Piscine Orthoreovirus Infection Research Articles

Sleeping With the Enemy? The Current Knowledge of Piscine Orthoreovirus (PRV) Immune Response Elicited to Counteract Infection.

Piscine orthoreovirus (PRV) is a virus in the genus Orthoreovirus of the Reoviridae family, first described in 2010 associated with Heart and Skeletal Muscle Inflammation (HSMI) in Atlantic salmon (Salmo salar). Three phases of PRV infection have been described, the early entry and dissemination, the acute dissemination phase, and the persistence phase. Depending on the PRV genotype and the host, infection can last for life. Mechanisms of immune response to PRV infection have been just beginning to be studied and the knowledge in this matter is here revised. PRV induces a classical antiviral immune response in experimental infection of salmonid erythrocytes, including transcriptional upregulation of ifn-α, rig-i, mx, and pkr. In addition, transcript upregulation of tcra, tcrb, cd2, il-2, cd4-1, ifn-γ, il-12, and il-18 has been observed in Atlantic salmon infected with PRV, indicating that PRV elicited a Th1 type response probably as a host defense strategy. The high expression levels of cd8a, cd8b, and granzyme-A in PRV-infected fish suggest a positive modulatory effect on the CTL-mediated immune response. This is consistent with PRV-dependent upregulation of the genes involved in antigen presentation, including MHC class I, transporters, and proteasome components. We also review the potential immune mechanisms associated with the persistence phenotype of PRV-infected fish and its consequence for the development of a secondary infection. In this scenario, the application of a vaccination strategy is an urgent and challenging task due to the emergence of this viral infection that threatens salmon farming.

Inactivation of Piscine orthoreovirus.

Piscine orthoreovirus infects various salmonid fish species, and the infection is associated with diseases such as heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). There are no vaccines available or genetically selected resistant hosts that can efficiently control piscine orthoreovirus (PRV) infection. Currently, the only prophylactic measure against PRV is general biosecurity measures aiming to break the transmission cycle. Methods to eradicate infectious virus from contaminated facilities are desirable, but the knowledge on how to inactivate PRV is lacking. A major bottleneck for inactivation studies is the lack of ability to propagate PRV in cell culture. Therefore, in this study we developed an in vivo model for detection of infectious PRV particles after treatment of the virus with inactivation tools such as heat, pH, iodine, UV and commercially available disinfectants. The results show that standard iodine treatment is efficient in inactivation of the virus, and similarly are high and low pH extremes and treatment with Virocid, a commercially available disinfectant. A UV dose of at least 50mJ/cm2 is required for inactivation, and the virus has high resistance against heat treatment.

Inflammatory reactions and melanogenesis in salmon during virus infection

Abstract Melanised skeletal muscle changes (black spots) are a serious problem for the salmon industry world-wide, causing extensive production losses. Piscine orthoreovirus (PRV) is associated with the condition. The initial cause for the focal changes remains obscure. Early changes start as muscle bleedings and extensive muscle necrosis, and so far, these changes cannot be explained, However, from this acute phase, the changes may progress into melanised focal changes appearing as chronic granulomatous reactions. Granulomatous melanised changes have been consistently observed in PRV infected fish, and in serious changes, the tissue persistence of virus dominate the changes despite the inflammatory changes. Here, we address the pathogenesis of PRV infection and phases of melanised focal changes development. Fluorescent in situ hybridisation (FISH) assay confirmed the colocalisation of iNOS2 transcripts (M1 activated macrophages) with PRV in red spot whereas both M1 and M2 (Arg2 transcripts) macrophages localized at PRV infected clusters in black spots. Melano-macrophages were found positive with Arg2 transcripts demonstrating their role in tissue repair mechanism. In PRV negative fish low expression of M1 and M2 specific transcripts suggest PRV act as an accelerator for the melanisation in these focal changes. Transcripts specific for melanin production were observed in non-melanised cells in early stages of the condition and in melano-macrophages in late stages. The nature of the melano-macrophages is disputed and the rationale for their melanin production is not understood. Our investigations reveal new characteristics both of the pathogenesis of focal melanised changes as well as of the melano-macrophages involved.

