a rat model of human t lymphocyte virus type i

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A R a t M o d e l o f H u m a n T L y m p h o c y te V i ru s T y p e I

HT LV-I) In fec tion . 1 . H um oral An t ibod y Respon se ,

Provirus Integrat ion, and HTLV-I-associated

Myelopathy/Tropica l Spast ic Parapares is - l ike

M yelopa thy in Seronega t ive HT LV-I Carr ier Rats

B y N o b u h i s a I s h ig u r o ,* ~ M a s a k a z u A b e ,* K a z u t o s h i S e to ,*

H i r o h a r u S a k u ra i ,* H i t o s h i I k ed a ,* A k e m i W a k i s a k a , *

T a k e h i r o T o g a s h i ,~ M a s a t o s h i T a t e n o , S a n d T a k a s h i Y o s h i k i *

Fr o m th e D ep a r tm en t s o f Pa th o lo g y a n d I Ped ia tr i c s, H o leka id o U n iver s it y S ch o o l o f M e~ c in e ;

a n d th e S D ep a r tm en t o f Pa th o lo g y , S a p p o r o C i t y G en er a l H o s p i ta l, S a p p o r o 0 6 0 , Ja p a n

S t l m m ~ ' y

H u m an T l y m p h o cy t e v i ru s t y p e I (HTLV-I ) can b e t r an sm i t t ed i n to sev era l i n b red s t ra ins o f

n ew b o rn an d ad u l t r a t s b y i n o c u l a t i n g n ew ly est abl ish ed H TLV -I - im m o r t a l i zed r a t T ce ll li n es

or the h um an T cel l l ine MT -2 . Th e t ransm ission eff iciency exceeds 80 , regard less o f s train

d i f ferences or the age at t ransmission . The product ion of an t i -HTLV-I an t ibodies s ign i f ican t ly

d if f er s am o n g th e s t r ain s an d d ep en d s o n t h e ag e a t t h e t im e o f t r an sm i ss io n . Ra t s n eo n a t a l l y

inoculated w i th HTL V-I-posit ive rat o r hu m an cel l s generally beco me seronegat ive HT LV-I carr iers

t h ro u g h o u t t h e i r l iv es , wh ereas a d u l t r a t s i n o cu l a t ed wi th HTLV-I -po s it iv e ce ll s a t 1 6 w k o f

ag e b eco m e se ro p o s i ti v e HTLV-I ca r ri e rs . T h e HTLV-I p ro v i ru s g e n o m e i s p resen t i n a lm o s t

al l organs, regard less o f w he the r the carr iers are seronegat ive or seroposi t ive. Acc ord ing to a n t ibody

t i ters to HTLV -I , there are th ree group s of inbred rat s t rains: ACI, F344 , an d SDJ (h igh responders) ;

W K A , B U F , a n d L E J ( i n t e rm e d i a t e r e s p on d e rs ) ; a n d L E W ( l o w r e sp o n d e r ). T h r e e o f th r e e 1 6 -

m o -o ld se ro n eg a t iv e HTLV-I ca r t ie r r a ts o f th e W K s t r a in d ev e lo p ed sp ast ic p a rap ares is o f t h e

h in d l eg s . Ne u ro p a th o lo g i ca l ex am in a t i o n s r ev ea l ed t h a t t h e l e s io n s we re co n f in ed p r im ar i l y t o

the lateral and a n ter io r fun icu l i o f the sp inal cord . B oth m yel in and axons w ere ex tensively dam aged

in a sy m m et r i ca l f a sh io n , an d i n f i l t r a t i o n wi th m ass iv e fo am y m acro p h ag es was ev id en t . Th e

m o s t sev ere l e sio ns w ere a t l ev e l s o f t h e t h o rac i c co rd an d c o n t i n u ed f ro m th e ce rv ica l t o t h e

lum bar area. Th ese h is top atho log ical features as wel l as clin ical sym ptom s largely para l ld f ind ings

in h u m an s wi t h HT LV-I -assoc i at ed m y e lo p a th y / t ro p i ca l sp ast ic p a rap ares is (H A M /TS P ) . Th ese

HTLV-I ca r r i e r r at s , i n p a r t i cu l a r t h e W K A ra t s d esc r ib ed ab ov e , can serv e as a u se fu l an im al

m o d e l fo r i n v es t i g a t i n g v i ru s -h o s t i n t e r ac t i o n s i n t h e e t i o p a th o g en es i s o f HTLV-I - r e l a t ed

i m m u n o l o g i c a l d i se a se s , p a r ti c u la r l y H A M / T S P .

TLV -I , w hic h was f i rs t iso lated an d characterized f rom

a p a t i en t w i th c u t an eo u s T ce ll l y m p h o m a (1 ) , i s d i s-

t i n c t f ro m o th er h u m an v i ru ses an d f ro m an im al r et ro v iru ses ,

in nucleic acids (2) , m ajor c ore pro teins (3) , an d reverse t ran-

scr ip tase (4). Fur th er ex tensive s tu dy show ed tha t HTLV -I

i s t h e o n co g en i c ag en t o f ad u l t T ca ll l eu k em ia (ATL) 1 (5 ).

I t was r ep o r t ed t h a t HTLV-I i n fec t i o n i s a l so r e l a t ed wi th

im m u n o lo g i ca l diseases such as H TLV -I-associatedm y d o p a t h y /

1 Abbreviations used in th is paper: A T L , a d u l t T c e l l l e uk e m i a; B r d U r d ,

5-bromo-2'-deoxyuridine; CN S, central nervous system; CSF , cerebrospinal

f lu id ; HAM /TS P, HT LV-I-associa tedmyelopathy/tropical spastic parapatesis.

t r o p i ca l sp as t i c p a rap ares i s (HAM/TS P ) (6 -9 ) , HTLV-I -

assoc i at ed b ro n ch o p n eu m o n o p a th y HA B) (1 0 ), HTLV-I -asso-

d a t ed a r t h ro p a th y (H AA P ) (1 1) , an d S j6 g ren sy n d ro m e (1 2 ).

To an a ly ze m e ch an i sm s i n v o lv ed i n t h e d ev e lo p m en t o f th ese

d i seases , an an im al m o d e l wo u ld b e u se fu l HTLV-I h as b een

rep o r t ed t o h av e im m o r t a l i z i n g e f fec ts o n h u m an (1 3 ), s im ian

(14) , cat (15), an d rabbi t (16) lym phoc ytes . Yoshik i and co l -

l eag u es (1 7 , 1 8) fo u n d t h a t H TLV-I can i n fec t an d im m o r -

talize rat T cel ls in v i t ro . Yoshik i e t a l . (19 ) and S uga et a l .

