Samples from SAV3-infected Atlantic salmon were obtained from an in vivo SAV3 challenge trial described in detail elsewhere (34). Briefly, rSAV3 (referring to SAV3 virus recovered from cells transfected with a plasmid containing the entire SAV3 genome (prSAV3; originally cloned from the wild-type SAV3 isolate H20/03 (DQ149204, AY604236)) was used. The same prSAV3 plasmid was used as a backbone for the construction of SAV3 infectious strains with targeted mutations, two of which (SAV3-E2_319A and rSAV3-Cap_NLS) were included in this study. SAV3-E2_319A has a mutation in the predicted N-linked glycosylation motif in the E2 protein while rSAV3-CapNLS has a mutation in the subcellular localization signals of the capsid protein (35). Both attenuated SAV3 strains are infectious, transmittable to naïve cohabitant fish and induce innate anti-viral responses (34).

The in vivo experiments were conducted at the Aquaculture Research Station, Tromsø, Norway. The challenge trials were approved by the Norwegian Animal Research Authority (NFDA) according to the European Union Directive 2010/63/EU (permit numbers 16409 and 19014) and were performed in accordance with the recommendations of the current animal welfare regulations: FOR-1996-01-15-23 (Norway). Briefly, non-vaccinated Atlantic salmon, pre-smolts, strain NLA, were reared in a hatchery at the Aquaculture Research Station, Tromsø, Norway and confirmed free of the salmon pathogens ISAV, SAV, PRV and IPNV by RT-RT-qPCR. Fish were kept in running freshwater at 10°C, exposed to continuous light and fed with commercial dry feed and starved for 24 hours prior to handling and sampling. The fish were randomly selected for immunization, anesthetized by bath immersion in benzocaine chloride (0.5 g/10 L) for 2–5 min, labelled (tattoo) and intramuscularly (i.m.) injected. In this study, samples from control fish i.m. injected with PBS and fish i.m. infected with 0.2 ml cell culture medium containing 10² TCID50 virus [rSAV3, rSAV3-E2_319A or rSAV3-Cap_NLS (34)] or with a vaccine based on inactivated SAV3 with water-in-oil adjuvant (36) (kindly supplied by PHARMAQ AS) were analyzed. Shedder fish injected with SAV3 H20/03/2 at week 7 [as described in (34)], were added after sampling at week 8. Samples from 6-fish/time point were collected through a 12-week sampling period. Organs (heart, spleen, head kidney and gill) from virus-challenged and control fish were aseptically collected at 2, 4, 6-, 8-, 10- and 12-weeks post infection and kept in RNA-later. The organs were used for gene expression analyses by RT-qPCR following RNA isolation and subsequent cDNA synthesis as described below.

SSP-9 cells derived from Atlantic salmon head kidney (HK) (37), were kindly provided by Dr. S. Perez-Prieto (CSIC, Madrid, Spain). Chinook salmon embryo (CHSE-214) cells were purchased from American Type Culture Collection. Cell lines were maintained as monolayers in Leibovitz’s medium with L-glutamine (L-15) (Life Technologies) supplemented with antibiotics (10 U/ml penicillin, 10 μg/ml streptomycin) and 8% fetal bovine serum (FBS) at 20°C. For all in vitro infection trials Salmonid alphavirus subtype 3 (SAV3) (PDV-H10-PA3, provided by Professor Øystein Evensen, Norwegian University of Life Sciences) was used. The SAV3 virus was propagated in CHH-1 cells in L-15+ with 5% FBS at 15°C. Virus titer was determined by the TCID₅₀ method. Infectious pancreatic virus (IPNV) [strain N1, serotype Sp (38)] were propagation in CHSE-214 cells and titrated as described in (39).

SSP9 and CHSE-214 cells were seeded in 6-well plates at a density of approx. 2,5 × 10⁵ cells/ml and grown until 70% confluency. Culture media was removed, and cells were washed two times with 2 ml sterile PBS followed by addition of serum and antibiotic free medium containing SAV3 or IPNV (MOI = 0,1, MOI= 1 or MOI= 5). After allowing the virus to be absorbed for 3 h, the medium was replaced with L-15+ supplemented with 2% FBS and cells were incubated for 1, 3, 5, 7, 9, 10 and 12 days following SAV3 challenge and 6, 12, 24, 48 and 72 h following IPNV infection. Variable time points for RNA isolation were based on documented differences in SAV3 and IPNV replication kinetics in CHSE-214 cells wherein in for IPNV, viral specific RNA synthesis peaks 8-10 hours after infection (40) typically resulting in significant (>70%) CPE by 48 hours post infection rendering expression analysis at later time points challenging and largely uninformative (41, 42). In contrast, SAV3 infection of CHSE typically result in detectable viral transcripts around 2 dpi with minor CPE observed at 6 dpi (41, 42). Following respective incubation time points, RNA was isolated for cDNA synthesis and RT-qPCR (described in detail below).

