دانلود رایگان مقاله مهار آزمایشگاهی تب خوکی آفریقایی ویروس توپوایزومراز II

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.

4. Discussion

Recently it was shown that ASFV encodes for a protein (ORF P1192R) that co-localizes with cytoplasmic viral factories at intermediate and late phases of infection, being able to complement a Saccharomyces cerevisiae Top2A temperature-sensitive mutant (Coelho et al., 2015). Although, phylogenetic studies revealed that ORF P1192R shares high sequence homology (Forterre et al., 2007) and functional motifs and domains with bacterial topoisomerases (Coelho et al., 2015), no further studies have been conducted to explore the role of ASFV-Topo II in ASFV infection. Using a wellestablished in vitro model of infection we showed that ASFV-Topo II mRNA levels continuously increase from 2 hpi to 16 hpi, similarly to other late ASFV genes (reviewed in Rodríguez and Salas, 2013). Most probably, the transcription kinetics of ASFV-Topo II indicates its need during the stage of accumulation of viral genomes that serve as templates for DNA replication and transcription, thus, showing an increasing number of topological complexities (e. g. knots, tangles and catenanes) that must be solved (reviewed in Rodríguez and Salas, 2013). Still, we cannot reject the possibility that ASFV-Topo II activity may also be required for other mechanisms like genome unpacking after virus entry or genome compaction before viral egress. Further, our siRNA experiments show that a partial depletion of transcripts encoding ASFVTopo II reduces the viral-induced CPE, the ASFV progeny, the number of infected cells and also the number of viral factories per cell suggesting that viral transcription activity is diminished. Indeed, Vero cells transfected with siRNAs showed a reduction in mRNA levels of ASFV genes VP32 and VP72, early and late transcripts, respectively (Gil et al., 2008; Zhang et al., 2010).