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Abstract
Pancreas disease (PD) of Atlantic salmon is an emerging disease caused by Salmonid alphavirus (SAV) which mainly affects salmonid aquaculture in Western Europe. Although genome structure of SAV has been characterized and each individual viral protein has been identified, the role of 6K protein in viral replication and infectivity remains undefined. The 6K protein of alphaviruses is a small and hydrophobic protein which is involved in membrane permeabilization, protein processing and virus budding. Because these common features are shared across many viral species, they have been named viroporins. In the present study, we applied reverse genetics to generate SAV3 6K-deleted (Δ6K) variant and investigate the role of 6K protein. Our findings show that the 6K-deletion variant of salmonid alphavirus is non-viable. Despite viral proteins of Δ6K variant are detected in the cytoplasm by immunostaining, they are not found on the cell surface. Further, analysis of viral proteins produced in Δ6K cDNA clone transfected cells using radioimmunoprecipitation (RIPA) and western blot showed a protein band of larger size than E2 of wild-type SAV3. When Δ6K cDNA was co-transfected with SAV3 helper cDNA encoding the whole structural genes including 6K, the infectivity was rescued. The development of CPE after co-transfection and resolved genome sequence of rescued virus confirmed full-length viral genome being generated through RNA recombination. The discovery of the important role of the 6K protein in virus production provides a new possibility for the development of antiviral intervention which is highly needed to control SAV infection in salmonids.
Highlights
Salmonid alphavirus (SAV) is the causative agent of pancreas disease (PD) and sleeping disease in Atlantic salmon and rainbow trout, respectively
Genome sequencing except for the 59 and 39 end was performed with primers based on known sequences from other SAV3 strains deposited in GenBank
Viral proteins were expressed within pSAV3HHD6K transfected cells, viral envelope proteins were not detected on the cell surface and no viable virus was released from transfected cells that could spread to neighboring cells
Summary
Salmonid alphavirus (SAV) is the causative agent of pancreas disease (PD) and sleeping disease in Atlantic salmon and rainbow trout, respectively. Salmon pancreas disease virus (SPDV or SAV1) was first found in Ireland and Scotland in farmed Atlantic salmon [2]. The third subtype of SAV (SAV3) is so far exclusively found in Norway affecting both Atlantic salmon and rainbow trout [4]. Another three discrete subtypes (SAV4–6) have been identified in Scotland and Ireland based on partial sequence (nsP3 and E2) analysis [5], and a marine SAV2-related virus is found in PD outbreaks in mid-Norway and Scotland [6]. SAV 1–3 are fully sequenced, with a nucleotide identity of the three SAVs being above 90% over the entire genome
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