Phylogeny of the BRSV G gene

Phylogeny showing the relationship among 31 selected nucleotide sequences based on a 472 nt part from nucleotide number 178 to 649 of the G gene of BRSV. The sequences from this study, labelled with “*”, originated from 10 different farms with outbreaks of respiratory disease in Norway between 2009 and 2011, and one isolate from an outbreak in 1976. Included are also representatives of the six genetic subgroups of BRSV and recent published Swedish and Danish strains. The genetic subgroups (I-VI) and the strain type of the Norwegian sequences (A or B) are indicated on the right. The strains are indicated by the isolate name with GenBank Accession numbers AF188585, AF188586, AF188588, AF188604, JN619431, JN619439, JN619446, L27802, M58307, Y08717, Y08718, U92098, U92102, U92103, U24714-U24716, U33539 and U57823. The GenBank accession numbers of the Norwegian strains are: KF501149-KF501155, KF501159, KF501160 and KF501170-KF501172. The bootstrap values are shown next to the branches. The trees are drawn to scale, with branch lengths measured in the number of substitutions per site

Amino acid signatures found mostly or entirely in the internal proteins of Eurasian and American H13 and H16 AIVs from gulls and shorebirds, validated against a comprehensive set of AIV sequences in GenBank.

1<p>Geographical origin of the AIV segments is based on analyses by Wille et al. (2011) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063270#pone.0063270-Wille1" target="_blank">[5]</a>.</p>2<p>Signatures in brackets were only found in one or a few H13 or H16 sequences.</p

Host Restrictions of Avian Influenza Viruses: <i>In Silico</i> Analysis of H13 and H16 Specific Signatures in the Internal Proteins

<div><p>Gulls are the primary hosts of H13 and H16 avian influenza viruses (AIVs). The molecular basis for this host restriction is only partially understood. In this study, amino acid sequences from Eurasian gull H13 and H16 AIVs and Eurasian AIVs (non H13 and H16) were compared to determine if specific signatures are present only in the internal proteins of H13 and H16 AIVs, using a bioinformatics approach. Amino acids identified in an initial analysis performed on 15 selected sequences were checked against a comprehensive set of AIV sequences retrieved from Genbank to verify them as H13 and H16 specific signatures. Analysis of protein similarities and prediction of subcellular localization signals were performed to search for possible functions associated with the confirmed signatures. H13 and H16 AIV specific signatures were found in all the internal proteins examined, but most were found in the non-structural protein 1 (NS1) and in the nucleoprotein. A putative functional signature was predicted to be present in the nuclear export protein. Moreover, it was predicted that the NS1 of H13 and H16 AIVs lack one of the nuclear localization signals present in NS1 of other AIV subtypes. These findings suggest that the signatures found in the internal proteins of H13 and H16 viruses are possibly related to host restriction.</p></div

Table_1_Transcriptomics of early responses to purified Piscine orthoreovirus-1 in Atlantic salmon (Salmo salar L.) red blood cells compared to non-susceptible cell lines.xlsx

Piscine red blood cells (RBC) are nucleated and have been characterized as mediators of immune responses in addition to their role in gas exchange. Salmonid RBC are major target cells of Piscine orthoreovirus-1 (PRV-1), the etiological agent of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). PRV-1 replicates in RBC ex vivo, but no viral amplification has been possible in available A. salmon cell lines. To compare RBC basal transcripts and transcriptional responses to PRV-1 in the early phase of infection with non-susceptible cells, we exposed A. salmon RBC, Atlantic salmon kidney cells (ASK) and Salmon head kidney cells (SHK-1) to PRV-1 for 24 h. The RNA-seq analysis of RBC supported their previous characterization as pluripotent cells, as they expressed a wide repertoire of genes encoding pattern recognition receptors (PRRs), cytokine receptors, and genes implicated in antiviral activities. The comparison of RBC to ASK and SHK-1 revealed immune cell features exclusively expressed in RBC, such as genes involved in chemotactic activity in response to inflammation. Differential expression analysis of RBC exposed to PRV-1 showed 46 significantly induced genes (≥ 2-fold upregulation) linked to the antiviral response pathway, including RNA-specific PRRs and interferon (IFN) response factors. In SHK-1, PRV induced a more potent or faster antiviral response (213 genes induced). ASK cells showed a differential response pattern (12 genes induced, 18 suppressed) less characterized by the dsRNA-induced antiviral pathway. Despite these differences, the RIG-I-like receptor 3 (RLR3) in the family of cytosolic dsRNA receptors was significantly induced in all PRV-1 exposed cells. IFN regulatory factor 1 (IRF1) was significantly induced in RBC only, in contrast to IRF3/IRF7 induced in SHK-1. Differences in IRF expression and activity may potentially affect viral propagation.</p

