Abstract P-28: Protocol of Tick-Borne Encephalitis Virus Sample Preparation for Structural Studies

Fedotov, Anton; Vorovitch, Mikhail F.; Tuchinskaya, K. K.; Grishin, Konstantin V; Konyushko, O. I.; Osolodkin, Dmitry I.; Ишмухаметов, А. А.; Егоров, А.М.

doi:10.21103/ijbm.9.suppl_1.p28

Cited by 3 publications

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“…The virus titer was determined via the plaque-forming method and expressed as the logarithm of the number of plaque-forming units per ml (PFU/mL). Methylcellulose-coated titration was performed as described previously [ 12 , 13 ], the number of plaques in a cell was counted, and the TBEV titer was calculated according to the Reed and Muench method.…”

Section: Methodsmentioning

confidence: 99%

Size Distribution of Inactivated Tick-Borne Encephalitis Virus Particles Revealed by a Comprehensive Physicochemical Approach

et al. 2022

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Tick-borne encephalitis virus (TBEV) is an enveloped RNA virus, a member of the genus Flavivirus (family Flaviviridae). Here, we provide a detailed analysis of the size and structure of the inactivated TBEV vaccine strain Sofjin-Chumakov. Four analytical methods were used to analyze individual TBEV particles—negative staining TEM, cryo-EM, atomic force microscopy (AFM), and nanoparticle tracking analysis (NTA). All methods confirmed that the particles were monodisperse and that their mean size was ~50 nm. Cryo-EM data allowed us to obtain a 3D electron density model of the virus with clearly distinguishable E protein molecules. STEM-EELS analysis detected phosphorus in the particles, which was interpreted as an indicator of RNA presence. Altogether, the described analytical procedures can be valuable for the characterization of inactivated vaccine virus samples.

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Section: Methodsmentioning

confidence: 99%

Size Distribution of Inactivated Tick-Borne Encephalitis Virus Particles Revealed by a Comprehensive Physicochemical Approach

et al. 2022

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“…The virus titer was determined by the plaque-forming method and expressed as the logarithm of the number of plaque-forming units in ml (PFU/ml). Methylcellulose-coated titration was performed as described previously [12,13], the number of plaques in a cell was counted, and the TBEV titer was calculated according to the Reed and Muench method.…”

Section: Obtaining Inactivated Virions Of the Tbevmentioning

confidence: 99%

Size Distribution of Inactivated Tick-borne Encephalitis Virus Particles Revealed by a Comprehensive Physical-chemical Approach

Moiseenko¹,

Bagrov²,

Vorovich³

et al. 2022

Preprint

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Tick-borne encephalitis virus (TBEV) is a RNA-containing enveloped virus, a member of the Flaviviridae family. Here we describe a detailed analysis of the size and structure of inactivated TBEV (the Sofyin-Chumakov TBEV strain, used in vaccines). Four analytical methods were used to analyze individual TBEV particles—negative staining TEM, cryo-EM, AFM, and NTA. All methods confirmed that the particles were monodisperse, and their mean size was ~50 nm. Cryo-EM data were used to obtain a 3D electron density model of the virus with clearly distinguishable E-proteins. STEM-EELS analysis detected phosphorous in the particles, which was interpreted as the RNA signal. Altogether, the described analytical procedures can be valuable for the further analysis of vaccine inactivated virus samples.

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“…Here we present an approach based on the use of analytical electron microscopy (EM) to quantify the heterogeneity of TBEV particles in purified samples for further single-particle imaging (SPI) on X-ray free electron lasers (XFEL), which provide a unique opportunity to study the time-resolved solution structures of large macromolecular assemblies, such as viruses. The requirements for a virus sample for SPI study at XFELs include high concentration of particles, knowledge of particle size distribution, and absence of aggregates [2]. We established a methodological workflow for such characterization of inactivated TBEV samples.…”

Section: Introductionmentioning

confidence: 99%

Estimation of the structural heterogeneity of Tick-Borne Encephalitis vaccine particles

Moiseenko¹,

Zaitsev²,

Vorovitch³

et al. 2021

Microsc Microanal

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Abstract P-28: Protocol of Tick-Borne Encephalitis Virus Sample Preparation for Structural Studies

Abstract: Conclusion: The purification protocol based on the combination of tangential flow filtration and two steps of ultracentrifugation, one of them through the sucrose gradient, was the most efficient for obtaining high concentrated, non-aggregated virus suspension suitable for XFEL studies.

Cited by 3 publications

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Size Distribution of Inactivated Tick-Borne Encephalitis Virus Particles Revealed by a Comprehensive Physicochemical Approach

Size Distribution of Inactivated Tick-Borne Encephalitis Virus Particles Revealed by a Comprehensive Physicochemical Approach

Size Distribution of Inactivated Tick-borne Encephalitis Virus Particles Revealed by a Comprehensive Physical-chemical Approach

Estimation of the structural heterogeneity of Tick-Borne Encephalitis vaccine particles

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