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Title: Development of molecular virology techniques and functional analysis of the mechanisms leading to virulence acquisition in Infectious Salmon Anaemia Virus (ISAV)
Author: Fourrier, Mickael C. S.
ISNI:       0000 0004 5370 7341
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 2015
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Infectious salmon anaemia (ISA) is a viral disease which causes high mortality in farmed Atlantic salmon and has resulted in substantial economic losses to the Aquaculture industry. ISAV is an enveloped orthomyxovirus with a genome consisting of 8 RNA segments. Segments 5 and 6 encode two surface proteins, the fusion protein (F) and haemagglutinin-esterase (HE) respectively. Segment 6 is characterised by a highly polymorphic region (HPR) which corresponds to the near membrane domain of the HE protein stalk. Since the discovery of a putative non-virulent variant (HPR0) with a "full length" HPR and the fact that all currently characterised pathogenic ISAV isolates have deletions in the HPR, many studies have speculated on the importance of this region and its associated deletions in influencing the severity of the disease. However, the function of the HPR and how deletions within this region shape virulence is unknown. To address this issue, this project set out to develop and apply several molecular virology techniques to understand the functional role of the HE HPR deletions, as well as certain F protein mutations. In the long term, this would help to evaluate the risk associated with HPR0 variants in leading to the reemergence of virulent HPR deleted (HPR del.) ISAV strains in farmed Atlantic salmon. Two fusion assays based on the haemagglutination of erythrocyte ghost (EGs) were developed and used to assess the functional effect of engineered mutations. This first study (chapter 2) permitted to generate several HPR del. HE mutants from HPR0 variant sequences. Results demonstrated that in ISAV, the HPR is involved in viral fusion regulation and that deletions in this region enhanced this process, by promoting the activation of the F protein by the HE. This work also clearly indicated that the amino acid (aa) composition of the associated F protein is a critical factor influencing the fusion process in conjunction with HE HPR deletions. In chapter 3, combinations of both mutated HE and F proteins, were tested together to understand the interaction between the two surface glycoproteins in more detail. This approach demonstrated that improved F protein activation by an "HPR del." HE either facilitated proteolytic cleavage by an ubiquitous host cell protease and/or promoted subsequent post-cleavage refolding steps in the F protein. In addition this work identified K276 as the true proteolytic cleavage site of the ISAV F protein, and showed that aa substitutions and insertions upstream of this site increased fusion activity and occasionally proteolytic cleavage. In other viruses, such as Influenza and Newcastle disease viruses (NDV), mutations that enhance viral fusion have been linked to the acquisition of a higher virulence. This project also attempted to develop a reverse genetics system for ISAV, a technique that would permit the production of recombinant viruses and therefore allow direct assessment of the effect of specific mutations on viral replication. To achieve this goal, key ISAV and Atlantic salmon sequences had to be acquired. The work described in chapter 4 succeeded in characterising the 3' and 5' untranslated regions (UTRs) of the 8 ISAV genes. This was done by adapting an RNA segment self-circularisation method previously used with influenza virus with the addition of a critical dephosphorylation step. These sequences were essential for the production of RNA segments with ends that would interact correctly with ISAV polymerase complexes responsible for viral replication. The work described in chapter 5 attempted to characterise the polymerase I (Pol I) promoter of Atlantic salmon using gene walking and sequencing. Such a promoter would have been essential to develop a reverse genetics system based on a salmon cell line and produce recombinant ISAV. An alternative approach, relying on a plasmid bearing mouse Pol I promoter /terminator sequences and expression in baby hamster kidney cells (BHK-21) was also developed. This, combined with some of the 3' and 5' UTRs identified in chapter 4 permitted the expression of chimeric ISAV RNA segments expressing either green fluorescence protein (GFP) or firefly luciferase (FF).
Supervisor: Not available Sponsor: Scottish Government ; Marine Scotland Science
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available