A circular single-stranded DNA mycovirus infects plants and confers broad-spectrum fungal resistance.

Circular single-stranded DNA (ssDNA) viruses have been rarely found in fungi, and the evolutionary and ecological relationships among ssDNA viruses infecting fungi and other organisms remain unclear. In this study, a novel circular ssDNA virus, tentatively named Diaporthe sojae circular DNA virus 1 (DsCDV1), was identified in the phytopathogenic fungus Diaporthe sojae isolated from pear trees. DsCDV1 has a monopartite genome (3185 nt in size) encapsidated in isometric virions (21-26 nm in diameter). The genome comprises seven putative open reading frames encoding a discrete replicase (Rep) split by an intergenic region, a putative capsid protein (CP), several proteins of unknown function (P1-P4), and a long intergenic region. Notably, the two split parts of DsCDV1 Rep share high identities with the Reps of Geminiviridae and Genomoviridae, respectively, indicating an evolutionary linkage with both families. Phylogenetic analysis based on Rep or CP sequences placed DsCDV1 in a unique cluster, supporting the establishment of a new family, tentatively named Gegemycoviridae, intermediate to both families. DsCDV1 significantly attenuates fungal growth and nearly erases fungal virulence when transfected into the host fungus. Remarkably, DsCDV1 can systematically infect tobacco and pear seedlings, providing broad-spectrum resistance to fungal diseases. Subcellular localization analysis revealed that DsCDV1 P3 is systematically localized in the plasmodesmata, while its expression in trans-complementation experiments could restore systematic infection of a movement-deficient plant virus, suggesting that P3 is a movement protein. DsCDV1 exhibits unique molecular and biological traits not observed in other ssDNA viruses, serving as a link between fungal and plant ssDNA viruses and presenting an evolutionary connection between ssDNA viruses and fungi. These findings contribute to expanding our understanding of ssDNA virus diversity and evolution, offering potential biocontrol applications for managing crucial plant diseases.

Genomoviruses in Liver Samples of Molossus molossus Bats.

CRESS-DNA encompasses a broad spectrum of viruses documented across diverse organisms such as animals, plants, diatoms, fungi, and marine invertebrates. Despite this prevalence, the full extent of these viruses' impact on the environment and their respective hosts remains incompletely understood. Furthermore, an increasing number of viruses within this category lack detailed characterization. This investigation focuses on unveiling and characterizing viruses affiliated with the Genomoviridae family identified in liver samples from the bat Molossus molossus. Leveraging viral metagenomics, we identified seven sequences (MmGmV-PA) featuring a circular DNA genome housing two ORFs encoding replication-associated protein (Rep) and capsid protein (Cap). Predictions based on conserved domains typical of the Genomoviridae family were established. Phylogenetic analysis revealed the segregation of these sequences into two clades aligning with the genera Gemycirculavirus (MmGmV-06-PA and MmGmV-07-PA) and Gemykibivirus (MmGmV-01-PA, MmGmV-02-PA, MmGmV-03-PA, MmGmV-05-PA, and MmGmV-09-PA). At the species level, pairwise comparisons based on complete nucleotide sequences indicated the potential existence of three novel species. In summary, our study significantly contributes to an enhanced understanding of the diversity of Genomoviridae within bat samples, shedding light on previously undiscovered viral entities and their potential ecological implications.

First Insights into the Occurrence of Circular Single-Stranded DNA Genomes in Asian and African Cattle.

Circular replicase-encoding single-stranded (CRESS) DNA viruses and other circular DNA agents are increasingly found in various samples and animals. A specific class of these agents-termed bovine meat and milk factors (BMMF)-has been supposed to act as a factor in indirect carcinogenesis in humans. Initial observations attributed the BMMF to European cattle breeds and foodstuffs produced thereof. In the present study, blood and fecal samples from African and Asian cattle were examined. BMMF molecules and genomoviruses were detected in all bovids under study. The majority (79%) of the 29 circular elements could be assigned to BMMF groups 1 and 2, whereas CRESS viruses of the family Genomoviridae accounted for the smaller part (21%). Two genomoviruses belong to the genus Gemykibivirus and one to the genus Gemykrogvirus. The remaining three might be considered as novel species within the genus Gemycircularvirus. The majority of all isolated molecules originated from fecal samples, whereas only three derived from blood. The results from this study expand our knowledge on the diversity and presence of circular DNA in different ruminants that serve for food production in many countries over the world.

