Establishment of persistent infection with non-cytopathic bovine viral diarrhoea virus in cattle is associated with a failure to induce type I interferon

B. Charleston1, M. D. Fray1, S. Baigent1, B. V. Carr1 and W. I. Morrison1

Institute for Animal Health, Compton, Newbury, Berkshire RG20 7NN, UK1

Author for correspondence: Bryan Charleston. Fax +44 1635 577263. e-mail bryan.charleston{at}bbsrc.ac.uk


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
The establishment of persistent infections with non-cytopathic bovine virus diarrhoea virus (ncpBVDV) is crucial for the maintenance of BVDV in cattle populations. Also, super-infection of persistently infected individuals with antigenically homologous cytopathic BVDV (cpBVDV) results in fatal mucosal disease. Persistent infection with ncpBVDV is established by infection of the foetus during the first trimester of pregnancy. It has been shown previously that foetal infection with cpBVDV does not result in persistent infection. Infection of cells in vitro has demonstrated that cpBVDV induces type I interferon (IFN), whereas ncpBVDV fails to induce IFN. In this study we demonstrate that foetal challenge with cpBVDV results in IFN production, whereas ncpBVDV does not. These findings strongly suggest that the ability of ncpBVDV to inhibit the induction of type I IFN has evolved to enable the virus to establish persistent infection in the early foetus.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Bovine viral diarrhoea virus (BVDV) is a pestivirus belonging to the family Flaviviridae. Pestiviruses are positive-stranded RNA viruses whose genome comprises a single open reading frame of approximately 12·5 kb. Two biotypes of BVDV, cytopathic (cp) and non-cytopathic (ncp), can be differentiated by their effect in cell culture (Zhang et al., 1996 ). Infection of bovine foetuses with ncpBVDV during the first 120 days of pregnancy can result in the birth of persistently infected (PI) offspring that are immunotolerant to the virus.

Superinfection of PI animals with a strain of cpBVDV that is antigenically homologous to the persistent ncp virus results in fatal mucosal disease. (Brownlie et al., 1984 ). In contrast, an experimental study in which bovine foetuses were challenged with cpBVDV between 63 and 107 days of gestation failed to produce persistent infection (Brownlie et al., 1989 ). These results indicated that cpBVDV is not able to establish persistent infections.

It has been shown that ncpBVDV isolates do not induce type I interferons (IFNs) in vitro (Diderholm & Dinter, 1966 ; Nakamura et al., 1995 ; Adler et al., 1997 ) and block the induction of IFN by other activators, namely double-stranded RNA (dsRNA) and viruses (Rossi & Kiesel, 1980 ). However, cpBVDV has been shown to induce type I IFN in vitro. Adler et al. (1997) proposed that the different capabilities of cp and ncpBVDV to establish persistent infections are related to the difference in their ability to induce IFN. The principal aims of the present study were to compare the capacities of ncp and cpBVDV to induce IFN in the bovine foetus during the first trimester of pregnancy and to determine whether or not the IFN response induced by ncpBVDV in utero is different from that observed in the dam.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Virus.
The experiments utilized an homologous pair of ncp and cp viruses (Pe515nc and Pe515c) originally isolated from a case of mucosal disease; Pe515c is the cytopathic BVDV isolate that was used in a previous experimental study of infection in bovine foetuses (Brownlie et al., 1989 ), which demonstrated that cpBVDV does not establish persistent infection. The viruses were propagated in a primary cell line derived from calf testis, as previously described (Brownlie et al., 1984 ). Virus titres of the challenge material (TCID50) were determined on calf testis cells (Brownlie et al., 1984 ).

{blacksquare} Virus isolation and neutralizing antibody titration.
Virus isolations were conducted on calf testis cells cultured on coverslips, as previously described (Brownlie et al., 1984 ). Five replicate coverslips were used for each sample. Virus neutralizing antibody titres were determined using a microtitre-based assay, as previously described (Brownlie et al., 1984 ).

{blacksquare} In utero infection.
Nine BVDV antibody-negative cows were presented for in utero infection at approximately 60 days of pregnancy (range 58–70 days). A laparotomy was performed on each of the cows as described previously (Fray et al., 2000 ) and 10 ml of amniotic fluid was removed by aspiration using an 18 gauge needle. Five ml of the appropriate challenge material was injected directly into the amniotic fluid. Three animals were injected with 5x106 TCID50 of ncpBVDV, three with 5x106 TCID50 of cpBVDV and three with mock-infected cell culture supernatant.

{blacksquare} Post-mortem samples.
One animal from each group was killed on days 3, 5 and 7 post-challenge. Samples of amniotic fluid, foetal spleen and maternal serum were harvested at post-mortem examination and stored at -70 °C.

