In vitro antiviral activity of fluoroquinolones against African swine fever virus

Abstract

African swine fever (ASF) is a viral swine disease against which neither an effective vaccine nor a treatment is available. The antiviral effect of thirty fluoroquinolones on the infectivity of African swine fever virus (ASFV) was screened in vitro. There was a severe reduction of the cytopathic effect in ASFV-infected Vero cells when exposed to six independent fluoroquinolones, or to some of their combinations, from an early phase of infection. Moreover, after 7-day treatments, ASFV genome could not be detected by PCR, and the culture supernatants were unable to infect new cell cultures. Pulsed field gel electrophoresis (PFGE) analysis revealed a diminished viral DNA replication without identifiable genome fragmentation in cells exposed to fluoroquinolones. In parallel, altered patterns of viral protein synthesis were observed from early infection. The overall results indicate that bacterial topoisomerase inhibitors interfere with the ASFV replication cycle probably by targeting a putative ASFV-topoisomerase II, opening a new window for antiviral treatments.

Introduction

African swine fever (ASF) is one of the most threatening diseases of domestic pigs. It is endemic in most of Sub-Saharan African countries and has been reintroduced to the European continent via Georgia in 2007. Since then it has spread through neighboring countries, drastically changing the European ASF situation. Apart from the implications for the affected countries the close proximity puts the European Union (EU) at serious risk of ASF introduction. The control of ASF in the Russian Federation and neighboring areas has been hampered by the absence of a coordinated control program, the abundance of backyard pig units with low or no biosecurity and the traditional use of swill feeding. Under this scenario, the ASF situation in Eastern Europe poses a constant risk of ASF entry into the EU, especially via routes that are difficult to control, such as wild boar movements, illegal movement of animals and animal products and, contaminated vehicles or other fomites. Despite the intense effort to discover a vaccine, none is yet available and the control of the disease relies on the early diagnosis and application of strict sanitary measures, including stamping out, which present severe economic and social hazards for affected countries (Sánchez-Vizcaíno et al., in press). The etiologic agent, the African swine fever virus (ASFV), is a large nucleocytoplasmic DNA virus (NCLDV), the sole member of the Asfiviridae family (Dixon et al., 2005). ASFV encodes for a topoisomerase type II enzyme (ASFV-TopoII) (Baylis et al., 1992, Garcia-Beato et al., 1992), which is a unique feature among mammal-infecting viruses. Phylogenetic studies revealed that ASFV-TopoII does not branch with the host cellular type II topoisomerases (Gadelle et al., 2003) and shares about 25% overall identity with the bacterial DNA gyrases and topoisomerase IV (Liu, 1994), raising the possibility of using antibacterial topoisomerase inhibitors (e.g. fluoroquinolones) to interfere with ASFV replication.

Fluoroquinolones exhibit a potent antibacterial activity by trapping the DNA gyrases and topoisomerase IV on DNA and promoting the formation of drug-enzyme-DNA cleavage complexes, that induce disruption of DNA replication and trigger cell-death mechanisms (e.g. oxidative stress and genomic fragmentation) (Drlica and Malik, 2003, Malik et al., 2007, Drlica et al., 2008). While DNA gyrases are responsible for relieving torsional stress ahead of the replication fork, topoisomerase IV shows an effective decatenating activity essential for de novo separation of genomes.

Recent studies have shown that chemical modifications in the fluoroquinolone molecular structure are responsible for their new “nonclassical” antiviral properties against both RNA (Witvrouw et al., 1998, Shi et al., 2009, Ahmed and Daneshtalab, 2012) and DNA viruses (Ebert et al., 1990, Ali et al., 2007, Sharma et al., 2011). Considering that ASFV encodes for a topoisomerase type II enzyme, we aimed at analyzing the potential inhibitory activity of those antibiotics on ASFV-DNA replication and on viral protein synthesis using Vero cell cultures infected with Ba71V isolate. Considering that neither a vaccine nor a treatment are available against ASF, the discovery of new antivirals and their application to pigs in farms located next to the outbreak focus to create a security ring around the outbreak, may be very useful in controlling the spread of the infection and giving time to the authorities to implement countermeasures.