Abstract
BACKGROUND/OBJECTIVES: Understanding virulence determinants can inform safer and more efficacious live attenuated vaccine design. However, applying this knowledge across related viruses does not always result in conserved phenotypes from similar mutants. METHODS: Using Usutu virus (USUV), an emerging orthoflavivirus spreading through Europe, we assessed whether the attenuating effect of the mutations described for related orthoflaviviruses is conserved. Candidate attenuating mutations were selected based on previous studies in other orthoflaviviruses and incorporated into USUV. RESULTS: Nine variants, with mutations in the USUV envelope, non-structural (NS) proteins NS1, NS2A, or NS4B were stable and selected for further characterisation. The variants with an attenuating phenotype in cell culture were then compared to the wild-type virus in an Ifnar(-/-) mouse model. Mutations of the envelope glycosylation sites and glycosaminoglycan binding sites, which were recognised as more-conserved mechanisms of orthoflavivirus attenuation, were attenuating in USUV as well. However, not all the mutations explored in the USUV non-structural proteins exhibited an attenuated phenotype. Instead, the attenuation was either less pronounced, or there was no change in phenotype relative to the wild-type virus at all. CONCLUSIONS: In addition to improving our understanding of USUV virulence determinants, these results add to a growing body of literature highlighting the most promising mechanisms to target for the design of safe live attenuated vaccines against emerging orthoflaviviruses.