Experimental Epizotology of Low-Virulent Variants of African Swine Fever Virus

Abstract

African swine fever (ASF) remains an urgent problem of pig farming in Ukraine, the solution of which is possible only on the basis of deep scientific knowledge about the specific driving forces of the epizootic in its specific nozoareal. This is necessary in order to target anti-epizootic measures on the most vulnerable link of the epizootic chain in a specific nozoareal. The aim of the work was to develop a low-budget methodological base for experimental epizootology of low-virulent ASFV variants in Ukraine, in particular, to study the mechanisms of the formation of enzootic areas, quality control of anti-epizootic measures, and evaluation of the effectiveness of the antiviral drugs against them in Ukraine. Methods. Experimental and epizootological studies in the adaptation of suckling piglets to keeping in the biosecurity-level BSL-3 for laboratory animals (Patent UA No. 133248 dated 03/25/2019) were conducted at the laboratory base of the Odesa branch of NSC «IECVM». All procedures with infectious active biological materials in the current order were carried out in the BSL-3 module, built and certified with the assistance of the US Government in UAPRI (Odesa). The ASF agent strain «IECVM/Ternopil/2017» (infectious activity 4.0—7.5lg HAdU50/cm 3) circulating in the Ternopil region in 2017—2020 was used as a test virus. The presence of low-virulence variants of the ASFV pathogen in the studied samples was determined by a bioassay on suckling piglets, followed by three consecutive passages on a stable Vero line of the baby green monkey kidney cells. The isolated ASF virus was identified according to the methods and reagents recommended by the OIE Manual. Results. Intermittent passages «by the founder’s method» of dilutions 10-1 and 10-2 of the ASF virus strain «IECVM/Ternopil/2017» on piglets (n=20) and the culture of porcine alveolar macrophages («ASFVPAM») allowed us to identify highly-, moderately-, and low-virulent variants/clones in its composition. Verifi cation by bioassay on suckling piglets (n=5) of low-virulent clones of the agent, which were stabilized in Vero cell culture («ASFVVero»), showed that after intraperitoneal infection at a dose of 4.25 lgHAdU50/cm 3, they are capable of causing only a non-lethal (within 2 weeks) viral infection with a maximum daily rectal temperature of 39.4±0.22 °C and duration of fever on average 1.6±0.14 days (5 of 5 piglets). Clones with greater virulence («ASFVPAM») under similar conditions were able to cause a lethal infection with a maximum temperature of 40.7±0.37°C and duration of fever on average 3.9±0.27 days (17 of 20 piglets). Low-virulent clones were revealed by direct immunofluorescence in pulmonary and spleen smears of clinically healthy piglets on days 14 and 17 post-infection (p.i.); their antigens were visualized in Vero cells by indirect immunoperoxidase method after 48 h p.i. at dose about 0.01 lg HAdU50/cm 3. They caused «crumbly» hemadsorption of infected Vero cells and their virions had typical for Asfarvirus view and size (210—220 nm). The obtained data served as the basis for analysis of the mechanism of rooting ASF agents in West Podillia enzootic foci, as well as for implication of the concept of low-budget quality control of anti-epizootic measures and evaluation of antiviral drugs’ activities against ASF. Conclusions. Low-cost operational procedures have been developed that allow one to use a vivarium of laboratory animals for ASF bioassay and meet principal requirements for science-based research in important aspects of experimental ASF epizootology. With their help, confirmation of the heterogeneity of the population of the ASF virus circulating in endemic foci of the Ukrainian Western Podillia was obtained (p<0.05, n=25)><0.05, n=25). The developed methodological approach is suitable for the study of fundamental issues of ASF epizootology, as well as for the quality control of anti-epizootic measures against ASF. In particular, it is advisable to use it to improve the biosecurity of agricultural export programs in Ukraine, a country that is disadvantaged by ASF.