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Mycoviruses are viruses that infect fungi and are widespread across all major fungal taxa, exhibiting great biological diversity. Since their discovery in the 1960s, researchers have observed a myriad of fungal phenotypes altered due to mycoviral infection. In this review, we examine the nuanced world of mycoviruses in the context of the medically and agriculturally important fungal genus, Aspergillus. The advent of RNA sequencing has revealed a previous underestimate of viral prevalence in fungi, in particular linear single-stranded RNA viruses, and here we outline the diverse viral families known to date that contain mycoviruses infecting Aspergillus. Furthermore, we describe these novel mycoviruses, highlighting those with peculiar genome structures, such as a split RNA dependent RNA polymerase gene. Next, we delineate notable mycovirus-mediated phenotypes in Aspergillus, in particular reporting on observations of mycoviruses that affect their fungal host's virulence and explore how this may relate to virus-mediated decreased stress tolerance. Furthermore, mycovirus effects on microbial competition and antifungal resistance are discussed. The factors that influence the manifestation of these phenotypes, such as temperature, fungal life stage, and infection with multiple viruses, among others, are also evaluated. In addition, we attempt to elucidate the molecular mechanisms that underpin these phenotypes, examining how mycoviruses can be targets, triggers, and even suppressors of RNA silencing and how this can affect fungal gene expression and phenotypes. Finally, we highlight the potential therapeutic applications of mycoviruses and how, in an approach analogous to bacteriophage therapy, their ability to produce hypovirulence in Aspergillus might be used to attenuate invasive aspergillosis infections in humans.

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Infection with Aspergillus fumigatus polymycovirus 1 (AfuPmV-1) affects Aspergillus fumigatus Af293's growth in vitro, iron metabolism, resistance in intermicrobial competition with Pseudomonas aeruginosa, resistance to osmotic stress, and resistance to the chitin synthase inhibitor nikkomycin Z. Here, we show that response to high temperature, Congo Red-induced stress, and hydrogen peroxide are also dependent on the viral infection status of A. fumigatus. AfuPmV-1- infected Af293 was more susceptible than virus-free Af293 to growth inhibition by high temperature, hydrogen peroxide, Congo Red exposure, and nutrient restriction. Increased resistance of virus-free fungus was observed when cultures were started from conidia but, in the case of high temperature and hydrogen peroxide, not when cultures were started from hyphae. This indicates that the virus impairs the stress response during the growth phase of germination of conidia and development into hyphae. In conclusion, our work indicates that AfuPmV-1 infection in A. fumigatus impairs host responses to stress, as shown by exposure to high temperature, oxidative stress such as hydrogen peroxide, and some cell wall stresses, as shown by exposure to Congo Red (in agreement with our previous observations using nikkomycin Z) and nutrient restriction.

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Metarhizium anisopliae is a well-studied entomopathogenic fungus that is widely used in biological control programs. The presence of polymycoviruses in this fungus is common, but their effects on fungal development and stress tolerance are not well understood. In this study, we report the discovery of a novel double-stranded RNA virus, named Metarhizium anisopliae polymycovirus 1 (MaPmV1), which comprises four dsRNAs ranging from 2.4 to 1.4 kbp in length. Phylogenetic analysis revealed that MaPmV1 belongs to the Polymycoviridae family. Biological comparison between MaPmV1-infected (Vi) and -free (Vf) isogenic lines showed that MaPmV1 remarkably enhances the growth rate and conidiation of the host fungus. The upregulation of growth- and conidiation-related genes in Vi strains supports this finding. In addition, MaPmV1 increases the sensitivity of the host to UV-B irradiation, which is evidenced by the downregulation of DNA damage repair genes in Vi strains. However, MaPmV1 does not appear to have any significant impact on the virulence of M. anisopliae. Furthermore, overexpression of individual viral proteins in M. anisopliae did not result in any significant phenotypic alterations, indicating that MaPmV1-mediated changes are not related to a single viral protein. Overall, our findings suggest that mycoviruses can be exploited to enhance fungal development in entomopathogenic fungi, which may lead to improved conidium production on a large scale.

