Preparation and Characterization of Inactivated Tick-Borne Encephalitis Virus Samples for Single Particle Imaging at European XFEL

Abstract

X-ray imaging of virus particles at European XFEL could eventually allow solving their complete structure, potentially approaching resolution of other structural virology methods. To achieve this ambitious goal with today's technologies, several mL of purified virus suspension containing at least 10 to the 12 particles per mL are required. Such large amounts of concentrated suspension have never before been obtained for enveloped viruses. Tick-borne encephalitis virus (TBEV) represents an attractive model system for the development of enveloped virus purification and concentration protocols, given the availability of large amounts of inactivated virus material provided by vaccine manufacturing facilities. Here we present the development of a TBEV vaccine purification and concentration scheme combined with a quality control protocol allowing substantial amounts of highly concentrated non-aggregated suspension to be obtained. Preliminary single particle imaging experiments were performed for this sample at European XFEL, showing distinct diffraction patterns.

Structural diversity of tick-borne encephalitis virus particles in the inactivated vaccine based on strain Sofjin

“…The cryo-EM study was performed for the formaldehyde inactivated TBEV virions of the Sofjin-Chumakov strain, on which a commercial tick-borne encephalitis vaccine is based. The purified sample [ 18 , 19 ] consisted of mostly spherical virions with an approximate diameter of 50 nm. Sample also contained a minute fraction of distorted particles, which were visually different from the normal particles.…”

“…TBEV strain Sofjin-Chumakov (Genbank KC806252) is a prototype strain of the Far-Eastern subtype and used to produce commercial inactivated TBE vaccines (FSASI “Chumakov FSC R&D IBP RAS” (Institute of Poliomyelitis), Moscow, Russia) [ 17 ]. Reproduction, inactivation, and purification of TBEV was performed as described in [ 18 , 19 ]. Virus was inactivated by 0.02% formaldehyde and stored in TNE/5 buffer solution (20 mM Tris-HCl, 25 mM NaCl, 1 mM EDTA, pH 7.8).…”