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Volume 29 Issue 5
October 2014
Article Contents
Citation: Tetiana Krupodorova, Svetlana Rybalko, Victor Barshteyn. Antiviral activity of Basidiomycete mycelia against influenza type A (serotype H1N1) and herpes simplex virus type 2 in cell culture [J].VIROLOGICA SINICA, 2014, 29(5) : 284-290.  http://dx.doi.org/10.1007/s12250-014-3486-y

Antiviral activity of Basidiomycete mycelia against influenza type A (serotype H1N1) and herpes simplex virus type 2 in cell culture

  • Corresponding author: Victor Barshteyn, barmash14@gmail.com
  • Received Date: 03 July 2014
    Accepted Date: 15 October 2014
    Published Date: 24 October 2014
  • In this study, we investigated the in vitro antiviral activity of the mycelia of higher mushrooms against influenza virus type A (serotype H1N1) and herpes simplex virus type 2 (HSV-2), strain BH. All 10 investigated mushroom species inhibited the reproduction of influenza virus strain A/FM/1/47 (H1N1) in MDCK cells reducing the infectious titer by 2.0-6.0 lg ID50. Four species, Pleurotus ostreatus, Fomes fomentarius, Auriporia aurea, and Trametes versicolor, were also determined to be effective against HSV-2 strain BH in RK-13 cells, with similar levels of inhibition as for influenza. For some of the investigated mushroom species—Pleurotus eryngii, Lyophyllum shimeji, and Flammulina velutipes—this is the first report of an anti-influenza effect. This study also reports the first data on the medicinal properties of A. aurea, including anti-influenza and antiherpetic activities. T. versicolor 353 mycelium was found to have a high therapeutic index (324.67), and may be a promising material for the pharmaceutical industry as an anti-influenza and antiherpetic agent with low toxicity. Mycelia with antiviral activity were obtained in our investigation by bioconversion of agricultural wastes (amaranth flour after CO2 extraction), which would reduce the cost of the final product and solve some ecological problems.
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    1. Amoros M, Boustie J, Py M-L, Hervé V, Robin V, Girre L. 1997. Antiviral activity of Homobasidiomycetes: evaluation of 121 Basidiomycetes extracts on four viruses. Int J Pharmacognosy, 35:255-260.
        doi: 10.1076/phbi.35.4.255.13308

    2. Awadh A N A, Mothana R A A, Lesnau A, Pilgrim H, Lindequist U. 2003. Antiviral activity of Inonotus hispidus. Fitoterapia, 74:483-485.
        doi: 10.1016/S0367-326X(03)00119-9

    3. Bruggemann R, Orlandi J M, Benati F J, Faccin L C. 2006. Antiviral activity of Agaricus blazei murrill ss. heinem extract against human and bovine herpes viruses in cell culture. Braz J Microbiol, 37:561-565.

    4. Buchalo A S, Mitropolskay N Yu, Mukchaylova O B. 2011. Culture collections of mushrooms IBK. Kyiv: Altpress, p100. (In Ukrainian)

    5. Саrdoso F T G S, Camelini C M, Mascarello A, Rossi M J, Nunes R J, Barardi C R M, Mendonзa M M, Simхes C M 2011. Anti-herpetic activity of a sulfated polysaccharide from Agaricus brasiliensis mycelia. Antivir Res, 92:108-114.
        doi: 10.1016/j.antiviral.2011.07.009

    6. Corey L, Whitley R J, Stone E F, Whitley R J, Mohan K. 1988. Difference between herpes simplex virus type 1 and type 2 neonatal encephalitis in neurological outcome. Lancet, 331:1-4.
        doi: 10.1016/S0140-6736(88)90997-X

    7. Dawood F S, Iuliano A D, Reed C, Meltzer M I, Shay D K, Cheng P-Y, Bandaranayake D, Breiman R F, Brooks W A, Buchy P, Feikin D R, Fowler K B, Gordon A, Hien N T, Horby P, Huang Q S, Katz M A, Krishnan A, Lal R, Montgomery J M, Mølbak K, Pebody R, Presanis A M, Razuri H, Steens A, Tinoco Y O, Wallinga J, Yu H, Vong S, Bresee J, Widdowson M-A. 2012. Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: a modelling study. Lancet Infect Dis, 12:687-695.
        doi: 10.1016/S1473-3099(12)70121-4