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In fungi, viral infections frequently remain cryptic causing little or no phenotypic changes. It can indicate either a long history of coevolution or a strong immune system of the host. Some fungi are outstandingly ubiquitous and can be recovered from a great diversity of habitats. However, the role of viral infection in the emergence of environmental opportunistic species is not known. The genus of filamentous and mycoparasitic fungi Trichoderma (Hypocreales, Ascomycota) consists of more than 400 species, which mainly occur on dead wood, other fungi, or as endo- and epiphytes. However, some species are environmental opportunists because they are cosmopolitan, can establish in a diversity of habitats, and can also become pests on mushroom farms and infect immunocompromised humans. In this study, we investigated the library of 163 Trichoderma strains isolated from grassland soils in Inner Mongolia, China, and found only four strains with signs of the mycoviral nucleic acids, including a strain of T. barbatum infected with a novel strain of the Polymycoviridae and named and characterized here as Trichoderma barbatum polymycovirus 1 (TbPMV1). Phylogenetic analysis suggested that TbPMV1 was evolutionarily distinct from the Polymycoviridae isolated either from Eurotialean fungi or from the order Magnaportales. Although the Polymycoviridae viruses were also known from Hypocrealean Beauveria bassiana, the phylogeny of TbPMV1 did not reflect the phylogeny of the host. Our analysis lays the groundwork for further in-depth characterization of TbPMV1 and the role of mycoviruses in the emergence of environmental opportunism in Trichoderma. IMPORTANCE Although viruses infect all organisms, our knowledge of some groups of eukaryotes remains limited. For instance, the diversity of viruses infecting fungi-mycoviruses-is largely unknown. However, the knowledge of viruses associated with industrially relevant and plant-beneficial fungi, such as Trichoderma spp. (Hypocreales, Ascomycota), may shed light on the stability of their phenotypes and the expression of beneficial traits. In this study, we screened the library of soilborne Trichoderma strains because these isolates may be developed into bioeffectors for plant protection and sustainable agriculture. Notably, the diversity of endophytic viruses in soil Trichoderma was outstandingly low. Only 2% of 163 strains contained traces of dsRNA viruses, including the new Trichoderma barbatum polymycovirus 1 (TbPMV1) characterized in this study. TbPMV1 is the first mycovirus found in Trichoderma. Our results indicate that the limited data prevent the in-depth study of the evolutionary relationship between soilborne fungi and is worth further investigation.

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Infection with Aspergillus fumigatus polymycovirus 1 (AfuPmV-1) weakens the resistance of biofilms of common A. fumigatus reference strain Af293 in intermicrobial competition with Pseudomonas aeruginosa, and sensitizes A. fumigatus for antifungal effects of nikkomycin Z. We compared the sensitivity of two virus-infected (VI) and one virus-free (VF) Af293 strains to hypertonic salt. Salt stress impairs the growth of VI and VF at all times; VF control growth always exceeds VI, and VF growth in salt always exceeds VI. Since VF growth exceeds VI in the presence and absence of salt, we also examined growth in salt as a percentage of control growth. Initially, as a percentage of control, VI exceeded VF, but at 120 h VF began to exceed VI consistently even by this measure; thus, at that time the growth of VF in salt surges in relation to control growth, or, alternatively, its growth in salt persists compared to the relative inhibition of VI. In summary, virus infection impairs the response of A. fumigatus to several different stresses, including hypertonic salt.

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Polymycoviridae is a recently established family of mycoviruses. Beauveria bassiana polymycovirus 4 (BbPmV-4) was previously reported. However, the effect of the virus on host fungus B. bassiana was not clarified. Here, a comparison between virus-free and virus-infected isogenic lines of B. bassiana revealed that BbPmV-4 infection of B. bassiana changes morphology and could lead to decreases in conidiation and increases in virulence against Ostrinia furnacalis larvae. The differential expression of genes between virus-free and virus-infected strains was compared by RNA-Seq and was consistent with the phenotype of B. bassiana. The enhanced pathogenicity may be related to the significant up-regulation of genes encoding mitogen activated protein kinase, cytochrome P450, and polyketide synthase. The results enable studies of the mechanism of interaction between BbPmV-4 and B. bassiana.

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Infection with Aspergillus fumigatus polymycovirus 1 (AfuPmV-1) weakens resistance of Aspergillus fumigatus common reference strain Af293 biofilms in intermicrobial competition with Pseudomonas aeruginosa. We compared the sensitivity of two infected and one virus-free Af293 strains to antifungal drugs. All three were comparably sensitive to drugs affecting fungal membranes (voriconazole, amphotericin) or cell wall glucan synthesis (micafungin, caspofungin). In contrast, forming biofilms of virus-free Af293 were much more resistant than AfuPmV-1-infected Af293 to nikkomycin Z (NikZ), a drug inhibiting chitin synthase. The IC50 for NikZ on biofilms was between 3.8 and 7.5 µg/mL for virus-free Af293 and 0.94-1.88 µg/mL for infected strains. The IC50 for the virus-free A. fumigatus strain 10AF was ~2 µg/mL in most experiments. NikZ also modestly affected the planktonic growth of infected Af293 more than the virus-free strain (MIC 50%, 2 and 4 µg/mL, respectively). Virus-free Af293 biofilm showed increased metabolism, and fungus growing as biofilm or planktonically showed increased growth compared to infected; these differences do not explain the resistance of the virus-free fungus to NikZ. In summary, AfuPmV-1 infection sensitized A. fumigatus to NikZ, but did not affect response to drugs commonly used against A. fumigatus infection. Virus infection had a greater effect on NikZ inhibition of biofilm than planktonic growth.

