Archives of Virology

Archives of ~rlrology 61, 13--21 (1979)

© b y Springer-Verlag 1979

Isolation and Cell Culture Propagation of Rotaviruses From Turkeys and Chickens By M. S. McNvLT¥, G. M. ALLAN, D. TODD, a n d J . B. McFER~AN Veterinary l~eseareh Laboratories, Stormont, Belfast, Northern Ireland W i t h 3 Figures Accepted March 14, 1979

Summary R o t a v i r u s e s were d e t e c t e d b y electron m i c r o s c o p y in t h e faeces of t u r k e y p o u l t s a n d broiler chickens w i t h diarrhoea. A p p a r e n t l y s y m p t o m l e s s infection was also o b s e r v e d in broilers. T h e a v i a n r o t a v i r u s e s could n o t be isolated in cell cultures b y c o n v e n t i o n a l techniques, b u t were a d a p t e d to serial g r o w t h in chick cell cultures following t r y p s i n t r e a t m e n t of t h e virus a n d t h e cells. I m m u n o f l u o r e s c e n c e studies showed t h a t t h e a v i a n a n d m a m m a l i a n r o t a v i r u s e s are a n t i g e n i c a l l y related. A n t i b o d i e s to r o t a v i r u s were w i d e s p r e a d in sera collected from t u r k e y s , chickens a n d ducks.

Introduetion W h i l e i t is well e s t a b l i s h e d t h a t r o t a v i r u s e s can cause e n t e r i t i s in a wide v a r i e t y of m a m m a l i a n species (6, t5), t h e r e are o n l y a few r e p o r t s of r o t a v i r u s infection in a v i a n species (3, 8, 16). The p u r p o s e of t h i s p a p e r is to describe t h e i s o l a t i o n in cell cultures of r o t a v i r u s e s from chickens a n d t u r k e y s a n d to r e p o r t some serological studies w i t h these viruses.

Materials and Methods Preparation o/Faeces Specimens ]or Examination by Electron Microscopy Approximately 15 per cent suspensions of faeces were made in 0.15 • NaC1, 0.01 sodium phosphate buffer, p H 7.2 (PBS). An equal volume of the fluorocarbon Arcton 113 (Arklone P, ICI Ltd., l~uncorn, Cheshire) was a d d e d to the suspension and an emulsion formed b y thorough mixing. Following centrifugation a t 3000 × g for 20 minutes at 4 ° C, the upper aqueous phase was removed. This was ultracentrifuged for 1 hour a t 4 ° C at 23,000 r p m (91,000×g) using a Beckman SW41 swing-out rotor. Pellets were resuspended in a few drops of water or 1 per cent ammonium acetate, mounted on 400-mesh carbon-coated grids and stained with raethylamine tungstate (EM scope Laboratories, London). Grids were examined in a Philips 301 electron microscope at an accelerating voltage of 80 kV.

0304-8608/79/0061/0013/$ 01.80

14

M.S.

N[cNuLTY,

G. M. ALLASr, D. TODD,

and J. B. McFE~I~ASr:

