Piscine Orthoreovirus: HSMI Reference

Overview and Taxonomy of Piscine Orthoreovirus: HSMI Reference

Piscine orthoreovirus (PRV) represents a highly significant emerging pathogen within the family Reoviridae, constituting a distinct and unambiguous new genus that has fundamentally altered the virological landscape of global salmonid aquaculture [12, 32]. Since its initial molecular characterization in association with heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar) during the early 2010s, PRV has been the subject of intensive investigation, revealing a complex taxonomy comprising three distinct genotypes (PRV-1, PRV-2, and PRV-3) that exhibit striking host species specificity, differential pathogenicity, and a remarkable capacity for long-term persistence within infected populations [4, 11, 27]. The virus is recognized by the World Organisation for Animal Health (WOAH) as an economically critical pathogen of salmonids, and its international distribution pattern, coupled with its high environmental stability, has raised substantial concerns regarding transboundary disease spread, particularly into regions with nascent or expanding salmon aquaculture industries [24, 34, 35].

Taxonomic Position and Genomic Architecture

Phylogenetic analyses unequivocally demonstrate that PRV constitutes a deeply branched lineage within the subfamily Spinareovirinae, sharing a common ancestor with members of the genera Orthoreovirus and Aquareovirus but displaying sufficient genetic divergence to warrant classification as a separate, novel genus [12, 32]. Early comparative genomic studies by Markussen et al. [12] provided the foundational sequence analysis of the PRV genome, demonstrating that while amino acid identities between PRV and prototype mammalian orthoreoviruses (MRV T3D) or aquareoviruses (GCRV-873) are generally low for most gene segments, numerous critical protein motifs and key functional amino acid residues are evolutionarily conserved. Specifically, the inner core-forming proteins λ1 and σ2 exhibit predicted structural similarities suggesting a conserved core architecture, while the outer capsid proteins μ1, σ1, and σ3 display markedly lower amino acid identities, indicating substantial divergence in mechanisms of cellular interaction and host tropism [12]. This genomic architecture comprises ten discrete segments of double-stranded RNA (dsRNA), classified by size into three large (L1, L2, L3), three medium (M1, M2, M3), and four small (S1, S2, S3, S4) segments, each encoding at least one primary open reading frame (ORF) [12, 27]. Critically, the genome demonstrates polycistronic potential, with the S1 segment encoding not only the outer capsid protein σ3 but also an additional 124-amino-acid protein (p13) localized to intracellular Golgi-like structures, while the S2 and L2 segments are predicted to encode small accessory proteins of 71 (p8) and 98 (p11) amino acids, respectively [12]. This genomic complexity, particularly the presence of these auxiliary ORFs, distinguishes PRV from typical aquareoviruses and aligns it more closely with the orthoreovirus lineage, supporting the designation of a new genus within the family Reoviridae [12, 32].

The virion architecture, inferred from electron microscopic studies and sequence-based structural predictions, reveals a non-enveloped, icosahedral particle approximately 70-80 nm in diameter, characterized by a double-layered capsid that is typical of reoviruses [12, 18, 30]. The outer capsid is composed of three major proteins: μ1, which undergoes proteolytic cleavage and myristoylation, processes essential for endosomal membrane penetration during infection, with conserved cleavage sites identified in the homologous PRV protein; σ1, the cell-attachment protein that mediates viral binding to sialic acid residues on host cells, with critical amino acid residues for receptor binding being partially or wholly conserved; and σ3, which contains a conserved zinc finger motif implicated in dsRNA-binding and modulation of the host interferon response [12]. The inner core, comprising λ1, λ2, and σ2, houses the viral RNA-dependent RNA polymerase (λ3) and the guanylyltransferase (μ2), which are central to viral transcription and replication within cytoplasmic inclusion bodies, or "viral factories" [12, 22, 25]. The multifunctional μ2 protein is of particular note, as in mammalian orthoreoviruses it plays a pivotal role in the formation and structural organization of viral inclusion bodies and can modulate interferon-β signaling and the induction of myocarditis, a conserved function that may have direct relevance to the pathogenesis of HSMI [12].

Genotypic Classification and Phylogenetic Relationships

The taxonomic framework for PRV has evolved considerably since its initial description, and the current classification recognizes three distinct genotypes, PRV-1, PRV-2, and PRV-3, which are differentiated primarily by host species preference, disease phenotype, and phylogenetic analysis of the S1 genome segment [4, 11, 20, 27]. Genotype 1 (PRV-1) is the most extensively characterized and is the etiological agent of HSMI in Atlantic salmon, representing the archetypal member of the genus [1, 13, 18, 19]. Within PRV-1, whole-genome and S1-based phylogenetic analyses have resolved two major sub-genotypes, designated Ia and Ib, which exhibit distinctive geographic distributions and are associated with differential virulence profiles [19, 32]. Sub-genotype Ia is predominantly found in Norway, Canada, and the North Atlantic region, while sub-genotype Ib is more prevalent in Chile and certain European populations [26, 29, 32]. A landmark study by Kibenge et al. [32] performed whole-genome analysis on PRV isolates from both western Canada and Chile, establishing that the Canadian PRV strains matched sub-genotype Ia and Chilean PRV strains matched sub-genotype Ib. Molecular clock analyses suggested that Canadian PRV diverged from Norwegian sub-genotype Ia around 2007 ± 1, while Chilean PRV diverged from Norwegian sub-genotype Ib around 2008 ± 1, indicating relatively recent introductions into these regions through aquaculture translocation [32].

Genotype 2 (PRV-2) was first molecularly characterized by Takano et al. [27] in coho salmon (Oncorhynchus kisutch) suffering from erythrocytic inclusion body syndrome (EIBS) in Japan. Full-genome sequencing revealed that PRV-2 possesses the same ten-segment dsRNA genome organization as PRV-1, but phylogenetic analyses based on the S1 and λ3 genes demonstrated that it forms a distinct, deeply branching cluster clearly separated from PRV-1 [27]. The nucleotide identity between PRV-1 and PRV-2 across coding regions is approximately 80%, with the encoded amino acid identities ranging from 96.7% for the highly conserved λ1 core protein to as low as 79.1% for the outer capsid protein σ3 [20, 27]. This genetic distance, combined with the distinct clinical presentation of EIBS, characterized by severe anemia and the formation of cytoplasmic inclusion bodies in erythrocytes, supports the designation of PRV-2 as a separate viral species within the genus [27].

Genotype 3 (PRV-3) was initially detected in association with an HSMI-like disease in farmed rainbow trout (Oncorhynchus mykiss) in Norway in 2013, with subsequent identification in Denmark, where retrospective testing of archived material confirmed its presence since at least 1995 [5, 10, 16]. The molecular characterization by Dhamotharan et al. [20] demonstrated that the complete coding sequences of PRV-3 share approximately 80.1% nucleotide identity with PRV-1, and the amino acid identities of the predicted proteins range from 96.7% for λ1 to 79.1% for σ3, mirroring the divergence seen between PRV-1 and PRV-2. Phylogenetic analysis unequivocally placed PRV-3 in a separate cluster, equidistant from both PRV-1 and PRV-2 [20]. Despite these genetic differences, rabbit antisera raised against purified PRV-1 or recombinant PRV-1 proteins cross-react with PRV-3, indicating that several antigenic and structural properties are conserved between the two genotypes, a finding with significant implications for serological surveillance and vaccine development [20].

Host Specificity and Differential Pathogenicity

The three PRV genotypes exhibit a well-defined pattern of host species specificity and disease association, which is a central feature of their taxonomy and epidemiology [4, 11, 23]. PRV-1 primarily infects and causes disease in Atlantic salmon, although it can also infect other salmonid species, including Chinook salmon (Oncorhynchus tshawytscha), coho salmon, rainbow trout, and brown trout (Salmo trutta), albeit with variable outcomes [7, 15, 17, 28]. Critically, experimental challenges have demonstrated that while PRV-1 can replicate in Pacific salmonid species, it does not induce the severe cardiac pathology characteristic of HSMI in these hosts, indicating that host-specific factors, including differences in immune recognition and viral receptor expression, are key determinants of disease outcome [15, 28]. In contrast, PRV-2 is specifically associated with EIBS in coho salmon and Chinook salmon, a disease characterized by erythrocytic inclusion bodies, hemolytic anemia, and jaundice, and has not been consistently associated with the myocarditis that defines HSMI [27]. PRV-3 displays a preference for rainbow trout, causing an HSMI-like disease with heart and red muscle inflammation, anemia, and, in some cases, jaundice syndrome [5, 16, 23]. Atlantic salmon can be infected with PRV-3 but typically remain clinically unaffected, although PRV-3 infection in this species can induce cross-protective immunity against subsequent PRV-1 challenge and HSMI development [4, 11].

The molecular basis for this host species specificity is incompletely understood but is believed to involve interactions between the viral attachment protein σ1 and sialic acid-containing receptors on host erythrocytes, as well as differences in the capacity of the virus to modulate host antiviral responses [2, 4, 12]. Transcriptomic analyses by Tsoulia et al. [4] revealed that PRV-1 and PRV-3 both replicate efficiently in Atlantic salmon blood cells and induce the typical innate antiviral responses triggered by dsRNA viruses. However, PRV-3 elicited a significantly stronger and more rapid antiviral response than PRV-1 at two weeks post-infection, despite similar levels of viral RNA replication, suggesting that the timing and magnitude of early innate immune activation are critical determinants of viral dissemination and disease progression [4]. This difference in the kinetics of antiviral responses may provide PRV-1 with an evolutionary advantage in Atlantic salmon, facilitating its dissemination to cardiac tissue, a critical step in the pathogenesis of HSMI [4].

Evolutionary Dynamics and the Emergence of Virulent Lineages

One of the most compelling aspects of PRV biology is the evidence that the virus has undergone significant evolutionary change, particularly within the PRV-1 lineage, that is temporally linked to the emergence of HSMI as a disease entity [13, 19]. Heart and skeletal muscle inflammation was first diagnosed in Norwegian farmed Atlantic salmon in 1999, yet retrospective analyses have demonstrated that PRV has been present in Norwegian aquaculture since at least the late 1980s, and in Chile since at least 1994, with the latter identified through archaeovirological examination of formalin-fixed, paraffin-embedded heart tissues [9, 13, 19]. This temporal disconnect between viral presence and disease emergence strongly suggests that ancestral PRV-1 strains were of low virulence and that genetic changes occurring over time led to the evolution of more pathogenic variants [13, 19].

A landmark study by Dhamotharan et al. [19] performed phylogenomic analyses on 31 PRV-1 genomes collected over a 30-year period from fish with known disease status, grouping the viral sequences into two monophyletic clusters: one significantly associated with HSMI and the other with low-virulent isolates. A PRV-1 strain from Norway sampled in 1988, a decade before the first HSMI outbreak, grouped within the low-virulent cluster, providing direct evidence for the temporal evolution of virulence [19]. The two clusters were most clearly resolved for segments S1 and M2, with only a limited number of amino acid substitutions unique to the HSMI-associated cluster, all mapping to the proteins encoded by these two segments, σ3 and μ2, respectively [19]. The co-evolution of the S1-M2 pair coincided temporally with the emergence of HSMI in Norway, potentially arising through the accumulation of mutations or segment reassortment events, and this highly virulent genotype has remained remarkably stable in Norwegian aquaculture since approximately 1997 [19]. In contrast, PRV-1 strains from the North American Pacific Coast and the Faroe Islands have not undergone this evolutionary transition and remain more closely related to the ancestral, low-virulence PRV-1 strains [13, 14, 19, 28].

Experimental confirmation of these virulence differences was provided by Wessel et al. [13], who performed a dose-standardized challenge trial comparing six PRV-1 isolates: two Norwegian field isolates from 2018, three historical Norwegian isolates predating HSMI, and one Canadian isolate. The Norwegian 2018 isolates induced lower viral protein loads in blood cells but significantly higher plasma viremia, followed by the development of histopathological lesions consistent with HSMI. In stark contrast, all three historical Norwegian isolates and the Canadian isolate induced only mild cardiac lesions that did not meet the diagnostic criteria for HSMI [13]. This phenotypic difference was unequivocally linked to viral proteins encoded by segments S1, M2, L1, L2, and S4, providing the first definitive evidence that PRV-1 isolates differ in their intrinsic virulence and that these differences have a genetic basis [1, 13]. Subsequent studies by Vatne et al. [1] extended this work by sequencing 37 Norwegian PRV-1 isolates and identifying eight distinct genogroups based on combinations of the five putatively virulence-associated segments. Two groups were classified as high-virulent and two as low-virulent based on comparison with reference isolates, with the remaining four groups designated as of unknown virulence. Notably, the geographic distribution revealed a higher frequency of high-virulent isolates in mid- and northern Norway, regions that have historically experienced the highest incidence of HSMI outbreaks [1, 33].