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Talaromyces amestolkiae is an important fungal species owing to its ubiquity in soils, plants, air, and food. In this study, we identified a novel six-segmented polymycovirus, Talaromyces amestolkiae polymycovirus 1 (TaPmV-1). Each of the double-stranded (ds) RNA segments of TaPmV-1 contained a single open reading frame, and the proteins encoded by dsRNA1, dsRNA2, dsRNA3, and dsRNA 5 shared significant amino acid identities of 56, 40, 47, and 43%, respectively, with the corresponding proteins of Aspergillus fumigatus polymycovirus-1(AfuPmV-1). DsRNA1, dsRNA3, and dsRNA5 of TaPmV-1 encoded an RNA-dependent RNA polymerase (RdRp), a viral methyltransferase, and a PAS-rich protein, respectively. The functions of the proteins encoded by dsRNA2, dsRNA4, and dsRNA6 have not been elucidated. Comparison of the virus-infected strain LSH3 with virus-cured strain LSHVF revealed that infection with TaPmV-l may reduce the production of red pigments and induce the clustering of fungal sclerotia. Furthermore, transcriptomic analyses demonstrated that infection with TaPmV-l downregulated the expression of transcripts related to metabolism, and may correlate with the reduced production of red pigments and clustering of sclerotia in T. amestolkiae. These results of this study provide novel insights into the mechanism of fungal gene regulation by polymycovirus infections at the transcriptome level, and this study is the first to report a novel polymycovirus of T. amestolkiae.

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Members of the family Polymycoviridae are small viruses with multi-segmented and non-conventionally encapsidated double-stranded (ds) RNA genomes. Typically, polymycoviruses have four genomic segments, although some have up to eight. The genus Polymycovirus includes several species whose members infect fungi (ascomycetes and basidiomycetes), and oomycetes, altering host morphology, sporulation, growth and virulence. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Polymycoviridae, which is available at ictv.global/report/polymycoviridae.

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In this study, a novel positive-sense single-stranded RNA (+ssRNA) mycovirus, tentatively named Colletotrichum fructicola RNA virus 1 (CfRV1), was identified in the phytopathogenic fungus Colletotrichum fructicola. CfRV1 has seven genomic components, encoding seven proteins from open reading frames (ORFs) flanked by highly conserved untranslated regions (UTRs). Proteins encoded by ORFs 1, 2, 3, 5, and 6 are more similar to the putative RNA-dependent RNA polymerase (RdRp), hypothetical protein (P2), methyltransferase, and two hypothetical proteins of Hadaka virus 1 (HadV1), a capsidless 10- or 11-segmented +ssRNA virus, while proteins encoded by ORFs 4 and 7 showed no detectable similarity to any known proteins. Notably, proteins encoded by ORFs 1 to 3 also share considerably high similarity with the corresponding proteins of polymycoviruses. Phylogenetic analysis conducted based on the amino acid sequence of CfRV1 RdRp and related viruses placed CfRV1 and HadV1 together in the same clade, close to polymycoviruses and astroviruses. CfRV1-infected C. fructicola strains demonstrate a moderately attenuated growth rate and virulence compared to uninfected isolates. CfRV1 is capsidless and potentially encapsulated in vesicles inside fungal cells, as revealed by transmission electron microscopy. CfRV1 and HadV1 are +ssRNA mycoviruses closely related to polymycoviruses and astroviruses, represent a new linkage between +ssRNA viruses and the intermediate double-stranded RNA (dsRNA) polymycoviruses, and expand our understanding of virus diversity, taxonomy, evolution, and biological traits. IMPORTANCE A scenario proposing that dsRNA viruses evolved from +ssRNA viruses is still considered controversial due to intergroup knowledge gaps in virus diversity. Recently, polymycoviruses and hadakaviruses were found as intermediate dsRNA and +ssRNA stages, respectively, between +ssRNA and dsRNA viruses. Here, we identified a novel +ssRNA mycovirus, Colletotrichum fructicola RNA virus 1 (CfRV1), isolated from Colletotrichum fructicola in China. CfRV1 is phylogenetically related to the 10- or 11-segmented Hadaka virus 1 (HadV1) but consists of only seven genomic segments encoding two novel proteins. CfRV1 is naked and may be encapsulated in vesicles inside fungal cells, representing a potential novel lifestyle for multisegmented RNA viruses. CfRV1 and HadV1 are intermediate +ssRNA mycoviruses in the linkage between +ssRNA viruses and the intermediate dsRNA polymycoviruses and expand our understanding of virus diversity, taxonomy, and evolution.

