African Swine Fever Virus Infection in the Argasid Host,Ornithodoros porcinus porcinus

The pathogenesis of African swine fever virus (ASFV) infection in Ornithodoros porcinus porcinus was examined in nymphal ticks infected with the ASFV isolate Chiredzi/83/1. At times postinfection (p.i.) ranging from 6 h to 290 days, ticks or dissected tick tissues were titrated for virus and examined ultrastructurally for evidence of virus replication. The ASFV infection rate in ticks was 100% in these experiments, and virus infection was not associated with a significant increase in tick mortality. Initial ASFV replication occurred in phagocytic digestive cells of the midgut epithelium. Subsequent infection and replication of ASFV in undifferentiated midgut cells was observed at 15 days p.i. Generalization of virus infection from midgut to other tick tissues required 2 to 3 weeks and most likely involved virus movement across the basal lamina of the midgut into the hemocoel. Secondary sites of virus replication included hemocytes (type I and II), connective tissue, coxal gland, salivary gland, and reproductive tissue. Virus replication was not observed in the nervous tissue of the synganglion, Malpighian tubules, and muscle. Persistent infection, characterized by active virus replication, was observed for all involved tick tissues. After 91 days p.i., viral titers in salivary gland and reproductive tissue were consistently the highest detected. Successful tick-to-pig transmission of ASFV at 48 days p.i. correlated with high viral titers in salivary and coxal gland tissue and their secretions. A similar pattern of virus infection and persistence in O. porcinus porcinus was observed for three additional ASFV tick isolates in their associated ticks.African swine fever (ASF) is a highly lethal disease of domestic pigs for which animal slaughter and area quarantine are the only methods of disease control. African swine fever virus (ASFV), the causative agent of ASF, is a large double-stranded DNA virus which is the only member of an unnamed family of viruses. ASFV is the only known DNA arbovirus (4, 6, 12). The natural arthropod host for ASFV is Ornithodoros porcinus porcinus (Walton) ticks (40). Some confusion exists in earlier reports since ticks that should be classified as O. porcinus porcinus are often referred to as either O. moubata porcinus or simply O. moubata (59).ASFV can infect hosts through either a sylvatic cycle or a domestic cycle. In the sylvatic cycle, ASFV infects warthogs (Phacochoerus aethiopicus) and bushpigs (Potamochoerus spp.) as well as ticks of the genus Ornithodoros (7–10, 36, 55). In sub-Saharan Africa, warthogs occupy burrows which are frequently infested with large numbers of O. porcinus porcinus ticks (38, 45, 57, 58), and a correlation, though not absolute, has been established between ASFV infection of warthogs and the presence of O. porcinus porcinus ticks in burrows (57). In ASFV-enzootic areas, adult warthogs are typically nonviremic, although most are seropositive (28, 41, 46, 53, 58), and virus can usually be isolated only from lymph nodes (28, 41). Young warthogs, which are confined to the burrow for the first months of life, are most likely to be infected through feeding of infected O. porcinus porcinus ticks. Infection in young warthogs is subclinical, with viremic titers ranging from 2 to 3 log₁₀ 50% hemadsorption dose (HAD₅₀)/ml (56, 57), a level sufficient to infect a low percentage of naive ticks (42, 58, 30). The sylvatic ASFV cycle is further maintained by transovarial (43) and venereal (44) transmission in ticks. In burrows containing ASFV-infected ticks, infection rates are typically low (<2%), with the highest rate occurring in adult females (40, 45, 57, 65). The mechanism of ASFV transmission from the sylvatic cycle in Africa to the domestic cycle is most likely through feeding of infected ticks on pigs (41, 58), since direct contact between infected warthogs and domestic pigs has failed to result in transmission (36, 10, 28, 58), except in a single case (8). The virus may be transmitted between domestic pigs by either direct or indirect contact (33).Various characteristics of ASFV infection have been studied in a number of Ornithodoros spp. ticks. The first association of ASFV with a tick was made by Sanchez-Botija (50), who reported isolation of ASFV from O. erraticus, a tick native to the Iberian peninsula and later considered important to maintenance of ASFV in an enzootic cycle in that region (51). In the first experimental infection, striking differences were found in the percentage of O. moubata porcinus ticks infected by two different ASFV isolates, a low infectious dose for ticks (ranging from of 0.9 to 4 log₁₀ HAD₅₀) was demonstrated, and transmission out to 469 days postinfection (p.i.) was successful with single ticks (42). Experimental ASFV infection and transmission to pigs has been demonstrated for O. savignyi, a tick found in Africa (34), O. coriaceus (23, 25) and O. turicata (25), ticks indigenous to the United States, and O. puertoricensis (25, 14), a tick indigenous to the Caribbean. A 40% mortality rate was found in infected O. coriaceus (25) and O. puertoricensis ticks (15). O. marocanus, which was formerly referred to as O. erraticus, transmitted ASFV out to 588 days p.i., although 73% mortality was reported for infected ticks (16, 17). A number of reports have not found significant virus-induced mortality in O. moubata porcinus ticks (22, 40–44). In contrast, mortality rates were 35% higher in infected O. moubata porcinus females in the only study to examine mortality during the gonotrophic cycle (26).Specific aspects of ASFV infection in the natural host remain poorly understood. Greig (22) experimentally infected O. moubata porcinus ticks with pathogenic ASFV isolates and used virus titration and immunofluorescence of dissected tissues to determine that the midgut was the initial site of viral replication and the site of longest persistence. Several other tissues were also found to have detectable levels of virus, although the midgut was the only tissue which was consistently positive. The presence of ASFV has been demonstrated in hemocytes of infected O. coriaceus ticks by electron microscopy and immunofluorescence studies, but the presence or nature of virus replication was not addressed (13).Here we describe the pathogenesis and persistence of ASFV infection in O. porcinus porcinus ticks. Our data indicate that initial ASFV replication occurs in phagocytic digestive cells of the midgut epithelium, with secondary replication occurring in undifferentiated midgut cells at later times p.i. Generalization of virus infection from the midgut to other tick tissues required 2 to 3 weeks. Secondary sites of virus replication include hemocytes (type I and II), coxal gland, salivary gland, connective tissue, and reproductive tissue. Successful tick-to-pig transmission correlated with relatively high viral titers in salivary and coxal glands. Persistent infection in the tick involves continuous viral replication in several tissues and is associated with minimal cytopathology.