Heart inflammation and piscine orthoreovirus genotype-1 in Pacific Canada Atlantic salmon net-pen farms: 2016-2019.

Piscine orthoreovirus genotype-1 (PRV-1) is a virus commonly associated with Atlantic salmon aquaculture with global variability in prevalence and association with disease. From August 2016 to November 2019, 2,070 fish sampled at 64 Atlantic salmon net-pen farm sites during 302 sampling events from British Columbia, Canada, were screened for PRV-1 using real-time qPCR. Nearly all populations became PRV-1 positive within one year of seawater entry irrespective of location, time of stocking, or producer. Cohorts became infected between 100-300 days at sea in > 90% of repeatedly sampled sites and remained infected until harvest (typically 500-700 days at sea). Heart inflammation, which is sometimes attributed to PRV-1, was also assessed in 779 production mortalities from 47 cohorts with known PRV status. Mild heart inflammation was common in mortalities from both PRV + and PRV- populations (67% and 68% prevalence, respectively). Moderate and severe lymphoplasmacytic heart inflammation was rare (11% and 3% prevalence, respectively); however, mainly arose (66 of 77 occurrences) in populations with PRV-1. Detection of PRV-1 RNA was also accomplished in water and sediment for which methods are described. These data cumulatively identify that PRV-1 ubiquitously infects farmed Atlantic salmon in British Columbia during seawater production but only in rare instances correlates with heart inflammation.

Innate antiviral defense demonstrates high energetic efficiency in a bony fish.

BACKGROUND: Viruses can impose energetic demands on organisms they infect, in part by hosts mounting resistance. Recognizing that oxygen uptake reliably indicates steady-state energy consumption in all vertebrates, we comprehensively evaluated oxygen uptake and select transcriptomic messaging in sockeye salmon challenged with either a virulent rhabdovirus (IHNV) or a low-virulent reovirus (PRV). We tested three hypotheses relating to the energetic costs of viral resistance and tolerance in this vertebrate system: (1) mounting resistance incurs a metabolic cost or limitation, (2) induction of the innate antiviral interferon system compromises homeostasis, and (3) antiviral defenses are weakened by acute stress. RESULTS: IHNV infections either produced mortality within 1-4 weeks or the survivors cleared infections within 1-9 weeks. Transcription of three interferon-stimulated genes (ISGs) was strongly correlated with IHNV load but not respiratory performance. Instead, early IHNV resistance was associated with a mean 19% (95% CI = 7-31%; p = 0.003) reduction in standard metabolic rate. The stress of exhaustive exercise did not increase IHNV transcript loads, but elevated host inflammatory transcriptional signaling up to sevenfold. For PRV, sockeye tolerated high-load systemic PRV blood infections. ISG transcription was transiently induced at peak PRV loads without associated morbidity, microscopic lesions, or major changes in aerobic or anaerobic respiratory performance, but some individuals with high-load blood infections experienced a transient, minor reduction in hemoglobin concentration and increased duration of excess post-exercise oxygen consumption. CONCLUSIONS: Contrary to our first hypothesis, effective resistance against life-threatening rhabdovirus infections or tolerance to high-load reovirus infections incurred minimal metabolic costs to salmon. Even robust systemic activation of the interferon system did not levy an allostatic load sufficient to compromise host homeostasis or respiratory performance, rejecting our second hypothesis that this ancient innate vertebrate antiviral defense is itself energetically expensive. Lastly, an acute stress experienced during testing did not weaken host antiviral defenses sufficiently to promote viral replication; however, a possibility for disease intensification contingent upon underlying inflammation was indicated. These data cumulatively demonstrate that fundamental innate vertebrate defense strategies against potentially life-threatening viral exposure impose limited putative costs on concurrent aerobic or energetic demands of the organism.

Evaluation of histopathology, PCR, and qPCR to detect Mikrocytos mackini in oysters Crassostrea gigas using Bayesian latent class analysis.

