In this study we show that SAV-3 infection of Atlantic salmon cause pathology in target organs alongside high viral replication despite high expression levels of IFNα mRNA and interferon-stimulated genes, ISG-15 and Mx, at early time points post challenge. Type 1 interferons are well known for the establishment of an antiviral state in neighboring uninfected cells following viral invasion in vertebrates [11, 13]. This is most important during the early stages of an infection, prior to the onset of the adaptive immune response. The increase in viral loads over time in target organs (pancreas, heart and skeletal muscles) and the progression of pathology in the pancreas and heart despite the up-regulation of IFN-α, Mx and ISG-15 (Figure 3) suggest that the onset of the innate response comes too late to limit virus replication. This fits well with a previous report where treatment of cells with IFN-α at the same time as SAV-3 infection failed to protect the cells against CPE in an in-vitro model .
All tissues examined in the present study contained SAV-3 and the kinetics of viral loads were in general consistent with the trends of expression of IFN-α, in common with reports of previous studies done in Atlantic salmon-derived cells  and also with other viruses . The anticipation is that SAV-3 was sensed by host cells via pattern recognition receptors such as MDA-5 and LGP2  leading to the expression and induction of IFN-α and consequently ISG . The trends of induction of Mx and ISG-15 by IFN-α were, on average, consistent with previous reports [24, 25] while the relationship between the expression profiles was not always proportional and for ISG-15, a somewhat different expression pattern was observed in some tissues of the IM group. In one study using an in-vitro model to assess the induction of ISG by IFN-α, similar inconsistencies were observed . These findings probably reflect the complexity of the interferon signaling pathways or the diversity in fish since most fish ISG are often duplicated , as well as the effect of IFN-α independent stimulation of ISG [38, 39].
In higher vertebrates, down-stream effects of IFN-α/β induction include the increased expression of MHC I molecules  and activation of NK cells [41, 42]. From the genes examined in the present study (Table 2), MHC I was one of the earliest genes to be induced in each organ following increased virus expression and up-regulation of IFN-α. This was consistent with previous reports where a strong association was found between IFN induction and the transcription of MHC I gene . MHC I is expressed in all nucleated cells and its transcription is elevated during viral infections as a result of IFN-α/β induction and more especially, IFN-γ . It is noteworthy that in the present study, MHC I was also induced in almost all tissues where IFN-γ was up-regulated (Table 2), suggesting an association between the two genes in SAV infections in Atlantic salmon as also reported by others .
IFN-γ is a powerful pro-inflammatory cytokine produced by cells of the lymphocyte lineage and is required for the control of intracellular pathogens . Its target cells are mainly those of the monocytic origin but CD4+ Th1 cells are also activated [46, 47]. In the present study, the expression of IFN-γ at 2 and/or 4 wpi in all organs analyzed suggests the involvement/activation of NK-like cells as part of the innate response since at these time points, there was no accompanying expression of T cell-related genes (CD4+, CD8+, TCR, CD3ε) (Table 2). This was consistent with the report that NK cells are the primary source of IFN-γ during the innate immune response [11, 48]. However at 8 wpi, the expression of different genes (MHC I, CD8, TCR, MHC II, IL-12, CD4, TCR and CD3ε genes as well as the augmentation of IFN-γ) in the heart suggests a combined cytotoxic and Th1 mediated response. The pathological changes observed and the infiltration of inflammatory cells (Figure 1) fit very well with the expression of TNF-α and IL-8. It is noteworthy that the up-regulation of genes suggestive of a Th1/cytotoxic response was associated with inflammation/pathology at 8 weeks, with the reaction in the pancreas being greatly down-scaled suggesting a contraction phase. Even though IFN-γ has been shown to have a mild direct effect on SAV-3 , it appears to play an important role in shaping the cell mediated response or possibly contributes to the pathology seen in the target organs.
In conformity with the latter notion, it has been shown from studies of higher vertebrates that the expression of IFN-γ requires tight control since it can lead to immunopathology . Furthermore, it has also been demonstrated that IFN-γ producing cells are suppressed by IL-10. IL-10 on the other hand, is itself produced by a large number of immune cells including regulatory and IFN-γ producing T cells [45, 50, 51]. In the present study IL-10 was consistently induced alongside IFN-γ, with the two genes showing similar trends (Table 2) that also rhymed with viral loads in individual tissues. These findings suggest the conservation of the regulation of these genes in vertebrates.
