Probing the dynamics of Usutu virus infection, replication, and spread using an in vitro cell culture model

Abstract

Viral outbreaks such as those caused by H1N1 influenza, Zika, Ebola viruses, and most recently, SARS-CoV-2, are unpredictable and potentially devastating to global heath. Yet many of these emerging/re-emerging pathogens lack research and public health prioritization. One such pathogen, Usutu virus (USUV) is a mosquito-borne, positive-sense single-stranded RNA virus in the Flaviviridae family. Despite its discovery over 60 years ago and reports of human neuroinvasive infection, the mechanisms of USUV replication, virulence, and pathogenicity are not well understood. As USUV exhibits a wide host range with varying degrees of pathogenesis, we examined its replication in relevant reservoir, host, and vector cell lines. Here, we show USUV replicated differentially across human, murine, avian, and mosquito cell lines, and induced the most severe cytopathic effects in human Huh7 hepatoma cells. Given the little genetic diversity among USUV strains, we hypothesize other isolates demonstrate similar replication kinetics across these cell lines. To facilitate interrogation of the USUV genome, we generated an infectious complementary DNA clone and recombinant USUV reporter virus. RNA transcribed from our infectious clone produced USUV proteins but exhibited low replication in cell culture. Characterization of its replicative ability in comparison to USUV isolates is still needed. Additionally, recombinant USUV expressing Gaussia luciferase did not yield fluorescent activity in cell culture. During replication, the flaviviral polyprotein is processed by a virally-encoded protease, which also targets host proteins. We established a cell culture expression system to investigate the role of the USUV putative protease in species-specific antagonism of host innate immune responses during infection, and expect USUV to possess a similar mechanism to that of its flavivirus relatives, cleaving proteins required for an 10 intracellular antiviral response. Overall, this study represents steps toward reducing knowledge gaps in USUV molecular virology crucial to future surveillance, diagnosis, and antiviral development.