Antiviral Agents From Fungi : Diversity, Mechanisms and Potential Applications

This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Title: Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY 4.0 https://creativecommons.org/licenses/by/4.0/ Antiviral Agents From Fungi : Diversity, Mechanisms and Potential Applications © 2018 Linnakoski, Reshamwala, Veteli, Cortina-Escribano, Vanhanen and Marjomäki Published version Linnakoski, Riikka; Reshamwala, Dhanik; Veteli, Pyry; Cortina-Escribano, Marta; Vanhanen, Henri; Marjomäki, Varpu Linnakoski, R., Reshamwala, D., Veteli, P., Cortina-Escribano, M., Vanhanen, H., & Marjomäki, V. (2018). Antiviral Agents From Fungi : Diversity, Mechanisms and Potential Applications. Frontiers in Microbiology, 9, Article 2325. https://doi.org/10.3389/fmicb.2018.02325 2018 fmicb-09-02325 October 1, 2018 Time: 17:2 # 1 REVIEW published: 02 October 2018 doi: 10.3389/fmicb.2018.02325 Edited by: Juan-Carlos Saiz, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Spain Reviewed by: Ulrike Lindequist, University of Greifswald, Germany Taisuke Izumi, Henry M. Jackson Foundation, United States *Correspondence: Riikka Linnakoski [email protected] Specialty section: This article was submitted to Virology, a section of the journal Frontiers in Microbiology Received: 03 July 2018 Accepted: 11 September 2018 Published: 02 October 2018 Citation: Linnakoski R, Reshamwala D, Veteli P, Cortina-Escribano M, Vanhanen H and Marjomäki V (2018) Antiviral Agents From Fungi: Diversity, Mechanisms and Potential Applications. Front. Microbiol. 9:2325. doi: 10.3389/fmicb.2018.02325 Antiviral Agents From Fungi: Diversity, Mechanisms and Potential Applications Riikka Linnakoski1*, Dhanik Reshamwala2, Pyry Veteli1, Marta Cortina-Escribano3, Henri Vanhanen3and Varpu Marjomäki2 1Natural Resources Institute Finland (Luke), Helsinki, Finland, 2Division of Cell and Molecular Biology, Department of Biological and Environmental Science, Nanoscience Center, University of Jyväskylä, Jyväskylä, Finland, 3Natural Resources Institute Finland (Luke), Joensuu, Finland Viral infections are amongst the most common diseases affecting people worldwide. New viruses emerge all the time and presently we have limited number of vaccines and only few antivirals to combat viral diseases. Fungi represent a vast source of bioactive molecules, which could potentially be used as antivirals in the future. Here, we have summarized the current knowledge of fungi as producers of antiviral compounds and discuss their potential applications. In particular, we have investigated how the antiviral action has been assessed and what is known about the molecular mechanisms and actual targets. Furthermore, we highlight the importance of accurate fungal species identification on antiviral and other natural products studies. Keywords: antiviral agents, antiviral mechanisms, endophytes, fungal secondary metabolites, medicinal mushrooms, natural products INTRODUCTION Viruses cause serious outbreaks in all continents leading to difficult symptoms and mortality, and enormous economic burden for society. In addition, the constant emergence of new serotypes in virus groups that have a high mutation rate and low fidelity for viral replication adds challenges in combatting against these viruses. Viruses can be divided into those containing a lipid envelope and those whose genome is only covered by a protein shell. Enveloped viruses are less stable and more prone to degradation when treated with lipid solvents. Their infection mechanisms are usually based on the presence of fusogenic peptides in the lipid envelope leading to a merge of viral and cellular membranes. The non-enveloped viruses are much more stable and may stay active in wastewaters and on surfaces from several weeks to months. The non-enveloped viruses such as Noro viruses and enteroviruses are therefore causing outbreaks that are difficult to handle. In addition, they show little sensitivity to chemical disinfectants (Wutzler and Sauerbrei, 2004;Chan and Abu Bakar, 2005). Thus, there is a need for both vaccines and antivirals to encounter viral infections. However, the development of vaccines against a wide range of newly emerging virus serotypes is challenging, and currently vaccines are available only against a handful of viruses. In addition, vaccination cannot help if the infection is already present in the system. The antiviral drugs inhibit the virus infection either by specifically targeting the viral proteins or the host cellular factors that the viruses exploit for their reproduction (Clercq, 2002). However, the problem