Short communication: Evaluation of charged membrane filters and buffers for concentration and recovery of infectious salmon anaemia virus in seawater

Abstract

Infectious salmon anaemia virus (ISAV) is the cause of an important waterborne disease of farmed Atlantic salmon. Detection of virus in water samples may constitute an alternative method to sacrificing fish for surveillance of fish populations for the presence of ISA-virus. We aimed to evaluate different membrane filters and buffers for concentration and recovery of ISAV in seawater, prior to molecular detection. One litre each of artificial and natural seawater was spiked with ISAV, followed by concentration with different filters and subsequent elution with different buffers. The negatively charged MF hydrophilic membrane filter, combined with NucliSENS® lysis buffer, presented the highest ISAV recovery percentages with 12.5 ± 1.3% by RT-qPCR and 31.7 ± 10.7% by RT-ddPCR. For the positively charged 1 MDS Zeta Plus® Virosorb® membrane filter, combined with NucliSENS® lysis buffer, the ISAV recovery percentages were 3.4 ± 0.1% by RT-qPCR and 10.8 ± 14.2% by RT-ddPCR. The limits of quantification (LOQ) were estimated to be 2.2 x 103 ISAV copies/L of natural seawater for both RT-qPCR and RT-ddPCR. The ISAV concentration method was more efficient in natural seawater.

Infectious Salmon Anemia Virus Shedding from Infected Atlantic Salmon (Salmo salar L.)—Application of a Droplet Digital PCR Assay for Virus Quantification in Seawater

“…ISAV prevalence in seawater tanks and in the gill and heart samples at the peak of infection followed the same progression. In our previous laboratory experiments, limit of detection (LOD) of virus for the concentration method A was calculated to be 1.1 × 10 4 ISAV RNA copies/L of natural seawater [ 20 ], which is similar to the average virus detection found in the present study. Nevertheless, in field condition, reliable detection of ISAV, which may be present in the seawater at very low concentrations, may require some further optimization of the method.…”

“…A comparison of the performance of the five different concentration methods showed that the use of the method of applying the electronegatively charged filter together with NucliSENS ® lysis buffer for elution resulted in detection of significantly higher levels of ISAV RNA from seawater by RT-ddPCR compared to the other four methods. The higher ISAV RNA concentration detected from seawater with the MF - /buffer 1 method (A) correlates with results from an in vitro study carried out with seawater samples spiked with ISAV [ 20 ], as well as from recent equivalent studies with salmonid alphavirus in seawater [ 20 , 26 ].…”

“…The same primer and probe used for RT-qPCR assays were used for RT-ddPCR. The annealing temperature and primer/probe concentrations have been optimized as described previously [ 20 ]. The RT-ddPCR was performed in 20 μL volume, with 1.8 μL of RNA extracted from the seawater concentrates, and with primers and probe at final concentrations of 900 nM and 250 nM, respectively.…”

“…Recently, we have developed a method for detection of Salmonid alphavirus (SAV) in seawater for surveillance purposes [ 19 , 20 ]. In order to also implement this methodology for ISAV, in vitro experiments for concentrating virus in seawater have been carried out [ 20 ].…”

“…Recently, we have developed a method for detection of Salmonid alphavirus (SAV) in seawater for surveillance purposes [ 19 , 20 ]. In order to also implement this methodology for ISAV, in vitro experiments for concentrating virus in seawater have been carried out [ 20 ]. The study involved spiking of ISAV in seawater, followed by concentration of virus by adsorption to charged membrane filters and virus elution, before molecular detection.…”