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African swine fever (ASF) caused by ASF virus (ASFV) is an infectious transboundary animal disease notifiable to the World Organization for Animal Health causing high mortality in domestic pigs and wild boars threatening the global domestic pig industry. To date, twenty-four ASFV genotypes have been described and currently genotypes II, IX, X, XV and XVI are known to be circulating in Tanzania. Despite the endemic status of ASF in Tanzania, only one complete genome of ASFV from the country has been described. This study describes the first complete genome sequence of ASFV genotype XV. In addition, the first Tanzanian complete genome of ASFV genotype IX and three ASFV strains belonging to genotype II collected during ASF outbreaks in domestic pigs in Tanzania were determined in this study using Illumina sequencing and comparative genomics analysis. The generated ASFV complete genome sequences ranged from 171,004 to 184,521 base pairs in length with an average GC content of 38.53% and encoded 152 to 187 open reading frames. The results of this study provide insights into the genomic structure of ASFV and can be used to monitor changes within the ASFV genome and improve our understanding of ASF transmission dynamics.

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.

African swine fever virus (ASFV) remains a threat to global pig populations. Infections with ASFV lead to a hemorrhagic disease with up to 100% lethality in Eurasian domestic and wild pigs. Although myeloid cells are the main target cells for ASFV, T cell responses are impacted by the infection as well. The complex responses remain not well understood, and, consequently, there is no commercially available vaccine. Here, we review the current knowledge about the induction of antiviral T cell responses by cells of the myeloid lineage, as well as T cell responses in infected animals, recent efforts in vaccine research, and T cell epitopes present in ASFV.

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.

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 (ASF) poses a major threat to pig populations and food security worldwide. The disease is endemic to Africa and Eastern Europe and is rapidly emerging into Asia, where it has led to the deaths of millions of pigs in the last 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 characterizing the interactions between ASFV and sncRNAs. Although comparatively modest changes to host sncRNA abundances were observed upon ASFV infection, we discovered and characterized a novel functional ASFV-encoded sncRNA. The results from this study add important insights into ASFV host-pathogen interactions. This knowledge may be exploited to develop more effective ASFV vaccines that take advantage of the sncRNA system., African swine fever virus (ASFV) causes a lethal hemorrhagic disease of domestic pigs, against which no vaccine is available. ASFV has a large, double-stranded DNA genome that encodes over 150 proteins. Replication takes place predominantly in the cytoplasm of the cell and involves complex interactions with host cellular components, including small noncoding RNAs (sncRNAs). A number of DNA viruses are known to manipulate sncRNA either by encoding their own or disrupting host sncRNA. To investigate the interplay between ASFV and sncRNAs, a 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 an up to 1-log reduction in ASFV growth, indicating that ASFV utilizes a virus-encoded small RNA to disrupt its own replication. IMPORTANCE African swine fever (ASF) poses a major threat to pig populations and food security worldwide. The disease is endemic to Africa and Eastern Europe and is rapidly emerging into Asia, where it has led to the deaths of millions of pigs in the last 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 characterizing the interactions between ASFV and sncRNAs. Although comparatively modest changes to host sncRNA abundances were observed upon ASFV infection, we discovered and characterized a novel functional ASFV-encoded sncRNA. The results from this study add important insights into ASFV host-pathogen interactions. This knowledge may 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.

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.

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

African swine fever virus (ASFV) is a large icosahedral DNA virus which replicates predominantly in the cytoplasm of infected cells. The ASFV double-stranded DNA genome varies in length from about 170 to 193 kbp depending on the isolate and contains between 150 and 167 open reading frames. These are closely spaced and read from both DNA strands. The virus genome termini are covalently closed by imperfectly base-paired hairpin loops that are present in two forms that are complimentary and inverted with respect to each other. Adjacent to the termini are inverted arrays of different tandem repeats. Head to head concatemeric genome replication intermediates have been described. A similar mechanism of replication to Poxviruses has been proposed for ASFV. Virus genome transcription occurs independently of the host RNA polymerase II and virus particles contain all of the enzymes and factors required for early gene transcription. DNA replication begins in perinuclear factory areas about 6h post-infection although an earlier stage of nuclear DNA synthesis has been reported. The virus genome encodes enzymes required for transcription and replication of the virus genome and virion structural proteins. Enzymes that are involved in a base excision repair pathway may be an adaptation to enable virus replication in the oxidative environment of the macrophage cytoplasm. Other ASFV genes encode factors involved in evading host defence systems and modulating host cell function. Variation between the genomes of different ASFV isolates is most commonly due to gain or loss of members of multigene families, MGFs 100, 110, 300, 360, 505/530 and family p22. These are located within the left terminal 40kbp and right terminal 20kbp. ASFV is the only member of the Asfarviridae, which is one of the families within the nucleocytoplasmic large DNA virus superfamily.

