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Pancreas disease (PD) caused by salmonid alphavirus subtype 3 (SAV3) is a serious disease with large economic impact on farmed Norwegian Atlantic salmon production despite years of use of oil-adjuvanted vaccines against PD (OAVs). In this study, two commercially available PD vaccines, a DNA vaccine (DNAV) and an OAV, were compared in an experimental setting. At approximately 1040° days (dd) at 12 °C post immunization, the fish were challenged with SAV3 by cohabitation 9 days after transfer to sea water. Sampling was done prior to challenge and at 19, 54, and 83 days post-challenge (dpc). When compared to the OAV and control (Saline) groups, the DNAV group had significantly higher SAV3 neutralizing antibody titers after the immunization period, significantly lower SAV3 viremia levels at 19 dpc, significantly reduced transmission of SAV3 to naïve fish in the latter part of the viremic phase, significantly higher weight gain post-challenge, and significantly reduced prevalence and/or severity of SAV-induced morphologic changes in target organs. The DNAV group had also significantly higher post-challenge survival compared to the Saline group, but not to the OAV group. The data suggest that use of DNAV may reduce the economic impact of PD by protecting against destruction of the pancreas tissue and subsequent growth impairment which is the most common and costly clinical outcome of this disease., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Salmonid alphavirus (SAV) is the etiological cause of pancreas disease (PD) in Atlantic salmon (Salmo salar). Several vaccines against SAV are in use, but PD still cause significant mortality and concern in European aquaculture, raising the need for optimal tools to monitor SAV immunity. To monitor and control the distribution of PD in Norway, all salmonid farms are regularly screened for SAV by RT-qPCR. While the direct detection of SAV is helpful in the early stages of infection, serological methods could bring additional information on acquired SAV immunity in the later stages. Traditionally, SAV antibodies are monitored in neutralization assays, but they are time-consuming and cumbersome, thus alternative assays are warranted. Enzyme-linked immunosorbent assays (ELISAs) have not yet been successfully used for anti-SAV antibody detection in aquaculture. We aimed to develop a bead-based immunoassay for SAV-specific antibodies. By using detergent-treated SAV particles as antigens, we detected SAV-specific antibodies in plasma collected from both a SAV challenge trial and a field outbreak of PD. Increased levels of SAV-specific antibodies were seen after most fish had become negative for viral RNA. The bead-based assay is time saving compared to virus neutralization assays, and suitable for non-lethal testing due to low sample size requirements. We conclude that the bead-based immunoassay for SAV antibody detection is a promising diagnostic tool to complement SAV screening in aquaculture., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Infectious pancreatic necrosis virus (IPNV) infection has been a major problem in salmonid aquaculture. Marker-assisted selection of individuals with resistant genotype at the major IPN quantitative trait locus (IPN-QTL) has significantly reduced mortality in recent years. We have identified host miRNAs that respond to IPNV challenge in salmon fry that were either homozygous resistant (RR) or homozygous susceptible (SS) for the IPN-QTL. Small RNA-sequenced control samples were compared to samples collected at 1, 7, and 20 days post challenge (dpc). This revealed 72 differentially expressed miRNAs (DE miRNAs). Viral load (VL) was lower in RR vs. SS individuals at 7 and 20 dpc. However, analysis of miRNA expression changes revealed no differences between RR vs. SS individuals in controls, at 1 or 7 dpc, while 38 "high viral load responding" miRNAs (HVL-DE miRNAs) were identified at 20 dpc. Most of the HVL-DE miRNAs showed changes that were more pronounced in the high VL SS group than in the low VL RR group when compared to the controls. The absence of differences between QTL groups in controls, 1 and 7 dpc indicates that the QTL genotype does not affect miRNA expression in healthy fish or their first response to viral infections. The miRNA differences at 20 dpc were associated with the QTL genotype and could, possibly, contribute to differences in resistance/susceptibility at the later stage of infection. In silico target gene predictions revealed that 180 immune genes were putative targets, and enrichment analysis indicated that the miRNAs may regulate several major immune system pathways. Among the targets of HVL-DE miRNAs were IRF3, STAT4, NFKB2, MYD88, and IKKA. Interestingly, TNF-alpha paralogs were targeted by different DE miRNAs. Most DE miRNAs were from conserved miRNA families that respond to viral infections in teleost (e.g., miR-21, miR-146, miR-181, miR-192, miR-221, miR-462, miR-731, and miR-8159), while eight were species specific. The miRNAs showed dynamic temporal changes implying they would affect their target genes differently throughout disease progression. This shows that miRNAs are sensitive to VL and disease progression, and may act as fine-tuners of both immediate immune response activation and the later inflammatory processes., (Copyright © 2020 Woldemariam, Agafonov, Sindre, Høyheim, Houston, Robledo, Bron and Andreassen.)

