Environment by environment interactions (ExE) differ across genetic backgrounds (ExExG).

While the terms "gene-by-gene interaction" (GxG) and "gene-by-environment interaction" (GxE) are commonplace within the field of quantitative and evolutionary genetics, "environment-by-environment interaction" (ExE) is a term used less often. However, in this study, we find that environment-by-environment interactions are common and differ for different genotypes (ExExG). To reach this conclusion, we analyzed a large dataset of roughly 1,000 mutant yeast strains with varying degrees of resistance to different antifungal drugs. Many researchers endeavor to predict combinations of drugs that are more lethal than either single drug. But we show that the effectiveness of a drug combination, relative to the effectiveness of single drugs, often varies across different drug resistant mutants. Even mutants that differ by only a single nucleotide change can have dramatically different drug x drug (ExE) interactions. Studying how ExE interactions change across genotypes (ExExG) is not only important when modeling the evolution of pathogenic microbes. High throughput screens of GxG and GxE have taught us about the basic cell biology and gene regulatory networks underlying genetic interactions. ExExG has been omitted but stands to impart similar lessons about the architecture of living systems. In this study, we call attention to ExExG, measure its prevalence, introduce a new framework that in some instances better predicts its direction and magnitude, and make the case for further study of this type of genetic interaction.

An ultra high-throughput, massively multiplexable, single-cell RNA-seq platform in yeasts.

Yeasts are naturally diverse, genetically tractable, and easy to grow such that researchers can investigate any number of genotypes, environments, or interactions thereof. However, studies of yeast transcriptomes have been limited by the processing capabilities of traditional RNA sequencing techniques. Here we optimize a powerful, high-throughput single-cell RNA sequencing (scRNAseq) platform, SPLiT-seq (Split Pool Ligation-based Transcriptome sequencing), for yeasts and apply it to 43,388 cells of multiple species and ploidies. This platform utilizes a combinatorial barcoding strategy to enable massively parallel RNA sequencing of hundreds of yeast genotypes or growth conditions at once. This method can be applied to most species or strains of yeast for a fraction of the cost of traditional scRNAseq approaches. Thus, our technology permits researchers to leverage "the awesome power of yeast" by allowing us to survey the transcriptome of hundreds of strains and environments in a short period of time and with no specialized equipment. The key to this method is that sequential barcodes are probabilistically appended to cDNA copies of RNA while the molecules remain trapped inside of each cell. Thus, the transcriptome of each cell is labeled with a unique combination of barcodes. Since SPLiT-seq uses the cell membrane as a container for this reaction, many cells can be processed together without the need to physically isolate them from one another in separate wells or droplets. Further, the first barcode in the sequence can be chosen intentionally to identify samples from different environments or genetic backgrounds, enabling multiplexing of hundreds of unique perturbations in a single experiment. In addition to greater multiplexing capabilities, our method also facilitates a deeper investigation of biological heterogeneity, given its single-cell nature. For example, in the data presented here, we detect transcriptionally distinct cell states related to cell cycle, ploidy, metabolic strategies, and so forth, all within clonal yeast populations grown in the same environment. Hence, our technology has two obvious and impactful applications for yeast research: the first is the general study of transcriptional phenotypes across many strains and environments, and the second is investigating cell-to-cell heterogeneity across the entire transcriptome.

Using source-associated mobile genetic elements to identify zoonotic extraintestinal E. coli infections.

A one-health perspective may provide new and actionable information about Escherichia coli transmission. E. coli colonizes a broad range of vertebrates, including humans and food-production animals, and is a leading cause of bladder, kidney, and bloodstream infections in humans. Substantial evidence supports foodborne transmission of pathogenic E. coli strains from food animals to humans. However, the relative contribution of foodborne zoonotic E. coli (FZEC) to the human extraintestinal disease burden and the distinguishing characteristics of such strains remain undefined. Using a comparative genomic analysis of a large collection of contemporaneous, geographically-matched clinical and meat-source E. coli isolates (n = 3111), we identified 17 source-associated mobile genetic elements - predominantly plasmids and bacteriophages - and integrated them into a novel Bayesian latent class model to predict the origins of clinical E. coli isolates. We estimated that approximately 8 % of human extraintestinal E. coli infections (mostly urinary tract infections) in our study population were caused by FZEC. FZEC strains were equally likely to cause symptomatic disease as non-FZEC strains. Two FZEC lineages, ST131-H22 and ST58, appeared to have particularly high virulence potential. Our findings imply that FZEC strains collectively cause more urinary tract infections than does any single non-E. coli uropathogenic species (e.g., Klebsiella pneumoniae). Our novel approach can be applied in other settings to identify the highest-risk FZEC strains, determine their sources, and inform new one-health strategies to decrease the heavy public health burden imposed by extraintestinal E. coli infections.

