UDC 636.4:619:616.98:578:577.2.08:51-76

doi: 10.15389/agrobiology.2015.6.785eng

Supported by Russian Foundation for Basic Research, science project «Mol-a-ved» (15-34-20995).


K.A. Mima, G.S. Burmakina, I.A. Titov, A.S. Malogolovkin

All-Russian Institute of Veterinary Virology and Microbiology, Federal Agency of Scientific Organizations,
Pokrov, Petushinskii Region, Vladimir Province, 601120 Russia,
e-mail mima89@ya.ru

Received June 30, 2015

The 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 post-translational 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. 

Keywords: African swine fever, glycoprotein, variability, glycosylation, transient expression, immunotypes.


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