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African swine fever (ASF) is a highly contagious and deadly viral disease affecting domestic and wild pigs of all ages. African swine fever virus (ASFV) has spread rapidly through Eastern and Southeastern Asia first appearing in Vietnam in 2019.Molecular typing of African swine fever virus (ASFV) in Vietnam has identified two principal variants circulating based on the sequencing of the intergenic region (IRG) between the I73R and I329L genes. Identification of additional genetic markers would enable higher resolution tracing of outbreaks within the country.Sequence analysis suggested the IRG between the A179L and A137R genes may also exhibit variability, PCR primers were designed and samples from Vietnam were subject to Sanger sequencing.We developed a novel method for sub-grouping of ASFV based on the IRG between the A179L and A137R genes of ASFV. Our results demonstrated that the finding of the insertion or deletion of an 11- nucleotide sequence (GATACAATTGT) between the A179L-A137R genes.The sub-grouping method may provide useful insights into the evolution of genotype II ASFV as well as providing evidence of a relationship between geographically separated outbreaks.

Deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase) of African swine fever virus (ASFV) is an essential enzyme required for efficient virus replication. Previous crystallography data have indicated that dUTPase (E165R) may serve as a therapeutic target for inhibiting ASFV replication, however, the specificity of the targeting site(s) in ASFV dUTPase remains unclear. In this study, 19 mouse monoclonal antibodies (mAbs) were produced, in which four mAbs showed inhibitory reactivity against E165R recombinant protein. Epitope mapping studies indicated that E165R has three major antigenic regions: 100–120 aa, 120–140 aa, and 140–165 aa. Three mAbs inhibited the dUTPase activity of E165R by binding to the highly conserved 149–RGEGRFGSTG–158 amino acid sequence. Interestingly, 8F6 mAb specifically recognized ASFV dUTPase but not Sus scrofa dUTPase, which may be due to structural differences in the amino acids of F151, R153, and F154 in the motif V region. In summary, we developed anti-E165R-specific mAbs, and identified an important antibody-binding antigenic epitope in the motif V of ASFV dUTPase. Our study provides a comprehensive analysis of mAbs that target the antigenic epitope of ASFV dUTPase, which may contribute to the development of novel antibody-based ASFV therapeutics.

African swine fever is a devastating disease of domestic swine and wild boar caused by a large double-stranded DNA virus that encodes for more than 150 open reading frames. There is no licensed vaccine for the disease and the most promising current candidates are modified live viruses that have been attenuated by deletion of virulence factors. Like many viruses African swine fever virus significantly alters the host cell machinery to benefit its replication and viral genes that modify host pathways represent promising targets for development of gene deleted vaccines. Autophagy is an important cellular pathway that is involved in cellular homeostasis, innate and adaptive immunity and therefore is manipulated by a number of different viruses. Autophagy is regulated by a complex protein cascade and here we show that African swine fever virus can block formation of autophagosomes, a critical functional step of the autophagy pathway through at least two different mechanisms. Interestingly this does not require the A179L gene that has been shown to interact with Beclin-1, an important autophagy regulator.

African swine fever virus (ASFV) is a highly contagious pathogen which causes a lethal haemorrhagic fever in domestic pigs and wild boar. The large, double-stranded DNA virus replicates in perinuclear cytoplasmic replication sites known as viral factories. These factories are complex, multi-dimensional structures. Here we investigated the protein and membrane compartments of the factory using super-resolution and electron tomography. Click IT chemistry in combination with stimulated emission depletion (STED) microscopy revealed a reticular network of newly synthesized viral proteins, including the structural proteins p54 and p34, previously seen as a pleomorphic ribbon by confocal microscopy. Electron microscopy and tomography confirmed that this network is an accumulation of membrane assembly intermediates which take several forms. At early time points in the factory formation, these intermediates present as small, individual membrane fragments which appear to grow and link together, in a continuous progression towards new, icosahedral virions. It remains unknown how these membranes form and how they traffic to the factory during virus morphogenesis.