Evolution of anelloviruses from a circovirus-like ancestor through gradual augmentation of the jelly-roll capsid protein.

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 ß-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.

Extreme Sensitivity of Fitness to Environmental Conditions: Lessons from #1BigBatch.

The phrase "survival of the fittest" has become an iconic descriptor of how natural selection works. And yet, precisely measuring fitness, even for single-celled microbial populations growing in controlled laboratory conditions, remains a challenge. While numerous methods exist to perform these measurements, including recently developed methods utilizing DNA barcodes, all methods are limited in their precision to differentiate strains with small fitness differences. In this study, we rule out some major sources of imprecision, but still find that fitness measurements vary substantially from replicate to replicate. Our data suggest that very subtle and difficult to avoid environmental differences between replicates create systematic variation across fitness measurements. We conclude by discussing how fitness measurements should be interpreted given their extreme environment dependence. This work was inspired by the scientific community who followed us and gave us tips as we live tweeted a high-replicate fitness measurement experiment at #1BigBatch.

Novel polyomaviruses identified in fecal samples from four carnivore species.

Polyomaviruses are oncogenic viruses that are generally thought to have co-evolved with their hosts. While primate and rodent polyomaviruses are increasingly well-studied, less is known about polyomaviruses that infect other mammals. In an effort to gain insight into polyomaviruses associated with carnivores, we surveyed fecal samples collected in the USA from bobcats (Lynx rufus), pumas (Puma concolor), Canada lynxes (Lynx canadensis), and grizzly bears (Ursus arctos). Using a viral metagenomic approach, we identified six novel polyomavirus genomes. Surprisingly, four of the six genomes showed a phylogenetic relationship to polyomaviruses found in prey animals. These included a putative rabbit polyomavirus from a bobcat fecal sample and two possible deer-trophic polyomaviruses from Canada lynx feces. One polyomavirus found in a grizzly bear sample was found to be phylogenetically distant from previously identified polyomaviruses. Further analysis of the grizzly bear fecal sample showed that it contained anelloviruses that are known to infect pigs, suggesting that the bear might have preyed on a wild or domestic pig. Interestingly, a polyomavirus genome identified in a puma fecal sample was found to be closely related both to raccoon polyomavirus 1 and to Lyon-IARC polyomavirus, the latter of which was originally identified in human saliva and skin swab specimens but has since been found in samples from domestic cats (Felis catus).

Challenges and potential solutions for studying the genetic and phenotypic architecture of adaptation in microbes.

All organisms are defined by the makeup of their DNA. Over billions of years, the structure and information contained in that DNA, often referred to as genetic architecture, have been honed by a multitude of evolutionary processes. Mutations that cause genetic elements to change in a way that results in beneficial phenotypic change are more likely to survive and propagate through the population in a process known as adaptation. Recent work reveals that the genetic targets of adaptation are varied and can change with genetic background. Further, seemingly similar adaptive mutations, even within the same gene, can have diverse and unpredictable effects on phenotype. These challenges represent major obstacles in predicting adaptation and evolution. In this review, we cover these concepts in detail and identify three emerging synergistic solutions: higher-throughput evolution experiments combined with updated genotype-phenotype mapping strategies and physiological models. Our review largely focuses on recent literature in yeast, and the field seems to be on the cusp of a new era with regard to studying the predictability of evolution.

Novel adenovirus associated with common tern (Sterna hirundo) chicks.

