Piscine orthoreovirus subtype 3 (PRV-3) causes heart inflammation in rainbow trout (Oncorhynchus mykiss)

Abstract

Piscine orthoreovirus (PRV) mediated diseases have emerged throughout salmonid aquaculture. Three PRV subtypes are currently reported as causative agents of or in association with diseases in different salmonid species. PRV-1 causes heart and skeletal muscle inflammation (HSMI) in Atlantic salmon (Salmo salar) and is associated with jaundice syndrome in farmed chinook salmon (Oncorhynchus tshawytscha). PRV-2 causes erythrocytic inclusion body syndrome (EIBS) in coho salmon in Japan. PRV-3 has recently been associated with a disease in rainbow trout (Oncorhynchus mykiss) characterized by anaemia, heart and red muscle pathology; to jaundice syndrome in coho salmon (Oncorhynchus kisutch). In this study, we conducted a 10-week long experimental infection trial in rainbow trout with purified PRV-3 particles to assess the causal relationship between the virus and development of heart inflammation. The monitoring the PRV-3 load in heart and spleen by RT-qPCR shows a progressive increase of viral RNA to a peak, followed by clearance without a measurable change in haematocrit. The development of characteristic cardiac histopathological findings occurred in the late phase of the trial and was associated with increased expression of CD8+, indicating cytotoxic T cell proliferation. The findings indicate that, under these experimental conditions, PRV-3 infection in rainbow trout act similarly to PRV-1 infection in Atlantic salmon with regards to immunological responses and development of heart pathology, but not in the ability to establish a persistent infection.

Figure 1

Purified PRV-3 particles. A Transmission electron microscopy (TEM) of purified PRV-3 viral particles. B Western blot analysis of proteins from purified PRV-3 particles using antiserum raised against PRV-1 σ1, σ3 and µ1 proteins. Observed bands correspond to predicted full length proteins of 35 kDa, 37 kDA and 74 kDa, respectively.

Figure 2

PRV-3 infection kinetics. RT-qPCR of PRV-3 segment S1 in spleen and heart. Colored dots indicate Ct values of individual fish while horizontal bars show the median Ct value. Samples were considered virus positive at Ct levels below 35 and suspected between Ct 35-40 (grey shaded area); Ct values above 40 are negative. A Shedders (Blue) and cohabitants (red) in tank challenged with purified PRV-3 particles. B Shedders (green) and cohabitants (orange) in tank challenged with PRV-3 infected blood.

Figure 3

Histopathological findings and T-cell markers in the hearts of PRV-3 infected fish. A, B Rainbow trout infected with PRV-3 purified particles Epicarditis (long arrows) and inflammation in stratum compactum (outer layer) and stratum spongiosum (internal layer) of heart ventricle (arrow heads) (H&E). C Relative expression of the T-cell marker CD4 in the heart. The Ct values were normalized against control fish from 0 wpc. Significant differences compared to control are shown in asterisks *P < 0.05 **P < 0.001 and ***P < 0.0001. D Relative expression of the T-cell marker CD8 in the heart. The Ct values were normalized against control fish from 0 wpc. Significant differences compared to control are shown in asterisks *P <0.05 **P <0.001 and ***P <0.0001.

Figure 4

PRV-3 proteins in blood cells and anti-PRV-3 antibodies in plasma. A Western blot analysis of blood cell pellets from cohabitants to purified PRV-3 injected fish. B Multiplex analysis of PRV-3 µ1c specific antibodies in cohabitant fish measured in Mean fluorescence intensity (MFI). Six fish were tested per sampling and group.

Figure 5

Innate antiviral immune gene expression in spleen of PRV-3 challenged fish. Relative expression of IFN1, IFNγ, Mx, and Viperin in controls, pure virus injected fish, blood pellet injected fish and their respective cohabitant groups (n = 6). The Ct values were normalized against control fish from 0 wpc. Significant differences compared to control are shown in asterisks *P <0.05 **P <0.01, ***P <0.001, ****P <0.0001.