Circular Single-stranded DNA Viruses Research Articles

Molecular characterization of canine circovirus based on the Capsid gene in Thailand

BackgroundCanine circovirus (CanineCV) is a single-stranded circular DNA virus that infects domestic and wild canids in many countries. CanineCV is associated with gastroenteritis and diarrhea, respiratory disease, and generalized vasculitis leading to a fatal event. The Capsid protein (Cap) is a structural protein of the virus which has high genetic variability and plays a role in the canine immune response. In this study, we cloned the full-length CanineCV Capsid gene (Cap). In-silico analyses were used to explore the genomic and amino acid variability and natural selection acting on the Cap gene. The immune relevance for T-cell and B-cell epitopes was predicted by the immunoinformatic approach.ResultsAccording to the Cap gene, our results showed that CanineCV was separated into five phylogenetic groups. The obtained CanineCV strain from this study was grouped with the previously discovered Thai strain (MG737385), as supported by a haplotype network. Entropy analyses revealed high nucleotide and amino acid variability of the Capsid region. Selection pressure analysis revealed four codons at positions 24, 50, 103, and 111 in the Cap protein evolved under diversifying selection. Prediction of B-cell epitopes exhibited four consensus sequences based on physiochemical properties, and eleven peptide sequences were predicted as T-cell epitopes. In addition, the positive selection sites were located within T-cell and B-cell epitopes, suggesting the role of the host immune system as a driving force in virus evolution.ConclusionsOur study provides knowledge of CanineCV genetic diversity, virus evolution, and potential epitopes for host cell immune response.

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.

Porcine circovirus type 2 infection promotes the SUMOylation of nucleophosmin-1 to facilitate the viral circular single-stranded DNA replication.

The mechanism of genome DNA replication in circular single-stranded DNA viruses is currently a mystery, except for the fact that it undergoes rolling-circle replication. Herein, we identified SUMOylated porcine nucleophosmin-1 (pNPM1), which is previously reported to be an interacting protein of the viral capsid protein, as a key regulator that promotes the genome DNA replication of porcine single-stranded DNA circovirus. Upon porcine circovirus type 2 (PCV2) infection, SUMO2/3 were recruited and conjugated with the K263 site of pNPM1's C-terminal domain to SUMOylate pNPM1, subsequently, the SUMOylated pNPM1 were translocated in nucleoli to promote the replication of PCV2 genome DNA. The mutation of the K263 site reduced the SUMOylation levels of pNPM1 and the nucleolar localization of pNPM1, resulting in a decrease in the level of PCV2 DNA replication. Meanwhile, the mutation of the K263 site prevented the interaction of pNPM1 with PCV2 DNA, but not the interaction of pNPM1 with PCV2 Cap. Mechanistically, PCV2 infection increased the expression levels of Ubc9, the only E2 enzyme involved in SUMOylation, through the Cap-mediated activation of ERK signaling. The upregulation of Ubc9 promoted the interaction between pNPM1 and TRIM24, a potential E3 ligase for SUMOylation, thereby facilitating the SUMOylation of pNPM1. The inhibition of ERK activation could significantly reduce the SUMOylation levels and the nucleolar localization of pNPM1, as well as the PCV2 DNA replication levels. These results provide new insights into the mechanism of circular single-stranded DNA virus replication and highlight NPM1 as a potential target for inhibiting PCV2 replication.

Characterization of the genomic sequence of a circo-like virus and of three chaphamaparvoviruses detected in mute swan (Cygnus olor).

We report the complete genomes of four ssDNA viruses: a circular replication-associated protein-encoding single-stranded DNA virus belonging to a clade previously detected only in mammals, and three chaphamaparvoviruses, which were detected by viromic surveillance of mute swan (Cygnus olor) fecal samples from the United Kingdom.

Metagenomic Identification of Novel Eukaryotic Viruses with Small DNA Genomes in Pheasants.

