Characterization of pathogenic and non-pathogenic African swine fever virus isolates from Ornithodoros erraticus inhabiting pig premises in Portugal.

Ten African swine fever virus isolates from the soft tick Ornithodoros erraticus collected on three farms in the province of Alentejo in Portugal were characterized by their ability to cause haemadsorption (HAD) of red blood cells to infected pig macrophages, using restriction enzyme site mapping of the virus genomes and by experimental infection of pigs. Six virus isolates induced haemadsorption and four were non-haemadsorbing (non-HAD) in pig macrophage cell cultures. The restriction enzyme site maps of two non-HAD viruses, when compared with a virulent HAD isolate, showed a deletion of 9.6 kbp in the fragment adjacent to the left terminal fragment and of 1.6 kbp in the right terminal fragment and an insertion of 0.2 kbp in the central region. The six HAD viruses isolated were pathogenic and produced typical acute African swine fever in pigs and the four non-HAD isolates were non-pathogenic. Pigs that were infected with non-HAD viruses were fully resistant or had a delay of up to 14 days in the onset of disease, after challenge with pathogenic Portuguese viruses. Non-HAD viruses could be transmitted by contact but with a lower efficiency (42-50 %) compared with HAD viruses (100 %). The clinical differences found between the virus isolates from the ticks could have implications for the long-term persistence of virus in the field because of the cross-protection produced by the non-pathogenic isolates. This may also explain the presence of seropositive pigs in herds in Alentejo where no clinical disease had been reported.

Transovarial transmission of African swine fever virus in the argasid tick Ornithodoros moubata.

The aim of this study was to determine filial infection prevalence of experimentally infected colony Ornithodoros moubata Walton (Ixodoidea: Argasidae) ticks for African swine fever virus (ASFV). Three groups of ticks were used: an uninfected control group, one group orally infected with the VIC T90/1 isolate and another group orally infected with the LIV 13/33 isolate of ASFV. The results show that filial infection prevalences were not constant but were highly variable between egg batches from different ticks and between successive egg batches from the same tick. Filial infection prevalences ranged from 1.8% to 31.8% for ticks infected with the VICT90/1 isolate and from 1.2% to 35.5% for ticks infected with the LIV 13/33 isolate. A similar pattern was noted after the third feed. Immunohistochemisty showed that virus replicates in the developing larval cells and not in the yolk sac cells or within the outer layers of the eggs. The results show that ASFV can replicate to a high titre (10(5.1)log10HAD50) within the larval cells of the developing egg.

Effects of infection of the tick Ornithodoros moubata with African swine fever virus.

The effects of infection with African swine fever virus (ASFV) on adult and nymphal Ornithodoros moubata Murray (Ixodoidea, Argasidae) ticks were examined. Three groups of ticks were used, an uninfected control group, one group infected with the VIC T90/1 isolate of ASFV and another group infected with the LIV 13/33 isolate of ASFV. Infection with ASFV did not affect the oviposition rates of infected ticks when compared with uninfected ticks. There was no difference between infected and uninfected ticks in progeny hatching rates and first nymphal stage feeding rates. Feeding rates of infected adult ticks were also unaffected. However, a significant increase in mortality rates was observed amongst the adult ticks that fed on an infective bloodmeal compared to ticks fed on an unifected bloodmeal.

Pilot scale thermal treatment of pig slurry for the inactivation of animal virus pathogens.

This paper describes a pilot scale treatment plant that has been designed and built for the thermal inactivation in pig slurry of two viruses that infect pigs--African swine fever virus (ASFV) and swine vesicular disease virus (SVDV). The plant treats pig slurry continuously at a rate of up to 100 litres/hour and functions by heating the slurry, maintaining at least 99.99% of the slurry at the required temperature for a minimum period of 5 minutes, and then recovering the heat to raise the temperature of the incoming slurry. Results obtained indicated that SVDV was inactivated in pig slurry to below detectable levels with an alkaline pH (pH 7.5 to 8, as is usually the case) at a temperature of between 50 and 55 degrees C. In acidified slurry (pH 6.4), inactivation occurred between 55 and 60 degrees C. The difference in inactivation temperatures was probably due to the presence of free ammonia in the unacidified slurry. ASFV was inactivated by operating the plant at a temperature of 53 degrees C at a pH of 8.

