Deletion of the gene for the African swine fever virus BCL-2 family member A179L increases virus uptake and apoptosis but decreases virus spread in macrophages and reduces virulence in pigs

The African swine fever virus (ASFV) encodes inhibitors of innate immune responses, including type I interferon and apoptosis. The BCL-2 family A179L gene was deleted from the virulent genotype I isolate Benin 97/1. In infected macrophages, this resulted in increased caspase 3 and 7 activities, Annexin V binding to surface phosphatidylserine, and DNA fragmentation, measured by terminal deoxynucleotidyl transferase nick-end labeling, compared to Benin 97/1 expressing A179L or mock-infected macrophages. These results confirmed that apoptosis was induced earlier in macrophages infected with viruses from which A179L was deleted. Increased cell entry of the A179L gene-deleted virus was indicated since the number of infected cells at early times, as measured by fluorescent protein expression, was much higher than infection with the wild-type virus. This increase in entry was reduced following blocking of the virus by binding to Annexin V, suggesting that entry was mediated by phosphatidylserine receptors. Yields of infectious viruses were similar over a single cycle but significantly lower for the A179L gene-deleted virus over a multistep growth cycle. Pigs immunized with the Benin Delta A179L virus showed no clinical signs and a weak cellular response to ASFV. The immunized pigs were not protected against challenge with the virulent parental virus Benin 97/1 although viremia was lower at 3 d post-challenge than the control nonimmune pigs. Thus, reduced levels of virus replication in pigs limited the induction of a protective immune response. The results show an important role for the A179L protein in virus replication in macrophages and virulence in pigs. IMPORTANCE African swine fever virus (ASFV) causes a lethal disease of pigs with high economic impact in affected countries in Africa, Europe, and Asia. The virus encodes proteins that inhibit host antiviral defenses, including the type I interferon response. Host cells also activate cell death through a process called apoptosis to limit virus replication. We showed that the ASFV A179L protein, a BCL-2 family apoptosis inhibitor, is important in reducing apoptosis in infected cells since deletion of this gene increased cell death and reduced virus replication in cells infected with the A179L gene-deleted virus. Pigs immunized with the Benin Delta A179L virus showed no clinical signs and a weak immune response but were not protected from infection with the deadly parental virus. The results show an important role for the A179L protein in virus replication in macrophages and virulence in pigs and suggest manipulation of apoptosis as a possible route to control infection.