Salmon Erythrocytes Sequester Active Virus Particles in Infectious Salmon Anaemia

Abstract

Infectious salmon anaemia virus (ISAV) binds circulating Atlantic salmon erythrocytes, but the relevance of this interaction for the course of infection and development of disease remains unclear. We here characterise ISAV-erythrocyte interactions in experimentally infected Atlantic salmon and show that ISAV-binding to erythrocytes is common and precedes the development of disease. Viral RNA and infective particles were enriched in the cellular fraction of blood. While erythrocyte-associated ISAV remained infectious, erythrocytes dose-dependently limited the infection of cultured cells. Surprisingly, immunostaining of blood smears revealed expression of ISAV proteins in a small fraction of erythrocytes in one of the examined trials, confirming that ISAV can be internalised in this cell type and engage the cellular machinery in transcription and translation. However, viral protein expression in erythrocytes was rare and not required for development of disease and mortality. Furthermore, active transcription of ISAV mRNA was higher in tissues than in blood, supporting the assumption that ISAV replication predominantly takes place in endothelial cells. In conclusion, Atlantic salmon erythrocytes bind ISAV and sequester infective virus particles during infection, but do not appear to significantly contribute to ISAV replication. We discuss the implications of our findings for infection dynamics and pathogenesis of infectious salmon anaemia.

Destruction of the vascular viral receptor in infectious salmon anaemia provides in vivo evidence of homologous attachment interference

“…Detection of viral proteins by immunostaining has identified that the main target cells of infection are endothelial cells, lining the vasculature of all organs [ 5 ]. In addition, circulating erythrocytes sequester active viral particles in infected fish [ 5 , 6 ].…”

The infectious salmon anaemia virus esterase prunes erythrocyte surfaces in infected Atlantic salmon and exposes terminal sialic acids to lectin recognition

“…Cell pellets were prepared from full blood or density-purified erythrocytes and stored at −80 °C before preparation of membrane-enriched fractions as previously described [24]. Briefly, 100 μL cell pellets were lysed by 1:10 dilution in ice cold water with 1% protease inhibitor (P8340, Sigma-Aldrich, 10 min, on ice).…”

“…To map virus-binding sites in Atlantic salmon tissues and membrane-enriched cell fractions, we used virus antigen preparations as primary probes as previously described [21,23,24]. Deparaffinised formalin-fixed tissue sections were incubated with 100 µL ISAV antigen (512 HAU/mL, 60 min, RT), washed (PBS × 3), quenched with peroxidase block (5 min, RT), treated with blocking buffer (Background sniper, Biocare Medical, 30 min, RT), incubated with mouse IgG 1 specific to ISAV HE (clone 3H6F8 [52], hybridoma supernatants diluted 1:100, 60 min, RT), washed (PBS × 3), and signal was visualised by the MACH2 HRP polymer-DAB (Biocare Medical) system, following manufacturer's instructions.…”

“…Consequently, any surface modulation would be independent of the cellular expression of viral proteins. Second, prior to the onset of sepsislike clinical signs and mortality, ISAV-infected fish typically develop anaemia [24,25]. In general, sialic acids and their 9-O-acetylation are involved in regulating the circulating half-life of erythroid-lineage cells in other species [26][27][28][29]; hence, a modulation of erythrocyte sialic acids could potentially contribute to the pathogenesis of ISA.…”

“…Several lines of reasoning made us curious if erythrocyte surfaces were modulated in a similar manner in infected fish: First, nucleated fish erythrocytes are targeted by ISAV, but show limited, if any, permissiveness to infection [24]. Consequently, any surface modulation would be independent of the cellular expression of viral proteins.…”