title: In vitro inhibition of African swine fever virus-topoisomerase II disrupts viral replication.
abstract: African swine fever virus (ASFV) is the etiological agent of a highly-contagious and fatal disease of domestic pigs, leading to serious socio-economic impact in affected countries. To date, neither a vaccine nor a selective anti-viral drug are available for prevention or treatment of African swine fever (ASF), emphasizing the need for more detailed studies at the role of ASFV proteins involved in viral DNA replication and transcription. Notably, ASFV encodes for a functional type II topoisomerase (ASFV-Topo II) and we recently showed that several fluoroquinolones (bacterial DNA topoisomerase inhibitors) fully abrogate ASFV replication in vitro. Here, we report that ASFV-Topo II gene is actively transcribed throughout infection, with transcripts being detected as early as 2 hpi and reaching a maximum peak concentration around 16 hpi, when viral DNA synthesis, transcription and translation are more active. siRNA knockdown experiments showed that ASFV-Topo II plays a critical role in viral DNA replication and gene expression, with transfected cells presenting lower viral transcripts (up to 89% decrease) and reduced cytopathic effect (-66%) when compared to the control group. Further, a significant decrease in the number of both infected cells (75.5%) and viral factories per cell and in virus yields (up to 99.7%, 2.5 log) was found only in cells transfected with siRNA targeting ASFV-Topo II. We also demonstrate that a short exposure to enrofloxacin during the late phase of infection (from 15 to 1 hpi) induces fragmentation of viral genomes, whereas no viral genomes were detected when enrofloxacin was added from the early phase of infection (from 2 to 16 hpi), suggesting that fluoroquinolones are ASFV-Topo II poisons. Altogether, our results demonstrate that ASFV-Topo II enzyme has an essential role during viral genome replication and transcription, emphasizing the idea that this enzyme can be a potential target for drug and vaccine development against ASF.
authors: CIISA, Faculdade de Medicina Veterinária, Universidade de Lisboa, Av. da Universidade Técnica, 1300-477, Lisboa, Portugal.
authors: Ferdinando B
authors: FB
authors: Freitas
authors: CIISA, Faculdade de Medicina Veterinária, Universidade de Lisboa, Av. da Universidade Técnica, 1300-477, Lisboa, Portugal.
authors: Gonçalo
authors: G
authors: Frouco
authors: CIISA, Faculdade de Medicina Veterinária, Universidade de Lisboa, Av. da Universidade Técnica, 1300-477, Lisboa, Portugal.
authors: Carlos
authors: C
authors: Martins
authors: CIISA, Faculdade de Medicina Veterinária, Universidade de Lisboa, Av. da Universidade Técnica, 1300-477, Lisboa, Portugal.
authors: Alexandre
authors: A
authors: Leitão
authors: CIISA, Faculdade de Medicina Veterinária, Universidade de Lisboa, Av. da Universidade Técnica, 1300-477, Lisboa, Portugal. Electronic address: fernandof@fmv.ulisboa.pt.
authors: Fernando
authors: F
authors: Ferreira
mesh_terms: D000357:African Swine Fever / Q000821:virology; D000358:African Swine Fever Virus / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics / Q000502:physiology; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D002522:Chlorocebus aethiops; D004250:DNA Topoisomerases, Type II / Q000187:drug effects*; D000077422:Enrofloxacin; D024841:Fluoroquinolones / Q000494:pharmacology; D016679:Genome, Viral / Q000187:drug effects; D034622:RNA Interference; D060888:Real-Time Polymerase Chain Reaction; D013552:Swine; D059005:Topoisomerase II Inhibitors / Q000494:pharmacology*; D014709:Vero Cells; D014779:Virus Replication / Q000187:drug effects* / Q000235:genetics
chemical_list: D000998:Antiviral Agents; D024841:Fluoroquinolones; D059005:Topoisomerase II Inhibitors; D000077422:Enrofloxacin; D004250:DNA Topoisomerases, Type II
keywords: ASFV-Topoisomerase II; Antiviral therapy; Vaccine development; qPCR; siRNA