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African swine fever virus encodes proteins that inhibit apoptosis including one member of the BCL-2 family, A179L. Deletion of the A179L gene from the virulent genotype I isolate Benin 97/1 compared to Benin 97/1 expressing A179L or mock-infected macrophages, resulted in increased Caspase 3 and 7 activity, annexin V binding to surface phosphatidyl serine and DNA fragmentation, measured by terminal deoxynucleotidyl transferase nick-end labelling. These results confirmed that apoptosis was induced earlier in macrophages infected with the BeninΔA179L virus. Increased cell entry of the A179L gene-deleted virus was indicated at early times since up to double the numbers of cells expressed fluorescent protein from the virus genome. Yields of infectious virus were similar over a single cycle but were significantly lower for the A179L gene-deleted virus over a multi-step growth cycle. Pigs immunised and boosted with the BeninΔA179L virus showed no clinical signs, although a weak cellular response to ASFV was observed showing that the virus had replicated. The immunised pigs were not protected against challenge with the virulent parental virus Benin 97/1 although viremia was lower at 3 days post-challenge compared to the control non-immune pigs. The reduced levels of virus replication in macrophages probably limited induction of a protective immune response. The results show an important role for the A179L protein in virus replication in macrophages and virulence in pigs.IMPORTANCEAfrican swine fever virus (ASFV) causes a lethal disease of pigs that has spread extensively in Africa, Europe and Asia. The virus codes for more than 150 proteins, many of which help the virus to evade the host’s defences following infection. We investigated the effect of deleting one of these genes, A179L, from the genome of an ASFV isolate that causes death of infected pigs. A179L belongs to the BCL-2 protein family, consisting of members which promote or inhibit apoptosis with A179L belonging to the latter. Deleting the A179L gene reduced ASFV replication and spread between macrophages, its main target cells. This was correlated with an increase in cell death. Pigs infected with the virus with A179L gene deleted did not show signs of disease and no virus replication was detected in blood. A low immune response was generated but the immunised pigs were not protected when challenged with the parental deadly virus. The results show that the A179L gene is important for ASFV to replicate efficiently in cells and in animals.

Primary cultures represent the most reliable method to isolate and propagate field isolates of African swine fever virus (ASFV ). Within the pig ASFV predominantly targets the reticuloendothelial system for replication; therefore, primary macrophage cell cultures are commonly used to isolate, propagate, and study the virus life cycle in the laboratory. In this chapter we will describe methods for the direct isolation of pulmonary alveolar macrophages by lung lavage and the culture of monocyte-derived macrophages from pig blood. We also include a method for the positive selection of CD14+ monocytes as a source for monocyte-derived macrophages from pig blood using microbeads.

Real-time polymerase chain reaction (PCR) for the detection of African swine fever virus (ASFV) is the tool of choice for the diagnostic laboratory and is a robust and easily scalable method for the researcher analyzing viral replication both in vitro and in vivo. In this chapter, we describe protocols for both quantitative real-time polymerase chain reactions (qPCR) and non-quantitative real-time polymerase chain reactions (real-time PCR) for the detection of African swine fever virus genome in a range of samples.

The main target cells for African swine fever virus (ASFV) replication in pigs are of monocyte macrophage lineage and express markers typical of the intermediate to late stages of differentiation. The lack of a porcine cell line, which accurately represents these target cells, limits research on virus host interactions and the development of live-attenuated vaccine strains. We show here that the continuously growing, growth factor dependent ZMAC-4 porcine macrophage cell line is susceptible to infection with eight different field isolates of ASFV. Replication in ZMAC-4 cells occurred with similar kinetics and to similar high titres as in primary porcine bone marrow cells. In addition we showed that twelve passages of an attenuated strain of ASFV, OURT88/3, in ZMAC-4 cells did not reduce the ability of this virus to induce protection against challenge with virulent virus. Thus, the ZMAC-4 cells provide an alternative to primary cells for ASFV replication.

