Trans R Soc Trop Med Hyg-1952-Dick-509-20

7/25/2019 Trans R Soc Trop Med Hyg-1952-Dick-509-20

http://slidepdf.com/reader/full/trans-r-soc-trop-med-hyg-1952-dick-509-20 1/12

TRANSACTIONSOF THE ROYAL SOCIETY OF

TROPICAL MEDICINE AND HYGIENE.

Vo l. 46. No. 5. Septem ber, 1952.

coMMUNIC TIONS

509

Z I K A V I R U S

I). I S O L A T I O N S A N D S E R O L O G I C A L S P E C I F I C I T Y

Y

G. W . A . DIC K,

The N at ional Ins t i tu te for M edical Research , London

S . F . K I T C H E N ,

Formerly s taf f me m ber of the Division of Medicine an d Publ ic H eal th, The Rockefel ler

Foundation , New York , U . S . A .

ND

A. J . HADDOW ,

Formerly s taf f me mb er of Internat ional Hea l th Division, The Rockefel ler Foundat ion, N ew

Y o r k , U . S . A .

From the Virus Research Institute, Entebbe,

Uganda.

T h e i so l at io n o f f il te r a b le v ir u s e s f r o m m o s q u i t o e s t a k e n i n U g a n d a h a s a l r e a d y

b e e n r e c o r d e d o n se v e r al o c c as io n s . T w o o f t h e a g e n t s s o r e c o v e r e d , a l t h o u g h

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

U ga nd a , v iz . ye l lo w fev er v i rus MAI~AVFYet al., 1942 ; SMITI~BURN a n d HADDOW,

1946 ; SMITHBVRNet al. , 1949) an d R ift Val ley fev er v iru s SMITHBURNet al., 1948).

A th i rd w h ich was ca ll ed M en g o en cep h a lo m y e l i t i s DIC K e t a l., 1 9 48 ) n o w k n o w n

t o b e i d en t ic a l w i t h C o l u m b i a S K , M M a n d e n c e p h a l o m y o c a r d i t i s v i r u se s D IC K ,

1949 ; WARREN e t a l., 1949), has be en i so la ted on severa l occa s ions f r om

T a e n i o r -

h y n c h u s

spp . DIC K e t a l ., loc . c i t. , DICK an d HADDOW, unp ub l i sh ed)) . GILLETT

a n d D I C K u n p u b l i s h e d ) h a v e , h o w e v e r , fa i le d to t r a n s m i t th i s a g e n t i n t h e l a b o r -

a t o r y b y t h r e e s p e ci e s o f T a e n i o r h y n c h u s . T h e i so l at io n o f t h r e e h i t h e r t o u n k n o w n ,

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

v iz . B u n y a m w e r a v i r u s SMITHBURNet al . , 1946), Sem liki Fo re st v ir us SMITHBURN

an d HADDOW, 1944), N ta ya v iru s SMITHBURN an d HADDOW, 1951) ; th e d esc rip t ion

o f a fo u r th , Ug a n d a S v i ru s , is t o b e p u b l i s h ed DIC K an d HADDOW ). T h e p u rp o s e

o f th i s c o m m u n i c a t i o n is t o d e s c r i b e t h e i so l at io n o f y e t a n o t h e r v i r u s w h i c h i s

b e l ie v e d t o b e h i t h e r t o u n r e c o r d e d . P a r t i c u l a r i n t e r e s t a t ta c h e s t o t h e r e c o v e r y

o f th i s v i r us , p a r t l y b e c a u s e i t w a s e n c o u n t e r e d o n t w o o c c a si o n s s e p a r a t e d b y

b y g u e s t onF e b r u a r y4 ,2 0 1 6

h t t p : / / t r s t mh . oxf or d j o ur n a l s . or

g /

D o wnl o a d e d

f r om

http://trstmh.oxfordjournals.org/





































510 ZlKA vm us

9 m o n t h s , a n d p a r t l y b e c a u s e i t w a s i s o la t e d i n d e p e n d e n t l y b y G . W . A . D . a n d l a t e r

b y S . F . K . e m p l o y i n g d if f e re n t m e t h o d s , b o t h o f w h i c h w e r e b e i n g u s e d p r im a r i ly

i n t h e s ea r ch f o r y e l l o w f ev e r v i r u s i n n a t u r e . Th i s v i r u s h a s b e en ca l led Z i k a

v i r u s a f t e r t h e g eo g r ap h i ca l n a m e o f t h e a r ea f r o m w h ek e t h e i s o l a t io n s w e r e ma d e .

MATER IALS AND METHODS

T h e m i c e u s e d i n a l l e x p e r i m e n t s w e r e d e s c e n d e d f r o m t h e s t o c k o f S w i s s

m i c e fr o m C a r w o r t h F a r m s , N e w Y o r k . A g r o u p o f m i c e c o n s is t e d o f s ix , a n d

u n l e s s o t h e r w i s e s t a t e d a l l m i c e u s e d i n t h e e x p e r i m e n t s r e c o r d e d i n t h i s p a p e r

w e r e 35 t o 4 2 d ay s o l d . T h e q u a n t i t y o f t h e i n tr ace r eb r a l i n o c u l u m f o r all m i ce

w a s 0 . 0 3 m l .

