痘苗病毒天坛株感染AG6小鼠脑病模型的建立与初步分析

doi:10.12300/j.issn.1674-5817.2025.070

实验动物与比较医学 ›› 2026, Vol. 46 ›› Issue (1): 3-10.DOI: 10.12300/j.issn.1674-5817.2025.070

痘苗病毒天坛株感染AG6小鼠脑病模型的建立与初步分析

杨琳¹^,², 金梦², 吴汗青¹^,², 李顺²(), 周晓辉²()()

^1.上海师范大学生命科学学院, 上海 200233
^2.上海市公共卫生临床中心实验动物部, 上海 201508

收稿日期:2025-05-13 修回日期:2025-08-19 出版日期:2026-02-25 发布日期:2026-02-14
作者简介:杨琳（1996—），女，硕士研究生，研究方向：感染性动物模型。E-mail：1016358073@qq.com
LI Shun (ORCID: 0000-0002-9887-8879), E-mail: lishun86@126.com
李顺，博士，副研究员。博士毕业于德国慕尼黑工业大学，并获极优等博士学位（Magna Cum Laude）。在Cell Death Differ、PLoS Pathog、Emerg Microbes Infect等杂志上发表论文30余篇，参编专著2部，申请国家发明专利4项。主持完成国家自然科学基金资助项目、上海市人才发展资金项目、上海市科委实验动物专项、上海市卫生和计划生育委员会项目和复旦大学附属公共卫生临床中心院内重点项目共8项，参与科技部国家重点研发计划和国家自然科学基金面上项目共5项，并获得2019年日本实验动物学会国际奖、2019年ICLAS-CALAS国际青年奖等荣誉。现任Military Medical Research期刊科学编辑，Animal Models and Experimental Medicine、《广州医科大学学报》等期刊编委，hLife、iMeta、Medicine Advance、Bio Integration等期刊青年编委，Front Oncol、Front Genet、World J Gastroentero等杂志的审稿专家。入选上海市科学技术委员会专家人才库、国家自然科学基金青年项目评审专家、上海市实验动物学会教育与科普专业委员会副主任委员。（1986—），男，博士，副研究员，研究方向：人类疾病动物模型构建及应用研究。E-mail： lishun86@126.com。ORCID：0000-0002-9887-8879
周晓辉，博士，研究员，复旦大学附属上海市公共卫生临床中心实验动物部主任，上海市新发与再现传染病研究所副所长、感染动物模型课题组组长。兼任全国实验动物标准化技术委员会委员，中国实验动物学会理事、动物实验生物安全委员会副主任委员、实验动物标准化委员会委员，中国合格评定国家认可委员会生物安全制度与技术专家，上海市实验动物学会理事、生物安全专业委员会主任委员，上海市实验动物标准化技术委员会委员，以及Anim Model Exp Med、Front Microbiol和《中国比较医学杂志》副主编，《实验动物与比较医学》常务编委兼终审专家。主要从事感染性动物模型构建与感染免疫机制研究。至今在Nature、Cell Death Differ、PLoS Pathog、Emerg Microbes Infect等国内外专业期刊上发表SCI论文60余篇，申请国家发明专利9项（授权2项），获上海市优秀发明大奖赛银奖3项。主持国家自然科学基金面上项目、上海市白玉兰科技人才基金、上海市自然科学基金、教育部回国留学人员启动基金、上海市科学技术委员会实验动物专项等项目，参与承担国家973项目、国家传染病重大专项、国家重点研发计划等多项研究。参与制定实验动物相关国家标准3项，参与编著全国八年制及“5+3”一体化临床医学专业规划教材《实验动物学》，参与撰写实验动物相关专著3本。（1973—），男，博士，研究员，研究方向：新发传染病动物模型构建与免疫机制研究。E-mail： zhouxiaohui@shaphc.org。ORCID： 0000-0002-7491-628X
ZHOU Xiaohui (ORCID: 0000-0002-7491-628X), E-mail: zhouxiaohui@shaphc.org;
基金资助:
国家重点研发计划-基础科研条件与重大科学仪器设备研发专项“单/多病因传染病动物模型的研制及应用”(2023YFF0724800);国家重点研发计划-基础科研条件与重大科学仪器设备研发专项“单/多病因传染病动物模型的研制及应用”(2023YFF0724801);上海市公共卫生临床中心项目“流感相关脑病发生机制研究”(KY-GW-2025-22)

Establishment and Preliminary Analysis of an AG6 Mouse Encephalopathy Model Induced by Vaccinia Virus Tiantan Strain Infection

