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

Two viruses, infectious salmon anaemia (ISA) virus and a novel togavirus-li ke virus, were isolated from ISA disease outbreaks that were first reported as a new syndrome, haemorrhagic kidney syndrome (HKS) affecting farmed Atl antic salmon Salmo salar L. on the East coast of Canada. Laboratory confirm ation of ISA diagnosis was initially complicated by isolation of only the t ogavirus-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 rea ction (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 con sistent with ISA virus infection in experimentally inoculated Atlantic salm on parr (similar to 35 g) in freshwater, beginning 12 d post inoculation. I SA virus was detected by virus isolation from kidney and liver tissues of a ll dead or moribund fish tested. A comparison of virus isolation, 1-step pr ocedure RT-PCR and RNA dot-blot hybridization for detection of ISA virus (I SAV) in fish tissues showed virus isolation to have 100% sensitivity, follo wed by RT-PCR (66 and 28% sensitivity in kidney and liver, respectively), w ith RNA dot-blot hybridization as the least sensitive method (20 and 10% se nsitivity in kidney and liver, respectively). No togavirus-like virus was d etected in these samples by virus isolation. Moreover, another togavirus-li ke virus isolate grown in CHSE-214 cells in the absence of any other detect able pathogen was non-pathogenic in experimentally inoculated fish. This st udy confirms that the original ISA outbreaks in New Brunswick, Canada, were caused solely by ISAV.