Classical swine fever virus induces proinflammatory cytokines and tissue factor expression and inhibits apoptosis and interferon synthesis during the establishment of long-term infection of porcine vascular endothelial cells
Emmanuelle Bensaude1,
Jane L. E. Turner1,
Philip R. Wakeley1,
David A. Sweetman2,
Claire Pardieu2,
Trevor W. Drew1,
Thomas Wileman2 and
Penelope P. Powell2
1 Department of Virology, Veterinary Laboratories Agency, Weybridge, Surrey KT15 3NB, UK
2 Department of Immunology and Pathology, BBSRC Institute for Animal Health, Ash Road, Pirbright, Surrey GU24 0NF, UK
Correspondence
Penelope P. Powell
penny.powell{at}bbsrc.ac.uk
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ABSTRACT
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Infection with virulent strains of classical swine fever virus (CSFV) results in an acute haemorrhagic disease of pigs, characterized by disseminated intravascular coagulation, thrombocytopenia and immunosuppression, whereas for less virulent isolates infection can become chronic. In view of the haemorrhagic pathology of the disease, the effects of the virus on vascular endothelial cells was studied by using relative quantitative PCR and ELISA. Following infection, there was an initial and short-lived increase in the transcript levels of the proinflammatory cytokines interleukins 1, 6 and 8 at 3 h followed by a second more sustained increase 24 h post-infection. Transcription levels for the coagulation factor, tissue factor and vascular endothelial cell growth factor involved in endothelial cell permeability were also increased. Increases in these factors correlated with activation of the transcription factor NF-
B. Interestingly, the virus produced a chronic infection of endothelial cells and infected cells were unable to produce type I interferon. Infected cells were also protected from apoptosis induced by synthetic ouble-stranded RNA. These results demonstrate that, in common with the related pestivirus bovine viral diarrhoea virus, CSFV can actively block anti-viral and apoptotic responses and this may contribute to virus persistence. They also point to a central role for infection of vascular endothelial cells during the pathogenesis of the disease, where a proinflammatory and procoagulant endothelium induced by the virus may disrupt the haemostatic balance and lead to the coagulation and thrombosis seen in acute disease.
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INTRODUCTION
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The innate cellular response against virus infection includes production of inflammatory and antiviral cytokines, as well as induction of cell death through apoptosis. Many viruses have evolved mechanisms that inhibit inflammation and prevent apoptosis and, as a consequence, are able to establish chronic infections. Classical swine fever virus (CSFV) is a member of the Pestivirus genus in the Flaviviridae family and causes classical swine fever, an OIE List A disease of pigs. Strains of low to moderate virulence can persist in vivo, whereas strains of high virulence cause an acute disease with high mortality rates, characterized by haemorrhagic fever, thrombocytopenia and disseminated intravascular coagulation (Thiel et al., 1996
; Moennig & Plagemann, 1992
). Interestingly, CSFV replicates in macrophages and vascular endothelial cells in pigs (Trautwein, 1988
). Vascular endothelial cells maintain the haemostatic balance by providing a quiescent, anti-thrombotic barrier. However, they are rapidly activated by pathogens to express a proinflammatory and procoagulant phenotype to eliminate infection (Bierhaus & Nawroth, 2003
). If this is not controlled, factors produced by infected endothelial cells may disrupt the haemostatic balance and cause pathological damage. Indeed, endothelial cell involvement for several viral haemorrhagic diseases, such as African swine fever, Ebola and dengue virus infections, has been demonstrated (Vallee et al., 2001
; Avirutnan et al., 1998
; Yang et al., 1998
). In each case, it includes the appearance of microthrombi, disseminated intravascular coagulation, lymphocytopenia and fibrinolysis (Summerfield et al., 2000
; van Oirschot, 1988
). The changes in haemostatic balance are thought to be caused by proinflammatory and antiviral factors, cell adhesion molecules and blood coagulation factors induced in endothelial cells. A central regulator of the expression of these proinflammatory genes is the transcription factor NF-
B and many viruses manipulate the NF-
B pathway, resulting in suppression of antiviral responses or prevention of apoptosis (Hiscott et al., 2001
; Tait et al., 2000
).
