Cell-adapted African swine fever virus Pig/HLJ/18 is highly attenuated but fails to induce immune protection against a challenge with its parental virus

▸ A cell-adapted virus, named HLJ18/BK33, was successfully obtained by serially passaging the ASFV Pig/HLJ/18 in BK2258 cells. ▸ ASFV HLJ18/BK33 exhibited clear CPE and efficient replication in BK2258 cells. ▸ ASFV HLJ18/BK33 was highly attenuated with significantly reduced biosafety risks. African swine fever (ASF) is an acute, hemorrhagic disease caused by the African swine fever virus (ASFV), with a mortality up to 100%. The disease poses a seriously threat to the global swine industry, yet no commercial vaccines or antiviral drugs are available other than in Vietnam. ASFV attenuation through serial passages is a key approach for vaccine development. In this study, a cell-adapted virus, named HLJ18/BK33, was successfully generated by serially passaging the ASFV Pig/HLJ/18 in wild boar kidney cells (BK2258). This adapted virus exhibited clear cytopathic effects (CPE) and replicated stably and efficiently in BK2258 cells and porcine alveolar macrophages. Whole-genome sequence analysis revealed that, compared with the Pig/HLJ/18 virus, HLJ18/BK33 had a large deletion of 6162 bp from sites 181,027 to 187,188, and four single nucleotide deletions that led to frameshift mutations, resulting in the truncated expression of three open reading frames (ORFs) (ASFV_G_ACD_00120, ASFV_G_ACD_00350, and A179L), and the fusion expression of two ORFs (MGF_110-14L and MGF_110-11L). Additionally, four genes exhibited missense mutations, leading to single amino acid changes. Five pigs intramuscularly inoculated with 10 6 TCID 50 of HLJ18/BK33 remained healthy with normal body temperatures and no clinical signs, indicating a high attenuation of virulence for HLJ18/BK33 in pigs. Upon challenge with the parental Pig/HLJ/18 virus, four of the five inoculated pigs developed persistent high fever and ASF-related clinical signs and died within 13 days of the challenge; the remaining pig developed transient fever but survived until the end of the observation period. These results indicate that the HLJ18/BK33 virus is highly attenuated but cannot induce protection against the parental virulent virus. Even though the HLJ18/BK33 virus is not a good vaccine candidate, its stable replication and distinct CPE in BK2258 cells as well as its low biosafety risk make it a valuable resource for studies on virus-host interactions, antiviral drug screening, diagnostic methods, and biological characteristics.