21 Pàg., African swine fever virus multigene family (MGF) 360 and 505 genes have roles in suppressing the type I interferon response and in virulence in pigs. The role of the individual genes is poorly understood. Different combinations of these genes were deleted from the virulent genotype II Georgia 2007/1 isolate. Deletion of five copies of MGF 360 genes, MGF360-10L, -11L, -12L, -13L, and -14L, and three copies of MGF505-1R, -2R, and -3R reduced virus replication in macrophages and attenuated virus in pigs. However, only 25% of the immunized pigs were protected against challenge. Deletion of MGF360-12L, -13L, and -14L and MGF505-1R in combination with a negative serology marker, K145R (GeorgiaΔK145RΔMGF(A)), reduced virus replication in macrophages and virulence in pigs, since no clinical signs or virus genome in blood were observed following immunization. Four of six pigs were protected after challenge. In contrast, deletion of MGF360-13L and -14L, MGF505-2R and -3R, and K145R (GeorgiaΔK145RΔMGF(B)) did not reduce virus replication in macrophages. Following immunization of pigs, clinical signs were delayed, but all pigs reached the humane endpoint. Deletion of genes MGF360-12L, MGF505-1R, and K145R reduced replication in macrophages and attenuated virulence in pigs since no clinical signs or virus genome in blood were observed following immunization. Thus, the deletion of MGF360-12L and MGF505-1R, in combination with K145R, was sufficient to dramatically attenuate virus infection in pigs. However, only two of six pigs were protected, suggesting that deletion of additional MGF genes is required to induce a protective immune response. Deletion of MGF360-12L, but not MGF505-1R, from the GeorgiaΔK145R virus reduced virus replication in macrophages, indicating that MGF360-12L was most critical for maintaining high levels of virus replication in macrophages. IMPORTANCE African swine fever has a high socioeconomic impact and no vaccines to aid control. The African swine fever virus (ASFV) has many genes that inhibit the host's interferon response. These include related genes that are grouped into multigene families, including MGF360 and 505. Here, we investigated which MGF360 and 505 genes were most important for viral attenuation and protection against genotype II strains circulating in Europe and Asia. We compared viruses with deletions of MGF genes. Deletion of just two MGF genes in combination with a third gene, K145R, a possible marker for vaccination, is sufficient for virus attenuation in pigs. Deletion of additional MGF360 genes was required to induce higher levels of protection. Furthermore, we showed that the deletion of MGF360-12L, combined with K145R, impairs virus replication in macrophages in culture. Our results have important implications for understanding the roles of the ASFV MGF genes and for vaccine development., We acknowledge funding from BBSRC Grant numbers BBS/E/1/00007039, 7031, 7038 and 7034 and support from the Pirbright flow cytometry and sequencing facilities

19 Pág., This research was funded by Biotechnology and Biological Sciences Research Council, grant number BBS/E/I/00007039/7031/7034 with support by the Pirbright flow cytometry and sequencing facilities. Some parts of the quoted research were funded in part by UK Aid from the UK Government through Global Alliance for Livestock Veterinary Medicines (GALVmed) (grant number and FCDO. The findings and conclusions contained within are those of the authors and do not necessarily reflect positions or policies of GALVmed.

The limited knowledge on the role of many of the approximately 170 proteins encoded by African swine fever virus restricts progress towards vaccine development. In this study we investigated the effect of deleting combinations of different genes from a previously attenuated virus, BeninΔDP148R on: virus replication in macrophages, virus persistence and clinical signs post immunization, and induction of protection against challenge. Deletion of either EP402R or EP153R genes individually or in combination from BeninΔDP148R did not reduce virus replication in vitro. However, deletion of EP402R dramatically reduced viral persistence in vivo, whilst maintaining high levels of protection against challenge. The additional deletion of EP153R (BeninΔDP148RΔEP153RΔEP402R) further attenuated the virus and no viremia or clinical signs were observed post immunization. This was associated with decreased protection and detection of moderate levels of challenge virus in blood. Interestingly, the deletion of EP153R alone from BeninΔDP148R did not result in further virus attenuation and a slight increase in virus genome copies in blood was observed at different times post immunization when compared with BeninΔDP148R. These results show that EP402R and EP153R have a synergistic role in promoting viremia, however EP153R alone does not seem to have a major impact on virus levels in blood.