This study has shown that CSFV can replicate in endothelial cells in vitro, where it activates NF-
B and increases expression of proinflammatory and procoagulant factors. If this were to occur in vivo, these changes in gene expression could contribute to the haemorrhagic pathogenesis of the disease. Interestingly, endothelial cells infected with CSFV did not die and a long-term virus infection was maintained in tissue culture. In addition, CSFV infection in cell culture did not result in type I interferon (IFN) production and suppressed both IFN-
production and apoptosis induced by double-stranded RNA (dsRNA). These findings suggest that CSFV persistence in vitro involves active inhibition of cellular responses to the viral replicative intermediate, dsRNA.
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METHODS
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Reagents.
Virus antigen was stained using monoclonal antibody (mAb) WH303 against glycoprotein E2 (Edwards et al., 1988
). Interleukin (IL)-6 was quantified in a Quantikine P porcine IL-6 ELISA (P6000; R&D Systems). IL-8 was quantified by ELISA using capture antibody anti-porcine IL-8 (MAB 535) and biotinylated detection antibody (BAF 535) from R&D Systems. IFN-
was quantified using capture antibody anti-porcine IFN-
K9 mAb (27100-1) and biotinylated detection antibody F17 mAb (27105-1) from R&D Systems. Polyinosinicpolycytidylic acid sodium salt (pIpC) was from Sigma.
Virus and cells.
The CSFV strain Alfort 187 (Ruggli et al., 1996
) was kindly provided by the CSFV Community Reference Laboratory (Hanover, Germany). The virus was propagated in the pig kidney cell line PK15 (Paton et al., 1995
). Porcine aortic endothelial cells (PAECs) were isolated from aorta (Vallee et al., 2001
) and cultured in RPMI with 2 mM glutamine, 1 mM sodium pyruvate, 50 IU penicillin ml-1 and 50 µg streptomycin ml-1 plus 10 % foetal calf serum (FCS) demonstrated to be bovine viral diarrhoea virus (BVDV)- and antibody-free. The cells were maintained on gelatin-coated flasks.
Virus infection and titration.
Before infection, PAECs were washed twice with PBS. At time 0, the viral inoculum (Alfort 187 at an m.o.i. of of 2 TCID50 per cell) or an equal amount of uninfected PK15 cell lysate or PBS for control mock-infected cells was added and adsorbed for 2 h at 37 °C. The inoculum was then removed and replaced with complete media. Cells were passaged after reaching confluence at 3, 10, 16 and 23 days post-infection (p.i.). Cumulative totals of virus secreted into the medium between passages were titrated on days 1, 2, 3, 6, 7, 8, 9, 10, 13, 16, 20 and 23. PAECs were fixed and stained using anti-CSFV E2 antibody WH303, and an aliquot of each cell culture supernatant was stored at -70 °C for titration of infectious virus. Cells were fixed in acetone and virus was detected using mAb WH303, followed by a secondary peroxidase-conjugated rabbit anti-mouse antibody using 3-amino-9-ethylcarbazole as the enzyme substrate (Katz et al., 1987
). Virus was titrated on PK15 cells and stained as described above.
RNA isolation and relative quantitative PCR.
PAECs were infected with an m.o.i. of 2 TCID50 per cell of CSFV Alfort 187 as described above. For control experiments, cells were treated either with PBS or with an inoculum from uninfected PK15 cells, prepared by an identical procedure to virus-infected cell inoculum (mock infection). At 1, 2, 3, 5, 8, 12, 18, 24, 30, 48 and 72 h p.i., cells were lysed in 1 ml TRIzol (Invitrogen). Total RNA was extracted, resuspended in water and reverse transcribed using Moloney murine leukaemia virus reverse transcriptase (Promega) and random hexamer primers at 42 °C for 1 h, followed by 10 min at 94 °C. For amplification of cytokine DNA by PCR, the primers in Table 1
were used with Taq polymerase (Promega) and the products were separated by agarose gel electrophoresis and visualized with ethidium bromide staining. Relative quantitative PCR was carried out using Sybr Green reagent (Applied Biosystems). For all amplifications, the cycle conditions were 50 °C for 2 min and 95 °C for 10 min, followed by 50 cycles of 94 °C for 15 s, 55 °C for 30 s and 72 °C for 1 min. For each sample, cDNA corresponding to the
-actin gene, as well as the gene of interest, was amplified. PCR reactions were set up in triplicate. The Ct values were used to compare each sample with time 0 from that experiment. Ct (cycle threshold) represents the PCR cycle at which an increase in reporter fluorescence above a baseline signal can first be detected. cDNA was quantified relative to
-actin as the endogenous control; the relative concentration of target gene mRNA was equal to 2-
Ct. The primers listed in Table 1
were designed from porcine sequences in GenBank.
