Lee Eun Gyeong, Kim Ki Hong
Department of Aquatic Life Medicine, Pukyong National University, Busan, 48513, South Korea.
Department of Aquatic Life Medicine, Pukyong National University, Busan, 48513, South Korea.
Fish Shellfish Immunol. 2023 Mar;134:108617. doi: 10.1016/j.fsi.2023.108617. Epub 2023 Feb 14.
The replication of viral hemorrhagic septicemia virus (VHSV) in appropriate host cells depends on environmental factors and the host cell's immunity. The dynamics of each VHSV RNA strand (vRNA, cRNA, and mRNA) in different conditions can provide a clue on the viral replication strategies, which can be a base for the development of efficient control measures. As VHSV is known to be sensitive to temperature and type I interferon (IFN) responses, in this study, we analyzed the effect of temperature difference (15 °C and 20 °C) and IRF-9 gene knockout on the dynamics of the three VHSV RNA strands in Epithelioma papulosum cyprini (EPC) cells using a strand-specific RT-qPCR. The tagged primers designed in this study successfully worked to quantify the three strands of VHSV. In the results of the temperature effect, the higher speed in viral mRNA transcription and the significantly higher (more than 10 times at 12-36 h) copy number of cRNA at 20 °C compared to those at 15 °C suggested the positive effect of high temperature on VHSV replication. In the results of the IRF-9 gene knockout effect, although IRF-9 gene knockout did not bring a dramatic effect on VHSV replication compared to the temperature effect, the increase of mRNA in IRF-9 KO cells was faster than normal EPC cells, which was reflected in the copy numbers of cRNA and vRNA. The IRF-9 gene knockout effect was not dramatic even in the replication of rVHSV-ΔNV-eGFP that harbors eGFP gene ORF instead of NV gene ORF. These results suggest that VHSV may be highly susceptible to pre-activated type I IFN responses but not highly susceptible to post-infection-mediated type I IFN responses or lowered type I IFN before infection. In both experiments of temperature effect and IRF-9 gene knockout effect, the copy number of cRNA never exceeded the copy number of vRNA at all assay times, suggesting that the binding efficiency of the RNP complex to the 3' end of cRNA might be lower than that to the 3' end of vRNA. Further research is needed to elucidate the regulatory mechanism that limits the amount of cRNA at an appropriate level during VHSV replication.
病毒性出血性败血症病毒（VHSV）在合适的宿主细胞中的复制取决于环境因素和宿主细胞的免疫力。不同条件下各VHSV RNA链（vRNA、cRNA和mRNA）的动态变化可为病毒复制策略提供线索，这可为制定有效的控制措施奠定基础。由于已知VHSV对温度和I型干扰素（IFN）反应敏感，在本研究中，我们使用链特异性RT-qPCR分析了温度差异（15℃和20℃）和IRF-9基因敲除对鲤上皮瘤（EPC）细胞中三条VHSV RNA链动态变化的影响。本研究设计的标记引物成功用于定量VHSV的三条链。在温度效应的结果中，与15℃相比，20℃时病毒mRNA转录速度更快，cRNA拷贝数显著更高（在12 - 36小时时超过10倍），这表明高温对VHSV复制有积极影响。在IRF-9基因敲除效应的结果中，尽管与温度效应相比，IRF-9基因敲除对VHSV复制没有产生显著影响，但IRF-9基因敲除细胞中mRNA的增加速度比正常EPC细胞快，这反映在cRNA和vRNA的拷贝数上。即使在携带eGFP基因ORF而非NV基因ORF的rVHSV-ΔNV-eGFP的复制中，IRF-9基因敲除效应也不显著。这些结果表明，VHSV可能对预激活的I型IFN反应高度敏感，但对感染后介导的I型IFN反应或感染前降低的I型IFN不高度敏感。在温度效应和IRF-9基因敲除效应的两个实验中，在所有检测时间点cRNA的拷贝数从未超过vRNA的拷贝数，这表明RNP复合物与cRNA 3'端的结合效率可能低于与vRNA 3'端的结合效率。需要进一步研究以阐明在VHSV复制过程中将cRNA量限制在适当水平的调控机制。
