High-throughput sequencing of small RNAs allowed the identification of a novel DNA virus in a Chinese mulberry tree affected by a disease showing mosaic and dwarfing symptoms. Rolling-circle amplification and PCR with specific primers, followed by sequencing of eleven independent full-length clones, showed that this virus has a monopartite circular DNA genome (∼2.95 kb) containing ORFs in both polarity strands, as reported previously for geminiviruses. A field survey showed the close association of the virus with diseased mulberries, so we tentatively named the virus mulberry mosaic dwarf-associated virus (MMDaV). The MMDaV genome codes for five and two putative proteins in the virion-sense and in the complementary-sense strands, respectively. Although three MMDaV virion-sense putative proteins did not share sequence homology with any protein in the databases, functional domains [coiled-coil and transmembrane (TM) domains] were identified in two of them. In addition, the protein containing a TM domain was encoded by an ORF located in a similar genomic position in MMDaV and in several other geminiviruses. As reported for members of the genera Mastrevirus and Becurtovirus, MMDaV replication-associated proteins are expressed through the alternative splicing of an intron, which was shown to be functional in vivo. A similar intron was found in the genome of citrus chlorotic dwarf-associated virus (CCDaV), a divergent geminivirus found recently in citrus. On the basis of pairwise comparisons and phylogenetic analyses, CCDaV and MMDaV appear to be closely related to each other, thus supporting their inclusion in a putative novel genus in the family Geminiviridae.

There are today significant knowledge gaps on phytovirus populations and on the drivers impacting them but also on the comparative performance-methodological approaches for their study. We used and compared two viral sequence enrichment approaches, double-stranded RNAs (dsRNA) and virion-associated nucleic acids (VANA), for phytovirome description in complex pools representative of the most prevalent plant species in unmanaged and cultivated ecosystems. Viral richness was assessed by determining operational taxonomic units (OTU) following the clustering of conserved viral domains. There is some limited evidence of an impact of cultivation on viral populations. These results provide data allowing us to reason the methodological choices in virome studies. For researchers primarily interested in RNA viruses, the dsRNA approach is recommended because it consistently provided a more comprehensive description of the analyzed phytoviromes, but it understandably underrepresented DNA viruses and bacteriophages.

Viral metagenomics relies on high-throughput sequencing and on bioinformatic analyses to access the genetic content and diversity of entire viral communities. No universally accepted strategy or tool currently exists to define operational taxonomy units (OTUs) and evaluate viral alpha or beta diversity from virome data. Here we present a new bioinformatic resource, the VirAnnot (automated viral diversity estimation) pipeline, which performs the automated identification of OTUs. Reverse-position-specific BLAST (RPS-Blastn) is used to detect conserved viral protein motifs. The corresponding contigs are then aligned and a clustering approach is used to group in the same OTU contigs sharing more than a set identity threshold. A 10% threshold has been validated as producing OTUs that reasonably approach, in many families, the International Committee for the Taxonomy of Viruses taxonomy and can therefore be used as a proxy to viral species.

Cherry virus A (CVA) infection appears to be prevalent in cherry plantations worldwide. In this study, the diversity of CVA isolates from 31 cherry samples collected from different orchards around Bohai Bay in northeastern China was analyzed. The complete genome of one of these isolates, ChYT52, was found to be 7,434 nt in length excluding the poly (A) tail. It shares between 79.9–98.7% identity with CVA genome sequences in GenBank, while its RdRp core is more divergent (79.1–90.7% nt identity), likely as a consequence of a recombination event. Phylogenetic analysis of ChYT52 genome with CVA genomes in Genbank resulted in at least 7 major clusters plus additional 5 isolates alone at the end of long branches suggesting the existence of further phylogroups diversity in CVA. The genetic diversity of Chinese CVA isolates from 31 samples and GenBank sequences were analyzed in three genomic regions that correspond to the coat protein, the RNA-dependent RNA polymerase core region, and the movement protein genes. With few exceptions likely representing further recombination impact, the trees various trees are largely congruent, indicating that each region provides valuable phylogenetic information. In all cases, the majority of the Chinese CVA isolates clustering in phylogroup I, together with the X82547 reference sequence from Germany. Statistically significant negative values were obtained for Tajima’s D in the three genes for phylogroup I, suggesting that it may be undergoing a period of expansion. There was considerable haplotype diversity in the individual samples and more than half samples contained genetically diverse haplotypes belonging to different phylogroups. In addition, a number of statistically significant recombination events were detected in CVA genomes or in the partial genomic sequences indicating an important contribution of recombination to CVA evolution. This work provides a foundation for elucidation of the epidemiological characteristics and evolutionary history of CVA populations.

The cucurbit vegetable chieh-qua (Benincasa hispida var. chieh-qua How) is an important crop in South China and southeast Asian countries. Viral diseases cause substantial loss of chieh-qua yield. To identify the viruses that affect chieh-qua in China, ribosomal RNA-depleted total RNA sequencing was performed using chieh-qua leaf samples with typical viral symptoms. The virome of chieh-qua comprises four known viruses (melon yellow spot virus (MYSV), cucurbit chlorotic yellows virus (CCYV), papaya ringspot virus (PRSV) and watermelon silver mottle virus (WSMoV) and two novel viruses: cucurbit chlorotic virus (CuCV) in the genus Crinivirus and chieh-qua endornavirus (CqEV) in the genus Alphaendornavirus. The complete genomes of the two novel viruses in chieh-qua and three other isolates of CuCV in pumpkin, watermelon and cucumber were determined and the recombination signals of pumpkin and watermelon isolates of CuCV were detected. A reverse transcriptase PCR indicated that the dominant viruses of chieh-qua in Hainan are MYSV (66.67%) and CCYV (55.56%), followed by CuCV (27.41%), WSMoV (7.41%), cucumber mosaic virus (8.15%), zucchini yellow mosaic virus (6.67%), PRSV (6.67%) and CqEV (35.56%). Our findings support diagnostic and prevalence studies of viruses infecting chieh-qua in China, enabling sustainable control strategies for cucurbit viruses worldwide.