( 20 ) s u g g e s t e d t h a t H T L V - I c a n b e t r a n s m i t t e d in t o W K A

and F344 rats in v ivo .

W e have no w t ransm it ted HTL V-I in to several nbre d s t rains

o f n ew b o rn a n d ad u l t r at s , an d s t r a in d i ff e ren ces i n h u m o ra l

an t i b o d y r esp o nses ag a in s t HTLV -I an d t i s su e d i s t r i b u t i o n

9 8 1

J. Exp . Med. 9 The Rockefe l ler Un iversi ty Press 9 0 0 2 2 - 1 0 0 7 /9 2 /1 0 /0 9 8 1 /0 9 $2.00

V o l u m e 1 76 O c t o b e r 1 9 9 2 9 8 1 -9 8 9

Published October 1, 1992


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o f t h e H T L V - I p r o v i ru s g e n o m e w e r e i n v e s t ig a t e d . D e v e l o p -

m e n t o f H A M / T S P - l i k e m y e l o p a t h y i n H T L V - I c a rr ie r ra ts

w a s a l s o m o n i t o r e d . T h i s i s t h e f i rs t d e s c r i p t i o n o f a n a n i m a l

m o d e l f o r t h e H A M / T S P t h a t o c c u rs i n h u m a n s 9

ater ia l s and ethod s

Cel l L ines . Fou r HT L V - I - imm or ta l i ze d r a t ce ll li nes, L ewis -S1 ,

W K A - S 1 , F 3 4 4- $1 , a n d A C I - S 1 , w e r e n e w l y e s t a b l is h e d b y c o c u l -

t i v a t i n g s p l e e n c e l l s o f f o u r i n b r e d r a t s o f L E W / H k m ( L E W ) ,

W K A H / H k m ( W K A ) , F 34 4/ SI c( F3 44 ), an d A C I / H k m ( A C I )

s t ra i n s w i t h A T L c e ll s f r o m a t y p i c a l A T L p a t i e n t a f t e r t r e a t i n g

t h e m w i t h 5 - b r o m o - 2 ' - d e o x y u r i d i n e ( B r d U r d ) , a s d e s c r ib e d e ls e -

w h e r e ( 1 7 ). T h e c e ll s w e r e m a i n t a i n e d i n R P M I 1 6 4 0 s u p p l e m e n t e d

wi th 10% hea t - inac t iva t ed FC S, pen ic i l l i n (100 IU/ml ) , and s t r ep -

t o m y c i n ( 1 0 0 / z g / m l ) , a n d w e r e f r e e f r o m e x o g e n o u s I 1 . - 2 .

T h e H T L V - I p roducer h um an ce l l l ine , M T -2 , was k ind ly p rov ided

b y D r . I . M i y o s h i ( D e p a r t m e n t o f M e d i c i n e , K o c h i M e d i c a l C o l -

l ege , Koch i , J apan) ( 13) .

Antibodies. R T H - 7 , R 1 - 3 B 3 , a n d R1 -1 0 B5 w e r e m A b s t h a t

r e ac t ed w i t h t h e r a t h o m o l o g u e s o f C D 4 , C D 5 , a n d C D S , r e sp e c -

t iv e ly , a n d w e r e g e n e r o u s l y p r o v i d e d b y D r s . A , M a t s u u r a a n d K .

Kik uch i (Sapporo M edica l C o l l ege , Sappo ro , J apan ) ( 21 , 22) . Ant i -

L e u - 3a (B e c t on D i c k i n s o n & C o . , M o u n t a i n V i ew , C A ) a n d A R T

1 8 ( 2 3 ) ( B o e h r i n g e r M a n n h e i m , M a n n h e i m , G e r m a n y ) w e r e t h e

mA bs us ed aga ins t hu m an C D 4 and r a t IL -2 r ecep to r , r espec tive ly .

Po lyclona l an t ibod ies aga ins t r a t and hum an Ig were purchas ed f rom

O r g a n o n T e k n i k a C o . ( D u r h a m , N C ) .

Animals and Transmiss ion o f H TLV I . I n b r e d f e m a l e W K A ,

L E W , A C I , S D J / H k m ( S D J ) , B U F / H k m ( B U F ) , a n d L E J / H k m

( L EJ ) r a ts w e r e o b t a i n e d f r o m t h e I n s t i t u t e f o r A n i m a l E x p e r i m e n t

(Ho kka id o Unive r s i ty Schoo l o f M edic ine , Sapporo , J apan) . I nb red

female F344 r a t s were purchas ed f ro m SL C J apan (Sh izuoka, J apan ) .

107 L ewis -S1 and W KA -S1 ce l l s, wh ich w ere t r ea t ed wi t h

M i t o m y c i n C ( M M C ; K y o w a H a k k o , C o . , T o ky o , J a p a n ) , a t a c o n -

c e n t r a t i o n o f 2 5 r a g / m 1 f o r 3 0 m i n a t 3 7 ~ w e r e in o c u l a t e d i n t o

t h e p e ri to n e a l c a v i ty o f n e w b o r n L E W a n d W K A r a ts w i t h i n 2 4 h

af t e r b i r th , r espec t ive ly . 107 M T -2 ce ll s were inocu la t ed in to the

p e r it o n e al c a v it y o f n e w b o r n L E W , W K A , F 3 4 4 , a n d A C I r a ts

wi th in 24 h a f t e r b i r th . 107 M T -2 ce ll s were in j ec t ed in to the t a i l

v e in o f 1 6 -w k - o ld L E W , W K A , F 3 4 4 , A C I , S D J , B U F , a n d L E J

ra t s twice , a t 2 -w k in t erva ls . HT L V- I - in f ec t ed r a t s were ma in ta ined

under t he P3 l eve l .

T o i d e n t i fy t h e a n t i - H T L V - I a n t i b o d y a n d H T L V - I p r o v i r u s g e -

h o m e , p e r i p h e r a l b l o o d w a s t a k e n f r o m t h e t a i l v e i n .

