基于MS2噬菌体的西尼罗病毒装甲RNA质控品的制备

doi:10.16431/j.cnki.1671-7236.2025.06.028

Abstract

Abstract: 【Objective】 The absence of secure and stable RNA reference materials for the nucleic acid assays of West Nile virus (WNV) at home and abroad was compromising the precision and credibility of the testing outcomes.This study was conducted to develop a safe and stable WNV nucleic acid testing quality control product to provide technical support for the monitoring of West Nile fever.【Method】 The WNV nucleic acid detection target gene,the MS2 bacteriophage coat protein CP,and the maturation enzyme protein A gene sequences were inserted into an expression vector to obtain the recombinant plasmid pET-CPA-WN.After transformation,expression and purification,a WNV armored RNA quality control material was prepared and subjected to Real-time quantitative PCR and digital PCR quantification,homogeneity and stability to assess its potential as a reference material.【Result】 PCR detection,double enzyme digestion and gene sequencing results all confirmed the successful construction of the recombinant plasmid pET-CPA-WN.After expression and purification,uniform virus-like particles with a diameter of 23-28 nm were obtained.Following nuclease digestion and Real-time quantitative PCR detection,the particle solution showed almost no residual nucleic acid and encapsulated the target gene in the form of armored RNA.The determined value was 8.80×10⁹ copies/mL.Stability experiments demonstrated that this armored RNA could remain stable for up to 20 days at 37 ℃.Random sampling of 10 samples for homogeneity testing confirmed good uniformity.【Conclusion】 The armored RNA prepared based on MS2 bacteriophage had high copy number,good uniformity and stability,which could provide a safe and stable reference sample for WNV molecular detection.

Key words: West Nile virus (WNV); MS2 bacteriophage; armored RNA

CLC Number:

S852.65

LIU Dan, GAO Jianshuai, ZHANG Boyuan, LI Huitong, JIANG Hui, FAN Xuezheng, ZHANG Guangzhi, DING Jiabo, XIONG Tao, SHEN Qingchun. Preparation of West Nile Virus Armored RNA Quality Control Product Based on MS2 Bacteriophage[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(6): 2762-2771.