I S O L A T I O N O F V I R U S

Loca l i t y

A b o u t 7 m i l e s n o r t h e a s t o f t h i s I n s t i t u t e t h e r e i s a f o r e s t e d a r e a c a l le d Z ik a . T h i s

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

o f l a r g e tr e es . I t li es a l o n g t h e e d g e o f a l o n g a r m o f L a k e V i c t o r ia f r o m w h i c h i t i s s e p a r a t e d

b y a p a p y r u s s w a m p . T h e g r e a t e r p a r t o f t h e f o r e st r u n s p a ra l l e l w i t h t h e E n t e b b e - K a r n p a l a

r o a d ; t h e r e is a n a r r o w s t r e t c h o f g r a s s l a n d b e t w e e n t h e f o r e s t a n d t h e ro a d . T h e f o r e s t a t n o

p l a c e i s m o r e t h a n 5 0 0 y a r d s w i d e . I t i s a b o u t 1 m i l e in l e n g t h a n d i s a l m o s t c o n t i n u o u s

w i t h s c a t t er e d f o r e s t w h i c h i n t u r n j o i n s th e f o r e s t at B u j u k o o n t h e K a m p a l a - F o r t P o r t a l

r o a d . A d e t a i le d d e s c r i p t i o n o f t h i s f o r e s t h a s re c e n t l y b e e n p r e p a r e d b y B UX TO N i n p r e s s) .

O w i n g t o t h e r e la t iv e l y h i g h i n c i d e n c e o f i m m u n i t y t o y e ll o w f e v e r a m o n g t h e m o n k e y s

o n t h e E n t e b b e p e n i n s u l a , Z i k a w a s c h o s e n as o n e o f t h e y e l l o w f e v e r s t u d y a r ea s . A s a n

i m p l e m e n t a t i o n o f t h e s en t in e l rh e s us m o n k e y p r o g r a m m e c o m m e n c e d i n B w a m b a i n 1 94 6

H AD D OW e t a l. , 1 94 8 ), r h e s u s m o n k e y s w e r e l o c a t e d i n c ag e s o n w o o d e n p l a t f o r m s i n t h e

c a n o p y o f t re e s t h r o u g h o u t t h e l e n g t h o f th e Z i k a F o r e s t.

First Isolation.

I n A p r i l 19 47 , s i x s en t i n e l p l a t f o r ms w er e in u s e a t Z i k a . T h e t e m p er a t u r e s

o f t h e r h e s u s mo n k ey s o n t h e p l a t f o r ms w er e tak en d a i ly . O n 1 8 t h A p r i l , 1 94 7,

i t w a s r e p o rt e d t h a t th e t e m p e r a t u r e o f o n e o f t h e s e m o n k e y s - - R h e s u s 7 6 6 - - w a s

3 9 .7 ° C . F i g . 1 ). O n 1 9 t h A p r i l it s t em p er a t u r e w as r eco r d ed a s 4 0 ° C .

O n t h e fo l lo w i n g d a y i t w a s b r o u g h t t o t h e l a b o r a t o r y a t E n t e b b e . I t w a s k e p t

u n d e r o b s e r v a t i o n f o r 3 0 d a y s b u t s h o w e d n o a b n o r m a l i t y o t h e r t h a n t h e s l i g h t

p y r ex i a r eco r d ed i n F i g . 1 .

A s a m p l e o f b l o o d w a s t a k e n f ro m R h e s u s 7 6 6 o n 2 0 th A p r i l, t h e 3 r d d a y o f

f ev e r . T h e s e r u m w as i n j ec t ed i n t r ace r eb r a l l y 0 .0 3 ml . ) an d i n t r ap e r i t o n ea l l y

0 . 0 6 m l . ) in t o g r o u p s o f m i c e a n d i n t o a n o t h e r m o n k e y , R h e s u s 771 0.75 ml .

s u b c u t a n e o u s l y ) . T h e m i c e w h i c h w e r e i n o c u la t e d in t r a p e ri to n e a l l y w i t h t h e

s e r u m s h o w e d n o a b n o r m a l i t y d u r i n g a n o b s e r v a t i o n p e r i o d o f 3 0 d a y s . A l l o f

t h o s e i n o cu l a t ed i n t r ace r eb r a l l y s h o w ed s i g n s o f s i ck n es s o n t h e 1 0 t h d ay a f t e r

i n o cu l a t io n . A fi l te r ab l e t r an s mi s s i b l e ag en t w as i s o la t ed f r o m t h e b r a i n s o f t h e s e

s ic k m i c e . T h e f u r t h e r a d a p t a ti o n to m i c e o f t h is a g e n t i so l a te d f r o m t h e s e r u m

o f R h es u s 7 6 6 i s d e s c r i b ed i n t h e n ex t co m m u n i ca t i o n D I CK , 1 9 5 2 ).



g /

D o wnl o a d e d

f r om






































G . W . A . D I C K S . F . K I T C H E N A N D A . J . H A D D O W 5

T h e r h e s u s m o n k e y ( 7 7 1 ) w h i c h w a s in o c u l a t e d w i t h s e r u m o f R h e s u s 7 6 6

s h o w e d n o e le v a t io n o f t e m p e r a t u r e a b o v e it s n o r m a l r a n g e , a n d n o o t h e r a b n o r m -

a l it ie s d u r i n g an o b s e r v a t i o n p e r i o d o f 2 3 d ay s .

A s w i l l b e d e s c r i b e d l a t e r ; t h e a g e n t i s o l a t e d f r o m t h e s e r u m o f R h e s u s 7 6 6

w h i ch w i l l b e r e f e r r ed t o a s Z i k a v i r u s ( 7 6 6 s t r a i n ) w as n eu t r a l i z ed ( a ) b y co n v a l -

e s c e n t s e r u m t a k e n f r o m R h e s u s 7 6 6 , 1 m o n t h ( 2 0 th M a y ) a f t e r t h e f e b r il e e p i s o d e

( F i g . 1 ) , an d ( b ) b y s e r u m t ak en f r o m R h es u s 7 7 1 , 3 5 d ay s a f t e r i n o cu l a t i o n w i t h

R H F _ 3 U S

7

o C

°

39

38

i

I0 15

APRIL

1947

T E M P E R A T U R E

i I I

20 25 30

RE ORD

F I G I

t h e s e r u m o f R h e s u s 7 6 6 , b u t n o t b y ( c ) p r e - i n f e c ti o n s e r a fr o m t h e s e m o n k e y s .

