Mycovirus therapy for invasive pulmonary aspergillosis?

With the current revived interest in the use of bacteriophages for the treatment of bacterial infections, the study of mycoviruses as novel therapeutic solutions for invasive aspergillosis is the logical next step. Although ssRNA, dsRNA, and ssDNA mycoviruses have been identified, the majority of characterised mycoviruses have dsRNA genomes. Prevalence of dsRNA mycoviruses in Aspergillus spp. varies, and mycoviruses can have different effects on their fungal hosts: hypovirulence, hypervirulence, or a killer phenotype. Therapeutically, extracellular transmission of the mycovirus is essential. DsRNA mycoviruses lack an extracellular phase; however, a single ssDNA mycovirus with homologues in Aspergillus genomes has been described with an extracellular mode of transmission. Mycoviruses can induce hypovirulence or a killer phenotype, and both can be exploited therapeutically. Mycoviruses inducing hypovirulence have been used to control chestnut blight, however for aspergillosis no such mycovirus has been identified yet. Mycovirus encoded killer toxins or anti-idiotypic antibodies and killer peptides derived from these have been demonstrated to control fungal infections including aspergillosis in animals. This indicates that mycoviruses inducing both phenotypes could be exploited therapeutically as long as the right mycovirus has been identified.

Analysis of the codon usage pattern of the RdRP gene of mycovirus infecting Aspergillus spp.

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.

A novel chrysovirus from a clinical isolate of Aspergillus thermomutatus affects sporulation.

A clinical isolate of Aspergillus thermomutatus (Teleomorph: Neosartorya pseudofischeri) was found to contain ~35 nm isometric virus-like particles associated with four double-stranded (ds) RNA segments, each of which coded for a single open reading frame. The longest dsRNA element (3589 nt) encodes a putative RNA-dependent RNA polymerase (1114 aa), the second longest dsRNA element (2772 nt) encodes a coat protein (825 aa), and the other two dsRNAs (2676 nt, 2514 nt) encode hypothetical proteins of 768 aa and 711 aa, respectively. Phylogenetic analysis of the amino acid sequences showed 41-60% similarity to the proteins coded by the dsRNAs of the most closely related virus, Penicillium janczewskii chrysovirus 2, indicating that it is a new species based on the International Committee on Taxonomy of Viruses criteria for the genus Chrysovirus. This is the first virus reported from A. thermomutatus and was tentatively named Aspergillus thermomutatus chrysovirus 1. A virus free line of the fungal isolate, cured by cycloheximide treatment, produced large numbers of conidia but no ascospores at both 20°C and 37°C, whereas the virus infected line produced ten-fold fewer conidia at 20°C and a large number of ascospores at both temperatures. The effects of the virus on fungal sporulation have interesting implications for the spread of the fungus and possible use of the virus as a biological control agent.

The Effect of Aspergillus Thermomutatus Chrysovirus 1 on the Biology of Three Aspergillus Species.

This study determined the effects of Aspergillus thermomutatus chrysovirus 1 (AthCV1), isolated from Aspergillus thermomutatus, on A. fumigatus, A. nidulans and A. niger. Protoplasts of virus-free isolates of A. fumigatus, A. nidulans and A. niger were transfected with purified AthCV1 particles and the phenotype, growth and sporulation of the isogenic AthCV1-free and AthCV1-infected lines assessed at 20 °C and 37 °C and gene expression data collected at 37 °C. AthCV1-free and AthCV1-infected A. fumigatus produced only conidia at both temperatures but more than ten-fold reduced compared to the AthCV1-infected line. Conidiation was also significantly reduced in infected lines of A. nidulans and A. niger at 37 °C. AthCV1-infected lines of A. thermomutatus and A. nidulans produced large numbers of ascospores at both temperatures, whereas the AthCV1-free line of the former did not produce ascospores. AthCV1-infected lines of all species developed sectoring phenotypes with sclerotia produced in aconidial sectors of A. niger at 37 °C. AthCV1 was detected in 18% of sclerotia produced by AthCV1-infected A. niger and 31% of ascospores from AthCV1-infected A. nidulans. Transcriptome analysis of the naturally AthCV1-infected A. thermomutatus and the three AthCV1-transfected Aspergillus species showed altered gene expression as a result of AthCV1-infection. The results demonstrate that AthCV1 can infect a range of Aspergillus species resulting in reduced sporulation, a potentially useful attribute for a biological control agent.