References

[1] SCHWARZ E R,LONG M T.Comparison of West Nile virus disease in humans and horses:Exploiting similarities for enhancing syndromic surveillance[J].Viruses,2023,15(6):1230.
[2] SIMONIN Y.Circulation of West Nile virus and Usutu virus in Europe:Overview and challenges[J].Viruses,2024,16(4):599.
[3] ANGELOU A,KIOUTSIOUKIS I,STILIANAKIS N I.A climate-dependent spatial epidemiological model for the transmission risk of West Nile virus at local scale[J].One Health,2021,13:100330.
[4] PARÉ J,MOORE A.West Nile virus in horses—What do you need to know to diagnose the disease?[J].The Canadian Veterinary Journal,2018,59(10):1119-1120.
[5] HADFIELD J,BRITO A F,SWETNAM D M,et al.Twenty years of West Nile virus spread and evolution in the Americas visualized by Nextstrain[J].PLoS Pathogens,2019,15(10):e1008042.
[6] CHANCEY C,GRINEV A,VOLKOVA E,et al.The global ecology and epidemiology of West Nile virus[J]. BioMed Research International,2015,2015:376230.
[7] 王淑娟,王莹,徐天刚,等.西尼罗热国内外疫情形势与防控[J].中国动物检疫,2023,40(9):82-90.WANG S J,WANG Y,XU T G,et al.The epidemiological situation and prevention and control of West Nile fever at home and abroad[J].China Animal Quarantine,2023,40(9):82-90.(in Chinese)
[8] WHITNEY K,MCCORMICK S.Comparing West Nile virus and COVID-19[J].Sociology of Health & Illness,2020,42(8):e16-e18.
[9] BAKONYI T,HAUSSIG J M.West Nile virus keeps on moving up in Europe[J].Eurosurveillance,2020,25(46):2001938.
[10] LU L,ZHANG F,OUDE MUNNINK B B,et al.West Nile virus spread in Europe:Phylogeographic pattern analysis and key drivers[J]. PLoS Pathogens,2024,20(1):e1011880.
[11] 陈伯权,刘琴芝,周国芳.我国一些地区人血清的虫媒病毒抗体调查[J].中华流行病学杂志,1983,4(5):263-266.CHEN B Q,LIU Q Z,ZHOU G F.Survey of arbovirus antibodies in human sera from some regions in China[J].Chinese Journal of Epidemiology,1983,4(5):263-266.(in Chinese)
[12] 梁国栋.我国西尼罗病毒和Tahyna病毒的发现与流行[J].微生物与感染,2016,11(2):66-71.LIANG G D.Discovery and epidemiology of West Nile virus and Tahyna virus in China[J]. Microbes and Infection,2016,11(2):66-71.(in Chinese)
[13] 梁国栋.中国急需加强对西尼罗病的研究[J].中华实验和临床病毒学杂志,2006,3:285-286.LIANG G D.China urgently needs to strengthen research on West Nile disease[J].Chinese Journal of Experimental and Clinical Virology,2006,3:285-286.(in Chinese)
[14] CENDEJAS P M,GOODMAN A G.Vaccination and control methods of West Nile virus infection in equids and humans[J].Vaccines,2024,12(5):485.
[15] BUSCH M P,KLEINMAN S H,TOBLER L H,et al.Virus and antibody dynamics in acute West Nile virus infection[J].The Journal of Infectious Diseases,2008,198(7):984-993.
[16] LUSTIG Y,SOFER D,BUCRIS E D,et al.Surveillance and diagnosis of West Nile virus in the face of Flavivirus cross-reactivity[J].Frontiers in Microbiology,2018,9:2421.
[17] BRIEN J D,LAZEAR H M,DIAMOND M S.Propagation,quantification,detection,and storage of West Nile virus[J]. Current Protocols in Microbiology,2013,31:15D.3.1-15D.3.18.
[18] ZHANG W,WU J,LI Y,et al.Rapid and accurate in vitro assays for detection of West Nile virus in blood and tissues[J].Transfusion Medicine Reviews,2009,23(2):146-154.
[19] LUSTIG Y,MANNASSE B,KOREN R,et al.Superiority of West Nile virus RNA detection in whole blood for diagnosis of acute infection[J].Journal of Clinical Microbiology,2016,54(9):2294-2297.
[20] ZAMBENEDETTI M R,PAVONI D P,DALLABONA A C,et al.Internal control for Real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics[J]. Memórias do Instituto Oswaldo Cruz,2017,112(5):339-347.
