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African swine fever (ASF) is a highly impactful infectious disease in the swine industry, leading to substantial economic losses globally. The causative agent, African swine fever virus (ASFV), possesses intricate pathogenesis, warranting further exploration. In this study, we investigated the impact of ASFV infection on host gene transcription and organelle changes through macrophage transcriptome sequencing and ultrastructural transmission electron microscopy observation. According to the results of the transcriptome sequencing, ASFV infection led to significant alterations in the gene expression pattern of porcine bone marrow derived macrophages (BMDMs), with 2404 genes showing upregulation and 1579 genes downregulation. Cytokines, and chemokines were significant changes in the expression of BMDMs; there was significant activation of pattern recognition receptors such as Toll-like receptors and Nod-like receptors. According to the observation of the ultrastructure, mitochondrial damage and mitochondrial autophagy were widely present in ASFV-infected cells. The reduced number of macrophage pseudopodia suggested that virus-induced structural changes may compromise pathogen recognition, phagocytosis, and signal communication in macrophages. Additionally, the decreased size and inhibited acidification of secondary lysosomes in macrophages implied suppressed phagocytosis. Overall, ASFV infection resulted in significant changes in the expression of cytokines and chemokines, accompanied by the activation of NLR and TLR signaling pathways. We reported for the first time that ASFV infection led to a reduction in pseudopodia numbers and a decrease in the size and acidification of secondary lysosomes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

African swine fever (ASF) is a highly contagious viral disease that affects domestic and wild pigs. The causative agent of ASF is African swine fever virus (ASFV), a large double-stranded DNA virus with a complex virion structure. Among the various proteins encoded by ASFV, A137R is a crucial structural protein associated with its virulence. However, the structure and molecular mechanisms underlying the functions of A137R remain largely unknown. In this study, we present the structure of A137R determined by cryogenic electron microscopy single-particle reconstruction, which reveals that A137R self-oligomerizes to form a dodecahedron-shaped cage composed of 60 polymers. The dodecahedron is literally equivalent to a T = 1 icosahedron where the icosahedral vertexes are located in the center of each dodecahedral facet. Within each facet, five A137R protomers are arranged in a head-to-tail orientation with a long N-terminal helix forming the edge through which adjacent facets stitch together to form the dodecahedral cage. Combining structural analysis and biochemical evidence, we demonstrate that the N-terminal domain of A137R is crucial and sufficient for mediating the assembly of the dodecahedron. These findings imply the role of A137R cage as a core component in the icosahedral ASFV virion and suggest a promising molecular scaffold for nanotechnology applications., Importance: African swine fever (ASF) is a lethal viral disease of pigs caused by African swine fever virus (ASFV). No commercial vaccines and antiviral treatments are available for the prevention and control of the disease. A137R is a structural protein of ASFV that is associated with its virulence. The discovery of the dodecahedron-shaped cage structure of A137R in this study is of great importance in understanding ASFV pathogenicity. This finding sheds light on the molecular mechanisms underlying the functions of A137R. Furthermore, the dodecahedral cage formed by A137R shows promise as a molecular scaffold for nanoparticle vectors. Overall, this study provides valuable insights into the structure and function of A137R, contributing to our understanding of ASFV and potentially opening up new avenues for the development of vaccines or treatments for ASF., Competing Interests: The authors declare no conflict of interest.

African swine fever virus (ASFV), the cause of a highly contagious hemorrhagic and fatal disease of domestic pigs, has a complex multilayer structure. The inner capsid of ASFV located underneath the inner membrane enwraps the genome-containing nucleoid and is likely the assembly of proteolytic products from the virally encoded polyproteins pp220 and pp62. Here, we report the crystal structure of ASFV p150 △NC , a major middle fragment of the pp220 proteolytic product p150. The structure of ASFV p150 △NC contains mainly helices and has a triangular plate-like shape. The triangular plate is approximately 38 Å in thickness, and the edge of the triangular plate is approximately 90 Å long. The structure of ASFV p150 △NC is not homologous to any of the known viral capsid proteins. Further analysis of the cryo-electron microscopy maps of the ASFV and the homologous faustovirus inner capsids revealed that p150 or the p150-like protein of faustovirus assembles to form screwed propeller-shaped hexametric and pentametric capsomeres of the icosahedral inner capsids. Complexes of the C terminus of p150 and other proteolytic products of pp220 likely mediate interactions between the capsomeres. Together, these findings provide new insights into the assembling of ASFV inner capsid and provide a reference for understanding the assembly of the inner capsids of nucleocytoplasmic large DNA viruses (NCLDV). IMPORTANCE African swine fever virus has caused catastrophic destruction to the pork industry worldwide since it was first discovered in Kenya in 1921. The architecture of ASFV is complicated, with two protein shells and two membrane envelopes. Currently, mechanisms involved in the assembly of the ASFV inner core shell are less understood. The structural studies of the ASFV inner capsid protein p150 performed in this research enable the building of a partial model of the icosahedral ASFV inner capsid, which provides a structural basis for understanding the structure and assembly of this complex virion. Furthermore, the structure of ASFV p150 △NC represents a new type of fold for viral capsid assembly, which could be a common fold for the inner capsid assembly of nucleocytoplasmic large DNA viruses (NCLDV) and would facilitate the development of vaccine and antivirus drugs against these complex viruses., Competing Interests: The authors declare no conflict of interest.

African swine fever virus (ASFV) is a complex nucleocytoplasmic large DNA virus that causes African swine fever, a lethal hemorrhagic disease that currently threatens the pig industry. Recent studies have identified the viral structural proteins of infectious ASFV particles. However, the functional roles of several ASFV structural proteins remain largely unknown. Here, we characterized the function of the ASFV structural protein H240R (pH240R) in virus morphogenesis. pH240R was identified as a capsid protein by using immunoelectron microscopy and interacted with the major capsid protein p72 by pulldown assays. Using a recombinant ASFV, ASFV-ΔH240R, with the H240R gene deleted from the wild-type ASFV (ASFV-WT) genome, we revealed that the infectious progeny virus titers were reduced by approximately 2.0 logs compared with those of ASFV-WT. Furthermore, we demonstrated that the growth defect was due to the generation of noninfectious particles with a higher particle-to-infectious titer ratio in ASFV-ΔH240R-infected primary porcine alveolar macrophages (PAMs) than in those infected with ASFV-WT. Importantly, we found that pH240R did not affect virus-cell binding, endocytosis, or egress but did affect ASFV assembly; noninfectious virions containing large aberrant tubular and bilobulate structures comprised nearly 98% of all virions observed in ASFV-ΔH240R-infected PAMs by electron microscopy. Notably, we demonstrated that ASFV-ΔH240R infection induced high-level expression of inflammatory cytokines in PAMs. Collectively, we show for the first time that pH240R is essential for ASFV icosahedral capsid formation and infectious particle production. Also, these results highlight the importance of pH240R in ASFV morphogenesis and provide a novel target for the development of ASF vaccines and antivirals. IMPORTANCE African swine fever is a lethal hemorrhagic disease of global concern that is caused by African swine fever virus (ASFV). Despite extensive research, there exist relevant gaps in knowledge of the fundamental biology of the viral life cycle. In this study, we identified pH240R as a capsid protein that interacts with the major capsid protein p72. Furthermore, we showed that pH240R was required for the efficient production of infectious progeny virions as indicated by the H240R- deleted ASFV mutant (ASFV-ΔH240R). More specifically, pH240R directs the morphogenesis of ASFV toward the icosahedral capsid in the process of assembly. In addition, ASFV-ΔH240R infection induced high-level expression of inflammatory cytokines in primary porcine alveolar macrophages. Our results elucidate the role of pH240R in the process of ASFV assembly, which may instruct future research on effective vaccines or antiviral strategies.

