culu m was 5 p.f.u, p er cell. Cells were washed twice and resuspe ndedat a con cen tratio n of 2 x 105 cells per ml in RP M I 1640 con taining 10% FBS. Cells werestained for the presence of dengue-2 viral antigen by indirect immun ofluorescence (IF) at24, 48, and 72h as previously described [21]. The first antibody was the murine hyper-imm une ascitic fluid to dengue-2 virus and the second a ntibod y was a fluorescein isothio-cyanate (FITC)-conjugated sheep ant i -mouse immunoglobul in G ant ibody (Cappel Lab-oratories, Malvern, PA, U.S.A.).

Titration of dengue-2 virusThe am oun t o f infectious virus prod uced b y each cell l ine was determ ined by plaque assayin Vero cells. Intracellular virus was assayed after three freeze-thaw cycles with cells atconcen tration o f 1 x 106/ml [35, 36]. O ne-tenth m l o f serially 10-fold-diluted culture su-pern atant f luid was placed on Vero cell mon olayers in 24-well plates (Costar, Cam bridge,MA , U .S.A.) and incubated at 37 C for 2 h. The s uperna tant f luid was then remov ed, thecells were washed once with M EM containing 2% FBS, and 1 ml 1% agar (Difco Labo-ratories, Detroit , MI, U.S.A.) containing R PM I 1640, 10% FBS, 0.5% n on-essential aminoacids, 0.5% essential vitamins and 0.3% DE AE -dex tran was used as an overlay. The plateswere incubated at 37 C for 7 days, stained with neutral red a nd the plaques cou nted [22].

Establishment o f persistent infectionCells were infected with dengu e-2 virus at a m.o.i , o f 5 p.f .u./ml in the absence of an tibodyas described above an d were cultured at 2 105/ml in RP M I containing 10% FBS. Cellswere resuspended at a concen tration of 2 105/ml every 3 or 4 days. The percent o f dengueantigen-positive cells was examined weekly using IF.

Establishment o f dengue-2 virus antigen-positive Raft cell clonesDengue-2 antigen-positive Raji cell clones were established using a limiting dilution tech-nique. Persistently infected Raji cultures, 90% of which h ad dengue-2 virus antigen, werecultured at 0.5 cell/well (1 cell/2 wells) in 0.2 ml RPMI containing 10% FBS in 96 well flatbo ttom plates (Costar, Cam bridge, MA , U.S.A.). Cells were cultured for 3 weeks. Grow ingcells were exam ined for cytoplasmic dengue-2 virus antigen using IF.

ResultsAc ute infection o f huma n m ononuctear cell l ines with dengue-2 virus

Te n m ye l o m on oc y t i c c e ll li nes , e i gh t B c e l l l ine s , a nd f ive T c e l l l ine s we r e us e di n t he e xpe r i me n t s . A l l t he c e l l l i ne s c ou l d be i n f e c t e d w i t h de ngue - 2 v i r us i nt h e a b s en c e o f a n t i b o d y ( T a b l e 1). A n t i b o d y t o d e n g u e - 2 v i ru s a u g m e n t e d d e n -g u e - 2 v i r u s in f e c t i o n o f m y e l o m o n o c y t i c c el l l in e s d e t e r m i n e d b y I F a n d v i r u sa s sa y s . H o w e v e r , a n t i b o d y d i d n o t a u g m e n t i n f e c t i o n o f B o r T c el l l in e s. I nm y e l o m o n o c y t i c c el l l in es K 5 6 2 , H E L 9 2 - 1 - 7 , J O S K - I , a n d J O S K - M c el ls c o n -t a i n e d a h i g h p e r c e n t a g e o f a n t ig e n - p o s i t i v e c el ls , w h i l e H L - 6 0 , K G - 1 , a n dT H P - 1 c o n t a i n e d f e w e r a n t i g e n - p o s i t i v e ce ll s. I n B c e ll l in e s J i y o y e , A R H - 7 7 ,a n d I M - 9 c o n t a i n e d a h i g h p e r c e n t a g e o f a n t ig e n - p o s i t i v e ce ll s, w h i l e R a m o s ,D a u d i a n d C A 4 6 c o n t a i n e d f e w e r a n t i g e n - p o s i t i v e ce ll s. In T c e ll l in e s J u r k a ta n d C E M c o n t a i n e d a h i g h p e r c e n t a g e o f a n t i g e n - p o s i t i v e c el ls .


