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SID 5 Research Project Final Report

SID 5 (Rev. 3/06) Page 1 of 14

NoteIn line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The SID 5 (Research Project Final Report) is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra website. A SID 5 must be completed for all projects.

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Project identification

1. Defra Project code SE0785

2. Project title

Classical swine fever evasion of innate immune defenses

3. Contractororganisation(s)

Veterinary laboratories agency Department of virologyNew HawSurreyKT15 3NB

54. Total Defra project costs £ 573,371(agreed fixed price)

5. Project: start date................ 01 April 2006

end date................. 31/3/2009


6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...................................................................................YES NO (a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They

should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow.Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer.In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.

(b) If you have answered NO, please explain why the Final report should not be released into public domain

Executive Summary7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the

intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.

Classical swine fever is a haemorrhagic disease that poses one of the greatest disease threats to the

swine industry. Better methods to control this disease would be beneficial.

Recently interest in methods that exploit or modulate the host’s immune response to provide protection

against disease has increased. However, as viruses and their hosts have evolved together, viruses have

developed a multitude of ways to evade the host’s immune system. The effective design of interventions

based on modulation of the immune system will require an understanding of the mechanisms used by

viruses to evade the host’s responses.

The objectives of this project were therefore to increase our understanding of the molecular mechanisms

used by classical swine fever virus (CSFV) to evade the host’s innate immune defenses.

The first objective was to examine how this virus prevents cells from undergoing apoptosis. Apoptosis, or

programmed cell death, is an important component of the host’s innate immunity by which viral infected

cells, and hence the infecting virus, are eliminated. Prevention of this response may allow the virus to

establish infection and persist within the host. To examine how the virus may prevent cell death we

stimulated infected and uninfected porcine endothelial cells to undergo apoptosis using dsRNA, a viral

replication intermediate that is one of the signals cells use to recognise viral infection. We then

interrogated various steps within the apoptotic pathway to establish where the virus may be targeting the

pathway. The molecular events that lead to apoptosis are complex and, as unwanted cell death is to be

avoided, have to be carefully controlled. Our previous work has indicated that the virus is able to block

multiple steps in dsRNA-induced apoptosis. One of the major points of apoptotic control is at the

mitochondria. If the balance of interactions between numerous pro- and anti-apoptotic proteins tips in

favour of cell death the membrane potential of the mitochondria is lost, which leads to a sequence of

events that commit the cell to die. We have confirmed that CSFV infection is able to prevent loss of the


mitochondrial membrane potential, indicating that the inhibition is prior to this point. Interestingly, this

inhibition was specific for dsRNA stimulated apoptosis, CSFV infection was not able to prevent apoptosis

induced by a signal used by cytotoxic T cells to initiate cell killing (FasL), indicating that CSFV infected

cells would be susceptible to this host defence mechanism.

We have also investigated if CSFV targets the pro- and anti-apoptotic mitochondrial proteins that control

loss of the membrane potential. These proteins have to be activated or induced to control their activity.

The induction of transcription of one of these pro-apoptotic proteins, NoxA, in response to dsRNA is

reduced by CSFV infection. The extent that this contributes to CSFV inhibition of apoptosis requires

further investigation. We also know that CSFV inhibits an early step in the dsRNA pathway, so we

investigated if this may be the formation of a dsRNA death-inducing signalling complex or DISC, however

it is currently unclear if this is a step targeted by the virus.

Our second objective was to investigate the role of the viral proteins in virus pathogenesis and virulence.

The first protein encoded by the viral genome, Npro, is known to be involved in pathogenesis. Using the

yeast two-hybrid method a number of host proteins that interact with Npro were identified. Two of these

have been characterised in detail. The first, IB, is an inhibitor of the transcription factor NFB that

controls the expression of many host immune responses. We have detected that when over-expressed, by

transfection of cells with a plasmid, the Npro protein results in a transient accumulation of IB in the

nucleus of cells stimulated with tumour necrosis factor alpha (TNF). We hypothesise that this IB:Npro

interaction modulates the expression of NFB regulated genes in response to TNF.

The second npro interacting protein, which we have characterised, is Hax1. This protein has been

reported to have a number of functions, one of which is in cell survival. We have confirmed that Hax1 is

relocalised within the cell during CSFV infection and, although the role of this interaction has yet to be

determined, we hypothesise that it may somehow assist CSFV infected cells survive.

The final objective was to analyse the effect of interferon (IFN) on CSFV infection, to prove the principle

that CSFV may be tractable to interventions based on treatment with components of the host’s immune

system.

