Archives of Virology

Archives of Virology 62, 229--240 (1979)

© by Springer Vertag 1979

Non-Productive Paramyxovirus Infection: Nariva Virus Infection in Hamsters By R. P, Roos 1 and R. WOLLMANN2 Departments of Neurology L and Pathology 2, University of Chicago, Division of the Biological Sciences and Pritzker School of Medicine, Chicago, Illinois, U.S.A. With 8 Figures Accept.ed June 29, 1979

Summary The pathogenesis of infection with Nariva virus ( N V ) - - r e e e n t l y classified as a p a r a m y x o v i r u s - - w a s studied in the hamster, an animal closely related to the natural host. Intracranial inoculation of suckling hamsters produces an acute necrotizing encephalitis with large amounts of infectious virus and virus antigen in the brain. I n contrast, weanling hamsters have only small amounts of infectious virus and only early in the disease, when they are well; later, when clinically ill, they have a non-productive infection with continuing evidence of viral antigen, but no detectable infectious virus. Weanlings die later t h a n sucklings with less cerebral parenchymal necrosis. The integrity of the immune system affects the expression of NV since brain tissue from anti-lymphocyte serum (ALS) treated infected weanling hamsters have more infectious virus, and for longer periods, t h a n brain tissue from untreated infected weanling hamsters. Changing susceptibility of the host's neural cells m a y also be involved in determining the course of the illness and expression of the virus since: 1) ALS treatment does not influence the clinical course of the disease or pathology, 2) ALS treated weanlings still have much lower levels of infectious virus than sucklings, 3) infected weanling and suckling hamsters have a similar time course of development of NV neutralizing antibody.

Introduction Paramyxoviruses cause a broad spectrum of neurological disease in man, from acute self-limited meningitis to the chronic encephalitis of subacute sclerosing panencephMitis (SSPE). The factors responsible for development of diseases such as SSPE are being elucidated partly through systematic studies of paramyxovirus nervous system infections naturally found in laboratory animals. I t has become clear from such studies t h a t both the outcome of illness and expression of the 16"

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R . P . R o o s and R. WOLLSIANN:

virus are influenced b y t h e virus s t r a i n e m p l o y e d , t h e m a t u r i t y of t h e host i m m u n e s y s t e m a n d t h e v a r y i n g susceptibilities of h o s t n e r v o u s s y s t e m cell p o p u l a t i o n s of different ages ( 1 - - 3 , 6, 9 - - 1 1 , 13). N a r i v a virus (NV), r e c e n t l y classified as a p a r a m y x o v i r u s (4, 15), was i s o l a t e d in 1962 from w e l l - a p p e a r i n g T r i n i d a d i a n rats, Zygoclontomys b. brevicauda (14); Zygodontomys is a m e m b e r of t h e C t g I C E T I D A E f a m i l y of r o d e n t s , as is t h e h a m s t e r . T h e classification of N V as a p a r a m y x o v i r u s is b a s e d on: h e m a g g t u t i n a t i o n properties, w i t h r e d blood cell r e c e p t o r i n s e n s i t i v i t y to r e c e p t o r d e s t r o y i n g e n z y m e (8) ; p r o d u c t i o n of s y n c y t i a in tissue culture (8) ; u l t r a s t r u c t u r a l a p p e a r a n c e c h a r a c t e r i s t i c of p a r a m y x o v i r u s (15). E x c e p t for a single r e p o r t (14) t h a t i n t r a cerebral (i. c.) i n o c u l a t i o n of N V kills suckling, b u t n o t weanling mice, t h e a g e n t ' s in vivo p r o p e r t i e s are u n e x p l o r e d . T h e p r e s e n t s t u d y i n v e s t i g a t e s cerebra] infection w i t h N V in t h e h a m s t e r , a n a n i m a l closely r e l a t e d t o t h e n a t u r a l host. T h e e x p e r i m e n t s d e l i n e a t e t h e p a t h o genesis of t h e disease i n d u c e d following i.e. i n o c u l a t i o n of N V w i t h r e g a r d t o t h e d i s t r i b u t i o n a n d t i m e course of d e v e l o p m e n t of: 1) h i s t o p a t h o l o g i e a l lesions, 2) in. feetious v i r u s a n d 3) v i r u s antigen. T h e n a t u r e of a n d t h e reasons for t h e age d e p e n d e n c e of disease s u s c e p t i b i l i t y are e x a m i n e d .

