Novel Partitivirus Enhances Virulence of and Causes Aberrant Gene Expression in Talaromyces marneffei

Abstract

Talaromyces marneffei is the most important thermal dimorphic fungus causing systemic mycosis in Southeast Asia. We report the discovery of a novel partitivirus, Talaromyces marneffeipartitivirus-1 (TmPV1). TmPV1 was detected in 7 (12.7%) of 55 clinical T. marneffei isolates. Complete genome sequencing of the seven TmPV1 isolates revealed two double-stranded RNA (dsRNA) segments encoding RNA-dependent RNA polymerase (RdRp) and capsid protein, respectively. Phylogenetic analysis showed that TmPV1 occupied a distinct clade among the members of the genus Gammapartitivirus Transmission electron microscopy confirmed the presence of isometric, nonenveloped viral particles of 30 to 45 nm in diameter, compatible with partitiviruses, in TmPV1-infected T. marneffei Quantitative reverse transcription-PCR (qRT-PCR) demonstrated higher viral load of TmPV1 in the yeast phase than in the mycelial phase of T. marneffei Two virus-free isolates, PM1 and PM41, were successfully infected by purified TmPV1 using protoplast transfection. Mice challenged with TmPV1-infected T. marneffei isolates showed significantly shortened survival time (P < 0.0001) and higher fungal burden in organs than mice challenged with isogenic TmPV1-free isolates. Transcriptomic analysis showed that TmPV1 causes aberrant expression of various genes in T. marneffei, with upregulation of potential virulence factors and suppression of RNA interference (RNAi)-related genes. This is the first report of a mycovirus in a thermally dimorphic fungus. Further studies are required to ascertain the mechanism whereby TmPV1 enhances the virulence of T. marneffei in mice and the potential role of RNAi-related genes in antiviral defense in T. marneffeiIMPORTANCETalaromyces marneffei (formerly Penicillium marneffei) is the most important thermal dimorphic fungus in Southeast Asia, causing highly fatal systemic penicilliosis in HIV-infected and immunocompromised patients. We discovered a novel mycovirus, TmPV1, in seven clinical isolates of T. marneffei TmPV1 belongs to the genus Gammapartitivirus of the family Partitiviridae We showed that TmPV1 enhanced the virulence of T. marneffei in mice, with shortened survival time and higher fungal burden in the organs of mice challenged with TmPV1-infected T. marneffei isolates than in those of mice challenged with virus-free isogenic isolates. Transcriptomics analysis showed that TmPV1 altered the expression of genes involved in various cellular processes in T. marneffei, with upregulation of potential virulence factors and suppression of RNAi machinery which may be involved in antiviral defense. This is the first report of a mycovirus in a thermal dimorphic fungus. The present results offer insights into mycovirus-fungus interactions and pathogenesis of thermal dimorphic fungi.

Keywords: Talaromyces marneffei; dimorphic; fungus; novel; partitivirus.

FIG 1

(A) dsRNA extracted from Talaromyces marneffei. (B) Schematic representation of the genome organization of TmPV1 dsRNA-1 and dsRNA-2. (C and D) Conserved 5′-UTR (C) and 3′-UTR (D) sequence elements in TmPV1 dsRNA-1 (top rows) and dsRNA-2 (bottom rows). (E) Transmission electron microscopy of TmPV1 purified from T. marneffei isolate PM40. (A) Lanes 1 to 7, two dsRNA bands were observed in 7 T. marneffei isolates; lanes 9 to 16, no dsRNA bands were observed in the remaining 48 T. marneffei isolates (7 selected isolates are shown); lane M, lambda DNA digested by Eco471 (AvaII). Sizes are indicated as kilobase pairs (left). (B) Open bars represent open reading frames (ORFs) encoding the putative RNA-dependent RNA polymerase or capsid protein. The 5′ and 3′ untranslated regions are indicated as single lines. The nucleotide positions of the initiation and termination codons are indicated below the border of the ORF. Nucleotides in the 5′-UTR (C) and 3′-UTR (D) of dsRNA-1 and dsRNA-2 that are identical are indicated with reverse highlighting. (E) Isometric, nonenveloped viral particles of 30 to 45 nm in diameter, compatible with partitiviruses, are indicated by white arrows. Bar = 200 nm.

