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Archiv fiir die gesamte Virusforschung 35, 183--193 (1971) (~ by Springer-Verlag 1971

Z i k a V i r u s I n f e c t i o n o f t h e Central N e r v o u s S y s t e m o f Mice

By

T. M. BELL, E. J. FIELD, and H. K. NARANG

Medical Research Council, Demyelinating Diseases Unit, Newcastle General Hospital, Newcastle upon Tync, England

With 8 Figures

Received February 10, 1971

Summary

Intracerebral inoculation of newborn and 5-week-old mice with Zika virus resulted in an early and m a r k e d enlargement of astroglial cells with p a t c h y destruction of the pyriform cells of A m m o n ' s horn. Replication of the virus was demonstrated in b o t h neuroncs and astroglial cells. New virions appeared to be formed within networks of endoplasmic reticulum. The similarity of these ultra- structural observations to those obtained from in vivo studies of other group B arboviruses is contrasted with the widely differing findings from in vitro studies.

1. Introduction

Zika virus is a group B arbovirus prevalent in central Africa. Originally isolated from a sentinel rhesus m o n k e y in the Zika forest, near Entebbe, U g a n d a (1), it has since been recovered frequently from mosquitoes of the A edes (Stegomyia) africanus (Theobald) species in the same area (2, 3, 4). All isolates have been readily a d a p t e d to intracerebral passage, initially in 5- to 6-week-old adult mice (5), b u t latterly in 1- t o 5-day-old suckling mice (2). Pathological changes arc confined to the central nervous system in older animals, b u t myocarditis and skeletal myositis are also found in the y o u n g animals (2). I n this s t u d y pathological changes in brain are correlated with growth of the virus as demonstrated in the electron microscope and the general p a t t e r n of replication of the group B arbo- viruses is discussed.

2. Materials and Methods 2.1. V i r u s

One ampoule of the MP 1751 strain of Zika virus was kindly supplied by the Director, East African Virus Research Institute, Entebbe, Uganda. This strain was isolated from a pool of 50 A. (S.) a]rican~es collected at the Zika forest on 1.11.1962 and passaged intracerebrally once in baby mice before lyophilization. The freeze-dried powder was rehydrated with sterile double-distilled water and diluted 1 : 10 in Eagle's BME (Wellcome TC 48).

1 3 "

184 T . M . B E L L , E. J. F I E L D , a n d H. K. NARANG:

2 . 2 . I n o c u l a t i o n of M i c e 0 n o - d a y - o l d W e b s t e r Swiss (WS) white mice were inoculated i n t r a c e r e b r a l l y with

0.03 ml of the r e c o n s t i t u t e d virus a n d observed daily. On the seventh post-inoculation d a y all animals fell sick, a n d the b r a i n s were harvested. A 10% suspension of b r a i n was prepared a n d used as a pool to inoculate f u r t h e r one-day-old WS mice. These animals fell sick on d a y six a n d were either perfused with a formal, methanol, acetic acid fixative, or the b r a i n s were fixed in formal a m m o n i u m bromide or glutaraldehyde. Five-week-old mice were also inoculated i n t r a c e r e b r a l l y a n d a p p r o x i m a t e l y 20~/o became obviously sick between days 14 a n d 20. The b r a i n s were fixed in formal a m m o n i u m bromide. Histological a n d electron microscopical e x a m i n a t i o n s were carried out as previously described (6).

2 . 3 . C o n f i r m a t i o n of I d e n t i t y of V i r u s The mouse b r a i n pool of Zika virus was t i t r a t e d in s t a t i o n a r y t u b e cultures of

secondary rhesus m o n k e y k i d n e y (RMK) cells a n d the TCDso calculated. Reference a n t i s e r u m prepared against the p r o t o t y p e s t r a i n of Zika virus was o b t a i n e d from the Director, E.A.V.R.I., E n t e b b e . A n e u t r a l i z a t i o n test using serial 2.fold dilutions of this serum against 100 TCDs0 of the MP 1751 strain of Zika virus was set up in mono- layer cultures of RMK cells.

