INFLUENCE OF siRNA TARGETING CYHV-2 ORF57 GENE ON REPLICATION OF CYHV-2 IN CSC CELLS
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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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Keywords:
- Cyprinid herpesvirus 2 /
- RNA interference /
- ORF57 /
- siRNA /
- CSC cell line /
- TCID50
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图 1 正常细胞与病变细胞状态图
A. 正常细胞; B. 病变后细胞。标尺为500 μm
Figure 1. Normal cells and pathological cells
A. normal cells; B. the cells with CPE. The bar in figure represent 500 μm
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图 2 在不同条件下FAM-β-actin-siRNA转染效果
A和B转染浓度是40 nmol/L, C和D转染浓度是80 nmol/L, E和F转染浓度是120 nmol/L; A、C、E为转染6h后换液, B、D、F为转染24h后换液。标尺为200 μm
Figure 2. Effect of different concentrations of FAM-β-actin- siRNA transfection
The transfection concentrations of A and B are 40 nmol/L, C and D are 80 nmol/L, E and F are 120 nmol/L; A, C and E are 6h after transfection, the fluid changed, while B, D and F are 24h after transfection, the fluid changed. The bar in figure represent 200 μm
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图 3 转染后24h、48h、72h和120h荧光信号
A—D分别为24h、48h、72h和120h观察结果(标尺为200 μm)
Figure 3. Observation of fluorescence effect at 24h, 48h, 72h and 120h
A—D are the observation results at 24h, 48h, 72h and 120h respectively (the bar in figure represent 200 μm)
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图 4 ORF57-siRNAs在120h干扰CyHV-2致CSC细胞病变效果
A. ORF57-siRNA-1组; B. ORF57-siRNA-2组; C. ORF57-siRNA-3组; D. 阴性siRNA组; E. Mock对照组; F. 空白组 (标尺500 μm)
Figure 4. Effect of ORF57-siRNAs on CyHV-2-induced CSC CPE at 120h
A.the emergence of CPE of CyHV-2-ORF57-1-siRNA group at 120h after infection; B.the emergence of CPE of CyHV-2-ORF57-2-siRNA group at 120h after infection; C.the emergence of CPE of CyHV-2-ORF57-3-siRNA group at 120h after infection; D.the emergence of CPE of Negative siRNA control group at 120h after infection; E.the emergence of CPE of Mock group at 120h after infection; F.the emergence of CPE of Blank group at 120h after infection. (the bar in figure represent 500 μm)
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图 5 ORF57-siRNAs处理后48h ORF57相对表达量
*表示差异显著, P<0.05; **表示差异极显著, P<0.01; 下同
Figure 5. Relative expression of ORF57 at 48h after treatment with ORF57-siRNAs
*the difference is significant, P<0.05; **the difference is extremely significant, P<0.01. The same applies below
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图 6 不同时间点ORF57-siRNA-2对ORF57转录的抑制作用
Figure 6. Inhibitory effect of ORF57-siRNA-2 on transcription of ORF57 at different times
表 1 siRNA和PCR引物序列信息
Table 1 Sequences of siRNA and primers
组别Group 序列Sequence (5′—3′) ORF57-siRNA-1 Sense GCAAGAAGUCGUUCGAUAATT Antisense UUAUCGAACGACUUCUUGCTT ORF57-siRNA-2 Sense GGUACUUUCCAACGGCCAATT Antisense UUGGCCGUUGGAAAGUACCTT ORF57-siRNA-3 Sense GGAGCCACUGGAACAUCAATT Antisense UUGAUGUUCCAGUGGCUCCTT FAM-β-actin-siRNA Sense FAM-GGGAUGACAUGGAGAAGAUTT Antisense AUCUUCUCCAUGUCAUCCCTT siRNA-