Isolation o / A v i a n Rotaviruses in Cell Cultures C h i c k e m b r y o l i v e r (CEL) a n d c h i c k k i d n e y (CK) cell c u l t u r e s were p r e p a r e d a n d g r o w n as d e s c r i b e d p r e v i o u s l y (10). A p p r o x i m a t e l y 15 p e r c e n t s u s p e n s i o n s of faeces were m a d e i n E a g l e ' s ( B H K ) m e d i u m c o n t a i n i n g 1000 U of penicillin a n d 1000 ,ug of s t r e p t o m y c i n p e r m l (ESA). T h e s e were e x t r a , t e d w i t h f l u o r o c a r b o n as d e s c r i b e d a b o v e a n d t h e a q u e o u s p h a s e w a s u s e d to i n f e c t cell c u l t u r e s . P r i o r to i n o c u l a t i o n , t h e a q u e o u s p h a s e was i n c u b a t e d for 1 h o u r a t 37 ° C w i t h t r y p s i n , w h i c h was a d d e d to give a final c o n c e n t r a t i o n of 5 ~g/ml. T h e e n z y m e was p r e p a r e d as 1 p e r c e n t s t o c k c r y s t a l l i n e t r y p s i n in 1 m ~ HC1 (1), a n d was d i l u t e d w i t h E S A i m m e d i a t e l y before use. T h e m e d i u m was r e m o v e d f r o m c o n f l u e n t m o n o l a y e r s of C E L or C K cells o n 13 m m 2 r o u n d coverslips c o n t a i n e d i n plastic b i j o u - t y p e b o t t l e s (Sterilin L t d . , T e d d i n g t o n , Middlesex) a n d 0.5 m l of i n o e u l u m was a d d e d . T h i s was c e n t r i f u g e d o n t o t h e m o n o l a y e r for 1 h o u r a t 2500 × g a t r o o m t e m p e r a t u r e (4). A further 1 ml of ESA containing 5 ~g trypshl per ml was then added and the cultures incubated at 37 ° C. Coverslips were harvested and processed for ilranunofluorescent staining as described below. If the trypsin removed most of the monolayer, the cells were r e c o v e r e d f r o m t h e m e d i u m b y c e n t r i f u g a t i o n , r e s u s p e n d e d in a s m a l l v o l u m e of P B S a n d s m e a r e d o n a glass m i c r o s c o p e slide. S m e a r s were a i r - d r i e d a t r o o m t e m p e r a t u r e , then. fixed a n d s t a i n e d . C u l t u r e m a t e r i a l for serial p a s s a g e was h e l d a t - - 7 0 ° C u n t i l fresh cell c u l t u r e s were available, t h e n t h a w e d a n d i n o c u l a t e d as described above. Cell c u l t u r e s were p r e p a r e d for t h i n - s e c t i o n e l e c t r o n m i c r o s c o p y as d e s c r i b e d p r e v i o u s l y (11).

Immunofluoreseenee Indirect Stmning F r e s h l y t r y p s i n i z e d P K t 5 cells were i n f e c t e d w i t h l a m b r o t a v i r u s as p r e v i o u s l y d e s c r i b e d (i3), seeded o n t o d e g r e a s e d M u I t i s p o t T e f l o n - c o a t e d m i c r o s c o p e slides (C. A. H e n d l e y a n d Co., E s s e x ) a n d h a r v e s t e d a f t e r 18 h o u r s i n c u b a t i o n a t 37 ° C. A l t e r n a t i v e l y , M D B K cells were i n f e c t e d w i t h isolate 7 5 - - 4 4 7 of b o v i n e r o t a v i r u s (14) a n d h a r v e s t e d 48 h o u r s a f t e r infection. I n f e c t e d cell c u l t u r e s were fixed in a c e t o n e for 10 m i n u t e s a t r o o m t e m p e r a t u r e p r i o r t o o v e r n i g h t s t a i n i n g a t 4 ° C w i t h t u r k e y a n d c h i c k e n sera, F o l l o w i n g w a s h i n g in P B S , c o u n t e r s t a i n i n g was p e r f o r m e d w i t h e i t h e r F I T C - e o n j u g a t e d a n t i - c h i c k e n I g G p r e p a r e d i n r a b b i t s or a n t L t u r k e y I g G p r e p a r e d i n goats (Nordic I m m u n o l o g i c a l L a b o r a t o r i e s , M a i d e n h e a d , Berks). A f t e r a f u r t h e r w a s h i n P B S , t h e m a t e r i a I was m o u n t e d i n b u f f e r e d glycerol a n d e x a m i n e d using ultra-violet illumination. Control experiments showed that antibody to rotavirus was present in both FITC e o n j u g a t e s t o a t i t r e of a p p r o x i m a t e l y 1/30. H o w e v e r , t h i s w a s n o l o n g e r d e t e c t a b l e w h e n t h e y w e r e t e s t e d a t t h e i r w o r k i n g d i l u t i o n s of 1/80 t o 1/100. S u b s e q u e n t w o r k h a s s h o w n t h a t in s o m e b a t c h e s of c o n j u g a t e t h e t i t r e of t h e a n t i b o d y t o r o t a v i r u s was h i g h e r t h a n t h a t of t h e a n t i - s p e c i e s a n t i b o d y . S p e c i m e n s of sera were collected f r o m 15 t u r k e y s w h i c h h a d e x p e r i e n c e d r o t a v i r u s - a s s o e i a t e d d i a r r h o e a 2 - - 3 weeks p r e v i o u s l y . E q u a l v o l u m e s of t h e s e sera were pooled to p r o v i d e a pooled c o n v a l e s c e n t t u r k e y s e r u m . M a n y of t h e c h i c k e n a n d t u r k e y field sera h a d to b e a d s o r b e d w i t h b o v i n e liver p o w d e r to e l i m i n a t e non-speeifie s t a i n i n g . L i v e r p o w d e r was a d d e d to t h e sera a t a p p r o x i m a t e l y 0.05 g p e r m I a n d t h e t w o were m i x e d o v e r n i g h t a t 4 o C. F o l l o w i n g c e n t r i f u g a t i o n a t 3000 × g for 30 m i n u t e s t h e s u p e r n a t a n t w a s r e m o v e d . T h i s process w a s r e p e a t e d before t h e sera were e x a m i n e d for a n t i b o d y t o r o t a v i r u s . Direct Staining I t y p e r i m m l m e sera t o c~lf, l a m b a n d p i g r o t a v i r u s e s were p r e p a r e d in r a b b i t s , c o n j u g a t e d w i t h F I T C a n d u s e d as d e s c r i b e d p r e v i o u s l y (13).