Recombinant Atlantic salmon IFNa1 (GenBank accession no. DQ354152.1), IFNc (GenBank accession no. JX524153) and IFNb (GeneBank accession no. JX524152) were produced by transfection of sub confluent HEK-293 cells with IFN expression plasmids as previously described (25). Rainbow trout (Oncorhynchus mykiss) IFNγ was produced in E. coli (43) and protein concentrations were measured with a QuickStart Bradford protein assay kit (Bio-Rad) with bovine serum albumin as a standard. JAK I inhibitor was obtained from Calbiochem (CAS 457081-03-7).

Total leukocyte populations from Head-kidney (HK) were isolated on Percoll (GE Healthcare) gradients following previously established protocols (44). Briefly, HK were sampled aseptically and kept in ice-cold transport medium (L-15 medium with 10 U/ml penicillin, 10 μg/ml streptomycin, 2% fetal bovine serum, 20 U/ml heparin) until homogenization on 100 μm cells strainers (Falcon). The resulting cell suspensions were layered on 25/54% discontinuous Percoll gradients and centrifuged at 400 × g for 40 min at 4°C. Cells at the interface were collected and washed twice in L-15 medium. Cells were counted using an automatic cell counter (Countess II Automated cell counter, Thermo Fisher, cat. nr. AMQAF1000). A total of 3 x 10⁶ cells/ml were seeded in 1 ml L-15+ media supplemented with 8% FBS in 24 well plates (Nunclon Delta Surface, Thermo Scientific), stimulated with 500U of rIFNa, rIFNb, rIFNc or 10ng/ml IFNγ and cultivated at 16°C for 6, 12, 24 or 48 hours. Cells cultured in L15+/8% FBS media alone and harvested at the corresponding time points were used as controls. For inhibitory studies HKLs were isolated and stimulated as described above and incubated for 12 hours in the absence or presence of 15 nmol/ml of JAK I inhibitor, cells with inhibitor alone were included as controls. Following respective incubation time points, RNA was isolated for cDNA synthesis and RT-qPCR (described in detail below).

SSP-9 cells (7× 10⁵ cells/well) and CHSE cells (7 x 10⁵ cells/well) were seeded in 1 ml culture media in 6 well culture plates. Cells in triplicate wells were stimulated with 1000 U/ml rIFNa, 1000 U/ml rIFNc or 10 ng/ml rIFNγ and harvested at different time points. For inhibitory studies SSP9 cells were stimulated as described above and incubated for 12 hours in the absence or presence of 15 nmol or 150 nmol of JAK I inhibitor, cells with inhibitor alone were included as controls. Following respective incubation time points, RNA was isolated for cDNA synthesis and RT-qPCR (described in detail below).

SSP-9 cells grown in 6 well plates as described above were stimulated with 10 ng/ml rIFNγ in the presence or absence of JAK I inhibitor were harvested at 12 hps, washed and lysed in M-PER (Mammalian protein extraction buffer) supplemented with HALT Protease inhibitor (Thermo fisher) and subjected to SDS-PAGE using NuPAGE Novex Bis-Tris 4–12% gels (Life Technologies). Proteins were transferred to PVDF membrane and incubated in blocking agent (Intercept Blocking Buffer, Licor) in TBS-T overnight at 4°. The membranes were incubated with either a polyclonal rabbit antibody prepared against Atlantic salmon Mx1 diluted 1/3000 (v/v) followed by an incubation with goat anti-rabbit Ig (H+L) secondary antibody conjugated to Alexa fluor 680 diluted 1/10000 (v/v) for 1 h at room temperature or actin 1/1000 (v/v) followed by incubation with a goat-anti-mouse Ig (H+L) secondary antibody conjugated to Alexa fluor 680 diluted 1/10000 (v/v) for 1 h at room temperature. After washing the membrane was imaged using Odyssey CLX infrared imaging system.

