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Abstract: We sought to advance our understanding of the genetic organization and biological significance of double-stranded RNA (dsRNA) viruses infecting Agaricus bisporus. Widespread in commercial hybrid strains is a seemingly benign virus composed of three dsRNAs [L (>13 kbp), M (5.2 kbp) and S (2.4 kbp)] that are packaged as naked molecules in fungal membrane vesicles. A cDNA clone representing ca. 97% of S-RNA was sequenced and found to lack both a major open reading frame (ORF) and significant sequence homology with any known gene. This cDNA hybridized to S, M and L, suggesting all three dsRNAs belong to a single virus. Thus, S appears to be a defective RNA composed of sequences derived from L and M, but without a coding function. L, M and S did not hybridize with L1-L5, M1 and M2 dsRNAs (3.8 to 1.7 kbp) associated with the putatively pathogenic La France isometric virus (LIV). The complete ORF sequences of L1, L3, M1 and M2 of North American and European isolates of LIV shared > 93% homology at the nucleotide and amino acid levels. Using an Agrobacterium-mediated gene delivery method, various constructs of the LIV ORF sequences are being transformed into A. bisporus in an effort to evaluate their effect on viral replication and host phenotype.
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