A panel of intestinal samples collected from common pheasants (Phasianus colchicus) between 2008 and 2017 was used for metagenomic investigation using an unbiased enrichment protocol and different bioinformatic pipelines. The number of sequence reads in the metagenomic analysis ranged from 1,419,265 to 17,507,704 with a viral sequence read rate ranging from 0.01% to 59%. When considering the sequence reads of eukaryotic viruses, RNA and DNA viruses were identified in the samples, including but not limited to coronaviruses, reoviruses, parvoviruses, and CRESS DNA viruses (i.e., circular Rep-encoding single-stranded DNA viruses). Partial or nearly complete genome sequences were reconstructed of at least three different parvoviruses (dependoparvovirus, aveparvovirus and chaphamaparvovirus), as well as gyroviruses and diverse CRESS DNA viruses. Generating information of virus diversity will serve as a basis for developing specific diagnostic tools and for structured epidemiological investigations, useful to assess the impact of these novel viruses on animal health.

Dynamics and Conformations of a Full-Length CRESS-DNA Replicase.

Circular Rep-encoding single-stranded DNA (CRESS-DNA) viruses encode for a Replicase (Rep) that is essential for viral replication. Rep is a helicase with three domains: an endonuclease, an oligomeric, and an ATPase domain (ED, OD, and AD). Our recent cryo-EM structure of the porcine circovirus 2 (PCV2) Rep provided the first structure of a CRESS-DNA Rep. The structure visualized the ED to be highly mobile, Rep to form a homo-hexamer, bound ssDNA and nucleotides, and the AD to adopt a staircase arrangement around the ssDNA. We proposed a hand-over-hand mechanism by the ADs for ssDNA translocation. The hand-over-hand mechanism requires extensive movement of the AD. Here, we scrutinize this mechanism using all-atom Molecular Dynamics (MD) simulation of Rep in three states: (1) Rep bound to ssDNA and ADP, (2) Rep bound to ssDNA, and (3) Rep by itself. Each of the 700 nsec simulations converges within 200 nsec and provides important insight into the dynamics of Rep, the dynamics of Rep in the presence of these biomolecules, and the importance of ssDNA and ADP in driving the AD to adopt the staircase arrangement around the ssDNA. To the best of our knowledge, this is the first example of an all-atom MD simulation of a CRESS-DNA Rep. This study sets the basis of further MD studies aimed at obtaining a chemical understanding of how Rep uses nucleotide binding and hydrolysis to translocate ssDNA.

Development and application of a self-assembling split-fluorescent protein toolkit to monitor geminiviral movement and infection in plant

Geminiviruses are a group of circular single-stranded DNA viruses that cause severe diseases in many crop plants. However, there is still no fluorescent protein tag suitable for labeling viral proteins endogenously due to the limited genomic space and structure of geminiviruses for foreign gene fragment insertion. Here, we established a split super-folder green fluorescent protein (sfGFP)-based imaging system that provides a platform to visualize the subcellular localization of geminiviral proteins in Nicotiana benthamiana. A short fragment of the GFP- coding sequence (GFP11) was inserted into a specific locus of the geminiviral genome, while the remainder of the GFP (GFP1–10) was transiently or constitutively expressed in N. benthamiana. Consequently, complementation fluorescence enables the examination of the subcellular localization of viral proteins in particular cells. Using this split sfGFP system, we examined the subcellular localization of the coat protein and BV1 protein of tomato golden mosaic virus (TGMV) and further monitored its intercellular and long-distance movement in N. benthamiana during viral infection. This approach allows us to study endogenously the subcellular localization and viral movement of geminiviruses in N. benthamiana. This new split sfGFP system may also hold the potential to provide orthogonal fluorescent proteins usable for geminiviral genome tagging in plants.

Quantification of Virion-Sense and Complementary-Sense DNA Strands of Circular Single-Stranded DNA Viruses.