Quantification of cytokines by ELISA.
PAECs were infected with Alfort 187 (m.o.i. of 2 TCID50 per cell) as described above and overlaid with 2 ml culture medium with 10 % FCS. At time points 0, 30 and 72 h p.i., cell culture supernatants were harvested and stored at -70 °C. For IL-6 detection, the Quantikine kit was used according to the manufacturer's instructions. For IL-8 and IFN-
detection, microtitre plates were coated overnight with capture antibodies diluted in PBS, then blocked for 1 h with 5 % BSA. Samples were incubated for 2 h, prior to detection with a biotinylated secondary antibody for 1 h, followed by peroxidase-conjugated Extravidin (Sigma) for 1 h. Peroxidase activity was measured using the substrate tetramethylbenzidine at A450.
Quantification of dsRNA-induced apoptosis and IFN-
production.
For apoptosis quantification, following 30 h of treatment with pIpC, cells were lysed and tested using a nucleosome ELISA kit (CN Biosciences). This technique allows the quantification of apoptotic cells in vitro by nucleosome-free DNA affinity-mediated capture, followed by an anti-histone detection step. The test was performed according to the manufacturer's instructions. Apoptotic indices were calculated as ratios of the A450595 for treated and untreated cells. For IFN-
quantification, PK15 cells were incubated with complete medium containing GeneJuice transfection reagent (Novagen) and pIpC. After 8 h, cell culture supernatants were stored at -70 °C before IFN-
concentration was measured by ELISA.
Quantification of NF-
B p65 DNA-binding activity.
Following infection with Alfort 187 (m.o.i. of 2) or treatment with TNF-
, the level of p65 activity in PAECs was measured using the NF-
B TransAM kit (Active Motif) according to the manufacturer's instructions. Briefly, cells were lysed at 4 °C and protein concentrations were measured using the Bradford assay (Bio-Rad). Lysates (20 µg total proteins) were incubated in ELISA wells coated with the oligonucleotide motif recognized by active p65. p65 was then detected using a specific antibody, followed by a secondary antibody conjugated to peroxidase. The colorimetric reaction was measured at A450, with 620 nm acting as the reference wavelength. The experiment was repeated three times and a representative figure is shown in the results.
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RESULTS
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CSFV infection of PAECs in vitro
Since in vivo studies have shown that vascular endothelial cells are host cells for CSFV (Trautwein, 1988
), this study used primary vascular endothelial cell cultures to study phenotypic changes following infection. To characterize the nature of the infection, PAECs were infected with CSFV Alfort 187, fixed after 24 h and immunostained with an antibody against viral glycoprotein E2. E2 was localized in the cytoplasm and on the cell surface of over 90 % of the cells (Fig. 1
A and FACs analysis data not shown). As in continuous cell cultures (e.g. PK15 cells), CSFV Alfort 187 produced no cytopathic effect in these primary cells. CSFV-infected PAECs were maintained in culture for 4 weeks and the cells were passaged on the days indicated in Fig. 1(B)
. Cell-culture supernatants were sampled throughout the time course and cumulative totals of virus secreted into the medium between passages were determined. Virus production fell slightly over the first 10 days p.i. and then maintained a steady titre over the next 3 weeks. Virus secretion increased after passage, suggesting that there was an increased rate of virus replication in proliferating cells. Cell number was compared between infected and uninfected cells. Cell number did not alter between the two over a 2 week period, indicating that infection did not alter the kinetics of cell proliferation. Infection with an m.o.i. of 2 led to 100 % of the cells being infected, as detected by E2 staining at 24 h p.i. The percentage of cells infected remained at 100 % over the time course of the experiment.

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Fig. 1. CSFV infection of PAECs. (A) PAECs were infected with CSFV Alfort 187 and fixed at 24 h p.i. Virus was stained with an anti-E2 mAb (WH303) and visualized by immunohistochemistry. (B) Infected PAECs were maintained in culture for 4 weeks and passaged on days 3, 6, 10, 16 and 23 p.i. (dashed lines). Aliquots of cell culture supernatant were harvested throughout the experiment and infectious virus was titrated.