in using viral proteins as drug targets is the high rate at which viruses produce mutant resistant strains against them (De Palma et al., 2008). Cellular factors exploited by viruses also serve Frontiers in Microbiology | www.frontiersin.org 1October 2018 | Volume 9 | Article 2325 fmicb-09-02325 October 1, 2018 Time: 17:2 # 2 Linnakoski et al. Antiviral Agents From Fungi as potential drug targets. However, they cannot be considered automatically as reliable targets, since viruses may deviate from their original pathway and still cause an effective infection (Van der Linden et al., 2015). Also, targeting cellular factors might have an adverse effect on normal functioning of the host cells. Furthermore, the mechanisms of non-enveloped viruses to break the host cell membrane barrier is less well known, which forms an additional challenge in developing strategies against these viruses. An antiviral drug has to fulfill a set of prerequisites when undergoing preclinical and clinical trials. A vital requirement is that the drug should be effective in inhibiting the virus infection without causing any cytotoxicity and with minimal side effects to the host cells. In addition, a drug should be able to completely inhibit the virus infection, partial inhibition leads to the generation of drug resistant mutant strains. Due to these prerequisites, only a handful of synthetic antiviral drugs have made it past the clinical phase. Until today, the successful ‘one bug–one drug’ approach has been used for antiviral drug development. However, today the focus has shifted toward designing broad-spectrum antivirals, which can act on multiple viruses by targeting a common but essential viral function (Vigant et al., 2015). Combinatorial chemistry is nowadays a preferred approach adapted by major drug companies for discovering pharmacologically significant compounds (Strobel and Daisy, 2003). Although combinatorial chemistry approach has proven successful in optimizing structures of drug compounds, only one de novo new chemical entity (NCE) has been approved as a drug [sorafenib (Nexavar) acting as anti-tumor] in these 25 plus years from this method (Cragg and Newman, 2007). On the other hand, bioactive compounds isolated from natural biological sources offer a vast and unexplored diversity of chemical structures, unmatched by even the biggest combinatorial databases (Strobel and Daisy, 2003). Since thousands of years, natural products have served as traditional medicine and still provide the most affordable treatment for diseases in many developing countries (Amzat and Razum, 2018). Around 40% of modern drugs and 49% of new chemical products registered by the United States Food and Drug Administration (FDA) are based on natural products or their derivatives (Brewer, 2000). Bioactive compounds are naturally derived metabolites and/or by-products from microorganisms, plants, or animals (Baker et al., 2000). Since the past 25 years, bioactive compounds from many traditional medicinal plants have been screened for their antiviral activity by various research groups in Asia, Far East, Europe, and America (Jassim and Naji, 2003). Particular importance for novel drug discoveries has been bioactive molecules of fungal origin. Especially fungi growing in unique environments such as endophytic and marine fungi are being constantly explored for their antibacterial and antifungal potential. During the past decade, many novel bioactive natural products possessing cytotoxic, anticancer, antibacterial or antifungal activities have been discovered from marine fungi (Mayer et al., 2013;Cheung et al., 2014;Singh et al., 2015). Fungi potentially contain and/or produce several effective molecules that could also be used as antivirals for other hosts. The discovery and characterization of fungal compounds having antiviral activities is an emerging field of research, and several compounds have already been identified as promising. In this review, we go through the present knowledge of fungi-derived extracts and other bioactive agents against viral infection. We especially focus on how the antiviral action has been assessed and how much is known about the mechanisms of action and actual targets. FUNGI AS A SOURCE OF ANTIVIRAL AGENTS - AN OVERVIEW The kingdom Fungi represents a rich source of various biologically active compounds. During the past decades, thousands of compounds with diverse biological activities have been recognized and continue to be investigated. Fungal compounds with antiviral activities are less extensively studied, but also number of these investigations is on