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Potential of small-molecule fungal metabolites in antiviral chemotherapy

Various viral diseases, such as acquired immunodeficiency syndrome, influenza, and hepatitis, have emerged as leading causes of human death worldwide. Scientific endeavor since invention of DNA-dependent RNA polymerase of pox virus in 1967 resulted in better understanding of virus replication and development of various novel therapeutic strategies. Despite considerable advancement in every facet of drug discovery process, development of commercially viable, safe, and effective drugs for these viruses still remains a big challenge. Decades of intense research yielded a handful of natural and synthetic therapeutic options. But emergence of new viruses and drug-resistant viral strains had made new drug development process a never-ending battle. Small-molecule fungal metabolites due to their vast diversity, stereochemical complexity, and preapproved biocompatibility always remain an attractive source for new drug discovery. Though, exploration of therapeutic importance of fungal metabolites has started early with discovery of penicillin, recent prediction asserted that only a small percentage (5–10%) of fungal species have been identified and much less have been scientifically investigated. Therefore, exploration of new fungal metabolites, their bioassay, and subsequent mechanistic study bears huge importance in new drug discovery endeavors. Though no fungal metabolites so far approved for antiviral treatment, many of these exhibited high potential against various viral diseases. This review comprehensively discussed about antiviral activities of fungal metabolites of diverse origin against some important viral diseases. This also highlighted the mechanistic details of inhibition of viral replication along with structure–activity relationship of some common and important classes of fungal metabolites.


Viral diseases: A therapeutic challenge
Disease caused by pathogenic viral infections afflicted whole human race with high mortality rates 1,2 and remained as one of the prime causes of human death worldwide. 3 Human immunodeficiency virus (HIV), influenza, and hepatitis C virus (HCV) are three most perilous viral diseases which cause maximum human death. Discovery of effective vaccines though have somewhat helped in eradication of some important viral pathogens, such as smallpox, polio, mumps, etc. but proved ineffective in combating these viral diseases with high mortality rate. 4 A virus is a RNA/DNA-containing unique pathogen which requires utilization of host cell machinery and environment for its replication. Due to this unique feature, it is very difficult to make viruses or their replication as a therapeutic target, without exerting any adverse effect to the host cells. 5 Earlier days viral replication was thought to be carried out by cellular enzymes and thus the scope for selective inhibition of viral replication was very discouraging. 6 Active research on antiviral drug development started with discovery of first viral enzyme DNA-dependent RNA polymerase of pox virus in 1967. 7 Subsequent research resulted in discovery of several other enzymes and their biochemical function in various stages of viral replication. Common viral replication cycles involve several stages, which include attachment and entry to the host cell, transcription of viral mRNA, replication of viral genome, assembly and budding as progeny virus particles. Viral reverse transcriptase (RT) or RNAdependent RNA polymerase does not have a proofreading mechanism. 8 Therefore, viruses containing RNA genomes such as HIV, HCV, and influenza are genetically susceptible to easy mutation. Accumulated mutation in viral genome is one of the main reasons behind the emergence of drug-resistant strain. [9][10][11] Although several new antiviral drugs have been approved in last two decades, rapid emergence of drugs resistance viral strains and adverse health effects, which arises due to prolonged use of these drugs, made continuous search for new antivirals inevitable. Thus, designing or discovery of safe drugs which is less susceptible to drug resistance still remains a serious challenge to synthetic and natural product chemist.

Current therapeutic approaches and requirement for novel drug
The arsenal of present-day antiviral may be divided into two broad categories 12 according to their targets. Directly acting antivirals, that directly affect viral proteins or viral genomes, are generally more specific, and therefore, less toxic but poses a higher risk of creating resistant viruses. That is why these single point targeted drugs are frequently ineffective, unsafe, and detrimental to therapeutic response. Whereas, host-acting antivirals, which modify host cellular factors or arouse immune response components to affect virus life cycles. These drugs may possess a broader antiviral activity spectrum and less risk of developing virus resistance but may be more toxic to the host cell. Ideally, combinations of effective therapeutic agents that target multiple stages in the viral replication cycle with little or no toxicity to the host are desirable. Therefore, currently, a combined therapy (or highly active antiretroviral therapy, HAART) is used, 13,14 which employ a cocktail of antivirals which target different biological process of viral replication cycle. 12 Due to the therapeutic synergy, it effectively suppresses viral replication and significantly prolongs the life of virus-infected patients. Therefore, there remains a pressing need for developing new antivirals with novel modes of action. 15 Fungal metabolites as source of novel drug: Past history, present status, and future potential Small-molecule secondary metabolites from natural resources traditionally played an important role in drug discovery and proved to be great savior against various dreaded diseases. 16,17 Due to their structural diversity, stereochemical complexity, 18,19 and evolutionarily optimized biocompatibility 17,20 these compounds still remained as indomitable attraction in medicinal chemist community. Metabolites of fungal origin being source and inspiration of many blockbuster drugs and drug leads are one of the major contributors to this success story. Enthusiastic search for bioactive metabolites 20,21 triggered by the success of penicillin resulted in many other natural and semisynthetic antimicrobials, such as griseofulvin 22 and cephalosporins, 23 etc. Cyclosporine, an immunosuppressive cyclic peptide, was first discovered from soilderived fungus, Tolypocladium inflatum (Beauveria nivea) and Cylindrocarpon lucidum 24 has changed started a new era in organ transplantation. 25 Subsequently, cyclosporine A has been reported from a variety of different common organisms, including many strains of T. inflatum as well as Acremonium, Beauvaria, Fusarium, Paecillomyces, and Verticillium species. 26 Presently there are more than 15 fungal metabolites that are in use as FDA-approved drugs and some of these are still dominating the drug market. 27 For example, mevastatin, lovastatins along with some synthetic analogs, like, atorvastatin had 15.5 billion sales in 2004, antibiotics market dominated by two fungal metabolites penicillin and cephalosporin had 15 billion dollar sales only in 2002, fungi-derived oral antifungal agent griseofulvin had 31.1 million sales in 2007, and antibiotic amoxicillin and immunosuppressive fungal metabolites cyclosporine had 1.4 and 1.7 billion dollars sales, respectively, during the period of 2004-2008. 27 In last 50 years a large number of structurally diverse metabolites were isolated from numerous fungal species. Some of these finally came out as lifesaving drug 27 and many of these are at different stages of drug development process. According to the recent estimation [28][29][30] so far only a small fraction of fungal species have been identified and much less have been scientifically investigated for bioactive metabolites. Thus, there is plenty of scope for finding many other potential drug lead through exploration of new fungal metabolites and their bioactivity study. Another big advantage of isolating compounds from fungal species is that those can be cultivated or cultured so that large amount of the species can be generated at a reasonable time and cost. Development of efficient and automated isolation techniques and enhanced capability of carrying out bioassay of large number of compounds (through high throughput screening) can accelerate the exploration of more and more new fungal metabolites. One of the main problems in antiviral metabolite research is the accessibility of the bioassay facility. Many countries with abundance of fungal species are not having sophisticated and comprehensive antiviral screening facilities. Therefore, major portions of the isolated metabolites not ever been tasted against any viral strain. Recently, many countries opened their antiviral screening facility for international synthetic and natural product compounds under certain terms and conditions. Enthusiastic exploration of much more new fungal species, development of easy but effective metabolite isolation techniques, and easing the access to sophisticated and comprehensive screening facilities will surely expedite the antiviral fungal metabolite exploration process.