Allergic and Noninvasive Infectious Pulmonary Aspergillosis Syndromes.

Aspergillus spp are ubiquitous in the environment, and inhalation of Aspergillus spores is unavoidable. An intact immune system, with normal airway function, protects most people from disease. Globally, however, the toll from aspergillosis is high. The literature has largely focused on invasive aspergillosis, yet the burden in terms of chronicity and prevalence is higher for noninvasive Aspergillus conditions. This article discusses allergic aspergilloses and provides an update on the diagnosis and management of allergic bronchopulmonary aspergillosis, including in patients with cystic fibrosis, and an update on severe asthma with fungal sensitization. In addition, the presentation, investigation, and management of noninvasive infectious aspergilloses are reviewed.

A novel dsRNA element isolated from the Aspergillus foetidus mycovirus complex.

Aspergillus foetidus virus (AfV) contains at least two icosahedral particle types named AfV-fast (-F) and AfV-slow (-S), based on relative electrophoretic mobility. AfV-F is a quadripartite double-stranded RNA (dsRNA) virus, and AfV-S contains AfV-S1, which is a member of the genus Victorivirus in the family Totiviridae, and AfV-S2, which may be a satellite RNA or satellite virus and is described here. Analysis of the complete AfV-S2 nucleotide sequence reveals it to be significantly similar to an unclassified RNA from the fungus Rosellinia necatrix and distantly related to the RNA-dependent RNA polymerases of several single-stranded RNA genomes.

Sequence determination of a quadripartite dsRNA virus isolated from Aspergillus foetidus.

Virus infection of Aspergillus foetidus was documented over 40 years ago and was one of the first mycovirus infections described in a filamentous fungus. The virus, named Aspergillus foetidus virus (AfV), contains at least two types of icosahedral particles, called AfV-fast (-F) and AfV-slow (-S) virions, based on their relative electrophoretic mobilities. Here, we report the complete nucleotide sequence of the AfV-F genome isolated from virions purified from the prototype isolate of the fungus. The AfV-F double-stranded (ds) RNA genome is tetra-segmented, and the plus strands of each of the four segments, but not the minus strands, are polyadenylated. The organisation and sequences of the four AfV-F dsRNAs are similar to those described for Alternaria alternata virus 1, which we propose is a member of an emerging mycovirus genus ("Alternavirus") and family ("Alternaviridae"), which also includes AfV-F.

The complete nucleotide sequence of a totivirus from Aspergillus foetidus.

Virus infection of Aspergillus foetidus was first described in the 1970s, and the purified virus, named Aspergillus foetidus virus (AfV), contains at least two types of icosahedral particles, called AfV-fast (-F) and AfV-slow (-S) virions, based on their relative electrophoretic mobilities. AfV-S consists of a mixture of two viruses, the larger of which, called AfV-S1, is a dsRNA-containing member of the genus Victorivirus in the family Totiviridae, and its complete nucleotide sequence is described here.

Aspergilosis invasiva: reporte de un caso y revisión de literatura / Invasive aspergillosis: a case report and literature review

La aspergilosis invasiva es una enfermedad infecciosa producida por un hongo del género Aspergillus. La infección se adquiere generalmente por vía respiratoria al inhalar las esporas que se encuentran en altas concentraciones en el aire, el suelo y sobre todo en la materia orgánica en descomposición. El diagnóstico es difícil realizarlo, por tanto el tratamiento muchas veces se instaura de manera tardía; una vez instaurada la infección el pronóstico es muy malo. Se presenta el caso de un paciente masculino de 9 años de edad, con desnutrición severa y pancitopenia, atendido en el Hospital Materno Infantil de Tegucigalpa, a quien en la autopsia se le demostró aspergilosis invasiva. No se encontró publicaciones locales sobre este tema...