African swine fever virus(ASFV) causes a lethal haemorrhagic disease of domestic pigs, to which there is no vaccine available. ASFV has a large, double-stranded DNA genome that encodes over 150 proteins. Replication takes place in the cytoplasm of the cell and involves complex interactions with host cellular components including small non-coding RNAs (sncRNAs). A number of DNA viruses are known to manipulate sncRNA either by encoding their own or disrupting host sncRNA. In order to investigate the interplay between ASFV and sncRNAs, study of host and viral small RNAs extracted from ASFV-infected primary porcine macrophages (PAMs) was undertaken. We discovered that ASFV infection had only a modest effect on host miRNAs, with only 6 miRNAs differentially expressed during infection. The data also revealed 3 potential novel small RNAs encoded by ASFV, ASFVsRNA1-3. Further investigation of ASFVsRNA2 detected it in lymphoid tissue from pigs with ASF. Overexpression of ASFVsRNA2 led to up to a 1 log reduction in ASFV growth indicating that ASFV utilises a virally-encoded small RNA to disrupt its own replication. This study describes the modest impact of ASFV on host sncRNAs and the identification of a functional ASFV-encoded sncRNA.ImportanceAfrican swine fever (ASF) poses a major threat to pig populations and food security worldwide. The disease is endemic in Africa and Eastern Europe and rapidly emerging into Asia where it has led to the deaths of millions of pigs in the past 12 months. The development of safe and effective vaccines to protect pigs against ASF has been hindered by lack of understanding of the complex interactions between ASFV and the host cell. We focused our work on characterising the interactions between ASFV and sncRNAs. We found only modest changes to host sncRNA abundance after ASFV infection, and discovered a functional ASFV-encoded sncRNA. The knowledge from this study can be exploited to develop more effective ASFV vaccines that take advantage of the sncRNA system.

African swine fever (ASF) is a devastating viral disease of pigs and wild boar, and it threatens global food security. We aimed to identify suitable sample matrices for use in ASF surveillance programs. Six pigs inoculated with ASFV were sampled at postmortem. Blood, bone marrow, ear biopsies, and oral, nasal, and rectal swabs were taken from all pigs. All samples were analyzed using 3 real-time PCR (rtPCR) assays and a LAMP assay. ASFV was detected at > 107 genome copies/mL in blood; bone marrow was found to provide the highest viral load. Ct values provided by the rtPCR assays were correlated, and ASFV was detected in all oral, nasal, and rectal swabs and in all ear biopsy samples irrespective of the location from which they were taken. The LAMP assay had lower sensitivity, and detected ASFV in 54 of 66 positive samples, but delivered positive results within 17 min. We identified additional sample matrices that can be considered depending on the sampling situation: bone marrow had a high probability of detection, which could be useful for decomposed carcasses. However, ear biopsies provide an appropriate, high-throughput sample matrix to detect ASFV and may be useful during surveillance programs.

© 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.

This study compares different combinations of doses and routes of immunisation of pigs with low virulent African swine fever virus (ASFV) genotype I isolate OURT88/3, including the intramuscular and intranasal route, the latter not previously tested. Intranasal immunisations with low and moderate doses (103 and 104 TCID50) of OURT88/3 provided complete protection (100%) against challenge with virulent genotype I OURT88/1 isolate. Only mild and transient clinical reactions were observed in protected pigs. Transient moderate virus genome levels were detected in blood samples after challenge that decreased, but persisted until the end of the experiment in some animals. In contrast, pigs immunised intramuscularly with low and moderate doses (103 and 104 TCID50) displayed lower percentages of protection (50–66%), and low or undetectable levels of virus genome were detected in blood samples throughout the study. In addition, clinical courses observed in protected pigs were asymptomatic. In pigs that were not protected and developed acute ASF, an exacerbated increase of IL-10 sometimes accompanied by an increase of IFNγ was observed before euthanasia. These results showed that factors including delivery route and dose determine the outcome of immunisation with the naturally attenuated isolate OURT88/3., Highlights • Protection induced by combinations of doses and routes of immunisation of pigs with ASFV isolate OUR T88/3 is described. • Intranasal immunisations with low and moderate doses provided complete protection with persistent low virus genome levels. • Intramuscular immunisations with low and moderate doses provided lower protection with undetectable virus genome levels. • Increased IL-10 serum concentrations, sometimes together with increased IFNγ, was observed before death in not-protected pigs. • Changes in serum concentrations of TNFα, IFNγ, IL-4 and IL-10 were not detected in none of immunised protected pigs.

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.