3. Results

3 . 1 . t t i s t o p a t h o l o g y

T h e m o s t s t r i k i n g changes were f o u n d i n A m m o n ' s horn~ where t h e r e were localized s e g m e n t s of necrosis i n t h e b a n d of p y r i f o r m cells (Fig. 1 a, b, e, d). M u c h

F i g . 1 a. N o r m a l A m m o n ' s h o r n of m o u s e 7 - d a y s - o l d . N o t e r e g u l a r a r r a n g e m e n t of p y r i f o r m cells. H . E . • 56

s c a t t e r e d h y p e r e h r o m a t i c n u c l e a r d e b r i s was p r e s e n t a n d t h e t i s s u e p r e s e n t e d a m o t h - e a t e n a p p e a r a n c e . M o d e r a t e p e r i v a s c u l a r cuffing was o c c a s i o n a l l y seen. A s t r o e y t e h y p e r t r o p h y was p r o m i n e n t (Fig. 2 a , b). T h e r e l a t i v e l y p l u m p cells of t h e n e w b o r n a n i m a l were e n l a r g e d a n d p r e s e n t e d m o r e b r a n c h e d processes. Microglial cells were n o t p r o m i n e n t . I n t h e 5-week-old m o u s e (where t h e disease

Zika Virus Infection of the Central Nervous System of Mice 185

t o o k a b o u t 14 d a y s t o develop) astroglial changes were 'again r e m a r k a b l e (Fig. 3). T h e n o r m a l a n i m a l a t eight weeks has w i s p y a n d p o o r l y stainable astroglial cells in t h e Cajal p r e p a r a t i o n . Astroglial changes in t h e b a b y mouse were l i m i t e d to A m m o n ' s h o r n b u t in t h e 5-week-old a n i m a l s infected astroglia was p r o m i n e n t t h r o u g h o u t t h e cortex.

F i g . 1 b. A m m o n ' s h o r n of Z i k a i n f e c t e d m o u s e 7 - d a y s - o l d . N o t e d i s o r g a n i z a t i o n of p y r i f o r m c e l l l a y e r a n d d i l a t a t i o n of v e s s e l s i n w h i t e m a t t e r w i t h o n l y v e r y s l i g h t p e r i v a s e u l a r c u f f i n g , t t . E . x 56

F i g . l c . N o r m a l p y r i f o r m c e l l s : 7 - d a y s - o l d m o u s e . H . E . x 560

I n t h e cortex of t h e b a b y mouse t h e r e were m a n y n e r v e cells w i t h " e m p t y " vesicular nuclei a n d some i n t e r s t i t i a l l y s c a t t e r e d nuclear e h r o m a t i n particles (Fig. 4a, b). P e r i v a s c u l a r cuffing was n o t seen.

186 T . M . BELL, E. J. FIELD, and H. K. NARANG:

F i g . l d . S a m e a r e a i n 7 - d a y s o l d Z i k a i n f e c t e d m o u s e . H . E . • 560

3 . 2 . E l e c t r o n M i c r o s c o p y

E x a m i n a t i o n of t h e c o r t e x a n d A m m o n ' s h o r n of 7 - d a y - o l d mice i n j e c t e d i n t r a c e r e b r a l l y w h e n 1 - d a y - o l d w i t h t h e M P 1751 s t r a i n of Zika v i r u s r e v e a l e d

F i g . 2 a . A s t r o c y t e s i l l A m m o l l ' S h o r n o f n o r m a l 7 - d ~ y s - o l d m o u s e . N o t e p l u m p c e l l s w i t h r e l a t i v e l y s i m p l e p r o c e s s

n u m e r o u s cells c o n t a i n i n g i n t r a c y t o p l a s m i c inclusions or " v i r u s f a c t o r i e s " (Figs. 5, 6 a n d 7). B o t h glial cells a n d n e u r o n s were a f f e c t e d a n d m o r e t h a n one " f a c t o r y " could s o m e t i m e s be seen i n a single n e u r o n e process (Fig. 5). I n t h o s e cell profiles

