A variety of highly divergent eukaryotic ssDNA viruses in sera of pigs

Over the last decade, viral metagenomics has been established as a non-targeted approach for identifying viruses in stock animals, including pigs. This has led to the identification of a vast diversity of small circular ssDNA viruses. The present study focuses on the investigation of eukaryotic circular Rep-encoding single-stranded (CRESS) DNA viral genomes present in serum of commercially reared pigs from southern Brazil. Several CRESS DNA viral genomes were detected, including representatives of the families Smacoviridae (n=5), Genomoviridae (n=3), Redondoviridae (n=1), Nenyaviridae (n=1) and other yet unclassified genomes (n=9), plus a circular DNA molecule, which probably belongs to the phylum Cressdnaviricota. A novel genus within the family Smacoviridae, tentatively named ‘Suismacovirus’, comprising 21 potential new species, is proposed. Although the reported genomes were recovered from pigs with clinical signs of respiratory disease, further studies should examine their potential role as pathogens. Nonetheless, these findings highlight the diversity of circular ssDNA viruses in serum of domestic pigs, expand the knowledge on CRESS DNA viruses’ genetic diversity and distribution and contribute to the global picture of the virome of commercially reared pigs.

A Mechanically Transmitted DNA Mycovirus Is Targeted by the Defence Machinery of Its Host, Botrytis cinerea

Eukaryotic circular single-stranded DNA (ssDNA) viruses were known only to infect plants and vertebrates until the discovery of the isolated DNA mycovirus from the fungus Sclerotinia sclerotiorum. Similar viral sequences were reported from several other sources and classified in ten genera within the Genomoviridae family. The current study reports two circular ssDNA mycoviruses isolated from the phytopathogen Botrytis cinerea, and their assignment to a newly created genus tentatively named Gemydayirivirus. The mycoviruses, tentatively named botrytis gemydayirivirus 1 (BGDaV1) and BGDaV2, are 1701 and 1693 nt long and encode three and two open reading frames (ORFs), respectively. Of the predicted ORFs, only ORF I, which codes for a replication initiation protein (Rep), shared identity with other proteins in GenBank. BGDaV1 is infective as cell-free purified particles and confers hypovirulence on its natural host. Investigation revealed that BGDaV1 is a target for RNA silencing and genomic DNA methylation, keeping the virus at very low titre. The discovery of BGDaV1 expands our knowledge of the diversity of genomoviruses and their interaction with fungal hosts.

Geminiviruses: Taxonomic Structure and Diversity in Genomic Organization

Background: Geminiviridae is one of the best-characterized and hence, one of the largest plant-virus families with the highest economic importance. Its members characteristically have a circular ssDNA genome within the encapsidation of twinned quasi-icosaheadral virions (18-38 nm size-range). Objective: Construction of a narrative review on geminiviruses, to have a clearer picture of their genomic structure and taxonomic status. Methods: A thorough search was conducted for papers and patents regarding geminiviruses, where relevant information was used to study their genomic organization, diversity and taxonomic structure. Results: Geminiviruses have been classified into nine genera (viz., genus Begomovirus, Mastrevirus, Curtovirus, Topocuvirus, Becurtovirus, Turncurtovirus, Capulavirus, Eragrovirus and Grablovirus) having distinct genomic organizations, host ranges and insect vectors. Genomic organization of all genera generally shows the presence of 4-6 ORFs encoding for various proteins. For now, Citrus chlorotic dwarf-associated virus (CCDaV), Camellia chlorotic dwarf-associated virus (CaCDaV) and few other geminiviruses are still unassigned to any genera. The monopartite begomoviruses (and few mastreviruses) have been found associated with aplhasatellites and betasatellites (viz., ~1.3 kb circular ssDNA satellites). Recent reports suggest that deltasatellites potentially reduce the accumulation of helper-Begomovirus species in host plants. Some patents have revealed the methods to generate transgenic plants resistant to geminiviruses. Conclusion: Geminiviruses rapidly evolve and are a highly diverse group of plant-viruses. However, research has shown new horizons in tackling the acute begomoviral diseases in plants by generating a novel bio-control methodology in which deltasatellites can be used as bio-control agents and generate transgenic plants resistant to geminiviruses.