Serum Neutralisation Test ]or Antibody to Cal/ J~otavirus D o u b l i n g d i l u t i o n s of t u r k e y a n d c h i c k e n sera were m a d e i n E a g l e ' s ( B t t K ) m e d i u m , b u f f e r e d w i t h 33 m ~ b i c a r b o n a t e a n d c o n t a i n i n g 100 U of penicillin a n d

In vitro Cultivation of Avian l~otaviruses

15

100 ~g of streptomycin per ml (EBA). Serum dilutions (0.5 ml) were added to equal volumes of EBA containing 100 TCIDs0 of isolate 75--447 of cMf rotavirus (14). The serum-virus mixtures were incubated overnight at 4° C ansi then inoculated into roller tube cultm'es of MDBK cells. Two tubes were used for each serum dilution. The tubes were rolled at 37° C for I hour to allow-the virus to adsorb to the cells before the cultures were refed with EBA. The cultures were incubated at 37° C in a roller apparatus and examined daily for 7 days for evidence of a eytopathic effect.

Results Detection o] Rotaviruses in Avian Faeces by Electron Microscopy We have previously reported the detection by electron microscopy of rotavirus particles in the faeces of 5 week old turkey poults with diarrhoea (16). More recently, we have observed large numbers of rotavirus particles in the caecal contents of 3 to 7 day old poults affected with a syndrome charactcrised clinically b y diarrhoea, inflammation of the vent and vent pecking. I n a search for rotaviruses in chickens, specimens of pooled caecal and large intestinal contents from 54 broiler chickens aged between 19 and 36 days were examined b y electron microscopy. These broilers were from flocks free from obvious disease and the intestinal contents were normal in volume, consistency and colour. Rotaviruses were observed in very small numbers in 6 of these specimens. Rotavirus particles have been observed in greater numbers in the faeces of 42 day old birds from a broiler flock with a diarrhoea problem. The avian rotaviruses were morphologically indistinguishable from those which infect mammalian species. I n t a c t double-shelled particles averaged 66 nm in diameter. Single-shelled particles were about 10 nm smaller (Fig. 1),

