Archives of Virology 48, 261--269 (1975) © by Springer-Verlag ~975
Structural and Growth Characteristics of Two Avian Reoviruses By H. ~NICK, D. CURSIEFE~-, and H. BECHT Institut ffir Virologie, Justus Liebig-Universit~$, Giel3en, Federal Republic of Germany With 6 Figures Accepted April 15, 1975
Summary Two virus strains which had been suspected to be the etiological agents of infectious bursitis (Gumboro disease) and of inclusion body hepatitis of chickens were characterized by their morphology, their peptide composition and the segmented genome of their double-stranded RNA to be typical reoviruses. Although the 2 avian strains did not clearly differ :in their serological behaviour, the size of some of their I~NA segments were not identical Both strains replicated in tissue cultures prepared from the chorioallantoic membrane of embryonated eggs with growth characteristics of reoviruses.
Introduction A virus had been isolated from the bursa of Fabricius of chickens affected with infectious bursitis (Gumboro disease). The virus was 650 A in diameter, the capsid was constructed as a double shell and it had a density of 1.34 g/m1 and a I~NA content of about 10 per cent (6). Another virus which had been suspected to be of some etiological significance for inclusion body hepatitis of chickens was included in these studies. I t has been shown to be antigenically related to viruses which were supposed to be avian reoviruses (16). I n order to verify that these two viruses are true reoviruses a detailed analysis of their physico-chemical and serological properties was carried out and compared with the representative reovirus type 3, and attempts were made to grow the viruses in culture to define their growth ehracteristics.
Materials and Methods Viruses
The lt5th passage of strain 2207/68 in embryonated eggs was obtained from Dr. KSsters, Giei~en. This virus had been incriminated as being the causative agent of Gumboro disease (6). The 6th passage in e~nbryonated eggs of strain "Wi", which had 18"
262
H . NICK, D.
CURSIEFEN,a n d H . BECtlT:
b e e n alleged to b e t h e etiological a g e n t of infectious h e p a t i t i s of chickens, was o b t a i n e d f r o m Dr. K u s s m a u l , S t u t t g a r t . T h e s t r a i n h i s t o r y h a s b e e n d e s c r i b e d (15). T h e D e a r i n g s t r a i n of r e o v i r u s t y p e 3 s e r v e d as reference virus.
Production and Purification. of Virus T h e t w o a v i a n s t r a i n s were g r o w n in e m b r y o n a t e d eggs a f t e r 8 or 9 d a y s of c u l t i v a tion. W h e n t h e e m b r y o s h a d die(] 2 - - 4 d a y s a f t e r i n f e c t i o n t h e a l l a n t o i c fluid was h a r v e s t e d , coarse m a t e r i a l was r e m o v e d b y c e n t r i f u g a t i o n , a n d v i r u s p a r t i c l e s were s e d i m e n t e d i n a Spinco r o t o r 15 a t 15,000 r . p . m , a t 15 ° C for 2.5 h o u r s . T h e p e l l e t was r e s u s p e n d e d i n SSC b u f f e r (0.15 ~ NaC1, 0.015 M N a - c i t r a t e , p i t 7.2) b y b r i e f s o n i c a t i o n , m i x e d w i t h N o n i d e t N P - 4 0 a t a finM c o n c e n t r a t i o n of 1 p e r c e n t a n d centrif u g e d in a sucrose g r a d i e n t ( 2 0 - - 5 0 p e r cent) in a S W 2 7 r o t o r a t 25.000 r. p . m , a n d 15 ° C for 90 m i n u t e s . T h e v i r u s b a n d s were pooled, d i l u t e d a b o u t t e n f o l d , a n d t h e v i r u s was s e d i m e n t e d b y c e n t r i f u g a t i o n . T h e v i r u s pellet was r e s u s p e n d e d , 3.2 m l were m i x e d w i t h 1.8 m l of s a t u r a t e d CsC1 a n d c e n t r i f u g e d a t 50,000 r . p . m , i n a S%V65 r o t o r for 22 h o u r s a t 15 ° C. T h e v i r u s b a n d s were pooled again, d i l u t e d a n d c o n c e n t r a t e d b y centrifugation. l~eovirus t y p e 3 was g r o w n i n L-cells a n d p u r i f i e d e s s e n t i a l l y as d e s c r i b e d (3).
Growth o] Viruses in Tissue Culture B o t h a v i a n s t r a i n s were g r o w n in cells of t h e e h o r i o a l l a n t o i e m e m b r a n e of e m b r y o n a t e d c h i c k e n eggs (CAM cells) w h i c h were p r e p a r e d as r e c e n t l y d e s c r i b e d (4). 3 × 106 cells were seeded i n t o 5 c m p l a s t i c dishes a n d i n c u b a t e d w i t h 4 m l of m e d i u m 199 c o n t a i n i n g 10 p e r cent, fetal calf s e r u m . ~ h e n m o n o l a y e r s h a d f o r m e d a f t e r 2 d a y s t h e cells were i n o c u l a t e d w i t h 1 m l of infectious a l l a n t o i c fluid. A f t e r a n a d s o r p t i o n p e r i o d of one h o u r t h e cells were w a s h e d t h r e e t i m e s w i t h P B S , a n d i n c u b a t i o n was c o n t i n u e d w i t h 2 m l of m e d i u m 199 c o n t a i n i n g 2 p e r c e n t fetal calf s e r u m . A t d i f f e r e n t periods a f t e r i n f e c t i o n t h e m e d i u m w a s r e m o v e d a n d t h e cells processed b y freezing a n d t h a w i n g . P l a q u e t i t r a t i o n of m e d i u m a n d cell-associated infeetixdty w a s d o n e b y i n o e n l a t i n g CAM cells w i t h 0.2 mI of v i r u s d i l u t i o n s a n d c o v e r i n g t h e ceils w i t h 4 m l of 0.7 p e r c e n t p - a g a r in m e d i u m 199 s u p p l e m e n t e d w i t h 7 p e r c e n t fetal calf s e r u m . T h e cells were s t a i n e d w i t h t r y p a n blue a f t e r 2 ½ - - 4 days. F o r n e u t r a l i z a t i o n t e s t s a b o u t 100 p l a q u e f o r m i n g unit, s ( P F U ) were m i x e d w i t h a n t i s e r u m in a series of t w o f o l d d i l u t i o n s , k e p t a t r o o m t e m p e r a t u r e for I h o u r a n d i n o c u l a t e d o n t o CAM eells. T h e h i g h e s t s e r u m d i l u t i o n w M c h r e d u c e d t h e p l a q u e c o u n t b y m o r e t h a n 80 p e r c e n t was t a k e n as t h e end-point.