Detec tion o f An t ibod ies aga ins t H TL V I A n t igen s . T h e t i t e r o f

a n t i b o d y a g a i n s t H T L V - I a n t i g e n s i n t h e s e r u m a n d c e r e b r o sp i n a l

f luid (CSF) was determ ined by the par ticle agg lut inat io n tes t (Serodia

9 H T L V - I ; F u ji R e b i o I n c . , T o k y o , J a p a n ) .

PolyraeraseChain Reaction. D N A was i s o la t ed f rom F ico l l-Paque

s e p a r a t e d P B M C o r o r g a n t i s s u e s u s i n g S D S a n d p r o t e i n a s e K

methods (24) . PC R was ca r r i ed ou t as des c r ibed (25) . B r i e f ly , 0 . 5

# g o f e x t ra c t e d D N A w a s s u b j e c te d t o 3 5 c yc le s o f P C R a m p l i fi c a -

t i o n u s i n g T a q p o l y m e r a s e (T a k ar a S h u z o C o . , K y o t o , J a p a n ) a t

9 4 ~ f o r 1 m i n , 5 8 ~ f o r 2 r a i n , a n d 7 2 ~ f o r 3 m i n . T h e p r i m e r s

w e r e 5 ' - 2 3 2 8 G C C A A A C C C A A G A T C A C T T T A A G C 2 3 5 1 - 3 '

( se ns e) an d 3 '- 2 5 8 8 G G A A A T T T G G T C T T G C G G A G 2 6 0 7 -5 ' (a n -

t is ense) f o r 10ol r eg ion amp l i f i ca t ion (Syn the t i c Gen e t i cs I nc . , San

D i e g o , C A ) , a n d 5 '- 7 35 8 C G G A T A C C C A G T C T A C G T G T 7 3 7 7 -3 '

( se n se ) a n d 3 '- 75 1 6 G C T A C C ' I G C G C A A T A G C C G A G 7 4 96 -5 ' ( a n -

t i se n s e ) f o r p X r e g i o n a m p l i fi c a t io n ( 2 6 ). E a c h P C R p r o d u c t w a s

s ub jec t ed to e l ec t rophores i s on a 2% agaros e ge l and t r ans f e r r ed

t o n y l o n m e m b r a n e . T h e m e m b r a n e w a s h y b ri d iz e d w i t h r a di o ac -

t iv e ly h b e le d o l ig o n u c le o ti d e p ro b e s: 3 ' - 24 9 8 C G T T T T C C C G G C -

G G A C A T T A G A A C G G T T A T G T C C G C G G T C 2 5 3 7 -5 ' ( an ti se n se )

f o r p o l re g io n a n d 3 ' -7 4 4 7 A G G T G A T C T G A T G C T C T G G A C A G -

G T G G C C A G T A G C ~ C G 7 4 8 6 - 5' (a n ti se n se ) f o r p X r e g i o n . T h e

m e m b r a n e w a s w a s h e d in 2 x S S C, 0 . 2 % S D S a t r o o m t e m p e r a -

t u r e f o r 1 5 m i n , t w i c e , a n d i n 0 . 2 x S S C , 0 . 2 % S D S a t 5 5 ~ f o r

15 r a in , twice . T he s pec i f i c ampl i f i ed bands were de t ec t ed by ex-

p o s u r e t o K o d a k X A K f i lm a t - 7 0 ~ F o r t h e s t an d a rd co n t ro l ,

t h e d e t e c t i o n l im i t o f t h e P C K w a s u p t o 5 - 6 .

Lig ht Microsco~. I n a d d i t i o n t o r o u t i n e h e m a t o x y l i n a n d e o s in

(HE ) s t a in , L ux o l fas t b lue (L FB ) , B od ian , and H olze r s ta ins were

us ed .

R e s u l t s

C h a r a c te r iz a t io n o f N e w l y E s t ab l is h e d H T L V I i m m o r t a l i z e d

R a t T C e l l L i n e s. I n f o u r n e w l y e s t a b l i s h e d H T L V - I - i m -

m o r t a l i z e d r a t c e ll l i n es , it w a s c o n f i r m e d t h a t a t l e a s t o n e

f u ll le n g t h o f H T L V - I p r o v i r u s g e n o m e w a s i n t e g r a t e d , t r a n -

T a b l e 1 . Characterization o f HTLV I immor ta l i zed Ra t Ce l l L ines

H u m a n H T L V - I

R a t m a r k e r * m a r k e r *

P r o v i r u s P r o t e i n

C e l l l in e C D 5 C D 4 C D 8 I L - 2 R s lg C D 4 s lg g e n o m e m R N A s yn th es is ~

L ewis -S1 + + - + - - - + + +

F344-$1 + + - + - - - + + +

W K A - S 1 + + - + - - - + + +

A C I - S 1 + + - + - - - + N D +

* Cel l surface markers were detected by indirect immune f luorescence us ing FAC Scan (Becton Dickinson & Co. ) . Detai ls of ant ibodies are in Mater i -

als and M ethods .

* H TL V- I gag proteins were detected with an HTL V-specif ic mA b (Epi tope Inc. , Beaver ton, OR.) by W estern blo t analys is.

982 A R at M odel o f Hum an T L ymphocy te Vi rus T ype I I n fec t ion



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scr ibed, and expressed , us ing Sou thern , N or th ern , and Western

Bl o t s a n a ly s es , r e s p e c t iv e l y . An a l y s e s o f c e l l s u r f a c e ma r k e r s

s h o w e d t h e s e c e ll l in e s t o b e p o s i ti v e f o r ra t C D 4 a n d C D 5 ,

a n d t o b e n e g a t i v e f o r r a t C D 8 a n d h u m a n m a r k e r s . T h e y

a l s o e x p r e s s e d t h e r a t I L - 2 r e c e p t o r . T h e r e s u l t s a r e s u m ma -

r i z e d i n T a b l e 1 . M o r p h o l o g i c a l l y , n u c l e a r p o l y m o r p h i s m

r e s e m b l i n g A T L c el ls w a s s e e n in a l a rg e p r o p o r t i o n o f W K A -

1 a n d A CI - S I c e ll s. T h e o t h e r c e l l l i n e s a ls o c o n t a i n e d c e l ls

w i t h n u c l e a r p o l y m o r p h i s m , a l b e it t o a l e ss e r e x t e n t . O n l y

i n t h e A CI - S1 c e l l l i n e we r e e x t r a c e l l u l a r t y p e C v i r u s p a r t i -

c l e s s o me t i me s e v i d e n t , a s s e e n e l e c t r o n mi c r o s c o p i c a l l y .