YANG Lin¹^,², JIN Meng², WU Hanqing¹^,², LI Shun²(), ZHOU Xiaohui²()()

^1.College of Life Sciences, Shanghai Normal University, Shanghai 200233, China
^2.Experimental Animal Department, Shanghai Public Health Clinical Center, Shanghai 201508, China

Received:2025-05-13 Revised:2025-08-19 Published:2026-02-25 Online:2026-02-14

摘要/Abstract

摘要：

目的构建痘苗病毒天坛株（vaccinia virus Tiantan strain，VTT）感染AG6小鼠脑病动物模型。方法通过以0.01的感染复数（multiplicity of infection，MOI）感染Vero细胞，扩增VTT并进行浓缩和滴定，孵育72 h后，收集含病毒的细胞进行浓缩；将浓缩的病毒悬浮液连续稀释（10倍梯度）并加入到含有形成致密单层Vero细胞的6孔板中进行噬斑实验，计数每孔形成的空斑数目，并根据样品的稀释倍数计算病毒滴度。将14只5~6周龄的AG6小鼠（雌雄各半，并按性别分笼饲养）随机分为对照组（n=3，PBS）、低接种量组（n=6，1×10? PFU）和高接种量组（n=5，5×10? PFU）。将小鼠经异氟烷吸入麻醉后，进行滴鼻感染。每日观察各组小鼠精神状态，记录其体重及死亡情况，并在感染后第13天经尾静脉注射2%伊文思蓝（Evans Blue）（4 mL/kg体重）以评估各组小鼠血脑屏障（blood-brain barrier，BBB）的破损情况，然后收集小鼠脑组织样本进行星形胶质细胞和小胶质细胞活化的免疫荧光分析。结果纯化后的VTT滴度为1×10⁷PFU/mL。与对照组相比，低接种量组小鼠的体重无明显变化，且未出现死亡；高接种量组小鼠在感染后第5天开始体重显著减轻（P＜0.05），并伴有致死结果。感染后第13天，低接种量组小鼠的脑组织中未检测到伊文思蓝外渗，高接种量组小鼠的嗅球区域显示出明显的蓝色染色，提示BBB破裂。免疫荧光分析显示，感染后第13天，低接种量组小鼠嗅球区域星形胶质细胞和小胶质细胞没有明显增殖，高接种量组小鼠中可观察到明显的胶质细胞活化。结论成功建立VTT感染AG6小鼠的脑病动物模型，其特征是小鼠BBB损伤和特异性定位于嗅球区域的反应性胶质增生（表现为星形胶质细胞和小胶质细胞增生）。

关键词: 痘苗病毒天坛株, 脑病动物模型, 血脑屏障, 胶质细胞, AG6小鼠

Abstract:

Objective A mouse model of vaccinia virus Tiantan strain (VTT)-induced encephalopathy was developed using AG6 mice. Methods VTT was amplified by infecting Vero cells at a multiplicity of infection (MOI) of 0.01, followed by concentration and titration. After 72 h of incubation, virus-containing cells were collected and subjected to concentration. The concentrated viral suspension was serially diluted (10-fold dilutions) and added to 6-well plates containing confluent Vero cell monolayers for plaque assay. The number of plaques formed in each well was counted, and the virus titer was calculated based on the dilution factor. Fourteen 5-6-week-old AG6 mice (half male and half female, housed separately by sex) were randomly divided into a control group (n=3, PBS), a low-dose group (n=6, 1×10? PFU), and a high-dose group (n=5, 5×10? PFU). The mice were anesthetized by isoflurane inhalation and then infected via intranasal instillation. The mental state of the mice in each group was observed daily, and the body weight and mortality were recorded. On day 13 post-infection, 2% Evans Blue (4 mL/kg body weight) was administered via tail vein injection to assess blood-brain barrier (BBB) disruption. Subsequently, brain tissue samples were collected for immunofluorescence analysis to evaluate the activation of astrocytes and microglia. Results The titer of purified VTT was 1×10? PFU/mL. Compared with the control group, mice in the low-dose group showed no significant change in body weight, and no lethality was observed. In contrast, mice in the high-dose group exhibited significant weight loss starting on day 5 post-infection (P<0.05), accompanied by lethality. On day 13 post-infection, no Evans Blue extravasation was detected in the brain tissues of the low-dose group, while the olfactory bulb region of the high-dose group displayed distinct blue staining, indicating disruption of the BBB. Immunofluorescence analysis revealed no significant proliferation of astrocytes and microglia in the olfactory bulb region of the low-dose group on day 13 post-infection. In contrast, marked activation of glial cells was observable in the high-dose group. Conclusion An animal model of VTT-induced encephalopathy in AG6 mice is successfully established, characterized by BBB disruption and reactive gliosis specifically localized to the olfactory bulb region, manifested as astrocytic and microglial proliferation.

