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
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Abstract |
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Introduction |
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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.
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Methods |
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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
).
In utero infection.
Nine BVDV antibody-negative cows were presented for in utero infection at approximately 60 days of pregnancy (range 5870 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.
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.
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 ).
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 manufacturers 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 (AldrichSigma) at a dilution of 1:2500.
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Results |
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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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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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Discussion |
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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.
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References |
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Received 20 December 2000;
accepted 25 April 2001.