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A versatile fluorescent reporter for Usutu virus drug screening and infection tracking [viralRNAseq]

ABSTRACT: Usutu virus (USUV) is an emerging mosquito-borne flavivirus closely related to West Nile and Japanese encephalitis viruses. Despite its increasing prevalence in Europe and reports of neuroinvasive disease in humans, the molecular and immunological determinants of USUV infection remain poorly understood, largely due to the absence of tractable experimental tools. Here, we report the generation and characterization of the first full-length fluorescent reporter USUV expressing the red fluorescent protein mScarlet. Using a previously established infectious clone of the Vienna 2001 strain, we inserted the mScarlet gene into a permissive region of the viral genome, generating a stable and replication-competent reporter virus. USUV-mScarlet exhibited replication kinetics comparable to wild-type virus and maintained fluorescence over at least ten serial passages in human hepatoma (Huh7) cells. Application of this system identified interferon-alpha 2a (IFN-α2a) as a potent antiviral against USUV and revealed robust induction of the type I interferon response in infected and bystander mouse fibroblasts. In type I/III IFN signaling deficient (Ifnar-/- or Stat1-/-) mice, which are permissive for viral replication, USUV-mScarlet infection resulted in transient disease and reporter expression in multiple peripheral tissues. Among splenocytes, myeloid cells were highly enriched in reporter expression, suggesting their role as targets of viral infection. Collectively, these findings demonstrate that USUV-mScarlet faithfully recapitulates wild-type infection dynamics while enabling single-cell/quantitative visualization and sorting of infected cells to assess viral spread and host responses. This reporter platform provides a versatile tool for studying USUV biology and pathogenesis and will facilitate the development of targeted antivirals and vaccines against this emerging flavivirus.

ORGANISM(S): Homo sapiens

PROVIDER: GSE312629 | GEO | 2026/09/28

REPOSITORIES: GEO

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Project description:Flaviviruses (genus Orthoflavivirus) are arthropod-borne viruses which cause approximately 400 million annual global infections in humans. As obligate intracellular pathogens, flaviviruses must co-opt cellular machinery to facilitate their infection and spread. Notably, all flaviviruses replicate in association with the host endoplasmic reticulum (ER); genome replication, sequestered within virus-induced ER invaginations called viral replication organelles (vROs), is coupled to viral assembly, which occurs on ER membranes juxtaposed to sites of vRO formation. Owing to the ER’s incredible topological complexity, ER remodeling proteins exert considerable influence in the regulation of flavivirus infections. We previously reported that the cellular ER remodeling protein Atlastin-2 (ATL2), normally responsible for fusing homotypic ER tubules, is critical for the replication of multiple flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV). However, a mechanistic explanation for the function of ATL2 during infection was unclear. In this work, we report on the conserved role that ATL2 plays in facilitating the spatial organization of flavivirus replication. Immunofluorescence and electron microscopy indicated that ATL2 knockdown resulted in significant vRO spatial dysregulation in both DENV- and ZIKV-infected cells, with an accompanying decrease in virus production and increase in the activation of cellular innate immune responses as vRO formation was disrupted. Correlative light and electron microscopy demonstrated that ATL2 concentrated in areas of vRO formation during flavivirus infection. ATL2 mutational analysis showed that tethering-competent but fusion-defective ATL2 was sufficient to rescue DENV and ZIKV replication in ATL2-knockout cells. Finally, a synthetic peptide that inhibited ATL2 activity significantly reduced DENV replication in cell culture and in human primary cells. Taken together, these results show that ATL2 plays a critical and conserved role in flavivirus infection, with its membrane tethering activity functioning to organize membranes for vRO biogenesis. Importantly, this function can be targeted to inhibit viral replication.

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A versatile fluorescent reporter for Usutu virus drug screening and infection tracking [viralRNAseq]

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