Key words: Vaccinia virus Tiantan strain, Animal model of encephalopathy, Blood-brain barrier, Glial cells, AG6 mice

中图分类号:

Q95-33

杨琳,金梦,吴汗青,等. 痘苗病毒天坛株感染AG6小鼠脑病模型的建立与初步分析[J]. 实验动物与比较医学, 2026, 46(1): 3-10. DOI: 10.12300/j.issn.1674-5817.2025.070.

YANG Lin,JIN Meng,WU Hanqing,et al. Establishment and Preliminary Analysis of an AG6 Mouse Encephalopathy Model Induced by Vaccinia Virus Tiantan Strain Infection[J]. Laboratory Animal and Comparative Medicine, 2026, 46(1): 3-10. DOI: 10.12300/j.issn.1674-5817.2025.070.

图/表 3

图1 VTT的噬斑实验结果以及感染后小鼠的体重曲线和生存曲线注：A，痘苗病毒天坛株（VTT）的噬斑实验结果。从左至右，依次为PBS组、稀释105倍组、稀释106倍组、稀释107倍组、稀释108倍组和稀释109倍组。不同滴度VTT感染后AG6小鼠的体重变化曲线（B）和生存曲线（C）。??P＜0.01，???P＜0.001，????P＜0.000 1。

Figure 1 Plaque assay results， mouse weight change curves， and survival curves following VTT infectionNote： A， The plaque assay results of vaccinia virus Tiantan strain （VTT）. From left to right， the groups are PBS， 10?-fold dilution group， 10?-fold dilution group， 10?-fold dilution group， 10?-fold dilution group， and 10?-fold dilution group. Weight change curves （B） and survival curves （C） of AG6 mice following infection with VTT at different titers. ??P＜0.01， ???P＜0.001， ????P＜0.000 1.

图2 伊文思蓝法评价不同滴度VTT感染后AG6小鼠的血脑屏障通透性注：A，小鼠实验流程图，从左至右，依次为滴鼻感染，感染后第13天胶质纤维酸性蛋白（GFAP）、离子钙接头蛋白分子-1（IBA-1）的免疫荧光染色和伊文思蓝染色。B，从左至右，依次为滴鼻PBS组、1×105 PFU和5×105 PFU痘苗病毒天坛株（VTT）感染组的伊文思蓝染色。箭头指示为阳性的嗅球部位。

Figure 2 Evaluation of blood-brain barrier permeability in AG6 mice infected with different titers of VTT using the Evans Blue assayNote： A， Mouse experiment flowchart， from left to right， intranasal infection； glial fibrillary acidic protein （GFAP） and ionized calcium-binding adapter molecule 1（IBA-1） immunofluorescence staining and Evans Blue staining on day 13 post-infection. B， From left to right， intranasal administration of PBS group， 1×105 PFU and 5×105 PFU of vaccinia virus Tiantan strain （VTT） infected groups after injection of Evans Blue. An arrow points to the positive staining in the olfactory bulb.

图3 GFAP和IBA-1免疫荧光标记显示小鼠嗅球星形胶质细胞及小胶质细胞分布（×20）注：A，小鼠在第13天时嗅球组织的胶质纤维酸性蛋白（GFAP）免疫荧光染色。从左至右，依次为标记细胞核、标记星形胶质细胞、双通道叠加图；从上至下，依次为滴鼻PBS组、1×105 PFU和5×105 PFU痘苗病毒天坛株（VTT）感染组。B，小鼠在第13天时嗅球组织的离子钙接头蛋白分子1（IBA-1）免疫荧光染色。从左至右，依次为标记细胞核、标记小胶质细胞、双通道叠加图；从上至下，依次为滴鼻PBS组、1×105 PFU和5×105 PFU VTT感染组。

Figure 3 Immunofluorescence labeling of GFAP and IBA-1 showing the distribution of astrocytes and microglia in the olfactory bulb of mice（×20）Note： A， Immunofluorescence staining of glial fibrillary acidic protein （GFAP） in the olfactory bulb tissue of mice on day 13. From left to right， images of nuclei staining， glial fibrillary acidic protein （GFAP）， and the dual-channel merged image； From top to bottom， intranasal administration of PBS group， 1×105 PFU and 5×105 PFU of vaccinia virus Tiantan strain （VTT） infected groups. B， Immunofluorescence staining of ionized calcium-binding adapter molecule 1（IBA-1） in the olfactory bulb tissue of mice on day 13. From left to right， images of nuclei staining， ionized calcium-binding adapter molecule 1（IBA-1）， and the dual-channel merged image； From top to bottom， intranasal administration of PBS group， 1×105 PFU and 5×105 PFU of VTT infected groups.

