Archives of Virology
Archives of Virology 62, 341--346 (1979)
© by Springer-Verlag 1979
Properties of Border Disease Virus as Studied in a Sheep Cell Line By H. LAUDE and J. GELFI Station de Recherches de Virologic et d'Immunologie, I.N.R.A., Thiverval-Grignon, France With 1 Figure Accepted April 9, 1979
Summary A fetal lamb muscle cell line has been isolated in which two strains of Border disease virus replicate well and induce a clear-cut cytopathic effect, thus providing a sensitive and practical assay system for both the virus and neutralizing antibodies. This system enabled us to study some characteristics of Border disease virus in comparison with two other agents responsible for hog cholera and bovine viral diarrhea. Our results indicate t h a t the last virus and the Border disease agent are indistinguishable in vitro.
Introduction Border disease (BD) is a congenital disease of sheep, responsible for fetal and neonatal death. Affected newborn lambs usually exibit an abnormally hairy birth coat, tremor associated with defective myelination of the central nervous system, and low viability (see 6 and 14). Since the antibodies induced in infected animals have been shown to cross-react with bovine viral diarrhea (BVD) and hog cholera (HC) viruses (7, 11, 16), the virus has been included in the pestivirus genus within the Togaviridae family (12). However more details of its antigenieity and physical properties must be known before it is placed in a definite taxonomic group. So far only two succesfnl attempts to isolate BD-virus in tissue culture have been reported. The BD-strain isolated b y VANTSlS st al. (14) is able to induce a significant cytopathic effect (CPE) in lamb ceils, and the CPE-producing agent was neutralized by an antiserum to BVD-virus. On the other hand, HA~K~ESS et al. (6) reported that repeated attempts to demonstrate a CPE associated with the BD-agent have met with no success in any of the tissue culture systems that have been tested. The virus could be detected by immunofluorescence and was shown to be neutrahzed b y an antiserum to HC-virus. I n this paper we describe the use of a new sheep cell line in which both the BD-virus strains mentioned above induce a clear-cut CPE, thus providing a con-
0304-8608/79/0062/0341/$ 01.20
342
H. LAUDE and J. GELH:
v e n i e n t s y s t e m for a n in vitro a s s a y of t h i s a g e n t . W e also r e p o r t s o m e f i n d i n g s r e s u l t i n g f r o m t h e c o m p a r i s o n of B D - v i r u s w i t h o t h e r p e s t i v i r u s m e m b e r s .
Materials and Methods Viruses The two B D - v i r u s strains used were recovered from l a m b ' s infected brain a n d reproduced t h e disease w h e n inoculated into sheep (6, 14). The strain n a m e d as W e had received six passages in calf testis p r i m a r y tissue culture (6). The strain designated E d had undergone 25 passages in fetal lamb kidney secondary tissue culture (14).
Celt Cultures Three strains of fetal l a m b muscle cells were used, which h a d been s u b c u l t i v a t e d 15 times or less, in E L Y - m e d i u m s u p p l e m e n t e d w i t h 10 per r e n t sheep serum and antibiotics. A continuous cell line (FLM), recovered from one of these cultures, was e m p l o y e d between 30 and 70 subpassages. F e t M calf kidney secondary cultures were p r o p a g a t e d in Eagle's m i n i m u m essential m e d i u m (MEM) w i t h a 10 per cent. calf serum supplement. All infected cells were m a i n t a i n e d in M E M containing 5 - - 1 0 per r e n t fetal calf sermn which had been tested for the absence of anti-pestivirus antibodies.
Plaque Assay The procedure was sirnilar to the test published previously for HC-virus (9). Briefly, 24 hours old m o n o l a y e r F L M . c e l l cultures were obtained by inoculating 8 × 10 a cells per 30 m m container (Coster 6-wells trays), and were infected w i t h an a p p r o p r i a t e BI)-virus dilution. T h e n 3 ml M E M incorporated w i t h 4 per cent fetal CS and containing 0.6 per cent agar were added. After incubation at 38 ° C in a COs incubator for designated periods, cultures were e x a m i n e d against an indirect light source or after neutral red staining.
Cross-Neutralization Test The origins of the antisera and viruses listed in Table 1 h a v e been described elsewhere (2). Antisera to BD.viruses were prepared in pigs (see below). I n t h e plaque reduction n e u t r a l i z a t i o n test used, tenfold virus dilutions were m i x e d w i t h a c o n s t a n t dose of antiserum. After 1 h o u r at 37 ° C, 0.2 m l samples of this suspension were a d d e d to t h e F L M - m o n o l a y e r s for B D and BVD-viruses, and to PK1a-monolayers for HCvirus. The inoeula were r e m o v e d after 1 hour.
