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

doi:10.12300/j.issn.1674-5817.2024.178

Abstract

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

CLC Number:

Q95-33

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.

Figures/Tables 5

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	-	-	+	+	++	++	+++	+++	+++	+++	+++	++++

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.

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.

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

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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