the increase. We have compiled a list of fungal orders with reported positive antiviral activities (Table 1) and also mapped this information on illustrative phylogenetic trees (Figures 1–3). Fungal species with reported antiviral activities are given in Supplementary Table S1. These demonstrate that the previous studies have focused on the late-diverging fungal phyla (Ascomycota and Basidiomycota) and on rather limited taxonomic groups, while several remaining completely uninvestigated. Particularly well-studied for their biologically active compounds, including antivirals, are edible and medicinal mushrooms. Another group of fungi that has been a focus of interest are endophytic fungi, particularly those that grow in marine habitats. The biologically active compounds can be roughly divided into two major groups of molecules; the high-molecular weight compounds present in the extracts and products derived from the fruiting bodies of edible and medicinal mushrooms, and the small organic molecules (secondary metabolites) excreted by the endophytic and other fungi in a liquid culturing (fermentation) setups. Further rough division can be made when considering the repertoire of antiviral compounds found from different fungal taxonomic groups. Mapping the antiviral compounds on the larger phylogeny of Fungi (Figure 1) demonstrates that all the currently known secondary metabolites have been identified from Ascomycota and Basidiomycota. Ascomycota with antiviral activities includes endophytes and other microfungi restricted to limited number of orders (Figure 2), while the edible and medicinal mushrooms in the Agaricales and Polyporales (Basidiomycota) (Figure 3) are recognized as a source of high-molecular weight compounds. The increasing number of published fungal genome data combined with the traditional bioactivity screening methods has provided novel insights into the true capacity of fungi as producers of bioactive compounds (Bergmann et al., 2007;Khaldi et al., 2010; Brakhage, 2013;Clevanger et al., 2017). These studies indicate that differences exist between these two phyla in a number of secondary metabolites biosynthetic gene clusters and their dominance in their genomes; basidiomycetes typically having fewer compared to ascomycetes (Brakhage, 2013). However, the Frontiers in Microbiology | www.frontiersin.org 2October 2018 | Volume 9 | Article 2325 fmicb-09-02325 October 1, 2018 Time: 17:2 # 3 Linnakoski et al. Antiviral Agents From Fungi TABLE 1 | Fungal orders with positive antiviral activities. Phylum Order Virus∗Reference Ascomycota Amphisphaeriales EV711, HIV-I1Li et al., 2008;Wang J. et al., 2014;Jia et al., 2015 Capnodiales H1N11Peng et al., 2013;Wu et al., 2014 Chaetothyriales HIV-I4Ondeyka et al., 2003;Mlinaric et al., 2005 Diaporthales HIV-14, HSV-11Jayasuriya et al., 2003;Bunyapaiboonsri et al., 2010 Dothideales HSV-15Isaka et al., 2007 Eurotiales EV712, DENV3, H1N12, HIV-14, H3N22, JEV1, Zika virus2 Omura et al., 1993;Matsuzaki et al., 1995;Singh et al., 2003a;Shiomi et al., 2005;Sebastian et al., 2011;Zhang et al., 2011;Gao et al., 2013a;He et al., 2013;Bashyal et al., 2014;Fang et al., 2014;Peng et al., 2014;Wang J.-F. et al., 2014;Stierle and Stierle, 2015;Yu et al., 2016;Raekiansyah et al., 2017 Glomerellales HIV-14Mlinaric et al., 2005 Helotiales HSV-11Rowley et al., 2003 Hypocreales EV712, HIV-14, HSV-11, H1N11,4,H3N21,4 Hazuda et al., 1999;Yoshimoto et al., 1999;Minagawa et al., 2002;Singh et al., 2003a,b;Sawadjoon et al., 2004;Mlinaric et al., 2005;Jiang et al., 2011; Ma et al., 2013;Li et al., 2014;Zhao et al., 2017;Pang et al., 2018 Microascales HIV-14Mlinaric et al., 2005 Ophiostomatales HIV-14Mlinaric et al., 2005 Pezizales HIV-14Pérez et al., 2014 Pleosporales HIV-14, HSV-11Hazuda et al., 1999;Singh et al., 2002;Guo et al., 2009;Shushni et al., 2011; Bashyal et al., 2014;Zhang et al., 2015 Saccharomycetales HIV-14Mlinaric et al., 2005 Sordaliales HIV-14, influenza A and B4Mlinaric et al., 2005;Sacramento et al., 2015 Xylariales H1N12, HIV-14, HSV-11Hazuda et al., 1999;Pittayakhajonwut et al., 2005;Zhang et al., 2016 Basidiomycota Agaricales BoHV-11,3, H1N12, HCV5, HBV4,5, HCV5, HIV-12, HSV-11,2,3, HSV-21,2, influenza A2, polio2, RSV1,2, vaccinia1, VS1, VZV2, WEE2 Kandefer-Szersze ´ n et al., 1980;Amoros et al., 1997;Saboulard et al., 1998; Piraino and Brandt, 1999;Wang and Ng, 2000, 2001;Sorimachi et al., 2001; Lehmann et al., 2003;Chen et al., 2004;Mlinaric et al., 2005;Bruggemann et al., 2006;Grinde et al., 2006;Faccin et al., 2007;Razumov et al., 2010;Zhu et al., 2010;Cardozo et al., 2011, 2014;Gao et al., 