A new approach to identification of African swine fever virus proteins

African swine fever virus (ASFV) is the sole member of Asfaviridae family and it is responsible for African swine fever, which is highly contagious and fatal disease of pigs. Since the 1960s, inactivated vaccines, attenuated live vaccines, and subunit vaccines had been developed, but still there is no effective vaccine against it. In this study we used different methods of protein precipitation coupled with an RP-HPLC method to analyze the ASFV proteins. The aim of this work was to develop a sensitive reversed-phase HPLC method, applicable for the identification of specific proteins in the African swine fever vaccine. Two different stationary phases were used for protein separation and identification, i.e., C4 and C18. These columns present different hydrophobic properties, with an impact on protein separation. In the aim of protein separation by HPLC, some methods of protein precipitation were used, as follows: saline and acid precipitation, trypsin action, and disulfide bonds reduction by dithiothreitol. The saline and acid protein precipitation were used at a medium value of saturation percent of ammonium sulfate and trichloroacetic acid, respectively, in a reaction medium (30–60% ammonium sulfate saturation and 40% trichloroacetic acid saturation, respectively). For trypsin treatment of proteins, a 1% aqueous solution of trypsin was used, so that viral proteins would undenatured. In the case of dithiothreitol, a 100 mM aqueous solution was used, so that secondary structure of proteins is maintained. Some major polypeptides with molecular weights of 10,000 – 70,000 Da were found by using the protein precipitation methods coupled with the proposed HPLC method. The cell culture medium used for viral particle growing was treated in the same manner as viral suspension. The protein precipitation coupled to the HPLC method has shown that only viral suspension contains polypeptides with molecular weights of 10,000 – 70,000 Da. Protein precipitation coupled to the HPLC proposed method applied to cell culture medium has not shown specific viral polypeptides and proteins, and the chromatographic profile is different from that of viral suspension.