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The prevention and treatment of viral diseases remains an urgent problem in modern medicine. Influenza virus infection is one of a challenging global issues with regular widespread epidemics or pandemics resulting in high mortality worldwide. The type A viruses are the most virulent human pathogens among the three influenza types and cause the most severe disease. A total of 18,500 laboratory-confirmed deaths were caused by the 2009 influenza A (H1N1) pandemic in the period April 2009–August 2010, but estimates of respiratory and cardiovascular mortality associated with this pandemic were 15 times higher than reported laboratoryconfirmed deaths (Dawood F S, et al., 2012). Sexually transmitted diseases represent another global healthcare problem. Worldwide, many people are infected with herpes si-mplex virus type 2 (HSV-2; a sexually transmitted disease that can cause recurrent, painful genital ulcers). Furthermore, HSV-2 is potentially lethal for neonates (Corey L, et al., 1988) and may facilitate the transmission of the human immunodeficiency virus (Holmberg S D, et al., 1988). The importance of the search for effective antiviral medications, including those of natural origin, is obvious.
The biologically active substances of Basidiomycetes— their fruit bodies, vegetative mycelia (biomass), spores, or cultural liquids, deprived of side effects—may form the basis of such drugs. A number of mushroom species have been identified as potential sources of drugs, with antiviral action against the influenza virus (Mothana R A A, et al., 2003; Ohta Y, et al., 2007; Ibragimova Zh B, et al., 2012; Vlasenko V A, et al., 2012; Fillippova E I, et al., 2012, 2013) and HSV-2 (Amoros M, et al., 1997; Oh K.-W, et al,. 2000; Kostina N E, et al., 2013). Fruiting bodies have been the object of most studies of the antiviral activity of Basidiomycetes (Takehara M, et al., 1979; Amoros M, et al., 1997; El-Mekkawy S., et al., 1998; Eo S-K, et al,. 1999; Piraino F, et al., 1999; Wang H X, et al., 2000; Oh K.-W, et al,. 2000; Awarhdh A N A, et al., 2003; Mothana R A A, et al., 2003; Ngai P H K, et al., 2003; Stamets P, 2005; Bruggemann R, et al., 2006; Ohta Y, et al., 2007; Faccin L C, et al., 2007; Gu C Q, et al., 2007; Lv H, et al., 2009; Kabanov A S, et al., 2011; Ibragimova Zh B, et al., 2012; Vlasenko V A, et al., 2012; Fillippova E I, et al., 2012, Fillippova E I, et al., 2013; Kostina N E, et al., 2013). However, the vegetative mycelium of these mushrooms is not inferior to fruit bodies in terms of the content of antiviral active substances (Hirose K, et al., 1987; Eo S-K, et al,. 2000; Liu J, et al., 2004; Ng T B, et al., 2006; Cardozo FTGS, et al., 2011; Prozenko M A, et al., 2012; Teplyakova T V, et al., 2012) and has a number of advantages in the cultivation process. Vegetative mycelium has a constant qualitative and quantitative composition, and its cultivation requires much less time and significantly less energy. Another little-studied aspect is the influence of the substrate on the antiviral activity of mycelium. Only Teplyakova T V, et al.(2012) have used a non-standard substrate (oat-corn water) in investigating the antiviral activity of polyporoid mushroom mycelium.
The aim of the current work was to assess the antiviral activity of extracts from Basidiomycetes mycelium, cultivated on a natural substrate (amaranth flour after CO2 extraction) in in vitro assays against influenza virus type A and HSV-2.
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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 ℃.
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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 ℃.
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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 ℃.
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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.
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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.
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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.
Mushrooms
Extracts from mushroom mycelia (biomass)
Cell lines and viruses
Cytotoxicity assay
Antiviral assay
Data analysis
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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 cellsToxic dose of sample (mg/mL)
RK-13 cells50 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
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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.), lgInfectivity of the virus (VAF titer) in RK-13 cells
(ID50 in lgTCID50/mL)Neutralization index
(ID50 control – ID50 exp.), lgP. 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.
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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.
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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.
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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.