Preparation and characterization of inactivated tick-borne encephalitis virus samples for single-particle imaging at the European XFEL

Abstract

X-ray imaging of virus particles at the European XFEL could eventually allow their complete structures to be solved, potentially approaching the resolution of other structural virology methods. To achieve this ambitious goal with today's technologies, about 1 ml of purified virus suspension containing at least 1012 particles per millilitre is 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, the development of a TBEV vaccine purification and concentration scheme is presented combined with a quality-control protocol that allows substantial amounts of highly concentrated non-aggregated suspension to be obtained. Preliminary single-particle imaging experiments were performed for this sample at the European XFEL, showing distinct diffraction patterns.

The structure of inactivated mature tick-borne encephalitis virus at 3.0 Å resolution

“…Formaldehyde-inactivated TBEV strain Sofjin-Chumakov is produced in the industrial scale for the vaccine manufacturing process and represents its antigen. We prepared a purified and concentrated iTBEV suspension (Supplementary Figure S1) according to the previously optimized protocol [ 26 ] and used it for the structure determination.…”

“…iTBEV sample (strain Sofjin-Chumakov [ 49 ]) was obtained according to the method developed earlier [ 26 ]. Briefly, virus-containing cultural liquid was inactivated with 0.2% formaldehyde, iteratively centrifuged, resuspended, and centrifuged on the sucrose density gradient.…”

“…The residue was resuspended in the TNE/5 buffer, aliquoted, and kept at −70°C. TBEV concentration was assessed as described [ 26 ].…”

“…Data processing workflow was described in details earlier [ 26 ]. Briefly, we have collected a cryo-EM dataset of inactivated virus sample using an in-house cryo-TEM Titan Krios (Thermo-Fisher Scientific) equipped with the Falcon II detector at 300 kV, 0.863 Å pixel size and a total dose of 64 e - /Å/s.…”

“…TBEV was obtained according to the same protocol as iTBEV, excluding formaldehyde inactivation step. Antigen concentration in the TBEV and iTBEV samples was performed as described earlier [ 26 ]. Aliquots of TBEV and iTBEV (10 μL) were dissolved in 90 μL of TN buffer (0.1 M Tris–HCl, 0.13 M NaCl, рН 7.8) or in 90 μL of 0.23 M phosphate saline buffer (pH 6.0) and incubated for 30 min at 4°C.…”