To study the temporal progression of IPNV and SAV3 infections (up to 72 hours and five days, respectively) along with control (uninfected CHSE-214), 2,25 × 10⁵ CHSE-214 cells were seeded in 35mm confocal dishes and allowed to adhere overnight. Culture media was removed, and cells were washed two times with 2 ml sterile PBS followed by addition of serum- and antibiotic free medium containing SAV3 or IPNV (MOI= 1). After allowing the virus to be absorbed for 3 h, the medium was replaced with L-15+ supplemented with 2% FBS and cells were incubated for 1, 3, 5 and 7 days following SAV3 challenge and 6, 12, 24 and 48 h following IPNV infection. Following respective incubation time points, cells were fixed. For fixation, PFA was used for twenty minutes followed by three washes with PBS. The cell membranes were stained with CellMask™ orange followed by NucSpot^® Live direct dye for nuclear staining and stored until imaging. Images were acquired using a DeltaVision OMX V4 Blaze imaging system from GE Healthcare Life Sciences equipped with a 60X 1.42NA oil-immersion objective from Olympus; three sCMOS cameras; and lasers for excitation at wavelengths of 488 nm, 568 nm, and 642 nm. The exposure time and illumination power were adjusted to obtain a maximum of 10000 grayscale counts on the camera chip. A total of ten z-plane frames with z-steps of 100nm were collected on each imaging channel along the optical axis of the objective. The frames were automatically stitched into an 8×8 tile mosaic image by the built-in software package SoftWoRx.

Total RNA from tissues, primary leucocytes, SSP9 and CHSE cell lines were isolated using the RNeasy Mini Kit (Qiagen) following the manufacturer`s recommendation and RNA was quantified using NanoDrop (ND 1000 Spectrophotometer). For RNA isolated from tissues and primary leucocytes 1000 and 500 ng respectively was treated using DNase I to remove all residual genomic DNA. Twenty microliter cDNA reactions were synthesized using TacMan reverse transcription reagents (Applied Biosystems) using random hexamer primers under the following conditions: 25°C for 10 min, 37°C for 30 min and 95°C for 5 min. cDNA samples were diluted 1:2 and stored at -20°C until use. For SSP9 and CHSE cells cDNA was synthesized using the QuantiTect RT kit (Qiagen) according to the manufacturer`s instructions with 500ng of RNA per 20 µl of reaction. cDNA was diluted 1:2 and stored at -20°C until use. Quantitative PCR (RT-qPCR) was run as 10ul duplicate reactions on a 7500 Fast Real-Time PCR systems (Applied Biosystems) according to standard protocol. All primers were validated prior to use and primer sequences are available upon request. For each primer pair and tissue/cell a negative control (no template) and a no reverse transcriptase control RT (-) was performed. A threshold difference of at least 6 quantification cycles (Cq) between Rt (+) and RT (-) was used as a cut-off. Ct values >38 was rejected. Parameters were as follows: 2 min 95°C followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 min. Melt curve analysis were performed to ensure that there were no artifacts, and a single product was amplified. Relative quantitative PCR gene expression analysis was performed using the ΔΔCt method. Expression of individual genes was examined relative to the endogenous EF1-α controlled. Relative expression (zero-hour samples) was calculated using the 2-ΔCt method. For infected tissues and stimulated cells, fold change or alternatively log2 fold change was calculated against the appropriate controls.

Sasa-LGA1 and Sasa-LIA promoter regions containing 2000 bp upstream and 75 bp downstream of the transcriptional start site were extracted from the Salmo salar genome NC_027320, GCF_000233375.1. First, the transcription factor biding sites were identified using the online application PROMO which uses TRANSFAC database v8.3.0 and constructs positional weight matrices in a species or taxon using known transcription factor binding sites and then search for matches in query DNA sequences (45). Based on these results. The 2000 bp sequence was further processed to create different constructs for luciferase assay containing different promoters. All constructs were synthesized in PGL basic promoter by Twist Biosciences, San Francisco. The full 2000 bp upstream promoter was synthesized in PGL basic promoter for both Sasa-LGA and Sasa-LIA. For Sasa-LIA truncated promoter construct were designed to either including or exclude specific ISRE and GAS elements; -150/+75 including the ISRE and GAS elements, along with -700/+75 bp, -1200/+75 bp, -1200/-700 bp (including +75 at 5’). The mutated constructs were focused on only ISRE and GAS elements. The Sasa-LIA-150 + 75 bp construct was selected to mutate ISRE element GAAAGTGAAA to CCGAGTGACG and GAS- like element TTCAGAA to GCGAGCG. Similarly, for Sasa-LGA a systematic deletion of promoter was done with +75-200 including the IRF1/3 and ISRE element, +75-500 including STAT binding site, -1200/+75, and -1300/-1900 (including +75 at TSS) that included putative GAS like element but no proximal ISRE elements. For mutation, the ISRE element in the -200/+75 construct was mutated.