Diverse DNA virus genomes identified in fecal samples of Mexican free-tailed bats (Tadarida brasiliensis) captured in Chiricahua Mountains of southeast Arizona (USA).

Bats (order Chiroptera) are some of the most abundant mammals on earth and their species ecology strongly influences zoonotic potential. While substantial research has been conducted on bat-associated viruses, particularly on those that can cause disease in humans and/or livestock, globally, limited research has focused on endemic bats in the USA. The southwest region of the US is of particular interest because of its high diversity of bat species. We identified 39 single-stranded DNA virus genomes in the feces of Mexican free-tailed bats (Tadarida brasiliensis) sampled in the Rucker Canyon (Chiricahua Mountains) of southeast Arizona (USA). Twenty-eight of these belong to the virus families Circoviridae (n = 6), Genomoviridae (n = 17), and Microviridae (n = 5). Eleven viruses cluster with other unclassified cressdnaviruses. Most of the viruses identified represent new species. Further research on identification of novel bat-associated cressdnaviruses and microviruses is needed to provide greater insights regarding their co-evolution and ecology relative to bats.

Diverse single-stranded DNA viruses identified in New Zealand (Aotearoa) South Island robin (Petroica australis) fecal samples.

The South Island robin (Petroica australis) is a small passerine bird endemic to New Zealand (Aotearoa). Although its population has declined recently and it is considered 'at risk,' little research has been done to identify viruses in this species. This study aimed to survey the diversity of single-stranded DNA viruses associated with South Island robins in a small, isolated population on Nukuwaiata Island. In total, 108 DNA viruses were identified from pooled fecal samples collected from 38 individual robins sampled. These viruses belong to the Circoviridae (n = 10), Genomoviridae (n = 12), and Microviridae (n = 73) families. A number of genomes that belong to the phylum Cressdnaviricota but are otherwise unclassified (n = 13) were also identified. These results greatly expand the known viral diversity associated with South Island robins, and we identify a novel group of viruses most closely related genomoviruses.

Virome in Fecal Samples From Wild Giant Pandas (Ailuropoda Melanoleuca).

The giant panda (Ailuropoda melanoleuca) is one of the most endangered mammals in the world; anthropogenic habitat loss and poaching still threaten the survival of wild pandas. Viral infection has become one of the potential threats to the health of these animals, but the available information related to these infections is still limited. In order to detect possible vertebrate viruses, the virome in the fecal samples of seven wild giant pandas from Qinling Mountains was investigated by using the method of viral metagenomics. From the fecal virome of wild giant pandas, we determined six nearly complete genomes belonging to the order Picornavirales, two of which may be qualified as a novel virus family or genus. In addition, four complete genomes belonging to the Genomoviridae family were also fully characterized. This virological investigation has increased our understanding of the gut viral community in giant pandas. Whether these viruses detected in fecal samples can really infect giant panda needs further research.

Identification and Characterization of Circular Single-Stranded DNA Genomes in Sheep and Goat Milk.

In recent years, a variety of circular replicase-encoding single-stranded (CRESS) DNA viruses and unclassified virus-like DNA elements have been discovered in a broad range of animal species and environmental samples. Key questions to be answered concern their presence in the human diet and their potential impact on disease emergence. Especially DNA elements termed bovine meat and milk factors (BMMF) are suspected to act as co-factors in the development of colon and breast cancer. To expand our knowledge on the occurrence of these potential pathogens in human nutrition, a total of 73 sheep and 40 goat milk samples were assayed by combining rolling circle amplification (RCA), PCR and Sanger sequencing. The present study further includes retail milk from the aforementioned species. We recovered 15 single stranded (ss) circular genomes. Of those, nine belong to the family Genomoviridae and six are members of the unclassified group of BMMF. Thus, dairy sheep and goats add to dispersal of CRESS viruses and circular ssDNA elements, which enter the food chain via milk. The presence of these entities is therefore more widespread in Bovidae than initially assumed and seems to be part of the common human nutrition.