Materials and Methods Animals Syrian hamsters (Engle L a b o r a t o r y Animals, Inc., Farmersburg, Indiana), Mesocricetus auratus, and inbred BALB/e mice (Laboratory Supply Co., Inc., Indianapolis, Indiana) including suckling, weanling and pregnant animals with timed gestations were used. Virus NV which had been intracerebrally passed two times in suckling mice, was obtained from Dr. Robert E. Shope, Yale University. Stock virus was prepared as a 10 percent suspension of brains from suckling mice in H a n k s ' belanced salt solution (HBSS) five days following i.e. inoculation. The suspension was clarified b y centrifugation at 1000×g for 10 minutes, and aliquots were stored at --70 ° C. Stock virus contained 10~.I plaque-forming units (PFU) per g of brain on Vero cells (Industrial Biological Laboratories, P~ockville, 3/[aryland) and 105.~ 50 percent lethal doses (LDso) per g of brain b y i.e. inoculation of suckling hamsters. Pathogenesis Studies All i.c. inoculations were given in volumes of 0.02 ml for suckling animals and 0.03 ml for weanlings, t I a m s t e r s were inoculated with 10s.4 or 10 La P F U of the stock virus; control animals received a similar volume of a 10 percent suspension of normal mouse brain. A n additional t0 weanling hamsters were inoculated with 104.6 P F U of a tissue culture stock of NV that had been plaque purified three times. Two or three suckling or weanling hamsters were killed a t regular intervals starting two or three days after inoculation until no survivors remained or until the fourteenth d a y after inoculation. A t the time of sacrifice, animals were bled and serum or plasma was obtained. After cardiac perfusion with either phosphate buffered saline (PBS) or 10 percent buffered formalin, brain, liver, kidney, and spleen were removed for viral assay, immunofluoreseent and/or histologie studies. Viral Assay o] Tissue Tissue samples for viral assay were ground in chilled mortars ~nd prepared as 10 percent suspensions in t t B S S containing 2 percent fetal calf serum (FCS). Suspensions were clarified a t 1000×g for 10 minutes and stored a t --70 ° C. Ten-fold dilu-

Non-Productive Paramyxovirus Infection

231

tions were prepared and assayed by plaquing on Vero cells. Three 35-ram plastic dishes were inoculated with 0.2 ml of each dilution, incubated and rocked for 1 hour at 37 ° C, washed once, and overlaid with 3 ml of minimum essential medium (MEM) containing 0.5 percent agarose (Seakem Marine Colloids Inc., Springfield, New Jersey) and 5 percent FCS. Plaques were read at 10 days after staining overnight with an overlay containing neutral red (1:30,000) in 0.5 percent agarose and MEM. Inoculation of newborn mice or hamsters was used in some cases for virus recovery.

Histology Tissues were fixed in 10 percent buffered formalin, embedded in paraffin, cut at 8 pt, and stained with hematoxylin and eosin.

2~luorescent Antibody Staining Tissues were frozen directly on dry ice and stored at - - 7 0 ° C. Seven ~ sections were cut in a cryostat, fixed for 10 minutes in acetone, and stained for viral antigen by the indirect immunofluorescent technique. Imm~me serum was obtained from weanling mice which had been inoculated intraperitoneally (i.p.) three times at 2-week intervals with stock virus containing 10~-1 P F U and bled I0 days after the last injection. A control serum obtained from mice inoculated on a similar schedule with a 10 percent suspension of normal suckling mouse brain was also employed. Sera were used at a 1:40 dilution. Fhlorescein isothiocyanate-conjugated antimouse IgG (Huntingdon Research Center, Inc., Baltimore, Maryland) was used at a 1 : 20 dilution. The specificity of this fluorescence for infected tissue was tested by reacting infected and normal tissue with immune and control sera followed by the fluorescein isothiocyanateconjugated antimouse IgG.

