Construction of the First Russian Recombinant Live Attenuated Vaccine Strain and Evaluation of Its Protection Efficacy Against Two African Swine Fever Virus Heterologous Strains of Serotype 8

Abstract

Background/Objectives: The spread of African swine fever virus (ASFV) has led to major economic losses to pork worldwide. In Russia, there are no developed or registered vaccines against ASFV genotype II, which is associated with numerous ASFV outbreaks in populations of domestic pigs and wild boars in the country. Methods: We introduced deletions of the six MGF360 and MGF505 genes of the ASFV virulent Stavropol_01/08 strain, isolated in Russia in 2008. Results: We show here that this deletion did lead to full attenuation of the ASFV virulent Stavropol_01/08 strain. Animals intramuscularly inoculated with 10⁴ HAD₅₀ of ΔMGF360/505_Stav developed a strong immune response and short period of viremia (at 3-7 days post-inoculation). Recombinant ΔMGF360/505_Stav strain provides complete protection of pigs against the ASFV parental Stavropol_01/08 strain (10³ HAD₅₀). Therefore, in our experiment, we did not detect the genome of both the virulent and the recombinant strains in the blood and organs post-challenge with the Stavropol_01/08. In contrast, we found only partial protection (40%) of the ΔMGF360/505_Stav-immunized pigs against challenge with the ASFV heterologous Rhodesia strain. Additionally, the surviving animals had a prolonged fever, and their condition was depressed for most of the experiment. Conclusions: Thus, the ASFV recombinant ΔMGF360/505_Stav strain is the first live attenuated vaccine (LAV) in Russia that induces complete protection in pigs challenged with the highly virulent, epidemiologically relevant strains genotype II and serotype 8. However, this ASF LAV is not able to provide a high level of protection against other variants of serotype 8.

Keywords: African swine fever; African swine fever virus; MGF360; MGF505; genotype; live attenuated vaccine; serogroup; serotype.

Figure 1

Generation of the ASFV recombinant ΔMGF360/505_Stav strain. (A) Scheme of the location of the MGF360/MGF505 gene deletion in the ASFV Stavropol_01/08 genome. (B) The recombinant ΔMGF360/505_Stav genome coverage plot using Illumina reads mapped to the ASFV Stavropol_01/08 genome. (C) Fluorescence microscopy of the primary culture of porcine macrophages infected with the ASFV recombinant ΔMGF360/505_Stav strain is shown. Scale bar indicates 100 μm.

Figure 2

Comparative analysis of in vitro replication of the recombinant ΔMGF360/505_Stav strain (blue line) and the parental Stavropol_01/08 virus (red line) strain. The data are presented as the mean virus titer (expressed in log10 HAD₅₀/mL) ± standard deviation (SD) at each point (0, 24, 48, 72, 96, and 120 h post-infection (hpi)).

Figure 3

The safety and immunogenicity profile study of the ASFV recombinant ΔMGF360/505_Stav strain when inoculating pigs with 10⁴ HAD₅₀ per animal. The lethality kinetics (A), body temperatures (B), humoral immune response (C), daily viral loads in blood (D) in the pigs inoculated with the ASFV recombinant ΔMGF360/505_Stav strain (blue line) and control non-inoculated animals (red line). (B) The fever cutoff (40 °C) is marked as a gray dashed line. (C) The dashed lines represent the cut-off value, above which values were considered positive, below which were considered negative, and between them, there was a gray area where the values were considered doubtful. Data on body temperature, antibody response, and viral load are presented as individual values for each animal. dpi—days post-infection. ns—p > 0.05.

Figure 4

The efficiency of immunization with the ASFV recombinant ΔMGF360/505_Stav strain against the ASFV virulent parental Stavropol_01/08 strain when pigs were immunized with 10⁴ HAD₅₀ per animal and then challenged with 10³ HAD₅₀ per animal of the ASFV homologous Stavropol_01/08 strain (Experiment 2). The lethality kinetics (A), humoral immune response (B), body temperatures post-challenge (C), clinical signs post-challenge (D), daily viral loads in blood post-challenge (E), and post-mortem viral loads in organs (F) in pigs immunized with the ASFV recombinant ΔMGF360/505_Stav strain, infected with the Stavropol_01/08 strain (blue line), non-immunized animals, infected with the Stavropol_01/08 strain (red line), and control non-inoculated animals (green line). (B) The dashed lines represent the cut-off value, above which values were considered positive, below which were considered negative, and between them, there was a gray area where the values were considered doubtful. (C) The fever cutoff (40 °C) is marked as a gray dashed line. Data on body temperature, clinical assessment, antibody response, and viral load are presented as individual values for each animal. dpi—days post-immunization; dpc—days post-challenge. * p < 0.05.

Figure 5

The efficiency of immunization with the ASFV recombinant ΔMGF360/505_Stav strain against the ASFV virulent Rhodesia strain, when pigs were immunized with 10⁴ HAD₅₀ per animal and then challenged with 10³ HAD₅₀ per animal of the ASFV heterologous Rhodesia strain (Experiment 3). The lethality kinetics (A), humoral immune response (B), body temperatures post-challenge (C), clinical signs post-challenge (D), daily viral loads in blood post-challenge (E), and post-mortem viral loads in organs (F) in pigs immunized with the ASFV recombinant ΔMGF360/505_Stav strain, infected with the Rhodesia strain (blue line), non-immunized animals, infected with the Rhodesia strain (orange line), and control non-inoculated animals (green line). (B) The dashed lines represent the cut-off value, above which values were considered positive, below which were considered negative, and between them, there was a gray area where the values were considered doubtful. (C) The fever cutoff (40 °C) is marked as a gray dashed line. Data on body temperature, clinical assessment, antibody response, and viral load are presented as individual values for each animal. dpi—days post-immunization; dpc—days post-challenge. * p < 0.05; **** p < 0.0001.