Recovery and assay of African swine fever and swine vesicular disease viruses from pig slurry.

Assaying samples for infectious virus is more difficult when the sample is toxic to cells used in the assay, e.g. with samples of infected pig slurry. Various techniques were compared for the recovery of African swine fever virus (ASFV) and swine vesicular disease virus (SVDV) in pig slurry. Extraction with Freon led to 80-100% recovery of SVDV added to pig slurry. The assay sensitivity enabled undiluted, centrifuged sample to be put directly onto monolayers of IB-RS2 cells, allowing a minimum detection level of 100.7 pfu ml-1. ASFV was difficult to recover intact, and the best technique allowed a recovery of 60% with a minimum detectable level of 101.8 HAD50 ml-1, due to toxicity to the cells at low sample dilutions. Extraction with the addition of an equal volume of ox serum to inoculated slurry was best at recovering ASFV. Poor recoveries with the other techniques may have been due to the inactivation of the virus while in the slurry rather than as a result of the inability of the method to extract ASFV.

African swine fever virus infection of the bushpig (Potamochoerus porcus) and its significance in the epidemiology of the disease.

Warthog (Phacochoerus aethiopicus), giant forest hog (Hylochoerus meinertzhageni) and bushpig (Potamochoerus porcus) are known to be susceptible to infection with African swine fever (ASF) virus. Little however, is known about the ecology of the disease in the bushpig. This study has shown that the bushpig remains viraemic for between 35 and 91 days following infection during which time it is able to infect the tick vector O. moubata. These ticks were able to transmit the disease to pigs. The virus persists in the lymphatic tissues for less than 34 weeks. Bushpigs infected with LIL 20/l virus but not VIC T90/l virus transmitted infection to in-contact pigs. Infected domestic pigs did not transmit the infection to in-contact bushpigs. ASF virus was able to replicate in in vitro cultures of bushpig leucocytes and endothelial cells. Recovered bushpigs could be reinfected with some strains of virus but not others. While it has been demonstrated that bushpigs remain carriers of ASFV following infection a complete understanding of their significance in the epidemiology of the disease awaits further investigations of their association with O. moubata.

Thrombocytopenia associated with apoptotic megakaryocytes in a viral haemorrhagic syndrome induced by a moderately virulent strain of African swine fever virus.

A viral haemorrhagic syndrome was induced in 14 pigs by inoculation with an African swine fever (ASF) virus strain of moderate virulence, to determine changes in megakaryocyte (MK) numbers and morphology and thus to assess the role of these cells in the thrombocytopenia characteristic of subacute ASF. The strain tested induced changes in the proportion of different types of MK (typical nucleated MKs, apoptotic MKs and immature MKs); it also caused subcellular lesions over the first 7 days post-inoculation (dpi). At 7 dpi, severe thrombocytopenia was observed. There was a statistically significant increase in apoptotic MK numbers. The MKs showed three stages in the course of the disease: a compensatory stage, represented by cytoplasmic projections, a hypermaturity stage, represented by apoptotic MKs, and a regenerative stage, represented by clusters of immature MKs. These changes, especially the presence of numerous apoptotic MKs, may explain the early and transitory thrombocytopenia detected in subacute ASF. The large number of apoptotic MKs observed may be associated with the accelerated maturation of these cells, resulting from the action of cytokines, or peripheral platelet consumption, or both.

Double-labelling immunohistochemical study of megakaryocytes in African swine fever.