Adenoviruses have been identified in a wide variety of avian species, and in some species, they have been shown to cause disease and increase mortality. As part of an endeavor to investigate viruses associated with common terns (Sterna hirundo), a novel adenovirus was identified in fecal samples from two common terns on Gull Island, Lake Ontario, Canada. The coding-complete genome sequence of the new adenovirus is 31,094 bp, containing 28 putative genes, and this is the first adenovirus to be associated with terns. The virus was identified in two out of 13 fecal samples from tern chicks, and it was found to be most closely related to duck adenovirus 1, with the DNA polymerase sharing 58% amino acid sequence identity. Phylogenetic analysis based on DNA polymerase protein sequences showed that the new virus forms a distinct sub-branch within the atadenovirus clade and likely represents a new species in this genus.

Discovery of novel fish papillomaviruses: From the Antarctic to the commercial fish market.

Fish papillomaviruses form a newly discovered group broadly recognized as the Secondpapillomavirinae subfamily. This study expands the documented genomes of the fish papillomaviruses from six to 16, including one from the Antarctic emerald notothen, seven from commercial market fishes, one from data mining of sea bream sequence data, and one from a western gull cloacal swab that is likely diet derived. The genomes of secondpapillomaviruses are ∼6 kilobasepairs (kb), which is substantially smaller than the ∼8 kb of terrestrial vertebrate papillomaviruses. Each genome encodes a clear homolog of the four canonical papillomavirus genes, E1, E2, L1, and L2. In addition, we identified open reading frames (ORFs) with short linear peptide motifs reminiscent of E6/E7 oncoproteins. Fish papillomaviruses are extremely diverse and phylogenetically distant from other papillomaviruses suggesting a model in which terrestrial vertebrate-infecting papillomaviruses arose after an evolutionary bottleneck event, possibly during the water-to-land transition.

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.

Diverse Single-Stranded DNA Viruses Identified in Chicken Buccal Swabs.

High-throughput sequencing approaches offer the possibility to better understand the complex microbial communities associated with animals. Viral metagenomics has facilitated the discovery and identification of many known and unknown viruses that inhabit mucosal surfaces of the body and has extended our knowledge related to virus diversity. We used metagenomics sequencing of chicken buccal swab samples and identified various small DNA viruses with circular genome organization. Out of 134 putative circular viral-like circular genome sequences, 70 are cressdnaviruses and 26 are microviruses, whilst the remaining 38 most probably represent sub-genomic molecules. The cressdnaviruses found in this study belong to the Circoviridae, Genomoviridae and Smacoviridae families as well as previously described CRESS1 and naryavirus groups. Among these, genomoviruses and smacoviruses were the most prevalent across the samples. Interestingly, we also identified 26 bacteriophages that belong to the Microviridae family, whose members are known to infect enterobacteria.

A novel lineage of polyomaviruses identified in bark scorpions.

Polyomaviruses are non-enveloped viruses with circular double-stranded DNA genomes (~4-7 kb). Initially identified in mammals, polyomaviruses have now been identified in birds and a few fish species. Although fragmentary polyomavirus-like sequences have been detected as apparent 'hitchhikers' in shotgun genomics datasets of various arthropods, the possible diversity of these viruses in invertebrates remains unclear. Scorpions are predatory arachnids that are among the oldest terrestrial animals. Using high-throughput sequencing and traditional molecular techniques we determine the genome sequences of eight novel polyomaviruses in scorpions (Centruroides sculpturatus) from the greater Phoenix area, Arizona, USA. Analysis of Centruroides transcriptomic datasets elucidated the splicing of the viral late gene array, which is more complex than that of vertebrate polyomaviruses. Phylogenetic analysis provides further evidence of co-divergence of polyomaviruses with their hosts, suggesting that at least one ancestral species of polyomaviruses was circulating amongst the primitive common ancestors of arthropods and chordates.

Agricultural practices drive biological loads, seasonal patterns and potential pathogens in the aerobiome of a mixed-land-use dryland.

