2024 06 v.40 1263-1273
人偏肺病毒假病毒阳性标准品的制备与鉴定
基金项目(Foundation):
中国疾病预防控制中心病毒病预防控制所青年科学基金（项目号：IVDC-202304）,题目：基于铁蛋白的呼吸道合胞病毒和人偏肺病毒二联纳米颗粒疫苗研究~~
邮箱(Email):
mafl@ivdc.chi-nacdc.cn;zhengls@ivdc.chinacdc.cn;
DOI:
10.13242/j.cnki.bingduxuebao.004615
中文作者单位:
中国疾病预防控制中心病毒病预防控制所,传染病溯源预警和智能决策全国重点实验室,国家卫生健康委员会医学病毒与病毒病重点实验室;中国医科大学公共卫生学院;
摘要(Abstract):
为制备一种安全、有效的人偏肺病毒（Human metapneumovirus,HMPV）假病毒阳性标准品，并用于核酸检测的质量控制，本研究对335条HMPV全基因序列比对分析，选择HMPV基因表达量高且进化较为保守的N基因构建重组假病毒颗粒。合成全长1 185 bp的N基因序列并与CD513B载体进行重组连接，采用第三代慢病毒包装体系构建HMPV假病毒颗粒，超速离心浓缩后感染HEK293T细胞，RT-qPCR核酸检测N基因和透射电镜观察鉴定重组假病毒颗粒，确定假病毒阳性标准品使用浓度，进一步评价阳性标准品的热稳定性和均一性。研究结果显示，包装获得的HMPV假病毒颗粒能够感染HEK293T细胞，假病毒颗粒为圆形或椭圆形病毒样结构，直径在150～200 nm之间，并带有G蛋白刺突。HMPV假病毒稀释103倍后Ct值最适于进行核酸检测，并且均一性和稳定性可达到阳性标准品要求。本研究成功构建了含有HMPV N基因全长的假病毒颗粒，可作为核酸检测方法的阳性标准品，实现HMPV核酸提取和核酸检测过程的全程质量控制。
关键词(KeyWords):
人偏肺病毒;假病毒;阳性标准品
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参考文献
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[24]郑夔，袁帅，黎东红，等.新疆出血热病毒实时荧光RT-PCR检测方法的假病毒阳性对照品制备[J].华南预防医学，2016, 42(1):1-5. DOI:10. 13217/j.scjpm. 2016. 0001.
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[26]王浩，麻粉莲，王超，等.尼帕病毒Taqman qRT-PCR检测方法的建立[J].病毒学报，2020, 36(3):371-376. DOI:10. 13242/j. cnki. bingduxuebao. 003696.
[27]Ura T, Okuda K, Shimada M. Developments in viral vector-based vaccines[J]. Vaccines(Basel), 2014, 2(3):624-641. DOI:10. 3390/vaccines2030624.
[28]Zhu FC, Li YH, Guan XH, et al. Safety, tolerability,and immunogenicity of a recombinant adenovirus type-5vectored COVID-19 vaccine:a dose-escalation, openlabel, non-randomised, first-in-human trial[J]. Lancet,2020, 395(10240):1845-1854. DOI:10. 1016/S0140-6736(20)31208-3.
[29]伍政宇，罗楚铭，贾婷婷，等.新型冠状病毒假病毒的制备及血浆中和抗体检测[J].病毒学报，2022, 38(3):513-522. DOI:10. 13242/j. cnki.bingduxuebao. 004126.
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[2] van den Hoogen BG, de Jong JC, Groen J, et al. A newly discovered human pneumovirus isolated from young children with respiratory tract disease[J]. Nat Med, 2001, 7(6):719-724. DOI:10. 1038/89098.
[3] Williams JV, Harris PA, Tollefson SJ, et al. Human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children[J]. N Engl J Med, 2004, 350(5):443-450. DOI:10. 1056/nejmoa025472.
[4] Li ZJ, Zhang HY, Ren LL, et al. Etiological and epidemiological features of acute respiratory infections in China[J]. Nat Commun, 2021, 12:5026. DOI:10. 1038/s41467-021-25120-6.
[5] Shinoda D, Tsukagoshi H, Komuro K, et al. Detection of respiratory viruses during the early phase of the coronavirus disease 2019 pandemic in ibaraki and Gunma prefectures, Japan[J]. Jpn J Infect Dis, 2022, 75(5):530-532. DOI:10. 7883/yoken. jjid. 2022. 061.