Latent class analysis (LCA) is a common method to evaluate the diagnostic sensitivity (DSe) and specificity (DSp) for pathogen detection assays in the absence of a perfect reference standard. Here we used LCA to evaluate the diagnostic accuracy of 3 tests for the detection of Mikrocytos mackini in Pacific oysters Crassostrea gigas: conventional polymerase chain reaction (PCR), real-time quantitative PCR (qPCR), and histopathology. A total of 802 Pacific oysters collected over 12 sampling events from 9 locations were assessed. Preliminary investigations indicated that standard LCA assumptions of test independence and constant detection accuracy across locations were likely unrealistic. This was mitigated by restructuring the LCA in a Bayesian framework to include test-derived knowledge about pathogen prevalence and load for categorizing populations into 2 classes of infection severity (low or high) and assessing separate DSe and DSp estimates for each class. Median DSp estimates were high (>96%) for all 3 tests in both population classes. DSe estimates varied between tests and population classes but were consistently highest for qPCR (87-99%) and lowest for histopathology (21-51%). Acknowledging that detection of M. mackini may be fitted to multiple diagnostic and management purposes, qPCR had the highest DSe while maintaining similar DSp to both conventional PCR and histopathology and thus is generally well-suited to most applications.

Piscine orthoreovirus: Biology and distribution in farmed and wild fish.

Piscine orthoreovirus (PRV) is a common and widely distributed virus of salmonids. Since its discovery in 2010, the virus has been detected in wild and farmed stocks from North America, South America, Europe and East Asia in both fresh and salt water environments. Phylogenetic analysis suggests three distinct genogroups of PRV with generally discrete host tropisms and/or regional patterns. PRV-1 is found mainly in Atlantic (Salmo salar), Chinook (Oncorhynchus tshawytscha) and Coho (Oncorhynchus kisutch) Salmon of Europe and the Americas; PRV-2 has only been detected in Coho Salmon of Japan; and PRV-3 has been reported primarily in Rainbow Trout (Oncorhynchus mykiss) in Europe. All three genotypes can establish high-load systemic infections by targeting red blood cells for principal replication. Each genotype has also demonstrated potential to cause circulatory disease. At the same time, high-load PRV infections occur in non-diseased salmon and trout, indicating a complexity for defining PRV's role in disease aetiology. Here, we summarize the current body of knowledge regarding PRV following 10 years of study.

Factors associated with severity of naturally occurring piscirickettsiosis in netpen- and tank-reared juvenile Atlantic salmon at a research aquarium in western Canada.

The opportunistic examination of factors associated with an outbreak of piscirickettsiosis (SRS) is described in Atlantic salmon Salmo salar post-smolts held in an open netpen or in tanks supplied with raw sea water at a research aquarium in western Canada. During the outbreak, seawater temperature was significantly higher and salinity significantly lower in the netpen compared with the tanks. Mortality in the netpen began approximately 3 weeks prior to that in the tanks, and cumulative mortality in the netpen (34%) was significantly higher than in the tanks (12%). Piscirickettsia salmonis was confirmed by qPCR in tissues from moribund and dead fish and from colonies grown on enriched blood agar medium. Neither P. salmonis nor SRS were observed in salmon held concurrently in UV-irradiated sea water. The elevated mortality was curtailed by treatment with oxytetracycline. These observations further indicate warmer, less saline and periodically hypoxic seawater are risk factors for SRS. UV irradiation of sea water is shown to be a tool for SRS management in fish-holding facilities.

Consequences of Piscine orthoreovirus genotype 1 (PRV-1) infections in Chinook salmon (Oncorhynchus tshawytscha), coho salmon (O. kisutch) and rainbow trout (O. mykiss).