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.

The African swine fever virus (ASFV) is the deadly pathogen of African swine fever (ASF) that induces high mortality, approaching 100% in domestic pigs, causes enormous losses to the global pig industry, and threatens food security. Currently, there is no effective treatment or preventive countermeasure. dUTPases (deoxyuridine 5'-triphosphate pyrophosphatases) are ubiquitous enzymes that are essential for the hydrolysis of dUTP and prevent the misincorporation of dUTP into newly synthesized DNA. Here, we present the crystal structures of the ASFV dUTPase in complex with the product dUMP and cofactor Mg 2+ at a resolution of 2.2 Å. We observed that a unique "turning point" at G125 plays an unexpected critical role in the swapping region of the C-terminal segment, which is further stabilized by the interactions of the last C-terminal β strand with the β1 and β2 strands, thereby positioning the catalytic motif 5 into the active site of its own subunit instead of into a third subunit. Therefore, the ASFV dUTPase employs a novel two-subunit active site that is different than the classic trimeric dUTPase active site, which is composed of all three subunits. Meanwhile, further results confirmed that the configuration of motifs 1 to 5 has high structural homology with and a catalytic mechanism similar to that of the known trimeric dUTPases. In general, our study expands the information not only on the structural diversity of the conserved dUTPase family but also on the details needed to utilize this dUTPase as a novel target in the treatment of ASF. IMPORTANCE African swine fever virus (AFSV), a large enveloped double-stranded DNA virus, causes a deadly infection in domestic pigs. In addition to Africa, Europe, and South America, countries in Asia, such as China, Vietnam, and Mongolia, have suffered the hazards posed by ASFV outbreaks in recent years. Until now, there has been no vaccine for protection from ASFV infection or effective treatments to cure ASF. Here, we solved the crystal structure of the ASFV dUTPase-dUMP-Mg 2+ complex. The ASFV dUTPase displays a noncanonical folding pattern that differs from that of the classic homotrimeric dUTPase, in which the active site is composed of two subunits. In addition, several nonconserved residues within the 3-fold axis channel play a vital role in ASFV dUTPase homotrimer stability. Our finding on these unique structural features of the ASFV dUTPase could be explored for the design of potential specific inhibitors that target this unique enzyme., (Copyright © 2020 American Society for Microbiology.)

African swine fever virus (ASFV) is a complex nucleocytoplasmic large DNA virus (NCLDV) that causes a devastating swine disease currently present in many countries of Africa, Europe, and Asia. Despite intense research efforts, relevant gaps in the architecture of the infectious virus particle remain. Here, we used single-particle cryo-EM to analyze the three-dimensional structure of the mature ASFV particle. Our results show that the ASFV virion, with a radial diameter of ∼2,080 Å, encloses a genome-containing nucleoid surrounded by two distinct icosahedral protein capsids and two lipoprotein membranes. The outer capsid forms a hexagonal lattice (triangulation number T = 277) composed of 8,280 copies of the double jelly-roll major capsid protein (MCP) p72, arranged in trimers displaying a pseudo-hexameric morphology, and of 60 copies of a penton protein at the vertices. The inner protein layer, organized as a T = 19 capsid, confines the core shell, and it is composed of the mature products derived from the ASFV polyproteins pp220 and pp62. Also, an icosahedral membrane lies between the two protein layers, whereas a pleomorphic envelope wraps the outer capsid. This high-level organization confers to ASFV a unique architecture among the NCLDVs that likely reflects the complexity of its infection process and may help explain current challenges in controlling it., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Andrés et al.)

African swine fever virus (ASFV) is a large double-stranded DNA virus with an icosahedral multilayered structure. ASFV causes a lethal swine hemorrhagic disease and is currently responsible for widespread damage to the pork industry in Asia. Neither vaccines nor antivirals are available and the molecular characterization of the ASFV particle is outstanding. Here, we describe the cryogenic electron microscopy (cryo-EM) structure of the icosahedral capsid of ASFV at 4.6-Å. The ASFV particle consists of 8,280 copies of the major capsid protein p72, 60 copies of the penton protein, and at least 8,340 minor capsid proteins, of which there might be 3 different types. Like other nucleocytoplasmic large DNA viruses, the minor capsid proteins form a hexagonal network below the outer capsid shell, functioning as stabilizers by "gluing" neighboring capsomers together. Our findings provide a comprehensive molecular model of the ASFV capsid architecture that will contribute to the future development of countermeasures, including vaccines., (Copyright © 2019 Elsevier Inc. All rights reserved.)

African swine fever virus (ASFV) is a giant and complex DNA virus that causes a highly contagious and often lethal swine disease for which no vaccine is available. Using an optimized image reconstruction strategy, we solved the ASFV capsid structure up to 4.1 angstroms, which is built from 17,280 proteins, including one major (p72) and four minor (M1249L, p17, p49, and H240R) capsid proteins organized into pentasymmetrons and trisymmetrons. The atomic structure of the p72 protein informs putative conformational epitopes, distinguishing ASFV from other nucleocytoplasmic large DNA viruses. The minor capsid proteins form a complicated network below the outer capsid shell, stabilizing the capsid by holding adjacent capsomers together. Acting as core organizers, 100-nanometer-long M1249L proteins run along each edge of the trisymmetrons that bridge two neighboring pentasymmetrons and form extensive intermolecular networks with other capsid proteins, driving the formation of the capsid framework. These structural details unveil the basis of capsid stability and assembly, opening up new avenues for African swine fever vaccine development., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The continuing spread of African swine fever (ASF) outside Africa in Europe, the Russian Federation, China and most recently to Mongolia and Vietnam, has heightened awareness of the threat posed by this devastating disease to the global pig industry and food security. In this review we summarise what we know about the African swine fever virus (ASFV), the disease it causes, how it spreads and the current global situation. We discuss current control methods in domestic and wild pigs and prospects for development of vaccines and other tools for control., (Copyright © 2019. Published by Elsevier B.V.)