[6] Kapandji N, Darmon M, Valade S, et al. Clinical significance of human metapneumovirus detection in critically ill adults with lower respiratory tract infections[J]. Ann Intensive Care, 2023, 13(1):21. DOI:10. 1186/s13613-023-01117-w.
[7]陈其娴，罗经伟，许少坚，等. 1起人偏肺病毒引起的急性呼吸道感染暴发疫情调查[J].热带医学杂志，2020, 20(2):257-260. DOI:10. 3969/j. issn. 1672-3619. 2020. 02. 028.
[8] Korsun NS, Angelova SG, Trifonova IT, et al. The prevalence and genetic characterization of human metapneumovirus in Bulgaria, 2016-2019[J].Intervirology, 2021, 64(4):194-202. DOI:10. 1159/000516821.
[9]麻粉莲，王超，陈爱珺，等.人偏肺病毒在传代细胞和人呼吸道上皮细胞中分离和鉴定[J].病毒学报，2022, 38(2):313-321. DOI:10. 13242/j. cnki.bingduxuebao. 004103.
[10]麻粉莲，郑丽舒.人偏肺病毒感染研究进展[J].病毒学报，2024, 40(1):133-139. DOI:10. 13242/j. cnki.bingduxuebao. 004438.
[11]Zhang L, Li Q, Liu Q, et al. A bioluminescent imaging mouse model for Marburg virus based on a pseudovirus system[J]. Hum Vaccin Immunother, 2017, 13(8):1811-1817. DOI:10. 1080/21645515. 2017. 1325050.
[12]黄益曼，宋敬东，韩辉，等.尼帕病毒N基因核酸检测方法对假病毒阳性标准品的制备及鉴定[J].疾病监测，2022, 37(8):1010-1014. DOI:10. 3784/jbjc. 202204070135.
[13]Li Q, Liu Q, Huang W, et al. Current status on the development of pseudoviruses for enveloped viruses[J].Rev Med Virol, 2018, 28(1):e1963. DOI:10. 1002/rmv. 1963.
[14]葛慧敏，蒋雯玲，方中岳，等.假病毒包装系统发展及新兴应用[J].病毒学报，2024, 40(3):631-640.DOI:10. 13242/j. cnki. bingduxuebao. 004522.
[15]Wang Y, Zhou Z, Wu X, et al. Pseudotyped viruses[J]. Adv Exp Med Biol, 2023, 1407:1-27. DOI:10. 1007/978-981-99-0113-5_1.
[16]Russell CJ, Penkert RR, Kim S, et al. Human metapneumovirus:a largely unrecognized threat to human health[J]. Pathogens, 2020, 9(2):E109.DOI:10. 3390/pathogens9020109.
[17]Piyaratna R, Tollefson SJ, Williams JV. Genomic analysis of four human metapneumovirus prototypes[J].Virus Res, 2011, 160(1-2):200-205. DOI:10. 1016/j. virusres. 2011. 06. 014.
[18]Wang X, Li Y, Deloria-Knoll M, et al. Global burden of acute lower respiratory infection associated with human metapneumovirus in children under 5 years in2018:a systematic review and modelling study[J].Lancet Glob Health, 2021, 9(1):e33-e43. DOI:10. 1016/s2214-109x(20)30393-4.
[19]Mattiuzzo G, Ashall J, Doris KS, et al. Development of lentivirus-based reference materials for Ebola virus nucleic acid amplification technology-based assays[J].PLoS One, 2015, 10(11):e0142751. DOI:10. 1371/journal. pone. 0142751.
[20]Sun Y, Jia T, Sun Y, et al. External quality assessment for Avian Influenza A(H7N9)Virus detection using armored RNA[J]. J Clin Microbiol,2013, 51(12):4055-4059. DOI:10. 1128/jcm. 02018-13.
[21]周冬根，李云鹏，罗洁，等.用于MERS-CoV核酸检测的假病毒阳性对照品制备及鉴定[J].中国国境卫生检疫杂志，2019, 42(4):233-238. DOI:10. 16408/j. 1004-9770. 2019. 04. 002.
[22]Cheng Y, Niu J, Zhang Y, et al. Preparation of Histagged armored RNA phage particles as a control for realtime reverse transcription-PCR detection of severe acute respiratory syndrome coronavirus[J]. J Clin Microbiol,2006, 44(10):3557-3561. DOI:10. 1128/jcm. 00713-06.
