Human 3D liver spheroids support productive infection of a novel tick-borne phenuivirus

The identification of novel tick-borne viruses, such as Mukawa virus (MKWV), underscores a growing need to assess their potential public health risks. In this study, we isolated the MKWV strain HLJ1 from Ixodes persulcatus ticks. While this initial isolate demonstrated limited replication in mammalian cell lines and mice, it productively infected human primary cell-derived 3D spheroids. Serial passaging in this model significantly enhanced viral titers, suggesting adaptive evolution. The resulting adapted strain exhibited increased virulence, causing pronounced cytopathic effects in Vero cells, infecting diverse mammalian cell types, and leading to 100% mortality in suckling mice, with associated liver inflammation and damage. These pathogenic outcomes were recapitulated in the 3D human liver spheroids, which showed impaired cellular synthetic functions, cell death, and heightened inflammatory responses following infection. Epidemiological screening of 145 serum samples from tick-bitten patients in Northeastern China revealed low but detectable exposure, with 1.4% positive for MKWV RNA, 4.8% for IgG antibodies, and 3.4% for neutralizing antibodies. Collectively, our findings integrate a novel human-relevant 3D culture system with field surveillance to highlight the potential risks of MKWV to human health and provide a model framework for evaluating emerging tick-borne viruses.