control [11]Sense UUCUCCGAACGUGUCACGUTT Antisense ACGUGACACGUUCGGAGAATT ORF57-1 Sense ATCATGGCAGAGTTTTTTACTGAGGAC Antisense GACGACATCGGTCTTTTTCTCTGC ORF57-2 Sense GCCCGCCGACTGGATAGAC Antisense AGGAACGCGAGGCTGTT ORF57-3 Sense AAGCCCAAGCGACTCACC Antisense TGGAGCTTTGGGTTTAGCGC β-actin Sense TCACCTCCCTTGCTCCTTCCAC Antisense CTCCTGCTTGCTGATCCACATCTG 
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表 2 加样配比表
Table 2 Sampling ratio table
分组
Group浓度组1
Concentration
group 1 (μL)浓度组2
Concentration
group 2 (μL)浓度组3
Concentration
group 3 (μL)阴性siRNA
对照组
siRNA-negative
control group (μL)Mock
对照
Mock control
group (μL)siRNA复合物
siRNA complexesFAM-β-actin-siRNA 1 2 3 2 0 Opti-MEMⅠ 49 48 47 48 50 Lipofectamine 复合物
Lipofectamine complexesLipofectamine 1 1 1 1 1 Opti-MEMⅠ 49 49 49 49 49 Opti-MEMⅠ 400 400 400 400 400
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表 3 siRNA抑制试验分组
Table 3 Grouping of transfection inhibition test
组别
Group转染试剂Transfection reagent siRNA CyHV-2 取样时间
Sampling timeBlank group - - + / Mock group + - + 24h、48h、72h和120h Negative siRNA + + + 24h、48h、72h和120h β-actin siRNA + + + 24h、48h、72h和120h ORF57-siRNA + + + 24h、48h、72h和120h 注: +. 含; -. 不含Note: + means added; - means not added
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表 4 TCID50病毒滴度测定
Table 4 Detection of TCID50 in different virus dilutions
组别
Group稀释梯度
Gradient dilution10–5 10–6 10–7 10–8 10–9 10–10 10–11 10–12 病变孔数
Number of wells with CPE7 7 6 1 1 0 0 0 未病变孔数
Number of wells without CPE1 1 2 7 7 8 8 8
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表 5 Image J 统计荧光点数量
Table 5 Number of fluorescent spots counted by Image J
siRNA终浓度
Final concentration
of siRNA (nmol/L)转染后维持6h
Transfection after 6h转染后维持24h
Transfection after 24h40 618±18 700±26 80 1063±101 1366±136 120 1266±111 1031±46
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表 6 在各转染方案下β-actin基因拷贝数
Table 6 Copy number of β-actin gene under each transfection scheme(copies/ng)
siRNA终浓度
Final concentration
of siRNA (nmol/L)转染后维持6h
Transfection after 6h转染后维持24h
Transfection after 24h40 8.423×104 1.258×104 80 1.147×104 3.375×103 120 4.985×104 4.685×104
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表 7 转染后24h、48h、72h和120h β-actin基因表达情况
Table 7 Expression of β-actin gene at 24h, 48h, 72h and 120h after transfection (copies/ng)
转染siRNA 后时间
Times after transfection (h)β-actin基因拷贝数
Copy number of β-actin gene24 3.403×103 48 3.315×103 72 1.900×103 120 3.001×103
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表 8 ORF57-siRNAs对CyHV-2 TCID50的影响
Table 8 Effect of ORF57-siRNA on CyHV-2 TCID50
组别Group TCID50 (mL) ORF57-siRNA-1 107.432 ORF57-siRNA -2 106.776 ORF57-siRNA -3 107.353 Negative siRNA 108.487 Mock 108
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[1] Doszpoly A, Benko M, Csaba G, et al. Introduction of the family Alloherpesviridae: The first molecular detection of herpesviruses of cyprinid fish in Hungary [J]. Magyar Allatorvosok Lapja, 2011, 133(3): 174-181.
[2] 王璐. 江苏地区鲫出血病病原的分离、鉴定及检测方法研究 [D]. 南京: 南京农业大学, 2012: 61. Wang L. The research on isolation and identification of pathogeny from the hermorrage disease of prussian carp (Carassius gibelio) in Jiangsu China and its detection method [D]. Nanjing: Nanjing Agricultural University, 2012: 61.