Table_3_Transcriptomics of early responses to purified Piscine orthoreovirus-1 in Atlantic salmon (Salmo salar L.) red blood cells compared to non-susceptible cell lines.xlsx

Piscine red blood cells (RBC) are nucleated and have been characterized as mediators of immune responses in addition to their role in gas exchange. Salmonid RBC are major target cells of Piscine orthoreovirus-1 (PRV-1), the etiological agent of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). PRV-1 replicates in RBC ex vivo, but no viral amplification has been possible in available A. salmon cell lines. To compare RBC basal transcripts and transcriptional responses to PRV-1 in the early phase of infection with non-susceptible cells, we exposed A. salmon RBC, Atlantic salmon kidney cells (ASK) and Salmon head kidney cells (SHK-1) to PRV-1 for 24 h. The RNA-seq analysis of RBC supported their previous characterization as pluripotent cells, as they expressed a wide repertoire of genes encoding pattern recognition receptors (PRRs), cytokine receptors, and genes implicated in antiviral activities. The comparison of RBC to ASK and SHK-1 revealed immune cell features exclusively expressed in RBC, such as genes involved in chemotactic activity in response to inflammation. Differential expression analysis of RBC exposed to PRV-1 showed 46 significantly induced genes (≥ 2-fold upregulation) linked to the antiviral response pathway, including RNA-specific PRRs and interferon (IFN) response factors. In SHK-1, PRV induced a more potent or faster antiviral response (213 genes induced). ASK cells showed a differential response pattern (12 genes induced, 18 suppressed) less characterized by the dsRNA-induced antiviral pathway. Despite these differences, the RIG-I-like receptor 3 (RLR3) in the family of cytosolic dsRNA receptors was significantly induced in all PRV-1 exposed cells. IFN regulatory factor 1 (IRF1) was significantly induced in RBC only, in contrast to IRF3/IRF7 induced in SHK-1. Differences in IRF expression and activity may potentially affect viral propagation.</p

MOESM1 of Piscine orthoreovirus infection in Atlantic salmon (Salmo salar) protects against subsequent challenge with infectious hematopoietic necrosis virus (IHNV)

Additional file 1. Grading of histopathological changes related to development of HSMI in A. salmon heart. This table displays histopathological grading of HSMI lesions in heart sections of A. salmon H&E stained. Median value per time point of each group is calculated

Table_2_Transcriptomics of early responses to purified Piscine orthoreovirus-1 in Atlantic salmon (Salmo salar L.) red blood cells compared to non-susceptible cell lines.xlsx

Piscine red blood cells (RBC) are nucleated and have been characterized as mediators of immune responses in addition to their role in gas exchange. Salmonid RBC are major target cells of Piscine orthoreovirus-1 (PRV-1), the etiological agent of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). PRV-1 replicates in RBC ex vivo, but no viral amplification has been possible in available A. salmon cell lines. To compare RBC basal transcripts and transcriptional responses to PRV-1 in the early phase of infection with non-susceptible cells, we exposed A. salmon RBC, Atlantic salmon kidney cells (ASK) and Salmon head kidney cells (SHK-1) to PRV-1 for 24 h. The RNA-seq analysis of RBC supported their previous characterization as pluripotent cells, as they expressed a wide repertoire of genes encoding pattern recognition receptors (PRRs), cytokine receptors, and genes implicated in antiviral activities. The comparison of RBC to ASK and SHK-1 revealed immune cell features exclusively expressed in RBC, such as genes involved in chemotactic activity in response to inflammation. Differential expression analysis of RBC exposed to PRV-1 showed 46 significantly induced genes (≥ 2-fold upregulation) linked to the antiviral response pathway, including RNA-specific PRRs and interferon (IFN) response factors. In SHK-1, PRV induced a more potent or faster antiviral response (213 genes induced). ASK cells showed a differential response pattern (12 genes induced, 18 suppressed) less characterized by the dsRNA-induced antiviral pathway. Despite these differences, the RIG-I-like receptor 3 (RLR3) in the family of cytosolic dsRNA receptors was significantly induced in all PRV-1 exposed cells. IFN regulatory factor 1 (IRF1) was significantly induced in RBC only, in contrast to IRF3/IRF7 induced in SHK-1. Differences in IRF expression and activity may potentially affect viral propagation.</p

DataSheet_1_Transcriptomics of early responses to purified Piscine orthoreovirus-1 in Atlantic salmon (Salmo salar L.) red blood cells compared to non-susceptible cell lines.docx