Infectious Transmission of H TL V I into Newborn and A dult

Ra ts . L e w i s -S 1 a n d W K A - S 1 c e ll s w e r e i n j e c te d i n t o n e w -

b o r n L E W a n d W K A r ats . P u ri fi ed D N A f r om P B M C w a s

s u b j e c t e d t o P C R a m p l i f ic a t io n , u s i n g b o t h p o l a n d p X

p r im e r s . S a m p l e s i n w h i c h P C R r e su l ts w e r e p o s i ti v e f o r b o t h

p o l a n d p X p r i m e r s w e r e c o n s i d e r e d t o b e p o s i t i v e . T h e

H T L V - I p r ov i ru s g e n o m e w a s e v i de n t in P B M C f r o m 1 6 o f

1 8 L E W r a ts , a n d al l o f th r e e W K A r a ts a t 4 - 6 m o a f t e r

b i r t h ( T a b le 2 ) . W e c o n s i d e r e d t h a t L e w is -S 1 a n d W K A - S 1

c e ll s p r o d u c e d H T L V - I v i ru s a n d h a d i n f e c ti v i t y p o t e n ti a l f o r

t h e n e w b o r n r a ts , h o w e v e r , a n ti b o d i e s a g a in s t H T L V - I w e r e

n e v e r d e t e c t e d in t h e s e r a t s a t 1 - 7 m o a f t e r b i r t h . W h e n MT - 2

c e ll s w e r e i n je c te d i n to n e w b o r n L E W , W K A , F 3 4 4 , a nd

A C I r a ts , t h e H T L V - I p r o v ir u s g e n o m e w a s d e t e c te d i n P B M C

f r o m a ll o f e i g h t L E W r a ts ( 1 0 0 % ) , e i g h t o f ni n e W K A r at s

( 8 9 % ) , 1 6 o f 2 0 F 3 4 4 ra t s ( 8 0 % ) , a n d s e ve n o f e i g h t A C I

r a ts ( 8 8 % ) . O n l y 2 o f 1 6 F 3 4 4 r at s s e r o c o n v e r te d f o r a n ti -

HT L V- I a n t i b o d i e s , t h e t i t e r s b e i n g b e t we e n 1 : 8 a n d 1 : 1 6 .

N o n e o f t h e o t h e r r a ts h a d a n t i -H T L V - I a n t i b o d i e s ( T ab le 2 ) .

W h e n M T o 2 c el ls w e r e i n j e c te d i n t r a v e n o u s l y i n t o s ev e n

d i f f e r e n t s t r a i n s o f a d u l t r a t s , a ll f iv e ( 1 0 0 % ) i n e a c h s t r a i n

( e x c e p t f o r F3 4 4 , s i n c e o n l y o n e r a t wa s t e s t e d ) c a r r i e d t h e

H T L V - I p r o v i r u s g e n o m e i n t h e i r D N A e x t r a c t e d f r o m

P B M C s 4 m o a f t e r i n j e c t i o n ( T a b l e 3 ) . A n t i - H T L V - I a n t i -

b o d i e s we r e d e t e c t e d i n a l l t h e s e r a t s a s e a r l y a s 3 wk a f t e r

i n j e c t i o n , a n d t h e t i t e r s r a n g e d b e t w e e n 1 : 8 a n d 1 :8 ,1 9 2 t h e r e -

a ft er . T h e s p e c i f ic i t y o f t h e s e a n t i b o d i e s a g a i n s t HT L V- I a n -

t i g e n w a s c o n f i r m e d b y e v i d e n c e o f H T L V - I - sp e c i fi c g a g p r o -

t e i n s ( p 1 9 , p 2 4 , p 5 3 ) , o b t a i n e d u s i n g We s t e r n B l o t s ( d a t a

n o t s h o w n ) .

Hum oral Immun e R esponse against HT LV I. H u m o r a l a n -

t i b o d y re s po n s es a g a i n st HT L V- I we r e c o mp a r e d . Ad u l t L E W ,

W K A , F 3 4 4 , A C I , S D J , B U F , a n d L E J r a ts w e r e g iv e n in -

t r a v e n o u s i n j e c t i o n s o f 1 0 7 M T - 2 c e ll s, t w i c e , a s d e s c r i b e d

a b o v e , a n d a n t i - HT L V - I a n t i b o d i e s i n t h e s e ra we r e me a s u r e d

a t 2 , 3 , 7 , 1 3 , 2 1 , 2 8 , 4 0 , a n d 4 9 w k a f t e r t h e f ir s t in j e c t i o n .

As s h o wn i n F i g . 1 , t h e a n t i b o d i e s we r e d e t e c t a b l e a s e a r l y

a s 2 - 3 we e k s a f t e r t h e i n j e c t i o n , a n d a p l a t e a u wa s r e a c h e d

a t 1 2 w k . A n t i b o d y t i t e rs t e n d e d t o d e c l in e s l i g h t ly a f t e r 2 8

w k b u t w e r e h i g h a t 4 9 w k . A C I a n d S D J ra t s w e r e c o n s i d-

e r e d t o b e h i g h r e s p o n d e r s , a s t h e t i t e r o f a n t i - HT L V- I a n t i -

b o d i e s w a s h ig h e r t h a n f o r o t h e r s t r ai n s, a n d L E W r a ts w e r e

l o w r e s p o n de r s , w i t h l o w a n t i -H T L V - I a n t ib o d i e s. F 3 4 4 r a ts

p r o d u c e d a h i g h l e v el o f a n ti - H T L V - I a n t i b o d i es f o r u p t o

1 3 w k a f t e r t h e f ir s t i n j e c t i o n . Fu r t h e r m e a s u r e me n t o f a n ti -

H T L V - I a n t i b o d y w a s d o n e i n o n l y o n e o f F 3 4 4 r a t s m a i n -

t a i n i n g a h i g h l e v el o f a n t i b o d y t i t e r o f 1 : 4 ,0 9 6 a t 2 8 wk .