Zika Virus Infection of the Central Nervous System of Mice 187

F i g . 2 b . A s t r o c y t e s i n A m m o n ' s h o r n of 7 - d a y - o l d m o u s e w i t h Z i k a i n f e c t i o n . N o t e i n c r e a s e d n u m b e r of a s t r o c y t e s w i t h p h l m p b o d i e s a n d l ~ r g e n u m b e r s of p r o c e s s e s . Caj a l g o l d c h l o r i d e i m p r e g n a t i o n • 560

F i g . 3. A m m o n ' s h o r n o f 5 0 - d a y - o l d m o u s e w i t h Z i k a i n f e c t i o n . N o t e l a r g e n u m b e r s of w e l l i m p r e g n a t e d a s t r o c y t e s . C a j a l g o l d c h l o r i d e i m p r e g n a t i o n • 140

w h i c h i n c l u d e d a s e c t i o n of t h e nucleus, t h e " v i r u s f a c t o r i e s " were u s u a l l y f o u n d i n close p r o x i m i t y t o it (Fig. 6).

T h e " f a c t o r i e s " were c o m p o s e d of a n e t w o r k of e n d o p l a s m i c r e t i c u l u m a n d large n u m b e r s of d i s t e n d e d e n d o p l a s m i c c i s t e r n a e (Fig. 8). T h e s e enclosed n u m e r o u s v e s i c u l a r bodies, 74 t o 78 m ~ in d i a m e t e r , w i t h i n w h i c h t h e r e w a s a fine r e t i c u l a r n e t w o r k . D e n s e - c o r e d particles, w i t h t h e t y p i c a l a p p e a r a n c e of

188 BELL et al.: Zika Virus Infection of the Central Nervous System of Mice

group B arboviruses, were found t h r o u g h o u t the "factories". These particles had an overall diameter of 40 to 43 m~z, the central core being 28 to 30 m~. The virions were frequently found in short chains within tubular elements of endoplasmie reticulum which appeared to be in continuity with the distended eisternae (Fig. 8).

The cytoplasm of infected cells appeared normal in areas removed from the "virus factories" and large numbers of mitochondria were present in these cells, m a n y adjacent t o the "factories".

F i g . 4 a. C e r e b r a l c o r t e x o f 7 - d a y - o l d n o r m a l m o u s e . N o t e w e l l s t a i n e d n u c l e i

Fig. ~ b. Same area in 7-day-old Zika infected mouse. Note margination of chromatin in neurones and nuclear debris. There is no perivasenlar cuffing. I-I.E. • 350

F i g . 5. A m m o n ' s h o r n of 7 - d a y - o l d m o u s ~ i n f e c t e d w i t h Z i k a v i r u s . P r o f i l e of d e n d r i t e e m b r a c e d b y a n a s t r o g l i a l cell ( A G N ) . T w o v i r u s f a c t o r i e s ( V F ) a r e v i s i b l e , iViitochondria a r e d a r k (M) x 9,800

F i g . 6. V i r u s f a c t o r y ( m o r e a d v a n c e d s t a g e t h a n f i g . 5) in c y t o p l a s m of a s t r o g l i a l cell. • 11,500

190 T . M . BELL, E. J. FIELD, a n d H. K. NARANG:

F i g . 7. A m m o n ' s h o r n 7 - d a y - o l d m o u s e i n f e c t e d w i t h Z i k a . V i r u s f a c t o r y eontainiug" i n d i v i d u a l v i s i o n ( V P ) p r e s e n t i n c y t o p l a s m of p y r i f o r m n e r v e cell. N e u r o t u b u l e s a r e p r o m i n e n t ( N T ) . E n d o p l a s m i c

r e t i e u h n n c o n t a i n s v e s i c l e s ( a r r o w s ) • 38,000

F i g . 8. T h a l a m u s n e u r o n e of 7 - d a y - o l d m o u s e w i t h Z i k a i n f e c t i o n . E n d o p l a s m i c r e t i c u l m n ( E R ) c o n t a i n s m a t u r e v i r u s p a r t i c l e s ( V P ) . V e s i c l e s ( a r r o w s ) seen i n a s s o c i a t i o n w i t h e n d o p l a s m i e r e t i c u h m l • 105,000

