Norwegian Veterinary Institute, P.O. Box 750 Sentrum, N-0106 Oslo, Norway.
Norwegian University of Life Sciences, Faculty of Veterinary Medicine, Virology Unit, P.O. Box 8146 Dep., N-0033 Oslo, Norway.
J Virol Methods. 2021 Jan;287:113990. doi: 10.1016/j.jviromet.2020.113990. Epub 2020 Oct 6.
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 × 10 and 2.0 × 10 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.
水媒病毒感染对鱼类健康构成重大威胁。对于许多病毒来说，了解病原体、宿主和环境之间的相互作用是传播的主要障碍。鲑鱼甲病毒（SAV）可感染并引起海水养殖鲑鱼的胰腺疾病（PD）。在感染过程中，SAV 从受感染的鱼中排泄到海水中。我们评估了两种类型的过滤器和四种不同的洗脱液，用于浓缩 SAV3。将 1 升海水与 SAV3 混合，然后进行过滤，从膜过滤器中洗脱病毒。对于带负电荷的 MF 亲水膜过滤器（MF-）与 NucliSENS®裂解缓冲液结合，SAV3 的回收率为 RT-ddPCR 39.5±1.8%和 RT-qPCR 25.9±5.7%。使用带正电荷的 1 MDS Zeta Plus®Virosorb®膜过滤器（MD+）与 NucliSENS®裂解缓冲液结合，回收率为 RT-ddPCR 19.0±0.1%和 RT-qPCR 13.3±3.8%。通过 RT-ddPCR 估计自然海水中 SAV3 的定量下限（LOQ）和检测下限（LOD）分别为 5.18×10 和 2.0×10 SAV3 拷贝/L。从少量海水中回收 SAV3，以及对标准实验室设备的要求，表明 MF 过滤器与 NucliSENS®裂解缓冲液结合将是在实验性试验中进一步验证的候选方法。