Geographic Distribution and Global Dissemination

Piscine orthoreovirus has achieved a global distribution that mirrors the international trade and translocation of live salmonids and their products, rendering it a truly transboundary pathogen of major economic significance [9, 10, 21, 26, 32, 34, 35]. The virus has been detected in virtually all major salmon-producing regions, including Norway, Scotland, Ireland, Iceland, the Faroe Islands, Canada (both Atlantic and Pacific coasts), the United States (Washington and Alaska), Chile, Japan, and Denmark [5, 9, 10, 21, 26, 27, 31, 34, 35]. The mechanisms of introduction and dissemination include the movement of live fish (particularly smolts from hatcheries), the transport of infected ova and milt (with evidence suggesting the possibility of vertical transmission), the use of well-boats for transport, and the natural migration of wild anadromous salmonids that can act as reservoirs [3, 6, 8, 21].

The situation in Chile is particularly illustrative of the global spread of PRV. The first HSMI outbreak in Chilean farmed Atlantic salmon was reported in 2011, and subsequent phylogenetic analysis grouped Chilean PRV-1 isolates within sub-genotype Ib [26]. However, Rozas-Serri et al. [9] performed a retrospective archaeovirological study on formalin-fixed, paraffin-embedded Atlantic salmon heart tissues from Chilean aquaculture dating back to 1992-1999, and detected PRV-1 RNA in a sample from 1994. This finding demonstrates that PRV-1 was present in Chile for at least 17 years before the first clinical HSMI outbreak, and the virus identified was a low-virulence genogroup, likely introduced through the importation of infected eggs or broodstock [9]. This temporal pattern, viral presence long preceding disease emergence, is remarkably similar to that observed in Norway, supporting the hypothesis that

Molecular Pathogenesis of PRV-1 and Induction of Heart and Skeletal Muscle Inflammation

The molecular pathogenesis of Piscine orthoreovirus genotype 1 (PRV-1) and its progression to heart and skeletal muscle inflammation (HSMI) in Atlantic salmon (Salmo salar) represents a paradigm of host–virus co-evolution where viral genetic determinants, cellular tropism, and immune dynamics converge to produce a disease of significant economic consequence for global salmon aquaculture. HSMI is classified as a notifiable disease on list 3 by the Norwegian Food Safety Authority, and the World Organisation for Animal Health (WOAH) recognizes it as an emerging transboundary disease of salmonids, underscoring its importance to international aquatic animal health governance [33, 34]. Understanding the molecular underpinnings of PRV-1 pathogenesis requires a dissection of viral entry, replication kinetics, cellular tropism, the host antiviral response, and the specific viral genetic elements that distinguish high-virulence from low-virulence isolates.

Viral Architecture and Genomic Determinants of Virulence

PRV-1 is a non-enveloped, double-stranded RNA (dsRNA) virus belonging to the family Reoviridae, genus Orthoreovirus, although phylogenetic analyses indicate that PRV represents a distinct new genus within this family, separate from both mammalian orthoreoviruses and aquareoviruses [12, 32]. The genome comprises ten segmented dsRNA molecules (L1, L2, L3; M1, M2, M3; S1, S2, S3, S4) encoding at least ten structural proteins (λ1, λ2, λ3, μ1, μ2, σ1, σ2, σ3, σNS, and the core protein μNS) and several non-structural proteins, including p13 encoded by the S1 segment [12, 40]. The segmented nature of the genome is critical to PRV-1 pathogenesis, as it enables reassortment, a mechanism that has been directly implicated in the emergence of virulent strains [19].

A landmark study by Dhamotharan et al. (2019) performed phylogenomic analysis of 31 PRV-1 genomes collected over three decades and identified two distinct monophylogenetic clusters: one associated with clinical HSMI and the other with low-virulent or asymptomatic infections [19]. Crucially, the HSMI-associated cluster was defined by co-evolution of segments S1 and M2, which encode the outer capsid protein σ1 and the non-structural protein μNS, respectively. The S1–M2 pair appears to have undergone selection pressure coinciding with the first emergence of HSMI in Norway in 1999, and this pair has remained remarkably conserved in Norwegian salmon aquaculture since 1997 [19]. Only a limited number of amino acid substitutions, all located in the S1- and M2-encoded proteins, were uniquely associated with the HSMI phenotype [19]. This finding has profound implications: it suggests that the acquisition of virulence was not a gradual accumulation of mutations across the genome but rather a discrete evolutionary event involving reassortment or co-adaptation of specific segment pairs.

Further corroboration comes from Wessel et al. (2020), who performed a dose-standardized challenge trial comparing six PRV-1 isolates, including two Norwegian field isolates from 2018 (high-virulence), three historical Norwegian isolates predating the first HSMI report (low-virulence), and one Canadian isolate (low-virulence) [13]. The 2018 isolates induced significantly higher plasma viremia and characteristic HSMI lesions, whereas the historical and Canadian isolates produced only mild cardiac inflammation. Phenotypic differences were linked to proteins encoded by segments S1, M2, L1, L2, and S4 [13]. The S1-encoded σ1 protein is the viral attachment protein, homologous to the mammalian orthoreovirus σ1, which mediates sialic acid binding and cellular entry [12]. In PRV-1, σ1 contains conserved residues critical for sialic acid binding, and its structural integrity is essential for infectivity [2, 12]. The M2-encoded μNS protein is the major non-structural protein that forms viral factories, cytoplasmic inclusion bodies where viral replication and assembly occur [12, 42]. Amino acid changes in μNS could alter factory morphology, replication efficiency, or the ability to subvert host antiviral responses.

Vatne et al. (2021) extended this work by sequencing five genomic segments (S1, S4, M2, L1, L2) from 37 Norwegian PRV-1 isolates and classifying them into eight genogroups, with two defined as high-virulent and two as low-virulent based on comparison with reference isolates of known virulence [1]. The geographic distribution revealed a higher frequency of high-virulent isolates in mid- and northern Norway, correlating with the epidemiological pattern of HSMI outbreaks [1, 33]. This regional clustering suggests that virulent strains may have emerged and spread within specific aquaculture networks, and that fallowing and biosecurity measures could reduce their prevalence [3].

Cellular Tropism: Erythrocytes as the Primary Replication Niche

A defining feature of PRV-1 pathogenesis is its tropism for erythrocytes, which are nucleated in salmonids and possess active immune functions [30, 36]. Finstad et al. (2014) first demonstrated that erythrocytes are the major target cells for PRV-1, with more than 50% of erythrocytes becoming PRV-positive in individual fish during peak infection [30]. Viral particles are concentrated in large cytoplasmic inclusions that resemble viral factories, as confirmed by immunofluorescence, confocal microscopy, and electron microscopy [30]. These inclusions contain reovirus-like particles and are analogous to the erythrocytic inclusion bodies described in erythrocytic inclusion body syndrome (EIBS) caused by PRV-2 in coho salmon [27, 30].

The ex vivo cultivation system developed by Wessel et al. (2015) demonstrated that PRV-1 replicates productively in Atlantic salmon erythrocytes, with a significant increase in viral load over time, formation of cytoplasmic inclusions containing dsRNA and viral protein, and successful passage to naïve erythrocytes [45]. This system has been instrumental in dissecting the early molecular events of infection. Transcriptomic analysis of PRV-1-exposed erythrocytes revealed a robust innate antiviral response, including upregulation of interferon-α (IFN-α), RIG-I, Mx, and PKR transcripts [45]. Importantly, the antiviral response in erythrocytes is long-lasting, persisting for weeks after the onset of infection, and involves upregulation of potential inhibitors of translation [25]. This may explain why PRV-1 protein production in erythrocytes halts while viral RNA persists for months, a mechanism that facilitates the establishment of persistent infection [22, 25].

Tsoulia et al. (2024) compared the transcriptomic responses of erythrocytes, Atlantic salmon kidney (ASK) cells, and salmon head kidney (SHK-1) cells to PRV-1 exposure [36]. Erythrocytes expressed a wide repertoire of pattern recognition receptors (PRRs), cytokine receptors, and antiviral genes, supporting their characterization as pluripotent immune cells. Notably, RIG-I-like receptor 3 (RLR3) was significantly induced in all cell types, but interferon regulatory factor 1 (IRF1) was induced only in erythrocytes, whereas IRF3 and IRF7 were induced in SHK-1 cells [36]. This differential IRF activation may influence viral propagation and the timing of the antiviral response, potentially giving PRV-1 an evolutionary advantage by delaying the onset of a fully effective interferon response in erythrocytes.

Dissemination from Blood to Target Tissues: The Transition to Cardiomyocytes

The transition from erythrocyte infection to cardiomyocyte infection is the critical step in HSMI pathogenesis. Dhamotharan et al. (2020) performed a detailed temporal analysis of PRV-1 dissemination in experimentally infected Atlantic salmon [41]. Viral RNA and protein were first detected in erythrocytes and plasma, peaking before the appearance of virus in cardiomyocytes and hepatocytes. Histopathological evaluation revealed that PRV-1 infection induced severe epicarditis and myocarditis with degeneration of cardiomyocytes, necrosis, and diffuse cellular infiltration [41]. The virus was subsequently cleared from regenerating heart tissue and hepatocytes but persisted in erythrocytes, indicating that erythrocytes serve as both the primary replication site and a reservoir for long-term persistence [22, 41].

The mechanism by which PRV-1 transfers from erythrocytes to cardiomyocytes remains incompletely understood, but several lines of evidence suggest that infected erythrocytes may adhere to or be phagocytosed by macrophages within the heart, facilitating viral delivery. Malik et al. (2021) used fluorescent in situ hybridization (FISH) to localize PRV-1 RNA in a subset of M1 (pro-inflammatory) macrophages in both heart and skeletal muscle, while M2 (anti-inflammatory) macrophages were widely scattered in the heart but did not contain PRV-1 RNA [38]. This suggests that M1 macrophages may be involved in viral dissemination or early inflammatory responses, whereas M2 macrophages may contribute to tissue repair and recovery following viral clearance [38, 41]. The presence of arginase-2-positive, macrophage-like cells in the heart further supports the role of M2 polarization in regenerative processes [41].

Immune Dynamics: The Double-Edged Sword of Antiviral and Inflammatory Responses

The host immune response to PRV-1 is a complex interplay between protective antiviral mechanisms and immunopathology. Vallejos-Vidal et al. (2022) comprehensively reviewed the immune response to PRV, describing three phases: early entry and dissemination, acute dissemination, and persistence [37]. During the acute phase, PRV-1 induces a classical antiviral response characterized by upregulation of IFN-α, RIG-I, Mx, and PKR in erythrocytes [25, 37]. Concurrently, a Th1-type adaptive response is elicited, with upregulation of T-cell receptor alpha and beta (TCRα, TCRβ), CD2, IL-2, CD4-1, IFN-γ, IL-12, and IL-18 [37]. The high expression of CD8α, CD8β, and granzyme A indicates a robust cytotoxic T lymphocyte (CTL) response, which is consistent with the upregulation of MHC class I, transporters, and proteasome components involved in antigen presentation [37, 38].

Malik et al. (2021) demonstrated that the immune response in heart tissue with HSMI lesions is characterized by CD8+ and MHC-I-expressing cells, with a strong cellular immune response targeting PRV-1 [38]. In skeletal muscle, MHC-I-expressing cells and CD8+ cells were dispersed between myocytes, but these cells did not stain for PRV-1, suggesting that the inflammatory response in muscle may be secondary to the cardiac pathology [38]. The CTL response appears to be effective in clearing the virus from heart tissue, as gene expression analysis confirmed a drop in PRV-1 levels following the cell-mediated immune response [38]. However, this immune clearance may also contribute to tissue damage, as the inflammatory infiltrate itself can cause myocyte degeneration and necrosis.

The timing and magnitude of the antiviral response are critical determinants of disease outcome. Tsoulia et al. (2024) compared the transcriptional responses in blood cells of Atlantic salmon infected with PRV-1, PRV-2, or PRV-3 [4]. PRV-1 and PRV-3 both replicated well and induced typical innate antiviral responses, but PRV-3 triggered a stronger antiviral response at two weeks post-infection, despite similar viral RNA replication levels. By five weeks, the responses were nearly equal [4]. The authors proposed that the difference in timing may give PRV-1 an evolutionary edge, facilitating its dissemination to the heart before a fully effective immune response is mounted [4]. This hypothesis is supported by the observation that PRV-3 infection completely blocks subsequent PRV-1 infection and HSMI development, likely due to the early and potent activation of innate immune responses that cross-protect against PRV-1 [11].

Host Genetic Factors and the Role of Co-Infections

The molecular pathogenesis of PRV-1 is not solely determined by viral genetics; host genotype plays a significant modulatory role. Polinski et al. (2025) demonstrated that the severity of PRV-1-associated heart inflammation varies significantly depending on the Atlantic salmon stock challenged, irrespective of the viral isolate used [46]. New Brunswick Tobique River Atlantic salmon developed less heart inflammation than Mowi-McConnell Atlantic salmon from Western Canada, which in turn developed less inflammation than Mowi Atlantic salmon from Scotland [46]. This indicates that host genetic factors can attenuate or exacerbate disease, potentially through differences in innate immune responsiveness, erythrocyte susceptibility, or the efficiency of CTL responses.