Piscine red blood cells (RBC) are nucleated and have been characterized as mediators of immune responses in addition to their role in gas exchange. Salmonid RBC are major target cells of Piscine orthoreovirus-1 (PRV-1), the etiological agent of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). PRV-1 replicates in RBC ex vivo, but no viral amplification has been possible in available A. salmon cell lines. To compare RBC basal transcripts and transcriptional responses to PRV-1 in the early phase of infection with non-susceptible cells, we exposed A. salmon RBC, Atlantic salmon kidney cells (ASK) and Salmon head kidney cells (SHK-1) to PRV-1 for 24 h. The RNA-seq analysis of RBC supported their previous characterization as pluripotent cells, as they expressed a wide repertoire of genes encoding pattern recognition receptors (PRRs), cytokine receptors, and genes implicated in antiviral activities. The comparison of RBC to ASK and SHK-1 revealed immune cell features exclusively expressed in RBC, such as genes involved in chemotactic activity in response to inflammation. Differential expression analysis of RBC exposed to PRV-1 showed 46 significantly induced genes (≥ 2-fold upregulation) linked to the antiviral response pathway, including RNA-specific PRRs and interferon (IFN) response factors. In SHK-1, PRV induced a more potent or faster antiviral response (213 genes induced). ASK cells showed a differential response pattern (12 genes induced, 18 suppressed) less characterized by the dsRNA-induced antiviral pathway. Despite these differences, the RIG-I-like receptor 3 (RLR3) in the family of cytosolic dsRNA receptors was significantly induced in all PRV-1 exposed cells. IFN regulatory factor 1 (IRF1) was significantly induced in RBC only, in contrast to IRF3/IRF7 induced in SHK-1. Differences in IRF expression and activity may potentially affect viral propagation.</p

PRV RNA levels in blood and heart.

<p>PRV RNA levels (Ct values) in blood (A) and heart (B) from naïve fish sampled at Day 0 (white dots), non-infected controls (Ctrl, grey dots), PRV-infected (PRV, black dots) and PRV-infected fish exposed to periodic hypoxic stress (PRV-H, red dots), at each time-point during the infection trial. Weeks post-infection (WPI) are indicated on the x-axis. Ct value ≥ 37.0 indicates no virus RNA detected. A non-parametric Mann-Whitney unpaired rank test was performed between the groups at all time-points detecting no significant differences between the groups (<i>p</i> > 0.05).</p

Hypoxia tolerance and responses to hypoxic stress during heart and skeletal muscle inflammation in Atlantic salmon (<i>Salmo salar</i>)

<div><p>Heart and skeletal muscle inflammation (HSMI) is associated with <i>Piscine orthoreovirus</i> (PRV) infection and is an important disease in Atlantic salmon (<i>Salmo salar)</i> aquaculture. Since PRV infects erythrocytes and farmed salmon frequently experience environmental hypoxia, the current study examined mutual effects of PRV infection and hypoxia on pathogenesis and fish performance. Furthermore, effects of HSMI on hypoxia tolerance, cardiorespiratory performance and blood oxygen transport were studied. A cohabitation trial including PRV-infected post-smolts exposed to periodic hypoxic stress (4 h of 40% O₂; PRV-H) at 4, 7 and 10 weeks post-infection (WPI) and infected fish reared under normoxic conditions (PRV) was conducted. Periodic hypoxic stress did not influence infection levels or histopathological changes in the heart. Individual incipient lethal oxygen saturation (ILOS) was examined using a standardized hypoxia challenge test (HCT). At 7 WPI, i.e. peak level of infection, both PRV and PRV-H groups exhibited reduced hypoxia tolerance compared to non-infected fish. Three weeks later (10 WPI), during peak levels of pathological changes, reduced hypoxia tolerance was still observed for the PRV group while PRV-H performed equal to non-infected fish, implying a positive effect of the repeated exposure to hypoxic stress. This was in line with maximum heart rate (<i>f</i>_Hmax) measurements, showing equal performance of PRV-H and non-infected groups, but lower <i>f</i>_Hmax above 19°C as well as lower temperature optimum (<i>T</i>_opt) for aerobic scope for PRV, suggesting reduced cardiac performance and thermal tolerance. In contrast, the PRV-H group had reduced hemoglobin-oxygen affinity compared to non-infected fish. In conclusion, Atlantic salmon suffering from HSMI have reduced hypoxia tolerance and cardiac performance, which can be improved by preconditioning fish to transient hypoxic stress episodes.</p></div