Detection of Salmonid IgM Specific to the Piscine Orthoreovirus Outer Capsid Spike Protein Sigma 1 Using Lipid-Modified Antigens in a Bead-Based Antibody Detection Assay.

Bead-based multiplex immunoassays are promising tools for determination of the specific humoral immune response. In this study, we developed a multiplexed bead-based immunoassay for the detection of Atlantic salmon (Salmo salar) antibodies against Piscine orthoreovirus (PRV). Three different genotypes of PRV (PRV-1, PRV-2, and PRV-3) cause disease in farmed salmonids. The PRV outer capsid spike protein σ1 is predicted to be a host receptor binding protein and a target for neutralizing and protective antibodies. While recombinant σ1 performed poorly as an antigen to detect specific antibodies, N-terminal lipid modification of recombinant PRV-1 σ1 enabled sensitive detection of specific IgM in the bead-based assay. The specificity of anti-PRV-1 σ1 antibodies was confirmed by western blotting and pre-adsorption of plasma. Binding of non-specific IgM to beads coated with control antigens also increased after PRV infection, indicating a release of polyreactive antibodies. This non-specific binding was reduced by heat treatment of plasma. The same immunoassay also detected anti-PRV-3 σ1 antibodies from infected rainbow trout. In summary, a refined bead based immunoassay created by N-terminal lipid-modification of the PRV-1 σ1 antigen allowed sensitive detection of anti-PRV-1 and anti-PRV-3 antibodies from salmonids.

Melanized focal changes in skeletal muscle in farmed Atlantic salmon after natural infection with Piscine orthoreovirus (PRV).

Melanized focal changes in skeletal muscle of farmed Atlantic salmon (Salmo salar) are a major quality problem. The aetiology is unknown, but infection with Piscine orthoreovirus (PRV) has been associated with the condition. Here, we addressed the pathogenesis of red and melanized focal changes and their association with PRV. First, a population of farmed fish (PRV‐negative prior to sea transfer) was sequentially investigated throughout the seawater period. The fish were autopsied and tested for PRV infection. Muscular changes were described by macroscopy and histology, and a classification system was established. Second, in an experimental infection trial, PRV was injected intramuscularly to induce changes. The farmed fish was gradually infected with PRV. Red focal changes occurred throughout the observation period with a low prevalence regardless of PRV status. Melanized changes were highly diverse and their prevalence increased during the trial. Changes of low macroscopic grade and histological category were more prevalent in PRV‐negative fish. Diffuse granulomatous melanized changes only occurred after PRV infection. No muscular changes were observed in the experimentally challenged fish. Our studies do not indicate that PRV infection causes red focal changes, but seems important in the development of granulomatous melanized changes.

Antiviral Responses and Biological Concequences of Piscine orthoreovirus Infection in Salmonid Erythrocytes.

Salmonid red blood cells are the main target cells for Piscine orthoreovirus (PRV). Three genotypes of PRV (PRV-1,2,3) infect Atlantic salmon (Salmo salar), Chinook salmon (Onchorhynchus tshawytscha), Coho salmon (Oncorhynchus kisutch), rainbow trout (Onchorhynchus mykiss) and brown trout (Salmo trutta), and can cause diseases like heart and skeletal muscle inflammation (HSMI), jaundice syndrome, erythrocyte inclusion body syndrome (EIBS) and proliferative darkening syndrome (PDS). Purified PRV administrated to fish has proven the causality for HSMI and EIBS. During the early peak phase of infection, salmonid erythrocytes are the main virus-replicating cells. In this initial phase, cytoplasmic inclusions called “virus factories” can be observed in the erythrocytes, and are the primary sites for the formation of new virus particles. The PRV-infected erythrocytes in Atlantic salmon mount a strong long-lasting innate antiviral response lasting for many weeks after the onset of infection. The antiviral response of Atlantic salmon erythrocytes involves upregulation of potential inhibitors of translation. In accordance with this, PRV-1 protein production in erythrocytes halts while virus RNA can persist for months. Furthermore, PRV infection in Coho salmon and rainbow trout are associated with anemia, and in Atlantic salmon lower hemoglobin levels are observed. Here we summarize and discuss the recently published findings on PRV infection, replication and effects on salmonid erythrocytes, and discuss how PRV can be a useful tool for the study of innate immune responses in erythrocytes, and help reveal novel immune functions of the red blood cells in fish.