Co-infections with other pathogens can also influence PRV-1 pathogenesis. Lund et al. (2016) showed that experimental PRV-1 infection protects against subsequent salmonid alphavirus (SAV) infection and pancreas disease, suggesting that PRV-1-induced antiviral responses can cross-protect against other viruses [43]. Conversely, co-infection with Paranucleospora theridion or Kudoa thyrsites has been observed in HSMI-affected fish, but PRV-1 alone was statistically correlated with the occurrence and severity of heart lesions [31]. The potential for synergistic interactions between PRV-1 and other pathogens, such as infectious salmon anemia virus (ISAV), has been suggested in the context of hemorrhagic kidney syndrome, where both viruses were detected in renal lesions [52].

Metabolic Consequences and Physiological Impact

The molecular pathogenesis of PRV-1 extends to systemic metabolic disturbances. Metabolomic analysis of plasma from PRV-1-infected Atlantic salmon revealed substantial disruption of lipid homeostasis, particularly affecting lysophosphatidylcholines, ceramides, and triglycerides [49]. These metabolic changes may reflect altered membrane dynamics in infected erythrocytes, oxidative stress, or the systemic inflammatory response. The combination of metabolomics with proteomics identified biomarkers that are representative of HSMI progression, offering potential tools for early diagnosis and monitoring [49].

The physiological consequences of PRV-1 infection are profound. Lund et al. (2017) demonstrated that Atlantic salmon with HSMI have reduced hypoxia tolerance and cardiac performance, as measured by incipient lethal oxygen saturation and maximum heart rate [50]. Interestingly, preconditioning fish to transient hypoxic stress episodes improved their tolerance, suggesting that adaptive responses to hypoxia can mitigate some of the cardiorespiratory impairments caused by HSMI [50]. However, Zhang et al. (2019) caution that high-load PRV-1 infections do not necessarily imply physiological impairment, as a low-virulence strain from Pacific Canada did not affect oxygen affinity or carrying capacity of erythrocytes despite severe viremia [51]. This underscores the critical distinction between infection and disease, and highlights the importance of viral strain and host factors in determining the clinical outcome.

Viral Persistence and the Role of Erythroid Progenitors

A hallmark of PRV-1 infection is its ability to establish long-term persistence. Malik et al. (2019) demonstrated that PRV-1 establishes a productive, persistent infection in Atlantic salmon, with viral genomic dsRNA detectable in plasma throughout an 18-week trial, indicating continuous production and release of viral particles [22]. The highest level of PRV-1 RNA in the persistent phase was found in kidney, and in situ hybridization confirmed that PRV-1 RNA was present in erythroid progenitor cells, erythrocytes, macrophages, and melano-macrophages [22]. The presence of virus in erythroid progenitor cells is particularly significant, as it provides a mechanism for maintaining infection through erythropoiesis, ensuring that newly formed erythrocytes are already infected [22]. This may explain why PRV-1 can persist for the lifetime of the fish, and why the virus is so difficult to eradicate from farmed populations.

Kannimuthu et al. (2023) conducted a long-term challenge experiment in juvenile Atlantic salmon and found that PRV-1 infection persisted for at least 65 weeks post-challenge, with high viral loads in whole blood, spleen, and head kidney [6]. Despite achieving high viremia, no mortality was observed, and heart lesions typical of HSMI resolved after the acute phase [6]. This indicates that the persistent phase is characterized by immune regulation and tissue repair, rather than ongoing pathology. The ability of PRV-1 to persist without causing continuous disease is a key factor in its widespread prevalence in aquaculture, as infected fish can serve as asymptomatic carriers that shed virus into the environment [6, 44].

Species Specificity and the Molecular Basis of Host Range

The molecular pathogenesis of PRV-1 is also influenced by host species. While PRV-1 causes severe HSMI in Atlantic salmon, it replicates with lower efficiency and causes minimal pathology in brown trout (Salmo trutta), Chinook salmon (Oncorhynchus tshawytscha), coho salmon (O. kisutch), and rainbow trout (O. mykiss) [7, 15, 23]. Kannimuthu et al. (2023) demonstrated that all life stages of Atlantic salmon and brown trout can be infected with PRV-1, but brown trout showed lower infection prevalence and only mild infections without pathological changes [7]. This species-specific susceptibility is likely due to differences in cellular receptors, innate immune responses, or the ability of the virus to replicate in erythrocytes of different species.

The molecular basis for this host range restriction may lie in the σ1 protein, which mediates viral attachment. PRV-1 σ1 shares homology with mammalian orthoreovirus σ1, which binds to sialic acids and junctional adhesion molecule-A (JAM-A) [12]. However, the specific receptor for PRV-1 in salmonid erythrocytes has not been identified. Differences in receptor expression or glycosylation patterns between species could explain the differential susceptibility. Additionally, the antiviral response of erythrocytes may vary between species, with Atlantic salmon erythrocytes being more permissive to PRV-1 replication than those of other salmonids [25].

Implications for Vaccine Development and Disease Control

The molecular pathogenesis of PRV-1 presents unique challenges for vaccine development. The inability to propagate PRV-1 in continuous cell lines has been a major bottleneck, as it precludes the production of traditional inactivated or live-attenuated vaccines [39, 47]. However, the ex vivo cultivation system in erythrocytes and the development of virus-like particle (VLP) vaccines offer promising alternatives [45, 48]. Galleguillos-Becerra et al. (2025) developed a VLP-based vaccine expressing the six structural proteins of PRV-1 using a baculovirus expression system, which significantly reduced viral loads in challenged Atlantic salmon [48]. DNA vaccines expressing the non-structural protein μNS and the attachment protein σ1 have also shown moderate protective effects, likely due to enhanced intracellular expression and antigen presentation [2

Genetic Diversity and Virulence Determinants of PRV-1 Genogroups

The genetic architecture of Piscine orthoreovirus genotype 1 (PRV-1) represents a complex tapestry of viral evolution, host adaptation, and phenotypic divergence that has profound implications for both the pathogenesis of heart and skeletal muscle inflammation (HSMI) and the development of rational control strategies. Unlike many viral pathogens of aquaculture where a single virulent lineage predominates, PRV-1 exhibits a striking spectrum of virulence phenotypes that are intimately linked to specific constellations of genomic segments, creating a paradigm where genetic diversity directly translates into differential disease outcomes. Understanding the molecular determinants that separate high-virulence from low-virulence PRV-1 genogroups is therefore not merely an academic exercise but a critical prerequisite for epidemiological surveillance, vaccine antigen selection, and the implementation of genogroup-based risk mitigation strategies in Atlantic salmon aquaculture.

Phylogenetic Architecture and Genogroup Classification

The seminal work by Vatne et al. established the foundational framework for PRV-1 genogroup classification through comprehensive sequencing of five genomic segments, S1, S4, M2, L1, and L2, that had been previously implicated in virulence determination [1]. By analyzing 37 Norwegian PRV-1 isolates and comparing them to reference isolates with experimentally validated virulence phenotypes, the investigators delineated eight distinct genogroups based on combinatorial segment analysis. Critically, two genogroups were classified as high-virulent, two as low-virulent, and the remaining four as unknown virulence status, highlighting the complexity of associating specific genetic signatures with pathogenic potential [1]. The geographic distribution of these genogroups revealed a non-random pattern, with high-virulent isolates occurring at significantly higher frequencies in mid- and northern Norwegian aquaculture regions, suggesting possible directional selection or founder effects operating within distinct aquaculture zones [1, 33].

This classification system was subsequently validated and refined through experimental challenge trials that provided the first definitive demonstration of virulence differences between PRV-1 isolates. Wessel et al. conducted dose-standardized comparisons of six isolates, including two Norwegian field isolates from 2018, three historical Norwegian isolates predating the first HSMI description in 1999, and one Canadian isolate, and demonstrated that the 2018 Norwegian isolates induced histopathological lesions consistent with HSMI, while the historical Norwegian and Canadian isolates produced only mild cardiac inflammation [13]. The phenotypic divergence was directly linked to viral proteins encoded by segments S1, M2, L1, L2, and S4, establishing these five segments as the primary determinants of PRV-1 virulence [13]. This experimental confirmation transformed our understanding of PRV-1 diversity from a purely descriptive phylogenetic exercise into a functionally relevant framework for predicting disease outcomes.

Genomic Segments Encoding Virulence: The S1-M2 Axis

Among the five virulence-associated segments, the S1 and M2 gene segments have emerged as the most phylogenetically informative and functionally significant determinants of PRV-1 pathogenicity. Dhamotharan et al. conducted comprehensive phylogenetic and sequence analyses of 31 PRV-1 genomes collected over a 30-year period, revealing that Norwegian viral sequences cluster into two distinct monophylogenetic groups, one associated with HSMI and the other comprising low-virulent isolates [19]. Notably, a PRV-1 strain from Norway sampled in 1988, a full decade before the first HSMI description in 1999, grouped with the low-virulent cluster, indicating that ancestral PRV-1 strains were likely benign and that virulence emerged through subsequent evolutionary events [19]. The two monophylogenetic clusters were particularly evident for segments S1 and M2, with only a limited number of amino acid substitutions uniquely associated with the HSMI phenotype, all of which localized to the S1- and M2-encoded proteins [19]. The observed co-evolution of the S1-M2 segment pair coincided temporally with the emergence of HSMI in Norway, suggesting that these segments evolved through cumulative mutation accumulation and/or segment reassortment events that conferred a selective advantage for virulence [19].

The functional importance of the S1-encoded σ1 protein cannot be overstated. As the outer capsid protein mediating viral attachment, σ1 dictates host cell tropism and is the primary target of neutralizing antibody responses [2, 12]. Markussen et al. demonstrated that despite low overall amino acid identity with mammalian orthoreovirus σ1 proteins, key residues critical for sialic acid binding are partially or wholly conserved in PRV-1 σ1, suggesting functional conservation of the receptor-binding mechanism [12]. However, the antigenic properties of σ1 are highly variable; recombinant σ1 expressed in conventional systems exhibits poor antibody recognition, and only structural modifications such as lipidation or fusion with molecular chaperones can improve epitope exposure [2]. This antigenic variability likely reflects the genetic diversity among PRV-1 genogroups, as the S1 segment exhibits the highest nucleotide divergence among all genomic segments, particularly between sub-genotypes Ia and Ib [32, 49].

The M2-encoded μ1 protein, which mediates endosomal membrane penetration during reovirus entry, also harbors critical virulence determinants. Markussen et al. identified conserved cleavage sites and myristoylation motifs in PRV-1 μ1 that are essential for the membrane penetration function, mirroring the mechanisms employed by mammalian orthoreoviruses [12]. More importantly, the μ2 protein, encoded by the M1 segment, contains a proline residue that in mammalian orthoreoviruses plays a central role in both the formation and structural organization of viral inclusion bodies and the modulation of interferon-β signaling and myocarditis induction [12]. The presence of this conserved residue in PRV-1 μ2 suggests that analogous mechanisms of host immune modulation and tissue pathology may operate during PRV-1 infection, although the specific contributions of μ2 to PRV-1 virulence remain to be experimentally dissected.

Evolutionary Origins and Global Dissemination of Virulent Genogroups

The evolutionary trajectory of PRV-1 virulence is intimately connected to the virus’s global dissemination history and the selective pressures imposed by intensive Atlantic salmon aquaculture. Kibenge et al. conducted comprehensive whole-genome analyses demonstrating that PRV-1 represents an unambiguous new genus within the family Reoviridae, distantly related to both Orthoreovirus and Aquareovirus genera [32]. Phylogenetic analysis of S1 sequences grouped Norwegian PRV-1 strains into Genotype I with two sub-genotypes, Ia and Ib, with Canadian strains matching sub-genotype Ia and Chilean strains matching sub-genotype Ib [32]. The molecular clock analysis indicated that Canadian PRV-1 diverged from Norwegian sub-genotype Ia around 2007±1, while Chilean PRV-1 diverged from Norwegian sub-genotype Ib around 2008±1 [32], coinciding with the intensification of global Atlantic salmon aquaculture and the movement of live fish and ova between production regions.

Critically, the emergence of high-virulence PRV-1 genogroups appears to be a Norwegian phenomenon, with isolates from the North American Pacific Coast and the Faroe Islands remaining more closely related to the ancestral, low-virulence PRV-1 strains that have not undergone the S1-M2 co-evolution associated with clinical HSMI [19]. This geographic restriction of virulent genogroups is supported by extensive experimental and field evidence. Polinski et al. demonstrated that PRV-1 from Pacific Canada, regardless of source fish health status, induces only high-load viremia with minor focal heart inflammation that lacks the characteristic transcriptional induction of inflammatory cytokines seen with Norwegian isolates [14]. Similarly, Garver et al. showed that western North American PRV-1 is highly transmissible to both Atlantic and Sockeye salmon but fails to induce HSMI pathology, even with persistent infection lasting up to 59 weeks post-challenge [28]. The virus replicates efficiently, but the host response is characterized by only a minor transcriptional induction of the antiviral Mx gene, lacking the robust inflammatory cascade that typifies HSMI pathogenesis [28].