CHSE-214 were seeded into 96-well culture plates at a density of 1.6 × 10⁴ cells/well in 100ul of L-15 medium supplemented with 8% FBS and grown to 50% confluence overnight at 20 °C. The cells were transiently transfected by adding a transfection mix consisting of 10 µl Opti-MEM^® (Life Technologies) solution containing 90 ng of promoter reporter (firefly luciferase) construct, 10 ng Renilla luciferase vector (Promega- Madison WI), and 0.3 µl TransIT-LT1 per well. Atlantic salmon LIA and LGA1 promoter constructs were investigated, while pGL3-basic was included as empty vector control. All promoter fragments and mutants were synthesized by Twist bioscience and cloned into pGL3-basic. 24 hours post transfection, cells were stimulated with 600 U/ml of recombinant (r)Type I rIFNa or rIFNc or 30 ng/ml of rINFγ. Luciferase activity was measured 48 h post stimulation using the Dual-Luciferase^® Reporter Assay System (Promega, Madison, WI) according to the manufacturer’s protocol. The constitutively expressing Renilla luciferase construct provided an internal control value to which the expression of the experimental firefly luciferase was normalized. The firefly and Renilla luciferase activity was measured in a Luminoscan RT luminometer, in which all samples for the luciferase assay were set up in four parallels for each treatment and the results are given as relative light units (RLU). The results are presented as fold change in relative light units (RLU) by dividing the RLU of the stimulated samples by the average RLU of the corresponding non-stimulated samples.

For in vivo studies all quantitative data were based on duplicated measurements from a minimum of four fish n ϵ (46) and were analyzed in GraphPad Prism 8. For primary cells and cell lines all quantitative data were based on triplicated samples undergoing duplicated measurements. All in vitro experiments were repeated a minimum of three times in independent experiments. Statistical evaluations were performed using Tukey`s multiple comparisons test following a significant one-way ANOVA. Correlation among MHC class I L lineage expression, pathogen load and interferon expression were determined using the Pearson Correlation coefficiency (p=0.05) calculated from the relative expression of each gene normalized to EF1-alpha(B). For all analysis a p value < 0.05 was considered significant.

Genome searches were performed using previously identified Atlantic salmon MHC class I L lineage LIA and LGA1 sequences and blasted against annotated salmonid genomes available in NCBI using mega blast. Genomic regions identified through these searches were screened for LIA and LGA1 leader sequences based on Atlantic salmon LIA and LGA1 cDNA sequences and the upstream proximal (-500) promoter regions were extracted. Genomes used in this study were as follows: Oncorhyncus gorbuscha GCA_021184085.1 (pink salmon), Oncorhyncus keta GCA_023373465.1 (chum salmon), Oncorhynchus kisutch GCA_002021735.2 (coho salmon), Oncorhynchus mykiss GCA_013265735.3 (rainbow trout), Oncorhynchus nerka GCA_006149115.2 (sockeye salmon), Oncorhynchus tshawytscha GCA_018296145.1 (Chinook salmon), Salmo salar GCA_905237065.2 (Atlantic salmon), Salmo trutta.

GCA_901001165.1 (brown trout), Salvelinus GCA_002910315.2 (unclassified species in the genus Salvelinus), Salvelinus fontinalis GCA_029448725.1 (brook trout) and Salvelinus namaycush GCA_016432855.1 (lake trout). For promoter regions, all evolutionary analyses were conducted in MEGA7. Sequences were aligned using ClustalX with manual corrections for some predicted sequences and bootstrapped phylogenetic trees were constructed using the Maximum Likelihood method. The percentage of trees in which the associated taxa clustered together are shown next to the branches. The trees are drawn to scale, with branch lengths measured in the number of substitutions per site. Gene models were predicted based on expressed LIA and LGA1 sequences in Atlantic salmon.