Animal Experiments Pigs (50 kg) which h a d been previously checked for t h e absence of antibodies against pestiviruses were given 1 m l i n t r a m u s c u l a r injection of 1 × 106 P F U of BDvirus; booster injections were given 3 weeks later. Three weeks after the 2nd injection the four pigs (two for each strain) and two u n i m m u n i z e d pigs were challenged using a fully pathogenic strain of IIC-virus.
Results
Propagation o~ BD-Virus in FLM-Celt8 T h e E d - s t r a i n i n d u c e d a s i g n i f i c a n t C P E in F L M - c e l l s f r o m t h e 2 n d passage, l e a d i n g to e x t e n s i v e cell d e a t h a f t e r 4 - - 5 d a y s of i n c u b a t i o n . I n c o n t r a s t , no C P E was o b s e r v e d on s e c o n d a r y calf k i d n e y cells a f t e r 3 b l i n d passages. T h e W e - s t r a i n d i d not, p r o d u c e n o t i c e a b l e C P E b e f o r e a 6 serial p a s s a g e on F L M - c e l l s . W e obs e r v e d t h a t t h e C P E p r o d u c e d b y t h e W e - s t r a i n was a l w a y s s l o w e r t o a p p e a r t h a n w i t h t h e E d - s t r a i n . F u r t h e r m o r e , it was n o t so clear in c e r t a i n F L M - c e l l s t r a i n s as in t h e e s t a b l i s h e d F L M - c e l l s . S u b s e q u e n t l y , since t h e line g r e w r e a d i l y
In vitro Properties of Border Disease Agent
343
and was easy to handle, it was retained for assay and propagation of BD-viruses. The replication of BVD-virus in the FLM-line was studied in the same way. All the nine BVD-strains assayed (10) readily multiphed in FLM cells without any preliminary adaptation, inducing rapid destruction of the cell sheet.
Virus Plaque Titration Using FLM-line monotayers under agar overlay, it was possible to obtain countable plaques 2 to 3 days p.i. by both Ed- and We-strain of BD-virus (Fig. 1) and to use these for quantitative titration of BD-virus. Both BD-virus strains reached total infectivity titres of 5 × 106 plaques forming units (PFU) per ml if infected cultures were frozen when the CPE started to develop at about 48 hours p.i.
Fig. 1. Appearance of plaques formed by BD- and BVD-viruses on the FLM-eell line at 72 hours p.i. a BD-strain Weybridge. b BD-strain Edimburgh. c BVD-strain NADL
Speci/icity o/the Cytopathic E/]ect Several results indicate t h a t the detected CPE is specific for BD-virus. 1) CPE never appeared in mock-infected cultures passed serially like BD-infected cultures. 2) Ultracentrifugation showed that the two cytopathic agents possess hydrodynamical characteristics of the pestiviruses namely Sw 2° value of 138 and buoyant density in sucrose of 1.12 mg/ml (10). No CPE was detected from the gradient fractions apart from the peak. 3) BD-infeeted cell monolayers were treated 24 hours p.i. with anti-HC-virus conjugate and showed a discrete cytoplasmic fluorescence, which was not observed in mock-infected cell sheets. 4) The cytopathie agents present either in We- or Ed-strain are both antigenieally related to HC- and BVD-vimses.
Studies on Antigenicity From the results presented in Table l, it appears t h a t the external antigens of BD-virus are more closely related to those of BVD-virus than to those of HCvirus. The 331-strain, a variant responsible for chronic forn~ls of tIC, was included in this study because it is already known t h a t it is antigenically closer to BVD-
344
H. LAUDE and J. GELFI:
Table i. Cross-neutralization tests on B D - , B VD- and HC-viruses and their respective antisera a
Antiserum
BD-strains Ed We
BVDV NADL
331
Ed We NADL 331 ~fort
4.1 1.8 1.7 1.4 1.7
1.7 1.4 3.7 N.D. 0.6
1.2 1 2 4 5.4
2.2 1.9 2.2 1.8 1.45
~[CV-strains Alfort~o 0.25 0.5 0.45 N.D. 7.9
a Logi0 plaque reduction neutralization-indices using immune sera diluted 1 : 20
virus than are fully virulent HC-virus strains (2). I t can be noticed that the 331 strains also cross-react with BD-virus more intensively than the Alfort-strain, Animal Experiment8
BD-virus inoculation into pigs did not produce any clinical disease. Immunological responses of varying degree were observed. Homologous neutralization indices of 4.1. and 3.5 were found in sera from pigs immunized with Ed-strain material, whereas indices of 1.8 and 1.1 were encountered in sera from We-strain material immunized pigs. After HCV-challenge the former pigs recovered after showing mild symptoms, whereas the latter succumbed at 11 days p.i. with symptoms commonly associated with hog cholera, as in the control group. These results indicate that BD-immunized pigs could be cross-protected against HCvirus, as it has also been reported for pigs immunized with BVD-virus.