[21] 于龙,郭惠琳,田洁.基于噬菌体MS2的装甲RNA技术及其在病毒核酸检测中的应用[J].中国国境卫生检疫杂志,2024,47(4):426-430.YU L,GUO H L,TIAN J.Phage MS2-based armored RNA technology and its application in viral nucleic acid testing[J].China Frontier Health and Quarantine,2024,47(4):426-430.(in Chinese)
[22] GARMANN R F,GOLDFAIN A M,MANOHARAN V N.Measurements of the self-assembly kinetics of individual viral capsids around their RNA genome[J]. Proceedings of the National Academy of Sciences of the United States of America,2019,116(45):22485-22490.
[23] LE D T,MVLLER K M.In vitro assembly of virus-like particles and their applications[J].Life,2021,11(4):334.
[24] WANG S,LIU Y,LI D,et al.Preparation and evaluation of MS2 bacteriophage-like particles packaging Hepatitis E virus RNA[J]. FEMS Microbiology Letters,2016,363(20):fnw221.
[25] QI L,ZHANG Z,WANG M,et al.One-plasmid double-expression system for preparation of MS2 virus-like particles packaging SARS-CoV-2 RNA[J].Frontiers in Cellular and Infection Microbiology,2023,13:1238543.
[26] 王国强,马红芳,曾华辉,等.基于MS2噬菌体病毒样颗粒的RT-PCR检测猪流行性腹泻病毒质控品的制备[J].西南农业学报,2020,33(11):2686-2692.WANG G Q,MA H F,ZENG H H,et al.Preparation of RT-PCR detection quality control for Porcine epidemic diarrhea virus based on MS2 bacteriophage virus-like particles[J].Southwest Agricultural Journal,2020,33(11):2686-2692.(in Chinese)
[27] KLINGELHÖFER D,BRAUN M,KRAMER I M,et al.A virus becomes a global concern:Research activities on West-Nile virus[J].Emerging Microbes & Infections,2023,12(2):2256424.
[28] SEWGOBIND S,MCCRACKEN F,SCHILLING M.JMM profile:West Nile virus[J].Journal of Medical Microbiology,2023,72(7):10.1099.
[29] 乔彩霞,张鹤晓,高志强,等.动物病毒核酸检测质控品和标准物质研究进展[J].中国预防兽医学报,2014,36(7):582-584.QIAO C X,ZHANG H X,GAO Z Q,et al.Research progress on quality control products and reference materials for animal virus nucleic acid testing[J].Chinese Journal of Preventive Veterinary Medicine,2014,36(7):582-584.(in Chinese)
[30] LIMA S M,PEABODY D S,SILVA J L,et al.Mutations in the hydrophobic core and in the protein-RNA interface affect the packing and stability of Icosahedral viruses[J].European Journal of Biochemistry,2004,271:135-145.
[31] 常乐,张括,张瑞,等.含轮状病毒NSP3基因病毒样颗粒的构建和表达[J].北京大学学报(医学版),2012,44(5):737-741.CHANG L,ZHANG K,ZHANG R,et al.Construction and expression of virus-like particles containing the Rotavirus NSP3 gene[J].Journal of Peking University (Medical Edition),2012,44(5):737-741.(in Chinese)
[32] SHULMAN L M,HINDIYEH M,MUHSEN K,et al.Evaluation of four different systems for extraction of RNA from stool suspensions using MS-2 coliphage as an exogenous control for RT-PCR inhibition[J].PLoS One,2012,7:e39455.
[33] ZHAN S,LI J,XU R,et al.Armored long RNA controls or standards for branched DNA assay for detection of Human immunodeficiency virus type 1[J].Journal of Clinical Microbiology,2009,47:2571-2576.
[34] 马思杰,胡群,童淑梅.西尼罗病毒装甲RNA质控品的制备[J].中国国境卫生检疫杂志,2022,45(1):5-7.MA S J,HU Q,TONG S M.Preparation of West Nile virus armored RNA quality control product[J].China Frontier Health and Quarantine,2022,45(1):5-7.(in Chinese)
[35] 程巧珍,陈春,应婷,等.含五种RNA病毒核酸假病毒的制备及特性研究[J].中国计量大学学报,2022,33(2):281-287.CHENG Q Z,CHEN C,YING T,et al.Preparation and characterization of pseudoviruses containing nucleic acids of five RNA viruses[J].Journal of China Jiliang University,2022,33(2):281-287.(in Chinese)
[36] 窦敏,张国广,于广福,等.含口蹄疫病毒IRES RNA病毒样颗粒表达载体的构建[J].中国生物工程杂志,2007,9:31-35.DOU M,ZHANG G G,YU G F,et al.Construction of virus-like particle expression vectors containing Foot-and-mouth disease virus IRES RNA[J]. China Biotechnology Journal,2007,9:31-35.(in Chinese)

