A novel mycovirus named Aspergillus fumigatus tetramycovirus-1 (AfuTmV-1) was discovered and characterized in the human pathogenic fungus, A. fumigatus clinical isolate Af293. The virus reveals several unique features not previously found in double-stranded RNA (dsRNA) viruses and represents the first dsRNA that is infectious both as a purified entity and a naked dsRNA. The AfuTmV-1 is an unencapsidated dsRNA mycovirus comprised of four genomic segments, ranging in size from 1.1 to 2.4 kbp. The largest component encodes a putative viral RNA-dependent RNA polymerase (RdRP) where the sequence of the most highly conserved motif changes from GDDX to GDNQ. The third largest dsRNA encodes an S-adenosyl methionine-dependent methyltransferase (SAM) capping enzyme and the smallest dsRNA encodes a proline-alanine rich protein. Short interspersed nuclear elements (SINEs) were also identified in the fungal genome. Identification of the elements revealed tRNA-related and 5S rRNA-related SINE families which showed variation in transcription activity and copy number. AfuTmV-1 sequences together with SINEs were subsequently exploited to develop alternative tools for silencing genes in A. fumigatus. A truncated AfuTmV-1 based vector was successfully constructed and used as a prototype vector for generating a recombinant virus-induced gene silencing (VIGS) vector. Transcriptional fusion SINE-derived vectors were also developed to silence an ALB1/PKSP gene responsible for conidial pigmentation. With anticipation that the development will provide a powerful reverse genetic tool for functional genomics studies to identify key elements involved in fungal pathogenicity and also provide a medical benefit in exploiting mycoviruses as a future therapeutic agent against fungal infections.