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Interference activity of bovine herpes virus 1 strains against Aujeszky's disease virus in cell cultures
~
Pergamon
Comp. lmmun. Microbiol. in/ect. Dis. Vol. 20, No. 2, pp. I71 175, 1997
PII:S 0 1 4 7 - 9 5 7 1 ( 9 6 ) 0 0 0 2 3 - 9
Published by Elsevier Science Ltd Printed in Great Britain o147-9571,/97 $17.00 + o.o0
I N T E R F E R E N C E A C T I V I T Y OF B O V I N E H E R P E S VIRUS 1 STRAINS AGAINST AUJESZKY'S DISEASE V I R U S IN CELL C U L T U R E S R. P E S H E V , *
R. B O S T A N D J I E V A
a n d H. H A R A L A M B 1 E V
Central Veterinary Research Institute, Sofia-1606, P. Slaveikov, 15A Blvd, Bulgaria
(Recei~,ed.for publication 16 July 1996) A b s t r a c t - - T h e interference-inducing activity of different low- and high-virulence strains of bovine herpes virus 1 (BHV-1) against Aujeszky's disease virus was studied by a parallel titration on permissive and non-permissive cell culture. The low-virulence BHV-1 strains possessed better interference-inducing activity than the high-virulence strains. An interference blocking test (IBT) was developed for the detection of BHV-I antibodies in bovine sera. Comparative results of IBT and the virus neutralisation reaction did not show statistically reliable differences. Published by Elsevier Science Ltd
Key words: Interference-inducing activity, bovine herpes virus 1, interferon, interference blocking test R ~ s u m ~ L ' a c t i v i t 6 interf~rente par induction des diffdrents Herpesvirus 1 des boeufs (HVB-I) de basse et haute virulence a 6t6 6tudide par titrage parall61e sur des cultures cellulaires permissives et non permissives. Les HVB de basse virulence avaient une activit6 inductrice interf6rente meilleure que les HVB I de haute virulence. Un test de blocage de l'interf6rence (TBI) a 6t~ d6velopp6 pour la d&ection d'anticorps HVB-I dans le serum bovin. La comparaison entre TBI et le test de neutralisation du virus ne d6montre pas de diffdrence statistique. Published by Elsevier Science Ltd
Mots-clqLs': Activit6 interfdrente par induction, herpervirus 1 des boeufs, interfdron, test de blocage de l'interf6rence
INTRODUCTION Many authors [1 3] established interferon-mediated early resistance in cattle after treatment with attenuated bovine herpes virus 1 (BHV-I) strains. Interferon in the serum, nasal and vaginal secretion of cattle inoculated with BHV-1 has been detected [5 9]. The antiviral effect of bovine interferon on the replication of bovine respiratory viruses has been studied [10]. Treatment with bovine interferon reduced viral yield for each of the viruses in comparison with that of control cultures. The results of several studies suggest that bovine interferon can protect against, or at least reduce, the clinical severity of infection with respiratory viruses [4, 5, 11]. The strong resistance against superinfection with cytopathic strains of Aujeszky's disease virus (ADV) was observed after inoculation of BHV-1 in non-permissive cultures of chicken embryonic fibroblasts (CEF)[12]. In our study, a quantitative comparison of interference-inducing activity between different strains of BHV-I and ADV was studied. *Author for correspondence. 171