Immunological Studies Serum neutralizing antibody was measured by mixing 2-fold dilutions of serum with an equal volume of diluent containing 50 P F U of NV per 0.I ml. The Nariva virus used was a tissue culture stock prepared by passing the suckling mouse brain stock three times in Veto cells. After incubation at room temperature for 60 minutes, Vero celt monolayers were inoculated with 0.2 ml of the virus serum mixture and plaque assayed as described above. Neutralizing antibody titers are reported as the highest dilution of serum causing 50 percent or greater plaque reduction. Sera from mice immunized with stock virus and with control tissue, as described above, were included in all tests. The N V hyperimmune serum had a neutralizing antibody titer of 1:640; control serum was < 1:10. A]I sera were heated to 56 ° C for 30 minutes prior to testing. For irnmunosuppression, 16 weanling hamsters were treated with rabbit antiserum to hamster lymphocytes (Microbiological Associates, Bethesda, Maryland). The anti-lymphocyte serum (ALS) was given i.p. (1.25 ml per 100 g) 1 day before and 1, 3, 5, 7, 9 and 11 days after NV i.c. inoculation. The serum is certified for potency in eytotoxicity tests and in skin allograft prolongation (DBA/2f skin grafts survive over 22 days when grafted on to C 5 7 B L / 6 f mice).

Results

Clinical Studies T h e m o r t a l i t y of suckling a n d of weanling h a m s t e r s following i.e. i n o c u l a t i o n is s u m m a r i z e d in F i g u r e 1. Three d a y s after i n o c u l a t i o n of 103.4 P F U or 101.4 P F U of N V , n e w b o r n animals s t o p p e d suckling welt; those receiving i03-4 P F U died 4 t o 6 d a y s a f t e r i n o c u l a t i o n while those receiving I01-4 P F U died 4 t o 9 d a y s a f t e r inoculation.

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R . P . Roos and R. WOLn~IA~:

Weanling hamsters became sick 5 or 6 days after inoculation at either dilution of stock virus, Fifty percent of ill weanlings developed intermittent generalized or continuous focal seizures. AnimMs became thin and died with lethargy or seizures 7 to t l days after inoculation of 10 3-4 and 8 to 13 days after inoculation of 101'4 PFU.

100-

80/t

/

60" 40 20

.

1

2

3

4

5

6

7

8

.

9

.

.

.

.

10 11 12 13 14

DAYS POST INOCULATION

Fig. 1. Mortality of suckling ( ) and weanling hamsters (. . . . . . . . . ) after inoculation with 10a.4 PFU (o) or 101.4 PFU (.) of Nariva virus

Pathological Studies Suckling animals developed a meningitis and ependymitis by day 2 after infection. The cortex became disrupted and a severe neerotizing paneneephalitis developed by day 5 to 6 (Fig. 2). Multinucleated giant cells were observed in the ependyma and neuropil (Fig. 3). Weanling animals had evidence of ependymitis and meningitis by day 4 after infection. Perivaseular and subependymal monouuclear cell infiltrates (Figs. 4, 5) appeared on day 5 and were present in animals dying or sacrificed through day 14. In contrast to infected suckling animals, weanlings rarely exhibited multinueleated giant cells or necrosis. The only visceral lesions found were gastric ulcers or bronchopneumonia noted in occasional wea.nlings.

Virological Studies The results of plaque assays on brain homogenates of suckling hamsters inoculated with the two dilutions of NV are summarized in Figure 6. Sucklings had evidence of infectious virus early with the development of high titers b y day 2 (inoculated with 103.4 PFU) or day 4 (inoculated with l0 L4 PFU); high titers were maintained until death. Virus antigen, as demonstrated by fluorescence, was present two days after inoculation of sucklings, becoming abundant on day 3 and thereafter. Antigen appeared first in the inoculation site, and spread rapidly to ependyma, choroid plexus and meninges. Antigen then appeared in focal areas of the cortex, and finally became extensively and diffusely distributed (Fig. 7).

