Development of a method for assessing the structural heterogeneity of a population of different strains of tick-borne encephalitis virus

Вирус клещевого энцефалита (ВКЭ) является возбудителем тяжелого неврологического заболевания человека и широко распространен на территории Евразии. При репродукции флавивирусов помимо инфекционных вирионов накапливается набор неинфекционных вирусных структур: незрелые формы вирионов, пустые формы (не содержащие геном вирусные частицы), а также агрегаты поверхностного белка Е, способные оказывать влияние на иммунный ответ, и патогенез. Штаммы ВКЭ могут различаться по соотношению этих форм в инфекционном материале, т.е. по характеру структурной гетерогенности. Цель: подобрать комплекс методов, способных выявить данные различия. Методы. Общую концентрацию белка Е определяли методом ИФА, число частиц, содержащих геном (ГСЧ) - ПЦР в реальном времени, а для выявления инфекционных вирусных частиц - титрование в культуре клеток. Результаты. Разработан метод оценки структурной гетерогенности популяции ВКЭ. Было показано, что повышенное содержание неинфекционных вирусных частиц, содержащих геном, и несвязанных с ними белка Е не зависит от подтипа вируса. Выводы. Штаммы ВКЭ отличились по соотношению общего числа ГСЧ к числу инфекционных вирионов и по содержанию белка Е, связанного и несвязанного с ГСЧ. Tick-borne encephalitis virus (TBEV) is widespread in Europe and Asia and causes severe neurological disease in humans. It has been established that the reproduction of flaviviruses leads to the accumulation of a whole set of non-infectious viral structures aside from infectious virions. These structures include immature virions, empty forms (containing no genome) and aggregates of the surface protein E. These structures, despite being non-infectious, are able to influence the immune response and, consequently, the pathogenesis of TBEV infection. The aim of this work was to select a set of methods which can be implemented to identify these differences. Methods. Virus samples were analised for protein E concentration, number of genome-containing particles and infectivity. The total concentration of protein E in samples was evaluated using ELISA. The number of genome-containing particles was determined by a real-time PCR, and to assess the number of infectious virus particles titration in PEK cell culture was used. Results. An assay for total concentration of protein E in culture fluid of cells infected with different strains of TBEV based on the commertially available ELISA kit was developed. TBEV strains used in the study varied by the ratio of genome-containing particles to infectious virions. The amount of protein E not associated with genome-containing virions was calculated as a difference between total content of protein E and the amount of protein E bound to genome-containing particles. This amount was also different for studied samples of TBEV strains. Conclusion. No correlation was observed between the increased content of non-infectious genome-containing particles or the amount of residual protein E and TBEV subtypes.

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Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus

Journal of Virology ◽

10.1128/jvi.79.18.11813-11823.2005 ◽

2005 ◽

Vol 79 (18) ◽

pp. 11813-11823 ◽

Cited By ~ 22

Author(s):

Sigrid Elshuber ◽

Christian W. Mandl

Keyword(s):

Structural Integrity ◽

Incubation Temperature ◽

Surface Protein ◽

Normal Growth ◽

Growth Conditions ◽

Encephalitis Virus ◽

Furin Cleavage ◽

Tick Borne Encephalitis ◽

Substitution Mutations ◽

Tick Borne Encephalitis Virus

ABSTRACT Cleavage of the viral surface protein prM by the proprotein convertase furin is a key step in the maturation process of flavivirus particles. A mutant of tick-borne encephalitis virus (TBEV) carrying a deletion mutation within the furin recognition motif of protein prM (changing R-T-R-R to R-T-R) was previously shown to be noninfectious in BHK-21 cells. We now demonstrate how natural selection can overcome this lethal defect in two different growth systems by distinct resuscitating mutations. In BHK-21 cells, a spontaneous codon duplication created a minimal furin cleavage motif (R-R-T-R). This mutation restored infectivity by enabling intracellular prM cleavage. A completely different mutation pattern was observed when the mutant virus was passaged in mouse brains. The “pr” part of protein prM, which is removed by cleavage, contains six conserved Cys residues. The mutations selected in mice changed the number of Cys residues to five or seven by substitution mutations near the original cleavage site, probably causing a major perturbation of the structural integrity of protein prM. Although viable in mice, such Cys mutants could not be passaged in BHK-21 cells under normal growth conditions (37°C), but one of the mutants exhibited a low level of infectivity at a reduced incubation temperature (28°C). No evidence for the cleavage of protein prM in BHK-21 cells was obtained. This suggests that under certain growth conditions, the structural perturbation of protein prM can restore the infectivity of TBEV by circumventing the need for intracellular furin-mediated cleavage. This is the first example of a flavivirus using such a molecular mechanism.

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Spectrum of antiviral activity of 4-aminopyrimidine N-oxides against a broad panel of tick-borne encephalitis virus strains

Antiviral Chemistry and Chemotherapy ◽

10.1177/2040206620943462 ◽

2020 ◽

Vol 28 ◽

pp. 204020662094346

Author(s):

Evgenia V Dueva ◽

Ksenia K Tuchynskaya ◽

Liubov I Kozlovskaya ◽

Dmitry I Osolodkin ◽

Kseniya N Sedenkova ◽

...