DNA vaccine expressing the non-structural proteins of Piscine orthoreovirus delay the kinetics of PRV infection and induces moderate protection against heart -and skeletal muscle inflammation in Atlantic salmon (Salmo salar)

Piscine orthoreovirus (PRV) causes heart- and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). Erythrocytes are the main target cells for PRV. HSMI causes significant economic losses to the salmon aquaculture industry, and there is currently no vaccine available. PRV replicates and assembles within cytoplasmic structures called viral factories, mainly organized by the non-structural viral protein µNS. In two experimental vaccination trials in Atlantic salmon, using DNA vaccines expressing different combinations of PRV proteins, we found that expression of the non-structural proteins µNS combined with the cell attachment protein σ1 was associated with an increasing trend in lymphocyte marker gene expression in spleen, and induced moderate protective effect against HSMI.

Piscine orthoreovirus infection in Atlantic salmon (Salmo salar) protects against subsequent challenge with infectious hematopoietic necrosis virus (IHNV)

Infectious hematopoietic necrosis virus (IHNV) is endemic in farmed rainbow trout in continental Europe and in various salmonid fish species at the Pacific coast of North America. IHN has never occurred in European Atlantic salmon (Salmo salar) farms, but is considered as a major threat for the European salmon industry. Another virus, Piscine orthoreovirus (PRV), is widespread in the sea phase of Atlantic salmon, and is identified as the causative agent of heart and skeletal muscle inflammation. The aim of this study was to investigate the interactions between a primary PRV infection and a secondary IHNV infection under experimental conditions. A PRV cohabitation challenge was performed with Atlantic salmon. At peak of PRV viremia the fish were challenged by immersion with an IHNV genogroup E isolate. Clinical signs and morbidity were monitored. Target organs were sampled at selected time points to assess viral loads of both pathogens. Antiviral immune response and presence of histopathological findings were also investigated. Whereas the PRV-negative/IHNV positive group suffered significant decrease in survival caused by IHNV, the PRV infected groups did not suffer any morbidity and showed negligible levels of IHNV infection. Antiviral response genes were induced, as measured in spleen samples, from PRV infected fish prior to IHNV challenge. In conclusion, PRV-infection protects Atlantic salmon against IHNV infection and morbidity, most likely by inducing a protective innate antiviral response.

The effect of exposure to farmed salmon on piscine orthoreovirus infection and fitness in wild Pacific salmon in British Columbia, Canada.

The disease Heart and Skeletal Muscle Inflammation (HSMI) is causing substantial economic losses to the Norwegian salmon farming industry where the causative agent, piscine orthoreovirus (PRV), is reportedly spreading from farmed to wild Atlantic salmon (Salmo salar) with as yet undetermined impacts. To assess if PRV infection is epidemiologically linked between wild and farmed salmon in the eastern Pacific, wild Pacific salmon (Oncorhynchus sp.) from regions designated as high or low exposure to salmon farms and farmed Atlantic salmon reared in British Columbia (BC) were tested for PRV. The proportion of PRV infection in wild fish was related to exposure to salmon farms (p = 0.0097). PRV was detected in: 95% of farmed Atlantic salmon, 37–45% of wild salmon from regions highly exposed to salmon farms and 5% of wild salmon from the regions furthest from salmon farms. The proportion of PRV infection was also significantly lower (p = 0.0008) where wild salmon had been challenged by an arduous return migration into high-elevation spawning habitat. Inter-annual PRV infection declined in both wild and farmed salmon from 2012–2013 (p ≤ 0.002). These results suggest that PRV transfer is occurring from farmed Atlantic salmon to wild Pacific salmon, that infection in farmed salmon may be influencing infection rates in wild salmon, and that this may pose a risk of reduced fitness in wild salmon impacting their survival and reproduction.