Although CSFV infection prevents IFN induction in many cells, when cells were treated with porcine IFN,

the interferon stimulated gene MxA was induced, indicating that CSFV does not inhibit IFN signalling

pathways and that the virus will be sensitive to IFN treatment. Consistent with this, CSFV replication was

reduced by prior treatment of cells with both porcine IFN and IFN. Further work to address the relative

efficacy of candidate immunomodulatory molecules, including those of the IFN family, against CSFV is

warranted to facilitate the development of future novel intervention methods.

Project Report to Defra8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with

details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the scientific objectives as set out in the contract; the extent to which the objectives set out in the contract have been met;


details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer).

Objective 1: How does CSFV inhibit apoptosis?

Objective 1.1 (VLA)Does CSFV infection prevent the loss of the mitochondrial potential induced by double-stranded RNA?

Objective 1.2 (VLA)Does CSFV prevent formation of the dsRNA-triggered death-inducing signalling complexes?

Objective 1.3 (VLA)Generation of antibodies recognising porcine cellular proteins involved in the apoptotic pathway

Objective 1.4 (VLA)Does CSFV inhibit the double-stranded RNA protein kinase PKR?

Objective 1.5 (VLA +IAH)Does CSFV affect the mitochondrial regulators of the Bcl2 family

Objective 2: Investigation of the role of viral proteins in pathogenesis and virulence?Objective 2.1: (IAH)Construction of a cDNA library from porcine macrophage cells

Objective 2.2: (IAH)Screen of the porcine macrophage library for proteins interacting with Npro and one other CSFV protein

Objective 2.3 (IAH)Confirmation and characterisation of the identified protein-protein interactions

Objective 3: Analysis of the effects of IFN on CSF infectionObjective 3.1: (VLA)Investigation of the inhibition of CSFV by different Type I IFNs

The overall aim of this project is to enhance our understanding of how CSFV interacts with the innate immune

system of the host. This knowledge will ultimately assist the scientific community in designing new and more

effective intervention strategies

Objective 1: How does CSFV inhibit apoptosis?One of the key features of CSFV infection is dysregulation of the innate immune system. Infection of many cell

types in culture is able to prevent those cells from undergoing controlled cell death or apoptosis. Apoptosis is

employed by multicellular organisms to eliminate damaged, aberrant or infected cells. It is hypothesised that the

ability of CSFV to prevent cells undergoing apoptosis enables the virus to establish infection and persist. A

characteristic of apoptotic cell death is the activation of the caspase family of proteases. Caspases that function

near the apex of cell death cascades are designated initiators and include caspases-8 and -9, whereas those

involved in the terminal stages of cell death, such as caspases -3 and -7, are termed effector caspases. Bcl-2

family proteins are also key regulators of cell death pathways and can be anti-apoptotic, such as Bcl-2, Bcl-XL and

Mcl-1 or pro-apoptotic, such as Bax, Bak NoxA and Bid.

Cell death can be triggered by signals categorized as extrinsic, if extracellular, or intrinsic if originating from within

the cell. The prototypal cell death pathway is that of the Fas receptor and its extrinsic cell death ligand, FasL.

FasL induces oligomerization of the Fas receptor and formation of a death-inducing signaling complex, or DISC,


which results in activation of caspase-8. Caspase-8 cleaves Bid to produce active, truncated Bid (tBid), which

then recruits pro-apoptotic Bcl-2 proteins to the mitochondria. Mitochondria undergo specific changes associated

with propagation of the cell death signal. These changes include loss of inner membrane potential (m) and

release of proteins from the intermembrane space including cytochrome c. Cytochrome c forms a complex with

caspase-9 that in turn activates caspases-3 and -7 thereby committing a cell to apoptosis. Intrinsic cell death

processes frequently activate the cascade at the level of mitochondria.

The aims of this objective were to identify what points within the apoptosis pathway CSFV is able to abrogate this

immune defence. We therefore investigated the effect of CSFV infection on apoptotic signalling.

Objective 1.1 Does CSFV infection prevent the loss of the mitochondrial potential induced by double-stranded RNA?

