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.

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.

Annual Recurrences of Viral Hemorrhagic Septicemia Epizootics in Age 0 Pacific Herring Clupea pallasii Valenciennes, 1847.

Throughout a 20 year biosurveillance period, viral hemorrhagic septicemia virus was isolated in low titers from only 6/7355 opportunistically sampled adult Pacific herring, reflecting the typical endemic phase of the disease when the virus persists covertly. However, more focused surveillance efforts identified the presence of disease hot spots occurring among juvenile life history stages from certain nearshore habitats. These outbreaks sometimes recurred annually in the same temporal and spatial patterns and were characterized by infection prevalence as high as 96%. Longitudinal sampling indicated that some epizootics were relatively transient, represented by positive samples on a single sampling date, and others were more protracted, with positive samples occurring throughout the first 10 weeks of the juvenile life history phase. These results indicate that viral hemorrhagic septicemia (VHS) epizootics in free-ranging Pacific herring C. pallasii are more common than previously appreciated; however, they are easily overlooked if biosurveillance efforts are not designed around times and locations with high disease potential.

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.

Screening of Fish Cell Lines for Piscine Orthoreovirus-1 (PRV-1) Amplification: Identification of the Non-Supportive PRV-1 Invitrome.

Piscine reovirus (PRV) is the causative agent of heart and skeletal muscle inflammation (HSMI), which is detrimental to Atlantic Salmon (AS) aquaculture, but so far has not been cultivatable, which impedes studying the disease and developing a vaccine. Homogenates of head kidney and red blood cells (RBC) from AS in which PRV-1 had been detected were applied to fish cell lines. The cell lines were from embryos, and from brain, blood, fin, gill, gonads, gut, heart, kidney, liver, skin, and spleen, and had the shapes of endothelial, epithelial, fibroblast, and macrophage cells. Most cell lines were derived from the Neopterygii subclass of fish, but one was from subclass Chondrostei. Cultures were examined by phase contrast microscopy for appearance, and by quantitative polymerase chain reaction (qPCR) for PRV-1 RNA amplification and for the capacity to transfer any changes to new cultures. No changes in appearance and Ct values were observed consistently or transferable to new cultures. Therefore, 31 cell lines examined were unable to support PRV-1 amplification and are described as belonging to the non-supportive PRV-1 invitrome. However, these investigations and cell lines can contribute to understanding PRV-1 cellular and host tropism, and the interactions between virus-infected and bystander cells.

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.

Author Correction: Piscine orthoreovirus demonstrates high infectivity but low virulence in Atlantic salmon of Pacific Canada.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Corrigendum: High-Load Reovirus Infections Do Not Imply Physiological Impairment in Salmon.

[This corrects the article DOI: 10.3389/fphys.2019.00114.].

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.

Synergistic osmoregulatory dysfunction during salmon lice (Lepeophtheirus salmonis) and infectious hematopoietic necrosis virus co-infection in sockeye salmon (Oncorhynchus nerka) smolts.

While co-infections are common in both wild and cultured fish, knowledge of the interactive effects of multiple pathogens on host physiology, gene expression and immune response is limited. To evaluate the impact of co-infection on host survival, physiology and gene expression, sockeye salmon Oncorhynchus nerka smolts were infected with the salmon louse Lepeophtheirus salmonis (V-/SL+), infectious hematopoietic necrosis virus (IHNV; V+/SL-), both (V+/SL+), or neither (V-/SL-). Survival in the V+/SL+ group was significantly lower than the V-/SL- and V-/SL+ groups (p = 0.024). Co-infected salmon had elevated osmoregulatory indicators and lowered haematocrit values as compared to the uninfected control. Expression of 12 genes associated with the host immune response was analysed in anterior kidney and skin. The only evidence of L. salmonis-induced modulation of the host antiviral response was down-regulation of mhc I although the possibility of modulation cannot be ruled out for mx-1 and rsad2. Co-infection did not influence the expression of genes associated with the host response to L. salmonis. Therefore, we conclude that the reduced survival in co-infected sockeye salmon resulted from the osmoregulatory consequences of the sea lice infections which were amplified due to infection with IHNV.

