Fosse, J. H.; Andresen, A. M. S.; Ivanova, L.; Ploss, F. B.; Heffernan, I. A.; Weli, S. C.; Patel, S. C.; Petersen, P. E.; Dahl, M. M.; Christiansen, D. H.; Furnesvik, L.; Falk, K.

Sialic acid-binding viruses often encode receptor-destroying enzymes that modulate infection through host glycan alteration. Isavirus salaris (ISAV), the aetiological agent of infectious salmon anaemia, encodes a haemagglutinin esterase with 4-O-acetylesterase activity. The ISAV esterase removes the virus-targeted epitope and causes homologous attachment interference, but its broader biological roles remain poorly understood. Several aspects of ISAV receptor engagement and release remain unresolved: Although ISAV agglutinates erythrocytes from both rainbow trout and Atlantic salmon, only rainbow trout erythrocytes elute from this interaction. By contrast, Atlantic salmon erythrocytes remain persistently bound, despite the presence of an active esterase. Here, we used ISAV-erythrocyte interactions to dissect receptor destruction and dissociation across viral genotypes and strains. We uncovered substantial functional diversity in viral permissiveness for Atlantic salmon erythrocyte elution. Common variants of residues 229 and 230 of the haemagglutinin-esterase, located at the distal rim of the P1 esterase pocket, were key determinants of elution permissiveness. Elution further required an active esterase catalytic triad. In vivo, Atlantic salmon infected with ISAV carrying the elution-permissive 229N variant showed earlier loss of erythrocyte Neu4,5Ac2 than fish infected with an elution-restrictive strain. The loss of Neu4,5Ac2 preceded the reduction in ISAV-bound erythrocytes by several days, indicating that receptor removal alone was insufficient to trigger virion release. Targeted sialic acid analyses revealed similar levels of Neu4,5Ac2 on Atlantic salmon and rainbow trout erythrocytes. Atlantic salmon erythrocytes additionally expressed di-O-acetylated Neu4,5,9Ac3. By contrast, brown trout erythrocytes expressed little or no Neu4,5Ac2, consistent with their reported inability to support ISAV haemagglutination. Together, our findings give insight into viral and host determinants of ISAV receptor interactions. We demonstrate functional diversity within the ISAV esterase and link virion binding and release to salmonid erythrocyte sialic acid composition. The delay between receptor loss and virion dissociation further supports the existence of additional erythrocyte attachment factors.