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Activation of vascular endothelial cells following infection with CSFV
Expression of mRNA for the proinflammatory cytokines IL-1
, IL-6 and IL-8 was studied by isolation of total cellular RNA, reverse transcription and relative quantitative PCR (Fig. 2
A). The fold increase in proinflammatory mRNA was relative to the zero time point and normalized against
-actin mRNA levels. Mock-infected PAECs, treated with either uninfected PK15 cell lysates or with PBS, showed no increase in cytokine gene expression throughout the 72 h time course. Cells infected with CSFV showed a biphasic increase in mRNA for IL-1
, IL-6 and IL-8. An initial rapid increase was detected between 1 and 3 h p.i., with a maximum at 2 h p.i. (when the virus inoculum was removed). This increase was short lived, with mRNA levels returning to normal by 4 h p.i. The decline suggested that there was a rapid removal or inhibition of the signal causing the increase in cytokine transcription and also that the cytokine transcripts were degraded very rapidly after synthesis. In addition, uninfected cell lysates did not cause the same increase in cytokine synthesis as infected cell lysates, indicating that endothelial activation was due to the virus in the preparation. The second peak in cytokine transcription occurred between 48 and 72 h p.i. (Fig. 2A
). For IL-6, the second peak showed a 72-fold increase, for IL-8 a 282-fold increase and for IL-1
a 265-fold increase. Cytokines secreted from infected cells into the media were measured by ELISA (Fig. 2B
). Both IL-6 and IL-8 increased in the culture supernatant, accumulating over 72 h p.i. to a concentration of 300 pg ml-1 and 30 ng ml-1, respectively. Protein synthesis and secretion of these factors, therefore, mirrored the increase in mRNA levels induced by the virus. A capture ELISA to measure porcine IL-1
secreted from cells was unable to detect any increase in this cytokine in the supernatants of infected endothelial cells (data not shown). IL-1
has no signal sequence and is not normally secreted from cells except after death (Dinarello, 1998
). However, it can act as a potent intracellular autocrine factor, either by directly binding to the nucleus or by binding to an intracellular pool of IL-1 receptors. The increased IL-1
mRNA in infected cells may correlate with increased intracellular protein. The expression of several endothelial cell-specific genes that might play a role in the pathogenesis of the disease was investigated. Tissue factor (TF) is an initiator of the blood coagulation cascade and vascular endothelial cell growth factor (VEGF) controls vascular permeability, while E-selectin is a cell-surface adhesion molecule that recruits leukocytes to the site of infection. Expression of mRNA for these genes was also studied by relative quantitative PCR (Fig. 3
). In control uninfected cells, the levels of all three mRNAs were low. In contrast, after CSFV infection, the mRNA levels of all three genes showed a similar biphasic increase to that seen for IL synthesis. An initial rapid and short-lived increase was followed by a more sustained increase, beginning 24 h p.i. The increase for each gene was lower than for the proinflammatory cytokines, possibly reflecting their function as membrane-bound regulatory molecules rather than signalling molecules. E-selectin mRNA, for example, had increased only 10-fold, VEGF mRNA 12-fold and TF mRNA 40-fold by 48 h p.i.

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Fig. 2. CSFV infection increases IL-1 , IL-8 and IL-6 mRNA levels and IL-6 and IL-8 protein secretion. PAECs were infected with CSFV Alfort 187 (open circles) or mock-infected (filled circles). (A) Relative quantitative PCR was used to quantitate mRNA levels over 72 h p.i. Error bars show the maximal and minimal fold increase in mRNA (experiments carried out in triplicate). (B) IL-6 and IL-8 secretion into the medium of PAECs over 72 h p.i. was measured by ELISA.
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Fig. 3. CSFV infection increases TF, VEGF and E-selectin mRNA levels. PAECs were infected with CSFV Alfort 187 (open circles) or mock-infected (filled circles). Relative quantitative PCR was used to measure mRNA levels. Error bars show the maximal and minimal fold increase in mRNA (experiments carried out in triplicate).
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The rapid activation of endothelial cells, characterized by the production of proinflammatory cytokines and cellular adhesion molecules, is controlled by the transcription factor NF-
B (Ghosh et al., 1998
). To investigate whether these changes in gene expression following CSFV infection were associated with activation of NF-
B, an assay was carried out to measure the activity of the 65 kDa subunit of NF-
B. Total cell lysates of infected cells were added to ELISA plates coated with an oligonucleotide sequence recognized by NF-
B p65. After washing, bound p65 protein was detected using an anti-p65 antibody (Fig. 4
). NF-
B activity increased early in infection, maximally 1 h after addition of virus, and had fallen by 5 h p.i. A second increase in NF-
B activity was seen at 18 h p.i., which was sustained for up to 72 h p.i. These times of increase in NF-
B activity coincided with the times post-infection when IL secretion and E-selectin, TF and VEGF mRNA levels were greatest.