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94 I. Kur ane et al.Table 1. Dengue-2 virus infection of human mononuclear cell tines*

Cell line Ant ib ody ** % dengue-2 antigen-positive cells24 h 48 h 72 h

Virus titer(p.f.u./ml)at 48 h

Myelomonocyticcell lines

K562

HEL92-1-7

JOSK-I

JOSK-M

JOSK-S

JOSK-K

U937

THP-1KG-1

HL-60

+

+

+

90.8 95.9 99.0 5.057.8 76.5 99.0 4.535.8 35.4 41.8 2.322.8 26.5 37.2 1.537.5 34.3 18.5 5.0

7.7 4.1 1.7 5.7+ 28.6 23.0 8.4 2.3- 16.0 11.0 3.5 2.3+ 16.1 9.5 6.4 1.6- 6.3 3.8 3.4 1.5+ 15.1 14.1 3.9 4.0- 3.6 3.7 3.3 1.6+ 15.7 17.4 4.1 3.8- 0.6 1.3 0.9 4.2+

+7.5 6.8 9.8 1.10.4 0.5 0.5 2.02.4 1.7 0.9 1.31.4 1.3 0.5 3.0

+ 1.7 2.9 1.6 1.4- 0.2 0.4 0.3 3.5

x 106x 106x 10 5x 10 5x l0 sx 104x lOsx 10 5x 105x 105x 105x 105x 104x 103x 105x 104x 104x 103x 103x 102

B cell linesJiyoye

ARH-77

IM-9

Raji

HS-Sultan

CA46

+

+

+

+

+

+

82.6 81.4 81.7 4.082.4 86.4 85.0 5.028.4 45.7 30.8 5.425.0 30.9 31.4 3.922.6 22.7 17.2 3.530.3 25.3 13.1 3.110.2 6.6 11.7 8.57.8 9.6 13.7 9.58.8 6.9 8.3 4.04.8 5.6 7.5 3.01 . 5 1 . 5 1 . 3 4.02.5 2.2 1.0 2.0

x 104x 104x 105 105x 105x 105x 103x 103x 103 103X 103X 103


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Deng ue-2 virus infection of hu ma n cell lines 95Table I contmued

Cell l ine Antib ody ** % dengue-2 antigen-positive cells24h 48h 72h

Virus titer(p.f.u./ml)at 48 h

D au di + 1.1 1.1 0.8 1.0 x 10 2- 0.5 1.8 0.8 1.0 x 102

Ra mo s + 0.8 0.5 0.4 8.0 x 10- 0.5 0.5 0.3 5.0 x 10

T cell linesJu rk at + 50.0 39.2 39.7 6.6 x 104- 48.7 44,5 34.5 6.1 10 4C EM + 36.3 27.0 21.3 1.3 105- 34.0 23.4 18.5 1.5 x 105HS B-2 + 13.8 24.4 81.7 1.0 105- 19.5 28.5 84.0 3.0 x 105,M olt 4 + 8.2 16.8 41.7 1.1 x 105

- 7.6 15.3 44.4 1.7 x 105M ol t3 + 4.0 12.9 t4.0 7.5 x 10 3

- 8.1 12.0 21.3 1.3 x 104* Cells were infected with dengue-2 virus at a m.o.i, of 5 p.f.u./cell in the presence orabsence of antibody as described in Materials and methods** Anti-dengue-2 antibody was used at f inal dilution of 1 : 10,000. + Presence of antibody ,- absence of ant ibody

