African swine fever virus (ASFV) encodes multiple copies of MGF360 and MGF530/505 gene families. These genes have been implicated in the modulation of the type I interferon (IFN) response. We investigated the effect of modulating the IFN response on virus attenuation and induction of protective immunity by deleting genes MGF360 (MGF360-10L, 11L, 12L, 13L, 14L) and MGF530/505 (MGF530/505-1R, 2R and 3R) and interrupting genes (MGF360-9L and MGF530/505-4R) in the genome of the virulent ASFV isolate Benin 97/1. Replication of this deletion mutant, BeninΔMGF, in porcine macrophages in vitro was similar to that of the parental virulent virus Benin 97/1 and the natural attenuated isolate OURT88/3, which has a similar deletion of MGF360 and 530/505 genes. Levels of IFN-β mRNA in macrophages infected with virulent Benin 97/1 isolate were barely detectable but high levels were detected in macrophages infected with OURT88/3 and intermediate levels in macrophages infected with BeninΔMGF. The data confirms that these MGF360 and MGF530/505 genes have roles in suppressing induction of type I IFN. Immunisation and boost of pigs with BeninΔMGF showed that the virus was attenuated and all pigs (5/5) were protected against challenge with a lethal dose of virulent Benin 97/1. A short transient fever was observed at day 5 or 6 post-immunisation but no other clinical signs. Following immunisation and boost with the OURT88/3 isolate 3 of 4 pigs were protected against challenge. Differences were observed in the cellular and antibody responses in pigs immunised with BeninΔMGF compared to OURT88/3. Deletion of IFN modulators is a promising route for construction of rationally attenuated ASFV candidate vaccine strains.

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The transcription factor NFAT (nuclear factor of activated T cells) controls the expression of many immunomodulatory proteins. African swine fever virus inhibits proinflammatory cytokine expression in infected macrophages, and a viral protein A238L was found to display the activity of the immunosuppressive drug cyclosporin A by inhibiting NFAT-regulated gene transcription in vivo. This it does by binding the catalytic subunit of calcineurin and inhibiting calcineurin phosphatase activity.

cDNA cassettes encoding the foot-and-mouth disease virus (FMDV) structural protein precursor (P1-2A) together with the 3C protease, which cleaves this molecule to lAB, 1C and 1D, were constructed. These cassettes were introduced into vaccinia virus (VV) transfer vectors. Attempts to isolate recombinant Ws constitutively expressing these cassettes were unsuccessful. However, when the P1-2A-3C cassette was placed under the control of the bacteriophage T7 promoter, stable VV/FMDV recombinants were isolated. Co-infection with recombinant VV vTF7-3 (which expresses T7 RNA polymerase) led to the production of correctly processed FMDV capsid proteins. Analysis by sucrose gradient centrifugation showed that material which co-sedimented with natural empty capsid particles (70S) was formed. Electron microscopy revealed empty capsid-like particles with diameters of about 30 nm. Studies using monoclonal antibodies specific for conformational epitopes indicated that the antigenicity of the synthetic particles was similar to whole virions and natural empty capsid particles. Surprisingly, merely the modification of a single amino acid residue within the myristoylation consensus sequence at the N terminus of P 1-2A allowed the isolation of a recombinant VV which constitutively expressed the correctly processed proteins. However, the capsid proteins expressed from this mutant cassette failed to assemble into 70S empty particles.

Foot-and-mouth disease virus (FMDV) manifests an extreme sensitivity to acid, which is thought to be important for entry of the RNA genome into the cell. We have compared the low-pH-induced disassembly in vitro of virions and natural empty capsids of three subtypes of serotype A FMDV by enzyme-linked immunosorbent assay and sucrose gradient sedimentation analysis. For all three subtypes (A22 Iraq 24/64, A10 61 , and A24 Cruzeiro), the empty capsid was more stable by 0.5 pH unit on average than the corresponding virion. Unexpectedly, in the natural empty capsids used in this study, the precursor capsid protein VP0 was found largely to be cleaved into VP2 and VP4. For picornaviruses the processing of VP0 is closely associated with encapsidation of viral RNA, which is considered likely to play a catalytic role in the cleavage. Investigation of the cleavage of VP0 in natural empty capsids failed to implicate the viral RNA. However, it remains possible that these particles arise from abortive attempts to encapsidate RNA. Empty capsids expressed from a vaccinia virus recombinant showed essentially the same acid lability as natural empty capsids, despite differing considerably in the extent of VP0 processing, with the synthetic particles containing almost exclusively uncleaved VP0. These results indicate that it is the viral RNA that modulates acid lability in FMDV. In all cases the capsids dissociate at low pH directly into pentameric subunits. Comparison of the three viruses indicates that FMDV A22 Iraq is about 0.5 pH unit more sensitive to low pH than types A10 61 and A24 Cruzeiro. Sequence analysis of the three subtypes identified several differences at the interface between pentamers and highlighted a His-␣-helix dipole interaction which spans the pentamer interface and appears likely to influence the acid lability of the virus.