{blacksquare} Type I interferon assay.
Levels of biologically type I IFN were assayed in duplicate in samples of serum and amniotic fluid using a chloramphenicol acetyltransferase (CAT) reporter assay (Fray et al., 2001 ).

{blacksquare} Western blot.
Homogenates of foetal spleens were prepared and the protein concentration of each sample determined (BCA protein assay kit; Pierce). An equivalent quantity of protein from each sample was suspended in 15 µl of electrophoresis sample buffer, resolved under reducing conditions, and transferred to ECL-nitrocellulose membrane as described previously for the preparation of nitrocellulose-bound antigen (Collen et al., 2000 ). After blocking the membrane with 5% (w/v) semi-skimmed milk in PBS containing 0·1% (v/v) NP40, the membranes were probed and processed for ECL visualization of proteins according to the manufacturer’s instructions (Amersham). Mx protein was detected using a rabbit antiserum raised against human MxA (Serum no. 49; a gift from P. Staeheli, Freiburg, Germany) at a dilution of 1:800, and horseradish peroxidase-conjugated anti-rabbit IgG (Aldrich–Sigma) at a dilution of 1:2500.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Virology
The results of virus isolation from foetal and maternal samples are summarized in Table 1. All nine pregnant animals used in this study were negative for BVDV antibodies and virus at the time of foetal infection. Samples of amniotic fluid taken at the time of challenge were also all negative for virus, as were samples of amniotic fluid and foetal spleen from mock-challenged pregnancies at all time points. Virus was not detected in samples of amniotic fluid from any of the three animals challenged with cpBVDV, but was detected in samples of foetal spleen taken on days 5 and 7 after infection (5/5 coverslips positive). By contrast, the amniotic fluid from ncpBVDV-challenged pregnancies was positive for virus infection on days 5 and 7 after infection (3/5 and 1/5 positive coverslips, respectively). Virus was also detected in foetal spleens on days 3, 5 and 7 (2/5, 5/5 and 5/5 coverslips positive, respectively) after challenge with ncpBVDV.


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Table 1. Virus isolation results from foetal and maternal tissue

 
Virus was detected in the blood of dams challenged with ncpBVDV on days 5 and 7 (2/5 and 5/5 positive coverslips, respectively) and in dams challenged with cpBVDV on day 7 (5/5 positive coverslips). BVDV was not detected in the serum of mock-infected dams.

Type I IFN biological assay
Type I IFN was not detected in samples of serum or amniotic fluid from any of the animals at the time of challenge or in samples from mock-infected dams post-challenge (Table 2). Similarly, IFN was not detected in the amniotic fluid of the ncpBVDV-infected foetuses following challenge. However, IFN was detected in the amniotic fluid of the cpBVDV-infected pregnancies on day 5 and day 7 post-infection, but was undetectable on day 3 (Table 2).


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Table 2. Type I IFN bioactivity in foetal amniotic fluid and maternal serum

 
IFN was detected in the serum of dams carrying foetuses infected with ncpBVDV from day 4 onwards, i.e. in the two dams killed on days 5 and 7 but not in the dam killed on day 3 (Table 2). The serum from the dams carrying foetuses infected with cpBVDV did not contain IFN, except in a sample from one animal on the sixth day after foetal infection, which contained low levels of IFN activity.

Western blot analysis of Mx protein
Mx protein is detected as a protein band of approximately 78 kDa by Western blotting. A single band of the expected size was detected in the positive control sample, primary cultures of calf testis cells stimulated with dsRNA. Non-specific protein bands with a molecular mass of less than 75 kDa were detected in all of the other samples. No Mx-specific bands were detected in the samples from the mock-infected pregnancies (Fig. 1). A strongly staining Mx-specific band was present in the samples from foetal spleens harvested 5 days and 7 days after challenge with cpBVDV, but was absent from the day 3 sample (Fig. 1). Faint Mx-specific bands were present in all the samples from the ncp BVDV-challenged pregnancies. The bands in the day 5 and day 7 samples were of equivalent intensity, but only a very faint band was present in the day 3 sample.



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Fig. 1. Western blot analysis of foetal spleen homogenates probed with anti-Mx protein antibody. Foetuses were either mock-challenged, or challenged with ncp or cpBVDV. Post-mortem examinations were performed 3, 5 and 7 days after challenge. Lysate harvested from calf testis cells, stimulated with dsRNA, was used as a positive control. Mx protein was detected as a discrete band of approximately 78 kDa.

