Three new methods for identifying IHNV in fish tissue samples have been developed. These methods are based on the initisl growth of the virus in cells in culture and the subsequent analysis of the viral proteins by gel electrophoresis. These methods permit positive identification of IHNV infection as well as strain typing quickly.
The virion protein patterns of 72 isolates of infectious hematopoietic necrosis virus (IHNV) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE) of 35S-methionine labeled virus. This analysis led to the classification of these virus isolates into 5 types according to the apparent molecular weights of the virion N and G proteins. Type 1 virus is characterized by a nucleocapsid protein with an approximate molecular weight of 40,500 daltons. Types 2 and 3 viruses have N proteins of 42,800 and 43,250 respectively. Virus belonging to the Type 2 classification was responsible for the recent outbreaks of IHN disease among fish in the lower Columbia River. The Type 3 virus isolates were obtained from California. In addition to Type 3 virus, California has a different IHN virus type at Coleman hatchery on the Sacramento River. This Type 4 virus is characterized by a slower migrating G protein with an apparent molecular weight of 70,000 daltons. All other virus isolates have G proteins of molecular weight of 67,000 daltons. The Type 5 IHN virus category contains isolates not sufficiently distinct to warrant their classification as a separate type. These findings have been useful in determining (1) a particular virus type is characteristic for a geographic area and will infect many different salmonid species in that area and (2) the same type isolated from patental fish is responsible for the subsequent outbreak of the disease in the progeny fish.
The specific radioactive labeling of virus proteins in the infected cell has also been used to study the intra-cellular synthesis of viral proteins. Using techniques originally developed for diagnosing an IHNV infection, we were able to characterize the sequence of events leading to the synthesis of viral proteins. The temporal synthesis of the viral polypeptides suggest that they were derived from the translation of independently transcribed monocistronic mRNAs.
Polyvalent antisera to purified IHNV was used to develop two additional methods for detecting IHNV infection in cells. Antibody to IHNV was measured by solid phase direct binding assays with radioiodinated Protein A or with immunoperoxidase staining. The high binding antibody titer of rabbit anti-IHNV sera made possible the development of two immunolgical tests for IHNV. Both immunological methods were highly specific, sensitive to less than 10 ng of virus protein and represented new methods of characterizing different strains of IHNV.