Archives of Microbiology

, Volume 200, Issue 6, pp 961–970 | Cite as

Polyunsaturated fatty acids from Phyllocaulis boraceiensis mucus block the replication of influenza virus

  • Ana Rita de Toledo-Piza
  • Maria Isabel de Oliveira
  • Giuseppina Negri
  • Ronaldo Zucatelli Mendonça
  • Cristina Adelaide Figueiredo
Original Paper


Influenza viruses cause worldwide outbreaks and pandemics in humans and animals every year with considerable morbidity and mortality. The molecular diversity of secondary metabolites extracted from mollusks is a good alternative for the discovery of novel bioactive compounds with unique structures and diverse biological activities. Phyllocaulis boraceiensis is a hermaphroditic slug that exudes mucus, in which was detected hydroxy polyunsaturated fatty acids that exhibited potent antiviral activity against measles virus. The objective of this study was to evaluate this property against Influenza viruses. Cell viability and toxicity of the mucus were evaluated on Madin–Darby canine kidney (MDCK) cells by MTT assay. Antiviral activity from mucus against influenza viruses was carried out by determination of the virus infection dose and by immunofluorescence assays. The crude mucus and its fractions exhibited low cytotoxicity on MDCK cells. A significant inhibition of viral replication, reduced by the order of eight times, was observed in influenza-induced cytopathic effect. In immunofluorescence assay was observed a decrease of more than 80% of the viral load on infected MDCK cell treated with mucus and its fractions. The viral glycoproteins hemagglutinin and neuraminidase located on the surface of the virus are crucial for the replications and infectivity of the influenza virus. Some authors demonstrated that lipids, such as, polyunsaturated fatty acids exhibited multiple roles in antiviral innate and adaptive responses, control of inflammation, and in the development of antiviral therapeutics. As corroborated by other studies, hydroxy polyunsaturated fatty acids interfered with the binding of influenza virus on host cell receptor and reduced viral titers. The results obtained indicated that polyunsaturated fatty acids from P. boraceiensis crude mucus and fractions 39 exerted antiviral activity against influenza virus.


Fatty acids Antiviral Influenza Mollusks 



This study was funded by São Paulo Research Foundation (FAPESP—2012/22906-9 and 2012/22555-1).

Compliance with ethical standards

Conflict of interest

The authors declare that there are no conflicts of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.