Bone marrow samples from pigs infected with the highly virulent Malawi'83 or moderately virulent Dominican Republic (DR'78) isolates of African swine fever virus were studied by means of a double labelling immunohistochemical technique which stained the major structural protein VP73 of the virus and megakaryocytes simultaneously. In pigs infected with the highly virulent Malawi'83 isolate, 2.2 per cent of megakaryocytes were VP73+ five days after inoculation, and at six and seven days 2.5 and 9.5 per cent of megakaryocytes were VP73+. Some infected and uninfected megakaryocytes showed pyknosis and karyorrhexis, particularly at seven days after inoculation. However, in comparison with uninfected pigs, the number of megakaryocytes decreased only at seven days after inoculation. In pigs infected with the moderately virulent DR'78 isolate, only 0.2 per cent of megakaryocytes were VP73+ at eight days after inoculation. However, at eight, nine and 10 days after inoculation the total number of megakaryocytes was significantly lower (P < 0.01) than in control uninfected pigs, and the majority of the megakaryocytes showed signs of cell death such as pyknosis and karyorrhexis. The fact that this greater destruction of megakaryocytes was associated with the lower rate of infection of this cell type suggests that indirect damage to megakaryocytes is an additional mechanism of thrombocytopenia in acute and subacute African swine fever.

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 virus infection of bone marrow: lesions and pathogenesis.

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.

Development of microscopic lesions in splenic cords of pigs infected with African swine fever virus.

Acute forms of African swine fever are characterized by hemorrhagic lesions in the lymphoid organs. This paper reports the evolution of lesions in the splenic cords of pigs inoculated with African swine fever (ASF) virus (strain Malawi'83). Ultrastructural examination of the splenic cords of the infected pigs revealed numerous macrophages attached to the muscle cells harboring virus replication center and cytopathic effects at 3 dpi (days post-infection). From 5 dpi, the splenic cords contained a large number of erythrocytes associated with abundant fibrin deposits, mainly arranged around the muscle cells, from which macrophages had disappeared. It is likely that the ASF virus replication, and consequent cytopathic effects, observed in the fixed macrophages of splenic cords, may be responsible for the fibrin deposition.

Subcellular changes in the tonsils of pigs infected with acute African swine fever virus.

A study of the pathogenesis of acute African swine fever (ASF) was carried out in pigs inoculated with a highly virulent strain of ASF virus to determine the sequential development of the subcellular changes in a particular lymphoepithelial organ, the tonsil. The apoptosis of the lymphocytes and the inhibition of lymphocyte proliferation were the main changes that occurred in the tonsillar lymphoid structures. This may explain the early lymphopenia observed in acute ASF. Moreover, vascular changes, consisting of increased vascular permeability, activation of endothelial cells and loss of these cells, might have been the cause of the characteristic haemorrhages found in the lymphoid organs during this disease. Virus replication has been observed in the epithelial cells, fibroblasts and reticular cell beginning on day 5 post-infection. The activation of the endothelial cells, apoptosis of lymphocytes, decreased lymphocyte mitosis and virus replication in non-mononuclear phagocyte system (MPS) cells all occurred after an intense proliferation and activation of the tonsillar macrophages and coincide with virus replication, which occurs in the macrophages 5 days post infection.

The pathogenic role of pulmonary intravascular macrophages in acute African swine fever.

Recent studies of pulmonary intravascular macrophages have led to the re-examination of the mechanisms giving rise to alveolar oedema. A highly virulent isolate of African swine fever virus was replicated in pulmonary intravascular macrophages, interstitial and alveolar macrophages, fibroblasts and neutrophils. The alveolar oedema-characteristic of acute forms of African swine fever-and the vascular changes observed, which consisted of the formation of fibrin microthrombi in septal capillaries and the vacuolisation of endothelial cells, may have been due, however, to the activation of pulmonary intravascular macrophages, and not to the cytopathic effect subsequent to the replication of the African swine fever virus. Furthermore, it was observed that virus replication in cells not belonging to the mononuclear phagocyte system-such as fibroblasts and neutrophils-occurred earlier than in cells belonging to that system.

Subcellular changes in platelets in acute and subacute African swine fever.

