Abstract Talaromyces marneffei (Tm) is one of seven dimorphic human fungal pathogens that are causative agents of substantial global morbidity and mortality in both immunocompetent and immunocompromised patients. Tm is endemic throughout Southeast Asia in a geographic region that encompasses more than half of the global population, and is a frequent cause of infection and death in HIV/AIDS patients. The organism is present in the environment and in association with bamboo rat species and there is substantial risk of frequent and repeated exposure. Treatment options are limited and suboptimal, diagnostic modalities remain to be optimized, and our understanding of virulence determinants is limited. Although studies have shown that Tm is amenable to transformation and gene deletion via homologous recombination through Agrobacterium mediated trans-kingdom (AMT) conjugation, little investigation has been done to elucidate virulence factors in Tm. A recent study reported the presence of a novel dsRNA mycovirus in some clinical isolates, and that presence of this virus is correlated with enhanced virulence in animal models and the down regulation of the expression of genes involved in RNAi, a process which can target and degrade dsRNAs. In our proposal, we seek to capitalize upon these advances in order to dissect the mycoviral and genetic determinants of Tm virulence. In aim 1, we will analyze the presence or absence of the recently discovered mycovirus in a large collection (N=293) of well-validated Tm human clinical isolates with robust mycological and clinical outcome data and for which whole-genome data is available, and to assess correlations of the presence of this mycovirus determinant with disease severity and patient outcomes. We will develop approaches to cure and reintroduce this mycovirus in order to construct congenic strains with and without the virus for direct laboratory analyses of virulence. In aim 2, we will determine if the supression of RNAi caused by the mycovirus results in a hypermutator phenotype in virus-infected strains. Using a transposon trap assay, we will screen the Tm patient sample collection for isolates that produce a larger number of drug-resistant colonies, and determine if drug resistance is due to the insertion of transposable elements into the gene responsible for resistance. We will then assess any correlations of the hypermutator phenotype with virus-infected and virus-uninfected Tm patient samples. We will use AMT to delete components of the RNAi pathway and we will determine the consequences of these mutations on hypermutation, viral load, and if virulence is altered. These studies will advance our understanding of Tm mycoviral and genetic virulence determinants and provide insights with possible implications for patient prognosis and treatment.