  1. Arita M (2012) Mediator lipidomics in acute inflammation and resolution. J Biochem 152:313–319CrossRefPubMedGoogle Scholar
  2. Benkendorff K (2010) Molluscan biological and chemical diversity: secondary metabolites and medicinal resources produced by marine molluscs. Biol Rev 85:757–775. PubMedCrossRefGoogle Scholar
  3. Benkendorff K, Rudd D, Nongmaithem BD et al (2015) Are the traditional medical uses of Muricidae molluscs substantiated by their pharmacological properties and bioactive compounds? Mar Drugs 13:5237–5275CrossRefPubMedPubMedCentralGoogle Scholar
  4. Bollinger JG, Rohan G, Sadilek M, Gelb MH (2013) LC/ESI-MS/MS detection of FAs by charge reversal derivatization with more than four orders of magnitude improvement in sensitivity. J Lipid Res 54:3523–3530CrossRefPubMedPubMedCentralGoogle Scholar
  5. Boriskin YS, Leneva IA, Pécheur E-I, Polyak SJ (2008) Arbidol: a broad-spectrum antiviral compound that blocks viral fusion. Curr Med Chem 15:997–1005. CrossRefPubMedGoogle Scholar
  6. Dang VT, Benkendorff K, Green T, Speck P (2015) Marine snails and slugs: a great place to look for antiviral drugs: table 1. J Virol 89:8114–8118. CrossRefPubMedPubMedCentralGoogle Scholar
  7. Desbois A, Lawlor K (2013) Antibacterial activity of long-chain polyunsaturated fatty acids against Propionibacterium acnes and Staphylococcus aureus. Mar Drugs 11:4544–4557. CrossRefPubMedPubMedCentralGoogle Scholar
  8. Ding Y, Cao Z, Cao L et al (2017) Antiviral activity of chlorogenic acid against influenza A (H1N1/H3N2) virus and its inhibition of neuraminidase. Sci Rep 7:45723. CrossRefPubMedPubMedCentralGoogle Scholar
  9. Dolin R (2011) Resistance to neuraminidase inhibitors. Clin Infect Dis 52:438–439. CrossRefPubMedGoogle Scholar
  10. Fiers W, De Filette M, Birkett A et al (2004) A universal human influenza A vaccine. Virus Res 103:173–176. CrossRefPubMedGoogle Scholar
  11. García-Sastre A (2013) XLessons from lipids in the fight against influenza. Cell. PubMedCrossRefGoogle Scholar
  12. Gioula G, Melidou A, Exindari M et al (2011) Oseltamivir-resistant influenza A pandemic (H1N1) 2009 virus in northern Greece. Hippokratia 15:272–274PubMedPubMedCentralGoogle Scholar
  13. Hsu F-F, Turk J (2008) Elucidation of the double-bond position of long-chain unsaturated fatty acids by multiple-stage linear ion-trap mass spectrometry with electrospray ionization. J Am Soc Mass Spectrom 19:1673–1680. CrossRefPubMedPubMedCentralGoogle Scholar
  14. Hulbert AJ, Kelly MA, Abbott SK (2014) Polyunsaturated fats, membrane lipids and animal longevity. J Comp Physiol B Biochem Syst Environ Physiol 184:149–166CrossRefGoogle Scholar
  15. Hurt AC, Chotpitayasunondh T, Cox NJ et al (2012) Antiviral resistance during the 2009 influenza A H1N1 pandemic: public health, laboratory, and clinical perspectives. Lancet Infect Dis 12:240–248. CrossRefPubMedGoogle Scholar
  16. Jin XW, Mossad SB (2012) 2012–2013 influenza update: hitting a rapidly moving target. Clevel Clin J Med 79:777–784. CrossRefGoogle Scholar
  17. Kiefer J, Noack K, Bartelmess J et al (2010) Vibrational structure of the polyunsaturated fatty acids eicosapentaenoic acid and arachidonic acid studied by infrared spectroscopy. J Mol Struct 965:121–124CrossRefGoogle Scholar
  18. Li D, Graham LD (2007) Epidermal secretions of terrestrial flatworms and slugs: Lehmannia valentiana mucus contains matrilin-like proteins. Comp Biochem Physiol B Biochem Mol Biol 148:231–244CrossRefPubMedGoogle Scholar
  19. Longtin J, Patel S, Eshaghi A et al (2011) Neuraminidase-inhibitor resistance testing for pandemic influenza A (H1N1) 2009 in Ontario, Canada. J Clin Virol 50:257–261. CrossRefPubMedGoogle Scholar