Piscine orthoreovirus genotype 1 (PRV-1) is the causative agent of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar L.). The virus has also been found in Pacific salmonids in western North America, raising concerns about the risk to native salmon and trout. Here, we report the results of laboratory challenges using juvenile Chinook salmon, coho salmon and rainbow trout injected with tissue homogenates from Atlantic salmon testing positive for PRV-1 or with control material. Fish were sampled at intervals to assess viral RNA transcript levels, haematocrit, erythrocytic inclusions and histopathology. While PRV-1 replicated in all species, there was negligible mortality in any group. We observed a few erythrocytic inclusion bodies in fish from the PRV-1-infected groups. At a few time points, haematocrits were significantly lower in the PRV-1-infected groups relative to controls, but in no case was anaemia noted. The most common histopathological finding was mild, focal myocarditis in both the non-infected controls and PRV-1-infected fish. All cardiac lesions were judged mild, and none were consistent with those of HSMI. Together, these results suggest all three species are susceptible to PRV-1 infection, but in no case did infection cause notable disease in these experiments.

Sockeye salmon demonstrate robust yet distinct transcriptomic kidney responses to rhabdovirus (IHNV) exposure and infection.

Aquatic rhabdoviruses are globally significant pathogens associated with disease in both wild and cultured fish. Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus that causes the internationally regulated disease infectious hematopoietic necrosis (IHN) in most species of salmon. Yet not all naïve salmon exposed to IHNV become diseased, and the mechanisms by which some individuals evade or rapidly clear infection following exposure are poorly understood. Here we used RNA-sequencing to evaluate transcriptomic changes in sockeye salmon, a keystone species in the North Pacific and natural host for IHNV, to evaluate the consequences of IHNV exposure and/or infection on host cell transcriptional pathways. Immersion challenge of sockeye salmon smolts with IHNV resulted in approximately 33% infection prevalence, where both prevalence and viral kidney load peaked at 7 days post challenge (dpc). De novo assembly of kidney transcriptomes at 7 dpc revealed that both infected and exposed but noninfected individuals experienced substantial transcriptomic modification; however, stark variation in gene expression patterns were observed between exposed but noninfected, infected, and unexposed populations. GO and KEGG pathway enrichment in concert with differential expression analysis identified that kidney responses in exposed but noninfected fish emphasised a global pattern of transcriptional down-regulation, particularly for pathways involved in DNA transcription, protein biosynthesis and macromolecule metabolism. In contrast, transcriptomes of infected fish demonstrated a global emphasis of transcriptional up-regulation highlighting pathways involved in antiviral response, inflammation, apoptosis, and RNA processing. Quantitative PCR was subsequently used to highlight differential and time-specific regulation of acute phase, antiviral, inflammatory, cell boundary, and metabolic responsive transcripts in both infected and exposed but noninfected groups. This data demonstrates that waterborne exposure with IHNV has a dramatic effect on the sockeye salmon kidney transcriptome that is discrete between resistant and acutely susceptible individuals. We identify that metabolic, acute phase and cell boundary pathways are transcriptionally affected by IHNV and kidney responses to local infection are highly divergent from those generated as part of a disseminated response. These data suggest that primary resistance of naïve fish to IHNV may involve global responses that encourage reduced cellular signaling rather than promoting classical innate antiviral responses.

Parallel studies confirm Francisella halioticida causes mortality in Yesso scallops Patinopecten yessoensis.

Francisella halioticida is a marine bacterium originally described as the causative agent of mass mortality among giant abalone Haliotis gigantea. Recent field studies in Canada and Japan have suggested that this bacterium is also the cause of adductor muscle lesions and high mortality of Yesso scallops Patinopecten yessoensis, although a causal relationship has not been established. In the present study, the pathogenicity of F. halioticida in Yesso scallops was assessed in both Canada and Japan using bacteria isolated from diseased Yesso scallops in each respective country. Independent laboratory experiments revealed that scallops challenged with F. halioticida via bath exposure resulted in high mortality and histological lesions characterized by massive haemocyte infiltration. The presence of F. halioticida was confirmed using PCR, and F. halioticida was re-isolated from a portion of dead and surviving specimens. These results fulfill Koch's classic criteria for establishing disease causation and provide conclusive evidence that F. halioticida causes adductor muscle lesions and high mortality in Yesso scallops.

Seawater detection and biological assessments regarding transmission of the oyster parasite Mikrocytos mackini using qPCR.

