CHARACTERIZATION OF THE SMALL OPEN READING FRAME ON GENOME SEGMENT-A OF INFECTIOUS PANCREATIC NECROSIS VIRUS

The genome of infectious pancreatic necrosis virus (IPNV) is composed of two segments of dsRNA. The larger segment contains a small ORF part ly overlapping the 5' end of the polyprotein reading frame. Yet very l ittle is known about this possible new gene, which presumably codes fo r a 17 kDa polypeptide (VP5). The region of the viral genome which enc ompasses the small ORF was reverse-transcribed and amplified by PCR be fore cloning and sequencing. Analysis of the sequences obtained from f ive different virus strains revealed that the small ORF is not found o n one of them, and that it is truncated on two others. Moreover, the d educed amino acid sequences did not appear to be well conserved. Despi te the large variations between IPNV strains at the genomic level, all predicted VP5 are arginine-rich basic polypeptides. To verify whether the small ORF is translated into protein in fish cells, the 17 kDa po lypeptide of the VR-299 strain was expressed as a fusion protein in a prokaryotic expression vector and used to produce a specific antiserum . This antiserum reacted with concentrated virus in an immunodot assay , indicating that VP5 is synthesized in infected cells, but probably o nly in small quantities. When tested with 12 other IPNV strains, resul ts were less conclusive than those obtained with strain VR-299. Nevert heless, three of the 12 viruses gave a clearly negative signal in the immunodot assay, suggesting that possibly more than one viral strain l acks the small ORF.