172
R. Peshev et al.
For detection of BHV-I antibodies in cattle sera, interference blocking tests (IBT) in non-permissive cell cultures were developed. MATERIALS AND METHODS Viruses BHV-1 strain 'Ozirovo' was isolated from calf with respiratory disorders without nasal discharges. Strain ~Tchervena voda' was isolated from pregnant heifer with infectious rhinotracheitis symptoms. The BHV-1 strains 'Podgumer' and 'Vrana' were recently isolated from calves with respiratory disorders, high temperature, conjunctivitis and red nose. Primary bovine cell culture and permanent cell line calf trachea (CTr) were used for BHV-1 cultivation. The strains were freeze-dried with infectious titres between 105~ and 106~ TCIDs0/ml. Freeze-dried A D V strain 'Bartha' with an infectious titre of 104.5 TCID~0/ml was used as a challenge-indicator virus. The highest dilution of strains causing interference in cell cultures was marked as the interference titre. Cell cultures Primary culture of C E F was cultivated in Hank's balanced salt solution with 0.5% lactoalbumin hydrolysate and 7% horse serum. The primary bovine cell and permanent cell line CTr were cultivated in Eagle's minimal essential medium. The media was supplemented with 10% foetal calf serum, 0.075% sodium bicarbonate, 30 mM Hepes buffer, 0.2 M L-glutamin and antibiotics, penicillin 100 1U/ml and streptomycin 100 #g/ml. Determination of inte~J~rence In each well of a sterile tissue-culture plate were added 100/~1 cell suspension containing 5 x 104 cell per ml. Plates were incubated in a humidified CO2 incubator at 3 7 C until confluent cell monolayers were formed. The medium was removed 24 h later by inverting the plates on to sterile gauze pads. BHV-I strains or their dilution were added in four wells per dilution in 50 #1 amounts. Challenge A D V 'Bartha' strains were added 48 h later. Titration of investigated BHV-1 strains, challenge virus and back titration of challenge virus were performed. Virus positive controls and negative cell culture controls were also included. The numbers of wells showing cytopathic effect and those without cytopathic effect were determined. BHV-I and A D V titres were calculated by a recognised method [ 13]. Per[ormance o[ I B T test Thirty-three bovine sera originating from infected farms were parallel tested by IBT and the virus neutralisation reaction (VNR) test. The IBT was performed in plastic microplates as follows. Two-fold dilutions of tested serum were neutralised with a constant amount of BHV-I (104 TCIDs0/ml). A C E F suspension containing 2 x 104 cells per ml was added. Twenty-four hours later, after incubation at 37'C, the cells were superinfected with a constant amount 103 TC1Ds0/ml of A D V strain 'Bartha'. The cytopathic effects were observed after 24-48h incubation at 37:'C. The interference blocking titre was measured as the highest dilution of the tested sera with BHV 1 and A D V in which the cytopathic effects caused by ADV were observed. The V N R test was performed as described previously [14]. Linear regression analyses and t-tests were used to compare the sensitivity of the two reactions.
Interference activity of bovine herpes
173
RESULTS A quantitative comparison between interference-inducing activity of BHV- 1 strains was performed by parallel titration of every strain in a permissive cell line CTr and non-permissive cultures of CEF. After superinfection with 103 TCIDs0 of 'Bartha' strain, the interference activity of every strain was obtained by determining the difference between the infectious titre in CTr and the interference titre in CEF. Comparative results between the strains are presented in Fig. 1. Evidently the strains 'Ozirovo' and 'Tchervena voda' possess better interference-inducing activity. Their minimal interference dose is between 102 and 102.5TC1Ds0/ml. The strains 'Podgumer' and 'Vrana' have a significantly higher interference dose between 1036 and 1046 TCIDs0/ml (Fig. 1). An IBT for the detection of BHV-1 antibodies in cattle sera was developed using the established interference activity of BHV-1 in CEF cells. Comparable results were obtained with IBT and the micro-VNR test when parallel testing of 33 cattle sera originating from infected farms was performed. A total of 82% positive samples was obtained for both tests. No significant differences between IBT and the VNR test were found using linear regression analyses (Fig. 2) and the t-test (Fig. 3). DISCUSSION The decrease in ADV titre provides evidence that interference exists between bovine herpes viruses and the Aujeszki's disease virus. Bovine herpes virus strains with different activity were used by us for quantitative evidence of this interference. Different BHV-1 strains have different interferon-inducing activities. After a great number of passages in cell cultures, viral strains 'Ozirovo' and 'Tcervena voda' are partially attenuated and their interferon-inducing activity is lower than 'Podgumer' and 'Vrana' viral strains. These results show that the high passaged BHV-1 strains have higher interference activity than the low passaged strains. This information can be used to explain the higher ADV titre and to give quantitative assessment of interference.