Establishment o f cell lines persistently infected with dengue-2 virusW e d e t e r m i n e d t h e a b i l i ty o f c e r ta i n c e ll s t o b e c o m e p e r s i s t e n t ly i n f e c t e d w i t hd e n g u e - 2 v i ru s . K 5 6 2 , R a j i, a n d H S B - 2 c el l l in e s w e r e i n f e c te d w i t h d e n g u e - 2v i r u s a t m . o .i , o f 5 p . f .u . / ce l l i n t h e a b s e n c e o f a n t i b o d y t o d e n g u e - 2 v i r us , a n dw e r e c u l t u r e d f o r 2 5 w e ek s . C e ll s w e r e r e s u s p e n d e d a t a c o n c e n t r a t i o n o f 2 x 105/m l t w i c e a w e e k . T h e p e r c e n t o f d e n g u e - 2 v i r u s a n t i g e n - p o s i t i v e ce ll s w a s a l m o s t100% one w e e k a f t e r i n f e c t i o n o f K5 62 ( F i g. 1) a nd HS B - 2 c e ll s ( F i g . 3 ), a ndt h r e e w e e k s a f t e r i n f e c ti o n o f R a j i c e ll s ( F ig . 2 ). A h i g h p e r c e n t a g e ( m o r e t h a n7 0 % ) o f d e n g u e - 2 v i r u s a n t i g e n - p o s i t i v e ce ll s w a s o b s e r v e d f o r 2 5 w e e k s . T h e s er e s u l t s d e m o n s t r a t e t h a t p e r s i s t e n t d e n g u e - 2 v i r u s i n f e c t i o n s w e r e r e a d i l y e s -t a b l i s h e d i n m y e l o m o n o c y t i c , B a n d T c el l l in e s.

W e m e a s u r e d d e n g u e - 2 v i r u s t i t e r s i n c u l t u r e s u p e r n a t a n t f l u i d s 2 2 w e e k sa f t e r t he i n f e c t i on . I n f e c t i ou s de ng ue - 2 v i r us a t t he t i t e r o f 2 . 6 x 102 p . f . u . / m l ,1 .8 x 10 ~ p . f . u . / m l , a n d 1 .0 x 102 p . f . u . / m l we r e de t e c t e d i n t h e s u pe r n a t a n tf l u i ds o f pe r s i s t e n t l y i n f e c t e d K 562 , HS B- 2 , a n d Ra j i , r e s pe c t i ve ly . I n t r a c e l l u l a r


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96 I. Ku ran e et al.100"

~ 80"

60 ".,>oc "~ 40-

0" t 3 c , ,

2010 !

K562

i i i ~ i L i i i i i i0 2 4 6 8 10 12 14 16 18 20 22 24 26Time af ter infect ion (weeks)

Fig. 1. Persistent infection of K562 cells infected with dengue-2 virus at a m.o.i, of 5 p.f.u./cell. Cells were resuspended at 2 x t05/ml every 3 or 4 days. Percentage of dengue-2 virusantigen-positive cells was det ermine d using IF

100 -

~_~ 80-

6 0

~- m 40o O

2 0 -Raji

t t , , i i i i ~ I0 2 4 6 8 10 12 14 16 18 20Time af ter infect ion (weeks)

3--0----,0

2'2 ' '4 26

Fig. 2. Persistent infection of Raji cells infected with dengue-2 virus at a m.o.i, of 5 p.f.u./cell. Cells were resuspended at 2 x 105/ml every 3 or 4 days. Percentage of dengue-2 virusantigen-positive cells was deter mined using IF