Isolation o/Tur/cey and Chic/sen Rotaviruses in Cell Cultures Faecal material from 6 day old turkeys with diarrhoea was treated with trypsin and inoculated into cultures of CK cells which were maintained on trypsincontaining medium. Electron microscopic examination of the faecal material showed it contained numerous rotavirus particles. Viral replication was followed in early passages by indirect immunofluorescent staining using the pooled convalescent turkey serum. Later passages were stained with an FITC-eonjugated antiserum to calf rotavirus. Following an initial passage of 24 hours duration, tess than 1 per cent of CK cells contained cytoplasmic antigen which reacted with the pooled convalescent serum. However, the number of infected cells increased with successive 24 hour passages (Fig. 2). After 10 serial passages in CK cells, the turkey virus was passaged twice in CEL cells. A stock pool of the virus was then prepared in CEL cells. A working pool was grown up from the stock pool in CEL cells maintained in 25 sq em plastic bottles. All passages of the virus in CEL celts were of approximately 48 hours duration. When the working pool was used to infect CEL cells, about 70 per cent of the cells consistently contained viral antigen when harvested 48 hours after infection (Fig. 2 d). Faecal material containing rotavirus particles from broiler chickens with diarrhoea was similarly inoculated into CEL cultures and passagcd at 48 hourly inter-

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M.S. McNu~TY, G. M. AT,L~N, D. TODD, and J. B. McFE~m~N:

vals. Synthesis of viral antigen was again followed b y immunofluoreseence using the antisera to the t u r k e y and calf rotaviruses. As with the t u r k e y virus, the number of infected cells increased with each successive passage. At the 4th passage about 50 per cent of the cells in the monolayer contained rotavirus antigen (Fig. 2 e). A t t e m p t s to isolate the t u r k e y and chicken rotaviruses from the faecal preparations used above were unsuccessful when t r y p s i n t r e a t m e n t was not employed. Synthesis of viral antigen was demonstrated initially, b u t the number of fluorescing cells markedly decreased with each passage and fluorescence was not observed after 4 serial passages.

Fig. 1. Negative contrast electron microscopy of avian rotaviruses. Bar represe~ts 100 nm. a Double-shelled rotavirus particles from turkey faeces, b Single-shelled particles from turkey faeees, c Single- and double-shelled particles from CEL eells infected wigh chicken rotavirus

I n vitro C u l t i v a t i o n of A v i a n l~otaviruses

17

After 10 passages in CK cells and then 5 passages in CEL cells, the turkey virus produced what appeared to be a cytopathie effect in CEL cells. Cells in infected cultures became granular and detached from the glass. This change was not observed in uninfected trypsin-treated cultures. Electron microscopic examination of CEL cells infected with the working pool of turkey rot,avirus revealed the presence of numerous rotavirus particles

Fig. 2. I m m u n o f l u o r e s c e n c e . a P K 15 cells infected w i t h I a m b r o t a v i r u s , s t a i n e d w i t h pooled convalescent t u r k e y s e r u m , b C K cells i n f e c t e d w i t h 9th p a s s a g e t u r k e y r o t a virus, s t a i n e d w i t h pooled convalescent t u r k e y s e r u m , c CEL cells infected w i t h 4th passage chicken rotavirus, stained w i t h a n t i s e r u m t o cMf r o t a v i r u s , d CEL cells infected w i t h w o r k i n g pool of t u r k e y r o t a v i r u s , s t a i n e d w i t h a n t i s e r u m to l a m b r o t a virus. (All × 200) 2 Arch.Virol. 61/1--2

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M.S. M e N u r ~ , G. M. ALLAN,D. TODD, and J. B. MeFEmcAN:

within dilated eisternae of the rough endoplasmie reticulum (Fig. 3). Rotavirus particles were also detected by negative contrast electron microscopy in lysates of CEL cultures infected with either the turkey or chicken rotavirus isolate (Fig. 1 c).

Antigenic Relationship Between Avian and Mammalian Rotaviruses The antigenic relationship between avian and mammalian rotaviruses was investigated by immunofluorescenee. The pooled turkey convalescent serum reacted with turkey, chicken, calf and lamb rotavirns antigens in infected cell cultures. Similarly, turkey and chicken rotavirns antigens were stained by FITCconjugated hyperimmune sera to calf, lamb and pig rotaviruses. Some of these reactions are illustrated in Figure 2. Fluorescence was not detected when uninfected cell cultures were used.

