Identification of Inhibitory Compounds Against Singapore Grouper Iridovirus Infection by Cell Viability-Based Screening Assay and Droplet Digital PCR
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Singapore grouper iridovirus (SGIV) is one of the major causative agents of fish diseases and has caused significant economic losses in the aquaculture industry. There is currently no commercial vaccine or effective antiviral treatment against SGIV infection. Annually, an increasing number of small molecule compounds from various sources have been produced, and many are proved to be potential inhibitors against viruses. Here, a high-throughput in vitro cell viability-based screening assay was developed to identify antiviral compounds against SGIV using the luminescent-based CellTiter-Glo reagent in cultured grouper spleen cells by quantificational measurement of the cytopathic effects induced by SGIV infection. This assay was utilized to screen for potential SGIV inhibitors from five customized compounds which had been reported to be capable of inhibiting other viruses and 30 compounds isolated from various marine organisms, and three of them [ribavirin, harringtonine, and 2-hydroxytetradecanoic acid (2-HOM)] were identified to be effective on inhibiting SGIV infection, which was further confirmed with droplet digital PCR (ddPCR). In addition, the ddPCR results revealed that ribavirin and 2-HOM inhibited SGIV replication and entry in a dose-dependent manner, and harringtonine could reduce SGIV replication rather than entry at the working concentration without significant toxicity. These findings provided an easy and reliable cell viability-based screening assay to identify compounds with anti-SGIV effect and a way of studying the anti-SGIV mechanism of compounds.
KeywordsSingapore grouper iridovirus Antiviral drug screening Compound Cell viability-based assay Digital droplet PCR
This work was supported by the National Natural Science Foundation of China (31502195, 31602191), the Zhuhai Scholar Professor Program (2015), the Guangdong Natural Science Foundation (2015A030308012), and the Science and Technology Planning Project of Guangdong Province (2017A030303010).
Compliance with Ethical Standards
Conflict of Interest
The authors declare that they have no conflict of interest.
- Goodwin S, Tuthill TJ, Arias A, Killington RA, Rowlands DJ (2009) Foot-and-mouth disease virus assembly: processing of recombinant capsid precursor by exogenous protease induces self-assembly of pentamers in vitro in a myristoylation-dependent manner. J Virol 83(21):11275–11282CrossRefPubMedPubMedCentralGoogle Scholar
- Harvey R, Brown K, Zhang Q, Gartland M, Walton L, Talarico C, Lawrence W, Selleseth D, Coffield N, Leary J, Moniri K, Singer S, Strum J, Gudmundsson K, Biron K, Romines KR, Sethna P (2009) GSK983: a novel compound with broad-spectrum antiviral activity. Antivir Res 82(1):1–11CrossRefPubMedGoogle Scholar
- Islam MK, Baudin M, Eriksson J, Oberg C, Habjan M, Weber F, Overby AK, Ahlm C, Evander M (2016) High-throughput screening using a whole-cell virus replication reporter gene assay to identify inhibitory compounds against Rift Valley fever virus infection. J Biomol Screen 21(4):354–362CrossRefPubMedGoogle Scholar
- Moya J, Pizarro H, Jashes M, De Clercq E, Sandino AM (2000) In vivo effect of EICAR (5-ethynyl-1-beta-D-ribofuranosylimidazole-carboxamide) on experimental infected rainbow trout (Oncorhynchus mykiss) and coho salmon (Onchorhynchus kisutch) fry with infectious pancreatic necrosis virus. Antivir Res 48(2):125–130CrossRefPubMedGoogle Scholar
- Mueller NH, Pattabiraman N, Ansarah-Sobrinho C, Viswanathan P, Pierson TC, Padmanabhan R (2008) Identification and biochemical characterization of small-molecule inhibitors of West Nile virus serine protease by a high-throughput screen. Antimicrob Agents Chemother 52(9):3385–3393CrossRefPubMedPubMedCentralGoogle Scholar