Discussion Fetal lamb muscle cell-cultures appear to be a very useful system for assaying BD virus. On the one hand it was previously reported that Ed-strain CPE induced on secondary fetal lamb kidney cells did not appear before 5 days p.i. (14). On the other hand the We-strain was considered not to be cytopathic and so, a conjugated anti-HCV was employed for its detection (6). The present work shows that a FLM cell line is highly susceptible to BD-virus and that the replication of both strains leads to a significant CPE within 2--8 days p.i. In addition the system provides a convenient plaque assay for measuring virus infectivity, and satisfactory titres are obtained. Probably FLI~I-cells would also constitute a convenient tool for the recovery of other BD-viruscs from field specimens. The FLM cell-line might also provide an appropriate system for the comparison of BD, BVD and l:IC-viruses since the three types of virus replicate well in these cells (these results and 8). Both BD- and BVD-viruses woduced clear-cut CPE in infected FLM-cells, whereas HC-virus did not. In the study of antigenic properties, it appears that BD-virus exhibits a closer relationship with BVD-virus than with tiC-virus. I t must be pointed out, however, that the residual infectivity of the neutralized HC-virus samples was titrated on porcine cells, whereas the neutralization tests for both BVD- and BD-virus
I n vitro Properties of Border Disease Agent
345
were p e r f o r m e d in FLM-cells. I n t h e same way, in all previous studies on antigenic relationships b e t w e e n HC- a n d B V D - v i r u s e s t h e n e u t r a l i z a t i o n indices were perf o r m e d on a s e p a r a t e cell s y s t e m for each virus. N o w t h e a m o u n t of n e u t r a l i z a t i o n o b s e r v e d in v i r u s - a n t i b o d y m i x t u r e s is c o n s i d e r a b l y influenced b y t h e h o s t cell s y s t e m in which r e s i d u a l virus i n f e c t i v i t y is a s s a y e d (see 3). T h u s i t w o u l d be b e t t e r to h a v e a c o m m o n cell s y s t e m in which t o s t u d y t h e t h r e e agents. Unf o r t u n a t e l y we could n o t do i t because of t h e low efficacy of p l a t i n g of I I C - v i r u s on our F L M - l i n e (8). T h e results of cross-protection in pigs, suggest t h a t t h e r e are i m m u n o l o g i c a l differences b e t w e e n BD- a n d HO-viruses a n d t h u s agree with t h e n e u t r a l i z a t i o n findings. W h e n c o m p a r i n g B D a n d BVD-viruses, it is i m p o r t a n t to notice t h a t t h e serological differences f o u n d are no m o r e p r o n o u n c e d t h a t those a l r e a d y r e p o r t e d b e t w e e n different strains of B V D - v i r u s (2). So it a p p e a r s t h a t B D a n d BVDviruses c a n n o t be clearly d i f f e r e n t i a t e d in vitro. I n vivo it is k n o w n t h a t m e m b e r s of t h e pestivirus genus do n o t possess a b s o l u t e host specificity (4) a n d further, t h a t t h e y all e x h i b i t t e r a t o g e n i c a c t i o n (16). Significantly enough, i t has been r e p o r t e d t h a t some B V I ) - s t r a i n s are able t o induce in sheep species p a t h o l o g i c a l f e a t u r e s ressembling t h a t oeuring a f t e r a B D - i n f e c t i o n (1, 13, 15). Conversely, B D - v i r u s was f o u n d t o be as p a t h o g e n i c for t h e calf fetus as B V D - v i r u s (5). I n s u m we conclude t h a t , in light of t h e evidence available, B D - a n d B V D - s h o u l d n o t be considered to be t w o s e p a r a t e v i r a l entities.
Aeknowledgments It is a pleasure to record our thanks to Dr. J. T. Vantsis (Moredun Research Institute, Edinburgh) and J. W. I-larkness (Central Veterinary Laboratory, Weybridge) for giving us their BD-strain. We are also grateful to Dr. E. Plateau (Laboratoire Central V6t~rinaire, Maisons-Alfort) with help on how virulent hog cholera challenge could be performed, and to Dr. M. Launais (Laboratoires Cogla, Libourne) for providing primary lamb cells.
