Anelloviruses are highly prevalent in diverse mammals, including humans, but so far have not been linked to any disease and are considered to be part of the 'healthy virome'. These viruses have small circular single-stranded DNA (ssDNA) genomes and encode several proteins with no detectable sequence similarity to proteins of other known viruses. Thus, anelloviruses are the only family of eukaryotic ssDNA viruses currently not included in the realm Monodnaviria. To gain insights into the provenance of these enigmatic viruses, we sequenced more than 250 complete genomes of anelloviruses from nasal and vaginal swab samples of Weddell seal (Leptonychotes weddellii) from Antarctica and a fecal sample of grizzly bear (Ursus arctos horribilis) from the USA and performed a comprehensive family-wide analysis of the signature anellovirus protein ORF1. Using state-of-the-art remote sequence similarity detection approaches and structural modeling with AlphaFold2, we show that ORF1 orthologs from all Anelloviridae genera adopt a jelly-roll fold typical of viral capsid proteins (CPs), establishing an evolutionary link to other eukaryotic ssDNA viruses, specifically, circoviruses. However, unlike CPs of other ssDNA viruses, ORF1 encoded by anelloviruses from different genera display remarkable variation in size, due to insertions into the jelly-roll domain. In particular, the insertion between β-strands H and I forms a projection domain predicted to face away from the capsid surface and function at the interface of virus-host interactions. Consistent with this prediction and supported by recent experimental evidence, the outermost region of the projection domain is a mutational hotspot, where rapid evolution was likely precipitated by the host immune system. Collectively, our findings further expand the known diversity of anelloviruses and explain how anellovirus ORF1 proteins likely diverged from canonical jelly-roll CPs through gradual augmentation of the projection domain. We suggest assigning Anelloviridae to a new phylum, 'Commensaviricota', and including it into the kingdom Shotokuvirae (realm Monodnaviria), alongside Cressdnaviricota and Cossaviricota.

We describe the complete capsid of a genotype C1-like Enterovirus A71 variant recovered from wastewater in a neighborhood in the greater Tempe, Arizona area (Southwest United States) in May 2020 using a pan-enterovirus amplicon-based high-throughput sequencing strategy. The variant seems to have been circulating for over two years, but its sequence has not been documented in that period. As the SARS-CoV-2 pandemic has resulted in changes in health-seeking behavior and overwhelmed pathogen diagnostics, our findings highlight the importance of wastewater-based epidemiology (WBE ) as an early warning system for virus surveillance.

The discovery of cruciviruses revealed the most explicit example of a common protein homologue between DNA and RNA viruses to date. Cruciviruses are a novel group of circular Rep-encoding single-stranded DNA (ssDNA) (CRESS-DNA) viruses that encode capsid proteins that are most closely related to those encoded by RNA viruses in the family Tombusviridae The apparent chimeric nature of the two core proteins encoded by crucivirus genomes suggests horizontal gene transfer of capsid genes between DNA and RNA viruses. Here, we identified and characterized 451 new crucivirus genomes and 10 capsid-encoding circular genetic elements through de novo assembly and mining of metagenomic data. These genomes are highly diverse, as demonstrated by sequence comparisons and phylogenetic analysis of subsets of the protein sequences they encode. Most of the variation is reflected in the replication-associated protein (Rep) sequences, and much of the sequence diversity appears to be due to recombination. Our results suggest that recombination tends to occur more frequently among groups of cruciviruses with relatively similar capsid proteins and that the exchange of Rep protein domains between cruciviruses is rarer than intergenic recombination. Additionally, we suggest members of the stramenopiles/alveolates/Rhizaria supergroup as possible crucivirus hosts. Altogether, we provide a comprehensive and descriptive characterization of cruciviruses.IMPORTANCE Viruses are the most abundant biological entities on Earth. In addition to their impact on animal and plant health, viruses have important roles in ecosystem dynamics as well as in the evolution of the biosphere. Circular Rep-encoding single-stranded (CRESS) DNA viruses are ubiquitous in nature, many are agriculturally important, and they appear to have multiple origins from prokaryotic plasmids. A subset of CRESS-DNA viruses, the cruciviruses, have homologues of capsid proteins encoded by RNA viruses. The genetic structure of cruciviruses attests to the transfer of capsid genes between disparate groups of viruses. However, the evolutionary history of cruciviruses is still unclear. By collecting and analyzing cruciviral sequence data, we provide a deeper insight into the evolutionary intricacies of cruciviruses. Our results reveal an unexpected diversity of this virus group, with frequent recombination as an important determinant of variability.

