In the last decade, the number of emerging Flaviviruses described worldwide has increased considerably. cell death and anti-viral response. Our results highlight the need to better characterize the physiopathology related to USUV infection in order to anticipate the potential threat of USUV emergence. Author summary Usutu virus (USUV) is an African mosquito-borne virus closely related to West Nile virus and belongs to the Japanese encephalitis virus serogroup in the genus. Recently several neurological disorders such as encephalitis, meningitis and meningoencephalitis were Veliparib associated with USUV-infection in immunocompromised and immunocompetent patients. The goal of our work was to study the ability of USUV to infect neuronal cells and to characterize the effects of USUV infection in these cells. We have shown that USUV can infect efficiently several neuronal cells (mature neurons, astrocytes, microglia, IPSc-derived human neuronal stem cells (NSCs)). Interestingly, USUV replicates in human astrocytes more efficiently than Veliparib another mosquito-borne flavivirus, Zika virus, reduces cell proliferation and induces strong anti-viral response. Moreover, USUV induces caspase-dependent apoptosis in NSCs. Our results suggest that USUV infection may lead to encephalitis and/or meningoencephalitis via neuronal toxicity and inflammatory response. Introduction The recent Zika virus (ZIKV) outbreak has reminded us that the emergence of new viruses depends on multiple factors and is therefore extremely difficult to predict. Among potential emerging viruses, Usutu virus (USUV) has recently focused attention. USUV is an African mosquito-borne virus closely related to West Nile virus (WNV) that belongs to the Japanese encephalitis virus (JEV) serogroup in the genus (family) [1]. USUV was discovered in 1959 from a mosquito of the species in South Africa and isolated by intracerebral inoculation of newborn mice [2]. The USUV genome is a positive, single-stranded RNA genome of 11,064C11,066 nucleotides with one open-reading frame encoding a 3434-amino-acid-residue polyprotein, which is subsequently cleaved into three structural (core, membrane, and envelope) and eight nonstructural (NS1, NS2A, NS2B, NS3, NS4A, 2K, NS4B, and NS5) proteins [3C5]. USUV natural life cycle is similar to WNV: it involves birds as reservoirs and ornithophilic mosquitoes as vectors like the common common blackbirds (and [40,42]. To PLAT monitor viral replication in the murine central nervous system (CNS), we first used acute hippocampus slices prepared from dissected brains Veliparib from 6C7 day-old wild type (WT) mice. Two days post-isolation, USUV was applied (3×105 cells tradition infective dose 50% (TCID50) per slice) on top of the slices, which were further managed in tradition. 4 days post-infection (dpi), slices were fixed, astrocytes, microglial cells and neurons labeled by GFAP, Iba1 and NeuN staining respectively and USUV antigens were observed using a pan-flavivirus antibody (4G2) that recognizes the package protein of several flavivirus [43]. Fig 1A shows that in mock-treated slices, no pan-flavivirus marking was observed, whereas USUV-infected samples showed strong pan-flavivirus staining, indicating an efficient USUV illness. Co-labeling with neuronal- (NeuN), astrocyte- (GFAP) and microglial- (Iba1) specific Veliparib antibodies with the pan-flavivirus antibody showed a broad tropism of USUV for mind cells (Fig 1B and 1C). Fig 1 USUV infects efficiently organotypic murine mind slices. To confirm these observations OR (RIG-I), (LGP2) or were also upregulated by both viruses, whereas was only modulated by USUV (Fig 5C). Importantly, in all cases, the upregulation of these antiviral genes was stronger following USUV than ZIKV illness, up to 100 instances for the chemokines and for the (Fig 5C). Additional genes, such as the chemokine and the transcription factors and were specifically upregulated in USUV infected astrocytes. Curiously, additional variations in the cellular reactions induced by USUV ZIKV were observed, in particular concerning the MAPK pathway (and users of the inflammasome pathway such as and that were preferentially modulated by ZIKV (Fig 5C). Fig 5 Anti-viral reactions in USUV- and ZIKV-infected human being astrocytes. These data focus on the strong induction of an antiviral response by USUV and suggest considerable variations in the cellular response process against flaviviruses in astrocytes. Human being IPSc-derived NSCs are undergoing apoptosis under USUV illness The recent ZIKV epidemic highlighted that developing brains can become highly sensitive to flavivirus illness [45C48,53,54]. Moreover, in the adult mind, specific niches such as the hippocampus are involved in adult neurogenesis and are potential focuses on.