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A virus-free (VF) A. fumigatus isolate has been shown to be resistant in competition with Pseudomonas as compared to the isogenic line infected with Aspergillus fumigatus polymycovirus 1 (AfuPmV-1), and this phenotype was apparently related to alterations in iron metabolism. Here we investigated further the mechanisms underpinning this phenotype. The extracellular siderophore profiles of five isogenic VF and virus-infected (VI) strains were sampled at 24, 31, 48, 54, and 72 h in submerged cultures and quantitatively examined by liquid chromatography and mass spectrometry. Intracellular profiles of conidia and cultures at the stationary growth phase were defined. VF A. fumigatus demonstrated the best fitness represented by the fastest onset of its exponential growth when grown on an iron-limited mineral medium. The exponential phase and transitional production phase of the extracellular triacetylfusarinine C (TafC) were achieved at 24 and 31 h, respectively, contrary to VI strains, which acted more slowly. As a result, the TafC reservoir was consumed sooner in the VF strain. Additionally, the VF strain had lower ferricrocin and higher hydroxyferricrocin content in the pellet during the stationary phase. All of these differences were significant (Kruskal-Wallis, p < 0.01). In our study, the siderophore reservoir of a VF strain was consumed sooner, improving the fitness of the VF strain in competition with P. aeruginosa.

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The genus Polymycovirus of the family Polymycoviridae accommodates fungal RNA viruses with different genomic segment numbers (four, five, or eight). It is suggested that four members form no true capsids and one forms filamentous virus particles enclosing double-stranded RNA (dsRNA). In both cases, viral dsRNA is associated with a viral protein termed "proline-alanine-serine-rich protein" (PASrp). These forms are assumed to be the infectious entity. However, the detailed molecular characteristics of PASrps remain unclear. Here, we identified a novel five-segmented polymycovirus, Penicillium janthinellum polymycovirus 1 (PjPmV1), and characterized its purified fraction form in detail. The PjPmV1 had five dsRNA segments associated with PASrp. Density gradient ultracentrifugation of the PASrp-associated PjPmV1 dsRNA revealed its uneven structure and a broad fractionation profile distinct from that of typical encapsidated viruses. Moreover, PjPmV1-PASrp interacted in vitro with various nucleic acids in a sequence-non-specific manner. These PjPmV1 features are discussed in view of the diversification of genomic segment numbers of the genus Polymycovirus.

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The search for viruses infecting fungi, or mycoviruses, has extended our knowledge about the diversity of RNA viruses, as exemplified by the discovery of polymycoviruses, a phylogenetic group of multisegmented RNA viruses with unusual forms. The genomic RNAs of known polymycoviruses, which show a phylogenetic affinity for animal positive-sense single-stranded RNA [(+)RNA] viruses such as caliciviruses, are comprised of four conserved segments with an additional zero to four segments. The double-stranded form of polymycovirus genomic RNA is assumed to be associated with a virally encoded protein (proline-alanine-serine-rich protein [PASrp]) in either of two manners: a capsidless colloidal form or a filamentous encapsidated form. Detailed molecular characterizations of polymycoviruses, however, have been conducted for only a few strains. Here, a novel polymyco-related virus named Hadaka virus 1 (HadV1), from the phytopathogenic fungus Fusarium oxysporum, was characterized. The genomic RNA of HadV1 consisted of an 11-segmented positive-sense RNA with highly conserved terminal nucleotide sequences. HadV1 shared the three conserved segments with known polymycoviruses but lacked the PASrp-encoding segment. Unlike the known polymycoviruses and encapsidated viruses, HadV1 was not pelleted by conventional ultracentrifugation, possibly due to the lack of PASrp. This result implied that HadV1 exists only as a soluble form with naked RNA. Nevertheless, the 11 genomic segments of HadV1 have been stably maintained through host subculturing and conidiation. Taken together, the results of this study revealed a virus with a potential novel virus lifestyle, carrying many genomic segments without typical capsids or PASrp-associated forms.IMPORTANCE Fungi collectively host various RNA viruses. Examples include encapsidated double-stranded RNA (dsRNA) viruses with diverse numbers of genomic segments (from 1 to 12) and capsidless viruses with nonsegmented (+)RNA genomes. Recently, viruses with unusual intermediate features of an infectious entity between encapsidated dsRNA viruses and capsidless (+)RNA viruses were found. They are called polymycoviruses, which typically have four to eight dsRNA genomic segments associated with one of the virus-encoded proteins and are phylogenetically distantly related to animal (+)RNA caliciviruses. Here, we identified a novel virus phylogenetically related to polymycoviruses, from the phytopathogenic fungus Fusarium oxysporum The virus, termed Hadaka virus 1 (HadV1), has 11 (+)RNA genomic segments, the largest number in known (+)RNA viruses. Nevertheless, HadV1 lacked a typical structural protein of polymycoviruses and was not pelleted by standard ultracentrifugation, implying an unusual capsidless nature of HadV1. This study reveals a potential novel lifestyle of multisegmented RNA viruses.