    8. El-Mekkawy S, Meselhy M R, Nakamura N, Tezuka Y, Hattory M, Kakiuci N. 1998. Anti-HIV-1-protease substances from Ganoderma lucidum. Phytochemistry, 49:1651-1657.
        doi: 10.1016/S0031-9422(98)00254-4

    9. Eo S-K, Kim Y-S, Lee C-K, Han S-S. 1999. Antiviral activities of various water and methanol soluble substances isolated from Ganoderma lucidum. J Ethnopharmacol, 68:129-136.
        doi: 10.1016/S0378-8741(99)00067-7

    10. Eo S-K, Kim Y-S, Lee C-K, Han S-S. 2000. Possible mode of antiviral activity of acidic protein bound polysaccharide isolated from Ganoderma lucidum on herpes simplex viruses. J Ethnopharmacol, 72:475-481.
        doi: 10.1016/S0378-8741(00)00266-X

    11. Faccin L C, Benati F, Rinca V P, Mantovani M S, Soares S A, Gon-zaga M L, Nozawa C, Carvalho Linhares R E. 2007. Antiviral activity of aqueous and ethanol extracts and of an isolated poly-saccharide from Agaricus brasiliensis against poliovirus type 1. Lett Appl Microbiol, 45:24-28.
        doi: 10.1111/j.1472-765X.2007.02153.x

    12. Filippova E I, Mazurkova N A, Kabanov A S, Teplyakova T V, Ibragimova Z B, Makarevich E V, Mazurkov O Y, Shishkina L N. 2012. Antiviral properties of aqueous extracts isolated from higher Basidiomycetes as respect to pandemic influenza virus A (H1N1)2009. Mod Probl Sci Educ, 2: 1-7.(In Russian)

    13. Filippova E I, Kabanov A S, Skarnovich M O, Mazurkov O Yu, Teplyakova T V, Kosogova T A, Makarevich E V, Ibragimova Zh B, Troshkova G P, Shishkina L N, Mazurkova N A. 2013. Extracts of Basidiomycetes suppress reproduction of the virus of bird flu A (H5N1) in experiments in vitro and in vivo. Mod Probl Sci Educ, 5: 1-9.(In Russian)

    14. French A. 2007. Corious versicolor supplementation for recurrent herpes simplex. Clin J Mycol. 2:11-12.

    15. Gu C Q, Li J W, Chao F, Jin M, Wang X W, Shen Z Q. 2007. Isolation, identification and function of a novel anti-HSV-1 protein from Grifola frondosa. Antivir Res, 75:250-257.
        doi: 10.1016/j.antiviral.2007.03.011

    16. Hirose K, Hakozaki M, Kakuchi J, Matsunaga K, Yoshikumi C, Takahashi M, Tochikura T S, Yamamoto N. 1987. A biological response modifier, PSK, inhibits reverse transcriptase in vitro. Biochem Biophys Res Commun, 149:562-567.
        doi: 10.1016/0006-291X(87)90404-9

    17. Holmberg S D, Stewart J A, Gerber A R, Byers R H, Lee F K, O'Malley P M, Nahmias A J. 1988. Prior herpes simplex virus type 2 infection as a risk factor for HIV infection. J Am Med Ass, 259: 1048-1050.
        doi: 10.1001/jama.1988.03720070048033

    18. Ibragimova Zh B, Makarevich E V, Kosogova T A, Mazurkov O Yu, Teplyakova T V, Mazurkova N A. 2012. Anti-influenza virus activity of aqueous extract of macro- and micromycetes in experiments in vitro and in vivo. Mod Probl Sci Educ, 4:1-11.(In Russian)

    19. Kabanov A S, Kosogova T A, Shishkina L N, Teplyakova T V, Skarnobich M O, Mazurkova N A, Puchkova L I, Malkova E V, Stavsky E A, Drozdov I G. 2011. Study of antiviral activity of extracts obtained from basidial fungi against influenza viruses of different subtypes in experiments in vitro and in vivo. J Microbiol Epidemiol Immunobiol, 1: 40-43.(In Russian)

    20. Kostina N E, Ibragimova Zh B, Protsenko M A, Makarevich E V, Skarnovich M A, Philippova E I, Gorbunova I A, Vlasenko V A, Troshkova G P, Mazurkova N A, Shishkina L N. 2013. Isolation, characteristic and antiviral properties of biologically active agents of the highest mushrooms of Western Siberia. Mod Probl Sci Educ, 3:1-8.(In Russian)

