HAVBRUK2-Stort program for havbruksforskning

Pancreas disease (PD), caused by salmonid alphavirus (SAV) is a major challenge in Norwegian aquaculture. Diseased fish shows extensive damage in pancreas and inflammation in the heart and skeletal muscle resulting in reduced fish welfare and increased costs for the industry due to reduced growth and loss due to mortality. Disease outbreaks in Norway might be caused by either the SAV3 or SAV2 virus genotypes of which SAV3 caused outbreaks are considered to have a higher impact on fish health and welfare and the industry. There is a general separation in geographical distribution of the two genotypes, but both are also found within the same county in the overlap between these geographical regions and might also be seen together in single individuals of PD outbreaks. This causes a concern to whether these can recombine and cause a new genotype of higher virulence. We have shown earlier that SAV3 can change its genome through recombination and that this can result in new virus variants or on the other side, in defect virus genomes due to deletions of important genomic components. In this project we will study how this recombination happen and characterize both viable and defect outcomes. This will also include studies describing possibilities of and characterization of outcome in a SAV3/SAV2 recombination event. The presence of defect genomes through various disease phases and the salmon’s immune response in relation to this will also be studied for both genotypes. In general, we will develop new knowledge on the potential of SAV3 and SAV2 to recombine and the importance for resulting disease. New knowledge will also be achieved on recombination mechanisms and give significant insight into the role and importance of eventually resulting defect virus genomes, a product of viral replication previously overlooked in virological research, but increasing evidence now points towards an important role in a multifactorial virus system. The project is led by Associate professor Aase B. Mikalsen, NMBU, Norway. Collaborating partner is Dr. Marco Vignuzzi, ASTAR Labs, Singapore (previous Pasteur Institute, France). Other personnel involved is researcher/PhD Turhan Markussen, Professor Øystein Evensen (NMBU) and we have recently hired MSc Fabian Kropp for a 3-year PhD on a parallel related project. In the first years of the project, we have focused on basic studies to gain increased knowledge of the SAV2 genotype by full genome sequencing and replication studies in cell culture. Subsequently, we have established optimized tools and procedures for later cell culture and fish challenge experiments including both SAV3 and SAV2. Reverse genetics vector plasmids for production of both SAV3 and SAV2 is now available in the lab. The available SAV3 vector has been used to produce a clean full length starting point for serial passaging in cell culture followed by microscopy observations and titration at specific time points at NMBU. Virus harvested at selected passages has been analyzed for presence of defect viral genomes (DVGs), i.e. SAV3 RNA including deletions. We have identified abundant DVGs characterized by specific deletion patterns that manage to carry over several passages. This suggests that they can compete with full-length genomes, which is an interesting and important progress in project results. DVG candidates with potential inhibiting characteristics have been selected and testing of their effect in virus replication is in progress. We have also in the first years of the project, initiated studies on how the presence of defective RNA harboring deletions may affect the outcome of diagnostic real-time PCR. Several sets of primers specific to SAV genomic regions with low prevalence of deletions as well as to candidate regions with high prevalence of deletions (i.e. deletion hot spots) have been designed and efficiency validated. Real-time PCR using the sets of primers with various specificity for full-length RNA/RNA incl. deletion have been tested on examples of RNA originating in cell culture or from tissue samples available in the lab.

Pancreas disease (PD) is a major disease in Norwegian salmon farming. It is a notifiable disease and Norwegian Food Safety Authorities have established national regulations in order to limit its spread. Despite so and the use of commercial vaccines, number of reported PD cases remains high. The disease causes substantial economic losses every year and has also a welfare impact on the fish and quality of the final product (flesh quality). SAV3 has recently been shown to occur a co-infection with sub-type 2 in Norway. The focus of this project builds on previous results describing RNA recombination of SAV3 in salmon. Recombination events during infection has raised concern that SAV3 can recombine with SAV2 RNA, when infecting the same individual, the outcome being unpredictable. So we will explore possible recombination events between SAV3 and SAV2 and study and characterise the outcome. We have previously shown that recombination is imprecise, which results in high levels of defective RNA which until recently was regarded as not useful for virus replication. Recent research on mammalian viruses indicates that such defect RNA might be relevant in a multi-component viral system. Through collaboration with Dr. Vignuzzi's group at Pasteur Institute who is in the forefront of this research, we will explore the impact of defective RNA on virus replication, host responses and implications for diagnosis. We will include studies on mechanisms resulting in the deletions, i.e. role of RNA secondary structures. New knowledge will shed light on SAV infection in salmon, provide more details on immunological reactions to the infection, and a particular novel and ambitious aspect is use of defective genomes as a therapeutic approach to cure infection and disease, mechanisms not studied in any target host for any virus previously. This will provide a basis for health control and reduce disease and mortality related to SAV infection and will provide comparative knowledge.