The historical presence of PRV-1 in Chile, as revealed by archaeovirological analysis of formalin-fixed, paraffin-embedded heart tissues from 1992-1999, identified PRV-1 as a low-virulence genogroup as early as 1994, predating the first HSMI outbreak in Chile by 17 years [9]. This finding underscores that PRV-1 was present in Chilean aquaculture for nearly two decades before the emergence of clinically significant disease, suggesting that the introduction of high-virulence genogroups, rather than the virus itself, was the critical event precipitating HSMI outbreaks in Chile. The coho salmon in Chile harbor even greater genetic diversity, with PRV strains grouping in both sub-genotypes Ia and Ib as well as forming a distinct phylogenetic cluster designated Genotype II that includes the Norwegian PRV-3-related virus [26].

Host Genetics and the Modulation of Virulence Expression

While viral genogroup is a primary determinant of HSMI outcome, emerging evidence indicates that host genetic background exerts a powerful modulatory effect on virulence expression, complicating the simple equation of genogroup equals disease severity. Polinski et al. conducted controlled laboratory challenges using three PRV-1 isolates, PRV-1a from the eastern Pacific, PRV-1a from the western Atlantic, and PRV-1b from the Norwegian Sea, and demonstrated that virus replication dynamics, host recognition, and PRV-1-associated heart inflammation were discrete relative to the Atlantic salmon stock challenged, irrespective of the viral isolate used [46]. Specifically, New Brunswick Tobique River Atlantic salmon exhibited significantly less heart inflammation compared to Mowi-McConnell Atlantic salmon of Western Canada, which in turn showed less inflammation than Mowi Atlantic salmon of Scotland when cumulatively considering all three PRV-1 isolates [46]. This host genotype effect indicates that the presence of PRV-1a or PRV-1b alone is insufficient to reliably predict disease and highlights a potential mechanism, selective breeding for reduced HSMI susceptibility, for mitigating disease severity even in the presence of high-virulence genogroups.

The World Organisation for Animal Health (WOAH) recognizes HSMI as an important emerging disease of Atlantic salmon, and the genetic diversity of PRV-1 genogroups presents both challenges and opportunities for disease surveillance and control. The development of genogroup-specific RT-qPCR assays targeting the S1 genomic segment, as described by Siah et al., enables differentiation between PRV-1 variants associated with HSMI and those that are not, providing a powerful tool for risk-based surveillance in regions where both high- and low-virulence genogroups circulate [54]. However, the application of such assays must be interpreted within the context of host genetics, as demonstrated by the differential susceptibility of Atlantic salmon stocks to PRV-1-induced pathology [46]. Furthermore, the complete absence of clinical HSMI in British Columbia despite high PRV-1 prevalence in farmed Atlantic salmon, documented through longitudinal farm studies correlating PRV-1 load with histopathological lesion development [31], suggests that additional environmental or management factors, including oxygen saturation, temperature, and co-infection status, contribute to the complex etiology of HSMI.

Mechanistic Basis of Genogroup-Specific Virulence

The molecular mechanisms by which specific PRV-1 genogroups cause differential virulence are beginning to be elucidated through comparative transcriptomics and proteomics. Tsoulia et al. compared the transcriptional responses in blood cells of Atlantic salmon infected with three PRV genotypes, revealing that PRV-1 and PRV-3 both replicate well in blood cells and induce typical innate antiviral responses triggered by dsRNA viruses [4]. However, PRV-3 triggered stronger antiviral responses than PRV-1 two weeks post-infection despite similar viral RNA replication levels, with the responses converging by five weeks [4]. This difference in the timing of antiviral responses may give PRV-1 an evolutionary edge, facilitating its dissemination from erythrocytes to heart tissue, a critical step for HSMI development [4]. The PRV-1 genogroups that successfully establish high-level viremia without triggering early, potent antiviral responses may therefore be better positioned to achieve the cardiac infection necessary for HSMI pathogenesis.

The cellular dynamics of PRV-1 infection further illuminate genogroup-specific virulence determinants. Malik et al. demonstrated that PRV-1 infection in heart tissue is characterized by a strong cellular immune response with increased MHC-I expression and a high number of cytotoxic CD8+ granzyme-producing cells that target PRV-1-infected cells [38]. M1 macrophages, which are pro-inflammatory, do not contribute to the initial development of HSMI, while M2 macrophages, which are anti-inflammatory, reside in the heart in large numbers and may contribute to the subsequent recovery following PRV-1 clearance [38]. High-virulence genogroups may be those that either evade or subvert these cellular immune responses more effectively, or that induce more severe tissue damage through the dysregulation of the M1/M2 macrophage balance. The observation that PRV-1 establishes persistent, productive infection in erythrocyte progenitor cells in the kidney [22] suggests that the ability to maintain a long-term reservoir in hematopoietic tissues may also be a determinant of genogroup virulence, as virus continuously produced from this reservoir could sustain cardiac inflammation over extended periods.

The functional significance of the non-structural protein μNS in virulence determination should not be overlooked. Haatveit et al. demonstrated that DNA vaccines expressing μNS combined with the cell attachment protein σ1 delayed PRV-1 infection kinetics and induced moderate protective effects against HSMI [42]. The μNS protein is the primary organizer of viral factories, the cytoplasmic structures where PRV-1 replication and assembly occur. Genogroup-specific differences in μNS sequence could therefore influence the efficiency of viral factory formation, the rate of virus production, or the subcellular localization of replication complexes, all of which could impact virulence. The identification of additional open reading frames encoding small proteins, p13 from the S1 segment, p8 from S2, and p11 from L2 [12], suggests that the PRV-1 genome may be more complex than initially appreciated, with these small proteins potentially playing roles in host immune modulation or cellular pathogenesis that differ between genogroups.

Implications for Disease Management and Future Research Directions

The delineation of PRV-1 genogroups and the identification of the genomic segments encoding virulence determinants have direct implications for disease management in Atlantic salmon aquaculture. The observation that high-virulence genogroups are more prevalent in mid- and northern Norwegian aquaculture regions [1] suggests that spatially targeted interventions, such as synchronized fallowing, as demonstrated by Vatne et al. [3], could be effective in reducing the circulation of virulent variants within specific geographic zones. Furthermore, the finding that exposure to PRV-1 at the freshwater stage can serve as a source of introduction to marine sites [3], combined with the documented persistence of PRV-1 infection from fry to parr stages without causing mortality [6], underscores the importance of screening and managing broodstock and hatchery populations to prevent the introduction of high-virulence genogroups into seawater production sites.

The development of a virus-like particle (VLP)-based vaccine by Galleguillos-Becerra et al., which expresses the six structural proteins of PRV-1 (λ1, λ2, μ1, σ1, σ2, σ3) and incorporates a c-myc epitope tag to enable differentiating infected from vaccinated animals (DIVA) strategies [48], represents a promising approach for controlling high-virulence genogroups. If such vaccines can be formulated to include antigens derived from the specific genogroups circulating in a given production region, they could provide targeted protection while allowing surveillance for the introduction of novel virulent variants. The inactivated PRV-1 vaccine developed by Wessel et al., which demonstrated significant reduction in viral loads and histopathological lesions following experimental challenge [53], provides further proof-of-concept that genogroup-specific vaccine formulations could be efficacious. However, the variable immunogenicity of the σ1 protein across genogroups [2] remains a significant challenge that must be addressed through rational antigen design.

The extensive genetic diversity of PRV-1 genogroups, their differential geographic distribution, and the complex interplay between viral genotype and host genetic background underscore the need for continued genomic surveillance and the development of genogroup-specific diagnostic tools. The WOAH listing of HSMI as a notifiable disease in several salmon-producing nations reflects the economic importance of this pathogen, and the ability to differentiate high-virulence from low-virulence genogroups is essential for risk assessment and regulatory decision-making. Future research must focus on elucidating the functional consequences of the specific amino acid substitutions that differentiate high- and low-virulence PRV-1 genogroups, particularly those in the S1- and M2-encoded proteins, and on understanding how these molecular differences translate into altered virus-host interactions at the cellular and tissue levels. Only through such mechanistic understanding can we hope to develop the next generation of control strategies that effectively mitigate the impact of HSMI while accounting for the remarkable genetic plasticity of Piscine orthoreovirus-1.

Epidemiology and Geographic Distribution of PRV-1 in Farmed Atlantic Salmon

Piscine orthoreovirus genotype 1 (PRV-1) represents one of the most economically significant viral pathogens confronting global Atlantic salmon aquaculture, with its distribution intricately linked to the international trade in live fish, eggs, and the movement of stock between production zones. As the aetiological agent of heart and skeletal muscle inflammation (HSMI), a disease that can cause cumulative mortality rates approaching 30% with morbidity reaching 100% in affected cohorts [34], PRV-1 has become endemic in virtually every major Atlantic salmon-producing region. However, the virus exhibits a perplexing epidemiological paradox: its presence correlates poorly with disease expression, a phenomenon that has driven intensive investigation into the genetic, environmental, and host determinants that govern the transition from subclinical infection to fulminant HSMI. This section synthesizes the current understanding of PRV-1 epidemiology and geographic distribution, drawing upon molecular epidemiological surveys, experimental challenge studies, and long-term surveillance programmes conducted across Norway, Canada, Chile, Scotland, and the Faroe Islands.

Global Distribution and the Norwegian Epicentre

Norway, as the world’s largest producer of farmed Atlantic salmon, represents the epicentre of both PRV-1 prevalence and HSMI disease outbreaks. The virus has been present in Norwegian aquaculture since at least the late 1980s, predating the first description of HSMI in 1999 by over a decade [13, 19]. Molecular phylogenetic analyses of archival material have demonstrated that PRV-1 sequences from 1988 cluster with low-virulence lineages, suggesting that a discrete evolutionary event, likely involving co-evolution of genomic segments S1 and M2, was necessary for the emergence of virulent strains capable of inducing the characteristic cardiac pathology of HSMI [19]. This temporal framework is critical for understanding the current epidemiological landscape, as it indicates that PRV-1 circulated asymptomatically in Norwegian salmon populations for at least a decade before acquiring the genetic determinants necessary for pathogenicity.

Contemporary surveillance reveals that PRV-1 is ubiquitous in Norwegian salmon aquaculture, with prevalence estimates approaching 95–100% in farmed populations during the marine phase of production [1, 3, 34]. However, the distribution of virulent versus low-virulent genogroups is spatially structured. Vatne and colleagues [1] conducted a comprehensive genotyping study of 37 Norwegian PRV-1 isolates across five genomic segments (S1, S4, M2, L1, and L2) putatively linked to virulence, identifying eight distinct genogroups. Among these, two genogroups were classified as high-virulent and two as low-virulent based on comparison with reference isolates of known pathogenic potential [1]. Critically, the geographic distribution revealed a striking latitudinal gradient: high-virulent isolates occurred with significantly greater frequency in mid- and northern Norway, while low-virulent variants were more prevalent in southern production zones [1, 33]. This spatial pattern aligns with historical risk-mapping studies conducted by Kristoffersen and colleagues [33], who demonstrated that the probability of HSMI outbreak at the cohort level was substantially higher in Mid-Norway compared to southern regions, and highly variable but generally elevated in Northern Norway. Their logistic regression model identified infection pressure, cohort lifespan, and cohort size as significant risk factors, suggesting that the geographic clustering of disease reflects both viral genogroup distribution and farm-level management practices [33].

The introduction and dissemination dynamics of PRV-1 within Norwegian aquaculture have been further elucidated through intensive field studies in geographically isolated production zones. Vatne and colleagues [3] tracked PRV-1 over a complete production cycle across five sites in a restricted Norwegian fjord system, sequencing 32 virus isolates and performing genogroup analysis. Their findings revealed multiple independent introductions of the virus to the area, yet a surprising degree of genetic homogeneity among co-circulating variants. Importantly, at three sites where fallowing, the practice of leaving sites empty between production cycles, was synchronously implemented, the PRV-1 variants present in the new generation differed from those in the previous cycle, indicating that coordinated fallowing can effectively break the cycle of viral persistence and re-introduction [3]. This observation has profound implications for disease management, as it suggests that area-wide management strategies can disrupt the transmission network that sustains PRV-1 within endemic regions. The study also identified exposure to PRV-1 during the freshwater stage as a potential source of introduction, highlighting the vulnerability of the production cycle at its earliest phases [3].

Genotypic Structuring and Virulence Determinants

The molecular epidemiology of PRV-1 is fundamentally shaped by the phylogenetic division of the virus into two major sub-genotypes within Genotype I: sub-genotype Ia and sub-genotype Ib [32]. This genotypic classification, originally established by Kibenge and colleagues [32] based on S1 gene sequences, has proven remarkably stable across geographic regions and time. Sub-genotype Ia encompasses isolates from Norway, Canada, Iceland, and the Faroe Islands, while sub-genotype Ib is predominantly associated with Chilean isolates [32]. However, the relationship between sub-genotype and virulence is not straightforward, as experimental challenge studies have conclusively demonstrated that phenotype cannot be reliably predicted from genotype alone.

