Marine Biotechnology

, Volume 20, Issue 1, pp 35–44 | Cite as

Identification of Inhibitory Compounds Against Singapore Grouper Iridovirus Infection by Cell Viability-Based Screening Assay and Droplet Digital PCR

  • Kuntong Jia
  • Yongming Yuan
  • Wei Liu
  • Lan Liu
  • Qiwei QinEmail author
  • Meisheng YiEmail author
Original Article


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.


Singapore grouper iridovirus Antiviral drug screening Compound Cell viability-based assay Digital droplet PCR 


Funding Information

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.

Supplementary material

10126_2017_9785_MOESM1_ESM.docx (601 kb)
Table S1 (DOCX 600 kb)


  1. Abdelmohsen UR, Balasubramanian S, Oelschlaeger TA, Grkovic T, Pham NB, Quinn RJ, Hentschel U (2016) Potential of marine natural products against drug-resistant fungal, viral, and parasitic infections. Lancet Infect Dis 17(2):e30–e41CrossRefPubMedGoogle Scholar
  2. Andrés G, García-Escudero R, Salas ML, Rodríguez JM (2002) Repression of African swine fever virus polyprotein pp220-encoding gene leads to the assembly of icosahedral core-less particles. J Virol 76(6):2654–2666CrossRefPubMedPubMedCentralGoogle Scholar
  3. Beaucourt S, Vignuzzi M (2014) Ribavirin: a drug active against many viruses with multiple effects on virus replication and propagation. Molecular basis of ribavirin resistance. Curr Opin Virol 8:10–15CrossRefPubMedGoogle Scholar
  4. Belnap DM, Filman DJ, Trus BL, Cheng N, Booy FP, Conway JF, Curry S, Hiremath CN, Tsang SK, Steven AC, Hogle JM (2000) Molecular tectonic model of virus structural transitions: the putative cell entry states of poliovirus. J Virol 74(3):1342–1354CrossRefPubMedPubMedCentralGoogle Scholar
  5. Capul AA, Perez M, Burke E, Kunz S, Buchmeier MJ, De La Torre JC (2007) Arenavirus Z-glycoprotein association requires Z myristoylation but not functional RING or late domains. J Virol 81(17):9451–9460CrossRefPubMedPubMedCentralGoogle Scholar
  6. Cordo SM, Candurra NA, Damonte EB (1999) Myristic acid analogs are inhibitors of Junin virus replication. Microbes Infect 1(8):609–614CrossRefPubMedGoogle Scholar
  7. Farazi TA, Waksman G, Gordon JI (2001) The biology and enzymology of proteinN-myristoylation. J Biol Chem 276(43):39501–39504CrossRefPubMedGoogle Scholar
  8. Feld JJ, Hoofnagle JH (2005) Mechanism of action of interferon and ribavirin in treatment of hepatitis C. Nature 436(7053):967–972CrossRefPubMedGoogle Scholar
  9. Gardner TJ, Cohen T, Redmann V, Lau Z, Felsenfeld D, Tortorella D (2015) Development of a high-content screen for the identification of inhibitors directed against the early steps of the cytomegalovirus infectious cycle. Antivir Res 113:49–61CrossRefPubMedGoogle Scholar
  10. 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
  11. Göttlinger HG, Sodroski JG, Haseltine WA (1989) Role of capsid precursor processing and myristoylation in morphogenesis and infectivity of human immunodeficiency virus type 1. Proc Natl Acad Sci U S A 86(15):5781–5785CrossRefPubMedPubMedCentralGoogle Scholar
  12. Graci JD, Cameron CE (2006) Mechanisms of action of ribavirin against distinct viruses. Rev Med Virol 16(1):37–48CrossRefPubMedGoogle Scholar
  13. Harper DR, Gilbert RL, Blunt C, McIlhinney RAJ (1993) Inhibition of varicella-zoster virus replication by an inhibitor of protein myristoylation. J Gen Virol 74(6):1181–1184.CrossRefPubMedGoogle Scholar
  14. 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
  15. Hirayama J, Ikebuchi K, Abe H, Kwon KW, Ohnishi Y, Horiuchi M, Shinagawa M, Ikuta K, Kamo N, Sekiguchi S (1997) Photoinactivation of virus infectivity by hypocrellin A. Photochem Photobiol 66(5):697–700CrossRefPubMedGoogle Scholar
  16. Holopainen R, Ohlemeyer S, Schütze H, Bergmann SM, Tapiovaara H (2009) Ranavirus phylogeny and differentiation based on major capsid protein, DNA polymerase and neurofilament triplet H1-like protein genes. Dis Aquat Org 85(2):81–91CrossRefPubMedGoogle Scholar