To investigate whether the apoptotic regulatory steps of mitochondrial membrane potential (m) loss and

cytochrome c release are inhibited, CSFV-infected or uninfected endothelial cells were treated with the following

apoptosis stimulators: pIpC (a synthetic dsRNA) for 1, 3 or 5h, staurosporine (STS) for 4.5h, or human

membrane-bound FasL for 22h. Cells were labelled with the mitochondrion-specific, potentiometric dye

tetramethyl-rhodamine-methyl-ester (TMRM). Fluorescence was analyzed by flow cytometry. Loss of m,

reflected by the reduction in the percentage of TMRM-positive cells, was evident following pIpC addition to

uninfected but not to CSFV-infected cells (Figure 1A). In contrast, m loss was universally high following FasL or

staurosporine treatment (Figure 1B).

Fig 1 CSFV inhibits loss of the mitochondrial membrane potential in cells stimulated to undergo apoptosis by dsRNA but not by other apoptotic stimuli

In parallel, the release of cytochrome c from mitochondria into the cytosol was monitored, by western blotting of

cytosolic fractions, following differential digitonin lysis. Using antibodies directed against cytochrome c, and viral

Npro as a marker for infection, the appearance of cytochrome c in the cytosolic fraction was clearly detected in

uninfected but not CSFV-infected cells (Figure 2). In contrast, infected cells were not protected against

cytochrome c translocation following FasL or staurosporine treatment. Equivalent protein loading was assessed

using an anti-tubulin antibody.


Fig 2 CSFV inhibits the release of cytochrome c in cells stimulated to undergo apoptosis by dsRNA but not by other apoptotic stimuli

These results confirm that CSFV infection inhibits apoptosis at points either at or upstream of the mitochondria.

Also the inhibition is specific for apoptosis stimulated by dsRNA, other inducers were not inhibited. Host cells

recognise dsRNA as a signal of intracellular virus infection, whereas FasL induced apoptosis is a mechanism

used by cytotoxic T cells to induce death in cells expressing foreign antigens. These results indicate that CSFV

has a mechanism to prevent cells in which it is replicating from cell death but that those infected cells would be

susceptible to T cell-mediated killing.

Objective 1.2 Does CSFV prevent formation of dsRNA-triggered death-inducing signalling complexes?

Our earlier work has indicated that CSFV is able to inhibit dsRNA-induced apoptosis, at multiple points in the

signalling pathway. One of these points is upstream of caspase 8, the key initiator caspase in the death receptor

pathway. During death receptor induced apoptosis binding of a ligand results in death receptors aggregating and

forming death-inducing-signalling-complexes or DISCs. These recruit multiple procaspase 8 molecules, via

adapter molecules such as FADD, and results in cleavage and activation of caspase 8. We therefore aimed to

investigate if CSFV infection disrupts formation of a dsRNA-induced DISC. To this end various commercially

available antibodies were screened for cross reactivity with porcine versions of the proteins that we expect might

form this dsRNA DISC, namely FADD (Fas associated protein with death domain), TRADD (TNFRSF1A-

associated via death domain), caspase 8 and TLR3 (Toll like receptor 3). A commercial antibody that recognises

active porcine caspase 8 at a low level was identified, but none tested recognised porcine FADD. We therefore

raised polyclonal sera against a peptide based on the porcine FADD sequence. Using these sera Western

analysis confirmed that the level and size of FADD protein is unchanged in infected compared to uninfected cells,

indicating that the inhibition of apoptosis is not due to a direct loss or cleavage of this protein. Our next aim was to

investigate if CSFV infection blocks formation of a multi-protein complex. Using a magnetic bead based pull down

system we confirmed that our polyclonal sera pulled-down the FADD protein. However, using the caspase 8


antibody, we were unable to detect formation of a complex of FADD with caspase 8 in either uninfected or

infected cells. This may be due to the low level of binding of the anti-caspase 8 antibody to the porcine version of

caspase 8 or the conditions used may not have maintained the integrity of any multi-protein complexes. It is

therefore unknown if CSFV interacts with a dsRNA induced DISC and this area warrants further investigation.

Objective 1.3 Generation of antibodies recognising porcine cellular proteins involved in the apoptotic pathway

Studies of animal diseases can be hampered due to a lack of species-specific reagents compared to the wealth of

reagents available for human and rodent researchers. To provide reagents for this and other objectives the

following commercially available reagents were screened for cross-reaction with porcine proteins by confocal

microscopy and western analysis.