PAECs infected with CSFV do not produce IFN
PAECs were either mock-infected or infected with CSFV at an m.o.i. of 2. Expression of type I IFN genes (IFN-
and -
) was studied by relative quantitative PCR. There was no significant difference between the levels of mRNA in infected and mock-infected cells (Fig. 5
A). This finding was supported by ELISA data showing no detectable IFN-
in culture supernatants over 72 h p.i. (Fig. 5B
). A positive control, indicating that endothelial cells produce abundant IFN-
and -
mRNA when stimulated with IFN-
, is shown in Fig. 5(C)
. RNA samples were treated with RNase-free DNase to ensure that samples were not contaminated with genomic DNA. In a further experiment, PK15 cells were treated with the synthetic dsRNA pIpC, a potent inducer of IFN-
. Secretion of IFN-
into the supernatant of these cells was measured by ELISA (Fig. 5D
). Addition of pIpC to cells in the presence of the transfection reagent GeneJuice further stimulated the secretion of IFN-
to over 1000 ng ml-1. Interestingly, cells chronically infected with CSFV did not produce detectable amounts of IFN-
after pIpC treatment, even in the presence of transfection reagent (Fig. 5D
).
CSFV inhibits dsRNA-induced apoptosis of infected PAECs
As shown above, endothelial cells became chronically infected with CSFV after 2472 h p.i. There was an activation of the inflammatory pathway and an inhibition of IFN mRNA and protein synthesis after treatment with dsRNA. Therefore, experiments were performed to investigate whether CSFV could also block dsRNA-induced apoptosis. Cell death was induced in control uninfected PAECs using the synthetic dsRNA pIpC (Fig. 6
A). The apoptotic index increased to 9·6 with increasing concentrations of pIpC ranging from 25 to 500 µg ml-1. In contrast, when pIpC was added to CSFV-infected cells, cell death was inhibited, even at the highest concentration of pIpC tested (500 µg ml-1).

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Fig. 6. CSFV suppresses dsRNA-induced apoptosis in PAECs and requires viral protein synthesis. (A) Confluent PAECs, either persistently infected with CSFV (open circles) or uninfected (filled circles), were treated with increasing concentrations of pIpC (0500 µg ml-1). After 30 h, cells were lysed and apoptosis was quantified using a nucleosome ELISA. (B) PAECs were infected at an m.o.i. of 2 with CSFV Alfort 187 and subsequently treated at 0, 6, 12 or 24 h p.i. with 25 µg pIpC ml-1. After 30 h, cells were lysed and apoptosis was quantified using a nucleosome ELISA.
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In addition, viral proteins were shown to be necessary for the protection against apoptosis (Fig. 6B
). PAECs were infected with CSFV for various times from 0 to 24 h and then treated with pIpC. CSFV did not protect the cells from apoptosis when added at the same time as pIpC, but the apoptotic indices decreased with increasing times post-infection, demonstrating a relationship between virus replication and protection from pIpC-induced apoptosis.
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DISCUSSION
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In this study, we have shown that CSFV activates endothelial cell proinflammatory responses but at the same time suppresses IFN production and apoptotic pathways. Eventually, CSFV establishes a long-term infection of endothelial cells with virus replication sustained over 4 weeks. Activation of endothelial cells was indicated by increased IL-1
, IL-6 and IL-8 mRNA levels. IL-1 showed the highest initial mRNA peak and may have played a role in the secondary induction of the other endothelial cell cytokines, IL-6, IL-8 and VEGF. We were unable to show the secretion of IL-1
protein from infected endothelial cells by ELISA. This cytokine has no signal sequence and in many cases remains cell-associated until cells are lysed after death. Increased intracellular IL-1 is a potent autocrine activator of several endothelial cell genes involved in vascular function. In contrast to our finding that there is no IL-1 secreted from infected endothelial cells, another study has shown that IL-1 is released from CSFV-infected macrophages (Knoetig et al., 1999
). This led to secretion of prostaglandins, also important mediators of vascular tone and function, and suggests that there may be a significant paracrine activation of vascular cells by infected macrophages in vivo. IL secretion is also necessary to stimulate immune cell responses. IL-8 is, for example, an important chemoattractant for immune cells, while IL-1 and IL-6 prime B- and T-cell responses against infected cells. The immediate activation of the proinflammatory response seen at 2 h p.i. was transient and probably due to virus binding, but both were virus-specific, since the initial increase and the secondary increase after 24 h p.i. were not seen with uninfected cell lysates. We showed increased binding of NF-
B, indicating transcriptional activation of these genes. Cytokine mRNAs have a short half-life after synthesis and the rapid reduction in mRNA levels a few hours after addition of virus suggests rapid intracellular cytokine mRNA degradation. Virus replication and protein synthesis has been shown to commence 1218 h p.i. (Mittelholzer et al., 2000
), suggesting that the second increase in cytokine mRNA levels after 24 h p.i. was due to the presence of replicating virus. The second increase in cytokine synthesis at 1872 h p.i. was more sustained and occurred together with an increase in the synthesis of TF, VEGF and E-selectin mRNA, molecules involved in vascular permeability and coagulation. Both initial and secondary peaks correlated with an activation of the 65 kDa/RelA subunit of NF-
B. However, p65 activation was highest following initial binding at 2 h p.i., while cytokine synthesis shows a more sustained increase 24 h p.i. NF-
B is an important modulator of the apoptotic pathway, controlling the synthesis of several anti-apoptotic genes such as bcl2 and bcl-xl (Karin & Lin, 2002
) and these proteins may play a protective role against apoptosis following CSFV infection.
