Effect of mutations upstream of the proteolytic cleavage site.

<p>Fusion activities of five mutated F proteins co-expressed with the WT HE-SK779/06 were measured with a content mixing assay (A). Proteolytic cleavage was examined by western blot (B). Blot intensity was assessed using the software Image Studio Digits 4.0 and results expressed as percentages relative to the WT F protein. Percentages illustrate the effect of F protein mutations on the proteolytic cleavage. (_***) corresponds to P < 0.001.</p

Fusion activity of HE and F-Nevis protein with addition of calcium ionophore A23187 to the culture media.

<p>Fusion activity of the WT HE and F-Nevis protein was measured as described before in the presence of various concentrations of calcium ionophore A23187 in calcium free (A) and calcium containing media (B) to examine if the activating protease was calcium dependent.</p

Quantification of glycoprotein surface expression.

<p>The expression levels of each mutant glycoprotein were measured using dual antibody staining on living cells, fluorescent microscopy and image analysis. Results included measurements from cell monolayers. They were obtained from triplicate measurements, from three separate experiments and expressed as intensity mean values for green and red pixels. Expression levels of each mutated protein was compared with that of the corresponding wild type (WT) protein (in bold). Differences in surface expression levels were not statistically significant (P > 0.05).</p

Primers used to generate a deletion and mutations in the SK779/06 HE and F proteins respectively.

<p>Mutated codons are in bold and underlined in each DNA sequence. This table also provides information on the two sequences which were synthesised by Geneart (Invitrogen) and inserted in the F-SK779/06 (in bold and underlined). The position of the HPR deletion generated in the HE is also presented in the first row.</p

Schematic illustration of the structure of ISAV surface glycoproteins.

<p>The F protein contains the predicted transmembrane domain (TM), heptad repeat (HR) and fusion peptide (FP) which is also highlighted in grey in the sequence (A). The HE contains a Highly Polymorphic Region (HPR) (B). HRs positions were predicted using the program LearnCoil-VMF (<a href="http://cis.poly.edu/~jps/matcher.html" target="_blank">http://cis.poly.edu/~jps/matcher.html</a>) (data no shown). Arrows indicate important aa positions in the proteins, while substitutions and insertions introduced into the mutant F proteins (A) and the deletion in the mutant HE (B) are in bold and underlined.</p

Fusion activity and proteolytic cleavage of mutant HE and F protein combinations under different culture conditions.

<p>Fusion activities of combinations of glycoproteins containing either a full length HPR HE (A and C, grey bars) or a deleted HPR HE (A and C, white bars) co-expressed with mutated F proteins were measured with a content mixing assay under two culture conditions: with trypsin and low pH treatments applied to the cell monolayer (A) and under normal culture conditions (C). Proteolytic cleavage of mutant HE and F proteins under the two same culture conditions was examined by western blot (B and D). Percentages under each pair of blots were obtained by comparing the blot intensity of the group containing a deleted HPR HE and the one from the WT HE SK779/06 with a full length HPR. Therefore, percentages illustrate the effect of the HPR deletion on the proteolytic cleavage. (_***) corresponds to P < 0.001 and (_**) to P < 0.01.</p

Primers used to generate aa substitutions in the Nevis F protein.

<p>These mutations aimed at identifying the cleavage site from the two proposed. Mutated codons are in bold and underlined in each DNA sequence.</p

Infection with purified <i>Piscine orthoreovirus</i> demonstrates a causal relationship with heart and skeletal muscle inflammation in Atlantic salmon

<div><p>Viral diseases pose a significant threat to the productivity in aquaculture. Heart- and skeletal muscle inflammation (HSMI) is an emerging disease in Atlantic salmon (<i>Salmo salar</i>) farming. HSMI is associated with <i>Piscine orthoreovirus</i> (PRV) infection, but PRV is ubiquitous in farmed Atlantic salmon and thus present also in apparently healthy individuals. This has brought speculations if additional etiological factors are required, and experiments focusing on the causal relationship between PRV and HSMI are highly warranted. A major bottleneck in PRV research has been the lack of cell lines that allow propagation of the virus. To bypass this, we propagated PRV in salmon, bled the fish at the peak of the infection, and purified virus particles from blood cells. Electron microscopy, western blot and high-throughput sequencing all verified the purity of the viral particles. Purified PRV particles were inoculated into naïve Atlantic salmon. The purified virus replicated in inoculated fish, spread to naïve cohabitants, and induced histopathological changes consistent with HSMI. PRV specific staining was demonstrated in the pathological lesions. A dose-dependent response was observed; a high dose of virus gave earlier peak of the viral load and development of histopathological changes compared to a lower dose, but no difference in the severity of the disease. The experiment demonstrated that PRV can be purified from blood cells, and that PRV is the etiological agent of HSMI in Atlantic salmon.</p></div

Summary cohabitant group.

<p>(A) PRV RNA in blood cells and heart measured by RT-qPCR, shown as individual and mean Ct-values from 2 to 8 weeks post challenge (wpc). (B) Amount of σ1- and μNS -protein measured by flow cytometry, shown as mean fluorescence intensity (MFI) for individual fish and group mean. (C) Histopathological score in the heart (total cardiac score) and red skeletal muscle shown as individual and group mean from 4 to 8 wpc. (D) Light microscopic images at 8 wpc in PRV-Low; (D) Heart lesions in the ventrical including severe epicarditis (arrowhead) and inflammation in the compact (*) and spongy myocardium (**) consistent with HSMI. (E) Mild inflammation in the red skeletal muscle (arrowhead), presence of melanin (*). Scale bar 20 μm. Color coding: PRV-High (red), PRV-Low (green), positive control (black) and negative control (grey). n = 6.</p

Primers and probe used in this study.

<p>Primers and probe used in this study.</p