Acute African Swine Fever Research Articles

Clinicopathological and ultrastructural study of African swine fever outbreak in North-East India

The present investigation focuses on examining the clinical, histopathological, and ultrastructural changes that occurred in pig, during an outbreak of African swine fever (ASF) in 2022 in Assam, India. The disease initially manifested as a per-acute case with high mortality but without any evident clinical signs. Subsequently, some animals exhibited an acute form of the disease characterized by high fever (104–106 °F), anorexia, vomiting, respiratory distress, and bleeding from the anal and nasal orifices. During acute African swine fever virus (ASFV) infections, elevated levels of pro-inflammatory IL-1α, IL-1β, IL-6, TNF, CCL2, CCL5, and CXCL10 were detected in the palatine tonsil, lymph nodes, spleen, and kidney using qPCR assay. These molecular changes were associated with haemorrhages, edemas, and lymphoid depletion. Postmortem examinations revealed prominent features such as splenomegaly with haemorrhages, haemorrhagic lymphadenitis, severe petechial haemorrhage in the kidney, pneumonia in the lungs, and necrotic palatine tonsil. Histopathological analysis demonstrated lymphocyte depletion in lymphoid organs, multi-organ haemorrhages, and interstitial pneumonia in the lungs. Scanning electron microscopy (SEM) further confirmed lymphocyte depletion in lymphoid organs through lymphocyte apoptosis and kidney damage with distorted tubules due to red blood cell destruction. Transmission electron microscopy reaffirmed lymphocyte apoptosis by observing chromatin condensation and nucleus margination in lymphocytes of lymphoid organs. These findings provide comprehensive insights into the clinical, histopathological, and ultrastructural aspects of ASF outbreak in pigs. Understanding the pathological changes associated with ASF can contribute to improved diagnosis, prevention, and control measures for this highly contagious and economically devastating viral disease.

Transcriptomic analysis reveals upregulated host metabolisms and downregulated immune responses or cell death induced by acute African swine fever virus infection.

The African swine fever virus is a virulent and communicable viral disease that can be transmitted by infected swine, contaminated pork products, or soft tick vectors. Nonstructural proteins encoded by ASFV regulate viral replication, transcription, and evasion. However, the mechanisms underlying the host response to ASFV infection remain incompletely understood. In order to enhance comprehension of the biology and molecular mechanisms at distinct time intervals (6, 12, 24 h) post infection, transcriptome analyses were executed to discern differentially expressed genes (DEGs) between ASFV and mock-infected PAMs. The transcriptomic analysis unveiled a total of 1,677, 2,122, and 2,945 upregulated DEGs and 933, 1,148, and 1,422 downregulated DEGs in ASFV- and mock-infected groups at 6, 12, and 24 h.p.i.. The results of the transcriptomic analysis demonstrated that the infection of ASFV significantly stimulated host metabolism pathways while concurrently inhibiting the expression of various immune responses and cell death pathways. Our study offers crucial mechanistic insights into the comprehension of ASFV viral pathogenesis and the multifaceted host immune responses. The genes that were dysregulated may serve as potential candidates for further exploration of anti-ASFV strategies.

Recombinant porcine interferon cocktail delays the onset and lessens the severity of African swine fever

African swine fever (ASF) is a highly contagious and deadly disease that affects domestic and wild pigs. No commercial vaccine or antiviral is currently available against ASF. The control of ASF primarily relies on implementing effective biosecurity measures during the breeding process. Here, we evaluated the preventive and therapeutic potential of the interferon (IFN) cocktail (a mixture of recombinant porcine IFN α and γ) on ASF. The IFN cocktail treatment delayed the onset of ASF symptoms and ASF virus (ASFV) replication for approximately one week. However, IFN cocktail treatment could not prevent the death of the pigs. Further analysis showed that IFN cocktail treatment increased the expression of multiple IFN-stimulated genes (ISGs) in porcine peripheral blood mononuclear cells in vivo and in vitro. Additionally, IFN cocktail modulated the expression of pro- and anti-inflammatory cytokines and reduced tissue injury in the ASFV-infected pigs. Collectively, the results suggest that the IFN cocktail restricts the progression of acute ASF by inducing high levels of ISGs, contributing to the pre-establishment of antiviral status, and modulating the balance of pro- and anti-inflammatory mediators to lessen cytokine storm-mediated tissue damage.