Non-Productive Paramyxovirus Infection

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Fig. 2. MeningStis and neerotizing encephalitis in suckling hamster 6 days afLer inoculation with 101-4PFU. Hematoxylin and eosin; × 160

t~ig. 3. lVIultinucleated giant cells in temporM lobe of suckling hamster 5 days after inoculation with l01.4 PFU. Hematoxylin and eosin; × 500

234

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Fig. 4. Ependymitis, ependymal disruption and mononuclear cell perivascular infiltrates in weanling hamster I0 days after inoculation with 108.4 PFU. Hematoxylin and cosin; x 63

Fig. 5. Mononuclear cell perivascular in~ilt,rates in weanling hamster brain 10 days after inoculation with 103.4 P F U . Hematoxylin and eosin; × 250

Non-Productive Paramyxovirus Infection

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Brain homogenates from only 8 of 29 weanling hamsters inoculated with 10a-4 P F U and 2 of 23 weanling hamsters inoculated with 101.4 P F U of NV had evidence of infectious virus (Table 1). The infectivity of the brain homogenates from ~hese 10 weanlings was relatively low compared to homogenates of sucklings; the highest titer was 104.5 P F U per g of brain. All 10 animals were clinically well and had been inoculated less than 10 days previously. None of the 16 animals studied after day 9 and only 2 of 17 studied on days 7 to 9 had infectious virus. Only 1 of an additional 10 weanling animals which had been inoculated with plaque-purified virus had infectious virus; this animal, sacrificed 4 days after inoculation had 104.3 P F U per g of brain. Virus antigen, as seen by fluorescence, regularly appeared in the brains of weanling hamsters by day 3 and continued through day 13 after inoculation with either dilution of XV (Table 1). This antigen was consistently present in brains of animals that contained no infectious virus. The antigen usually appeared first in the inoculation site and then spread to ependyma, choroid plexus and meninges. The antigen subsequently appeared subependymally and in the cortex, but usually remained more focal and limited than in infected suckling hamsters (Fig. 8).

1

2

3

4

5

DAYS AFTER[~Ig~E~EEt~P.LIHOCULATi~N

Fig. 6. Growth of v ~ s

in brain of suckling hamsters after inoculation of IO~.4PFU (o) and 101.4 P F U (°) of N a r i v a vL~s

Table t. In/ectious virus and fluorescent antibody staining in weanling hamsters a/ter

Nariva v~rus inoculation Days post inoculation

Infectious virus~ 103.4 P F U i.c. 101.4 P F U i.e.

Fluorescent antibody stainingb

2, 3 4---6 7--9 10--12 13, 14

2/3~ 5/7 1/10 0/6 o/a

+ +-~-~ + +

0/3 I/6 1/7 0/5 0/2

R a t i o of n u m b e r of b r a i n h o m o g e n a t e s from weanling h a m s t e r s w i t h infectious virus o v e r t h e n u m b e r of b r a i n h o m o g e n a t e s t e s t e d b + , + + , A m o u n t of fluorescent a n t i b o d y s t a i n i n g in b r a i n to N a r i v a virus a f t e r inoculation of weanling h a m s t e r s a t e i t h e r dilution

236

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Roos

and 1~. WOLLIVrANN:

Fig. 7. Viral antigen focally collected in ependyma and choroid plexus and diffusely collected lateral to lateral ventricle in suckling hamster 5 days after inoculation with 101.4 PFU. Fluorescent antibody stain; × 160

Fig. 8. Viral antigen in subependymal focus in weanling hamster 6 days after inoculation with 101.4 P F U . Fluorescent antibody stain; × 400