African swine fever virus (ASFV) is a large and complex DNA virus that causes a highly lethal swine disease for which there is no vaccine available. The ASFV particle, with an icosahedral multilayered structure, contains multiple polypeptides whose identity is largely unknown. Here, we analyzed by mass spectroscopy the protein composition of highly purified extracellular ASFV particles and performed immunoelectron microscopy to localize several of the detected proteins. The proteomic analysis identified 68 viral proteins, which account for 39% of the genome coding capacity. The ASFV proteome includes essentially all the previously described virion proteins and, interestingly, 44 newly identified virus-packaged polypeptides, half of which have an unknown function. A great proportion of the virion proteins are committed to the virus architecture, including two newly identified structural proteins, p5 and p8, which are derived from the core polyproteins pp220 and pp62, respectively. In addition, the virion contains a full complement of enzymes and factors involved in viral transcription, various enzymes implicated in DNA repair and protein modification, and some proteins concerned with virus entry and host defense evasion. Finally, 21 host proteins, many of them localized at the cell surface and related to the cortical actin cytoskeleton, were reproducibly detected in the ASFV particle. Immunoelectron microscopy strongly supports the suggestion that these host membrane-associated proteins are recruited during virus budding at actin-dependent membrane protrusions. Altogether, the results of this study provide a comprehensive model of the ASFV architecture that integrates both compositional and structural information. IMPORTANCE African swine fever virus causes a highly contagious and lethal disease of swine that currently affects many countries of sub-Saharan Africa, the Caucasus, the Russian Federation, and Eastern Europe and has very recently spread to China. Despite extensive research, effective vaccines or antiviral strategies are still lacking, and many basic questions on the molecular mechanisms underlying the infective cycle remain. One such gap regards the composition and structure of the infectious virus particle. In the study described in this report, we identified the set of viral and host proteins that compose the virion and determined or inferred the localization of many of them. This information significantly increases our understanding of the biological and structural features of an infectious African swine fever virus particle and will help direct future research efforts., (Copyright © 2018 American Society for Microbiology.)

African swine fever (ASF) is an acute and often fatal disease affecting domestic pigs and wild boar, with severe economic consequences for affected countries. ASF is endemic in sub-Saharan Africa and the island of Sardinia, Italy. Since 2007, the virus emerged in the republic of Georgia, and since then spread throughout the Caucasus region and Russia. Outbreaks have also been reported in Belarus, Ukraine, Lithuania, Latvia, Estonia, Romania, Moldova, Czech Republic, and Poland, threatening neighboring West European countries. The causative agent, the African swine fever virus (ASFV), is a large, enveloped, double-stranded DNA virus that enters the cell by macropinocytosis and a clathrin-dependent mechanism. African Swine Fever Virus is able to interfere with various cellular signaling pathways resulting in immunomodulation, thus making the development of an efficacious vaccine very challenging. Inactivated preparations of African Swine Fever Virus do not confer protection, and the role of antibodies in protection remains unclear. The use of live-attenuated vaccines, although rendering suitable levels of protection, presents difficulties due to safety and side effects in the vaccinated animals. Several African Swine Fever Virus proteins have been reported to induce neutralizing antibodies in immunized pigs, and vaccination strategies based on DNA vaccines and recombinant proteins have also been explored, however, without being very successful. The complexity of the virus particle and the ability of the virus to modulate host immune responses are most likely the reason for this failure. Furthermore, no permanent cell lines able to sustain productive virus infection by both virulent and naturally attenuated African Swine Fever Virus strains exist so far, thus impairing basic research and the commercial production of attenuated vaccine candidates., (© 2018 Elsevier Inc. All rights reserved.)

African swine fever (ASF) is a highly contagious viral disease of swine which causes high mortality, approaching 100%, in domestic pigs. ASF is caused by a large, double stranded DNA virus, ASF virus (ASFV), which replicates predominantly in the cytoplasm of macrophages and is the only member of the Asfarviridae family, genus Asfivirus . The natural hosts of this virus include wild suids and arthropod vectors of the Ornithodoros genus. The infection of ASFV in its reservoir hosts is usually asymptomatic and develops a persistent infection. In contrast, infection of domestic pigs leads to a lethal hemorrhagic fever for which there is no effective vaccine. Identification of ASFV genes involved in virulence and the characterization of mechanisms used by the virus to evade the immune response of the host are recognized as critical steps in the development of a vaccine. Moreover, the interplay of the viral products with host pathways, which are relevant for virus replication, provides the basic information needed for the identification of potential targets for the development of intervention strategies against this disease.

Collective evidence argues that two members of the nucleocytoplasmic large DNA viruses (NCLDVs) acquire their membrane from open membrane intermediates, postulated to be derived from membrane rupture. We now study membrane acquisition of the NCLDV African swine fever virus. By electron tomography (ET), the virion assembles a single bilayer, derived from open membrane precursors that collect as ribbons in the cytoplasm. Biochemically, lumenal endoplasmic reticulum (ER) proteins are released into the cytosol, arguing that the open intermediates are ruptured ER membranes. ET shows that viral capsid assembles on the convex side of the open viral membrane to shape it into an icosahedron. The viral capsid is composed of tiny spikes with a diameter of ∼5 nm, connected to the membrane by a 6 nm wide structure displaying thin striations, as observed by several complementary electron microscopy imaging methods. Immature particles display an opening that closes after uptake of the viral genome and core proteins, followed by the formation of the mature virion. Together with our previous data, this study shows a common principle of NCLDVs to build a single internal envelope from open membrane intermediates. Our data now provide biochemical evidence that these open intermediates result from rupture of a cellular membrane, the ER., (© 2015 John Wiley & Sons Ltd.)