Circular ssDNA viruses are ubiquitous and can be found in both prokaryotes and eukaryotes. To understand the interaction of ssDNA viruses with their hosts, it is important to characterize the dynamics of viral sense (VS) and complementary-sense (CS) viral strands during the infection process. Here, we present a simple and rapid protocol that allows sensitive and accurate determination of the VS and CS strands generated during viral infection.The method consists of a two-step qPCR in which the first step uses a strand-specific (CS or VS) labeled primer and T4 DNA polymerase that lacks strand displacement activity and makes a single copy per VS or CS strand. Next, the T4 DNA polymerase and unincorporated oligonucleotides are removed by a silica membrane spin column. Finally, the purified VS or CS strands are quantified by qPCR in a second step in which amplification uses a tag primer and a specific primer. Absolute quantification of VS and CS strands is obtained by extrapolating the Cq data to a standard curve of ssDNA, which can be generated by phagemid expression. Quantification of VS and CS strands of two geminiviruses in infections of Solanum lycopersicum (tomato) and Nicotiana benthamiana plants using this method is shown.

Circular replication-associated protein-encoding single-stranded DNA virus with risk of spillover is widely prevalent in domestic animals in China

Circular replication-associated protein (Rep)-encoding single-stranded (CRESS) DNA viruses are highly diverse and have a broad range of hosts. In this study, we report the detection of Bo-Circo-like virus AH20-1 in the feces of diarrheal cattle. The virus has a circular genome of 3,912 nucleotides, three major putative open reading frames, and encodes a Rep gene of 310 amino acids. We found that the virus is closely related to the Bo-Circo-like virus CH strain, which belongs to the novel Kirkoviridae family. Furthermore, we conducted a nationwide surveillance program and found that the virus is prevalent in China (23.6%, 205/868), with the BCLa subtype being the predominant strain. Our findings suggest that the virus can infect sheep, highlighting the potential for cross-species transmission. Our pressure analysis indicates that the CRESS-DNA Kirkoviridae family has broad host adaptation, and that selection pressure played an important role in the evolution of its Rep genes. Our study underscores the need for continued epidemiological surveillance of this virus due to its widespread prevalence in our ruminant population and potential for cross-species transmission.

Torque Teno Virus plasma DNA load: a novel prognostic biomarker in CAR-T therapy.

Torque Teno Virus (TTV) is a single-stranded circular DNA virus which has been identified as a surrogate marker of immune competence in transplantation. In this study we investigated the dynamics of plasma TTV DNAemia in 79 adult patients undergoing chimeric antigen receptor T-cell (CAR-T) therapy for relapsed or refractory large B-cell lymphoma, also evaluating the impact of TTV on immunotoxicities, response and survival outcomes. After lymphodepleting therapy, TTV DNA load was found to decrease slightly until reaching nadir around day 10, after which it increased steadily until reaching maximum load around day 90. TTV DNA load < 4.05 log10 copies/ml at immune effector cell-associated neurotoxicity syndrome (ICANS) onset identified patients at risk of progressing to severe forms of ICANS (OR 16.68, P = 0.048). Finally, patients who experienced falling or stable TTV DNA load between lymphodepletion and CAR-T infusion had better progression-free survival than those with ascending TTV DNA load (HR 0.31, P = 0.006). These findings suggest that TTV monitoring could serve as a surrogate marker of immune competence, enabling predictions of CAR-T efficacy and toxicity. This could pave the way for the development of TTV-guided therapeutic strategies that modulate clinical patient management based on plasma TTV load, similar to suggested strategies in solid organ transplant recipients.

Anelloviridae taxonomy update 2023.

The family Anelloviridae comprises negative single-stranded circular DNA viruses. Within this family, there are 30 established genera. Anelloviruses in the genus Gyrovirus have been identified infecting various avian species, whereas those in the remaining 29 genera have been found primarily infecting various mammal species. We renamed the 146 anellovirus species with binomial species names, as required by the International Committee on Taxonomy of Viruses (ICTV) using a "genus + freeform epithet" format.

