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.

Wastewater surveillance uncovers regional diversity and dynamics of SARS-CoV-2 variants across nine states in the USA.

Wastewater-based epidemiology (WBE) is a non-invasive and cost-effective approach for monitoring the spread of a pathogen within a community. WBE has been adopted as one of the methods to monitor the spread and population dynamics of the SARS-CoV-2 virus, but significant challenges remain in the bioinformatic analysis of WBE-derived data. Here, we have developed a new distance metric, CoVdist, and an associated analysis tool that facilitates the application of ordination analysis to WBE data and the identification of viral population changes based on nucleotide variants. We applied these new approaches to a large-scale dataset from 18 cities in nine states of the USA using wastewater collected from July 2021 to June 2022. We found that the trends in the shift between the Delta and Omicron SARS-CoV-2 lineages were largely consistent with what was seen in clinical data, but that wastewater analysis offered the added benefit of revealing significant differences in viral population dynamics at the state, city, and even neighborhood scales. We also were able to observe the early spread of variants of concern and the presence of recombinant lineages during the transitions between variants, both of which are challenging to analyze based on clinically-derived viral genomes. The methods outlined here will be beneficial for future applications of WBE to monitor SARS-CoV-2, particularly as clinical monitoring becomes less prevalent. Additionally, these approaches are generalizable, allowing them to be applied for the monitoring and analysis of future viral outbreaks.

Leveraging an established neighbourhood-level, open access wastewater monitoring network to address public health priorities: a population-based study.

BACKGROUND: Before the COVID-19 pandemic, the US opioid epidemic triggered a collaborative municipal and academic effort in Tempe, Arizona, which resulted in the world's first open access dashboard featuring neighbourhood-level trends informed by wastewater-based epidemiology (WBE). This study aimed to showcase how wastewater monitoring, once established and accepted by a community, could readily be adapted to respond to newly emerging public health priorities. METHODS: In this population-based study in Greater Tempe, Arizona, an existing opioid monitoring WBE network was modified to track SARS-CoV-2 transmission through the analysis of 11 contiguous wastewater catchments. Flow-weighted and time-weighted 24 h composite samples of untreated wastewater were collected at each sampling location within the wastewater collection system for 3 days each week (Tuesday, Thursday, and Saturday) from April 1, 2020, to March 31, 2021 (Area 7 and Tempe St Luke's Hospital were added in July, 2020). Reverse transcription quantitative PCR targeting the E gene of SARS-CoV-2 isolated from the wastewater samples was used to determine the number of genome copies in each catchment. Newly detected clinical cases of COVID-19 by zip code within the City of Tempe, Arizona were reported daily by the Arizona Department of Health Services from May 23, 2020. Maricopa County-level new positive cases, COVID-19-related hospitalisations, deaths, and long-term care facility deaths per day are publicly available and were collected from the Maricopa County Epidemic Curve Dashboard. Viral loads of SARS-CoV-2 (genome copies per day) measured in wastewater from each catchment were aggregated at the zip code level and city level and compared with the clinically reported data using root mean square error to investigate early warning capability of WBE. FINDINGS: Between April 1, 2020, and March 31, 2021, 1556 wastewater samples were analysed. Most locations showed two waves in viral levels peaking in June, 2020, and December, 2020-January, 2021. An additional wave of viral load was seen in catchments close to Arizona State University (Areas 6 and 7) at the beginning of the fall (autumn) semester in late August, 2020. Additionally, an early infection hotspot was detected in the Town of Guadalupe, Arizona, starting the week of May 4, 2020, that was successfully mitigated through targeted interventions. A shift in early warning potential of WBE was seen, from a leading (mean of 8·5 days [SD 2·1], June, 2020) to a lagging (-2·0 days [1·4], January, 2021) indicator compared with newly reported clinical cases. INTERPRETATION: Lessons learned from leveraging an existing neighbourhood-level WBE reporting dashboard include: (1) community buy-in is key, (2) public data sharing is effective, and (3) sub-ZIP-code (postal code) data can help to pinpoint populations at risk, track intervention success in real time, and reveal the effect of local clinical testing capacity on WBE's early warning capability. This successful demonstration of transitioning WBE efforts from opioids to COVID-19 encourages an expansion of WBE to tackle newly emerging and re-emerging threats (eg, mpox and polio). FUNDING: National Institutes of Health's RADx-rad initiative, National Science Foundation, Virginia G Piper Charitable Trust, J M Kaplan Fund, and The Flinn Foundation.

Complete Coding Sequences of Rhinovirus Types A46, A39, C56, and C48.