This review summarizes recent structural and molecular biology studies related to the morphogenesis of African swine fever virus (ASFV). ASFV possesses icosahedral morphology and is constituted by four concentric layers: the central nucleoid, the core shell, the inner envelope and the icosahedral capsid. The extracellular virus acquires an external envelope by budding through the plasma membrane. The genes coding for 19 of the 54 structural proteins of the ASFV particle are known and the localization within the virion of 18 of these components has been identified. ASFV morphogenesis occurs in specialized areas in the cytoplasm, named viral factories, which are proximal to the microtubule organization center near the nucleus. Investigations of the different steps of morphogenesis by immunocytochemical and electron microscopy techniques, as well as molecular biology and biochemical studies, have shed light on the formation of the different domains of the virus particle, including the recognition of endoplasmic reticulum membranes as the precursors of the virus inner envelope, the progressive formation of the capsid on the convex face of the inner envelope and the simultaneous assembly of the core shell on the concave side of the envelope, with the pivotal contribution of the virus polyproteins and their proteolytic processing by the virus protease for the development of this latter domain. The use of ASFV inducible recombinants as a tool for the study of the individual function of structural and nonstructural proteins has been determinant to understand their role in virus assembly and has provided new insights into the morphogenetic process., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Novel findings and concepts in the field of virology particularly regarding virosphere and giruses--a group of large nuclear-cytoplasmic deoxyriboviruses are briefly summarized. In the context of novel understanding the major taxonomic features and virus pathogenicity including African swine plague are interpreted.

Infection of cells with African swine fever virus (ASFV) can lead to the formation of zipper-like stacks of structural proteins attached to collapsed endoplasmic reticulum (ER) cisternae. We show that the collapse of ER cisternae observed during ASFV infection is dependent on the viral envelope protein, J13Lp. Expression of J13Lp alone in cells is sufficient to induce collapsed ER cisternae. Collapse was dependent on a cysteine residue in the N-terminal domain of J13Lp exposed to the ER lumen. Luminal collapse was also dependent on the expression of J13Lp within stacks of ER where antiparallel interactions between the cytoplasmic domains of J13Lp orientated N-terminal domains across ER cisternae. Cisternal collapse was then driven by disulphide bonds between N-terminal domains arranged in antiparallel arrays across the ER lumen. This provides a novel mechanism for biogenesis of modified stacks of ER present in cells infected with ASFV, and may also be relevant to cellular processes., (© 2011 John Wiley & Sons A/S.)

The mechanisms involved in the construction of the icosahedral capsid of the African swine fever virus (ASFV) particle are not well understood at present. Capsid formation requires protein p72, the major capsid component, but other viral proteins are likely to play also a role in this process. We have examined the function of the ASFV structural protein pB438L, encoded by gene B438L, in virus morphogenesis. We show that protein pB438L associates with membranes during the infection, behaving as an integral membrane protein. Using a recombinant ASFV that inducibly expresses protein pB438L, we have determined that this structural protein is essential for the formation of infectious virus particles. In the absence of the protein, the virus assembly sites contain, instead of icosahedral particles, large aberrant tubular structures of viral origin as well as bilobulate forms that present morphological similarities with the tubules. The filamentous particles, which possess an aberrant core shell domain and an inner envelope, are covered by a capsid-like layer that, although containing the major capsid protein p72, does not acquire icosahedral morphology. This capsid, however, is to some extent functional, as the filamentous particles can move from the virus assembly sites to the plasma membrane and exit the cell by budding. The finding that, in the absence of protein pB438L, the viral particles formed have a tubular structure in which the icosahedral symmetry is lost supports a role for this protein in the construction or stabilization of the icosahedral vertices of the virus particle.

African swine fever virus (ASFV) protein pB602L has been described as a molecular chaperone for the correct folding of the major capsid protein p72. We have studied the function of protein pB602L during the viral assembly process by using a recombinant ASFV, vB602Li, which inducibly expresses the gene coding for this protein. We show that protein pB602L is a late nonstructural protein, which, in contrast with protein p72, is excluded from the viral factory. Repression of protein pB602L synthesis inhibits the proteolytic processing of the two viral polyproteins pp220 and pp62 and leads to a decrease in the levels of protein p72 and a delocalization of the capsid protein pE120R. As shown by electron microscopy analysis of cells infected with the recombinant virus vB602Li, the viral assembly process is severely altered in the absence of protein pB602L, with the generation of aberrant "zipper-like" structures instead of icosahedral virus particles. These "zipper-like" structures are similar to those found in cells infected under restrictive conditions with the recombinant virus vA72 inducibly expressing protein p72. Immunoelectron microscopy studies show that the abnormal forms generated in the absence of protein pB602L contain the inner envelope protein p17 and the two polyproteins but lack the capsid proteins p72 and pE120R. These findings indicate that protein pB602L is essential for the assembly of the icosahedral capsid of the virus particle.

When exiting the cell vaccinia virus induces actin polymerization and formation of a characteristic actin tail on the cytosolic face of the plasma membrane, directly beneath the extracellular particle. The actin tail acts to propel the virus away from the cell surface to enhance its cell-to-cell spread. We now demonstrate that African swine fever virus (ASFV), a member of the Asfarviridae family, also stimulates the polymerization of actin at the cell surface. Intracellular ASFV particles project out at the tip of long filopodia-like protrusions, at an average rate of 1.8 microm min(-1). Actin was arranged in long unbranched parallel arrays inside these virus-tipped projections. In contrast to vaccinia, this outward movement did not involve recruitment of Grb2, Nck1 or N-WASP. Actin polymerization was not nucleated by virus particles in transit to the cell periphery, and projections were not produced when the secretory pathway was disrupted by brefeldin A treatment. Our results show that when ASFV particles reach the plasma membrane they induce a localized nucleation of actin, and that this process requires interaction with virus-encoded and/or host proteins at the plasma membrane. We suggest that ASFV represents a valuable new model for studying pathways that regulate the formation of filopodia.

Recently, we reported that African swine fever virus (ASFV) multigene family (MGF) 360 and 530 genes are significant swine macrophage host range determinants that function by promoting infected-cell survival. To examine the function of these genes in ASFV's arthropod host, Ornithodoros porcinus porcinus, an MGF360/530 gene deletion mutant (Pr4Delta35) was constructed from an ASFV isolate of tick origin, Pr4. Pr4Delta35 exhibited a significant growth defect in ticks. The deletion of six MGF360 and two MGF530 genes from Pr4 markedly reduced viral replication in infected ticks 100- to 1,000-fold. To define the minimal set of MGF360/530 genes required for tick host range, additional gene deletion mutants lacking individual or multiple MGF genes were constructed. The deletion mutant Pr4Delta3-C2, which lacked three MGF360 genes (3HL, 3Il, and 3LL), exhibited reduced viral growth in ticks. Pr4Delta3-C2 virus titers in ticks were significantly reduced 100- to 1,000-fold compared to control values at various times postinfection. In contrast to the parental virus, with which high levels of virus replication were observed in the tissues of infected adults, Pr4Delta3-C2 replication was not detected in the midgut, hemolymph, salivary gland, coxal gland, or reproductive organs at 15 weeks postinfection. These data indicate that ASFV MGF360 genes are significant tick host range determinants and that they are required for efficient virus replication and generalization of infection. The impaired virus replication of Pr4Delta3-C2 in the tick midgut likely accounts for the absence of the generalized infection that is necessary for the natural transmission of virus from ticks to pigs.

