INFLUENCE OF siRNA TARGETING CYHV-2 <i>ORF57</i> GENE ON REPLICATION OF CYHV-2 IN CSC CELLS

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

doi:10.7541/2021.2020.296

Volume 46 Issue 4

May 2022

Turn off MathJax

Article Contents

Abstract

References

ACTA HYDROBIOLOGICA SINICA > 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:

PDF (1902 KB)

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

1.
School of Fisheries and Life, Shanghai Ocean University, Shanghai 201200, China
2.
Ministry of Agriculture and Rural Areas Key Laboratory of Healthy Freshwater Aquaculture, Key Laboratory of Fish Health and Nutrition of Zhejiang Province, Zhejiang Institute of Freshwater Fisheries, Huzhou 313001, China
3.
Key Laboratory of Fishery Drug Development, Ministry of Agriculture and Rural Affairs, Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510380, China

Funds: Supported by National Key R & D Program of China (2019YFD0900103); Key R & D Program Projects of Zhejiang Province (2019C02058)]

Received Date: January 06, 2021
Rev Recd Date: June 23, 2021
Available Online: April 07, 2022
Published Date: April 13, 2022

Graphical Abstract

Abstract

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 10^8.487/mL to 10^6.776/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.
- Cyprinid herpesvirus 2,
- RNA interference,
- ORF57,
- siRNA,
- CSC cell line,
- TCID₅₀

FullText(HTML)

References (28)

References

[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.

Cited By

Get Citation

PDF

XML

Article views PDF downloads

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

Abstract

References

Catalog

Related

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

Abstract

References

Catalog

Related

Export File

Citation

Format

Content