Aquaculture Pathology Laboratory, School of Animal and Comparative Biomedical Sciences, University of Arizona, Tucson, AZ 85721, USA.
Viruses. 2020 Sep 16;12(9):1030. doi: 10.3390/v12091030.
Taura syndrome is a World Organization for Animal Health (OIE)-listed disease of marine shrimp that is caused by Taura syndrome virus (TSV), a single-stranded RNA virus. Here we demonstrate the utility of using 15-year-old archived Davidson's-fixed paraffin-embedded (DFPE) shrimp tissues for TSV detection and phylogenetic analyses. Total RNA was isolated from known TSV-infected DFPE tissues using three commercially available kits and the purity and ability to detect TSV in the isolated RNA were compared. TSV was successfully detected through RT-qPCR in all the tested samples. Among the TSV-specific primers screened through RT-PCR, primer pair TSV-20 for the RNA-dependent RNA polymerase (RdRp), primers TSV-15 and TSV-16 for the capsid protein gene VP2 and primers TSV-5 for the capsid protein gene VP1 amplified the highest number of samples. To assess the phylogenetic relation among different TSV isolates, the VP1 gene was amplified and sequenced in overlapping segments. Concatenated sequences from smaller fragments were taken for phylogenetic analyses. The results showed that the TSV isolates from this study generally clustered with homologous isolates from the corresponding geographical regions indicating RNA derived from DFPE tissues can be used for pathogen detection and retrospective analyses. The ability to perform genomic characterization from archived tissue will expedite pathogen discovery, development of diagnostic tools and prevent disease spread in shrimp and potentially other aquaculture species worldwide.
桃拉综合征是世界动物卫生组织(OIE)列出的一种海洋虾类疾病,由桃拉综合征病毒(TSV)引起,这是一种单链 RNA 病毒。在这里,我们展示了使用 15 年前存档的 Davidson 固定石蜡包埋(DFPE)虾组织进行 TSV 检测和系统发育分析的实用性。使用三种市售试剂盒从已知 TSV 感染的 DFPE 组织中分离总 RNA,并比较了分离 RNA 的纯度和检测 TSV 的能力。通过 RT-qPCR 在所有测试样本中均成功检测到 TSV。在通过 RT-PCR 筛选的 TSV 特异性引物中,用于 RNA 依赖性 RNA 聚合酶(RdRp)的引物对 TSV-20、用于衣壳蛋白基因 VP2 的引物 TSV-15 和 TSV-16 以及用于衣壳蛋白基因 VP1 的引物 TSV-5 扩增了最多数量的样本。为了评估不同 TSV 分离株之间的系统发育关系,通过 RT-PCR 扩增并测序了 VP1 基因的重叠片段。来自较小片段的拼接序列用于系统发育分析。结果表明,本研究中的 TSV 分离株通常与相应地理区域的同源分离株聚类,表明源自 DFPE 组织的 RNA 可用于病原体检测和回顾性分析。从存档组织中进行基因组特征分析的能力将加速病原体的发现、诊断工具的开发,并防止虾类和全球潜在其他水产养殖物种中的疾病传播。
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