The early phenotypic changes described here suggest that endothelial cells may have a direct involvement in the pathogenesis of classical swine fever in vivo. Secretion of proinflammatory cytokines, growth factors and reactive oxygen species from infected endothelial cells and other factors, such as platelet activating factor from infected macrophages, disrupts the haemostatic balance, leading to thrombocytopenia and platelet aggregation followed by haemorrhagic fever (Knoetig et al., 1999
; van Oirschot, 1988
). In addition, CSFV infection causes immunosuppression and lymphocyte depletion, due to bystander apoptosis observed in non-infected cells, particularly B and T lymphocytes (Summerfield et al., 1998
). The production of inflammatory cytokines by infected endothelial cells could play a role in this immunosuppression, as well as facilitating virus dissemination by attracting monocytic cells. This study was carried out using the virulent Alfort 187 strain and it will be important in future studies to compare these changes with those induced by an attenuated virus, such as the C strain, which does not cause haemorrhagic fever.
In common with the related pestivirus BVDV (Schweizer & Peterhans, 2001
), throughout CSFV infection of cells in vitro there was no up-regulation of IFN synthesis in response to virus or to synthetic dsRNA. Cells were also protected from dsRNA-induced apoptosis and this protection appeared to be dependent on viral protein synthesis. An interesting recent study (Ruggli et al., 2003
) has shown that the resistance of CSFV-infected cells to dsRNA-induced apoptosis and IFN production can be abrogated by the deletion of the N-terminal protease, Npro. This indicates a novel function for this protease in inhibition of the cellular innate immune system, but its role remains to be established. Recent work has implicated IFNs as important regulators of viral-induced apoptosis (Tanaka et al., 1998
). IFN synthesis is controlled through the combined action of the transcription factor NF-
B and IFN regulatory factors (IRFs), which together form part of the enhanceosome complex on the IFN-
promoter. Interestingly, and pertinent to this study, BVDV has been shown to inhibit IFN-
transcription by preventing IRF3 binding to DNA (Baigent et al., 2002
). IRF3 is constitutively present in the cytoplasm of all cells and activated by phosphorylation to translocate to the nucleus and mediate gene expression (Schafer et al., 1998
). IRF3 has been shown to be involved in regulating cellular apoptosis for several virus infections, such as Sendai virus (Heylbroeck et al., 2000
). The importance of IRF3 in innate immune defences is illustrated by the number of virally encoded proteins that have been found specifically to target this transcription factor. These include the influenza virus NS1 protein (Talon et al., 2000
) and the vaccinia virus protein E3L (Smith et al., 2001
). Significantly, there is also a viral IRF3 encoded by human herpesvirus 8 (Lubyova & Pitha, 2000
). Because of its similarity to BVDV, it is likely that CSFV also targets IRF3.
Commitment to apoptosis is thought to depend on IFN production (Tanaka et al., 1998
) and viruses must evade this response before establishing persistence. However, it is often difficult to link this to virus pathogenicity. For BVDV, it has been shown that, whereas non-cytopathic biotypes fail to produce IFN in vitro (Schweizer & Peterhans, 2001
), strong IFN responses were induced in calves in vivo (Charleston et al., 2002
). However in the foetus, IFN was not produced by non-cytopathic BVDV and the virus persisted (Charleston et al., 2001
). Transmission to the foetus is a very important step in viral evasion for both BVDV and CSFV, leading to persistently infected neonatal animals (Thiel et al., 1996
). Elucidation of the viral mechanisms of activation and inhibition of signal transduction pathways in vitro will provide strategies to control the persistence of these diseases in vivo.