Log~0 7/ 6/
QInfectioustiter
5./ 4./
[] Interference titer i5 A
!
/
1
3- /
2. / 1-/ 01, ,
Ozirovo Tchervena Podgumer Vrana voda Fig. 1. Interference-inducing activity of strains of BHV-1 in cell culture CEF against Aujesky's disease virus.
174
R. Peshev et al.
IBT vs. VNR Y = 0.15639 + 0.95962"X Correlation: r = 0.88562 i
5 rr z >
.............
.................
.
: s_:i
. ,.
..,.,o
' . 7 . . . . . ° ..........
,4 " _ . s .-."
3
.fr~"
s" " -
e" : J-,--" t ; - " ' 6 " iJ - : -1
" :: ........... . . . . . . . . . . . . . . . . . . . . . . . . .
w q.:'~: -1
,
.
i
1
.
.
3
.
.
................. .
5
"-'o,,,. Regression 95% confid.
.
7
9
IBT Fig. 2. Regression anlyses between IBT and V N R test.
Box & Whisker Plot
i-I
tl
_o
-T-
Min-Max 25%-75%
IBT
=
VNR
Mean
SEM
IB T
5.03
0.492
VNR
4.98
0.533
Median value
T
p
0.188
0.85
Fig. 3. Statistical comparison between IBT and V N R test by t-test.
Interference activity of bovine herpes
175
T h e i n t e r f e r e n c e t e s t u s e d in t h i s s t u d y g a v e a b e t t e r p o s s i b i l i t y o f t e s t i n g B H V - 1 v i r u l e n c e t h a n o t h e r k n o w n tests. T h e c h a n g e s o f A D V t i t r e o b t a i n e d w i t h t h e I B t e s t a l s o gave quantitative estimation of interference and confirmed these phenomena. This test can b e u s e d in t h e d i a g n o s i s o f B H V - 1 i n f e c t i o n in t h e l a b o r a t o r y w i t h o u t p e r m a n e n t cell cultures because regression analyses and t-test differences between IBT and VNR tests were not observed. T h e a i m o f o u r f u t u r e i n v e s t i g a t i o n s will b e t o c o m p a r e t h e i n t e r f e r e n c e r e s i s t a n c e o f attenuated and virulent strains of Aujeszky's disease virus. Acknowledgement--This work was supported by Grant CC 227 of the 'Science Foundation' of Ministry of Science and Education, R. Bulgaria.
REFERENCES 1. Ahl R. and Straub O. C. (1971) Die lokale interferonbildung im respirations und genitaltrakt experimenteller infection mit Rhinotracheitis (IBR)-und Blaschenausschlag (IPV) virus. Dtsch. Tieraerztl. Wochenschr. 78, 653 655. 2. Todd J.+ Volenec F. and Paton I. (1971) lntranasal vaccination against IBR: Studies on early onset of protection and use of the vaccine in pregnant cows. J. Am. Vet. Med. Ass. 159, 1370 1374. 3. Haralambiev H. Viral respiratory infections in calves. Doctor's Thesis, Sofia (1976). 4. Cummins J. M. and Rosenquist B. D+ (1980) Protection of calves against rhinovirus infection by nasal secretion interferon induced by infectious bovine rhinotracheitis virus. Am. J. Vet. Res. 41, 161-165. 5. Cummins J. M. and Rosenquist B. D. (1982) Partial protection of calves against parainfluenza-3 virus infection by nasal secretion interferon induced by infectious bovine rhinotracheitis virus Am. J. Vet. Res. 43, 1334~ 1338. 6. Rosenquist B. D. and Loan R. W. (1969) Interferon induction in the bovine species by infectious bovine rhinotracheitis virus. Am. J. Vet. Res. 30, 1293-1303. 7. Savan M., Angulo A. B. and Derbyshire J. B. (1979) Interferon, antibody responses and protection induced by an intranasal infectious bovine rhinotracheitis vaccine. Can. Vet. J. 20, 207 210. 8. Todd J. D., Volenec F. J. and Paton I. M. (1972) Interferon in nasal secretions and sera of calves after intranasal administration of avirulent infectious bovine rhinotracheitis virus: association of interferon in nasal secretion with early resistance to challenge with virulent virus, ln/i, ct. lmmunol. 5, 699 706. 9. Zygraich N., Lobmann M.+ Vascobinic E., Berge E. and Huygelen C. (1974) ht vi+~oand in t'itro properties of a temperature sensitive mutant of infecious bovine rhinotracheitis virus. Res. Vet. Sci. 16, 328 335. 