  20. Lorizate M, Kräusslich HG (2011) Role of lipids in virus replication. Cold Spring Harb Perspect Biol 3:1–20CrossRefGoogle Scholar
  21. Miletic I, Miric M, Lalic Z, Sobajic S (1991) Composition of lipids and proteins of several species of molluscs, marine and terrestrial, from the Adriatic Sea and Serbia. Food Chem 41:303–308. CrossRefGoogle Scholar
  22. Morita M, Kuba K, Ichikawa A et al (2013) The lipid mediator protectin D1 inhibits influenza virus replication and improves severe influenza. Cell 153:112–125. CrossRefPubMedGoogle Scholar
  23. Murphy RC (2014) Tandem mass spectrometry of lipids. Royal Society of Chemistry, CambridgeGoogle Scholar
  24. Nachbagauer R, Krammer F (2017) Universal influenza virus vaccines and therapeutic antibodies. Clin Microbiol Infect 23:222–228CrossRefPubMedPubMedCentralGoogle Scholar
  25. Nitsch-Osuch A, Brydak LB (2014) Influenza viruses resistant to neuraminidase inhibitors. Acta Biochim Pol 61:505–508PubMedGoogle Scholar
  26. Novoa B, Romero A, Álvarez ÁL et al (2016) Antiviral activity of myticin C peptide from mussel: an ancient defense against herpesviruses. J Virol 90:7692–7702. CrossRefPubMedPubMedCentralGoogle Scholar
  27. Pakarinen E (1994) The Importance of mucus as a defence against carabid beetles by the slugs Arion fasciatus and Deroceras reticulatum. J Molluscan Stud 60:149–155CrossRefGoogle Scholar
  28. Pielak RM, Chou JJ (2010) Flu channel drug resistance: a tale of two sites. Protein Cell 1:246–258. CrossRefPubMedPubMedCentralGoogle Scholar
  29. Renaud C, Kuypers J, Englund JA (2011) Emerging oseltamivir resistance in seasonal and pandemic influenza A/H1N1. J Clin Virol 52:70–78. CrossRefPubMedGoogle Scholar
  30. Ruangrung K, Suptawiwat O, Maneechotesuwan K et al (2016) Neuraminidase activity and resistance of 2009 pandemic H1N1 influenza virus to antiviral activity in bronchoalveolar fluid. J Virol 90:4637–4646. CrossRefPubMedPubMedCentralGoogle Scholar
  31. Samji T (2009) Influenza A: understanding the viral life cycle. Yale J Biol Med 82:153–159PubMedPubMedCentralGoogle Scholar
  32. Schoggins JW, Randall G (2013) Lipids in innate antiviral defense. Cell Host Microbe 14:379–385. CrossRefPubMedGoogle Scholar
  33. Shankaran S, Bearman GML (2012) Influenza virus resistance to neuraminidase inhibitors: implications for treatment. Curr Infect Dis Rep 14:155–160. CrossRefPubMedGoogle Scholar
  34. Shapaval V, Afseth NK, Vogt G, Kohler A (2014) Fourier transform infrared spectroscopy for the prediction of fatty acid profiles in Mucor fungi grown in media with different carbon sources. Microb Cell Fact 13:86CrossRefPubMedPubMedCentralGoogle Scholar
  35. Sheu TG, Deyde VM, Okomo-Adhiambo M et al (2008) Surveillance for neuraminidase inhibitor resistance among human influenza A and B viruses circulating worldwide from 2004 to 2008. Antimicrob Agents Chemother 52:3284–3292. CrossRefPubMedPubMedCentralGoogle Scholar
  36. Shi Y, Zhang B, Lu Y et al (2017) Antiviral activity of phenanthrenes from the medicinal plant Bletilla striata against influenza A virus. BMC Complement Altern Med 17:273. CrossRefPubMedPubMedCentralGoogle Scholar
  37. Simopoulos AP (2008) The importance of the omega-6/omega-3 fatty acid ratio in cardiovascular disease and other chronic diseases. Exp Biol Med (Maywood) 233:674–688. CrossRefGoogle Scholar
  38. Son K-N, Liang Z, Lipton HL (2015) Double-stranded RNA Is detected by immunofluorescence analysis in RNA and DNA virus infections, including those by negative-stranded RNA viruses. J Virol 89:9383–9392. CrossRefPubMedPubMedCentralGoogle Scholar
