Emergence of Salmonid Alphavirus Genotype 2 in Norway—Molecular Characterization of Viral Strains Circulating in Norway and Scotland

Publication history: Accepted - 29 July 2021; Published online - 6 August 2021.Pancreas disease (PD) and sleeping disease (SD), caused by an alphavirus, are endemic in European salmonid aquaculture, causing significant mortality, reduced growth and poor flesh quality. In 2010, a new variant of salmonid alphavirus emerged in Norway, marine salmonid alphavirus genotype 2 (SAV2). As this genotype is highly prevalent in Scotland, transmission through well boat traffic was hypothesized as one possible source of infection. In this study, we performed full-length genome sequencing of SAV2 sampled between 2006 and 2012 in Norway and Scotland, and present the first comprehensive full-length characterization of Norwegian marine SAV2 strains. We analyze their relationship with selected Scottish SAV2 strains and explore the genetic diversity of SAV. Our results show that all Norwegian marine SAV2 share a recent last common ancestor with marine SAV2 circulating in Scotland and a higher level of genomic diversity among the Scottish marine SAV2 strains compared to strains from Norway. These findings support the hypothesis of a single introduction of SAV2 to Norway sometime from 2006–2010, followed by horizontal spread along the coast.This research was funded by Norwegian Seafood Research Fund (FHF) grant 90079

Atlantic Salmon Reovirus Infection Causes a CD8 T Cell Myocarditis in Atlantic Salmon (Salmo salar L.)

Heart and skeletal inflammation (HSMI) of farmed Atlantic salmon (Salmo salar L.) is a disease characterized by a chronic myocarditis involving the epicardium and the compact and spongious part of the heart ventricle. Chronic myositis of the red skeletal muscle is also a typical finding of HSMI. Piscine reovirus (PRV) has been detected by real-time PCR from farmed and wild salmon with and without typical changes of HSMI and thus the causal relationship between presence of virus and the disease has not been fully determined [1]. In this study we show that the Atlantic salmon reovirus (ASRV), identical to PRV, can be passaged in GF-1 cells and experimental challenge of naïve Atlantic salmon with cell culture passaged reovirus results in cardiac and skeletal muscle pathology typical of HSMI with onset of pathology from 6 weeks, peaking by 9 weeks post challenge. ASRV replicates in heart tissue and the peak level of virus replication coincides with peak of heart lesions. We further demonstrate mRNA transcript assessment and in situ characterization that challenged fish develop a CD8+ T cell myocarditis

Characterization of a novel calicivirus causing systemic infection in atlantic salmon (Salmo salar L.): proposal for a new genus of caliciviridae.

The Caliciviridae is a family of viruses infecting humans, a wide range of animals, birds and marine fish and mammals, resulting in a wide spectrum of diseases. We describe the identification and genetic characterization of a novel calicivirus replicating in Atlantic salmon. The virus has a high prevalence in farmed salmon and is found in fish suffering from several diseases and conditions and also in presumable healthy fish. A challenge and vaccination trial shows that the calicivirus replicates in Atlantic salmon and establishes a systemic infection, which can be reduced by vaccination with formalin-inactivated virus preparation. The virus, named Atlantic salmon calicivirus (ASCV), is found in two genetically distinct variants, a cell culture isolated and a variant sequenced directly from field material. The genomes are 7,4 kb and contain two open reading frames where typical conserved amino acid motifs and domains predict a gene order reminiscent of calicivirus genomes. Phylogenetic analysis performed on extracted capsid amino acid sequences segregated the two ASCV variants in a unique cluster sharing root with the branch of noroviruses infecting humans and the unassigned Tulane virus and St-Valérien like viruses, infecting rhesus monkey and pig, respectively, with relatively large distance to the marine calicivirus subgroup of vesiviruses. Based on the analyses presented, the ASCV is predicted to represent a new genus of Caliciviridae for which we propose the name Salovirus

Expression of virus genome and Mx at different time post challenge.

<p>Relative expression of ASRV and Mx following injection challenge from 4 to 10 weeks post challenge (wpc). a) Relative replication level (log₁₀) of ASRV in heart specimens was increased by 6 wpc and peaked at 9 wpc, which was the time point significantly different from all other samplings. b) Mx mRNA expression in heart tissue show peak expression at same time as virus replication. Different letters denote significant difference (p<0.05).</p

CPE in GF-1 cells.

<p>GF-1 cell cultures infected with sterile filtered heart tissue homogenate supernatant collected from a) a clinical outbreak of HSMI and b) from non-related healthy salmon (controls).</p

Immunohistochemistry for CD3 and CD8 positive cells.

<p>In situ detection of CD3- and CD8-positive T cells infiltrating the epicardium and underlying compact layer at 9 and 10 weeks post challenge. Positive cells are displayed with reddish/brownish color. All sections are counterstained with Mayer’s hematoxylin. A) CD3-positive cells infiltrating the epicardium, 9 WPC; B) CD3 positive cells with infiltration of the epicardium (top) and in the compact layer of the myocardium, 10 WPC; C) Overview of CD8-positive cells of the compact layer of the myocard (C) and epicardium (E), 9 WPC; D) Detail of CD8-positive cells infiltrating into the compact layer of the epicardium and into compact layer. Large proportion of lymphocytis cells are CD8-positive, 9 WPC. Original magnifications; x2.5 (A) and x40 (B–D).</p

Histopathological changes in heart.

<p>Sequential histopathological of heart epicardium and the compactum of the ventricle at 4 to 10 weeks post challenge (WPC). All sections are counterstained with hematoxylin and eosin. A) 4 WPC with normal thin, epicardium (E) and normal compact layer (C) of heart ventricle; B) 7 WPC. Infiltration with lymphocytic cells in the epicardium located around small blood vessels (BV); C) 9 WPC. Epicardial infiltration extending into underlying compactum (arrow); D) 10 WPC. Epicardium (right) with infiltration of inflammatory cells along small vascular structures (arrows); E) 9 WPC and focal, infiltration of inflammatory cells in compactum of the heart ventricle; and F) 10 WPC showing myocardial necrosis (arrow) and moderate inflammation. x10 (A) and x40 (B–F), original magnification.</p

Histoscores at different time post challenge.

<p>Inflammation score following injection challenge examined at 0 to 10 weeks post challenge (wpc). Scores are 0 = no pathological changes, 1 = mild pathological changes, 2 = moderate pathological changes, 3 = severe pathological changes (for details, see text). Asterisk (*) indicates significant difference (p<0.05) to results from sampling at time 0 (all negative).</p