The A179L Gene of African Swine Fever Virus Suppresses Virus-Induced Apoptosis but Enhances Necroptosis.

A179L, a non-structural protein of African swine fever virus (ASFV), is capable of suppressing apoptosis by binding the BH3 domain of the pro-apoptotic Bcl-2 family proteins via a conserved ligand binding groove. Our present study aims to determine if A179L affects necroptosis, the second form of programmed cell death induced by DNA and RNA viruses. Here we report that A179L enhanced TNF-α or TSZ (TNF-α, Smac, and Z-Vad)-induced receptor-interacting protein kinase (RIPK1), RIPK3, and mixed lineage kinase domain like peudokinase (MLKL) phosphorylation in L929 cells, a murine fibrosarcoma cell line. Sytox green staining revealed that A179L significantly increased the number of necroptotic cells in TSZ-treated L929 cells. Using human herpes simplex virus 1 (HSV-1) to model DNA virus-induced cell death, we found that A179L blocked the HSV-1-induced cleavage of poly (ADP-ribose) polymerase (PARP), caspase 8, and caspase 3 and decreased the number of apoptotic cells in HSV-1-infected IPEC-DQ cells, a porcine intestinal epithelial cell line. In contrast, A179L transfection of IPEC-DQ cells enhanced HSV-1-induced RIPK1, RIPK3, and MLKL phosphorylation and increased the number of necroptotic cells. Consistently, A179L also suppressed apoptosis but enhanced the necroptosis induced by two RNA viruses, Sendai virus (SeV) and influenza virus (IAV). Our study uncovers a previously unrecognized role of A179L in regulating cell death and suggests that A179L re-directs its anti-apoptotic activity to necroptosis.

African Swine Fever Virus CD2v Protein Induces ß-Interferon Expression and Apoptosis in Swine Peripheral Blood Mononuclear Cells.

African swine fever (ASF) is a hemorrhagic disease of swine characterized by massive lymphocyte depletion in lymphoid tissues due to the apoptosis of B and T cells, a process likely triggered by factors released or secreted by infected macrophages. ASFV CD2v (EP402R) has been implicated in viral virulence and immunomodulation in vitro; however, its actual function(s) remains unknown. We found that CD2v expression in swine PK15 cells induces NF-κB-dependent IFN-ß and ISGs transcription and an antiviral state. Similar results were observed for CD2v protein treated swine PBMCs and macrophages, the major ASFV target cell. Notably, treatment of swine PBMCs and macrophages with CD2v protein induced apoptosis. Immunoprecipitation and colocalization studies revealed that CD2v interacts with CD58, the natural host CD2 ligand. Additionally, CD58 knockdown in cells or treatment of cells with an NF-κB inhibitor significantly reduced CD2v-mediated NF-κB activation and IFN-ß induction. Further, antibodies directed against CD2v inhibited CD2v-induced NF-κB activation and IFN-ß transcription in cells. Overall, results indicate that ASFV CD2v activates NF-κB, which induces IFN signaling and apoptosis in swine lymphocytes/macrophages. We propose that CD2v released from infected macrophages may be a significant factor in lymphocyte apoptosis observed in lymphoid tissue during ASFV infection in pigs.

African Swine Fever: Disease Dynamics in Wild Boar Experimentally Infected with ASFV Isolates Belonging to Genotype I and II.

After the re-introduction of African swine fever virus (ASFV) genotype II isolates into Georgia in 2007, the disease spread from Eastern to Western Europe and then jumped first up to Mongolian borders and later into China in August 2018, spreading out of control and reaching different countries of Southeast Asia in 2019. From the initial incursion, along with domestic pigs, wild boar displayed a high susceptibility to ASFV and disease development. The disease established self-sustaining cycles within the wild boar population, a key fact that helped its spread and that pointed to the wild boar population as a substantial reservoir in Europe and probably also in Asia, which may hinder eradication and serve as the source for further geographic expansion. The present review gathers the most relevant information available regarding infection dynamics, disease pathogenesis and immune response that experimental infections with different ASFV isolates belonging to genotype I and II in wild boar and feral pigs have generated. Knowledge gaps in areas such as disease pathogenesis and immune response highlights the importance of focusing future studies on unravelling the early mechanisms of virus-cell interaction and innate and/or adaptive immune responses, knowledge that will contribute to the development of efficacious treatments/vaccines against ASFV.

