Macrophage Transcriptional Responses following In Vitro Infection with a Highly Virulent African Swine Fever Virus Isolate

Abstract

We used a porcine microarray containing 2,880 cDNAs to investigate the response of macrophages to infection by a virulent African swine fever virus (ASFV) isolate, Malawi LIL20/1. One hundred twenty-five targets were found to be significantly altered at either or both 4 h and 16 h postinfection compared with targets after mock infection. These targets were assigned into three groups according to their temporal expression profiles. Eighty-six targets showed increased expression levels at 4 h postinfection but returned to expression levels similar to those in mock-infected cells at 16 h postinfection. These encoded several proinflammatory cytokines and chemokines, surface proteins, and proteins involved in cell signaling and trafficking pathways. Thirty-four targets showed increased expression levels at 16 h postinfection compared to levels at 4 h postinfection and in mock-infected cells. One host gene showed increased expression levels at both 4 and 16 h postinfection compared to levels in mock-infected cells. The microarray results were validated for 12 selected genes by quantitative real-time PCR. Levels of protein expression and secretion were measured for two proinflammatory cytokines, interleukin 1␤ and tumor necrosis factor alpha, during a time course of infection with either the virulent Malawi LIL20/1 isolate or the OUR T88/3 nonpathogenic isolate. The results revealed differences between these two ASFV isolates in the amounts of these cytokines secreted from infected cells. African swine fever virus (ASFV) causes inapparent persistent infections in its natural hosts, warthogs (Phacochoerus aethiopicus), bushpigs (Potamochoerus porcus), and soft ticks (Ornithodoros moubata), which inhabit warthog burrows. In contrast, the virus causes an acute hemorrhagic fever with high mortality rates in pigs, although some low-virulence isolates have been reported (6,33,50,52,53).The virus replicates in cells of the mononuclear phagocytic system and reticulo-endothelial cells of lymphoid tissues and organs. Widespread apoptotic cell death occurs in both T and B lymphocytes of lymphoid tissues (9, 21) and in arteriolar and capillary endothelial cells (10,(15)(16)(17)22). Disseminated intravascular coagulation develops during the late phase of acute infections, and this may lead to the characteristic hemorrhagic syndrome (20,22,41).ASFV is a large, icosahedral, double-stranded DNA virus which replicates in the cytoplasm of infected cells and has been classified as the only member of a new virus family (13), the Asfarviridae. The genome encodes between 160 and 175 proteins, including a number that interfere with host defense systems. These include proteins such as the A238L protein, which inhibits both the activation of the host NF-B transcription factor and calcineurin phosphatase activity. The latter inactivation results in the inhibition of calcineurin-dependent pathways, such as activation of the nuclear factor of activated T cell transcription factors (23,24,37,38,44,47). Members of ASFV multigene families 360 and 53...

Species-Specific Variation in RELA Underlies Differences in NF-κB Activity: a Potential Role in African Swine Fever Pathogenesis

“…In a study of macrophage transcription profiles following ASFV infection in vitro, several factors within the NF-B signaling pathway displayed elevated expression (i.e., NFKB1, NFKBIA); however, RELA expression was not observed to be altered (56). The impact of ASFV infection on the warthog RELA expression level is not known, since this study used only domestic pig macrophages.…”

“…Effect of RELA sequence variation on induced NF-B activity. To determine if the ability to induce NF-B activity was altered by the different pig RELA proteins, we stimulated transiently transfected RELA Ϫ/Ϫ MEFs with known inducers of NF-B signaling (9,14,15,16,(42)(43)(44)56). Induction with tumor necrosis factor alpha (TNF-␣) nearly doubled (90% increase) warthog RELA activity, whereas domestic pig RELA showed a 70% increase in activity (Fig.…”

“…ASFV is highly infectious, with virulent isolates causing an acute, rapidly fatal hemorrhagic fever in domestic pigs (Sus scrofa) (10,34). This is thought, in part, to be the result of a proinflammatory cytokine storm driven by infected macrophages (9,15,16,(42)(43)(44)56). Initiation of a systemic inflammatory response results in severe hematological and vascular perturbations, ultimately leading to cardiovascular collapse in a manner not dissimilar to septic shock (3,17,18,20,21,40,52,53).…”

“…Various groups have initiated transcription profiling of host genes implicated in ASFV infection (15,16,(42)(43)(44)56). These studies identified numerous upregulated host genes, but to date, all are limited to analysis in domestic pig cells.…”

Transcriptional immunoresponse of tissue-specific macrophages in swine after infection with African swine fever virus

“…These included genes expected to be activated as part of the host response to infection, encoding proinflammatory cytokines, chemokines, cell surface proteins, components of signalling pathways, and stress response proteins. The complex study was conducted by Zhang et al (34), who wanted to explore the broadest spectrum of factors on which the impact of ASFV infection might be visible. The range of distinctness of immune response transcriptional profile in blood monocytes/blood-macrophages was expressed as: cytokine and chemokine, surface protein, intracellular signalling proteins, and proteins activated by stress.…”