Development and evaluation of a method for concentration and detection of salmonid alphavirus from seawater

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.

Keywords: Droplet digital PCR; Membrane filters; Method development; Seawater; Virus concentration.