[3] 袁雪梅, 潘晓艺, 郝贵杰, 等. 一例异育银鲫(Carassius auratus gibelio)暴发性出血病病原分析 [J]. 海洋与湖沼, 2019, 50(4): 913-920. Yuan X M, Pan X Y, Hao G J, et al. Analysis of pathogen in an explosive hemorrhage disease of Carassius auratus gibelio [J]. Oceanologia et Limnologia Sinica, 2019, 50(4): 913-920.
[4] Thangaraj R S, Nithianantham S R, Dharmaratnam A, et al. Cyprinid herpesvirus-2 (CyHV-2): a comprehensive review [J]. Reviews in Aquaculture, 2020(13): 796-821.
[5] 张奇亚. 淡水生态系统中几种大DNA病毒研究概述 [J]. 水生生物学报, 2020, 44(5): 961-975. doi: 10.7541/2020.112 Zhang Q Y. An overview on several large DNA viruses in freshwater ecosystems [J]. Acta Hydrobiologica Sinica, 2020, 44(5): 961-975. doi: 10.7541/2020.112
[6] Napoli C, Lemieux C, Jorgensen R. Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans [J]. Plant Cell, 1990, 2(4): 279-289. doi: 10.2307/3869076
[7] Fire A. RNA-triggered gene silencing [J]. Trends in Genetics, 1999, 15(9): 358-363. doi: 10.1016/S0168-9525(99)01818-1
[8] 宋华丽, 孙效迎, 孔祥会, 等. RNA干扰技术在水产动物抗病毒和抗寄生虫研究中的应用研究进展 [J]. 生物技术通报, 2020, 36(2): 193-205. Song H L, Sun X Y, Kong X H, et al. Application of RNA interference technology in antiviral and antiparasitic research of aquatic animals [J]. Biotechnology Bulletin, 2020, 36(2): 193-205.
[9] 黄桂菊, 喻达辉, 柳明, 等. 石斑鱼神经坏死病毒RNA干扰的转染条件优化与效果分析 [J]. 华南农业大学学报, 2011, 32(2): 93-98. doi: 10.3969/j.issn.1001-411X.2011.02.022 Huang G J, Yu D H, Liu M, et al. Preliminary study on RNA interference against red-spotted grouper nervous necrosis virus-optimization of transfection condition and comparison of interference effect based on FHM cell [J]. Journal of South China Agricultural University, 2011, 32(2): 93-98. doi: 10.3969/j.issn.1001-411X.2011.02.022
[10] 魏钰娟, 潘晓艺, 蔺凌云, 等. 异育银鲫(Carassius auratus gibelio)脊髓组织细胞系的建立及对CyHV-2的敏感性 [J]. 海洋与湖沼, 2020, 51(5): 1232-1238. doi: 10.11693/hyhz20200300071 Wei Y J, Pan X Y, Lin L Y, et al. Establishment of spinal cord cell line of Carassius auratus gibelio and its sensitivity to CyHV-2 [J]. Oceanologia et Limnologia Sinica, 2020, 51(5): 1232-1238. doi: 10.11693/hyhz20200300071
[11] Wang C, Li N, Liu X, et al. A novel endogenous human CaMKII inhibitory protein suppresses tumor growth by inducing cell cycle arrest via p27 stabilization [J]. Journal of Biological Chemistry, 2008, 283(17): 11565-11574. doi: 10.1074/jbc.M800436200
[12] Reed L J, Muench H. A simple method of estimating fifty percent endpoints [J]. American Journal of Epidemiology, 1938, 27(3): 493-497. doi: 10.1093/oxfordjournals.aje.a118408
[13] 陈芸, 朱作言. RNA干扰在抗病毒研究中的应用 [J]. 水生生物学报, 2006, 30(3): 356-359. doi: 10.3321/j.issn:1000-3207.2006.03.018 Chen Y, Zhu Z Y. Application of RNA interference (RNAi) in virus resistance [J]. Acta Hydrobiologica Sinica, 2006, 30(3): 356-359. doi: 10.3321/j.issn:1000-3207.2006.03.018
[14] 陈芸. 用RNA干扰(RNAi)抗草鱼出血病病毒的初步研究 [D]. 武汉: 中国科学院水生生物研究所, 2005: 106. Chen Y. Study of disease resistant on grass carp reovirus by RNA interference (RNAi) [D]. Wuhan: Institute of Hydrobiology, Chinese Academy of Sciences, 2005: 106.