Fungal metabolite inhibitors for various viral diseases
Though no fungal metabolite has been approved so far for the treatment of viral drug, many of these metabolites exhibited extraordinary activity against various strains, especially against HIV and influenza viruses. This paper critically reviewed the bioactivity of different classes of fungal metabolites against various viral diseases. In many cases same fungal metabolites were obtained from various other fungal species. To maintain the focus, only the fungal species from where its bioactivity is first detected is mentioned here, though in some cases references have been given for the initial isolation of these metabolites. Many of these antiviral fungal metabolites also exhibited other important biological activities but those are not mentioned here unless their mechanisms of action influence the antiviral activity. Most of the cases the structure and stereochemistry of the fungal metabolites have been determined by using various spectroscopic techniques such as NMR, MS, UV, IR, single crystal X-ray, polarimetry, and CD spectrometry and are not mentioned individually in the text. Where structure confirmation of metabolites needed extra measures such as, synthetic modification or derivatization, only those cases are mentioned individually in the text. Bioactivities of some already reviewed [31][32][33] fungal metabolites is also included here for a further in-depth discussion of their important structural features and to make this review more comprehensive.

Inhibitors of HIV
Acquired immunodeficiency syndrome (AIDS), caused by HIV, is one of the major contributors to an increase in infectious disease mortality. 34,35 Since first reported official cases of AIDS in 1981 it has erupted as pandemic for last four decades. According to 2011 estimate, more than 34 million people were living with HIV infection and number of newly infected cases and number of deaths in the same year reached more than 2.5 million and 1.7 million, respectively. 36 Due to its increasing extension and the high associated mortality rate, undoubtedly AIDS represents a global health threat. Fighting this disease remains one of the major challenges for chemotherapy in the 21st century.
The causative agent, HIV is a structurally unique member of Retroviridae family which contains two copies of positive single-stranded RNA and a conical capsid. HIV has the potential to infect pivotal cells of the human immune system such as dendritic cells, macrophages, and mainly T helper cells. Though there are two major types of this virus existing as HIV-1 and HIV-2, the former type is the major cause of the epidemic of AIDS worldwide. Four decades of intensive research from industry and academia resulted in 26 clinically approved drugs till date. These antiretroviral drugs can be classified into six different groups according to their biological targets, namely entry inhibitors (fusion inhibitors and chemokine receptors CCR5 and CXCR4), RT inhibitors (nucleoside or nonnucleoside based), integrase inhibitors, and protease inhibitors 37 ( Figure 1). These drugs are mainly directed against three key viral enzymes required for replication, RT, protease and integrase, one viral envelope glycoprotein gp41, and a coreceptor (CCR5) of the host cell. 38 A major concern in antiretroviral drug therapy is rapid emergence of resistant strain. 39 That is why single point targeted drugs are frequently ineffective, unsafe, and detrimental to therapeutic response. The current combined therapy (HAART) with synergistic effect of antivirals of different target points effectively suppress viral replication and significantly prolonged the life of AIDS patients. 35 Thus, the continual emergence of drug resistance viral strains has created a pressing need for continuous search and development of potent anti-HIV drugs with new modes of action. 40 This resulted in discovery of a number of potential metabolites from various natural sources. 12 In this portion HIV inhibitory activity of various fungal metabolites is presented and their mode of inhibition has been discussed.
Entry inhibitors. Blocking of HIV entry to host human cell is also one of the most important targets for HIV therapies. [41][42][43] The viral spike of HIV-1 is composed of three gp120 envelope glycoproteins attached noncovalently to three gp41 transmembrane molecules. Entry of HIV to host cell begins with highly specific binding of the HIV glycoprotein gp120 envelope protein with a CD4 molecule, on the surface of most susceptible cells. Viral entry is initiated by binding to the CD4 receptor on the cell surface, which induces large conformational changes in gp120 which trigger the entry of viral materials to the host cell. Several studies demonstrated that chemokine receptors CCR5 and CXCR4 are essential as coreceptors for gp120 and CD4 binding. Inhibition of such binding can prohibit the entry of viral material to the host cell and thus can prevent further replication. This is a very effective alternative target for HIV therapeutics. The molecule which inhibits fusion of viral and host cell through specific binding with glycoprotein is called fusion inhibitor. Other types of entry inhibition take place through specific binding with the coreceptors CCR5 and CXCR4. Several fungal metabolites with viral entry inhibitory activity are discussed here.
Two tetramic acid type fungal metabolites, Sch 210971 [11] and Sch 210972 [12] isolated from a culture of Chaetomium globosum, demonstrated very strong CCR-5 inhibitory activity 52 with IC 50 values of 1.2 mM and 79 nM, respectively. Compound [11] which is an epimer of [12] showed over 15-fold decrease in it inhibition potency. Their high specificity also confirmed by CCR-2 (not bind even at a very high concentration>100 mM) binding assay. Two compounds pericoannosin A [13] and periconiasins F [14] isolated from endophytic fungus Periconia sp. showed low anti-HIV activity with IC 50 s of 69.6 and 29.2 mM, respectively against efavirenz standard with an IC 50 of 1.4 nM. 53 Anibamine [15], a novel pyridine quaternary alkaloid as a TFA salt (plausibly an artifact of the isolation), from Aniba sp. 54 ; ophiobolin C from fermentation extracts of fungi Mollisia sp.; and 19,20-epoxycytochalasin Q from Xylaria sp. 55 have shown to exhibit considerable HIV-1 inhibition. The identity of the natural counterion of Anibamine [15] is unknown. AnibamineTFA competed for the binding of 125 I-gp120 to human CCR5 with an IC 50 of 1 mM. Ophiobolin C [16] and 19,20-epoxycytochalasin Q [17] exhibited binding IC 50 values of 40 and 60 mM, respectively.
RT inhibitors. RT, a critical enzyme in the HIV life cycle, generates complementary DNA from an RNA template. As this process does not occur in normal cell, it has become one of the main targets of anti-HIV chemotherapeutics. There are two functionally distinct classes of RT inhibitors: nucleoside RT inhibitors and nonnucleoside RT inhibitors (NNRTIs). 56 NNRTIs are important components of highly active antiretroviral therapy (HAART). So far there are five NNRTIs nevirapine (NVP), efavirenz (EFV), etravirine (ETR), rilpivirine (RPV), and delavirdine (DLV) approved by FDA for HIV treatment. 57,58 All of these drugs bind to the hydrophobic pocket in the palm domain, adjacent to the thumb domain on the RT p66 subunit, in blocking allosteric sites of RT. [59][60][61][62] Drug resistance is the most important factor of the failure of HAART. NNRTI-resistant mutations exist widely in patients since NVP, EFV, and DLV have been used in clinical settings over the past decade.
Both clinical and in vitro studies have shown that E138K is a resistant mutation to ETR and RPV, which were approved by the FDA in 2008 and 2012, respectively. 63,64 Therefore, there remains a strong demand for the development of new NNRTIs that can effectively inhibit NNRTI-resistant viruses.