参考文献 24

[1]	ZHANG Z L, DONG L L, ZHAO C, et al. Vaccinia virus-based vector against infectious diseases and tumors[J]. Hum Vaccin Immunother, 2021, 17(6):1578-1585. DOI:10.1080/21645515.2020.1840887 .
[2]	QIN L, LIANG M, EVANS D H. Genomic analysis of vaccinia virus strain TianTan provides new insights into the evolution and evolutionary relationships between Orthopoxviruses[J]. Virology, 2013, 442(1):59-66. DOI:10.1016/j.virol.2013.03.025 .
[3]	THÈVES C, CRUBÉZY E, BIAGINI P. History of smallpox and its spread in human populations[J]. Microbiol Spectr, 2016, 4(4):1-10. DOI:10.1128/microbiolspec.poh-0004-2014 .
[4]	HENDERSON D A. The eradication of smallpox: an overview of the past, present, and future[J]. Vaccine, 2011, 29(): D7-D9. DOI:10.1016/j.vaccine.2011.06.080 .
[5]	LI E T, GUO X P, HONG D X, et al. Duration of humoral immunity from smallpox vaccination and its cross-reaction with Mpox virus[J]. Signal Transduct Target Ther, 2023, 8(1):350. DOI:10.1038/s41392-023-01574-6 .
[6]	孙雅洁. 抗天花病毒药物筛选模型的建立及药效评价[D]. 北京: 中国人民解放军军事医学科学院, 2007. DOI: 10.7666/d.Y1201420 .
	SUN Y J. Establishment of a screening model for anti-smallpox virus drugs and evaluation of their efficacy[D]. Beijing: Academy of Military Medical Sciences, 2007. DOI: 10.7666/d.Y1201420 .
[7]	GRANT R, NGUYEN L L, BREBAN R. Modelling human-to-human transmission of monkeypox[J]. Bull World Health Organ, 2020, 98(9):638-640. DOI:10.2471/BLT.19.242347 .
[8]	FINE P E, JEZEK Z, GRAB B, et al. The transmission potential of monkeypox virus in human populations[J]. Int J Epidemiol, 1988, 17(3):643-650. DOI:10.1093/ije/17.3.643 .
[9]	ARTENSTEIN A W, JOHNSON C, MARBURY T C, et al. A novel, cell culture-derived smallpox vaccine in vaccinia-naive adults[J]. Vaccine, 2005, 23(25):3301-3309. DOI:10.1016/j.vaccine. 2005.01.079 .
[10]	MARRIOTT K A, PARKINSON C V, MOREFIELD S I, et al. Clonal vaccinia virus grown in cell culture fully protects monkeys from lethal monkeypox challenge[J]. Vaccine, 2008, 26(4):581-588. DOI:10.1016/j.vaccine.2007.10.063 .
[11]	OSBORNE J D, SILVA M DA, FRACE A M, et al. Genomic differences of Vaccinia virus clones from Dryvax smallpox vaccine: the Dryvax-like ACAM2000 and the mouse neurovirulent Clone-3[J]. Vaccine, 2007, 25(52):8807-8832. DOI:10.1016/j.vaccine.2007.10.040 .
[12]	YOU Q, WU J, LIU Y, et al. HMGB1 release induced by EV71 infection exacerbates blood-brain barrier disruption via VE-cadherin phosphorylation[J]. Virus Res, 2023, 338:199240. DOI:10.1016/j.virusres.2023.199240 .
[13]	ZHANG R, FU Y X, CHENG M, et al. sEVs(RVG) selectively delivers antiviral siRNA to fetus brain, inhibits ZIKV infection and mitigates ZIKV-induced microcephaly in mouse model[J]. Mol Ther, 2022, 30(5):2078-2091. DOI:10.1016/j.ymthe. 2021. 10.009 .
[14]	WANG M, YANG F, HUANG D N, et al. Anti-idiotypic antibodies specific to prM monoantibody prevent antibody dependent enhancement of dengue virus infection[J]. Front Cell Infect Microbiol, 2017, 7:157. DOI:10.3389/fcimb. 2017. 00157 .
[15]	LIU J Y, LIU Y, NIE K X, et al. Flavivirus NS1 protein in infected host sera enhances viral acquisition by mosquitoes[J]. Nat Microbiol, 2016, 1(9):16087. DOI:10.1038/nmicrobiol.2016.87 .
[16]	AMIR A, MEHMOOD QADRI H, SAFFI J, et al. Neuroinvasive potential of monkeypox virus: a 25-year systematic review[J]. Cureus, 2025, 17(1): e77924. DOI:10.7759/cureus.77924 .
[17]	LEE P Y, CRON R Q. The multifaceted immunology of cytokine storm syndrome[J]. J Immunol, 2023, 210(8):1015-1024. DOI:10.4049/jimmunol.2200808 .
[18]	MISHRA M K, DUTTA K, SAHEB S K, et al. Understanding the molecular mechanism of blood-brain barrier damage in an experimental model of Japanese encephalitis: correlation with minocycline administration as a therapeutic agent[J]. Neurochem Int, 2009, 55(8):717-723. DOI:10.1016/j.neuint. 2009.07.006 .
[19]	ROE K, KUMAR M, LUM S, et al. West Nile virus-induced disruption of the blood-brain barrier in mice is characterized by the degradation of the junctional complex proteins and increase in multiple matrix metalloproteinases[J]. J Gen Virol, 2012, 93(Pt 6):1193-1203. DOI:10.1099/vir.0.040899-0 .
[20]	ROTHHAMMER V, QUINTANA F J. Control of autoimmune CNS inflammation by astrocytes[J]. Semin Immunopathol, 2015, 37(6):625-638. DOI:10.1007/s00281-015-0515-3 .
[21]	GIOVANNONI F, QUINTANA F J. The role of astrocytes in CNS inflammation[J]. Trends Immunol, 2020, 41(9):805-819. DOI:10.1016/j.it.2020.07.007 .
[22]	CHEN Z Z, ZHONG D, LI G Z. The role of microglia in viral encephalitis: a review[J]. J Neuroinflammation, 2019, 16(1):76. DOI:10.1186/s12974-019-1443-2 .
[23]	CHEN C J, OU Y C, CHANG C Y, et al. Glutamate released by Japanese encephalitis virus-infected microglia involves TNF-α signaling and contributes to neuronal death[J]. Glia, 2012, 60(3):487-501. DOI:10.1002/glia.22282 .
[24]	LUO D, MIAO Y J, KE X L, et al. Baculovirus surface display of zika virus envelope protein protects against virus challenge in mouse model[J]. Virol Sin, 2020, 35(5):637-650. DOI:10.1007/s12250-020-00238-x .

