The A segment of infectious pancreatic necrosis virus (IPNV) is expressed as a polyprotein encoding three primary gene products, VP2, NS and VP3, from a large open reading frame. The nucleotide sequence for the A segment of the Sp isolate of IPNV was determined. The NS protein is the putative autocatalytic proteinase responsible for the cleavage of the polyprotein. The functional boundaries of the NS proteinase were mapped by plasmid deletion analysis and examined in an La vitro, translation system. The NS proteolytic activity was determined to lie within the EcoRI and Nsil restriction sites. Characterization of the NS proteinase also was approached by use of proteinase inhibitors and site-directed mutagenesis of the putative catalytic and cleavage sites. Eight proteinase inhibitors, representative of all four proteinase classes, were tested and all failed to inhibit the NS enzyme. Mutagenesis of a putative aspartyl proteinase catalytic motif, DTG, to VTG did not affect proteolytic processing. Additionally, the mutagenesis of the predicted N-terminal cleavage site did not alter processing, however, altered processing was observed when the predicted C-terminal cleavage site was mutated. The major capsid protein, VP2, was mapped with polyclonal and monoclonal antisera. The VP2 gene was digested with Sau3A and subcloned into the pATH expression vector. The trpE-fusion proteins were characterized with polyclonal and monoclonal antisera. Two immunoreactive regions were identified with anti IPNV-Sp sera. A common immunoreactive region, B10, was reactive with antisera to three serotypes of IPNV as well as a neutralizing monoclonal antibody, AS-1. A serotype specific immunoreactive region, A43, also was identified, being recognized only by anti IPNV-Sp sera. The B segment of IPNV encodes the putative RNA-dependent RNA polymerase (RdRp), VP1. The nucleotide sequence for the B segment of the Sp isolate was determined and the deduced amino acid sequences were compared to other polymerases. Concensus sequences associated with GTP-binding proteins and RdRps were identified in the VP1 sequence. However, unlike RdRps associated with single-stranded RNA viruses, the IPNV VP1 proteins lack the Gly-Asp-Asp motif characteristic of this enzyme family. Additionally, the VP1 protein was expressed in a bacterial system and polyclonal antisera was raised against the protein. / Graduation date: 1993

Three closely related isolates belonging to the A��� serotype of infectious pancreatic necrosis virus (IPNV) were selected for comparison, to provide insight into the nature of variation in the virulence of IPN viruses. Brook trout fry (Salvelinus fontinalis) were experimentally infected with the three isolates by immersion. Cumulative mortalities over a 62 day period for the three isolates were 67%, 78%, and 93%. The negative control was 3%. Virus titers from whole fish homogenates sampled at peak mortality for each isolate were statistically similar, indicating that quantity of virus does not account for virulence differences. For the two least virulent isolates, the virus titer was inversely correlated with fish weight, whereas for the most virulent isolate, no correlation was observed. Amino acid sequences of the viral capsid protein VP2 were determined using the reverse transcriptase polymerase chain reaction (RT-PCR). There were two amino acid changes at residue 217 and 288 between the two least virulent isolates and the most virulent isolate. These changes might provide a specific molecular basis for the variations in virulence among isolates. The progression of IPN virus infection in the experimentally infected fry was followed using histopathology, in situ cDNA hybridization, and alkaline phosphatase immunohistochemistry (APIH). While microscopic lesions were limited almost exclusively to necrosis of the pyloric caeca and pancreas, positive reactions with in situ hybridization and APIH were observed in tissues throughout infected fish. An IPNV infection appeared to be established in the fish by two routes: by entering the skin/lateral line and diffusing through the muscle, and from the oral region into the gastrointestinal tract by ingestion. In a second experiment, within a group of experimentally infected brook trout fry, external and behavioral signs of IPN disease in moribund fish disappeared, with the fish becoming healthy in appearance. Several of these fish were sampled, along with dead, moribund, and asymptomatic fish (never showed signs of IPN disease). Very few differences were observed among the fish sampled, using histopathology and in situ hybridization. Fish that appeared to recover after displaying signs of IPN disease died within a 2 week period. / Graduation date: 1997