Ma et al. isolated 11 phenylspirodrimanes along with stachybotrins D [18] from the fermented broth of fungus Stachybotrys chartarum 65 and studied their anti-HIV against both wild-type HIV-1 and five NNRTI-resistant strains. Only active compound [18] exhibited an inhibitory effect on HIV-1 replication with an EC 50 value of 8.4 mM with no cytotoxicity at 10 mM. In-depth mechanistic study revealed that compound [18] inhibits the RT RNA-dependent DNA polymerase activity in a dose-dependent manner with an EC 50 66 Inactivity of structurally related metabolite helotialins C [21] from the same extract in assay suggested that the C-13/C-14 olefin is important for their anti-HIV activity.
Bioactivity-guided isolation of ethyl acetate extract of a fermentation broth of endophytic fungi Alternaria tenuissima QUE1Se and spectroscopic identification resulted in two new metabolites, altertoxins V [25] and VI [26] 67 along with three known altertoxins I [22], II [23], and III [24]. 68,69 Among these, altertoxins I [22], II [23], III [24] and altertoxins V [25] completely inhibited HIV-1 viral replication in A3.01 cells at concentrations of 0.50, 2.20, 0.30, and 1.50 mM, respectively, compared to standard positive control AZT at 20 mM. Altertoxins VI [26] showed reduced effectiveness probably due to its instability in the cell culture medium. Altertoxin V [25] displayed the lowest IC 50 value of 0.09 mM whereas altertoxins I [22], II [23], and III [24] exhibited higher IC 50 values of 1.42, 0.21, and 0.29 mM. All the active compounds are insignificantly cytotoxic at these concentrations. From their IC 50 and cytotoxicity data therapeutic index of [22], [23], [24], and [25] were determined to be 3, 6.5, 15, and 50 mM, respectively. Their therapeutic index provides a narrow window for further drug development study but surely provide a good platform for the development of synthetically modified nontoxic anti-HIV drug.
HIV integrase inhibitors. HIV-1 integrase is one of the three enzymes that are critical for viral replication. HIV DNA is inserted into the host genome by a specialized DNA recombination reaction in which the viral integrase is the key player. 70,71 The integration reaction is composed of three steps, 3 0 -processing, strand transfer, and gap filling, and integrase catalyzes the first and second steps. The third step is thought to be catalyzed by cellular enzymes. It catalyzes three essential steps that include assembly, endonucleolytic cleavage (3 0 -end processing) of the viral DNA, and strand transfer of the viral DNA into the host cell DNA. 70,72,73 The absence of HIV integrase enzyme in the host cells and its indispensability for HIV replication cycle makes it an excellent target for the development of a nontoxic antiretroviral therapeutic agent. The inhibitors of the other two key enzymes, HIV RT and HIV protease, have led to many approved drugs that continue to have enormous impact on the control of the spread of HIV-1 infection. But, inhibitors for HIV integrase inhibitors, with only three recently approved drugs, 57 dolutegravir, elvitegravir, and raltegravir (raltegravir potassium) are much underexplored and possess enormous potential to be a vital component in combination therapy (HAART). Also combating future drug resistance for HIV integrase inhibitors 74 requires a sufficiently strong drug discovery pipeline. Thus, research toward development of new HIV integrase inhibitor drugs bears huge importance. [75][76][77] Two enantiomeric homologous fungal metabolites equisetin [27] and phomasetin [28], isolated from the marine fungi Fusarium heterosporum and Phoma sp. respectively, 78 exhibited strong in vitro HIV-1 integrase inhibitory activity. These fungal metabolites and other two structurally related metabolites integric acid [29], isolated from Xylaria sp. 78 and oteromycin [30], isolated from two different strains of unidentified fungus, 79 are structural homologs that comprise a structurally and functionally unique class of integrase inhibitors. A detailed in vitro inhibition assay of these metabolites carried out bySingh et al. 80 revealed that integric acid is the most active in inhibiting three different types of activity of HIV-1 integrase enzyme, e.g. 3 0 end processing, strand transfer, and disintegration with IC 50 values 5-10 mM. Despite being enantiomers equisetin and phomasetin showed almost equal inhibitory activity for both 3 0 end processing and strand transfer reaction with IC 50 value 7 and 15 mM and 15-20 mM, respectively. 78 Oteromycin exhibited much reduced inhibition compared to the other three structural homologs. 80 Integrasone [63], 81 a dihydroxy bicyclic epoxy lactone polyketide fungal metabolite, isolated by same group from an unidentified mycelium, which moderately inhibited the strand transfer reaction of HIV-1 integrase with an IC 50 value of 41 mM. The aliphatic chain may play a significant role in the activity, as was observed in the case of integric acid. 82 Two polyketides, cytosporic acid [32] isolated from fermentation broth of the filamentous fungus Cytospora sp., 83 and Australifungin [33], isolated from the coprophilus fungus Sporormiella australis, 84 almost equally inhibited strand transfer reaction of HIV-1 integrase with an IC 50 of 20 mM in an in vitro assay. But, Australifungin 84 [34] did not show any inhibition even at 200 mM. The lack of activity of [34] indicates that the a-keto aldehyde of [33] and the carboxyl group of [32] are critical for the activity.
Funalenone [35], originally isolated from mycelium of Aspergillus niger FO-5904, 85 Erabulenol B [36] isolated from Penicillium sp. FO-5637, 86 and atrovenetinone methyl acetal [37] was isolated from a culture broth of Penicillium sp. FKI-1463 87 was evaluated for their integrase inhibition and anti-HIV activity. 87 All these metabolites showed considerably strong in vitro anti-HIV activity with IC 50 values 1.7, 17, and 6.7 mM, respectively. They also inhibited HIV-1 integrase enzyme with IC 50 values 10, 7.5, and 19 mM, respectively. Compound [35] and [36] are found to be better integrase inhibitors with much less cytotoxicity (IC 50 87 and 230 mM, respectively) and high selectivity compared to [37] (cytotoxicity IC 50 13 mM). Compound [37] is a methyl acetal derivative of Atrovenetinone [38], which was initially obtained from oxidation of atrovenetin produced by Penicillium sp. 88 and was lately isolated from a culture broth of Gremmeniella abietina. 89 All these phenalenone compounds reported to possess many other favorable biological activities. 90 Integracide A [39], four tetracyclic triterpenoids isolated from the fermentation broth of a Fusarium sp. together with other three analogs. 91 Integracide A, a sulfated ester, exhibited significant inhibitory activity in both coupled and strand transfer reactions of recombinant HIV-1 integrase with IC 50 values of 4 and 9 mM, respectively. However, due to their high toxicity (IC 50 25 mM), it is not much useful for further therapeutic consideration. The other metabolic analogs, integracide B, C, and D, showed very weak or no inhibitory activity.Integrastatins A [40] and integrastatins B [41] are two novel aromatic [6/6/6/6]-ring system, isolated from fermented extract of unidentified fungus from Mexico, inhibits HIV-1 integrase in coupled and strand transfer assays with IC50 values of 0.6 and 1.1 mM for [40] and 1.04 and 2.5 mM for [41], respectively. 92 Three dimeric alkyl aromatics of polyketide origin, integracins A [42], B [43], and C [44], isolated from Cytonaema sp., 93 inhibited coupled reaction of recombinant HIV-1 integrase with an IC 50 value of 3.2, 6.1, and 3.5 mM, respectively. But these are 10-30-fold less active in strand transfer inhibition with IC 50 values of 32, 17, and 88 mM, respectively. The monomeric metabolite [77] from same species was not active even at 100 mM. 93 Four unsymmetrical bis-naphtho-g-pyrone; isochaetochromin B1 [46], isochaetochromin B2 [47], isochaetochromin D1 [48], and oxychaetochromin B [49] isolated from the culture broth of Fusarium sp., inhibited both the coupled and strand transfer activity of HIV-1 integrase with IC 50 values of 1-3 and 4-12 mM, respectively. 94 In in vitro bioassay the dehydro compound, [48] exhibited most inhibition activity with IC 50 values of 1 and 4 mM in coupled and strand transfer assays, respectively. Compounds [46] and [47] showed similar bioactivity with IC 50 values of 2 and 12 mM in corresponding assays. Oxychaetochromin B [49] also exhibited nearly equal activity with corresponding IC 50 values of 3 and 9 mM, respectively. Semisynthetic modification of hydroxyl group of [47] failed to improve the bioactivity, suggesting the necessity of free -OH for bioactivity.
