Family Genomoviridae Research Articles

Novel plant-associated genomoviruses from the Brazilian Cerrado biome.

The discovery rate of new plant viruses has increased due to studies involving high-throughput sequencing (HTS), particularly for single-stranded DNA viruses of the family Genomoviridae. We carried out an HTS-based survey of genomoviruses in a wide range of native and exotic trees grown in the Brazilian Cerrado biome, and the complete genome sequences of two novel members of the family Genomoviridae from two distinct genera were determined. Specific primers were designed to detect these genomoviruses in individual samples. A new gemykolovirus (Tecoma stans associated gemykolovirus) was detected in Tecoma stans, and a new gemykibivirus (Ouratea duparquetiana associated gemykibivirus) was detected in Ouratea duparquetiana. A gemykrogvirus related to Gila monster associated gemykrogvirus (80% pairwise identity) was also detected in foliar samples of Trembleya parviflora. Our pilot study paves the way for a better characterization of this diverse collection of genomoviruses as well as their interactions with the associated tree species.

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.

Characterization of Transcriptional Responses to Genomovirus Infection of the White Mold Fungus, Sclerotinia sclerotiorum.

Soybean leaf-associated gemygorvirus-1 (SlaGemV−1) is a CRESS-DNA virus classified in the family Genomoviridae, which causes hypovirulence and abolishes sclerotia formation in infected fungal pathogens under the family Sclerotiniaceae. To investigate the mechanisms involved in the induction of hypovirulence, RNA-Seq was compared between virus-free and SlaGemV−1-infected Sclerotinia sclerotiorum strain DK3. Overall, 4639 genes were differentially expressed, with 50.5% up regulated and 49.5% down regulated genes. GO enrichments suggest changes in integral membrane components and transmission electron microscopy images reveal virus-like particles localized near the inner cell membrane. Differential gene expression analysis focused on genes responsible for cell cycle and DNA replication and repair pathways, ubiquitin proteolysis, gene silencing, methylation, pathogenesis-related, sclerotial development, carbohydrate metabolism, and oxalic acid biosynthesis. Carbohydrate metabolism showed the most changes, with two glycoside hydrolase genes being the most down regulated by −2396.1- and −648.6-fold. Genes relating to pathogenesis showed consistent down regulation with the greatest being SsNep1, SsSSVP1, and Endo2 showing, −4555-, −14.7-, and −12.3-fold changes. The cell cycle and DNA replication/repair pathways were almost entirely up regulated including a putative cyclin and separase being up regulated 8.3- and 5.2-fold. The oxalate decarboxylase genes necessary for oxalic acid catabolism and oxalic acid precursor biosynthesis genes and its metabolism show down regulations of −17.2- and −12.1-fold changes. Sclerotial formation genes also appear differentially regulated including a melanin biosynthesis gene Pks1 and a sclerotia formation gene Sl2 with fold changes of 3.8 and −2.9.

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.

Family Genomoviridae: 2021 taxonomy update.

The family Genomoviridae (phylum Cressdnaviricota, class Repensiviricetes, order Geplafuvirales) includes viruses with circular single-stranded DNA genomes encoding two proteins, the capsid protein and the rolling-circle replication initiation protein. The genomes of the vast majority of members in this family have been sequenced directly from diverse environmental or animal- and plant-associated samples, but two genomoviruses have been identified infecting fungi. Since the last taxonomic update of the Genomoviridae, a number of new members of this family have been sequenced. Here, we report on the most recent taxonomic update, including the creation of one new genus, Gemytripvirus, and classification of ~420 new genomoviruses into 164 new species. We also announce the adoption of the "Genus + freeform epithet" binomial system for the naming of all 236 officially recognized species in the family Genomoviridae. The updated taxonomy presented in this article has been accepted by the International Committee on Taxonomy of Viruses (ICTV).

