This article summarizes the study results on the generation of attenuated strains of African swine fever virus (ASFV) of seroimmunotypes I–VIII and the creation of live vaccines for temporary protection of pigs during a period of epizootics in the surveillance zone (a zone adjacent to the area of outbreak). These studies were initiated at the Federal Research Center for Virology and Microbiology (FRCVM, formerly VNIIVViM) at the time of introduction of the pathogen to the Iberian Peninsula in the middle of the 20th century. The developed experimental vaccines against ASFV seroimmunotypes I–V provided protection against virulent strains of homologous seroimmunotypes by day 14 after vaccination, lasting at least four months.
This article is devoted to the development and evaluation of the immunoblotting test system for serological diagnosis of African swine fever (ASF), based on the highly purified recombinant p30 of ASF virus (ASFV) strain Stavropol 01/08 (Stavropol 2008), representative of the ASFV currently circulating in the Russian Federation. The main project stages are as follows: (i) cloning of the central hydrophilic region of the ASFV gene CP204L (p30) into a prokaryotic vector; (ii) expression and chromatographic purification of the recombinant product p30 with thioredoxin and poly-histidine site (p30e1_TrxA_6xHis); (iii) development of the immunoblotting test system (Rec p30-IB) using the highly purified recombinant p30; and (iv) evaluation of Rec p30-IB using sera and organ samples from domestic pigs and wild boars experimentally or naturally infected by ASFV. Testing of the Rec p30-IB showed the diagnostic specificity and sensitivity of the assay to be 98.75% and 100.00%, respectively. High sensitivity of the Rec p30-IB allowed the detection of ASFV-specific antibodies in samples of organs of the immune system and blood sera, collected from domestic pigs and wild boars, starting from 6 to 8 days post-infection, regardless of virus virulence, seroimmunotype and geographic origin of the samples (East Europe, South Europe, West Europe, Central and south-east Africa).
A b s t r a c tThe capability of causing haemadsorption at African swine fever (ASF) virus (ASFV) reproduction in swine bone marrow cell cultures, leukocytes or continuous cells in the presence of swine erythrocytes is characteristic of the majority of the virus isolates (W.A. Malmquist, D. Hay, 1960). This trait is used for ASF diagnosis based on autohaemadsorption in porcine blood, the virus titration in cell culture, and selection of its attenuated variants in vitro (A.D. Sereda et al., 2014). The haemadsorption inhibition assay (HIA) in tandem with the bioassay using the disease-resistant pigs is applied for seroimmunotype-based classification of ASFV isolates (N.I. Mitin et al., 1985). The heterogeneity of an ASFV population for quantitative haemadsorption characteristic (like «dense», «moderate» or «loose») is a phenotypic trait of ASFV isolates, strains and/or variants (V. Makarov et al., 2016). Also, the proportion of the circumference of red blood cells as observed at their contact with infected macrophages serves as another quantifiable feature of haemadsorption. Some quantitative differences in HIA activity levels of swine blood sera are determined in the assays carried out using virulent reference variants and their attenuated derivatives, and the obtained results require some interpretation. The loss of ability to induce haemadsorption is not critical for ASFV reproduction and often accompanied by a decrease in the pathogen virulence levels. Hence, as a rule, attenuated ASFV variants are prepared through a selection by limiting dilution from populations of virulent isolates of the virus clones that are characterized by a reduced potential to induce haemadsorption (D.V. Kolbasov et al., 2014). In the course of the virus reproduction, haemadsorption precedes the exocytosis. Virions do not play a significant role in the mechanism of haemadsorption, nevertheless, their interaction with erythrocyte membranes promotes the virus dissemination throughout the swine organism and more effective introduction into the gut cells of ticks (L.K. Dixon et al., 2004). ASFV haemadsorbing potentiality is determined by highly glycosylated transmembrane protein CD2v (J.M. Rodríguez et al., 1993). Probably, nonhaemadsorbing avirulent isolates emerge as a result of some shift of the open reading frames for EP402R and EP153R encoding the CD2v and lectin-like proteins, respectively (D. A. Chapman et al., 2008). An assumption is made that the haemadsorption phenomenon is due to an interaction between carbohydrate residues of glycoproteins of ASFV oligosaccharides and lectin-like receptors of swine red blood cells.
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