3 . 3 . C o n f i r m a t i o n o f I d e n t i t y

T h e 1 0 % m o u s e b r a i n p o o l of Z i k a v i r u s t i t r e d 10 ̀ 5 TCDs0 p e r 0.1 m l i n m o n o - l a y e r s of s e c o n d a r y R M K cells. 100 TCD~o of t h i s v i r u s w a s n e u t r a l i z e d b y t h e r e f e r e n c e a n t i s e r u m t o a d i l u t i o n of 1 : 320.

Zika Virus Infection of the Central Nervous System of Mice 191

4. Discussion

Zika virus a p p e a r s to h a v e a n e a r l y effect u p o n astroglial cells p r o d u c i n g e n l a r g e m e n t a n d extension of t h e i r processes. Microglial cells do n o t a p p e a r to be excited. I n a d d i t i o n t h e virus affects t h e p y r i f o r m cells of A m m o n ' s h o r n in a p a t c h y m a n n e r . T h e r e is no especial predilection for lesions in t h e p e r i v e n t r i c u l a r areas (as in neuro-influenzal lesions [6]). Arboviruses in general h a v e been claimed to s t i m u l a t e astroglia electively (7) b u t this m a y be because (in t h e mouse) this e l e m e n t of t h e glia is m o r e r e a c t i v e a n d certainly m o r e r e a d i l y stainable t h a n microglia (6). Moreover t h e s a m e precocious change in astroglia has been f o u n d in measles encephalitis in t h e m o u s e ( B E L L , NARANG a n d FIELD - - unpublished) where microglia r e m a i n e d unaffected.

I n t h e electron microscope Zika virus was found to replicate b o t h in astroglial cells a n d in neurones. Microglial cells were n o t encountered w i t h a n y g r e a t e r f r e q u e n c y t h a n normal. R e p l i c a t i o n in neurones indicates t h a t t h e i r destruction, so clearly seen in t h e light microscope, is due specifically t o t h e Zika virus infection. H o w e v e r , in m o s t cases t h e infected neurons do n o t show gross d a m a g e especially in areas of t h e c y t o p l a s m r e m o t e f r o m t h e " v i r u s factories". I t , therefore, a p p e a r s possible t h a t t h e a c t u a l cell d e s t r u c t i o n is due, in p a r t a t least, t o a n i m m u n o - logical elimination m e c h a n i s m as p r o p o s e d b y R o o k a n d WEBB (8) for L a n g a t virus encephalitis.

T h e " v i r u s f a c t o r i e s " a p p e a r t o be v e r y similar t o c y t o p l a s m i c inclusions o b s e r v e d in mouse brains infected w i t h J a p a n e s e B encephalitis ( J B E ) virus (9, 10), central E u r o p e a n encephalitis (CEE) virus (11) a n d E n t e b b e b a t s a l i v a r y gland (EBSG) virus (12). These are all g r o u p B arboviruses a n d r e p r e s e n t m e m b e r s t r a n s m i t t e d b y mosquitoes ( J B E a n d Zika), b y ticks (CEE) a n d b y t h e bite of t h e infected host (EBSG) (13). YASVZUMI a n d his colleagues (9) o b s e r v e d 20 m ~ particles in t h e nucleus a n d suggested t h a t t h e complete virus was f o r m e d in t h e nucleus, a l t h o u g h some precursors were f o r m e d in t h e cytoplasm. T h e i n t r a n u c l e a r particles h a v e n o t been o b s e r v e d in t h e o t h e r p r e p a r a t i o n s a l t h o u g h t h e c y t o p l a s m i c inclusions are n o r m a l l y f o u n d in close p r o x i m i t y to t h e nucleus. Mitoehondria are also p r e v a l e n t in t h e c y t o p l a s m a d j a c e n t t o t h e inclusion. I t , therefore, a p p e a r s t h a t these " v i r u s f a c t o r i e s " are indeed t h e sites of viral replication, a l t h o u g h precursors (possibly t h e ribonucleoprotein) m a y be f o r m e d w i t h i n t h e nucleus. T h r o u g h o u t t h e factories m a t u r e virions can be seen w i t h i n sacs or t u b u l e s f o r m e d f r o m e x p a n d e d endoplasmic r e t i c u l u m a n d it is believed t h a t this is t h e a c t u a l site of f o r m a t i o n of new virus particles. T h e m o d e of release of t h e virus particles f r o m t h e cell has n o t y e t been d e m o n s t r a t e d in vivo.