We report the coding-complete sequences of rhinovirus types C48, A46, A39, and C56, determined from nasopharyngeal swabs from three individuals with influenza-like symptoms in the United States. One sample showed a coinfection of rhinovirus types A46 and C48.

Molecular identification and phylogenetic characterization of A-strain isolates of maize streak virus from western Ethiopia.

The A-strain of maize streak virus (MSV) causes maize streak disease (MSD), which is a major biotic threat to maize production in sub-Saharan Africa. Previous studies have described different MSV strains of economic importance from southern and eastern African countries and how eastern African regions are hubs for MSV diversification. Despite these efforts, due to a lack of extensive sampling, there is limited knowledge about the MSV-A diversity in Ethiopia. Here, field sampling of maize plants and wild grasses with visible MSD symptoms was carried out in the western Ethiopian regions of Gambela, Oromia, and Benishangul-Gumuz during the maize-growing season of 2019. The complete genomes of MSV isolates (n = 60) were cloned and sequenced by the Sanger method. We used a model-based phylogenetic approach to analyse 725 full MSV genome sequences available in the GenBank database together with newly determined genome sequences from Ethiopia to determine their subtypes and identify recombinant lineages. Of the 127 fields accessed, MSD prevalence was highest, at 96%, in the Gambela region and lowest in Oromia, at 66%. The highest mean symptom severity of 4/5 (where 5 is the highest and 1 the lowest) was observed in Gambela and Benishangul-Gumuz. Our results show that these newly determined MSV isolates belong to recombinant lineage V of the A1 subtype, with the widest dissemination and greatest economic significance in sub-Saharan Africa and the adjacent Indian Ocean islands.

Genome Sequences of Anelloviruses, Genomovirus, and Papillomavirus Isolated from Nasal Pharyngeal Swabs.

The genome sequences of three anelloviruses (genus Alphatorquevirus), a genomovirus (genus Gemykolovirus), and an unclassified papillomavirus were identified in four human nasopharyngeal swabs, and one was positive for influenza A and one for influenza B virus. The influenza B virus-positive sample had a coinfection with an anellovirus and a papillomavirus.

A parasite outbreak in notothenioid fish in an Antarctic fjord.

Climate changes can promote disease outbreaks, but their nature and potential impacts in remote areas have received little attention. In a hot spot of biodiversity on the West Antarctic Peninsula, which faces among the fastest changing climates on Earth, we captured specimens of two notothenioid fish species affected by large skin tumors at an incidence never before observed in the Southern Ocean. Molecular and histopathological analyses revealed that X-cell parasitic alveolates, members of a genus we call Notoxcellia, are the etiological agent of these tumors. Parasite-specific molecular probes showed that xenomas remained within the skin but largely outgrew host cells in the dermis. We further observed that tumors induced neovascularization in underlying tissue and detrimentally affected host growth and condition. Although many knowledge gaps persist about X-cell disease, including its mode of transmission and life cycle, these findings reveal potentially active biotic threats to vulnerable Antarctic ecosystems.

Virion-Associated Nucleic Acid-Based Metagenomics: A Decade of Advances in Molecular Characterization of Plant Viruses.

Over the last decade, viral metagenomic studies have resulted in the discovery of thousands of previously unknown viruses. These studies are likely to play a pivotal role in obtaining an accurate and robust understanding of how viruses affect the stability and productivity of ecosystems. Among the metagenomics-based approaches that have been developed since the beginning of the 21st century, shotgun metagenomics applied specifically to virion-associated nucleic acids (VANA) has been used to disentangle the diversity of the viral world. We summarize herein the results of 24 VANA-based studies, focusing on plant and insect samples conducted over the last decade (2010 to 2020). Collectively, viruses from 85 different families were reliably detected in these studies, including capsidless RNA viruses that replicate in fungi, oomycetes, and plants. Finally, strengths and weaknesses of the VANA approach are summarized and perspectives of applications in detection, epidemiological surveillance, environmental monitoring, and ecology of plant viruses are provided. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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.

Novel circular DNA virus identified in Opuntia discolor (Cactaceae) that codes for proteins with similarity to those of geminiviruses.