2013b;Yamamoto et al., 2013;Krupodorova et al., 2014 Boletales HIV-14, HSV-15, vaccinia1, VS1Kandefer-Szersze ´ n et al., 1980;Kanokmedhakul et al., 2003;Mlinaric et al., 2005 Cantharellales HIV-14, vaccinia1Kandefer-Szersze ´ n et al., 1980;Mlinaric et al., 2005 Gomphales vaccinia1Kandefer-Szersze ´ n et al., 1980 Hymenochaetales influenza A and B4Ichimura et al., 1998;Awadh Ali et al., 2003; Polyporales BoHV-11, EBV-A3, EV712, H1N12, H3N22, HCV2, HHV-12,4, HIV4, HSV-11,2,4, HSV-21,2, influenza A2, MCMV1,2, measles2, mumps2, polio1,2,3, PV-11, VSV2, WEE2, EMCV2,4 Hirose et al., 1987;Okada and Minamishima, 1987;Tochikura et al., 1987, 1988;Suzuki et al., 1989;Sorimachi et al., 1990;Sarkar et al., 1993;Amoros et al., 1997;Collins and Ng, 1997;El-Mekkawy et al., 1998;Min et al., 1998; Eo et al., 1999a,b, 2000;Kim et al., 2000;Iwatsuki et al., 2003;Mothana et al., 2003;Ngai and Ng, 2003;Singh et al., 2003a;Mlinaric et al., 2005; Niedermeyer et al., 2005;Gu et al., 2007;El Dine et al., 2008;Sato et al., 2009; Razumov et al., 2010;Rincão et al., 2012;Teplyakova et al., 2012; Krupodorova et al., 2014;Zhang et al., 2014;Matsuhisa et al., 2015; Mizerska-Dudka et al., 2015 Russulales HIV-14, vaccinia1, VS1Kandefer-Szersze ´ n et al., 1980;Mlinaric et al., 2005;Wang et al., 2007 Categories for antiviral methods used in the studies: 1plague reduction assay; 2CPE (cytopathic effect) inhibition assay; 3microscope immunofluorescent assay; 4Specific protease assay; 5other. ∗WEE, Western equine encaphilitis; VZV, Varicella zoster; RSV, respiratory syncytial virus; HCV, hepatitis C virus; HBV, hepatitis B virus; MCMV, murine cytomegalovirus; VSV, vesicular stomatitis virus; EBV, Epstein-Barr virus; PV-1, poliovirus 1; WNV, west nile virus; HHV, human herpes virus; EMCV, encephalomyocarditis virus; DENV, Dengue Virus; JEV, Japanese encephalitis virus. reported differences between Ascomycota and Basidiomycota reflect also to the bias from the different methods that have been commonly used in screening their biologically active compounds, not differences in their true arsenals of bioactive compounds. The most recent estimates predicting fungal species diversity indicate that only 3–8% of existing fungal species are discovered and described (Hawksworth and Lücking, 2017). Therefore, the fungi investigated and found to have potential positive antiviral activities thus far represent only a minute fraction of these organisms and their potential. Edible and Medicinal Mushrooms Mushrooms have been an important part of our diet for centuries due to their nutritional properties. Their rich content in proteins, carbohydrates, minerals, vitamins, unsaturated fatty acids and low values of fat and energy content makes them a valuable food Frontiers in Microbiology | www.frontiersin.org 3October 2018 | Volume 9 | Article 2325 fmicb-09-02325 October 1, 2018 Time: 17:2 # 4 Linnakoski et al. Antiviral Agents From Fungi FIGURE 1 | A tree illustrating the larger phylogeny of Fungi shows that the origin of presently known fungal-derived antiviral agents (highlighted) is restricted to the late-diverging fungal phyla (Ascomycota and Basidiomycota). The figure is constructed based on phylogenetic relationships of Fungi on Tree of Life Web Project (http://tolweb.org). This tree is illustrative and does not represent real phylogenetic data. Dashed lines: The group may not be monophyletic, or phylogenetic position of the group is uncertain. source (Barros et al., 2007, 2008;Ça˘ glarırmak, 2007;Kalaˇ c, 2009; Ouzouni et al., 2009;Reis et al., 2012). Some species producing conspicuous fruiting bodies have a long history of medicinal use. Bioactive compounds of the fungal genera which have had an important role in traditional medicine, such as Ganoderma, have been subject to extensive research. However, there is a broad number of other edible and medicinal species from different genera considered to be potential antiviral precursors (Supplementary Table S1 and Figure 3). The antiviral activity of these mushrooms is associated mainly to the presence of polysaccharides in mycelium and fruiting bodies, and synthesis of triterpenoid secondary metabolites (Chen et al., 2012;Rincão et al., 2012). However, large number of other potentially bioactive compounds and/or genes involved in their synthesis has been reported (Shiao, 2003;Chen et al., 2012), indicating that the full potential of mushroom and medicinal fungi as a source of bioactive compounds remains only partially understood. Previous study has reported considerable differences in the contents of bioactive compounds produced at different