( T a b l e I ). T h e r e w a s t h u s c o n c l u s i v e e v i d e n c e t h a t th e a g e n t is o l at e d i n m i c e

h a d c o m e f ro m t h e s e r u m o f t h e s e n t in e l R h e s u s m o n k e y ( 76 6 ).

Second Isolation

I n J a n u a r y 1 94 8, i n an a t t em p t t o i s o l a te y e l l o w f ev e r v i ru s f r o m m o s q u i t o e s ,

a s e ri es o f 2 4 - h o u r c a t c h e s w a s m a d e i n Z i k a F o r e s t . T h e t r e e - p l a t f o r m s r e f e r r e d

t o a b o v e w e r e e m p l o y e d i n m a k i n g t h e s e m o s q u i t o c a tc h e s u s in g m e t h o d s w h i c h

h av e a l r ead y b e en d es c r i b ed (H A D DO W e t al., 1 9 4 7 ). T h e m o s q u i t o e s t ak en each



g /

D o wnl o a d e d

f r om






































512 ZlKA VIRUS

day were bro ugh t to the laboratory in Entebb e. After identification, the following

gro upin g acc ording to species or genera was made :

1. Aedes Stegomyia) africanus

Theobald.

2. Other arboreal Aedes spp.

3. Non-arboreal

Aedes

spp.

4. Taeniorhynchus

spp.

5. Eretmapoditesspp.

6. Other mosquitoes.

Because of its suspe cted impl icat ion in the forest cycle of yello w fever HADI)OW

et al., 1948), A. a f r i canus was treated individually as a species. After the groupin g

of the mosquitoes had bee n effected, the mosquitoes were render ed inactive by

placing the tubes in which they were contained in a refrigerator at approximately

4°C.

From 5th to 20th January, nine lots of A. afr icanus were secured, and each was ground

up in 10 per cent. serum-saline. The quantity of the serum-saline diluent employed was

the minimum quanti ty which would make a satisfactory suspension of each lot of mosquitoes.

These suspensions were centrifuged at approximately 2,000 r.p.m, for 15 minutes. An

aliquot of each suspension was passed through a Seitz E.K. pad, and groups of mice were

inoculated intracerebrally with the unfiltered supernates and with the Seitz filtrates.

This account is particularly concerned with the mosquito catch of 11 th to 12th January,

1948. Eighty-six A . africanus lot E/1/48) were taken, and were treated as described above.

All of the six mice which were inoculated with the unfiltered supernate of this lot of mos-

quitoes appeared inactive on the 7th day after inoculation. Of those inoculated with the

Seitz filtrate one mouse died on the 6th day and one appeared sick on the 14th day. Suc-

cessful transfers were made with brain suspensions from mice inozulated with the unfiltered

and with the Seitz-filtered suspensions of A. a fr icanus . Evidence will be presented which

demonstrates that the virus isolated from the suspensims of the E/1/48 lot of A. afr icanus

was a strain E/1 strain) of Zika virus. The his tory of the adaptation of this strain of virus

is described later DICK, 1952).

After the groups of mice had been inoculated with the suspensions of each lot of A.

africanus taken in the January, 1948, mosqulto-catches, the remaining portions of the

unfiltered supernates and Seitz filtrates of all lots of A. afr icanus were inoculated subcut-

aneously into Rhesus 758. Between 5th and 20th January this monkey received nine

inoculations of the remainder of suspensions of A. africanus totalling 607 specimens. Th e

number of mosquitoes in each lot and the days of inoculation are indicated in Fig. 2. The

total quantities of the unfiltered and Seitz-filtered su~pens ons inoculated into Rhesus 758

were 3.22 ml. and 0.61 ml. respectively. Th e quantities of the suspensions of the E/1/48

lot of mosquitoes available for inoculation on 13th January were 0.4 ml. of the unfiltered

supernate and 0.1 ml. of the Seitz filtrate.

Rhesus 758 showed no abnormality as a result of the inoculation of the sus-

pensions of A. a f r i canus . Th e slight elevations of tem per atu re recorded on

24th Janu ary and 5th Feb ru ar y Fig. 2) are not significant. Because of the ap-

pearance of sickness on the 7th day 20th January) in mice inoculate d with the

unfiltered supernate of the E/1/48 lot of

A. a f r i canus ,

a sample of blood was

withdrawn from Rhesus 758 on the 21st, 22nd and 23rd January, i.e. on the

8th, 9th and 10th days after inoculation with the E/1/48 lot of A. a f r i canus . T h e

sera from these samples of bloo d were inoculated in tracerebrally into g roups o f

mice. Of the six mice inoculat ed with ser um taken on 21st Januar y, one was sick



g /

D o wnl o a d e d

f r om






































G W A D I C K ~ S F K I T C H E N A N D A J H A D D O W 5 3

on the 10th day and two were found dead on the 19th and 20th days. Successful

transfers were made with a Seitz-filtered suspension of the brain of the sick mouse.

Of the six mice inoculated intracerebrally with serum taken from Rhesus 758 on

the 22nd January, one had quefitionable signs of sickness on the 12th day after

inoculation and was found dead on the 13th day ; another mouse was paralysed

on the 20th day. Successful transfers were made with a Seitz-filtered suspension

of the brain of the dead mouse. (The paralysed mouse proved to be an example

of Theiler s encephalomyelitis.) All the mice inoculated with serum taken from

Rhesus 758 on 23rd January remained well.