T h e F 3 4 4 s t ra i n w a s t h u s c o n s i d e re d t o b e a h i g h r e s p o n d e r ,

l i k e t h e AC I a n d S DJ s t ra in s . Ra t s o f t h e o t h e r t h r e e s t r a in s

p r o d u c e d a n t i - H T L V - I a n t i b o d i e s w i t h i n t e r m e d i a t e t i t e r s ,

a n d we r e c l a s s e d a s i n t e r me d i a t e r e s p o n d e r s . T h e d i f f e r e n c e

i n h u m o r a l r e s p o n s e a g a i n s t HT L V- I i s s t a t is t i ca l l y s i g n i fi c a n t

(s tudent ' s t t es t ) .

Tissue Distribution of the HT LV I Provirus Ge nom e. Five fe-

ma l e F3 4 4 r a t s in j e c t e d w i t h MT - 2 c el ls i n t h e n e o n a t a l p e r i o d

w e r e k i ll ed a t 5 0 w k . D N A s e x t ra c te d f r o m P B M C a n d ti ss ue s

f r o m e a c h o r g a n w e r e s u b j e c te d t o P C K a m p l i f ic a t io n , u s i n g

p X p r i m e rs . E a c h D N A s a m p l e w a s P C K a n a ly z e d m o r e t h a n

t w i c e , a n d t h e s a m p l e i n w h i c h t h e H T L V - I p r o v i ru s g e n o m e

w a s r e p r o d u c i b l y d e m o n s t r a t e d w a s j u d g e d a s p o s i ti v e . T h e

H T L V - I p r o v i r u s g e n o m e w a s d e t e c t e d i n a l m o s t a ll o r g an s

e x a m i n e d , i n c l u d in g t h e t h y m u s ( 5 / 5 ) , k i d n e y ( 5 / 5 ) , s p l e e n

( 4 / 5 ) , s u b m a n d i b u l a r g l a n d ( 4 / 5 ) , l y m p h n o d e s ( 5 / 5 ) , a n d

P B M C ( 5 / 5 ) ( T a b l e 4 ) . T h e P C R r e s u l t i s s h o w n i n F ig . 2 .

Development o f HA M /TS P I ike Myetopathy.

E i g h t o f n i n e

W K A r a ts n e o n a t a ll y i n o c u l a te d w i t h M T - 2 ce ll s b e c a m e

HT L V- I c ar ri er s y e t h a d n o d e t e c t a b l e a n t i- HT L V - I a n t i b o d i e s

T a b l e

2 . Transmission of H TL V I into Newborn Rats

Ra t M H C Ce l ls i n je c te d An t i - HT L V- I P r es e nc e o f HT L V - I

Strain RT 1 n (107) antibodies* provirus genom e*

L E W 1 1 8 L E W - S1 0 / 1 8 ( 0 % ) 1 6 / 1 8 ( 8 9 % )

W K A k 3 W K A - S 1 0 / 3 ( 0 % ) 3 / 3 ( 10 0 % )

L E W 1 8 M T - 2 0 / 8 ( 0 % ) 8 / 8 ( 1 0 0% )

W K A k 9 M T - 2 0 / 9 ( 0 % ) 8 / 9 ( 8 9 % )

F3 4 4 I v1 2 0 MT - 2 2 / 2 0 ( 1 0 %) 1 6 / 2 0 ( 8 0 %)

A C I a v l 8 M T - 2 0 / 8 ( 0 % ) 7 / 8 ( 8 8 % )

Anti-HTL V-I antibodies we re measured at 1 and 7 mo after birth.

* Presence of the HTL V-I provirus genome was determined 4--6 mo after birth.

983 Ish igu ro t al .



4/9

able 3 . Transmissionof HTL V I into Adul t Rats

R a t M H C A g e a t f ir st A n t i - H T L V - I P r es e nc e o f H T L V - I

S t r a in R . T 1 n M T - 2 i n j e c ti o n * a n t i b o d ie s p r o v i r u s g e n o m e *

w k

L E W 1 5 1 6 5 / 5 5 / 5

W K A k 5 1 6 5 / 5 5 / 5

F 3 4 4 l v l 5 1 6 5 / 5 1 / 1

A C I a v l 5 1 6 5 / 5 5 / 5

S D J u 5 1 6 5 / 5 5 / 5

B U F b 5 1 6 5 / 5 5 / 5

LEJ j 5 16 5 /5 5 /5

* 107 ce l l s o f MT-2 were in jec ted in to the ta i l ve in twice a t 2 -wk in tervals .

t Puri f ied DNA from PBMC was subjec ted to PCR ampl i f ica t ion a t 4 mo after in jec t ion .

T a b l e 2 ) . F o u r r a t s w e r e k i l l e d a t 4 m o , a n d t i s s u e d i s tr i b u -

t i o n o f p r o v i r u s g e n o m e s w a s e a am i n e d . O n e o f t h e re m a i n i ng

f o u r m a l e r a ts d i e d a t 1 2 m o o f u n k n o w n c a u s es a n d a n a u -

t o p s y w a s n o t d o n e . A t 1 6 m o , t w o o f th r e e r a ts w e r e f ir s t

s e e n t o h a v e s p a s ti c pa r ap a re s is o f t h e h i n d l e g s , a n d 2 w k

la te r , the remain ing had s imi la r sym ptom s. Th e spas t ic parapa-

r e s i s p r o g r e s s e d w i t h i n 3 t o o , a n d a l l t h r e e r a t s c o u l d h a r d l y

m o v e b e c a u s e o f t h e m u s c u l a r a t r o p h y o f th e h i n d l e gs . A

m o s t s e v e r el y a f fe c te d r a t, W K A n o . 5 4 , a n d a m o d e r a t e l y

a f f e c te d r a t , W K A n o . 5 7 , w e r e h is t o l o g i c a l l y e x a m i n e d .

P r o v i r u s g e n o m e s i n t h e c e n t r a l n e r v o u s s y s t e m C N S ) t is s ue s ,

a s w e l l a s i n t h e P B M C , a n d a n t i - H T L V - I a n t i b o d i e s i n t h e

s e r u m a n d C S F , w e r e a l so e x am i n e d . T h e r e m a i n i n g o n e w a s

f o u n d d e a d a n d a n a u t o p s y w a s n o t d o n e . M a c r o sc o p i c al ly ,

t h e b r a in s o f b o t h r a ts a p p e a r ed n o r m a l . T h e s p in a l c o rd o f

W K A n o . 5 4 s h o w e d s l i g h t a tr o p h y , p a r ti c u l a rl y i n th e t h o -

r a c ic r e g i o n , w h i l e t h a t o f n o . 5 7 a p p e a r e d t o b e n o r m a l .