  39. Storms AD, Gubareva LV, Su S et al (2012) Oseltamivir-resistant pandemic (H1N1) 2009 virus infections, United States, 2010–11. Emerg Infect Dis 18:308–311. CrossRefPubMedPubMedCentralGoogle Scholar
  40. Tabakaeva OV, Tabakaev AV (2017) Lipids and fatty acids from soft tissues of the Bivalve Mollusk Spisula sachalinensis. Chem Nat Compd 1–5Google Scholar
  41. Tam VC, Quehenberger O, Oshansky CM et al (2013) XLipidomic profiling of influenza infection identifies mediators that induce and resolve inflammation. Cell. PubMedPubMedCentralCrossRefGoogle Scholar
  42. Taubenberger JK, Reid AH, Janczewski TA, Fanning TG (2001) Integrating historical, clinical and molecular genetic data in order to explain the origin and virulence of the 1918 Spanish influenza virus. Philos Trans R Soc B Biol Sci 356:1829–1839. CrossRefGoogle Scholar
  43. Teissier E, Zandomeneghi G, Loquet A et al (2011) Mechanism of inhibition of enveloped virus membrane fusion by the antiviral drug arbidol. PLoS One. CrossRefPubMedPubMedCentralGoogle Scholar
  44. Thomas MC, Kirk BB, Altvater J et al (2014) Formation and fragmentation of unsaturated fatty acid [M–2H+Na]-ions: stabilized carbanions for charge-directed fragmentation. J Am Soc Mass Spectrom 25:237–247. CrossRefPubMedGoogle Scholar
  45. Toledo-Piza AR, Maria DA, De Toledo-Piza AR et al (2013) Angiogenesis enhanced by Phyllocaulis boraceiensis mucus in human cells. FEBS J 280:5118–5127. CrossRefPubMedGoogle Scholar
  46. Toledo-Piza AR de, Figueiredo CA, Oliveira MI de et al (2016) The antiviral effect of mollusk mucus on measles virus. Antiviral Res 134:172–181. CrossRefPubMedGoogle Scholar
  47. Trufelli H, Famiglini G, Termopoli V, Cappiello A (2011) Profiling of non-esterified fatty acids in human plasma using liquid chromatography-electron ionization mass spectrometry. Anal Bioanal Chem 400:2933–2941. CrossRefPubMedGoogle Scholar
  48. Veit M, Engel S, Thaa B et al (2013) Lipid domain association of influenza virus proteins detected by dynamic fluorescence microscopy techniques. Cell Microbiol 15:179–189CrossRefPubMedGoogle Scholar
  49. Wilson J, Gobble C, Chickos J (2015) Vaporization, sublimation, and fusion enthalpies of some saturated and unsaturated long chain fatty acids by correlation gas chromatography. J Chem Eng Data 60:202–212. CrossRefGoogle Scholar
  50. Wu D, He Y (2014) Potential of spectroscopic techniques and chemometric analysis for rapid measurement of docosahexaenoic acid and eicosapentaenoic acid in algal oil. Food Chem 158:93–100CrossRefPubMedGoogle Scholar
  51. Xie Y, Li G, You J et al (2012) A novel labeling reagent of 2-(12-benzo[b]acridin-5-(12H)-yl)-acetohydrazide for determination of saturated and unsaturated fatty acids in traditional chinese herbs by HPLC-APCI-MS. Chromatographia 75:571–583. CrossRefGoogle Scholar
  52. Zhong J, Cui X, Shi Y et al (2013) Antiviral activity of Jinchai capsule against influenza virus. J Tradit Chin Med 33:200–204. CrossRefPubMedGoogle Scholar
  53. Zhu N, Dai X, Lin DS, Connor WE (1994) The lipids of slugs and snails: Evolution, diet and biosynthesis. Lipids 29:869–875. CrossRefPubMedGoogle Scholar
  54. Zhukova NV (2014) Lipids and fatty acids of nudibranch mollusks: potential sources of bioactive compounds. Mar Drugs 12:4578–4592. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Ana Rita de Toledo-Piza
    • 1
  • Maria Isabel de Oliveira
    • 2
  • Giuseppina Negri
    • 3
  • Ronaldo Zucatelli Mendonça
    • 1
  • Cristina Adelaide Figueiredo
    • 2
  1. 1.Laboratory of ParasitologyButantan InstituteSão PauloBrazil
  2. 2.Respiratory Infectious DiseasesAdolfo Lutz InstituteSão PauloBrazil
  3. 3.Department of Preventive MedicineFederal University of São PauloSão PauloBrazil

Personalised recommendations