The infection of mammalian cells by many viruses induces apoptosis and a variety of signal transduction pathways either to promote cell survival or to enhance the cell death. BTV infection of mammalian cells also triggers apoptosis. There are two common pathways for the induction of apoptosis and it appears that BTV may trigger both intrinsic and extrinsic pathways [9–12]. The extrinsic pathway is primarily initiated by virus attachment to receptors, while the intrinsic pathway is mediated by damage to the mitochondria. In this report, we have undertaken a series of stepwise experiments to examine the cellular activations of various caspases and thereby induction of apoptosis during BTV infection. Further, we investigated if the caspase activities via intrinsic and extrinsic pathways during BTV infection are interdependent. Data obtained in this report conclusively confirmed the activation of caspase-8 in BTV infected cells by two different methods. The cleavage of caspase-8 was observed from 12 h p.i. by western analysis and the active, cleaved form was visualised by confocal microscopy. As the primary activation of caspase-8 is due to receptor binding, it was expected that caspase-8 activation would occur relatively early after infection with BTV. However, we noted that the caspase-8 activation in BTV infection was somewhat delayed compared to other viruses (e.g., reoviruses). Although we have not investigated further, it can be hypothesised that it is regulated by c-FLIP, an apoptosis inhibitor, as in reovirus infection . The control of caspase-8 by c-FLIP could be regulated by the activity of MAPK p38 [31, 32], which has been shown to play a role in BTV-induced apoptosis and endothelial damage [33, 34].
Our data also demonstrated the involvement of the intrinsic pathway in BTV infection as documented by the disruption of the mitochondria and release of cytochrome C which is known to be responsible for activating a number of events that leads to the cleavage of caspase-9. Thus, our data supports the recent report that cytochrome C is released from the mitochondria during an early stage of the BTV replication cycle . The direct evidence of caspase-9 cleavage was confirmed by western analysis. Moreover, the timing of mitochondrial damage precedes the cleavage of caspase-9 as presented in this report. Although the activation of an apoptotic response by the initiator caspase-8, -9 and executioner caspase-3 in response to BTV replication has been reported previously, to date the relationship between the two pathways remains unknown.
In this study, the relationship between the intrinsic and extrinsic pathways was determined using a series of assays including cell markers, pharmacological inhibitors and knock-out cell lines. Unlike reoviruses , there was no cleavage of BID by caspase-8 in BTV infected cells. Therefore, the extrinsic pathway is unlikely to induce the mitochondrial damage as observed in our studies. Furthermore, the use of a chemical inhibitor and the caspase-9 deficient cell line (Jurkat ΔC9) clearly confirmed that caspase-8 activation was also independent of caspase-9 during BTV infection of mammalian cells. Thus, the activation of each caspase pathway during BTV-1 infection appears to be independent.
In addition to the activation of executioner caspase-3, we found that BTV infection also induces the activation of caspase-7, another executioner caspase which is closely related to caspase-3, sharing similar structure and substrates, but less promiscuous . Caspase-3 and -7 share a number of target proteins and both recognise caspase death substrate PARP. We investigated PARP cleavage in BTV infection and the cleavage of PARP was detected in response to BTV infection of mammalian cells, adding further support that BT clinical signs are mainly mediated by apoptosis.
DeMaula et al. [5, 6] have hypothesised that the main component of BT clinical signs and lesions in endothelial cells is due to cellular necrosis. Their hypothesis was based on the observation at the late stage of BTV infection. In this study, we examined cellular necrosis by using a cellular marker HMGB-1. When necrosis is induced it causes the loss of cellular membrane integrity and HMGB-1 is translocated from nucleus to cytoplasm and rapidly released into the extracellular space, which results in inflammation and tissue damage [22, 37, 38]. When we examined the cytosolic and nuclear fractions of BTV infected HeLa at 48 h p.i., there was no HMGB-1 translocation to the cytosol. Therefore, the cell death and damage observed in BTV infection of mammalian cells is not due to necrosis but probably due to the very late timing of events and the cell disruption caused by the inflammatory response.