We used a porcine microarray containing 2,880 cDNAs to investigate the response of macrophages to infection by a virulent African swine fever virus (ASFV) isolate, Malawi LIL20/1. One hundred twenty-five targets were found to be significantly altered at either or both 4 h and 16 h postinfection compared with targets after mock infection. These targets were assigned into three groups according to their temporal expression profiles. Eighty-six targets showed increased expression levels at 4 h postinfection but returned to expression levels similar to those in mock-infected cells at 16 h postinfection. These encoded several proinflammatory cytokines and chemokines, surface proteins, and proteins involved in cell signaling and trafficking pathways. Thirty-four targets showed increased expression levels at 16 h postinfection compared to levels at 4 h postinfection and in mock-infected cells. One host gene showed increased expression levels at both 4 and 16 h postinfection compared to levels in mock-infected cells. The microarray results were validated for 12 selected genes by quantitative real-time PCR. Levels of protein expression and secretion were measured for two proinflammatory cytokines, interleukin 1␤ and tumor necrosis factor alpha, during a time course of infection with either the virulent Malawi LIL20/1 isolate or the OUR T88/3 nonpathogenic isolate. The results revealed differences between these two ASFV isolates in the amounts of these cytokines secreted from infected cells. African swine fever virus (ASFV) causes inapparent persistent infections in its natural hosts, warthogs (Phacochoerus aethiopicus), bushpigs (Potamochoerus porcus), and soft ticks (Ornithodoros moubata), which inhabit warthog burrows. In contrast, the virus causes an acute hemorrhagic fever with high mortality rates in pigs, although some low-virulence isolates have been reported (6,33,50,52,53).The virus replicates in cells of the mononuclear phagocytic system and reticulo-endothelial cells of lymphoid tissues and organs. Widespread apoptotic cell death occurs in both T and B lymphocytes of lymphoid tissues (9, 21) and in arteriolar and capillary endothelial cells (10,(15)(16)(17)22). Disseminated intravascular coagulation develops during the late phase of acute infections, and this may lead to the characteristic hemorrhagic syndrome (20,22,41).ASFV is a large, icosahedral, double-stranded DNA virus which replicates in the cytoplasm of infected cells and has been classified as the only member of a new virus family (13), the Asfarviridae. The genome encodes between 160 and 175 proteins, including a number that interfere with host defense systems. These include proteins such as the A238L protein, which inhibits both the activation of the host NF-B transcription factor and calcineurin phosphatase activity. The latter inactivation results in the inhibition of calcineurin-dependent pathways, such as activation of the nuclear factor of activated T cell transcription factors (23,24,37,38,44,47). Members of ASFV multigene families 360 and 53...

African swine fever virus (ASFV) causes an acute haemorrhagic disease of domestic pigs against which there is no effective vaccine. The attenuated ASFV strain OUR T88/3 has been shown previously to protect vaccinated pigs against challenge with some virulent strains including OUR T88/1. Two genes, DP71L and DP96R were deleted from the OUR T88/3 genome to create recombinant virus OUR T88/3ΔDP2. Deletion of these genes from virulent viruses has previously been shown to reduce ASFV virulence in domestic pigs. Groups of 6 pigs were immunised with deletion virus OUR T88/3ΔDP2 or parental virus OUR T88/3 and challenged with virulent OUR T88/1 virus. Four pigs (66%) were protected by inoculation with the deletion virus OUR T88/3ΔDP2 compared to 100% protection with the parental virus OUR T88/3. Thus the deletion of the two genes DP71L and DP96R from OUR T88/3 strain reduced its ability to protect pigs against challenge with virulent virus.

The African swine fever virus protein A238L inhibits activation of NFAT transcription factor by binding calcineurin and inhibiting its phosphatase activity. NFAT controls the expression of many immunomodulatory proteins. Here we describe a 14-amino-acid region of A238L that is needed and sufficient for binding to calcineurin. By introducing mutations within this region, we have identified a motif (PxIxITxC/S) required for A238L binding to calcineurin; a similar motif is found in NFAT proteins. Peptides corresponding to this domain of A238L bind calcineurin but do not inhibit its phosphatase activity. Binding of A238L to calcineurin stabilizes the A238L protein in cells. Although A238L-mediated suppression of NF-B-dependent gene expression occurs by a different mechanism, the A238L-calcineurin interaction may be required to stabilize A238L.Many large DNA viruses encode proteins that help the virus to evade the host immune response (1, 9, 24). African swine fever virus (ASFV), the prototypic member of the African swine fever-like virus genus (10), encodes a potent immunosuppressive protein, A238L. A238L inhibits activation of the NF-B transcription factor (25,28,32) and the activity of the calcium/calmodulin-regulated phosphatase calcineurin (CaN) (23). The virus, therefore, has the potential to inhibit transcriptional activation of immunomodulatory genes dependent on these pathways in infected macrophages.NF-B is retained in the cytoplasm in a complex with the inhibitor protein, IB, in resting cells. Following activation, IB is phosphorylated at two key Ser residues (Ser32 and Ser36 in IB-␣) and is then ubiquitinated and targeted for degradation by the 26S proteasome (for a review, see reference 16). This process exposes nuclear localization signals within nuclear factor B (NF-B) which subsequently translocates to the nucleus and binds to promoters with the appropriate DNA recognition sequence. A238L coprecipitates with the p65 subunit of NF-B (32) and therefore might function as an IB mimic which does not respond to signal-induced degradation.The second function of A238L, inhibition of CaN phosphatase activity, is mediated by direct binding to the catalytic subunit of CaN [CaN(A)] (23). CaN is activated following calcium release and binding of calmodulin, which results in displacement of the autoinhibitory (AI) domain from the enzyme active site. CaN regulates a number of different pathways, including activation of the NFAT family of transcription factors, which are present in a phosphorylated form in the cytoplasm in resting cells. Activation of CaN results in dephosphorylation and nuclear translocation of NFAT factors which, in cooperation with other transcription factors, play an essential role in transcriptional activation of cytokine and other immunomodulatory genes (8,29,30). The immunosuppressive drugs cyclosporin A (CsA) and FK506 bind to CaN in a complex with the immunophilin proteins cyclophilin A (CypA) and FKBP12, respectively (34); this drug-immunophilin complex