Analysis of an Intrinsic Mycovirus Associated With Reduced Virulence of the Human Pathogenic Fungus Aspergillus fumigatus

Abstract

Aspergillus fumigatus is an airborne fungal pathogen that causes severe infections with invasive growth in immunocompromised patients. Several mycoviruses have recently been isolated from A. fumigatus strains, but there are presently no reports of mycoviralmediated reduction or elimination of fungal pathogenicity in vertebrate models. Here, we report the biological features of a novel mycovirus, A. fumigatus chrysovirus 41362 (AfuCV41362), isolated from the hypovirulent A. fumigatus strain IFM 41362. The AfuCV41362 genome is comprised of four dsRNAs, each of which contains a single ORF (ORF1-4). ORF1 encodes a protein with sequence similarity to RNA-dependent RNA polymerases of viruses in the family Chrysoviridae, while ORF3 encodes a putative capsid protein. Viral RNAs are expressed primarily during the germination stage, and RNA-seq analysis of virus-infected A. fumigatus at the germination stage suggested that the virus suppressed expression of several pathogenicity-associated host genes, including hypoxia adaptation and nitric oxide detoxification genes. In vitro functional analysis revealed that the virus-infected strain had reduced tolerance to environmental stressors. Virus-infected A. fumigatus strain IFM 41362 had reduced virulence in vivo compared to the virus-free strain in a mouse infection model. Furthermore, introduction of the mycovirus to a natively virus-free KU A. fumigatus strain induced virus-infected phenotypes. To identify mycovirus genes responsible for the reduced virulence of A. fumigatus, each viral ORF was ectopically expressed in the virus-free KU strain. Ectopic expression of the individual ORFs only nominally reduced virulence of the host fungus in a mouse infection model. However, we found that ORF3 and ORF4 reduced tolerance to environmental stresses in in vitro analysis. Based on these results, we suggest that the AfuCV41362 mycovirus ORF3 and ORF4 reduce fungal virulence by suppressing stress tolerance together with other viral genes, rather than alone.

Phenotypic and Molecular Biological Analysis of Polymycovirus AfuPmV-1M From Aspergillus fumigatus: Reduced Fungal Virulence in a Mouse Infection Model

“…However, previous studies have reported that the introduction of AfuCV (Jamal et al, 2010) or AfuPV-1 virus (Bhatti et al, 2011) to host fungi provoked no significant alterations of pathogenicity in murine infection models. Two exceptions are: (1) infection with AfuPmV-1 virus showed slight potentiation in pathogenicity in an insect infection model (Kanhayuwa et al, 2015), and (2) our previous report of chrysovirus, a different viridae from 1 https://talk.ictvonline.org/taxonomy/ the present work, showed reduced fungal virulence in a mouse model (Takahashi-Nakaguchi et al, 2020). Therefore, further mycoviruses that reduce A. fumigatus pathogenicity in a murine infection model remain to be detected (Özkan and Coutts, 2015).…”

“…Furthermore (4), the growth of fungal hyphae and formation of conidia were retarded in the virus-infected strains in comparison with the virus-free strain during in vitro experiments. Incidentally, effects of AfuPmV-1M mycovirus infection on A. fumigatus were re-confirmed by introducing the mycovirus into the originally virus-free KU strain (AfS35, FGSC A1159, akuA:loxP) via the protoplast fusion method (Kanematsu et al, 2004;Lee et al, 2011;Takahashi-Nakaguchi et al, 2020). Tolerance to oxidative and osmotic stresses was decreased even in the whole virusintroduced strain (Supplementary Figure S8).…”

“…In that report we examined expression levels of viral RNA in host filamentous fungi at each developmental stage (to the best of our knowledge for the first time). The time course of AfuPmV-1M dsRNA expression was different from that of AfuCV41362 dsRNAs during host fungal development, as examined by real-time PCR (Takahashi-Nakaguchi et al, 2020). Similarly to AfuCV41362 dsRNAs, the expression levels of AfuPmV-1M dsRNAs increased as germination of the host fungus A. fumigatus proceeded.…”

“…We have previously reported a double-stranded mycovirus, AfuCV41362, isolated from a different origin, of A. fumigatus strain IMF 41362 (Takahashi-Nakaguchi et al, 2020). AfuCV41362 consists of four dsRNA fragments and belongs to the Chrysoviridae.…”

Understanding Mucormycoses in the Age of “omics”

“…Beyond providing novel information on potential symbioses, mycovirus therapy presents a novel therapeutic solution for IFI, especially as resistance to anti-fungal treatments are on the rise ( Van De Sande et al, 2010 ). Success in the use of bacteriophage to treat respiratory bacterial infections suggest that parallel results could occur through the use of mycoviruses, which has been explored for Aspergillus therapy ( van de Sande and Vonk, 2019 ; Takahashi-Nakaguchi et al, 2020 ). Further discovery and characterizations of novel mycoviruses in Mucorales through -omics based approaches will not only offer a more complete biology of these fungi but may identify potential use of mycovirus therapy for mucormycoses.…”