High-Load Reovirus Infections Do Not Imply Physiological Impairment in Salmon.

The recent ubiquitous detection of PRV among salmonids has sparked international concern about the cardiorespiratory performance of infected wild and farmed salmon. Piscine orthoreovirus (PRV) has been shown to create substantial viremia in salmon by targeting erythrocytes for principle replication. In some instances, infections develop into heart and skeletal muscle inflammation (HSMI) or other pathological conditions affecting the respiratory system. Critical to assessing the seriousness of PRV infections are controlled infection studies that measure physiological impairment to critical life support systems. Respiratory performance is such a system and here multiple indices were measured to test the hypothesis that a low-virulence strain of PRV from Pacific Canada compromises the cardiorespiratory capabilities of Atlantic salmon. Contrary to this hypothesis, the oxygen affinity and carrying capacity of erythrocytes were unaffected by PRV despite the presence of severe viremia, minor heart pathology and transient cellular activation of antiviral response pathways. Similarly, PRV-infected fish had neither sustained nor appreciable differences in respiratory capabilities compared with control fish. The lack of functional harm to salmon infected with PRV in this instance highlights that, in an era of unprecedented virus discovery, detection of viral infection does not necessarily imply bodily harm and that viral load is not always a suitable predictor of disease within a host organism.

Piscine orthoreovirus demonstrates high infectivity but low virulence in Atlantic salmon of Pacific Canada.

Piscine orthoreovirus (PRV) is ubiquitous in farmed Atlantic salmon and sometimes associated with disease - most notably, Heart and Skeletal Muscle Inflammation (HSMI). However, PRV is also widespread in non-diseased fish, particularly in Pacific Canada, where few cases of severe heart inflammation have been documented. To better understand the mechanisms behind PRV-associated disease, this study investigated the infection dynamics of PRV from Pacific Canada and the potential for experimental passage of putatively associated heart inflammation in Pacific-adapted Mowi-McConnell Atlantic salmon. Regardless of the PRV source (fish with or without HSMI-like heart inflammation), infections led to high-load viremia that induced only minor focal heart inflammation without significant transcriptional induction of inflammatory cytokines. Repeated screening of PRV dsRNA/ssRNA along with histopathology and gene expression analysis of host blood and heart tissues identified three distinct phases of infection: (1) early systemic dissemination and replication without host recognition; (2) peak replication, erythrocyte inclusion body formation and load-dependent host recognition; (3) long-term, high-load viral persistence with limited replication or host recognition sometimes accompanied by minor heart inflammation. These findings contrast previous challenge trials with PRV from Norway that induced severe heart inflammation and indicate that strain and/or host specific factors are necessary to initiate PRV-associated disease.

Differential Effects of Adult Salmon Lice Lepeophtheirus salmonis on Physiological Responses of Sockeye Salmon and Atlantic Salmon.

The salmon louse Lepeophtheirus salmonis, a type of sea lice (family Caligidae), is enzootic in marine waters of British Columbia and poses a health risk to both farmed Atlantic Salmon Salmo salar and wild Pacific salmon Oncorhynchus spp. At the adult stage, sea lice infections can often result in severe cutaneous lesions in their salmonid hosts. To evaluate and compare the physiological consequences of adult L. salmonis infections, smolts of Atlantic Salmon and Sockeye Salmon O. nerka were exposed to 2 (low), 6 (medium), or 10 (high) adult female lice/fish. Mean lice abundance decreased over time in all groups. Skin disruption due to parasite infection was observed in both species. Plasma samples were collected from infected fish and uninfected controls at 1, 3, 5, and 7 d postinfection and measured for indicators of osmoregulatory function and stress. Sockeye Salmon, regardless of L. salmonis exposure level, showed a rapid onset of elevated osmolality and sodium and chloride ion concentrations which were sustained until 7 d postinfection when values returned to levels comparable with the unexposed controls. Conversely, these effects were not measured in Atlantic Salmon. Additionally, differential host effects in blood glucose levels were observed, with Sockeye Salmon displaying immediate elevation in glucose. Relative to Atlantic Salmon, infection with L. salmonis caused a profound physiological impact to Sockeye Salmon characterized by loss of osmoregulatory integrity and a stress response. This work provides the first comprehensive report of the physiological consequences of infections with adult L. salmonis in Sockeye Salmon smolts and helps to further define the mechanisms of susceptibility in this species.