Mikrocytos mackini is an intracellular parasite of oysters and causative agent of Denman Island disease in Pacific oysters Crassostrea gigas. Although M. mackini has been investigated for decades, its natural mode of transmission, mechanism for host entry, and environmental stability are largely unknown. We explored these biological characteristics of M. mackini using a recently described quantitative PCR (qPCR) assay. We detected M. mackini in the flow-through tank water of experimentally infected oysters and during disease remission in host tissues following 6 wk of elevated water temperature. Waterborne exposure of oysters to M. mackini further confirmed the potential for extracellular seawater transmission of this parasite and also identified host gill to have the highest early and continued prevalence for M. mackini DNA compared to stomach, mantle, labial palps, or adductor muscle samples. However, infections following waterborne challenge were slow to develop despite a substantial exposure (>106 M. mackini l-1 for 24 h), and further investigation demonstrated that M. mackini occurrence and infectivity severely declined following extracellular seawater incubation of more than 24 h. This study demonstrates a potential for using qPCR to monitor M. mackini in wild or farmed oyster populations during periods of disease remission or from environmental seawater samples. This work also suggests that gill tissues may provide a primary site for waterborne entry and possibly shedding of M. mackini in oysters. Further, although extracellular seawater transmission of M. mackini was possible, poor environmental stability and infection efficiency likely restricts the geographic transmission of M. mackini between oysters in natural environs and may help to explain localized areas of infection.

De novo assembly of Sockeye salmon kidney transcriptomes reveal a limited early response to piscine reovirus with or without infectious hematopoietic necrosis virus superinfection.

BACKGROUND: Piscine reovirus (PRV) has been associated with the serious disease known as Heart and Skeletal Muscle Inflammation (HSMI) in cultured Atlantic salmon Salmo salar in Norway. PRV is also prevalent in wild and farmed salmon without overt disease manifestations, suggesting multifactorial triggers or PRV variant-specific factors are required to initiate disease. In this study, we explore the head kidney transcriptome of Sockeye salmon Oncorhynchus nerka during early PRV infection to identify host responses in the absence of disease in hopes of elucidating mechanisms by which PRV may directly alter host functions and contribute to the development of a disease state. We further investigate the role of PRV as a coinfecting agent following superinfection with infectious hematopoietic necrosis virus (IHNV) - a highly pathogenic rhabdovirus endemic to the west coast of North America. RESULTS: Challenge of Sockeye salmon with PRV resulted in high quantities of viral transcripts to become present in the blood and kidney of infected fish without manifestations of disease. De novo transcriptome assembly of over 2.3 billion paired RNA-seq reads from the head kidneys of 36 fish identified more than 320,000 putative unigenes, of which less than 20 were suggested to be differentially expressed in response to PRV at either 2 or 3 weeks post challenge by DESeq2 and edgeR analysis. Of these, only one, Ependymin, was confirmed to be differentially expressed by qPCR in an expanded sample set. In contrast, IHNV induced substantial transcriptional changes (differential expression of > 20,000 unigenes) which included transcripts involved in antiviral and inflammatory response pathways. Prior infection with PRV had no significant effect on host responses to superinfecting IHNV, nor did host responses initiated by IHNV exposure influence increasing PRV loads. CONCLUSIONS: PRV does not substantially alter the head kidney transcriptome of Sockeye salmon during early (2 to 3 week) infection and dissemination in a period of significant increasing viral load, nor does the presence of PRV change the host transcriptional response to an IHNV superinfection. Further, concurrent infections of PRV and IHNV do not appear to significantly influence the infectivity or severity of IHNV associated disease, or conversely, PRV load.

Effects of single and repeated infections with Neoparamoeba perurans on antibody levels and immune gene expression in Atlantic salmon (Salmo salar).