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ACKNOWLEDGEMENTS
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The work was funded by UK Defra grant SE0764. Our thanks go to M. Bailey for discussions and to G. Ibata and C. Watson, VLA Weybridge, for cell culture and virus production and J. Seekings and S. Eagle for technical assistance.
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REFERENCES
|
---|
Avirutnan, P., Malasit, P., Seliger, B., Bhakdi, S. & Husmann, M. (1998). Dengue virus infection of human endothelial cells leads to chemokine production, complement activation and apoptosis. J Immunol 161, 63386346.[Abstract/Free Full Text]
Baigent, S. J., Zhang, G., Fray, M. D., Flick-Smith, H., Goodbourn, S. & McCauley, J. W. (2002). Inhibition of beta interferon transcription by noncytopathogenic bovine viral diarrhea virus is through an interferon regulatory factor 3-dependent mechanism. J Virol 76, 89798988.[Abstract/Free Full Text]
Bierhaus, A. & Nawroth, P. P. (2003). Modulation of the vascular endothelium during infection the role of NF-kappa B activation. In Host Response Mechanisms in Infectious Disease. Contributions to Microbiology, vol. 10, pp. 86105. Basel: Karger.
Charleston, B., Fray, M. D., Baigent, S., Carr, B. V. & Morrison, W. I. (2001). Establishment of persistent infection with non-cytopathic bovine viral diarrhoea virus in cattle is associated with a failure to induce type I interferon. J Gen Virol 82, 18931897.[Abstract/Free Full Text]
Charleston, B., Brackenbury, L. S., Carr, B. V., Fray, M. D., Hope, J. C., Howard, C. J. & Morrison, W. I. (2002). Alpha/beta and gamma interferons are induced by infection with noncytopathic bovine viral diarrhea virus in vivo. J Virol 76, 923927.[Abstract/Free Full Text]
Dinarello, C. A. (1998). Interleukin-1. In The Cytokine Handbook, 3rd edn, pp. 3572. San Diego, CA: Academic Press.
Edwards, S., Sands, J. J. & Harkness, J. W. (1988). The application of monoclonal antibody panels to characterize pestivirus isolates from ruminants in Great Britain. Arch Virol 102, 197206.[Medline]
Ghosh, S. M., May, J. & Kopp, E. B. (1998). NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu Rev Immunol 16, 225260.[CrossRef][Medline]
Heylbroeck, C., Balachandran, S., Servant, M. J., DeLuca, C., Barber, G. N., Lin, R. & Hiscott, J. (2000). The IRF-3 transcription factor mediates Sendai virus-induced apoptosis. J Virol 74, 37813792.[Abstract/Free Full Text]
Hiscott, J., Kwon, H. & Genin, P. (2001). Hostile takeovers: viral appropriation of the NF-kappaB pathway. J Clin Invest 107, 143151.[Free Full Text]
Karin, M. & Lin, A. (2002). NF-kappaB at the crossroads of life and death. Nat Immunol 3, 221227.[CrossRef][Medline]
Katz, J. B., Ludemann, L., Pemberton, J. & Schmerr, M. J. (1987). Detection of bovine virus diarrhea virus in cell culture using an immunoperoxidase technique. Vet Microbiol 13, 153157.[CrossRef][Medline]
Knoetig, S. M., Summerfield, A., Spagnuolo-Weaver, M. & McCullough, K. C. (1999). Immunopathogenesis of classical swine fever: role of monocytic cells. Immunology 97, 359366.[CrossRef][Medline]
Lubyova, B. & Pitha, P. M. (2000). Characterisation of a novel human herpesvirus 8-encoded protein, vIRF-3, that shows homology to viral and cellular interferon regulatory factors. J Virol 74, 81948201.[Abstract/Free Full Text]
Mittelholzer, C., Moser, C., Tratschin, J. D. & Hofmann, M. A. (2000). Analysis of classical swine fever virus replication kinetics allows differentiation of highly virulent from avirulent strains. Vet Microbiol 74, 293308.[CrossRef][Medline]
Moennig, V. & Plagemann, P. G. W. (1992). The pestiviruses. Adv Virus Res 41, 5398.[Medline]