Exophillic acid [75], a novel dimeric 2,4-dihydroxy alkyl benzoic acid, derived from fungus Exophiala pisciphila, 98 and structurally related to another fungal metabolite aquastatin A [76], isolated from Fusarium aquaeductuum, 99 moderately inhibit the strand transfer reaction of HIV-1 integrase with IC 50 values of 68 and 50 mM, respectively. No HIV-1 integrase inhibitory data are available for two other structurally similar fungal metabolites, KS-502 [77], isolated from Sporothrix sp. 100 and TPI-1 [78] from Hypomyces sp. and Nodulisporium sp. Bioassay-guided isolation from the fermentation broth of a soil-derived fungal culture of Aspergillus flavipes led to the isolation of aspochalasin L [79] 101 and the known cyclohexapeptide WIN66306 [80]. 102,103 Compounds [79] and [80] inhibited HIV integrase with IC 50 values of 71.7 and 32.1 mM, respectively, and exhibited CC 50 values of 70 and 75 mM, respectively. 101 In HIV-1 replication inhibition assays in HuT78 cells, none of these has shown any effect at concentrations less than their CC 50 s.
HIV-1 protease (HIV-1 PR) inhibitors. HIV-1 PR is one of the important enzymes necessary for the replication of HIV. This enzyme belongs to the aspartyl protease class and functions as a dimer of 99 amino acids each. This enzyme cleaves the HIV gag and gag-pol polyprotein backbone at nine specific cleavage sites to produce shorter, functional proteins. 104 Protease inhibitors and RT inhibitors form the basis of HAART, which has been successful in improving survival rates and quality of life for HIV-infected individuals. 105 Intense research from academia and industry for around two decades resulted in eight commercially available HIV-1 PR inhibitors which include saquinavir, nelfinavir, ritonavir, indinavir, amprenavir, lopinavir, atazanavir, and fosamprenavir. 57,106 The loss of sensitivity to protease inhibitors usually occurs because the resistant viral strains encode for protease molecules containing specific amino acid mutations that lower the affinity for the inhibitors yet maintains sufficient affinity for the substrate and sufficient catalytic efficiency. 107 The development of more powerful inhibitors with a lower susceptibility to mutations and reduced side effects remains a major challenge. Searching for HIV-1 PR inhibitors from natural sources has thus become a promising approach in this respect. Secondary metabolites of fungal origin with considerable HIV protease activity are described here.
Triterpenes are most prevalent and therapeutically important class of compounds present in various ganoderma species. Many of these have exhibited remarkable inhibitory activity against HIV-1 PR.A group of triterpenoids isolated from Ganoderma colossum by Dine et al. [108][109][110] called colossolactones, characterized by the presence of a six-membered a, bunsaturated d-lactone group in their side chain with or without a seven-membered lactone ring as ring A. Most of the compounds from this mushrooms inhibited HIV-1 PR in a dose-dependent manner. Compounds colossolactone I [81] and colossolactone II [82] 50 116) and other analogs with 3 -b-hydroxy groups (IC 50 > 200 mM). Among the ganoderma alcohols, compounds having unsaturation at 24 (25) were more inhibitory than 24-hydroxy compounds. The 23-oxo ganoderic acids (the major compounds of G. lucidum) were failed to show any activity against HIV PR. Intermolecular hydrogen bonding between a 23-oxo group and a neighboring 26-carboxylic acid in the side chain, which could prevent interaction with amino acid residues of enzyme molecules, might be responsible for this inactivity. Thus, substituents (free hydroxy and oxo groups) at C-3 in the A ring and a terminal functional group in the side chain seem to have critical roles in their anti-HIV-1 PR activity. Ganoderiol F [95], 20-hydroxylucidenic acid N [96], ganodermadiol [98], and 20(21)-dehydrolucidenic acid N [97] were comparatively better inhibitors against HIV-1 PR, with IC 50 values of 22, 25, 29, and 48 mM, respectively.
Two farnesyl hydroquinones, ganomycin I [99] and ganomycin B [100], isolated from the chloroform extract of the same mushroom by the same group 110 showed HIV-1 PR inhibition with IC 50 values of 7.5 and 1.0 mg/ml, respectively. Through kinetics and virtual docking study, it has been found that compound [100] competitively inhibits the active site of the enzyme whereas the tetracyclic triterpene schisanlactone A [90], was a dimerization inhibitor, 108 with an IC 50 value of 5.0 mg/ml. Bisalkylated 2,5-dihydroxybenzoquinones or asteriquinones are a class of fungal metabolites very prevalent among various fungal species. 113,114 Many of these asteriquinones found to exhibit remarkable inhibitory activities when tested against HIV-1-protease. Isocochliodinol 115 [101] and didemethylasterriquinone D [102] initially isolated from Chaetomium sp. and later isolated from Chrysosporium merdarium 60 together with semicochliodinol A [103] and B [104] and tested for their HIV-1 PR inhibitory activity. All these asteriquinones showed very strong inhibitory activity against HIV-1 PR with mean IC 50 values of 0.24, 0.18, 0.37, and 0.5 mM, respectively. Molecular modeling of the HIV-1 PR inhibitor complexes showed hydrogen bonding between the dihydroxybenzoquinone moiety of [101] and [102] to both active site aspartic acids (Asp25/Asp25 0 ) of the protease and the indole parts of the inhibitors filling the P2 and P2 0 pockets of the protease. Hinnuliquinone, a C2-symmetric dimeric bisindolylquinone, initially isolated as pigment from fungus Nodulisporium hinnuleum, 116 again recently isolated from an unidentified fungus and found to show significant HIV-1 PR activity with an IC 50 of 2.5 mM. It also showed remarkable inhibition against highly resistant mutant enzyme A44 117 with an IC 50 120 it has been found that symmetric dimeric structure of hinnuliquinone and no substitution at 5, 6, and 7 positions are two major structural features behind its high HIV-1 PR inhibitory activity.