Salmonid alphavirus (SAV) is the OIE-listed, viral cause of pancreas disease (PD) in farmed Atlantic salmon. SAV is routinely detected by PCR-methods while typical histopathological lesions are additionally used to confirm the diagnosis. Field evaluation of diagnostic test performance is essential to ensure confidence in a test's ability to predict the infection or disease status of a target animal. For most tests used in aquaculture, characteristics like sensitivity (Se) and specificity (Sp) at the analytical level may be known. Few tests are, however, evaluated at the diagnostic level according to the OIE standard. In the present work, we estimated diagnostic test sensitivity (DSe) and diagnostic test specificity (DSp) for five laboratory tests used for SAV detection. As there is no gold standard, the study was designed using Bayesian latent class analysis. Real-time RT-PCR, cell culture, histopathology, virus neutralization test, and immunohistochemistry were compared using samples taken from three different farmed Atlantic salmon populations with different infection status; one population regarded negative, one in an early stage of infection, and one in a later stage of infection. The average fish weight in the three populations was 2.0, 1.6, and 1.5 kg, respectively. The DSe and DSp of real-time RT-PCR is of particular interest due to its common use as a screening tool. The method showed high DSe (≥0.977) and moderate DSp (0.831) in all 3-populations models. The results further suggest that a follow-up test of serum samples in real-time RT-PCR negative populations may be prudent in cases where epidemiological information suggest a high risk of infection and where a false negative result is of high consequence. This study underlines the need to choose a test appropriate for the purpose of the testing. In the case of a weak positive PCR-result, a follow-up test should be conducted to verify the presence of SAV. Cell culture showed high DSe and DSp and may be used to verify viral presence., (Copyright © 2019 Jansen, Guarracino, Carson, Modahl, Taksdal, Sindre, Brun and Tavornpanich.)

The occurrence of infectious salmon anemia (ISA) outbreaks in marine farmed Atlantic salmon constitutes a recurring challenge in Norway. Here, we aim to identify risk factors associated with ISA outbreaks with an unknown source of infection (referred to as primary ISA outbreaks). Primary ISA outbreaks are here defined by an earlier published transmission model. We explored a wide range of possible risk factors with logistic regression analysis, trying to explain occurrence of primary ISA with available data from all Norwegian farm sites from 2004 to June 2017. Explanatory variables included site latitude and a range of production and disease data. The mean annual risk of having a primary outbreak of ISA in Norway was 0.7% during this study period. We identified the occurrence of infectious pancreatic necrosis (IPN), having a stocking period longer than 2 months, having the site located at high latitude and high fish density (biomass per cage volume) in the first six months after transfer to sea site as significant risk factors ( p < 0.05). We have identified factors related to management routines, other disease problems, and latitude that may help to understand the hitherto unidentified drivers behind the emergence of primary ISA outbreaks. Based on our findings, we also provide management advice that may reduce the incidence of primary ISA outbreaks.