We determine the presence and diversity of rhinoviruses in nasopharyngeal swab samples from 248 individuals who presented with influenza-like illness (ILI) at a university clinic in the Southwest United States between October 1, 2020 and March 31, 2021. We identify at least 13 rhinovirus genotypes (A11, A22, A23, A25, A67, A101, B6, B79, C1, C17, C36, and C56, as well a new genotype [AZ88**]) and 16 variants that contributed to the burden of ILI in the community. We also describe the complete capsid protein gene of a member (AZ88**) of an unassigned rhinovirus A genotype.

Single-stranded (ss) DNA viruses are a major component of the earth virome. In particular, the circular, Rep-encoding ssDNA (CRESS-DNA) viruses show high diversity and abundance in various habitats. By combining sequence similarity network and phylogenetic analyses of the replication proteins (Rep) belonging to the HUH endonuclease superfamily, we show that the replication machinery of the CRESS-DNA viruses evolved, on three independent occasions, from the Reps of bacterial rolling circle-replicating plasmids. The CRESS-DNA viruses emerged via recombination between such plasmids and cDNA copies of capsid genes of eukaryotic positive-sense RNA viruses. Similarly, the rep genes of prokaryotic DNA viruses appear to have evolved from HUH endonuclease genes of various bacterial and archaeal plasmids. Our findings also suggest that eukaryotic polyomaviruses and papillomaviruses with dsDNA genomes have evolved via parvoviruses from CRESS-DNA viruses. Collectively, our results shed light on the complex evolutionary history of a major class of viruses revealing its polyphyletic origins.

Although millions of distinct virus species likely exist, only approximately 9000 are catalogued in GenBank's RefSeq database. We selectively enriched for the genomes of circular DNA viruses in over 70 animal samples, ranging from nematodes to human tissue specimens. A bioinformatics pipeline, Cenote-Taker, was developed to automatically annotate over 2500 complete genomes in a GenBank-compliant format. The new genomes belong to dozens of established and emerging viral families. Some appear to be the result of previously undescribed recombination events between ssDNA and ssRNA viruses. In addition, hundreds of circular DNA elements that do not encode any discernable similarities to previously characterized sequences were identified. To characterize these 'dark matter' sequences, we used an artificial neural network to identify candidate viral capsid proteins, several of which formed virus-like particles when expressed in culture. These data further the understanding of viral sequence diversity and allow for high throughput documentation of the virosphere.

Numerous metagenomic studies have uncovered a remarkable diversity of circular replication-associated protein (Rep)-encoding single-stranded (CRESS) DNA viruses, the majority of which are uncultured and unclassified. Unlike capsid proteins, the Reps show significant similarity across different groups of CRESS DNA viruses and have conserved domain organization with the N-terminal nuclease and the C-terminal helicase domain. Consequently, Rep is widely used as a marker for identification, classification and assessment of the diversity of CRESS DNA viruses. However, it has been shown that in certain viruses the Rep nuclease and helicase domains display incongruent evolutionary histories. Here, we systematically evaluated the co-evolutionary patterns of the two Rep domains across classified and unclassified CRESS DNA viruses. Our analysis indicates that the Reps encoded by members of the families Bacilladnaviridae, Circoviridae, Geminiviridae, Genomoviridae, Nanoviridae and Smacoviridae display largely congruent evolutionary patterns in the two domains. By contrast, among the unclassified CRESS DNA viruses, 71% appear to have chimeric Reps. Such massive chimerism suggests that unclassified CRESS DNA viruses represent a dynamic population in which exchange of gene fragments encoding the nuclease and helicase domains is extremely common. Furthermore, purging of the chimeric sequences uncovered six monophyletic Rep groups that may represent new families of CRESS DNA viruses.