10. Fulton R. W.+ Downing M. M. and Cummins J. M. (1984) Antiviral effects of bovine interferons on bovine respiratory tract viruses. J. Clin. Microbiol. 19, 4, 492497. 11. MacLachlan N. J. and Rosenquist B. D. (1982) Duration of protection of calves against rhinovirus challenge exposure by infectious bovine rhinotracheitis virus-induced interferon in nasal secretion. Am. J. ~2,t. Res. 43, 289-294. 12+ Haralambiev H., Simeonov K., Mintschev V., Todorova U. and Ivanova V. (1991) lnterferenz zwischen dem bovinen Herpes virus typ I (BHV 1) und dem Herpes virus suis typ I (SHV 1) in zell kulturen yon Huhuerembryo-Fibroblasten. Therarzt. Umschau 46, 18-24. 13. Reed L. J. and Muench H. (1938) A simple method of estimating fifty per cent end points. An+. J. ttvg. 27, 493-497. 14. Carbrey E. A. (1972) Recommended standart laboratory techniques for diagnosing infectious bovine rhinotracheitis, bovine virus diarrhea, and shipping fever. Proc. U. S. Anita. Health Assoc. 75, 629 648.
Pergamon
Comp. lmmun. Microbiol. in/ect. Dis. Vol. 20, No. 2, pp. I71 175, 1997
PII:S 0 1 4 7 - 9 5 7 1 ( 9 6 ) 0 0 0 2 3 - 9
Published by Elsevier Science Ltd Printed in Great Britain o147-9571,/97 $17.00 + o.o0
I N T E R F E R E N C E A C T I V I T Y OF B O V I N E H E R P E S VIRUS 1 STRAINS AGAINST AUJESZKY'S DISEASE V I R U S IN CELL C U L T U R E S R. P E S H E V , *
R. B O S T A N D J I E V A
a n d H. H A R A L A M B 1 E V
Central Veterinary Research Institute, Sofia-1606, P. Slaveikov, 15A Blvd, Bulgaria
(Recei~,ed.for publication 16 July 1996) A b s t r a c t - - T h e interference-inducing activity of different low- and high-virulence strains of bovine herpes virus 1 (BHV-1) against Aujeszky's disease virus was studied by a parallel titration on permissive and non-permissive cell culture. The low-virulence BHV-1 strains possessed better interference-inducing activity than the high-virulence strains. An interference blocking test (IBT) was developed for the detection of BHV-I antibodies in bovine sera. Comparative results of IBT and the virus neutralisation reaction did not show statistically reliable differences. Published by Elsevier Science Ltd
Key words: Interference-inducing activity, bovine herpes virus 1, interferon, interference blocking test R ~ s u m ~ L ' a c t i v i t 6 interf~rente par induction des diffdrents Herpesvirus 1 des boeufs (HVB-I) de basse et haute virulence a 6t6 6tudide par titrage parall61e sur des cultures cellulaires permissives et non permissives. Les HVB de basse virulence avaient une activit6 inductrice interf6rente meilleure que les HVB I de haute virulence. Un test de blocage de l'interf6rence (TBI) a 6t~ d6velopp6 pour la d&ection d'anticorps HVB-I dans le serum bovin. La comparaison entre TBI et le test de neutralisation du virus ne d6montre pas de diffdrence statistique. Published by Elsevier Science Ltd
Mots-clqLs': Activit6 interfdrente par induction, herpervirus 1 des boeufs, interfdron, test de blocage de l'interf6rence
INTRODUCTION Many authors [1 3] established interferon-mediated early resistance in cattle after treatment with attenuated bovine herpes virus 1 (BHV-I) strains. Interferon in the serum, nasal and vaginal secretion of cattle inoculated with BHV-1 has been detected [5 9]. The antiviral effect of bovine interferon on the replication of bovine respiratory viruses has been studied [10]. Treatment with bovine interferon reduced viral yield for each of the viruses in comparison with that of control cultures. The results of several studies suggest that bovine interferon can protect against, or at least reduce, the clinical severity of infection with respiratory viruses [4, 5, 11]. The strong resistance against superinfection with cytopathic strains of Aujeszky's disease virus (ADV) was observed after inoculation of BHV-1 in non-permissive cultures of chicken embryonic fibroblasts (CEF)[12]. In our study, a quantitative comparison of interference-inducing activity between different strains of BHV-I and ADV was studied. *Author for correspondence. 171