Hypoxia tolerance and responses to hypoxic stress during heart and skeletal muscle inflammation in Atlantic salmon (Salmo salar).

Heart and skeletal muscle inflammation (HSMI) is associated with Piscine orthoreovirus (PRV) infection and is an important disease in Atlantic salmon (Salmo salar) aquaculture. Since PRV infects erythrocytes and farmed salmon frequently experience environmental hypoxia, the current study examined mutual effects of PRV infection and hypoxia on pathogenesis and fish performance. Furthermore, effects of HSMI on hypoxia tolerance, cardiorespiratory performance and blood oxygen transport were studied. A cohabitation trial including PRV-infected post-smolts exposed to periodic hypoxic stress (4 h of 40% O2; PRV-H) at 4, 7 and 10 weeks post-infection (WPI) and infected fish reared under normoxic conditions (PRV) was conducted. Periodic hypoxic stress did not influence infection levels or histopathological changes in the heart. Individual incipient lethal oxygen saturation (ILOS) was examined using a standardized hypoxia challenge test (HCT). At 7 WPI, i.e. peak level of infection, both PRV and PRV-H groups exhibited reduced hypoxia tolerance compared to non-infected fish. Three weeks later (10 WPI), during peak levels of pathological changes, reduced hypoxia tolerance was still observed for the PRV group while PRV-H performed equal to non-infected fish, implying a positive effect of the repeated exposure to hypoxic stress. This was in line with maximum heart rate (fHmax) measurements, showing equal performance of PRV-H and non-infected groups, but lower fHmax above 19°C as well as lower temperature optimum (Topt) for aerobic scope for PRV, suggesting reduced cardiac performance and thermal tolerance. In contrast, the PRV-H group had reduced hemoglobin-oxygen affinity compared to non-infected fish. In conclusion, Atlantic salmon suffering from HSMI have reduced hypoxia tolerance and cardiac performance, which can be improved by preconditioning fish to transient hypoxic stress episodes.

Viral Protein Kinetics of Piscine Orthoreovirus Infection in Atlantic Salmon Blood Cells.

Piscine orthoreovirus (PRV) is ubiquitous in farmed Atlantic salmon (Salmo salar) and the cause of heart and skeletal muscle inflammation. Erythrocytes are important target cells for PRV. We have investigated the kinetics of PRV infection in salmon blood cells. The findings indicate that PRV causes an acute infection of blood cells lasting 1–2 weeks, before it subsides into persistence. A high production of viral proteins occurred initially in the acute phase which significantly correlated with antiviral gene transcription. Globular viral factories organized by the non-structural protein µNS were also observed initially, but were not evident at later stages. Interactions between µNS and the PRV structural proteins λ1, µ1, σ1 and σ3 were demonstrated. Different size variants of µNS and the outer capsid protein µ1 appeared at specific time points during infection. Maximal viral protein load was observed five weeks post cohabitant challenge and was undetectable from seven weeks post challenge. In contrast, viral RNA at a high level could be detected throughout the eight-week trial. A proteolytic cleavage fragment of the µ1 protein was the only viral protein detectable after seven weeks post challenge, indicating that this µ1 fragment may be involved in the mechanisms of persistent infection.

