Identification, molecular and biological characterization of two novel secovirids in wild grass species in Belgium.

High throughput sequencing was performed on virion-associated nucleic acids (VANA) from a pool of fifty asymptomatic rough bluegrasses (Poa trivialis L.) collected in a Belgian grazed pasture. Bioinformatics analyses produced some contigs presenting similarities with secovirid genomes, in particular nepoviruses and waikaviruses. Three distinct positive-sense single-stranded RNAs including 5' and 3' UTR were reconstructed and they represented two novel viruses infecting rough bluegrass, for which the provisional names poaceae Liege nepovirus A (PoLNVA, 7298 nt for RNA1 and 4263 nt for RNA2) and poaceae Liege virus 1 (PoLV1, 11,623 nt) were proposed. Compared to other Secoviridae members, the highest amino acid identity reached 90.7 % and 66.7 % between PoLNVA and nepoviruses for the Pro-Pol and CP regions respectively, while PoLV1 presented the highest amino acid identity with waikaviruses but with lower identities, i.e. 41.2 % for Pro-Pol and 25.8 % for CP regions, far below the ICTV demarcation criteria for novel secovirid. Based on sequence identity and phylogenetic analyses, PoLNVA was proposed to belong to the genus Nepovirus and PoLV1 as an unclassified secovirids. Detection of the two novel viruses was confirmed in high prevalence in rough bluegrass and ten other wild Poaceae species (Agropyron repens, Agrostis capillaris, Apera spica-venti, Anthoxanthum odoratum, Cynosorus cristatus, Festuca rubra, Holcus lanatus, Lolium perenne, Phleum bertolini and Phleum pratense) by RT-PCR and Sanger sequencing, revealing a diverse host range within Poaceae for these novel secovirids. Seed transmission was evaluated and confirmed for PoLNVA.