[23]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]. J Clin Microbiol, 2009, 47(8):2571-2576. DOI:10. 1128/jcm. 00232-09.
[24]郑夔，袁帅，黎东红，等.新疆出血热病毒实时荧光RT-PCR检测方法的假病毒阳性对照品制备[J].华南预防医学，2016, 42(1):1-5. DOI:10. 13217/j.scjpm. 2016. 0001.
[25]王浩，郑丽舒.尼帕病毒检测方法研究进展[J].病毒学报，2019, 35(6):948-955. DOI:10. 13242/j. cnki.bingduxuebao. 003611.
[26]王浩，麻粉莲，王超，等.尼帕病毒Taqman qRT-PCR检测方法的建立[J].病毒学报，2020, 36(3):371-376. DOI:10. 13242/j. cnki. bingduxuebao. 003696.
[27]Ura T, Okuda K, Shimada M. Developments in viral vector-based vaccines[J]. Vaccines(Basel), 2014, 2(3):624-641. DOI:10. 3390/vaccines2030624.
[28]Zhu FC, Li YH, Guan XH, et al. Safety, tolerability,and immunogenicity of a recombinant adenovirus type-5vectored COVID-19 vaccine:a dose-escalation, openlabel, non-randomised, first-in-human trial[J]. Lancet,2020, 395(10240):1845-1854. DOI:10. 1016/S0140-6736(20)31208-3.
[29]伍政宇，罗楚铭，贾婷婷，等.新型冠状病毒假病毒的制备及血浆中和抗体检测[J].病毒学报，2022, 38(3):513-522. DOI:10. 13242/j. cnki.bingduxuebao. 004126.
[30]胡敏昊，王玮，吴小珉，等. H7N9亚型禽流感假病毒的制备与验证[J].病毒学报，2023, 39(5):1227-1234. DOI:10. 13242/j. cnki. bingduxuebao. 004381.
[31]Dai S, Min YQ, Li Q, et al. Interactome profiling of Crimean-Congo hemorrhagic fever virus glycoproteins[J]. Nat Commun, 2023, 14:7365. DOI:10. 1038/s41467-023-43206-1.
[32]Sun H, Xu J, Zhang G, et al. Developing pseudovirusbased neutralization assay against Omicron-included SARS-CoV-2 variants[J]. Viruses, 2022, 14(6):1332. DOI:10. 3390/v14061332.
[33]Xiang Q, Li L, Wu J, et al. Application of pseudovirus system in the development of vaccine, antiviral-drugs,and neutralizing antibodies[J]. Microbiol Res, 2022,258:126993. DOI:10. 1016/j. micres. 2022. 126993.
[34]Page KA, Landau NR, Littman DR. Construction and use of a human immunodeficiency virus vector for analysis of virus infectivity[J]. J Virol, 1990, 64(11):5270-5276. DOI:10. 1128/jvi. 64. 11. 5270-5276. 1990.
[35]Wiltshire TD, Milosevic D, Jacob EK, et al. Sensitive detection of integrated and free transcripts in chimeric antigen receptor T-cell manufactured cell products using droplet digital polymerase chain reaction[J].Cytotherapy, 2021, 23(5):452-458. DOI:10. 1016/j.jcyt. 2020. 12. 012.
[36]Ou X, Liu Y, Lei X, et al. Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV[J]. Nat Commun, 2020, 11:1620. DOI:10. 1038/s41467-020-15562-9.
[37]Zufferey R, Nagy D, Mandel RJ, et al. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo[J]. Nat Biotechnol, 1997, 15(9):871-875. DOI:10. 1038/nbt0997-871.
基本信息:
DOI：10.13242/j.cnki.bingduxuebao.004615
中图分类号:R373.1
引用信息:
[1]郑诗源,黄益曼,孟小草等.人偏肺病毒假病毒阳性标准品的制备与鉴定[J].病毒学报,2024,40(06):1263-1273.DOI:10.13242/j.cnki.bingduxuebao.004615.
基金信息:
中国疾病预防控制中心病毒病预防控制所青年科学基金（项目号：IVDC-202304）,题目：基于铁蛋白的呼吸道合胞病毒和人偏肺病毒二联纳米颗粒疫苗研究~~
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