【目的】登革病毒（dengue virus，DENV）是引起登革热和重症登革热的病原体，主要在热带和亚热带地区传播。登革病毒感染可导致多样的疾病表现，但目前致病机制尚未完全清楚，也无有效的疫苗和药物。现有的动物模型感染DENV后缺乏登革热临床症状或免疫反应受到干预，使得疾病机制、疫苗与药物研究受阻。因此，建立能模拟人类疾病表现的有效DENV动物模型是一个亟待解决的问题。本研究将探究树鼩不同组织细胞对DENV的易感性和感染特性，建立DENV感染性树鼩细胞模型；同时，构建正常免疫反应的登革病毒急性感染模型，为DENV的致病机制和药物研发提供有效工具。

【方法】登革病毒以MOI = 0.02体外感染8种树鼩细胞，以常用细胞作为阳性参考，每12 h收集细胞样本用RT-qPCR测定载量、观察细胞病变，用噬斑法检测滴度，比较各胞感染6天内病变效应、增殖动力学及子代感染性的差异，对易感树鼩细胞应用潜力进行评估。以尾静脉联合腹腔注射6×10⁵ PFU登革病毒构建感染树鼩模型，观察动物造模后7天内每日行为、状态、体温体重等表现，RT-qPCR检测血清病毒载量；造模后2、4、6、28天取样血液、组织，RT-qPCR检测病毒组织分布和载量，HE染色检查组织病理变化，测定血常规；造模后1、2、3、4周取血清测定中和抗体水平，分析树鼩感染后与人类表现的异同。利用外周血细胞转录组测序分析病毒感染引起树鼩免疫水平的变化，通过差异分析和富集分析筛选可能发挥作用的关键基因和信号通路，对部分基因进行RT-qPCR验证。

【结果】除TRMECs 外，DENV对7种树鼩细胞易感，随感染时间的变化，几种细胞均出现变圆、脱落、坏死及裂解等明显的细胞病变；pTRECs、TBMECs、TASMCs、TAECs、THs5种细胞的病毒载量与阳性对照细胞相当，且均产生感染性子代病毒，其中TAECs、pTRECs和THs病毒载量与滴度较高，作为候选细胞模型。较高滴度感染的树鼩产生了持续7天的病毒血症，部分个体产生二次病毒血症，并且感染后2、4、6天在多种组织中检测到病毒载量；大部分树鼩心、肝、脾、肾、脑组织表现轻度病变，并检测到病毒E蛋白表达；树鼩在感染后2、4、6天检测到中性粒细胞下降和淋巴细胞升高，感染后1、2、3、4周维持的中和抗体。转录组测序检测到大量显著差异表达基因，富集分析结果提示了树鼩感染DENV后可能在第6天恢复免疫状态。选择HIF1A、THBS1、UBXN7、ROCK1进行验证，其变化趋势与测序结果一致。

【结论】多种树鼩细胞对DENV易感且支持病毒复制，可产生感染活性的子代，扩充了DENV易感细胞库，其中TAECs、pTRECs和THs最适合成为DENV病毒感染细胞模型；高滴度体内感染DENV的树鼩模拟了人类登革热的部分表现，包括病毒血症、组织损伤、淋巴细胞和中性粒细胞变化，个别动物产生了疑似重症表型，证明了该模型的潜力，有待进一步研究；探讨了树鼩感染DENV后的免疫特征，识别了关键基因，为后续研究提供思路。

【Objectives】Dengue virus (DENV) is the pathogen of dengue fever and severe dengue fever, which is mainly transmitted in tropical and subtropical regions. Dengue virus infection can lead to various disease manifestations, but the mechanism of pathogenesis is not yet fully understood, and there is no effective vaccine or drug. Therefore, the establishment of effective DENV animal models that can simulate human disease manifestations is an urgent problem. This study aimed to construct an acute infection model of dengue virus with normal immune response using tree shrews to provide a usable candidate platform for relevant researches; meanwhile, to explore the susceptibility and infection characteristics of tree shrew cells to DENV and to provide an emerging species basis for in vitro studies of this virus.

【Methods】Dengue virus infected 8 kinds of tree shrew cells in vitro with MOI = 0.02, using commonly used cells as a positive reference, cell samples were collected every 12 h to determine the load by RT-qPCR, observe the cell lesions, and detect the titer by by plaque assay, comparing the differences in lesion effects, proliferation kinetics and offspring infectivity within 6 days of infection, and evaluating the potential of application of the susceptible tree shrew cells. Infected tree shrew cell model was constructed by intravenous combined intraperitoneal injection of 6×10⁵ PFU of dengue virus, observing the daily behavior, status, temperature and body weight within 7 days after modeling The infected tree shrew model was constructed by intravenously and intraperitoneally injecting 6×10⁵ PFU dengue virus, and the animals were observed for 7 days after modeling for daily behaviors, status, body temperature and weight, and serum viral load was detected by RT-qPCR; blood and tissue samples were taken at 2, 4, 6, and 28 days after modeling for detection of viral distribution and viral load in tissues, and histopathological changes were examined by HE staining and blood tests were performed; the potential application of susceptible tree shrew cells was evaluated. Serum samples were taken at 1, 2, 3, and 4 weeks after modeling to determine the neutralizing antibody level and analyze the similarities and differences between tree shrews and humans after infection. Changes in the immune level of tree shrews caused by viral infection were analyzed using sequencing, and key genes and signaling pathways that might be involved in the virus infection were screened by differential analysis and enrichment analysis, and some genes were verified by RT-qPCR.

【Results】DENV was susceptible to seven kinds of tree shrew cells except TRMECs, and several kinds of cells showed obvious cytopathic lesions such as rounding, detachment, necrosis and lysis with the change of infection time; the viral loads of five kinds of cells, namely pTRECs, TBMECs, TASMCs, TAECs, and THs, were comparable to that of the positive control cells and all of them produced infectious progeny viruses.  Among them, TAECs, pTRECs and THs with high viral loads and titers were used as candidate cell models. Higher-titer infected tree shrews developed viraemia lasting up to 7 days, with some individuals developing secondary viraemia, , and viral loads were detected in a variety of tissues on days 2, 4, and 6 post-infection; most of the tree shrew heart, liver, spleen, kidney, and brain tissues exhibited mild lesions and viral E protein expression was detected; and the tree shrews detected a decrease in neutrophils and an elevation in lymphocytes on days 2, 4, and 6 post-infection, and neutralising antibodies that were maintained at weeks 1, 2, 3, and 4 post-infection. Transcriptome sequencing detected a large number of significantly differentially expressed genes, and the results of enrichment analyses suggested that tree shrews might return to immune status on day 6 after DENV infection. HIF1A, THBS1, UBXN7 and ROCK1 were selected for validation, and their trends were consistent with the sequencing results.

【Conclusions】Various species of tree shrew cells are susceptible to DENV and support viral replication, which can produce infection-active progeny, expanding the DENV-susceptible cell pool, of which TAECs, pTRECs and THs are the most suitable to be DENV virus-infected cell models; High-titer in vivo infection of DENV in tree shrews mimicked some of the manifestations of human dengue fever, including viremia, tissue damage, lymphocyte and neutrophil changes, and individual animals produced a suspected severe disease phenotype, proving the potential of this model, and it needs to be further studied; The immune characteristics of tree shrews infected with DENV were explored, and key genes were identified to provide ideas for follow-up studies.