The morphological changes in platelets in acute and subacute African swine fever (ASF) and their relationship to pathogenesis were studied. Eight pigs were inoculated with a highly virulent strain of African swine fever (Malawi '83) and 14 with a moderately virulent strain (Dominican Republic '78) for ultrastructural study of platelets, monocyte/macrophages and vascular structures in the liver, spleen, lymph node, bone marrow, lung and kidney. Both viruses produced activation and degranulation of platelets from day 3 after inoculation onwards, coinciding with activation of the mononuclear phagocyte system and virus replication in monocyte/macrophages. Platelet aggregation and viscous metamorphosis of platelets were observed at 5 and 7 days after inoculation with the highly virulent strain, coinciding with endothelial alterations, but platelet aggregation was less prevalent and there was no sign of viscous metamorphosis in animals inoculated with the moderately virulent strain. Virions within platelets were observed at the final stage of acute ASF and at 5-7 days after inoculation in subacute ASF. This suggests that platelets assist in disseminating ASF virus within the body, especially in subacute infections.

Apoptosis in lymph nodes in acute African swine fever.

This paper reports apoptosis of lymph-node lymphocytes in swine experimentally inoculated with a virulent African swine fever (ASF) virus isolate (Malawi '83). Apoptosis was observed in both compartments of cortical tissue, but was more intense in diffuse lymphoid tissue (T area). Lymphopenia detected in peripheral blood was associated with T-lymphocyte depletion. No evidence of ASF virus replication was observed in lymphocytes in the lymph nodes studied. This finding, together with the high rate of virus replication recorded in macrophages in diffuse lymphoid tissue as compared with the low rate recorded for lymphoid follicles, suggests a mechanism for the induction of apoptosis related to virus replication in cells of the mononuclear phagocyte system.

Structural and ultrastructural study of glomerular changes in African swine fever.

The pathological effect of haemorrhagic fever viruses on the kidney have not been clearly documented. This study reports glomerular lesions in African swine fever. In the acute form of the disease there was an acute diffuse proliferative glomerulonephritis, which was believed to be related to virus replication in circulating monocytes and glomerular mesangial cells, and to the presence of abundant circulating cell debris resulting from viral replication at other sites. In the subacute form, the proliferative mesangial glomerulonephritis observed may have been associated with systemic immune-mediated phenomena, and with subendothelial and mesangial deposits of immunoglobulins and complement components.

In vivo replication of African swine fever virus (Malawi '83) in neutrophils.

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.

Virus association with lymphocytes in acute African swine fever.

This paper reports the presence of mature viral particles within the lymphocytes of samples taken from pigs inoculated with a highly virulent African swine fever (ASF) virus isolate (Malawi 83), and the adhesion of the lymphocytes to macrophages containing the virus replication sites. Virus replication in lymph-node medullar tissue macrophages was observed from 3 days post inoculation (pi). At 3 days pi, transmission electron microscopy revealed hemadsorption in some infected macrophages. At 5 and 7 days pi, a number of macrophages with virus replication were surrounded by a ring of lymphocytes. In such cases, mature viral particles were observed in membrane evaginations of the infected cell that corresponded to invaginations of the lymphocyte membrane. Also at 5 and 7 days pi, mature virions were observed within the cytoplasm of some lymphocytes. However, incomplete virions and African swine fever virus replication sites were not observed within the lymphocytes.

Experimental African swine fever: apoptosis of lymphocytes and virus replication in other cells.

In order to determine the cause of cellular death of lymphocytes in pigs with acute African swine fever and the relationships between African swine fever virus (ASFV) and interstitial cells, ten pigs were inoculated with a highly virulent strain of ASFV (Malawi '83) and samples taken for ultrastructural study of hepatic and renal interstitial tissues. We demonstrated death by apoptosis of lymphocytes and virus replication in fibroblasts, smooth muscle cells and endothelial cells in the interstitial tissues of pigs inoculated with ASFV. From day 5 onwards, apoptotic lymphocyte and intense virus replication in hepatic interstitial macrophages and fibroblasts were observed. By day 7, apoptotic lymphocytes and virus replication in macrophages, interstitial capillary endothelial cells and fibroblasts in the kidney were observed. Virus replication was also seen in smooth muscle cells of hepatic and renal arterioles and venules. Our results suggest that mononuclear phagocyte system (MPS) cell activation, and the resulting release of cytokines, could induce apoptosis of lymphocytes and virus replication in non-MPS cells.