Paton, D. J., Sands, J. J., Lowings, J. P., Smith, J. E., Ibata, G. & Edwards, S. (1995). A proposed division of the pestivirus genus using monoclonal antibodies, supported by cross-neutralisation assays and genetic sequencing. Vet Res 26, 92109.[Medline]
Ruggli, N., Tratschin, J. D., Mittelholzer, C. & Hofmann, M. A. (1996). Nucleotide sequence of classical swine fever virus strain Alfort/187 and transcription of infectious RNA from stably cloned full-length cDNA. J Virol 70, 34783487.[Abstract]
Ruggli, N., Tratschin, J.-D., Schweizer, M., McCullough, K., Hofmann, M. A. & Summerfield, A. (2003). Classical swine fever virus interferes with cellular antiviral defences: evidence for a novel function of Npro. J Virol 77, 76457654.[Abstract/Free Full Text]
Schafer, S. L., Lin, R., Moore, P. A., Hiscott, J. & Pitha, P. M. (1998). Regulation of type I interferon gene expression by interferon regulatory factor-3. J Biol Chem 273, 27142720.[Abstract/Free Full Text]
Schweizer, M. & Peterhans, E. (2001). Noncytopathic bovine viral diarrhea virus inhibits double-stranded RNA-induced apoptosis and interferon synthesis. J Virol 75, 46924698.[Abstract/Free Full Text]
Smith, E. J., Marie, I., Prakash, A., Garcia-Sastre, A. & Levy, D. E. (2001). IRF3 and IRF7 phosphorylation in virus-infected cells does not require double-stranded RNA-dependent protein kinase R or Ikappa B kinase but is blocked by vaccinia virus E3L protein. J Biol Chem 276, 89518957.[Abstract/Free Full Text]
Summerfield, A., Knoetig, S. M. & McCullough, K. C. (1998). Lymphocyte apoptosis during classical swine fever: implication of activation-induced cell death. J Virol 72, 18531861.[Abstract/Free Full Text]
Summerfield, A., Knoetig, S. M., Tschudin, R. & McCullough, K. C. (2000). Pathogenesis of granulocytopenia and bone marrow atrophy during classical swine fever involves apoptosis and necrosis of uninfected cells. Virology 272, 5060.[CrossRef][Medline]
Tait, S. W. G., Reid, E. B., Greaves, D. R., Wileman, T. E. & Powell, P. P. (2000). Mechanism of inactivation of NF-
B by a viral homologue of I
B
: signal induced release of I
B
results in binding of the viral homologue to NF-
B. J Biol Chem 275, 3465634664.[Abstract/Free Full Text]
Talon, J., Horvath, C. M., Polley, R., Basler, C. F., Muster, T., Palese, P. & Garcia-Sastre, A. (2000). Activation of interferon regulatory factor 3 is inhibited by the influenza A virus NS1 protein. J Virol 74, 79897996.[Abstract/Free Full Text]
Tanaka, N., Sato, M., Lamphier, M. S., Nozawa, H., Oda, E. N., Oguchi, S., Schreiber, R. D., Tsujimoto, Y. & Taniguchi, T. (1998). Type 1 interferons are essential mediators of apoptotic death in virally infected cells. Genes Cells 3, 2937.[Abstract/Free Full Text]
Thiel, H. J., Plagemann, P. G. W. & Moennig, V. (1996). Pestiviruses. In Fields Virology, 3rd edn. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia: LippincottRaven.
Trautwein, G. (1988). Pathology and pathogenesis of the disease. In Classical Swine Fever and Related Viral Infections. Edited by B. Liess. Dordrecht: Martinus Nijhoff.
Vallee, I., Tait, S. W. G. & Powell, P. P. (2001). African swine fever virus infection of porcine aortic endothelial cells leads to inhibition of inflammatory responses, activation of the thrombotic state and apoptosis. J Virol 75, 1037210382.[Abstract/Free Full Text]
van Oirschot, J. T. (1988). Description of the virus infection. In Classical Swine Fever and Related Viral Infections. Edited by B. Liess. Dordrecht: Martinus Nijhoff.
Yang, Z., Delgardo, R., Xu, L., Todd, R. F., Nabel, E. G., Sanchez, A. & Nabel, G. J. (1998). Distinct cellular interactions of secreted and transmembrane Ebola virus glycoproteins. Science 279, 983984.[Free Full Text]
Received 9 September 2003;
accepted 22 December 2003.