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.

Diverse cressdnaviruses and an anellovirus identified in the fecal samples of yellow-bellied marmots.

Over that last decade, coupling multiple strand displacement approaches with high throughput sequencing have resulted in the identification of genomes of diverse groups of small circular DNA viruses. Using a similar approach but with recovery of complete genomes by PCR, we identified a diverse group of single-stranded viruses in yellow-bellied marmot (Marmota flaviventer) fecal samples. From 13 fecal samples we identified viruses in the family Genomoviridae (n = 7) and Anelloviridae (n = 1), and several others that ware part of the larger Cressdnaviricota phylum but not within established families (n = 19). There were also circular DNA molecules identified (n = 4) that appear to encode one viral-like gene and have genomes of <1545 nts. This study gives a snapshot of viruses associated with marmots based on fecal sampling.

Characterization of a novel genomovirus in the phytopathogenic fungus Botrytis cinerea.

This study characterized a single-stranded circular DNA virus in Botrytis cinerea-namely, Botrytis cinerea genomovirus 1 (BcGV1). The genome of BcGV1 was 1710 nucleotides (nts) long, possessing two ORFs, encoding a putative replication initiation protein (Rep) and a hypothetical protein. The Rep contained seven conserved motifs. The two ORFs were separated by two intergenic regions; the large intergenic region (LIR) contained 259 nts while the small intergenic region (SIR) contained 95 nts. A nonanucleotide, TAACAGTAC, in the LIR was predicted to be associated with the initiation of viral replication. Based on the phylogenetic tree constructed by Reps, BcGV1 belongs to the family Genomoviridae, forming an independent branch, indicating that BcGV1 may belong to a new genus. BcGV1 could be detected in 6.7% of tested B. cinerea strains, suggesting that BcGV1 may be widely distributed in the Chinese B. cinerea population.

Viral diversity in oral cavity from Sapajus nigritus by metagenomic analyses.

Sapajus nigritus are non-human primates which are widespread in South America. They are omnivores and live in troops of up to 40 individuals. The oral cavity is one of the main entry routes for microorganisms, including viruses. Our study proposed the identification of viral sequences from oral swabs collected in a group of capuchin monkeys (n = 5) living in a public park in a fragment of Mata Atlantica in South Brazil. Samples were submitted to nucleic acid extraction and enrichment, which was followed by the construction of libraries. After high-throughput sequencing and contig assembly, we used a pipeline to identify 11 viral families, which are Herpesviridae, Parvoviridae, Papillomaviridae, Polyomaviridae, Caulimoviridae, Iridoviridae, Astroviridae, Poxviridae, and Baculoviridae, in addition to two complete viral genomes of Anelloviridae and Genomoviridae. Some of these viruses were closely related to known viruses, while other fragments are more distantly related, with 50% of identity or less to the currently available virus sequences in databases. In addition to host-related viruses, insect and small vertebrate-related viruses were also found, as well as plant-related viruses, bringing insights about their diet. In conclusion, this viral metagenomic analysis reveals, for the first time, the profile of viruses in the oral cavity of wild, free ranging capuchin monkeys.

A Preliminary Study of the Virome of the South American Free-Tailed Bats (Tadarida brasiliensis) and Identification of Two Novel Mammalian Viruses.