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Because weanling h a m s t e r s a p p e a r e d to h a v e a n o n - p r o d u c t i v e infection, several a t t e m p t s to rescue virus were m a d e . B r a i n h o m o g e n a t e s of weanling h a m s t e r s d y i n g 6 a n d 11 d a y s a f t e r i n o c u l a t i o n d i d n o t cause clinical disease when i n j e c t e d into suckling h a m s t e r s . Virus was recovered in vitro b y e x p l a n t or b y e o c u l t i v a t i o n with Vero cells from t h e b r a i n of sick weanling h a m s t e r s 7 a n d 12 d a y s after inoculation, a l t h o u g h b r a i n h o m o g e n a t e s h a d no infectious virus. No virus could be r e c o v e r e d b y p l a q u e assays on Veto cells from t h e blood or visceral tissue, t a k e n a t several t i m e periods, f r o m suckling a n d weanling animals. I m m u n o f l u o r e s c e n t studies of visceral tissue of d y i n g a n i m a l s were negative. Immunological Studies

P o o l e d sera from i n o c u l a t e d suckling h a m s t e r s a n d from 1 or 2 weanling h a m sters a t several t i m e points were a s s a y e d for virus n e u t r a l i z a t i o n a c t i v i t y (Table 2). F i v e d a y s after i n o c u l a t i o n of 103.4 P F U , b o t h suckling a n d weanling h a m s t e r s h a d a n e u t r a l i z i n g a n t i b o d y t i t e r of 1 : 160. Table 2. Neutralizing antibody determina~ons after inoculation o/Nariva virus at two di]]erentdilu~ons Neutralizing antibody Days post inoculation

Suckling

Weanling

4 5 6 7 8 9 10 tl 12 13

1:80 1:160 ~b ~ ~ ~ ~ ~ ~ t

ND a 1:320; >1:320; >1:320; ND > 1:320 >1:320 >1:320; >1:320 >1:320

ND, b ~,

103.4 P F U i.e.

1:160 >1:320 > 1 : 3 2 0 ; 1:40

1:40

levels 101.4 P F U i.e. Suckling

Weanling

ND ND 1:40 ~ ~ ~ ~ ~ ~ ~

ND ND 1:40 > 1 : 3 2 0 ; 1:40 >1:320 ND ND 1:40 > 1 : 3 2 0 ; 1:320 ND

not done no survivors

Table 3. In]ectious virus in, weanling hamste~:~ after Nariva virus inoculation and injection with either saline or anti-lymphocyte serum ( A L S ) Infectious virus Days post, inoculation

Saline i.p.

ALS i.p.

5 7 8

++, +÷~ +,--

÷÷, ++ ++,-~ +,-++, ++,++

9

--

a

,

@ @ ~

brain

,

~ ÷ ,

--

103 tO 104.7 P F U / g brain; -~ = 101-7to 103 P F U / g brain; -- = < 101.7 P F U / g

238

I~. P. l~oos and g . W o L ~ I . ~ :

I n an effort to elucidate the role of the immune response in infected weanling hamsters, the inoculated animals were treated with ALS; ALS treated animals had little or no NV neutralizing antibody at the time of sacrifice. Weanling hamsters infected with NV and treated with ALS had a similar mortality and pathology to those infected and not treated. I n an experiment with ALS summarized in Table 3, ALS treated animals had evidence of persistent, infectious virus on days 8 and 9, when infectious virus could no longer be detected in untreated inoculated animals. Discussion