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.

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.

To the Editor: African swine fever virus (ASFV) causes a notifiable disease in domestic pigs for which no treatment or vaccine is available, resulting in a mortality rate of 5 years after the last confirmed case in an outbreak (3). These ticks can feed on alternative hosts, evade eradication attempts (such as acaricide application and flamethrowers), and survive for up to 15 years (1). Although Ornithodoros species have been reported in the Caucasus region, their distribution is not well known (1). It is also not known if the Georgia 2007/1 ASFV strain responsible for continuing outbreaks in the Caucasus region can replicate in ticks. Thus, we conducted a study to determine whether the Georgia 2007/1 isolate of ASFV can replicate in Ornithodoros ticks. O. erraticus ticks from Alentejo, Portugal (provided by Fernando Boinas, Universidade Tecnica de Lisboa in Lisbon, Portugal) were sorted into groups of 10 adults or fifth-instar nymphs, placed into 60-mL containers covered with nylon cloth (16-cm mesh), and maintained at 85% relative humidity and 27°C for 18 months without feeding. Heparinized pig blood containing antibacterial drugs and fungicide (10 µL of streptomycin [10,000 IU/mL], 10 µL of amphotericin B [250 µg/mL], and 5 µL of neomycin [10 mg/mL 0.9% NaCl]/mL of blood) was mixed with the Georgia 2007/1 isolate (4) or the OUR T88/1 isolate (5) as a positive control to obtain virus titers of 4 log10 or 6 log10 50% hemadsorbing doses (HAD50)/mL blood. These titers were within the observed range in naturally infected pigs (6), and thus simulated the field situation. Ticks were fed infected blood by using a Hemotek membrane-feeding system (Discovery Workshops, Accrington, UK). Meal reservoirs were covered with stretched Parafilm that was wiped with a thin film of uninfected blood to encourage feeding. Pots of ticks were placed on the membrane and allowed to feed for 20 minutes. Immediately after and 3, 6, 9, and 12 weeks after feeding, 10 ticks from each feeding group were killed by freezing in dry ice. After being washed with a detergent solution and phosphate-buffered saline, ticks were placed individually in tubes with 200 µL of RPMI medium (Sigma-Aldrich Company Ltd., Gillingham, UK), a 3 mm-diameter stainless steel ball (Dejay Distribution Ltd., Launceston, UK), and 1-mm silicon carbide particles (Stratech Scientific Ltd, Newmarket, UK). They were then homogenized by shaking for 5 cycles of 3 minutes at 25-Hz frequency using a TissueLyser (QIAGEN, Valencia, CA, USA). To complete a 1-mL volume, 800 µL of RPMI medium was added to the tubes after centrifuging 2× for 30 seconds at 2,000 rpm. Supernatants were transferred to fresh tubes and centrifuged for 5 minutes at 1,000 × g. Virus titers were estimated on porcine bone marrow cells (7) and expressed as log10 HAD50 per tick. Previous studies suggest that it takes 3–4 weeks for ticks to completely digest and clear ingested blood and that virus isolated after this period is due to viral replication (5,6). A general linear model, fitted via maximum likelihood, was used to assess the effects of isolate, dose, time after feeding, and interaction between isolate and time after feeding on the viral titer in the tick. Confidence intervals were calculated by profile likelihood. Results showed that the Georgia 2007/1 strain can replicate in the O. erraticus tick. We recovered virus titers of 9.8 log10 HAD50 per tick. Ticks that fed on blood containing 6 log10 HAD50 ASFV on average had virus titers 2.15 log10 HAD50 higher than those for ticks that fed on blood containing 4 log10 HAD50/mL. Over time, the average titer for both isolates increased at an estimated rate of 0.65 log10 HAD50/week, indicating replication. Statistical analysis suggested that immediately after feeding, ticks fed on the Georgia 2007/1 strain contained 1.36 log10 HAD50 less virus than those fed on the OUR T88/1 isolate, but we detected no statistically significant difference in the replication rates of the 2 isolates. Parameter estimates are shown in the Table and the model fit is shown in the Figure. Table General linear model of the effects of different parameters on the titer of ASFV in experimentally infected Ornithodoros erraticus ticks* Figure Predicted regression for each isolate–dose combination is shown. A) Ticks fed on African swine fever virus (ASFV) strain OUR T88/1 at 4 log10 50% hemadsorbing doses (HAD50)/mL. B) Ticks fed on ASFV strain OUR T88/1 at 6 log10 HAD50/mL. C) Ticks ... The whole-tick titers reported in this study are consistent with those from previous studies (5,6). However, >9 weeks after the ticks fed, we observed higher titers than those reported (5), and many results showed >9.8 log10 HAD50 per tick. The TissueLyser may have been more effective at releasing ASFV from tick tissues than previously used methods. Our results suggest that virus replication within the ticks began by 3 weeks after feeding on infected blood; this timing is consistent with that in previous studies (5,8,9). We observed high viral titers for >12 weeks after infection. Previous studies showed that ASFV can persist at high titers for 20 weeks after infection (10). We demonstrated that ASFV Georgia 2007/1 isolate can replicate efficiently in ticks. This finding highlights the importance of clarifying the distribution of Ornithodoros species ticks in the Russian Federation and Caucasus region and the relationship of these ticks to species susceptible to ASFV.