Cytochalasins are another important class of fungal secondary metabolites containing highly substituted isoindolone ring with a benzyl group at the C-3 position and fused to an 11 -to 14-membered macrocyclic ring. So far, more than 80 cytochalasins, isolated from various fungal species, including Aspergillus, Phomopsis, Penicillium, Zygosporium, Chaetomium, Phoma, Xylaria, Hypoxylon, and Rhinocladiella sp. 121,122 These compounds exhibit a broad spectrum of bioactivities. 123 Ondeyka et al. 124 [107] showed fast, specific, and competitive HIV-1 PR inhibitory activity with an IC 50 of 3 mM and the mode of inhibition was competitive with respect to substrate (apparent Ki ¼ 1 mM). 126 The inhibition of [107] was also independent of the HIV-1 PR concentration. Two other structurally related cytochalasins isolated from same fungus, [108] and [109] failed to show any such activity. The striking difference in protease inhibitory activity of [107] with other structurally similar compound [108] and [109] suggests that hydrogen bonding capability C-7 hydroxyl group of [107] might have a critical role to play in such activity. Protease activity assay with other commercially available cytochalasin showed that only cytochalasin A, containing 14-membered lactone ring showed almost similar activity with compound [107] containing an 11-membered carbocyclic ring. Seven new cytochalasin type of metabolites have been isolated from cultures of an isolate of Stachybotrys charatum. 127 [118], isolated from fruiting bodies of the xylariaceous ascomycete Daldinia concentrica, 130 showed HIV-1 inhibitory effect with an EC 50 value of 0.31 mg/ml in cytopathic inhibition assay with a high (TI ¼ 247) therapeutic index. Further investigation revealed that concentricolide [118] blocks syncytium formation between HIV-1 infected cells and normal cells with an EC 50 value of 0.83 mg/ml. A b-carboline compound, flazin [119], isolated from Suillus granulatus 131 has been shown weak anti-HIV-1 activity but its synthetic amido analog flazinamide 132 [120]

Inhibitors of influenza virus
Influenza is a viral respiratory disease that affects mainly the nose, throat, bronchi and, sometimes lungs. The causative agent influenza A virus (IAV), being a genus of the Orthomyxoviridae family, reproduces rapidly, mutates frequently, and occasionally crosses species barriers. 133 Emergence of a new mutated influenza virus with fatal pathogenicity also are able to unleash a major pandemic with a greater morbidity and mortality. The worldwide spread of the swine-originated influenza virus A (H1N1) in human in 2009 had attracted great attention all over the world. At present antiviral drugs that block the activities of M2 protein and neuraminidase (NA), such as adamantanes (M2 protein), zanamivir, and oseltamivir (NA) can stop the infection effectively. 134,135 Despite these successes, the emergence of drug resistance viral strain and toxicity of present drugs remain as major concerns. 136 Therefore, development of novel antiviral agent with high efficacy and low toxicity bears a great importance.
Two novel compounds with pentacyclic moiety containing cis-fused decalin, stachyflin [121] and acetylstachyflin [122], are isolated through solid-state fermentation of Stachybotrys sp. 137 Their in vitro antiviral activities against IAV and cytotoxicities were tested using MDBK cells and compared with two known drug, amantadine and zanamivir. Stachyflin [121] exhibited about 77 times more antiviral activity against influenza A/W SN/33 (H1N1) virus with an IC 50 value of 0.003 mM than acetylstachyflin [122] with an IC 50 value of 0.23 mM. Structural correlation with bioactivity suggested that the hydroxyl group at the C-3 position might be important for the antiviral activity. Inhibitory activity stachyflin [121] was around 1760 and 250 times more than that of two known antiinfluenza drug amantadine (IC 50 of 5.3 mM) and zanamivir (IC 50 of 0.75 mM), respectively. Stachyflin being a fusion inhibitor 138,139 between the viral envelope and the endosome constituting the cell membrane is mechanistically different from amantadine (NA inhibitor) and zanamivir (ion channel inhibitor). The cytotoxicities of [121] and [122] against MDBK cells were 65 and 44 mM, respectively.
Neosartoryadins A [123] and B [124], together with three biogenetically related compounds were isolated from the endophytic fungus Neosartorya udagawae. 140 Both these compounds displayed activity against influenza virus A (H1N1) with IC 50 values of 66 and 58 mM, respectively, versus positive control ribavirin with IC 50 ¼ 94 mM. Among 14 different compounds isolated from ethyl acetate extract of the culture of mangrovederived fungus Cladosporium sp. 141 oxoglyantrypine [125], norquinadoline A [126], deoxynortryptoquivaline [127], deoxytryptoquivaline [128], tryptoquivaline [129], and quinadoline B [130]  Isolation of six new isoindolones derivatives along with six known compound is reported from endophytic fungus Emericella sp. (HK-ZJ) of mangrove plant Aegiceras corniculatum. 142 Among these only compounds emerimidine A [131] and emerimidine B [132] showed moderate inhibitory activity against IAV (H1N1) with IC 50 values of 42.07 and 62.05 mg/ml, respectively, in cytopathic effect (CPE) inhibition using MDCK cells (ribavirin used as a positive control, IC 50 24.60 mg/ml). Cladosins C [133] along with four new other hybrid polyketides were isolated from the deep-sea-derived fungus Cladosporium sphaerospermum. 143 The structures and stereochemistry of all these compounds were determined by spectroscopic data, chemical conversion using both Mosher's and Marfey's methods. Cladosins C exhibited activity against influenza A H1N1 virus with an IC 50 ¼ 276 mM in comparison to ribavirin as a positive control, IC 50 131 mM. A cyclic tetrapeptide, asperterrestide A [134] isolated along with two new and 10 other known compounds, from fermentation broth of the marine-derived fungus Aspergillus terreus. 144 Their structures were elucidated by spectroscopic analysis, and the absolute configuration of [134] was determined by the Mosher ester technique and analysis of the acid hydrolysates using a chiral phase HPLC column. Compound [134] showed inhibitory effects on the influenza virus strains A/WSN/33 (H1N1) and A/Hong Kong/8/68 (H3N2) with IC 50 values of 15 and 8.1 mM.
One new butenolide isoaspulvinone E [135] and two known butenolides aspulvinone E [136] and pulvic acid [137] have recently been isolated by Gao et al. 145 from the marine-derived fungus, A. terreus Gwq-48. All these compounds were moderately active against influenza A H1N1 virus with IC 50 values of 32.3, 56.9, and 29.1 mg/ ml, respectively, versus two positive controls ribavirin and zanamivir with IC 50 ¼ 24.6 mg/ml and 28.4 ng/ml, respectively. Among these only compound [135] showed significant H1N1 viral NA inhibition activity. Docking experiment with active site of enzyme with these compounds showed that trans orientation of aryl group is necessary for this activity.
