African swine fever virus (ASFV) is the causal agent of a highly-contagious and fatal disease of domestic pigs, leading to serious socio-economic consequences in affected countries. Once, neither an anti-viral drug nor an effective vaccines are available, studies on new anti-ASFV molecules are urgently need. Recently, it has been shown that ASFV type II topoisomerase (ASFV-topo II) is inhibited by several fluoroquinolones (bacterial DNA topoisomerase inhibitors), raising the idea that this viral enzyme can be a potential target for drug development against ASFV. Here, we report that genistein hampers ASFV infection at non-cytotoxic concentrations in Vero cells and porcine macrophages. Interestingly, the antiviral activity of this isoflavone, previously described as a topo II poison in eukaryotes, is maximal when it is added to cells at middle-phase of infection (8 hpi), disrupting viral DNA replication, blocking the transcription of late viral genes as well as the synthesis of late viral proteins, reducing viral progeny. Further, the single cell electrophoresis analysis revealed the presence of fragmented ASFV genomes in cells exposed to genistein, suggesting that this molecule also acts as an ASFV-topo II poison and not as a reversible inhibitor. No antiviral effects were detected when genistein was added before or at entry phase of ASFV infection. Molecular docking studies demonstrated that genistein may interact with four residues of the ATP-binding site of ASFV-topo II (Asn-144, Val-146, Gly-147 and Leu-148), showing more binding affinity (-4.62 kcal/mol) than ATP (-3.02 kcal/mol), emphasizing the idea that this viral enzyme has an essential role during viral genome replication and can be a good target for drug development against ASFV.

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Rigid amphipathic fusion inhibitors (RAFIs) are a family of nucleoside derivatives that inhibit the infectivity of several enveloped viruses by interacting with virion envelope lipids and inhibiting fusion between viral and cellular membranes. Here we tested the antiviral activity of two RAFIs, 5-(Perylen-3-ylethynyl)-arabino-uridine (aUY11) and 5-(Perylen-3-ylethynyl)uracil-1-acetic acid (cm1UY11) against African swine fever virus (ASFV), for which no effective vaccine is available. Both compounds displayed a potent, dose-dependent inhibitory effect on ASFV infection in Vero cells. The major antiviral effect was observed when aUY11 and cm1UY11 were added at early stages of infection and maintained during the complete viral cycle. Furthermore, virucidal assay revealed a significant extracellular anti-ASFV activity for both compounds. We also found decrease in the synthesis of early and late viral proteins in Vero cells treated with cm1UY11. Finally, the inhibitory effect of aUY11 and cm1UY11 on ASFV infection in porcine alveolar macrophages was confirmed. Overall, our study has identified novel anti-ASFV compounds with potential for future therapeutic developments.

BackgroundAfrican swine fever virus (ASFV) is the causative agent of African swine fever (ASF) that is the significant disease of domestic pigs. Several studies showed that ASFV can influence on porcine blood cells in vitro. Thus, we asked ourselves whether ASFV infection results in changes in porcine blood cells in vivo. A series of experiments were performed in order to investigate the effects of ASFV infection on porcine peripheral white blood cells. Nine pigs were inoculated by intramuscular injection with 104 50% hemadsorbing doses of virus (genotype II) distributed in Armenia and Georgia. The total number of fifteen cell types was calculated during experimental infection.ResultsAlthough band-to-segmented neutrophils ratio became much higher (3.5) in infected pigs than in control group (0.3), marked neutropenia and lymphopenia were detected from 2 to 3 days post-infection. In addition to band neutrophils, the high number of other immature white blood cells, such as metamyelocytes, was observed during the course of infection. From the beginning of infection, atypical lymphocytes, with altered nuclear shape, arose and became 15% of total cells in the final phase of infection. Image scanning cytometry revealed hyperdiploid DNA content in atypical lymphocytes only from 5 days post-infection, indicating that DNA synthesis in pathological lymphocytes occurred in the later stages of infection.ConclusionFrom this study, it can be concluded that ASFV infection leads to serious changes in composition of white blood cells. Particularly, acute ASFV infection in vivo is accompanied with the emergence of immature cells and atypical lymphocytes in the host blood. The mechanisms underlying atypical cell formation remain to be elucidated.