Norwegian School of Veterinary Science, Department of Basic Sciences and Aquatic Medicine, PO Box 8146 Dep., N-0033 Oslo, Norway.
Fish Shellfish Immunol. 2010 May-Jun;28(5-6):845-53. doi: 10.1016/j.fsi.2010.02.001. Epub 2010 Feb 10.
The mechanisms of viral persistence of infectious pancreatic necrosis virus (IPNV) are not well understood. In this study we have used a model of IPNV persistently infected CHSE (Chinook salmon embryonic) cells as correlate of persistent infection in fish focusing on differentially expressed genes using subtractive hybridization (SSH). Selected genes were also analyzed by quantitative real-time PCR (qPCR) in persistently infected parr of Atlantic salmon. Persistent infection was established by growing CHSE cells surviving an IPNV infection. Infection in rescued cells was non-lytic with a virus yield of 10(3)-10(5) TCID(50)/ml of supernatant, resembling what can be found during a persistent infection in vivo. By comparing gene expression in persistently infected cell vs. non-infected cells we found an upregulation of genes involved in direct interaction or degradation of viral proteins, proteasome activating subunit 3, and of ATRX (X-linked alpha-thalassemia/mental retardation syndrome), a transcription repressor, which may indicate a repression of viral replication through reduced transcription. Further ephrin B1 (signal-transduction group) was found strongly up-regulated, and receptors for various ephrins are used for cell interaction and as entry points for other viruses in higher vertebrates. Endonuclease/reverse transcriptase 1 (RVT1) was also found highly up-regulated in persistently infected cells. The comparison of persistently infected cells to in vivo infected fish showed that the expression profiles found in CHSE cells give corresponding results for selected genes, as ATRX, ephrin B1 and RVT-1. We observed similar results by use of two independent methods (SSH and qPCR) for 8 out of 15 genes analyzed and the transcript profile of persistently IPNV-infected cells involve upregulation of genes encoding proteins involved in viral protein degradation and translation inhibition. The understanding is that this may contribute to keep the number of virus particles low during viral persistence.
传染性胰脏坏死病毒 (IPNV) 的持续性感染机制尚未得到很好的理解。在这项研究中，我们使用了 IPNV 持续感染的 CHSE（奇努克鲑鱼胚胎）细胞模型作为鱼类持续性感染的相关模型，重点关注差异表达基因，并使用消减杂交（SSH）进行分析。选择的基因也通过大西洋鲑幼鱼持续感染的定量实时 PCR（qPCR）进行了分析。通过让在 IPNV 感染中幸存的 CHSE 细胞生长，建立了持续性感染。在拯救的细胞中，感染是非裂解性的，上清液中的病毒产量为 10(3)-10(5)TCID(50)/ml，类似于体内持续性感染时的情况。通过比较持续感染细胞与未感染细胞的基因表达，我们发现与病毒蛋白的直接相互作用或降解有关的基因、蛋白酶体激活亚单位 3 和 ATRX（X 连锁的 α-地中海贫血/智力低下综合征）的表达上调，这可能表明通过减少转录来抑制病毒复制。进一步发现 Ephrin B1（信号转导组）强烈上调，高等脊椎动物中各种 Ephrin 的受体用于细胞相互作用和作为其他病毒的进入点。还发现内切核酸酶/逆转录酶 1（RVT1）在持续感染的细胞中高度上调。将持续感染的细胞与体内感染的鱼类进行比较，结果显示在 CHSE 细胞中发现的表达谱与选定基因（如 ATRX、Ephrin B1 和 RVT-1）的结果相对应。我们使用两种独立的方法（SSH 和 qPCR）对分析的 15 个基因中的 8 个进行了类似的观察，并且持续 IPNV 感染细胞的转录谱涉及编码参与病毒蛋白降解和翻译抑制的蛋白质的基因的上调。这表明，这可能有助于在病毒持续性感染期间保持病毒颗粒数量低。