Background: MicroRNAs (miRNAs) control multiple biological processes including the innate immune responses by negative post-transcriptional regulation of gene expression. As there were no studies on the role(s) of miRNAs in viral diseases in Atlantic salmon, we aimed to identify miRNAs responding to salmonid alphavirus (SAV) infection. Their expression were studied at different time points post infection with SAV isolates associated with different mortalities. Furthermore, the genome sequences of the identified miRNAs were analysed to reveal putative cis-regulatory elements, and, finally, their putative target genes were predicted., Results: Twenty differentially expressed miRNAs (DE miRNAs) were identified. The expression of the majority of these increased post infection with maximum levels reached after the viral load were stabilized or decreasing. On the other hand, some miRNAs (e.g. the miRNA-21 family) showed decreased expression at the early time points post infection. There were significant differences in the temporal expression of individual miRNA associated with different SAV isolates. Target gene prediction in SAV responsive immune network genes showed that seventeen of the DE miRNAs could target 24 genes (e.g. IRF3, IRF7). Applying the Atlantic salmon transcriptome as input 28 more immune network genes were revealed as putative targets (e.g. IRF5, IRF4). The majority of the predicted target genes promote inflammatory response. The upstream sequences of the miRNA genes revealed a high density of cis-regulatory sequences known as binding sites for immune network transcription factors (TFs). A high expression in the late phase could therefore be due to increased transcription promoted by immune response activated TFs. Based on the in silico target predictions, we discuss their putative roles as early promotors or late inhibitors of inflammation. We propose that the differences in expressions associated with different SAV isolates could contribute to their differences in mortality rates., Conclusions: This study represents the first steps in exploring miRNAs important in viral-host interaction in Atlantic salmon. We identified several miRNAs responding to SAV infection. Some likely to prohibit harmful inflammation while other may promote an early immune response. Their predicted functions need to be validated and further studied in functional assays to fully understand their roles in immune homeostasis.

Heart and skeletal muscle inflammation (HSMI) and pancreas disease (PD) cause substantial losses in Atlantic salmon (Salmo salar) aquaculture. The respective causative agents, Piscine orthoreovirus (PRV) and Salmonid alphavirus (SAV), are widespread and often concurrently present in farmed salmon. An experimental infection in Atlantic salmon was conducted to study the interaction between the two viruses, including the immunological mechanisms involved. The co-infected fish were infected with PRV four or ten weeks before they were infected with SAV. The SAV RNA level and the PD specific lesions were significantly lower in co-infected groups compared to the group infected by only SAV. The expression profiles of a panel of innate antiviral response genes and the plasma SAV neutralization titers were examined. The innate antiviral response genes were in general upregulated for at least ten weeks after the primary PRV infection. Plasma from co-infected fish had lower SAV neutralizing titers compared to the controls infected with only SAV. Plasma from some individuals infected with only PRV neutralized SAV, but heat treatment removed this effect. Field studies of co-infected fish populations indicated a negative correlation between the two viruses in randomly sampled apparently healthy fish, in line with the experimental findings, but a positive correlation in moribund or dead fish. The results indicate that the innate antiviral response induced by PRV may temporary protect against a secondary SAV infection., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Uncultivable HPR0 strains of infectious salmon anaemia viruses (ISAVs) infecting gills are non-virulent putative precursors of virulent ISAVs (vISAVs) causing systemic disease in farmed Atlantic salmon (Salmo salar). The transition to virulence involves two molecular events, a deletion in the highly polymorphic region (HPR) of the hemagglutinin-esterase (HE) gene and a Q266→L266 substitution or insertion next to the putative cleavage site (R267) in the fusion protein (F). We have performed ultra-deep pyrosequencing (UDPS) of these gene regions from healthy fish positive for HPR0 virus carrying full-length HPR sampled in a screening program, and a vISAV strain from an ISA outbreak at the same farming site three weeks later, and compared the mutant spectra. As the UDPS data shows the presence of both HE genotypes at both sampling times, and the outbreak strain was unlikely to be directly related to the HPR0 strain, this is the first report of a double infection with HPR0s and vISAVs. For F amplicon reads, mutation frequencies generating L266 codons in screening samples and Q266 codons in outbreak samples were not higher than at any random site. We suggest quasispecies heterogeneity as well as RNA structural properties are linked to transition to virulence. More specifically, a mechanism where selected single point mutations in the full-length HPR alter the RNA structure facilitating single- or sequential deletions in this region is proposed. The data provides stronger support for the deletion hypothesis, as opposed to recombination, as the responsible mechanism for generating the sequence deletions in HE.

