登革病毒对树鼩不同组织来源细胞的易感性及感染特性研究

doi:10.12300/j.issn.1674-5817.2024.178

实验动物与比较医学 ›› 2025, Vol. 45 ›› Issue (2): 229-238.DOI: 10.12300/j.issn.1674-5817.2024.178

• 实验动物资源开发与利用 • 上一篇下一篇

登革病毒对树鼩不同组织来源细胞的易感性及感染特性研究

刘欣¹, 杞梦迪¹, 王文广¹, 罕园园¹, 陆美丽¹^,², 李娜¹, 代解杰¹(), 陆彩霞¹()()

^1.北京协和医学院/中国医学科学院医学生物学研究所, 昆明 650118
^2.云南大学生命科学学院, 昆明 650500

收稿日期:2024-12-03 修回日期:2025-02-09 出版日期:2025-04-25 发布日期:2025-04-25
通讯作者: 代解杰（1961—），男，研究员，博士生导师，研究方向：人类疾病动物模型建立与比较医学。E-mail：djj@imbcams.com.cn。ORCID： 0000-0002-4242-9089；
陆彩霞（1979—），女，博士，主任技师，硕士生导师，研究方向：人类疾病动物模型建立。E-mail：lcx@imbcams.com.cn。ORCID：0000-0001-7186-7451
作者简介:刘欣（1999—），女，硕士研究生，研究方向：人类疾病动物模型建立及机制研究。E-mail：livxin@student.pumc.edu.cn
基金资助:
云南省科技计划项目“云南省眼科疾病防治研究重点实验室”(2017DG008);昆明市科技创新团队“树鼩细胞资源库建立及病毒性疾病动物模型研究创新团队”(2019-1-R-24483)

Study on Susceptibility and Infection Characteristics of Dengue Virus in Cells Sourced from Different Tissues of Tree Shrews

LIU Xin¹, QI Mengdi¹, WANG Wenguang¹, HAN Yuanyuan¹, LU Meili¹^,², LI Na¹, DAI Jiejie¹(), LU Caixia¹()()

^1.Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming 650118, China
^2.School of Life Sciences, Yunnan University, Kunming 650500, China

Received:2024-12-03 Revised:2025-02-09 Published:2025-04-25 Online:2025-04-25
Contact: DAI Jiejie (ORCID: 0000-0002-4242-9089), E-mail: djj@imbcams.com.cn;
LU Caixia (ORCID: 0000-0001-7186-7451), E-mail: lcx@imbcams.com.cn

摘要/Abstract

摘要：

目的探究登革病毒（dengue virus，DENV）对树鼩不同组织来源细胞的易感性及感染特征，为扩充DENV的树鼩易感细胞库提供依据。方法将DENV以感染复数（multiplicity of infection，MOI）为0.02的剂量分别接种于树鼩皮肤成纤维细胞（tree shrew skin fibroblasts，TSFs）、原代树鼩肾上皮细胞（primary tree shrew renal epithelial cells，pTRECs）、树鼩主动脉内皮细胞（tree shrew aortic endothelial cells，TAECs）、树鼩主动脉平滑肌细胞（tree shrew aortic smooth muscle cells，TASMCs）、树鼩肝细胞（tree shrew hepatocytes，THs）、树鼩角膜基质细胞（tree shrew corneal stromal cells，TCSCs）、树鼩脑微血管内皮细胞（tree shrew brain microvascular endothelial cells，TBMECs）及树鼩视网膜微血管内皮细胞（tree shrew retinal microvascular endothelial cells，TRMECs），用常规培养DENV的C6/36、Vero、A549及BHK-21细胞系作为阳性对照。在感染病毒后6 d内，每12 h用倒置显微镜观察各种细胞的病变效应，用实时荧光定量PCR检测细胞培养物中病毒核酸载量，绘制病毒增殖曲线，用噬斑实验检测病毒滴度。结果除TRMECs外，DENV对7种树鼩细胞均易感。随着感染时间的延长，7种树鼩细胞均出现变圆、脱落、坏死及裂解等明显的病变效应。在pTRECs、TBMECs、TASMCs、TAECs和THs这5种树鼩细胞的培养裂解物中检测到的病毒核酸载量与4种阳性对照细胞接近，大于4×10⁷ 拷贝/μL。以上5种树鼩细胞均能产生感染性子代病毒，其中3种树鼩细胞TAECs（3.13×10⁵PFU/mL）、THs（2.03×10⁵PFU/mL）、pTRECs（1.58×10⁵ PFU/mL）中的DENV滴度较接近阳性对照细胞 C6/36（3.85×10⁵ PFU/mL），且能更早收获病毒。结论 DENV对树鼩多种组织来源的细胞易感，且病毒增殖速度快于常见其他物种来源的传代细胞系，其中TAECs、THs和pTRECs尤其适于DENV增殖培养。同时，这些细胞可能参与DENV在树鼩体内的感染。