REPercussions: how geminiviruses recruit host factors for replication.

Circular single-stranded DNA viruses of the family Geminiviridae encode replication-associated protein (Rep), which is a multifunctional protein involved in virus DNA replication, transcription of virus genes, and suppression of host defense responses. Geminivirus genomes are replicated through the interaction between virus Rep and several host proteins. The Rep also interacts with itself and the virus replication enhancer protein (REn), which is another essential component of the geminivirus replicase complex that interacts with host DNA polymerases α and δ. Recent studies revealed the structural and functional complexities of geminivirus Rep, which is believed to have evolved from plasmids containing a signature domain (HUH) for single-stranded DNA binding with nuclease activity. The Rep coding sequence encompasses the entire coding sequence for AC4, which is intricately embedded within it, and performs several overlapping functions like Rep, supporting virus infection. This review investigated the structural and functional diversity of the geminivirus Rep.

Highly divergent CRESS DNA and picorna-like viruses associated with bleached thalli of the green seaweed Ulva.

Marine macroalgae (seaweeds) are important primary producers and foundation species in coastal ecosystems around the world. Seaweeds currently contribute to an estimated 51% of the global mariculture production, with a long-term growth rate of 6% per year, and an estimated market value of more than US$11.3 billion. Viral infections could have a substantial impact on the ecology and aquaculture of seaweeds, but surprisingly little is known about virus diversity in macroalgal hosts. Using metagenomic sequencing, we characterized viral communities associated with healthy and bleached specimens of the commercially important green seaweed Ulva. We identified 20 putative new and divergent viruses, of which the majority belonged to the Circular Rep-Encoding Single-Stranded (CRESS) DNA viruses [single-stranded (ss)DNA genomes], Durnavirales [double-stranded (ds)RNA], and Picornavirales (ssRNA). Other newly identified RNA viruses were related to the Ghabrivirales, the Mitoviridae, and the Tombusviridae. Bleached Ulva samples contained particularly high viral read numbers. While reads matching assembled CRESS DNA viruses and picorna-like viruses were nearly absent from the healthy Ulva samples (confirmed by qPCR), they were very abundant in the bleached specimens. Therefore, bleaching in Ulva could be caused by one or a combination of the identified viruses but may also be the result of another causative agent or abiotic stress, with the viruses simply proliferating in already unhealthy seaweed tissue. This study highlights how little we know about the diversity and ecology of seaweed viruses, especially in relation to the health and diseases of the algal host, and emphasizes the need to better characterize the algal virosphere. IMPORTANCE Green seaweeds of the genus Ulva are considered a model system to study microbial interactions with the algal host. Remarkably little is known, however, about viral communities associated with green seaweeds, especially in relation to the health of the host. In this study, we characterized the viral communities associated with healthy and bleached Ulva. Our findings revealed the presence of 20 putative novel viruses associated with Ulva, encompassing both DNA and RNA viruses. The majority of these viruses were found to be especially abundant in bleached Ulva specimens. This is the first step toward understanding the role of viruses in the ecology and aquaculture of this green seaweed.

Replication mechanisms of circular ssDNA plant viruses and their potential implication in viral gene expression regulation.

The replication of members of the two circular single-stranded DNA (ssDNA) virus families Geminiviridae and Nanoviridae, the only ssDNA viruses infecting plants, is believed to be processed by rolling-circle replication (RCR) and recombination-dependent replication (RDR) mechanisms. RCR is a ubiquitous replication mode for circular ssDNA viruses and involves a virus-encoded Replication-associated protein (Rep) which fulfills multiple functions in the replication mechanism. Two key genomic elements have been identified for RCR in Geminiviridae and Nanoviridae: (i) short iterative sequences called iterons which determine the specific recognition of the viral DNA by the Rep and (ii) a sequence enabling the formation of a stem-loop structure which contains a conserved motif and constitutes the origin of replication. In addition, studies in Geminiviridae provided evidence for a second replication mode, RDR, which has also been documented in some double-stranded DNA viruses. Here, we provide a synthesis of the current understanding of the two presumed replication modes of Geminiviridae and Nanoviridae, and we identify knowledge gaps and discuss the possibility that these replication mechanisms could regulate viral gene expression through modulation of gene copy number.

