The application of recombinant DNA technology to the study of the infectious pancreatic necrosis virus
Infectious pancreatic necrosis virus (IPNV), a birnavirus, is an economically important pathogen in the fish farming industry. The aim of this project was to apply the techniques of recombinant DNA technology to develop methods for the detection of IPNV nucleic acid and the classification of IPNV strains, and to the production of a recombinant vaccine. A polymerase chain reaction (PCR) protocol was developed to amplify cDNA encoding three regions of the IPNV/Sp strain genome: the VP2 and the VP3 genes, encoding the major and minor structural proteins respectively, and the hypervariable region of the VP2 gene (VP2var) encoding a strain-specific sequence. The last was used to prepare a cDNA probe to detect Sp strain nucleic acid in the kidney and pancreas from IPNV infected fish. From this preliminary study, it appeared that the kidney stromal cells were the main site of viral nucleic acid. The PCR and nucleotide sequence analysis were used to investigate IPNV strain variation. The VP2var regions of the indigenous Scottish Sp strain, a virulent field isolate from Shetland and the Norwegian N1 strain were isolated using the PCR and cloned into the bacteriophage M13mp18. Nucleotide sequence analysis of the VP2var showed that the strains belonged to the Sp serotype. The cDNA encoding the VP2 and VP3 genes was cloned into the phagemid pBluescript and the plasmid YEpGALPH2 for expression of recombinant viral proteins in Escherichia coli and Saccharomyces cerevisiae respectively. Although the VP2 and VP3 genes had been cloned in pBluescript in the correct orientation for expression, synthesis of recombinant viral proteins was not detected.