Preparation o/Antisera Chickens f r o m a flock free of G u m b o r o disease were i n j e c t e d s u b c u t a n e o u s l y w i t h a p u r i f i e d p r e p a r a t i o n of s t r a i n 2207/68 a t t h e age of 4 weeks. T h e a n i m M s were i n j e c t e d a s e e o n d t i m e 3 weeks l a t e r a n d bled a f t e r a n o t h e r week. A n t i s e r a against, s t r a i n " W i " w i t h c o m p a r a b l e p o t e n c i e s could b e p r e p a r e d b y i n j e c t i n g v i r u s - c o n t a i n i n g a l l a n t o i e fluid b y t h e s a m e route.
Gel Precipitation A n t i g e n s for gel-diffusion were e x t r a c t e d f r o m t h e c h o r i o - a l l a n t o i e m e m b r a n e s of i n f e c t e d eggs. Tissue h o m o g e n a t e s were u s e d as a n t i g e n a f t e r c r u d e debris h a d b e e n r e m o v e d b y low-speeed e e n t r i f u g a t i o n .
Immuno]luorescence CAM-cells g r o w n on eoverslips were s t a i n e d w i t h t h e g a m m a - g l o b u l i n f r a c t i o n of c h i c k e n a n t i s e r a w h i c h h a d b e e n c o n j u g a t e d w i t h fluorescein i s o t h i o c y a n a t e . T h e s a m e p r o c e d u r e was followed as i n (4).
Electron 2kIicroscopy A d r o p of v i r u s s u s p e n s i o n was m i x e d w i t h 2 p e r c e n t p h o s p h o t u n g s t i c acid o n a F o r m v a r - e o a t e d c o p p e r grid a n d e x a m i n e d i n a n E h n i s e o p e i01.
S t r c u t u r e a n d G r o w t h of T w o A v i a n I~eoviruses
263
Extraction o/ Viral R N A Purified virus in SSC was shaken twice w i t h an equal v o l u m e of w a t e r - s a t u r a t e d phenol for 10 m i n u t e s at r o o m t e m p e r a t u r e (12). R e s i d u a l phenol was r e m o v e d f r o m t h e aqueous phase w i t h ether, and R N A was p r e c i p i t a t e d w i t h two volumes of ethanol at - - 2 0 ° C o v e r n i g h t (2).
Melting Curve R N A dissolved in 0.1 × SSC was a d j u s t e d to an optical density of 1.05 at 260 nm. The sample was h e a t e d a n d t h e e x t i n c t i o n registered continously in a P y e Unlearn S p e e t r o p h o t o m e t e r SP 8000 b e t w e e n 40 ° and 95 ° C.
RNase Resistance o] Viral R N A CAM-cells were infected w i t h strain W i in allantoic fluid. After' an adsorption period of 1 h o u r t h e cells were washed and i n c u b a t e d w i t h m e d i u m 199. 50 fzCi of 3H-uridine were added to each 9 cm culture dish. A f t e r 20 hours of i n c u b a t i o n the m e d i u m was h a r v e s t e d , unlabeled virus was added as carrier and the p r e p a r a t i o n was purified b y g r a d i e n t c e n t r i f u g a t i o n as described above. R N A was e x t r a c t e d w i t h SDS and phenol (10), dissolved in 2 x SSC a n d d i v i d e d into two parts. 100 Bg of p a n c r e a t i c R N a s e was a d d e d to one half, and t h e m i x t u r e was i n c u b a t e d at r o o m t e m p e r a t u r e for 20 minutes. TCA-precipitable r a d i o a c t i v i t y was m e a s u r e d in t,he t r e a t e d a n d u n t r e a t e d p r e p a r a t i o n s b y liquid scintillation.
Polyacrylamide Gel Electrophoresis The conditions described (8) were employed. F o r the analysis of viral R N A the 7.5 per cent gels c o n t a i n e d 6 M urea. The buffer system h a d been described (7). Virus samples were dissociated in 2 ~ urea, 1 per cent SDS and 0.1 per cent m e r c a p t o e t h a n o l at 55 ° C for 60 minutes. Electrophoresis was carried out at 4 m A per gel for 24 hours. A f t e r the run the gels were r e m o v e d from t h e tube, rinsed in destilled w a t e r for 5 - - 6 h o u r s a n d stained w i t h m e t h y l e n e blue (9). The p r o t e i n composition was d e t e r m i n e d in 7.5 per cent gels which contained 5 5,I urea. The buffer was 0.1 5~ N a - p h o s p h a t pl~ 7.2 (14). The virus samples were dissociated in 6 ~ urea, t per c e n t SDS and 0. t per c e n t m e r e a p t o e t h a n o l in a boiling w a t e r b a t h for 2 minutes. Eleetrophoresis was carried out at 6 m A per gel for 20 hours. The gels were stained for 30 m i n u t e s in Coomassie brilliant blue at 37 ° C and destained w i t h 7.5 per cent acetic acid at 65 ° C.
Results
Morphology o~ Virion T h e v i r u s c o u l d be p u r i f i e d b y g r a d i e n t e e n t r i f n g a t i o n . B o t h a v i a n s t r a i n s h a d a n i d e n t i c a l s e d i m e n t a t i o n p a t t e r n in c e s i u m chloride. T h r e e b a n d s c o u l d be. o b s e r v e d . T h e i r d e n s i t y was 1.34, 1.30 a n d 1.275 g / m l (Fig. 1). T h e r e f e r e n c e reov i r u s 3 h a d t h e e x p e c t e d d e n s i t y of 1.36 g/ml. I n t h e e l e c t r o n m i c r o s c o p e t h e p a r t i c l e s w i t h a d e n s i t y of 1.34 g / m l w e r e spherical, u n i f o r m in size w i t h a d i a m e t e r of a b o u t 65 n m (Figs. 2 a, b) in a c c o r d a n c e w i t h p r e v i o u s o b s e r v a t i o n s (6) for s t r a i n 2207/68. T h e e a p s o m e r e s h a d t h e appearan.ee of h o l l o w cylinders. I t c o u l d be a f f i r m e d t h a t t r e a t m e n t w i t h t 0 0 fxg/ml of e h y m o t r y p s i n c h a n g e d t h e size of t h e p a r t i c l e s to a b o u t 50 n m a n d t h e d e n s i t y to 1.42 g / m l . T h e m o r p h o l o g i c a l l y d i s t i n c t c a p s o m e r e s h a d d i s a p p e a r e d a n d n o special m o r p h o l o g i c a l e n t i t i e s w e r e d i s c e r n i b l e o n t h e s u r f a c e of s u c h p a r t i c l e s . V i r u s p a r t i c l e s w i t h a d e n s i t y of 1.30 c o n s i s t e d of e m p t y shells (Fig. 2 e), a n d t h e m a t e r i a l b a n d i n g a t a d e n s i t y of 1.275 g / m l c o n t a i n e d b r e a k d o w n p r o d u e t s , single e a p s o m e r e s a n d a m o r p h o u s masses.