Preparation of West Nile Virus Armored RNA Quality Control Product Based on MS2 Bacteriophage

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	HUANG Xiaojiu, LEI Lei, PENG Xiaoye, WANG Kaixin, CHEN Yingyi, WANG Jixian, WANG Yuge, DUAN Deyong, YANG Yi, WANG Aibing. Construction of a IPEC-J2 Cell Line Stably Overexpressing NM-ⅡA Tail and Its Effect on Porcine Epidemic Diarrhea Virus Infection [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(5): 2243-2252.
[2]	WU Peng, YUAN Yang, YANG Yunyun, FENG Yi, WANG Yan, YANG Ying, CHEN Jiangfeng, JIANG Haibo, WEN Ming. Diagnosis and Pathogen Identification of a Case of Mixed Infection of Duck Plague Virus and Pasteurella multocida in Breeding Goose [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(5): 2253-2265.
[3]	WU Yue, SHI Xingya, ZHANG Shuai, ZHAO Yunhuan, GUO Liming, ZUO Yuzhu, FAN Jinghui. Isolation and Identification of Porcine Pseudorabies Virus and Genetic Evolution Analysis of gE and gC Genes [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(5): 2266-2277.
[4]	JIAO Xianqin, MA Xiao, TIAN Runbo, LIU Ying, MA Shijie, CHEN Hongying. Construction of Recombinant Porcine Pseudorabies Virus Expressing Cap Protein of Porcine Circovirus Type 2d [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(5): 2278-2286.
[5]	LIU Min, CHENG Huai, ZHENG Zhenzhen, ZHANG Mengsi, ZHANG Hewei, REN Jingqiang. Eukaryotic Expression and Polyclonal Antibody Preparation of N Protein of Canine Parainfluenza Virus [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(5): 2287-2294.
[6]	PAN Xiangying, ZENG Zhiyong, LIANG Haiying, TANG Deyuan, WANG Bin, YE Ni, TIAN Hongli, BIAN Mengting, LIU Jiajia, HUANG Shu. Truncated Expression of Porcine Rotavirus VP6 Protein and Establishment of an Indirect ELISA Antibody Detection Method [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(3): 1231-1240.
[7]	LIN Weishan, MA Doudou, LEI Mengtong, WEI Bin, LI Guocai, WANG Guanghua, WANG Geping, JIAN Yingna, LI Xiuping. Epidemiological Investigation and Genetic Evolution Analysis of 5'-UTR of Bovine Viral Diarrhea Virus in Yaks in Haibei，Qinghai Province [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(3): 1241-1249.
[8]	DING Ding, DING Xiaojun, MUSA Rehman, ZHEN Jie, SALAMAITI Srajiding, LI Na, SUN Yawei, CHEN Rulong, YAO Gang, MA Xuelian. Analysis on Viral Pathogen Detection and Epidemic Characteristics of Calf Diarrhea in Some Large-scale Cattle Farms in Xinjiang [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(3): 1272-1280.
[9]	MAO Fuchao, ZHAI Chongkai, TIAN Wenjing, WANG Conghui, SONG Minjie, WANG Yingxian, ZHANG Hewei. An Updated Review of Porcine Deltacoronavirus in Terms of Infection and Anti-infection Research [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(3): 1281-1291.
[10]	LI Huanhuan, YU Chenmin, TIAN Xiaorong, LI Rui, LI Zongyun, ZHANG Yanyan, ZHAO Di, WANG Lei, HOU Yongqing, WU Tao. Protective Effect of Catechu Extract on the Colon of Young Piglets Infected with Porcine Epidemic Diarrhea Virus [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(3): 1360-1369.
[11]	WANG Xiaoge, SI Xuanying, YAN Zhiwei, WANG Fei, YOU Longqi, LIU Geng, CAI Mao, LIANG Juncheng, LIANG Yuxiu, DU Yongkun, ZHANG Gaiping. Preparation of Monoclonal Antibody Against African Swine Fever Virus p22 Protein and Identification of the Antigenic Epitope [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(2): 781-791.
[12]	YANG Li, DU Xiaomei, LIU Mengyuan, WU Shenglong, BAO Wenbin, WU Zhengchang. Screening and Functional Verification of Key lncRNA Affecting Porcine Epidemic Diarrhea Virus Replication [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(2): 792-800.
[13]	LI Yuanyuan, YUAN Qian, FENG Xiaoran, DENG Si, LI Na, YAO Lunguang, QIN Yinghui. Isolation and Identification of a Strain of Snakehead Fish Vesiculovirus [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(2): 811-822.
[14]	ZOU Chang, HU Yue, LI Haihua, QIAO Jiayun. Genetic Evolution Analysis of VP2 Gene of Feline Panleukopenia Virus in Baoding Area [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(2): 823-831.
[15]	BAI Yihan, WANG Dongliang. Research Progress on Genetic and Host Diversity of Porcine Circoviruses [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(2): 832-843.