    21. Liu J, Yang F, Ye L B, Yang X J, Timani K A, Zheng Y I, Wang Y H. 2004. Possible mode of action of antiherpetic activities of a proteoglycan isolated from mycelia of Ganoderma lucidum in vitro. J Ethnopharmacol, 95:265-272.
        doi: 10.1016/j.jep.2004.07.010

    22. Lv H, Kong Y, Yao Q, Zhang B, Leng FW, Bian H J, Balzarini J, Van Damme E, Bao J K.. 2009. Nebrodeolysin, a novel hemolytic protein from mushroom Pleurotus nebrodensis with apoptosis-inducingandanti-HIV-1effects. Phytomedicine, 16:198-205.
        doi: 10.1016/j.phymed.2008.07.004

    23. Mothana R A A, Awadh A N A, Jansen R, Wegner U, Mentel R, Lindequist U. 2003. Antiviral lanostanoid triterpenes from the fungus Ganoderma pfeifferi. Fitoterapia, 74:177-180.
        doi: 10.1016/S0367-326X(02)00305-2

    24. Ng T B, Wang H, Wan D C C. 2006. Polysaccharopeptide from the turkey tail fungus Trametes versicolor (L.:Fr.) Pilát inhibits human immunodeficiency virus type 1 reverse transcriptase and protease. Int J Med Mushr, 8:39-43.
        doi: 10.1615/IntJMedMushr.v8.i1

    25. Ngai P H K, Ng T B. 2003. Lentin, a novel and potent antifungal protein from shitake mushroom with inhibitory effects on ac-tivity of human immunodeficiency virus-1 reverse tran-scriptase and proliferation of leukemia cells. Life Sci, 73:3363-3374.
        doi: 10.1016/j.lfs.2003.06.023

    26. Oh K-W, Lee C-K, Kim Y-S, Eo S-K, Han S-S. 2000. Antiherpetic activities of acidic protein bound polysaccharide isolated from Ganoderma lucidum alone and in combinations with acyclovir and vidarabine. Ethnopharmacol, 72:221-227.
        doi: 10.1016/S0378-8741(00)00254-3

    27. Ohta Y, Lee J-B, Hayashi K, Fujita A, Park D K, Hayashi T. 2007. In vivo anti-influenza virus activity of an immunomodulatory acidic polysaccharide isolated from Cordyceps militaris grown on germinated soybeans. J Agr Food Chem, 55: 10194-10199.
        doi: 10.1021/jf0721287

    28. Piraino F, Brandt C R. 1999. Isolation and partial characterization of an antiviral, RC-183, from the edible mushroom Rozites caperata. Antivir Res, 43: 67-78.
        doi: 10.1016/S0166-3542(99)00035-2

    29. Prozenko M A, Berdasheva A V, Skarnovich M A, Kostina H E, Kosogova T A, Teplyakova T V, Troshkova G P. 2012. Com-parison of aqueous extracts of cultured mycelium and fruiting body of Fomes fomentarius. Int J Appl Fundam Res, 7:135-136.(In Russian)

    30. Stamets P. 2005. Medicinal polypores indigenous to the Pacific Northwest old growth forests of North America: screening for novel antiviral activity. In: Mushrooms Biology and Mushrooms Products. Proc 5th Intl Con, Shanghai: China, 431-439.

    31. Takehara M, Kuida K, Mori K. 1979. Antiviral activity of virus-like particles from Lentinus edodes (Shiitake). Arch Virol, 59:269-274.
        doi: 10.1007/BF01317423

    32. Teplyakova T V, Psurtseva N V, Kosogova T A, Mazurkova N A, Khanin V A, Vlasenko V A. 2012. Antiviral activity of Po-lyporoid mushrooms (higher Basidiomycetes) from Altai Mo-untains (Russia). Int J Med Mushr, 14: 37-45.
        doi: 10.1615/IntJMedMushr.v14.i1.40

    33. Vlasenko V A, Teplyakova T V, Mazurkova N A, Kosogova T A, Berdasheva A V, Psurtseva N V. 2012. Study of antiviral activity of medicinal fungi of the genus Phellinus s.l. in West Siberia. Bull Altai State Agr Univ, 4:29-31.(In Russian)