Bats provide important ecosystem services as pollinators, seed dispersers, and/or insect controllers, but they have also been found harboring different viruses with zoonotic potential. Virome studies in bats distributed in Asia, Africa, Europe, and North America have increased dramatically over the past decade, whereas information on viruses infecting South American species is scarce. We explored the virome of Tadarida brasiliensis, an insectivorous New World bat species inhabiting a maternity colony in Rosario (Argentina), by a metagenomic approach. The analysis of five pooled oral/anal swab samples indicated the presence of 43 different taxonomic viral families infecting a wide range of hosts. By conventional nucleic acid detection techniques and/or bioinformatics approaches, the genomes of two novel viruses were completely covered clustering into the Papillomaviridae (Tadarida brasiliensis papillomavirus type 1, TbraPV1) and Genomoviridae (Tadarida brasiliensis gemykibivirus 1, TbGkyV1) families. TbraPV1 is the first papillomavirus type identified in this host and the prototype of a novel genus. TbGkyV1 is the first genomovirus reported in New World bats and constitutes a new species within the genus Gemykibivirus. Our findings extend the knowledge about oral/anal viromes of a South American bat species and contribute to understand the evolution and genetic diversity of the novel characterized viruses.

Virus Discovery in Desert Tortoise Fecal Samples: Novel Circular Single-Stranded DNA Viruses.

The Sonoran Desert tortoise Gopherus morafkai is adapted to the desert, and plays an important ecological role in this environment. There is limited information on the viral diversity associated with tortoises (family Testudinidae), and to date no DNA virus has been identified associated with these animals. This study aimed to assess the diversity of DNA viruses associated with the Sonoran Desert tortoise by sampling their fecal matter. A viral metagenomics approach was used to identify the DNA viruses in fecal samples from wild Sonoran Desert tortoises in Arizona, USA. In total, 156 novel single-stranded DNA viruses were identified from 40 fecal samples. Those belonged to two known viral families, the Genomoviridae (n = 27) and Microviridae (n = 119). In addition, 10 genomes were recovered that belong to the unclassified group of circular-replication associated protein encoding single-stranded (CRESS) DNA virus and five circular molecules encoding viral-like proteins.

Single Stranded DNA Viruses Associated with Capybara Faeces Sampled in Brazil.

Capybaras (Hydrochoerus hydrochaeris), the world's largest rodents, are distributed throughout South America. These wild herbivores are commonly found near water bodies and are well adapted to rural and urban areas. There is limited information on the viruses circulating through capybaras. This study aimed to expand the knowledge on the viral diversity associated with capybaras by sampling their faeces. Using a viral metagenomics approach, we identified diverse single-stranded DNA viruses in the capybara faeces sampled in the Distrito Federal, Brazil. A total of 148 complete genomes of viruses in the Microviridae family were identified. In addition, 14 genomoviruses (family Genomoviridae), a novel cyclovirus (family Circoviridae), and a smacovirus (family Smacoviridae) were identified. Also, 37 diverse viruses that cannot be assigned to known families and more broadly referred to as unclassified circular replication associated protein encoding single-stranded (CRESS) DNA viruses were identified. This study provides a snapshot of the viral diversity associated with capybaras that may be infectious to these animals or associated with their microbiota or diet.

Unravelling the Single-Stranded DNA Virome of the New Zealand Blackfly.

Over the last decade, arthropods have been shown to harbour a rich diversity of viruses. Through viral metagenomics a large diversity of single-stranded (ss) DNA viruses have been identified. Here we examine the ssDNA virome of the hematophagous New Zealand blackfly using viral metagenomics. Our investigation reveals a plethora of novel ssDNA viral genomes, some of which cluster in the viral families Genomoviridae (n = 9), Circoviridae (n = 1), and Microviridae (n = 108), others in putative families that, at present, remain unclassified (n = 20) and one DNA molecule that only encodes a replication associated protein. Among these novel viruses, two putative multi-component virus genomes were recovered, and these are most closely related to a Tongan flying fox faeces-associated multi-component virus. Given that the only other known multi-component circular replication-associated (Rep) protein encoding single-stranded (CRESS) DNA viruses infecting plants are in the families Geminiviridae (members of the genus Begomovirus) and Nanoviridae, it appears these are likely a new multi-component virus group which may be associated with animals. This study reiterates the diversity of ssDNA viruses in nature and in particular with the New Zealand blackflies.

Low prevalence of Gemycircularvirus DNA in immunocompetent and immunocompromised subjects.

