Department of Molecular and Biomedical Sciences, Aquaculture Research Institute, University of Maine, Orono, ME, USA.
J Fish Dis. 2013 Jan;36(1):35-44. doi: 10.1111/j.1365-2761.2012.01412.x. Epub 2012 Sep 7.
Infectious salmon anaemia (ISA), caused by ISA virus (ISAV), is a serious disease of farmed Atlantic salmon, Salmo salar L. Recently, molecular- and immunofluorescent-based techniques have become powerful diagnostic tools for ISAV detection, but culture-based techniques remain the gold standard. A disadvantage of ISAV culture is that the incubation time required before cytopathic effect (CPE) is observed in cell monolayers. To decrease time until CPE is observed, a low-speed centrifugation technique was applied to existing standard operating procedures for ISAV culture in three fish cell lines. Time until CPE observation was compared in CHSE, SHK and ASK cells, treated or not treated with low-speed centrifugation after inoculation with ISAV. Low-speed centrifugation treatment significantly enhanced observable cell infection. Compared to control cells, the length of time until ISAV CPE observation decreased in centrifuged ASK and CHSE cells. Low-speed centrifugation was also incorporated into a modified clinical shell vial assay. At 48 h post-inoculation with approximately 20 viral particles, ISAV was detected by an immunofluorescence antibody test in treated ASK and SHK1 cells but not in control cells. Finally, this enhanced viral adsorption assay performed in ASK cells demonstrated higher sensitivity than a real-time RT-PCR assay performed on RNA isolated from ISAV-spiked salmon kidney homogenates.
传染性鲑鱼贫血病（ISA）由传染性鲑鱼贫血病毒（ISAV）引起，是一种严重的养殖大西洋鲑鱼（Salmo salar L.）疾病。最近，基于分子和免疫荧光的技术已成为 ISAV 检测的有力诊断工具，但基于培养的技术仍然是金标准。ISAV 培养的一个缺点是，在细胞单层中观察到细胞病变效应（CPE）之前需要的孵育时间。为了缩短观察到 CPE 的时间，在三种鱼类细胞系中对 ISAV 培养的现有标准操作程序应用了低速离心技术。在接种 ISAV 后，用或不用低速离心处理的 CHSE、SHK 和 ASK 细胞中，比较观察到 CPE 的时间。低速离心处理显著增强了可观察到的细胞感染。与对照细胞相比，在离心的 ASK 和 CHSE 细胞中，观察到 ISAV CPE 的时间缩短了。低速离心也被纳入改良的临床壳瓶检测中。在接种约 20 个病毒颗粒后 48 小时，通过免疫荧光抗体试验在处理过的 ASK 和 SHK1 细胞中检测到 ISAV，但在对照细胞中未检测到。最后，在 ASK 细胞中进行的这种增强的病毒吸附测定比从 ISAV 感染的鲑鱼肾脏匀浆中分离的 RNA 进行的实时 RT-PCR 测定具有更高的灵敏度。
