Z-FA-FMK demonstrates differential inhibition of aquatic orthoreovirus (PRV), aquareovirus (CSRV), and rhabdovirus (IHNV) replication

Benzyloxycarbonyl-phenylalanyl-alanyl-fluoromethyl ketone (Z-FA-FMK) is a protease inhibitor that has been shown to strongly inhibit mammalian orthoreovirus replication. Here we explore the ability of Z-FA-FMK to inhibit three important yet genetically discrete aquatic fish viruses: chum salmon aquareovirus (CSRV), piscine orthoreovirus (PRV), and the rhabdovirus infectious hematopoietic necrosis virus (IHNV). Z-FA-FMK significantly attenuated CSRV in vitro transcription and infectious yield following low-dose (2-20 μM) exposure, yet a relatively high dose (200 μM) was required to completely block CSRV replication. For PRV and IHNV, no significant attenuation of in vitro viral transcription was observed following low-dose (2-20 μM) exposure; and although high dose (200 μM) exposure significantly attenuated both PRV and IHNV transcription, neither was completely inhibited. These transcriptional results were similarly reflected in IHNV infectious titre observed at 7 days post exposure. PRV titre is currently undeterminable in vitro; however, in vivo intra-peritoneal injection of PRV into juvenile Atlantic salmon (Salmo salar) in conjunction with 1.5 mg/kg Z-FA-FMK did not affect PRV replication as measured by blood associated viral transcripts at 14 days post challenge. These results indicate that aquatic ortho- and aqua-reoviruses appear to possess resilience to Z-FA-FMK relative to mammalian orthoreoviruses and suggest that environmental parameters or alternative mechanisms for viral replication may affect the efficacy of Z-FA-FMK as an antireoviral compound. Further, as Z-FA-FMK has been shown to irreversibly inhibit cysteine proteases such as cathepsins B and L in vitro at concentrations of ≤100 μM, continued replication of IHNV (and possibly PRV) at 200 μM Z-FA-FMK suggests that replication of these viruses can occur in a cathepsin-independent manner whereas CSRV likely requires cathepsins or similar cysteine proteases for successful replication.