Aspergilosis invasiva: reporte de un caso y revisión de literatura / Invasive aspergillosis: a case report and literature review

RESUMEN. La aspergilosis invasiva es una enfermedad infecciosa producida por un hongo del género Aspergillus. La infección se adquiere generalmente por vía respiratoria al inhalar las esporas que se encuentran en altas concentraciones en el aire, el suelo y sobre todo en la materia orgánica en descomposición. El diagnóstico es difícil realizarlo, por tanto el tratamiento muchas veces se instaura de manera tardía; una vez instaurada la infección el pronóstico es muy malo. Se presenta el caso de un paciente masculino de 9 años de edad, con desnutrición severa y pancitopenia, atendido en el Hospital Materno Infantil de Tegucigalpa, a quien en la autopsia se le demostró aspergilosis invasiva. No se encontró publicaciones locales sobre este tema...(AU)

Dynamics of dsRNA mycoviruses in black Aspergillus populations.

Approximately 10% of all examined 668 representatives of black Aspergillus species, independent of worldwide location, were infected with double-stranded RNA (dsRNA) mycoviruses. These isometric viruses (25-40 nm diameter) contained a variety of often multiple segments of different dsRNA sizes ranging from 0.8 to 4.4 kb in size. In one strain the virus shows clear visible effects on its host with non-sporulating sectors. We quantified the fitness costs of these and more 'cryptic' virus infections on mycelial growth rate and spore production, and on competitive ability with respect to other strains under different growth conditions. Mycovirus infection proved detrimental in all these measures. The reduced success in interference competition due to mycovirus infection belies co-evolution of mycovirus and host to a mutually beneficial symbiosis, like in killer virus systems in yeast and smut and agrees more to recent infections. For a stable virus infection frequency in the black Aspergillus population, fitness costs and spontaneous loss should be balanced with new infections. Implications of even small viral fitness effects combined with the observed transmission limits for host and mycovirus are discussed.

Mycotoxin production and evolutionary relationships among species of Aspergillus section Clavati.

Aspergillus clavatus is a commonly encountered fungus in the environment, producing a number of mycotoxins including patulin, kojic acid, cytochalasins and tremorgenic mycotoxins. A. clavatus belongs to Aspergillus section Clavati together with six other species, all of which possess clavate-shaped vesicles. Patulin production was analysed by thin layer chromatography and high performance liquid chromatography, while a primer pair developed for the detection of an iso-epoxydon dehydrogenase gene involved in the biosynthesis of patulin in penicillia was used to detect the ability of patulin production in the isolates examined. A good correlation was observed between patulin producing properties, and the presence of an iso-epoxydon dehydrogenase gene fragment among the isolates tested. A. longivesica was found for the first time to produce patulin. Ribotoxin production was also examined using a PCR-based approach. Ribotoxins were detected for the first time in an A. pallidus and a Hemicarpenteles acanthosporus isolate. A phylogenetic analysis of intergenic transcribed spacer sequence data indicated that most isolates belong to two main clades that have also been identified earlier based on 26 S rDNA sequence data. A. pallidus isolates clustered together with A. clavatus strains. Although A. clavatus isolates produced highly homogeneous random amplified polymorphic DNA profiles, phylogenetic analysis of these data let us cluster A. clavatus isolates into distinct clades. Correlations were not observed between either patulin or ribotoxin production, and the taxonomic position of the isolates tested, indicating that patulin and ribotoxin producing abilities were lost several times during evolution of Aspergillus section Clavati. Although patulin was earlier found to inhibit mycovirus replication, one of the mycovirus carrying isolates also produced patulin, and both carried the iso-epoxydon dehydrogenase gene.

Double-stranded rna elements and virus-like particles in Aspergilli.