O n m i c r o s c o p i c e x a m i n a t i o n o f n o . 5 4 , b o t h m y e l i n a n d a x o n s

i n t h e l a te r a l a n d a n t e r i o r f u n i c u l i w e r e e x t e n s i v e l y d a m a g e d

i n a s y m m e t r i c a l f a s h i o n , a n d w e r e i n f i l t r a t e d w i t h a b u n d a n t

f o a m y m a c r o p h a g e s F i g. 3 , A-D . M a r k e d v a c u o l ar c h a n g e s

w e r e o b s e r v e d , h o w e v e r , s l i g h t v a c u o l a r c h a n g e s w e r e a l s o

o b s e r v e d in t h e a g e - m a t c h e d u n t r e a t e d c o n t r o l s . T h e l e s i o n

d e v e l o p e d p r e f e r e n t i a l l y i n t h e l a t e r a l a n d a n t e r i o r f u n i c u l i ,

a n d a p p a r e n t l y t o a l e s s e r e x t e n t i n t h e p o s t e r i o r f u n i c u l i .

T h e m o s t s e v e r e l e s i o n s w e r e a t l e v e l s o f t h e t h o r a c i c c o r d ,

w h e r e t h e y c o n t i n u e d f r o m t h e c e r v i c a l t o t h e l u m b a r c o r d

a n d w e r e l o c a l iz e d in t h e o u t e r p o r t i o n o f t h e f u n i c u li . T h e

s c h e m a t i c d i s t r i b u t i o n o f t h e a f f e c t e d l e s i o n i n t h e s p in a l c o r d

i s s h o w n i n F i g. 3 E . T h e i n n e r p o r t i o n o f t h e f u n ic u l i a n d

c e n t r a l g r a y m a t t e r a p p e a r e d in t a c t . L y m p h o c y t i c in f i l t ra t i o n

w a s v i r t u a l ly a b s e n t i n t h e w h i t e a n d g r a y m a t t e r , o r i n t h e

p e r i v a s c u l a r a r e a s t h r o u g h o u t t h e e n t i r e s p i n a l c o r d . F i b e r s

o f t h e a n t e r i o r a n d p o s t e r i o r r o o t s o f t h e s e v e r e l y a f f e c te d

c o r d a l so s h o w e d l o s s o f m y e l in a n d a x o n s w i t h i n f il t ra t io n

o f f o a m y m a c r o p h a g e s. T h e r e a p p e a r ed t o b e n o n e u r o n a l

t itre of

antibod

1 2 -

1 0 ( 2 n )

8

4

2 -

o

l

0 10 20 30 40 50 wee ks af ter in fect ion

J ,

~ ~ l F 3 44 ( 5

WKA (5)

LEW (5)

A C t 5 )

SDJ (5)

BUF (5)

= LEJ (5)

F ig ur e 1. Strain differences in anti-HTLV -I

antibody pro duction in rats. Results represent

mean titer _+ SD. Differences in response

against HTLV-I are statistically significant

(student's t test). * p


5/9

T a b l e 4 .

TissueDistribution of H TL V I Provirus Genome in F344 Rats

Tissues examined

Submandibular

Rat no. Cer ebr um Cerebellu m Thymus Heart Lung Li ve r Ki dn ey Spleen gland Lymph nodes PBM C

3 6 - - + - + - + + + + +

12 8 - - + + + + + + + + +

12 9 + - + - - + + + - + +

13 0 + + + + + - + + + + +

13 1 + - + - - - + - + + +

degeneration in the anterior hor n cells, ceils of Clark's column,

or any nuclei.

Loss of myelin or axonal degeneration was no t seen in th e

cerebrum, th e cerebellum, or the brain stem above the level

of th e medulla oblongata. Perivascular cufflngs with lymph o-

cytes were n ot observed. Atypical lym phocytes were absent

in the brain and spinal cord. Muscle of the h ind legs showed

severe group atro phy with out inflamm atory reaction. Oth er

organs were histologically normal. Pathological findings of

W KA no. 57 were essentially the same as those in no. 54,

wi th loss of myelin and axons and infiltration of foamy mac-

rophages in th e hteral and anterior funiculi. Alth oug h severity

of the affected lesions was less pronounced than that of no.

54, levels of the thoracic cord wer e preferentially dam aged

(Fig. 3 E). Lymphocytic nfiltration was not observed. Anti-

HTLV-I antibodies in the sera of nos. 54 and 57 and CSF

of no. 54 were negative. CSF of no. 57 could not be col-

lected. T he HTL V-I provirus geno me was evident in the

PBMC , cerebrum, and spinal cord of no. 57 by PC R amplifi-

cation. The spinal cord of no. 54 was no t examined.

F i g u r e 2 . T i s s u e d i s t r i b u t i o n o f t h e H T L V - I p r o v i r u s g e n o m e , u s i n g

P C R a n d p X p r i m e r s i n F 3 4 4 r a t n o . 1 3 0 ) . T h e p o s i t i v e c o n t r o l i s c l o n e d

L e w i s - S 1 ce l ls , a n d t h e n e g a t i v e c o n t r o l i s P B M C o f a n o r m a l r a t .

i s cuss ion

Several observations presented in this paper lead to the con-

clusion that the inbred rat provides a useful animal model

for HTLV-I infection in humans. In 1984, we reported t hat

HTLV-I infected and imm ortalized rat T cells of th e spleen,

lymp h nodes, and thymus, wh en cocultivated wit h BrdU rd-

treated ATL ceils (17). In the present study, by inoc ulating

new ly established rat T cell lines or a hum an T cell line, MT-2,

we succeeded in establishing H TLV-I carriers in several in-

bred strains of newbo rn and adult rats, as determined by virus

genome integration. We found no strain differences in the

susceptibility to HTLV-I virus infection in neonates, and trans-

mission efficiency xceeded80 . A dult rats of several different

strains are equally susceptible to HTLV -I infection. Ne wb orn

HTLV-I carriers, in general, sho w an unresponsiveness in hu-

moral antibody response to HTLV-I through out their life span.

However, this unresponsiveness can be easily broken down,

since a secondary challenge of MT -2 cells at 7 mo leads to

the production o f anfi-HTLV-I antibodies wi th antibod y titers

of 1:256 to 1:512, as tested 1 mo later (our unp ublished results).