172
R. Peshev et al.
For detection of BHV-I antibodies in cattle sera, interference blocking tests (IBT) in non-permissive cell cultures were developed. MATERIALS AND METHODS Viruses BHV-1 strain 'Ozirovo' was isolated from calf with respiratory disorders without nasal discharges. Strain ~Tchervena voda' was isolated from pregnant heifer with infectious rhinotracheitis symptoms. The BHV-1 strains 'Podgumer' and 'Vrana' were recently isolated from calves with respiratory disorders, high temperature, conjunctivitis and red nose. Primary bovine cell culture and permanent cell line calf trachea (CTr) were used for BHV-1 cultivation. The strains were freeze-dried with infectious titres between 105~ and 106~ TCIDs0/ml. Freeze-dried A D V strain 'Bartha' with an infectious titre of 104.5 TCID~0/ml was used as a challenge-indicator virus. The highest dilution of strains causing interference in cell cultures was marked as the interference titre. Cell cultures Primary culture of C E F was cultivated in Hank's balanced salt solution with 0.5% lactoalbumin hydrolysate and 7% horse serum. The primary bovine cell and permanent cell line CTr were cultivated in Eagle's minimal essential medium. The media was supplemented with 10% foetal calf serum, 0.075% sodium bicarbonate, 30 mM Hepes buffer, 0.2 M L-glutamin and antibiotics, penicillin 100 1U/ml and streptomycin 100 #g/ml. Determination of inte~J~rence In each well of a sterile tissue-culture plate were added 100/~1 cell suspension containing 5 x 104 cell per ml. Plates were incubated in a humidified CO2 incubator at 3 7 C until confluent cell monolayers were formed. The medium was removed 24 h later by inverting the plates on to sterile gauze pads. BHV-I strains or their dilution were added in four wells per dilution in 50 #1 amounts. Challenge A D V 'Bartha' strains were added 48 h later. Titration of investigated BHV-1 strains, challenge virus and back titration of challenge virus were performed. Virus positive controls and negative cell culture controls were also included. The numbers of wells showing cytopathic effect and those without cytopathic effect were determined. BHV-I and A D V titres were calculated by a recognised method [ 13]. Per[ormance o[ I B T test Thirty-three bovine sera originating from infected farms were parallel tested by IBT and the virus neutralisation reaction (VNR) test. The IBT was performed in plastic microplates as follows. Two-fold dilutions of tested serum were neutralised with a constant amount of BHV-I (104 TCIDs0/ml). A C E F suspension containing 2 x 104 cells per ml was added. Twenty-four hours later, after incubation at 37'C, the cells were superinfected with a constant amount 103 TC1Ds0/ml of A D V strain 'Bartha'. The cytopathic effects were observed after 24-48h incubation at 37:'C. The interference blocking titre was measured as the highest dilution of the tested sera with BHV 1 and A D V in which the cytopathic effects caused by ADV were observed. The V N R test was performed as described previously [14]. Linear regression analyses and t-tests were used to compare the sensitivity of the two reactions.