264
H. :NIe:K, D. CU~SIEFE~, and I-I. BE(JIlT:
Fig. 1. Cesium chloride density gradient of strain 2207/68, Virus which had been concentrated from allantoie fluid and purified on a sucrose density gradient was mixed with a saturated solul~ion of cesimn chloride and centrifuged in a Spinco S~W65 rotor at 50,000 r . p . m , for 22 hours at 15 ° C. The t,hree visible bands from top to b o t t o m have a density of 1.275, 1.30 and 1.34 g/ml
Fig. 2. Electron micrographs of purified virus preparations stained with phosphotungstie acid a) Whole virus particles of strain Wi; density 1.34 g/ml b) Whole virus particles of strain 2207/68 ; density 1.34 g/ml c) E m p t y particles of strain 2207/68; density 1.30 g/ml Magnification was 120,000 fold
Structure o/ Viral R N A I t could be clearly shown b y a c i d consists of d o u b l e - s t r a n d e d sharp t r a n s i t i o n b e t w e e n 81 ° a n d shows t h a t m o s t of t h e v i r a l R N A
two i n d e p e n d e n t m e t h o d s t h a t the viral nucleic R N A . The profile of tile m e l t i n g curve shows a 88 ° C (Fig. 3) w i t h a T i n - v a l u e of 83 ° C. T a b l e t is g N a s e - r e s i s t ~ n t . I t is possible t h a t some of t h e
Structure and Growth of Two Avian Reoviruses
265
digested material is labeled R N A of cellular origin. Under identical experimental conditions more than 99 per cent of the 3H-uridine incorporated into singlestranded R N A is rendered acid-soluble (10).
K 1,z c
S 1,3 (M ~d 1.2 C3
o
lJ 1,0~
I/ ;s 7; 7's
do
oc
Tm Fig. 3. Melting curve of viral RNA. I~NA extracted from purified virus particles of strain 2207/68 was heated and its absorbance registered continuously at 260 nm
Table 1. RNase Resistance of Viral R N A Treatment of sample
dpm aH-uridine
None
1270
l~Nase
695
Phenol-extracted RNA of virus grown in CAMcells was treated with i00 ,ug/ml of ]~Nase at room temperature for 20 minutes. ]~adioaetivity precipitable by TCA was counted
In polyacrylamide gels the g N A could be separated into 10 distinct pieces (Fig. 4). The most striking difference between the avian strains and reovirus type 3 is segment 7 which is much larger in avian viruses. Figure 4 shows furthermore that besides the very obvious differences in the R N A patterns of reovirus 3 and the avian strains there are definite differences in size in some corresponding R N A segments of the two avian strains too. In strain Wi segment L3 could never be clearly separated from segment L2, and the 3 smallest pieces of group S are shghtly smaller in strain 2207/68 as compared to strain Wi. The molecular weights of the R N A pieces in these 2 strains were calculated from the migration distances in the gels of Figure ~ in comparison with the known values for reovirus 3 (13). They were listed in Table 2.
Polypeptide Composition 7 structural proteins could regularly be detected by aerylamide gel analysis of the 2 avian strains (Fig. 5), which can be classified into three size groups as for reovirus 3 (14), and they have comparable molecular weights. However, in the gels of the avian strains a third faint band with a molecular weight of approximately 75,000 could reproducibly be demonstrated in the medium size class. A third polypeptide in the smallest class, on the other hand, was only irregularly and never clearly visible even when the gels were heavily loaded.
266
H. NICK, D. CURSIEFEN,and H. BECI-IT:
Fig. 4
Fig. 5
Fig. 4L Polyacrylamide gel analysis of viral tRNA. The gels contained 7.5 per cent aerylamide. Eleetrophoresis was carried ou~ at 4 mA per gel for 24, hours. Tile gels were stained with methylene blue From left to right: reovirus type 3, avian strain W'i, avian strain 2207/68 Fig. 5. Polyacrylamide gel electrophoresis of viral proteins. The gels contained 7.5 per cent aerylamide. Electrophoretie runs were for 20 hours at 6 mA per gel. The gels were stained with Coomassie brilliant blue. From 1eft to right : reovirus type 3, avian strain Wi, avian strain 2207/68
Serological Behaviour I n gel diffusion tests antisera agMnst the two a v i a n strains produced one precipitation line which fused completely when the antigens or antisera were placed into a d j a c e n t wells of the agar gel. No precipitation could be n o t e d between antiW i serum or anti-2207/68 serum a n d a cell extract from L-cells infected with reovirus t y p e 3. Antisera against, both a v i a n strains neutralized the i n f e c t i v i t y effieiently, a n d the reciprocal titers were almost identical. Neither a n t i s e r u m neutralized reovirus type 3 (Table 3).
Structure and Growth of Two Avian Reoviruses
267
Table 2. Molecular Weights o / R N A Segments o] the Two Avian Reoviruses ( Daltons × 10G) Segment
Reovirus t y p e 3
Strain Wi
Strain 2207/68
L1 L2 L3 M1 M2 M3 S1 $2 S3 $4
2.50 2.40 2.30 1.60 1.60 1.40 0.92 0.76 0.64 0.61
2.25 2.20 2.17 1.65 1.50 1.39 1.05 0.70 0.66 0.63
2.25 2.20 2.15 1.65 1.45 1.33 1.05 0.65 0.59 0.56
The molecular weights were computed from the relative migration distances in a 7.5 per cent aerylamide gel. The values published for reovirus type 3 (13) and listed in the first row were taken as standard references. The designation of the segments followed the proposal of SHATKIN (13)
Table 3. ±~eutralization o/ the Avian Reoviruses Strain 2207/68 and Wi Virus strain
Reciprocal neutralization titers of antisera Anti-Wi
Anti-2207
Normal serum
2207 Wi
512 1024
1024 512
none none
Reo type 3
none
none
none
Neutralization assays of the 2 avian strains were carried out on CAM-cells, of reovirus type 3 on L-cells. A reduction of plaque counts by more than 80 per cent was taken as end-points. Normal serum was from specific pathogen free birds.