Development of a sensitive, high-throughput extraction protocol for qPCR detection of African swine fever virus in formalin-fixed, paraffin-embedded tissues.

African swine fever (ASF) causes fatal disease in pigs and is an escalating threat to the global swine industry. ASF has re-emerged from Africa as a transcontinental epidemic spreading through the Caucasus into Europe, Russia, China, numerous Asian countries, and the Caribbean. ASF virus (ASFV) is a U.S. select agent requiring handling in high-containment biosafety level 3 (BSL-3) laboratories for pathogen work. Formalin-fixation eliminates infectivity and preserves the genome, providing noninfectious specimens for BSL-2 work. Recovery of DNA from formalin-fixed, paraffin-embedded tissue (FFPET) is challenging and cumbersome. A reliable and easy-to-perform method for DNA recovery from FFPET would facilitate surveillance. To meet this objective, we developed a high-throughput protocol for the recovery of ASFV DNA from FFPET. Deparaffinization, tissue lysis, and reversal of cross-linking were performed in a single tube, followed by DNA purification via automated magnetic bead extraction. Quantitative PCR (qPCR) detection was used to determine the copy number of the B646L gene that encodes for the ASFV p72 protein in tissues (5 pigs, 4 tissues) from pigs with lesions consistent with acute ASF. Copy numbers obtained from FFPET were within one log of copy numbers obtained from fresh tissue, thus enabling ASF qPCR surveillance from formalin-inactivated and preserved tissues at BSL-2 at diagnostic sensitivity similar to fresh tissues tested at BSL-3.

Experimental Infection of Domestic Pigs with African Swine Fever Virus Isolated in 2019 in Mongolia.

African swine fever (ASF) is an infectious viral disease caused by African swine fever virus (ASFV), that causes high mortality in domestic swine and wild boar (Sus scrofa). Currently, outbreaks are mitigated through strict quarantine measures and the culling of affected herds, resulting in massive economic losses to the global pork industry. In 2019, an ASFV outbreak was reported in Mongolia, describing a rapidly progressing clinical disease and gross lesions consistent with the acute form of ASF; the virus was identified as a genotype II virus. Due to the limited information on clinical disease and viral dynamics within hosts available from field observations of the Mongolian isolates, we conducted the present study to further evaluate the progression of clinical disease, virulence, and pathology of an ASFV Mongolia/2019 field isolate (ASFV-MNG19), by experimental infection of domestic pigs. Intramuscular inoculation of domestic pigs with ASFV-MNG19 resulted in clinical signs and viremia at 3 days post challenge (DPC). Clinical disease rapidly progressed, resulting in the humane euthanasia of all pigs by 7 DPC. ASFV-MNG19 infected pigs had viremic titers of 108 TCID50/mL by 5 DPC and shed virus in oral secretions late in disease, as determined from oropharyngeal swabs. Whole-genome sequencing confirmed that the ASFV-MNG19 strain used in this study was a genotype II strain highly similar to other regional strains. In conclusion, we demonstrate that ASFV-MNG19 is a virulent genotype II ASFV strain that causes acute ASF in domestic swine.

Chronological expression and distribution of African swine fever virus p30 and p72 proteins in experimentally infected pigs

African swine fever virus (ASFV), the causative agent of contagious hemorrhagic disease in domestic pigs and wild boars, has temporally regulated gene expression kinetics. The p30 and p72 major structural proteins are involved in viral entry each with different expression kinetics, but neither of their chronological expressions and distribution have been identified in virus-infected animals. Here, we found that both transcription and translation levels of p30 were significantly higher than those of p72 in target organs during the earlier infection-phase. Lymphocyte apoptosis/necrosis and angiectasia were observed as signs of early infection with acute African swine fever. These results show that the chronologically differential expression of ASFV structural proteins tends to be prominent in infected animals, and the p30 protein could play a role in the indication of acute lesions during early infection compared to the late-expressed p72 protein. In conclusion, we propose to consider the chronological expression dynamics of ASFV structural proteins in infected animals to understand virus pathogenesis and antigen targeting for vaccine development.

