Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Sat, 02 Feb 2019 18:52:21 Rigid amphipathic fusion inhibitors demonstrate antiviral activity against African swine fever virus Astghik Hakobyan, 1 Inmaculada Galindo, 2 Almudena Nañez, 2 Erik Arabyan, 1 Zaven Karalyan, 3 Alexey A. Chistov, 4 Philipp P. Streshnev, 4 Vladimir A. Korshun, 4 Covadonga Alonso 2 and Hovakim Zakaryan 1, * Abstract 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. INTRODUCTION Due to its devastating impact on pig farming, African swine fever (ASF) is one of the most important viral diseases of domestic pigs, causing huge socio-economic losses in affected countries. Depending on host characteristics and the circulating viral isolate, clinical signs may vary from the highly lethal form with 100 % mortality to sub-clinical and aclinical forms [1, 2]. In the highly lethal form, clinical signs may include high fever, severe depression, anorexia, bloody diarrhoea, cyanosis and haemorrhagic lesions. Clinical signs are accompanied by severe thrombocytopenia and lympho- penia [3, 4]. Animals usually die within 10–14 days of infec- tion. Although ASF is widespread in sub-Saharan Africa, it also circulates on the European continent following a single introduction into Georgia in the Caucasus [5]. To date, there is no effective vaccine for ASF and the control of this disease is totally reliant on early diagnosis and the application of quarantine, including slaughter of affected pigs [6, 7]. Therefore, ASF poses a serious and constant risk for all European countries. The causative agent of ASF is African swine fever virus (ASFV), a large, enveloped, double-stranded DNA virus of approximately 190 kbp. It is the sole member of the Asfarviri- dae family and the only known DNA arbovirus infecting dif- ferent species of soft ticks (Ornithodoros spp.). In infected pigs, ASFV replicates in cells of the mononuclear phagocyte system, mostly in monocytes and macrophages, although other cell types can also be infected in later stages of the dis- ease [8, 9]. The ASFV life cycle starts with viral attachment and entry into the cells. ASFV enters the host cells by exploit- ing different entry mechanisms such as clathrin-mediated endocytosis and micropinocytosis [10, 11]. The decapsidation and disassembly of virions occur at late endosomal compart- ments. Once decapsidated, ASFV particles expose the inner envelope which allows the fusion of this viral membrane with the membrane of the endosomes, and naked cores can be released into cytosol in order to start replication [12, 13]. Viral DNA replication involves the nuclear phase, when rela- tively small DNA fragments are synthesized, and the cyto- plasmic phase, occurring in perinuclear cytoplasmic viral factories [14]. In these factories newly synthesized virions are Received 26 October 2017; Accepted 4 December 2017 Author affiliations: 1 Group of Antiviral Defense Mechanisms, Institute of Molecular Biology of NAS RA, 0014, Yerevan, Armenia; 2 Department of Biotechnology, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, INIA, Ctra. de la Coruña Km 7.5, 28040 Madrid, Spain; 3 Laboratory of Cell Biology and Virology, Institute of Molecular Biology of NAS RA, 0014, Yerevan, Armenia; 4 Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia. *Correspondence: Hovakim Zakaryan, h_zakaryan@mb.sci.am Keywords: African swine fever virus; antivirals; antiviral therapy; nucleoside analogues. Abbreviations: ANOVA, analysis of variance; ASFV, African swine fever virus; aUY11, 5-(Perylen-3-ylethynyl)-arabino-uridine; CC 50 , the 50% cytotoxic concentration; cm1UY11, 5-(Perylen-3-ylethynyl)uracil-1-acetic acid; DMEM, Dulbecco’s Modified Eagle’s Medium; EMEM, Eagle’s minimum essential medium; HADU, hemadsorption unite; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; OD, optical density; RAFI, rigid amphipathic fusion inhibitors. RESEARCH ARTICLE Hakobyan et al., Journal of General Virology 2018;99:148–156 DOI 10.1099/jgv.0.000991 000991 ã 2018 The Authors 148