References 1. ACLA2gD, H. 1Vi., GARD, G. P., PLANW, J. W. : Infection of sheep with a mucosal disease virus. Austr. Vet. J. 48, 70 (1972). 2. CORTEIEI~, G., AYNAUD, J. M., GALICI-IEll., C., GELFI, J . : Activit6 antig6nique eomparde de deux togavirus: le virus de Ia peste porcine et le virus de la maladie des muqueuses. Ann. Reeh. Vet. 5, 373--393 (1974). 3. DET.LA-PO~TA, A. J., ~TESTAW~kY,E. G. : A multi-hit model for the neutralization of animal viruses. J. gen. Virol. 38, 1 - - i 9 (1977). 4. FaENCR, E. L., SzvowDoN, W. A.: The infection of pigs with bovine mucosal disease virus. European eomlnunities seminar on hog cholera and african swine fever, 249--261. Hannover: 1976. 5. GIBBONS, D. F., WINKLER, C. E., SHAN, I. G., TERLECKI, S., I:LIOttARDSON, C., DONE, J. : Pathogenicity of the Border disease agent for the bovine fetus. Brit. vet. J. 130, 357 (1974). 6. HA~K~ESS, J. W., KISrG, A. A., TEaLECKI, S., SANDS, J. J. : Border disease of sheep : Isolation of the virus in tissue culture and experimental reproduction of the disease. Vet. Rec. 100, 71---72 (1977). 7. HUCK, I~. A., EvA/qs, D. H., WooDs, D. G., KING, A. A., ST[tAR% P., SEAN, I. G.: Border disease of sheep. Comparison of the results of serological testing using complement fixation, immunodiffusion, neutralization and immunoftuoreseent techniques. Brit. vet. J. 131, 427--437 (1975). 24a Arch.Virol.62[4
346
H. LAUDE and J. GELFI: I n vitro Properties of Border Disease Agent
8. LAUDE, H. : Virus de la peste porcine classique : interf@rence avec le VSV et titrage par le pi'oc@d6 des plages inverses. Arch. Virol. 56, 273 277 (1978). 9. LAUDE, H. : A direct plaque assay for hog cholera virus. J. gen. Virol. 40, 225--228 (1978). 10. LAUDE, i . : N o n a r b o - t o g a v i r i d a e : coinparative h y d r o d y n a m i c properties of the pestivirus genus. Arch. Virol. 62, 347--352 (1979) 11. OSBURN, B. I., CLARKE, G. L., STEV~TART,W. C., SAWYER,M. : Border disease--like s y n d r o m e in l a m b s : antibodies to hog cholera and bovine viral diarrhea viruses. J. amer. vet. ined. Ass. 163, 1165--1167 (1973). 12. PO~TERFIELD, J. S., CASALS,J., CHU1V~AKOV,M. P., GAIDA1VIOVIOI-I,S. Y., HANNOUN, C., HOLMES, I. H., HORZINEK,M. C., MUSSGA¥, M., OKER-BLOlVl,N., RUSSEL, P. K., TRENT, D . W . : Togaviridae. I n t e r v i r o l o g y 9, 129--148 (1978). 13. S~OWDON, W. A., PARSONSON, I. M., BROU~, M. L. : The reaction of p r e g n a n t ewes to inoculation w i t h Inucosal disease virus of bovine origin. J. coinp. Pathol. 85, 241--251 (1975). 14. VANTSIS,J. T., BARLOW, R. M., FRASER, J., RENNIE, J. C., MOULD, D. L. : E x p e r i m e n t in Border disease V I I I . P r o p a g a t i o n and properties of a c y t o p a t h i e virus. J. coinp. Pathol. 86, 111--120 (1976). 15. WARD, G. M. : E x p e r i m e n t a l infection of p r e g n a n t sheep w i t h bovine viral diarrhoea mucosal disease virus. Cornel. Vet. 61, 179--191 (1971). 16. WRATHAL,A. E., BAILEY, J., DONE, J. T., SHAN, I. G., WINKLER, C. E., GIBBONS, D. F., I:)ATTEI~SON,D. S. P., SWEASEY, D. : Effects of experimental Border disease infection in the p r e g n a n t sow. Zbh Vet. Med. B 25, 62--69 (1978). A u t h o r s ' address: Dr. H. LAUDE, Station de Virologie, L a b o r a t o i r e de Pathologie porcine, 78850 Thiverval-Grignon, France. R e c e i v e d April 9, 1979
Archives of Virology 62, 341--346 (1979)
© by Springer-Verlag 1979
Properties of Border Disease Virus as Studied in a Sheep Cell Line By H. LAUDE and J. GELFI Station de Recherches de Virologic et d'Immunologie, I.N.R.A., Thiverval-Grignon, France With 1 Figure Accepted April 9, 1979
Summary A fetal lamb muscle cell line has been isolated in which two strains of Border disease virus replicate well and induce a clear-cut cytopathic effect, thus providing a sensitive and practical assay system for both the virus and neutralizing antibodies. This system enabled us to study some characteristics of Border disease virus in comparison with two other agents responsible for hog cholera and bovine viral diarrhea. Our results indicate t h a t the last virus and the Border disease agent are indistinguishable in vitro.