Growth in C A M Cells
14 to 16 hours p o s t infection n u m e r o u s cells infected w i t h s t r a i n W i showed vacuoles which g r a d u a l l y increased in size a n d were e v e n t u a l l y visible in all cells of t h e layer. 1 to 2 hours l a t e r t h e cells a p p e a r e d g r a n u l a t e d , t h e y c o n t r a c t e d to form large clusters, a n d t h e y f i n a l l y d e t a c h e d from the culture dish. I n t r a c e l l u l a r virus titers s t a r t e d to increase 4 8 hours p o s t infection a n d r a p i d l y r e a c h e d a c o n s t a n t level a b o u t 24 hours p o s t infection. E x t r a c e ] l u l a r virus could be demons t r a t e d a p p r o x i m a t e l y 16 hours p o s t infection when c y t o p a t h i c effects were r e a d i l y visible. The titers increased to r e a c h a m a x i m u m of a p p r o x i m a t e l y 107 to 10 s P F U / m l 1 0 - - 1 2 hours l a t e r (Fig. 6). P r o d u c t i o n of virus-specific antigens could first be d e m o n s t r a t e d b y immunofluorescence w i t h c o n j u g a t e d a n t i - W i s e r u m b e t w e e n 6 a n d 8 hours p o s t infection as a fine g r a n u l a r fluorescence in t h e c y t o p l a s m of epitheloid cells. A t l a t e r periods b r i l l i a n t l y fluorescing granules a n d p a t c h e s were d i s t r i b u t e d in t h e whole c y t o p l a s m , b u t no fluorescence could be seen in the nuclei. As r e p o r t e d p r e v i o u s l y (4) m a n y fibroblasts which were n e v e r s t a i n e d w i t h t h e c o n j u g a t e d serum r e m a i n e d a t t a c h e d to t h e coverslip in a loose a n d irregular netlike a r r a n g e m e n t even when all epitheloid cells h a d been d e s t r o y e d . The l a t e n t p e r i o d a n d t h e a p p e a r a n c e of c y t o p a t h i e changes of s t r a i n 2207/68 were u s u a l l y d e l a y e d b y 2 - - 3 hours; the m o r p h o l o g i c a l a p p e a r a n c e of the cytop a t h i c changes was identical to t h e p i c t u r e described for s t r a i n Wi.
H, NICK, D. CUIISIEFEN,and H. BECHT:
268 10s
~ ~ --i-
"~--A --
extra ,I, ~ - - -
10 7
ill / E •. ~
intra 10 5
#/// LL EL
10 t'
~.A//
/I
10 3
t
102
I 2
I 4
I 6
l,,I I I 8 lO 12 14
I I 17 19
I I 24 26
I 1 29 31
I
I
1
36 3 8 40
I
I
I
46 4 8 50
hours Fi. Fig. 6. Growth curve of the avian reovirus strain Wi in CAi~Cl-eells. Monolayers were infected with allantoie fluid. Individual plates were withdrawn at different times after infection and assayed for infectivity in the medium and in the cells after freezing and thawing. Plaque titrations were done in CAlVi-eetls Discussion
The data obtained in this study permit the definite classification of the avian virus strains 2207/68 and Wi as reoviruses. I t could be shown that the RNA is double-stranded b y its RNase resistance and the typical melting curve with a Tm value of 83°C in 0.1 ×SSC which is almost identical with the melting point of 84 ° C determined for reoviruses (2). Polyacrylamide gel analysis showed furthermore that the genome of both avian strains consists of the ten segments which are typical for reovirnses (13) and that there are marked differences in the RNA patterns between the avian and mammalian reoviruses. Minor differences, however, could also be noted between the 2 avian strains which were reproducibly obtained in repeated runs with different virus preparations. The serological data available do not indicate any significant differences in the antigenic structure of the two avian viruses, and according to the gel-patterns the size of their structural proteins seems to be identical. Even though tile serological techniques applied indicate a very close relatedness or even identity of two viruses, the size distribution of the I~NA segments represents specific markers for each strain, which may be used for their identification. The avian virus strains which have been described as reoviruses (5, 11) are obviously larger and have a much higher density, The structure of their RNA and of their polypeptides have not been analyzed. A comparison with our data is therefore not possible. The pathogenic significance of the strains 2207/68 and Wi must still be considered as open for discussion, particularly with respect to the etiology of infectious bursitis. Electron micrographs of immune precipitates showed virus particles in the bursa of Fabrieius of infected birds which did not have the morphology of reoviruses (1). No precipitation could be noted in agar gels when extracts from such bursal material diffused against anti-Wi or anti-2207/68 antiserum (I5,
Structure and Growth
of Two
Avian Reoviruses
269
NICK, u n p u b l i s h e d results). The birds which were injected for the p r e p a r a t i o n of the antisera used in the experiments described in this paper did n o t show a n y clinical signs or pathological lesions of G u m b o r o disease. Acknowledgments The work was supported by the Sonderforschungsbereich 47 (Virologic).
Referenees 1. AL~IEIDA,J. D., MORRIS, R. : Antigenically-relatedviruses associated with infectious bursal disease. J. gen. Virol. 20, 369--375 (1973). 2. BEI~LAM¥, A. ]~., SHAPIRO, L., AUGUST, J. T., JOKLIK, YV. K. : Studies on reovirus RNA. Characterization of reovirus genome RNA. J. reel. Biol. 29, 1--17 (1967). 3. BELLAMY, A. R., HOLE, L. V., BAGUI,EY, B. C.: Isolation of the trinucleotide pppGpCpU from reovirus. Virology 42, 415--420 (1970). 4. @URS~EFEN, D., BECttT, H. : I n vitro cultivation of cells from the chorioallantoic membrane of chick embryos. 5~[ed. MicrobioL Immunol. 161, 3--10 (1975). 5. KAWA~IU~A, i . , SHIMIZU, F., MAEDA, M., TSUBA~A~A, H.: Avian reovirus: Its properties and serological classification. Nat. Inst. Anita. mlth. Quart. 5, 115--124 (1965). 6. KOSTEI~S, J., BEC~T, H., RUDOLPh, R.: Properties of the infectious bursal agent of chicken (IBA). Med. Microbio]. Immunol. 157, 291--298 (1972). 7. LOE~rl~:G, U. E. : The fractionation of high-molecular weight ribonucleic acid by polyacrylamide-gel electrophoresis. Biochem. J. 102, 251--257 (1967). 8. MARTIN, S. A., ZWEERINK~ H. J. : Isolation and characterization of two types of b]uetongue virus particles. Virology 50, 495--506 (1972). 9. PEACOCK, A. C., DI~'G~AN, C. W. : Resolution of multiple ribonucleic acid species by polyacrylamide gel electrophoresis. Biochemistry 6, 1818--1827 (1967). 10. SC~OLT~SSEK, C., ROTT, R.: Hybridization studies with influenza virus RNA. Virology 39, 400--407 (1969). 11. SEKIGUC~I, K., KOIDE, F., KAWA~[UI~, H. : Physico-chemical properties of avian reovirus and its nucleic acid. Arch. ges. Virusforsch. 24, 123--136 (1968). 12. SHAT:KIN, A. J. : Inactivity of purified reovirus RNA as a template for E. coli polymerases in vitro. Prec. Nat. Acad. Sci. U.S. 54, 1721--1728 (1965). 13. SIIATKIN, A. J., SIP]~, J. D., Loll, P. : Separation of ten reovirus genome segments by polyacrylamide gel electrophoresis. J. Virol. 2, 986--991 (1968). 14. SI~IITH, R. E., ZxcVEERINK,t{. J., JOKI~I~K,W. K. • Polypeptide components of virions, top component, and cores of reovirus type 3. ViroIogy 39, 791---810 (1969). 15. V~rOERNLE, i . , BI~UNNEI~, A., KUSSS~AVL, K.-F. : Naehweis avi/i,rer Reo-Viren im Agar-Gel-Prgzipitationstest. Tier/~rztl. Umsch. 29, 307--312 (1974). Authors' address: H. BECHT, I n s t i t u t ffir Virologie, Justus-Liebig-Universitgt, Frankfurter Stral3e 107, /)-6300 Gief~en, Federal Republic of Germany. Received March 14, 1975
Structural and Growth Characteristics of Two Avian Reoviruses By H. ~NICK, D. CURSIEFE~-, and H. BECHT Institut ffir Virologie, Justus Liebig-Universit~$, Giel3en, Federal Republic of Germany With 6 Figures Accepted April 15, 1975
Summary Two virus strains which had been suspected to be the etiological agents of infectious bursitis (Gumboro disease) and of inclusion body hepatitis of chickens were characterized by their morphology, their peptide composition and the segmented genome of their double-stranded RNA to be typical reoviruses. Although the 2 avian strains did not clearly differ :in their serological behaviour, the size of some of their I~NA segments were not identical Both strains replicated in tissue cultures prepared from the chorioallantoic membrane of embryonated eggs with growth characteristics of reoviruses.