    34. Wang H X, Ng T B. 2000. Isolation of a novel ubiquitin-like protein from Pleurotus ostreatus mushroom with anti-human immu-nodeficiency virus, translation-inhibitory, and ribonuclease activities. Biochem Biophys Res Commun, 276: 587-593.
        doi: 10.1006/bbrc.2000.3540

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    Antiviral activity of Basidiomycete mycelia against influenza type A (serotype H1N1) and herpes simplex virus type 2 in cell culture

      Corresponding author: Victor Barshteyn, barmash14@gmail.com
    • 1. Institute of Food Biotechnology and Genomics of the National Academy of Sciences of Ukraine, Kyiv 04123, Ukraine
    • 2. Institute of Epidemiology and Infectious Diseases of the Academy of Medical Sciences of Ukraine, Kyiv 03038, Ukraine

    Abstract: In this study, we investigated the in vitro antiviral activity of the mycelia of higher mushrooms against influenza virus type A (serotype H1N1) and herpes simplex virus type 2 (HSV-2), strain BH. All 10 investigated mushroom species inhibited the reproduction of influenza virus strain A/FM/1/47 (H1N1) in MDCK cells reducing the infectious titer by 2.0-6.0 lg ID50. Four species, Pleurotus ostreatus, Fomes fomentarius, Auriporia aurea, and Trametes versicolor, were also determined to be effective against HSV-2 strain BH in RK-13 cells, with similar levels of inhibition as for influenza. For some of the investigated mushroom species—Pleurotus eryngii, Lyophyllum shimeji, and Flammulina velutipes—this is the first report of an anti-influenza effect. This study also reports the first data on the medicinal properties of A. aurea, including anti-influenza and antiherpetic activities. T. versicolor 353 mycelium was found to have a high therapeutic index (324.67), and may be a promising material for the pharmaceutical industry as an anti-influenza and antiherpetic agent with low toxicity. Mycelia with antiviral activity were obtained in our investigation by bioconversion of agricultural wastes (amaranth flour after CO2 extraction), which would reduce the cost of the final product and solve some ecological problems.

    • The mushroom species Auriporia aurea 5048 (Peck) Ryvarden, Flammulina velutipes 1878 (Curtis) Singer, Fomes fomentarius 355 (L.) Fr., Ganoderma lucidum 1900 (Curtis) P. Karst., Lentinus edodes 502 (Berk.) Singer, Lyophyllum shimeji 1662 (Kawam.) Hongo, Pleurotus eryngii 2015 (DC.) Quel., Pleurotus ostreatus 551 (Jacq.) P. Kumm., Schizophyllum commune 1768 Fr., and Trametes versicolor 353 (L.) Lloyd. were kindly supplied by the Culture Collection of Mushrooms (IBK) of the M.G. Kholodny Institute of Botany of the National Academy of Sciences of Ukraine (Buchalo A S, et al., 2011).

      The waste of flour from Amaranthus hybridusL . (variety “Ultra”; Mykolaiv Region, Ukraine, 2011) grains after CO2 extraction was used as the base of the culture medium. CO2 extraction conditions were as follows: pressure 7.2 MPa; temperature 24 ℃; time of extraction 2 h. Mycelial cultures were initially grown in Petri dishes (90 mm diameter) on culture medium with pH 6.0, composed of (g/L): 20.0 glucose, 3.0 yeast extract, 2.0 peptone, 1.0 K2HPO4 , 1.0 K2HPO4 , and 0.25 MgSO4•7H2 O. The liquid culture medium (substrate-60 g amaranth flour in 1 L distilled water) was sterilized by autoclaving for 20 min at 121 ℃. Flasks (250 mL) with 50 mL liquid medium were inoculated with three mycelial plugs of 8 mm diameter cut from the Petri dishes using a sterile borer at the stage of actively growing mycelia. Mycelia were grown as static cultures in flasks for 14 days at 26 ± 2 ℃.

    • The samples used in experiments were of mycelial extracts. Biomass was separated from the culture liquid, thoroughly washed with distilled water, dried with highvacuum freeze drying with a Cryodos-50 freeze dryer (Terrasa, Spain), and pounded in a porcelain mortar. A total of 300 mg biomass was suspended in 3 mL sterile 0.9 % NaCl solution and sonicated using an MSE-100 W sonifier (MSE, London, UK) for 20 min at amplitude 24 μm. The precipitate was separated using a Beckman Coulter J2-21 centrifuge (Beckman Coulter, Inc, Brea, CA, USA) for 20 min at 10,000 rpm, and the supernatant was used for studies. The extracts (samples) were refrigerated at -20 ℃.

