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Two viruses, infectious salmon anaemia (ISA) virus and a novel togavirus-like virus, were isolated from ISA disease outbreaks that were first reported as a new syndrome, haemorrhagic kidney syndrome (HKS) affecting farmed Atlantic salmon Salmo salar L. on the East coast of Canada. Laboratory confirmation of ISA diagnosis was initially complicated by isolation of only the togavirus-like agent using the CHSE-214 cell line. Here we demonstrate that a clinical sample from a disease outbreak of ISA contained a mixture of ISA virus and togavirus-like virus. Reverse transcriptase-polymerase chain reaction (RT-PCR) confirmed the presence of both viruses during serial passage of cultures in SHK-1 and CHSE-214 cells. Virus harvested at passage level 3 in both cell lines caused high mortalities and severe gross pathology consistent with ISA virus infection in experimentally inoculated Atlantic salmon parr (approximately 35 g) in freshwater, beginning 12 d post inoculation. ISA virus was detected by virus isolation from kidney and liver tissues of all dead or moribund fish tested. A comparison of virus isolation, 1-step procedure RT-PCR and RNA dot-blot hybridization for detection of ISA virus (ISAV) in fish tissues showed virus isolation to have 100% sensitivity, followed by RT-PCR (66 and 28% sensitivity in kidney and liver, respectively), with RNA dot-blot hybridization as the least sensitive method (20 and 10% sensitivity in kidney and liver, respectively). No togavirus-like virus was detected in these samples by virus isolation. Moreover, another togavirus-like virus isolate grown in CHSE-214 cells in the absence of any other detectable pathogen was non-pathogenic in experimentally inoculated fish. This study confirms that the original ISA outbreaks in New Brunswick, Canada, were caused solely by ISAV.

A dual infection of infectious salmon anaemia (ISA) virus and a togavirus-like virus in ISA of Atlantic salmon Salmo salar in New Brunswick, Canada