Introduction Border disease (BD) is a congenital disease of sheep, responsible for fetal and neonatal death. Affected newborn lambs usually exibit an abnormally hairy birth coat, tremor associated with defective myelination of the central nervous system, and low viability (see 6 and 14). Since the antibodies induced in infected animals have been shown to cross-react with bovine viral diarrhea (BVD) and hog cholera (HC) viruses (7, 11, 16), the virus has been included in the pestivirus genus within the Togaviridae family (12). However more details of its antigenieity and physical properties must be known before it is placed in a definite taxonomic group. So far only two succesfnl attempts to isolate BD-virus in tissue culture have been reported. The BD-strain isolated b y VANTSlS st al. (14) is able to induce a significant cytopathic effect (CPE) in lamb ceils, and the CPE-producing agent was neutralized by an antiserum to BVD-virus. On the other hand, HA~K~ESS et al. (6) reported that repeated attempts to demonstrate a CPE associated with the BD-agent have met with no success in any of the tissue culture systems that have been tested. The virus could be detected by immunofluorescence and was shown to be neutrahzed b y an antiserum to HC-virus. I n this paper we describe the use of a new sheep cell line in which both the BD-virus strains mentioned above induce a clear-cut CPE, thus providing a con-
0304-8608/79/0062/0341/$ 01.20
342
H. LAUDE and J. GELH:
v e n i e n t s y s t e m for a n in vitro a s s a y of t h i s a g e n t . W e also r e p o r t s o m e f i n d i n g s r e s u l t i n g f r o m t h e c o m p a r i s o n of B D - v i r u s w i t h o t h e r p e s t i v i r u s m e m b e r s .
Materials and Methods Viruses The two B D - v i r u s strains used were recovered from l a m b ' s infected brain a n d reproduced t h e disease w h e n inoculated into sheep (6, 14). The strain n a m e d as W e had received six passages in calf testis p r i m a r y tissue culture (6). The strain designated E d had undergone 25 passages in fetal lamb kidney secondary tissue culture (14).
Celt Cultures Three strains of fetal l a m b muscle cells were used, which h a d been s u b c u l t i v a t e d 15 times or less, in E L Y - m e d i u m s u p p l e m e n t e d w i t h 10 per r e n t sheep serum and antibiotics. A continuous cell line (FLM), recovered from one of these cultures, was e m p l o y e d between 30 and 70 subpassages. F e t M calf kidney secondary cultures were p r o p a g a t e d in Eagle's m i n i m u m essential m e d i u m (MEM) w i t h a 10 per cent. calf serum supplement. All infected cells were m a i n t a i n e d in M E M containing 5 - - 1 0 per r e n t fetal calf sermn which had been tested for the absence of anti-pestivirus antibodies.
Plaque Assay The procedure was sirnilar to the test published previously for HC-virus (9). Briefly, 24 hours old m o n o l a y e r F L M . c e l l cultures were obtained by inoculating 8 × 10 a cells per 30 m m container (Coster 6-wells trays), and were infected w i t h an a p p r o p r i a t e BI)-virus dilution. T h e n 3 ml M E M incorporated w i t h 4 per cent fetal CS and containing 0.6 per cent agar were added. After incubation at 38 ° C in a COs incubator for designated periods, cultures were e x a m i n e d against an indirect light source or after neutral red staining.
Cross-Neutralization Test The origins of the antisera and viruses listed in Table 1 h a v e been described elsewhere (2). Antisera to BD.viruses were prepared in pigs (see below). I n t h e plaque reduction n e u t r a l i z a t i o n test used, tenfold virus dilutions were m i x e d w i t h a c o n s t a n t dose of antiserum. After 1 h o u r at 37 ° C, 0.2 m l samples of this suspension were a d d e d to t h e F L M - m o n o l a y e r s for B D and BVD-viruses, and to PK1a-monolayers for HCvirus. The inoeula were r e m o v e d after 1 hour.