Gemycircularviruses (GemyCV) are a vast array of viruses belonging to the Genomoviridae family. Prevalence and pathogenesis in humans are still poorly understood. Different GemyCV species were investigated in 661 Italian subjects by species-specific PCRs. Only the GemyCV-C1c species was detected, with low prevalence and the highest rate in HIV immunosuppressed patients.

Genomoviruses associated with mountain and western pine beetles.

Genomoviruses are circular single-stranded DNA viruses (∼2 kb in size) classified into nine genera, they are highly diverse and have been identified in a variety of samples ranging from fungi to animal sera. Here we identify five genomoviruses belonging to the Gemycircularvirus genus and one to the Gemykibivirus genus from mountain pine beetle and western pine beetle sampled in Arizona. Collectively these six viral genomes share <77% genome-wide pairwise identity and hence represent six new species of genomoviruses. Four of the gemycircularviruses from the mountain pine beetles are recombinant, with one having a recombinant region that spans the entire capsid protein. Pine beetles have a symbiotic relationship with certain tree pathogenic fungi. Therefore given that Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1, a gemycircularvirus, induces hypovirulence in the plant pathogenic fungus Sclerotinia sclerotiorum and infects the mycophagous insect Lycoriella ingenua, it is possible that the six genomoviruses identified here may be directly associated with the pine beetle fungal symbionts and/or with the insects themselves.

Identification of circular single-stranded DNA viruses in faecal samples of Canada lynx (Lynx canadensis), moose (Alces alces) and snowshoe hare (Lepus americanus) inhabiting the Colorado San Juan Mountains.

The San Juan Mountains of southern Colorado provide subalpine habitat for a suite of mammalian species including Canada lynx (Lynx canadensis), moose (Alces alces) and snowshoe hare (Lepus americanus). In the winter field season of 2016 five faecal samples from lynx, and one each from moose and snowshoe hare were collected to identify small single-stranded DNA viruses associated with these three prominent species. Thirty-two novel viruses were identified and classified as members of two well established ssDNA families Genomoviridae (n = 22) and Microviridae (n = 10) and one recently proposed new family, Smacoviridae (n = 1). In addition one highly novel circular ssDNA virus was identified which at present does not group with any known family. A high level of genomovirus diversity was identified from faeces collected between and across the three mammal species, with full genome-wide pairwise comparisons showing 57%-97% identity. Twenty genomoviruses can be assigned to the genus Gemycircularvirus and represent 11 species, and two into a distinct species in the genus Gemykolovirus. The single smacovirus identified from moose also represents a distinct smacovirus species. Ten microviruses, seven from moose, one from snowshoe hare and two from lynx, all are part of the Gokushovirinae subfamily. The two from lynx are highly similar to a microvirus previously detected in domestic cat (sharing 88%-90% genome-wide identity), indicating this may be a common felid gut microbiome associated virus. Our findings highlight the broad range of diverse ssDNA viruses present in three mammals inhabiting the San Juan Mountains.

Identification of an anellovirus and genomoviruses in ixodid ticks.

Ticks are blood-feeding arachnids that are vectors of several important human and animal pathogens. Little is known about single-stranded DNA (ssDNA) viruses that are associated with these ectoparasites. As part of a pilot study to identify ssDNA viruses present in ticks, female American dog ticks (Dermacentor variabilis) and blacklegged ticks (Ixodes scapularis) were collected in eastern Pennsylvania (USA), and the extracted viral DNA was analyzed using viral metagenomics approaches. Three genomoviruses were recovered from pooled samples of D. variabilis (n = 2) and I. scapularis (n = 1): two belonging to the genus Gemycircularvirus, sharing < 63% pairwise identity with other members within the genus; and the third belonging to the genus Gemykolovirus, sharing < 70% pairwise identity to other gemykoloviruses. Furthermore, a genome of an anellovirus belonging to the sharing 62-65% nucleotide identity with torque teno canis viruses (genus Thetatorquevirus) was also recovered from a D. variabilis pooled sample.