FIG 2

Phylogenetic tree constructed from the amino acid sequences of the RdRp (A) and capsid proteins (B) of TmPV1 and other members of Partitiviridae. Totals of 360 and 801 amino acid positions were included in the analysis, respectively. Phylogenetic analysis was performed by maximum likelihood based on the Le_Gascuel_2008 model with gamma distributions (A) and the General Reverse Transcriptase with Frequency model, gamma distributions (B), using MEGA 6. Bootstrap values were calculated as percentages from 100 replicates, and only values of ≥70% are shown. The scale bars indicate the estimated number of substitutions per 100 amino acids. Accession numbers are given as cited in the GenBank database.

FIG 3

(A and B) Survival of mice challenged with 8 × 10⁶ conidia of TmPV1-free T. marneffei or TmPV1-infected T. marneffei isolate PM41 (A) and PM1 (B). (C and D) Fungal loads in the liver, spleen, kidney, and lung of mice challenged with isolate PM41 (C) and PM1 (D). (E and F) Survival of mice challenged with 4 × 10⁶ conidia of TmPV1-free T. marneffei or TmPV1-infected T. marneffei isolate PM41 (E) and PM1 (F). For survival analysis, groups of 10 BALB/c female mice were challenged intravenously with 8 × 10⁶ conidia and survival was recorded daily. The Kaplan-Meier method and the log-rank test were performed for statistical analysis. ***, P < 0.0001. For fungal load analysis, groups of 6 BALB/c female mice were challenged intravenously with 8 × 10⁶ conidia. Mice were sacrificed and necropsies were performed at day 12 (PM1) or day 7 (PM41) postinoculation. Statistical analysis was performed for comparisons between mice inoculated with TmPV1-free spore and TmPV1-infected spore of each organ using Student’s t test. *, P < 0.05; **, P < 0.001; N.S., not significant.

FIG 4

Histopathological sections of lung tissues of mice challenged with TmPV1-infected and TmPV1-free T. marneffei. Formalin-fixed paraffin-embedded lung tissue sections were stained using (A to D) hematoxylin and eosin (H&E) and (E to H) Grocott’s methenamine silver (GMS), respectively (magnification, ×200). More-severe inflammation and higher fungal loads were observed in lungs of mice challenged with TmPV1-infected T. marneffei.

FIG 5

(A) Volcano plot of the RNA-seq data. (B) Classification of differentially expressed genes in TmPV1-infected T. marneffei isolate PM1 compared to isogenic TmPV1-free isolate. A total 16 genes were differentially expressed as indicated by red dots in the volcano plot, among which 11 genes were upregulated and 5 genes were downregulated. Classification of genes was based on the gene ontology terms as determined by UniProt and manual annotation. The number of genes is denoted next to each category designation.

FIG 6

mRNA levels of three differentially expressed genes in TmPV1-free and TmPV1-infected T. marneffei isolates PM1 and PM41 in the yeast phase. (A and B) Relative mRNA levels in fragments per kilobase of transcript per million fragments mapped (FPKM) obtained from RNA-seq data for the GABA transaminase gene (A) and for the nitrite reductase and nitrate transporter fusion genes (B) in isolate PM1. (C to E) Relative mRNA expression levels of GABA transaminase (C), nitrite reductase (D), and nitrate transporter (E) in isolates PM1 and PM41 obtained by qRT-PCR with four independent experimental replicates. Statistical analysis was performed using Student’s t test for comparisons between TmPV1-free and TmPV1-infected isogenic isolates. *, P < 0.05; **, P < 0.001; ***, P < 0.0001. Error bars indicate standard errors of the means.

FIG 7

Relative mRNA expression levels of dcl-1 (A), dcl-2 (B), and qde-2 (C) in TmPV1-free and TmPV1-infected T. marneffei isolate PM1 and PM41 in the yeast phase and of dcl-1 (D), dcl-2 (E), and qde-2 (F) in the mycelial phase. Results were obtained from four independent experimental replicates. Statistical analysis was performed using Student’s t test for comparisons between TmPV1-free and TmPV1-infected isogenic isolates. *, P < 0.05; **, P < 0.001; ***, P < 0.0001; N.S., not significant. Error bars indicate standard errors of the means.