stages of fungal life cycle (Chen et al., 2012), implying that antiviral studies need to take into account the phenotypic variation and growth conditions of the fungal material. Endophytes, Marine Fungi and Plant Pathogens Endophytic fungi that inhabit above-ground tissues of healthy plant at least part of their life cycle are highly diverse in terms of species richness. These primarily ascomycetous (Ascomycota) fungi common in all terrestrial habitats are considered to have important ecological roles in the terrestrial plant communities. Their interactions with host plants and cross-talk with other endophytic microorganisms colonizing the same plant are complex and dynamic (Kusari et al., 2012). Endophytic fungi have been recognized as a rich source of secondary metabolites, which role in the natural habitat likely include chemical signaling, defense against other microorganism, and establishment of symbiosis with host plant (Schulz and Boyle, 2005;Yim et al., 2007;Khaldi et al., 2010). Some also mimic plant defense compounds, and can, therefore, protect host plants against herbivores and pathogens (Kusari et al., 2012). These secondary metabolites are known to have great chemical variety and numerous biological activities with pharmaceutical and biotechnological potential. It has been hypothesized that extreme habitats harbor greater changes for novel drug discovery (Thatoi et al., 2013;Chávez et al., 2015). Interestingly, rich fungal species diversity inhabits extreme environments such as deep-sea sediments and mangrove ecosystems (Kumaresan and Suryanarayanan, 2001;Mahé et al., 2013). Many of ascomycetous species found in these habitats have been discovered having antiviral and other biological activities (Desmukh et al., 2018). The extreme conditions are thought to shape the secondary metabolite patterns of fungi, and these fungi are recognized as a particularly promising source of diverse and structurally unprecedented novel compounds, which some have already been structurally characterized and several been discovered to constitute of novel carbon skeletons (Saleem et al., 2007). However, also already relatively well-known fungi should not be overlooked. Less intensively investigated fungi for their bioactivities include tree-pathogens that also seem promising Frontiers in Microbiology | www.frontiersin.org 4October 2018 | Volume 9 | Article 2325 fmicb-09-02325 October 1, 2018 Time: 17:2 # 5 Linnakoski et al. Antiviral Agents From Fungi FIGURE 2 | In phylum Ascomycota, antiviral agents have been mainly identified from endophytes and other microfungi restricted to limited number of orders. Higher red color intensity indicates higher number of reports in literature. The figure is constructed based on phylogenetic relationships of Fungi on Tree of Life Web Project (http://tolweb.org). This tree is illustrative and does not represent real phylogenetic data. IA, indole alkaloids; NRPS, non-ribosomal peptides; PKS, polyketides; NRPS-PKS, hybrids; T, terpenoids; N/A, information not available. Dashed lines: The group may not be monophyletic, or phylogenetic position of the group is uncertain. source of antiviral agents. A previous study has detected a number of plant pathogenic fungi with various ecological roles (whiterot fungi, soft-rot fungi, blue-stain fungi and insect-symbionts) having antiviral activities (Mlinaric et al., 2005). Antiviral Research and Fungal Taxonomy Accurate organism identification is the basis for any biological research and its applications. This is particularly important for bioactive compounds aimed for pharmaceutical products. When the physical material used is reported with a misapplied name, the reproducibility of the study is very low. Unfortunately, in the literature on bioactivity and mechanisms of action of isolated compounds or crude extracts of fungal origin, reporting on the methods used to identify fungal materials reveals insensitivity to the relevant taxonomic discussion. Methodologically, only a minority of studies have included Frontiers in Microbiology | www.frontiersin.org 5October 2018 | Volume 9 | Article 2325 fmicb-09-02325 October 1, 2018 Time: 17:2 # 6 Linnakoski et al. Antiviral Agents From Fungi FIGURE 3 | Antiviral agents reported from the phylum Basidiomycota. Higher red color intensity indicates higher number of reports in literature. The figure is constructed based on phylogenetic relationships of Fungi on Tree of Life Web Project (http://tolweb.org). This tree is illustrative and does not represent real phylogenetic data. L, lignin derivative; PS, polysaccharides; P, proteins; C, polysaccharide-protein/amino