© 2020 by the authors., Classical approaches to African swine fever virus (ASFV) vaccine development have not been successful; inactivated virus does not provide protection and use of live attenuated viruses generated by passage in tissue culture had a poor safety profile. Current African swine fever (ASF) vaccine research focuses on the development of modified live viruses by targeted gene deletion or subunit vaccines. The latter approach would be differentiation of vaccinated from infected animals (DIVA)-compliant, but information on which viral proteins to include in a subunit vaccine is lacking. Our previous work used DNA-prime/vaccinia-virus boost to screen 40 ASFV genes for immunogenicity, however this immunization regime did not protect animals after challenge. Here we describe the induction of both antigen and ASFV-specific antibody and cellular immune responses by different viral-vectored pools of antigens selected based on their immunogenicity in pigs. Immunization with one of these pools, comprising eight viral-vectored ASFV genes, protected 100% of pigs from fatal disease after challenge with a normally lethal dose of virulent ASFV. This data provide the basis for the further development of a subunit vaccine against this devastating disease., C.L.N., L.C.G. and L.K.D. were supported by Department for Food, Environment and Rural Affairs (DEFRA) grants SE1514, SE1515 and SE1516 as well Biotechnology and Biological Sciences Research Council (BBSRC) grants, BBS/E/I/00007030, BBS/E/I/00007031, BBS/E/I/00007034, BBS/E/I/00007035, BBS/E/I/00007036, BBS/E/I/00007037, BBS/E/I/00007038 and BBS/E/I/00007039. G.T. was also supported by SE1514 and SE1515 and M.M. was supported by SE1515. A.L.R and R.P. were supported by DEFRA grants SE1515 and SE1516 respectively. H.G. was supported by DEFRA grant SE1516. G.L.S. was supported by BBSRC grant BBS/E/I/00002120.

African swine fever (ASF) is a lethal haemorrhagic disease of domestic pigs for which there is no vaccine. Strains of the virus with reduced virulence can provide protection against related virulent strains of ASFV, but protection is not 100% and there are concerns about the safety profile of such viruses. However, they provide a useful tool for understanding the immune response to ASFV and previous studies using the low virulent isolate OUR T88/3 have shown that CD8+ cells are crucial for protection. In order to develop a vaccine that stimulates an effective anti-ASFV T-cell response we need to know which of the >150 viral proteins are recognized by the cellular immune response. Therefore, we used a gamma interferon ELIspot assay to screen for viral proteins recognized by lymphocytes from ASF-immune pigs using peptides corresponding to 133 proteins predicted to be encoded by OUR T88/3. Eighteen antigens that were recognized by ASFV-specific lymphocytes were then incorporated into adenovirus and MVA vectors, which were used in immunization and challenge experiments in pigs. We present a systematic characterization of the cellular immune response to this devastating disease and identify proteins capable of inducing ASFV-specific cellular and humoral immune responses in pigs. Pools of viral vectors expressing these genes did not protect animals from severe disease, but did reduce viremia in a proportion of pigs following ASFV challenge.

Summary The attenuated African swine fever virus genotype I strain OURT88/3 has previously been shown to induce protection of European breeds of domestic pigs against challenge with virulent isolates. To determine whether protective immune responses could also be induced in indigenous breeds of pigs from the Kinshassa region in Democratic Republic of Congo, we immunized a group of eight pigs with OURT88/3 strain and challenged the pigs 3 weeks later with virulent genotype I strain OURT88/1. Four of the pigs were protected against challenge. Three of the eight pigs died from African swine fever virus and a fourth from an unknown cause. The remaining four pigs all survived challenge with a recent virulent genotype I strain from the Democratic Republic of Congo, DRC 085/10. Control groups of non-immune pigs challenged with OURT88/1 or DRC 085/10 developed signs of acute ASFV as expected and had high levels of virus genome in blood.