Antibody Company Cat n. Confocal Western Bcl-2 BD 610538 - +Bax Transduction Lab 610982 +Bad BD 610391 -

Cytochrome C Ab-cam ab 13575-100 +FADD Stressgen AAP-210 -FADD Cell Signalling 2782 -FADD Abcam ab24533-100 - -

TRADD Neomarkers RB-1529-PO - -Noxa (p53AIP-1) Zymed 52-3587 + /-

HAX-1 BD 610824 Only if over-expressed +IRF3 Abcam ab11978-50 +

Active Caspase-3 Ab-cam ab 13847-100 + +Uncleaved Caspase-3 BD 610322 + -

Cleaved caspase 3 Cell signalling 9664 +Caspase 8 MBL M032-3 - -Caspase 8 Abcam ab15552 +Caspase-8 BD 551234 + -

Table 1 Assessment of antibody reactivity with porcine proteins

As none of the FADD antibodies reacted with the porcine version of the protein we identified a potentially

immunogenic peptide within the FADD protein and raised rabbit polyclonal sera to this peptide. Screening of this

anti peptide serum confirmed it reacted specifically with porcine FADD by Western analysis and in pull down

assays.

In addition to generating antibodies recognising porcine proteins we have produced reagents to classical swine

fever proteins, in particular we have raised polyclonal sera against both peptides and recombinant protein

preparations of CSFV Npro.

Objective 1.4 Does CSFV inhibit the double-stranded RNA protein kinase PKR?

Our focus on other aspects of this objective has, unfortunately, prevented us from examining this sub-objective.

PKR (dsRNA-dependent protein kinase) is induced by interferon and activated by autophosphorylation upon

binding to dsRNA. Once activated PKR inhibits protein translation by phosphorylating the eukaryotic translation

factor 2 (eIF2). This restricts viral protein translation and replication. PKR also modulates cellular events

including induction of apoptosis through FADD mediated death signalling. Other researchers working with the

related pestivirus BVDV have indicated that cytopathic (cp) BVDV induces PKR activation, whereas non-

cytopathic (ncp) BVDV prevents PKR activation by super infection with another virus (Gil et al 2006 Virus


research 116, 69-77). The extent of the involvement, if any, of PKR in CSFV inhibition of apoptosis still warrants

investigation.

Objective 1.5

Does CSFV affect the mitochondrial regulators of the Bcl2 family

When cells are stimulated to undergo apoptosis caspase 8 mediates cleavage of Bid to the truncated form tBid.

This activates its pro-death activity and results in tBid translocation to the mitochondria, where it promotes loss of

the mitochondrial membrane potential and cytochrome c release. Previously we have identified that CSFV

infection is able to inhibit apoptosis induced by over-expression of tBid, indicating that the virus is able to block

apoptosis somewhere downstream of the activation of tBid as well as upstream of caspase 8. We therefore

investigated CSFV’s interaction with mitochondrial regulators of apoptosis. The balance of interactions which

occur in the mitochondria between pro- and anti-apoptotic members of the Bcl2 family of proteins largely

determine if a cell lives or dies. One candidate Bcl2 protein that CSFV may be affecting is the pro-apoptotic

protein Noxa. Bcl2 proteins are restrained from causing unwanted cell death by a variety of control mechanisms.

In the case of Noxa regulation is at the transcriptional level. In human cells Noxa mRNA is induced by p53 in

response to DNA damage but is also induced by infection with ssRNA viruses or dsRNA. The transcription factor

IRF3, but not p53, is required for this viral mediated induction. CSFV infection results in a loss of the transcription

factor IRF3 from cells so we examined if Noxa mRNA is affected in CSFV infected cells. Quantitative PCR

confirmed that NoxA mRNA is indeed induced in porcine aveolar macrophages in response to dsRNA but that this

induction is reduced by CSFV infection (Fig 3).

Fig 3 Induction of transcription of the pro apoptotic NoxA is reduced in CSFV infected cells NoxA promotes apoptosis by binding to a subset of anti-apoptotic mitochondrial proteins to prevent their action.

On it’s own NoxA is a weak inducer of apoptosis and, although likely to contribute, the extent to which this

reduction in NoxA transcription contributes to the prevention of apoptosis is yet to be determined.

In our previous studies we identified another mitochondrial protein, Hax1, as a binding partner for the CSFV

protein Npro using the yeast 2-hybrid system. Hax 1 is an interesting protein that appears to have numerous

functions, possibly due to the fact that it is expressed in a number of variant forms. It has homology with the Bcl-2

proteins and a growing body of evidence suggests that Hax-1 promotes cell survival. The interaction between

Npro and Hax1 was confirmed to occur in porcine cells by co-precipitation (pull down) assays (Fig 4) and a region

in the C- terminus of Npro that has similarities to a proposed Hax 1 binding consensus sequence has been

identified. We confirmed that this region of Npro is involved in Hax1 binding and that relocalisation of Hax1 occurs


in cells transfected with CSFV Npro and upon CSFV infection. The precise role of this interaction with the Hax 1

protein during CSFV infection is yet to be determined but the interaction may somehow promote the cell survival

activity of the Hax1 protein, thereby assisting the virus to persist.

