Institute of Veterinary Virology, University of Berne, Laenggass-Str. 122, CH-3012 Berne, Switzerland1
Author for correspondence: Ernst Peterhans. Fax +41 31 631 2534. e-mail ernst.peterhans{at}ivv.unibe.ch
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bovine viral diarrhoea (BVD) virus is a pestivirus of the family Flaviviridae (Baker, 1987 ; Nettleton & Entrican, 1995
). This virus is of particular interest in studies of virus-induced apoptosis because of the existence of closely related pairs of cytopathic (cp) and non-cytopathic (ncp) biotypes (Weiss et al., 1994
; Paton, 1995
). The two biotypes of such a pair differ only in one non-structural protein, NS23, which is cleaved into NS2 and NS3 in cp, but not ncp, biotypes (Brownlie, 1990
; Meyers & Thiel, 1996
). Moreover, both biotypes of BVD virus are associated with mucosal disease, a lethal form of infection with BVD virus. It is observed in animals infected persistently with ncp BVD virus from the early period of their intrauterine development. The cp biotype of BVD virus detected in animals with mucosal disease may be the result of a mutation of the ncp biotype (hence the term virus pair), or may be due to superinfection. The cp biotype is believed to kill these immunotolerant animals because their immune systems fail to control virus multiplication (Brownlie, 1990
; Meyers & Thiel, 1996
). Cells infected with cp BVD virus have been shown to undergo apoptosis (Zhang et al., 1996
), which is associated with cleavage of poly(ADP-ribose) polymerase (Hoff & Donis, 1997
; Schweizer & Peterhans, 1999
) and can be prevented by selected antioxidants (Schweizer & Peterhans, 1999
). In addition to inducing apoptosis in its host cells, BVD virus may also influence the viability of uninfected cells, via factors released from infected cells. An example of such factors are interferons (IFNs), which have been shown to have anti- as well as pro-apoptotic properties (Rodriguez-Villanueva & McDonnell, 1995
; Yanase et al., 1998
). Specifically, IFNs have been shown to prime for apoptosis via increased synthesis of 2',5'-oligoadenylate (2-5A) and its activation of RNase L (Der et al., 1997
; Diaz-Guerra et al., 1997
; Castelli et al., 1998
) or via inhibition of protein synthesis by PKR (Jagus et al., 1999
). Apoptosis of uninfected NIH3T3 cells was observed when RNase L was overexpressed by means of an inducible promoter or activated by 2-5A or double-stranded RNA (Castelli et al., 1998
). In contrast, inhibition of the 2',5'-oligoadenylate synthetase/RNase L system prevented apoptosis in response to double-stranded RNA (Rivas et al., 1998
). Pre-treatment of mice with IFN-
was shown to enhance lipopolysaccharide (LPS)-triggered apoptosis markedly in thymocytes, whereas the simultaneous injection of anti-IFN-
antibody and LPS prevented apoptosis (Kato et al., 1997
). In vitro, human immunodeficiency virus type 1 (HIV-1)-infected macrophages (M
) enhanced dramatically the proportion of cells undergoing apoptosis of uninfected but not infected T cells (Herbein et al., 1998
). These authors postulate an indirect mechanism of apoptosis in uninfected T cells mediated by antigen-presenting cells. Furthermore, in porcine reproductive and respiratory syndrome virus infection, it is mainly uninfected bystander cells that undergo apoptosis, rather than infected cells. In vivo studies showed the apoptotic cells to consist predominantly of mononuclear cells (Sirinarumitr et al., 1998
).
Supernatants of M infected with cp BVD virus that prime uninfected M
for apoptosis were found to contain IFN (Adler et al., 1997
). Moreover, recombinant bovine (rbo) IFN-
I.1 primes for apoptosis (Adler et al., 1995
). Therefore, we investigated whether it is the IFNs that are responsible for priming by supernatants of cp BVD virus-infected M
. We show that priming was due to a factor(s) distinct from, or in addition to, IFNs. The presence of such a factor(s) in supernatants of M
infected with different strains of cp BVD virus and bovine herpesvirus-1 (BHV-1), a cp DNA virus, suggests that priming for activation-induced apoptosis may be of more general significance in virushost interactions.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cells.