Antarctic cryoconite holes, or small melt-holes in the surfaces of glaciers, create habitable oases for isolated microbial communities with tightly linked microbial population structures. Viruses may influence the dynamics of polar microbial communities, but the viromes of the Antarctic cryoconite holes have yet to be characterized. We characterize single-stranded DNA (ssDNA) viruses from three cryoconite holes in the Taylor Valley, Antarctica, using metagenomics. Half of the assembled metagenomes cluster with those in the viral family Microviridae (n = 7), and the rest with unclassified circular replication associated protein (Rep)-encoding single-stranded (CRESS) DNA viruses (n = 7). An additional 18 virus-like circular molecules encoding either a Rep, a capsid protein gene, or other unidentified but viral-like open reading frames were identified. The samples from which the genomes were identified show a strong gradient in microbial diversity and abundances, and the number of viral genomes detected in each sample mirror that gradient. Additionally, one of the CRESS genomes assembled here shares ~90% genome-wide pairwise identity with a virus identified from a freshwater pond on the McMurdo Ice Shelf (Antarctica). Otherwise, the similarity of these viruses to those previously identified is relatively low. Together, these patterns are consistent with the presence of a unique regional virome present in fresh water host populations of the McMurdo Dry Valley region.

Pigeon circovirus (PiCV) infects pigeon populations worldwide and has been associated with immunosuppression in younger pigeons. Recombination is a common mechanism of evolution that has previously been shown in various members of the Circoviridae family, including PiCV. In this study, three groups of pigeons acquired from separate lofts were screened for PiCV, and their genome sequence was determined. Following this, they were housed in a single loft for 22 days, during which blood and cloacal swab samples were taken. From these blood and cloacal swabs, PiCV genomes were determined with the aim to study the spread and recombination dynamics of PiCV in the birds. Genome sequences of PiCV were determined from seven pigeons (seven tested PiCV positive) before they were housed together in a loft (n = 58 sequences) and thereafter from the ten pigeons from blood and cloacal swabs (n = 120). These 178 PiCV genome sequences represent seven genotypes (98% pairwise identity genotype demarcation), and they share >88% genome-wide pairwise identity. Recombination analysis revealed 13 recombination events, and a recombination hotspot spanning the 3' prime region, the replication-associated protein (rep) gene and the intergenic region. A cold spot in the capsid protein-coding region of the genome was also identified. The majority of the recombinant regions were identified in the rep coding region. This study provides insights into the evolutionary dynamics of PiCV in pigeons kept under closed rearing systems.

Brazil is one of the major passion fruit producers worldwide. Viral diseases are among the most important constraints for passion fruit production. Here we identify and characterize a new passion fruit infecting-virus belonging to the family Geminiviridae: passion fruit chlorotic mottle virus (PCMoV). PCMoV is a divergent geminivirus unlike previously characterized passion fruit-infecting geminiviruses that belonged to the genus Begomovirus. Among the presently known geminiviruses, it is most closely related to, and shares ~62% genome-wide identity with citrus chlorotic dwarf associated virus (CCDaV) and camelia chlorotic dwarf associated virus (CaCDaV). The 3743 nt PCMoV genome encodes a capsid protein (CP) and replication-associated protein (Rep) that respectively share 56 and 60% amino acid identity with those encoded by CaCDaV. The CPs of PCMoV, CCDaV, and CaCDaV cluster with those of begomovirus whereas their Reps with those of becurtoviruses. Hence, these viruses likely represent a lineage of recombinant begomo-like and becurto-like ancestral viruses. Furthermore, PCMoV, CCDaV, and CaCDaV genomes are ~12-30% larger than monopartite geminiviruses and this is primarily due to the encoded movement protein (MP; 891-921 nt) and this MP is most closely related to that encoded by the DNA-B component of bipartite begomoviruses. Hence, PCMoV, CCDaV, and CaCDaV lineage of viruses may represent molecules in an intermediary step in the evolution of bipartite begomoviruses (~5.3 kb) from monopartite geminiviruses (~2.7-3 kb). An infectious clone of PCMoV systemically infected Nicotiana benthamina, Arabidopsis thaliana, and Passiflora edulis.