Interference activity of bovine herpes
173
RESULTS A quantitative comparison between interference-inducing activity of BHV- 1 strains was performed by parallel titration of every strain in a permissive cell line CTr and non-permissive cultures of CEF. After superinfection with 103 TCIDs0 of 'Bartha' strain, the interference activity of every strain was obtained by determining the difference between the infectious titre in CTr and the interference titre in CEF. Comparative results between the strains are presented in Fig. 1. Evidently the strains 'Ozirovo' and 'Tchervena voda' possess better interference-inducing activity. Their minimal interference dose is between 102 and 102.5TC1Ds0/ml. The strains 'Podgumer' and 'Vrana' have a significantly higher interference dose between 1036 and 1046 TCIDs0/ml (Fig. 1). An IBT for the detection of BHV-1 antibodies in cattle sera was developed using the established interference activity of BHV-1 in CEF cells. Comparable results were obtained with IBT and the micro-VNR test when parallel testing of 33 cattle sera originating from infected farms was performed. A total of 82% positive samples was obtained for both tests. No significant differences between IBT and the VNR test were found using linear regression analyses (Fig. 2) and the t-test (Fig. 3). DISCUSSION The decrease in ADV titre provides evidence that interference exists between bovine herpes viruses and the Aujeszki's disease virus. Bovine herpes virus strains with different activity were used by us for quantitative evidence of this interference. Different BHV-1 strains have different interferon-inducing activities. After a great number of passages in cell cultures, viral strains 'Ozirovo' and 'Tcervena voda' are partially attenuated and their interferon-inducing activity is lower than 'Podgumer' and 'Vrana' viral strains. These results show that the high passaged BHV-1 strains have higher interference activity than the low passaged strains. This information can be used to explain the higher ADV titre and to give quantitative assessment of interference.
Log~0 7/ 6/
QInfectioustiter
5./ 4./
[] Interference titer i5 A
!
/
1
3- /
2. / 1-/ 01, ,
Ozirovo Tchervena Podgumer Vrana voda Fig. 1. Interference-inducing activity of strains of BHV-1 in cell culture CEF against Aujesky's disease virus.
174
R. Peshev et al.
IBT vs. VNR Y = 0.15639 + 0.95962"X Correlation: r = 0.88562 i
5 rr z >
.............
.................
.
: s_:i
. ,.
..,.,o
' . 7 . . . . . ° ..........
,4 " _ . s .-."
3
.fr~"
s" " -
e" : J-,--" t ; - " ' 6 " iJ - : -1
" :: ........... . . . . . . . . . . . . . . . . . . . . . . . . .
w q.:'~: -1
,
.
i
1
.
.
3
.
.
................. .
5
"-'o,,,. Regression 95% confid.
.
7
9
IBT Fig. 2. Regression anlyses between IBT and V N R test.
Box & Whisker Plot
i-I
tl
_o
-T-
Min-Max 25%-75%
IBT
=
VNR
Mean
SEM
IB T
5.03
0.492
VNR
4.98
0.533
Median value
T
p
0.188
0.85
Fig. 3. Statistical comparison between IBT and V N R test by t-test.