Age-related viral load and severity of systemic pathological lesions in acute naturally occurring African swine fever virus genotype II infections

African swine fever (ASF) causes a contagious hemorrhagic disease in all ages of pigs without sex predilections. The objective of this study was to determine the age-related viral loads and severity of systemic pathological lesions among three different swine group ages (weaned pigs, fattening pigs, and sows) during a recent outbreak of acute ASF in Vietnam. Age-related viral loads were determined in 5 major organs (lung, liver, spleen, kidney, and lymph node) by immunohistochemistry as well as in the blood by real-time polymerase chain reaction (PCR). Age-related systemic pathological lesions were analyzed in the listed organs among three age groups.Weaned pigs had significantly (p < 0.05) higher levels of viral loads in their lung, liver, lymph nodes and blood than in those of fattening pigs and sows. Fattening pigs had significantly (p < 0.05) higher scores of macroscopic lung and lymphoid lesions, and microscopic liver lesions compared with those of weaned pigs and sows. The results of this study demonstrated that viral loads were age-related in acute naturally occurring ASF but the severity of pathological lesions was not correlated with the level of viral loads in the five major organs.

Structure of African Swine Fever Virus and Associated Molecular Mechanisms Underlying Infection and Immunosuppression: A Review.

African swine fever (ASF) is an acute, highly contagious, and deadly infectious disease. The mortality rate of the most acute and acute ASF infection is almost 100%. The World Organization for Animal Health [Office International des épizooties (OIE)] lists it as a legally reported animal disease and China lists it as class I animal epidemic. Since the first diagnosed ASF case in China on August 3, 2018, it has caused huge economic losses to animal husbandry. ASF is caused by the African swine fever virus (ASFV), which is the only member of Asfarviridae family. ASFV is and the only insect-borne DNA virus belonging to the Nucleocytoplasmic Large DNA Viruses (NCLDV) family with an icosahedral structure and an envelope. Till date, there are still no effective vaccines or antiviral drugs for the prevention or treatment of ASF. The complex viral genome and its sophisticated ability to regulate the host immune response may be the reason for the difficulty in developing an effective vaccine. This review summarizes the recent findings on ASFV structure, the molecular mechanism of ASFV infection and immunosuppression, and ASFV-encoded proteins to provide comprehensive proteomic information for basic research on ASFV. In addition, it also analyzes the results of previous studies and speculations on the molecular mechanism of ASFV infection, which aids the study of the mechanism of clinical pathological phenomena, and provides a possible direction for an intensive study of ASFV infection mechanism. By summarizing the findings on molecular mechanism of ASFV- regulated host cell immune response, this review provides orientations and ideas for fundamental research on ASFV and provides a theoretical basis for the development of protective vaccines against ASFV.

Long‐term follow‐up of convalescent pigs and their offspring after an outbreak of acute African swine fever in Vietnam

African swine fever (ASF) is a contagious haemorrhagic disease in pigs and has become endemic in several Vietnam provinces since the first outbreak in 2019. The presence of carriers and the recurrence of disease in the surviving swine herd after an ASF outbreak has not previously been properly evaluated. In this study, pigs naturally infected with an acute form of ASF were allowed to recover from the disease. A serological follow-up was conducted for more than 14 months with 14 convalescent gilts and their offspring. All convalescent animals had long lasting high serum antibody levels without persistent viremia. They also did not excrete virus via nasal discharge post-recovery. These convalescent pigs could partially perform as replacement gilts despite the fact that ASF affected reproductive performance. Here, we confirmed that there were neither the carriers of nor recurrence of disease in the convalescent pigs and their offspring following the outbreak of acute ASF. These findings may facilitate efforts to design a new farming model in ASF endemic provinces in Vietnam where there is a lack of a repopulation strategy due to the limited funding received from the local regulatory authorities.

Deletion of the K145R and DP148R Genes from the Virulent ASFV Georgia 2007/1 Isolate Delays the Onset, but Does Not Reduce Severity, of Clinical Signs in Infected Pigs.

