Characterization of a novel Gammapartitivirus, named TmPV1, infecting thermal dimorphic human pathogenic fungus Talaromyces marneffei (formerly Penicillium marneffei)

Abstract

Talaromyces marneffei (Penicillium marneffei) is the most important thermal dimorphic fungus causing respiratory, skin and systemic mycosis in Southeast Asia. However, the existence of mycoviruses in T. marneffei and their potential biological role is largely unknown. This study reports the discovery and the biological characterization of a novel member in the family Partitiviridae, Talaromyces marneffei partitivirus-1 (TmPV1). Two distinct bands of dsRNA were isolated from seven of 55 T. marneffei isolates. Complete genome sequencing of the seven TmPV1 isolates revealed the typical genome organization of the family Partitiviridae, with two dsRNA segments, dsRNA1 and dsRNA2, each containing a single open reading frame encoding for RNA-dependent RNA polymerase (RdRp) and capsid protein respectively. Phylogenetic analysis showed that TmPV1 were most closely related to the members in the genus Gammapartitivirus, with sequences from the seven T. marneffei isolates forming a distinct clade within the genus. Transmission electron microscopy confirmed the presence of isometric, non-enveloped viral particles of 30-45 nm in diameter, compatible with partitiviruses, in a TmPV1-infected T. marneffei strain. While TmPV1 can be detected in both yeast and mycelial phases of T. marneffei, qRT-PCR showed that the viral load of TmPV1 in yeast phase was significantly higher than that in mycelial phase. TmPV1 represents a novel species of the genus Gammaparitivirus under the family Partitiviridae. Isogenic TmPV1-infected and TmPV1-free T. marneffei strains PM1 and PM41 were constructed using protoplast transfection. TmPV1 did not induce significant morphological changes in both mycelial and yeast phases in T. marneffei. TmPV1 also did not induce change of mycelial growth rate in T. marneffei. However, TmPV1 induced fungal hypervirulence in vivo in a murine model. Fungal burden of TmPV1-infected T. marneffei in mice organs, liver, spleen, kidney and lung, was higher compared to those inoculated with TmPV1-free T. marneffei. There was no change in the intracellular survival in vitro of TmPV1-infected T. marneffei demonstrated in murine cell line J774 and hPBMC derived macrophage. Transcriptomic study between TmPV1-infected and TmPV1-free T. marneffei revealed that TmPV1 induced changes in the expression profiles of 78 genes in yeast phase. Three potential virulence factors, GABA transaminase, succinate semialdehyde dehydrogenase (SSDH) and chitin deacetylase were up-regulated in TmPV1 T. marneffei yeast phase. Further studies are needed to elucidate their roles in TmPV1-induced hypervirulence. The expression of the RNAi-related genes, dcl-1, dcl-2 and qde-2 were suppressed in transcriptional level in both mycelial and yeast phases in TmPV1-infected T. marneffei. This indicated that TmPV1 may suppress the RNAi machinery in T. marneffei, and suggested that the RNAi machinery may be involved in anti-viral defense in T. marneffei. This is the first report of a mycovirus in a thermal dimorphic fungus. The enhancement of fungal virulence and alteration of gene expression profiles of T. marneffei by TmPV1 offer insights into mycovirus-fungus interaction and pathogenesis of thermally dimorphic fungi. 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 anti-viral defense in T. marneffei.