UNLABELLED: Tick-borne encephalitis virus (TBEV) is a neurotropic flavivirus that causes thousands of human infections annually. Viral tropism in the brain is determined by the presence of necessary receptors, entry factors, and the ability of the virus to overcome host defenses. The viral structural proteins, pre-membrane (prM), and envelope (E) play an important role in receptor binding, membrane fusion, particle maturation, and antibody neutralization. To understand how these proteins influence virus distribution and tropism in the brain, we generated a chimeric virus harboring the prM and ectodomain of E from TBEV in the background of the low-pathogenic Langat virus (LGTV). We solved the atomic structures of both the chimeric virus and LGTV to compare them to the known TBEV structure. We show that this chimeric virus remains low-pathogenic, while being structurally and antigenically similar to TBEV. Using 3D optical whole brain imaging combined with immunohistochemistry, we found that both LGTV and the chimeric virus primarily infect the cerebral cortex, with no significant differences in their localization or tropism. In contrast, TBEV shows high infection of the cerebellum and a strong preference toward Purkinje cells, indicating that factors other than the prM and E proteins are important for determining TBEV tropism in the brain. Together, this provides new insights into the roles of the structural and non-structural proteins of tick-borne flaviviruses. IMPORTANCE: Although an effective vaccine exists, there is no treatment for those infected by the tick-borne encephalitis virus (TBEV). This study aimed to better understand how the virus's surface proteins influence viral tropism and pathogenicity. We created a chimeric virus with prM and E proteins of TBEV in the genetic background of the low-pathogenic Langat virus (LGTV). The chimeric virus remained low pathogenic, similar to LGTV. Both viruses infected similar brain regions, while TBEV showed a strong preference for the cerebellum and Purkinje cells. This means that other parts of the virus, such as non-structural proteins or NCR, likely decide how the virus behaves in the brain. This study also presents the first cryogenic electron microscopy structure of LGTV, the first whole-brain imaging of TBEV infection in mouse brain, and a new model system to study surface proteins in tick-borne flaviviruses-laying groundwork for future studies on viral tropism, antibody cross-reactivity, and virus-receptor interaction.

Department of Clinical Microbiology Sahlgrenska University Hospital Gothenburg Sweden

Department of Clinical Microbiology Umeå University Umeå Sweden

Department of Infectious Diseases Institute of Biomedicine at the Sahlgrenska Academy University of Gothenburg Gothenburg Sweden

Department of Medical and Translational Biology Umeå University Umeå Sweden

Department of Medical Biosciences Umeå University Umeå Sweden

Faculty of Biological and Environmental Sciences Molecular and Integrative Bioscience Research Programme University of Helsinki Helsinki Finland

Faculty of Science Masaryk University Brno Czech Republic

Faculty of Veterinary Medicine University of Veterinary Sciences Brno Brno Czech Republic

Helsinki Institute of Life Sciences Institute of Biotechnology University of Helsinki Helsinki Finland

Institute of Bioengineering University of Tartu Tartu Estonia

Laboratory of Arbovirology Institute of Parasitology Biology Centre of the Czech Academy of Sciences Ceske Budejovice Czech Republic

Laboratory of Emerging Viral Infections Veterinary Research Institute Brno Czech Republic

Okinawa Institute of Science and Technology Graduate University Okinawa Japan

Proteomics Core Facility Sahlgrenska Academy University of Gothenburg Gothenburg Sweden

The Laboratory for Molecular Infection Medicine Sweden Umeå Sweden

Umeå Center for Microbial Research Umeå Sweden

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