[15] 马杰. RNA基因干扰技术抑制草鱼呼肠孤病毒复制的研究 [D]. 武汉: 华中农业大学, 2013: 94-95. Ma J. Study on the inhibition of replication of grass carp reovirus by RNA interference [D]. Wuhan: Huazhong Agricultural University, 2013: 94-95.
[16] Wu Y, Lü L, Yang L S, et al. Inhibition of white spot syndrome virus in Litopenaeus vannamei shrimp by sequence-specific siRNA [J]. Aquaculture, 2007, 271(1-4): 21-30. doi: 10.1016/j.aquaculture.2007.06.029
[17] Fouad A M, Soliman H, Abdallah E S H, et al. In-vitro inhibition of spring viremia of carp virus replication by RNA interference targeting the RNA-dependent RNA polymerase gene [J]. Journal of Virological Methods, 2018(263): 14-19.
[18] Zenke K, Nam Y K, Kim K H. Development of siRNA expression vector utilizing rock bream β-actin promoter: a potential therapeutic tool against viral infection in fish [J]. Applied Microbiology Biotechnology, 2010, 85(3): 679-690. doi: 10.1007/s00253-009-2177-3
[19] 牟维豪, 周燕, 耿毅, 等. RNA干扰对大鲵蛙病毒主要功能基因表达及其增殖的影响 [J]. 南方水产科学, 2017, 13(4): 80-86. doi: 10.3969/j.issn.2095-0780.2017.04.010 Mou W H, Zhou Y, Geng Y, et al. Effect on main functional genes expression and replication of Chinese giant Salamander ranavirus (CGSRV) by RNA interference [J]. South China Fisheries Science, 2017, 13(4): 80-86. doi: 10.3969/j.issn.2095-0780.2017.04.010
[20] van Beurden S J, Bossers A, Voorbergen-Laarman M H A, et al. Complete genome sequence and taxonomic position of anguillid herpesvirus 1 [J]. Journal of General Virology, 2010(91): 880-887.
[21] Afonso C L, Tulman E R, Delhon G, et al. Genome of crocodilepox virus [J]. Journal of Virology, 2006(80): 4978-4991.
[22] Boutier M, Gao Y, Vancsok C, et al. Identification of an essential virulence gene of cyprinid herpesvirus 3 [J]. Antiviral Research, 2017(145): 60-69.
[23] Michel B, Leroy B, Stalin Raj V, et al. The genome of cyprinid herpesvirus 3 encodes 40 proteins incorporated in mature virions [J]. Journal of General Virology, 2010(91): 452-462.
[24] Gao W, Wen H, Wang H, et al. Identification of structure proteins of cyprinid herpesvirus 2 [J]. Aquaculture, 2020(523): 735184.
[25] Tang R, Lu L, Wang B, et al. Identification of the immediate-early genes of cyprinid herpesvirus 2 [J]. Viruses, 2020, 12(9): 994. doi: 10.3390/v12090994
[26] Tseng Y C, Mozumdar S, Huang L. Lipid-based systemic delivery of siRNA [J]. Advanced Drug Delivery Reviews, 2009, 61(9): 721-731. doi: 10.1016/j.addr.2009.03.003
[27] 杨光, 曹国军, 李洁, 等. siRNA抑制SARS冠状病毒感染Vero E6细胞 [J]. 医学分子生物学杂志, 2004, 1(5): 270-273. doi: 10.3870/j.issn.1672-8009.2004.05.003 Yang G, Cao G J, Li J, et al. Inhibition of SARS-CoV infection in Vero-E6 cells by siRNAs [J]. Journal of Medical Molecular Biology, 2004, 1(5): 270-273. doi: 10.3870/j.issn.1672-8009.2004.05.003
[28] 李兵. RNA干扰抑制草鱼呼肠孤病毒复制的细胞模型 [D]. 武汉: 华中农业大学, 2009: 26-29. Li B. Cellular model for inhibition of grass carp reovirus replication mediated by RNA interference [D]. Wuhan: Huazhong Agricultural University, 2009: 26-29.
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