acid complex; NRPS, non-ribosomal peptides; PKS, polyketides; T, terpenoids; N/A, information not available. Dashed lines: The group may not be monophyletic, or phylogenetic position of the group is uncertain. a combination of morphological and molecular methods for species identification (Raja et al., 2017). Given the factual diversity of kingdom Fungi, and the resulting difficulties in delimitating species and genera, as well as constant discoveries of species new to science (Hawksworth and Lücking, 2017), transparency in this matter is paramount. Long lasting debates among taxonomists, whether to accept new names, splitting of an old species into many new, or combinations of old names are an everyday affair in the field. This has in some cases resulted in considerable nomenclatural stratification, highlighting the need to engage taxonomists also in the study of applications. To illustrate this problem, we evaluated literature on one of the most commonly reported name appearing in fungal antiviral research, ‘Ganoderma lucidum,’ as well as other species in the genus Ganoderma Karst. The poroid, saprotrophic fungal species G. lucidum (W. Curt. : Fr.) Karst is an concise example of the broader issue. The traditional medicinal use of Ganoderma spp. in East Asia, South-East Asia, and Africa has promoted interest in studying the bioactivity of these fungi, with ‘G. lucidum’ often cited as the species of the material. However, exact delimitation of the species concept for G. lucidum, with a European type locality, has been difficult due to lack of a holotype specimen (Steyaert, 1972;Moncalvo and Ryvarden, 1997). After morphological and Frontiers in Microbiology | www.frontiersin.org 6October 2018 | Volume 9 | Article 2325 fmicb-09-02325 October 1, 2018 Time: 17:2 # 7 Linnakoski et al. Antiviral Agents From Fungi molecular phylogenetic studies on the diversity of the genus in the past decades (Moncalvo et al., 1995;Cao et al., 2012;Zhou et al., 2015), the consensus in the taxonomic literature is that the industrially cultivated “Linghzi” and “Reishi” do not represent the G. lucidum s. str, but in fact other species (Wang et al., 2009; Cao et al., 2012). Therefore, careful consideration is required when identifying such samples under this name. Here, we listed the reported methods of acquisition and identification used in each antiviral study on Ganoderma (Supplementary Table S2). As a summary, out of the 13 studies, only four used material that we can safely assume to represent the species declared, as a fungal taxonomist was being consulted. In eight cases it seems unlikely given the sourcing of the materials, but could in principle be verified to the contrary, assuming access to the original material in herbaria. In one case, the experimental set-up is likely not reproducible due to vague description of the material used, and apparent lack of any preserved specimens. No studies reported sequence data accession numbers, nor morphological criteria used for species determination. Various forms of authorship, including outdated and erroneous, were present with the name G. lucidum. Whether fungal material is in fact correctly identified, has consequences to the independent reproducibility of the study, and reflects also to understanding the species characteristics (i.e., requirements and phenotypic variation in artificial cultivation settings). There is yet a limited amount of comparative work on the differences between species and strains of the composition in the bioactive compounds within Ganoderma. The publications available at the moment indicate that differences may be considerable (Welti et al., 2015;Hennicke et al., 2016), though assessments into the extent of occurrence of compounds of interest within the genus is again convoluted by the nontransparent reporting of materials (Richter et al., 2015). In conclusion, given the likelihood of misapplied names in the literature, citing studies not reporting identification criteria as evidence on the antiviral potential of G. lucidum s. str. needs to take this ambiguity into account. The misidentification of species and even genera is even more likely with microscopic fungi (such as endophytes) containing minute and overlapping morphological characteristics, and of which taxonomy and diversity remains widely uninvestigated. Therefore, we highlight the importance for transparency in reporting of used nomenclature, physical fungal material and method of identification, which is paramount to the advancement of research on antivirals from fungi. Furthermore, we encourage the