Air carries a diverse load of particulate microscopic biological matter in suspension, either aerosolized or aggregated with dust particles, the aerobiome, which is dispersed by winds from sources to sinks. The aerobiome is known to contain microbes, including pathogens, as well as debris or small-sized propagules from plants and animals, but its variability and composition has not been studied comprehensibly. To gain a dynamic insight into the aerobiome existing over a mixed-use dryland setting, we conducted a biologically comprehensive, year-long survey of its composition and dynamics for particles less than 10 µm in diameter based on quantitative analyses of DNA content coupled to genomic sequencing. Airborne biological loads were more dependent on seasonal events than on meteorological conditions and only weakly correlated with dust loads. Core aerobiome species could be understood as a mixture of high elevation (e.g. Microbacteriaceae, Micrococcaceae, Deinococci), and local plant and soil sources (e.g. Sphingomonas, Streptomyces, Acinetobacter). Despite the mixed used of the land surrounding the sampling site, taxa that contributed to high load events were largely traceable to proximal agricultural practices like cotton and livestock farming. This included not only the predominance of specific crop plant signals over those of native vegetation, but also that of their pathogens (bacterial, viral and eukaryotic). Faecal bacterial loads were also seasonally important, possibly sourced in intensive animal husbandry or manure fertilization activity, and this microbial load was enriched in tetracycline resistance genes. The presence of the native opportunistic pathogen, Coccidioides spp., by contrast, was detected only with highly sensitive techniques, and only rarely. We conclude that agricultural activity exerts a much stronger influence that the native vegetation as a mass loss factor to the land system and as an input to dryland aerobiomes, including in the dispersal of plant, animal and human pathogens and their genetic resistance characteristics.

Identification of the Begomoviruses Squash Leaf Curl Virus and Watermelon Chlorotic Stunt Virus in Various Plant Samples in North America.

Geminiviruses are a group of plant-infecting viruses with single-stranded DNA genomes. Within this family, viruses in the genus Begomovirus are known to have a worldwide distribution causing a range of severe diseases in a multitude of dicotyledonous plant species. Begomoviruses are transmitted by the whitefly Bemisia tabaci, and their ssDNA genomes can be either monopartite or bipartite. As part of a viral survey, various plants including those in the families Alliaceae, Amaranthaceae, Apiaceae, Asteraceae, Brassicaceae, Cactaceae, Cucurbitaceae, Lamiaceae, Lauraceae, Malvaceae, Oleaceae and Solanaceae were sampled and screened for begomoviruses using both a high-throughput sequencing and a begomovirus-specific primer pair approach. Based on the sequences derived using these approaches, the full-length genome of various begomoviruses were amplified from plants using abutting primers. Squash leaf curl virus (SLCV) and watermelon chlorotic stunt virus (WCSV) were identified in Cactaceae (n = 25), Solanaceae (n = 7), Cucurbitaceae (n = 2) and Lamiaceae (n = 1) samples. WCSV is an Old World bipartite begomovirus that has only recently been discovered infecting watermelons in the Americas. Our discovery of WCSV in the USA is the first indication that it has reached this country and indicates that this virus might be widespread throughout North America. Phylogenetic analysis suggests WCSV was introduced to the New World twice. The detection of begomoviruses in cactus plants suggests possible spillover events from agricultural areas into native vegetation. Since WCSV and SLCV have previously been found in mixed infections, pseudo-recombination infection experiments were conducted. We demonstrate that WCSV DNA-B is successfully trans-replicated by SLCV DNA-A despite very low degree of similarity between the replication-associated iterative sequences present in their common region, an essential feature for binding of the replication associated protein. This study highlights the importance of viral surveys for the detection of spillover events into native vegetation, but also suggests the need for more surveillance of WCSV in the USA, as this virus is a serious threat to watermelon cultivation in the Middle East.

Circular DNA viruses identified in short-finned pilot whale and orca tissue samples.

Members of the Delphinidae family are widely distributed across the world's oceans. We used a viral metagenomic approach to identify viruses in orca (Orcinus orca) and short-finned pilot whale (Globicephala macrorhynchus) muscle, kidney, and liver samples from deceased animals. From orca tissue samples (muscle, kidney, and liver), we identified a novel polyomavirus (Polyomaviridae), three cressdnaviruses, and two genomoviruses (Genomoviridae). In the short-finned pilot whale we were able to identify one genomovirus in a kidney sample. The presence of unclassified cressdnavirus within two samples (muscle and kidney) of the same animal supports the possibility these viruses might be widespread within the animal. The orca polyomavirus identified here is the first of its species and is not closely related to the only other dolphin polyomavirus previously discovered. The identification and verification of these viruses expands the current knowledge of viruses that are associated with the Delphinidae family.