RHESU3 758

= C ,

4 0 u

3 9 ~

T E M P E R A T U R E R E C O R D

A N D S U M M A R Y O F I N O C U L A T I O N S

I

3

, .TAN

1 9 4 8

- - - + . . . . R E S U L T 5 ~N M I C E

1 I ' 1~ '1 ' I~ '~ '1 '1 ' ~ N U M B E R S O F A E B E 5 A F R I C A N U 5 I N O C U L A T E D

~ ~ ~ ~ -~ ~ ~ ~ NrO RHESUS 7S8 • M~CE

I I I 1 I I I

8 1 3 I S 2 3 2 . 8 , 2 7

F E B

1 9 4 6

F I G Z

It was subsequently shown by neutralization tests that Rhesus 758 had

developed neutralizing antibody to (a) the agent isolated from its serum, (b) the

E/1 strain of virus isolated directly in mice from A afr ieanus and (c) to the 766

strain of Zika virus. These tests, which will be described in detail, indicated that

a strain of Zika virus (Zika virus 758 strain) was isolated from the serum of

Rhesus 758 which had been inoculated with suspensions of A afr icanus (Fig. 2).

SEROLOGICAL STUDIES

Neu tra l i za t ion Tes t s

Preliminary investigations, which will not be discussed here in detail, showed

that adult mice were relatively insusceptible to intraperitoneal inoculations of



g /

D o wnl o a d e d

f r om






































51 4 ZlKA VIRUS

Z i k a v i ru s . A n i n t r a c e r e b r a l n e u t r a l i z a t i o n t e s t b a s e d o n t h e m e t h o d d e s c r i b e d

b y T H E IL E R ( 1 9 33 ) w a s a c c o r d i n g l y u s e d f o r n e u t r a l i z a t i o n t e s t s w i t h Z i k a v i r u s .

T h e t e c h n i q u e e m p l o y e d i n a l l n e u t r a l iz a t i o n te s ts , e x c e p t i n t h e c a s e o f a f ew t e s t s

m a d e i n 1 9 4 8, w a s a s f o ll o w s : t e n - f o l d d i l u ti o n s o f th e l i g h t ly c e n t ri f u g e d s u p e m a t e o f a

1 0 p e r c e n t . s u s p e n s i o n o f i n f e c t e d m o u s e b r a i n s w e r e m a d e i n s e r u m - s a l i n e . T o 0 . 3 m l .

q u a n t i t ie s o f t h e s e r a to b e t e s t e d w e r e a d d e d 0 . 3 m l . o f o n e o f t h e d i l u t i o n s o f m o u s e b r a i n

v i r u s a s i n d i c a t e d i n T a b l e s I t o I V . T h e s e r u m v i r u s m i x t u r e s w e r e i n c u b a t e d a t 3 7 . 5 ° C .

f o r 2 h o u r s p r i o r t o t h e i n t r a c e r e b r a l in o c u l a t i o n o f g r o u p s o f m i c e w i t h e a c h m i x t u r e .

I n t h e o t h e r n e u t r a l iz a t i o n t e s t te c h n i q u e , u n c e n t r i f u g e d v i r u s s u s p e n s i o n s o f i n f e c te d

m o u s e b r a i n s w e r e d i l u t e d t o 1 0 -2 a n d 0 . 1 5 m l . o f th i s w a s m i x e d w i t h 0 . 3 m l . o f t h e s e r a

t o b e t e s te d . T h e f u r t h e r p r o c e d u r e s o f i n c u b a t i o n a n d i n o c u l a t i o n w e r e a s d e s c r i b e d

a b o v e .

Demons t ra t ion o f an t ibody in sera o f i n f ec t ed o r i nocu la t ed rhesus monkeys

T h e d e v e l o p m e n t o f a n t i b o d y i n t h e c o n v a l e s c e n t s e r u m o f R h e s u s 7 6 6 a n d

i n t h e s e r u m o f R h e s u s 7 71 w a s d e m o n s t r a t e d b y t h e n e u t r a l i z a t i o n t e s t d o c u -

m e n t e d i n T a b l e I . I n t h a t t e s t t h e f i r s t n e u t r a l i z a t i o n t e c h n i q u e d e s c r i b e d w a s

e m p l o y e d u s i n g i n f e c t e d b r a i n s o f t h e n i n t h p a s s a g e o f t h e 7 6 6 s t r a i n o f v i r u s .

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

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

t h e v i r u s t i t r e s . I t w i l l b e s e e n t h a t n e u t r a l i z a t i o n in d i c e s o f o v e r 8 , 00 0 a n d o v e r

T A BL E I . I n t r a c e r e b r a l n e u t r a l i z a t i o n t e s t s w i t h s e r a o f R h e s u s 7 6 6 a n d 7 7 1 .

V i r u s

S e r u m

R h e s u s D a t e D e s c r i p t io n s

N o . b l e d

7 6 6 1 8 . I I . 4 7 P r i o r t o g o i n g t o

Z i k a a s s e n t i n e l

7 6 6 2 0 . V . 4 7 3 1 d a y s a f t e r o n -

s e t o f p y r e x i a

7 71 8 . I V . 4 7 P r e - i n o c u l a t i o n

w i t h R h e s u s 7 66

s e r u m

7 71 2 5 . V I . 4 7 3 6 d a y s a f t e r

i n o c u l a t i o n w i t h

R h e s u s 7 6 6 s e r u m

M o r b i d i t y r a ti o s

o f m i c e i n o c u -

l a t e d w i t h s e r u m

+ v i r u s d i l u t i o n

10-1 10-2 10-3 10-4

6 /6 6 /6 6 /6 6 /6

1 / 6 0 / 6 0 / 6 0 / 6

6 /6 6 /6 6 /6 6 /6

0 . 5 o r <

M D s o e t L o g u n it s

n e u t r a l i z e d

4 . 5 o r >

0 . 6 o r <

4 . 5 o r >

0 / 6 0 / 6 0 / 6 0 / 6

3 . 9 o r >

4 . 0 o r >

* ( 1) 5 0 p e r c e n t . m o r b i d i t y e n d p o i n t .