African swine fever virus (ASFV) causes an acute hemorrhagic fever in domestic pigs, with high socioeconomic impact. No vaccine is available, limiting options for control. Although live attenuated ASFV can induce up to 100% protection against lethal challenge, little is known of the antigens which induce this protective response. To identify additional ASFV immunogenic and potentially protective antigens, we cloned 47 viral genes in individual plasmids for gene vaccination and in recombinant vaccinia viruses. These antigens were selected to include proteins with different functions and timing of expression. Pools of up to 22 antigens were delivered by DNA prime and recombinant vaccinia virus boost to groups of pigs. Responses of immune lymphocytes from pigs to individual recombinant proteins and to ASFV were measured by interferon gamma enzyme-linked immunosorbent spot (ELISpot) assays to identify a subset of the antigens that consistently induced the highest responses. All 47 antigens were then delivered to pigs by DNA prime and recombinant vaccinia virus boost, and pigs were challenged with a lethal dose of ASFV isolate Georgia 2007/1. Although pigs developed clinical and pathological signs consistent with acute ASFV, viral genome levels were significantly reduced in blood and several lymph tissues in those pigs immunized with vectors expressing ASFV antigens compared with the levels in control pigs. IMPORTANCE The lack of a vaccine limits the options to control African swine fever. Advances have been made in the development of genetically modified live attenuated ASFV that can induce protection against challenge. However, there may be safety issues relating to the use of these in the field. There is little information about ASFV antigens that can induce a protective immune response against challenge. We carried out a large screen of 30% of ASFV antigens by delivering individual genes in different pools to pigs by DNA immunization prime and recombinant vaccinia virus boost. The responses in immunized pigs to these individual antigens were compared to identify the most immunogenic. Lethal challenge of pigs immunized with a pool of antigens resulted in reduced levels of virus in blood and lymph tissues compared to those in pigs immunized with control vectors. Novel immunogenic ASFV proteins have been identified for further testing as vaccine candidates.

Highlights • Immunised pigs with BeninΔMGF were protected against parental virulent ASFV strain. • To improve safety and efficacy new doses and routes of immunisation were tested. • Intramuscular immunisation of high doses showed the best percentage of protection. • A new ELISA detected specific IgM antibodies at early stages after ASFV infection. • Early induction of IFNγ and IL-10 in vaccinated pigs probably critical to protection., A live attenuated African swine fever virus (ASFV) vaccine candidate, produced by deletion of several genes belonging to multi-gene families MGF360 and 505 from virulent Benin 97/1 strain (BeninΔMGF), induces protection in pigs against parental virulent strain. In order to better define the safety and efficacy of this attenuated vaccine candidate and to understand protective mechanisms, we extended previous studies by intramuscular immunisation of pigs with the deletion mutant BeninΔMFG at different doses (102, 103, 104 TCID50), together with intranasal immunisation at the 103 dose. Results demonstrated a strong correlation between both doses and routes of immunisation of BeninΔMFG and the percentage of protection achieved, the onset of clinical signs, the viremia levels reached and the onset of death in non-protected pigs. The results show that the intramuscular route using high doses (104 TCID50) is the best option for immunisation. Only transient increase in temperature associated with a peak of virus genome levels was observed in most pigs after immunisation. Then, virus genome levels progressively decreased throughout the experiment until reaching low or undetectable levels in those protected pigs that survived after challenge. The IgM antibody responses following immunisation were detected between day 7–10 post-immunisation and remained at elevated levels for 10–18 days in most pigs before dropping. IgG was detected from day 15 to 21 post-immunisation and maintained at increased levels for the remainder of the experiment in most pigs. Induction of IFNγ and IL-10 was detected by ELISA in sera from some pigs immunised with 103 TCID50 by intramuscular or intranasal route at early times post-immunisation. IL-10 was also detected in serum from some non-protected pigs included in these groups after challenge.