This paper report on the lesions occurred in the thymus in experimental acute African swine fever (ASF). Twenty-one pigs were inoculated with the highly virulent ASF virus (ASFV) isolate Spain-70. Animals were slaughtered from 1 to 7 days post infection (dpi). Three animals with similar features were used as controls. Thymus samples were fixed in 10% buffered formalin solution for histological and immunohistochemical study and in 2.5% glutaraldehyde for ultrastructural examination. For immunohistochemical study, the avidin-biotin-peroxidase complex (ABC) technique was used to demonstrate viral protein 73 and porcine myeloid-histiocyte antigen SWC3 using specific monoclonal antibodies. Cell apoptosis was evaluated by the TUNEL assay. Blood samples were taken daily from all pigs and were used for leukocyte counts. The results of this study show a severe thymocyte apoptosis not related to the direct action of ASFV on these cells, but probably to a quantitative increase in macrophages in the thymus and their activation. A decrease in the percentage of blood lymphocytes was observed at the same time No significant vascular changes were observed in the study. With these results we suggest that ASFV infection of the thymus does not seem to play a critical role in the acute disease. Although severe apoptosis was observed, animals died because of the severe lesions found in the other organs.

High-pressure freezing is applicable to both morphological and immunocytochemical studies. We are investigating the morphogenesis of foot-and-mouth disease virus and African swine fever virus by the use of high-pressure freezing of infected cells. Foot-and-mouth disease virus particles are not detected in sections of conventionally immersion-fixed infected cells, but when the cells are prepared by high-pressure freezing, newly formed virions are readily seen throughout the cell. We report two methods for high-pressure freezing of virally infected cells: first, two sapphire discs frozen 'face to face' with a narrow spacer to prevent cell damage and, second, a fibrous filter substrate that can be easily cut into discs to fit into the freezing planchettes. Cells readily adhere to the fibres in vitro, and the complete disc can be rapidly transferred to the planchettes for freezing. Immunolabelling studies of the microneme proteins of the parasite Eimeria tenella indicate that high-pressure freezing followed by freeze-substitution in acetone with uranyl acetate allows high-sensitivity immunolabelling for these proteins.

To determine, in the acute form of African swine fever (ASF), the relationship between the appearance of pulmonary oedema and viral replication and expression of cytokines by pulmonary intravascular macrophages (PIMs), 14 pigs were inoculated intramuscularly with ASF virus (strain España'70) and killed in pairs on days 1-7 post-inoculation. Samples of lung were examined immunohistochemically and ultrastructurally. The immunohistochemical study was carried out with antibodies against interleukin-1 alpha (IL-1alpha), tumour necrosis factor-alpha (TNF-alpha), viral antigen of ASF (Vp73) and a myeloid marker (SWC3). Viral replication was observed mainly in PIMs, which at the same time showed intense activation, accompanied by the expression of IL-1alpha and TNF-alpha. The occurrence of interstitial oedema, neutrophil sequestration and fibrin microthrombi in septal capillaries coincided with high degrees of cytokine expression by infected PIMs. Alveolar macrophages did not show a significant change in cytokine expression as a result of ASF infection, and viral replication was detected in only a low percentage of these cells., (Copyright Harcourt Publishers Ltd.)

This report examines the role of African swine fever virus (ASFV) structural protein pE120R in virus replication. Immunoelectron microscopy revealed that protein pE120R localizes at the surface of the intracellular virions. Consistent with this, coimmunoprecipitation assays showed that protein pE120R binds to the major capsid protein p72. Moreover, it was found that, in cells infected with an ASFV recombinant that inducibly expresses protein p72, the incorporation of pE120R into the virus particle is dependent on p72 expression. Protein pE120R was also studied using an ASFV recombinant in which E120R gene expression is regulated by the Escherichia coli lac repressor-operator system. In the absence of inducer, pE120R expression was reduced about 100-fold compared to that obtained with the parental virus or the recombinant virus grown under permissive conditions. One-step virus growth curves showed that, under conditions that repress pE120R expression, the titer of intracellular progeny was similar to the total virus yield obtained under permissive conditions, whereas the extracellular virus yield was about 100-fold lower than in control infections. Immunofluorescence and electron microscopy demonstrated that, under restrictive conditions, intracellular mature virions are properly assembled but remain confined to the replication areas. Altogether, these results indicate that pE120R is necessary for virus dissemination but not for virus infectivity. The data also suggest that protein pE120R might be involved in the microtubule-mediated transport of ASFV particles from the viral factories to the plasma membrane.

During the cytoplasmic maturation of African swine fever virus (ASFV) within the viral factories, the DNA-containing core becomes wrapped by two shells, an inner lipid envelope and an outer icosahedral capsid. We have previously shown that the inner envelope is derived from precursor membrane-like structures on which the capsid layer is progressively assembled. In the present work, we analyzed the origin of these viral membranes and the mechanism of envelopment of ASFV. Electron microscopy studies on permeabilized infected cells revealed the presence of two tightly apposed membranes within the precursor membranous structures as well as polyhedral assembling particles. Both membranes could be detached after digestion of intracellular virions with proteinase K. Importantly, membrane loop structures were observed at the ends of open intermediates, which suggests that the inner envelope is derived from a membrane cisterna. Ultraestructural and immunocytochemical analyses showed a close association and even direct continuities between the endoplasmic reticulum (ER) and assembling virus particles at the bordering areas of the viral factories. Such interactions become evident with an ASFV recombinant that inducibly expresses the major capsid protein p72. In the absence of the inducer, viral morphogenesis was arrested at a stage at which partially and fully collapsed ER cisternae enwrapped the core material. Together, these results indicate that ASFV, like the poxviruses, becomes engulfed by a two-membraned collapsed cisterna derived from the ER.

African swine fever virus (ASFV) is a large complex icosahedral double-stranded DNA virus that replicates in the cytoplasm of susceptible cells. Assembly of new virus particles occurs within the perinuclear viroplasm bodies known as virus factories. Two types of virus particle are routinely observed: "fulls," which are particles with an electron-dense DNA-containing nucleoid, and "empties," which consist of the virus protein and membrane icosahedral shell but are without the incorporation of the virus genome. The objective of this study was to understand ASFV morphogenesis by determining the distribution of intracellular viral DNA in the virus factory and during virus particle assembly. The ultrastructural localisation of DNA within ASFV-infected cells was achieved using two complementary methods: with an ASFV-specific DNA probe to the major capsid protein (p73) gene (B646L) hybridised in situ or through detection of all forms of DNA (viral and cellular) with gold-labelled DNase. Conditions for in situ hybridisation at the electron microscopic level were optimised for infected cells in two Lowicryl resins (K4M and HM20) and using two nonradioactive probe labels (digoxygenin and biotin). The morphological data indicate that the viral DNA, perhaps from specialised storage sites within the factory, begins to condense into a pronucleoid and is then inserted, at a single vertex, into an "empty" particle. Further maturation of the viral particle, including closure of the narrow opening in the icosahedron, gives rise to "intermediate" particles, where the nucleoprotein core undergoes additional consolidation to produce the characteristic mature or "full" virions. The site of particle closure may represent a "weak point" at one vertex, but the mechanisms and structures involved in the packaging and release of the virus genome via such a port are yet to be determined., (Copyright 1998 Academic Press.)