    • MDCK (Madin–Darby canine kidney) and RK-13 (rabbit kidney) cells were grown in medium containing 90% RPMI 1640 medium (R8758, Sigma-Aldrich, Schnelldorf, Germany) supplemented with 10% fetal bovine serum (F7524, Sigma-Aldrich), penicillin (100 U/mL), streptomycin (100 U/mL), and kanamycin (50 U/mL). Cell cultures were maintained at 37 ℃ in a humidified atmosphere with 5% CO2 . The test viruses included influenza virus strain A/FM/1/47 (H1N1) (the infectious titer in MDCK cells was 106 median tissue culture infective dose [TCID50]/mL, hemagglutination titer 1:256) and HSV-2 strain BH (infectious titer 106 TCID50/mL) from the D.I. Ivanovsky Institute of Virology of the Russian Academy of Medical Sciences (Moscow, Russia). Stocks of influenza virus strain A/FM/1/47 (H1N1) and HSV-2 were stored at -70 ℃.

    • MDCK and RK-13 cells were plated onto 96-well plates and incubated at 37 ℃ in a humidified atmosphere with 5% CO2. After 48 h, the monolayered cells were incubated in the presence of a variety of concentrations (range 0.77–50 mg/mL) of the test samples. Plates were incubated for 5 days under the same conditions. The cytopathic effect in the cell monolayer was monitored daily by the cells’ morphology. Maximum tolerated concentrations (i.e., the maximal non-toxic concentrations) were determined by evaluating the cytopathic effect.

    • The effects of the studied mushroom mycelium extracts on the process of virus multiplication were investigated in MDCK cells (for influenza virus A/FM/1/47 [H1N1]) and RK-13 cells (for herpes virus type 2). Both cell cultures were pretreated with dilutions of the clarified extract (range 0.77–50 mg/mL) for 30–60 min at 37 ℃ and then infected with the viruses. The cells infected with virus-containing fluid were incubated at 37 ℃ for 3 days. The infectious titers of viruses, presence of virus-specific antigens, and hemagglutinin levels were assayed in culture liquid. The infectious titer was evaluated using a series of 10-fold dilutions of virus-containing culture liquid. The half maximal effective concentration (EC50) inhibiting viral reproduction was calculated.

    • All experiments were confirmed in three independent replicates. The antiviral activity of the mycelium was determined as the reduction factor (log10) of the viral titer by comparison with untreated controls. The standard deviation in the reduction of virus titer was about 0.5 log10. Mycelium was defined as active if the viral yield decreased by ≥2 log10 at the maximum tolerated concentration.

    • As the first step in screening antiviral activity, the cytotoxicity of various concentrations of mycelial extracts was evaluated using an inhibition assay in MDCK and RK-13 cell plaques (with similar cytopathic effect). A maximum tolerated concentration of 25.0 mg/mL was determined for four species (A. aurea, F. fomentarius, F. velutipes, and T. versicolor), while the extracts of the other species, particularly two edible mushrooms (P. eryngii and L. edodes), were more toxic (Table 1).

