Please cite this article in press as: Olavarría, V.H., et al., ISA virus regulates the generation of reactive oxygen species and p47phox expression in a p38 MAPK-dependent manner in Salmo salar. Mol. Immunol. (2014), http://dx.doi.org/10.1016/j.molimm.2014.07.016 ARTICLE IN PRESS G Model MIMM-4457; No. of Pages 8 Molecular Immunology xxx (2014) xxx–xxx Contents lists available at ScienceDirect Molecular Immunology j ourna l ho me pa ge: www.elsevier.com/locate/molimm ISA virus regulates the generation of reactive oxygen species and p47phox expression in a p38 MAPK-dependent manner in Salmo salar Víctor H. Olavarría a,∗ , Sharin Valdivia a , Boris Salas a , Melina Villalba a , Rodrigo Sandoval a , Harold Oliva c , Samuel Valdebenito c , Alejandro Ya˜ nez a,b,∗ a Facultad de Ciencias, Instituto de Bioquímica y Microbiología, Universidad Austral de Chile, Campus Isla Teja S/N, Valdivia, Chile b Interdisciplinary Center for Aquaculture Research (INCAR), Víctor Lamas 1290, PO Box 160-C, Concepción, Chile c Veterquímica, Lonquén10.387, Maipú, Santiago Chile a r t i c l e i n f o Article history: Received 23 April 2014 Received in revised form 1 July 2014 Accepted 5 July 2014 Available online xxx Keywords: ISA virus MAPK ROS production Innate immunity a b s t r a c t Several viruses, including Orthomyxovirus, utilize cellular reactive oxygen species (ROS) for viral genomic replication and survival within host cells. However, the role of ROS in early events of viral entry and signal induction has not been elucidated. Here, we show that ISA virus (ISAV) induces ROS production very early during infection of CHSE-214 and SHK-1Ycells, and that production is sustained over the observed 24 h post-infection. The mitogen-activated protein kinase (MAPK) family is responsible for important signaling pathways. In this study, we report that ISAV activates ERK and p38 in Salmo salar. In salmonid macrophages, while ERK was required for SOD, GLURED, p47phox expression, p38 regulated the ROS production by the NADPH oxidase complex activation. These results, together with the presence of several consensus target motifs for p38 MAPK in the promoter of the S. salar p47phox gene, suggest that p38 MAPK regulates p47phox gene expression in fish through the activation of this key transcription factor. © 2014 Elsevier Ltd. All rights reserved. 1. Introduction Phagocytic cells generate reactive oxygen species (ROS) as a mechanism to eliminate invading pathogens. Oxygen-containing free radicals such as superoxide (O 2- ), the hydroxyl radical (OH), and hydrogen peroxide (H 2 O 2 ), are highly reactive molecules due to the presence of unpaired valence shell electrons. Additionally, these molecules mediate important roles in cell signaling, homeo- stasis, and immunity. The imbalance between the generation of ROS and the cell’s ability to detoxify these same mediators produces a state known as oxidative stress (Valko et al., 2007). The infectious salmon anemia virus (ISAV) is an aquatic Orthomyxovirus (genus Isavirus) that causes a multisystemic disease characterized by high mortality with pale gills, severe anemia, decreased hematocrit value and severe hemorrhagic necrosis of internal organs (Kibenge et al., 2004; Rimstad and Mjaaland, 2002). Several ISAV isolates with different virulence properties and pathogenicity in vivo are described as acute or protracted variants (Kibenge et al., 2006). However, in vitro cor- relation between replication properties of different ISAV isolates and the development of cytopathic effect (CPE) is less clear ∗ Corresponding authors. Tel.: +56632221465. E-mail addresses: volavarria@uach.cl (V.H. Olavarría), ayanez@uach.cl (A. Ya˜ nez). (Mjaaland et al., 2002). Therefore, in vitro studies are important for understanding mechanisms of pathogenesis and resistance to ISAV at the cellular level, particularly on oxidative stress caused by enhanced production of ROS and reduced capacity for their neutralization in virus infected cells (Hasnain et al., 2003). Recently, it was shown that during ISAV infection, several genes that are directly or indirectly involved in metabolism of ROS were down-regulated and there was a reduced level of glutathione as well (Schiøtz et al., 2008). ROS are generated by the activity of the NADPH oxidase family of enzymes in the membrane of phagocytic cells. These NADPH oxidases generate ROS by carrying electrons across membranes from NADPH in the cytosol to an electron acceptor (i.e., oxygen) in the extracellular space or phagosome (Bedard and Krause, 2007). The NADPH activation occurs after the phosphorylation of p47phox by different types of protein kinases, such as PKC  (Olavarría et al., 2010) and MAPK, among others. Particularly, mitogen-activated protein kinase (MAPK) pathways are central to the control of cellular responses including host defense, inflammatory responses and cell death. In fact, MAPKs can be activated by a wide variety of different stimuli, but in general, ERK1 and ERK2 are preferentially activated in response to growth factors and phorbol esters, while p38 kinase is more responsive to stress stimuli ranging from osmotic shock and ionizing radiation to cytokine stimulation (Pearson et al., 2001). Once activated, MAPKs phosphorylate target substrates on serine or threonine http://dx.doi.org/10.1016/j.molimm.2014.07.016 0161-5890/© 2014 Elsevier Ltd. All rights reserved.