Fig. 3. Thin section of part of cytoplasm of CEL cell infected with working pool of turkey rotavirus. Note virus particles within dilated eisternae of rough endoplasmic retieulum. Bar represents 200 nm

Survey o/Avian Sera /or Antibody to Rotavirus The antigenic relationship between mammalian and avian rotaviruses enabled us to carry out a survey of avian sera for antibody to rotavirus, using indirect immunofluorescent staining of P K 1 5 celts infected with lamb rotavirus. Sera were collected from parent turkey and chicken flocks when the birds were approximately 20 weeks old. I n the initial screening only 1 serum per farm was examined, and sera were tested at a dilution of 1/20. Antibody to rotavirus was detected in 9 of 26 (35 per cent) turkey sera and in 14 of 35 chicken sera (40 per cent) examined. Of 59 turkey sera collected from 5 farms identified as antibody-

I n vitro Cultivation of Avian Rotaviruses

19

positive b y the initial screening procedure, 53 (90 per cent) possessed antibody. Similarly, a n t i b o d y was detected in 25 of 42 (59 per cent) chicken sera from 5 positive farms. I~o a n t i b o d y was detected in sera collected from the laboratory's specific pathogen free chicken flock. This flock is maintained in positive pressure filtered air isolation houses and is regularly monitored for infection with a n u m b e r of avian pathogens. This has included direct electron microscopic examination of faeces and rotaviruses have not been observed. Indirect immunofluorescence a n t i b o d y titres were relatively high in both t u r k e y and chicken sera. Of the 23 t u r k e y and chicken sera identified as antibodypositive when screened at a dilution of 1/20, 22 were still positive when tested at a dilution of 1/100. The indirect immunoftuorescence titre of the pooled convalescent t u r k e y serum was 640. The geometric m e a n titre of 10 r a n d o m l y selected positive chicken sera was 394. We have also examined sera from adult ducks b y indirect immunofluorescence, 10 of 21 sera screened at a dilution of t/20 h a d a n t i b o d y to rotavirus. Chicken and t u r k e y sera with varying indirect immunofluoreseence a n t i b o d y titres were tested for neutralising a n t i b o d y to isolate 75--447 of bovine rotavirus. Significant levels of neutralising activity were found in m a n y sera with indirect immunofluorescence titres in excess of 40. Some of the results are shown in Table 1. Generally speaking, neutralisation titres were lower t h a n indirect immunofluorescence titres, b u t some sera, e.g. t u r k e y serum 8, were exceptions to this rule.

Table 1. Comparison o/ rotavirus antibody titres o/ avian sera measure/1 by indirect immunojluorescenee and neutralisation tests

Pooled turkey convalescent serum Turkey serum 4 Turkey serum 8 Turkey serum 11 Chicken serum 2 Chicken serum 4 Chicken serum 6 Chicken serum 11

Indirect FA titre

Neutralisation titre

640 ~20 160 160 320 ~ 640 160 ~ 20

60 ~20 ~ 640 80 20 ~ 640 30 30

Discussion Rotaviruses have been detected in avian species only comparatively recently (3, 8, 16). As far as we are aware, this is the first account of the isolation of these viruses from turkeys a n d chickens. Like their m a m m a l i a n counterparts, avian rotaviruses are difficult to isolate a n d grow in cell cultures using conventional techniques. However, m a m m a l i a n rotaviruses can be serially p r o p a g a t e d in celt cultures if the virus inoculum is t r e a t e d with proteolytic enzymes (2, 18), or if trypsin is incorporated into the m e d i u m after infection (1). However, it is n o t 2*