To further delineate gene specific SAV3 inducible responses among Atlantic salmon L lineage genes, tissue specific transcriptional induction and gene expression kinetics of six functionally expressed L lineage genes (Sasa-LIA, Sasa-LGA1, Sasa-LHA, Sasa-LDA, Sasa-LFA and Sasa-LCA) (15) were analyzed over a 12-week experimental immunization challenge using a recombinant SAV3 infectious strain. Accordingly, fish were i.m. infected with an infectious recombinant (r)SAV3 strain and subsequently challenged on week 8 by co-habitation with SAV3 (H20/03) infected shedder fish as previously described (34). At different times post-infection (2-, 4-, 6-, 8-, 10- and 12-weeks post infection [wpi]), log2 fold change in expression of the various L lineage genes in the heart (main site of viral replication), head-kidney, spleen and gill were determined ( Figure 1 ; Supplementary Figure 1 ). Consistent with previous reports (15) Sasa-LIA and Sasa-LGA1 gene expressions were markedly upregulated at the fist, 2 wpi, time point. For both Sasa-LIA and Sasa-LGA1 the highest level of induction was observed in the heart, with an average fold increase of 46 (SD ± 10) and 25 (SD ± 3.3) respectively, followed by head-kidney and to a lesser extent spleen, with little to no transcriptional induction observed in the gill ( Figures 1B, C ). Notably, Sasa-LIA gene expression in the heart, despite the continuous detection of viral specific transcripts, was transient ( Figures 1B, C ). Across all examined tissues Sasa-LIA gene expression peaked at the 2-week time point followed by a return to near baseline levels by 4 wpi. In stark contrast, Sasa-LGA1 expression in the heart remained elevated through the course of infection, albeit with a gradual decrease from 2 to 8 wpi. Similarly, 2 weeks after exposure to SAV3 infected shedders a moderate but transient induction of Sasa-LIA was observed in the heart and head-kidney with expression returning to baseline levels by the next sampling point. Comparably, post challenge Sasa-LGA1 expression in the heart was significantly upregulated compared to PBS controls at both the 10- and 12-week time points. Neither Sasa-LHA nor Sasa-LDA were upregulated with a >2 log2-fold increase following SAV3 infection in any of the tissues examined with the exception of heart where significant upregulation of both genes was apparent at 4 wpi ( Figure 1E , Supplementary Figure 1 ). For Sasa-LCA significant upregulation was observed in the heart at 4- and 6-wpi, which was reciprocated 4 weeks after addition of shedder fish ( Figure 1E . Supplementary Figure 1 ). Consistent with previous results indicating a highly tissue specific expression pattern for some L lineage genes (15) Sasa-LFA expression was only detectable above threshold levels in the gills and showed no significant transcriptional response to SAV3 challenge. In the same sample set expression patterns of IFNa, IFNc and IFNγ were examined ( Figure 1E , Supplementary Figure 1 ) revealing, as expected, a strong induction of type I IFNa, IFNc and type II IFNγ in response to SAV3 infection. Similar to what was observed for Sasa-LIA, IFNa expression in the heart peaked at 2 wpi, was significantly reduced by 4 wpi and continued to decline at the 6-, and 8 wpi time points. In contrast, elevated expression levels of both IFNc and IFNγ were maintained at the 2-, 4- and 6-wpi time points followed by a marginal reduction at 8 wpi.

To investigate the factors governing discrete Sasa-LIA and Sasa-LGA1 transcriptional induction patterns we examined the expression of these genes in fish i.m. infected with two genetically modified infectious strains of rSAV3 or with a vaccine based on inactivated SAV3 with water-in-oil adjuvants (36) ( Figure 2A ). These rSAV3 strains, which have previously been shown to actively infect and replicate in Atlantic salmon (34) are attenuated in either the envelope protein E2 (rSAV3-E2_319A) or the capsid protein nuclear localization signal (rSAV3-Cap_NLS). Similar to rSAV3, at 4 wpi both attenuated strains, as assessed by mRNA expression of select interferon stimulated genes, induce comparable anti-viral responses in the heart (34). Congruent with these observations, comparable levels of viral transcripts were detected in hearts isolated from fish infected with WT-rSAV3, rSAV3-E2_319A or rSAV3-Cap_NLS while no viral transcripts were detected in fish injected with the inactivated SAV3 ( Figure 2D ). While inactivated SAV3 failed to induce Sasa-LIA gene expression, significantly increased mRNA levels were detected at 2 wpi in response to WT-rSAV3, rSAV3-E2_319A and rSAV3-Cap_NLS ( Figure 2B ). However, Sasa-LIA expression in fish infected with rSAV3-E2_319A was significantly lower compared to both rSAV3-Cap_NLS and WT- rSAV3. As seen in Figure 2C Sasa-LGA1 was upregulated in response to all three viral strains and, albeit not statistically significant, by 4 wpi inactivated SAV3 infection resulted in elevated Sasa-LGA1 expression comparable to that of fish infected with actively replicating SAV3 strains. As a comparison no significant difference in expression of type I IFNa, type II IFN-y or the interferon inducible gene Mx1/2 were observed between rSAV3, rSAV3-E2_319A or rSAV3-Cap_NLS at 2 wpi ( Figures 2E-G ). However, at 4 wpi expression of IFNa in the rSAV3-E2_319A was lower compared to the other viral strains and did not reach significant induction levels compared to fish injected with inactivated SAV3.

To further assess the effects of viral infection on the regulation of L lineage gene expression, specifically LIA and LGA1, we analyzed the temporal dynamics of these genes in response to SAV3 infection in SSP-9 cells (37). Similar to in vivo observations Sasa-LIA gene expression was upregulated during the early stage of infection (1 dpi) with infection using a higher virus MOI resulting in higher Sasa-LIA induction ( Figure 3A ). Further, despite highest detection of SAV nsp1 RNA at 3 dpi, Sasa-LIA transcriptional levels returned to near baseline by this time point and remained at this level throughout the course of the infection ( Figures 3A, C ). A similar gene expression pattern was observed for IFNa which was strongly correlated with that of Sasa-LIA ( Figure 3D , Supplementary Figure 2 ). In contrast to what was observed following in vivo SAV3 challenge Sasa-LGA1 was not significantly induced in response to SAV3 infection in SSP-9 cells ( Figure 3B ).