      Sample Concentration
      (mg/mL)
      Toxic dose of sample (mg/mL)
      MDCK cells
      Toxic dose of sample (mg/mL)
      RK-13 cells
      50 25 12.5 6.2 3.1 1.55 0.77 50 25 12.5 6.2 3.1 1.55 0.77
      Substrate* 10/10 0/10 0/10 0/10 0/10 0/10 0/10 10/10 0/10 0/10 0/10 0/10 0/10 0/10
      F. fomentarius 10/10 0/10 0/10 0/10 0/10 0/10 0/10 10/10 0/10 0/10 0/10 0/10 0/10 0/10
      F. velutipes 10/10 0/10 0/10 0/10 0/10 0/10 0/10 10/10 0/10 0/10 0/10 0/10 0/10 0/10
      A. aurea 10/10 0/10 0/10 0/10 0/10 0/10 0/10 10/10 0/10 0/10 0/10 0/10 0/10 0/10
      T. versicolor 10/10 0/10 0/10 0/10 0/10 0/10 0/10 10/10 0/10 0/10 0/10 0/10 0/10 0/10
      P. ostreatus 10/10 10/10 0/10 0/10 0/10 0/10 0/10 10/10 10/10 0/10 0/10 0/10 0/10 0/10
      S. commune 10/10 10/10 0/10 0/10 0/10 0/10 0/10 10/10 10/10 0/10 0/10 0/10 0/10 0/10
      G. lucidum 10/10 10/10 10/10 0/10 0/10 0/10 0/10 10/10 10/10 10/10 0/10 0/10 0/10 0/10
      L. shimeji 10/10 10/10 10/10 10/10 0/10 0/10 0/10 10/10 10/10 10/10 10/10 0/10 0/10 0/10
      P. eryngii 10/10 10/10 10/10 10/10 10/10 0/10 0/10 10/10 10/10 10/10 10/10 10/10 0/10 0/10
      L. edodes 10/10 10/10 10/10 10/10 10/10 0/10 0/10 10/10 10/10 10/10 10/10 10/10 0/10 0/10
      0/10: no cytopathic effect; 10/10: cytopathic effect (complete destruction of monolayer cells). *: 60 g amaranth in 1 L distilled water (the liquid culture medium). All experiments were confirmed in three independent replicates.

      Table 1.  Cytotoxicity of mycelial extracts from of MDCK and RK-13 cells

      The investigated mycelia had different potential antiviral activities against influenza virus strain A/FM/1/47 (H1N1), with inhibition of infectious titers ranging from 2.0 to 6.0 lg ID50 . Antiviral activity according to the inhibition of infectious titer increased in the following order: A. aurea=F. fomentarius > P. ostreatus=L. shimeji=L. edodes > P. eryngii=F. velutipes=G. lucidum > S. commune > T. versicolor. G. lucidum and T. versicolor generated the strongest antiviral effects, with T. versicolor showing the highest activity (Table 2). The anti-influenza activities of P. eryngii, L. shimeji, F. velutipes, and A. aurea have not previously been presented in the literature.

      Sample MTC
      (mg/mL)
      EC50
      (mg/mL)
      Therapeutic index
      (MTC/EC50)
      P. eryngii 1.55 5 0
      L. shimeji 3.1 0.62 5.0
      P. ostreatus 12.5 2.5 6.0
      S. commune 12.5 0.62 20.16
      L. edodes 1.55 0.077 20.12
      F. velutipes 25 1.25 20.0
      F. fomentarius 25 0.62 40.32
      A. aurea 25 0.62 40.32
      G. lucidum 0.2 0.077 80.5
      T. versicolor 25 0.077 324.67
      EC50: half maximal effective concentration; MTC: maximum tolerated concentration. All experiments were confirmed in three independent replicates

      Table 2.  Antiviral activity of samples in MDCK cells infected with influenza virus strain A/FM/1/47 (H1N1)

      Only four of the 10 studied species demonstrated activity against HSV-2: mycelial extracts of P. ostreatus, F. fomentarius, A. aurea and T. versicolor significantly inhibited HSV-2 replication in RK-13 cells (Table 3). The highest therapeutic indices (selectivity indices) were identified for A. aurea and T. versicolor, at 161.29 and 324.67, respectively. This study is the first to demonstrate the activity of A. aurea mycelium against HSV-2.

      Sample MTC
      (mg/mL)
      EC50
      (mg/mL)
      Therapeutic index
      (MTC/EC50)
      S. commune 12.5 0 0
      F. velutipes 25 0 0
      P. eryngii 1.55 0 0
      L. shimeji 3.1 0 0
      L. edodes 1.55 0 0
      G. lucidum 6.2 0 0
      F. fomentarius 25 0.62 40.32
      P. ostreatus 12.5 0.155 80.64
      А. aureа 25 0.155 161.29
      T. versicolor 25 0.077 324.67
      EC50: half maximal effective concentration; MTC: maximum tolerated concentration. All experiments were confirmed in three independent replicates.