Fig 4 The full length CSFV Npro protein, but not the N terminal region lacking the Hax1 binding sequence, interacts with Hax1 causing the protein to be captured from cell lysates using a GST pull-down assay.

Objective 2: Investigation of the role of viral proteins in pathogenesis and virulence?

The Npro protein prevents the induction of interferon by targeting the transcription factor IRF3 (La Rocca et al J.

Virology 7239-47, 2005). This transcription factor is lost from host cells from 18h post infection. Initial assays

indicated that this was due to an inhibition of transcription of the IRF3 gene. However, we have investigated the

mechanism behind the loss of this transcription factor further and shown that, like the related pestivirus BVDV,

this involves the Npro protein targeting IRF3 for degradation by proteasomes and thus preventing IRF3 activating

transcription from the IFN- promoter (Seago et al. J Gen Virol. 2007 3002-6.)

The role of the Npro protein in the inhibition of apoptosis has also been investigated. Infectious clones of CSFV

that lack the Npro protein have been obtained from a collaborator. Treatment of cells infected with these mutant

CSFV with pIpC results in the production of active caspase 3, whereas the appearance of the active form of this

caspase is reduced in cells infected with the wild type version of this infectious clone. The extent to which Npro is

responsible for CSFV inhibition of apoptosis, or if other viral proteins, such as the Erns proteins, contribute

requires further investigation.

Objective 2.1: (IAH)Construction of a cDNA library from porcine macrophage cellsObjective 2.2: (IAHScreen of the porcine macrophage library for proteins interacting with Npro and one other CSFV protein

In our previous work we exploited a human liver cDNA library to identify host proteins that interact with viral

proteins to try and elicidate their role in pathogenesis. Screening this library using the yeast 2-hybrid system

identified the proteins Hax1 and IκBα as interaction partners for the viral protein Npro. However, it was likely that

the use of a human library would not have identified interacting porcine proteins with low homology to human

counterparts and those proteins that are not expressed in liver. A second yeast-2-hybrid screen using a pig cDNA

library constructed from a macrophage cell line was therefore completed. Fifty-four clones encoding putative

SID 5 (Rev. 3/06) Page 10 of 14

interacting proteins have been sequenced. Analysis of the sequences revealed a number of known ‘false-

postives’. Some of the putative interaction partners have roles in regulating apoptosis, whereas many have no

characterised link to either apoptosis or the innate immune system. The implications of what role these putative

interactions have during apoptosis and other aspects of the virus’s infection of the host require further

investigations.

Objective 2.3 (IAH)Confirmation and characterisation of the identified protein-protein interactions

Two of the identified Npro interacting proteins have been characterised in detail, namely Hax1, which is described

above, and IB

IB is the inhibitor of NF-κB, a transcription factor involved in the control of apoptosis, immune response and

interferon production. The NF-κB transcription factor is normally sequestered in the cytoplasm as a latent complex

by a family of proteins, including IB. When activated by phosphorylation in response to stimulation, for example

by a viral pathogen, IIB is targeted for proteasomal degradation. This releases, and activates, NF-κB which

translocates to the nucleus where it stimulates the expression of numerous proteins that participate in immune

responses, as well as in oncogenesis and apoptosis. The inhibitor IB is rapidly resynthesised, it then

translocates to the nucleus, disassociates NF-κB from DNA and subsequently transports the transcription factor

back to its sequestered state in the cytoplasm.

The significance of the Npro/IB interaction was investigated after confirmation of the interaction using further

yeast-2-hybrid analysis and additional co-precipitation assays. We identified that Npro localises to both the

cytoplasmic and nuclear compartments in stably transfected cells and CSFV infected cells, indicating that this

protein has the potential to interact with IB in either or both compartments. Following TNF-α stimulation, PK-15

cell lines over expressing His-tagged Npro exhibit a transient nuclear accumulation of IB one hour after TNF-α

treatment (Fig 5)

Fig 5 Transient nuclear accumulation of IB in a cell line stably expressing CSFV Npro.