Immunological interactions between Piscine orthoreovirus and Salmonid alphavirus infections in Atlantic salmon

Heart and skeletal muscle inflammation (HSMI) and pancreas disease (PD) cause substantial losses in Atlantic salmon (Salmo salar) aquaculture. The respective causative agents, Piscine orthoreovirus (PRV) and Salmonid alphavirus (SAV), are widespread and often concurrently present in farmed salmon. An experimental infection in Atlantic salmon was conducted to study the interaction between the two viruses, including the immunological mechanisms involved. The co-infected fish were infected with PRV four or ten weeks before they were infected with SAV. The SAV RNA level and the PD specific lesions were significantly lower in co-infected groups compared to the group infected by only SAV. The expression profiles of a panel of innate antiviral response genes and the plasma SAV neutralization titers were examined. The innate antiviral response genes were in general upregulated for at least ten weeks after the primary PRV infection. Plasma from co-infected fish had lower SAV neutralizing titers compared to the controls infected with only SAV. Plasma from some individuals infected with only PRV neutralized SAV, but heat treatment removed this effect. Field studies of co-infected fish populations indicated a negative correlation between the two viruses in randomly sampled apparently healthy fish, in line with the experimental findings, but a positive correlation in moribund or dead fish.The results indicate that the innate antiviral response induced by PRV may temporary protect against a secondary SAV infection.

A bead based multiplex immunoassay detects Piscine orthoreovirus specific antibodies in Atlantic salmon (Salmo salar)

Future growth in aquaculture relies strongly on the control of diseases and pathogens. Vaccination has been a successful strategy for obtaining control of bacterial diseases in fish, but for viral diseases, vaccine development has been more challenging. Effective long-term protection against viral infections is not yet fully understood for fish, and in addition, optimal tools to monitor adaptive immunity are limited. Assays that can detect specific antibodies produced in response to viral infection in fish are still in their early development.Multiplex bead based assays have many advantages over traditional assays, since they are more sensitive and allow detection of multiple antigen-specific antibodies simultaneously in very small amounts of plasma or serum.In the present study, a bead based assay have been developed for detection of plasma IgM directed against Piscine orthoreovirus (PRV), the virus associated with the disease Heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon. Using recombinant PRV proteins coated on beads, antibodies targeting the structural outer capsid protein μ1 and the non-structural protein μNS were detected. Results from a PRV cohabitation challenge trial indicated that the antibody production was initiated approximately two weeks after the peak phase of PRV infection, coinciding with typical HSMI pathology. Thereafter, the antibody production increased while the epicardial inflammation became less prominent.In conclusion, the novel assay can detect PRV-specific antibodies that may play a role in viral defence. The bead-based immunoassay represents a valuable tool for studies on HSMI and possibly other diseases in aquaculture.

Experimental Piscine orthoreovirus infection mediates protection against pancreas disease in Atlantic salmon (Salmo salar).

Viral diseases are among the main challenges in farming of Atlantic salmon (Salmo salar). The most prevalent viral diseases in Norwegian salmon aquaculture are heart and skeletal muscle inflammation (HSMI) caused by Piscine orthoreovirus (PRV), and pancreas disease (PD) caused by Salmonid alphavirus (SAV). Both PRV and SAV target heart and skeletal muscles, but SAV additionally targets exocrine pancreas. PRV and SAV are often present in the same locations and co-infections occur, but the effect of this crosstalk on disease development has not been investigated. In the present experiment, the effect of a primary PRV infection on subsequent SAV infection was studied. Atlantic salmon were infected with PRV by cohabitation, followed by addition of SAV shedder fish 4 or 10 weeks after the initial PRV infection. Histopathological evaluation, monitoring of viral RNA levels and host gene expression analysis were used to assess disease development. Significant reduction of SAV RNA levels and of PD specific histopathological changes were observed in the co-infected groups compared to fish infected by SAV only. A strong correlation was found between histopathological development and expression of disease related genes in heart. In conclusion, experimentally PRV infected salmon are less susceptible to secondary SAV infection and development of PD.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-016-0389-y) contains supplementary material, which is available to authorized users.

Transcriptome analyses of Atlantic salmon (Salmo salar L.) erythrocytes infected with piscine orthoreovirus (PRV).