      Table 3.  Antiviral activity of samples in RK-13 cells infected with herpes simplex virus type 2, strain BH

    • Recently, the search for natural substances as raw materials for the pharmaceutical industry has revived interest in medicinal and edible mushrooms. While most studies have isolated therapeutically active substances from the fruiting bodies, the use of mycelium makes it possible to obtain products of consistent quality more quickly and at lower cost. Data regarding the absence of toxicity of various products have been obtained from cultivation, mainly on synthetic or semisynthetic substrates. The mycelium toxicity (non-critical) obtained in our studies (Table 1) can be related to the natural substrate (its toxicity), has been used by us.

      The influenza and herpes viruses have been a particular focus of research. The antiviral activity of mushroom preparations has been evaluated by researchers using different indicators, including the index of neutralization (Amoros M, et al., 1997; Ibragimova Zh D, et al., 2012; Teplyakova T V, et al., 2012; Filippova E I, et al., 2012; Kostina N E, et al., 2013) and the therapeutic index (Eo S-K, et al,. 1999; Oh K-W, et al,. 2000; Liu J, et al., 2004; GuC-Q, et al., 2007; Cardoso FTGS, et al., 2011). In the current study, we have shown that the magnitude of the neutralization index is not directly proportional to the magnitude of the therapeutic index (Table 2 to 4). This is to be expected, since the therapeutic index is the ratio of the minimal effective dose of a chemotherapeutic agent to the maximal tolerated dose, and this ratio can be very small (even if the neutralization index is large). So, water- and methanol-soluble substances isolated from the carpophores of G. lucidum have shown antiviral activity against influenza A virus A/Equine/2/Miami/1/63 strain, but the therapeutic index was zero (Eo S-K, et al,. 1999). In contrast, the current study found a high therapeutic index value (80.5) for G. lucidum mycelial activity against influenza virus strain A/FM/1/47 (H1N1). According to our data, F. fomentarius mycelium shows similar activity (Table 4) to preparations from F. fomentarius 11-72, which has been reported to inhibit influenza virus A/Aichi/2/68 in MDCK cell culture at 2.4 lg (Ibragimova Zh D, et al., 2012). The fruiting bodies of T. versicolor have been reported to slightly inhibit influenza virus A/Chicken/Kurgan/05/2005 (H5N1) in vivo (Ibragimova Zh B, et al., 2012). Our results (Table 4) for T. versicolor 353 mycelium were significantly higher (6.0 lg) than those reported in similar studies: water extracts of T. versicolor 2263 mycelium have been reported to repress viruses A/Chicken/Kurgan/05/2005 (H5N1)(2.5 lg) and A/Aichi/2/68 (H3N2)(0.5 lg) on MDCK cells (Teplyakova TV, et al., 2012). In the current study, L. shimeji, L. edodes and P. ostreatus mycelia showed neutralization indices for influenza virus strain A/FM/1/47 (H1N1) that were similar to the values obtained by Filippova EI, et al.(2012) for fruiting bodies of Ganoderma applanatum, Inonotus obliquus and Laetiporus sulphureus against pandemic influenza virus A/Moscow/226/2009 (H1N1).

      Sample virus H1N1, strain A/FM/1/47 virus HSV-2, strain BH
      Infectivity of the virus (VAF titer) in MDCK cells
      (ID50 in lgTCID50/mL)
      Neutralization index
      (ID50 control – ID50 exp.), lg
      Infectivity of the virus (VAF titer) in RK-13 cells
      (ID50 in lgTCID50/mL)
      Neutralization index
      (ID50 control – ID50 exp.), lg
      P. eryngii 2.0 4.0 6.0 0
      L. shimeji 3.0 3.0 6.0 0
      P. ostreatus 3.0 3.0 3.5 2.5
      S. commune 1.0 5.0 6.0 0
      L. edodes 3.0 3.0 6.0 0
      F. velutipes 2.0 4.0 6.0 0
      F. fomentarius 4.0 2.0 3.0 3.0
      A. aurea 4,0 2.0 4.0 2.0
      G. lucidum 2.0 4.0 6.0 0
      T. versicolor 0 6.0 0 6.0
      Substratea 6.0 0 6.0 0
      Control (virus) 6.0b 6.0c
      Note: HSV-2, herpes simplex virus type 2; TCID50, median tissue culture infective dose. The standard deviation for the reduction of virus titer was approximately 0.5 log10. a: 60 g amaranth in 1 L distilled water (the liquid culture medium). b: virus H1N1, strain A/FM/1/47; c: virus HSV-2, strain BH. All experiments were confirmed in three independent replicates.