Adult HTLV-I carriers continuously produce humo ral anti-

bodies to HTLV-I similar to those in humans. According io

antib ody titers against HTLV-I, there are three groups o f in-

bred rat strains: ACI, F344, and SDJ (high responders); WK A,

BUF, and LEJ (intermediate responders); and LEW (low

responders). In human carriers, there are high and low

responders to HTLV-I; as it is possibly related to particular

HL A haplotypes, the forme r is likely to beaome HA M/ TS P

(27). Therefore, strain difference in th e hum oral anti body re-

sponse to HTLV-I in the rat provides useful information for

future research.

Three of three new born HTLV-I carriers of the W KA strain

developed spastic paraparesis of th e hin d legs, at 16 too. Clinical

symptom s and neuropathological findings generally mimi c

those of HA M/T SP seen in humans (6-9, 28-30). The le-

sions are prima rily confined to the lateral and anterior funiculi

of t he spinal cord. The most severe lesions are levels of th e

thoracic cord, and contin ue from the cervical to th e lumba r

cord. Both myelin and axons are extensively damaged, in a

symme trical fashion, and are infiltrated wi th massive foamy

macrophages. Newborn and adult carriers of other strains

9 8 5 I s h i g u r o e t a l .



6/9

Fi gu re 3. Histopathologica l findings and schematic distributio n of the affected lesion in the spinal cord of HAM rats. A and B) Symmetrical distribu-

tion of the damage in the white matte r is shown, as a whole view of the affected thoracic cord no. 54, Th7) x 30). C and D) Vacuolar degeneration,

demye lination , and infiltrati on of foamy macrophages are evident in the enclosed area of A x 140). Distri buti on of affected lesions in spinal cords

of nos. 54 and 57 are represented in the schematic fig ure E). Th e so lid area shows severe damage and the hatch ed area a lesser degree of damage.

No marked change was found in the blank area, as compared with age-matched untreated control. A and C) HE stain; B and D) LFB stain.

986 A Rat Modal of Huma n T Lymphocyte Virus Type I Infection



7/9

have no t d evelop ed neuro log ical d isease cl in ically an d path o-

log i ca l ly . T h u s , HTLV-I ca r ri e rs o f t h e W K A s t r a in a r e su it -

a b le m o d el s f o r t h e H A M / T S P i n h u m an s . T h e W K A H / H k m

ra ts , f o rm er ly ca l led Wis t a r -K in g -A p tek m an r a ts , a r e ag o u t i

A / A ) , co a t co lo r B / B ) , and alb ino c/c), an d h av e b een t y p ed

fo r t h e fo l l o win g p o ly m o rp h i c l o c i : A k p - 1 ~ , A I I ~ , C s b , E s -

1 o , Es -2 ~ , Es -3 ~, Es .4 b , Es -7 b, E s -8 s, Es .9 ~ , Es - l O ~ , Ess i b , F h.1 b ,

Gst - 1 b

H a o - 1 ~ H b b ~, M d l - I ~ , P g d b , R T I k , S v p - I a , T a m - 1b

(31,

3 2 ). S in c e H A M / T S P o c c u r s w i t h i n c re a se d f r e q u e n c y w i t h

c e r ta i n H L A h a p l o ty p e s ( 27 ), t h e g e n e t i c b a c k g r o u n d o f

W K A , e sp e ci al ly t h e R T / k h a p l o ty p e o f r a t M H C , m a y b e

one of cr i t ical host factors d ete rm in in g d isease suscep tib i li ty .

Fur ther invest igat ions are necessary to clar i fy th is po in t .

Re t ro v i ru s - i n d u ced n eu ro lo g i ca l d i seases h av e b een r eco g -

n i zed fo r m an y y ears i n an im als . Am o n g th e m , r e tro v ir a l sp o n -

g i f o r m p o l i o e n c e p h a lo m y e l o p a t h y n w i l d m i c e ( 3 3 -3 5 ) a n d

v i sn a i n sh eep (3 6 -3 8 ) h av e b een m o s t ex ten s iv e ly s t u d i ed .

From an imal s tud ies , there i s ev idence that ret rov i ral in fec-

t i o n c a n d a m a g e t h e C N S t is s u e v i a e it h e r i m m u n o l o g i c a l

m e c h a n i s m s o r t h r o u g h a d i r e c t n e u r o t r o p ic e ff ec t o f t h e

ret rov i rus . T he in ten se lymp hoc yt ic in f i l t rat ion in the af fected

lesion and per ivascu lar areas o f the sp inal cord , the presence

o f I g G a n d I g M o l i go c l o n a l b a n d s i n t h e C S F , s o m e o f w h i c h

are d i rec t ed ag a in s t HTLV-I , t h e h ig h t i t e r o f an t i b o d i es t o

HTLV -I , and the therapeu t ic ef fectivenessof h igh-dose s tero ids

a re a l l c o n s i s te n t w i t h a n i m m u n e h y p o t h e s i s o f t h e C N S

t is s u e d a m a g e i n H A M / T S P ( 7 - 9 , 2 8 - 3 0 ) . H o w e v e r , t h e

m e c h a n i s m o f C N S t is su e d a m a g e i n H A M / T S P s ti ll r em a in s

v i r t u a l l y u n k n o w n . T h ere a r e a t l eas t t h r ee es sen ti a l p o in t s

to s t ress about these d iseased rats , ten tat ively designated as

H A M r a t s F i rs t , H A M r a ts p r o d u c e n o d e te c t a bl e a n ti -

b o d i es t o HTLV-I i n t h e CS F o r i n t h e se ru m . As a l r ead y

m e n t i o n e d , H A M / T S P p a t i e n ts a r e u su a l ly a ss oc ia te d w i t h

h ig h - t i te r ed an ti b od i es t o HTLV -I i n b o th t h e CS F a n d se ru m

(7 , 9 ). H u m o ra l an t i b o d y r esp o n se t o HTLV-I m ay n o t p l ay

a m ajo r ro l e , i f an y , i n t h e p a th o g en es i s o f HAM-TS P in

h u m a n s . P r o d u c t i o n o f h u m o r a l a n t ib o d i e s t o H T L V - I i n t h e

C N S o f H A M / T P S p a t i e n ts m i g h t b e a s ti r -p r o t e c ti n g r ea c -

t i o n o r m e r e l y a s e c o n d a r y p h e n o m e n o n . S e c o n d , i n c o n -

t rast to the af fected sp inal cord in H A M /T SP pat ien ts (28-30) ,

H A M r a ts s h o w n o l y m p h o c y t i c i n f il t ra t i o n i n t h e w h i t e o r

g ray m at t e r , o r i n t h e p e r iv ascu l ar a r eas t h ro u g h o u t t h e w h o le

sp in a l co rd . F o am y m acro p h ag es p red o m in an t ly i nf il tr a te t h e

affected les ion . Visna i s a s low ret rov i rus in fect ion of sheep

th a t p ro d u ces b o th i n f l am m ato ry an d d em y el in a t i n g le s io n s

o f t h e C N S . A l t h o u g h t h e p a t h o g e n es i s o f t h e C N S l es io n s

is s t i l l unclear , host factors in v i rus suscep t ib i l i ty and im-

m un e me chanism s und oub ted ly p lay pathog enic ro les (37--41).