I n NV il~feetion of hamsters, the age of the host animal determines the course of the illness as well as the expression of the virus. Suckling hamsters inoculated with NV develop ependymitis followed b y a fatal neerotizing paneneephalitis with multinneleated giant cells; these features have been described in other paramyxovirus infections of animals (7, 16). Brains of sucklings given NV contain large amounts of infectious virus and viral antigen. I n contrast to suckling animals, weanling hamsters develop a non-productive infection with evidence of virus antigen but without easily recoverable infectious virus. The infectious virus is present in only small amounts and only early in the course of clinical disease. Despite the lack of infectious virus the weanlings die with an encephalitis--with less parenchymal necrosis and inflammation than sucklings. The virus cannot be recovered from brains of dying weanlings b y i.c. inoculation into suckling hamsters, but can be rescued in vitro following explant of the infected brain tissue or b y cocultivation of the tissue with Veto cells. I t is of interest t h a t there was no evidence of visceral infection b y NV in either sucklings or weanling animals, raising questions regarding the means of spread ia nature. Perhaps biological differences between the hamsters and the natural host as well as the use of mouse-passaged material has changed the normal pathogenesis. Other paramyxoviruses have shown changes similar to NV in the virus-host relationship with age, but with varying mechanisms. Two measIes s t r a i n s - - a hamster neurotropic and a mouse-adapted s t r a i n - - p r o d u c e a conventional productive encephalitis in suckling mice, but a defective infection in weanlings. This age-dependent change in virus expression is thought related to the m a t u r i t y of the cells of the central nervous system (6, 11). I n contrast, the ability of two SSPE measles virus isolates to cause a non-productive infection in monkeys and hamsters is thought to depend upon the maturity of the host's immune response (1, 3). I n h u m a n SSPE, infection at an early age with primary measle.3 infection appears important in the development of a later defective subaeute encephalitis (5), but the relative roles of the m a t u r i t y of the host's immune system v i s a vis changing susceptibilities of the host's neural cells remain unclear. The non-productive infection of weanling hamsters with NV appears to be related both to the m a t u r i t y of the host's immune system and to age-related changes in susceptibility of the host's neural cells. ALS treated infected weanling hamsters have evidence of infections virus for a longer time t h a n untreated, infected weanlings suggesting t h a t interference with the integrity of the mature immune system can convert the non-productive infection to a more productive

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239

one similar to t h e suckling's infection. I m m u n e factors, however, do n o t seem to be t h e o n l y d e t e r m i n a n t of t h e v i r u s - h o s t r e l a t i o n s h i p f o u n d in weanlings since: A L S t r e a t e d a n i m a l s do n o t h~ve levels of v i r u s as high as suckling a n i m a l s ; A L S t r e a t e d a n i m a l s die a f t e r a clinical course a n d w i t h c e r e b r a l p a t h o l o g y similar t o u n t r e a t e d , i n f e c t e d weanlings; N V a n t i b o d i e s in suckling a n d w e a n l i n g a n i m a l s a p p e a r a t similar t i m e s a f t e r infection a n d a t c o m p a r a b l e levels. P e r h a p s , ager e l a t e d changes in h o s t cell s u s c e p t i b i l i t y are also responsible for t h e n o n - p r o d u c t i v e infection. These a g e - r e l a t e d h o s t cell factors could i n v o l v e changes of cell surface receptors or of cellular e n z y m e s n e c e s s a r y for v i r a l replication. I n N V infection t h e n e u r a l cells of b o t h suckling a n d w e a n l i n g h a m s t e r s p r e s u m a b l y h a v e virus surface r e c e p t o r s since v i r a l a n t i g e n is f o u n d in t h e same cell p o p u l a t i o n s . C e r t a i n proteases (12) h a v e been f o u n d to be essential for infectious virus p r o d u c t i o n in o t h e r p a r a m y x o v i r u s infections. P e r h a p s proteases, or o t h e r critical enzymes, a r e less a v a i l a b l e in t h e m a t u r e cells of t h e weanling a n i m a l s t h a n in t h e less well diff e r e n t i a t e d cells of sucklings. E l u c i d a t i o n of these h o s t m e c h a n i s m s m a y h a v e to a w a i t in vitro models.

Aeknowledgments This investigation was supported b y a grant from the National ~ u l t i p l e Sclerosis Society (I%Gl130-A-14), the Basil O'Connor Starter l~eseareh grant from March of Dimes (~=5-i62), and grants from Otho S.A. Sprague F o u n d a t i o n and Polish-American Police Association. The technical assistance of Kevin Lee and guidance of Dr. B. G. W. Arnason is gratefully acknowledged.