Two new rubrolides, rubrolides R [138] and S [139] were isolated from the fermentation broth of the marine-derived fungus A. terreus OUCMDZ-1925 showed anti-influenza A (H1N1) virus activity with IC 50 values 221.6, 87.1 mM, respectively, compared to positive control ribavirin with IC 50 value of 118.8 mM in CPE inhibition assay. 146 Wang et al. 147 isolated total of nine compounds from ethyl acetate extract of an acid-tolerant fungus, Penicillium purpurogenum, and tested their activity against influenza virus. Among these compounds purpurquinones B [140], purpurquinones C [141], purpuresters B [142], and TAN-931 [143]  Recent bioassay-guided isolation of an Antarctic soil-derived fungus, Aspergillus ochraceopetaliformis by Wang et al. 148 resulted in isolation of six new and two known metabolites. Among these compounds, chraceopone A [144], isoasteltoxin [145], and asteltoxin [146] exhibited antiviral activities against the H1N1 and H3N2 influenza viruses with IC 50 values of > 20.0/12.2, 0.23/0.66, and 0.54/0.84 mM, respectively. Sorbicatechols A [147] and B [148], two new sorbicillinoids, together with other two known compounds, protocatechuic acid methyl ester and caffeic acid methyl ester were isolated from the culture of the marine sediment-derived fungus Penicillium chrysogenum PJX-17. 149 Their structures were assigned by spectroscopic data and TDDFT ECD calculations. Compounds [147] and [148] exhibited activities against influenza virus A (H1N1), with IC 50 values of 85 and 113 mM, respectively. Two nitrobenzoyl sesquiterpenoids, 6b,9a-dihydroxy-14-p-nitrobenzoylcinnamolide [149], and a known analog, insulicolide A [150] were isolated from extracts of the culture of marine-derived fungus Aspergillus ochraceus Jcma1F17. 150 The new compound [150] also exhibited moderate inhibitory activity against H3N2 viruses, with IC 50 values of 17.0 mM, but structurally related compound [150] failed to show any activity. Three triterpenes, ganodermadiol [98], lucidadiol [151], and applanoxidic acid G [152] isolated by Mothana et al. 151 from the European Basidiomycete Ganoderma pfeifferi showed antiviral activity against influenza virus type A in dye uptake assay. 152,153 These compounds showed activity against influenza type A virus infection on MDCK cells with ED 50 values > 0.22, 0.22, and 0.19 mM, respectively.

Inhibitors for herpes viruses
Herpes simplex viruses (HSVs) inhibitors. HSVs are very prevalent human pathogens that belong to the Herpesviridae family. There are two serotypes of human HSVs, namely HSV-1 and HSV-2. These are characterized by four main constituents, namely an icosapentahedral capsid, an amorphous layer of proteins around the capsid, an envelope, and an electron-dense core with large double-stranded DNA. HSV-1 is the main causative agent for facial infections with visible cold sores or fever blisters and HSV-2, in general, is associated with genital infection and causes genital herpes, 155,156 but both strains can cause infection in either area. 157 HSV can have both lytic and latent infection cycles. After initial infection, HSV-1 and HSV-2 are transported along sensory nerves to the sensory nerve cell bodies, where they establish lifelong latency of the human host. HSV exits latency periodically and is transported to the body surface where recurrent infection occurs. 158 Though several viral genes critical for HSV replication have been identified, 159,160 HSV treatment is generally limited to few synthetic antivirals as acyclovir, penciclovir, and famciclovir. 161,162 Thus, the search for new antivirals with novel modes of action remains unavoidable.
Six new naphthalenone derivatives, balticols A-F [155-160] and a known metabolite altechromone A 163 were isolated from the ethyl acetate extract of the culture broth of Ascomycota fungal strain 222 obtained from driftwood collect from the coast of the Greifswalder Bodden, Baltic Sea, Germany. 164 All isolated balticols A-F [155][156][157][158][159][160] exhibited strong anti-HSV-1 activity with IC 50 values 0.01-1 mM/ml. Balticol E [159] exhibited 10 times more activity against Herpes type I viruses with an IC 50 value of 0.01 mg/ml in comparison to the standard aciclovir (IC 50 0.1 mM). However, balticol D [158] and balticol F [160] exhibited similar activity with the standard. Balticol C [157], balticol D [158], and balticol F [160] also showed considerable activity against influenza viruses with IC 50 values 1, 0.1, and 1 mg/ml, respectively, compared to the positive standard amantadine sulfate with IC 50 15 mg/ml. But the activity of balticol E [159] is only very specific to HSV-1 and showed no activity against influenza virus. Another 12-membered macrolide, balticolid [161], from same strain displayed anti-HSV-1 activity with an IC 50 value of 0.45 mM compared to positive control aciclovir with IC 50 value of 0.44 mM. 165 Among four sterols and 10 triterpenes were isolated from the fruiting bodies of Ganoderma pfeifferi by Niedermeyer et al., 166  and applanoxidic acid G [152] simultaneously isolated together from same species failed to show any such activity. Hesseltin A [167], a novel compound of mixed polyketide-terpenoid origins was isolated from the filamentous fungus Penicillium hesseltinei. 168 It exhibited 25-50% inhibition of HSV-1 viral growth at 300 mg/ml, but more significantly [167] did not show any cytotoxicity at that concentration. 168 Monorden [168], tetrahydromonorden [169], several resorcylic acid lactones pochonins A-E [170][171][172][173][174] were isolated from cultures of the clavicipitaceous hyphomycete Pochonia chlamydosporia var. catenulata strain P 0297. 169 Fermentation of P0297 in bromide containing culture media yielded monocillins II [175] and III [176] as major metabolites and monorden [168] as well as monocillins F [156] as minor metabolites. In the HSV1 replication assay, monorden [168] showed inhibitory activity in the nM (IC 50 0.2-0.8 mM) range accompanied by cytostatic effects. The other metabolites with epoxide moieties, [169], [170], [171], and [176] exhibited bioactivities with IC 50 values 1.5, 2, 10, and 0.4 mM, respectively. Monocillin III [176], without any chlorine substituent, showed inhibitory activity in the nanomolar range, which suggest that the chlorine substituent is not essential for the antiviral activity. Pochonins E and monocillin F [177] with an allyl alcohol moiety showed moderate inhibitory activity. Inhibition of HSV was generally accompanied by weak cytostatic effects on the host cell, as observed microscopically. This results in rather low tolerability in vitro described by the selectivity indices (SIs).
A series of lypophilic linear peptides, halovirs A-E [182][183][184][185][186], isolated from saline fermentation of a marine-derived fungus Scytidium sp. 171 have displayed potent antiviral activity against HSV-1 with ED 50 values 1.1, 3.5, 2.2, 2.0, and 3.1 mM, respectively, in virus-induced CPE assay on Vero cell. 171 Halovir A [182] exhibited equal inhibition toward replication of both HSV-1 and HSV-2 with an ED 50 value of 280 nm in standard plaque reduction assay but did not show any cytotoxicity toward the host cell even at 0.85 mM. Further mechanistic studies revealed that halovirs kill the virus in direct contact and in timedependent manner before it can affect the host cell. Though the mechanism of virucidal activity of halovirs was not clear but it is presumed to happen through membrane destabilization of virus.

Cytomegalovirus (CMV) inhibitors.
