Genome-wide target enriched viral sequencing reveals extensive ‘hidden’ salmonid alphavirus diversity in farmed and wild fish populations

Genome Sequencing of SAV3 Reveals Repeated Seeding Events of Viral Strains in Norwegian Aquaculture

“…As several of the samples sequenced in this study were from regions where both SAV2 and SAV3 have been detected, the effectiveness of this sequencing method at detecting subtypelevel co-infections was determined. This rationale also follows our recent work, which provided evidence for SAV subtype-level infections in the same samples using a short-read sequencing approach (Gallagher et al, 2020). In the current study, Nanopore reads from samples of confirmed single-subtype infections were individually mapped to a structural polyprotein reference sequence of the relevant SAV subtype using MiniMap2 (Li, 2018) with default parameters.…”

“…Finally, as several of the samples used in this study were from regions that have had outbreaks of both SAV2 and SAV3 in the past, our sequencing method was tested on the sensitivity and accuracy of detecting both subtypes in the same sample. Our previous study has shown that multiple SAV subtypes are commonly co-circulating on the same farm, and may also be found as co-infections within individual fish (Gallagher et al, 2020). At an error rate of less than 0.3%, our results show that sequencing ∼2 kb amplicons and sequencing on the MinION platform enables highly accurate detection of such co-infections, even when the second subtype is at a relatively low titer (as low as 5% of the total SAV reads).…”

“…Combining Nanopore reads from single-subtype infections sequenced using the above approach allowed us to test this method's ability to correctly detect SAV subtype level coinfections by mapping reads simultaneously to multiple reference genomes. A similar method has been used recently to provide evidence of SAV co-infections using high accuracy Illumina data (Gallagher et al, 2020), but its applicability to error-prone longreads was not previously established. Reads from SAV2 and SAV3 were combined in a range of ratios, producing bioinformatic mimics of co-infection scenarios.…”

Emergence of Salmonid Alphavirus Genotype 2 in Norway—Molecular Characterization of Viral Strains Circulating in Norway and Scotland

“…The distribution of these genotypes is, to a certain degree, geographically structured with SAV2 and 3 found in Norway [ 8 , 9 ], SAV1, 2, 4, 5 in Scotland and SAV 1, 2, 5, 6 in Ireland [ 10 ]. There is evidence for mixed genotype infection in salmon at the farm level and within genotype strain diversity within the individual fish [ 11 ]. Besides infecting Atlantic salmon and rainbow trout, an increasing number of studies have demonstrated SAV infections in non-salmonid fish and it is suggested that these may represent wild reservoirs of SAV.…”

“…Besides infecting Atlantic salmon and rainbow trout, an increasing number of studies have demonstrated SAV infections in non-salmonid fish and it is suggested that these may represent wild reservoirs of SAV. The non-salmonid fish species include flatfish [ 11 , 12 , 13 ] and Ballan wrasse [ 14 , 15 ], the latter being the source of a newly identified SAV7 genotype [ 14 ]. Although each of these various genotypes have mostly been identified in individual fish, sequence analyses of SAV derived from a number of wild flatfish [ 11 ] have shown the presence of two genotypes within a single fish, indicating that mixed infections with various SAV genotypes can occur.…”