The inactivation dynamics of infectious haematopoietic necrosis virus (IHNV) by b-propiolactone (BPL), binary ethylenimine (BEI), formaldehyde or heat and the antigenic and immunogenic properties of the inactivated vaccines were evaluated. Chemical treatment of IHNV with 2.7 mm BPL, 1.5 mm BEI or 50 mm formaldehyde abolished virus infectivity within 48 h whereas heat treatment at 50 or 100 degrees C rendered the virus innocuous within 30 min. The inactivated IHNV vaccines were recognized by rainbow trout, Oncorhynchus mykiss, IHNV-specific antibodies and were differentially recognized by antigenic site I or antigenic site II IHNV glycoprotein-specific neutralizing monoclonal antibodies. The BPL inactivated whole virus vaccine was highly efficacious in vaccinated rainbow trout challenged by waterborne exposure to IHNV 7, 28, 42 or 56 days (15 degrees C) after immunization. The formaldehyde inactivated whole virus vaccine was efficacious 7 or 11 days after vaccination of rainbow trout but performed inconsistently when tested at later time points. The other vaccines tested were not efficacious.

To assess the risk associated with processed rainbow trout (Oncorhynchus mykiss) produced in an area where infectious haematopoietic necrosis (IHN) virus is endemic, 240 fish weighing between 225 grams (g) and 500 g which exhibited spiral curvature or spinal compression deformity types were tested by virus isolation and polymerase chain reaction (PCR) techniques. Rainbow trout aged approximately one year which exhibited spiral deformities were considered to have a high likelihood of previously being infected with IHN virus (IHNV). The fish were gutted and degilled by automated machines and kept cool while specimens were collected for testing. Since internal organs and gills are removed during fish processing for food products, portions of the skin and muscle were collected from the area of the deformity, as well as brain tissue from each fish. Tissue homogenates were tested on epithelioma papulosum cyprini and chinook salmon embryo (CHSE)-214 cell lines which had been pre-treated with polyethylene glycol, using standard methods. Samples on CHSE-214 cells were incubated for 21 days at 15degreesC, then blind passaged for another incubation period of 21 days. Nested reverse transcription PCR (RT-PCR) used the central 1231 base pair portion of the glycoprotein gene to detect IHNV. All brain and skin-muscle homogenates gave negative results for the presence of IHNV by virus isolation and nested RT-PCR. To assess virus clearance experimentally, groups of 100 specific-pathogen-free rainbow trout (mean weight, 100 g) were either intraperitoneally injected with IHNV or sham infected and held separately in 385-litre aquaria in virus-free water at 15degreesC. In all, 33% of the infected fish died. Ten days after the last mortality or 34 days post infection, 6 fish from each treatment were sacrificed at weekly intervals for 6 weeks. Kidney and brain homogenates from individual fish were tested by virus isolation and nested RT-PCR using the nucleoprotein gene. All tissue homogenates gave negative results for IHNV. These results provide scientific information which can be used to assess the risk associated with the movement of processed rainbow trout from an area where IHNV is endemic.

The authors assessed the survival of an aquatic animal virus under different environmental conditions, using infectious haematopoietic necrosis (IHN) virus as an example. Three IHN virus (IHNV) isolates which exhibited antigenic differences were diluted either in fresh water collected from a spring, after this water had passed through a fish farm, or in river water from the river which received water from the fish farm. Each treatment was incubated at 15degreesC in a water bath and samples were removed at hourly and daily intervals. Virus concentrations were determined by plaque assay. Virus suspended in spring water survived longer than virus incubated in water obtained from a fish farm or the river. Virus suspected in river water exhibited a 99% reduction in virus concentration in 24 h. Survival of IHNV was also evaluated at different temperatures over a two-hour period. A 1982 isolate appeared to be less sensitive to temperature that isolates collected in 1990. The concentrations of IHNV in brain and kidney tissue obtained from adult rainbow trout which had been injected with IHNV and died were tested by plaque assay both before freezing and seven or fourteen days after freezing at -20degreesC. Negligible changes in virus concentration were detected. A preliminary study was also conducted to determine the genetic similarity of IHNV isolates from the state of Idaho, United State of America (USA), to isolates from other areas of North America where IHNV is endemic. Isolates were analysed using the ribonuclease (RNase) protection assay (RPA) and by nucleotide sequencing of reverse transcription polymerase chain reaction (RT-PCR) products of specific isolates. By RPA, a high level of genetic heterogeneity was found in Idaho compared to isolates from other locations. A phylogenetic analysis indicated that the isolates from Idaho could be grouped separately from all other IHNV isolates from across the Pacific Northwest, the USA and Canada. These results suggest that the Idaho, IHNV lineages may be phylogenetically distinct. These studies illustrate the complexity of evaluating virus survival and trafficking and the importance of developing this type of information for use in risk assessment.