Introduction A virus had been isolated from the bursa of Fabricius of chickens affected with infectious bursitis (Gumboro disease). The virus was 650 A in diameter, the capsid was constructed as a double shell and it had a density of 1.34 g/m1 and a I~NA content of about 10 per cent (6). Another virus which had been suspected to be of some etiological significance for inclusion body hepatitis of chickens was included in these studies. I t has been shown to be antigenically related to viruses which were supposed to be avian reoviruses (16). I n order to verify that these two viruses are true reoviruses a detailed analysis of their physico-chemical and serological properties was carried out and compared with the representative reovirus type 3, and attempts were made to grow the viruses in culture to define their growth ehracteristics.
Materials and Methods Viruses
The lt5th passage of strain 2207/68 in embryonated eggs was obtained from Dr. KSsters, Giei~en. This virus had been incriminated as being the causative agent of Gumboro disease (6). The 6th passage in e~nbryonated eggs of strain "Wi", which had 18"
262
H . NICK, D.
CURSIEFEN,a n d H . BECtlT:
b e e n alleged to b e t h e etiological a g e n t of infectious h e p a t i t i s of chickens, was o b t a i n e d f r o m Dr. K u s s m a u l , S t u t t g a r t . T h e s t r a i n h i s t o r y h a s b e e n d e s c r i b e d (15). T h e D e a r i n g s t r a i n of r e o v i r u s t y p e 3 s e r v e d as reference virus.
Production and Purification. of Virus T h e t w o a v i a n s t r a i n s were g r o w n in e m b r y o n a t e d eggs a f t e r 8 or 9 d a y s of c u l t i v a tion. W h e n t h e e m b r y o s h a d die(] 2 - - 4 d a y s a f t e r i n f e c t i o n t h e a l l a n t o i c fluid was h a r v e s t e d , coarse m a t e r i a l was r e m o v e d b y c e n t r i f u g a t i o n , a n d v i r u s p a r t i c l e s were s e d i m e n t e d i n a Spinco r o t o r 15 a t 15,000 r . p . m , a t 15 ° C for 2.5 h o u r s . T h e p e l l e t was r e s u s p e n d e d i n SSC b u f f e r (0.15 ~ NaC1, 0.015 M N a - c i t r a t e , p i t 7.2) b y b r i e f s o n i c a t i o n , m i x e d w i t h N o n i d e t N P - 4 0 a t a finM c o n c e n t r a t i o n of 1 p e r c e n t a n d centrif u g e d in a sucrose g r a d i e n t ( 2 0 - - 5 0 p e r cent) in a S W 2 7 r o t o r a t 25.000 r. p . m , a n d 15 ° C for 90 m i n u t e s . T h e v i r u s b a n d s were pooled, d i l u t e d a b o u t t e n f o l d , a n d t h e v i r u s was s e d i m e n t e d b y c e n t r i f u g a t i o n . T h e v i r u s pellet was r e s u s p e n d e d , 3.2 m l were m i x e d w i t h 1.8 m l of s a t u r a t e d CsC1 a n d c e n t r i f u g e d a t 50,000 r . p . m , i n a S%V65 r o t o r for 22 h o u r s a t 15 ° C. T h e v i r u s b a n d s were pooled again, d i l u t e d a n d c o n c e n t r a t e d b y centrifugation. l~eovirus t y p e 3 was g r o w n i n L-cells a n d p u r i f i e d e s s e n t i a l l y as d e s c r i b e d (3).
Growth o] Viruses in Tissue Culture B o t h a v i a n s t r a i n s were g r o w n in cells of t h e e h o r i o a l l a n t o i e m e m b r a n e of e m b r y o n a t e d c h i c k e n eggs (CAM cells) w h i c h were p r e p a r e d as r e c e n t l y d e s c r i b e d (4). 3 × 106 cells were seeded i n t o 5 c m p l a s t i c dishes a n d i n c u b a t e d w i t h 4 m l of m e d i u m 199 c o n t a i n i n g 10 p e r cent, fetal calf s e r u m . ~ h e n m o n o l a y e r s h a d f o r m e d a f t e r 2 d a y s t h e cells were i n o c u l a t e d w i t h 1 m l of infectious a l l a n t o i c fluid. A f t e r a n a d s o r p t i o n p e r i o d of one h o u r t h e cells were w a s h e d t h r e e t i m e s w i t h P B S , a n d i n c u b a t i o n was c o n t i n u e d w i t h 2 m l of m e d i u m 199 c o n t a i n i n g 2 p e r c e n t fetal calf s e r u m . A t d i f f e r e n t periods a f t e r i n f e c t i o n t h e m e d i u m w a s r e m o v e d a n d t h e cells processed b y freezing a n d t h a w i n g . P l a q u e t i t r a t i o n of m e d i u m a n d cell-associated infeetixdty w a s d o n e b y i n o e n l a t i n g CAM cells w i t h 0.2 mI of v i r u s d i l u t i o n s a n d c o v e r i n g t h e ceils w i t h 4 m l of 0.7 p e r c e n t p - a g a r in m e d i u m 199 s u p p l e m e n t e d w i t h 7 p e r c e n t fetal calf s e r u m . T h e cells were s t a i n e d w i t h t r y p a n blue a f t e r 2 ½ - - 4 days. F o r n e u t r a l i z a t i o n t e s t s a b o u t 100 p l a q u e f o r m i n g unit, s ( P F U ) were m i x e d w i t h a n t i s e r u m in a series of t w o f o l d d i l u t i o n s , k e p t a t r o o m t e m p e r a t u r e for I h o u r a n d i n o c u l a t e d o n t o CAM eells. T h e h i g h e s t s e r u m d i l u t i o n w M c h r e d u c e d t h e p l a q u e c o u n t b y m o r e t h a n 80 p e r c e n t was t a k e n as t h e end-point.