Wessel and colleagues [13] provided the first definitive demonstration of virulence differences among PRV-1 isolates through a dose-standardized challenge trial comparing six isolates: two Norwegian field isolates from 2018, three historical Norwegian isolates predating the first report of HSMI, and one Canadian isolate. The two contemporary Norwegian isolates induced histopathological lesions consistent with HSMI, while all three historical Norwegian isolates and the Canadian isolate induced only mild cardiac pathology [13]. Importantly, these phenotypic differences were linked to viral proteins encoded by segments S1, M2, L1, L2, and S4, the same segments that formed the basis of the genogroup classification system developed by Vatne and colleagues [1]. This concordance between molecular markers and biological phenotype validates the use of multi-segment genotyping as a tool for virulence surveillance.

The evolutionary trajectory of PRV-1 virulence appears to involve the co-evolution of the S1 and M2 genomic segments, which encode the outer capsid protein σ1 and the inner core protein μ2, respectively. Dhamotharan and colleagues [19] performed comprehensive phylogenetic and sequence analyses of 31 PRV-1 genomes collected over a 30-year period, identifying two main monophylogenetic clusters: one associated with HSMI and the other with low-virulent isolates. The HSMI-associated cluster was characterized by a limited number of unique amino acid substitutions in the S1- and M2-encoded proteins, and phylogenetic dating indicated that this co-evolutionary event occurred coincident with the emergence of HSMI in Norway in the late 1990s [19]. Furthermore, PRV-1 strains from the North American Pacific Coast and the Faroe Islands, regions where HSMI has been documented only rarely or with mild clinical expression, have not undergone this evolutionary transition and remain closely related to the precursor strains that do not cause clinical disease [19, 28]. This evolutionary model posits that the acquisition of virulence determinants in S1 and M2 represented a necessary, but not sufficient, condition for the emergence of HSMI as a clinical entity.

Regional Epidemiology: Canada, Chile, and Emerging Production Zones

The epidemiological landscape of PRV-1 in Canada presents a striking contrast to the Norwegian situation. Despite high prevalence rates, often exceeding 95% in farmed Atlantic salmon populations, HSMI has only been sporadically documented in British Columbia, and when present, typically manifests as mild pathology without the elevated mortality characteristic of Norwegian outbreaks [14, 28, 31]. Experimental challenge studies using PRV-1 isolates from Pacific Canada have repeatedly failed to reproduce the severe cardiac inflammation observed with Norwegian isolates, even when using identical host genetic backgrounds and challenge protocols [13, 14, 28]. Polinski and colleagues [14] demonstrated that regardless of whether the PRV-1 source originated from fish with or without HSMI-like heart inflammation, infections led to high-load viremia but induced only minor focal heart inflammation without significant transcriptional induction of inflammatory cytokines. This consistent finding strongly supports the hypothesis that North American PRV-1 isolates belong to a low-virulence lineage that lacks the genetic determinants necessary for inducing HSMI [13, 14, 19].

However, the Canadian situation is more nuanced than simple viral avirulence. Di Cicco and colleagues [31] conducted a longitudinal farm study in British Columbia that documented the development, peak, and recovery phases of HSMI at the population level, with histopathological lesion prevalence reaching 80–100% in sampled fish despite the absence of elevated farm-level mortality [31]. Statistical correlation analyses identified PRV-1 as the only infectious agent significantly associated with the occurrence and severity of heart lesions, and immunohistochemistry localized viral antigen within the inflammatory foci. This study provided the first definitive evidence that HSMI can occur in Canadian farmed salmon, albeit with a milder clinical phenotype than typically observed in Norway [31]. Critically, the same research group subsequently demonstrated that PRV-1 isolates involved in both HSMI in Atlantic salmon and jaundice/anemia syndrome in Chinook salmon in British Columbia were genetically indistinguishable, with no consistent differences identified in the viral genome sequences associated with the two disease phenotypes [17]. This observation suggests that factors beyond viral genetics, likely including host genetic background, environmental stressors, and co-infections, modulate the clinical expression of PRV-1 infection in the Canadian context.

The host genotype effect has recently been substantiated through controlled experimental challenges. Polinski and colleagues [46] compared the outcomes of three PRV-1 isolates (PRV-1a from the eastern Pacific, PRV-1a from the western Atlantic, and PRV-1b from Norwegian waters) across three different Atlantic salmon stocks: New Brunswick Tobique River salmon, Mowi-McConnell salmon of Western Canada, and Mowi salmon of Scotland. Their results demonstrated that virus replication dynamics, host recognition, and PRV-1-associated heart inflammation were distinctly modulated by the Atlantic salmon stock challenged, irrespective of the viral isolate used. Specifically, New Brunswick Tobique River salmon exhibited significantly less heart inflammation than Mowi-McConnell salmon, which in turn showed less inflammation than Scottish Mowi salmon, across challenges with all three viral isolates [46]. This finding represents a paradigm shift in our understanding of HSMI epidemiology, establishing that host genotype is at least as important as viral genotype in determining disease outcome and suggesting that selective breeding programmes may offer a viable strategy for mitigating HSMI impact in regions where virulent PRV-1 strains circulate.

In Chile, the epidemiological history of PRV-1 follows yet another trajectory. HSMI was first described in Chilean farmed Atlantic salmon in 2011, but archaeovirological investigations using formalin-fixed, paraffin-embedded heart tissues from the 1990s have demonstrated that PRV-1 has been present in Chilean aquaculture since at least 1994, 17 years before the first clinical outbreak [9]. The Chilean PRV-1 isolates belong predominantly to sub-genotype Ib, with phylogenetic analyses indicating divergence from Norwegian sub-genotype Ib around 2008 ± 1 year [32]. Interestingly, the archival material from 1994 contained a low-virulence genogroup, consistent with the observation that Chilean PRV-1 strains have historically caused less severe pathology than contemporary Norwegian isolates [9]. However, the Chilean situation is complicated by the co-circulation of multiple PRV genotypes. Godoy and colleagues [26] identified a distinct phylogenetic cluster, designated Genotype II (now recognized as PRV-3), in coho salmon (Oncorhynchus kisutch) with HSMI-like lesions, and demonstrated that Chilean PRV strains from coho salmon were more genetically diversified than those from Atlantic salmon, grouping in both sub-genotypes Ia and Ib as well as the novel Genotype II cluster [26]. The presence of PRV-3 in Chilean rainbow trout has also been confirmed, with sequences closely related to both PRV-Ss (salmon) and PRV-Om (rainbow trout) variants [29], indicating a complex multi-genotype epidemiological landscape that requires ongoing surveillance.

Transmission Dynamics and the Role of Freshwater Stages

A critical dimension of PRV-1 epidemiology that has received increasing attention is the role of freshwater production stages in viral maintenance and dissemination. Historically, HSMI was considered a disease exclusively of the marine phase, with clinical outbreaks typically occurring 4–8 months after seawater transfer [33, 34]. However, longitudinal surveillance and experimental challenge studies have fundamentally revised this understanding. Kannimuthu and colleagues [6] conducted a long-term challenge experiment examining PRV-1 kinetics in Atlantic salmon from the fry to parr stage, demonstrating that the virus can establish persistent infections in freshwater that last for at least 65 weeks post-challenge. Viral loads peaked at 2–4 weeks post-challenge in heart and muscle tissues, and PRV-1 was detected at relatively high levels in whole blood, spleen, and head kidney throughout the study period [6]. Despite achieving high viremia, the infection failed to cause mortality, and heart lesions typical of HSMI that appeared at 6–8 weeks post-challenge subsequently resolved, consistent with the observation that HSMI pathology in freshwater is typically mild or absent [6].

The epidemiological significance of freshwater infection lies in its potential for vertical transmission and broodstock-mediated dissemination. Kibenge and colleagues [21] demonstrated that eggs from PRV-1-infected broodstock were positive for the virus, raising the possibility of vertical transfer and the global spread of PRV-1 through the international trade in Atlantic salmon eggs. This finding is particularly concerning given the high prevalence of PRV-1 in broodstock populations, a study of escaped farmed Atlantic salmon in Washington State and British Columbia following a containment failure found that 95% or more of the source farm population was PRV-positive, with eggs also testing positive [21]. The detection of PRV-1 in broodstock and eggs suggests that hatchery biosecurity measures, including routine screening of broodstock and disinfection of eggs, are essential for preventing the introduction of the virus into naive freshwater facilities.

The potential for horizontal transmission within freshwater systems has also been investigated. Hauge and colleagues [44] demonstrated that PRV-1 can establish infection through the gastrointestinal tract, with anal administration of virus leading to systemic infection and subsequent shedding in faeces. This finding suggests that the faecal–oral route may be an important transmission pathway, particularly in recirculating aquaculture systems (RAS) where water reuse can amplify infectious load. Indeed, in Denmark, where PRV-3 (a related genotype) is prevalent in rainbow trout RAS, disease outbreaks have been observed exclusively in RAS facilities, with flow-through systems showing lower prevalence [10]. Although PRV-1 has not been associated with RAS outbreaks to the same extent as PRV-3, the shared biology of these viruses suggests that similar transmission dynamics may operate, warranting heightened surveillance in RAS facilities that culture Atlantic salmon.

Wild Fish Interactions and Environmental Dissemination

The interface between farmed and wild salmonid populations represents a critical dimension of PRV-1 epidemiology, with implications for conservation biology and the management of wild fish stocks. Multiple lines of evidence indicate that PRV-1 transmission occurs from farmed to wild salmonids, and that wild fish can become infected with the virus. Morton and colleagues [56] conducted a comprehensive survey of wild Pacific salmon (Oncorhynchus species) from regions designated as high or low exposure to salmon farms in British Columbia, finding that the proportion of PRV infection in wild fish was significantly related to proximity to farms. PRV was detected in 95% of farmed Atlantic salmon, 37–45% of wild salmon from regions highly exposed to salmon farms, and only 5% of wild salmon from regions furthest from farms [56]. Furthermore, inter-annual PRV infection prevalence declined in both wild and farmed salmon from 2012 to 2013, suggesting that infection rates in wild populations are dynamically linked to those in farmed reservoirs [56].

In Norway, Madhun and colleagues [55] examined returning adult Atlantic salmon captured at sea, finding that 15.8% of 419 fish tested positive for PRV-1. Critically, scale reading revealed that 10% of the sampled fish had escaped from farms, and the prevalence of PRV-1 in wild salmon (8%) was significantly lower than in farm escapees (86%) [55]. Sequencing of the S1 gene from infected fish revealed a mix of genotypes, and the observed increase in PRV prevalence with fish age (a proxy for cumulative exposure risk) suggested that virus transmission may occur on shared oceanic feeding grounds [55]. Similarly, Madhun and colleagues [8] investigated migrating salmon post-smolts in western Norwegian fjord systems, detecting PRV-1 in 4.6% of 651 sampled fish. The prevalence varied among fjord systems, with lowest prevalence (2.3%) in Hardangerfjorden, the system with highest fish farming intensity, suggesting that factors such as water dilution, smolt origin (wild versus hatchery-released), and temporal variation in viral shedding from farms influence exposure risk at the local scale [8].

The susceptibility of non-salmonid species and Pacific salmonids to PRV-1 infection further complicates the epidemiological picture. Experimental challenges have demonstrated that PRV-1 can replicate in Chinook salmon (Oncorhynchus tshawytscha), coho salmon (O. kisutch), and rainbow trout (O. mykiss), although infection does not typically cause notable disease in these species [15]. Brown trout (Salmo trutta) show lower infection prevalence and only mild infections without pathological changes in target organs, while all life stages of Atlantic salmon develop heart lesions characteristic of HSMI [7]. This species-specific susceptibility pattern suggests that brown trout may serve as a reservoir host capable of maintaining PRV-1 in the environment without suffering clinical disease, while Pacific salmonids represent spillover hosts that can become infected but generally do not amplify the virus to levels sufficient to sustain transmission [7, 15]. The implications for disease management are significant: wild salmonid populations in areas with intensive Atlantic salmon farming may experience ongoing exposure to PRV-1, with potential consequences for fitness, migration success, and population viability that warrant continued monitoring [34, 56].

Environmental Persistence and Implications for Geographic Spread

The ability of PRV-1 to persist in the environment and maintain infectivity under varied conditions is a critical determinant of its geographic distribution and the success of biosecurity measures. Wessel and colleagues [24] systematically evaluated the inactivation of PRV-1 using heat, pH extremes, iodine, ultraviolet (UV) radiation, and commercially available disinfectants. They found that standard iodine treatment, high and low pH extremes, and the disinfectant Virocid were effective at inactivating the virus, while a UV dose of at least 50 mJ/cm² was required to achieve inactivation [24]. Notably, PRV-1 demonstrated high resistance to heat treatment, requiring temperatures and exposure times that would pose challenges for

Diagnostic Approaches for PRV-1 Detection and Genotyping

The accurate detection and genotypic characterization of Piscine orthoreovirus-1 (PRV-1) represent a cornerstone of heart and skeletal muscle inflammation (HSMI) control in Atlantic salmon aquaculture. The diagnostic landscape for this pathogen is uniquely challenging, primarily because PRV-1 has resisted all attempts at propagation in continuous cell lines [2, 39, 47]. This fundamental biological constraint has forced the research community to develop and refine a suite of molecular, serological, and histopathological tools that operate in the absence of viral isolation. The diagnostic armamentarium must therefore address not only the presence of the virus but also the critical differentiation between high- and low-virulence genogroups, a distinction that carries profound implications for disease management and biosecurity.

