Identification of a novel waikavirus infecting Pittosporum tobira in China.

A novel waikavirus, tentatively named "Pittosporum tobira waikavirus" (PtWV), was identified in Pittosporum tobira plants exhibiting mosaic and ringspot symptoms on foliage in Yunnan, China. The full-length genomic sequence was determined by high-throughput sequencing and rapid amplification of cDNA ends. The genome of PtWV is 12,709 nt in length and has a large open reading frame (ORF) of 11,010 nt, encoding a polyprotein, and a small ORF that encodes a 13.2-kDa bellflower vein chlorosis virus (BVCV)-like protein. Phylogenetic analysis and sequence alignment revealed that PtWV is closely related to actinidia yellowing virus 1 (AcYV1), which shares the highest amino acid (aa) sequence similarity (50.1% identity) in the Pro-RdRp region. To the best of our knowledge, this is the first report of a novel waikavirus in P. tobira.

Occurrence and Distribution of Actinidia Viruses in Shaanxi Province of China.

Kiwifruit (Actinidia spp.) is an economically important fruit crop globally. China is the largest kiwifruit-growing country in the world, and Shaanxi Province is the major kiwifruit-growing region in China. A systematic survey detected various symptoms in kiwifruit plants grown in a commercial kiwifruit field in Shaanxi Province. Samples were collected from kiwifruit plants showing symptoms and used for virus detection by high-throughput sequencing. In addition to 10 known kiwifruit viruses, three new viruses were detected and tentatively named Actinidia yellowing ringspot virus (AYRSpV), Actinidia yellowing virus 1 (AcYV1), and Actinidia yellowing virus 2 (AcYV2). The genome sequences of the three new viruses and four known viruses were determined. Based on the demarcation criteria of the International Committee on Taxonomy of Viruses, AYRSpV might be a new member of the genus Ilarvirus in the family Bromoviridae, AcYV1 might be a new virus of the genus Waikavirus in the family Secoviridae, and AcYV2 might be a novel virus in the family Tombusviridae. Spherical viral particles were found in the samples infected with AYRSpV, AcYV1, and AcYV2 by transmission electron microscopy. Further analysis showed that all 13 viruses can infect both Actinidia deliciosa and A. chinensis but the incidences of these infections vary among different kiwifruit cultivars in different regions. These results provide valuable information for understanding the virome of kiwifruit in China.

Broadening the host range and genetic diversity of waikaviruses.

Waikaviruses are monopartite, positive sense, single-stranded RNA viruses that cause economically important plant diseases. Despite their importance, waikaviruses are poorly understood and only ten members are currently recognized. The present study on Sequence Read Archive (SRA)-based data-driven virus discovery (DDVD) identified 22 putative new waikaviruses, nearly doubling the number of known waikaviruses, in SRA libraries of diverse plant species, from ferns to trees. Besides, a highly divergent secoviral sequence with distinct genome features was identified in a wheat transcriptome. Other significant findings of the study include identification of a new waikavirus in a library derived from diseased water chestnut sample wherein a caulimovirus was reported, prediction of coiled-coils in hypothetical protein region of waikaviral polyprotein alignment and phylogenetic clustering of tree-infecting waikaviruses. The study not only reiterates the importance of DDVD in unveiling hitherto hidden viral sequences in plant SRA libraries but also deepens our understanding of waikaviral diversity.

Critical residues for proteolysis activity of maize chlorotic dwarf virus (MCDV) 3C-like protease and comparison of activity of orthologous waikavirus proteases.

Maize chlorotic dwarf virus (MCDV) encodes a 3C-like protease that cleaves the N-terminal polyprotein (R78) as previously demonstrated. Here, we examined amino acid residues required for catalytic activity of the protease, including those in the predicted catalytic triad, amino acid residues H2667, D2704, and C2798, as well as H2817 hypothesized to be important in substrate binding. These and other residues were targeted for mutagenesis and tested for proteolytic cleavage activity on the N-terminal 78 kDa MCDV-S polyprotein substrate to identify mutants that abolished catalytic activity. Mutations that altered the predicted catalytic triad residues and H2817 disrupted MCDV-S protease activity, as did mutagenesis of a conserved tyrosine residue, Y2774. The protease activity and R78 cleavage of orthologs from divergent MCDV isolates MCDV-Tn and MCDV-M1, and other waikavirus species including rice tungro spherical virus (RTSV) and bellflower vein chlorosis virus (BVCV) were also examined.

Identification of a maize chlorotic dwarf virus silencing suppressor protein.

Maize chlorotic dwarf virus (MCDV), a member of the genus Waikavirus, family Secoviridae, has a 11784 nt (+)ssRNA genome that encodes a 389kDa proteolytically processed polyprotein. We show that the N-terminal 78kDa polyprotein (R78) of MCDV acts as a suppressor of RNA silencing in a well-established assay system. We further demonstrate that R78 is cleaved by the viral 3C-like protease into 51 and 27kDa proteins (p51 and p27), and that p51 is responsible for silencing suppressor activity. Silencing suppressor activity of R78 is conserved in three divergent MCDV strains (MCDV-Severe, MCDV-M1, and MCDV-Tennessee), as well as the waikavirus Bellflower vein chlorosis virus, but was not detected for orthologous protein of Rice tungro spherical virus (RTSV-A) or the similarly-positioned protein from the sequivirus Parsnip yellow fleck virus (PYFV). This is the first identification of a virus suppressor of RNA silencing encoded by a waikavirus.