New World Cactaceae Plants Harbor Diverse Geminiviruses.

The family Cactaceae comprises a diverse group of typically succulent plants that are native to the American continent but have been introduced to nearly all other continents, predominantly for ornamental purposes. Despite their economic, cultural, and ecological importance, very little research has been conducted on the viral community that infects them. We previously identified a highly divergent geminivirus that is the first known to infect cacti. Recent research efforts in non-cultivated and asymptomatic plants have shown that the diversity of this viral family has been under-sampled. As a consequence, little is known about the effects and interactions of geminiviruses in many plants, such as cacti. With the objective to expand knowledge on the diversity of geminiviruses infecting cacti, we used previously acquired high-throughput sequencing results to search for viral sequences using BLASTx against a viral RefSeq protein database. We identified two additional sequences with similarity to geminiviruses, for which we designed abutting primers and recovered full-length genomes. From 42 cacti and five scale insects, we derived 42 complete genome sequences of a novel geminivirus species that we have tentatively named Opuntia virus 2 (OpV2) and 32 genomes of an Opuntia-infecting becurtovirus (which is a new strain of the spinach curly top Arizona virus species). Interspecies recombination analysis of the OpV2 group revealed several recombinant regions, in some cases spanning half of the genome. Phylogenetic analysis demonstrated that OpV2 is a novel geminivirus more closely related to viruses of the genus Curtovirus, which was further supported by the detection of three recombination events between curtoviruses and OpV2. Both OpV2 and Opuntia becurtoviruses were identified in mixed infections, which also included the previously characterized Opuntia virus 1. Viral quantification of the co-infected cactus plants compared with single infections did not show any clear trend in viral dynamics that might be associated with the mixed infections. Using experimental Rhizobium-mediated inoculations, we found that the initial accumulation of OpV2 is facilitated by co-infection with OpV1. This study shows that the diversity of geminiviruses that infect cacti is under-sampled and that cacti harbor diverse geminiviruses. The detection of the Opuntia becurtoviruses suggests spill-over events between viruses of cultivated species and native vegetation. The threat this poses to cacti needs to be further investigated.

Genome Sequences of Microviruses Identified in Gila Monster Feces.

The complete genome sequences of 33 microviruses were determined from fecal samples collected from 14 Arizona-dwelling Gila monsters using high-throughput sequencing. These microviruses with genomes 4,383 to 6,782 nucleotides (nt) long were broadly distributed across the 14 samples.

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.

Identification and Distribution of Novel Cressdnaviruses and Circular molecules in Four Penguin Species in South Georgia and the Antarctic Peninsula.

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.

Novel Circoviruses Detected in Feces of Sonoran Felids.

Sonoran felids are threatened by drought and habitat fragmentation. Vector range expansion and anthropogenic factors such as habitat encroachment and climate change are altering viral evolutionary dynamics and exposure. However, little is known about the diversity of viruses present in these populations. Small felid populations with lower genetic diversity are likely to be most threatened with extinction by emerging diseases, as with other selective pressures, due to having less adaptive potential. We used a metagenomic approach to identify novel circoviruses, which may have a negative impact on the population viability, from confirmed bobcat (Lynx rufus) and puma (Puma concolor) scats collected in Sonora, Mexico. Given some circoviruses are known to cause disease in their hosts, such as porcine and avian circoviruses, we took a non-invasive approach using scat to identify circoviruses in free-roaming bobcats and puma. Three circovirus genomes were determined, and, based on the current species demarcation, they represent two novel species. Phylogenetic analyses reveal that one circovirus species is more closely related to rodent associated circoviruses and the other to bat associated circoviruses, sharing highest genome-wide pairwise identity of approximately 70% and 63%, respectively. At this time, it is unknown whether these scat-derived circoviruses infect felids, their prey, or another organism that might have had contact with the scat in the environment. Further studies should be conducted to elucidate the host of these viruses and assess health impacts in felids.