Watching Usutu Virus Infect in Real Time
Review written by: Megan Mauriello
Usutu virus (USUV) (Orthoflavivirus usutuense) is a +ssRNA, mosquito-transmitted orthoflavivirus closely related to West Nile Virus (WNV, Orthoflavivirus nilense), Zika Virus (ZIKV, Orthoflavivirus zikaense), and Japanese encephalitis virus (JEV, Orthoflavivirus japonicum). USUV’s primary host is Eurasian blackbirds and the primary vector is Culex pipiens, but other zoonotic “dead-end” hosts are known. Since its first isolation from Eswatini in 1959, it has been documented in various parts of Africa and throughout much of Europe. While most human infections are asymptomatic, it can cause neuroinvasive disease in immunocompromised humans as well as in birds and mice. The molecular and immunological factors governing USUV infection dynamics are poorly understood, and no antiviral therapies for USUV infection in any hosts are currently approved.
The authors developed USUV-mScarlet by inserting mScarlet-I into the genome of a USUV Strain TC508 strain (from Vienna in 2001) cDNA infectious clone. The infectious Strain TC508 clone (USUV-WT) was developed via a circular polymerase extension reaction of 9 overlapping fragments. m-Scarlet-I was inserted into a TOPO vector with fragment 2. A native viral proteolytic cleavage site was inserted upstream of m-Scarlet-I, and the envelope transmembrane domain from Dengue virus DENV-1 was inserted downstream for polyprotein folding and reporter stability.
USUV-mScarlet had similar replication kinetics to wild-type (WT) USUV in Huh7 human hepatoma cells for 1-4 days post inoculation in vitro, but mice infected in vivo all recovered, demonstrating that USUV-mScarlet causes a milder infection than the wild-type virus. USUV-mScarlet maintained fluorescence over at least 10 serial passages in Huh7 cells. Insertion of mScarlet into the genome was also stable over at least 10 passages, as verified by next-generation sequencing of cDNAs directly upstream of NS1. Remdesivir and IFN-⍺2a inhibited USUV-mScarlet in L929 mouse fibroblasts, while sofosbuvir, ribavirin, and JNJ-A07 did not. In human Huh7 cells, all but JNJ-A07 exhibited antiviral activity, with ribavirin and IFN-⍺2a exhibiting the strongest antiviral activity. Importantly, IFN-⍺2a of USUV-WT was similar, suggesting that USUV-mScarlet is a reliable model of antiviral activity. However, remdesivir was cytotoxic to Huh7 cells at high concentrations. USUV-mScarlet infection induced type I IFN responses in L929 mouse fibroblasts and caused transient disease, viral expression, and virion presence in multiple peripheral tissues in mice. In particular, viral load was high in myeloid splenic cells, suggesting these cells are an early reservoir for the virus.
Development of USUV-mScarlet as a tractable, stable, replication-competent , full-length reporter virus facilitates visualization of viral infection in vitro and in vivo. USUV-mScarlet is a useful tool for developing antivirals and vaccines against USUV, as demonstrated by experiments with various known antiviral compounds. Orthovirus spp. are generally intolerant of heterologous gene insertion, so the success of this reporter virus could be an important step in the development of similar reporters in other species such as WNV, ZIKV, or JEV which are otherwise unamenable to such approaches.
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READ MORE: Amy N Nelson, Aaron E Lin, Saloni Sinha, Sydney J Mullin, Jianche Liu, Alexandra G Bell, Sebastian S Carver, Jason Sethiadi, Jiayu Zhang, Brigitte L Heller, Cameron Myhrvold, Robert E Schwartz, Alexander Ploss, A versatile fluorescent reporter for Usutu virus drug screening and infection tracking, PNAS Nexus, Volume 5, Issue 10, October 2026, pgag328, https://doi.org/10.1093/pnasnexus/pgag328