M were obtained from the blood of Red Holstein cows as described previously (Jungi et al., 1997
). Briefly, peripheral bovine mononuclear cells were isolated by using FicollHypaque centrifugation and M
were permitted to differentiate by culturing the cells for 7 days under non-adherent conditions in Teflon bags at a cell concentration of 4x106 cells/ml in Iscoves modified Dulbeccos minimum essential medium (MEM) supplemented with HEPES (10 mM), nonessential amino acids for MEM (Life Technologies, 1%), vitamins for MEM (Life Technologies, 1%), streptomycin (100 µg/ml), penicillin (100 IU/ml), amphotericin B (2·5 µg/ml), L-glutamine (2 mM), 2-mercaptoethanol (50 µM) and 20% FCS. Madin Darby bovine kidney (MDBK) cells and Sf9 insect cells were obtained from the American type culture collection (Manassas, VA, USA). MDBK cells of passages 120 to 140 were used for IFN type I assay. BVD and BHV-1 viruses were grown and titrated on bovine embryonic turbinate cells. These cells were prepared from foetuses obtained from a local abattoir and were maintained in MEM supplemented with 2% FCS, L-glutamine (2 mM), penicillin (100 IU/ml) and streptomycin (100 µg/ml) at 37 °C in a humidified 5% CO2 atmosphere. All batches of cells used in our experiments were found to be free of BVD virus by immunofluorescence testing.
Viruses.
BHV-1 (Colorado strain) was originally obtained from V. Bitsch (Aarhus, Denmark). The recombinant baculovirus vector expressing B18R and the vector control were kindly provided by G. L. Smith (Sir William Dunn School of Pathology, Oxford, UK) and have been described elsewhere (Alcamí & Smith, 1992 ; Symons et al., 1995
). The BVD virus strains used included NADL, TGAC (Ridpath et al., 1991
) and SuwaCP (all cp) and TGAN (Ridpath et al., 1991
) and SuwaNCP (both ncp). The TGAC and TGAN virus strains were kindly provided by V. Moennig (Hannover, Germany), whereas SuwaCP and SuwaNCP are a virus pair isolated from peripheral blood of an animal suffering from mucosal disease. Separation and biological cloning of the two Suwa BVD virus biotypes was performed by the plaque formation method using a methyl cellulose overlay medium consisting of 1·5% methyl cellulose in MEM supplemented with penicillin (100 IU/ml) and 2% FCS (Nakamura et al., 1993
).
The genetic relationship of SuwaCP and SuwaNCP BVD viruses was determined by sequencing using standard techniques and an ABI PRISM 310 Genetic analyser (Perkin Elmer). The following primers were used for amplification of BVD virus cDNA: 5'-UTR, 5' GAGGCTAGCCATGCCCTTAG 3' (sense) and 5' TCAACTCCATGTGCCATGTAC 3' (antisense); E2, 5' GACAGGGACTGTGAGCTGTA 3' (sense) and 5' GGCCCCTCACTTGATATGAT 3' (antisense); and NS23, 5' GAAGTCTACGGCATGCCAAA 3' (sense) and 5' ACTGGGGCTCTGGGTGTGGT 3' (antisense). Analysis of the sequences revealed that, with the exception of four point mutations in the NS23 region, the nucleotide sequences of the two virus genomes were identical. One of these mutations was silent, whereas the other three led to changes in the amino acid sequence. Northern blotting of genomic viral RNA performed by hybridization with virus strain-specific DIG-labelled RNA probes revealed that the genomes of SuwaCP and SuwaNCP BVD viruses were both approximately 12·5 kb in size (results not shown).
Induction of apoptosis.