  17. Huang YC, Han YS (2014) Determining anti-betanodavirus compounds through a GF-1 cell-based screening platform. Antivir Res 105:47–53CrossRefPubMedGoogle Scholar
  18. Huang Y, Huang X, Yan Y, Cai J, Ouyang Z, Cui H, Wang P, Qin Q (2011) Transcriptome analysis of orange-spotted grouper (Epinephelus coioides) spleen in response to Singapore grouper iridovirus. BMC Genomics 12(1):556CrossRefPubMedPubMedCentralGoogle Scholar
  19. Huang YC, Lin TS, Peng C, Chan NL, Han YS (2016) Strong inhibition of betanodavirus replication by ribavirin targeting RNA-dependent RNA polymerase. J Fish Dis 39(5):619–623CrossRefPubMedGoogle Scholar
  20. 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
  21. Jashés M, Gonzalez M, López-Lastra M, De Clercq E, Sandino A (1996) Inhibitors of infectious pancreatic necrosis virus (IPNV) replication. Antivir Res 29(2-3):309–312CrossRefPubMedGoogle Scholar
  22. Kaur P, Thiruchelvan M, Lee RCH, Chen H, Chen KC, Ng ML, Chu JJH (2013) Inhibition of chikungunya virus replication by harringtonine, a novel antiviral that suppresses viral protein expression. Antimicrob Agents Chemother 57(1):155–167CrossRefPubMedPubMedCentralGoogle Scholar
  23. Kim Y, Lee C (2013) Ribavirin efficiently suppresses porcine nidovirus replication. Virus Res 171(1):44–53CrossRefPubMedGoogle Scholar
  24. Kim YS, Ke F, Lei XY, Zhu R, Zhang QY (2010) Viral envelope protein 53R gene highly specific silencing and iridovirus resistance in fish cells by amiRNA. PLoS One 5(4):e10308CrossRefPubMedPubMedCentralGoogle Scholar
  25. Kiselinova M, Pasternak AO, De Spiegelaere W, Vogelaers D, Berkhout B, Vandekerckhove L (2014) Comparison of droplet digital PCR and seminested real-time PCR for quantification of cell-associated HIV-1 RNA. PLoS One 9(1):e85999CrossRefPubMedPubMedCentralGoogle Scholar
  26. Li Q, Maddox C, Rasmussen L, Hobrath JV, White LE (2009) Assay development and high-throughput antiviral drug screening against Bluetongue virus. Antivir Res 83(3):267–273CrossRefPubMedPubMedCentralGoogle Scholar
  27. Lin F, Liu Q, Yuan Y, Hong Y (2015) Development of retroviral vectors for insertional mutagenesis in medaka haploid cells. Gene 573(2):296–302CrossRefPubMedGoogle Scholar
  28. Liu Q, Xiong H, Lu L, Liu Y, Luo F, Hou W, Yang Z (2013) Antiviral and anti-inflammatory activity of arbidol hydrochloride in influenza A (H1N1) virus infection. Acta Pharmacol Sin 34(8):1075–1083CrossRefPubMedPubMedCentralGoogle Scholar
  29. Marroquí L, Estepa A, Perez L (2007) Assessment of the inhibitory effect of ribavirin on the rainbow trout rhabdovirus VHSV by real-time reverse-transcription PCR. Vet Microbiol 122(1-2):52–60CrossRefPubMedGoogle Scholar
  30. Martin KH, Grosenbach DW, Franke CA, Hruby DE (1997) Identification and analysis of three myristylated vaccinia virus late proteins. J Virol 71(7):5218–5226PubMedPubMedCentralGoogle Scholar
  31. Maurer-Stroh S, Eisenhaber F (2004) Myristoylation of viral and bacterial proteins. Trends Microbiol 12(4):178–185CrossRefPubMedGoogle Scholar
  32. McNamara CR, Degterev A (2011) Small-molecule inhibitors of the PI3K signaling network. Future Med Chem 3(5):549–565CrossRefPubMedPubMedCentralGoogle Scholar
  33. Mondal R, Koev G, Pilot-Matias T, He Y, Ng T, Kati W, Molla A (2009) Development of a cell-based assay for high-throughput screening of inhibitors against HCV genotypes 1a and 1b in a single well. Antivir Res 82(1):82–88CrossRefPubMedGoogle Scholar
  34. 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
  35. 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
  36. Noah JW, Severson W, Noah DL, Rasmussen L, White EL, Jonsson CB (2007) A cell-based luminescence assay is effective for high-throughput screening of potential influenza antivirals. Antivir Res 73(1):50–59CrossRefPubMedGoogle Scholar
