# New Insights into the Biology of the Emerging Tembusu Virus
## 4 Clinical Features and Pathogenicity
### Innate Immune Response and Viral Evasion
Several pathways and genes specifically related to nervous system and innate immune responses were modulated, providing evidence on the neuro-immune interactions in TMUV infection [113]. Despite efforts to shed light on the molecular mechanisms triggered by TMUV infection, intimate modalities of host responses to the infection remain incompletely known, and further studies will be necessary to uncover host-pathogen interactions. Flaviviruses have developed different immune evasion strategies [114]. Viral proteins, particularly nonstructural (NS) proteins, antagonize crucial signaling pathways, such asvirus recognition pathways and the IFN pathway, needed for an effective response [115]. Several studies also reported immune evasion mechanisms developed by TMUV. Interferon treatment of TMUV-infected avian cells has shown the absence of impact on virus replication, contrary to IFN treatment on mammalian TMUV-infected cells [95]. This observation suggests specific cellular mechanisms allowing TMUV to overcome IFN-mediated effects in avian cells specifically. Involvement of TMUV NS1 in the inhibition of RLR receptor signaling by impairment of the interaction of RIG-I and MDA5 with IPS1 was reported using reporter assay experiments. The NS1 protein interacted with the CARD domain of RLR adaptors, impeding recognition and association with virus molecule adaptors. Disruption of this interaction led to a suppression of RLR-mediated IFN-\(\beta\) production to finally facilitate immune evasion [98]. In 2019, [PERSON] et al. also infected HEK293T to study the impact of the viral protein NS2B and reported an interaction with actors of the RIG-I pathway, resulting in suppression of the type I interferon response. The TMUV NS2B3 viral protease may also inhibit IFN-\(\beta\) production. Contrary to TMUV-NS1, the NS2B3 protease acts directly on the mitochondrial duck STING (duSTING) protein, which is a key intermediate in the RLR pathway, to inhibit signal transduction to decrease the production of IFN-\(\beta\). In this context, synthesis rates of a subset of ISG-related proteins (Mx1 and OASL) are also downregulated. Interestingly, ZIKV and DENV NS2B3 present the same enzyme cleavage site as TMUV NS2B3 on duSTING [16]. In fact, ZIKV and DENV NS2B3 are able to hydrolyze duSTING, while TMUV NS2B3 is reciprocally able to cleave human STING, suggesting a potential relevance in the case of spillover of the virus to humans. A study by the same research group on the role of the NS2A protein found similar results, with an alteration in STING protein activity in RLR signal transduction. In the same way as NS2B, the binding of NS2A on STING blocks the interaction with TBK1, decreasing its phosphorylation and leading to the inhibition of IFN-\(\beta\) production [116]. A recent study reported that the NS4B protein can inhibit IFN-\(\beta\) production as previously observed with DENV. Thus, the TMUV NS4B protein has been identified as a major inhibitor of the RLR pathway decreasing the expression of RIG-I, MDA5, MAVS, STING and also TBK1, unlike NS2-A and -B. Moreover, specific mutations in the NS4B protein modifying interactions with TBK1 lead to phenotypic changes decreasing the pathogenicity of TMUV. Thus, NS4B appears to strongly interact with TBK1 and inhibit its recruitment by STING, resulting in a blockage of the signaling pathway [117]. These studies confirm the importance of TBK1 in the implementation of an efficient antiviral response, as reported by [PERSON] et al. [109], and strategies developed by TMUV to overcome the immune response. Finally, flavivirus nonstructural protein 5 (NS5) is an RNA-dependent RNA polymerase (RdRp) that catalyzes the replication of the viral RNA genome in the replication complex [118]. Little is known about the molecular mechanisms of replication with TMUV; however, NS5 also appears to play an important role in infection. Indeed, specific amino acid substitution in the NS5 protein decreases infectivity both in vitro in BHK21 and DEF cells and in vivo in ducklings.