Graduate School of Integrated Bioindustry, Sejong University, Seoul, 05006, South Korea.
Department of Aquatic Life Medicine, Pukyong National University, Busan, 48513, South Korea.
Fish Shellfish Immunol. 2018 Dec;83:443-448. doi: 10.1016/j.fsi.2018.09.025. Epub 2018 Sep 20.
Viral hemorrhagic septicemia virus (VHSV) has been a notorious pathogen in freshwater and marine fish. Due to the lack of effective treatment measures against VHSV disease, the development of prophylactic vaccines has been required, and methods that can produce high-titered viruses would be advantageous in producing cost-effective vaccines. Type I interferon (IFN) responses are the key elements of vertebrates' antiviral activities, and IFN-stimulated gene factor 3 (ISGF3) complex formed through type I IFNs up-regulates the expression of IFN-stimulated genes (ISGs). IFN regulatory factor 9 (IRF9) is a key component of ISGF3, so the inhibition of IRF9 would compromise host's type I IFN responses, which would weaken host antiviral activity. In this study, to increase the replication of VHSV, we generated IRF9 knockout Epithelioma papulosum cyprini (EPC) cells using a CRISPR/Cas9 vector that contains an EPC cell's U6 promoter-driven guide RNA cassette (targeting IRF9 gene) and a Cas9 expressing cassette. In the clones of IRF9 knockout EPC cells, there were no increase in ISG15 gene by poly I:C, and in Mx1 gene by both poly I:C and VHSV. Interestingly, although the increased folds were conspicuously lower than control EPC cells, the expression of ISG 15 gene in all the IRF9 knockout clones was significantly increased by VHSV infection. Control EPC cells pre-treated with poly I:C did not show any CPE when infected with VHSV, however, IRF9 knockout EPC cells showed CPE by VHSV infection in spite of being pretreated with poly I:C. The replication of VHSV in IRF9 knockout EPC cells was significantly faster and higher than that in control EPC cells indicating that the IRF9 knockout-mediated decrease of type I IFN responses allowed VHSV to replicate efficiently. Considering an economical aspect for the production of fish vaccines, the present IRF9 knockout EPC cells can be used to get higher-titered VHSV.
病毒性出血性败血症病毒 (VHSV) 一直是淡水和海水鱼类的一种恶名昭彰的病原体。由于缺乏针对 VHSV 疾病的有效治疗措施，因此需要开发预防性疫苗，而能够产生高滴度病毒的方法在生产高性价比疫苗方面将具有优势。I 型干扰素 (IFN) 反应是脊椎动物抗病毒活性的关键要素，I 型 IFNs 形成的 IFN 刺激基因因子 3 (ISGF3) 复合物上调 IFN 刺激基因 (ISGs) 的表达。干扰素调节因子 9 (IRF9) 是 ISGF3 的关键组成部分，因此抑制 IRF9 会损害宿主的 I 型 IFN 反应，从而削弱宿主的抗病毒活性。在这项研究中，为了增加 VHSV 的复制，我们使用包含 EPC 细胞 U6 启动子驱动的向导 RNA 盒（靶向 IRF9 基因）和 Cas9 表达盒的 CRISPR/Cas9 载体生成了 IRF9 敲除鲤鱼上皮瘤细胞 (EPC) 细胞。在 IRF9 敲除 EPC 细胞的克隆中，poly I:C 不会引起 ISG15 基因的增加，而 poly I:C 和 VHSV 都不会引起 Mx1 基因的增加。有趣的是，尽管增加的倍数明显低于对照 EPC 细胞，但所有 IRF9 敲除克隆中的 ISG15 基因在 VHSV 感染后表达均显著增加。用 poly I:C 预处理的对照 EPC 细胞在感染 VHSV 时不会出现任何 CPE，但 IRF9 敲除 EPC 细胞在先用 poly I:C 预处理的情况下感染 VHSV 时会出现 CPE。IRF9 敲除 EPC 细胞中 VHSV 的复制速度和滴度明显高于对照 EPC 细胞，表明 IRF9 敲除介导的 I 型 IFN 反应降低允许 VHSV 有效地复制。考虑到鱼类疫苗生产的经济性，本研究中的 IRF9 敲除 EPC 细胞可用于获得更高滴度的 VHSV。
