Abstract
Waterborne viral infections represent a major threat to fish health. For many viruses, understanding the interplay between pathogens, host and environment presents a major hurdle for transmission. Salmonid alphavirus (SAV) can infect and cause pancreas disease (PD) in farmed salmonids in seawater. During infection, SAV is excreted from infected fish to the seawater. We evaluated two types of filters and four different eluents, for concentration of SAV3. One L of seawater was spiked with SAV3, followed by filtration and virus elution from membrane filters. For the negatively charged MF hydrophilic membrane filter (MF-) combined with NucliSENS® lysis buffer the SAV3 recovery was 39.5 ± 1.8 % by RT-ddPCR and 25.9 ± 5.7 % by RT-qPCR. The recovery using the positively charged 1 MDS Zeta Plus® Virosorb® membrane filter (MD+), combined with NucliSENS® lysis buffer was 19.0 ± 0.1 % by RT-ddPCR and 13.3 ± 3.8 % by RT-qPCR. The limits of quantification (LOQ) and detection (LOD) were estimated to be 5.18 × 103 and 2.0 × 102 SAV3 copies/L of natural seawater, by RT-ddPCR. SAV3 recovery from small volumes of seawater, and the requirement for standard laboratory equipment, suggest the MF-filter combined with NucliSENS® lysis buffer would be a candidate for further validation in experimental trials.
Highlights
Salmonid alphavirus (SAV) is a positive-sense single-stranded RNA virus that belongs to the family Togaviridae, genus Alphavirus and en codes structural (E1-E3 and capsid) (Fringuelli et al, 2008), and non-structural proteins nsP1–nsP4 (Weston et al, 1999)
Two different types of membrane filters and four different buffers were evalu ated for their capacity to concentrate and elute SAV3 from 1 L seawater samples
The MF- filter/buffer 1 and MD + filter/buffer 1 methods produced the best SAV3 recoveries from natural seawater with 39.5 ± 1.8 % and 19.0 ± 0.1 %, when the samples were analysed by RT-ddPCR (Table 1)
Summary
Salmonid alphavirus (SAV) is a positive-sense single-stranded RNA virus that belongs to the family Togaviridae, genus Alphavirus and en codes structural (E1-E3 and capsid) (Fringuelli et al, 2008), and non-structural proteins nsP1–nsP4 (Weston et al, 1999). According to revised legislation from 2017, the control of PD requires monthly sampling of fish from all marine farming sites holding salmonid fish for testing by polymerase chain reaction (PCR). This surveillance method leads to sacrificing thousands of fish every year, and is problematic both in relation to aquaculture economy, as well as to animal welfare. For this reason, detection of the virus excreted into the seawater surrounding the fish populations would be more cost effective than sampling of fish, and potentially yield data on virus presence earlier in the course of the infection. RT-ddPCR has reduced sensitivity to inhibitors and provides direct quantification of the target (Burns et al, 2010; Lui and Tan, 2014; Racki et al, 2014)
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