关键词: 树鼩细胞, 登革病毒, 细胞病变效应, 病毒增殖曲线, 病毒感染

Abstract:

Objective To investigate the susceptibility and infection characteristics of dengue virus (DENV) in cells derived from diverse tissues of tree shrews and to provide a basis for expanding the repertoire of DENV-permissive cell models in this species. Methods DENV was inoculated at a multiplicity of infection (MOI) of 0.02 into tree shrew skin fibroblasts (TSFs), primary tree shrew renal epithelial cells (pTRECs), tree shrew aortic endothelial cells (TAECs), tree shrew aortic smooth muscle cells (TASMCs), tree shrew hepatocytes (THs), tree shrew corneal stromal cells (TCSCs), tree shrew brain microvascular endothelial cells (TBMECs), and tree shrew retinal microvascular endothelial cells (TRMECs). C6/36, Vero, A549, and BHK-21 cells (commonly used for DENV propagation) were used as positive controls. Over 6 days post-infection, cellular cytopathic effects were monitored at 12-hour intervals using an inverted microscope, viral RNA loads in cell lysates were quantified by real-time fluorescent quantitative PCR to generate proliferation curves, and viral titers were determined by plaque assay. Results Seven types of tree shrew cells, except TRMECs, were susceptible to DENV. Prolonged infection induced pronounced cytopathic effects, including cell rounding, detachment, necrosis, and lysis, across all susceptible cells. The viral RNA loads detected in lysates of pTRECs, TBMECs, TASMCs, TAECs and THs, approached those of positive controls (≥4×10? copies/μL). Infectious progeny viruses were produced by these five cell types, with three (TAECs, 3.13×10⁵ PFU/mL; THs, 2.03×10⁵ PFU/mL; pTRECs, 1.58×10⁵ PFU/mL) exhibiting titers comparable to C6/36 (3.85×10? PFU/mL) and earlier viral harvests. Conclusion DENV exhibits broad susceptibility to tree shrew cells of multiple tissue origins, with proliferation rates surpassing those of conventional cell lines sourced from other species. TAECs, THs, and pTRECs are particularly suitable for large-scale DENV proliferation, suggesting their potential involvement in in vivo infection.

Key words: Tree shrew cells, Dengue virus, Cytopathic effect, Viral proliferation curves, Viral infection

中图分类号:

Q95-33

刘欣,杞梦迪,王文广,等. 登革病毒对树鼩不同组织来源细胞的易感性及感染特性研究[J]. 实验动物与比较医学, 2025, 45(2): 229-238. DOI: 10.12300/j.issn.1674-5817.2024.178.

LIU Xin,QI Mengdi,WANG Wenguang,et al. Study on Susceptibility and Infection Characteristics of Dengue Virus in Cells Sourced from Different Tissues of Tree Shrews[J]. Laboratory Animal and Comparative Medicine, 2025, 45(2): 229-238. DOI: 10.12300/j.issn.1674-5817.2024.178.

图/表 5

表1 登革病毒感染不同细胞后的细胞病变效应变化

Table 1 Changes of cytopathic effect (CPE) in different cell types after dengue virus (DENV) infection

细胞 Cell	接种后时间/h Time post-infection/h
细胞 Cell	12	24	36	48	60	72	84	96	108	120	132	144
TAECs	-	-	+	++	+++	++++	++++	++++
TASMCs	-	-	+	++	+++	++++	++++	++++
THs	-	-	+	+	++	+++	++++	++++
pTRECs	-	±	+	++	+++	++++	++++	++++
TSFs	±	+	++	+++	+++	++++	++++	++++
TCSCs	-	-	±	+	+	++	+++	+++	++++	++++
TBMECs	-	-	-	±	+	++	+++	+++	++++	++++
TRMECs	-	-	-	±	±	+	+	+	+	+
Vero	-	-	-	±	+	+	+	+	++	++	+++	++++
BHK-21	-	-	-	+	++	+++	+++	++++	++++	++++	++++
A549	-	-	±	+	+	++	++	+++	+++	+++	++++	++++
C6/36	-	-	+	+	++	++	+++	+++	+++	+++	+++	++++

图1 倒置显微镜下不同来源的树鼩细胞和阳性对照细胞感染登革病毒前后的形态学变化（×200）注：TAECs为树鼩主动脉内皮细胞；TASMCs为树鼩主动脉平滑肌细胞；pTRECs为原代树鼩肾上皮细胞；THs为树鼩肝细胞；TBMECs为树鼩脑微血管内皮细胞；TCSCs为树鼩角膜基质细胞；TSFs为树鼩皮肤成纤维细胞；TRMECs为树鼩视网膜微血管内皮细胞；C6/36、Vero、A549及BHK-21作为阳性对照细胞。