  37. Ou-yang Z, Wang P, Huang X, Cai J, Huang Y, Wei S, Ji H, Wei J, Zhou Y, Qin Q (2012) Immunogenicity and protective effects of inactivated Singapore grouper iridovirus (SGIV) vaccines in orange-spotted grouper, Epinephelus coioides. Dev Comp Immunol 38(2):254–261CrossRefPubMedGoogle Scholar
  38. Paige LA, Zheng G, DeFrees SA, Cassady JM, Geahlen RL (1990) Metabolic activation of 2-substituted derivatives of myristic acid to form potent inhibitors of myristoyl CoA: protein N-myristoyltransferase. Biochemistry 29(46):10566–10573CrossRefPubMedGoogle Scholar
  39. Patel B, Das S, Prakash R, Yasir M (2010) Natural bioactive compound with anticancer potential. Int J Adv Pharm Sci 1(1):32–41CrossRefGoogle Scholar
  40. Perez M, Greenwald DL, De JC, Torre L (2004) Myristoylation of the RING finger Z protein is essential for arenavirus budding. J Virol 78(20):11443–11448CrossRefPubMedPubMedCentralGoogle Scholar
  41. Phillips T, Jenkinson L, McCrae C, Thong B, Unitt J (2011) Development of a high-throughput human rhinovirus infectivity cell-based assay for identifying antiviral compounds. J Virol Methods 173(2):182–188CrossRefPubMedGoogle Scholar
  42. Picazo E, Giordanetto F (2015) Small molecule inhibitors of ebola virus infection. Drug Discov Today 20(2):277–286CrossRefPubMedGoogle Scholar
  43. Prange R, Clemen A, Streeck RE (1991) Myristylation is involved in intracellular retention of hepatitis B virus envelope proteins. J Virol 65(7):3919–3923PubMedPubMedCentralGoogle Scholar
  44. Qin QW, Wu TH, Jia TL, Hegde A, Zhang RQ (2006) Development and characterization of a new tropical marine fish cell line from grouper, Epinephelus coioides susceptible to iridovirus and nodavirus. J Virol Methods 131(1):58–64CrossRefPubMedGoogle Scholar
  45. Rivas-Aravena A, Vallejos-Vidal E, Cortez-San Martin M, Reyes-Lopez F, Tello M, Mora P, Sandino AM, Spencer E (2011) Inhibitory effect of a nucleotide analog on infectious salmon anemia virus infection. J Virol 85(16):8037–8045CrossRefPubMedPubMedCentralGoogle Scholar
  46. Song WJ, Qin QW, Qiu J, Huang CH, Wang F, Hew CL (2004) Functional genomics analysis of Singapore grouper iridovirus: complete sequence determination and proteomic analysis. J Virol 78(22):12576–12590CrossRefPubMedPubMedCentralGoogle Scholar
  47. Spearman P, Wang JJ, Vander Heyden N, Ratner LEE, Vander Heyden N (1994) Identification of human immunodeficiency virus type 1 Gag protein domains essential to membrane binding and particle assembly. J Virol 68(5):3232–3242PubMedPubMedCentralGoogle Scholar
  48. White RA, Quake SR, Curr K (2012) Digital PCR provides absolute quantitation of viral load for an occult RNA virus. J Virol Methods 179(1):45–50CrossRefPubMedGoogle Scholar
  49. Whitley DS, Yu K, Sample RC, Sinning A, Henegar J, Norcross E, Chinchar VG (2010) Frog virus 3 ORF 53R, a putative myristoylated membrane protein, is essential for virus replication in vitro. Virology 405(2):448–456CrossRefPubMedGoogle Scholar
  50. Yasuhara-Bell J, Lu Y (2010) Marine compounds and their antiviral activities. Antivir Res 86(3):231–240CrossRefPubMedGoogle Scholar
  51. Zhang J-H (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4(2):67–73CrossRefPubMedGoogle Scholar
  52. Zhang J, Yang PL, Gray NS (2009) Targeting cancer with small molecule kinase inhibitors. Nat Rev Cancer 9(1):28–39CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2017

Authors and Affiliations

  1. 1.Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, Zhuhai Key Laboratory of Marine Bioresources and Environment, School of Marine SciencesSun Yat-sen UniversityGuangzhouChina
  2. 2.Department of Biological SciencesNational University of SingaporeSingaporeSingapore
  3. 3.College of Marine SciencesSouth China Agricultural UniversityGuangzhouChina

Personalised recommendations