Many of the approximately 165 proteins encoded by the African swine fever virus (ASFV) genome do not have significant similarity to known proteins and have not been studied experimentally. One such protein is DP148R. We showed that the DP148R gene is transcribed at early times postinfection. Deletion of this gene did not reduce virus replication in macrophages, showing that it is not essential for replication in these cells. However, deletion of this gene from a virulent isolate, Benin 97/1, producing the BeninΔDP148R virus, dramatically reduced the virulence of the virus in vivo . All pigs infected with the BeninΔDP148R virus survived infection, showing only transient mild clinical signs soon after immunization. Following challenge with the parental virulent virus, all pigs immunized by the intramuscular route (11/11) and all except one immunized by the intranasal route (5/6) survived. Mild or no clinical signs were observed after challenge. As expected, control nonimmune pigs developed signs of acute African swine fever (ASF). The virus genome and infectious virus were observed soon after immunization, coincident with the onset of clinical signs (∼10 6 genome copies or 50% tissue culture infective doses/ml). The levels of the virus genome declined over an extended period up to 60 days postimmunization. In contrast, infectious virus was no longer detectable by days 30 to 35. Gamma interferon (IFN-γ) was detected in serum between days 4 and 7 postimmunization, and IFN-γ-producing cells were detected in all pigs analyzed following stimulation of immune lymphocytes with whole virus. ASFV-specific antibodies were first detected from day 10 postimmunization. IMPORTANCE African swine fever (ASF) is endemic in Africa, parts of the Trans Caucasus, the Russian Federation, and several European countries. The lack of a vaccine hinders control. Many of the ASF virus genes lack similarity to known genes and have not been characterized. We have shown that one of these, DP148R, is transcribed early during virus replication in cells and can be deleted from the virus genome without reducing virus replication. The virus with the gene deletion, BeninΔDP148R, caused mild clinical signs in pigs and induced high levels of protection against challenge with the parental virulent virus. Therefore, deletion of this gene can provide a target for the rational development of vaccines.

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.

Autophagy is an intracellular pathway that can contribute to innate antiviral immunity by delivering viruses to lysosomes for degradation or can be beneficial for viruses by providing specialized membranes for virus replication. Here, we show that the picornavirus foot-and-mouth disease virus (FMDV) induces the formation of autophagosomes. Induction was dependent on Atg5, involved processing of LC3 to LC3II, and led to a redistribution of LC3 from the cytosol to punctate vesicles indicative of authentic autophagosomes. Furthermore, FMDV yields were reduced in cells lacking Atg5, suggesting that autophagy may facilitate FMDV infection. However, induction of autophagosomes by FMDV appeared to differ from starvation, as the generation of LC3 punctae was not inhibited by wortmannin, implying that FMDV-induced autophagosome formation does not require the class III phosphatidylinositol 3-kinase (PI3-kinase) activity of vps34. Unlike other picornaviruses, for which there is strong evidence that autophagosome formation is linked to expression of viral nonstructural proteins, FMDV induced autophagosomes very early during infection. Furthermore, autophagosomes could be triggered by either UV-inactivated virus or empty FMDV capsids, suggesting that autophagosome formation was activated during cell entry. Unlike other picornaviruses, FMDV-induced autophagosomes did not colocalize with the viral 3A or 3D protein. In contrast, ∼50% of the autophagosomes induced by FMDV colocalized with VP1. LC3 and VP1 also colocalized with the cellular adaptor protein p62, which normally targets ubiquitinated proteins to autophagosomes. These results suggest that FMDV induces autophagosomes during cell entry to facilitate infection, but not to provide membranes for replication.

Highlights ► Assembly of replicase proteins of (+) strand RNA virus occurs on the cytoplasmic face of membrane-bound organelles. ► Replicase assembly leads to membrane invaginations called spherules or double membraned vesicles. ► Replicase assembly can be facilitated by Arf1 dependent recruitment of phosphatidylinositol-4-kinase-III. ► Large DNA viruses replicate and/or assemble in perinuclear factories that resemble cellular aggresomes generated in response to protein aggregation. ► Virus factories segregate viral DNA and RNA and appear to be distinct entities within the cell., Many viruses reorganise cellular membrane compartments and the cytoskeleton to generate subcellular microenvironments called virus factories or ‘viroplasm’. These create a platform to concentrate replicase proteins, virus genomes and host proteins required for replication and also protect against antiviral defences. There is growing interest in understanding how viruses induce such large changes in cellular organisation, and recent studies are beginning to reveal the relationship between virus factories and viroplasm and the cellular structures that house them. In this review, we discuss how three supergroups of (+)RNA viruses generate replication sites from membrane-bound organelles and highlight research on perinuclear factories induced by the nucleocytoplasmic large DNA viruses.