The data presented in our paper demonstrates that BTV infection of mammalian cells induces caspase cascade, resulting in apoptosis. These results indicate that apoptosis is a major cause of cellular damage in the host animal, supporting previous reports [9, 12]. However, it will be imperative to investigate the caspase activation and the role of apoptosis in BT disease in susceptible sheep.
Previously, we reported the activation of NF-κB in BTV infected cells  and hypothesised that it has a role in BTV-induced apoptosis similar to that of reovirus T3 . However, here we found that the inhibition of NF-κB had no effect in virus-induced apoptosis. Indeed, our data clearly demonstrated that chemical inhibition of the p50 subunit of NF-κB enhanced the early onset of visible cytopathic effect in BTV infected cells, and thus contradicted the report of Mortola and Larsson .
Therefore, it was necessary to further investigate the response of NF-κB to BTV infection. The type of response by NF-κB is related to the phosphorylation and degradation of the IκB complex, an inhibitory protein complex, which masks the nuclear translocation signal of NF-κB. Our data showed the degradation of IκBα but not IκBβ in response to BTV infection, which indicates that the classical NF-κB pathway is activated resulting in a controlled transient response. Furthermore, we examine the activation of NF-κB by using a NF-κB dependent firefly luciferase reporter assay. Our data showed an initial early period of NF-κB activation that was not sustained, as the NF-κB activity appeared to be inhibited at later stages of virus replication. The NF-κB response observed by BTV infection was similar to that of the T3 strain of reovirus .
From our preliminary data, we postulate that the transient nature of the NF-κB response was due to BTV proteins suppression of NF-κB activation, which in turn allowed efficient virus replication. BTV NS1 and NS2 limited NF-κB activation when the response was stimulated by poly I:C. Both of these proteins have the ability to bind RNA and therefore could act by sequestering dsRNA and inhibiting the pathway, or could interact with cellular proteins to inhibit the cascade (i.e. rotavirus ), or inhibit mRNA export from the nucleus (i.e. rotavirus NSP3 ).
Thus, our data showed that activation of NF-κB by BTV infection had a minimal role, if any, in the induction of apoptosis. However, it may play a role in initiating an antiviral state through the induction of the innate immune response. To this end, we investigated the effect of NF-κB activation on BTV replication and generation of infectious virions. In the presence of an NF-κB inhibitor, BTV titres were higher at the early stages (up to 24 h p.i.) of virus replication than in the absence of inhibitor. This result would indicate that NF-κB activation acts to control virus replication. At the late times, virus titres were similar in both cases, which could be indicative of the virus proteins controlling the NF-κB response (i.e. NS1 and NS2). The induction of NF-κB response in controlling virus replication through the induction of an antiviral response needs to be investigated further, especially as a recent report demonstrated that BTV replication was significantly high in IFNα deficient mice resulting in rapid death of these animals .
BTV infection has been reported to produce strong cytokine responses [7, 43–45], however the mechanisms that trigger their production has not been investigated. NF-κB activation could be a candidate to initiate a cytokine response during BTV infection, although there are other pathways, including IRF that could induce cytokine response. In this report we were able to identify the translocation of IRF-3 to the nucleus in response to BTV infection, indicating the induction of IRF-3 by BTV infection. As well as IRF-3, which is sequestered in the cytosol, the production of IRF-7 and its subsequent translocation to the nucleus were also detectable in BTV infected cells. Moreover, using an IRF dependent firefly luciferase reporter assay we confirmed that IRF activation was stimulated during BTV infection. Interestingly, the kinetics of IRF activation mirrored NF-κB activation. This early response of IRF was suppressed over the course of BTV infection similar to that of NF-κB. Despite the activation of IRF and NF-κB, it is clear that BTV infection triggers robust apoptosis in mammalian cells, which most likely play a role in BTV pathogenesis.