Department of Aquatic Life Medicine, Pukyong National University, Busan 48513, the Republic of Korea.
Department of Aquatic Life Medicine, Pukyong National University, Busan 48513, the Republic of Korea.
J Virol Methods. 2022 Aug;306:114554. doi: 10.1016/j.jviromet.2022.114554. Epub 2022 May 24.
The iron flocculation method, which comprises the Fe-virus flocculate formation-filtration-resuspension steps, is extensively used to concentrate and precipitate viruses distributed in water. To apply this method to concentrate white spot syndrome virus (WSSV) in seawater, viral genomic and infective recovery yields were compared between polyethylene sulfone (PES) and polycarbonate (PC) membrane filters and two types of resuspension buffers (oxalate and ascorbate). Viral genome quantitation was determined above a 95 % limit of detection (11.48 viral DNA copies/μL) using quantitative real-time PCR. From WSSV-spiked seawater (10-10 viral DNA copies/mL), the viral genomic recovery yields of the PES-Oxalate, PC-Oxalate, PES-Ascorbate, and PC-Ascorbate conditions were 78.67 % ± 12.90 %, 84.53 % ± 24.30 %, 85.59 % ± 16.98 %, and 93.74 % ± 7.44 %, respectively. The detectable Fe-virus flocculates collected by the PC membrane were approximately 10 WSSV DNA copies/mL of seawater, a value more than 10-fold higher than that compared to the PES membrane filter (10 WSSV DNA copies/mL), regardless of the resuspension buffer types. WSSV resuspended with oxalate buffer caused mass mortality among whiteleg shrimp (Litopenaeus vannamei), inducing the expression of the virus envelope protein, VP28, similar to that of a native virus, suggesting stable viral activity during the resuspension process. Based on the PES-Ascorbate, WSSV particles could be successfully concentrated in seawater from shrimp farms with white spot disease outbreaks (approximately 10 WSSV DNA copies/mL). Collectively, these findings indicate that the simple and efficient method of iron flocculation is sufficient to concentrate WSSV in seawater and could be used as a non-invasive approach and one of the reasonable diagnostic processes for white spot disease surveillance.
铁絮凝法包括 Fe-病毒絮体形成-过滤-再悬浮步骤，广泛用于浓缩和沉淀分布在水中的病毒。为了将该方法应用于浓缩海水养殖虾白斑综合征病毒（WSSV），比较了聚醚砜（PES）和聚碳酸酯（PC）膜过滤器以及两种再悬浮缓冲液（草酸盐和抗坏血酸）之间的病毒基因组和感染性回收产量。使用定量实时 PCR 确定超过 95%检测限（11.48 个病毒 DNA 拷贝/μL）的病毒基因组定量。从 WSSV 污染的海水（10-10 个病毒 DNA 拷贝/mL）中，PES-Oxalate、PC-Oxalate、PES-Ascorbate 和 PC-Ascorbate 条件下的病毒基因组回收率分别为 78.67%±12.90%、84.53%±24.30%、85.59%±16.98%和 93.74%±7.44%。用 PC 膜收集的可检测到的 Fe-病毒絮体大约为 10 个海水 WSSV DNA 拷贝/mL，比 PES 膜过滤器（10 个 WSSV DNA 拷贝/mL）高 10 倍以上，无论再悬浮缓冲液类型如何。用草酸盐缓冲液悬浮的 WSSV 导致凡纳滨对虾（Litopenaeus vannamei）大量死亡，诱导病毒包膜蛋白 VP28 的表达，与天然病毒相似，表明在再悬浮过程中病毒具有稳定的活性。基于 PES-Ascorbate，可以成功地从爆发白斑病的虾养殖场海水中浓缩 WSSV（约 10 个 WSSV DNA 拷贝/mL）。总之，这些发现表明，铁絮凝的简单高效方法足以浓缩海水中的 WSSV，可以作为一种非侵入性方法和白斑病监测的合理诊断过程之一。