Molecular Detection: The Central Pillar of PRV-1 Diagnosis

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) has emerged as the gold standard for PRV-1 detection, with assays targeting various genomic segments depending on the diagnostic objective. The most widely deployed screening assays target the highly conserved L1 segment, which encodes the core RNA-dependent RNA polymerase, providing robust detection across all known PRV genotypes [26, 32]. This approach has been instrumental in large-scale epidemiological surveys, revealing PRV-1 prevalences exceeding 95% in some farmed populations and identifying the virus in historically archived formalin-fixed paraffin-embedded (FFPE) tissues dating back to 1994 in Chile [9] and as early as 1988 in Norway [19]. The World Organisation for Animal Health (WOAH) has recognized the economic importance of HSMI, and RT-qPCR-based surveillance programs have been central to understanding the global dissemination of this pathogen, which now threatens salmonid production across Norway, Scotland, Chile, Canada, and increasingly, the Russian Federation through its proximity to Norwegian aquaculture zones [34, 35].

However, the diagnostic challenge intensifies when the objective shifts from simple detection to virulence prediction. A landmark study by Vatne et al. [1] demonstrated that Norwegian PRV-1 isolates could be stratified into eight distinct genogroups based on the sequence analysis of five genomic segments (S1, S4, M2, L1, L2) putatively linked to virulence. This stratification showed that two genogroups were high-virulent, two were low-virulent, and four were of indeterminate virulence, with high-virulent isolates more frequently encountered in mid- and northern Norwegian aquaculture regions. This finding catalyzed the development of targeted RT-qPCR assays capable of discriminating between these pathotypes. Siah et al. [54] developed a real-time RT-qPCR assay specifically targeting the PRV-1 S1 segment sequences associated with HSMI, validated against 71 tissue samples from Norway, Chile, and both coasts of Canada. This assay demonstrated the potential to screen populations for carriage of potentially HSMI-causing variants, a capability that is critically important in regions like British Columbia, where PRV-1 is highly prevalent but HSMI has historically been less severe or absent [14, 28].

Genotyping and Phylogenetic Characterization

The genetic characterization of PRV-1 has revealed a complex evolutionary history that is tightly linked to disease emergence. Whole-genome sequencing of PRV-1 isolates has demonstrated that the virus belongs to a novel genus within the Reoviridae family, distinct from both Orthoreovirus and Aquareovirus [12, 32]. Phylogenetic analyses of the S1 segment, which encodes the outer capsid protein σ1 implicated in cell attachment and immunogenicity, have resolved two major sub-genotypes: Sub-genotype Ia, which includes Canadian and some Chilean isolates, and Sub-genotype Ib, which encompasses Norwegian and other Chilean strains [32]. Crucially, Dhamotharan et al. [19] showed that PRV-1 sequences cluster into two distinct monophyletic groups, one associated with HSMI outbreaks and another with low-virulent or asymptomatic infections. This divergence was most pronounced for the S1 and M2 segments, and the co-evolution of this segment pair coincided temporally with the emergence of HSMI in Norway during the late 1990s. The implications for diagnostics are profound: genotyping assays must now interrogate not just the presence of PRV-1 but the specific allelic variants of these critical virulence-associated segments.

The complexity of PRV-1 genotyping is further underscored by the discovery of additional genotypes. PRV-2, the causative agent of erythrocytic inclusion body syndrome (EIBS) in coho salmon, and PRV-3, associated with HSMI-like disease in rainbow trout, represent distinct viral species that can cross-infect Atlantic salmon and induce differential immune responses [4, 27]. PRV-3 has been shown to completely block subsequent PRV-1 infection in experimental settings, suggesting that non-pathogenic replicating PRV-3 could serve as a live-attenuated vaccine [11]. This cross-protective phenomenon necessitates that diagnostic protocols be capable of discriminating between PRV genotypes to avoid misinterpretation in vaccination or surveillance programs. Multiplex RT-qPCR assays targeting genotype-specific regions of S1 or the σ3-encoding segment provide this capability, as demonstrated in studies differentiating PRV-1 from PRV-3 in rainbow trout and Atlantic salmon [5, 10, 16, 23].

In Situ Detection and Cellular Localization

While molecular detection provides quantitative viral load data, it cannot reveal the spatial distribution of virus within tissues or the specific cell types supporting replication. In situ hybridization (ISH) and immunohistochemistry (IHC) have been instrumental in elucidating PRV-1 pathogenesis and refining diagnostic interpretation. Using ISH targeting PRV-1 RNA, Dhamotharan et al. [41] demonstrated that the virus initially replicates in erythrocytes, with peak viral plasma levels preceding the invasion of cardiomyocytes and hepatocytes. This temporal sequence is critical for diagnostic timing; blood sampling during the acute peak phase yields the highest viral loads, whereas heart tissue sampling is more informative during the later pathological phase. Malik et al. [38] employed fluorescent ISH (FISH) to localize PRV-1 RNA within M1 (pro-inflammatory) macrophages in both heart and skeletal muscle, while M2 (anti-inflammatory) macrophages, despite being abundant in heart tissue, did not harbor viral RNA. These findings indicate that macrophages play distinct roles in either harboring or clearing infection, and that ISH-based diagnostics can provide granularity beyond viral load quantification.

The detection of PRV-1 protein by IHC has been particularly valuable for confirming the causal relationship between virus and lesion. Wessel et al. [18] purified PRV-1 particles from infected erythrocytes and used these to demonstrate, via IHC, PRV-1-specific staining within pathological lesions of experimentally infected Atlantic salmon, fulfilling Koch's postulates. Finstad et al. [30] showed that PRV-1 could be detected in large cytoplasmic inclusions within erythrocytes using immunofluorescence and confocal microscopy, with more than 50% of erythrocytes positive in individual fish during peak infection. These cytoplasmic inclusions bear striking resemblance to those described in EIBS, highlighting the diagnostic need to differentiate PRV-1 from PRV-2 and other erythrocytic viruses [27, 30]. IHC has also proven useful in retrospective studies of archival FFPE tissues, confirming the presence of PRV-1 in heart tissues from Chile dating to 1994 [9] and in Canadian samples from the 1990s associated with haemorrhagic kidney syndrome, where dual infections with infectious salmon anaemia virus were identified [31, 52].

Cell Culture Limitations and Ex Vivo Systems

The inability to propagate PRV-1 in continuous cell lines remains the single greatest impediment to vaccine development and functional diagnostic assays [2, 39, 47]. Pham et al. [39] systematically screened 31 fish cell lines derived from embryos, brain, blood, fin, gill, gonads, gut, heart, kidney, liver, skin, and spleen, including cells with endothelial, epithelial, fibroblast, and macrophage morphologies. None supported consistent, transferable PRV-1 amplification, collectively termed the "non-supportive PRV-1 invitrome." Even beating heart cell cultures from rainbow trout, which replicate other cardiac viruses such as salmonid alphavirus and piscine myocarditis virus, failed to sustain PRV-1 or PRV-3 replication [47].

In response to this constraint, an ex vivo culture system using Atlantic salmon erythrocytes was developed. Wessel et al. [45] demonstrated that PRV-1 could be passaged to naïve erythrocytes using lysates from infected cells, with significant increases in viral load confirmed by RT-qPCR and flow cytometry. The formation of cytoplasmic inclusions containing both PRV protein and dsRNA, coupled with the upregulation of antiviral genes (IFN-α, RIG-I, Mx, PKR), confirmed active replication. This ex vivo system has proven invaluable for studying viral replication kinetics and antiviral immune responses, but it is not scalable for routine diagnostic use or vaccine production. More recently, virus-like particle (VLP) platforms have been developed that bypass the need for live virus culture entirely, expressing the six structural proteins (λ1, λ2, μ1, σ1, σ2, σ3) in baculovirus-infected insect cells [48]. These VLPs have demonstrated the ability to induce protective immunity and, importantly, incorporate epitope tags for DIVA (differentiating infected from vaccinated animals) strategies, representing a significant advance for both diagnostic and prophylactic applications.

Serological and Metabolomic Approaches

Serological detection of antibodies against PRV-1 has been explored but remains challenging due to the poor immunogenicity of key viral proteins. The outer capsid protein σ1, which is responsible for viral attachment, exhibits low antibody recognition when expressed in conventional recombinant systems [2]. However, structural modifications such as lipidation or fusion with molecular chaperones can improve epitope exposure, and DNA vaccines encoding σ1 have demonstrated partial efficacy in eliciting antibody responses [2, 42]. Importantly, cross-reactive antibodies have been detected following infection with PRV-3 but not after vaccination with inactivated PRV-1, suggesting that live or replicating vaccines may be necessary to induce a broad serological response suitable for DIVA applications [11]. The development of indirect ELISAs utilizing recombinant σ1 or σ3 proteins is ongoing, but the absence of standardized serological assays limits their current utility for routine surveillance.

Emerging metabolomic approaches offer a complementary diagnostic dimension. Ivanova et al. [49] employed targeted and untargeted metabolomics to identify plasma biomarkers associated with HSMI progression in PRV-1-infected Atlantic salmon. Distinct alterations in lipid metabolism, particularly in lyso-phosphatidylcholines, ceramides, and triglycerides, were observed during disease development, with multi-omics integration of metabolomics and proteomics data enabling the identification of metabolite-protein signatures that correlate with HSMI severity. While not yet validated for field deployment, these approaches hold promise for non-lethal diagnostic screening and for distinguishing between transient viremia and progressing disease.

Diagnostic Considerations for Persistent Infections and Coinfections

A particularly challenging aspect of PRV-1 diagnosis is the virus's ability to establish long-term persistent infections. Kannimuthu et al. [6] demonstrated that PRV-1 RNA remained detectable in blood, spleen, and head kidney for at least 65 weeks post-challenge, with plasma containing genomic dsRNA throughout the entire 65-week trial. During this persistent phase, viral protein production is halted while RNA persists at high levels, creating a diagnostic scenario where RT-qPCR may detect viral RNA in the absence of active replication or disease [22, 25]. This persistence is especially problematic in freshwater hatcheries and broodstock facilities, where PRV-1 can be transmitted vertically via infected eggs, as demonstrated by detection of PRV-1 in eggs from infected broodstock in Iceland [21]. The practical implication is that a single positive RT-qPCR result, particularly from blood or kidney, does not confirm active infection or predict disease risk. Sequential sampling and correlation with histopathological findings are essential for clinical diagnosis.

The ubiquity of PRV-1 and its frequent co-occurrence with other pathogens further complicates diagnosis. Coinfections with Paranucleospora theridion and Kudoa thyrsites are common in Atlantic salmon, and PRV-1 load has been statistically correlated with HSMI lesion severity in the presence of these agents [31]. Similarly, dual infections with salmonid alphavirus (SAV) have been documented, and experimental PRV-1 infection has been shown to partially protect against subsequent SAV infection, potentially masking pancreas disease symptoms [43]. The diagnostic workup for HSMI must therefore include a panel of assays for other relevant pathogens, including SAV, infectious pancreatic necrosis virus (IPNV), and piscine myocarditis virus (PMCV), to avoid attribution errors [29, 31, 47]. The recent detection of PRV-1 in wild salmonids and escaped farmed salmon in British Columbia and Norway also underscores the need for genotyping to trace transmission pathways and to assess the risk to wild populations [8, 21, 55, 56].

In summary, the diagnostic approaches for PRV-1 require a multi-pronged strategy integrating RT-qPCR for detection and genotyping, ISH and IHC for cellular localization and lesion confirmation, and increasingly, metabolomic and serological tools for functional assessment. The absence of cell culture isolation remains a critical gap, but advances in VLP technology and ex vivo erythrocyte culture are beginning to address this. For aquaculture practitioners and regulatory bodies, the key diagnostic objective has shifted from mere detection to virulence differentiation, enabling targeted management of high-risk genogroups and informed implementation of biosecurity measures such as synchronized fallowing, which has been shown to reduce inter-generational viral persistence [3]. Future diagnostic development should prioritize cost-effective, field-deployable genotyping assays, validated serological tests for DIVA-capable vaccines, and standardized protocols for distinguishing active replication from persistent viral RNA carriage.

Vaccine Development Strategies and Challenges for Piscine Orthoreovirus

The development of effective vaccines against Piscine orthoreovirus (PRV) represents one of the most formidable challenges in contemporary salmonid aquaculture vaccinology. Despite over two decades of research since the first description of heart and skeletal muscle inflammation (HSMI) in 1999 [34, 35], no commercial vaccine has been licensed for use against any PRV genotype. This immunological impasse stems from a confluence of biological, technical, and epidemiological obstacles that have collectively frustrated conventional vaccine development pipelines. The World Organisation for Animal Health (WOAH) has recognized HSMI as a significant transboundary disease of salmonids, and the Food and Agriculture Organization (FAO) has highlighted the economic threat posed by PRV to global aquaculture production. Understanding the multidimensional challenges and the innovative strategies being deployed to surmount them is essential for contextualizing the current state of PRV vaccinology.

