Tick-borne flaviviruses (TBF), including tick-borne encephalitis virus (TBEV) and Powassan virus (POWV), remain a global concern with high case fatality rates, long-term disabling sequela, and continued emergence. Structural analysis of TBFs by cryogenic electron microscopy (cryo-EM) single particle analysis, a critical method to understanding virion structure and antibody interactions, requires large quantities of infectious virus and appropriate biocontainment levels – difficult for BSL3-4 TBFs. We sought to investigate a new system to safely produce authentic TBFs virions through an insect-specific flavivirus (ISF) chimeric platform.
Chimeric flaviviruses were produced by exchanging the pre-membrane and envelope proteins of Binjari virus (an ISF) with the corresponding genes of the pathogenic flavivirus. They are unable to replicate in vertebrate cells, including human neuronal cell line. Over ten TBF chimeras, including mammalian (TBEV, POWV, Omsk haemorrhagic fever, Alkhurma, Kyasanur Forest disease, Langat (LGTV), Gadgets Gully virus (GGYV)) and seabird (Saumarez Reef virus; SREV) were constructed.
Chimeric TBF virions were gradient purified from infected mosquito cells (C6/36). SDS-PAGE and transmission-EM of these virions revealed expected protein sizes and virion morphology. bPOWV-LB, bLGTV and bSREV were imaged by cryo-EM and using single particle analysis and asymmetric reconstruction, the bPOWV-LB virion was resolved to 2.9 Å; bLGTV to 2.8 Å and bSREV to 3.0 Å. This is the first report of high-resolution structures of these TBFs. Multiple TBEV- and POWV-neutralising monoclonal antibodies were recombinantly produced and used to validate these antigens in ELISA and neutralisation assays. Two antibodies recognised all major mammalian TBFs tested, revealing a potential vaccine and therapeutic target. These antibodies also recognised bGGYV and bSREV, extending our antigenic knowledge on understudied TBFs from Australasia.
We demonstrate a safe and efficient platform to study structure and antibody interactions of TBFs in BSL2 conditions. This has resulted in multiple TBF virion structures resolved to near-atomic resolution.