Next, transcriptional responses of L lineage gene expression in response to viral infections in the chinook salmon embryonic cell line CHSE-214 were investigated. Here it should be noted that L lineage gene profiles in salmonids is complex, with a remarkably high degree of species-specific adaptations both in gene numbers and functionality (12). Thus, the L lineage profile of chinook salmon was contrasted with that of Atlantic salmon, revealing that while LIA is highly conserved and present as a bona-fide functionally expressed gene in both species, LGA1 has become pseudogenised in Chinook salmon ( Figure 4A , Supplementary Figure 3 ). Consistent with the genomic data no expression of Onts-LGA1 were observed in CHSE-214 cells (data not shown). Accordingly, only Onts-LIA was examined further. Compared to SSP-9 cells, for CHSE-214 exposed to SAV3 at MOIs of 1 and 5 viral kinetics was different likely reflecting the genetic differences inherent to these two cell types. In CHSE-214 cells, Onts-LIA transcript levels were not significantly induced until around 7 dpi reaching a peak of induction at 9 dpi before declining by 12 dpi ( Figure 4C ). However, similar to what was observed in SSP-9 cells Onts-LIA mRNA levels were highly correlated with nsP1 and IFNa transcript levels, as well as with all representative interferon stimulated genes (ISGs) tested (Mx1/2, Mx8 and CXCL10) while no significant correlation was manifested between Onts-LIA and IFNc transcripts ( Figure 4J , Supplementary Figure 4 ). When contrasted with the presence of SAV3 virus, as inferred from detection of nsp1 transcript levels, measured by RT-qPCR, viral transcripts could be detected already at day 3, reaching a peak at 7-9 dpi before declining at the 12 dpi time point ( Figure 4E ). Similarly, a gradual increase in CPE monitored via confocal microscopy on fixed cells, using cell mask and nuclear staining was detected for cells infected with 1 MOI SAV3 with signs of infection apparent as early as 1 dpi. Throughout the infection, compared to control cells, infected cells displayed distinct morphological changes including compromised cytoskeleton, cell membrane permeabilization, rounding and loss of cell adhesion ( Figures 4F-I ). In conclusion, these data indicate that while SAV3 infection leads to upregulation of Onts-LIA gene expression, transcriptional regulation appears to be tightly linked, possibly directly mediated by interferon stimulation rather than direct viral recognition.

In contrast to SAV3 which has been shown to induce the expression of type I IFN and ISGs both in vitro and in vivo IPNV, a dsRNA virus, inhibits IFN-induced responses in CHSE-214 cells (46). An interesting question was therefore how infections with IPNV would potentially influence the expression of L lineage genes. For CHSE-214 cells exposed to 1 MOI IPNV, CPE monitored via confocal microscopy showed a progressive loss of cell adhesion and compromised cell membrane and cytoskeleton along with increasing levels of lysed cell debris ( Figures 5B–E ). Signs of cell stress was apparent already at 6 hpi and progressively increased throughout the course of the infection. In concordance with the microscopic analysis Figure 5A shows IPNV VP2 transcript levels in the infected cells, which increased over time, indicating a productive infection. However, in stark contrast to SAV3 infections of the same cells, no significant induction of Onts-LIA was observed as compared to control cells. The mRNA expression of IFNa and Mx paralleled Onts-LIA expression and in general, expression of all three genes were very low throughout the study.