A method to study the function of individual African swine fever virus (ASFV) gene products utilizing the Escherichia coli lac repressor-operator system has been developed. Recombinant viruses containing both the lacI gene encoding the lac repressor and a strong virus late promoter modified by the insertion of one or two copies of the lac operator sequence at various positions were constructed. The ability of each modified promoter to regulate expression of the firefly luciferase gene was assayed in the presence and in the absence of the inducer isopropyl beta-D-thiogalactoside (IPTG). Induction and repression of gene activity were dependent on the position(s) of the operator(s) with respect to the promoter and on the number of operators inserted. The ability of this system to regulate the expression of ASFV genes was analyzed by constructing a recombinant virus inducibly expressing the major capsid protein p72. Electron microscopy analysis revealed that under nonpermissive conditions, electron-dense membrane-like structures accumulated in the viral factories and capsid formation was inhibited. Induction of p72 expression allowed the progressive building of the capsid on these structures, leading to assembly of ASFV particles. The results of this report demonstrate that the transferred inducible expression system is a powerful tool for analyzing the function of ASFV genes.

African swine fever (ASF) virus is a large DNA virus that shares the striking icosahedral symmetry of iridoviruses and the genomic organization of poxviruses. Both groups of viruses have a complex envelope structure. In this study, the mechanism of formation of the inner envelope of ASF virus was investigated. Examination of thin cryosections by electron microscopy showed two internal membranes in mature intracellular virions and all structural intermediates. These membranes were in continuity with intracellular membrane compartments, suggesting that the virus gained two membranes from intracellular membrane cisternae. Immunogold electron microscopy showed the viral structural protein p17 and resident membrane proteins of the endoplasmic reticulum (ER) within virus assembly sites, virus assembly intermediates, and mature virions. Resident ER proteins were also detected by Western blotting of isolated virions. The data suggested the ASF virus was wrapped by the ER. Analysis of the published sequence of ASF virus (R. J. Yanez et al., Virology 208:249-278, 1995) revealed a reading frame, XP124L, that encoded a protein predicted to translocate into the lumen of the ER. Pulse-chase immunoprecipitation and glycosylation analysis of pXP124L, the product of the XP124L gene, showed that pXP124L was retained in the ER lumen after synthesis. When analyzed by immunogold electron microscopy, pXP124L localized to virus assembly intermediates and fully assembled virions. Western blot analysis detected pXP124L in virions isolated from Percoll gradients. The packaging of pXP124L from the lumen of the ER into the virion is consistent with ASF virus being wrapped by ER cisternae: a mechanism which explains the presence of two membranes in the viral envelope.

African swine fever virus (ASFV) enters Vero cells by adsorptive endocytosis [Valdeira, M.L., Geraldes, A., 1985. Morphological study on the entry of African swine fever virus into cells, Biol Cell. 55, 35-40]. Electron microscopy of a lysosomotropic drug-controlled penetration indicated that this step takes place in the endosomes, after fusion between the viral envelope and the limiting membrane of the endosome. Inhibition studies with colcemid, cytochalasin B, sodium azide, dinitrophenol, lysosomotropic weak bases, and the ionophore monensin, showed that the virus uptake is largely independent of cytoskeletal and lysosomal function, but dependent on oxidative phosphorylation. Some protease inhibitors inhibited viral replication at an early step, indicating that the initiation of infection depends on a viral proteolytic cleavage.

The effects of African swine fever (ASF) virus infection on bone marrow hematopoiesis and microenvironment were determined by studying the sequential development of ultrastructural lesions of bone marrow and blood cell changes. Eight pigs (two pigs/infected group) were inoculated by intramuscular route with 10(5) 50% hemadsorbing doses (HAD50) of the Malawi'83 ASF virus isolate. Two uninfected pigs were used as controls. Ultrastructural changes developed by day 3 postinoculation (PI), persisted through day 7 PI, and were characterized by activation of macrophages. From day 5 PI, viral replication was observed in monocytes/macrophages, reticular cells, immature neutrophils, and promonocytes. Also viral replication was detected in megakaryocytes, endothelial cells, and pericytes at day 7 PI. Vascular alterations consisted of activation of sinusoidal endothelial cells, intravascular coagulation, and fibrin strands interspersed among microenvironment and hematopoietic cells. No significant changes were observed in total white blood cells counts, percentage of monocytes, and platelet counts; however, severe lymphopenia and neutrophilia were detected from day 3 PI. Results of this experiment indicate that there is increased hematopoiesis in bone marrow during acute ASF, coinciding with macrophage activation. Neither vascular changes nor viral replication in different bone marrow cell populations gave rise to impaired bone marrow function. Increased hematopoiesis would exert a positive influence by preventing the early onset of thrombocytopenia and would exert a negative influence by stimulating the spread of the virus via neutrophils. Increased hematopoiesis would be unable to compensate for the lymphopenia.

Polyprotein processing is a common strategy of gene expression in many positive-strand RNA viruses and retroviruses but not in DNA viruses. African swine fever virus (ASFV) is an exception because it encodes a polyprotein, named pp220, to produce several major components of the virus particle, proteins p150, p37, p34, and p14. In this study, we analyzed the assembly pathway of ASFV and the contribution of the polyprotein products to the virus structure. Electron microscopic studies revealed that virions assemble from membranous structures present in the viral factories. Viral membranes became polyhedral immature virions after capsid formation on their convex surface. Beneath the lipid envelope, two distinct domains appeared to assemble consecutively: first a thick protein layer that we refer to as core shell and then an electron-dense nucleoid, which was identified as the DNA-containing domain. Immunofluorescence studies showed that polyprotein pp220 is localized in the viral factories. At the electron microscopic level, antibodies to pp220 labeled all identifiable forms of the virus from the precursor viral membranes onward, thus indicating an early role of the polyprotein pp220 in ASFV assembly. The subviral localization of the polyprotein products, examined on purified virions, was found to be the core shell. In addition, quantitative studies showed that the polyprotein products are present in equimolar amounts in the virus particle and account for about one-fourth of its total protein content. Taken together, these results suggest that polyprotein pp220 may function as an internal protein scaffold which would mediate the interaction between the nucleoid and the outer layers similarly to the matrix proteins of other viruses.

