Presence of a full-length highly polymorphic region (HPR) in the ISAV haemagglutinin-esterase does not affect the primary functions of receptor binding and esterase activity.

The putatively avirulent infectious salmon anaemia virus (ISAV) HPR0 variant has key phenotypic differences to isolates from disease outbreaks in Atlantic salmon farms. It appears to not cause disease, potentially displays a different tissue tropism and has yet to be isolated in conventional ISAV-permissive cell lines. This study focussed on identifying the biological basis for the observed differences by examining the properties of the haemagglutinin-esterase (HE) proteins derived from NWM10 (HPR0), Nevis 390/98 (HPR7 pathogenic strain) and mutant combinations of the two. Using a transfection-based system and haemadsorption analysis in salmon cell lines, this study demonstrated for the first time that an HPR0 HE was fully functional in terms of receptor-binding and -destroying activity and also suggested that the presence of a full-length HPR alone did not appear to affect these functions.

Development of a real-time PCR assay for the identification of Gyrodactylus parasites infecting salmonids in northern Europe.

Gyrodactylus salaris is a monogenean freshwater parasite that causes high mortality in wild Atlantic salmon, and a number of countries employ monitoring programmes for its presence. A TaqMan-MGB (minor groove binding) probe real-time multiplex assay targeting the internal transcribed spacer ribosomal DNA (ITS rDNA) was developed to simultaneously identify G. salaris/G. thymalli and 2 other commonly occurring Gyrodactylus species infecting salmonids in northern Europe: G. derjavinoides and G. truttae. In addition, a Gyrodactylus genus-level assay was developed to assess parasite DNA quality. The species-specific real-time PCR method correctly identified target species from a wide geographical range and from a number of salmonid hosts. It did not amplify G. lucii or G. teuchis. These species were successfully amplified using the Gyrodactylus genus real-time assay. The species-specific real-time assay proved to be significantly faster than the currently employed molecular screening method of ITS rDNA PCR amplification followed by restriction fragment length polymorphism analyses (RFLP). However, as with ITS RFLP, the real-time method did not distinguish between G. salaris and the non-pathogenic G. thymalli, its principle advantage being a significant reduction in time to achieve an initial diagnostic screen before the employment of more in-depth analyses for those specimens giving a positive G. salaris/G. thymalli real-time result.

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.