Infectious pancreatic necrosis, an important problem of the salmon industry worldwide, is caused by Infectious pancreatic necrosis virus (IPNV). Fish surviving an IPNV infection become virus carriers, and the identification of infected fish is highly relevant to disease control. The target organ for IPNV diagnosis is the kidney, where the virus persists, usually with low virus loads. The current study documents a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assay that proved 100 times more sensitive than a conventional RT-PCR. Cell culture and real-time RT-PCR were compared for their ability to detect IPNV in carrier Atlantic salmon kidney samples after different preservation and storage procedures. Storage of whole tissue at -80°C for 1 month and storage of tissue homogenized in transport medium (TM) at +4°C for 1 week before investigation in cell cultures resulted in a marked reduction of virus infectivity. For detection by real-time RT-PCR, storage of whole tissue was suboptimal, whereas storage of tissue homogenized in TM did not affect virus detection. The results of the present study demonstrate that both cell culture and real-time RT-PCR are reliable tests for the detection of low amounts of IPNV in kidneys of carrier Atlantic salmon, and both methods are relatively robust against minor preservation and storage deviations, or both. Preservation of tissues in RNA stabilization solution seems only necessary when samples are to be shipped at ambient temperatures or when laboratory testing might be delayed. Independent of detection method, these results indicate that for long-term storage, samples are best kept at -80°C after homogenization in TM.

The early interactions between infectious pancreatic necrosis virus (IPNV) from Atlantic salmon and susceptible cell lines were studied using a virus overlay protein binding assay (VOPBA). Membrane preparations from four different cell lines, bluegill fry (BF)-2 cells, Chinook salmon embryo (CHSE)-214 cells, salmon head kidney (SHK)-1 cells and Atlantic salmon kidney (ASK) cells were separated by SDS-PAGE, blotted to nitrocellulose membranes and incubated with either a highly virulent IPNV field isolate or different recombinant IPNV strains exhibiting variations in virulence. Binding of virus to the respective membrane fractions was detected using either polyclonal or monoclonal antibodies. Independent of variations in virulence, IPNV bound in a specific manner to an approximately 220-kDa membrane protein from the salmonid cell lines CHSE-214, SHK-1 and ASK, whereas the size of the binding protein from the non-salmonid cell line BF-2 was approximately 190 kDa. Neutralization of IPNV prior to incubation with the blotted membrane fractions inhibited binding to the indicated proteins. Complete deglycosylation of the membrane proteins did not interfere with the binding of IPNV, suggesting that the interaction between virus and cells is not carbohydrate specific. The described binding proteins may represent putative cellular binding sites or receptors for IPNV.

Infectious pancreatic necrosis virus (IPNV), the causative agent of a highly infectious disease in salmonid fish, encodes a small non-structural protein designated VP5. This protein contains Bcl-2 homologous domains and inhibits apoptosis when expressed in cell culture. We have previously reported the generation of three VP5 mutants of IPNV-Sp serotype, using reverse genetics (Santi, N., Song, H., Vakharia, V.N., Evensen, Ø., 2005. Infectious pancreatic necrosis virus VP5 is dispensable for virulence and persistence. J. Virol. 79 (14), 9206-9216). The wild-type rNVI15 virus encodes a truncated 12-kDa VP5 protein, rNVI15-15K encodes a full-length 15-kDa VP5, whereas rNVI15-DeltaVP5 is deficient in VP5 expression. In the present report, the role of VP5 in apoptosis was assessed both in vitro and in vivo, using the recombinant IPNV strains. Apoptosis was observed in hepatocytes of Atlantic salmon post-smolts challenged with all three VP5 mutant viruses. Using a double-labeling technique to detect apoptotic cells and IPNV antigens, we found that viral antigen and apoptotic cells co-distributed. In addition, numerous double-positive cells were seen. The recombinant viruses also induced apoptosis in infected cell cultures, and the morphology and membrane integrity of infected cells at different time points was similar. In summary, these results indicate that IPNV induces apoptosis in infected cell cultures and in fish, independent of VP5 expression. However, substitutions of putative functionally important amino acids in the BH2 domain of VP5 of IPNV-Sp strains were identified, which might influence the anti-apoptosis effect of the protein, and partly explain the apparent absence of this specific function.