d e n g u e v i r u s w a s t h e n d e t e c t e d a f t e r t h r e e f r e e z e - t h a w c y c le s o f 1 x 10 6 i n f e c t e dce l ls . In t ra ce l lu la r v i rus t i te rs we re 2 .2 x 102 p . f .u . /m l a nd 4 .0 x 101 p . f .u . /m li n K 5 6 2 a n d H S B - 2 c el ls , r e s p ec t i v el y . I n t r a c e l l u l a r v i r u s w a s n o t d e t e c t e d ( < 10p . f . u . /m l ) i n p e r s i s t e n t ly i n f e c t e d R a j i c e l l s .Establishme nt of R aji cell clones persistently infected with dengue-2 virus usinga limiting dilution techniqueU s i n g a l i m i t in g d i l u t i o n t e c h n i q u e , w e t r ie d t o d e t e r m i n e w h e t h e r a s in g l ed e n g u e - 2 v i r u s a n t i g e n - p o s i t i v e c e ll c a n p r o l i f e r a t e t o p r o d u c e m u l t i p l e a n t i g e n -p o s i t i v e c el ls . P e r s i s t e n t ly i n f e c t e d R a j i c e l ls , 9 0 % o f w h i c h w e re a n t ig e n -p o s -


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Dengue-2 virus infection of human cetl lines 971 0 0

& 80-6 0 "

= ~ 40 'O

2 0 -

0

HSB-2

i i i i i ~ i i i i i i i0 2 4 6 8 10 12 14 16 18 20 22 24 26Time af ter infection" (weeks)

Fig. 3. Persistent infection of HSB-2 cells infected with dengue-2 virus at a m.o.i, of 5p.f.u./cell. Cells were resuspended at 2 x 10S/ml every 3 or 4 days. Percentage of dengue-2 virus antigen-positive cells was determined using IF

it ive, were cultured at a concentrat ion of 0.5 cel ls/well . The clonal l ines wereexamined for dengue-2 virus antigens after 3 weeks. 61 clones were established,54 (89%) of which con taine d 100% a ntigen-pos i t ive cel ls, and seven (11%) ofwhich c ontained no ant igen-posi tive cel ls .

DiscussionIn th is paper dengue-2 vi rus-human cel l in teract ions have been s tudied usinghum an mononu clear ce l l l ines. We have demons t ra t ed tha t a l l o f the 23 huma nmo non ucl ear cel l l ines including ten my elom ono cytic cel l l ines, eight B cel l l ines,and five T cel l l ines can be infected with dengue-2 virus. I t has been reportedthat h um an m onocy tes are the cel ls that predo mina nt ly suppor t infect ions wi thdengu e viruses [16]. I t has also been reported tha t B blast cells prep ared fromPBMC and Epstein-Barr v i rus (EBV)- t ransformed B lymphoblastoid cel l l inescould be infected with a dengue virus [35, 36], but that T blast cel ls preparedfrom P BM C or a T cel l l ine, Molt-4, could not be infected [36]. In our stud ywe exam ined f ive hum an T cell l ines , and al l could be infected wi th dengue-2virus. A h um an dengueviruses-specific T cel l clone, which was estab lished fromthe PBMC of a donor who had been previously infected wi th dengue-3 vi rusand has t he CD3 + , C D4 + , C D 8 - pheno type [24], cou ld be in fec t ed wi thdengue-2 virus (data n ot presented). Ou r success in infect ing hu m an T cel lsmay be due to the strain of dengue-2 virus or due to the higher m.o.i , we used.We used the New Guinea C st rain at a m.o. i , of 5 , whi le Theof i lopoulos et al .who reported that T cel ls could not be infected with dengue-2 virus used the16681 s t rain at a m.o. i, of 0 .05 [36] . Al th ough most rep or ts have s hown th atcell s infected wi th dengue vi rus have mono cyte- l ike morpho logy in vivo [3 , 4] ,