~ ( 2) A l l e n d p o i n t s a r e c a l c u l a te d b y t h e m e t h o d o f R e e d a n d M u e n c h ( 19 3 8) .

1 0 ,0 0 0 w e r e o b t a i n e d w i t h c o n v a l e s c e n t s e r u m f r o m R h e s u s 7 6 6 a n d 7 71 r e s p e c t -

i v e l y .

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

a 1 0 -2 s u s p e n s i o n o f t h e e i g h t h p a s s a g e o f t h e s t r a i n o f v i r u s i s o l a t e d f r o m t h e

s e r u m o f R h e s u s 7 5 8 ( t a k e n 2 1 s t J a n u a r y , 1 9 48 ) w a s c o m p l e t e l y n e u t r a l i z e d (a ) b y



g /

D o wnl o a d e d

f r om






































O. W. A. DICK S. F. KITCH EN AND A. J. HADDOW 515

s e r u m t a k e n f r o m R h e s u s 7 5 8 3 2 d a y s a f t e r i n o c u l a t i o n w i t h t h e s u s p e n s i o n o f

t h e E / 1 / 4 8 l o t o f

A. afr icanus

( b ) b y l a te c o n v a l e s c e n t s e r u m f r o m R h e s u s 7 6 6 , b u t

n o t b y ( c ) s e r u m t a k e n f r o m R h e s u s 7 5 8 o n 3 r d J a n u a r y , 1 94 8 , p r i o r to t h e i n o c u -

l a t io n s o f t h e s u s p en s i o n s o f

A. afr icanus

( F i g . 2 ). A s i mi la r ex p e r i m en t u s i n g t h e

s ev e n t h p as s ag e o f t h e E / 1 s t r a i n o f v i r u s s h o w e d t h a t a 1 0 -2 s u s p e n s i o n o f i n f ec t ed

m o u s e b ra i n w a s n e u tr a li z e d b y c o n v a le s c e n t s e r u m f r o m R h e s u s 7 58 . F r o m t h e s e

t e s t s i t w a s c o n c l u d e d t h a t R h e s u s 7 5 8 h a d d e v e l o p e d a n t i b o d y b o t h t o t h e s t r a i n

o f v i r u s i s o l a ted f r o m i ts s e r u m an d t o t h e s t r a i n i s o l a t ed d i r ec t l y i n mi ce f r o m t h e

E / 1 / 4 8 l o t o f

A. afr icanus.

Iden t i t y o f sen tinel monkey and A . a f r i can u s strains.

T h e i d e n t i ty o f t h e 7 6 6 a n d E / 1 s tr a in s o f Z ik a v i r u s w a s e s t a b l i s h e d b y t h e

n e u t r a li z a ti o n t e s t s o u t l i n e d in T a b l e I I . I n t h e s e t e s ts a n d i n a ll s u b s e q u e n t

n eu t r a l i z a t i o n t e s t s d e s c r i b ed i n t h i s co mmu n i ca t i o n , t h e f i r s t t e ch n i q u e d es c r i b ed

w a s e m p l o y e d .

I t w i ll b e s e e n f r o m T a b l e I I t h a t t h e r e w a s r e c i p ro c a l n e u tr a l iz a t io n o f t h e

E / 1 s t ra i n a n d 7 6 6 st ra i n o f v i r u s b y t h e l a te c o n v a l e s c e n t s e r u m o f b o t h R h e s u s

7 5 8 an d R h es u s 7 6 6 .

Serological specifidty.

C r o s s n e u t ra l i z a ti o n t e s ts w e r e m a d e w i t h Z i k a v ir u s , w i t h y e l l o w fe v e r a n d

H a w a i i d e n g u e v ir u se s a n d w i t h th e F A a n d G D V I I s tr ai ns o f T h e i le r s e n c e p h -

a l o my e l i ti s v i r u s . I n t h e ca s e o f o t h e r v ir u s e s , k n o w n an t i s e r a w e r e t e s t ed ag a i n st

d i l u t io n s o f Z i k a v i r u s o n l y .

Yel low F ever.

S o o n a f t e r t h e i s o l a ti o n o f t h e f ir s t s t r a in o f Z i k a v i r u s f r o m

R h e s u s 7 6 6 , it w a s s h o w n t h a t s a m p l e s o f s e r u m t a k e n f r o m R h e s u s 7 6 6 (a ) b e f o r e

i t w as s en t t o Z i k a , an d ( b ) d u r i n g co n v a l e s cen ce , h ad n o n eu t r a l i z i n g e f f ec t o n

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

i n t h e y e l l o w f ev e r n eu t r a l i z a t i o n t e s t a s mo d i f i ed b y S M ITH ~U R N 1 9 4 5 ). F u r t h e r -

m o r e , y e l l o w f e v e r h y p e r i m m u n e s e r u m f a il e d to n e u t r a l iz e e a r ly , pa s sa g e Z i k a

v i r u s ( 7 6 6 s t r a i n ) i n t e s t s emp l o y i n g t h e i n t r ace r eb r a l t e s t t e ch n i q u e a l r ead y

d es c r i b ed . S i mi l a rl y , p r e - i n o cu l a t i o n an d co n v a l e s cen t s e r a f r o m R h es u s 77 1

f a i led t o s h o w an y n e u t r a l i z a t io n o f y e l l o w f ev e r v i r u s an d t h e E / 1 s t r a i n o f v i r u s

w a s n o t ne u t ra l iz e d b y y e ll o w f e v e r h y p e r i m m u n e s e ru m . T h e r e w a s t h u s g o o d

ev i d en c e t h a t Z i k a v i ru s w as n o t r e l a t ed t o y e l l o w fev e r .

