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bioRxiv · 10.1101/2023.04.27.538639

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

Reis, A. L.·Rathakrishnan, A.·Goulding, L. V.·Goatley, L. C.·Barber, C.·Dixon, L. K.
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Abstract

African swine fever virus encodes proteins that inhibit apoptosis including one member of the BCL-2 family, A179L. Deletion of the A179L gene from the virulent genotype I isolate Benin 97/1 compared to Benin 97/1 expressing A179L or mock-infected macrophages, resulted in increased Caspase 3 and 7 activity, annexin V binding to surface phosphatidyl serine and DNA fragmentation, measured by terminal deoxynucleotidyl transferase nick-end labelling. These results confirmed that apoptosis was induced earlier in macrophages infected with the Benin{Delta}A179L virus. Increased cell entry of the A179L gene-deleted virus was indicated at early times since up to double the numbers of cells expressed fluorescent protein from the virus genome. Yields of infectious virus were similar over a single cycle but were significantly lower for the A179L gene-deleted virus over a multi-step growth cycle. Pigs immunised and boosted with the Benin{Delta}A179L virus showed no clinical signs, although a weak cellular response to ASFV was observed showing that the virus had replicated. The immunised pigs were not protected against challenge with the virulent parental virus Benin 97/1 although viremia was lower at 3 days post-challenge compared to the control non-immune pigs. The reduced levels of virus replication in macrophages probably limited 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. IMPORTANCEAfrican swine fever virus (ASFV) causes a lethal disease of pigs that has spread extensively in Africa, Europe and Asia. The virus codes for more than 150 proteins, many of which help the virus to evade the hosts defences following infection. We investigated the effect of deleting one of these genes, A179L, from the genome of an ASFV isolate that causes death of infected pigs. A179L belongs to the BCL-2 protein family, consisting of members which promote or inhibit apoptosis with A179L belonging to the latter. Deleting the A179L gene reduced ASFV replication and spread between macrophages, its main target cells. This was correlated with an increase in cell death. Pigs infected with the virus with A179L gene deleted did not show signs of disease and no virus replication was detected in blood. A low immune response was generated but the immunised pigs were not protected when challenged with the parental deadly virus. The results show that the A179L gene is important for ASFV to replicate efficiently in cells and in animals.

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Reis, A. L., Rathakrishnan, A., Goulding, L. V., Goatley, L. C., Barber, C., Dixon, L. K.. 2023-04-28. 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. https://doi.org/10.1101/2023.04.27.538639

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