African swine fever virus (ASFV) causes a lethal haemorrhagic disease of swine which can be transmitted through direct contact with infected animals and their excretions or indirect contact with contaminated fomites. The shedding of ASFV by infected pigs and the stability of ASFV in the environment will determine the extent of environmental contamination. The recent outbreaks of ASF in Europe make it essential to develop disease transmission models in order to design effective control strategies to prevent further spread of ASF. In this study, we assessed the shedding and stability of ASFV in faeces, urine and oral fluid from pigs infected with the Georgia 2007/1 ASFV isolate. The half‐life of infectious ASFV in faeces was found to range from 0.65 days when stored at 4°C to 0.29 days when stored at 37°C, while in urine it was found to range from 2.19 days (4°C) to 0.41 days (37°C). Based on these half‐lives and the estimated dose required for infection, faeces and urine would be estimated to remain infectious for 8.48 and 15.33 days at 4°C and 3.71 and 2.88 days at 37°C, respectively. The half‐life of ASFV DNA was 8 to 9 days in faeces and 2 to 3 days in oral fluid at all temperatures. In urine, the half‐life of ASFV DNA was found to be 32.54 days at 4°C decreasing to 19.48 days at 37°C. These results indicate that ASFV in excretions may be an important route of ASFV transmission. ISSN:1865-1674 ISSN:1865-1682

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., Highlights • Attenuated strain OUR T88/3 protects pigs against challenge with virulent strain OUR T88/1. • OUR T88/3 encodes virulence associated genes DP71L and DP96R. • Construction of recombinant virus OUR T88/3ΔDP2 with DP71L and DP96R genes deleted. • Growth of recombinant virus OUR T88/3ΔDP2 in vitro is similar to OUR T88/3. • Pigs inoculated with OUR T88/3ΔDP2 show reduced protection when challenged with OUR T88/1

The African swine fever virus DP71L protein recruits protein phosphatase 1 (PP1) to dephosphorylate the translation initiation factor 2α (eIF2α) and avoid shut-off of global protein synthesis and downstream activation of the pro-apoptotic factor CHOP. Residues V16 and F18A were critical for binding of DP71L to PP1. Mutation of this PP1 binding motif or deletion of residues between 52 and 66 reduced the ability of DP71L to cause dephosphorylation of eIF2α and inhibit CHOP induction. The residues LSAVL, between 57 and 61, were also required. PP1 was co-precipitated with wild type DP71L and the mutant lacking residues 52- 66 or the LSAVL motif, but not with the PP1 binding motif mutant. The residues in the LSAVL motif play a critical role in DP71L function but do not interfere with binding to PP1. Instead we propose these residues are important for DP71L binding to eIF2α., Highlights • The African swine fever virus DP71L protein recruits protein phosphatase 1 (PP1) to dephosphorylate translation initiation factor eIF2α (eIF2α). • The residues V16, F18 of DP71L are required for binding to the α, β and γ isoforms of PP1 and for DP71L function. • The sequence LSAVL downstream from the PP1 binding site (residues 57–61) are also important for DP71L function. • DP71L mutants of the LSAVL sequence retain ability to co-precipitate with PP1 showing these sequences have a different role to PP1 binding.