Preparation o/Antisera Chickens f r o m a flock free of G u m b o r o disease were i n j e c t e d s u b c u t a n e o u s l y w i t h a p u r i f i e d p r e p a r a t i o n of s t r a i n 2207/68 a t t h e age of 4 weeks. T h e a n i m M s were i n j e c t e d a s e e o n d t i m e 3 weeks l a t e r a n d bled a f t e r a n o t h e r week. A n t i s e r a against, s t r a i n " W i " w i t h c o m p a r a b l e p o t e n c i e s could b e p r e p a r e d b y i n j e c t i n g v i r u s - c o n t a i n i n g a l l a n t o i e fluid b y t h e s a m e route.
Gel Precipitation A n t i g e n s for gel-diffusion were e x t r a c t e d f r o m t h e c h o r i o - a l l a n t o i e m e m b r a n e s of i n f e c t e d eggs. Tissue h o m o g e n a t e s were u s e d as a n t i g e n a f t e r c r u d e debris h a d b e e n r e m o v e d b y low-speeed e e n t r i f u g a t i o n .
Immuno]luorescence CAM-cells g r o w n on eoverslips were s t a i n e d w i t h t h e g a m m a - g l o b u l i n f r a c t i o n of c h i c k e n a n t i s e r a w h i c h h a d b e e n c o n j u g a t e d w i t h fluorescein i s o t h i o c y a n a t e . T h e s a m e p r o c e d u r e was followed as i n (4).
Electron 2kIicroscopy A d r o p of v i r u s s u s p e n s i o n was m i x e d w i t h 2 p e r c e n t p h o s p h o t u n g s t i c acid o n a F o r m v a r - e o a t e d c o p p e r grid a n d e x a m i n e d i n a n E h n i s e o p e i01.
S t r c u t u r e a n d G r o w t h of T w o A v i a n I~eoviruses
263
Extraction o/ Viral R N A Purified virus in SSC was shaken twice w i t h an equal v o l u m e of w a t e r - s a t u r a t e d phenol for 10 m i n u t e s at r o o m t e m p e r a t u r e (12). R e s i d u a l phenol was r e m o v e d f r o m t h e aqueous phase w i t h ether, and R N A was p r e c i p i t a t e d w i t h two volumes of ethanol at - - 2 0 ° C o v e r n i g h t (2).
Melting Curve R N A dissolved in 0.1 × SSC was a d j u s t e d to an optical density of 1.05 at 260 nm. The sample was h e a t e d a n d t h e e x t i n c t i o n registered continously in a P y e Unlearn S p e e t r o p h o t o m e t e r SP 8000 b e t w e e n 40 ° and 95 ° C.
RNase Resistance o] Viral R N A CAM-cells were infected w i t h strain W i in allantoic fluid. After' an adsorption period of 1 h o u r t h e cells were washed and i n c u b a t e d w i t h m e d i u m 199. 50 fzCi of 3H-uridine were added to each 9 cm culture dish. A f t e r 20 hours of i n c u b a t i o n the m e d i u m was h a r v e s t e d , unlabeled virus was added as carrier and the p r e p a r a t i o n was purified b y g r a d i e n t c e n t r i f u g a t i o n as described above. R N A was e x t r a c t e d w i t h SDS and phenol (10), dissolved in 2 x SSC a n d d i v i d e d into two parts. 100 Bg of p a n c r e a t i c R N a s e was a d d e d to one half, and t h e m i x t u r e was i n c u b a t e d at r o o m t e m p e r a t u r e for 20 minutes. TCA-precipitable r a d i o a c t i v i t y was m e a s u r e d in t,he t r e a t e d a n d u n t r e a t e d p r e p a r a t i o n s b y liquid scintillation.
Polyacrylamide Gel Electrophoresis The conditions described (8) were employed. F o r the analysis of viral R N A the 7.5 per cent gels c o n t a i n e d 6 M urea. The buffer system h a d been described (7). Virus samples were dissociated in 2 ~ urea, 1 per cent SDS and 0.1 per cent m e r c a p t o e t h a n o l at 55 ° C for 60 minutes. Electrophoresis was carried out at 4 m A per gel for 24 hours. A f t e r the run the gels were r e m o v e d from t h e tube, rinsed in destilled w a t e r for 5 - - 6 h o u r s a n d stained w i t h m e t h y l e n e blue (9). The p r o t e i n composition was d e t e r m i n e d in 7.5 per cent gels which contained 5 5,I urea. The buffer was 0.1 5~ N a - p h o s p h a t pl~ 7.2 (14). The virus samples were dissociated in 6 ~ urea, t per c e n t SDS and 0. t per c e n t m e r e a p t o e t h a n o l in a boiling w a t e r b a t h for 2 minutes. Eleetrophoresis was carried out at 6 m A per gel for 20 hours. The gels were stained for 30 m i n u t e s in Coomassie brilliant blue at 37 ° C and destained w i t h 7.5 per cent acetic acid at 65 ° C.
Results
Morphology o~ Virion T h e v i r u s c o u l d be p u r i f i e d b y g r a d i e n t e e n t r i f n g a t i o n . B o t h a v i a n s t r a i n s h a d a n i d e n t i c a l s e d i m e n t a t i o n p a t t e r n in c e s i u m chloride. T h r e e b a n d s c o u l d be. o b s e r v e d . T h e i r d e n s i t y was 1.34, 1.30 a n d 1.275 g / m l (Fig. 1). T h e r e f e r e n c e reov i r u s 3 h a d t h e e x p e c t e d d e n s i t y of 1.36 g/ml. I n t h e e l e c t r o n m i c r o s c o p e t h e p a r t i c l e s w i t h a d e n s i t y of 1.34 g / m l w e r e spherical, u n i f o r m in size w i t h a d i a m e t e r of a b o u t 65 n m (Figs. 2 a, b) in a c c o r d a n c e w i t h p r e v i o u s o b s e r v a t i o n s (6) for s t r a i n 2207/68. T h e e a p s o m e r e s h a d t h e appearan.ee of h o l l o w cylinders. I t c o u l d be a f f i r m e d t h a t t r e a t m e n t w i t h t 0 0 fxg/ml of e h y m o t r y p s i n c h a n g e d t h e size of t h e p a r t i c l e s to a b o u t 50 n m a n d t h e d e n s i t y to 1.42 g / m l . T h e m o r p h o l o g i c a l l y d i s t i n c t c a p s o m e r e s h a d d i s a p p e a r e d a n d n o special m o r p h o l o g i c a l e n t i t i e s w e r e d i s c e r n i b l e o n t h e s u r f a c e of s u c h p a r t i c l e s . V i r u s p a r t i c l e s w i t h a d e n s i t y of 1.30 c o n s i s t e d of e m p t y shells (Fig. 2 e), a n d t h e m a t e r i a l b a n d i n g a t a d e n s i t y of 1.275 g / m l c o n t a i n e d b r e a k d o w n p r o d u e t s , single e a p s o m e r e s a n d a m o r p h o u s masses.
