Isolation and characterisation of segment 1 of the infectious salmon anaemia virus genome

The isolation and characterisation of the largest genomic segment of infectious salmon anaemia virus (ISAV) is reported. Following identification of ISAV-specific clones from a cDNA library, a rapid amplification of cDNA ends-PCR strategy was designed to obtain the sequence of the full length mRNA transcript. The full length open reading frame (ORF) of this gene was shown to be 2169 nucleotides in length, encoding a putative protein of 722 aa. This sequence was demonstrated by RT-PCR to be specific to ISAV-infected cell cultures. The start codon of this ORF was preceded by the ISAV consensus sequence 5' GCTAAGA 3' indicating the full 5' end of the gene to have been obtained. Based on protein size and amino acid composition, this protein was shown to be similar to the PB2 protein of other orthomyxoviruses. Furthermore, a bipartite nuclear localisation signal was identified in the C-terminus of the protein as is found on all of the influenza virus P proteins. Expression of the putative PB2 as a green fluorescent marker protein-fusion protein confirmed that this protein exhibited nuclear localisation in a fish cell line. Sequences of the ISAV segment 1 gene were obtained from Scottish, Norwegian and Canadian ISAV isolates. Analyses confirmed the close genetic relationship between Norwegian and Scottish ISAV and indicated that this segment was among the most conserved of the ISAV genes identified to date. Thus, this evidence strongly suggests that the genomic segment 1 of ISAV encodes a polymerase protein which is thought to be analagous in function to the PB2 protein of influenza viruses.