In the course of sobemovirus gene cloning the complete genome of Ryegrass mottle virus (RGMoV) was sequenced. Sequence analysis revealed differences including missing and extraneous nucleotides in comparison to the previously published sequence (Zhang, Toriyama, Takanashi, J. Gen. Plant Pathol. 67, 63 (2001)). A gene coding for a typical sobemovirus 3C-like serine protease was identified in ORF2a after multiple sequence alignment analysis. The newly identified 57-amino-acid stretch in ORF2a showed similarities ranging from 38.5 to 50.9% among sequenced genes of sobemovirus proteases. ORF analysis of the RGMoV polyprotein coding sequence demonstrated the arrangement of ORF2b coding for RNA-dependent RNA polymerase (RdRP) in the -1 frame in regard to ORF2a. The localization of conserved among sobemoviruses slippery sequence (UUUAAAC) at the 3'-end of ORF2a suggests the translation of RdRP via a -1 ribosomal frameshifting mechanism, allowing to include the RGMoV in the sobemovirus group with a Cocksfoot mottle virus-like (CfMV-like) genome organization.

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Expression and characterisation of the ryegrass mottle virus non-structural proteins The Ryegrass mottle virus (RGMoV) single-stranded RNA genome is organised into four open reading frames (ORF) which encode several proteins: ORF1 encodes protein P1, ORF2a contains the membrane-associated 3C-like serine protease, genome-linked protein VPg and a P16 protein gene. ORF2b encodes replicase RdRP and the only structural protein, coat protein, is synthesised from ORF3. To obtain the non-structural proteins in preparative quantities and to characterise them, the corresponding RGMoV gene cDNAs were cloned in pET- and pColdI-derived expression vectors and overexpressed in several E. coli host cells. For protease and RdRP, the best expression system containing pColdI vector and E. coli WK6 strain was determined. VPg and P16 proteins were obtained from the pET- or pACYC- vectors and E. coli BL21 (DE3) host cells and purified using Ni-Sepharose affinity chromatography. Attempts to crystallize VPg and P16 were unsuccessful, possibly due to non-structured amino acid sequences in both protein structures. Methods based on bioinformatic analysis indicated that the entire VPg domain and the C-terminal part of the P16 contain unstructured amino acid stretches, which possibly prevented the formation of crystals.

Background The highly contagious SARS‐CoV‐2 is mainly transmitted by respiratory droplets and aerosols. Consequently, people are required to wear masks and maintain a social distance to avoid spreading of the virus. Despite the success of the commercially available vaccines, the virus is still uncontained globally. Given the tropism of SARS‐CoV‐2, a mucosal immune reaction would help to reduce viral shedding and transmission locally. Only seven out of hundreds of ongoing clinical trials are testing the intranasal delivery of a vaccine against COVID‐19. Methods In the current study, we evaluated the immunogenicity of a traditional vaccine platform based on virus‐like particles (VLPs) displaying RBD of SARS‐CoV‐2 for intranasal administration in a murine model. The candidate vaccine platform, CuMV TT ‐RBD, has been optimized to incorporate a universal T helper cell epitope derived from tetanus‐toxin and is self‐adjuvanted with TLR7/8 ligands. Results CuMV TT ‐RBD vaccine elicited a strong systemic RBD‐ and spike‐IgG and IgA antibodies of high avidity. Local immune response was assessed, and our results demonstrate a strong mucosal antibody and plasma cell production in lung tissue. Furthermore, the induced systemic antibodies could efficiently recognize and neutralize different variants of concern (VOCs). Conclusion Our data demonstrate that intranasal administration of CuMV TT ‐RBD induces a protective systemic and local specific antibody response against SARS‐CoV‐2 and its VOCs.