Simple SummaryAfrican swine fever (ASF) causes serious economic losses to the global pig industry. Unfortunately, there is currently no effective and safe vaccine, although its development is actively continuing. To date, experimental vaccines based on the use of live attenuated strains of the ASF virus (ASFV) obtained by the deletion of viral genes responsible for virulence are the most effective. However, deletion of the same gene in different ASFV isolates can lead to different results. In our work, we have shown that, unlike some other isolates, the deletion of the CD2 gene from the genome of the virulent Congo strain does not lead to its weakening, although it affects the onset of ASF infection in domestic pigs.AbstractAfrican swine fever (ASF) is an infectious disease that causes the most significant losses to the pig industry. One of the effective methods for combating this disease could be the development of vaccines. To date, experimental vaccines based on the use of live attenuated strains of the ASF virus (ASFV) obtained by the deletion of viral genes responsible for virulence are the most effective. Deletion of the EP402R gene encoding a CD2-like protein led to the attenuation of various strains of the ASFV, although the degree of attenuation varies among different isolates. Here we have shown that the deletion of the EP402R gene from the genome of a high-virulent Congo isolate did not change either the virulence of the virus or its ability to replicate in the swine macrophage cell cultures in vitro. However, in vivo, animals infected with ΔCongo-v_CD2v had a delay in the onset of the disease and viremia compared to animals infected with the parental strain. Thus, deletion of the CD2 gene in different isolates of the ASFV has a different effect on the virulence of the virus, depending on its genetic background.