痘苗病毒天坛株感染AG6小鼠脑病模型的建立与初步分析

Establishment and Preliminary Analysis of an AG6 Mouse Encephalopathy Model Induced by Vaccinia Virus Tiantan Strain Infection

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 3

参考文献 24

相关文章 7

编辑推荐

Metrics

本文评价

[1]	刘佳, 叶岩荣, 沈赟, 唐启瑛, 陈梅卿, 易可慧, 陈少壮. 银杏内酯B通过调控脑内T细胞特性及与胶质细胞间相互作用促进缺血性脑卒中小鼠的神经功能恢复[J]. 实验动物与比较医学, 2024, 44(2): 139-148.
[2]	彭长庚, 富研, 朱冯婷, 夏瑞龙, 夏玮. 脊髓CD11c⁺小胶质细胞在神经病理性疼痛中的最新功能研究进展[J]. 实验动物与比较医学, 2022, 42(3): 171-176.
[3]	第五飞虎, 赵志靖, 邓毅恒. 高渗盐水保护血脑屏障减轻脑出血大鼠脑水肿的相关机制研究[J]. 实验动物与比较医学, 2021, 41(4): 327-332.
[4]	陆登成, 石安华, 陈帅, 韦姗姗. 小胶质细胞在焦虑症发病过程中作用及其机制研究进展[J]. 实验动物与比较医学, 2020, 40(1): 80-86.
[5]	关雅伦, 刘书华, 黄忠强, 李韵峰, 李雪娇, 李舸, 张钰. Tg2576小鼠的行为和病理特征观察[J]. 实验动物与比较医学, 2019, 39(4): 267-273.
[6]	王璇, 王文广, 李娜, 袁园, 张志成, 孙晓梅. 树鼩脊髓星形胶质细胞的分离鉴定[J]. 实验动物与比较医学, 2019, 39(1): 15-20.
[7]	王彦永, 张忠霞, 孙美玉, 王铭维. 慢性束缚应激对小鼠认知功能及海马不同亚区星形胶质细胞的影响[J]. 实验动物与比较医学, 2016, 36(3): 168-173.