Susceptibility and initial immune response of Tupaia belangeri cells to dengue virus infection

Dengue is an emerging disease of great public health significance worldwide. The lack of a suitable infection model has hampered dengue virus (DENV) pathogenesis study, and developing a suitable small animal model has been a long-standing challenge. The aim of this study was to develop a feasible experimental model of DENV infection using Tupaia belangeri. The susceptibility of tupaia to DENV infection and characteristics of its innate immune response were examined in vitro. We found that tupaia fibroblast cells support replication of DENV serotypes 1-4 with a linear increase in viral load 24-96 h post-infection in both cells and culture supernatants. DENV-2 resulted in the highest viral growth among all serotypes. To characterize the innate immune response in tupaia cells during the early phase of DENV infection, we first evaluated the evolutionary relationship between tupaia Toll-like receptors (TLR1-9) and those of other mammalian species. Phylogenetic analysis showed that tupaia TLRs are evolutionarily much closer to human than they are to rodent. We next established an innate immune response measurement system by assessing the mRNA expression of TLR1-9 and four cytokines in DENV-infected tupaia cells. All serotypes induced the upregulation of TLR8 mRNA expression in infected tupaia cells. Silencing of TLR8 led to an increase in viral replication, indicating the existence of antiviral response through TLR8 on DENV infection. Although upregulation of IFN-β and IL-6 expression was only observed in DENV-1 infected cells and a significant suppression of TNF-α was observed in DENV-2 infected cells alone, IL-8 was upregulated in all DENV-1-4. Thus, this study demonstrates for the first time the susceptibility of tupaia cells to DENV infections and the role of TLR8 in the anti-viral response of tupaia cells to DENV. These findings demonstrate the potential utility of tupaia as a model for DENV research in the future.