African swine fever virus (ASFV) can cause an acutely fatal haemorrhagic fever in domestic pigs although in its natural hosts, warthogs, bushpigs and the soft tick vector, Ornithodoros moubata, ASFV causes inapparent persistent infections. The virus is a large, cytoplasmic, double-stranded DNA virus which has a tropism for macrophages. As it is the only member of the Asfarviridae family, ASFV encodes many novel genes not encoded by other virus families. The ability of the virus to persist in its natural hosts and in domestic pigs, which recover from infection with less virulent isolates, shows that the virus has effective mechanisms to evade host defence systems. This review focuses on recent progress made in understanding the function of ASFV-encoded proteins, which are involved in modulating the host response to infection. Growing evidence suggests that a major strategy used by the virus is to modulate signalling pathways in infected macrophages, thus interfering with the expression of a large number of immunomodulatory genes. One potent immunomodulatory protein, A238L, inhibits both activation of the host NFkappaB transcription factor and inhibits calcineurin phosphatase activity. Calcineurin-dependent pathways, including activation of the NFAT transcription factor, are therefore inhibited. Another ASFV-encoded protein, CD2v, resembles the host CD2 protein, which is expressed on T cells and NK cells. This virus protein causes the adsorption of red blood cells around virus-infected cells and extracellular virus particles. Expression of the CD2v protein aids virus dissemination in pigs and the protein also has a role in impairing bystander lymphocyte function. This may be mediated either by a direct interaction of CD2v extracellular domain with ligands on lymphocytes or by an indirect mechanism involving interaction of the CD2v cytoplasmic tail with host proteins involved in signalling or trafficking pathways. Two ASFV proteins, an IAP and a Bcl2 homologue, inhibit apoptosis in infected cells and thus facilitate production of progeny virions. The prediction is that half to two-thirds of the approximately 150 genes encoded by ASFV are not essential for replication in cells but have an important role for virus survival and transmission in its hosts. These genes provide an untapped repository, and will be valuable tools for deciphering not only how the virus manipulates the host response to infection to avoid elimination, but also useful for understanding important host anti-viral mechanisms. In addition, they may provide leads for discovery of novel immunomodulatory drugs.

PCR analysis of the genomes of 18 different African swine fever virus (ASFV) isolates showed that the I14L open reading frame (ORF) was present as either a long form or short form in all of the isolates. Sequencing of the ORF from eight isolates confirmed that both forms of the ORF were well conserved. Antisera raised against the I14L protein identified the long form of the protein as a 21 kDa protein expressed late during ASFV infection. Immunofluorescent analysis of transiently expressed haemagglutinin-tagged forms of the I14L protein showed that the long form of the protein localized predominantly to the nucleus and within the nucleoli. In contrast, although the short form of the protein was also present predominantly in the nucleus, it did not localize to the nucleoli. Deletion of the N-terminal 14 amino acids from the long form of the I14L protein, which includes a high proportion of basic Arg/Lys residues, abolished the specific nucleolar localization of the protein, although the protein was still present in the nucleus. Addition of this 14 amino acid sequence to beta-galactosidase or replacement of the N-terminal 14 amino acids of the I14L short form with those from the long form directed both of these modified proteins to the nucleolus. This indicates that this 14 amino acid sequence contains all the signals required for nucleolar localization.

African swine fever virus is a large DNA virus which can cause an acute haemorrhagic fever in pigs resulting in high mortality. No vaccine is available, limiting options for control. The virus encodes up to 165 genes and virus particles are multi-layered and contain more than 50 proteins. Pigs immunised with natural low virulence isolates or attenuated viruses produced by passage in tissue culture and by targeted gene deletions can be protected against challenge with virulent viruses. CD8+ cells are required for protection induced by attenuated strain OURT88/3. Passive transfer of antibodies from immune to naive pigs can also induce protection. Knowledge of the genome sequences of attenuated and virulent strains and targeted gene deletions from virulent strains have identified a number of virus genes involved in virulence and immune evasion. This information can be used to produce rationally attenuated vaccine strains. Virus antigens that are targets for neutralising antibodies have been identified and immunisation with these recombinant proteins has been shown to induce partial protection. However knowledge of antigens which encode the dominant protective epitopes recognised by CD8+ T cells is lacking.

We have compared the complete genome sequences of the vaccine strain of rinderpest virus and the virulent strain from which it was derived. Only 87 bases differed between the two genomes (0.55%). Possibly significant differences in amino acid sequence were found in the N, P, F, H and L proteins. A number of differences were also found in the leader region (3' end of the genome), whilst the trailer region appears to be more conserved. In addition, the length of the genome was found in both cases to be 15882, an exact multiple of six, fulfilling predictions made earlier based on work with Sendai and measles viruses.