Tissue cultures infected w i t h t h r e e g r o u p B arboviruses h a v e been e x a m i n e d in t h e electron microscope. M u r r a y Valley encephalitis (MVE) virus grown in m o s q u i t o cell cultures produces virus factories similar to those described here (14). J B E virus however, replicates in t h e endoplasmie r e t i c u l u m of these cells w i t h o u t t h e f o r m a t i o n of a definite inclusion b o d y (14), while in porcine k i d n e y cells J B E virus a p p e a r s to f o r m b y b u d d i n g f r o m t h e surface of c y t o p l a s m i c vacuoles (15). Finally, Wesselsbron virus produces crystalline a n d " h o n e y - c o m b " p a t t e r n inclusion bodies in foetal l a m b k i d n e y cells w i t h m a t u r e particles present in endoplasmic eisternae (16). I n t h e tissue cultures large n u m b e r s of m a t u r e virus

192 T.M. BELL, E. J. FIELD, and H. K. NARANG:

p a r t i c l e s are p r e s e n t o u t s i d e t h e cells a n d release a p p e a r s t o be b y t h e m i g r a t i o n of v i r i o n c o n t a i n i n g v a c u o l e s t o t h e cell m e m b r a n e (15).

F r o m t h e s e o b s e r v a t i o n s i t is clear t h a t a l t h o u g h e x a m i n a t i o n of t h e g r o w t h of a r b o v i r u s e s i n t i s s u e c u l t u r e s is useful, it is n e c e s s a r y t o u s e t h e i n t a c t a n i m a l t o o b t a i n a p i c t u r e of t h e n a t u r a l p a t t e r n of e v e n t s .

A c k n o w l e d g e m e n t s

We wish to t h a n k Dr. G. W. Kafuko, Director, East African Virus Research h l s t i t u t e , E n t e b b e , Uganda, for s u p p l y i n g the Zika virus a n d antiserum.

The authors would also like to t h a n k Miss Greta Joyce, A.I.M.L.T. a n d Miss Joyce D avison, for histological p r e p a r a t i o n s a n d for help with electron microscopy respectively.

References

1. DICK, G. W. A., S. F. KITCHEN, a n d A. J. HADDOW: Zika Virus. (1) Isolations a n d serological specificity. Trans. roy. Soc. trop. Med. Hyg. 467 509--520 (1952).

2. WEINB~EN, M. P., a n d M. C. WILLIA~S : Zika Virus : F u r t h e r isolations in the Zika area, a n d some studies on the strains isolated. Trans. roy. Soc. trop. Med. Hyg. 52, 263--268 (1964).

3. WOODALL, J. P . : The viruses isolated from arthropods at the E a s t African Virus Research I n s t i t u t e in the 26 years e n d i n g December 1963. Proc. E. Afr. Acad. 2, 141-- 146 (1964).

4. HADDOW, A. J., M. C. WILLIAMS, J. P. WOODALL, D. I. H. SIMPSON, a n d L. K. H. GOM~_: Twelve isolations of Zika virus from Aedes (Stegomyia) a]ricanus (Theo- bald) t a k e n in a n d above a U g a n d a forest. Bull. W l d H l t h Org. 817 5 7 - - 6 9 (1964).