Heart and skeletal muscle inflammation (HSMI) is a widespread disease of farmed Atlantic salmon (Salmo salar L.) and is associated with piscine orthoreovirus (PRV) infection. PRV is detectable in blood long before development of pathology in cardiac- and skeletal muscle appear, and erythrocytes have been identified as important target cells for the virus. The effects of PRV infection on cellular processes of erythrocytes are not known, but haemolytic anemia or systemic lysis of erythrocytes does not seem to occur, even with high virus loads in erythrocytes. In this study, gene expression profiling performed with high-density oligonucleotide microarray showed that PRV infection of erythrocytes induced a large panel of virus responsive genes. These involved interferon-regulated antiviral genes, as well as genes involved in antigen presentation via MHC class I. PRV infection also stimulated negative immune regulators. In contrast, a large number of immune genes expressed prior to infection were down-regulated. Moderate reduction of expression was also found for many genes encoding components of cytoskeleton and myofiber, proteins involved in metabolism, ion exchange, cell-cell interactions as well as growth factors and regulators of differentiation. PRV did not affect expression of genes involved in heme biosynthesis, gas exchange or erythrocyte-specific markers, but some regulators of erythropoiesis showed decreased transcription levels. These results indicate that PRV infection activates innate antiviral immunity in salmon erythrocytes, but suppresses other gene expression programs. Gene expression profiles suggest major phenotypic changes in PRV infected erythrocytes, but the functional consequences remain to be explored.

Piscine orthoreovirus (PRV) replicates in Atlantic salmon (Salmo salar L.) erythrocytes ex vivo.

Piscine orthoreovirus (PRV) is a reovirus that has predominantly been detected in Atlantic salmon (Salmo salar L.). PRV is associated with heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon, and recently erythrocytes were identified as major target cells. The study of PRV replication and pathogenesis of the infection has been impeded by the inability to propagate PRV in vitro. In this study we developed an ex vivo cultivation system for PRV in Atlantic salmon erythrocytes. PRV was successfully passaged to naïve erythrocytes using lysates of blood cells from infected salmon. During cultivation a significant increase in viral load was observed by RT-qPCR and flow cytometry, which coincided with the formation of cytoplasmic inclusions. The inclusions resembled viral factories and contained both PRV protein and dsRNA. In addition, the erythrocytes generated an antiviral immune gene activation after PRV infection, with significant up-regulation of IFN-α, RIG-I, Mx and PKR transcripts. Supernatants from the first passage successfully transmitted virus to naïve erythrocytes. This study demonstrates that PRV replicates in Atlantic salmon erythrocytes ex vivo. The ex vivo infection model closely reflects the situation in vivo and can be used to study the infection and replication mechanisms of PRV, as well as the antiviral immune responses of salmonid erythrocytes.

Piscine orthoreovirus (PRV) infects Atlantic salmon erythrocytes

Piscine orthoreovirus (PRV) belongs to the Reoviridae family and is the only known fish virus related to the Orthoreovirus genus. The virus is the causative agent of heart and skeletal muscle inflammation (HSMI), an emerging disease in farmed Atlantic salmon (Salmo salar L.). PRV is ubiquitous in farmed Atlantic salmon and high loads of PRV in the heart are consistent findings in HSMI. The mechanism by which PRV infection causes disease remains largely unknown. In this study we investigated the presence of PRV in blood and erythrocytes using an experimental cohabitation challenge model. We found that in the early phases of infection, the PRV loads in blood were significantly higher than in any other organ. Most virus was found in the erythrocyte fraction, and in individual fish more than 50% of erythrocytes were PRV-positive, as determined by flow cytometry. PRV was condensed into large cytoplasmic inclusions resembling viral factories, as demonstrated by immunofluorescence and confocal microscopy. By electron microscopy we showed that these inclusions contained reovirus-like particles. The PRV particles and inclusions also had a striking resemblance to previously reported viral inclusions described as Erythrocytic inclusion body syndrome (EIBS). We conclude that the erythrocyte is a major target cell for PRV infection. These findings provide new information about HSMI pathogenesis, and show that PRV is an important factor of viral erythrocytic inclusions.