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

Abstract

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.

Experimental Assessment of Possible Factors Associated with Tick-Borne Encephalitis Vaccine Failure

“…The main direction of our research is emphasized by the fact that TBEV population is comprised of a wide variety of structurally different viral particles, which could affect both the development of the immune response during flavivirus infection [73] and the outcomes of the virus challenge studies. Moreover, the number of non-infectious viral particles in the studied TBEV strains exceeded the number of infectious viral particles 100 to 1000 times, as was shown before [47,74]. It is known for mosquito-borne flaviviruses that the number of non-infectious immature virus particles may depend on the reproduction system or individual characteristics of the strain [75].…”