      Table 4.  Antiviral activity of samples

      Our data demonstrating sufficiently high antiherpetic activity of P. ostreatus mycelium (neutralization index of 2.5 lg and therapeutic index of 80.64; Table 3 and 4) are in contrast to those indicating an absence of antiherpetic activity for P. ostreatus fruiting bodies in cell culture (Amoros M, et al., 1997). Our data also show significant antiherpetic activity for F. fomentarius mycelium (neutralization index 3.0 lg and therapeutic index 40.32; Table 3 and 4), in contrast to data showing the absence of such activity in fruiting bodies of this fungus in cell culture (Kostina N E, et al., 2013). Conversely, G. lucidum mycelium did not show antiherpetic action in our studies, unlike the results of other researchers who used polysaccharide–protein complexes isolated from G. lucidum fruiting bodies and mycelia, including APBP (activity against HSV-2 strain 233)(Oh KW, et al., 2000) and proteoglycan (activity against HSV-2 G strain ATCC VR-734) on Vero cells (Liu J. et al, 2004.). The selectivity index values for P. ostreatus, A. aurea, and T. versicolor mycelia were significantly higher in our studies than in similar experiments with G. lucidum mycelium against HSV-2 (Oh KW, et al., 2000; Liu J, et al., 2004). Our results indicate antiherpetic activity of T. versicolor mycelium, in contrast to other investigations with fruit bodies of this species (Amoros M, et al., 1997; Kostina NE, et al., 2013). Moreover, in the current study, T. versicolor showed the highest therapeutic index (324.67) among the studied fungi. A clinical trial in which a food additive (biomass) of T. versicolor reduced the frequency and even stopped outbreaks of HSV-2 in pregnant patients (French A, 2007) confirms the high antiherpetic activity of this fungus.

      It should be noted that the effective doses of P. ostreatus and A. aurea mycelium for HSV-2 neutralization were significantly lower than those for influenza virus A, with 13-fold higher antiherpetic activity than anti-influenza activity for P. ostreatus and fourfold higher antiherpetic activity for A. aurea. In contrast, the therapeutic indices of F. fomentarius and T. versicolor were identical for both viruses. Such differences may be caused by mushroom species specificity, variability in the biologically active substances, and different mechanisms of antiviral activity at the interaction between the mycelia and the virus. The high efficacy of viral replication inhibition might hint that the inhibitory activity of the tested substances occurs late in viral replication via impairment of viral protein synthesis.

      This study of the antiviral activity of the mycelia of 10 mushroom species suggests that A. aurea, F. velutipes, F. fomentarius, G. lucidum, L. edodes, L. shimeji, P. eryngii, P. ostreatus, S. commune, and T. versicolor have antiviral activity against influenza virus A/FM/1/47 (H1N1), while A. aurea, F. fomentarius, G. lucidum, and T. versicolor have antiviral activity against HSV-2, strain BH. For some of the investigated species, this is the first report of anti-influenza (P. eryngii, L. shimeji, F. velutipes, A. aurea) and antiherpetic (A. aurea) effects. To the best of our knowledge, there have been no previous reports on the potential medicinal properties of A. aurea.

      The wood-decaying medicinal Basidiomycete T. versicolor showed the highest therapeutic index (324.67 for both viruses). Therefore, T. versicolor 353 and its biologically active substances may be promising source materials for the pharmaceutical industry as antiinfluenza and antiherpetic agents with low toxicity. The use of products obtained in the biotechnological processing of agricultural waste (in this case, amaranth flour after CO2 extraction) and its conversion by fungi is one of the first steps in this direction of investigations, which deserves to be further explored. Further investigations will be needed to determine the most effective solvent for extracting biologically active mycelial substances; to study the qualitative and quantitative composition, antiviral activity, and mechanisms of antiviral activity of the extracted mycelial substances; and to confirm the effectiveness of T. versicolor mycelium in vivo.

    • The authors would like to thank Professor A.S. Buchalo for providing the fungal strains from the IBK Collection that we used in this study.

    • All the authors declare that they have no competing interest. This article does not contain any studies with human or animal subjects performed by any of the authors.

    • Tetiana Krupodorova and Victor Barshteyn conceived the study, cultivated mushrooms, obtained mycelia, participated in data analysis, and wrote the manuscript. Svetlana Rybalko carried out the antiviral assay in cell cultures and participated in data analysis.

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