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The myocytic necrosis is a common finding in all three investigated diseased hearts (Poppe and Ferguson, 2006). Together apoptosis and necrosis determine the final degree of lethal myocardial injury (Zhao and Vinten–

Johansen, 2002). In Paper IV, caspase 3 and TUNEL staining identified strong to moderate levels of apoptotic cells in the investigated diseased hearts and in agreement with significantly increased caspase 3 immunostaining identified in the teleost gills (Thalassoma pavo L.) exposed to cadmium (Brunelli et al., 2011).

Recently, caspase–3–mediated apoptosis has been shown in the regenerating spinal cord in teleost (Apteronotus leptorhynchus), suggesting its role in tissue replacement after injury (Sirbulescu and Zupanc, 2009). Additionally, pro–

apoptotic genes such as Bax and Bcl–x have been shown significantly upregulated in Atlantic salmon exposed to uranium (U) as compared to controls (Song et al., 2012). The increased presence of caspase 3⁺ cells has also been noted in the adult brain of brown ghost knifefish (Apteronotus leptorhynchus) affected with aneuploidy (Rajendran et al., 2008) and significantly increased caspase 3 staining identified in the intestine of soybean meal (SBM)–induced enteritis in Atlantic salmon (Bakke–McKellep et al., 2007). The above mentioned

studies support the increased presence of apoptotic cells (identified by caspase 3 and TUNEL) in the diseased hearts in the current study (Papers III and IV). The increased number of apoptotic cells (in CMS and PD–affected hearts) could be explained by the marked tissue regeneration (heart) capacity in Atlantic salmon affected with the investigated diseases (Ferguson et al., 1990; Kongtorp, 2009;

McLoughlin et al., 2002; Taksdal et al., 2007). As apoptosis is a highly ordered and energy demanding process, energy deprivation in cardiomyocytes could inhibit the terminal apoptotic events, and could lead to the programmed cell necrosis (Dorn II, 2009). CMS and PD were the cardiac diseases identified with more cardiac necrotic changes as compared to HSMI where mononuclear cells infiltration predominated (Papers II and IV) (Kongtorp et al., 2004, 2004a;

Grammes et al., 2012). This was reflected by the moderate to low levels of TUNEL and caspase 3⁺ cells in HSMI–affected hearts respectively (Paper III) and corroborated by low levels of caspase 3 immunostaining in HSMI–affected hearts (Grammes et al., 2012). Recently, a transcriptomic study of PMCV injected fish has shown the correlation of CMS–related lesions and upregulation of T cells and apoptotic genes at peak cardiac pathology/viral load (8 weeks post infection) in the hearts (Timmerhaus et al., 2011). This was in agreement with the increased presence of apoptotic cells in the CMS and PD–affected hearts (Paper IV).

As in mammals, fish viruses are also capable of inducing apoptosis in the hosts (Hay and Kannourakis, 2002; Silva et al., 2008). Phylogenetic analysis of piscine reovirus (PRV) associated with HSMI disease in Atlantic salmon revealed that PRV lies taxonomically in between the orthoreovirus and aquareovirus. PRV comprises 10 dsRNA genome segments and in line with orthoreoviruses as compared to aquareoviruses consisted of 11 segments (Palacios et al., 2010).

Although, the structural and functional properties of PRV are undetermined to date but sequence homologies suggested PRV closer to mammalian

orthoreoviruses (Finstad et al., 2012; Palacios et al., 2010). In mammals, reoviruses including orthoreoviruses induce apoptosis in a wide variety of cultured cells (in vitro) and in target tissues (in vivo) including the heart and CNS (Clarke et al., 2005). Reoviruses induce apoptosis by regulating several important genes (TNF ligand, Bid, Smac) related to extrinsic and intrinsic apoptosis pathways and apoptosis considered critical mechanism by which disease is triggered in the host (Clarke and Tyler, 2003; Clarke et al., 2005). As suggested by Kongtorp (2009), the HSMI–associated virus possesses the immunoregulatory properties. The cytopathic effect of chum salmon reovirus (CSV) has shown the apoptosis and syncytia (large multinucleated giant cells formed by the fusion of neighboring cells) formation in salmonid cell lines including epithelial–like CHSE–214, fibroblast–like RTG–2 and monocyte/macrophage–like RTS11 cell lines. The hemotypic aggregation was observed in RTS11 instead of syncytia formation that suggested the potential for CSV to modulate macrophage functions (DeWitte–Orr and Bols, 2007). Apart from giant cells, the above mentioned studies support the present observation of increased numbers of apoptotic cells in HSMI–affected hearts and suggest the similar cellular responses to those found in other orthoreoviruses (Paper III).

Mammalian alphaviruses such as Sindbis virus (SIN) and Semliki forestvirus (SFV) have shown to induce apoptosis in both cell cultures and target organs (Kiver, 2009), and further syncytia and apoptosis have also been shown in CHSE–214 cell culture by Norwegian salmonid alphavirs (SAV3) (Skotheim, 2009; Yousaf, 2008). Infectious pancreatic necrosis virus (IPNV) has been shown to regulate apoptosis and necrosis death pathways through the upregulation of TNFα in zebrafish cell culture (Wang et al., 2011) and it is likely that other fish RNA viruses including PRV, SAV or PMCV follow the similar patterns of cell death but it requires further assessment. Besides necrotic changes, apoptosis has also been reported in heart and pancreas tissues of Atlantic salmon in natural and

experimentally induced SAV3 infection by histopathology (Taksdal et al., 2007).

Recently, in vitro apoptosis has been shown in the chum salmon heart–1 cells (CHH–1) and CHSE–214 cells infected with SAV1 (Herath, 2010). The presence of apoptosis in heart cell line (CHH–1) supports the apoptosis identified in the hearts of the investigated diseases particularly PD. IPNV and viral nervous necrosis virus (VNNV) have been shown to induce apoptosis followed by secondary necrosis in cell cultures (Chen et al., 2006; Chen et al., 2010; Chiu et al., 2010; Hong et al., 1998; Su et al., 2009) and support the current findings of strong levels of both apoptosis (identified by caspase 3 and TUNEL) and necrosis (identified by H&E staining) cells in PD– and CMS–diseased hearts (Paper IV).

However, further evaluation is required to determine if apoptosis follows necrosis in the investigated diseases, although, apoptosis and necrosis were observed in the present study (Papers III and IV). Comparatively TUNEL identified more positive cells than caspase 3 immunostaining (Papers III and IV) due to the reason that TUNEL positivity might also indicate necrosis as suggested by Bianciardi et al. (2006).