M seeded in 25 cm2 culture flasks at a density of 5x106 cells per flask were infected with the appropriate strain of BVD virus or BHV-1 in 1·5 ml culture medium without FCS at an m.o.i. of 1 for 1 h at 37 °C. After adsorption of the virus, the inoculum was removed by washing the cells in culture medium without FCS prior to the addition of complete medium with 2% FCS. Supernatants were harvested after 48 h incubation at 37 °C and virus was inactivated by incubation with
-propiolactone at a 4000-fold dilution for 18 h at 4 °C followed by 2 h at 37 °C (Barrett et al., 1984
; Perler et al., 1999
). Virus inactivation was verified by titration on bovine turbinate cells. M
seeded in 24 well plates at a density of 5x105 cells per well were incubated with the inactivated supernatant (400 µl per well) and fresh culture medium with 2% FCS (400 µl per well) for 48 h at 37 °C. The inoculum was replaced by fresh culture medium containing 1 µg/ml LPS. Apoptosis was measured after 48 h incubation.
IFN assay.
Procedures for measuring biological activity of type I IFN in supernatants of virus-infected bovine monocyte-derived M were described previously. The assay used is based on the reduction of Sendai virus growth by IFN as determined by immunocytochemistry (Perler et al., 1999
).
Apoptosis assay.
The fragmentation of cellular DNA was analysed quantitatively by FACS analysis according to Cossarizza et al. (1995) . Detached cells were collected by centrifugation and the pellet was lysed with 500 µl lysis buffer (0·1 M sodium citrate, pH 6·5, 1% Triton X-100 and 25 µg/ml propidium iodide), resulting in the release of nuclei by nonadherent cells. The lysate was then added to the plate to release the nuclei of the remaining adherent cells. Nuclei were analysed with a FACScan flow cytometer (Becton Dickinson) after 30 min incubation at 4 °C in the dark. A minimum of 104 nuclei per sample was analysed.
Virus titration.
Viruses were passaged and titrated in bovine turbinate cells as described previously (Adler et al., 1994 ) and the titres of the virus stocks and supernatants were calculated according to Reed & Muench (1938)
.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
Characteristics of the priming activity
A set of experiments was performed to characterize further the factor(s) contained in supernatants of cp BVD virus-infected M responsible for priming for activation-induced apoptosis. The molecular mass was estimated by ultrafiltration of supernatants. Whereas filtration with membranes of an exclusion size of 100 kDa had no effect on the priming activity, a filter with a nominal exclusion size of 50 kDa eliminated the activity (not shown). Since many proteins with molecular masses of about 30 kDa are retained by the 50 kDa cut-off filters (Amicon), the molecular mass is between approximately 30 and 100 kDa. The priming activity of the supernatants was stable when exposed to pH 2 at 4 °C for 24 h, resisted heating at 56 °C for 30 min but not boiling for 15 min (Adler et al., 1997
). Significant differences in the virus load between the two BVD virus biotypes may influence the release of the apoptosis-priming factor(s). Distribution of cell-free and cell-associated virus of cp and ncp BVD virus grown in M
may affect the infected cell differentially. Titration of either cell-free or cell-associated cp (SuwaCP) and ncp (SuwaNCP) BVD virus grown in M
revealed no significant differences between the two biotypes. These virus strains, which are a virus pair, grew in M
to titres of 104 to 105 TCID50/ml.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
IFN type I was initially considered to be responsible for priming for apoptosis because, in infected M, only BHV-1 and cp, but not ncp, BVD viruses induced IFN type I, which, in its recombinant form, was shown to prime uninfected M
for activation-induced apoptosis (Adler et al., 1995
, 1997
). However, the mechanism of induction of IFN type I and of the priming factor(s) may be similar, which may explain the correlation between the ability of the different virus strains used to induce the formation of IFN type I and of the factor(s) that primes for LPS-induced apoptosis (Figs 1
and 3
).