A tripartite ssDNA mycovirus from a plant pathogenic fungus is infectious as cloned DNA and purified virions

Here, we describe a tripartite circular single-stranded (ss) DNA mycovirus, named Fusarium graminearum gemytripvirus 1 (FgGMTV1). The genome of FgGMTV1 comprises three circular ssDNA segments (DNA-A, DNA-B, and DNA-C). Sequence alignments and phylogenetic analyses showed that FgGMTV1 is nested within the family Genomoviridae. We also constructed the first infectious DNA clones of a DNA mycovirus. Our results show that DNA-A and DNA-B are mutually interdependent for their replication and are associated with severely reduced colony growth and hypovirulence. DNA-C relies on DNA-A and DNA-B for replication and is necessary for the recovery of abnormal fungal phenotypes. DNA-C also enhances the accumulation of viral DNA in infected fungi and permits stable colonization and easy transmission via conidia. This is the first multipartite DNA virus isolated from a fungus. Our phylogenetic analyses also suggest that the multipartite genome of FgGMTV1 may have evolved from a monopartite genome of an ancient genomovirus.

A New World begomovirus infecting Grapevine

Grapevine (Vitis L.), a deciduous woody vine, is a highly valuable agricultural crop. Grapevine, as other crops, is vulnerable to infectious pathogens. Several of them, including viruses, are a major threat to viticulture. Geminiviruses (family Geminiviridae) are insect transmitted, small non-enveloped viruses, with circular single-stranded DNA genomes, which are encapsidated in quasi-icosahedral geminated virions. There are only four geminiviruses associated to grapevine: two members of genus Grablovirus and two unassigned species. Here we present evidence of a novel begomovirus (genus Begomovirus) infecting grapevines from Argentina. Two circular ssDNA virus sequences were assembled from high-throughput sequencing data from Vitis vinifera cv Torrontes from Mendoza province, Argentina. Structural and functional annotation indicated that the virus sequences corresponded to complete DNA components A and B of a novel New World bipartite begomovirus. Genetic distance and evolutionary analyses support that the detected sequences correspond to a new virus, the first begomovirus reported to infect grapevine, a tentative prototype member of a novel species which we propose the name “grapevine begomovirus A” (GBVA).

Dynamic Genome Editing Using In Vivo Synthesized Donor ssDNA in Escherichia coli

As a key element of genome editing, donor DNA introduces the desired exogenous sequence while working with other crucial machinery such as CRISPR-Cas or recombinases. However, current methods for the delivery of donor DNA into cells are both inefficient and complicated. Here, we developed a new methodology that utilizes rolling circle replication and Cas9 mediated (RC-Cas-mediated) in vivo single strand DNA (ssDNA) synthesis. A single-gene rolling circle DNA replication system from Gram-negative bacteria was engineered to produce circular ssDNA from a Gram-positive parent plasmid at a designed sequence in Escherichia coli. Furthermore, it was demonstrated that the desired linear ssDNA fragment could be cut out using CRISPR-associated protein 9 (CRISPR-Cas9) nuclease and combined with lambda Red recombinase as donor for precise genome engineering. Various donor ssDNA fragments from hundreds to thousands of nucleotides in length were synthesized in E. coli cells, allowing successive genome editing in growing cells. We hope that this RC-Cas-mediated in vivo ssDNA on-site synthesis system will be widely adopted as a useful new tool for dynamic genome editing.

Efficient Homology-directed Repair with Circular ssDNA Donors

While genome editing has been revolutionized by the advent of CRISPR-based nucleases, difficulties in achieving efficient, nuclease-mediated, homology-directed repair (HDR) still limit many applications. Commonly used DNA donors such as plasmids suffer from low HDR efficiencies in many cell types, as well as integration at unintended sites. In contrast, single-stranded DNA (ssDNA) donors can produce efficient HDR with minimal off-target integration. Here, we describe the use of ssDNA phage to efficiently and inexpensively produce long circular ssDNA (cssDNA) donors. These cssDNA donors serve as efficient HDR templates when used with Cas9 or Cas12a, with integration frequencies superior to linear ssDNA (lssDNA) donors. To evaluate the relative efficiencies of imprecise and precise repair for a suite of different Cas9 or Cas12a nucleases, we have developed a modified Traffic Light Reporter (TLR) system [TLR-Multi-Cas Variant 1 (MCV1)] that permits side-by-side comparisons of different nuclease systems. We used this system to assess editing and HDR efficiencies of different nuclease platforms with distinct DNA donor types. We then extended the analysis of DNA donor types to evaluate efficiencies of fluorescent tag knock-ins at endogenous sites in HEK293T and K562 cells. Our results show that cssDNA templates produce efficient and robust insertion of reporter tags. Targeting efficiency is high, allowing production of biallelic integrants using cssDNA donors. cssDNA donors also outcompete lssDNA donors in template-driven repair at the target site. These data demonstrate that circular donors provide an efficient, cost-effective method to achieve knock-ins in mammalian cell lines.