Amoebic gill disease (AGD) is the main health problem for the salmon industry in Tasmania, Australia and is now reported in most salmon producing countries. Antibody and gene expression responses to the pathogen, Neoparamoeba perurans, have been studied independently following primary exposure; however, the effects of sequential reinfection, which can often occur during net-pen culture of salmon, remain unclear. The association between the transcription of immunoglobulin (Ig) and their systemic and mucosal antibody levels in regards to AGD is unknown. Herein, we assessed the antibody responses as well as Ig transcription in the gills of Atlantic salmon infected only once and also sequentially with N. perurans. After four successive AGD challenges, no significant differences in plasma or skin mucus levels of IgM were observed between AGD-naïve and challenged fish. However, IgM gene expression in gill lesions of AGD-affected fish increased up to 31 d after infection, while no changes in IgT, TCR and CD8 transcription were observed. Changes at IgM transcription level did not match the lack of antibody response in mucus, which is possibly explained by weak correlations existing between protein and mRNA abundances in cells and tissues. In the second experiment, which investigated Ig responses to AGD at the transcriptional as well as antibody production level in salmon after a single infection, the levels of serum or skin mucus IgM antibody were not affected and no changes in the IgM or IgT transcription were induced.

Transcriptional immune response of cage-cultured Pacific bluefin tuna during infection by two Cardicola blood fluke species.

Infections by two blood fluke species, Cardicola orientalis and Cardicola opisthorchis, currently present the greatest disease concern for the sea-cage culture of Pacific bluefin tuna (PBT) - a species of high global economic importance and ecological concern. In this study, we aimed to rapidly, quantitatively, and differentially identify infections by these two parasite species in cultured PBT as well as identify potential host immune responses. Using real-time qPCR, we were successful in quantitatively detecting parasite-specific DNA from within host blood, gill, and heart tissues; positively identifying parasitic infections 44 days earlier than microscopy methods previously employed. Both gill and heart became heavily infected by both parasite species in PBT within two months of sea-cage culture, which was only mitigated by the administration of anthelmintic praziquantel. Nevertheless, fish were observed to mount an organ specific transcriptive immune response during infection that mirrored the relative quantity of pathogenic load. In heart, significant (3-6 fold) increases in IgM, MHC2, TCRß, and IL-8 transcription was observed in infected fish relative to uninfected controls; whereas in the gills only IgM transcription was observed to be induced (11 fold) by infection. Interestingly, the relative quantity of IgM transcription was highly correlated to the relative abundance of C. orientalis but not C. opisthorchis DNA in the gill samples, even though this organ showed high prevalence of DNA from both parasite species. Taken together, these findings indicate that although ineffective at combating infection during primary exposure, a cellular immune response is mounted in PBT as a potential rejoinder to future Cardicola exposure, particularly against C. orientalis. Although future investigation into antibody effectiveness will be needed, this work provides valuable preliminary insight into host responsiveness to Cardicola infection as well as additional support for the need of anthelmintic treatment following primary parasite exposure during PBT culture.

Preliminary evidence of transcriptional immunomodulation by praziquantel in bluefin tuna and Atlantic salmon in vitro cultures.

Praziquantel (PZQ), long-used in veterinary and human medicine for the treatment of helminth parasites, is known to enhance humoral and cellular immune responsiveness in mammals but has unknown direct immunomodulatory capabilities in fish. In the present study, we examined the ability of PZQ to induce gene transcriptional changes in immune-competent primary tissue/organ cultures of two highly important yet evolutionarily discrete fish species--Southern bluefin tuna Thunnus maccoyii and Atlantic salmon Salmo salar. These cultures consisted of mixed blood cell population for both species, as well as intestinal explants from bluefin. Although expression profiles varied between species and tissue/organ type, PZQ induced both T-cell receptor (more than twofold) and IL-8 transcriptional expression (more than fourfold). Additionally, increased expression of other inflammatory cytokines including IL-1ß was detected in blood cell cultures from both species, and a general pattern of heightened antiviral signaling was observed. Specifically, elevated transcription of Type I (IFNα) and Type II (IFNγ) interferon in Atlantic salmon blood cultures along with elevated expression of MHC class I in blood cultures of both species. These findings provide preliminary evidence for direct immunomodulation by PZQ in fish and insight into its potential capacity as an immune stimulant/adjuvant in the rapidly expanding aquaculture industry.