Dengue Virus. T h e r e s u l t s o f c ro s s n eu t r a l iz a t i o n t e s t s w i t h Z i k a an d H aw a i i

d e n g u e v ir u s e s a r e p r e s e n t e d i n T a b l e I I I . I n t h e te s t u s i n g Z i k a v i ru s , t h e

i n tr a c e r e b r a l t e c h n i q u e a l r e a d y d e s c r i b e d w a s e m p l o y e d . A l l s e r a i n t h is a n d

s u b s eq u en t n eu t r a l i z a t i o n t e s t s t o b e d e s c r i b ed , w e r e i n ac t i v a t ed a t 5 6 ° C . f o r

3 0 m i n u t e s b e f o r e b e i n g t e st e d. T h e d e n g u e i m m u n e s e r u m ( M 4 8 9 7) w a s

s u p p l i e d b y D r . K . C . S M IT H BU R N ( D i v i s io n o f M e d i c i n e a n d P u b l i c H e a l t h



g /

D o wnl o a d e d

f r om






































T

A

C

o

n

a

z

o

e

s

w

i

h

h

E

1

a

7

s

a

n

o

Z

k

v

u

~

V

i

u

S

a

n

E

1

9

h

p

7

2

h

p

S

u

m

R

h

u

D

a

e

D

e

p

o

6

5

V

I

5

N

o

m

a

7

2

V

I

5

E

1

s

a

n

m

m

u

7

1

V

.

5

7

s

a

n

m

m

u

9

1

V

.

5

N

o

m

a

7

1

V

.

5

E

1

s

a

n

m

m

u

7

1

V

.

5

7

s

a

n

m

m

u

M

o

a

y

o

m

o

b

d

y

o

m

i

c

i

n

a

e

w

i

h

s

u

m

+

v

u

d

u

o

1

2

1

3

1

4

1

5

1

6

1

7

1

8

6

6

6

6

5

6

3

6

0

6

4

6

1

6

0

6

0

6

0

6

0

6

0

6

0

6

6

6

4

6

0

6

1

6

0

6

0

6

0

6

0

6

0

5

L

D

s

0

o

M

D

s

0

6

8

4

4

o

>

3

5

o

<

4

2

1

6

<

1

5

<

L

u

s

n

a

z

2

4

<

3

3

o

>

2

6

o

>

2

7

o

>

*

T

e

a

e

c

c

a

e

o

m

o

a

y

a

o

n

h

c

o

h

e

u

n

h

E

1

s

a

n

a

o

m

o

b

d

y

a

o

n

h

e

u

n

h

7

s

a

n

t

S

o

n

e

2

T

e

T

A

C

r

o

n

a

z

o

e

s

w

i

h

Z

k

a

H

a

w

a

d

v

u

V

i

u

Z

k

E

1

h

p

H

a

w

a

d

8

p

u

m

D

e

p

o

R

h

u

N

o

N

o

m

a

9

D

e

m

m

u

M

.

4

Z

k

m

m

u

7

Z

k

n

m

m

u

7

Z

k

m

m

u

7

D

e

m

m

u

8

8

S

v

v

a

o

o

m

i

c

n

a

e

w

i

h

s

u

m

p

u

v

u

d

u

o

1

3

1

4

1

5

1

6

1

7

1

8

0

6

0

5

2

6

5

6

5

6

0

6

0

6

1

6

5

6

6

6

6

6

6

6

0

8

0

8

0

8

5

8

7

8

0

8

0

8

0

8

5

8

8

8

7

8

8

8

8

8

L

D

o

~

6

6

7

0

o

>

3

5

<

5

9

o

>

5

8

2

6

<

L

U

n

s

n

a

z

3

1

o

>

0

1

o

>

3

3

o

>

I

S

o

n

e

2

T

e

by guest on February 4, 2016http://trstmh.oxfordjournals.org/ Downloaded from







G . W . A . D I C K So F . K I T C H E N A N D A . J . H A D D O W ~ 1 ~

Laboratories of Th e Rockefeller Foundation). It was demonstrated in this

laboratory that this serum neutralized 3.2 log units of 83rd-passage mouse brain

Hawaii dengue virus when tested intracerebrally in 7-day-old mice by the method

described.

It will be seen in Table III that the Hawaii dengue immune serum had no

neutralizing effect on 108th mouse brain passage Zika virus.

The strain of Hawaii dengue virus employed (Table I I I was supplied as a

lyophilized 5 per cent. mouse brain suspension of 77th mouse passage virus by

Dr. M. THEILER (Division of Medicine and Public Health Laboratories of The

Rockefeller Foundat ion). After five intracerebral passages in 7-day-old mice in

this laboratory, two brains of the 82nd mouse passage virus were used for a neutral-

ization test. The brains were emulsified in the whole serum of a dengue non-

immune monkey to make a 10 per cent. suspension. Serial 10-fold dilutions of

this suspension were made with that serum as indicated in Table I II . The dengue

anti-serum used in this tes t was supplied by Dr. A. B. SABIN (the Children s

Hospital, Cincinnati, Ohio) as lyophilized rhesus monkey serum. This serum

was rehydrated and inactivated immediately before use. The subsequent pro-

cedures were the same as in the neutralization tests already described except that

(a) the serum-virus mixtures were incubated for only 1 hour at 37.5°C., and

(b) the inoculations were made intracerebrally into 7-day-old mice. It may be

seen in Table III that Zika immune serum had no significant neutralizing effect

on 82nd-passage Hawaii dengue virus.

Theiler s encephalom yelitis virus. The FA and TO strains of Theiler s mouse

encephalomyelitis virus were received from Dr. M. THEILER and the GD VII

strain from Dr. H. A. HowE. Antiserum to the FA strain was prepared by

repeated inoculations of a rabbit (375) with mouse brain virus and antiserum to the

GD VII strain was prepared in cotton-rats by a similar method. It was shown

that while antisera to the FA and GD VII strains neutralized 1.4 and 1.6 log

units of these viruses respectively, they gave no significant neutralization of Zika

virus. Reciprocally it was demonstrated that there was no significant neutrali-

zation of the FA, GD VII or TO strains of virus by antiserum to Zika virus. In

general, neutralization tests with Thei ler s virus are unsatisfactory. Apart from

the immunological evidence, the pathogenicity of Zika virus indicates that it is

not identical with strains of Theiler s encephalomyelitis of mice.