Infectious salmon anemia (ISA) is a disease, caused by an orthomyxovirus, which has considerable economic impact on farming of Atlantic salmon. Here we describe the results of immunization against ISA using plasmids expressing the ISA virus hemagglutinin-esterase (HE). Immunized Atlantic salmon demonstrated moderate protection after challenge with ISA virus, with relative percent survival of 39.5 and 60.5 in two parallel groups. No protection was seen after immunization using a plasmid expressing the ISA virus nucleoprotein. Fish in the HE-immunized group had earlier onset of clearance of the virus than control fish. There was no detectable ISA virus specific humoral response after immunization. After challenge a specific humoral response could be demonstrated in the fish in all groups, but no correlation between this response and protection was found.

The tegument protein pp71 (UL82) of human cytomegalovirus (HCMV) has previously been shown to transactivate the major immediate-early enhancer-promoter of HCMV. Furthermore, this protein is able to enhance the infectivity of viral DNA and to accelerate the infection cycle, suggesting an important regulatory function during viral replication. To gain insight into the underlying mechanisms that are used by pp71 to exert these pleiotropic effects, we sought for cellular factors interacting with pp71 in a yeast two-hybrid screen. Here, we report the isolation of the human Daxx (hDaxx) protein as a specific interaction partner of HCMV pp71. hDaxx, which was initially described as an adapter protein involved in apoptosis regulation, has recently been identified as a nuclear protein that interacts and colocalizes with PML in the nuclear domain ND10. In order to assess whether pp71 can also be detected in ND10 structures, a vector expressing pp71 in fusion with the green fluorescent protein was used for transfection of human fibroblasts. This revealed a colocalization of pp71 with the ND10 proteins PML and Sp100. In addition, cotransfection of a hDaxx expression vector resulted in an enhanced recruitment of pp71 to ND10. Targeting of pp71 to nuclear dots could also be observed in infected human fibroblasts in the absence of de novo viral protein synthesis. Moreover, cotransfection experiments revealed that pp71-mediated transactivation of the major immediate-early enhancer-promoter was synergistically enhanced in the presence of hDaxx. These results suggest an important role of hDaxx for pp71 protein function.

Several studies have shown that human cytomegalovirus (HCMV) induces growth suppression of hematopoietic progenitors. In vitro studies have demonstrated that the HCMV-induced suppression is independent of viral protein production. Previous studies have indicated a link between HCMV infection and apoptosis in human cells. The purpose of our study was to investigate whether the observed inhibitory effect of HCMV on the human myeloid progenitors could be connected to the induction of apoptosis. The growth and cell death of the hematopoietic cell line MO7e was investigated following infection with HCMV virions and dense bodies. Both virions and dense bodies inhibited the growth of MO7e cells, and induced cell death measured by trypan blue staining. In addition, both HCMV virions and dense bodies caused an increased amount of apoptosis-characteristic DNA fragmentation in the MO7e cells compared to mock-treated cells. The HCMV virions were also able to induce an increased expression of phosphatidylserine on the cell surface, which is an early event in the initiation of apoptosis in most cell types. In conclusion, HCMV and HCMV dense bodies are able to induce apoptosis in the myeloid progenitor cell line MO7e.