CMV is a doublestranded DNA virus and is another member of the Herpesviridae family. It has the largest genome of the herpes viruses. Human CMV grows only in human cells and replicates best in human fibroblasts. CMV usually causes an asymptomatic infection or produces mild flulike symptoms; afterward, it remains latent throughout life and may reactivate. In immunocompromised individuals (by HIV infection, solid-organ transplantation, or bone marrow transplantation) symptomatic disease usually manifests as a mononucleosis syndrome. 172 Symptomatic CMV disease can affect almost every organ of the body, resulting in fever of unknown origin, pneumonia, hepatitis, encephalitis, myelitis, colitis, uveitis, retinitis, and neuropathy. Retinitis is the most common manifestation of CMV disease in patients who are HIV positive. In patients coinfected with HIV, CMV infection leads to progression to AIDS and eventually death, even in those receiving highly active antiretroviral therapy (HAART). 173 At least 60% of the US population has been exposed to CMV, 174 with a prevalence of more than 90% in high-risk groups (e.g. babies from infected mothers, people with HIV, male homosexuals etc.). 175,176 The prevailing age of infection varies worldwide. In developing countries, most infections are acquired during childhood, whereas, in developed countries, up to 50% of young adults are CMV seronegative. The incidence of CMV seropositivity rises with age and in a US-based study was reported to increase from 36% in children aged 6-11 years to 91% in individuals older than 80 years. 177 Other factors associated with CMV seropositivity include ethnicity (77% in Mexican Americans and 71% in blacks), 178 female sex, foreignborn status, and low socioeconomic status. 178 CMV is transmitted from person to person via close contact with an individual who is excreting the virus. It can be spread through the placenta, blood transfusions, organ transplantation, and breast milk. It can also be spread through sexual transmission. In the United States, congenital CMV transmission from a mother with acute infection during pregnancy is a significant cause of neurological abnormalities and deafness in approximately 8000 newborns annually. 179 The drug of choice for prevention of CMV disease in solidorgan transplant patients is valganciclovir. 180 Other than CMV retinitis, however, ganciclovir remains the mainstay of treatment, at least initially. Second-line treatments include foscarnet, cidofovir, or maribavir. Currently, there is no vaccine to prevent CMV infection.
A spiro compound having fused benzodihydrofuran and decalin moiety Sch 65676 [205] was isolated from the fermentation broth of an unidentified fungal culture. It exhibited in vitro inhibitory activity against the CMV protease with an IC 50 value of 9.8 mg/ml. 181 Inhibitors of Enterovirus 71 (EV71). EV71 is a small, nonenveloped, single-positive stranded, RNA virus from the Enterovirus genus of the Picornaviridae family. 182 It is a common causative agent in hand, foot, and mouth disease (HFMD) and sometimes provokes severe and fatal neurological complications in young children and infants, 183 which may result in acute flaccid paralysis and even death. 184,185 In 2014, the World Health Organization showed that over 2.8 million cases were diagnosed with HFMD in the Western Pacific Region, 186 with widespread occurrence in China. 187 Biochemical pathway of viral replication is explored in considerable detail 188 and other strategies have been employed to develop antiviral drugs on the basis of the molecular characteristics of the virus. 189 In spite of huge scientific effort so far, no direct targeting vaccines or antivirals are available to treat severe EV71 infections. Many natural products and their synthetic analogs and derivatives have exhibited interesting inhibitory activity. 190 Bioactivity of some secondary metabolites from fungal sources against EV71 has been described here.
Four new xanthone derivatives [188], [189], [190], and [191] along with 11 other new and known xanthones and terpenoid derivatives were isolated from the cultures of sponge-derived fungus Stachybotry sp. 191 Compounds [189], [190], and [191] displayed activities against intestinal virus EV71 with IC 50 values of 30.1, 50.0, and 40.3 mM, respectively. Compounds [188], [189], and [191] also exhibited significant inhibitory activity against cycloooxygenase (COX-2) with IC 50 values of 10.6, 8.9, and 34.3 mM, respectively. A new naphthalene derivative, vaccinal A [192], together with 12 other new and known phenol derivatives were isolated from a mangrove endophytic fungus Pestalotiopsis vaccinii. 192 Vaccinal A [192] exhibited activity against EV71 and COX-2 virus with an IC 50 value of 19.2 and 1.8 mM, respectively. Brefeldin A (BFA) [193] is a lactone antibiotic produced by fungal organisms Eupenicillium brefeldianum and later isolated from several other fungal species. 193,194 BFA, an inhibitor of COPI activity, has been shown to strongly inhibit viral RNA replication of poliovirus and EV11. 195,196 BFA inhibited EV71 replication in a dose-dependent fashion, without noticeable cytotoxicity. However, 20 ng/ml BFA inhibited EV71 replication effectively. Replication of EV71 fell substantially to 11% compared to the negative control, when 100 ng/ml BFA was used. 188 BFA inhibits protein transport from the endoplasmic reticulum to the Golgi apparatus indirectly by preventing the formation of COPI-mediated transport vesicles.

Inhibitors of respiratory syncytial virus (RSV)
Human RSV, a member of the genus Pneumovirus within the family Paramyxoviridae, is the leading cause of lower respiratory tract infection in infants and young children worldwide. 198 This negative (À) strand RNA virus with approximately 15 kilobases has 10 genes, which are responsible for the production of 11 viral proteins. Infection is mediated, in part, by an initial interaction between attachment protein (G) and a highly sulfated heparin-like glycosaminoglycan (Gag) located on the cell surface. Palivizumab and ribavirin are only therapeutic options available for treatment of RSV infections. Use of these drugs is also limited due to their toxicity in prolonged use and ineffectiveness against resistant strain. Therefore, there is a pressing need for development of new effective anti-RSV drugs 199 with less toxicity and novel mechanism of action.
Bioassay-guided separation of an extract of a gorgonian-derived fungus Aspergillus sp. led to isolation 22-O-(NMe-L-valyl)-21-epi-aflaquinolone B [195] along with one new epimer, 22-O-(NMe-L-valyl)-21-aflaquinolone B [196] and two other known analogs, aflaquinolones A [197] and D [198]. 200 Structure and stereochemistry of the new compounds were determined by spectroscopic methods, ECD spectra, chemical conversion, and Marfey's method. Compound [196] and [197] exhibited around 500 -and three-fold stronger anti-RSV activity with IC 50 value of 42 nM and 6.6 mM, respectively, compared to the positive control ribavirin (IC 50 ¼ 20 mM) in CPE assay. Outstanding anti-RSV activity with high therapeutic ratio (TC 50 /IC 50 ¼ 520) of compound [196] makes it a promising lead compound for anti-RSV drug discovery. Large increase of anti-RSV activity of [196] compared to [197] may be attributed to the presence of additional N-Me-L-Val residue. No antiviral activity of [195] and [197] suggest that the configuration of the cyclohexane unit play a critical role in anti-RSV activity. Agrocybone [199], a novel illudane-illudane bis-sesquiterpene, isolated from the basidiomycete Agrocybe salicacola, was found to exhibit weak antiviral activity against RSV with IC 50 value of 100 mM in CPE and plaque reduction assays. 201 Inhibitors of porcine reproductive and respiratory syndrome (PRRS) PRRS is one of the most economically devastating diseases affecting swine industry worldwide.The causative virus PRRS virus (PRRSV), like other members of the Arteriviridae family, has the ability to infect macrophages 202 and to persist in tissues for at least several months after the acute stage of infection subsides. This small, enveloped, positive (þ) strand RNA virus infects pigs and causes respiratory illness and a major failure in the reproduction of sows. Experimental infections have mirrored the clinical signs observed in natural outbreaks, including abortions, premature furrowing, mummified pigs, stillborn pigs, and elevated preweaning mortality. [203][204][205][206][207] By destroying macrophages it weakens a major part of the body's defense mechanism and allows bacteria and other viruses to proliferate and do damage. The high transmissibility of PRRSV 208 through a number of routes, including oral, intranasal, intramuscular, intraperitoneal, and vaginal, etc. increases the risk of its infection. Due to the complex epidemiologic profile, it has been especially difficult to control under the usual conditions of commercial swine production. An RNA genome of about 15 kilobases in size is responsible for the coding of two principal polyproteins, namely 1 a and 1ab. 209 PRRSV classifies into two subgroups, namely A and B, representing the North American and European strains, which have different level of virulence. PRRSV transmission can happen in many ways such as equipment, hands, needles, and footwear associated with mechanical transmitters, including flies and mosquitoes. Despite the prevalence of vaccines, control of the virus infection in usual swine conditions has always been a major hindrance to food industries.