References 1. ALBIgECHT, P., BURNSTEIN, T., KLUTCH, M. J., HICKS, J. T., EN~rIS, F. A. : Subacute sclerosing panencep:halitis: experimental infection in primates. Science 195, 64--66 (1977). 2. Br~RKS, J. S., NA~AYAN, O., McFARLAND, H. F., JO.XNSON, R. T. : Acute encephalop a t h y caused b y defective virus infection: I. Studies of Newcastle disease virus infections in newborn and adult mice. Neurology (Minneap.) 26, 584--588 (1976). 3. BYINGTON, D. P., JOHNSON, K. P. : Subaeute selerosing panencephalitis virus in immunosuppressed adult hamsters. Lab. Invest. 32, 91--97 (1975). 4. CHOPPIN, P. W., COMPPkNS, •. W. : I n : FR~N~_EL-Co~aAT, H., WAGNER, I~. I~. (eds.), Comprehensive Virology, 95---178. New York: Plenum Press 1975. 5. DETELS, t~., B~ODY, J. A., MoNEw, J., E D G e , A. H. : F u r t h e r epidemiological studies of subacute sclerosing panencephalitis. Lancet 2, 1 1 - - t 4 (1973). 6. GRIFFIN, D. E., MULLINIX, J., NARAYAN, O., JOHNSON, l~. T. : Age dependence of viral expression: comparative pathogenesis of two rodent-adapted strains of measles virus in mice. Infect. Immun. 9, 690--695 (1974). 7. IWASAKI, Y., KoPRows~cI, H. : Cell to cell transmission of virus in the central nervous system I. Subacute seIerosing panencephalitis. Lab. Invest. 3!, 187--197 (1974). 8. KARABA~SOS, N., BueKLEY, S. M., _A_~DOIN,P. : Nariva virus: further studies with particular reference to its hemadsorption and hemagglutinating properties. Prec. Soc. exp. Biol. Med. 130, 888--892 (1969). 9. I~AKOWA, S., KOESTNE~, A. : Age-related susceptibility to infection with canine distemper virus in gnotobiotic dogs. J. inf. Dis. 134, 629--632 (1976). 10. P~INCE, G. A., PORTE~, D. D. : The pathogenesis of respiratory syneytial virus in infant ferrets. Amer. J. PathoI. 82, 339--350 (1976).

240 g . P . t~oos and 1~. WOLLMANN: Non-Productive Paramyxovirus Infection 1 I. Roos, R. P., GRIFFIN, D. E., JOHNSON, I~. T. : Determinants of measles virus (HNT strain) replication in mouse brain. J. inf. Dis. 137, 722--727 (1978).

12. SGHEID,A., CHOP]PIN,P. W. : Identification of the biological activities of paramyxovirus glycoproteins. Activation of cell fusion, hemolysis, and infectivity by proteolytic cleavage of an inactive precursor protein of Sendal virus. Virology 57, 475 --490 (1974). 13. SH:I1VIOKATA,K., I~ISHIYAMA, Y., ITO, Y., KIMLIRA, I., NAGATA, M., IDA, M., SOBUE, I. : Pathogenesis of Sendal virus infection in the central nervous system in mice. Infect. I m m u n . 13, 1497--1502 (1976). 14. TIKASlNGH, E. S., JOZeKERS, A. I-I., SPE~0E, J., AI~rKEN, T. H. G. : Nariva virus, a hitherto undeseribed agent isolated from the Trinidadian rat, Zygodontomys b. brevicauda. Amer. J. trop. Med. Hyg. 15, 271--286 (1966). 15. WALDER, 1~..* Electron microscopic evidence of Nariva virus structure. J. gen. Virol. 11, 123--128 (1971). 16. WoLzzcs~zv, J. S., GII~D]~N, D. H., RORKE, L. B. : Experimental panencephalitis induced in suckling mice by parainfluenza 1 (6/94) virus. J. Neuropathoh exp. Neurol. 35, 271--286 (1976). Authors' address: R. P. Roos, M.D., Department of Neurology, The University of Chicago, 950 East 59th Street, Chicago, IL 60637, U.S.A. Received April 4, 1979