Initial events in infectious salmon anemia virus infection: Evidence for the requirement of a low-pH step

We have investigated the initial steps in the interaction between infectiou s salmon anemia virus (ISAV) and cultured cells from Atlantic salmon (SHK-1 cell line), Using radioactively or fluorescently labelled viral particles we have studied the binding and fusion kinetics and the effect of pH on bin ding, uptake, and fusion of ISAV to SHK-1 cells and liposomes, As pH in the medium was reduced from 7.5 to 4.5, the association of virus to the cells was nearly doubled. The same effect of pH was observed when fusion between ISAV and liposomes was analyzed. In addition, the binding of ISAV to intact SHK-1 cells and to cell membrane proteins blotted onto biters was neuramin idase sensitive. However, the increased binding induced by low pH was not n euraminidase sensitive, probably reflecting activation of a fusion peptide at low pH, By using confocal fluorescence microscopy, the increased fusion of fluorescently labelled ISAV with the plasma membrane due to low pH could be demonstrated. When vacuolar pH in the cells was raised during inoculati on with chloroquine or ammonium chloride, both electron and confocal micros copy showed accumulation of ISAV in endosomes and lysosomes, Production of infectious virus could be increased by lowering the extracellular pH during infection. Furthermore, chloro; quine present during virus inoculation als o caused a reduction in the synthesis of viral proteins in ISAV-infected ce lls as well as in the production of infective virus, These results indicate that ISAV binds to sialic acid residues on the cell surface and that the f usion between virus and cell membrane takes place in the acid environment o f endosomes, This provides further evidence for a high degree of similarity between ISAV and influenza virus and extends the basis for the classificat ion of this virus as a member of the Orthamyxoviridae family.