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金李萍, 潘晓艺, 蔺凌云, 姚嘉赟, 曹铮, 尹文林, 赖迎迢, 陶家发, 刘忆瀚, 沈锦玉. siRNA干扰对鲤疱疹病毒ORF57基因表达的影响[J]. 水生生物学报, 2022, 46(4): 537-544. DOI: 10.7541/2021.2020.296
引用本文: 金李萍, 潘晓艺, 蔺凌云, 姚嘉赟, 曹铮, 尹文林, 赖迎迢, 陶家发, 刘忆瀚, 沈锦玉. siRNA干扰对鲤疱疹病毒ORF57基因表达的影响[J]. 水生生物学报, 2022, 46(4): 537-544. DOI: 10.7541/2021.2020.296
JIN Li-Ping, PAN Xiao-Yi, LIN Ling-Yun, YAO Jia-Yun, CAO Zheng, YIN Wen-Lin, LAI Ying-Tiao, TAO Jia-Fa, LIU Yi-Han, SHEN Jin-Yu. INFLUENCE OF siRNA TARGETING CYHV-2 ORF57 GENE ON REPLICATION OF CYHV-2 IN CSC CELLS[J]. ACTA HYDROBIOLOGICA SINICA, 2022, 46(4): 537-544. DOI: 10.7541/2021.2020.296
Citation: JIN Li-Ping, PAN Xiao-Yi, LIN Ling-Yun, YAO Jia-Yun, CAO Zheng, YIN Wen-Lin, LAI Ying-Tiao, TAO Jia-Fa, LIU Yi-Han, SHEN Jin-Yu. INFLUENCE OF siRNA TARGETING CYHV-2 ORF57 GENE ON REPLICATION OF CYHV-2 IN CSC CELLS[J]. ACTA HYDROBIOLOGICA SINICA, 2022, 46(4): 537-544. DOI: 10.7541/2021.2020.296

siRNA干扰对鲤疱疹病毒ORF57基因表达的影响

INFLUENCE OF siRNA TARGETING CYHV-2 ORF57 GENE ON REPLICATION OF CYHV-2 IN CSC CELLS

  • 摘要: 研究通过在异育银鲫脊髓细胞系(Spinal cord tissue cell lines of Carassius auratus gibelio, CSC)中对鲤疱疹病毒Ⅱ型(Cyprinid herpesvirus 2, CyHV-2) ORF57进行RNA干扰, 以探究其对CyHV-2病毒复制的影响。首先, 以FAM标记的异育银鲫β-actin的siRNA进行CSC细胞转染条件的优化, 再将针对CyHV-2 ORF57基因设计的3条siRNA, 转染CSC细胞, 并进行病毒感染, 评估siRNA对病毒复制和致细胞病变的影响。转染条件优化结果显示, 在siRNA浓度为80 nmol/L, 转染液维持24h后更换维持液, β-actin基因表达量最低且观察到的荧光点数量最多。而ORF57-siRNAs干扰结果显示, ORF57-siRNA-2组表现出了较强的抑制效果, 在接毒48h时, ORF57-siRNA-2处理组的ORF57基因表达量降到相对于Mock组的33.55% (P<0.01), 并且各ORF57-siRNA组都表现出了延缓CyHV-2致细胞病变的时间和强度, 抑制时间可达120h。TCID50结果显示, 不同组的ORF57-siRNAs均能降低病毒滴度, 其中ORF57-siRNA-2将病毒原液TCID50 108.487/mL下降至106.776/mL。研究结果表明, 干扰ORF57的表达可大大降低CyHV-2的致细胞病变力和复制率, ORF57在CyHV-2复制与致细胞病变中起重要作用。本研究为CyHV-2基于siRNA技术的抗病毒治疗和弱毒株的改造提供了借鉴。

     

    Abstract: Cyprinid herpesvirus 2 (CyHV-2) is the pathogen of herpesviral hematopoietic necrosis of Carassius auratus. It is a double-stranded DNA virus that can infect goldfish, crucian carp and its varieties. This study investigated the effect of CyHV-2 ORF57 on viral replication in Spinal cord tissue cell lines of Carassius auratus gibelio (CSC). Lipofectamine 2000 was used to transfect siRNA. Three pairs of siRNAs of CyHV-2-ORF57 gene were transfected into CSC cells to evaluate the effect of ORF57 on viral replication and cytopathic effect (CPE). Real-time PCR analysis revealed that ORF57-siRNA-2 had the greatest suppression of ORF57 expression with a 33.55% (P<0.01) reduction at 48h associated with the strongest inhibitory effect. All three ORF57-siRNAs delayed the time and intensity of CyHV-2 cytopathogenic effect, with the inhibition time up to 120h. The results of TCID50 showed that ORF57-siRNAs reduced the titer of the virus, and ORF57-siRNA-2 decreased the virus stock solution TCID50 108.487/mL to 106.776/mL. This study show that ORF57 can mediate the CPE and replication rate of CyHV-2. This study provides a reference for the antiviral treatment based on siRNA technology and the construction of low virulent strain of CyHV-2.

     

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