Identification of diverse novel genomoviruses in gut of wild birds

Genomoviridae is a virus family belonging to circular replication associated protein encoding ssDNA (CRESS-DNA) viruses, which have diverse genomic architecture and are widely distributed among different ecosystems. In this study, we characterized 39 novel genomovirus genomes including 3 from wild egrets, 9 from wild cranes, as well as 27 from wild finches in three different types of cloacal swabs of wild bird sampled in the station of Xinqing bird ringing, Maoershan Nature Reserve in Heilongjiang Province, Changbai Mountain in Jilin Province, and Hangzhou Wetland Park in Zhejiang Province, China. Here, using a Rep sequence phylogeny-based analysis, we divided the 39 genomoviruses into 8 genera within the family Genomoviridae, including Gemycircularvirus (n=20), Gemykibivirus (n=3), Gemygorvirus (n=2), Gemyvongvirus (n=2), Gemykolovirus (n=3), Gemykrogvirus (n=6), Gemytondvirus (n=1), Gemyduguivirus (n=1) and one unclassified genomovirus. The 39 genomovirus genomes belong to 36 species (27 of which are new) based on the currently accepted genomovirus pairwise nucleotide sequence identity species demarcation threshold of 78%. Overall, the research enriches our knowledge of CRESS-DNA viral diversity in China and emphasizes the prevalence of genomoviruses in nature.

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.

Diverse genomoviruses representing twenty-nine species identified associated with plants.

Genomoviruses (family Genomoviridae) are circular single-stranded DNA viruses that have been mainly identified through metagenomics studies in a wide variety of samples from various environments. Here, we describe 98 genomes of genomoviruses found associated with members of 19 plant families from Australia, Brazil, France, South Africa and the USA. These 98 genomoviruses represent 29 species, 26 of which are new, in the genera Gemykolovirus (n = 37), Gemyduguivirus (n = 9), Gemygorvirus (n = 8), Gemykroznavirus (n = 6), Gemycircularvirus (n = 21) and Gemykibivirus (n = 17).

Plant flowers transmit various bio-agents through air

Growing air pollution, known as “April snow”, by fluffy catkins from plant flowering was frequently observed during the Spring time in China. Current efforts mainly focus on controlling catkin release, while their public health and environmental impacts are unknown. Here, using culturing, microscopic and sequence methods we found that the flying catkins carry a significant amount of bio-agents (bacteria, fungal, viruses, and pollens), e.g., up to 6.33×104 bacterial and 7.46×105 fungal cells per catkin. Abundant pollens from plants were also found riding on the flying catkins. For collected fluffy catkins, bacterial Paracoccus, Massilia, fungal Ophiocordyceps, Arthrocladiella, pollen Robina, Pickeringia, Pinus, and viral family Genomoviridae, to name a few, were found to be dominant. With infection and allergic potentials, the detected microbial structures on the fluffy catkins were different from those of local air, varying from one location to another. Being not just white pollution, flying plant fluffy catkins were discovered here also acting as a cloud of biological agents, and further actions need to be taken urgently to understand and limit their environmental and ecological consequences.

A New Method to Obtain the Complete Genome Sequence of Multiple-Component Circular ssDNA Viruses by Transcriptome Analysis.

Circular single-stranded DNA (ssDNA) viruses are widely distributed globally, infecting diverse hosts ranging from bacteria, archaea, and eukaryotes. Among these, the genome of Banana bunchy top virus (BBTV) comprises at least six circular, ssDNA components that are ∼1 kb in length. Its genome is usually amplified and obtained at the DNA level. However, RNA-based techniques to obtain the genome sequence of such multi-component viruses have not been reported. In this study, transcriptome sequencing analysis showed that the full-length of BBTV each genomic component was transcribed into viral mRNA (vmRNA). Accordingly, the near-complete genome of BBTV B2 isolate was assembled using transcriptome sequencing data from virus-infected banana leaves. Assembly analysis of BBTV-derived reads indicated that the full-length sequences were obtained for DNA-R, DNA-U3, DNA-S, DNA-M, DNA-N, NewS2, and Sat4 components, while two gaps (73 and 25 nt) missing in the DNA-C component which was further filled by reverse transcription-PCR (RT-PCR). The RT-qPCR analysis indicated that the vmRNA levels of coding regions were 3.19–103.53 folds higher than those of non-coding regions, implying that the integrity of genome assembly depended on the transcription level of non-coding region. In conclusion, this study proposes a new approach to obtain the genome of nanovirids, and provides insights for studying the transcriptional mechanism of the family Nanoviridae, Genomoviridae, and Geminiviridae.