Increasing evidence points to the importance of the interferon (IFN) response in determining the host range and virulence of African swine fever virus (ASFV). Infection with attenuated strains of ASFV leads to the upregulation of genes controlled by IFN pathways, including myxovirus resistance (Mx) genes that are potent effectors of the antiviral state. Mx gene products are known to inhibit the replication of many negative-sense single-stranded RNA viruses, as well as double-stranded RNA viruses, positive-sense single-stranded RNA viruses, and the reverse-transcribing DNA virus hepatitis B virus. Here, we provide data that extend the known range of viruses inhibited by Mx to include the large double-stranded DNA viruses. Stably transfected Vero cells expressing human MxA protein did not support ASFV plaque formation, and virus replication in these cells was reduced 100-fold compared with that in control cells. In contrast, ASFV replication in cells expressing MxB protein or a mutant MxA protein was similar to that in control Vero cells. There was a drastic reduction in ASFV late protein synthesis in MxA-expressing cells, correlating with the results of previous work on the effect of IFN on viral replication. Strikingly, the inhibition of ASFV replication was linked to the recruitment of MxA protein to perinuclear viral assembly sites, where the protein surrounded the virus factories. Interactions between ASFV and MxA were similar to those seen between MxA and different RNA viruses, suggesting a common inhibitory mechanism.

African swine fever virus (ASFV) is a member of a family of large nucleocytoplasmic DNA viruses that include poxviruses, iridoviruses, and phycodnaviruses. Previous ultrastructural studies of ASFV using chemical fixation and cryosectioning for electron microscopy (EM) have produced uncertainty over whether the inner viral envelope is composed of a single or double lipid bilayer. In this study we prepared ASFV-infected cells for EM using chemical fixation, cryosectioning, and high-pressure freezing. The appearance of the intracellular viral envelope was determined and compared to that of mitochondrial membranes in each sample. The best resolution of membrane structure was obtained with samples prepared by high-pressure freezing, and images suggested that the envelope of ASFV consisted of a single lipid membrane. It was less easy to interpret virus structure in chemically fixed or cryosectioned material, and in the latter case the virus envelope could be interpreted as having two membranes. Comparison of membrane widths in all three preparations indicated that the intracellular viral envelope of ASFV was not significantly different from the outer mitochondrial membrane ( P < 0.05). The results support the hypothesis that the intracellular ASFV viral envelope is composed of a single lipid bilayer.

African swine fever virus (ASFV) causes a lethal haemorrhagic disease of pigs. There are conflicting reports on the role of interferon in ASFV infection. We therefore analysed the interaction of ASFV with porcine interferon, in vivo and in vitro. Virulent ASFV induced biologically active IFN in the circulation of pigs from day 3-post infection, whereas low virulent OUR T88/3, which lacks genes from multigene family (MGF) 360 and MGF505, did not. Infection of porcine leucocytes enriched for dendritic cells, with ASFV, in vitro, induced high levels of interferon, suggesting a potential source of interferon in animals undergoing acute ASF. Replication of OUR T88/3, but not virulent viruses, was reduced in interferon pretreated macrophages and a recombinant virus lacking similar genes to those absent in OUR T88/3 was also inhibited. These findings suggest that as well as inhibiting the induction of interferon, MGF360 and MGF505 genes also enable ASFV to overcome the antiviral state., Highlights • Virulent strains of African swine fever virus induces interferon during infection. • Virulent, but not attenuated strains of ASFV are resistant to the antiviral effects of interferon in porcine macrophages. • Sensitivity to interferon is linked to genes within multigene family 360 and multigene family 530. • Dendritic cells are a potential source of the interferon detected in vivo.

Many viruses replicate and assemble in subcellular microenvironments called virus factories or ‘viroplasm.’ Virus factories increase the efficiency of replication and at the same time protect viruses from antiviral defenses. We describe how viruses reorganize cellular membrane compartments and cytoskeleton to generate these ‘mini-organelles’ and how these rearrangements parallel cellular responses to stress such as protein aggregation and DNA damage.