Effect of O. porcinus Tick Salivary Gland Extract on the African Swine Fever Virus Infection in Domestic Pig.

African swine fever is a haemorrhagic disease in pig production that can have disastrous financial consequences for farming. No vaccines are currently available and animal slaughtering or area zoning to restrict risk-related movements are the only effective measures to prevent the spread of the disease. Ornithodoros soft ticks are known to transmit the African swine fever virus (ASFV) to pigs in farms, following the natural epidemiologic cycle of the virus. Tick saliva has been shown to modulate the host physiological and immunological responses during feeding on skin, thus affecting viral infection. To better understand the interaction between soft tick, ASFV and pig at the bite location and the possible influence of tick saliva on pig infection by ASFV, salivary gland extract (SGE) of Ornithodoros porcinus, co-inoculated or not with ASFV, was used for intradermal auricular inoculation. Our results showed that, after the virus triggered the disease, pigs inoculated with virus and SGE presented greater hyperthermia than pigs inoculated with virus alone. The density of Langerhans cells was modulated at the tick bite or inoculation site, either through recruitment by ASFV or inhibition by SGE. Additionally, SGE and virus induced macrophage recruitment each. This effect was enhanced when they were co-inoculated. Finally, the co-inoculation of SGE and virus delayed the early local spread of virus to the first lymph node on the inoculation side. This study has shown that the effect of SGE was powerful enough to be quantified in pig both on the systemic and local immune response. We believe this model should be developed with infected tick and could improve knowledge of both tick vector competence and tick saliva immunomodulation.

[On the situation of African swine fever and the biological characterization of recent virus isolates]. / Zur Situation der Afrikanischen Schweinepest und der biologischen Charakterisierung aktueller Virusisolate.

African swine fever (ASF), a disease notifiable to the World Organization of Animal Health (OIE), is characterized by severe, unspecific clinical signs and high mortality rates. Hosts for ASF virus (ASFV) are only members of the family Suidae and soft ticks of the genus Ornithodoros. Currently, no vaccine is available and therefore, the control is primarily based on strict sanitary measures. The most important part is the early detection of the disease within affected animal holdings and the fast and reliable confirmation by laboratory diagnosis. Infections of domestic pigs and European wild boar with recent Armenian, Sardinian, Lithuanian or Kenyan ASFV isolates lead to severe, acute disease courses with the predominant symptom of high fever (> 41 degrees C) accompanied by further unspecific clinical signs such as lethargy, loss of appetite, diarrhoea, respiratory symptoms, and an increased bleeding tendency. In experimental infection studies the mortality rate reached 100%. The most prominent pathomorphological findings included ebony-colored gastrohepatic lymph nodes, lung oedema, petechiae in the renal cortex, and oedema of the gallbladder wall. In the light of the current epidemiological situation with endemic ASFV infections on Sardinia, outbreaks in Russia and several Eastern EU Member States there is a risk for an introduction in further, previously unaffected EU countries including Germany. Hence, appropriate sample materials (serum, blood, spleen) of domestic pigs with unspecific clinical symptoms or pathomorphological findings should be examined for both ASFV and classical swine fever virus.

A study of lymphoid organs and serum proinflammatory cytokines in pigs infected with African swine fever virus genotype II.

African swine fever virus (ASFV), the causative agent of one of the most important viral diseases of domestic pigs for which no vaccine is available, causes immune system disorders in infected animals. In this study, the serum levels of proinflammatory cytokines, as well as the histological and cellular constitution of lymphoid organs of pigs infected with ASFV genotype II were investigated. The results showed a high degree of lymphocyte depletion in the lymphoid organs, particularly in the spleen and lymph nodes, where ASFV infection led to a twofold decrease in the number of lymphocytes on the final day of infection. Additionally, ASFV-infected pigs had atypical forms of lymphocytes found in all lymphoid organs. In contrast to lymphocytes, the number of immature immune cells, particularly myelocytes, increased dramatically and reached a maximum on day 7 postinfection. The serum levels of TNF-α, IL-1ß, IL-6, and IL-8 were evaluated. Proinflammatory cytokines showed increased levels after ASFV infection, with peak values at 7 days postinfection, and this highlights their role in the pathogenesis of ASFV. In conclusion, this study showed that ASFV genotype II, like other highly virulent strains, causes severe pathological changes in the immune system of pigs.

