National Pathogen Collection Center for Aquatic Animals, Shanghai Ocean University, Shanghai, People's Republic of China.
Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture and Rural Affairs, Shanghai, People's Republic of China.
Virol J. 2018 May 24;15(1):92. doi: 10.1186/s12985-018-0993-8.
Grass carp (Ctenopharyngodon idella) hemorrhagic disease is caused by an acute infection with grass carp reovirus (GCRV). The frequent outbreaks of this disease have suppressed development of the grass carp farming industry. GCRV104, the representative strain of genotype III grass carp (Ctenopharyngodon idella) reovirus, belongs to the Spinareovirinae subfamily and serves as a model for studying the strain of GCRV which encodes an outer-fiber protein. There is no commercially available vaccine for this genotype of GCRV. Therefore, the discovery of new inhibitors for genotype III of GCRV will be clinically beneficial. In addition, the mechanism of GCRV with fiber entry into cells remains poorly understood.
Viral entry was determined by a combination of specific pharmacological inhibitors, transmission electron microscopy, and real-time quantitative PCR.
Our results demonstrate that both GCRV-JX01 (genotype I) and GCRV104 (genotype III) of GCRV propagated in the grass carp kidney cell line (CIK) with a typical cytopathic effect (CPE). However, GCRV104 replicated slower than GCRV-JX01 in CIK cells. The titer of GCRV-JX01 was 1000 times higher than GCRV104 at 24 h post-infection. We reveal that ammonium chloride, dynasore, pistop2, chlorpromazine, and rottlerin inhibit viral entrance and infection, but not nystatin, methyl-β-cyclodextrin, IPA-3, amiloride, bafilomycin A1, nocodazole, and latrunculin B. Furthermore, GCRV104 and GCRV-JX01 infection of CIK cells depended on dynamin and the acidification of the endosome. This was evident by the significant inhibition following prophylactic treatment with the lysosomotropic drug ammonium chloride or dynasore.
Taken together, our data have suggested that GCRV104 enters CIK cells through clathrin-mediated endocytosis in a pH-dependent manner. We also suggest that dynamin is critical for efficient viral entry. Additionally, the phosphatidylinositol 3-kinase inhibitor wortmannin and the protein kinase C inhibitor rottlerin block GCRV104 cell entry and replication.
草鱼出血病是由草鱼出血病病毒（GCRV）急性感染引起的。这种疾病的频繁爆发抑制了草鱼养殖业的发展。GCRV104 是基因型 III 草鱼呼肠孤病毒的代表株，属于 Spinareovirinae 亚科，是研究编码外纤蛋白的 GCRV 株的模型。目前还没有针对这种基因型 GCRV 的商业疫苗。因此，发现新型的基因型 III GCRV 抑制剂将具有临床意义。此外，GCRV 纤维进入细胞的机制仍知之甚少。
通过特定的药理学抑制剂、透射电子显微镜和实时定量 PCR 相结合的方法来确定病毒的进入。
我们的结果表明，草鱼肾细胞系（CIK）中 GCRV-JX01（基因型 I）和 GCRV104（基因型 III）均具有典型的细胞病变效应（CPE）。然而，GCRV104 在 CIK 细胞中的复制速度比 GCRV-JX01 慢。感染后 24 小时，GCRV-JX01 的滴度比 GCRV104 高 1000 倍。我们揭示氯化铵、dynasore、pistop2、氯丙嗪和rottlerin 抑制病毒进入和感染，但不抑制制霉菌素、甲基-β-环糊精、IPA-3、阿米洛利、巴弗洛霉素 A1、诺考达唑和 latrunculin B。此外，GCRV104 和 GCRV-JX01 感染 CIK 细胞依赖于胞质动力蛋白和内体酸化。这一点在通过溶酶体趋化性药物氯化铵或 dynasore 进行预防性治疗后显著抑制得到证实。
综上所述，我们的数据表明，GCRV104 通过网格蛋白介导的内吞作用以 pH 依赖性方式进入 CIK 细胞。我们还表明，胞质动力蛋白对病毒的有效进入至关重要。此外，磷酸肌醇 3-激酶抑制剂wortmannin 和蛋白激酶 C 抑制剂 rottlerin 阻断 GCRV104 的细胞进入和复制。