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98 I. Kurane et al.Scot t et al . isolated dengue virus f rom the non-adherent f ract ion of PBMCfrom patients [33]. Our results suggest that B and T cells may support thereplication of dengue-2 virus and pro bab ly oth er dengue viruses, especially ifthe B a nd T cells are ac tivated as they are in these cell l ines.I t has been repor ted that subneutral izing concentrat ions of ant ibody todengue viruses augment dengue virus infection of Fc~ receptor-positive cells[ 15]. This is due to the upta ke of viru s-antib ody complexes by Fc.~ receptor-posi t ive cells, and may contr ibute to the pathogenesis of severe complicat ionsof dengue [7, 13]. In the present stu dy all the myelo mo noc ytic cell l ines showedantibody -depend ent enhancem ent of dengue-2 virus infect ion, whereas we didno t ob serve enha nce me nt using B cell l ines or T cell l ines. I t has been repo rtedthat hum an B cells and some B cel l l ines have FcvR II s imilar to h um an m ono-cytes and m yelo mo noc ytic cell l ines [2] . Using K 562 cells, we have fou nd t hatFcvRII mediates ant ibody-dep enden t enhan ceme nt JR. Li t taua et al ., submit tedfor puN.] . However , i t has also been repor ted th at FcvRII o f B cell l ines maybe som ewhat dif ferent f rom FcvRII of monocytes and mye lomono cytic celll ines, because a mo noclo nal ant ibody to Fc rRII of K562 did no t react withFC rR II of B cel l l ines, Daudi and Raj i [25] . This may explain the absence ofant ibody-d epende nt enhance men t of dengue-2 virus infect ion using B cell lines .Studies of ant ibody-depe ndent enhancem ent have been mainly performed usingU937 cells [5] . Ou r results suggest tha t o ther m yelo mo noc yte cell l ines, suchas THP-1 and JOSK-I , are also useful for the s tudy of ant ibody-dependentenhan cem ent of v irus infect ion with dengue viruses and perhaps other f lavi-viruses.In this study we established persistent dengue-2 virus infections in a mye-lom ono cyti c cell l ine (K562), a B cell l ine (Raji) and a T cell l ine (HSB-2).Persistent dengue-2 virus infection of R aji cells has bee n reporte d [35], an d wepreviously used persistently infected R aji cells as targets in cell kill ing assays[22] and as inducer of in terferon ( IFN) f rom PB MC [23]. The present resul tsshow tha t h um an mye lomon ocytic and T cel ls also suppor t pers is tent dengue-2 virus infect ions . The in tera ct ion between dengue-2 virus and myelom onocytic ,T and B cells and the mechanism of virus persistence are interesting subjectsto be elucidated. Raji cell clones were established and the percent of antigen-posi tive and -negative clones was found to be s imilar to the f requency of dengue-2 virus antigen-positive and -negative cells in the original culture. All of thecells in these antigen-po sitive clones contin ued to con tain dengue-2 virus antigen,and all of the cells in the a ntigen-neg ative clones contin ued to be dengue-2 virusantigen-negative. These results suggest that dengue-2 virus antigen -positive cellsmay prol i ferate a nd produce ant igen-posi t ive progeny cells.

These experiments demonstrate that dengue-2 virus readily establishes per-sistent infections in hu m an T, B, and my elom ono cytic cell l ines in vitro. I t isconceivable that pers istent infect ions may occur in vivo a nd that infected cellsmay cont inue to s t imulate dengue virus-specif ic memory T lymphocytes toma intain dengue virus-specif ic immunity .


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Dengue-2 virus infection of human cell lines 99Acknowledgement

This work was supported by grants from the U.S. Army Medical Research and DevelopmentCommand (DAMD 17-86-C-6208), and from the National Institutes of Health (NIH-T 32-AI07272). The opinions contained herein are those of the authors and should not beconstrued as representing the official policies of the Department of Army or the Departmentof Defense.

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Den gue-2 virus infection of hu m an cell lines 10137. Tsuchiya S, Yamabe M, Yamaguchi Y, Kobayashi Y, Konno T, Tada K (1980)Establishment and characterization o f a hu ma n acute m onocy tic leukemia cell line(THP-1). Int J Cancer 26: 171 -t76

Auth ors' address: F. A. Ennis, M.D., D ivision of Infectious Diseases, Dep artm ent ofMedicine, University of Massachusetts Medical Center, Worcester, M A 01655, U.S.A.Received July 26, 1989

dengue-2 virus infection of human mononuclear cell lines

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