Only a few viruses, HIV (Finkel et al., 1995 ; Cottrez et al., 1997
; Chen et al., 1998
; Herbein et al., 1998
), murine cytomegalovirus (Koga et al., 1994
) and feline immunodeficiency virus (FIV) (Mizuno et al., 1997
), have been reported to date to prime uninfected cells for activation-induced apoptosis. The priming for activation-induced apoptosis seems to be due to different mechanisms. HIV has been shown to prime T cell apoptosis by a factor(s) released from monocytes (Chen et al., 1998
), but direct contact between infected and uninfected cells may also be involved (Cottrez et al., 1997
). The observations made in FIV infection point to soluble mediators released from infected cells. The factor involved has not been characterized in any of these studies. Several studies have attributed a role of soluble factors in priming for apoptosis to certain cytokines, among them IL-4 (Mangan et al., 1992
) and IFN-
(Munn et al., 1995
; Bingisser et al., 1996
). This raised the question as to whether a previously described cytokine mediates priming. IFN-
had to be considered, since in some of the blood-derived cultures both IFN-
mRNA and bioactivity were detected. However, this signal was tentatively attributed to lymphocytes rather than to M
, since it was not detected in bone marrow-derived cultures of M
infected in a similar manner (M. Schweizer, unpublished observation). This suggests that the M
-derived priming factor is distinct from IFN-
. Moreover, virus infection with both cp and ncp BVD viruses failed to induce detectable amounts of TNF-
, as determined by bioassay (Adler et al., 1996
). This argues against an important role of this cytokine in priming for activation-induced apoptosis.
It remains to be shown whether the M is the only cell type that produces a factor(s) capable of priming for apoptosis, and whether it is the only cell capable of being primed for activation-induced apoptosis. However, bovine turbinate cells produced neither IFN type I nor the priming factor in response to BVD virus or BHV-1 infection, nor could they be primed for LPS-induced apoptosis (results not shown). Recently, Lambot and co-workers showed that only the cp biotype of BVD virus induced apoptosis in bovine peripheral mononuclear cells. Directly relevant to this study is their observation that apoptosis in CD4+ and CD8+ T cells was enhanced significantly by the presence of monocytes (Lambot et al., 1998
). Together with the observations reported for BHV-1 (Hanon et al., 1996
; this work), HIV (Finkel et al., 1995
), FIV (Mizuno et al., 1997
) and murine cytomegalovirus (Koga et al., 1994
), this suggests that, during virus infection, mononuclear cells may play a role in regulating apoptosis in an array of other cell types. The concept that apoptosis of uninfected cells may require at least two signals, i.e. one for priming for and one for triggering apoptosis, opens up new perspectives regarding the study of the biochemistry involved.
It will be of particular interest to investigate whether apoptosis controlled by mononuclear phagocytes is related to lymphopoenia, a hallmark of most systemic virus infections. In addition, the increased susceptibility of cattle infected by BHV-1 to secondary bacterial infections, e.g. Pasteurella spp. (Bielefeldt Ohmann & Babiuk, 1985 ), may be related to activation-induced cell death. Moreover, priming may not be restricted to mediating sensitivity to activation-induced apoptosis, but may include other functional changes in uninfected cells. Priming by endogenously produced IFN type I and by exogenously added IFN-
down-regulated IFN-
plus TNF-
-induced nitric oxide production in mouse M
infected by tick-borne encephalitis virus and in uninfected M
, respectively (Kreil & Eibl, 1995
). The immunosuppression observed during measles is another example of such functional changes. It seems remarkable that an as yet unidentified factor(s) released from one B lymphocyte sufficed to impair antigen presentation by some 120 B lymphocytes (Fujinami et al., 1998
