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 partly overlapping the 5' end of the polyprotein reading frame. Yet very little is known about this possible new gene, which presumably codes for a 17 kDa polypeptide (VP5). The region of the viral genome which encompasses the small ORF was reverse-transcribed and amplified by PCR before cloning and sequencing. Analysis of the sequences obtained from five different virus strains revealed that the small ORF is not found on one of them, and that it is truncated on two others. Moreover, the deduced amino acid sequences did not appear to be well conserved. Despite 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 polypeptide of the VR-299 strain was expressed as 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 only in small quantities. When tested with 12 other IPNV strains, results were less conclusive than those obtained with strain VR-299. Nevertheless, three of the 12 viruses gave a clearly negative signal in the immunodot assay, suggesting that possibly more than one viral strain lacks the small ORF.