Test with other antisera. Neutralization tests were made with the antisera to

the viruses listed in Table IV and to Mengo encephalomyelitis and Uganda 8

viruses. Antisera to Bunyamwera, 8emliki Forest, Bwamba fever, Ntaya, Mengo

encephalomyelitis, Ilhdus and Uganda S viruses were prepared at this institute by

the inoculation of animals with mouse brain suspensions of these agents. Antisera

to Eastern equine, Western equine, St. Louis and Japanese B encephalitis viruses

were prepared in New York by Dr. K. C. SMITHBURN (Division of Medicine and

Public Health Laboratories, The Rockefeller Foundation) . All of these sera



g /

D o wnl o a d e d

f r om






































o

T

A

I

V

N

a

z

o

e

s

w

h

a

s

a

o

s

o

m

n

o

o

c

v

u

S

u

m

N

m

m

N

m

h

m

I

m

o

v

u

B

m

a

S

m

k

F

e

B

m

f

e

N

a

W

e

N

e

I

h

E

e

n

e

n

e

~

W

e

e

n

e

n

e

V

a

e

n

e

S

L

s

e

J

B

e

L

m

c

c

o

m

n

s

B

v

u

(

C

e

d

L

n

Z

k

S

v

v

r

a

o

o

m

c

n

a

e

S

c

o

s

u

m

p

u

v

u

d

u

o

s

u

m

R

*

A

J

H

R

4

e

h

i

o

p

s

2

6

R

2

R

4

R

8

R

M

.

4

R

M

.

4

G

n

g

2

R

M

.

4

R

M

.

4

G

n

g

R

1

H R

7

w

h

1

3

1

4

1

5

1

6

1

7

1

8

0

6

3

5

3

4

5

6

6

6

0

6

3

6

4

6

6

6

6

6

0

6

3

6

4

5

6

6

5

5

0

6

1

6

4

5

6

6

6

6

0

6

1

6

1

6

6

6

6

6

0

6

1

6

5

6

6

6

5

5

0

6

3

6

2

6

5

6

6

6

0

5

1

5

5

5

5

5

S

5

0

6

0

6

4

6

6

6

5

5

0

6

1

6

5

6

6

6

5

6

0

6

1

6

5

6

6

6

6

6

1

6

1

6

6

6

6

6

6

6

0

6

1

6

5

6

6

]

6

6

6

0

6

2

6

4

6

4

4

6

6

0

6

4

6

4

6

6

6

6

6

0

6

3

6

5

5

6

6

5

6

6

6

6

6

6

6

6

6

6

6

L

D

o

5

2

5

3

5

1

5

5

6

3

5

5

6

0

5

3

5

7

5

5

5

5

5

3

<

5

5

5

5

5

0

5

0

o

>

2

5

<

L

u

s

n

a

z

0

1

0

2

0

2

<

2

7

o

>

j

S

f

o

n

e

T

e

I

*

R

-

r

h

u

e

-

e

s

by guest on February 4, 2016http://trstmh.oxfordjournals.org/ Downloaded from







O. W. A. DICK S. F. KITCHEN AND A. J. HADDOW 51 9

n e u t r a li z e d m o r e t h a n 2 lo g s o f t h e h o m o l o g o u s v i ru s . L y m p h o c y t i c c h o ri o -

me n i n g i t is an t i se r a w e r e p r o v i d e d b y D r . F . O . M A C C A LLU M ( V i r u s R e f e r en c e

L a b o r a t o r y , C o l in d a le , L o n d o n ) a n d b y D r . T . P . H U GH E S ( f o r m e r s t a ff m e m b e r ,

T h e R o c k e f e l le r F o u n d a t i o n ) , V e n e z u e l a n e q u i n e e n c e p h a li ti s a n t i s e r u m b y D r . P .

K OLITSKy

( T h e R o c k e f e l l e r I n s t i t u t e f o r M e d i c a l R e s e a rc h , N e w Y o r k ), B . v i r u s

( C an f ie l d ) an t i s e r u m b y D r . A . B . S A B I N ( T h e C h i l d r en s H o s p i t a l , C i n c i n n a t i,

O h i o ) a n d l o u p i n g i l l an t i s e r u m b y D r . D . G . f t. ED WAR D ( T h e W el l co m e V e t e r -

i n a r y R e s e a r c h S t at io n , F r a n t , E n g l a n d ) . A c k n o w l e d g e m e n t is m a d e to a ll w h o

k i n d l y s u p p l i ed t h e s e s p ec i fi c n eu t r a l iz i n g an t is e r a .

A l l t h e an t i s e ra l i s t ed in T ab l e I V w er e t e s t ed ag a i n s t d i lu t i o n s o f t h e 9 9 t h

p a s s a ge o f t h e E / 1 s tr a i n o f Z i k a v i ru s . T h e m i c e e m p l o y e d in t h a t e x p e r i m e n t ,

w h i c h i s r e c o r d e d i n T a b l e I V , w e r e 4 2 to 4 5 d a y s o ld .

N e u t r a l iz a t i o n t e s t s w i t h M e n g o e n c e p h a l o m y e l i t is a n d U g a n d a S v i r u s

an t i s e ra g av e n o e v i d en ce o f an y s i g n if ican t n eu t r a l iz a t i o n .