Given that type I IFN classes are known to differ in their responses, we compared the ability of recombinant representatives of group 1 (rIFNa1) and group II (rIFNb and rIFNc) type I IFNs to modulate L lineage gene expression in primary head kidney leucocytes (HKLs, Figure 6 ), SSP-9 ( Figures 7A, B ) and CHSE-214 cells ( Figure 7C ). Further, to obtain additional insight into the interplay among type I and type II interferon induction and transcription regulation of L lineage genes, the modulating abilities of representative type I IFNs was contrasted with that of recombinant type II IFN (rIFNγ). For HKLs, expression of the interferon stimulated genes Mx1/2 and Mx8, which have been shown to differentially respond to IFNa and IFNγ stimulation (30) were included as controls and the transcriptional induction of IL-1, TNFα, IFNa1 and IFNγ was investigated in parallel ( Supplementary Figure ). In HKLs, 12 hours post stimulation, both Sasa-LIA and Sasa-LGA1 were strongly (~16 fold) upregulated in response to rINFγ stimulation ( Figures 6A, B ). Sasa-LIA expression was also significantly upregulated in response to type I rIFNa1 and rIFNc stimulation, while Sasa-LGA1 showed a modest induction in response to rIFNa1 and no significant induction in response to rIFNc stimulation at this time point. Comparably, while rIFNb stimulation induced expression of Mx1/2 and Mx8 at comparable levels to that of rIFNa1, no induction of L lineage gene expression was observed in response to rIFNb stimulation ( Figure 6 , Supplementary Figure 5 ). A modest induction of Sasa-LHA gene expression was observed in response to rINFγ with no significant induction in response to any of the type I IFNs tested. Sasa-LDA did not respond to type I IFNs or type II IFN stimulation and neither Sasa-LCA nor Sasa-LFA transcripts were detected in HKLs above threshold levels. Comparing the induction kinetics of Sasa-LIA and Sasa-LGA1 genes revealed that both L lineage genes responded rapidly, albeit with different magnitudes and distinct kinetics following stimulation with either rIFNa1, rIFNc or rIFNγ ( Supplementary Figure 5 ). Significantly elevated transcript levels were detected as early as 6 hps, peaking between 12 and 24 hps, followed by a return to baseline by 48hps. Induction kinetics were different depending on the stimuli, with rIFNa1 resulting in a somewhat bimodular upregulation peaking at 6 and 24hps. Comparably, highest transcription levels in response to INFc was detected at 6 hps for both Sasa-LIA and Sasa-LGA1 while IFNγ stimulation resulted in continuous increasing levels of Sasa-LIA up on till the 24h time point while Sasa-LGA1 transcription levels peaked at the 6-hour time point.

Similarly, in SSP-9 cells stimulated with rIFNγ we observed a rapid and marked upregulation of Sasa-LIA expression, a > 20-fold increase in gene expression was observed within 3 hps that peaked (>90-fold) at 24 h post-treatment and then declined somewhat at the last time point analyzed (72 h post-treatment, Figure 7A ). Similarly, rIFNa1, and to a lesser extent rIFNc stimulation upregulated Sasa-LIA expression, albeit at a significantly lower level compared to rIFNγ. These results were largely recapitulated in CHSE-214 cells where we observed marked induction of Onts-LIA in response to stimulation with both rIFNγ and rIFNa1 but not to rIFNc ( Figure 7C ).

In SSP-9 cells the highest level of Sasa-LGA1 induced expression, with a >10-fold induction compared to unstimulated cells, was observed in response to rIFNγ stimulation while rIFNc stimulation resulted in peak of Sasa-LGA1 expression at 6 hps that declined by the 24 h time-point ( Figure 7B ).

To further assess the impact of type I and type II IFN stimulation on L lineage gene induction, we analyzed Sasa-LIA and Sasa-LGA1 expression in SSP-9 cells in the presence or absence of a JAK inhibitor I (a reversible ATP-competitive inhibitor of Janus protein tyrosine kinases (JAKs)). As expected, inhibiting the JAK/STAT pathway resulted in a reduction in IFN type I induced Mx1/2 and IFNγ induced Mx8 gene expression and at a 10-fold higher dose the IFNγ-induced Mx protein levels were visibly affected ( Figures 8A-C ). Figures 8D, E shows that Sasa-LGA1 and Sasa-LIA transcript levels significantly decreased in rIFNγ -treated cells in the presence of the inhibitor compared to control cells. Reduction in Sasa-LIA transcript levels in the presence of the inhibitor were also apparent in rIFNa1-treated and, albeit not significant, in SSP-9 cells stimulated with rIFNc. Notably, no significant effect of the inhibitor was observed with regard to the basal expression of either Sasa-LIA or Sasa-LGA1 ( Supplementary Figure 6 ).

Similarly, following a 12-hour incubation with 15nM JAK I inhibitor a marked reduction in rIFNγ as well as rIFNa and rIFNc induced upregulation of both Sasa-LIA and Sasa-LGA1 was observed in HKLs ( Supplementary Figure 7 ). Collectively, these data indicate that Sasa-LIA and Sasa-LGA1 interferon induced transcription occurs, at least partially, in response to type I IFN and type II IFNγ induced JAK/STAT dependent signaling.