African swine fever virus is an icosahedral double-standed DNA virus which replicates in the cytoplasm of porcine monocytic cells. Progeny virus particles, like poxviruses and iridoviruses, are assembled in cytoplasmic inclusions, termed virus factories. The formation of these structures in susceptible cells infected in vitro with pathogenic and attenuated ASFV isolates has been studied by semiquantitative and qualitative electron microscopy. Recognizable virus factory elements were detected by B hr postinfection (hpi) and increased significantly in profile area between 12 and 18 hpi. The number of virus particles associated with the virus factories also increased significantly between 12 and 24 hpi. These included particles with ("full") and without ("empty") a nucleo-protein core. Similar results were obtained for both pathogenic (Malawi) and attenuated (Uganda) virus isolates, but the replication of pathogenic virus in the macrophage was more efficient than that of the attenuated virus in a porcine kidney cell line, where a relatively higher percentage of defective "empty" particles were observed. Analysis of virus factory formation was also done directly on lung and liver tissue from a pig infected with the highly pathogenic Malawi virus isolate. The in vivo data for virus factory area and particle number in both pulmonary intravascular macrophages and liver Kupffer cells at Day 4 postinfection were similar to those observed in vitro. As expected, using postembedding immunoelectron microscopy, DNA (both cellular and viral) was detected in the cell nucleus, cytoplasmic virus, and mature extracellular virus. More interestingly, DNA was absent in the cytoplasm, but readily observed in virus factories. This DNA, which we presume to be viral, was found in close association with membrane-like material and "empty" particles, suggesting that the virus DNA may enter a partially formed capsid which is then sealed in order to develope into a fully assembled particle. According to this hypothesis, ASFV morphogenesis involves the initial formation of "empty" particles within the virus factory. The adjacently formed nucleo-protein material is then inserted into the "empty" particles, as has been described for poxviruses. These particles then mature in to the "full" particles and leave the virus factory as a prelude to release from the cell by budding.

The presence of virus replication centers in neutrophils from pigs inoculated with a highly virulent strain of African swine fever virus is described for the first time in vivo. Virus antigens were observed from 3 days post-inoculation (dpi) onwards by means of an immunohistochemical technique. At this time (3 dpi), transmission electron microscopy studies revealed the presence of large amounts of neutrophils in the vascular lumens. At 5 and 7 dpi, neutrophils with phagosomes frequently contained virus particles. In addition, within the cytoplasm of some mature and immature neutrophils, both viral particles and virus replication centers were observed at 5 and 7 dpi.

Here we describe an African swine fever virus (ASFV) protein encoded by the open reading frame 5-AR that shares structural and functional similarities with the family of bacterial histone-like proteins which include histone-like DNA binding proteins, integration host factor, and Bacillus phage SPO1 transcription factor, TF1. The ASFV 5-AR gene was cloned by PCR and expressed in E. coli. Monospecific antiserum prepared to the 5-AR bacterial expression product specifically immunoprecipitated a protein of approximately 11.6 kDa from ASFV infected swine macrophages at late times post infection. Additionally, the 5-AR expression product was strongly recognized by ASFV convalescent pig serum, indicating its antigenicity during natural infection. Cloned p11.6 bound both double and single stranded DNA-cellulose columns. Consistent with a DNA binding function, immunoelectronmicroscopy localized p11.6 to the virion nucleoid, To our knowledge, p11.6 is the first bacterial histone-like DNA-binding protein found in an animal virus or eukaryotic cell system.

An antiserum was raised against the African swine fever virus (ASFV)-encoded ubiquitin-conjugating enzyme (UBCv1) and used to demonstrate by Western blotting (immunoblotting) and immunofluorescence that the enzyme is present in purified extracellular virions, is expressed both early and late after infection of cells with ASFV, and is cytoplasmically located. Antiubiquitin serum was used to identify novel ubiquitin conjugates present during ASFV infections. This antiserum stained virus factories late after infection, suggesting that virion proteins may be ubiquitinated. This possibility was confirmed by Western blotting, which identified three major antiubiquitin-immunoreactive proteins with molecular masses of 5, 18, and 58 kDa in purified extracellular virions. The 18-kDa protein was solubilized from virions at relatively low concentrations of the detergent n-octyl-beta-D-glucopyranoside, indicating that it is externally located and is possibly in the virus capsid. The 18-kDa protein was purified, and N-terminal amino acid sequencing confirmed that the protein was ubiquitinated and was ASFV encoded. The ASFV gene encoding this protein (PIG1) was sequenced, and the encoded protein expressed in an Escherichia coli expression vector. Recombinant PIG1 was ubiquitinated in the presence of E. coli expressed UBCv1 in vitro. These results suggest that PIG1 may be a substrate for UBCv1. The predicted molecular masses of the PIG1 protein and recombinant ubiquitinated protein were larger than the 18-kDa molecular mass of the ubiquitinated protein present in virions. Therefore, during viral replication, a precursor protein may undergo limited proteolysis to generate the ubiquitinated 18-kDa protein.

Highly purified skin-derived dendritic cells (SDDCs) isolated from swine skin by a simple novel method were cultured for 24 hours before independent or sequential inoculation with African swine fever virus (ASFV) and foot-and-mouth disease virus (FMDV). By avidin-biotin immunohistochemical staining, ASFV antigen was detected in 50% of SDDCs as early as 1.5 hours postinfection (HPI) and in 80% by 3 HPI when cytopathic effect was noted. Cell lysis was detected with FMDV infection as early as 8 HPI; immunostaining for FMDV antigen was found in 10% of the cells. African swine fever virus replication was detected by transmission electron microscopy in a high percentage of SDDCs by 11 HPI. Sequential infection with FMDV 3 hours after ASFV inoculation blocked FMDV infection. These findings demonstrate that both ASFV and FMDV infect dendritic cells of Langerhans cell type in vitro and ASFV interferes with FMDV infection.