natural product research community adopting the recently suggested set of standardized procedures for the identification of fungi (Raja et al., 2017). Overview of Methods Assessing Antiviral Activity The most widely used methods for the initial screening of fungal extracts to evaluate their antiviral activity are the plaque reduction assay (Zhu et al., 2004;Faccin et al., 2007;Rincão et al., 2012), cytopathic effect (CPE) assay (Liu et al., 2004;Zhang et al., 2011) and immunofluorescence assay (Faccin et al., 2007) (Table 2). In addition, various commercially available viability assays monitoring for, e.g., the cellular ATP levels have also been used. These assays are also used for performing the time of addition studies and investigating the direct virucidal activity of the fungal extracts (Liu et al., 2004;Faccin et al., 2007). All of these methods calculate in different ways the viability of the cells after virus action, and the antiviral activity is monitored as the rescue of the cells from the viral infection. The read out for the plaque reduction assay is the visual counting of the number of plaques formed [plaques forming unit (PFU)/ml] i.e., number of unstained “holes” in the culture plate after crystal violet staining of the cells that still adhere on the plate. This number is then used to calculate the percentage of viral inhibition (% V.I.) (Zhu et al., 2004). In immunofluorescence assay, the cells are observed under microscope and typically several hundreds of cells are scored. First, the number of infected cells is calculated from the number of cells showing high abundance of viral capsid proteins produced in the cell cytoplasm (Marjomäki et al., 2002). Then, from the obtained number, V.I. is calculated with respect to untreated infected cells (Faccin et al., 2007). In the case of CPE assay, the read out is based on the spectrophotometric absorbance reading of the stained viable cells, which is used to calculate the % V.I. (Liu et al., 2004). Typically, the viable cells left on the bottom of the culture plate and stained with crystal violet, are dissolved in the lysis buffer to provide a homogenous blue suspension that is easy to measure in the spectrophotometer (Schmidtke et al., 2001). The linear regression analysis of the plots of % V.I. is used to determine the 50% inhibitory concentration (IC50) which is used further to calculate the selectivity index (SI) (Rincão et al., 2012). The calculations are also given here as formulas: % VI calculated from the plaque reduction assay read-out = [1−(number of plaques in test/number of plaques in virus control)] ×100 (Rincão et al., 2012) % VI calculated from the CPE assay read-out = [(ODt)v−(ODc)v]/[(ODc)mock −(ODc)v] ×100 (Liu et al., 2004) where (ODt)vis the optical density (OD) of the cell, treated with virus and bioextract (test), (ODc)vis the OD of the cell, treated with virus (virus control) and (ODc)mock is the OD of the mock infected cell (cell control). SI =CC50/IC50 Where CC50 is 50% cytotoxic concentration, i.e., the concentration which caused a 50% reduction in the number of viable cells or in the optical density and IC50 is 50% inhibitory concentration, i.e., the concentration capable of reducing 50% PFU in relation to the controls. These above-mentioned methods only affirm the antiviral potential of bioactive compounds and do not reveal any information regarding their mechanism of action. Only few papers have progressed to evaluate the actual molecular targets. In order to study various viral or cellular targets of drug action, several approaches could be used. To study the direct effect on the Frontiers in Microbiology | www.frontiersin.org 7October 2018 | Volume 9 | Article 2325 fmicb-09-02325 October 1, 2018 Time: 17:2 # 8 Linnakoski et al. Antiviral Agents From Fungi TABLE 2 | Methods used to evaluate antiviral effects. To study Method Read out Reference Antiviral activity, Virucidal activity and CPE assay using crystal violet to stain viable cells OD values at 550–595 nm Schmidtke et al., 2001 Time of addition studies Plaque reduction assay No of plaques per well−>PFU/ml Rincão et al., 2012 Microscopy immunofluorescent assay to label newly synthetized capsid proteins % of infected cells with respect to untreated infected cells Faccin et al., 2007 Direct effect on virus Negative staining TEM Unstained, intact viruses vs. darkly stained, empty viruses Myllynen et al., 2016 Structural studies (e.g., x-ray crystallography or cryo-EM) Atomistical model exhibiting drug binding or virus opening De Colibus et al., 2014 Real-time spectroscopy using SYBR-Green Fluorescence