20

M.S. MONULT~, G. 1~[.ALL,~-, D. TODD, and J. B. McFEa~A-~:

clear from these studies whether the effect of the enzyme is on the virus or ca cellular receptors for the virus. Consequently we used both types of treatment in our isolation attempts. Rotaviruses were isolated from both chicken and turkey faeces using this approach, whereas no isolates were made without trypsin treatment. The isolation of these viruses in celt cultttres should greatly facilitate investigation of their biological and biochemical characteristics. I t is tempting to suggest that avian and mammalian rotaviruses have a similar level of pathogenicity. Indeed, previous reports of rotavirus infection in turkeys and hens have associated infection with outbreaks of diarrhoea (3, 8, t6). However, infection with rotavirus does not invariably result in illness in mammalian species (5, t2, 17). This would also appear to be the case with avian species, as shown by the symptomless infection of broiler chickens described in this paper. The excretion of very small numbers of virus particles in the faeces is apparently a common feature of asymptomatic rotavirus infection in chickens and human neonates (5). Mammalian rotaviruses possess a group antigen demonstrable by immunefluorescence or complement fixation tests (9, 19, 20). Our immunofluorescence studies indicate that this antigen is also present in the turkey and chicken rotaviruses. This was unexpected as most avian viruses do not cross-react by immunefluorescence with their mammalian counterparts. There was therefore a possibility that the viruses we isolated from turkeys and chickens represented a chance infection of these species with mammalian rotaviruses. However, the finding that antibody reacting with the mammalian rotavirus group antigen was widespread in turkey, chicken and duck sera argues against this. Furthermore, preliminary analysis of the I~NA segments of these avian viruses by polyacrylamide gel eleetrophoresis has revealed a feature not previously described for any of the mammalian rotaviruses (Tend and McNw,TY, unpublished observations). The serological suvey also showed that the percentage of birds with antibody to rotavirus on infected turkey farms was higher than that on chicken farms. This m a y be related to the system of poultry husbandry in Northern Ireland. Parent chicken flocks are derived from birds reared on a number of different farms and transferred to breeding farms at about 17 weeks of age. On the other hand, turkeys are more often reared and bred on the same farm. Assuming that rotavirus infections occur predominantly in young birds, one would expect infected parent turkey flocks to be almost t00 per cent positive for antibody, whereas the percentage of birds with antibody in the parent chicken flocks would depend on the proportions of birds supplied from sere-positive and sere-negative rearing farms. Given this situation, our figure of 40 per cent for parent chicken flocks with antibody is probably an underestimate as only one serum per farm was examined. Significant levels of neutralising activity against isolate 75--¢47 of bovine rotavirus were detected in some of the turkey and chicken sera (Table 1). This is probably due to the low level of cross-reaction which occurs between rotaviruses from different animal species (19). However, the considerable variation in the ratios of indirect immunofluorescenee and neutrMisation titres raises the possibility that turkeys and chickens can be infected with more than one serotype of rotavirus. This would not be surprising, as several serotypes of rotavirus capable of infecting humans have been shown to exist (7),

