A capsidless (+)RNA yadokarivirus hosted by a dsRNA virus is infectious as particles, cDNA, and dsRNA

Capsidless yadokariviruses (members of the order Yadokarivirales) with (+)RNA genomes divert the capsid of their partner icosahedral double-stranded RNA (dsRNA) viruses in different families of the order Ghabrivirales into the replication site. A yadokarivirus, AfSV2, has been reported from a German strain of the ascomycete fungus Aspergillus foetidus coinfected by two dsRNA viruses, a victorivirus (AfSV1, family Pseudototiviridae) and an alternavirus (AfFV, family Alternaviridae). Here, we identified AfSV1 as the partner of AfSV2 in a Japanese A. foetidus strain after showing the infectiousness of AfSV2 in three forms: virus particles (heterocapsid), transforming full-length complementary DNA (cDNA), and purified replicated form (RF) dsRNA that is believed to be inactive as a translational template. Virion transfection of virus-free A. foetidus protoplasts resulted in the generation of two strains infected either by AfSV1 alone or by both AfSV1 and AfSV2. Transformants with AfSV2 full-length cDNA launched AfSV2 infection only in the presence of AfSV1, but not those with AfSV2 RNA-directed RNA polymerase mutant cDNA. The purified fractions containing AfSV2 RF dsRNA also launched infection when transfected into protoplasts infected by AfSV1. Treatment with dsRNA-specific RNase III, but not with proteinase K, S1 nuclease, or DNase I, abolished the infectivity of AfSV2 RF dsRNA. Furthermore, we confirmed the infectiousness of gel-purified AfSV2 RF dsRNA in the presence of AfSV1. Taken together, our results show the unique infectious entity of AfSV2 and the expansion of yadokarivirus partners in the family Pseudototiviridae and provide interesting evolutionary insights.