 

   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
Infection with BVDV is common in the cattle populations of most countries worldwide. In the UK, it has been estimated that 95% of herds have serological evidence of current or previous infection with the virus (Paton et al., 1998 ). ncpBVDV is the most commonly isolated form of the virus. The widespread distribution of BVDV and the endemic state of infection within herds are in part attributable to the ability of ncpBVDV to establish lifelong persistent infection following exposure to virus in utero. Persistent infection occurs only if the foetus is exposed to virus prior to the onset of immune competence, at about 120 days of gestation, and persistently infected animals do not exhibit detectable antibody or T cell responses to the virus (Collen et al., 2000 ). Previous experimental studies, using the same isolate as the present study, have shown that in contrast to ncpBVDV, cpBVDV does not establish a persistent infection after foetal challenge during the first trimester of pregnancy (Brownlie et al., 1989 ). It was not possible to determine from these previous studies whether, as suggested by Brownlie et al. (1989) , the bovine foetus is refractory to infection with cpBVDV, or the virus is eliminated following a period of replication in the foetus. The results of the present study clearly indicate that cpBVDV does establish infection in the foetus. However, unlike ncpBVDV, which was detectable in both amniotic fluid and foetal spleen, cpBVDV was only detected in foetal spleen, suggesting that replication was more limited than with ncpBVDV. Hence there appears to be a mechanism within the foetus that is effective at controlling infection with cpBVDV but not ncpBVDV.

Previous studies of cells infected in vitro with BVDV have shown that type I IFN is induced following infection with cpBVDV but not ncpBVDV (Adler et al., 1997 ) and that ncpBVDV inhibits endogenous induction of type I IFN by other viruses (Nakamura et al., 1995 ). It has also been shown previously that cpBVDV induces IFN in the early foetus (Rinaldo et al., 1976 ). A major objective of this study was to seek evidence as to whether or not the difference between ncpBVDV and cpBVDV in their ability to establish persistent infections relates to their capacity to induce type I IFN in the bovine foetus. Type I IFN responses were evaluated by measuring IFN biological activity in amniotic fluid and by detecting expression of Mx protein in foetal spleen tissue. Mx proteins are synthesized in response to type I IFNs and dsRNA and have been used previously to indicate IFN bioactivity (Cella et al., 1999 ; Kim et al., 2000 ; Kracke et al., 2000 ). The foetal challenge model used in this study proved to be particularly suitable for investigating the induction of type I IFN, because there was no background IFN bioactivity in the pre-challenge samples or in samples from animals subjected to mock challenge with control culture medium. Infection with cpBVDV was associated with a strong type I IFN response, as indicated by the presence of biological activity in amniotic fluid and the detection of Mx protein in foetal spleen. By contrast, there was no detectable IFN activity in the amniotic fluid of ncpBVDV-challenged animals, despite the finding that ncp virus replicated to higher levels than cp virus. However, low levels of Mx protein were detected in the spleens of foetuses examined 5 and 7 days after infection with ncpBVDV. This likely reflects the greater sensitivity of Mx detection as an indicator of the IFN response, and suggests that ncpBVDV does not completely suppress IFN induction, although the possibility that low levels of Mx are induced by an IFN-independent pathway cannot be discounted, since IFN-independent induction of Mx has been reported in human monocytes infected with influenza virus (Ronni et al., 1995 ). Nevertheless, the findings indicate that type I IFN induction is substantially, if not completely, suppressed in foetuses infected with ncpBVDV as compared to those infected with cpBVDV.

The absence of measurable type I IFN in foetal infections with ncpBVDV contrasts with the detection of IFN in the serum of dams of foetuses infected with ncpBVDV in this study. The delay in detection of IFN in dams of foetuses infected with cpBVDV (day 6 as compared to day 4 with ncpBVDV) probably reflects the lower level of replication of cpBVDV in the foetus and a consequent delay in establishment of infection in the dams. The reason for the marked difference in the IFN response between the early foetus and immunocompetent animals is at present unclear. However, additional signals generated during the early stages of the adaptive immune response to the virus may play an important role in amplifying the IFN response in immunocompetent animals.

The detection of an IFN response in foetuses infected with cpBVDV but not in those infected with ncpBVDV suggests that this response may be involved in controlling foetal infections with cpBVDV. Exogenous type I IFN inhibits replication of BVDV in vitro (Bielefeldt-Ohmann & Babuik, 1988 ). However, whether such an innate response on its own would be able to eliminate the virus in vivo is unclear. An alternative explanation is that the induction of IFN prevents the development of immunological tolerance to the virus, so that elimination of the virus occurs when the foetus becomes immunologically competent.

In conclusion, the results of this study demonstrate that cpBVDV and ncpBVDV differ in their ability to induce type I IFN responses in the early bovine foetus and that, whereas ncpBVDV is able to stimulate strong type I IFN responses post-natally, it is unable to do so in the early foetus. Based on the findings of the present study, we propose that the capacity of the virus to inhibit IFN induction has evolved to enable the virus to establish persistent infection in the bovine foetus.


