Bowers Robert M, Lapatra Scott E, Dhar Arun K
Advanced BioNutrition Corporation, 7155-H Columbia Gateway Drive, Columbia, MD 21046, USA.
J Virol Methods. 2008 Feb;147(2):226-34. doi: 10.1016/j.jviromet.2007.09.003. Epub 2007 Nov 9.
Infectious pancreatic necrosis virus (IPNV) is a bisegmented double-stranded RNA virus belonging to the family Birnaviridae, genus Aquabirnavirus, which is a major viral pathogen of salmonid fish. The virus infects wild and cultured salmonids, causing high mortality in juvenile trout and salmon. A highly sensitive and specific real-time RT-PCR assay using the fluorogenic dye SYBR((R)) Green I was developed for the detection and quantitation of IPNV in rainbow trout (Oncorhynchus mykiss). Rainbow trout were infected experimentally with IPNV in the laboratory by injection or immersion and then pectoral fin, spleen, and head kidney samples were collected for analysis. The corresponding cDNA was synthesized using DNase I-treated total RNA and then real-time RT-PCR was performed using primers based on the IPNV non-structural protein gene, designated as either NS or VP4. Rainbow trout beta-actin and elongation factor 1alpha (EF-1alpha) genes were used as internal controls. Using real-time RT-PCR, the virus was successfully detected in pectoral fin, spleen, and head kidney tissue samples. The dissociation curves for each amplicon showed a single melting peak at 83, 81.5, and 84 degrees C for IPNV NS, trout beta-actin, and EF-1alpha genes, respectively. The amplicon size and nucleotide sequence was used to confirm the specificity of the products. Using a dilution series of in vitro transcribed RNA, IPNV was reliably detected down to 10 RNA copies and had a dynamic range up to 10(7) RNA copies. A time course assay, using immersion challenged samples, revealed that the virus could be detected in pectoral fin, spleen, and head kidney as early as 24h post-challenge. The average viral load in all three tissues increased over time, reaching its highest level at 21 days post-challenge, which was followed by a slight decrease at 28 days post-challenge. IPNV load in pectoral fin tissue was comparable to the viral load in spleen and head kidney tissues, indicating that pectoral fin could be used for the detection and quantification of IPNV. The development of a non-lethal detection method will be useful for the detection of IPNV and potentially other viruses of finfish in farmed and wild fish.
传染性胰腺坏死病毒（IPNV）是一种双链RNA病毒，其基因组由两个片段组成，属于双RNA病毒科水生双RNA病毒属，是鲑科鱼类的主要病毒病原体。该病毒可感染野生和养殖的鲑科鱼类，导致幼年鳟鱼和鲑鱼的高死亡率。本研究开发了一种使用荧光染料SYBR® Green I的高灵敏度和特异性实时RT-PCR检测方法，用于检测和定量虹鳟（Oncorhynchus mykiss）中的IPNV。在实验室中，通过注射或浸泡的方式使虹鳟感染IPNV，然后采集胸鳍、脾脏和头肾样本进行分析。使用经DNase I处理的总RNA合成相应的cDNA，然后使用基于IPNV非结构蛋白基因（命名为NS或VP4）的引物进行实时RT-PCR。虹鳟β-肌动蛋白和延伸因子1α（EF-1α）基因用作内对照。通过实时RT-PCR，在胸鳍、脾脏和头肾组织样本中成功检测到病毒。每个扩增子的解离曲线显示，IPNV NS、鳟鱼β-肌动蛋白和EF-1α基因的单一解链峰分别在83、81.5和84℃。扩增子大小和核苷酸序列用于确认产物的特异性。使用体外转录RNA的稀释系列，可靠地检测到低至10个RNA拷贝的IPNV，动态范围高达10(7)个RNA拷贝。使用浸泡攻击样本的时间进程分析表明，在攻击后24小时即可在胸鳍、脾脏和头肾中检测到病毒。所有三个组织中的平均病毒载量随时间增加，在攻击后21天达到最高水平，随后在攻击后28天略有下降。胸鳍组织中的IPNV载量与脾脏和头肾组织中的病毒载量相当，表明胸鳍可用于IPNV的检测和定量。开发一种非致死性检测方法将有助于检测养殖和野生鱼类中的IPNV以及潜在的其他鳍鱼类病毒。