Replitrons: A major group of eukaryotic transposons encoding HUH endonuclease

HUH endonucleases of the Rep (replication protein) class mediate the replication of highly diverse plasmids and viral genomes across all domains of life. HUH transposases have independently evolved from Reps, giving rise to three major transposable element groups: the prokaryotic insertion sequences IS200/IS605 and IS91/ISCR, and the eukaryotic Helitrons. Here, I present Replitrons, a second group of eukaryotic transposons encoding Rep HUH endonuclease. Replitron transposases feature a Rep domain with one catalytic Tyr (Y1) and an adjacent domain that may function in oligomerization, contrasting with Helitron transposases that feature Rep with two Tyr (Y2) and a fused helicase domain (i.e., RepHel). Protein clustering found no link between Replitron transposases and described HUH transposases, and instead recovered a weak association with Reps of circular Rep-encoding single-stranded (CRESS) DNA viruses and their related plasmids (pCRESS). The predicted tertiary structure of the transposase of Replitron-1, the founding member of the group that is active in the green alga Chlamydomonas reinhardtii, closely resembles that of CRESS-DNA viruses and other HUH endonucleases. Replitrons are present in at least three eukaryotic supergroups and reach high copy numbers in nonseed plant genomes. Replitron DNA sequences feature short direct repeats at, or potentially near, their termini. Finally, I characterize copy-and-paste de novo insertions of Replitron-1 using long-read sequencing of C. reinhardtii experimental lines. These results support an ancient and evolutionarily independent origin of Replitrons, in line with other major groups of eukaryotic transposons. This work expands the known diversity of both transposons and HUH endonucleases in eukaryotes.

Construction of an Agroinfectious Clone of a Korean Isolate of Sweet Potato Symptomless Virus 1 and Comparison of Its Infectivity According to Agrobacterium tumefaciens Strains in Nicotiana benthamiana

Sweet potato symptomless virus 1 (SPSMV-1) is a single-stranded circular DNA virus, belonging to the genus &lt;i&gt;Mastrevirus&lt;/i&gt; (family &lt;i&gt;Geminiviridae&lt;/i&gt;) that was first identified on sweet potato plants in South Korea in 2012. Although SPSMV-1 does not induce distinct symptoms in sweet potato plants, its co-infection with different sweet potato viruses is highly prevalent, and thus threatens sweet potato production in South Korea. In this study, the complete genome sequence of a Korean isolate of SPSMV-1 was obtained by Sanger sequencing of polymerase chain reaction (PCR) amplicons from sweet potato plants collected in the field (Suwon). An infectious clone of SPSMV-1 (1.1-mer) was constructed, cloned into the plant expression vector pCAMBIA1303, and agro-inoculated into &lt;i&gt;Nicotiana benthamiana&lt;/i&gt; using three &lt;i&gt;Agrobacterium tumefaciens&lt;/i&gt; strains (GV3101, LBA4404, and EHA105). Although no visual differences were observed between the mock and infected groups, SPSMV-1 accumulation was detected in the roots, stems, and newly produced leaves through PCR. The &lt;i&gt;A. tumefaciens&lt;/i&gt; strain LBA4404 was the most effective at transferring the SPSMV-1 genome to &lt;i&gt;N. benthamiana&lt;/i&gt;. We confirmed the viral replication in &lt;i&gt;N. benthamiana&lt;/i&gt; samples through strand-specific amplification using virion-sense- and complementary-sense-specific primer sets.

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.

Development of a DAS-ELISA for Gyrovirus Homsa1 Prevalence Survey in Chickens and Wild Birds in China.