Nonvirulent Infectious Salmon Anemia Virus (ISAV-HPR0) Not Detectable in Eggs or Progeny of Infected Captive Atlantic Salmon Brood.

The potential for infectious salmon anemia virus (ISAV)-an internationally regulated pathogen of salmon-to transmit vertically from parent to offspring is currently unclear. While the highly virulent ISAV phenotype known as ISAV-HPRΔ has been observed intra-ova, evidence for vertical transmission of the avirulent ISAV phenotype known as ISAV-HPR0 is lacking. In this study, we identified ISAV-HPR0-infected Atlantic salmon broodstock during spawning within a government research recirculating aquaculture facility using qPCR. Eggs and milt from infected brood were used to initiate 16 unique family dam-sire crosses from which 29-60 fertilized eggs per cross were screened for ISAV using qPCR (limit of detection ~100 virus genome copies/egg). A portion of eggs (~300) from one family cross was hatched and further reared in biosecure containment and periodically screened for ISAV by gill clipping over a 2-year period. ISAV was not detected in any of the 781 eggs screened from 16 family crosses generated by infected brood, nor in 870 gill clips periodically sampled from the single-family cohort raised for 2 years in biocontainment. Based on these findings, we conclude that ISAV-HPR0 has a limited likelihood for vertical parent-to-offspring transmission in cultured Atlantic salmon.

Rapid differentiation of infectious salmon anemia virus avirulent (HPR0) from virulent (HPRΔ) variants using multiplex RT-qPCR.

Infectious salmon anemia virus (ISAV; Isavirus salaris) causes an economically important disease of Atlantic salmon (Salmo salar L.). ISA outbreaks have resulted in significant losses of farmed salmon globally, often with a sudden onset. However, 2 phenotypically distinct variants of ISAV exist, each with divergent disease outcomes, associated regulations, and control measures. ISAV-HPRΔ, also known as ISAV-HPR deleted, is responsible for ISA outbreaks; ISAV-HPR0, is avirulent and is not known to cause fish mortality. Current detection methodology requires genetic sequencing of ISAV-positive samples to differentiate phenotypes, which may slow responses to disease management. To increase the speed of phenotypic determinations of ISAV, we developed a new, rapid multiplex RT-qPCR method capable of 1) detecting if a sample contains any form of ISAV, 2) discriminating whether positive samples contain HPRΔ or HPR0, and 3) validating RNA extractions with an internal control, all in a single reaction. Following assay development and optimization, we validated this new multiplex on 31 ISAV strains collected from North America and Europe (28 ISAV-HPRΔ, 3 ISAV-HPR0). Finally, we completed an inter-laboratory comparison of this multiplex qPCR with commercial ISAV testing and found that both methods provided equivalent results for ISAV detection.

Temperature-induced transcription of inflammatory mediators and the influence of Hsp70 following LPS stimulation of southern bluefin tuna peripheral blood leukocytes and kidney homogenates.

