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.
Piscine orthoreovirus (PRV) is the causative agent of heart and skeletal muscle inflammation (HSMI), an important emerging disease in farmed Atlantic salmon (Salmo salar L.) [1, 2]. HSMI is characterized by epi-, endo- and myocarditis, with infiltration of mononuclear CD8 positive cells, as well as myositis in red skeletal muscle [3, 4]. The mechanism by which PRV infection causes disease remains largely unknown. PRV has a segmented, double-stranded RNA (dsRNA) genome and belongs to the Reoviridae family [2]. Other fish reoviruses are grouped in the genus Aquareovirus, but phylogenetic analysis indicates that PRV branches off the common root of the genera Orthoreovirus and Aquareovirus, although it clusters more closely with the orthoreoviruses [5, 6]. PRV has 10 genomic segments, as do the orthoreoviruses, but the overall amino acid identity between the homologous proteins is very low, particularly for the surface-exposed and non-structural proteins. However, several amino acid motifs central to protein function are conserved for orthoreoviruses and PRV [6]. Unlike most orthoreoviruses, but similar to the mammalian orthoreoviruses (MRV), PRV is non-fusogenic [5]. This unique taxonomic placement of a fish virus within the Reoviridae family makes PRV particularly interesting. One genogroup and two sub-groups have been suggested after genomic analysis of PRV, but no specific sequence motifs have been found to be correlated with virulence [7, 8]. The lack of an in-vitro cultivation system has restricted the progress of the study of PRV.
Piscine
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HSMI was first described in Norway in 1999 [1]. Since then the number of outbreaks has increased and in 2012 there were 142 registered outbreaks [15]. The disease has also been reported in Scotland [16]. HSMI is mainly observed during the seawater grow-out phase of the fish, with morbidity close to 100% in affected cages, while cumulative mortality varies from negligible to 20% [17]. Typical gross pathologic changes in affected fish include signs of circulatory disturbance; pale heart, ascites, yellow liver, swollen spleen and petechiae in perivascular fat.
Diagnosis of HSMI is currently based on typical histopathological findings in heart and red skeletal muscle [3, 4]. Both the viral load in the heart, as measured by reverse transcription quantitative PCR (RT-qPCR) [4], and the presence of viral proteins in cardiomyocytes, as demonstrated by immunohistochemistry [18], correlate with the development of heart lesions, and indicate that PRV replicates in heart tissue. However, relatively high PRV loads have been detected in both farmed and wild salmon without presence of histopathological changes [8, 10].
PRV has been detected in the spleen and head kidney at higher loads than those of the heart [19], and inoculates originating from heart, liver, kidney/spleen and blood plasma from diseased fish have been used to reproduce disease [20]. The pathogenesis of PRV infection in salmon is largely unknown and possible consequences of PRV infection not related to HSMI should be further investigated. Interestingly, when immunohistochemistry was performed on heart sections from experimentally PRV-challenged fish, circulating cells were found to be PRV positive prior to detection of PRV in cardiomyocytes [18]. The PRV-positive blood cells were located in the cardiac lumen and in blood vessels and included both erythrocytes and leukocyte-like cells. Unfortunately, the available material of that experiment was not suitable for further characterization of these cells. The presence of viremia in PRV infection has not been studied.
We hypothesized that there is a cell-associated viremia in PRV infection that is of importance for the pathogenesis. In two consecutive PRV challenge experiments based on cohabitant transfer of virus, we assessed viral loads in blood, plasma and erythrocytes in the different phases of infection.
50 shedders and 50 cohabitants with an initial average weight of 48 g were used. The inoculum originated from a field outbreak of HSMI as determined by histopathological examination and RT-qPCR that showed high loads of PRV. Four fish from the outbreak, with a mean Ct-value 25.6 for PRV in the heart, were used. The fish were confirmed free of infectious salmon anemia virus (ISAV), salmonid alphavirus (SAV), piscine myocarditis virus (PMCV) and infectious pancreatic necrosis virus (IPNV) by RT-qPCR. The PRV inoculum was prepared by homogenization of the heart, spleen and head kidney. The latter two organs are known to contain higher PRV load than the heart [19].