The Fundamental Obstacle: Absence of a Permissive Cell Culture System

Perhaps the most intractable barrier to PRV vaccine development is the complete inability to propagate the virus in any continuous cell line [2, 39]. This deficiency has profound implications, as it precludes the production of inactivated whole-virus vaccines through traditional规模化 methods, impedes live-attenuated vaccine development, and severely constrains basic virological research. Comprehensive screening of 31 fish cell lines derived from embryos, brain, blood, fin, gill, gonads, gut, heart, kidney, liver, skin, and spleen, encompassing endothelial, epithelial, fibroblast, and macrophage morphologies, failed to identify any line capable of supporting PRV-1 amplification [39]. Even beating heart cell cultures derived from different developmental stages of rainbow trout, which might have been expected to provide a more physiologically relevant environment, proved refractory to infection with both PRV-1 and PRV-3 [47]. This universal non-permissiveness of available cell lines, termed the "non-supportive PRV-1 invitrome," has forced researchers to rely on in vivo propagation in live salmon, followed by laborious purification of viral particles from infected erythrocytes [18, 53]. While this approach has enabled experimental vaccine studies, it is entirely impractical for commercial-scale vaccine production, as it requires maintenance of infected fish populations, extensive blood collection, andultracentrifugation-based purification protocols that are neither scalable nor economically viable.

The Ex Vivo Paradigm and Its Limitations

The discovery that salmonid erythrocytes serve as the primary target cells for PRV replication [30, 45] opened an alternative avenue for virus propagation. An ex vivo cultivation system using Atlantic salmon red blood cells (RBCs) demonstrated that PRV could be passaged to naïve erythrocytes, with significant increases in viral load observed by RT-qPCR and flow cytometry coinciding with the formation of cytoplasmic inclusions resembling viral factories [45]. This system has proven invaluable for studying viral replication mechanisms and host antiviral responses [25, 36]. Crucially, however, the ex vivo system suffers from limited scalability, the requirement for continuous sourcing of primary erythrocytes from live fish, and the inability to achieve the high titers necessary for commercial vaccine formulation. Furthermore, the RBC system does not support indefinite passage, and the yield of purified virus particles remains modest compared to what would be required for an industrial vaccine production process.

Genotypic Diversity and Virulence Variation as Vaccine Design Challenges

The genetic heterogeneity of PRV presents another substantial obstacle to vaccine development. PRV-1 isolates circulating in Norwegian aquaculture cluster into at least eight distinct genogroups based on combinations of five genomic segments (S1, S4, M2, L1, and L2) [1]. Crucially, these genogroups differ dramatically in their virulence potential, with some classified as high-virulent and others as low-virulent or of unknown virulence [1, 13]. The geographic distribution of these variants is non-random, with high-virulent isolates occurring at higher frequency in mid- and northern Norway [1]. This diversity necessitates that any vaccine candidate must provide broad protection across multiple virulent genogroups, or alternatively, that vaccines are tailored to regional variant prevalence.

Phylogenetic analyses have revealed that PRV-1 strains from the North American Pacific Coast and Faroe Islands are more closely related to low-virulent precursor strains that predate the emergence of clinical HSMI in Norway [19]. The co-evolution of the S1-M2 segment pair, which encodes the outer capsid proteins σ1 and μ1 respectively, appears to have been pivotal in the emergence of high virulence [19]. This evolutionary trajectory complicates vaccine design, as protective immune responses must target epitopes that may vary between virulent and non-virulent strains. The development of RT-qPCR assays capable of differentiating HSMI-associated variants from low-virulence strains represents a critical diagnostic tool for targeted vaccination strategies [54], but does not directly address the challenge of inducing broad cross-protective immunity.

The Immunological Conundrum of the σ1 Protein

The outer capsid protein σ1, which mediates viral attachment to host cells, has been hypothesized to be the primary target of neutralizing antibodies based on homology with mammalian orthoreoviruses [2, 12]. However, recombinant σ1 expressed in conventional systems exhibits poor antibody recognition, suggesting that the native conformation is critical for epitope exposure [2]. Structural modifications such as lipidation or fusion with molecular chaperones have been shown to improve epitope presentation, indicating that antigen engineering will be essential for subunit vaccine development [2]. The σ1 protein may also mediate attachment to sialic acid residues on host cells, and conserved amino acid residues critical for this interaction have been identified in PRV σ1 [12], but the functional relevance of this binding for neutralization susceptibility remains incompletely understood.

Beyond σ1, the other surface proteins, μ1 and σ3, may contribute to protective immunity. The PRV μ1 protein is predicted to retain cleavage and myristoylation sites required for endosomal membrane penetration during infection [12], and antibodies targeting these functional domains could theoretically interfere with viral entry. The σ3 protein contains a conserved zinc finger motif [12], and while its antigenic properties are less well-characterized, it may contribute to the overall immune response. The non-structural protein μNS, which organizes viral factories [42], has emerged as an important vaccine antigen, particularly in DNA vaccine constructs where intracellular expression can drive potent cellular immune responses [2, 42].

Current Vaccine Strategies: Inactivated Whole Virus Approaches

The most direct vaccine strategy, inactivated whole-virus preparations, has been explored with partial success. A seminal study demonstrated that a single immunization with adjuvanted, formalin-inactivated PRV-1 purified from infected erythrocytes induced significant protection against HSMI in Atlantic salmon [53]. When fish were challenged by intraperitoneal injection, vaccinated animals showed markedly reduced viral loads and histopathological lesions compared to unvaccinated controls. However, when challenged by the more natural cohabitation route (i.e., exposure to infected shedder fish), protection was only moderate [53]. This discrepancy between injection and cohabitation challenge models has been observed consistently and suggests that the inactivated vaccine may be more effective at controlling systemic infection than at preventing mucosal entry or early replication.

The failure of inactivated PRV-1 vaccine to elicit robust antibody responses is noteworthy. In comparative studies, adjuvanted inactivated PRV-1 did not induce detectable antibodies against the σ1 protein [11], and the immune response was characterized by activation of genes involved in membrane trafficking and signaling pathways rather than the typical antiviral response associated with replicating virus [4]. This muted immunogenicity likely reflects the inability of inactivated particles to engage pattern recognition receptors effectively or to stimulate the full repertoire of innate immune pathways that replicating virus would activate [36, 37]. The lack of a robust adaptive response may also explain the inconsistent protection observed in field settings and underlines the need for more potent adjuvant systems or alternative vaccine platforms.

DNA Vaccines: Harnessing Intracellular Antigen Expression

DNA vaccines encoding PRV proteins have shown promise by leveraging the host's own cellular machinery for antigen production, thereby bypassing some of the limitations associated with inactivated vaccines. In two experimental trials, DNA vaccines expressing the non-structural protein μNS either alone or in combination with σ1 induced moderate protection against HSMI [42]. The combination vaccine was associated with an increasing trend in lymphocyte marker gene expression in the spleen, suggesting enhanced cellular immune activation [42]. The protective effect was characterized by delayed viral kinetics rather than complete prevention of infection, indicating that DNA vaccination may shift the balance in favor of the host immune response without achieving sterilizing immunity.

The mechanistic basis for DNA vaccine protection likely involves the induction of cytotoxic T lymphocyte (CTL) responses. PRV infection naturally elicits a strong CTL response characterized by upregulation of CD8α, CD8β, granzyme-A, and MHC class I expression [37, 38]. This cellular response is critical for viral clearance, particularly during the phase when PRV infects cardiomyocytes [38]. DNA vaccines are particularly adept at driving MHC class I-restricted antigen presentation, making them well-suited to augment this arm of the immune response. The finding that μNS, which organizes viral factories, is an effective DNA vaccine antigen [42] suggests that targeting non-structural proteins that are abundantly expressed during the active replication phase may focus the CTL response on infected cells.

The considerable variability in immune responses observed among individual fish following DNA vaccination [2] represents a significant hurdle for commercial application. This variability may stem from differences in plasmid uptake, expression kinetics, or genetic heterogeneity in immune response genes across fish populations. The identification of host genotype effects on PRV disease susceptibility [46] raises the possibility that vaccine responsiveness may also be genetically determined, necessitating tailored vaccination strategies for different salmonid stocks.

Cross-Protection and the Live-Attenuated Vaccine Paradigm

One of the most intriguing developments in PRV vaccinology is the demonstration that infection with non-pathogenic PRV genotypes can confer protection against virulent PRV-1 challenge. Specifically, primary infection with PRV-3, which causes mild or subclinical infection in Atlantic salmon, completely blocked subsequent PRV-1 infection and prevented HSMI development [11]. PRV-2 provided only partial protection, reducing infection levels and pathology in a subset of individuals [11]. The cross-protection mediated by PRV-3 appears to be driven by the induction of a potent and early innate antiviral response that overlaps with the response triggered by PRV-1, effectively pre-empting the pathogen's ability to establish infection in target tissues [4]. Transcriptomic analyses revealed that PRV-3 induced a stronger antiviral response than PRV-1 at two weeks post-infection, despite similar viral RNA replication levels, suggesting that the timing and magnitude of innate immune activation are critical determinants of protection [4].

This cross-protection phenomenon raises the possibility of developing live-attenuated vaccines based on naturally occurring low-virulence PRV strains or genetically engineered attenuated variants. The identification of low-virulent PRV-1 genogroups in Norwegian aquaculture [1] and the demonstration that historical PRV-1 isolates from the pre-HSMI era cause only mild pathology [13] provide potential starting points for live vaccine development. However, the use of live vaccines raises important safety concerns, including the potential for reversion to virulence, recombination with field strains, and environmental persistence. The long-term persistence of PRV-1 infection in Atlantic salmon, with viral RNA detectable in erythrocytes for months to years after initial infection [6, 22], further complicates the live vaccine approach, as vaccinated fish could become reservoirs for virus shedding.

Virus-Like Particle Based Vaccines and DIVA Strategies

A recent and technologically sophisticated approach involves the development of virus-like particles (VLPs) that mimic the structure of authentic PRV virions without containing infectious genetic material. A seminal study reported the production of recombinant VLPs by co-expressing the six structural proteins of PRV-1 (λ1, λ2, μ1, σ1, σ2, σ3) using a baculovirus-based expression system in insect cells [48]. This approach bypasses the requirement for cell culture propagation of the virus while presenting antigens in a multivalent, conformationally authentic format. The native VLP vaccine (VLP6n) significantly reduced viral loads in Atlantic salmon challenged with PRV-1, demonstrating proof-of-concept for this platform [48].

Critically, the VLP platform enables the implementation of DIVA (Differentiating Infected from Vaccinated Animals) strategies, which are essential for disease surveillance and eradication programs. By engineering a cmyc epitope tag into the σ1 protein, researchers created a VLP vaccine (VLP6c) that elicits antibodies against the heterologous tag, allowing serological discrimination between vaccinated fish and those naturally infected with wild-type PRV [48]. This innovation could facilitate the deployment of vaccination programs without compromising the sensitivity of surveillance systems for detecting circulating virus.

The Challenge of Mucosal Immunity and Early Infection

The natural route of PRV transmission appears to involve entry through the gastrointestinal tract, as demonstrated by experimental intubation studies [44]. Anal administration of PRV resulted in efficient infection, while oral administration led to slower viral kinetics and no evidence of HSMI [44]. The detection of PRV in feces with kinetics corresponding to blood infection suggests that fecal-oral transmission may be an important route of spread [44]. This route of entry poses particular challenges for vaccine development, as mucosal immunity at the intestinal epithelium may be required to block infection at the point of entry. Most current vaccine strategies (inactivated, DNA, VLP) are administered by intraperitoneal injection and may not efficiently induce mucosal immune responses. The development of oral or immersion vaccines capable of eliciting intestinal immunity could theoretically prevent initial infection, but such approaches remain nascent for PRV.

Antigenic Cross-Reactivity and Species Specificity

The three PRV genotypes exhibit distinct host species preferences and disease phenotypes: PRV-1 causes HSMI in Atlantic salmon, PRV-2 causes erythrocytic inclusion body syndrome in coho salmon, and PRV-3 induces HSMI-like disease in rainbow trout [4, 5, 16, 26, 27]. Despite these differences, there is substantial antigenic cross-reactivity between genotypes. Rabbit antisera raised against PRV-1 cross-react with PRV-3 proteins [20], and PRV-3 infection induces antibodies that recognize the PRV-1 σ1 protein [11]. This cross-reactivity is consistent with the high amino acid identity between genotypes, ranging from 79.1% for σ3 to 96.7% for λ1 [20]. The conservation of antigenic epitopes across genotypes provides a rationale for developing pan-genotype vaccines, but also raises the possibility that pre-existing immunity to one genotype could influence the outcome of infection with another, as demonstrated by PRV-3 cross-protection against PRV-1 [11].

