# New Insights into the Biology of the Emerging Tembusu Virus
## 4 Clinical Features and Pathogenicity
### Innate Immune Response and Viral Evasion  
Flaviviruses trigger a strong innate immune response, which induces the production of a broad range of complementary antiviral molecules. Innate immunity is initiated by recognition of specific viral components, named pathogen-associated molecular patterns (PAMPs), via cellular pattern recognition receptors (PRRs) and was largely investigated in mammals and in birds [100; 101; 102; 103]. Three classes of PRRs sense the presence of PAMPs in infected cells: Toll-like receptors (TLRs), retinoic acid-inducible gene I-like receptors (RLRs) and NOD-like receptors (NLRs). The first two are crucial for interferon (IFN) response and proinflammatory cytokine production. These molecules are both essential for eliminating viruses and for recruitment of innate and adaptive immune cells [102; 104]. Several studies investigated the innate immune response against TMUV both in vivo and in vitro in avian and mammalian species. After experimental infection of female ducks or ducklings, [PERSON] et al. and [PERSON] et al. showed a rapid multiplication of the virus in different organs (brain, spleen, kidney, heart, pancreas, thymus or bursa of Fabricius) associated with severe lesions. The infection triggers activation of the RLR and TLR pathways and overexpression of PRRs such as RIG-I or MDA-5. This activation leads to the upregulation of interferon-stimulated genes (ISGs) from the Mx and OAS families in the brain and spleen [105; 106]. Upregulation of the RLR genes RIG-I and MDA-5 induced an increase in type I IFN expression in the early phase of the infection in vivo [107]. A set of experimental infections in CEFs and in 293T cells have also shown the implication of the RLR and TLR pathways in the innate immune response to TMUV infection. Through activation of the molecular adaptors MDA5 and TLR-3, involved in the RLR and TLR signaling pathways, respectively, TMUV infection strongly increased the expression of a set of type I IFN genes and some critical ISGs (Mx1, OAS1, IFITM3 and OASL), thereby reducing virus replication [108]. [PERSON] et al. characterized the implication of TBK1, a molecule involved in several type I IFN signaling pathways, in an experimental duck model. Using overexpression and knockdown experiments, they suggested a key role of TBK1 in the antiviral innate immune response in DEFs via IFN-8 production [109]. TBK1 signaling in mammals is initiated by DDX3 interaction with TBK1/IKK\(\epsilon\), in order to activate ISG synthesis through IRF3/7 activation [110]. In TMUV-infected ducks, the duck-DDX3 (duDDX3) modulates the innate immune response, and duDDX3 overexpression inhibits TMUV. Nevertheless, albeit duDDX3 may influence TMUV replication, TMUV is able to inhibit duDDX3 expression, suggesting an underlying mechanism to evade the immune response [111]. In geese, TMUV infection triggers an immune response with an increase in proinflammatoryatory cytokines and interferons in several organs as well as in PMBCs. Interestingly, immunoblotting analysis presented co-localization of CD8+T cells and TMUV, associated with high cytokine expression, suggesting an activation cascade due to viral infection and leading to the establishment of an antiviral status [99]. Molecular mechanisms in specific tissues involved in the etiology of TMUV disease affecting the brain and female reproductive system were partially addressed. A global proteomic analysis of duck ovaries from infected animals revealed differential protein expression including proteins involved in cellular structure, RNA processing, innate immune response, protein biosynthesis and modification, vesicle transportation and signal transduction. Interestingly, some modulations of expression may be related to immune evasion strategies [112]. In TMUV-infected duck brains, a recent transcriptomic analysis provided information on molecular mechanisms engaged in neurovirulence and host responses.