African swine fever virus causes a frequently fatal disease of domestic pigs and wild boar that has a high economic impact across 3 continents. The large double-stranded DNA genome codes for approximately 160 proteins. Many of these have unknown functions and this hinders our understanding of the virus and host interactions. The purpose of the study was to evaluate the role of two virus proteins, K145R and DP148R, in virus replication in macrophages and virulence in pigs. To do this, the DP148R gene, alone or in combination with the K145R gene, was deleted from the virulent genotype II Georgia 2007/1 isolate. Neither of these deletions reduced the ability of the viruses to replicate in porcine macrophages compared to the parental wild-type virus. Pigs infected with GeorgiaΔDP148R developed clinical and post-mortem signs and high viremia, typical of acute African swine fever, and were culled on day 6 post-infection. The additional deletion of the K145R gene delayed the onset of clinical signs and viremia in pigs by 3 days, but pigs showed signs of acute African swine fever and were culled on days 10 or 13 post-infection. The results show that the deletion of DP148R did not attenuate the genotype II Georgia 2007/1 isolate, contrary to the results obtained with the genotype I Benin97/1 isolate. Additional deletion of the K145R gene delayed clinical signs, but infected pigs reached the humane endpoint. The deletion of additional genes would be required to attenuate the virus.

Genome Sequence of a Virulent African Swine Fever Virus Isolated in 2020 from a Domestic Pig in Northern Vietnam.

This study reports the genome sequence of an isolated African swine fever (ASF) virus (VNUA-ASFV-05L1/HaNam) obtained at the fourth passage on pulmonary alveolar macrophages. The virus was isolated during a typical acute ASF outbreak in pigs in a northern province of Vietnam in 2020.

Serum Concentrations of Vascular Endothelial Growth Factor, Stromal Cell-Derived Factor, Nitric Oxide and Endothelial DNA Proliferation in Development of Microvascular Pathology in Acute African Swine Fever

There is significant evidence that pathology of the microcirculation occurs in African swine fever (ASF); however, the mechanisms by which it develops are largely unknown. In the present experimental infection study, we show that an increase in vascular permeability in the initial stages of acute ASF is dependent on viraemia and elevation of the concentration of serum nitric oxide (NO). Macrophages activated by ASF virus (ASFV) are stimulated to produce NO and simultaneously to sensitize the endothelial cells through the action of vascular endothelial growth factor Β (VEGFΒ), which is followed by an increase in VEGF-mediated endothelial permeability. In the later stages of disease, the endothelial cells undergo DNA proliferation, which may additionally provoke capillary leakage, point haemorrhages and migration of blood cells into tissues. The possible mechanism of a shift in the cell cycle from the G1 to S and G2 stages could be a direct effect of ASFV. The terminal stages of disease are characterized by triggering of compensatory mechanisms such as stimulation of the synthesis of stromal cell-derived factor-1.

Association of hemophagocytic lymphohistiocytosis with kidney lesions in acute African swine fever virus infection

Glomerulonephritis due to African swine fever (ASF) is well documented. However, there is absence of good understanding of mechanisms involved in the development of pathology development. This study examines glomerulonephritis in association with acute infection induced by II genotype (Georgia 2007) of ASF virus. Taken together, the results of urinary analysis and the renal histological analysis led to the diagnosis of diffuse endocapillary proliferative glomerulonephritis with severe tubular injury associated with acute ASF (Georgia 2007). According to the pathogenesis, we have found that the diffuse endocapillary proliferative glomerulonephritis associated with the acute ASF develops with a delay of one to two days compared to development of hemophagocytic lymphohistiocytosis. The diagnosis of endocapillary proliferative glomerulonephritis confirms the characteristic of pathological changes in the composition of urine and urine sediment. The development of acute proliferative glomerulonephritis begins at 3 dpi, and finished at 4–6 dpi with the development of tubular necrosis. Our study demonstrates local macrophage proliferation. Local proliferation may be an important mechanism for amplifying macrophage-mediated renal injury. We have shown that the development of diffuse acute proliferative glomerulonephritis during ASF does not coincide with the presence of the virus in the blood or kidney tissues, but coincides with the developmental of ASFV derived hemophagocytic lymphohistiocytosis. The development of hemophagocytic lymphonocytosis also begins at least at 2–3 dpi and continues up to the terminal stage of the disease.

Deletion of the African Swine Fever Virus Gene DP148R Does Not Reduce Virus Replication in Culture but Reduces Virus Virulence in Pigs and Induces High Levels of Protection against Challenge.