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Adler, B., Adler, H., Pfister, H., Jungi, T. W. & Peterhans, E. (1997). Macrophages infected with cytopathic bovine viral diarrhoea virus release a factor(s) capable of priming uninfected macrophages for activation-induced apoptosis. Journal of Virology 71, 3255-3258.[Abstract]

Bielefeldt-Ohmann, H. & Babuik, L. A. (1988). Influence of interferons alpha II and gamma and tumour necrosis factor on persistent infection with bovine viral diarrhoea virus in vitro. Journal of General Virology 69, 1399-1403.[Abstract]

Brownlie, J., Clarke, M. C. & Howard, C. J. (1984). Experimental production of fatal mucosal disease in cattle. Veterinary Record 114, 535-536.[Medline]

Brownlie, J., Clarke, M. C. & Howard, C. J. (1989). Experimental infection of cattle in early pregnancy with a cytopathic strain of bovine virus diarrhoea virus. Research in Veterinary Science 46, 307-311.[Medline]

Collen, T., Douglas, A. J., Paton, D. J., Zhang, G. & Morrison, W. I. (2000). Single amino acid differences are sufficient for CD4+ T-cell recognition of a heterologous virus by cattle persistently infected with bovine viral diarrhea virus. Virology 276, 70-82.[Medline]

Cella, M., Salio, M., Sakakibara, Y., Langen, H., Julkunen, I. & Lanzavecchia, A. (1999). Maturation, activation, and protection of dendritic cells induced by double-stranded RNA. Journal of Experimental Medicine 189, 821-829.[Abstract/Free Full Text]

Diderholm, H. & Dinter, Z. (1966). Interference between strains of bovine virus diarrhea virus and their capacity to suppress interferon of a heterologous virus. Proceedings of the Society for Experimental Biology and Medicine 121, 976-980.

Fray, M. D., Supple, E. A., Morrison, W. I. & Charleston, B. (2000). Germinal centre localization of bovine viral diarrhoea virus in persistently infected animals. Journal of General Virology 81, 1669-1673.[Abstract/Free Full Text]

Fray, M. D., Mann, G. E. & Charleston, B. (2001). Validation of a Mx/CAT reporter gene assay for the quantification of bovine type-I interferon. Journal of Immunological Methods 249, 235-244.[Medline]

Kim, C. H., Johnson, M. C., Drennan, J. D., Simon, B. E., Thomann, E. & Leong, J. A. (2000). DNA vaccines encoding viral glycoproteins induce nonspecific immunity and Mx protein synthesis in fish. Journal of Virology 74, 7048-7054.[Abstract/Free Full Text]

Kracke, A., von Wussow, P., Al-Masri, A. N., Dalley, G., Windhagen, A. & Heidenreich, F. (2000). Mx proteins in blood leukocytes for monitoring interferon beta-1b therapy in patients with MS. Neurology 54, 193-199.[Abstract/Free Full Text]

Nakamura, S., Shimazaki, T., Sakamoto, K., Fukusho, A., Inoue, Y. & Ogawa, N. (1995). Enhanced replication of orbiviruses in bovine testicle cells infected with bovine viral diarrhoea virus. Journal of Veterinary Medical Science 57, 677-681.[Medline]

Paton, D. J., Christiansen, K. H., Alenius, S., Cranwell, M. P., Pritchard, G. C. & Drew, T. W. (1998). Prevalence of antibodies to bovine virus diarrhoea virus and other viruses in bulk tank milk in England and Wales. Veterinary Record 142, 385-391.[Medline]

Rinaldo, C. R., Isackson, D. W., Overall, J. C., Glasgow, L. A., Brown, T. T., Bistner, S. I., Gillespie, J. H. & Scott, F. W. (1976). Fetal and adult bovine interferon production during bovine viral diarrhea virus infection. Infection and Immunity 14, 1189-1194.

Ronni, T., Sareneva, T., Pirhonen, J. & Julkunen, I. (1995). Activation of IFN-alpha, IFN-gamma, MxA, and IFN regulatory factor 1 genes in influenza A virus-infected human peripheral blood mononuclear cells. Journal of Immunology 154, 2764-2774.[Abstract/Free Full Text]

Rossi, C. R. & Kiesel, G. K. (1980). Factors affecting the production of bovine type 1 interferon on bovine embryonic lung cells by polyriboinosinic-polyribocytidylic acid. American Journal of Veterinary Research 41, 557-560.[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 20 December 2000; accepted 25 April 2001.