Immune Evasion and the Persistence Problem

PRV has evolved sophisticated mechanisms to persist within the host, and these may subvert vaccine-induced immunity. The virus establishes a productive, persistent infection characterized by high-level viral RNA in erythroid progenitor cells and erythrocytes, but low-level protein production [22]. During the persistent phase, the host immune response returns to basal levels [6], suggesting that PRV actively suppresses or evades immune surveillance. The observation that PRV may interfere with antiviral signaling pathways [2] and that differences in interferon regulatory factor (IRF) expression between RBCs and other cell types may affect viral propagation [36] highlights the complexity of host-virus interactions that vaccines must overcome.

Future Directions and Rational Vaccine Design

The accumulated evidence points toward several priority areas for future vaccine development. First, the optimization of antigen presentation through structural modification of σ1, either by lipidation, chaperone fusion, or presentation on VLP scaffolds, is likely to enhance neutralizing antibody responses [2, 48]. Second, the combination of structural and non-structural antigens in multi-component vaccines may engage both humoral and cellular arms of the immune response, as suggested by the enhanced protection observed with μNS-σ1 DNA vaccines [42]. Third, the identification of host genetic factors that influence vaccine responsiveness [46] could enable the development of selectively bred salmon populations with improved vaccine take rates. Fourth, the development of scalable production systems, whether through VLP technology or other recombinant expression platforms, is essential for commercial viability. Fifth, the incorporation of appropriate adjuvants capable of driving the balanced Th1-type response characterized by IFN-γ, IL-12, and CTL activation [37] will be critical for vaccine efficacy.

The emerging picture is that no single vaccine platform will provide a universal solution to PRV infection. Instead, a portfolio approach combining inactivated vaccines for initial priming, DNA or VLP vaccines for boosting cellular immunity, and potentially live-attenuated vaccines for durable protection may be required. The success of PRV-3 cross-protection [11] suggests that carefully controlled exposure to non-pathogenic PRV strains could be integrated into management strategies, though the risks of viral persistence and shedding must be carefully evaluated. Continued investment in basic research on PRV replication mechanisms, immune evasion strategies, and host genetic determinants of resistance will provide the foundation for rational vaccine design. The economic imperative is clear: HSMI causes substantial mortality and morbidity in farmed Atlantic salmon [34], with cumulative mortality reaching 30% and morbidity approaching 100% in affected populations [33]. The development of effective vaccines is not merely a scientific challenge but a critical requirement for the sustainability of global salmon aquaculture.

Host Immune Response and Antiviral Mechanisms in PRV Infection

The host immune response to Piscine orthoreovirus (PRV) infection represents a complex, multi-phasic interplay between viral replication strategies and the salmonid immune system, encompassing both innate and adaptive arms. Understanding these mechanisms is critical for elucidating the pathogenesis of heart and skeletal muscle inflammation (HSMI) and for developing effective prophylactic strategies, particularly given the global economic significance of this disease in Atlantic salmon aquaculture, a sector closely monitored by organizations such as the World Organisation for Animal Health (WOAH) due to its transboundary implications [34, 35].

Innate Immune Recognition and the Interferon Response

The initial host defense against PRV hinges on the recognition of viral pathogen-associated molecular patterns (PAMPs), particularly double-stranded RNA (dsRNA), a hallmark of reovirus replication. Transcriptomic analyses have revealed that Atlantic salmon red blood cells (RBCs), the primary target cells for PRV, express a surprisingly broad repertoire of pattern recognition receptors (PRRs), including RIG-I-like receptors (RLRs) and Toll-like receptors (TLRs), positioning them as active immune sentinels rather than passive gas-exchange cells [25, 36]. Upon PRV-1 infection, a robust and sustained innate antiviral response is mounted within RBCs, characterized by the significant upregulation of genes encoding interferon (IFN)-α, RIG-I, Mx, and protein kinase R (PKR) [30, 36, 45]. This response is not merely transient; it persists for many weeks, long after the peak of viral replication, suggesting a sustained antiviral state within the erythrocyte population [25].

A critical finding is the differential activation of interferon regulatory factors (IRFs) across cell types. In PRV-1-exposed RBCs, IRF1 is significantly induced, whereas in head kidney cells (SHK-1), the response is dominated by IRF3 and IRF7 [36]. This divergence in IRF usage may have profound implications for viral propagation and the kinetics of the immune response. The RIG-I-like receptor 3 (RLR3), a cytosolic dsRNA sensor, is consistently induced across all susceptible cell types, underscoring its central role in PRV detection [36]. The antiviral state induced in RBCs includes the upregulation of potential inhibitors of translation, such as PKR, which may contribute to the observed halt in viral protein production even as viral RNA persists during the chronic phase [25].

The Role of Red Blood Cells as Immune Effectors

The discovery that salmonid RBCs are not only major targets but also active participants in the antiviral response has revolutionized our understanding of PRV pathogenesis. These nucleated cells are capable of mounting a pluripotent immune response, expressing genes for chemotactic factors, cytokine receptors, and a wide array of antiviral effectors [25, 36]. During the acute phase of infection, PRV-1 replicates extensively within erythrocytes, forming large cytoplasmic inclusions that serve as viral factories [30]. This phase is characterized by high viral loads in the blood, often with more than 50% of erythrocytes testing positive for the virus [30]. The infected RBCs then trigger a classical antiviral transcriptional program, including the upregulation of ifn-α, rig-i, mx, and pkr [37, 45].

The immune response in RBCs is not uniform across PRV genotypes. A comparative study demonstrated that PRV-3, which is non-pathogenic in Atlantic salmon, triggers a significantly stronger and earlier antiviral response in blood cells than PRV-1, despite achieving similar levels of viral RNA replication at two weeks post-infection [4]. This potent, early activation of the same innate immune pathways by PRV-3 is hypothesized to be the mechanism by which it confers cross-protection against subsequent PRV-1 infection and HSMI [4, 11]. In contrast, PRV-2 and inactivated PRV-1 vaccines fail to induce this typical antiviral signature, instead upregulating genes involved in membrane trafficking and signaling pathways [4]. This suggests that the timing and magnitude of the innate response are critical determinants of disease outcome, and that PRV-1 may have evolved to delay or subvert this response, giving it an evolutionary advantage in disseminating to the heart [4].

Adaptive Immune Responses: The Th1 and CTL Axis

As the infection progresses from the acute erythrocyte phase to the establishment of HSMI in the heart, the adaptive immune system becomes paramount. The histopathological hallmark of HSMI is a severe, diffuse mononuclear myocarditis and epicarditis, characterized by a massive infiltration of CD8+ cytotoxic T lymphocytes (CTLs) [16, 38, 41]. Gene expression analyses consistently show a strong upregulation of CD8α, CD8β, and granzyme-A in the hearts of infected fish, indicating a robust CTL-mediated immune response targeting PRV-infected cells [37, 38]. This cellular response is accompanied by increased expression of major histocompatibility complex class I (MHC-I) molecules, transporters associated with antigen processing (TAP), and proteasome components, all of which are essential for the presentation of viral peptides to CTLs [37].

The adaptive response is further characterized as a Th1-type response. Infected Atlantic salmon show transcriptional upregulation of T-cell receptor alpha and beta chains (TCRα, TCRβ), CD2, interleukin-2 (IL-2), CD4-1, interferon-gamma (IFN-γ), IL-12, and IL-18 [37]. This Th1 polarization is a classic host defense strategy against intracellular viral pathogens, promoting CTL activation and macrophage-mediated inflammation. The presence of CD8+ cells is directly correlated with the clearance of PRV-1 from heart tissue. In situ hybridization and immunohistochemistry have demonstrated that PRV-1 RNA and protein are present in cardiomyocytes and that the subsequent infiltration of CD8+ granzyme-producing cells is temporally associated with a decline in viral load and the resolution of inflammation [38, 41].

Macrophage Polarization and Tissue Repair

The role of macrophages in PRV infection is nuanced and biphasic. While initial hypotheses suggested that M1 (pro-inflammatory) macrophages might drive the early pathology, detailed studies have shown otherwise. In experimentally induced HSMI, M1 macrophages are present only in small numbers within heart lesions and do not appear to be the primary drivers of the initial inflammatory response [38]. Instead, the heart tissue is dominated by M2 (anti-inflammatory) macrophages, which are widely scattered and more abundant than M1 cells [38, 41]. These M2 macrophages do not contain PRV-1 RNA, suggesting they are not a major site of viral replication but rather are recruited to the site of injury to promote tissue repair and resolution of inflammation [38].

The shift towards an M2 phenotype is a critical step in recovery from HSMI. The presence of arginase-2-positive, macrophage-like cells in the heart indicates the onset of regenerative processes [41]. This is consistent with the clinical observation that HSMI lesions can resolve over time, with the heart tissue undergoing regeneration and the fish recovering [6, 38]. The rapid clearance of PRV-1 from heart tissue following the CTL response, coupled with the activity of M2 macrophages, allows for a relatively fast recovery following the peak of disease [38].

Viral Persistence and Immune Evasion

Despite a robust immune response, PRV-1 is notorious for establishing a persistent infection that can last for the lifetime of the fish [6, 22, 37]. The persistent phase is characterized by high levels of viral RNA but very low levels of viral protein production [22, 25]. The virus appears to find sanctuary in erythroid progenitor cells within the kidney, where it can maintain a productive, low-grade infection [22]. The presence of viral genomic dsRNA in the plasma throughout the persistent phase indicates that new viral particles are continuously produced and released, even in the absence of clinical disease [22].

The mechanisms by which PRV-1 evades immune clearance are not fully understood but are an active area of research. One hypothesis is that the virus interferes with antiviral signaling pathways [2]. The observation that PRV-1 induces a less potent and slower innate response compared to the non-pathogenic PRV-3 suggests that PRV-1 may have evolved mechanisms to dampen the host's early warning system [4]. Furthermore, the ability of the virus to persist in RBCs, which are long-lived cells with a limited capacity for antigen presentation, may allow it to avoid CTL-mediated killing [25]. The downregulation of viral protein production during the persistent phase also reduces the availability of targets for the adaptive immune response [22]. This long-term persistence has significant implications for disease management, as persistently infected fish can serve as a reservoir for transmission to naïve cohorts, even in the absence of clinical signs [6, 7].

Cross-Protection and Vaccine-Induced Immunity

The concept of cross-protection has emerged as a promising avenue for vaccine development. Primary infection with PRV-3, which is non-pathogenic in Atlantic salmon, completely blocks subsequent PRV-1 infection and prevents the development of HSMI [11]. This protection is mediated by the early and potent activation of the innate antiviral response, as well as the induction of cross-reactive antibodies against the PRV-1 σ1 protein [4, 11]. In contrast, PRV-2 provides only partial protection, and inactivated PRV-1 vaccines have shown inconsistent results, often failing to elicit robust antibody or T-cell responses [2, 11, 53].

The failure of inactivated vaccines to induce strong immunity is a major hurdle. Formalin-inactivated PRV-1 vaccines have demonstrated partial protection, particularly against injection challenge, but have been less effective against natural cohabitation challenge [53]. This is likely due to poor antigen presentation and the inability to induce a robust CTL response, which is crucial for clearing the virus from the heart [2]. DNA vaccines encoding the σ1 and μNS proteins have shown more promise, as they facilitate endogenous antigen expression and presentation via MHC class I, leading to a more comprehensive cellular immune response [2, 42]. The development of virus-like particle (VLP) vaccines represents another innovative strategy. A recent study demonstrated that a VLP vaccine expressing the six structural proteins of PRV-1 could significantly reduce viral loads in challenged fish and, when tagged with a c-myc epitope, could be used in a DIVA (Differentiating Infected from Vaccinated Animals) strategy [48].

The Influence of Host and Viral Genetics on the Immune Response

The outcome of PRV infection is not solely determined by the virus; host genetics play a critical role. Controlled challenge experiments have revealed that different Atlantic salmon stocks exhibit discrete levels of heart inflammation in response to the same PRV-1 isolate [46]. For instance, New Brunswick Tobique River salmon showed significantly less heart inflammation than Mowi-McConnell salmon from Western Canada, which in turn showed less inflammation than Mowi salmon from Scotland [46]. This indicates that host genotype is a major determinant of disease severity and that genetic selection for resistance could be a viable disease management strategy.

Concurrently, viral genetics are equally important. PRV-1 isolates differ markedly in their virulence, with high-virulent isolates from Norway capable of inducing severe HSMI, while low-virulent isolates from Canada and historical Norwegian samples cause only mild pathology [1, 13, 14, 19]. This difference in virulence is linked to specific genomic segments, particularly S1 and M2, which encode the outer capsid protein σ1 and the μ2 protein, respectively [13, 19]. The μ2 protein of mammalian orthoreoviruses is known to play a role in interferon-β signaling and the formation of viral inclusion bodies [12]. The co-evolution of the S1-M2 segment pair is temporally linked to the emergence of HSMI in Norway, suggesting that specific mutations in these proteins have allowed the virus to more effectively evade or modulate the host immune response [19]. The presence of a low-virulence PRV-1 genogroup in Chile since at least 1994, predating the first HSMI outbreak by 17 years, further supports the hypothesis that viral evolution is a key driver of disease emergence [9].

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