Interference activity of bovine herpes
175
T h e i n t e r f e r e n c e t e s t u s e d in t h i s s t u d y g a v e a b e t t e r p o s s i b i l i t y o f t e s t i n g B H V - 1 v i r u l e n c e t h a n o t h e r k n o w n tests. T h e c h a n g e s o f A D V t i t r e o b t a i n e d w i t h t h e I B t e s t a l s o gave quantitative estimation of interference and confirmed these phenomena. This test can b e u s e d in t h e d i a g n o s i s o f B H V - 1 i n f e c t i o n in t h e l a b o r a t o r y w i t h o u t p e r m a n e n t cell cultures because regression analyses and t-test differences between IBT and VNR tests were not observed. T h e a i m o f o u r f u t u r e i n v e s t i g a t i o n s will b e t o c o m p a r e t h e i n t e r f e r e n c e r e s i s t a n c e o f attenuated and virulent strains of Aujeszky's disease virus. Acknowledgement--This work was supported by Grant CC 227 of the 'Science Foundation' of Ministry of Science and Education, R. Bulgaria.
REFERENCES 1. Ahl R. and Straub O. C. (1971) Die lokale interferonbildung im respirations und genitaltrakt experimenteller infection mit Rhinotracheitis (IBR)-und Blaschenausschlag (IPV) virus. Dtsch. Tieraerztl. Wochenschr. 78, 653 655. 2. Todd J.+ Volenec F. and Paton I. (1971) lntranasal vaccination against IBR: Studies on early onset of protection and use of the vaccine in pregnant cows. J. Am. Vet. Med. Ass. 159, 1370 1374. 3. Haralambiev H. Viral respiratory infections in calves. Doctor's Thesis, Sofia (1976). 4. Cummins J. M. and Rosenquist B. D+ (1980) Protection of calves against rhinovirus infection by nasal secretion interferon induced by infectious bovine rhinotracheitis virus. Am. J. Vet. Res. 41, 161-165. 5. Cummins J. M. and Rosenquist B. D. (1982) Partial protection of calves against parainfluenza-3 virus infection by nasal secretion interferon induced by infectious bovine rhinotracheitis virus Am. J. Vet. Res. 43, 1334~ 1338. 6. Rosenquist B. D. and Loan R. W. (1969) Interferon induction in the bovine species by infectious bovine rhinotracheitis virus. Am. J. Vet. Res. 30, 1293-1303. 7. Savan M., Angulo A. B. and Derbyshire J. B. (1979) Interferon, antibody responses and protection induced by an intranasal infectious bovine rhinotracheitis vaccine. Can. Vet. J. 20, 207 210. 8. Todd J. D., Volenec F. J. and Paton I. M. (1972) Interferon in nasal secretions and sera of calves after intranasal administration of avirulent infectious bovine rhinotracheitis virus: association of interferon in nasal secretion with early resistance to challenge with virulent virus, ln/i, ct. lmmunol. 5, 699 706. 9. Zygraich N., Lobmann M.+ Vascobinic E., Berge E. and Huygelen C. (1974) ht vi+~oand in t'itro properties of a temperature sensitive mutant of infecious bovine rhinotracheitis virus. Res. Vet. Sci. 16, 328 335. 10. Fulton R. W.+ Downing M. M. and Cummins J. M. (1984) Antiviral effects of bovine interferons on bovine respiratory tract viruses. J. Clin. Microbiol. 19, 4, 492497. 11. MacLachlan N. J. and Rosenquist B. D. (1982) Duration of protection of calves against rhinovirus challenge exposure by infectious bovine rhinotracheitis virus-induced interferon in nasal secretion. Am. J. ~2,t. Res. 43, 289-294. 12+ Haralambiev H., Simeonov K., Mintschev V., Todorova U. and Ivanova V. (1991) lnterferenz zwischen dem bovinen Herpes virus typ I (BHV 1) und dem Herpes virus suis typ I (SHV 1) in zell kulturen yon Huhuerembryo-Fibroblasten. Therarzt. Umschau 46, 18-24. 13. Reed L. J. and Muench H. (1938) A simple method of estimating fifty per cent end points. An+. J. ttvg. 27, 493-497. 14. Carbrey E. A. (1972) Recommended standart laboratory techniques for diagnosing infectious bovine rhinotracheitis, bovine virus diarrhea, and shipping fever. Proc. U. S. Anita. Health Assoc. 75, 629 648.