We have cloned and sequenced the entire fusion (F) protein gene of the RBOK vaccine strain of rinderpest virus and the coding regions for the F genes of two mild field isolates of the virus from Africa. Analysis of the nucleotide and the predicted amino acid sequences showed that the vaccine virus was more than 99% identical in the protein coding region to the virulent Kabete O strain from which it was derived, whereas the field isolates differed by 10 to 12% from each other and from the vaccine strain. No changes were found in the F protein which could explain attenuation of the vaccine; however, each of the mild field isolates had amino acid changes in important functional areas which may be related to their attenuated phenotype.

The African swine fever virus (ASFV)-encoded CD2v transmembrane protein is required for the hemadsorption of red blood cells around infected cells and is also required for the inhibition of bystander lymphocyte proliferation in response to mitogens. We studied the expression of CD2v by expressing the gene with a V5 tag downstream from the signal peptide near the N terminus and a hemagglutinin (HA) tag at the C terminus. In ASFV-infected cells, a full-length glycosylated form of the CD2v protein, which migrated mainly as a 89-kDa product, was detected, as well as an N-terminal glycosylated fragment of 63 kDa and a C-terminal nonglycosylated fragment of 26 kDa. All of these forms of the protein were localized in the membrane fraction of cells. The 26-kDa C-terminal fragment was also produced in infected cells treated with brefeldin A. These data indicate that the CD2v protein is cleaved within the luminal domain and that this occurs in the endoplasmic reticulum or Golgi compartments. Confocal microscopy showed that most of the expressed CD2v protein was localized within cells rather than at the cell surface. Comparison of the localization of full-length CD2v with that of a deletion mutant lacking all of the cytoplasmic tail apart from the 12 membrane-proximal amino acids indicated that signals within the cytoplasmic tail are responsible for the predominant localization of the full-length and C-terminal 26-kDa fragment within membranes around the virus factories, which contain markers for the Golgi compartment. Processing of the CD2v protein was not observed in uninfected cells, indicating that it is induced by ASFV infection.

The virus epizootic which resulted in significant mortality in Siberian seals (Phoca sibirica) in Lake Baikal during 1987/88 was caused by canine distemper virus. Sequence analysis of the virus glycoprotein genes revealed that it was most closely related to recent European field isolates of canine distemper virus. This paper presents evidence that the same virus continued to circulate in seals in Lake Baikal after the initial epizootic. Three out of 45 brain tissue samples collected from seals culled in the spring of 1992 were positive for canine distemper virus-specific nucleic acid by the reverse transcription/polymerase chain reaction and the sequences were closely related to that of the original virus isolated in 1988.

The predicted extracellular domain of the CD2v protein of African swine fever virus (ASFV) shares significant similarity to that of the CD2 protein in T cells but has a unique cytoplasmic domain of unknown function. Here we have shown that CD2v is expressed as a glycoprotein of approximately 105 kDa in ASFV-infected cells. In the absence of an extracellular ligand, the majority of CD2v appears to localize to perinuclear membrane compartments. Furthermore, we have shown using the yeast two-hybrid system and by direct binding studies that the cytoplasmic tail of CD2v binds to the cytoplasmic adaptor protein SH3P7 (mAbp1, HIP55), which has been reported to be involved in diverse cellular functions such as vesicle transport and signal transduction. A cDNA clone encoding a variant form of SH3P7 could also be identified and was found to be expressed in a wide range of porcine tissues. Deletion mutagenesis identified proline-rich repeats of sequence PPPKPC in the ASFV CD2v protein to be necessary and sufficient for binding to the SH3 domain of SH3P7. In ASFV-infected cells, CD2v and SH3P7 co-localized in areas surrounding the perinuclear virus factories. These areas also stained with an antibody that recognizes a Golgi network protein, indicating that they contained membranes derived from the Golgi network. Our data provide a first molecular basis for the understanding of the immunomodulatory functions of CD2v in ASFV-infected animals.

