Persistent infection of Piscine orthoreovirus-1 (PRV-1) in Atlantic salmon (Salmo salar)

Abstract

This thesis focuses on infection kinetics, infected cell types, viral shedding, and specific immune responses in persistently Piscine orthoreovirus-1 (PRV-1) infected Atlantic salmon. The aim is to enhance the understanding of viral pathogenesis. PRV-1 is ubiquitous in Norwegian Atlantic salmon (Salmo salar) aquaculture. PRV-1 causes an acute infection of erythrocytes of Atlantic salmon and thereafter the virus spreads to cardiomyocytes which induce the disease of heart and skeletal muscle inflammation (HSMI). PRV-1 is not cleared by the Atlantic salmon immune system after acute infection and persists life-long in the host. In Atlantic salmon focal melanized changes (black spots) of white skeletal muscle tissues, which histologically appear as granulomatous structures, are commonly observed in the fillet and is associated with chronic PRV-1 infection. Even if PRV-1 associates with the development of melanized focal changes (black spots), the causal relationship is questionable.

Experimental challenge studies shows that PRV-1 establishes a productive and persistent infection at low level till the end of the trial. PRV-1 transcription level was high in blood cells in the acute phase and in the kidney during the persistent phase. PRV-1 caused plasma viremia that started in the acute phase and lasted for at least 18 weeks, i.e. the end of the experiment. In situ hybridization assays identified PRV-1 infection of Macrophage colony-stimulating factor receptor (MCSFR) positive macrophages in kidney and spleen and Erythropoietin receptor (EPOR) positive erythroid progenitor cells in the kidney, in both acute and persistent phases. The infected erythroid progenitor cells may represent a possible reservoir for PRV-1 as a continuous source of new generated erythrocytes.

Macrophage polarization response (M1/M2) and cell mediated immune response was assessed both in HSMI and black spots formation. M1 macrophages were dominant in the red spots and almost all co-stained for PRV-1. In spots assessed as “late phase” of red spots melanized M2 melano-macrophages appeared, indicating a transition phase to melanized spots. In the melanized spots M2 macrophages were highly abundant. The M2 melano-macrophages of the black spot generally co-stained for PRV-1. In the initial development of HSMI, macrophages were not abundant. Heart and skeletal muscle tissues with characteristic lesions of HSMI showed low numbers of M1 macrophages that only partly co-localized with PRV-1. M2 macrophages, on the other hand, dominated in the heart tissue and were abundant even at the peak in pathologic lesions, which is supportive of a good regenerating ability of the salmon heart. Specific cell mediated responses represented by CD8+, granzyme A (GzmA) and MHC-I expressing cells were identified in heart tissue of fish with HSMI. Massive staining of CD8+ cells with GzmA transcripts were detected in the heart compared to the skeletal muscle after the peak infection of PRV-1. PRV-1 was detected in multiple CD8+ and MHC-1 expressing cells in the heart. Skeletal muscle tissue had a relatively moderate immune response with low number of CD8+ and MHC-I cells and no co-localization with PRV-1. The FISH method revealed the close interplay between PRV-1 infected and cytotoxic cells.

A vaccination experiment was also performed by immunizing Atlantic salmon with the PRV subtypes PRV-2 or PRV-3 to study the protection potential against consecutive PRV-1 infection. This approach was also compared with an inactivated PRV-1 vaccine. The PRV-3 subtype cross-protected fish against secondary PRV-1 infection, while only partial protection was achieved by PRV-2 immunization and by the inactivated vaccine. Antibodies, cross reactive to PRV-1, were elevated in the PRV-3 immunized group, but low in PRV-2 immunized, and undetectable in the inactivated vaccine groups. Moreover, histopathological analysis showed no HSMI like heart lesions in the PRV-3 immunized group. The results provided sufficient evidence that PRV-3 can block the subsequent PRV-1 infection efficiently, at least for the ten-weeks period that the experiment lasted.

To conclude, our studies show that 1) PRV-1 establishes a productive and persistent infection with plasma viremia in Atlantic salmon. 2) Renal erythroid progenitor cells and macrophages could be long-term cellular reservoirs for PRV-1. 3) PRV-1 infection correlates with macrophage polarization in melanized focal changes of skeletal muscle. 4) M1 polarized macrophages do not correlate with initial development of HSMI however, M2 macrophages are associated with high level of PRV-1. 5) A strong activation of cellular immune response is triggered in heart, followed by a drop in PRV- 1 levels. And finally, 6) we demonstrated that the PRV-3 subtype can cross-protect against PRV-1 infection in Atlantic salmon.

Piscine orthoreovirus (PRV) er en meget vanlig infeksjon i havbruk av norsk Atlantisk laks (Salmo salar). Tre varianter av PRV har vært beskrevet, PRV-1 og PRV-2 og PRV-3, som hovedsakelig har vært assosiert med sykdommer i henholdsvis Atlantisk laks, coholaks (Oncorhynchus kisutch) og regnbueørret (O. mykiss). PRV-1 gir en akutt infeksjon av erytrocytter i Atlantisk laks og spres deretter til kardiomyocytter. Dette kan utløse sykdommen i hjerte- og skjelettmuskulær betennelse (HSMB). PRV-1 er også funnet å infisere andre organer og vev, men forholdet til sykdommer eller lidelser bortsett fra HSMI er vagt. PRV-1 infeksjon er persistent i Atlantiske laks. Fokale melaniserte forandringer i hvit skjelettmuskulatur, forandringene kan histologisk fremstå som granulomer, utvikler seg fra røde flekker som inneholder ekstravasale erytrocytter. Mange av betennelsescellene i melaniserte flekker er infisert med PRV-1. Selv om PRV-1 kan assosieres med utviklingen av melaniserte fokale endringer, har vi ikke funnet at PRV-1 er årsaken til at flekkene oppstår. I artiklene i denne tesis ble det fokusert på infeksjonskinetikk, infiserte celletyper, og immunresponser i persistent PRV-1-infisert Atlantisk laks, for å gi en bedre forståelse av PRV-1 sin eventuelle rolle i utvikling av flekker.