264
H. :NIe:K, D. CU~SIEFE~, and I-I. BE(JIlT:
Fig. 1. Cesium chloride density gradient of strain 2207/68, Virus which had been concentrated from allantoie fluid and purified on a sucrose density gradient was mixed with a saturated solul~ion of cesimn chloride and centrifuged in a Spinco S~W65 rotor at 50,000 r . p . m , for 22 hours at 15 ° C. The t,hree visible bands from top to b o t t o m have a density of 1.275, 1.30 and 1.34 g/ml
Fig. 2. Electron micrographs of purified virus preparations stained with phosphotungstie acid a) Whole virus particles of strain Wi; density 1.34 g/ml b) Whole virus particles of strain 2207/68 ; density 1.34 g/ml c) E m p t y particles of strain 2207/68; density 1.30 g/ml Magnification was 120,000 fold
Structure o/ Viral R N A I t could be clearly shown b y a c i d consists of d o u b l e - s t r a n d e d sharp t r a n s i t i o n b e t w e e n 81 ° a n d shows t h a t m o s t of t h e v i r a l R N A
two i n d e p e n d e n t m e t h o d s t h a t the viral nucleic R N A . The profile of tile m e l t i n g curve shows a 88 ° C (Fig. 3) w i t h a T i n - v a l u e of 83 ° C. T a b l e t is g N a s e - r e s i s t ~ n t . I t is possible t h a t some of t h e
Structure and Growth of Two Avian Reoviruses
265
digested material is labeled R N A of cellular origin. Under identical experimental conditions more than 99 per cent of the 3H-uridine incorporated into singlestranded R N A is rendered acid-soluble (10).
K 1,z c
S 1,3 (M ~d 1.2 C3
o
lJ 1,0~
I/ ;s 7; 7's
do
oc
Tm Fig. 3. Melting curve of viral RNA. I~NA extracted from purified virus particles of strain 2207/68 was heated and its absorbance registered continuously at 260 nm
Table 1. RNase Resistance of Viral R N A Treatment of sample
dpm aH-uridine
None
1270
l~Nase
695
Phenol-extracted RNA of virus grown in CAMcells was treated with i00 ,ug/ml of ]~Nase at room temperature for 20 minutes. ]~adioaetivity precipitable by TCA was counted
In polyacrylamide gels the g N A could be separated into 10 distinct pieces (Fig. 4). The most striking difference between the avian strains and reovirus type 3 is segment 7 which is much larger in avian viruses. Figure 4 shows furthermore that besides the very obvious differences in the R N A patterns of reovirus 3 and the avian strains there are definite differences in size in some corresponding R N A segments of the two avian strains too. In strain Wi segment L3 could never be clearly separated from segment L2, and the 3 smallest pieces of group S are shghtly smaller in strain 2207/68 as compared to strain Wi. The molecular weights of the R N A pieces in these 2 strains were calculated from the migration distances in the gels of Figure ~ in comparison with the known values for reovirus 3 (13). They were listed in Table 2.
Polypeptide Composition 7 structural proteins could regularly be detected by aerylamide gel analysis of the 2 avian strains (Fig. 5), which can be classified into three size groups as for reovirus 3 (14), and they have comparable molecular weights. However, in the gels of the avian strains a third faint band with a molecular weight of approximately 75,000 could reproducibly be demonstrated in the medium size class. A third polypeptide in the smallest class, on the other hand, was only irregularly and never clearly visible even when the gels were heavily loaded.
266
H. NICK, D. CURSIEFEN,and H. BECI-IT:
Fig. 4
Fig. 5
Fig. 4L Polyacrylamide gel analysis of viral tRNA. The gels contained 7.5 per cent aerylamide. Eleetrophoresis was carried ou~ at 4 mA per gel for 24, hours. Tile gels were stained with methylene blue From left to right: reovirus type 3, avian strain W'i, avian strain 2207/68 Fig. 5. Polyacrylamide gel electrophoresis of viral proteins. The gels contained 7.5 per cent aerylamide. Electrophoretie runs were for 20 hours at 6 mA per gel. The gels were stained with Coomassie brilliant blue. From 1eft to right : reovirus type 3, avian strain Wi, avian strain 2207/68
Serological Behaviour I n gel diffusion tests antisera agMnst the two a v i a n strains produced one precipitation line which fused completely when the antigens or antisera were placed into a d j a c e n t wells of the agar gel. No precipitation could be n o t e d between antiW i serum or anti-2207/68 serum a n d a cell extract from L-cells infected with reovirus t y p e 3. Antisera against, both a v i a n strains neutralized the i n f e c t i v i t y effieiently, a n d the reciprocal titers were almost identical. Neither a n t i s e r u m neutralized reovirus type 3 (Table 3).
Structure and Growth of Two Avian Reoviruses
267
Table 2. Molecular Weights o / R N A Segments o] the Two Avian Reoviruses ( Daltons × 10G) Segment
Reovirus t y p e 3
Strain Wi
Strain 2207/68
L1 L2 L3 M1 M2 M3 S1 $2 S3 $4
2.50 2.40 2.30 1.60 1.60 1.40 0.92 0.76 0.64 0.61
2.25 2.20 2.17 1.65 1.50 1.39 1.05 0.70 0.66 0.63
2.25 2.20 2.15 1.65 1.45 1.33 1.05 0.65 0.59 0.56
The molecular weights were computed from the relative migration distances in a 7.5 per cent aerylamide gel. The values published for reovirus type 3 (13) and listed in the first row were taken as standard references. The designation of the segments followed the proposal of SHATKIN (13)
Table 3. ±~eutralization o/ the Avian Reoviruses Strain 2207/68 and Wi Virus strain
Reciprocal neutralization titers of antisera Anti-Wi
Anti-2207
Normal serum
2207 Wi
512 1024
1024 512
none none
Reo type 3
none
none
none
Neutralization assays of the 2 avian strains were carried out on CAM-cells, of reovirus type 3 on L-cells. A reduction of plaque counts by more than 80 per cent was taken as end-points. Normal serum was from specific pathogen free birds.