Fresh leaf vegetables are a significant part of the Persian food. Following a survey for identification of nanoviruses and geminivirus infecting leaf vegetables, a novel geminivirus was identified in a diseased parsley sample showing upward marginal leaf curling, marginal leaf yellowing, dwarfing and reduced leaf size in south-eastern Iran. The genome was identified through combination of rolling circle amplification (RCA) and high throughput sequencing (HTS) approaches. The full-length genome (2779 nts) of the cloned geminivirus, parsley yellow leaf curl virus (PYLCV), shares <66 % genome-wide pairwise identity with all other known geminiviruses. The PYLCV genome has six open reading frames (ORFs) and appears to be a hybrid with the virion sense encoded proteins being most similar to those of becurtoviruses and curtoviruses, whereas the complementary sense encoded proteins are most similar to those of begomoviruses. In comparison with other geminivirus encoded capsid proteins (CPs) and replication associated proteins (Reps), the CP of PYLCV shares <56 % amino acid pairwise identity whereas the Rep shares <73 % amino acid pairwise identity. To demonstrate the pathogenicity of the geminivirus, a partial dimer infectious clone was constructed and used to agro-infect parsley as well as Nicotiana benthamiana, turnip, radish and tomato. The agro-inoculation resulted in infection with symptoms in 83.7 % (82/98) of the tested plant. Based on the similarity of the CP encoded by PYLCV to those of becurtoviruses and curtoviruses, it is likely that leafhoppers may be the primary transmission vector.

Circoviruses are circular single-stranded DNA (ssDNA) viruses that infect a variety of animals, both domestic and wild. Circovirus infection in birds is associated with immunosuppression and this in turn predisposes the infected animals to secondary infections that can lead to mortality. Farmed geese (Anser anser) in many parts of the world are infected with circoviruses. The majority of the current genomic information for goose circoviruses (GoCVs) (n = 40) are from birds sampled in China and Taiwan, and only two genome sequences are available from Europe (Germany and Poland). In this study, we sampled 23 wild and 19 domestic geese from the Gopło Lake area in Poland. We determined the genomes of GoCV from 21 geese; 14 domestic Greylag geese (Anser anser), three wild Greylag geese (A. anser), three bean geese (A. fabalis), and one white fronted goose (A. albifrons). These genomes share 83-95% nucleotide pairwise identities with previously identified GoCV genomes, most are recombinants with exchanged fragment sizes up to 50% of the genome. Higher diversity levels can be seen within the genomes from domestic geese compared with those from wild geese. In the GoCV capsid protein (cp) and replication associated protein (rep) gene sequences we found that episodic positive selection appears to largely mirror those of beak and feather disease virus and pigeon circovirus. Analysis of the secondary structure of the ssDNA genome revealed a conserved stem-loop structure with the G-C rich stem having a high degree of negative selection on these nucleotides.

Upon infection, DNA viruses can be sensed by pattern recognition receptors (PRRs), leading to the activation of type I and III interferons to block infection. Therefore, viruses must inhibit these signaling pathways, avoid being detected, or both. Papillomavirus virions are trafficked from early endosomes to the Golgi apparatus and wait for the onset of mitosis to complete nuclear entry. This unique subcellular trafficking strategy avoids detection by cytoplasmic PRRs, a property that may contribute to the establishment of infection. However, as the capsid uncoats within acidic endosomal compartments, the viral DNA may be exposed to detection by Toll-like receptor 9 (TLR9). In this study, we characterized two new papillomaviruses from bats and used molecular archeology to demonstrate that their genomes altered their nucleotide compositions to avoid detection by TLR9, providing evidence that TLR9 acts as a PRR during papillomavirus infection. Furthermore, we showed that TLR9, like other components of the innate immune system, is under evolutionary selection in bats, providing the first direct evidence for coevolution between papillomaviruses and their hosts. Finally, we demonstrated that the cancer-associated human papillomaviruses show a reduction in CpG dinucleotides within a TLR9 recognition complex. IMPORTANCE Viruses must avoid detection by the innate immune system. In this study, we characterized two new papillomaviruses from bats and used molecular archeology to demonstrate that their genomes altered their nucleotide compositions to avoid detection by TLR9, providing evidence that TLR9 acts as a PRR during papillomavirus infection. Furthermore, we demonstrated that TLR9, like other components of the innate immune system, is under evolutionary selection in bats, providing the first direct evidence for coevolution between papillomaviruses and their hosts.