Animal Experiments Pigs (50 kg) which h a d been previously checked for t h e absence of antibodies against pestiviruses were given 1 m l i n t r a m u s c u l a r injection of 1 × 106 P F U of BDvirus; booster injections were given 3 weeks later. Three weeks after the 2nd injection the four pigs (two for each strain) and two u n i m m u n i z e d pigs were challenged using a fully pathogenic strain of IIC-virus.
Results
Propagation o~ BD-Virus in FLM-Celt8 T h e E d - s t r a i n i n d u c e d a s i g n i f i c a n t C P E in F L M - c e l l s f r o m t h e 2 n d passage, l e a d i n g to e x t e n s i v e cell d e a t h a f t e r 4 - - 5 d a y s of i n c u b a t i o n . I n c o n t r a s t , no C P E was o b s e r v e d on s e c o n d a r y calf k i d n e y cells a f t e r 3 b l i n d passages. T h e W e - s t r a i n d i d not, p r o d u c e n o t i c e a b l e C P E b e f o r e a 6 serial p a s s a g e on F L M - c e l l s . W e obs e r v e d t h a t t h e C P E p r o d u c e d b y t h e W e - s t r a i n was a l w a y s s l o w e r t o a p p e a r t h a n w i t h t h e E d - s t r a i n . F u r t h e r m o r e , it was n o t so clear in c e r t a i n F L M - c e l l s t r a i n s as in t h e e s t a b l i s h e d F L M - c e l l s . S u b s e q u e n t l y , since t h e line g r e w r e a d i l y
In vitro Properties of Border Disease Agent
343
and was easy to handle, it was retained for assay and propagation of BD-viruses. The replication of BVD-virus in the FLM-line was studied in the same way. All the nine BVD-strains assayed (10) readily multiphed in FLM cells without any preliminary adaptation, inducing rapid destruction of the cell sheet.
Virus Plaque Titration Using FLM-line monotayers under agar overlay, it was possible to obtain countable plaques 2 to 3 days p.i. by both Ed- and We-strain of BD-virus (Fig. 1) and to use these for quantitative titration of BD-virus. Both BD-virus strains reached total infectivity titres of 5 × 106 plaques forming units (PFU) per ml if infected cultures were frozen when the CPE started to develop at about 48 hours p.i.
Fig. 1. Appearance of plaques formed by BD- and BVD-viruses on the FLM-eell line at 72 hours p.i. a BD-strain Weybridge. b BD-strain Edimburgh. c BVD-strain NADL
Speci/icity o/the Cytopathic E/]ect Several results indicate t h a t the detected CPE is specific for BD-virus. 1) CPE never appeared in mock-infected cultures passed serially like BD-infected cultures. 2) Ultracentrifugation showed that the two cytopathic agents possess hydrodynamical characteristics of the pestiviruses namely Sw 2° value of 138 and buoyant density in sucrose of 1.12 mg/ml (10). No CPE was detected from the gradient fractions apart from the peak. 3) BD-infeeted cell monolayers were treated 24 hours p.i. with anti-HC-virus conjugate and showed a discrete cytoplasmic fluorescence, which was not observed in mock-infected cell sheets. 4) The cytopathie agents present either in We- or Ed-strain are both antigenieally related to HC- and BVD-vimses.
Studies on Antigenicity From the results presented in Table l, it appears t h a t the external antigens of BD-virus are more closely related to those of BVD-virus than to those of HCvirus. The 331-strain, a variant responsible for chronic forn~ls of tIC, was included in this study because it is already known t h a t it is antigenically closer to BVD-
344
H. LAUDE and J. GELFI:
Table i. Cross-neutralization tests on B D - , B VD- and HC-viruses and their respective antisera a
Antiserum
BD-strains Ed We
BVDV NADL
331
Ed We NADL 331 ~fort
4.1 1.8 1.7 1.4 1.7
1.7 1.4 3.7 N.D. 0.6
1.2 1 2 4 5.4
2.2 1.9 2.2 1.8 1.45
~[CV-strains Alfort~o 0.25 0.5 0.45 N.D. 7.9
a Logi0 plaque reduction neutralization-indices using immune sera diluted 1 : 20
virus than are fully virulent HC-virus strains (2). I t can be noticed that the 331 strains also cross-react with BD-virus more intensively than the Alfort-strain, Animal Experiment8
BD-virus inoculation into pigs did not produce any clinical disease. Immunological responses of varying degree were observed. Homologous neutralization indices of 4.1. and 3.5 were found in sera from pigs immunized with Ed-strain material, whereas indices of 1.8 and 1.1 were encountered in sera from We-strain material immunized pigs. After HCV-challenge the former pigs recovered after showing mild symptoms, whereas the latter succumbed at 11 days p.i. with symptoms commonly associated with hog cholera, as in the control group. These results indicate that BD-immunized pigs could be cross-protected against HCvirus, as it has also been reported for pigs immunized with BVD-virus.