; Sun et al., 1998
). These findings suggest that priming may play an important role in the pathogenesis of virus infections.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adler, B., Adler, H., Jungi, T. W. & Peterhans, E. (1995). Interferon-alpha primes macrophages for lipopolysaccharide-induced apoptosis.Biochemical and Biophysical Research Communications 215, 921-927.[Medline]
Adler, H., Jungi, T. W., Pfister, H., Strasser, M., Sileghem, M. & Peterhans, E. (1996). Cytokine regulation by virus infection: bovine viral diarrhea virus, a flavivirus, downregulates production of tumor necrosis factor alpha in macrophages in vitro.Journal of Virology 70, 2650-2653.[Abstract]
Adler, B., Adler, H., Pfister, H., Jungi, T. W. & Peterhans, E. (1997). Macrophages infected with cytopathic bovine viral diarrhea virus release a factor(s) capable of priming uninfected macrophages for activation-induced apoptosis.Journal of Virology 71, 3255-3258.[Abstract]
Alcamí, A. & Smith, G. L. (1992). A soluble receptor for interleukin-1 encoded by vaccinia virus: a novel mechanism of virus modulation of the host response to infection.Cell 71, 153-167.[Medline]
Baker, J. C. (1987). Bovine viral diarrhea virus: a review.Journal of the American Veterinary Medical Association 190, 1449-1458.[Medline]
Barrett, A. D. T., Hunt, N. & Dimmock, N. J. (1984). A rapid method for the inactivation of virus infectivity prior to assay for interferons.Journal of Virological Methods 8, 349-351.[Medline]
Bielefeldt Ohmann, H. & Babiuk, L. A. (1985). Viralbacterial pneumonia in calves: effect of bovine herpesvirus-1 on immunologic functions.Journal of Infectious Diseases 151, 937-947.[Medline]
Bingisser, R., Stey, C., Weller, M., Groscurth, P., Russi, E. & Frei, K. (1996). Apoptosis in human alveolar macrophages is induced by endotoxin and is modulated by cytokines.American Journal of Respiratory Cell and Molecular Biology 15, 64-70.[Abstract]
Brownlie, J. (1990). Pathogenesis of mucosal disease and molecular aspects of bovine virus diarrhoea virus.Veterinary Microbiology 23, 371-382.[Medline]
Castelli, J., Wood, K. A. & Youle, R. J. (1998). The 2-5A system in viral infection and apoptosis.Biomedicine & Pharmacotherapy 52, 386-390.[Medline]
Chen, H., Yip, Y. K., George, I., Tyorkin, M., Salik, E. & Sperber, K. (1998). Chronically HIV-1-infected monocytic cells induce apoptosis in cocultured T cells.Journal of Immunology 161, 4257-4267.
Cossarizza, A., Franceschi, C., Monti, D., Salvioli, S., Bellesia, E., Rivabene, R., Biondo, L., Rainaldi, G., Tinari, A. & Malorni, W. (1995). Protective effect of N-acetylcysteine in tumor necrosis factor-alpha-induced apoptosis in U937 cells: the role of mitochondria.Experimental Cell Research 220, 232-240.[Medline]
Cottrez, F., Manca, F., Dalgleish, A. G., Arenzana-Seisdedos, F., Capron, A. & Groux, H. (1997). Priming of human CD4+ antigen-specific T cells to undergo apoptosis by HIV-infected monocytes. A two-step mechanism involving the gp120 molecule.Journal of Clinical Investigation 99, 257-266.
Der, S. D., Yang, Y. L., Weissmann, C. & Williams, B. R. (1997). A double-stranded RNA-activated protein kinase-dependent pathway mediating stress-induced apoptosis.Proceedings of the National Academy of Sciences, USA 94, 3279-3283.
Diaz-Guerra, M., Rivas, C. & Esteban, M. (1997). Activation of the IFN-inducible enzyme RNase L causes apoptosis of animal cells.Virology 236, 354-363.[Medline]
Evan, G. & Littlewood, T. (1998). A matter of life and cell death.Science 281, 1317-1322.
Finkel, T. H., Tudor-Williams, G., Banda, N. K., Cotton, M. F., Curiel, T., Monks, C., Baba, T. W., Ruprecht, R. M. & Kupfer, A. (1995). Apoptosis occurs predominantly in bystander cells and not in productively infected cells of HIV- and SIV-infected lymph nodes.Nature Medicine 1, 129-134.[Medline]
Fujinami, R. S., Sun, X., Howell, J. M., Jenkin, J. C. & Burns, J. B. (1998). Modulation of immune system function by measles virus infection: role of soluble factor and direct infection.Journal of Virology 72, 9421-9427.