In the present study, no samples were collected prior to day 14 in the IM group, therefore, data showing the initial distribution of virus before this time is lacking. However, the viral loads of cohabitants at 2 wpi represent infection at an earlier time point compared to 2 wpi in the IM group. These results suggest that the pancreas, heart, kidney and spleen are probably all infected about the same time although the virus ultimately replicates to different levels in the different organs, with the highest load being reached in the pancreas and heart in the cohabitant group (Figure 2b). In the IM group, the virus was administered via skeletal muscle injection and it is not unlikely that an initial replication of virus occurred at the injection site, probably followed by the “draining” of the virus to other organs. The association between high viral loads and pathology in the pancreas and heart (Figures 1 and 2) suggest that the virus threshold for pathology in these organs is just above 106 virus RNA copy numbers/μg of total RNA. The presence of SAV-3 in all tissues examined was consistent with previous reports that the virus has a wide range of tissue tropism in Atlantic salmon . The finding of the highest viral load and pathology in the pancreas at 2 wpi in the IM group compared to other tissues is interesting especially since the viral loads culminated in all organs except the skeletal muscle at 4 weeks. This suggests that the pancreas is the most preferred site of SAV-3 replication. Several other reports allude to the pancreas as the first organ in which pathology is observed following SAV infection [3, 33] and this fits with the definition of virus tropism, that being the ability of a virus to infect or cause damage to cells or tissues. On the contrary the slow and protracted increase in viral load in the skeletal muscles suggests that the organ is a site for viral persistence, in agreement with previous studies that have reported virus in this organ long after infection .
No lesions were observed in the skeletal muscles in the present study, in contrast with previous reports [3, 7]. The viral load during the final sampling of the study was on the increase suggesting that termination at 8 weeks was probably too early, which would explain the lack of lesions. For mice infected with Sindbis virus fatalities occur when the virus invades the neurons . For SAV-3 infections in Atlantic salmon, it is not clear which organs or the degree of pathology correlate with mortalities and should be a subject for further studies.
As already stated, a relationship exists between the viral load and tissue pathology, i.e. a viral load threshold has to be reached before pathology is caused. The delay in this threshold and also in the appearance of pathological changes in cohabitants in the present study compared to the IM group is consistent with a previous report where pathological changes in the former were not observed until 3 weeks following challenge . These findings demonstrate that SAV-3 can spread via water, making the cohabitation challenge a possibility. The IM route of infection for SAV-3 is not natural since it is expected that fish get infected either through vectors or the water itself. Challenge studies using the cohabitation model have previously been described although they have not performed according to expectations firstly because of the difficulty to induce mortalities experimentally for SAV in general [32, 34, 52] and secondly because the strength of virus challenge seems to be somewhat attenuated compared to IM challenge . In the present study, the presence of virus at low titers in cohabitants (Figure 2b, 2 and 4 wpi) probably allowed the fish to mount a protective immune response resulting in the delay/down regulation of pathology. Cohabitation challenge models for this virus should therefore aim to produce high quantities of infectious virus by shedders in order to enhance pathology in cohabitants or increase the number of shedders and thereby raise the infection pressure.
Finally, the rational development of vaccines offering protective immunity against pathogens relies on knowledge of basic immune responses to particular infections. This is not known in detail for SAV-3 infections in Atlantic salmon although a recent study performed by our group points to antibody responses playing a role . In the present study, we demonstrate that SAV-3 infections induce mRNA transcripts of genes including IFN-α and its stimulated genes (ISG) at early time, followed by IFN-γ, TNF-α, IL-12, IL-10, IL-8, CD3ε, CD4, CD8, TCR-α, MHC-I, and MHC-II as the infection progresses. This is similar to what has been observed in other alphavirus infections in higher vertebrates [12, 55], and suggests that the protection of fish against SAV-3 should be aimed at protocols that include eliciting both Th1 polarized and/or cytotoxic responses.