SID 5 (Rev. 3/06) Page 11 of 14

Cytoplasmic and nuclear fractions (confirmed by stain for the cytoplasmic and nuclear markers, alpha-tubulin

and histone H1) from TNF treated PK15 cell line expressing Npro (N) or transfected with a control plasmid

(C). In Npro transfected, but not control, cells IB accumulated in the nucleus 60 minutes after TNF

treatment

We propose a model of Npro binding to nuclear IB. In our investigations no obvious effect on NF-κB activation

has been observed, however, because of the transient nuclear accumulation of IB following TNF alpha

treatment, it is feasible that Npro regulates a specific subset of NF-κB "response" genes. This requires further

investigation.

Objective 3: Analysis of the effects of IFN on CSF infectionObjective 3.1: Investigation of the inhibition of CSF by different Type I IFNs

The aim for this objective was to analyse if different interferons (for example, in humans there are at least 12

functionally active IFN as well as IFN , and others) exhibit different activities towards CSFV, which may

provide information about future interventions to control the virus.

As CSFV prevents the induction of interferon in many cell types we first investigated if it is also able to prevent the

action of IFN. Treatment of cells with interferon stimulates the production of the antiviral protein MxA. Although

CSFV infection prevented induction of this protein in cells treated with dsRNA, due to the targeting of IFR3, CSFV

infection did not prevent MxA induction on treatment with porcine IFN. This indicates that CSFV does not target

IFN signalling pathways and thus should be sensitive to IFN treatment (Fig 6)

Fig 6 CSFV infection prevents the induction of IFN in response to dsRNA but does not prevent its action.

During this project were able to obtain preparations of one porcine IFN and porcine IFN. Extensive searches of

commercial sources indicated that other porcine IFNs are not yet commercially available. After standardising the

biological activity of the IFN preparation using a cell based Mx/Cat reporter assay, to allow comparison of

activity with other IFN preparations in the future, the activity of the porcine IFNs against replication of CSFV was

assessed in porcine endothelial cells. Both IFN preparations were able to inhibit CSFV replication, and in this cell

type the IFN preparation was more potent than IFN.

SID 5 (Rev. 3/06) Page 12 of 14

Fig 7 Treatment of cells with one porcine IFN subtype and IFN inhibits CSFV replication in endothelial cells

These results provide proof of principle that CSFV may be tractable to an IFN based intervention. However,

before such an intervention could be established further investigations are required. For example the recent

sequencing of the porcine genome has revealed there are 14 different IFN genes, as well as other more recently

identified interferons. The relative activities of these different proteins on CSFV replication and the existence of

any synergies requires further investigation to allow selection of most the promising candidate molecules to test in

vivo. This will require the production, characterisation and in vitro testing of biologically active preparations of

these candidate porcine immuno-modulatory molecules.

References to published material9. This section should be used to record links (hypertext links where possible) or references to other

published material generated by, or relating to this project.

SID 5 (Rev. 3/06) Page 13 of 14

The Npro product of classical swine fever virus interacts with IkappaBalpha, the NF-kappaB inhibitor.

Doceul V, Charleston B, Crooke H, Reid E, Powell PP, Seago J.

J Gen Virol. 2008 Aug;89(Pt 8):1881-9.

The Npro product of classical swine fever virus and bovine viral diarrhea virus uses a conserved mechanism to target interferon regulatory factor-3.

Seago J, Hilton L, Reid E, Doceul V, Jeyatheesan J, Moganeradj K, McCauley J, Charleston B, Goodbourn S.

J Gen Virol. 2007 Nov;88(Pt 11):3002-6.

Classical swine fever virus protects aortic endothelial cells from pIpC-mediated apoptosisBensaude E, Johns, H., La Rocca, A., Seago, J., Charleston, B., Steinbach, F., Drew, T., Everett, H. and

Crooke, H. 7th ESVV Pestivirus Symposium Uppsala, Sweden; 2008.

Classical swine fever virus infection protects aortic endothelial cells from pIpC mediated apoptosis.Bensaude, E., Johns, H., La Rocca, A., 1Seago, J., 2Charleston, B., Steinbach, F.,

Drew, T., Everett, H. and Crooke, H.

Oral presentation at Society for General Microbiology, Edinburgh March 2008

Role of classical swine fever virus in inhibiting apoptosisH.Everett, E.Bensaude, A.La Rocca, F.Steinbach, T.Drew, H.Crooke

Presentation 26th Annual meeting American society for Virology Oregon State University July 14-17th 2007

SID 5 (Rev. 3/06) Page 14 of 14

http://www.ncbi.nlm.nih.gov/pubmed/17947522?ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum



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