Hanon, E., Vanderplasschen, A., Lyaku, S., Keil, G., Denis, M. & Pastoret, P.-P. (1996). Inactivated bovine herpesvirus 1 induces apoptotic cell death of mitogen-stimulated bovine peripheral blood mononuclear cells.Journal of Virology 70, 4116-4120.[Abstract]
Herbein, G., Van Lint, C., Lovett, J. L. & Verdin, E. (1998). Distinct mechanisms trigger apoptosis in human immunodeficiency virus type 1-infected and in uninfected bystander T lymphocytes.Journal of Virology 72, 660-670.
Hoff, H. S. & Donis, R. O. (1997). Induction of apoptosis and cleavage of poly(ADP-ribose) polymerase by cytopathic bovine viral diarrhea virus infection.Virus Research 49, 101-113.[Medline]
Jagus, R., Joshi, B. & Barber, G. N. (1999). PKR, apoptosis and cancer.International Journal of Biochemistry and Cell Biology 31, 123-138.[Medline]
Jungi, T. W., Pfister, H., Sager, H., Fatzer, R., Vandevelde, M. & Zurbriggen, A. (1997). Comparison of inducible nitric oxide synthase expression in the brains of Listeria monocytogenes-infected cattle, sheep, and goats and in macrophages stimulated in vitro.Infection and Immunity 65, 5279-5288.[Abstract]
Kato, Y., Morikawa, A., Sugiyama, T., Koide, N., Jiang, G. Z., Lwin, T., Yoshida, T. & Yokochi, T. (1997). Augmentation of lipopolysaccharide-induced thymocyte apoptosis by interferon-gamma.Cellular Immunology 177, 103-108.[Medline]
Knipe, D. M. (1996). Virushost cell interactions. In Fields Virology, pp. 273-299. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia: LippincottRaven.
Koga, Y., Tanaka, K., Lu, Y. Y., Oh-Tsu, M., Sasaki, M., Kimura, G. & Nomoto, K. (1994). Priming of immature thymocytes to CD3-mediated apoptosis by infection with murine cytomegalovirus.Journal of Virology 68, 4322-4328.[Abstract]
Kreil, T. R. & Eibl, M. M. (1995). Viral infection of macrophages profoundly alters requirements for induction of nitric oxide synthesis.Virology 212, 174-178.[Medline]
Lambot, M., Hanon, E., Lecomte, C., Hamers, C., Letesson, J.-J. & Pastoret, P.-P. (1998). Bovine viral diarrhoea virus induces apoptosis in blood mononuclear cells by a mechanism largely dependent on monocytes.Journal of General Virology 79, 1745-1749.[Abstract]
Mangan, D. F., Robertson, B. & Wahl, S. M. (1992). IL-4 enhances programmed cell death (apoptosis) in stimulated human monocytes.Journal of Immunology 148, 1812-1816.
Meyers, G. & Thiel, H.-J. (1996). Molecular characterization of pestiviruses.Advances in Virus Research 47, 53-118.[Medline]
Mizuno, T., Momoi, Y., Endo, Y., Nishimura, Y., Goto, Y., Ohno, K., Watari, T., Tsujimoto, H. & Hasegawa, A. (1997). Apoptosis enhanced by soluble factor produced in feline immunodeficiency virus infection.Journal of Veterinary Medical Science 59, 1049-1051.[Medline]
Munn, D. H., Beall, A. C., Song, D., Wrenn, R. W. & Throckmorton, D. C. (1995). Activation-induced apoptosis in human macrophages: developmental regulation of a novel cell death pathway by macrophage colony-stimulating factor and interferon gamma.Journal of Experimental Medicine 181, 127-136.[Abstract]
Nakamura, S., Fukusho, A., Inoue, Y., Sasaki, H. & Ogawa, N. (1993). Isolation of different non-cytopathogenic bovine viral diarrhoea (BVD) viruses from cytopathic BVD virus stocks using reverse plaque formation method.Veterinary Microbiology 38, 173-179.[Medline]
Nettleton, P. F. & Entrican, G. (1995). Ruminant pestiviruses.British Veterinary Journal 151, 615-642.[Medline]
OBrien, V. (1998). Viruses and apoptosis.Journal of General Virology 79, 1833-1845.