Temperature is known to influence inflammatory signalling in mammals, but far less understood in fish. The aim of the present study was to explore the potential effects of temperature on innate immune signalling in head kidney and leukocyte populations of the economically important southern bluefin tuna through the identification and utilization of gene expression targets in vitro. Here, we identified the mRNA sequences of five potential inflammatory mediators - TNFα (1 and 2), IL-1ß, IL-8, and Cox2 - and demonstrate induction of four - TNFα (2), IL-1ß, IL-8, and Cox2 - following LPS stimulation of both peripheral blood leukocytes and head kidney homogenates in vitro by real-time quantitative PCR. Comparison of transcriptional expression in cultures held at 18 and 25 °C (both within the presumed natural temperature range of this heterothermic species) showed accelerated transcription of cytokines TNFα, IL-1ß and IL-8 following LPS stimulation at 25 °C in both tissue types. Peak induction reached comparable levels for each transcript at both temperatures during the 24 h test period with only limited (if any) protraction in expression resulting from cold temperature (18 °C) incubation. Partial mRNA sequences were also identified for both the constitutively expressed and heat inducible chaperone proteins Hsc70 and Hsp70, and 24 h incubation at 25 °C was sufficient to induce Hsp70 transcription in leukocyte but not in head kidney cell populations. Taken together these findings suggest that temperature exerts influence in the timing but not the degree of an innate inflammatory response in bluefin tuna and that different cell populations have differential responsiveness to heat shock in this heterothermic species. Further, LPS stimulation failed to induce Hsp70 at either incubation temperature in leukocytes; whereas 25 °C incubation caused Hsp70 up-regulation in leukocytes with or without the presence of LPS. This suggests that Hsp70 does not play a direct role in immune responsiveness for this species and that an environmental temperature of 25 °C in excess of 24 h initiates a cellular stress response in blood cells of this organism. Lastly, a strong correlation between Hsp70 and IL-8 transcriptional expression was observed following LPS/heat shock stimulation of leukocytes and five potential heat shock response elements were subsequently identified on the gene promoter region of IL-8 indicating that heat shock co-activation of this chemokine previously identified in mammals is also likely present in fish.

Pan-Piscine Orthoreovirus (PRV) Detection Using Reverse Transcription Quantitative PCR.

Piscine orthoreovirus (PRV) infects farmed and wild salmon and trout species in North America, South America, Europe, and East Asia. PRV groups into three distinct genotypes (PRV-1, PRV-2, and PRV-3) that can vary in distribution, host specificity, and/or disease potential. Detection of the virus is currently restricted to genotype specific assays such that surveillance programs require the use of three assays to ensure universal detection of PRV. Consequently, herein, we developed, optimized, and validated a real-time reverse transcription quantitative PCR assay (RT-qPCR) that can detect all known PRV genotypes with high sensitivity and specificity. Targeting a conserved region at the 5' terminus of the M2 segment, the pan-PRV assay reliably detected all PRV genotypes with as few as five copies of RNA. The assay exclusively amplifies PRV and does not cross-react with other salmonid viruses or salmonid host genomes and can be performed as either a one- or two-step RT-qPCR. The assay is highly reproducible and robust, showing 100% agreement in test results from an inter-laboratory comparison between two laboratories in two countries. Overall, as the assay provides a single test to achieve highly sensitive pan-specific PRV detection, it is suitable for research, diagnostic, and surveillance purposes.

Distribution and Pathogenicity of Two Cutthroat Trout Virus (CTV) Genotypes in Canada.

The sole member of the Piscihepevirus genus (family Hepeviridae) is cutthroat trout virus (CTV) but recent metatranscriptomic studies have identified numerous fish hepevirus sequences including CTV-2. In the current study, viruses with sequences resembling both CTV and CTV-2 were isolated from salmonids in eastern and western Canada. Phylogenetic analysis of eight full genomes delineated the Canadian CTV isolates into two genotypes (CTV-1 and CTV-2) within the Piscihepevirus genus. Hepevirus genomes typically have three open reading frames but an ORF3 counterpart was not predicted in the Canadian CTV isolates. In vitro replication of a CTV-2 isolate produced cytopathic effects in the CHSE-214 cell line with similar amplification efficiency as CTV. Likewise, the morphology of the CTV-2 isolate resembled CTV, yet viral replication caused dilation of the endoplasmic reticulum lumen which was not previously observed. Controlled laboratory studies exposing sockeye (Oncorhynchus nerka), pink (O. gorbuscha), and chinook salmon (O. tshawytscha) to CTV-2 resulted in persistent infections without disease and mortality. Infected Atlantic salmon (Salmo salar) and chinook salmon served as hosts and potential reservoirs of CTV-2. The data presented herein provides the first in vitro and in vivo characterization of CTV-2 and reveals greater diversity of piscihepeviruses extending the known host range and geographic distribution of CTV viruses.