There is growing interest in uncovering the viral diversity present in wild animal species. The remote Antarctic region is home to a wealth of uncovered microbial diversity, some of which is associated with its megafauna, including penguin species, the dominant avian biota. Penguins interface with a number of other biota in their roles as marine mesopredators and several species overlap in their ranges and habitats. To characterize the circular single-stranded viruses related to those in the phylum Cressdnaviricota from these environmental sentinel species, cloacal swabs (n = 95) were obtained from King Penguins in South Georgia, and congeneric Adélie Penguins, Chinstrap Penguins, and Gentoo Penguins across the South Shetland Islands and Antarctic Peninsula. Using a combination of high-throughput sequencing, abutting primers-based PCR recovery of circular genomic elements, cloning, and Sanger sequencing, we detected 97 novel sequences comprising 40 ssDNA viral genomes and 57 viral-like circular molecules from 45 individual penguins. We present their detection patterns, with Chinstrap Penguins harboring the highest number of new sequences. The novel Antarctic viruses identified appear to be host-specific, while one circular molecule was shared between sympatric Chinstrap and Gentoo Penguins. We also report viral genotype sharing between three adult-chick pairs, one in each Pygoscelid species. Sequence similarity network approaches coupled with Maximum likelihood phylogenies of the clusters indicate the 40 novel viral genomes do not fall within any known viral families and likely fall within the recently established phylum Cressdnaviricota based on their replication-associated protein sequences. Similarly, 83 capsid protein sequences encoded by the viruses or viral-like circular molecules identified in this study do not cluster with any of those encoded by classified viral groups. Further research is warranted to expand knowledge of the Antarctic virome and would help elucidate the importance of viral-like molecules in vertebrate host evolution.

Viruses encoding a replication-associated protein (Rep) within a covalently closed, single-stranded (ss)DNA genome are among the smallest viruses known to infect eukaryotic organisms, including economically valuable agricultural crops and livestock. Although circular Rep-encoding ssDNA (CRESS DNA) viruses are a widespread group for which our knowledge is rapidly expanding, biased sampling toward vertebrates and land plants has limited our understanding of their diversity and evolution. Here, we screened terrestrial arthropods for CRESS DNA viruses and report the identification of 44 viral genomes and replicons associated with specimens representing all three major terrestrial arthropod lineages, namely Euchelicerata (spiders), Hexapoda (insects), and Myriapoda (millipedes). We identified virus genomes belonging to three established CRESS DNA viral families (Circoviridae, Genomoviridae, and Smacoviridae); however, over half of the arthropod-associated viral genomes are only distantly related to currently classified CRESS DNA viral sequences. Although members of viral and satellite families known to infect plants (Geminiviridae, Nanoviridae, Alphasatellitidae) were not identified in this study, these plant-infecting CRESS DNA viruses and replicons are transmitted by hemipterans. Therefore, members from six out of the seven established CRESS DNA viral families circulate among arthropods. Furthermore, a phylogenetic analysis of Reps, including endogenous viral sequences, reported to date from a wide array of organisms revealed that most of the known CRESS DNA viral diversity circulates among invertebrates. Our results highlight the vast and unexplored diversity of CRESS DNA viruses among invertebrates and parallel findings from RNA viral discovery efforts in undersampled taxa.

Comprehensive inventories of plant viral diversity are essential for effective quarantine and sanitation efforts. The safety of regulated plant material exchanges presently relies heavily on techniques such as PCR or nucleic acid hybridisation, which are only suited to the detection and characterisation of specific, well characterised pathogens. Here, we demonstrate the utility of sequence-independent next generation sequencing (NGS) of both virus-derived small interfering RNAs (siRNAs) and virion-associated nucleic acids (VANA) for the detailed identification and characterisation of viruses infecting two quarantined sugarcane plants. Both plants originated from Egypt and were known to be infected with Sugarcane streak Egypt Virus (SSEV; Genus Mastrevirus, Family Geminiviridae), but were revealed by the NGS approaches to also be infected by a second highly divergent mastrevirus, here named Sugarcane white streak Virus (SWSV). This novel virus had escaped detection by all routine quarantine detection assays and was found to also be present in sugarcane plants originating from Sudan. Complete SWSV genomes were cloned and sequenced from six plants and all were found to share >91% genome-wide identity. With the exception of two SWSV variants, which potentially express unusually large RepA proteins, the SWSV isolates display genome characteristics very typical to those of all other previously described mastreviruses. An analysis of virus-derived siRNAs for SWSV and SSEV showed them to be strongly influenced by secondary structures within both genomic single stranded DNA and mRNA transcripts. In addition, the distribution of siRNA size frequencies indicates that these mastreviruses are likely subject to both transcriptional and post-transcriptional gene silencing. Our study stresses the potential advantages of NGS-based virus metagenomic screening in a plant quarantine setting and indicates that such techniques could dramatically reduce the numbers of non-intercepted virus pathogens passing through plant quarantine stations.