Significant differences in maternal carotenoid provisioning and effects on offspring fitness in Chinook salmon colour morphs.

In oviparous species, maternal carotenoid provisioning can deliver diverse fitness benefits to offspring via increased survival, growth and immune function. Despite demonstrated advantages of carotenoids, large intra- and interspecific variation in carotenoid utilization exists, suggesting trade-offs associated with carotenoids. In Chinook salmon (Oncorhynchus tshawytscha), extreme variation in carotenoid utilization delineates two colour morphs (red and white) that differ genetically in their ability to deposit carotenoids into tissues. Here, we take advantage of this natural variation to examine how large differences in maternal carotenoid provisioning influence offspring fitness. Using a full factorial breeding design crossing morphs and common-garden rearing, we measured differences in a suite of fitness-related traits, including survival, growth, viral susceptibility and host response, in offspring of red (carotenoid-rich eggs) and white (carotenoid-poor eggs) females. Eggs of red females had significantly higher carotenoid content than those of white females (6× more); however, this did not translate into measurable differences in offspring fitness. Given that white Chinook salmon may have evolved to counteract their maternal carotenoid deficiency, we also examined the relationship between egg carotenoid content and offspring fitness within each morph separately. Egg carotenoids only had a positive effect within the red morph on survival to eyed-egg (earliest measured trait), but not within the white morph. Although previous work shows that white females benefit from reduced egg predation, our study also supports a hypothesis that white Chinook salmon have evolved additional mechanisms to improve egg survival despite low carotenoids, providing novel insight into evolutionary mechanisms that maintain this stable polymorphism.

An alloherpesvirus infection of European perch Perca fluviatilis in Finland.

The order Herpesvirales includes viruses that infect aquatic and terrestrial vertebrates and several aquatic invertebrates (i.e. mollusks), and share the commonality of possessing a double-stranded DNA core surrounded by an icosahedral capsid. Herpesviruses of the family Alloherpesviridae that infect fish and amphibians, including channel catfish virus and koi herpesvirus, negatively impact aquaculture. Here, we describe a novel herpesvirus infection of wild European perch from lakes in Finland. Infected fish exhibited white nodules on the skin and fins, typically in the spring when prevalence reached nearly 40% in one of the sampled lakes. Transmission electron microscopic examination of affected tissues revealed abundant nuclear and cytoplasmic virus particles displaying herpesvirus morphology. Degenerate PCR targeting a conserved region of the DNA polymerase gene of large DNA viruses amplified a 520 bp product in 5 of 5 affected perch skin samples tested. Phylogenetic analysis of concatenated partial DNA polymerase and terminase (exon 2) gene sequences produced a well-supported tree grouping the European perch herpesvirus with alloherpesviruses infecting acipenserid, esocid, ictalurid, and salmonid fishes. The phenetic analysis of the European perch herpesvirus partial DNA polymerase and terminase nucleotide gene sequences ranged from 34.6 to 63.9% and 39.6 to 59.6% to other alloherpesviruses, respectively. These data support the European perch herpesvirus as a new alloherpesvirus, and we propose the formal species designation of Percid herpesvirus 2 (PeHV2) to be considered for approval by the International Committee on Taxonomy of Viruses.

