Characterization and comparison of the full 3′ and 5′ untranslated genomic regions of diverse isolates of infectious salmon anaemia virus by using a rapid and universal method

The 3′ and 5′ untranslated regions (UTRs) of the gene segments of orthomyxoviruses interact closely with the polymerase complex and are important for viral replication and transcription regulation. Despite this, the 3′ and 5′ RNA UTRs of the infectious salmon anaemia virus (ISAV) genome have only been partially characterized and little is known about the level of conservation between different virus subtypes. This report details for the first time, the adaptation of a rapid method for the simultaneous characterization of the 3′ and 5′ UTRs of each viral segment of ISAV. This was achieved through self circularization of segments using T4 RNA ligase, followed by PCR and sequencing. Dephosphorylation of 5′ ends using tobacco acid pyrophosphatase (TAP) proved to be a specific requirement for ligation of ISAV ends which was not essential for characterization of influenza virus in a similar manner. The development of universal primers facilitated the characterization of 4 genetically distinct ISAV isolates from Canada, Norway and Scotland. Comparison of the UTR regions revealed a similarity in organization and presence of conserved terminal sequences as reported for other orthomyxoviruses. Interestingly, the 3′ ends of ISAV segments including segments 1, 5 and 6, were shorter and 5′ UTRs generally longer than in their influenza counterparts.