The promoter regions of Sasa-LIA and Sasa-LGA1 possess distinct binding sites ( Figure 9 ). Within the proximal promoter region, spanning 500 bp upstream of the start codon canonical interferon response elements can be found in both Sasa-LIA and Sasa-LGA1. For Sasa-LIA, this region includes a putative STAT, two identical ISRE (GAAA-gt-GAAA), and a GAS-like (TTCAGAA) element while in the same region a single ISRE (GAAA-ga-GAAA) and a IRF1/3 site can be identified in Sasa-LGA1. Further, the distal promoter region (-2000/-500) of Sasa-LIA contains multiple putative STAT and IRF1/3 binding sites, the majority of which are concentrated in the -2000 to -1000bp region while the distal promoter region of Sasa-LGA1 contains a GAS-like element (TTCAGAA) and a concentration of STAT and IRF1/3 elements ( Figure 9 , Supplementary Figure 8 ). The promoter activity of Sasa-LIA and Sasa-LGA1 was analyzed through luciferase assay in CHSE-214 cells ( Figure 9 ). For Sasa-LIA, by stepwise truncating the promoter sequences it was apparent that the construct containing the two tandemly located ISRE and the GAS-like element (-150/75-Sasa-LIA) retained maximum luciferase activity, substantiating the assumption that ISRE and or GAS elements are essential for IFNγ, IFNa and IFNc induced expression of LIA in salmonids ( Figures 9A, B ). Thus, the (-150/75-Sasa-LIA) promoter construct was used in subsequence assays. Four mutated variants of Sasa-LIA -150/75 were synthesized, each containing mutations in either GAS (LIA-ΔGAS), ISRE1(LIA-ΔISRE1), ISRE2 (LIA-ΔISRE2), or double mutations for ISRE1/2 regions (LIA-ΔISRE1/ΔISRE2) ( Figure 10A ). Upon transient transfection into CHSE-214 cells and subsequent stimulation with rIFNa1, rIFNc or rIFNγ, all four mutated constructs exhibited significantly reduced luciferase activity compared to the unmutated -150/75-Sasa-LIA construct ( Figure 10B ). Following rIFNa1 and rIFNc stimulation luciferase activity was completely ablated in all four mutants when compared to -150/75-Sasa-LIA with no significant difference in activity observed across the different mutated constructs ( Figure 10C ). Comparably, following rIFNγ stimulation the luciferase activity of LIA-ΔGAS, LIA-ΔISRE1 and LIA-ΔISRE2 constructs remained elevated compared to controls but dropped significantly, reducing the activity on average to ~44%, 22%, and 20% respectively compared to the wild-type construct. In comparison the LIA-ΔISRE1/ΔISRE2 construct showed no detectable activity above controls. These findings demonstrate that while optimal IFNγ induced expression of Sasa-LIA likely involves all three promoter motifs it is critically dependent on the two ISRE motifs. In comparison in Sasa-LGA1 which has an arguably simpler active regulatory region, consisting of a single ISRE motif located 72 bp upstream of the start codon, no activity was observed for the LGA–ΔISRE1 construct indicating that the identified ISRE motif alone is essential for IFNγ–induced expression of Sasa-LGA1 ( Figures 10D, E ). Notably, while IFNγ stimulation significantly upregulated luciferase activity for the truncated Sasa-LGA1 -200/75 construct, Sasa-LGA1 constructs consisting of the extended distal promoter regions, despite containing an intact ISRE motif, showed significantly reduced or no luciferase activity ( Figures 9C, D ).

LIA represents an evolutionary old L lineage gene (12) and LIA orthologs can be found as single gene copies in all 11 salmonid species with annotated genome assemblies currently available in the NCBI databases (data not shown). In comparison LGA1 sequences, based on phylogenetic clustering with previously identified L lineage gene sequences, were identified in six of the eleven salmonid species analyzed and display variation both in gene copy number and degree of pseudogenization. Phylogeny of the LIA and LGA1 proximal promoter region sequence, with strong bootstrap values, cluster based on subgroups forming two clades that are further supported by identification of key promoter elements and conservation of gene structure ( Figure 11 ). The putative promoters of LIA all included a GAS-like element, with the consensus sequence TTCAGAA within 2-9 bp upstream of the ATG start codon and a minimum of two ISRE elements. In addition, a third putative ISRE sequence differing from the previously identified elements in the spacing nucleotides (GAAA-tg-GAAA compared to GAAA-gt-GAAA)) was identified in all six members of the Onchorhynchus genes but not in the Salmo nor Salvelinus LIA gene sequences ( Figure 11C ). Whether or not this is a functional ISRE element needs to be determined by functional assays. Other potential regulatory elements, including binding sites for the CCAAT/Enhancer Binding Protein β (C/EBPβ) in Sasa-LIA were not conserved across promoter regions identified in other species. Similarly, in all LGA1 promoter regions, with the exception of Onke-LGA1, a single consensus ISRE element was present. Collectively these data suggest that type I and type II IFN regulation of LIA and LGA1 gene expression is conserved across salmonids underpinning the roles of these non-classical MHC class I genes in anti-viral immunity.