Gyrovirus homsa1 (GyH1) is an emerging pathogenic single-stranded circular DNA virus that leads to immunosuppression, aplastic anemia, and multisystem damage in chickens. However, the prevalence of GyH1 infection in chickens and wild birds remains unknown. Here, we developed a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) to investigate GyH1 infection in 8 chicken species and 25 wild bird species. A total of 2258 serum samples from chickens (n = 2192) in 15 provinces, and wild birds (n = 66) in Jinan Wildlife Hospital were collected from 2017 to 2021 in China. The GyH1-positive rates in chickens and wild birds were 9.3% (203/2192) and 22.7% (15/66), respectively. GyH1 was present in all flocks in 15 provinces. From 2017 to 2021, the positive rate ranged from 7.93% (18/227) to 10.67% (56/525), and the highest positive rate was present in 2019. Upon chicken age, the highest positive rate (25.5%) was present in young chickens (14-35 days old). Moreover, the GyH1-positive rate in broiler breeders (12.6%, 21/167) was significantly higher than that in layer chickens (8.9%, 14/157). This study shows that GyH1 has spread in chicken flocks and wild birds, and the higher GyH1-positive rate in wild birds indicates the risk of spillover from wild birds to chickens. Our study expanded the GyH1 epidemiological aspects and provided a theoretical basis for GyH1 prevention.

Complete genome sequence analysis of a novel citlodavirus isolated from the leaves of Myrica rubra in Yunnan.

Through high-throughput sequencing, a novel citlodavirus, tentatively named "Myrica rubra citlodavirus 1" (MRV1,accession no. OP374189), was isolated from the leaves of Myrica rubra in Yunnan exhibiting narrow deformity of leaf tips, shrinkage, and chlorosis along the veins. The complete genome sequence was determined and analyzed using cloning and Sanger sequencing. MRV1 is a single-stranded circular non-enveloped DNA virus with a genome size of 3775 nucleotides and contains six open reading frames (ORFs). The virion-sense genome strand encodes a coat protein (CP, nt 750-1,493, 247 aa), two hypothetical movement proteins (V3, nt 382-666, 94 aa; and V2, nt 461-895, 144 aa), and one movement protein (MP, nt 1,527-2,438, 303 aa). The complementary strand of the genome encodes two replication proteins (RepA, nt 3,712-2,834, 292 aa; Rep, nt 2,867-2,553, 104 aa). The MRV1 genome contains the stem-loop motif 5'-TAATATTAC-3', which is a highly conserved nonanucleotide motif found in the origin of virion-strand replication in geminiviruses. Genome sequence alignment analysis showed that citrus chlorotic dwarf associated virus (CCDaV, accession no. JQ920490) shared the highest nucleotide sequence similarity (66.10% identity) with MRV1. Phylogenetic analysis showed that CCDaV is the closest known relative of MRV1, and that these viruses clustered in a single branch within a clade consisting of citlodaviruses. These results indicate that MRV1 should be regarded as a new species of the genus Citlodavirus in the family Geminiviridae.

Detection and genetic characterization of circoviruses in more than 80 bat species from eight countries on four continents

Several bat-associated circoviruses and circular rep-encoding single-stranded DNA (CRESS DNA) viruses have been described, but the exact diversity and host species of these viruses are often unknown. Our goal was to describe the diversity of bat-associated circoviruses and cirliviruses, thus, 424 bat samples from more than 80 species were collected on four continents. The samples were screened for circoviruses using PCR and the resulting amino acid sequences were subjected to phylogenetic analysis. The majority of bat strains were classified in the genus Circovirus and some strains in the genus Cyclovirus and the clades CRESS1 and CRESS3. Some strains, however, could only be classified at the taxonomic level of the order and were not classified in any of the accepted or proposed clades. In the family Circoviridae, 71 new species have been predicted. This screening of bat samples revealed a great diversity of circoviruses and cirliviruses. These studies underline the importance of the discovery and description of new cirliviruses and the need to establish new species and families in the order Cirlivirales.