Aquatic Animal Health Laboratory, Department of Pathobiology and Diagnostic Investigation, CVM & Department of Fisheries and Wildlife, CANR - Michigan State University, East Lansing, MI, 48824, USA.
Department of Biological Sciences, Wright State University, 235 Diggs Laboratory / 134 Oelman Hall, 3640 Colonel Glenn Hwy, Dayton, OH, 45435, USA.
Virol J. 2020 Jul 20;17(1):110. doi: 10.1186/s12985-020-01372-4.
Infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) are highly contagious, pathogenic Novirhabdoviruses affecting fish and are thusly notifiable diseases with the World Organization for Animal Health. This study assessed the relative capacities of IHNV and VHSV genes to modulate host general transcription and explores the abilities of specific IHNV genes to interfere with the interferon pathway in heterogenous teleost cell-lines.
Optimized protocols allowed for efficient transient transfections in EPC, BF-2, RTG-2 and RTgill-W1 cell lines of plasmids encoding IHNV (M genogroup) and VHSV (-IVb genotype) genes, including N, P, M, G and NV. Their impact on general cellular transcription was measured 48 hours post transfection (hpt) with luciferase constructs driven by a modified β-Actin promoter (pCAG). Their modulation of the innate antiviral immune response was characterized 72 hpt, using luciferase constructs measuring rainbow trout Type I IFN or MX-1 promoter augmentation, upon MAVS co-transfection.
M was generally confirmed as the strongest constitutive transcriptional suppressor while IHNV P, but not VHSV P, augmented constitutive transcription in fibroblastic cell types. Cell-specific effects were observed for viral G gene, with VHSV G exhibiting suppression of basal transcription in EPC and BF-2 but not in trout cells; while IHNV G was stimulatory in RTG-2, but inhibitory in RTgill-W1. NV consistently stimulated constitutive transcription, with higher augmentation patterns seen in fibroblastic compared to epithelial cells, and for IHNV NV compared to VHSV NV. The innate antiviral immune response, focusing on the IFN pathway, was silenced by IHNV M in all cell lines tested. IHNV N showed a dose-dependent suppression of type I IFN, but with minor effects on MX-1. IHNV P and G played minor IFN-inhibitory roles, consistent and dose-dependent only for G in rainbow trout cells. IHNV NV mediated a consistent stimulatory effect on either Type I IFN or MX-1, but much less pronounced in RTgill-W1.
This study extends our understanding of Novirhabdoviruses-host interaction, showing differential innate immune responses in heterogenous cell types. Viral regulators of innate immune signaling are identified, either as dose-dependent suppressors (such as M and N) or stimulators (mainly NV), indicating novel targets for the design of more efficient vaccination strategies.
传染性造血器官坏死病毒（IHNV）和病毒性出血性败血症病毒（VHSV）是高度传染性的致病性诺瓦克病毒，影响鱼类，因此是世界动物卫生组织通报的疾病。本研究评估了 IHNV 和 VHSV 基因调节宿主一般转录的相对能力，并探讨了特定 IHNV 基因在异质硬骨鱼细胞系中干扰干扰素途径的能力。
优化的方案允许在 EPC、BF-2、RTG-2 和 RTgill-W1 细胞系中高效瞬时转染，这些细胞系中编码 IHNV（M 基因群）和 VHSV（-IVb 基因型）基因的质粒，包括 N、P、M、G 和 NV。在转染后 48 小时（hpt），用改良的β-肌动蛋白启动子（pCAG）驱动的荧光素酶构建体测量其对一般细胞转录的影响。在共转染 MAVS 72 小时后，用测量虹鳟鱼 I 型 IFN 或 MX-1 启动子增强的荧光素酶构建体，测定其对先天抗病毒免疫反应的调节。
M 通常被确认为最强的组成型转录抑制因子，而 IHNV P 但不是 VHSV P 增强了成纤维细胞类型的组成型转录。在病毒 G 基因方面观察到细胞特异性效应，VHSV G 在 EPC 和 BF-2 中抑制基础转录，但在虹鳟鱼细胞中不抑制；而 IHNV G 在 RTG-2 中具有刺激作用，但在 RTgill-W1 中具有抑制作用。NV 一致地刺激组成型转录，在成纤维细胞中比在上皮细胞中具有更高的增强模式，并且与 VHSV NV 相比，IHNV NV 具有更高的增强模式。针对 IFN 途径的先天抗病毒免疫反应被所有测试的细胞系中的 IHNV M 沉默。IHNV N 表现出剂量依赖性的 I 型 IFN 抑制作用，但对 MX-1 的影响较小。IHNV P 和 G 发挥了较小的 IFN 抑制作用，仅在虹鳟鱼细胞中对 G 表现出一致和剂量依赖性的作用。IHNV NV 对 I 型 IFN 或 MX-1 均具有一致的刺激作用，但在 RTgill-W1 中的作用则不那么明显。
本研究扩展了我们对诺瓦克病毒与宿主相互作用的理解，显示了异质细胞类型中先天免疫反应的差异。鉴定了病毒先天免疫信号的调节因子，它们要么是剂量依赖性的抑制剂（如 M 和 N），要么是刺激剂（主要是 NV），这表明了设计更有效的疫苗策略的新靶标。
