Epizootic Hemorrhagic Disease Virus Induces and Benefits from Cell Stress, Autophagy, and Apoptosis

Abstract

The mode and timing of virally induced cell death hold the potential of regulating viral yield, viral transmission, and the severity of virally induced disease. Orbiviruses such as the epizootic hemorrhagic disease virus (EHDV) are nonenveloped and cytolytic. To date, the death of cells infected with EHDV, the signal transduction pathways involved in this process, and the consequence of their inhibition have yet to be characterized. Here, we report that the Ibaraki strain of EHDV2 (EHDV2-IBA) induces apoptosis, autophagy, a decrease in cellular protein synthesis, the activation of c-Jun N-terminal kinase (JNK), and the phosphorylation of the JNK substrate c-Jun. The production of infectious virions decreased upon inhibition of apoptosis with the pan-caspase inhibitor Q-VD-OPH (quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methyl ketone), upon inhibition of autophagy with 3-methyladenine or via the knockout of the autophagy regulator Atg5, or upon treatment of infected cells with the JNK inhibitor SP600125 or the cyclin-dependent kinase (CDK) inhibitor roscovitine, which also inhibited c-Jun phosphorylation. Moreover, Q-VD-OPH, SP600125, and roscovitine partially reduced EHDV2-IBA-induced cell death, and roscovitine diminished the induction of autophagy by EHDV2-IBA. Taken together, our results imply that EHDV induces and benefits from the activation of signaling pathways involved in cell stress and death.T he epizootic hemorrhagic disease virus (EHDV) is an arbovirus (genus orbiviruses) of the Reoviridae family that is transmitted by biting midges and infects ruminants. In recent years, outbreaks of epizootic hemorrhagic disease in cattle have been reported in Israel and Turkey (1, 2), suggesting that EHDV is an emerging threat to the cattle industry in Europe (3). EHDV presents structural and sequence similarities to the better-studied bluetongue virus (BTV), sharing its repertoire of infection targets and symptoms of disease (3, 4). However, in spite of structural similarities between these viruses, a recent study suggests that preexisting immunity to BTV does not protect against EHDV infection (5). The EHDV genome is organized in 10 double-stranded RNA (dsRNA) segments encoding seven structural proteins (VP1 to VP7) and the nonstructural (NS) proteins NS1 to NS3. Recently, an additional nonstructural protein, NS4, has been identified in BTV (6, 7), raising the possibility that the same protein occurs in EHDV. The present study mainly employs the Ibaraki strain of EHDV2 (EHDV2-IBA), originally isolated from infected cattle in 1959, in Ibaraki, Japan (8). Selected experiments were also carried out with EHDV7-Israel (EHDV7-ISR), isolated from infected cattle in 2006 in Israel (1).For different types of reoviruses, including BTV, apoptosis is integral to the cellular pathogenesis they induce (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Yet the molecular mechanisms that govern reovirus-mediated induction of apoptosis are a contentious matter (10,17,19,20). For orbiviruses in general and EHDV in partic...

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“…Here, we decided to pursue the attractive possibility of combining the advantages of both reoviruses and veterinary viruses. To this end, we chose the Ibaraki strain of the Epizootic Hemorrhagic Disease virus (EHDV2-IBA), as we previously showed that when infecting mammalian cells, EHDV2-IBA is cytolytic and induces apoptosis, necroptosis, autophagy and cell stress [34]. We hypothesized that serial passaging of EHDV2-IBA in LNCaP cells (schematically depicted in Figure 7A) may optimize its infection abilities of human cancer cells.…”

“…Cells were subsequently trypsinized, fixed in 2% paraformaldehyde and analyzed by fluorescence-activated cell sorting (FACS) to quantify the total number of GFP-positive cells. EHDV-TAU was generated by serial passaging of EHDV2-Ibaraki isolate [34] on LNCaP cells for 16 times. Each of the 16 repetitions included: (i) infection of LNCaP cells; (ii) lysis of infected cells by sonication; (iii) plaque assay of the viral progeny on naive LNCaP cells; (iv) plaque-purification of the virus from largest plaques, for re-infection of LNCaP cells.…”

“…Anti-NS3 antibodies were described in [34]. Rabbit anti-phospho-Tyr701-STAT1, rabbit anti-STAT1, rabbit anti-phospho-NF-κB p65 (Ser 536) and rabbit anti-NF-κB p65, diluted 1:1000 for western blot and 1:200 for immunofluorescence, were from Cell Signaling (Beverly, MA, USA).…”

“…As a cytolytic virus, we chose a variant of the epizootic hemorrhagic disease virus (EHDV), an orbivirus that naturally infects ruminants and is transmitted by biting midges [33]. When infecting mammalian cells, EHDV induces apoptosis, necrosis, autophagy and cell stress [34]. Notably, orbiviruses are strong inducers of the innate immunity/IFN response [35, 36], possibly due to their dsRNA genome.…”

Virus uncoating is required for apoptosis induction in cultured mammalian cells infected with African horse sickness virus

“…Microscopic examination of endothelial cells of the heart, lung, liver and spleen of AHSV-infected animals showed ultrastructural modifications that included the loss of intercellular junction integrity, the presence of cytoplasmic projections and condensation of the nucleus, thus suggesting that apoptosis may contribute to the pathogenesis of AHSV in the mammalian host (Laegreid et al, 1992;Gó mez-Villamandos et al, 1999). Indeed, for various members of the family Reoviridae, including Bluetongue virus (BTV), Epizootic haemorrhagic disease virus (EHDV) and others, apoptosis appears to be integral to the cellular pathogenesis induced by these viruses (DeMaula et al, 2001;Kominsky et al, 2002;Mortola et al, 2004;Clarke et al, 2005;O'Donnell et al, 2005;Danthi et al, 2008;Stewart & Roy, 2010;Shai et al, 2013). In the case of BTV, the prototype member of the genus Orbivirus, the outer capsid proteins VP2 and VP5 were shown to be sufficient for the induction of apoptosis in cultured mammalian cells (Mortola et al, 2004).…”