Ohmann, H. B., Lawman, M. J. & Babiuk, L. A. (1987). Bovine interferon: its biology and application in veterinary medicine.Antiviral Research 7, 187-210.[Medline]
Paton, D. J. (1995). Pestivirus diversity.Journal of Comparative Pathology 112, 215-236.[Medline]
Perler, L., Pfister, H., Schweizer, M., Peterhans, E. & Jungi, T. W. (1999). A bioassay for interferon type I based on inhibition of Sendai virus growth.Journal of Immunological Methods 222, 189-196.[Medline]
Reed, L. J. & Muench, H. (1938). A simple method for estimating fifty percent endpoints.American Journal of Hygiene 27, 493-497.
Ridpath, J. F., Lewis, T. L., Bolin, S. R. & Berry, E. S. (1991). Antigenic and genomic comparison between non-cytopathic and cytopathic bovine viral diarrhoea viruses isolated from cattle that had spontaneous mucosal disease.Journal of General Virology 72, 725-729.[Abstract]
Rivas, C., Gil, J., Melkova, Z., Esteban, M. & Diaz-Guerra, M. (1998). Vaccinia virus E3L protein is an inhibitor of the interferon (i.f.n.)-induced 2-5A synthetase enzyme.Virology 243, 406-414.[Medline]
Rodriguez-Villanueva, J. & McDonnell, T. J. (1995). Induction of apoptotic cell death in non-melanoma skin cancer by interferon-alpha.International Journal of Cancer 61, 110-114.
Sager, H., Brunschwiler, C. & Jungi, T. W. (1998). Interferon production by Theileria annulata-transformed cell lines is restricted to the beta family.Parasite Immunology 20, 175-182.[Medline]
Schweizer, M. & Peterhans, E. (1999). Oxidative stress in cells infected with bovine viral diarrhoea virus: a crucial step in the induction of apoptosis.Journal of General Virology 80, 1147-1155.[Abstract]
Sirinarumitr, T., Zhang, Y., Kluge, J. P., Halbur, P. G. & Paul, P. S. (1998). A pneumo-virulent United States isolate of porcine reproductive and respiratory syndrome virus induces apoptosis in bystander cells both in vitro and in vivo.Journal of General Virology 79, 2989-2995.[Abstract]
Sun, X., Burns, J. B., Howell, J. M. & Fujinami, R. S. (1998). Suppression of antigen-specific T cell proliferation by measles virus infection: role of a soluble factor in suppression.Virology 246, 24-33.[Medline]
Symons, J. A., Alcamí, A. & Smith, G. L. (1995). Vaccinia virus encodes a soluble type I interferon receptor of novel structure and broad species specificity.Cell 81, 551-560.[Medline]
Tschopp, J., Thome, M., Hofmann, K. & Meinl, E. (1998). The fight of viruses against apoptosis.Current Opinion in Genetics & Development 8, 82-87.[Medline]
Weiss, M., Hertig, C., Strasser, M., Vogt, H. R. & Peterhans, E. (1994). Bovine Virusdiarrhoe/Mucosal Disease: eine Übersicht.Schweizer Archiv für Tierheilkunde 136, 173-185.[Medline]
Yanase, N., Takada, E., Yoshihama, I., Ikegami, H. & Mizuguchi, J. (1998). Participation of Bax-alpha in IFN-alpha-mediated apoptosis in Daudi B lymphoma cells.Journal of Interferon and Cytokine Research 18, 855-861.[Medline]
Zhang, G., Aldridge, S., Clarke, M. C. & McCauley, J. W. (1996). Cell death induced by cytopathic bovine viral diarrhoea virus is mediated by apoptosis.Journal of General Virology 77, 1677-1681.[Abstract]
Received 14 June 1999;
accepted 15 December 1999.