I n vitro Cultivation of Avian l~otaviruses

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lleferenees 1. AL~IDA, ft. D., HALl,, T., BANAtt~rALA,J. E., TOTTERDELI,,B. M., CHRYSWIE,I. L. : The effect of ti3~psin on the growth of rotavirus. J. ten. Virol. 40, 213--218 (1978). 2. BABIUK, L. A., MOtIANIlVLED,K., SPENCE, L., FAIYVEL, M., PETRO, ]:~.: Rotavirus isolation and cultivation in the presence of trypsin. J. din. Microbiol. 6, 610--617 (1977). 3. BEI~GELA~D, M. E., MCADARAGH, J. ]9., STOTZ, I.: I~otaviral enteritis in turkey poults. ]groc. 26th Western Poultry Disease Conference, University of California, Davis, 129--130 (1977). 4. BRYDEN, A. S., DAVIES, i . A., TtIOULESS, M. E., F:LEWETT, T. H. : Diagnosis of rotavirus infection by cell culture. J. reed. Microbiol. 10, 121--125 (1977). 5. CHRYSTIE, I. L., TOTT~RDE~:L,M. B., BA>rA~VAI;A,J. E. : Asymptomatic endemic rotavirus infections in the newborn. Lancet i, 1176--1178 (1978). 6. F:LEWET% T. H., WOODE, G. N.: The rotaviruses. Arch. Virol. 57, 1---23 (1978). 7. FLEWETT, T. H., THOU:LESS, M. E., PINFOLD, J. N.~ BRYDEN, A. S., CANDEIAS, J. A. N. : More serotypes of human rotavirus. Lancet il, 632 (1978). 8. JO~ES, R. C., HUGHES, C. S., I~EI~-RY, l~. 1%. : Rotavirus infection in commercial laying hens. Vet. Rec. 1114, 22 (1979). 9. KAPIKIAlg, A. Z., CLINE, W. L., KIM, H. W., I4~LICA, A. R., WYATT, R. G., VAlVI¢IgK, D. H., C~zA~OC~;,R. M., JAMES, H. D., VA~rGH>T,A. L. : Antigenic relationships among five reovirus-like (RVL) agents b y complement fixation (CF) and development of new CF antigens for the h u m a n RVL agent of infantile gastroenteritis. Proc. Soe. exp. Biol. Med. 152, 535--539 (t976). i0. MCFERR.AN, J. B., McCRAcK~m', R. M., McKILLOP, E. R., 51CNULTY, M. S., COLLINS, D. S. : Studies on a depressed egg production syndrome in Northern Ireland. Avian Pathol. 7, 35--47 (1978). ii. McNu:LTY, M. S., CUR]nAN, W. L., McFERRAN, J. B.: The morphogenesis of a eytopathic bovine rotavirus in Madin-Darby bovine kidney cells. J. gen. Virol. 33, 503- -508 (1976). 12. McNv:LTY, M. S., MOFERRAN, J. B., BRYSON, D. G., LOGAN, E. F., CUI~ICAN,W. L. : Studies on rotavirus infection and diarrhoea in young calves. Vet. Bee. 99, 229 - - 2 3 0 (1976). 13. McNu:LwY, M. S., AI~LAN,G. M., PE~SOlV, G. R., McFERRA>r, J. B., CURRA>r,WL L., McCRAcKEN, B. M." t~eovirus-like agent (rotavirus) from lambs. Infect. I m m u n . 14, 1332 1338 (1976). 14. McNu:L~e¥, M. S., A:L:LA~r,G. M., MCFEI~IaAN, J. B." Cell culture studies with a cytopathic bovine rotavirus. Arch. Virol. 54, 201--209 (1977). 15. M c N u ~ Y , M. S.: ]~otaviruses. J. gen. Virol. 40, 1--18 (1978). 16. McNvLTw, M. S., AL:LA~~, G. M., STtrART,J. C. : lgotavirus infection in avian species. Vet. Bee. 103, 319--320 (1978). 17. S~ODGRASS, D. R., ~¥ES:LS, P. ~r. : The immunoprophylaxis of rotavirus infections in lambs. Vet. Rec. 102, 46--48 (1978). 18. T~EI~, K. W., Bo~:L, E. tt., Ae~c]~s, A. G.: Cell culture propagation of porcine rotavirus (reovirus-like agent). Amer. J. vet. l%es. 38, 1765 1768 (1977). 19. T~OULESS, M. E., BRYDEN, A. S., F:LEWETT, W. H., ~TOODE, G. N., BRIDGE:a, J. C., SNODG~ASS, D. i~., HERRING, J. A. : Serological relationships between rotaviruses from different species as studied by complement-fixation and neutralisation. Arch. Viro]. 53, 287--294 (1977). 20. WOODE, G. N., BRIDGER, 0-. C., JONES, J. M., FLEWETT, T. It., BttYDEN, A. S., DAVIES, H. A., Wm~E, G. B. B. : Morphological and antigenic relationships between viruses (rotaviruses) from acute gastroenteritis of children, calves, piglets, mice and foals. Infect. I m m u n . 14, 804--810 (t976). Authors' address: Dr. M. S. McNr~'Lw¥, Veterinary ]~esea.rch Laboratories, Stormont, Belfast, B T 4 3SD, Northern Ireland. Received February 19, 1979

Isolation and cell culture propagation of rotaviruses from turkeys and chickens.

Archives of Virology Archives of ~rlrology 61, 13--21 (1979) © b y Springer-Verlag 1979 Isolation and Cell Culture Propagation of Rotaviruses From...
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