Keyword(s):

Encephalitis Virus ◽

Northern Eurasia ◽

Virus Particles ◽

E Protein ◽

Tick Borne Encephalitis ◽

Tbev Strain ◽

Amino Phenol ◽

Tick Borne Encephalitis Virus ◽

Immature Virus

Tick-borne encephalitis is an important human arbovirus neuroinfection spread across the Northern Eurasia. Inhibitors of tick-borne encephalitis virus (TBEV) strain Absettarov, presumably targeting E protein n-octyl-β-d-glucoside (β-OG) pocket, were reported earlier. In this work, these inhibitors were tested in vitro against seven strains representing three main TBEV subtypes. The most potent compound, 2-[(2-methyl-1-oxido-5,6,7,8-tetrahydroquinazolin-4-yl)amino]-phenol, showed EC50 values lower than 22 µM against all the tested strains. Nevertheless, EC50 values for virus samples of certain strains demonstrated a substantial variation, which appeared to be consistent with the presence of E protein not only in infectious virions, but also in non-infectious and immature virus particles, protein aggregates, and membrane complexes.

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Comparative study of infectious and formaldehyde-inactivated tick-borne encephalitis virus particles

Archives of Virology ◽

10.1007/bf01310814 ◽

1984 ◽

Vol 79 (3-4) ◽

pp. 241-253 ◽

Cited By ~ 1

Author(s):

I. V. Krasilnikov ◽

L. B. Elbert ◽

M. K. Khanina ◽

S. G. Sobolev

Keyword(s):

Comparative Study ◽

Encephalitis Virus ◽

Virus Particles ◽

Tick Borne Encephalitis ◽

Tick Borne Encephalitis Virus

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Obtaining aptamers to a fragment of surface protein E of tick-borne encephalitis virus

Doklady Biochemistry and Biophysics ◽

10.1134/s1607672913010067 ◽

2013 ◽

Vol 448 (1) ◽

pp. 19-21 ◽

Cited By ~ 2

Author(s):

I. G. Kondratov ◽

M. A. Khasnatinov ◽

U. V. Potapova ◽

V. V. Potapov ◽

S. A. Levitskii ◽

...

Keyword(s):

Surface Protein ◽

Encephalitis Virus ◽

Tick Borne Encephalitis ◽

Tick Borne Encephalitis Virus

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Two Distinct Size Classes of Immature and Mature Subviral Particles from Tick-Borne Encephalitis Virus

Journal of Virology ◽

10.1128/jvi.77.21.11357-11366.2003 ◽

2003 ◽

Vol 77 (21) ◽

pp. 11357-11366 ◽

Cited By ~ 43

Author(s):

Steven L. Allison ◽

Yizhi J. Tao ◽

Gabriel O'Riordain ◽

Christian W. Mandl ◽

Stephen C. Harrison ◽

...

Keyword(s):

Mammalian Cells ◽

Size Class ◽

Encephalitis Virus ◽

E Proteins ◽

Virus Particles ◽

Mature Form ◽

Size Classes ◽

Tick Borne Encephalitis ◽

Tick Borne Encephalitis Virus ◽

Subviral Particles

ABSTRACT Flaviviruses assemble in the endoplasmic reticulum by a mechanism that appears to be driven by lateral interactions between heterodimers of the envelope glycoproteins E and prM. Immature intracellular virus particles are then transported through the secretory pathway and converted to their mature form by cleavage of the prM protein by the cellular protease furin. Earlier studies showed that when the prM and E proteins of tick-borne encephalitis virus are expressed together in mammalian cells, they assemble into membrane-containing, icosahedrally symmetrical recombinant subviral particles (RSPs), which are smaller than whole virions but retain functional properties and undergo cleavage maturation, yielding a mature form in which the E proteins are arranged in a regular T = 1 icosahedral lattice. In this study, we generated immature subviral particles by mutation of the furin recognition site in prM. The mutation resulted in the secretion of two distinct size classes of particles that could be separated by sucrose gradient centrifugation. Electron microscopy showed that the smaller particles were approximately the same size as the previously described mature RSPs, whereas the larger particles were approximately the same size as the virus. Particles of the larger size class were also detected with a wild-type construct that allowed prM cleavage, although in this case the smaller size class was far more prevalent. Subtle differences in endoglycosidase sensitivity patterns suggested that, in contrast to the small particles, the E glycoproteins in the large subviral particles and whole virions might be in nonequivalent structural environments during intracellular transport, with a portion of them inaccessible to cellular glycan processing enzymes. These proteins thus appear to have the intrinsic ability to form alternative assembly products that could provide important clues about the role of lateral envelope protein interactions in flavivirus assembly.

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TBE in South Korea

Tick-borne encephalitis - The Book ◽

10.33442/978-981-14-0914-1_12b-31 ◽

2019 ◽

Author(s):

Joon Young Song

Keyword(s):

South Korea ◽

Human Case ◽

Encephalitis Virus ◽

Surveillance Studies ◽

Tick Borne Encephalitis ◽

Tick Borne Encephalitis Virus

Although no human case of tick-borne encephalitis (TBE) has been documented in South Korea to date, surveillance studies have been conducted to evaluate the prevalence of tick-borne encephalitis virus (TBEV) in wild ticks.

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