5. DICK, G. W. A.: Zika virus .(II). P a t h o g e n i c i t y a n d physical properties. Trans. roy. Soc. trop. Med. Hyg. 467 521--534 (1952).

6. BELL, T. M., H. K. NARANG, a n d E. J. FIELD: I n f l u e n z a l encephalitis in mice: A histopathological a n d electron microscopical study. Arch. ges. Virusforsch. 347 158--167 (1971)

7. ZLOTNIK, I.: The reaction of astrocytes to acute virus infections of the central nervous system. Brit. J, exp. P a t h . 49~ 555 564 (1969).

8. ROOK, G. A. W., a n d H. E. WEBn : A n t i l y m p h o e y t e serum a n d tissue culture used to investigate role of cell-mediated response i n viral encephalitis in inice. Brit. reed. J. 4, 210--212 (1970).

9. YASUZUIN[I, G., I. TSUBO, R. SUGIItASA, a n d Y. N+~I;AI :Analysis of the development of J a p a n e s e ]3 encephalitis (JB E) virus. I. Electron microscopic studies of microglia infected with J B E virus. J. U l t r a s t r u c t . Res. l l 7 213--229 (1964).

10. OYANAGI, S., F. IKUTA, a n d E. R. Ross : Electron microscopic observations in mice infected with J a p a n e s e encephalitis. Acta n e u r o p a t h . (Berl.)13, 169--181 (1964).

11. BLI~ZI~GER, K., u n d W. MULLER: Elektronenmikroskopische B e o b a c h t u n g des Virus der Frtthsommer-Meningo-encephalitis (Stamin H Y P R ) in Nervenzellen y o n experimentell infizierten Albinoms Dtsch. Z. Nervenheilk. 1977 18--27 (1970).

12. P~AT, A., a n d T. M. BELL: E n t e b b e b a t salivary gland virus: Electron microscopic s t u d y of morphology a n d development in n e w b o r n mice. Arch. ges. Virusforseh. 317 230--236 (1970).

13. WIImlANS, M. C., D. I. H. SII~IPSON, R. C. SHEPHERD, J. P. O~SULLIVAN, J. C. CUNNINGHA)I, a n d M. LULE: Virus isolation from bats. Rep. E. Afr. Virus Res. Inst. 14, 42 (1965).

Z i k a Virus I n f e c t i o n of t h e C e n t r a l N e r v o u s S y s t e m of Mice 193

14. ]~ILSHIE, ]~. K., a n d J. REItACEK: S t u d i e s on t h e m o r p h o l o g y of M u r r a y Valley e n c e p h a l i t i s a n d J a p a n e s e e n c e p h a l i t i s g r o w i n g in c u l t u r e d m o s q u i t o cells. V i r o l o g y 34, 4 3 5 - - 4 4 3 (1968).

]5. OTA, Z. : E l e c t r o n m i c r o s c o p i c s t u d y of t h e developn~lent of J a p a n e s e B e n c e p h a l i t i s v i r u s in p o r c i n e k i d n e y s t a b l e (PS) cells. V i r o l o g y 25, 3 7 2 - - 3 7 8 (1965).

16. PARKER, J . R., a n d L. M. STANNARD: I n t r a c y t o p l a s m i c inclusions in f o e t a l l a m b k i d n e y cells i n f e c t e d w i t h Wesse]sbron virus. Arch. ges. Virusforsch. ~0, 4 6 9 - - 4 7 2 (1967).

A u t h o r s ' a d d r e s s : Dr. T. M. BELL, Medical R e s e a r c h Council, D e m y e l i n a t i n g Diseases U n i t , N e w c a s t l e General H o s p i t a l , W e s t g a t e R o a d , N e w c a s t l e u p o n Tyne, N E 4 6 BE, E n g l a n d .