The role of microtubules in intracellular transport of African swine fever virus (ASFV) and virus-induced inclusions was studied by immunofluorescence using anti-ASFV and anti-tubulin antibodies, by electron microscopy of infected Vero cells and by in vitro binding of virions to purified microtubules. MTC, a reversible colchicine analogue, was used to depolymerize microtubules. In cells treated with MTC multiple large inclusions containing ASFV antigens and particles were observed in the cytoplasm. Removal of the drug lead to migration and fusion of the inclusions at a perinuclear location. To study the effect of microtubule repolymerization on virus particle distribution, the particles were counted in thin sections of MTC treated cells and at different times after removal of the drug. In cells treated with MTC 6.8% and 3.6% of the virus particles were found respectively in the cytoplasm and at the cell membrane while 38% of the particles were located around the virosome. With reversal of the drug effect the number of virus particles around the virosomes progressively decreased to 10% at 2 h while the number of particles in the cytoplasm and at the cell membrane increased. At 2 h after removal of the drug 33.5% of the particles were found budding from the cell membrane. Virus particles were found closely associated with microtubules in cytoskeletons obtained by Triton X-100 extraction of taxol treated cells. The association of virus particles with microtubules was also observed in vitro using purified microtubules and virus particles.(ABSTRACT TRUNCATED AT 250 WORDS)

An inhibitory analysis of the role of glycosylation and glycoproteins in the manifestations of the most important properties of African swine virus was carried out using a set of strains and variants with contrasting characteristics. Glycoproteins were shown to realize some virus functions such as virulence and its variability, intracellular transport and exocytosis of virions, hemadsorption, but not immunological recognition of the infected cells by cytotoxic T-lymphocytes.

Light and electron microscopical studies were carried out after experimental induced and spontaneous infection with African swine fever virus. The experimental infection was performed in 18 pigs divided into two groups consisting of 9 animals. The pigs of group I were inoculated with virulent isolate E 70, those of group II with attenuated isolate E 75. Two infected pigs of group I and one control animal were killed on days 3, 5 or 7 p.i., two pigs of group II and one control animal were killed on days 9, 11 or 13 p.i. Additionally 19 spontaneous infected and seropositive animals and two seronegative pigs without clinical signs were examined. Infection with African swine fever virus resulted in slight morphological alterations of the intestine. The pathological findings showed a more intense involvement of the large intestine, especially the caecum and colon, than the small intestine, where the ileum was mostly affected. In all three groups edema and vacuolisation could be observed in endothelial cells. In spite of beginning degenerative signs, especially after spontaneous infection, the fenestrated structure of the endothelium was conserved in most of the cases. In animals infected with virulent isolate the vascular lumina contained aggregations of fibrin, which was severe pronounced in the pigs of the other groups. In the area of these alterations disturbance of permeability with extravasation could be found. In all groups single virions or virus aggregates could be identified in concave depressions of the erythrocyte membrane or free within the blood plasma, in some cases enveloped by a plasma-like material.(ABSTRACT TRUNCATED AT 250 WORDS)

The late cytoplasmic phases of African swine fever virus (ASFV) morphogenesis in monkey kidney cells have been studied by transmission electron microscopy, focusing attention on the synthesis of viral envelopes. Morphogenesis was studied after reversible cycloheximide blockage of monkey kidney cells infected with ASFV. ASFV appears to synthesize its external and internal envelopes within the cellular cytoplasm, at the same time as the capsid is formed, with intracellular and extracellular virions showing similar structure and polypeptide composition.

The virus capsomeres of the outer and inner layers of capsids were effectively released simultaneously from purified virions by lipase digestion and were purified by a linear gradient ultracentrifugation. The capsid consisted of an array of double layers of uniformly arranged individual capsomeres where a lipid(s) served as a matrix in between the capsomeres.

An acid phosphatase activity has been detected in purified preparations of African swine fever virus. Purified viral cores obtained after Nonidet-P40 and 2-mercaptoethanol treatment of the virus retained the activity as assayed with nitrophenyl phosphate as substrate at pH 5. Enzyme cytochemistry by electron microscopy showed that the acid phosphatase activity is localized mainly inside the core of the virion. The molecular weight and the isoelectric point of the virus acid phosphatase activity confirmed that it was distinct from the host cellular enzyme.

Seven African swine fever virus structural proteins were localized in the virion by immunoelectron microscopy. African swine fever virus-infected cells were incubated, before or after embedding and thin sectioning, with monoclonal antibodies specific for different structural proteins, and after labeling with protein A-gold complexes, the samples were examined in the electron microscope. Proteins p14 and p24 were found in the external region of the virion, proteins p12, p72, p17, and p37 were found in the intermediate layers, and protein p150 was found in the nucleoid and in one vertex. A monoclonal antibody that recognized protein p150 as well as p220, a virus-induced, nonstructural protein, could also bind to a component present in the nucleus of both uninfected and virus-infected cells.

Methods for the purification of African swine fever virus (ASFV) and its dissection into two fractions are described. The difficulties which have been encountered previously in attempts to purify the virus, namely contamination with large amounts of cellular constituents and aggregation of the virus particles, have been overcome by treatment with Tween 80 and by the use of 1 M-NaCl in the sucrose gradients. Five major polypeptides, mol. wt. 10(3) X 125 (VP1), 76 (VP2), 50 (VP3), 44 (VP4) and 39 (VP5) were found in the purified particles. The virus was dissected by treatment with Nonidet NP 40 into (a) a fraction which had the appearance of an empty capsid shell and capsid shell and contained the polypeptides VP2 and VP3 and (b) a structure containing VP1 and VP4. The location of VP5 was not ascertained.

Since the application of negative staining, preceded by fixation, prevents the disruption and distortion of the capsid of the African swine fever virus, improved contrast and evaluation of the appearance and size of virus particles in the electron microscope is possible and, in addition, the icosahedral shape of the virus is demonstrable. The mature virus particle contains at least 2 capsid layers and an outer envelope.

Cultures from pig bone marrow cells were infected with ASFV and the replication cycle was studied. In the cytoplasmic replication areas there are a differentiation of membrane segments. Some of them are polygonal, which give rise to virus particles. An over production of viral coded materials, including non-polygonal membranes seems to be an important feature of the replicative cycle of ASFV in this cell system. Viruses are released enveloped with a cellular membrane. Paracrystalline arrays of viruses are seldom seen in the cytoplasm. At no time did we see viruses in the nucleus. Infected cells showed marked cytopathic effect, beginning at early times post infection.

The sedimentation coefficient of the infective unit of African swine fever in tissue culture harvest fluids was measured in a preparative ultracentrifuge. The boundary locator method used also permitted making an estimate of heterogeneity. The sedimentation coefficient ranged from 3,000 to 8,000 Svedberg units, representing many classes of infective particles. Electron microscopy on culture fluids from infected cells showed many kinds of virus-containing units. Sucrose-CsCl gradient centrifugation was used to concentrate and to purify (partly) African swine fever virus for analytical ultracentrifugation. The optical patterns of the physical particles revealed a range of coefficients from 1,800 to 3,200 Svedberg units in tris-buffered saline solution at 20 C and buoyant densities from 1.19 to 1.24 g/ml in CsCl. The disparity of these values from those obtained by preparative ultracentrifugation indicates a change in the virus structure or a selection of viral populations on purification (or both).