Novel polymycoviruses are encapsidated in filamentous virions.

is a relatively new viral family that was established nearly 5 years ago, but their viral morphologies (naked or encapsidated) remain controversial since only one member namely, filamentous virus 1 (CcFV1), was identified as being encapsidated in filamentous virions. Here, three novel double-stranded RNA (dsRNA) viruses belonging to the family were identified in three phytopathogenic fungal strains and tentatively named -sinensis polymycovirus 1 (PcsPmV1), and polymycovirus 1 and 2 (PhcPmV1 and 2), respectively. PcsPmV1 and PhcPmVs have five or six genomic dsRNAs, ranging from 1,055 to 2,405 bp, encoding five or seven putative open reading frames (ORFs), of which ORF1 encodes an RNA-dependent RNA polymerase, ORF5 encodes a prolein-alanine-serine-rich (P-A-S-rich) protein behaving as coat protein (CP); and dsRNAs 4 and 6 encode putative proteins with unknown functions and share no detectable identities with known viral sequences. Upon examination under transmission electron microscopy after purification from fungal mycelia, PcsPmV1 and PhcPmVs were found to be encapsidated in filamentous particles, as was a known polymycovirus, RNA virus 1 (BdRV1), which was previously assumed to likely have no conventional virions. The morphology of PcsPmV1 was further supported by the observation that its particles could be decorated by polyclonal antibodies against its CP and bound by immuno-gold particles conjugated to the specific CP antibody. Together with CcFV1, BdRV1, PcsPmV1, and PhcPmVs, these provide strong evidence to support the notion that polymycoviruses are encapsidated in filamentous virions constituted by P-A-S-rich CPs. Moreover, their biological effects on their fungal hosts were assessed, suggesting that PcsPmV1 infection could enhance growth and virulence.IMPORTANCE, a recently established viral family, has raised questions about encapsidation. Here, we identify and characterize three novel polymycoviral double-stranded RNA (dsRNA) viruses in phytopathogenic fungal strains, tentatively named -sinensis polymycovirus 1, and polymycovirus 1 and 2, respectively. These polymycoviruses possess five or six genomic dsRNAs, ranging from 1,055 to 2,405 bp, with two encoding putative proteins of unknown functions and sharing no detectable identities with known viral sequences. Their morphologies indicate filamentous virions constituted by proline-alanine-serine-rich coat proteins, observed using immunosorbent electron microscopy combined with immune-gold labeling techniques. Additionally, RNA virus 1, previously assumed to lack conventional virions, is also shown to be encapsidated in filamentous particles. This study provides new evidence supporting the encapsidation of polymycoviruses into elongated and flexuous virions, significantly contributing to our understanding of the evolutionary particle architecture within the virosphere and expanding our knowledge of viral diversity and evolution. Moreover, this is the first report of a polymycovirus enhancing the virulence and growth of a phytopathogenic fungus.