The shedder fish were injected intraperitoneally with 0.1 mL inoculum, labeled by shortening the outer left maxilla, and placed in a tank containing 50 nave fish (cohabitants). The samples from challenge # 1 were collected from the cohabitant group; sampling six fish every second week starting at 6 weeks post challenge (wpc) and ending at 14 wpc. In addition, 3 cohabitant fish were sampled at 4 wpc to test for transmission of PRV. At each sampling, peripheral blood from the caudal vein was collected into heparinized vacutainers, and tissue from heart, skeletal muscle, spleen and head kidney were sampled in RNAlater (Life Technologies, Carlsbad, CA, USA) and used for RT-qPCR analysis. Parallel samples from the same organs were harvested in 10% phosphate-buffer formalin, embedded in paraffin, and used for histologic analysis.
At 9 wpc, blood from 15 cohabitant fish was sampled, pooled and centrifuged. After removing the plasma, the blood pellet was diluted 1:4 in phosphate-buffered saline (PBS) and stored at -80 C. The Ct-value of the diluted blood pellet was confirmed to contain high loads of PRV by RT-qPCR (Ct-value 19.9) and was used as challenge material in Challenge Experiment # 2. It was also confirmed free of other important viral pathogens of salmon, including ISAV, IPNV, SAV and PMCV.
Slides from the formalin fixed and paraffin embedded heart material collected from Challenge Experiments # 1 and 2 was stained with hematoxylin-eosin (HE) and evaluated by histopathological examination using conventional light microscopy.
RBC isolated from infected fish were fixed overnight at 4 C in PBS with 1.25% glutaraldehyde and 2% paraformaldehyde, washed twice in PBS, and three times in cacodylate buffer (0.1 M, pH 6.8) The cells were post-fixed with 1% OsO4 for 1 h at 4 C and washed with cacodylate buffer. The cells were then dehydrated through an ethanol series (70, 90, 96 and 100%) and embedded in LR-White resin. Thin sections were cut on an ultra microtome (LEICA EM UC 6). The sections were stained with 4% aqueous uranyl acetate and 1% KMNO4 for 10 min, then washed intensively in freshly distilled water. The sections were examined in a FEI MORGAGNI 268, and photographs were recorded using a VELETA camera.
The isolated RBC from Experimental Challenge # 2 were gated according to size and granularity to include only intact cells (Figure 3A). Samples from 0 wpc were used as negative controls, providing the background fluorescence signal from PRV-negative samples (Figure 3B). A PRV-positive erythrocyte population was first observed in two out of six individuals (F55, F56) at 5 wpc, as seen by a peak in fluorescence signal for both intracellular and surface staining. The majority of virus protein was detected intracellularly and a large, distinct population of PRV-positive RBC (up to 43% of cells PRV-positive in F56) could be observed (Figure 3C). As shown by RT-qPCR of RBC, the individual fish that were PRV-positive also contained distinctly higher viral loads compared with the rest of the group (Table 1). PRV was also detected on the surfaces of a small number of RBC of the same individuals. The surface staining data from Experimental Challenge # 2 is shown in additional file (Additional file 3).
At 7 wpc, PRV-positive erythrocytes from Experimental Challenge # 2 were observed in four out of five individual fish and a considerable proportion of isolated RBC (25 - 51%) were PRV-positive (Figure 3C). The majority of the virus was found intracellularly. In contrast with the 5 wpc samples, the PRV-positive cells at 7 wpc could be divided into two separate populations, showing low PRV-positive and high PRV-positive staining. The low PRV-positive population was comparable in intensity to the single PRV population detected at 5 wpc, indicating a development of cells containing high PRV levels over time (Figure 3C). At 8 wpc, high numbers of PRV-positive RBC were detected by intracellular staining in all six individuals. However, at 8 WPC the cells no longer clearly formed two populations of low and high PRV-positive staining. 152ee80cbc
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