Eleven new and seven known compounds were isolated from the culture broth and the mycelia of Alternaria sp. ZJ-2008003, a fungus obtained from a Sarcophyton sp. soft coral collected from the South China Sea. 210 Compounds [200], [201], and [202] exhibited antiviral activity against the PRRSV, with IC 50 values of 65, 22, and 39 mM, respectively.

Inhibitors of Molluscum contagiosum virus (MCV)
MCV is an unclassified pathogenic member of the Poxviridae, which replicate in human epidermis to create a common skin infection through augmentation of epidermal cell mitosis and disruption of cell differentiation. This linear, double-stranded DNA virus is characterized by lateral bodies, a core, an envelope, and a surface membrane.To escape from immune surveillance of it does not pass the basement membrane of the epidermis. Four different types (MCV I-VI) of this virus is identified. MCV I caused 96.6% of infections, and MCV II caused 3.4%, whereas MCV III and VI are very rare. 211,212 MCV infection in healthy people causes only small papules that are easily treated. In AIDS patients, however, MCV causes severe lesions that are essentially untreatable. 213,214 Cells near the surface of lesions become many times larger than normal, forming papules that become filled with a granular mass called ''molluscum bodies.'' Untreated papules in healthy people usually disappear spontaneously within several months, but in AIDS patients dense crops can persist, disfiguring infected patients. 215 A cyclic depsipeptide, sansalvamide A [203], isolated from a marine fungus Fusarium sp., exhibited in vitro MCV topoisomerase inhibitory activity in concentration-dependent fashion with an IC 50 of 124 mM. It is found to inhibit topoisomerase-catalyzed DNA relaxation and thus viral replication. 216

Inhibitors of tobacco mosaic virus (TMV)
TMV is a rod-like plant virus, with a complex capsid and positive (þ) strand RNA of approximately 6.4 kilobases. This virus does not usually kill the plant that is infected; it causes damage to flowers, leaves, and fruit through discoloration and mosaic mottling and stunts a plant's growth. 217,218 TMV is named for the first plant in which it was discovered but can infect over 400 different types of plants including vegetables, weeds, and flowers. The virus does not produce spores but spreads mechanically, entering plants via wounds. Till date prevention is the best control as there is no chemical treatment that effectively protects plants from TMV. [219][220][221] Therefore, a discovery of a naturally occurring chemical agent can be an important option for treatment of this virus.

Conclusion and perspective
Natural products with their great complexity and enormous diversity remain as inexhaustible source and inspiration of many drugs for various diseases. 17 Such products from fungal sources also played an important role to substantially enrich the present-day drug repertoire. 27 Current antiviral chemotherapy, driven by mainly synthetic drugs is gradually becoming ineffective due to rapid emergence of drug resistance viral strain 9,225 and their adverse side effect in prolonged use. [226][227][228] Thus, there is an urgent need for new compounds with novel mode action of which can target different therapeutic sites. 40,229 Due to gradual attrition rate in finding novel synthetic drug lead, 230,231 presentday drug industry once again increasingly being forced to rely on structurally diverse secondary metabolites from nature. 232 Fungal metabolites with great diversity and preapproved biocompatibility can be a potential source for new antiviral drug lead. Considering, very small fraction of fungal species has been discovered and only few percent of these extracts are tested for various viral diseases 233,234 there remains an enormous potential for finding new fungal metabolites as drug leads with novel mechanism of action. Thus, an energetic endeavor toward identification and collection of unknown fungal species and developing better culture methods is required to expedite the discovery process considerably.
In many cases, crude extracts of new fungal species have been studied but less effort has been expended for further isolation of their active metabolites. With better instrumentation and advanced database, characterization of new metabolites became comparatively easier and robotics also brought amazing efficiency in bioactivity screening. But, purification of metabolites from extracts still remained one of the most time-consuming steps in natural product drug discovery. Therefore, development of easy, inexpensive but effective and automated purification techniques for fungal metabolites is very crucial to accelerate bioactivity screening. 235 Due to lack of sophisticated and comprehensive viral screening facilities and lack of funding and stringent biodiversity rule in many countries, laboratories having access to various new fungal species hardly find opportunity to screen their metabolites against various viral diseases. Establishment of more and more sophisticated international antiviral screening facilities will be very helpful to deal such situation and will be a big boost to future antiviral research.
Most of the study of fungal metabolites reported so far mainly is done on in vitro level. Serious in vivo study of metabolites with high therapeutic index is essential to properly assess their strength and weaknesses in biological system. Bioactivity-guided gradual synthetic modification of new bioactive metabolites and subsequent structure-activity optimization study of generated analogs/derivatives is another area of crucial importance in drug discovery and has delivered many semisynthetic drugs in past. 17,236 Metabolites or their semisynthetic derivatives with appealing in vivo activity will be required in large amount for their clinical studies and further drug development process. In such case, exploration and engineering of biosynthetic pathway for efficient production of important metabolite will be another important area of research for mass production of metabolite with established bioactivity. 237 Though there are many fungal metabolite drugs being used for many other diseases, so far no antiviral drug has been discovered from fungal metabolites. One of the main reasons might be only a small fraction of the total isolated metabolites have been tested for antiviral screening due to lack of proper infrastructure for antiviral assay in many developing countries with high fungal biodiversity and fear associated with handling various viral strains. Therefore, besides identification and exploration of new fungal species and metabolites, effort should be devoted to comprehensive antiviral screening of many known but not-tested metabolites.
Reported metabolites with high in vitro therapeutic index should be taken up for in vivo screening and subsequent bioassay-guided structural optimization study. Promising lead metabolites can be taken up for further serious drug discovery research. Considering high antiviral activity of large number of reported fungal metabolites discussed in this review, enormity of estimated fungal biodiversity, number and structural diversity of untested fungal metabolites and gradual development in various facet of drug discovery process, eventual discovery of novel better antiviral drug from fungal metabolites should be a reality in near future. It is hoped that this review would be an inspiration and helpful resource for future research in the field of fungal metabolite-based antiviral drug discovery.