Journal of General Virology (2002), 83, 2693–2697. Printed in Great Britain .......................................................................................................................................................................................................... SHORT COMMUNICATION Characterization of the receptor-destroying enzyme activity from infectious salmon anaemia virus Marianne Kristiansen,† Marianne K. Frøystad, Anne Lise Rishovd and Tor Gjøen Department of Microbiology, School of Pharmacy, PO Box 1068, Blindern, 0316 Oslo, Norway Infectious salmon anaemia virus (ISAV) infects cells via the endocytic pathway and, like many other enveloped viruses, ISAV contains a receptor-de- stroying enzyme. We have analysed this ace- tylesterase activity with respect to substrate specificity, enzyme kinetics, inhibitors, temperature and pH stability. The ISAV acetylesterase was inhibited by di-isopropyl fluorophosphate (DFP) in a dose-dependent fashion but not by other known hydrolase inhibitors, suggesting that a serine resi- due is part of the active site. The pH optimum of the enzyme was in the range 75–80 and the enzymatic activity was lessened at temperatures above 40 C. The effect of DFP on agglutination/elution of erythrocytes by ISAV demonstrated that the acetylesterase activity is the bona fide receptor- destroying enzyme. A haemadsorption assay was used to analyse whether the esterase was active on the surface of infected cells or not. Infectious salmon anaemia virus (ISAV) causes an infectious disease of farmed Atlantic salmon, Salmo salar L. It has been diagnosed in Norway (Thorud & Djupvik, 1988), Canada (Mullins et al., 1998) and Scotland (Rodger et al., 1998). In addition, ISAV has been shown to replicate in brown trout (Salmo trutta L.) (Nylund et al., 1995), rainbow trout (On- chorhynchus mykiss, Walbaum) (Nylund et al., 1997) and herring (Clupea harengus) (Krossoy et al., 1999) without causing disease. It has also been suggested that ISAV may be transmitted by sea lice (Caligus elongatus and Lepeophtheirus salmonis) (Nylund et al., 1993), although it is not known whether ISAV replicates in the lice. Severe anaemia and high mortalities characterize ISAV-infected salmon. The ISAV genome consists of eight single-stranded RNA segments with negative polarity, suggesting that the virus is Author for correspondence : Tor Gjøen. Fax 47 22844944. e-mail tor.gjoenfarmasi.uio.no †Present address : AL Pharma, Harbitzalleen 3, PO Box 158 Skøyen, N-0112 Oslo, Norway. orthomyxovirus-like (Mjaaland et al., 1997). Comparison of the conserved motifs of the polymerase gene and the 5 mRNA ends with corresponding regions of other viruses with a segmented and negative-sense RNA genome shows a closer relationship with members of the Orthomyxoviridae (Krossoy et al., 1999 ; Sandvik et al., 2000). Recently, the complete sequence and genome organization of a Canadian ISAV isolate was reported (Clouthier et al., 2002). Other known members of this family are the influenza viruses, the Thogoto virus and the Dhori virus. The initial step of influenza infection is binding of the virus particles via their haemagglutinin (HA) to cell-surface sialic acids, and it has recently been demonstrated that the binding of ISAV to the cell surface is neuraminidase-sensitive (Eliassen et al., 2000). Many viruses express enzymatic activity on the surface, for example, the well-known virus sialidases present in influenza A and B, as well as in several para- myxoviruses. Sialidases remove sialic acids present on glyco- proteins or glycolipids and are designated RDEs (receptor- destroying enzymes). RDEs of influenza C, several corona- viruses and bovine toroviruses exhibit sialate O-acetylesterase as well as receptor-binding activity and are called haemag- glutinin esterases (HEs). It has been demonstrated that RDEs Table 1. Effect of various inhibitors on acetylesterase activity in ISAV An activity of 100 % in the control corresponds to 012 ΔA  min25 μg viral protein. Inhibitor Acetylesterase activity (%) Control 100 1 mM di-isopropyl fluorophosphate 90 1 mM physostigmine (eserine) 769 1 mM ethylendiaminetetra-acetic acid 886 1 mM -trans-epoxysuccinyl-leucylamido(4- guanido)butane 922 1 mM bis(p-nitrophenyl) phosphate 986 1 mm phenylmethylsulfonyl fluoride 922 1 mM 2,3-butanedione 1040 1 mgml mucin 976 1 mgml 5-N-acetyl-9-O-acetylneuraminic acid 994 1 mgml fetuin 982 0001-8477  2002 SGM CGJD