ABSTRACTMany of the approximately 165 proteins encoded by the African swine fever virus (ASFV) genome do not have significant similarity to known proteins and have not been studied experimentally. One such protein is DP148R. We showed that the DP148R gene is transcribed at early times postinfection. Deletion of this gene did not reduce virus replication in macrophages, showing that it is not essential for replication in these cells. However, deletion of this gene from a virulent isolate, Benin 97/1, producing the BeninΔDP148R virus, dramatically reduced the virulence of the virus in vivo. All pigs infected with the BeninΔDP148R virus survived infection, showing only transient mild clinical signs soon after immunization. Following challenge with the parental virulent virus, all pigs immunized by the intramuscular route (11/11) and all except one immunized by the intranasal route (5/6) survived. Mild or no clinical signs were observed after challenge. As expected, control nonimmune pigs developed signs of acute African swine fever (ASF). The virus genome and infectious virus were observed soon after immunization, coincident with the onset of clinical signs (∼106 genome copies or 50% tissue culture infective doses/ml). The levels of the virus genome declined over an extended period up to 60 days postimmunization. In contrast, infectious virus was no longer detectable by days 30 to 35. Gamma interferon (IFN-γ) was detected in serum between days 4 and 7 postimmunization, and IFN-γ-producing cells were detected in all pigs analyzed following stimulation of immune lymphocytes with whole virus. ASFV-specific antibodies were first detected from day 10 postimmunization.IMPORTANCE African swine fever (ASF) is endemic in Africa, parts of the Trans Caucasus, the Russian Federation, and several European countries. The lack of a vaccine hinders control. Many of the ASF virus genes lack similarity to known genes and have not been characterized. We have shown that one of these, DP148R, is transcribed early during virus replication in cells and can be deleted from the virus genome without reducing virus replication. The virus with the gene deletion, BeninΔDP148R, caused mild clinical signs in pigs and induced high levels of protection against challenge with the parental virulent virus. Therefore, deletion of this gene can provide a target for the rational development of vaccines.

Hemophagocytic lymphohistiocytosis in acute African swine fever clinic

IntroductionHemophagocytic lymphohistiocytosis (HLH) usually has been defined as the combination of a proliferation of cytologically benign, actively phagocytic macrophages in bone marrow, spleen, lymph nodes, etc. in association with fever, cytopenia, splenomegaly, and hypertriglyceridemia. HLH is often triggered by viral infection. The aim of this study was to ascertain the features of HLH involvement in African swine fever virus (ASFV) (genotype II) pathogenesis. MethodsThe serum levels of macrophage colony-stimulating factor (MCSF) and granulocyte-macrophage colony-stimulating factor (GMCSF), as well as the histological constitution (for hemophagocytic macrophages detection) of various organs of pigs infected with ASFV genotype II were investigated. The diagnosis of HLH was made according to universally accepted human criteria. ResultsThe association of fever, cytopenias, splenomegaly, and hemophagocytosis was present in 87.5% of the infected pigs (absence of hyperthermia in one of eight pigs). Marked hypertriglyceridemia was observed at 3–4days post infection. Previously it was shown that ASFV induced a significant decrease in the level of fibrinogen from day 5 till the end of experiment. Progression of the HLH coincided with a temporary increase in the serum levels of MCSF levels (early stage of disease) and GMCSF levels (2–3 pays post infection). ConclusionsHemophagocytic syndrome should be suspected in ASFV (genotypeII) infected pigs.

Evidence of hemolysis in pigs infected with highly virulent African swine fever virus.

Aim:The research was conducted to understand more profoundly the pathogenetic aspects of the acute form of the African swine fever (ASF).Materials and Methods:A total of 10 pigs were inoculated with ASF virus (ASFV) (genotype II) in the study of the red blood cells (RBCs), blood and urine biochemistry in the dynamics of disease.Results:The major hematological differences observed in ASFV infected pigs were that the mean corpuscular volume, mean corpuscular hemoglobin, and hematocrits were significantly decreased compared to controls, and the levels of erythropoietin were significantly increased. Also were detected the trends of decrease in RBC count at terminal stages of ASF. Analysis of blood biochemistry revealed that during ASF development, besides bilirubinemia significantly elevated levels of lactate dehydrogenase, and aspartate aminotransferase were detected. Analysis of urine biochemistry revealed the presence of bilirubinuria, proteinuria during ASF development. Proteinuria, especially at late stages of the disease reflects a severe kidney damage possible glomerulonefritis.Conclusion:The results of this study indicate the characteristics of developing hemolytic anemia observed in acute ASF (genotype II).

