Discovery and characterization of novel Aspergillus fumigatus mycoviruses

Abstract

In the last few years, increasing numbers of viruses infecting fungi have been identified. In this study, we used an in silico approach for the analysis of deep RNA sequencing data in order to discover and characterize putative genomic ssRNA or dsRNA mycovirus sequences in Aspergillus fumigatus. RNA sequencing reads of A. fumigatus strains were mapped against the A. fumigatus Af293 reference genome. Unmapped reads were collected for de novo assembly. Contigs were analyzed by Blastx comparison with a mycovirus protein database. Assembled viral genomes were used as template for remapping of RNA sequencing reads. In total, deep RNA sequencing results from 11 A. fumigatus strains were analyzed for the presence of mycoviral genomic RNAs. In 9 out of 11 strains, putative mycoviral RNA genomes were identified. Three strains were infected with two different mycovirus species. Two strains were infected with Aspergillus fumigatus polymycovirus type-1 (AfuPmV-1). Four strains contained fully recovered genomic RNA of unknown narna-like viruses designated as Aspergillus fumigatus narnavirus-1 and Aspergillus fumigatus narnavirus-2 (AfuNV-1 and AfuNV-2). Both viruses showed 38% amino acid sequence identity to Beihai narna-like virus-21. Three strains contained partially recovered genomic RNA of an unknown narna-like virus. Two strains contained fully recovered genomic RNAs of an unknown partitivirus designated as Aspergillus fumigatus partitivirus-2 (AfuPV-2) which showed 50% amino acid sequence identity to Alternaria alternata partitivirus-1. Finally, one strain contained fully recovered genomic RNA of an unknown mitovirus designated as Aspergillus fumigatus mitovirus-1 (AfuMV-1) which showed 34% amino acid sequence identity to Sclerotina sclerotiorum mitovirus. In silico analysis of deep RNA sequencing results showed that a majority of the A. fumigatus strains used here were infected with mycoviruses. Four novel A. fumigatus RNA mycoviruses could be identified: two different Aspergillus fumigatus narna-like viruses, one Aspergillus fumigatus partitivirus, and one Aspergillus fumigatus mitovirus.

Fig 1

(A) Schematic representation of the genomic organization of the two segments of the novel Aspergillus fumigatus partitivirus-2 (AfuPV2). (B) Alignment of the 5’-UTR regions of related partitiviruses. Conserved nucleotides found in the genomic segments of Aspergillus fumigatus partitiviruses-1 and -2, Botryosphaeria dothidea partivirus-1, and Alternaria alternata partitivirus-1 are indicated.

Fig 2. Phylogenetic relationships of the family partitiviridae.

An unrooted phylogenetic tree was calculated from a multiple alignment of the RdRp protein using the maximum likelihood method with 1000 bootstrap replicates. The various subgenera are indicated by colored boxes.

Fig 3. Schematic representation of the genomic organization of the novel narnaviridae Aspergillus fumigatus narnavirus-1 (AfuNV1), Aspergillus fumigatus narnavirus-2 (AfuNV2), and Aspergillus fumigatus mitovirus-1 (AfuMV).

Fig 4. Phylogenetic relationships of the family Narnaviridae.

An unrooted phylogenetic tree was calculated from a multiple alignment of the RdRp protein using the maximum likelihood method with 1000 bootstrap replicates. The various subgenera are indicated by colored boxes. The narnavirus and mitovirus genera are indicated.