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.

African swine fever virus (ASFV) infection usually results in an acute haemorrhagic disease with a mortality rate approaching 100% in domestic pigs. However, pigs can survive infection with less-virulent isolates of ASFV and may become chronically infected. Surviving animals are resistant to challenge with homologous or, in some cases, closely related isolates of the virus indicating that pigs can develop protective immunity against ASFV. During asymptomatic, non-virulent ASFV infections natural killer cell activity increases in pigs, suggesting this cell type plays a role in ASFV immunity. Furthermore, depletion of CD8(+) lymphocytes from ASFV immune pigs demolishes protective immunity against related virulent viruses. This suggests that ASFV specific antibody alone is not sufficient for protection against ASFV infection and that there is an important role for the CD8(+) lymphocyte subset in ASFV protective immunity. These results were supported by DNA immunization studies, demonstrating a correlation between the protection afforded against lethal challenge and the detection of a large number of vaccine-induced antigen-specific CD8(+) T-cells. Peripheral blood mononuclear cells (PBMCs) from ASF immune pigs protected from clinical disease show higher proportions of ASFV specific CD4(+)CD8(high+) double positive cytotoxic T cells than PBMCs from ASF immune but clinically diseased pig. The frequency of ASFV specific IFNγ producing T cells induced by immunization correlates to the degree of protection from ASFV challenge, and this may prove to be a useful indicator of any potential cross-protection against heterologous ASFV isolates.

Like most viruses African swine fever virus (ASFV) subsumes the host cell apparatus in order to facilitate its replication. ASFV replication is a highly orchestrated process with a least four stages of transcription, immediate-early, early, intermediate and late. As the infective cycle progresses through these stages most if not all of the organelles that comprise a nucleated cell are modified, adapted or in some cases destroyed. The entry of the virus is receptor-mediated, but the precise mechanism of endocytosis is a matter of keen, current debate. Once ASFV has exited from the endosomal-lysosomal complex the virus life-cycle enters into an intimate relationship with the microtubular network. Genome replication is believed to be initiated within the nucleus and ASFV infection completely reorders the structure of this organelle. The majority of replication and assembly occurs in discrete, perinuclear regions of the cell called virus factories and finally progeny virions are transported to the plasma membrane along microtubules where they bud out or are propelled away along actin projections to infect new cells. The generation of ASFV replication sites induces profound reorganisation of the organelles that comprise the secretory pathway and may contribute to the induction of cellular stress responses that ASFV modulates. The level of organisation and complexity of virus factories are not dissimilar to those seen in cellular organelles. Like their cellular counterparts the formation of virus factories, as well as virus entry and exit, are dependent on the various components of the cytoskeleton. This review will summarise these rearrangements, the viral proteins involved and their functional consequences.

We describe a method for high-pressure freezing and rapid freeze-substitution of cells in tissue culture which provides excellent preservation of membrane detail with negligible ice segregation artefacts. Cells grown on sapphire discs were placed 'face to face' without removal of tissue culture medium and frozen without the protection of aluminium planchettes. This reduction in thermal load of the sample/holder combination resulted in freezing of cells without visible ice-crystal artefact. Freeze-substitution at -90 degrees C for 60 min in acetone containing 2% uranyl acetate, followed by warming to -50 degrees C and embedding in Lowicryl HM20 gave consistent and clear membrane detail even when imaged without section contrasting. Preliminary data indicates that the high intrinsic contrast of samples prepared in this way will be valuable for tomographic studies. Immunolabelling sensitivity of sections of samples prepared by this rapid substitution technique was poor; however, reducing the uranyl acetate concentration in the substitution medium to 0.2% resulted in improved labelling. Samples substituted in this lower concentration of uranyl acetate also gave good membrane detail when imaged after section contrasting.

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.

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.