Growth in C A M Cells
14 to 16 hours p o s t infection n u m e r o u s cells infected w i t h s t r a i n W i showed vacuoles which g r a d u a l l y increased in size a n d were e v e n t u a l l y visible in all cells of t h e layer. 1 to 2 hours l a t e r t h e cells a p p e a r e d g r a n u l a t e d , t h e y c o n t r a c t e d to form large clusters, a n d t h e y f i n a l l y d e t a c h e d from the culture dish. I n t r a c e l l u l a r virus titers s t a r t e d to increase 4 8 hours p o s t infection a n d r a p i d l y r e a c h e d a c o n s t a n t level a b o u t 24 hours p o s t infection. E x t r a c e ] l u l a r virus could be demons t r a t e d a p p r o x i m a t e l y 16 hours p o s t infection when c y t o p a t h i c effects were r e a d i l y visible. The titers increased to r e a c h a m a x i m u m of a p p r o x i m a t e l y 107 to 10 s P F U / m l 1 0 - - 1 2 hours l a t e r (Fig. 6). P r o d u c t i o n of virus-specific antigens could first be d e m o n s t r a t e d b y immunofluorescence w i t h c o n j u g a t e d a n t i - W i s e r u m b e t w e e n 6 a n d 8 hours p o s t infection as a fine g r a n u l a r fluorescence in t h e c y t o p l a s m of epitheloid cells. A t l a t e r periods b r i l l i a n t l y fluorescing granules a n d p a t c h e s were d i s t r i b u t e d in t h e whole c y t o p l a s m , b u t no fluorescence could be seen in the nuclei. As r e p o r t e d p r e v i o u s l y (4) m a n y fibroblasts which were n e v e r s t a i n e d w i t h t h e c o n j u g a t e d serum r e m a i n e d a t t a c h e d to t h e coverslip in a loose a n d irregular netlike a r r a n g e m e n t even when all epitheloid cells h a d been d e s t r o y e d . The l a t e n t p e r i o d a n d t h e a p p e a r a n c e of c y t o p a t h i e changes of s t r a i n 2207/68 were u s u a l l y d e l a y e d b y 2 - - 3 hours; the m o r p h o l o g i c a l a p p e a r a n c e of the cytop a t h i c changes was identical to t h e p i c t u r e described for s t r a i n Wi.
H, NICK, D. CUIISIEFEN,and H. BECHT:
268 10s
~ ~ --i-
"~--A --
extra ,I, ~ - - -
10 7
ill / E •. ~
intra 10 5
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hours Fi. Fig. 6. Growth curve of the avian reovirus strain Wi in CAi~Cl-eells. Monolayers were infected with allantoie fluid. Individual plates were withdrawn at different times after infection and assayed for infectivity in the medium and in the cells after freezing and thawing. Plaque titrations were done in CAlVi-eetls Discussion
The data obtained in this study permit the definite classification of the avian virus strains 2207/68 and Wi as reoviruses. I t could be shown that the RNA is double-stranded b y its RNase resistance and the typical melting curve with a Tm value of 83°C in 0.1 ×SSC which is almost identical with the melting point of 84 ° C determined for reoviruses (2). Polyacrylamide gel analysis showed furthermore that the genome of both avian strains consists of the ten segments which are typical for reovirnses (13) and that there are marked differences in the RNA patterns between the avian and mammalian reoviruses. Minor differences, however, could also be noted between the 2 avian strains which were reproducibly obtained in repeated runs with different virus preparations. The serological data available do not indicate any significant differences in the antigenic structure of the two avian viruses, and according to the gel-patterns the size of their structural proteins seems to be identical. Even though tile serological techniques applied indicate a very close relatedness or even identity of two viruses, the size distribution of the I~NA segments represents specific markers for each strain, which may be used for their identification. The avian virus strains which have been described as reoviruses (5, 11) are obviously larger and have a much higher density, The structure of their RNA and of their polypeptides have not been analyzed. A comparison with our data is therefore not possible. The pathogenic significance of the strains 2207/68 and Wi must still be considered as open for discussion, particularly with respect to the etiology of infectious bursitis. Electron micrographs of immune precipitates showed virus particles in the bursa of Fabrieius of infected birds which did not have the morphology of reoviruses (1). No precipitation could be noted in agar gels when extracts from such bursal material diffused against anti-Wi or anti-2207/68 antiserum (I5,
Structure and Growth
of Two
Avian Reoviruses
269
NICK, u n p u b l i s h e d results). The birds which were injected for the p r e p a r a t i o n of the antisera used in the experiments described in this paper did n o t show a n y clinical signs or pathological lesions of G u m b o r o disease. Acknowledgments The work was supported by the Sonderforschungsbereich 47 (Virologic).
Referenees 1. AL~IEIDA,J. D., MORRIS, R. : Antigenically-relatedviruses associated with infectious bursal disease. J. gen. Virol. 20, 369--375 (1973). 2. BEI~LAM¥, A. ]~., SHAPIRO, L., AUGUST, J. T., JOKLIK, YV. K. : Studies on reovirus RNA. Characterization of reovirus genome RNA. J. reel. Biol. 29, 1--17 (1967). 3. BELLAMY, A. R., HOLE, L. V., BAGUI,EY, B. C.: Isolation of the trinucleotide pppGpCpU from reovirus. Virology 42, 415--420 (1970). 4. @URS~EFEN, D., BECttT, H. : I n vitro cultivation of cells from the chorioallantoic membrane of chick embryos. 5~[ed. MicrobioL Immunol. 161, 3--10 (1975). 5. KAWA~IU~A, i . , SHIMIZU, F., MAEDA, M., TSUBA~A~A, H.: Avian reovirus: Its properties and serological classification. Nat. Inst. Anita. mlth. Quart. 5, 115--124 (1965). 6. KOSTEI~S, J., BEC~T, H., RUDOLPh, R.: Properties of the infectious bursal agent of chicken (IBA). Med. Microbio]. Immunol. 157, 291--298 (1972). 7. LOE~rl~:G, U. E. : The fractionation of high-molecular weight ribonucleic acid by polyacrylamide-gel electrophoresis. Biochem. J. 102, 251--257 (1967). 8. MARTIN, S. A., ZWEERINK~ H. J. : Isolation and characterization of two types of b]uetongue virus particles. Virology 50, 495--506 (1972). 9. PEACOCK, A. C., DI~'G~AN, C. W. : Resolution of multiple ribonucleic acid species by polyacrylamide gel electrophoresis. Biochemistry 6, 1818--1827 (1967). 10. SC~OLT~SSEK, C., ROTT, R.: Hybridization studies with influenza virus RNA. Virology 39, 400--407 (1969). 11. SEKIGUC~I, K., KOIDE, F., KAWA~[UI~, H. : Physico-chemical properties of avian reovirus and its nucleic acid. Arch. ges. Virusforsch. 24, 123--136 (1968). 12. SHAT:KIN, A. J. : Inactivity of purified reovirus RNA as a template for E. coli polymerases in vitro. Prec. Nat. Acad. Sci. U.S. 54, 1721--1728 (1965). 13. SIIATKIN, A. J., SIP]~, J. D., Loll, P. : Separation of ten reovirus genome segments by polyacrylamide gel electrophoresis. J. Virol. 2, 986--991 (1968). 14. SI~IITH, R. E., ZxcVEERINK,t{. J., JOKI~I~K,W. K. • Polypeptide components of virions, top component, and cores of reovirus type 3. ViroIogy 39, 791---810 (1969). 15. V~rOERNLE, i . , BI~UNNEI~, A., KUSSS~AVL, K.-F. : Naehweis avi/i,rer Reo-Viren im Agar-Gel-Prgzipitationstest. Tier/~rztl. Umsch. 29, 307--312 (1974). Authors' address: H. BECHT, I n s t i t u t ffir Virologie, Justus-Liebig-Universitgt, Frankfurter Stral3e 107, /)-6300 Gief~en, Federal Republic of Germany. Received March 14, 1975