African swine fever virus infects macrophages, the natural host cells, via clathrin- and cholesterol-dependent endocytosis.

The main cellular target for African swine fever virus (ASFV) is the porcine macrophage. However, existing data about the early phases of infection were previously characterized in non-leukocyte cells such as Vero cells. Here, we report that ASFV enters the natural host cell using dynamin-dependent and clathrin-mediated endocytosis. This pathway is strongly pH-dependent during the first steps of infection in porcine macrophages. We investigated the effect of drugs inhibiting several endocytic pathways in macrophages and compared ASFV with vaccinia virus (VV), which apparently involves different entry pathways. The presence of cholesterol in cellular membranes was found to be essential for a productive ASFV infection while actin-dependent endocytosis and the participation of phosphoinositide-3-kinase (PI3K) activity were other cellular factors required in the process of viral entry. These findings improved our understanding of the ASFV interactions with macrophages that allow for successful viral replication.

The non-haemadsorbing African swine fever virus isolate ASFV/NH/P68 provides a model for defining the protective anti-virus immune response.

African swine fever virus ASFV/NH/P68 is a naturally occurring, non-haemadsorbing and non-fatal isolate. Longitudinal clinical and immunological studies on 31 pigs inoculated oronasally or intramuscularly with this isolate defined two discrete groups of animals: those developing ASF chronic type lesions and those remaining asymptomatic. Animals developing lesions had viraemia and fever late after infection, NK activity levels close to that of control animals and high levels of anti-ASFV specific antibodies together with a marked hypergammaglobulinaemia involving IgG1, IgG2, IgM and IgA immunoglobulin isotypes. Pigs remaining asymptomatic after infection, on the other hand, did not have viraemia or fever after day 14 post-infection and had elevated NK cell activity, but normal plasma Ig concentrations and relatively low specific anti-virus antibody concentrations throughout the duration of the experiments. Importantly, the latter group of pigs virus were resistant to subsequent challenge with the highly virulent ASFV/L60 isolate and survived with no major changes in any of the parameters examined and referred to above. Finally, lymphoproliferative responses to the mitogens concanavalin A, phytohaemagglutinin and pokeweed mitogen were not depressed in either of the two clinically defined groups of pigs. Thus further studies with this infection model may provide new insights on mechanisms of protective immunity to ASFV.

Ultrastructural changes related to the lymph node haemorrhages in acute African swine fever.

In order to determine the pathogenic mechanisms involved in lymph node haemorrhages in acute African swine fever (ASF), eight pigs were inoculated with ASF virus, strain Malawi'83. Lymph node haemorrhages were observed from three days post infection (dpi) onwards, coinciding with ASF virus replication in monocytes and macrophages adjacent to stimulated endothelial cells, phagocytic stimulation of capillary and small-vessel endothelial cells, increase in the number of fenestrations of endothelial cells, and endothelial cell loss, as well as clusters of blood cells and necrotic material beneath the endothelium. Vascular lumina were blocked by platelet plugs and fibrin microthrombi. These phenomena became more marked as the disease progressed. At five dpi, virus replication was also found in circulating neutrophils. At seven dpi, lesions were more intense and were accompanied by virus replication in sinus and capillary endothelial cells, and in other cell populations including pericytes, fibroblasts, smooth muscle fibres and reticular cells. The results obtained in this study suggest that lymph node haemorrhages are related to endothelial stimulation and the onset of disseminated intravascular coagulation. Virus replication in vessel wall cells occurs only in the final stages of the disease and plays a secondary role.

African swine fever interference with foot-and-mouth disease infection and seroconversion in pigs.