Mycoviruses with double-stranded RNA (dsRNA) genomes are frequently encountered in Aspergillus isolates. A detailed study of such dsRNA elements in black Aspergillus isolates collected worldwide was carried out, and the data were analysed. The results indicate that about 10% of black Aspergilli are infected. However, the geographic distribution of infected isolates exhibits large variations; 3-13% of the isolates collected from different continents were found to carry dsRNA elements. Hybridization experiments indicated that electrophoretic banding patterns are not reliable tools for estimating the diversity of these mycovirus genomes. Among strains representing other Aspergillus sections, dsRNA segments indicative of mycovirus infection were observed for the first time in 4 species (A. leporis, A. petrakii, A. fumigatus and A. primulinus). The latter species is able to reproduce sexually. This is the second report on the detection of naturally-occurring dsRNAs in sexually reproducing Aspergillus species. The presence of virus-like particles in these and other Aspergilli was also examined by electron microscopy. Most infected Aspergillus isolates examined were found to carry virus-like particles in the size range 36-40 nm.

Double-stranded RNA mycoviruses in species of Aspergillus sections Circumdati and Fumigati.

Isolates (178) belonging to Aspergillus sections Fumigati, Candidi, Clavati, and Circumdati were tested for the presence of double-stranded RNA (dsRNA) genomes. Altogether, 5.6% of the Aspergillus strains examined were infected with dsRNAs. dsRNA segments indicative of mycovirus infection were observed for the first time in Neosartorya hiratsukae, Neosartorya quadricincta, Petromyces alliaceus, and Aspergillus clavatus strains. Correlation was not observed between ochratoxin production and dsRNA content of the strains. This is the first report on the detection of naturally occurring dsRNAs in Aspergillus species that are able to reproduce sexually. The detection of dsRNA in sexual aspergilli gave us a chance to examine the transmission of these segments through ascospores. A Neosartorya hiratsukae strain transmitted the dsRNAs efficiently through sexual spores, while the stromata embedding the asci in Petromyces alliaceus did not transmit one of the dsRNA segments. The 0.6-kb dsRNA segment that was present in the single-stromatal cultures was found to be located in the mitochondrial fraction of this strain. This observation indicates that some mechanisms exist in aspergilli to exclude cytoplasmically located dsRNA molecules from stromatal structures.

Intra- and interspecies virus transfer in Aspergilli via protoplast fusion.

Intra- and interspecies transfer of dsRNA viruses between black Aspergilli and Aspergillus nidulans strains has been investigated using protoplast fusion. We found interspecies transfer of virus in all combinations of black Aspergillus and A. nidulans strains and vice versa. Using the same conditions, intraspecies virus transfer among heterokaryon incompatible strains was also tested. Whereas such transfer was always found among A. nidulans strains, transfer among black Aspergilli was frequently unsuccessful. The lack of virus transfer between black Aspergillus isolates was further investigated by using a mitochondrial oligomycin resistance marker as a positive control for cytoplasmic exchange. These experiments showed independent transfer of the oligomycin resistance and dsRNA viruses during protoplast fusion of heterokaryon incompatible black Aspergilli. The inefficient transfer of dsRNA viruses between black Aspergilli is not caused by absolute resistance to viruses but may be related to heterokaryon incompatibility reactions that operate intraspecifically. Consequences for the dynamics of mycoviruses in populations of black Aspergilli are discussed.

Heterokaryon incompatibility blocks virus transfer among natural isolates of black Aspergilli.

The extent of heterokaryon (also termed somatic or vegetative) incompatibility among black Aspergillus strains was examined using nitrate non-utilising mutants selected on chlorate medium. Pairings of complementary mutants showed that somatic compatibility between different strains is exceptional in natural populations of the asexual black Aspergilli. Mycoviruses are present in a considerable fraction of the sampled natural population, but surprisingly, horizontal transfer of mycoviruses only occurs-at least under laboratory conditions-between the (very rare) compatible combinations of strains. Thus, unlike other fungal species, somatic incompatibility in black Aspergilli efficiently blocks virus transfer. Viruses present in black Aspergillus isolates are highly efficiently transmitted to asexual progeny.