Viruses representing two new genomovirus species identified in citrus from Tunisia.

Using a high-throughput sequencing approach, we identified four genomoviruses (family Genomoviridae) associated with a sweet orange (Citrus sinensis) plant collected in Tunisia. The ssDNA genomes of these genomoviruses, which were amplified, cloned and Sangersequenced, range in size from 2156 to 2191 nt. Three of these viruses share > 99% full-genome pairwisesequence identity and are referred to as citrus Tunisia genomovirus 1 (CTNGmV-1). The CTNGmV-1 isolates share < 62% genome-wide pairwisenucleotide sequence identity with other genomoviruses and belong to the genus Gemykolovirus. The genome of the fourth virus, which was called CTNGmV-2, shares < 68% nucleotide sequence identity with other genomoviruses and belongs to the genus Gemycircularvirus. Based on the species demarcation criteria for members of the family Genomoviridae, CTNGmV-1 and -2 would each represent a new species. Although found associated with Citrus sp. plants, it is likely that these viruses infect fungi or other organisms associated with the plants.

A novel circular Rep-encoding single-stranded DNA virus detected in Agrotis ipsilon (Lepidoptera: Noctuidae) in China.

Long-distance migratory insects carry microorganisms that can potentially play a crucial role in the life cycles of their hosts. Here, we used Illumina and Sanger sequencing to determine the complete genome sequence of a novel circular Rep-encoding single-stranded (ss) DNA virus from an important migratory pest, Agrotis ipsilon (Hufnagel). The full genome of this new virus is about 2, 242 nt in length and shares 55-75% genome-wide pairwise sequence identity with members of the family Genomoviridae but 91% nucleotide sequence identity with finch-associated genomovirus 3 isolate S30P_D, which is tentatively abbreviated "FaGmV-3". Viral infection rates in A. ipsilon from Yantai, Langfang and Xinxiang were 4.5% (n = 88), 11.8% (n = 85) and 0% (n = 35), respectively. Phylogenetic analysis based on the deduced amino acid sequence of Rep indicated that the Agrotis ipsilon-associated virus is closely related to members of the genus Gemykibivirus, and we propose it to be a new member of this genus. Hence, it is tentatively named "Agrotis ipsilon-associated genomovirus" (AiGmV).

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.

Diverse single-stranded DNA viruses associated with honey bees (Apis mellifera)

Honey bees (Apis mellifera) research has increased in light of their progressive global decline over the last decade and the important role they play in pollination. One expanding area of honey bee research is analysis of their microbial community including viruses. Several RNA viruses have been characterized but little is known about DNA viruses associated with bees. Here, using a metagenomics based approach, we reveal the presence of a broad range of novel single-stranded DNA viruses from the hemolymph and brain of nurse and forager (worker divisions of labour) bees belonging to two honey bees subspecies, Italian (Apis mellifera linguistica) and New World Carniolan (Apis mellifera carnica). Genomes of 100 diverse viruses were identified, designated into three groupings; genomoviruses (family Genomoviridae) (n = 4), unclassified replication associated protein encoding single-stranded DNA viruses (n = 28), and microviruses (family Microviridae; subfamily Gokushovirinae) (n = 70). Amongst the viruses identified, it appears that nurses harbour a higher diversity of these viruses comparative to the foragers. Between subspecies, the most striking outcome was the extremely high number of diverse microviruses identified in the Italian bees comparative to the New World Carniolan, likely indicating an association to the diversity of the bacterial community associated with these subspecies.

Genome Sequence of a Single-Stranded DNA Virus Identified in Gila Monster Feces.

The Gila monster (Heloderma suspectum) is native to the Sonoran Desert. Metagenomic analyses of a Gila monster fecal sample revealed the presence of a small, circular, single-stranded DNA virus that is most closely related to a gemykrogvirus (family Genomoviridae) genome from caribou feces sharing 88% genome-wide pairwise identity.

