A b s t r a c tThe African swine fever virus (ASFV) is a unique representative of Asfaviridae family, which still remains the sole member of genus Asfarvirus. ASF virus is the causative agent of one of the most dangerous diseases of the animals from Suidae family, and moreover, it is capable of infecting soft ticks of the genus Ornithodoros. Genetic and phenotypic heterogeneity of ASF virus is one of the main reasons for the lack of vaccines against this dangerous transboundary disease. In this work we present the analysis of structure and functions of the most variable glycoproteins ASFV p54 and CD2v using bioinformatics analysis and recombinant constructs expressed in mammalian cell cultures, the African green monkey cell culture COS-I and the human embryonal kidney cell culture HEK-293. The index of variability of amino acid sequences for P54 and CD2v proteins was calculated by Simpson's method. The CD2v protein has variable region (N-terminal domain), which is highly glycosylated (28-30 sites) and located in the outer surface of the cell membrane. This region also contains immunoglobulin domain (amino acids at positions 1-225), which is responsible for CD2v interaction with antibodies. The revealed differences in post-translational modifications and genetic variations of CD2v protein might explain the diversity of the hemadsorption phenomenon among ASF virus isolates. In contrast, p54 protein has variable glycosylated extracellular and intracellular parts. High level of differences in the nucleotide sequences of p54gene (E183L) for various ASFV isolates may be the result of random mutations during virus evolution. Characteristic antigenic properties of ASF virus isolates can obviously be due to found peculiar posttranslational processing and genetic variations on СD2v protein. Herein we report the first bioinformatic analysis of post-traslation N-and O-glycosylation in most variable ASF virus proteins, p54 and СD2v. A transient expression of gene constructions used to obtain the recombinant products, p54-EGFP and CD2v-HA, allowed us to demonstrate the evidence for different localization of viral proteins p54-EGFP and CD2v-HA in the transfected cells. Particularly, the fluorescence caused by p54-EGFP was observed in the cytoplasm of the COS-I cells, transfected with recombinant plasmid р54-pEGFP-N1, whereas recombinant CD2v-НА protein was detected only in cell membrane. According to immunoblotting analysis, the CD2v molecular weight was 90 kDa against calculated 65 kDa indicating about 30 % of carbohydrate component in this surface glycoprotein. Moreover, 25 kDa and 90 kDa CD2v molecules, the probable differently glycosylated forms, were revealed in immonoblotting test that is in line with other published data. Thus, bioinformatic analysis and in vitro studies using transient expression in COS-I и HEK-293 cell cultures have shown that protein CD2v is the most likely candidate to define the interaction of ASF virus with the virus-specific antibodies.

Молекулярная эпидемиология вирусных инфекций традиционно основывается на анализе изменений отдельных генов или генетических маркеров. Анализ генов вируса африканской чумы свиней (АЧС), кодирующих иммуномодулирующие белки является важным инструментом изучения разнообразия и эволюции вируса. В данной работе мы провели структурный и филогенетический анализ иммуномодулирующих белков 5el (ген A238L), i14l (ген DP71L), k11l (ген I329L) вируса АЧС. Степень нуклеотидных замен конкатенированных генов A238L, I329L и DP71L вируса АЧС выявила очищающий (стабилизирующий) отбор на уровне нуклеотидных последовательностей. Характеристика вариабельности отобранной нами группы генов вируса АЧС представляет большой интерес для поиска генетических различий в иммуномодулирующих белках. Результаты секвенирования генов A238L, I329L и DP71L и их филогенетический анализ показали, что эти гены являются консервативными среди большой группы генов вируса АЧС. Ген I329L является генетическим маркером общности происхождения. Ген DP71L у восточноафриканских штаммов X генотипа имеет две формы: длинную (184 аминокислоты) и краткую (от 70 до 72 аминокислот) и образуется путем слияния 13L и 14L. Все российские изоляты вируса АЧС, выделенные в 2016-2017 гг., по изученным генам идентичны референтному штамму ASFV/Georgia/wb/2007. Характеристика вариабельности белков 5el, i14l, k11l может послужить выявлению таргетных участков в геноме вируса АЧС и для разработки вакцин. Полученные данные позволяют оценить генетическое разнообразие иммуномодулирующих белков вируса АЧС и динамику их эволюции, предсказать возможное участие генов A238L, I329L и DP71L в вирулентности различных штаммов вируса АЧС. Ключевые слова: вирус африканской чумы свиней, секвенирование, филогенетический анализ, иммуномодулирующие белки, анализ синонимичных и несинонимичных замен.

Molecular epidemiology of viral infections traditionally based on the analysis of changes in individual genes or genetic markers. The analysis of the African swine fever virus (ASFV) genes encoding immunomodulatory proteins is an important tool for studying the diversity and evolution of the virus. In this work, we carried out a structural and phylogenetic analysis of the ASF virus immunomodulatory proteins 5EL (A238l gene), I14L (Dp71l gene), K11L (I329l gene). The degree of nucleotide substitutions of the ASFV concatenated genes A238l, I329l and Dp71l revealed purifying (stabilizing) selection at the nucleotide sequences level. The variability characteristic of the selected group of ASFV genes is of great interest for the genetic differences search in immunomodulatory proteins. The sequencing results of the A238l, I329l and Dp71l genes and their phylogenetic analysis showed that these genes are 2 conservative among a large group of ASFV genes. The I329l gene is a genetic marker of common origin. The East African strains (Genotype X) of Dp71l gene have two forms: a long (184 amino acids) and a short (from 70 to 72 amino acids) and is formed by fusion of the 13L and 14L. All ASF virus Russian isolates isolated in 2016-2017 were identical to the reference strain ASFV/Georgia/wb/2007. Characterization of variability 5EL protein, I14L, K11L may be serve to identify target sites in the ASFV genome and to develop vaccines. The obtained data allow to evaluate the genetic diversity of the ASFV immunomodulatory proteins and the dynamics of their evolution, to predict the possible participation of the A238l, I329l and Dp71l genes in the virulence of various ASFV strains.

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Introduction. African swine fever virus (ASF) is a large DNA virus that is the only member of the Asfarviridae family. The spread of the ASF virus in the territory of the Russian Federation, Eastern Europe and China indicates the ineffectiveness of existing methods of combating the disease and reinforces the urgent need to create effective vaccines. One of the most significant antigens required for the formation of immune protection against ASF is a serotype-specific CD2v protein.The purpose of the study. This study presents the results of immuno-informatics on the identification of B- and T-cell epitopes for the CD2v protein of the ASF virus using in silico prediction methods.Material and methods. The primary sequence of the CD2v protein of the ASFV virus strain Georgia 2007/1 (ID-FR682468) was analyzed in silico by programs BCPred, NetCTLpan, VaxiJen, PVS and Epitope Conservancy Analysis.Results. Using the BCPred and VaxiJen programs, 4 major B-cell immunogenic epitopes were identified. Analysis of the secretory region of ASF virus CD2v protein in NetCTLpan revealed 5 T-cell epitopes from the 32nd to the 197th position of amino acids that cross-link from the 1st to the 13th allele of the MHC-I of pigDiscussion. This study presents the results in silico prediction to identify B- and T-cell epitopes of ASF virus CD2v protein. The soluble region of the CD2v protein can be included in the recombinant polyepitope vaccine against African swine fever.Conclusion. B- and T-cell epitopes in the secretory region of the CD2v protein (from 17 to 204 aa) of ASF virus were identified by in silico prediction. An analysis of the conservatism of the identified B- and T-cell epitopes allowed us to develop a map of the distribution of immune epitopes in the CD2v protein sequence.