Viral interference with secretory cargo is a common mechanism for pathogen immune evasion. Selective down regulation of critical immune system molecules such as major histocompatibility complex (MHC) proteins enables pathogens to mask themselves from their host. African swine fever virus (ASFV) disrupts the trans -Golgi network (TGN) by altering the localization of TGN46, an organelle marker for the distal secretory pathway. Reorganization of membrane transport components may provide a mechanism whereby ASFV can disrupt the correct secretion and/or cell surface expression of host proteins. In the study reported here, we used the tsO45 temperature-sensitive mutant of the G protein of vesicular stomatitis virus to show that ASFV significantly reduces the rate at which the protein is delivered to the plasma membrane. This is linked to a general reorganization of the secretory pathway during infection and a specific, microtubule-dependent disruption of structural components of the TGN. Golgin p230 and TGN46 are separated into distinct vesicles, whereupon TGN46 is depleted. These data suggest that disruption of the TGN by ASFV can slow membrane traffic during viral infection. This may be functionally important because infection of macrophages with virulent isolates of ASFV increased the expression of MHC class I genes, but there was no parallel increase in MHC class I molecule delivery to the plasma membrane.

Virus replication can cause extensive rearrangement of host cell cytoskeletal and membrane compartments leading to the “cytopathic effect” that has been the hallmark of virus infection in tissue culture for many years. Recent studies are beginning to redefine these signs of viral infection in terms of specific effects of viruses on cellular processes. In this chapter, these concepts have been illustrated by describing the replication sites produced by many different viruses. In many cases, the cellular rearrangements caused during virus infection lead to the construction of sophisticated platforms in the cell that concentrate replicase proteins, virus genomes, and host proteins required for replication, and thereby increase the efficiency of replication. Interestingly, these same structures, called virus factories, virus inclusions, or virosomes, can recruit host components that are associated with cellular defences against infection and cell stress. It is possible that cellular defence pathways can be subverted by viruses to generate sites of replication. The recruitment of cellular membranes and cytoskeleton to generate virus replication sites can also benefit viruses in other ways. Disruption of cellular membranes can, for example, slow the transport of immunomodulatory proteins to the surface of infected cells and protect against innate and acquired immune responses, and rearrangements to cytoskeleton can facilitate virus release.

Stress signaling from mitochondria and the endoplasmic reticulum (ER) leads to the induction of the proapoptotic transcription factor CHOP/GADD153. Many viruses use the ER as a site of replication and/or envelopment, and this activity can lead to the activation of ER stress and apoptosis. African swine fever virus (ASFV) is assembled on the cytoplasmic face of the ER and ultimately enveloped by ER membrane cisternae. The virus also recruits mitochondria to sites of viral replication and induces the mitochondrial stress protein hsp60. Here we studied the effects of ASFV on the induction of CHOP/GADD153 in infected cells. Interestingly, unlike other ER-tropic viruses, ASFV did not activate CHOP and was able to inhibit the induction of CHOP/GADD153 by a number of exogenous stimuli.

African swine fever virus (ASFV) is a large double-stranded DNA virus that replicates in discrete areas in the cytosol of infected cells called viral factories. Recent studies have shown that assembling virions acquire their internal envelopes through enwrapment by membranes derived from the endoplasmic reticulum (ER). However, the mechanisms that underlie the formation of viral factories and progenitor viral membranes are as yet unclear. Analysis of the published genome of the virus revealed a conserved multigene family that encodes proteins with hydrophobic signal sequences, indicating possible translocation into the ER lumen. Strikingly, two of these genes, XP124L and Y118L , encoded proteins with KDEL-like ER retention motifs. Analysis of XP124L and Y118L gene product by biochemical and immunofluorescence techniques showed that the proteins were localized to pre-Golgi compartments and that the KEDL motif at the C terminus of pXP124L was functional. XP124L expression, in the absence of other ASFV genes, had a dramatic effect on the contents of the ER that was dependent precisely on the C-terminal sequence KEDL. The normal subcellular distribution of a number of proteins resident to this important, cellular organelle was drastically altered in cells expressing wild-type XP124L gene product. PXP124L formed unusual perinuclear structures that contained resident ER proteins, as well as proteins of the ER-Golgi intermediate compartment. The data presented here hint at a role for MGF110 gene product in preparing the ER for its role in viral morphogenesis; this and other potential functions are discussed.

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.

African swine fever (ASF) is an acute haemorrhagic disease of domestic pigs for which there is currently no vaccine. We showed that experimental immunisation of pigs with the non-virulent OURT88/3 genotype I isolate from Portugal followed by the closely related virulent OURT88/1 genotype I isolate could confer protection against challenge with virulent isolates from Africa including the genotype I Benin 97/1 isolate and genotype X Uganda 1965 isolate. This immunisation strategy protected most pigs challenged with either Benin or Uganda from both disease and viraemia. Cross-protection was correlated with the ability of different ASFV isolates to stimulate immune lymphocytes from the OURT88/3 and OURT88/1 immunised pigs.

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.