Discussion Fetal lamb muscle cell-cultures appear to be a very useful system for assaying BD virus. On the one hand it was previously reported that Ed-strain CPE induced on secondary fetal lamb kidney cells did not appear before 5 days p.i. (14). On the other hand the We-strain was considered not to be cytopathic and so, a conjugated anti-HCV was employed for its detection (6). The present work shows that a FLM cell line is highly susceptible to BD-virus and that the replication of both strains leads to a significant CPE within 2--8 days p.i. In addition the system provides a convenient plaque assay for measuring virus infectivity, and satisfactory titres are obtained. Probably FLI~I-cells would also constitute a convenient tool for the recovery of other BD-viruscs from field specimens. The FLM cell-line might also provide an appropriate system for the comparison of BD, BVD and l:IC-viruses since the three types of virus replicate well in these cells (these results and 8). Both BD- and BVD-viruses woduced clear-cut CPE in infected FLM-cells, whereas HC-virus did not. In the study of antigenic properties, it appears that BD-virus exhibits a closer relationship with BVD-virus than with tiC-virus. I t must be pointed out, however, that the residual infectivity of the neutralized HC-virus samples was titrated on porcine cells, whereas the neutralization tests for both BVD- and BD-virus
I n vitro Properties of Border Disease Agent
345
were p e r f o r m e d in FLM-cells. I n t h e same way, in all previous studies on antigenic relationships b e t w e e n HC- a n d B V D - v i r u s e s t h e n e u t r a l i z a t i o n indices were perf o r m e d on a s e p a r a t e cell s y s t e m for each virus. N o w t h e a m o u n t of n e u t r a l i z a t i o n o b s e r v e d in v i r u s - a n t i b o d y m i x t u r e s is c o n s i d e r a b l y influenced b y t h e h o s t cell s y s t e m in which r e s i d u a l virus i n f e c t i v i t y is a s s a y e d (see 3). T h u s i t w o u l d be b e t t e r to h a v e a c o m m o n cell s y s t e m in which t o s t u d y t h e t h r e e agents. Unf o r t u n a t e l y we could n o t do i t because of t h e low efficacy of p l a t i n g of I I C - v i r u s on our F L M - l i n e (8). T h e results of cross-protection in pigs, suggest t h a t t h e r e are i m m u n o l o g i c a l differences b e t w e e n BD- a n d HO-viruses a n d t h u s agree with t h e n e u t r a l i z a t i o n findings. W h e n c o m p a r i n g B D a n d BVD-viruses, it is i m p o r t a n t to notice t h a t t h e serological differences f o u n d are no m o r e p r o n o u n c e d t h a t those a l r e a d y r e p o r t e d b e t w e e n different strains of B V D - v i r u s (2). So it a p p e a r s t h a t B D a n d BVDviruses c a n n o t be clearly d i f f e r e n t i a t e d in vitro. I n vivo it is k n o w n t h a t m e m b e r s of t h e pestivirus genus do n o t possess a b s o l u t e host specificity (4) a n d further, t h a t t h e y all e x h i b i t t e r a t o g e n i c a c t i o n (16). Significantly enough, i t has been r e p o r t e d t h a t some B V I ) - s t r a i n s are able t o induce in sheep species p a t h o l o g i c a l f e a t u r e s ressembling t h a t oeuring a f t e r a B D - i n f e c t i o n (1, 13, 15). Conversely, B D - v i r u s was f o u n d t o be as p a t h o g e n i c for t h e calf fetus as B V D - v i r u s (5). I n s u m we conclude t h a t , in light of t h e evidence available, B D - a n d B V D - s h o u l d n o t be considered to be t w o s e p a r a t e v i r a l entities.
Aeknowledgments It is a pleasure to record our thanks to Dr. J. T. Vantsis (Moredun Research Institute, Edinburgh) and J. W. I-larkness (Central Veterinary Laboratory, Weybridge) for giving us their BD-strain. We are also grateful to Dr. E. Plateau (Laboratoire Central V6t~rinaire, Maisons-Alfort) with help on how virulent hog cholera challenge could be performed, and to Dr. M. Launais (Laboratoires Cogla, Libourne) for providing primary lamb cells.