Fish viruses stored in RNAlater can remain infectious and even be temporarily protected from inactivation by heat or by tissue homogenates.

RNAlater is a commonly used transport and storage solution for samples collected for fish health investigations, particularly those potentially involving viruses. However, the infectivity of fish viruses after storage in RNAlater have not been determined. Nevertheless, knowledge of pathogen infectivity of preserved samples is crucial for ensuring safe transport and storage protocols. Therefore, the infectivity of three fish RNA viruses in RNAlater was examined at four temperatures: -80 °C, 4 °C, room temperature (RT, approximately 22 °C) and 37 °C. The viruses were viral hemorrhagic septicemia virus (VHSV), infectious pancreatic necrosis virus (IPNV) and chum salmon reovirus (CSV). Overall, three consistent outcomes were observed. First, all three viruses remained infectious in RNAlater at RT or lower. High log titres of these viruses remained over 30 d of storage in either RNAlater or PBS. Second, RNAlater delayed the thermal inactivation of these viruses when compared to PBS at 37 °C. For VHSV, the titre remained high in RNAlater after one day of incubation at 37 °C, but was inactivated to below threshold in PBS over the same period. For IPNV, the titre remained high in RNAlater after 30 d of incubation at 37 °C, but was inactivated to below threshold in PBS over the same period. For CSV, the titre was slightly higher in RNAlater than PBS at 37 °C over 7 d, and by day 30, only samples stored in RNAlater proved infectious at titres above the detection threshold. Third, RNAlater delayed the inactivation of these viruses when they were stored together with head kidney homogenates. For VHSV, infectious virus was recovered from samples stored at 4 °C in RNAlater by day 7 of incubation, whereas it was inactivated to below threshold in PBS over the same period. For both IPNV and CSV, infectious virus was recovered from samples stored at 37 °C in RNAlater for 7 d, but not so in PBS. In summary, fish viruses can remain infectious and are even temporarily protected from inactivation while in RNAlater. This makes RNAlater a potentially useful solution for the transport of fish viruses. At the same time, precautionary measures must be taken when transporting potentially infectious samples in RNAlater.

Transmission potential of infectious hematopoietic necrosis virus in APEX-IHN®-vaccinated Atlantic salmon.

Infectious hematopoietic necrosis virus (IHNV) outbreaks have had a significant negative impact on Atlantic salmon Salmo salar production in British Columbia, Canada, since the first outbreak was reported in 1992. In 2005, the APEX-IHN® vaccine was approved for use in Canada for prevention of IHN. The vaccine was proven to be safe and efficacious prior to approval; however, it is unknown as to whether APEX-IHN®-vaccinated Atlantic salmon infected with IHNV can support replication and virus shedding in sufficient quantities to provide an infectious dose to a nearby susceptible host. To determine whether vaccinated, infected fish are able to transmit an infectious dose of IHNV, vaccinated Atlantic salmon were injected with IHNV (104 plaque-forming units per fish) and cohabitated with either naïve Atlantic salmon or naïve sockeye salmon Oncorhynchus nerka. APEX-IHN®-vaccinated fish were significantly protected against IHNV with mortality occurring in only 2.6% of the population as opposed to 97% in unvaccinated controls. Vaccination in IHNV-infected Atlantic salmon completely abolished disease transmission to cohabitating naïve sockeye salmon and reduced virus spread among cohabitating naïve Atlantic salmon. At 7 mo post-vaccination, IHNV-neutralizing antibodies were detected in nearly all vaccinated fish (94%) with similar titer occurring between vaccinated, infected fish and vaccinated, uninfected fish, indicating APEX-IHN® vaccination induces a robust seroconversion response. Taken together, these results demonstrate that vaccination greatly reduces the infectious load and potential for IHNV transmission. As such, APEX-IHN® should be included in fish health management strategies when culturing Atlantic salmon in IHNV endemic areas.