Novel Virostatic Agents against Bluetongue Virus Linlin Gu1,2, Volodymyr Musiienko 3 , Zhijun Bai2,4, Aijian Qin1, Stewart W. Schneller3, Qianjun Li2* 1 Jiangsu Key Laboratory of Preventive Veterinary Medicine, Yangzhou University, Yangzhou, China, 2 Division of Infectious Diseases, Department of Medicine, University of Alabama at Birmingham, Alabama, United States of America, 3 Molette Laboratory for Drug Discovery, Department of Chemistry and Biochemistry, Auburn University, Auburn, Alabama, United States of America, 4 Guangzhou Center for Disease Control and Prevention, Guangdong, China Abstract Bluetongue virus (BTV), a member in the family Reoviridae, is a re-emerging animal disease infecting cattle and sheep. With its recent outbreaks in Europe, there is a pressing need for efficacious antivirals. We presented here the identification and characterization of a novel virostatic molecule against BTV, an aminothiophenecarboxylic acid derivative named compound 003 (C003). The virostatic efficacy of C003 could be improved via chemical modification, leading to a de novo synthesized compound 052 (C052). The 50% effective concentrations (EC50) of C003 and C052 were determined at 1.7660.73 mM and 0.2760.12 mM, respectively. The 50% cytotoxicity concentration (CC50) of C003 was over 100 mM and the CC50 of C052 was at 82.69 mM. Accordingly, the 50% selective index (SI50) of C003 and C052 against BTV was over 57 and 306, respectively. The inhibitory effect of C003/C052 on BTV-induced apoptosis was also confirmed via the inhibition of caspase-3/-7 activation post BTV infection. C003/C052 could inhibit BTV induced CPE even when added as late as 24 h.p.i., indicating that they might act at late stage of viral life-cycle. C003/C052 could reduce over two-logs of both the progeny virus production and the number of genomic viral RNA copies. Interestingly, both the activation of host autophagy and viral protein expression were inhibited post BTV infection when cells were treated with C003 and C052, suggesting that C003/C052 might act as virostatic agents via inhibiting host autophagy activation. Although further investigations might be needed to pin down the exact mechanism of C003/C052, our finding suggested that these compounds might be potent lead compounds with potential novel mechanism of action against BTV. Citation: Gu L, Musiienko V, Bai Z, Qin A, Schneller SW, et al. (2012) Novel Virostatic Agents against Bluetongue Virus. PLoS ONE 7(8): e43341. doi:10.1371/ journal.pone.0043341 Editor: Nupur Gangopadhyay, University of Pittsburgh, United States of America Received March 15, 2012; Accepted July 19, 2012; Published August 15, 2012 Copyright: 2012 Gu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This project was supported by grant 1R03MH08127-01 and 7R03MH08127-02 from NIH to Q. Li, and by the IMPACT funds from Department of Medicine at University of Alabama to Q. Li. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: liq@uab.edu Introduction Bluetongue virus (BTV), a double-strand RNA (dsRNA) virus, is the prototype virus in the genus Orbivirus within the family Reoviridae. The segmented BTV dsRNA genome encodes ten viral proteins (VP), including seven structural proteins (VP1-7) and three non-structural (NS) proteins, NS1, NS2 and NS3 [1,2,3]. As a member of arboviruses, BTV is transmitted by certain species of Culicoides biting midges, including C. variipennis and C. imocola [4,5]. Bluetongue disease is a non-contagious viral disease affecting domestic animals including sheep and cattle primarily, as well as wild ruminants such as buffalo, antelope, deer, elk and camels. BTV disease is one of the most important diseases of domestic livestock, causing $3 billion per year loss worldwide [6,7]. In sheep, the disease is acute, and mortality is accordingly high [8]. BTV is listed under the Office International des Epizooties (OIE) Terrestrial Animal Health Code –2009 [2]. Exotic BTV is also listed in the ‘‘USDA High Consequence Livestock Pathogens.’’ Due to its economic significance, BTV has been the subject of extensive molecular virology and structural biology studies [9,10,11]. The recent development of the BTV reverse genetics system facilitates our understanding toward the BTV viral life- cycle, the structural and functional interrelationship among viral proteins [12,13,14]. Hence, BTV is now one of the well- characterized viruses [6,9,15]. The main prevention and control measures in endemic areas include active surveillance programs, animal quarantine and movement restrictions, vaccination and insect control measures [2,16,17,18]. Vaccination is used as the most effective and practical measure to minimize losses related to BTV disease and to potentially interrupt the cycle from infected animal to vector [18,19,20]. However, due to the complexity of the virus, including the twenty-four different serotypes that have the ability of causing variable diseases, BTV is still endemic in many regions despite the high vaccine coverage in sheep and cattle [21,22,23,24]. The 1998–2001 outbreak of BTV-8 in the Mediterranean Basin is the greatest epizootic of the disease on record, showing that BTV has extended its range northwards into areas of Europe that were never affected before, and has since persisted in many of these locations [6,25,26,27]. In 2006, BTV-8 has reached three northern Europe countries, including the Netherlands, Belgium, and Germany [28,29], and further spread to surrounding countries, reaching Switzerland by the end of October 2007 [6,30,31,32]. Vaccination of individuals/animals during an outbreak can prove effective, however, protection of an individual/animal from the threat may not occur for two or more weeks after the initial vaccination [33]. Hence, only a drug can be offered as a therapeutic treatment of individuals/animals in an endemic area [33]. Surprisingly, there are no antiviral currently available against BTV disease. Recently, anti-BTV drug discovery has been PLOS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e43341