Figure 1 Morphological changes in tree shrew cells of different tissues and positive control cells before and after dengue virus （DENV） infection under inverted microscope （×200）Note： TAECs， tree shrew aortic endothelial cells； TASMCs， tree shrew aortic smooth muscle cells； pTRECs， primary tree shrew renal epithelial cells； THs， tree shrew hepatocytes； TBMECs， tree shrew brain microvascular endothelial cells； TCSCs， tree shrew corneal stromal cells； TSFs， tree shrew skin fibroblasts； TRMECs， tree shrew retinal microvascular endothelial cells； The C6/36， Vero， A549 and BHK-21 cells were employed as positive controls.

图2 实时荧光定量PCR检测感染不同来源的树鼩细胞后登革病毒的增殖曲线注：TAECs，树鼩主动脉内皮细胞；TASMCs，树鼩主动脉平滑肌细胞；THs树鼩肝细胞；pTRECs，原代树鼩肾上皮细胞；TSFs，树鼩皮肤成纤维细胞；TCSCs，树鼩角膜基质细胞；TBMECs，树鼩脑微血管内皮细胞；TRMECs，树鼩视网膜微血管内皮细胞；C6/36、Vero、A549及BHK-21作为阳性对照细胞。

Figure 2 Viral proliferation curves of tree shrew cells of different tissues after dengue virus （DENV） infection via real-time fluorescent quatitative PCRNote： TAECs， tree shrew aortic endothelial cells； TASMCs， tree shrew aortic smooth muscle cells； THs， tree shrew hepatocytes； pTRECs， primary tree shrew renal epithelial cells； TSFs， tree shrew skin fibroblasts； TCSCs， tree shrew corneal stromal cells； TBMECs， tree shrew brain microvascular endothelial cells； TRMECs， tree shrew retinal microvascular endothelial cells； The C6/36， Vero， A549 and BHK-21 cells were employed as positive controls.

表2 各种细胞感染后病毒载量峰值及达到峰值的时间

Table 2 Peak viral load and time-to-peak in different cell types after dengue virus (DENV) infection

细胞 Cell	病毒载量峰值/（拷贝·μL^-1） Peak viral load/ (copies·μL^-1)	时间/h Time/ h
pTRECs	(9.18±0.21)×10⁷	48
TBMECs	(7.43±0.75)×10⁷	72
TASMCs	(7.27±0.43)×10⁷	84
TAECs	(6.87±0.30)×10⁷	72
THs	(4.08±0.02)×10⁷	48
TCSCs	(1.11±0.05)×10⁷	36
TSFs	(1.50±0.05)×10⁶	84
Vero	(1.39±0.22)×10⁸	120
C6/36	(1.28±0.21)×10⁸	120
BHK-21	(6.73±0.13)×10⁷	48
A549	(1.13±0.05)×10⁷	60

图3 噬斑实验测定不同树鼩细胞中登革病毒的病毒滴度注：A，C6/36细胞中DENV增殖所形成的斑块；B，TAECs中DENV增殖所形成的斑块；C，TASMCs中DENV增殖所形成的斑块；D，THs中DENV增殖所形成的斑块；E，pTRECs中DENV增殖所形成的斑块；F，TBMECs中DENV增殖所形成的斑块。C6/36细胞作为阳性对照；红圈指示噬斑；10-3、10-4、10-5表示病毒液稀释倍数；control表示未感染病毒的对照孔。

Figure 3 Quantification of dengue virus （DENV） titer in tree shrew cells of different tissues via plaque assayNote： A， Plaques formed by DENV proliferating in C6/36 （positive control）； B， Plaques formed in tree shrew aortic endothelial cells （TAECs）； C， Plaques formed tree shrew aortic smooth muscle cells （TASMCs）； D， Plaques formed in tree shrew hepatocytes （THs）； E， Plaques formed by DENV proliferating in primary tree shrew renal epithelial cells （pTRECs）； F， Plaques in primary tree shrew brain microvascular endothelial cells （TBMECs）. The C6/36 cells were employed as the positive control. Red circles demarcate individual plaques. Viral dilutions are indicated as 10-3， 10-4， and 10-5. The “control” denotes uninfected wells， serving as a negative reference.

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登革病毒对树鼩不同组织来源细胞的易感性及感染特性研究

Study on Susceptibility and Infection Characteristics of Dengue Virus in Cells Sourced from Different Tissues of Tree Shrews

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