Complete genome sequences of two gemycircularviruses associated with non-cultivated plants in Brazil.

Gemycircularviruses (genus Gemycircularvirus, family Genomoviridae) are single-stranded DNA viruses that are spread around the world in association with several organisms and environments. In this work, we identified two gemycircularviruses associated with two non-cultivated plants in Brazil, Momordica charantia and Euphorbia heterophylla. Both viruses display the general genome structure of gemycircularviruses. The virus isolated from M. charantia showed the highest nucleotide sequence identity with Pteropus associated gemycircularvirus 5, and an atypical structure consisting of a hairpin embedded in the major stem-loop was observed in the intergenic region. The virus from E. heterophylla showed the highest nucleotide sequence identity with Odonata associated gemycircularvirus 1. Phylogenetic analysis groups the two new viruses together with other genomoviruses of the genus Gemycircularvirus.

Diverse replication-associated protein encoding circular DNA viruses in guano samples of Central-Eastern European bats.

Circular replication-associated protein encoding single-stranded DNA (CRESS DNA) viruses are increasingly recognized worldwide in a variety of samples. Representative members include well-described veterinary pathogens with worldwide distribution, such as porcine circoviruses or beak and feather disease virus. In addition, numerous novel viruses belonging to the family Circoviridae with unverified pathogenic roles have been discovered in different human samples. Viruses of the family Genomoviridae have also been described as being highly abundant in different faecal and environmental samples, with case reports showing them to be suspected pathogens in human infections. In order to investigate the genetic diversity of these viruses in European bat populations, we tested guano samples from Georgia, Hungary, Romania, Serbia and Ukraine. This resulted in the detection of six novel members of the family Circoviridae and two novel members of the family Genomoviridae. Interestingly, a gemini-like virus, namely niminivirus, which was originally found in raw sewage samples in Nigeria, was also detected in our samples. We analyzed the nucleotide composition of members of the family Circoviridae to determine the possible host origins of these viruses. This study provides the first dataset on CRESS DNA viruses of European bats, and members of several novel viral species were discovered.

Erratum to: Discovery of two small circular ssDNA viruses associated with the whitefly Bemisia tabaci.

The complete genome sequences of two novel small circular DNA viruses isolated from sweet-potato whiteflies collected in Central-West (AdDF) and Southeast (AdO) regions of Brazil were determined by Next Generation Sequencing (NGS), and confirmed by cloning and Sanger sequencing. The genomes are 2,199 and 2,211 nt-long, respectively, encoding a putative coat protein (CP) and a replication-associated protein (Rep) and showing a genomic organization typical of viruses from the family Genomoviridae. Phylogenetic analysis with deduced amino acid sequences of Rep indicates that the virus from AdO is closely related to other members of the genus Gemycircularvirus, while the virus from AdDF is related to those of the genus Gemyduguivirus. These new genomoviruses are tentatively named bemisia-associated genomovirus AdO and bemisia-associated genomovirus AdDF.

Discovery of two small circular ssDNA viruses associated with the whitefly Bemisia tabaci.

The complete genome sequences of two novel small circular DNA viruses isolated from sweet-potato whiteflies collected in central-West (AdDF) and Southeast (AdO) regions of Brazil were determined by Next Generation Sequencing (NGS), and confirmed by cloning and Sanger sequencing. The genomes are 2,199 and 2,211 nt-long, respectively, encoding a putative coat protein (CP) and a replication-associated protein (Rep) and showing a genomic organization typical of viruses from the family Genomoviridae. Phylogenetic analysis with deduced amino acid sequences of Rep indicates that the virus from AdO is closely related to other members of the genus Gemycircularvirus, while the virus from AdDF is distantly related to other genomovirus. It was thus classified in a putative new genus, for which the name "Gemybolavirus" is proposed. These new genomoviruses are tentatively named "Bemisia associated gemybolavirus AdDF", and "Bemisia associated gemycircularvirus AdO".