IL-23/IL-17/G-CSF pathway is associated with granulocyte recruitment to the lung during African swine fever

The interleukin (IL)-23/IL-17 pathway plays a crucial role in various forms of inflammation but its function in acute African swine fever (ASF) is not well understood. Thus, in this study, we aimed to find out whether IL-23/IL-17/G-CSF is released in acute ASF and what function it may have.The present study revealed that the production of IL-17 and IL-23 were significantly increased in the sera of ASFV infected pigs. Using ELISA, we found that the serum levels of IL-23 and IL-17 have overexpressed in ASF virus infected pigs compared with healthy controls. The levels of IL-17 and IL-23 increase in the early stages and the levels of G-CSF and C – reactive protein in the later stages of ASF. Simultaneously, with the increase of the levels of IL-23/IL-17 extravasation of granular leukocytes in the tissue (diapedesis) is observed. Diapedesis can explain the neutropenia that we identified previously in the terminal stages of ASF.The increase in serum levels of IL-23/IL-17 is preceded by enhanced migration of neutrophils in tissues, and the last one is preceded by neutropenia. The increase in serum levels of G-CSF has compensatory nature, directed on stimulation of proliferation of granulocytes. Taken together, our results revealed an overexpression of the IL-23/IL-17 axis in the ASF virus infected pigs, suggesting that it may be a crucial pathway in the diapedesis at ASF.

Lymphatic Function and Immune Regulation in Health and Disease

Lymphatic vessels are present throughout the body, except in the central nervous system and bone marrow. The lymph nodes (LNs), spleen, and other secondary lymphoid organs (e.g., Peyer's patches) are strategically located in anatomical sites where they collect foreign antigen and antigen presenting cells to activate antigen-specific lymphocytes efficiently.1 In the peripheral tissues, specialized lymphatic capillaries—called initial lymphatic vessels—allow soluble materials and cells to enter the lymphatic system easily. The collected fluid and cells form lymph, which is transported by smooth muscle-invested collecting lymphatic vessels to the draining lymph node. The lymph node provides a highly organized microarchitecture that supports optimal immune surveillance. The “filtered” lymph fluid, as well as naive and activated lymphocytes, exit the lymph node via efferent lymphatic vessels. Lymphatic capillaries, collecting lymphatic vessels, and lymph nodes together provide protective immunity for the body. Disruptions of lymphatic function compromise immune function and result in lymphedema. In this review, we will summarize the current knowledge of how lymphatic function is altered in inflammatory states, cancer, and infection.

An immunohistochemical study of the tonsils in pigs with acute African swine fever virus infection

An immunohistochemical study of the tonsils was carried out to gain further insight in the pathogenesis of acute African swine fever (ASF). Twenty-one pigs were inoculated by intramuscular route with a highly virulent isolate of ASF virus and painlessly killed at 1–7 dpi. Viral antigen was highly distributed in the tonsil from 3 to 4 dpi and an increase in the number of monocyte-macrophages was very evident at the same days post inoculation. This phenomenon was observed together with an increase of the expression of proinflammatory cytokines (Tumour necrosis factor alpha and Interleukin-1 alpha) and the apoptosis of lymphocytes studied by the terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) technique and haemorrhages. With these results, we can conclude that the tonsil is suffering similar lesions than those observed in other lymphoid organs in acute African swine fever, even when the route of inoculation is the intramuscular and not oral-nasal.

Proinflammatory Cytokines Induce Lymphocyte Apoptosis in Acute African Swine Fever Infection

Twenty-one pigs inoculated with a highly virulent isolate (E70) of African swine fever (ASF) virus were killed 1-7 days later; a further three animals served as uninfected controls. An early increase in TNF-alpha, IL-1alpha, IL-1beta and IL-6 expression was detected in lymphoid organs from infected animals, together with an increase in the serum concentrations of TNF-alpha and IL-1beta. These changes were accompanied by increased apoptosis of lymphocytes, and the presence of infected and uninfected macrophages showing changes indicative of secretory and phagocytic activation. The present study demonstrated an increase in the number of macrophages expressing TNF-alpha, IL-1 and IL-6 in proximity to lymphocytes undergoing apoptosis, supporting previous suggestions that in acute ASF proinflammatory cytokines induce lymphocyte apoptosis.