Naturally occurring mutations in envelope mediate virulence of Usutu virus

Usutu virus (USUV) is a mosquito-transmitted flavivirus that is closely related to West Nile virus. Recently, USUV emerged in Europe, where it has caused multiple bird die-off events and neuroinvasive disease in humans. Previously, we showed that USUV isolates from Africa cause significantly more severe disease than European isolates in mice. Sequence analysis revealed that the most virulent isolate (Uganda 2012) and the least virulent isolate (Netherlands 2016) differed by 21 amino acids across the viral polyprotein. Here, we sought to identify the viral determinants of and mechanisms for differential virulence. To accomplish this, we used our USUV reverse genetics system and bacteria-free cloning to generate chimeric viruses between Uganda 2012 and Netherlands 2016. Ifnar1^-/- mice infected with a Netherlands 2016 chimera containing all of the structural genes, or just the envelope gene, from Uganda 2012 had significantly higher mortality rates and viremia than those infected with wild-type Netherlands 2016. We were unable to identify a single amino acid in the envelope protein that resulted in significantly increased virulence compared to wild-type Netherlands 2016. These results indicate that multiple mutations in USUV envelope protein contribute to differential virulence between isolates. Through in vitro assays, we discovered that envelope mediates replication kinetics, with fusion occurring more slowly for wild-type Netherlands 2016 compared to viruses containing the envelope from Uganda 2012, suggesting a mechanism for envelope-mediated differential virulence of USUV. These studies provide insights into USUV pathogenic mechanisms, which could be used to evaluate the disease potential of related emerging viruses.IMPORTANCEUsutu virus (USUV) is currently emerging in Europe, where it has caused numerous mass bird die-off events and neuroinvasive disease in humans. Multiple strains of USUV are circulating throughout Europe, but only some of them have been associated with severe disease in humans. The USUV proteins responsible for and the mechanisms through which they cause severe disease are unknown; however, this information could be invaluable in evaluating disease potential of specific strains and the creation of anti-viral therapies. Here, we swapped genes between USUV strains that cause mild and severe disease and were able to identify a viral protein that mediates virulence. We also discovered that the mild strain of USUV takes significantly longer to complete fusion during viral entry into host cells than the severe strain. This delayed fusion could have impacts on cellular tropism, viral kinetics, susceptibility of the virus to immune responses, and, ultimately, disease severity.