S h eep ex p er im en ta l l y i n f ec t ed b y i n tr ace rebra l i n o cu l a t i o n o f

v i sn a v i ru s a r e ap p aren t l y h ea l th y , b u t a t au to p sy m an y sh o w

a n i n f la m m a t o r y e x u d a te t h r o u g h o u t t h e C N S a n d m e n i n g e s

w i th in week s (3 7 ) . I n t h e sp in al co rd , t h e re i s a m a rk ed h i s t o -

log ical d i f ference betwe en th e affected les ions of th e ear ly and

advanced s tages of the d isease (42) . At an ear ly s tage, the

m a in p a th o lo g i ca l ch an g es a re i n f l am m ato ry r eac t io n s co n -

s i s t in g o f ly m p h o c y t es , m acro p h ag e s , an d so m e p l asm a ce ll s.

In advanced les ions, dem yel inat ing foci c losely associated wi th

c l in ica l p a res is o ccu r a n d i n f l am m at io n i s m in im al o r ab sen t .

S in ce t h e i n i ti a l s tag e o f r a t HA M h as n o t b een ex am in ed ,

i t i s t o o ea r l y t o co n c lu d e wi t h ce r t a in ty t h a t l ack o f ly m -

pho cyt ic in f i l t rat ion in the af fected les ions i s a character i st ic

p a t h o l o g ic fe a t ur e o f r a t H A M . W h e t h e r t h e a b se n ce o f l y m -

phocyt ic in f i l t rat ion in the af fected les ion ref lects the s tage

of the d isease process , species d i f ference in th e ho st respo nse

b e t w e e n r a ts a n d h u m a n s , o r n o n i m m u n o l o g i c a l d e m ye li na -

t i o n i n t h e p a t h o g e n e s i s o f r a t H A M r e m a i n s t o b e d e t er -

m i n e d . T h i r d , H T L V - I p ro v i r u s g e n o m e w a s d e a r l y d e m o n -

s t r a t ed b y P CR in t h e sp in a l co rd an d ce reb ru m as we l l a s

P B M C o f a H A M r a t n o . 5 7. T h e p r ec is e lo c a l iz a t io n o f

p ro v i ru s g en o m e, wh e th e r i t l o ca l izes i n t h e d am ag ed sp in al

co rd t i ssu e , i n t h e i n f i l t r a t i n g m acro p h ag es , o r i n t h e b lo o d -

b o r n e o r h i d d e n l y m p h o c y t e s c a r r y i ng H T L V - I , w i l l r e m a i n

t o b e d e t e r m i n e d . N e i t h e r i n t e g r a t i o n o f p r o v ir u s g e n o m e ,

n o r ex p ress io n o f m K N A an d v ir a l p ro t e in s o f HTLV-I, h as

b e e n c o n f i r m e d i n t h e d i se a se d C N S t is su e s o f H A M / T S P

p a t i en t s (2 9, 4 3 ) . T h ere fo re , t h e re is n o d i r ec t p ro o f t h a t t h e

in f l am m at io n an d t i s su e d am ag e i n t h e sp in a l co rd o f

H A M / T S P p a t ie n t s i s a c o n se q u e n c e o f vi ra l i n f e c ti o n i n t h e

CN S t is sue . D e t e rm in a t i o n o f in s i t u l o ca l iza t i o n o f p ro v i ru s

g en o m e, m R N A , an d v i ra l p ro t e in s i n t h e a f f ect ed sp in a l co rd

o f t h e HAM ra t wo u ld b e o f c r i t i ca l im p o r t an ce fo r i n v es -

t i g a t i n g p a th o g en i c ro l es o f HTLV-I i n t h e d i sease m ech a-

n is m o f H A M / T S P i n h um a n s .

In co n c lu s io n , t h i s i s t h e f i rs t d esc r ip t i o n o f a r a t m o d e l

f o r H A M / T S P i n h u m a n s . T h i s m o d e l is e x p ec t e d t o c o n -

t r i b u te t o a b e t t e r u n d e r s t a n d i n g o f m e c h a n i s m s i n v o lv e d in

th e e t i o p a th o g en es i s o f HTLV-I -r e la t ed im m u n o lo g i ca l d is -

e a se s i n h u m a n s , p a r t i cu l a rl y H A M / T S P .

We thank D rs . I . M iyosh i, A. Matsuura, K. Kikuchi , and M. Hatanaka for p rov iding MT-2 cel ls , mA bs,

and H TLV-I probe; Ms. M . Yano and Dr . H. Ikeda for exam ination of the an t i-HTLV -I an t ibodies; Mr.

T. O sanai and the entire staff of the Insti tute for A nimal Experiment (H okkaido Un iversity School of

Me dicine) for m aintenance of infected rats; M s. C . Sudo for technical assistance of histopathological ex-

aminations; and Ms. M . Ishizaka for secretarial assistance. We also than k Dr. S. Matsum oto and M . Ohara

for crit ical reading of the m anuscript and Dr. K. Naga shima for useful discussionof the neuropathological

findings.

987 Ish igu ro t al.



8/9

This stud y was supported in part by grants from the M inistries of Education, Health and Welfare, and

Science and Technolog y of Japan.

Address correspondence to Takashi Yoshiki, Department of Pathology, Hokkaido University School of

Medicine, Kita-15 Nishi-7, Kita-ku, Sapporo 060, Japan.

Rece ived for publ icat ion 12 Ma y 1992 and in rev ised orm 30 June 1992.

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989 Ishig uro t al.


a rat model of human t lymphocyte virus type i

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