DISCUSSION

T h e p o s s i b le e x is t e n c e o f m i n o r a n t ig e n i c r e l a ti o n s hi p s b e t w e e n Z i k a v ir u s

a n d o t h e r v ir u s e s h a s n o t y e t b e e n c o m p l e t e l y e x p l o r e d in t h is l a b o ra t o ry . F r o m

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

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

t e s t ed , an d i s n o t r e l a t ed t o y e l l o w f ev e r , t h e H aw a i i s tr a i n o f d en g u e n o r t o t h e

G D V I I a n d F A s t r a in s o f T h e i l e r s v i r u s . I n a d d i t i o n to t h e s e ro l og i ca l t e s ts

w h i c h h a v e b e e n m a d e , t h e p a t h o g e n i c i t y a n d p r o p e r t i e s o f Z ik a v ir u s t e n d t o

d i f f e r en t i a t e i t f r o m o t h e r v i r u s e s , an d i t i s b e l i ev ed t h a t Z i k a v i r u s i s a h i t h e r t o

u n d e s c r i b e d v i r u s .

T h e o b s e r v a t i o n s r e c o r d e d i n t h i s p a p e r a r e d i s c u s s e d i n t h e p a p e r w h i c h

f o l lo w s , a l o n g w i t h s t u d i e s o n t h e p a t h o g en i c i t y an d p r o p e r t i e s o f Z i k a v ir u s .

SUMMARY

( 1) Th e i s o la t io n o f w h a t i s b e l i ev ed t o b e a h i th e r t o u n r ec o r d ed v i r u s i s

d e s c r i b e d . T h e f ir st i so l a ti o n w a s m a d e i n A p r i l 1 94 7 f r o m t h e s e r u m o f a p y r e x i a l

r h e s u s m o n k e y c a g e d i n t h e c a n o p y o f Z i k a F o r e s t . T h e s e c o n d is o la t io n w a s

m ad e f r o m a lo t o f ~ /. f r ic nus t ak en i n J an u a r y , 1 9 4 8, i n t h e s ame f o r e s t. T h e

v i r u s h a s b een ca l l ed Z i k a v i r u s a f t e r t h e l o ca l i t y f r o m w h er e t h e i s o l a t i o n s w e r e

m a d e .

(2 ) C r o s s n eu t r a l i z a t i o n t e s t s i n d i ca t e t h a t Z i k a v i r u s is n o t r e l a t ed t o y e l l o w

f e v e r, H a w a i i d e n g u e n o r t o th e F A a n d G D V I I s tr a in s o f T h e i l e r s m o u s e

en cep h a l o m y e l i t i s v i r u s . N e u t r a l i z a t i o n t e s t s w i t h Z i k a v i r u s an d t h e an t is e r a o f

s o m e o t h e r v i r u s e s w h i c h a r e n e u r o t r o p i c i n m i c e g a v e n o e v i d e n c e o f a n y i d e n t i ty

o f t h e s e w i t h Z i k a v i r u s .



g /

D o wnl o a d e d

f r om






































520 ZIK~ VIRUS

REFEREN ES

BUXTON,

A . P . ( in p r e s s) . O b s e r v a t i o n s o n th e d i u r n a l b e h a v i o u r o f t h e r e d t a i l m o n k e y

Cer cop i thecus a s can ius s chm id t i

M a t s c h i e ) i n a s m a l l f o r e s t i n U g a n d a .

D m K , G . W . A . , S M IT H B UR N , K . C . H A D D O W , A . J . ( 1 9 4 8 ).

B r i t . f t . e x p . P a t h . ,

29 , 547

- - , (1 94 9).

f t . I m m u n o l . ,

62 , 375 .

- - , (19 52).

T r a n s . R . S o c . t r o p . M e d . H y g . ,

46 , 521 .

, H A D DO W , A . J . ( i n p r e s s ) . U g a n d a S v i r u s . I . I s o l a t i o n a n d s e r o l o g i c a l

s p e c i f i c i t y .

HADDOW, A. J . , GILLETT, J . D . Hm HTO N, R. B . ( 1947) . B u l l . e n t . R e s . , 37 , 301 .

, SMITHBURN,

K . C . ,

D IC K , G . W .

A . ,

KITCHEN, S . F . LUMSDEN, W .

H . R . ( 1 9 4 8 ) .

A n n . t ro p . M e d . P a r a s i t . , 42 , 218 .

MAHAFFY, A . F . , SMITHBURN, K . C . , JACOBS, H . R . GILLETT, J . D . ( 1942) . T r a n s . R .

S o c . t r op . M e d . H y g . ,

36, 9 .

REED, L . J . MUENCH , H .

(1938) .

A m e r . J . H y g . ,

27 , 493 .

SMITHBURN, K . C . HADDO W, A . J .

(1944) .

J . I m m u n o l . ,

49 ,

141.

- - , (19 46 ). A m e r . J . t ro p . M e d . , 26, 261.

, , M A H m F ' ¢ , A . F . ( 1 9 4 6 ).

I b i d . ,

26 , 189 .

- - , - - ,

GILLETT, J .

D. (1948) .

B r i t . f t . e x p . P a t h . ,

29 , 107 .

- - , , LUM SDEN, W . H . R . (1 94 9 ) .

A n n . t r o p . M e d . P a r a s i t .

4 3 , 7 4 .

- - , (19 51 ). P r o c . S o c . e x p . B i o l . N . Y . , 77 , 130 .

THEILER, IVI. (1933).

A n n . t ro p . M e d . P a r a s i t. ,

27 , 57 .

WARREN, J . , SMADEL, J . E . Ru ss , S . B .

(1949) .

f t . I m m u n o l . ,

62 , 387 .



g /

D o wnl o a d e d

f r om




































Date post:	25-Feb-2018
Category:	Documents
Upload:	julio-martin
View:	215 times
Download:	0 times

Trans R Soc Trop Med Hyg-1952-Dick-509-20

Documents