Initial oral infection of pigs with either highly virulent (L-60) or moderately virulent (DR-2) African swine fever virus (ASFV), followed in 3 days with exposure to foot-and-mouth disease virus (FMDV) (tongue inoculation and contact), failed to cause FMDV infection or seroconversion in 18 of 22 L-60-infected pigs and 13 of 34 DR-2-infected pigs. Of the 13 DR-2-infected pigs remaining free of foot-and-mouth disease (FMD), 2 pigs survived to 24 days without antibody to FMDV, despite constant contact with clinically infected pigs with FMD. Three other DR-2-infected pigs never developed FMD lesions but did develop low levels of antibody to FMDV by day 17. A group of larger pig (in which DR-2 is less virulent) infected with DR-2 and then FMDV had a rapid but suppressed immune response to FMDV. Contact pigs introduced 3 days postinoculation and inoculated with FMDV only all became infected with ASFV by contact and died. This remarkably long lasting 1-way interference with FMD infection during acute and subacute African swine fever was not anticipated. Infection with ASFV may have blocked the initial target cells (possibly dendritic cells) necessary for establishment of FMDV infection.

Haemostatic abnormalities in African swine fever a comparison of two virus strains of different virulence (Dominican Republic '78 and Malta '78).

African swine fever (ASF) virus strains cause haemorrhage by producing a variety of defects, which vary in severity from strain to strain. To distinguish the main haemostatic defects leading to haemorrhage, two groups of pigs were infected with moderately virulent (Dominican Republic '78) and less virulent (Malta '78) ASF virus strains. Mortality rate and severity of clinical observations were greater in pigs infected with DR '78 virus compared with pigs infected with Malta '78 virus. The animals became febrile from day 3 to 4 onwards at a time when the viraemia was high (10(7) to 10(8) HAD50/ml). No difference was found during the period observed in their pattern of viraemia or pyrexia. Thrombocytopenia developed in both groups but with different kinetics, suggesting two different mechanisms of sequestration of platelets. When coagulation tests were performed, significant abnormalities were found, including evidence for disseminated intravascular coagulation. These abnormalities were much less pronounced in the group infected with Malta '78. Antithrombin III activity did not change significantly in either group. Decreased plasminogen activity was found in the early phase of disease in DR '78 infected pigs. These results indicate that when haemorrhage does occur in DR '78 infected pigs, it is a consequence of more pronounced degrees of haemostatic impairment probably due to a marked endothelial injury and/or generation of procoagulant activity.

Arachidonic acid metabolites in the pathophysiology of thrombocytopenia and haemorrhage in acute African swine fever.

Changes in the production of proaggregatory (thromboxane A2 and prostaglandin E2) and antiaggregatory (prostacyclin) prostaglandins by blood platelets, macrophages and endothelial cells during acute African swine fever caused by both a highly virulent virus and a less virulent virus were studied. No impairment in thromboxane A2 release by either platelets or macrophages could be detected but prostacyclin production by the endothelium was impaired. There was also a significant increase in prostaglandin E2 release by macrophages at the time when thrombocytopenia was most marked. However, the early event that causes primary aggregation remains obscure.

Coagulation changes in African swine fever virus infection.

Pigs were infected with highly virulent (Tengani '62), with moderately virulent (DR '79) African swine fever (ASF) virus, or with virulent hog cholera (HC) virus. Changes in platelet counts, selected coagulation assays and concentrations of factor VIII-related antigen (VIIIR:Ag) were monitored. Permeability of aortic endothelium was studied after the injection of Evan's blue dye on various days after infection with DR '79 ASF virus. Virulent ASF virus caused prolongation of the activated partial thromboplastin time (APTT), 1-stage prothrombin time, and thrombin clotting time as early as postinoculation day (PID) 4. These changes became progressively more severe until death. Both virulent HC and DR'79 viruses induced an increase APPT and thrombin clotting time at PID 3 to 4, only occasionally did the prothrombin time increased significantly (P less than 0.01). The APPT began to decrease on PID 7 and 8, but only DR'79-infected pigs lived long enough to regain a normal APTT. Infection by ASF viruses caused acute thrombocytopenia after PID 6 and platelet counts of HC virus-infected pigs decreased progressively from the onset of fever to levels of 1 to 2 X 10(5)/mm3 at PID 6 to 7. All ASF virus-infected pigs had an increase in VIIIR:Ag beginning at PID 3, with maximum increases at PID 6 to 7. Hog cholera virus infection did not cause consistent changes in levels of VIIIR:Ag. Pigs infected with DR'79 virus did not have increased vascular permeability to Evan's blue dye during infection; however, there was markedly decreased staining of the aorta after pigs became thrombocytopenic.