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BACKGROUND: Mycoviruses that infect fungi generally do not have a significant effect on the host and, instead, reduce the toxicity of the fungi. However, recent studies have shown that polymycovirus-1, a mycovirus that infects Aspergillus species known to cause disease in humans, is related to increased virulence of the fungus. METHODS: Comparative analysis was performed of RdRP gene codon usage patterns of Aspergillus fumigatus polymycovirus-1 (AfuPmV-1) and other mycoviruses known to infect Aspergillus spp. to examine the genetic characteristics of AfuPmV-1. In addition, codon usage analysis was performed to determine whether the nucleotide composition and codon usage characteristics of AfuPmV-1 were also present in other polymycoviruses and hypervirulence-related mycoviruses. Phylogenetic analysis was also performed to investigate their evolutionary relationship. RESULTS: Analysis of nucleotide composition indicated that AfuPmV-1 had the highest GC content among analyzed mycoviruses and relative synonymous codon usage analysis indicated that all of the codons preferred by AfuPmV-1 ended with C or G, while codons ending with A or U were not observed. Moreover, the effective number of codons, the codon adaptation index, and correspondence analysis showed that AfuPmV-1 had greater codon preference compared with other mycoviruses and that AfuPmV-1 had relatively high adaptability to humans and fungi. These results were generally similar among polymycoviruses. CONCLUSIONS: The codon usage pattern of AfuPmV-1 differs from other mycoviruses that infect Aspergillus spp. This difference may be related to the hypervirulence effect of AfuPmV-1. Analysis of AfuPmV-1 codon usage patterns could contribute to the identification and prediction of virulence effects of mycoviruses with similar genetic characteristics.

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The use of mycoviruses to manipulate the virulence of entomopathogenic fungi employed as biocontrol agents may lead to the development of novel methods to control attacks by insect pests. Such approaches are urgently required, as existing agrochemicals are being withdrawn from the market due to environmental and health concerns. The aim of this work is to investigate the presence and diversity of mycoviruses in large panels of entomopathogenic fungi, mostly from Spain and Denmark. In total, 151 isolates belonging to the genera Beauveria, Metarhizium, Lecanicillium, Purpureocillium, Isaria, and Paecilomyces were screened for the presence of dsRNA elements and 12 Spanish B. bassiana isolates were found to harbor mycoviruses. All identified mycoviruses belong to three previously characterised species, the officially recognised Beauveria bassiana victorivirus 1 (BbVV-1) and the proposed Beauveria bassiana partitivirus 2 (BbPV-2) and Beauveria bassiana polymycovirus 1 (BbPmV-1); individual B. bassiana isolates may harbor up to three of these mycoviruses. Notably, these mycovirus species are under distinct selection pressures, while recombination of viral genomes increases population diversity. Phylogenetic analysis of the RNA-dependent RNA polymerase gene sequences revealed that the current population structure in Spain is potentially a result of both vertical and horizontal mycovirus transmission. Finally, pathogenicity experiments using the Mediterranean fruit fly Ceratitis capitata showed no direct correlation between the presence of any particular mycovirus and the virulence of the B. bassiana isolates, but illustrated potentially interesting isolates that exhibit relatively high virulence, which will be used in more detailed virulence experimentation in the future.

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