References 1. ACLA2gD, H. 1Vi., GARD, G. P., PLANW, J. W. : Infection of sheep with a mucosal disease virus. Austr. Vet. J. 48, 70 (1972). 2. CORTEIEI~, G., AYNAUD, J. M., GALICI-IEll., C., GELFI, J . : Activit6 antig6nique eomparde de deux togavirus: le virus de Ia peste porcine et le virus de la maladie des muqueuses. Ann. Reeh. Vet. 5, 373--393 (1974). 3. DET.LA-PO~TA, A. J., ~TESTAW~kY,E. G. : A multi-hit model for the neutralization of animal viruses. J. gen. Virol. 38, 1 - - i 9 (1977). 4. FaENCR, E. L., SzvowDoN, W. A.: The infection of pigs with bovine mucosal disease virus. European eomlnunities seminar on hog cholera and african swine fever, 249--261. Hannover: 1976. 5. GIBBONS, D. F., WINKLER, C. E., SHAN, I. G., TERLECKI, S., I:LIOttARDSON, C., DONE, J. : Pathogenicity of the Border disease agent for the bovine fetus. Brit. vet. J. 130, 357 (1974). 6. HA~K~ESS, J. W., KISrG, A. A., TEaLECKI, S., SANDS, J. J. : Border disease of sheep : Isolation of the virus in tissue culture and experimental reproduction of the disease. Vet. Rec. 100, 71---72 (1977). 7. HUCK, I~. A., EvA/qs, D. H., WooDs, D. G., KING, A. A., ST[tAR% P., SEAN, I. G.: Border disease of sheep. Comparison of the results of serological testing using complement fixation, immunodiffusion, neutralization and immunoftuoreseent techniques. Brit. vet. J. 131, 427--437 (1975). 24a Arch.Virol.62[4
346
H. LAUDE and J. GELFI: I n vitro Properties of Border Disease Agent
8. LAUDE, H. : Virus de la peste porcine classique : interf@rence avec le VSV et titrage par le pi'oc@d6 des plages inverses. Arch. Virol. 56, 273 277 (1978). 9. LAUDE, H. : A direct plaque assay for hog cholera virus. J. gen. Virol. 40, 225--228 (1978). 10. LAUDE, i . : N o n a r b o - t o g a v i r i d a e : coinparative h y d r o d y n a m i c properties of the pestivirus genus. Arch. Virol. 62, 347--352 (1979) 11. OSBURN, B. I., CLARKE, G. L., STEV~TART,W. C., SAWYER,M. : Border disease--like s y n d r o m e in l a m b s : antibodies to hog cholera and bovine viral diarrhea viruses. J. amer. vet. ined. Ass. 163, 1165--1167 (1973). 12. PO~TERFIELD, J. S., CASALS,J., CHU1V~AKOV,M. P., GAIDA1VIOVIOI-I,S. Y., HANNOUN, C., HOLMES, I. H., HORZINEK,M. C., MUSSGA¥, M., OKER-BLOlVl,N., RUSSEL, P. K., TRENT, D . W . : Togaviridae. I n t e r v i r o l o g y 9, 129--148 (1978). 13. S~OWDON, W. A., PARSONSON, I. M., BROU~, M. L. : The reaction of p r e g n a n t ewes to inoculation w i t h Inucosal disease virus of bovine origin. J. coinp. Pathol. 85, 241--251 (1975). 14. VANTSIS,J. T., BARLOW, R. M., FRASER, J., RENNIE, J. C., MOULD, D. L. : E x p e r i m e n t in Border disease V I I I . P r o p a g a t i o n and properties of a c y t o p a t h i e virus. J. coinp. Pathol. 86, 111--120 (1976). 15. WARD, G. M. : E x p e r i m e n t a l infection of p r e g n a n t sheep w i t h bovine viral diarrhoea mucosal disease virus. Cornel. Vet. 61, 179--191 (1971). 16. WRATHAL,A. E., BAILEY, J., DONE, J. T., SHAN, I. G., WINKLER, C. E., GIBBONS, D. F., I:)ATTEI~SON,D. S. P., SWEASEY, D. : Effects of experimental Border disease infection in the p r e g n a n t sow. Zbh Vet. Med. B 25, 62--69 (1978). A u t h o r s ' address: Dr. H. LAUDE, Station de Virologie, L a b o r a t o i r e de Pathologie porcine, 78850 Thiverval-Grignon, France. R e c e i v e d April 9, 1979