African swine fever virus (ASFV) is a large DNA virus which replicates in the cytoplasm of infected cells and contains a long, linear, double-stranded DNA genome encoding ca. 150 open reading frames (ORFs). The virus causes a hemorrhagic fever in domestic pigs but no disease signs in its natural hosts, warthogs, bushpigs, and soft ticks of the Ornithodoros species Ornithodoros moubata and O. erraticus, which act as vectors for the virus. ASFV has a replication strategy similar to that of poxviruses, but its structure is different, and it is classified as the only member of a new virus family, the Asfarviridae (9). ASFV encodes enzymes and factors needed for replication and transcription of the virus genome, as well as proteins that may play a role in interactions with the host that are important for virus survival and transmission (5, 40). Among the latter class of genes are those related to cellular genes, including genes encoding proteins that inhibit apoptosis, such as proteins related to IAP and Bcl2 (6, 26, 32). The ASFV A238L protein prevents activation of NF-κB-dependent gene transcription (29, 33, 37) and also binds to and inhibits calcineurin phosphatase activity. Calcineurin-dependent pathways, such as the activation of NFAT transcription factor, are therefore inhibited (21, 22). The ASFV EP402R protein resembles the host cell surface protein CD2 and is required for the adsorption of red blood cells around virus-infected cells and for virus-induced inhibition of bystander lymphocyte proliferation in response to mitogens (4, 5, 34). The low level of amino acid similarity between ASFV-encoded proteins and other proteins makes predicting the function of virus-encoded proteins difficult. To facilitate this, we have identified host proteins that bind to virus-encoded proteins by using the yeast two-hybrid system. The ASFV j4R protein has no significant homology with other proteins in the database, and to date, no functional data on the j4R protein have been published. In the present study we show that j4R is expressed late after infection and is conserved in the genomes of different virus isolates. We have also shown that the α chain of nascent polypeptide-associated complex (i.e., αNAC) binds to ASFV j4R protein. The αNAC protein has roles in both translation and transcription. By binding to nascent polypeptide chains as they emerge from ribososmes, αNAC is proposed to prevent inappropriate targeting of polypeptides without signal sequences to the secretory pathway (38, 39, 43). αNAC has also been shown to act as a transcriptional coactivator potentiating transcription mediated by the c-Jun factor (23, 41). Here we demonstrate, by using recombinant proteins, that j4R binds directly to αNAC and, by coprecipitation, that j4R and αNAC are present in complexes in cells. Confocal microscopy suggested that a proportion of j4R and αNAC colocalizes in the cytoplasm in cells. By binding to αNAC the j4R protein may modulate either or both of these functions of αNAC.

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.

African swine fever virus (ASFV) is a complex DNA virus and the aetiological agent of a domestic pig disease that can assume different clinical forms, ranging from an acute haemorrhagic disease to unapparent infections. The complexity of the immune response has impaired the development of an effective vaccine against ASFV infection. Previous work has established that immunization of pigs with attenuated replication competent ASFV strains can induce good levels of protection against lethal challenge with virulent strains. However in some pigs adverse clinical signs occur following immunization. On the other hand, due to the highly acute nature of the disease following infection with the virulent strains, it is clear that the porcine innate response is insufficient to prolong the life of the animal until the serological and cellular adaptive immune responses provide immediate protection and, importantly, the generation of the immunological memory crucial to vaccine development. The type I interferon (IFN) response is the first line of innate immunity against viral infection. Most, if not all viruses have to overcome this host defense before they can establish productive infection. We are currently characterizing the induction of type I IFN in primary porcine macrophages infected with strains of different virulence. A better understating of how the IFN response is modulated by these different viruses, will provide a rational basis for the development of novel vaccine strategies, for example, further attenuation of the virus by deleting genes involved in the evasion of the IFN host system is in progress.