Immunobiological evaluation of the candidate vaccine strain MK-200 of the African swine fever virus

African swine fever (ASF, causative agent African swine fever virus, ASFV) is a fatal hemorrhagic viral infection of domestic pigs and Eurasian wild boars (Sus scrofa). Depending on the properties of isolates/strains, the course of the disease can be peracute, acute, subacute, chronic, or asymptomatic. The global epizootic of ASF has stimulated the development and study of candidate vaccine strains, primarily recombinant or attenuated, considered as the basis for first-generation vaccines. In this study, two cycles of viral DNA replication were identified in pigs, inoculated with the ASFV candidate vaccine strain MK-200. The first cycle occurred on days 17-21, and the second on days 56-70 post-inoculation. The aim of the study was the evaluation of the immunobiological characteristics of the candidate ASFV vaccine strain MK-200 based on observations of the clinical condition, the presence of virus-specific DNA, and virus-specific antibodies in samples obtained from the pigs. The study was conducted in 2022 at the Federal Research Center for Virology and Microbiology (FRCVM). Clinically healthy large white breed pigs (Sus domesticus) aged 2-3 months were used. ASFV virulent strains Mozambique-78 (III seroimmunotype, V genotype) and Stavropol 01/08 (VIII seroimmunotype, II genotype) and the attenuated strain MK-200 obtained by selection from the Mozambique-78 strain, with infectious titers of 107.0-108.0 HAU50/ml were obtained from the state collection of microorganisms of FRCVM. The primary culture of pig leukocyte cells (LC) was prepared according to GOST 28573-90. ASFV infectious titers were determined in LC cultures by hemadsorption assay and calculated by the method of B.A. Kerber and I.P. Ashmarin. The experimental design included intramuscular inoculation of 10 pigs with ASFV (strain MK-200) at a dose of 106.0 HAU50 on day 0. Samples for study were collected on days 0, 3, 5, 7, 14, 17, 21, 28, 35, 42, 49, 56, 63, 70, and 77 post-infection (p.i.). The body temperature of the experimental animals was measured rectally throughout the all observation period. Blood samples were collected by puncturing the jugular vein. To obtain saliva samples, pigs were given strands of rope to chew (LLC TD PROMT, Russia) for 30 min. The wet rope strands were squeezed into clean polyethylene bags (LLC TD PROMT, Russia), and the collected saliva was transferred to centrifuge tubes (Thermo Fisher Scientific, USA). Cheek swabs were collected using sterile polyester swabs with plastic applicators (MiniMed, Russia). The MagMAX™ Pathogen RNA/DNA Kit (Thermo Fisher Scientific, USA) was used to extract nucleic acids. Real-time PCR (qPCR) was performed using the IDEXX RealPCR ASFV DNA Mix test system (IDEXX, USA). To detect antibodies to ASFV proteins in pig serum, the ID Screen® African Swine Fever Competition test system (IDVet, France) was used. We revealed a linear relationship between Ct values and infectious titers in 10-fold dilutions of virus-containing suspensions obtained by infecting LC cultures with ASFV strains Mozambique-78 or Stavropol 01/08. This allowed the conversion of data obtained from further blood and oral cavity sample studies in qPCR into infectious titer values. Of the 10 animals inoculated with the MK-200 strain, only three showed a brief (1-2 days) increase in body temperature above the physiological norm (40.1-40.6 °С) between days 5 and 7 p.i. The body temperature of all remained animals was within physiological norms during the first 10 days. From days 8 to 77 p.i., in all animals no clinical signs of ASF were observed. Viral DNA was detected in the samples from days 3-5 p.i. The minimum Ct values (maximum amount of target matrix) in qPCR were observed from days 17-21 p.i., with the proportion of positive samples reaching 60-100 %. Calculated viremia values based on the linear relationship between ASFV infectious titers and Ct values did not exceed 104.0 HAU50/ml from days 0 to 63 p.i. and were below 101.9 HAU50/ml from day 70 p.i. Throughout the experiment, cyclic viremia and the potential for virus shedding through the oral cavity were observed. This indicates the risks of horizontal and vertical transmission of the vaccine strain to susceptible animals through direct or indirect contact. We recommend to the developers of candidate live vaccines against ASF to provide data on viremia and virus shedding studies for a period of 2.5 to 4 months post-vaccination.