intensity increase upon genome release Myllynen et al., 2016 Density gradient of radioactively labeled virus showing intact and uncoated viruses Radioactive counts (CPM) per each gradient fraction showing peaks of intact and empty viruses Marjomäki et al., 2002; Myllynen et al., 2016 Adsorption/receptor attachment Binding assay Radioactive counts (CPM) per each gradient fraction showing peaks of intact and empty viruses Marjomäki et al., 2002; Myllynen et al., 2016 Computational simulations (molecular docking) Binding energy upon drug binding (−kcal/mol) Zhang et al., 2014 Uncoating Density gradient of radioactively labeled virus showing intact and uncoated viruses Radioactive counts (CPM) per each gradient fraction showing peaks of intact and empty viruses Marjomäki et al., 2002; Myllynen et al., 2016 Real-time spectroscopy Structural studies (e.g., x-ray crystallography or cryo-EM) Fluorescence intensity increase upon genome release Myllynen et al., 2016 Hewat and Blaas, 2004; Levy et al., 2010 Replication intermediates (replication) and capsid protein production (translation) Immunolabeling and confocal microscopy Fluorescence intensity quantification of capsid or dsRNA production Martikainen et al., 2015 Specific viral proteases HIV-1 protease peptide cleavage assay Monitoring the fluorescence of the enzyme catalyzed reaction Singh et al., 2004 X-ray crystallography Atomistic details for binding Singh et al., 2004 scintillation proximity assay (SPA) Measuring radioactivity of the enzymatic reaction using radioactive biotinylated substrate and streptavidin tagged scintillant Guo et al., 2000 virus, there are several methods that could be employed. First of all, perhaps the easiest way to see gross effect on the virus particle is to negatively stain the virus samples and observe them under transmission electron microscope (TEM) (Myllynen et al., 2016). There is a characteristic feature to distinguish between intact viruses from empty particles in TEM imaging. The staining dye, e.g., 2% Uranyl acetate or 1% phosphotungstic acid cannot enter the capsid of intact viruses because of which the intact particles appear bright, i.e., unstained, in TEM images (due to the contrast of the dye). However, in case of empty viruses, since the capsid is open, the dye enters the capsid and stains the insides of the virus thus giving a dark appearance for empty virus particles in TEM images. Density gradient centrifugation of either radioactively labeled or non-labeled virus is also insightful in revealing the direct effect of the extract on the virus (Marjomäki et al., 2002; Myllynen et al., 2016). The read out of radioactive gradient fractionation is the radioactivity [counts per minute (CPM)] of various fractions from different densities showing peaks of more dense intact virus and less dense empty viruses or even smaller products like pentamers. Direct effects of bioactive agents should show clear changes in the fraction of intact versus empty viruses. The effect on the virus attachment on cellular receptors has been studied using binding assays. Binding is most sensitively studied using radioactively labeled virus and by performing binding assays in cold, hence eliminating the virus entry inside the cells by endocytosis (Marjomäki et al., 2002). Specific effects of molecules interfering with receptor binding have been also performed in silico by using molecular docking studies (Zhang et al., 2014). Whether the drug targets the virus uncoating in vitro or while the virus is inside cellular compartments, can be evaluated using real-time spectroscopy by using RNA/DNA binding fluorescent dyes (Myllynen et al., 2016) and using radioactive gradient fractionation studies, respectively. Radioactively labeled virus may be isolated from the cells for gradient fractionation which may reveal if there is a block in the viral genome release, thus leaving the virus as intact for longer periods. In order to assess the effect of bioextract on the efficiency of replication and viral translation, immunofluorescent labeling may be performed that reveals production of virus capsid proteins and specific replication intermediates, such as, e.g., dsRNA (Martikainen et al., 2015). Furthermore, qPCR to reveal new viral RNA production may be used. Frontiers in Microbiology | www.frontiersin.org 8October 2018 | Volume 9 | Article 2325 fmicb-09-02325 October 1, 2018 Time: 17:2 # 15 Linnakoski et al. 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The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Microbiology | www.frontiersin.org 18 October 2018 | Volume 9 | Article 2325