Viral metagenomic studies have enabled the discovery of many unknown viruses and revealed that viral communities are much more diverse and ubiquitous than previously thought. Some viruses have multiple genome components that are encapsidated either in separate virions (multipartite viruses) or in the same virion (segmented viruses). In this study, we identify what is possibly a novel bipartite plant-associated circular single-stranded DNA virus in a wild prickly pear cactus, Opuntia discolor, that is endemic to the Chaco ecoregion in South America. Two ~1.8 kb virus-like circular DNA components were recovered, one encoding a replication-associated protein (Rep) and the other a capsid protein (CP). Both of the inferred protein sequences of the Rep and CP are homologous to those encoded by members of the family Geminiviridae. These two putatively cognate components each have a nonanucleotide sequence within a likely hairpin structure that is homologous to the origins of rolling-circle replication (RCR), found in diverse circular single-stranded DNA viruses. In addition, the two components share similar putative replication-associated iterative sequences (iterons), which in circular single-stranded DNA viruses are important for Rep binding during the initiation of RCR. Such molecular features provide support for the possible bipartite nature of this virus, which we named utkilio virus (common name of the Opuntia discolor in South America) components A and B. In the infectivity assays conducted in Nicotiana benthamiana plants, only the A component of utkilio virus, which encodes the Rep protein, was found to move and replicate systemically in N. benthamiana. This was not true for component B, for which we did not detect replication, which may have been due to this being a defective molecule or because of the model plants (N. benthamiana) used for the infection assays. Future experiments need to be conducted with other plants, including O. discolor, to understand more about the biology of these viral components.

High-throughput sequencing of SARS-CoV-2 in wastewater provides insights into circulating variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) likely emerged from a zoonotic spill-over event and has led to a global pandemic. The public health response has been predominantly informed by surveillance of symptomatic individuals and contact tracing, with quarantine, and other preventive measures have then been applied to mitigate further spread. Non-traditional methods of surveillance such as genomic epidemiology and wastewater-based epidemiology (WBE) have also been leveraged during this pandemic. Genomic epidemiology uses high-throughput sequencing of SARS-CoV-2 genomes to inform local and international transmission events, as well as the diversity of circulating variants. WBE uses wastewater to analyse community spread, as it is known that SARS-CoV-2 is shed through bodily excretions. Since both symptomatic and asymptomatic individuals contribute to wastewater inputs, we hypothesized that the resultant pooled sample of population-wide excreta can provide a more comprehensive picture of SARS-CoV-2 genomic diversity circulating in a community than clinical testing and sequencing alone. In this study, we analysed 91 wastewater samples from 11 states in the USA, where the majority of samples represent Maricopa County, Arizona (USA). With the objective of assessing the viral diversity at a population scale, we undertook a single-nucleotide variant (SNV) analysis on data from 52 samples with >90% SARS-CoV-2 genome coverage of sequence reads, and compared these SNVs with those detected in genomes sequenced from clinical patients. We identified 7973 SNVs, of which 548 were "novel" SNVs that had not yet been identified in the global clinical-derived data as of 17th June 2020 (the day after our last wastewater sampling date). However, between 17th of June 2020 and 20th November 2020, almost half of the novel SNVs have since been detected in clinical-derived data. Using the combination of SNVs present in each sample, we identified the more probable lineages present in that sample and compared them to lineages observed in North America prior to our sampling dates. The wastewater-derived SARS-CoV-2 sequence data indicates there were more lineages circulating across the sampled communities than represented in the clinical-derived data. Principal coordinate analyses identified patterns in population structure based on genetic variation within the sequenced samples, with clear trends associated with increased diversity likely due to a higher number of infected individuals relative to the sampling dates. We demonstrate that genetic correlation analysis combined with SNVs analysis using wastewater sampling can provide a comprehensive snapshot of the SARS-CoV-2 genetic population structure circulating within a community, which might not be observed if relying solely on clinical cases.

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.

Genome characterization of parsley severe stunt-associated virus in Iran.

Parsley severe stunt-associated virus (PSSaV) is a recently identified nanovirus first reported in Germany. During a survey for identification of nanoviruses infecting apiaceous plants in south-eastern Iran, PSSaV was identified and characterized using a combination of rolling circle amplification (RCA) and high-throughput sequencing. Parsley plant samples were collected from vegetable production farms in Kerman province. From two symptomatic samples (39Ba and 40Ba), seven PSSaV components (DNA-C, -S, -M, -R, -N, -U1 and -U2) with two phylogenetically distinct variants of DNA-R (R1 and R2) were identified. In common with the German isolate of PSSaV, no DNA-U4 component was identified. In addition, associated alphasatellite molecules were identified in samples 39Ba [n = 6] and 40Ba [n = 5]. Sequence analyses showed that concatenated component sequences of the two Iranian PSSaVs share 97.2% nucleotide identity with each other and 82% to the German isolate. The coat proteins (CPs) of the PSSaV Iranian sequences share 97.2% amino acid identity and ~ 84% identity with that of the German isolate. Sequence and phylogenetic analyses of a total of 11 recovered alphasatellites from the two samples can be classified into the genera Fabenesatellite [n = 2], Milvetsatellite [n = 1], Mivedwarsatellite [n = 2], Subclovsatellite [n = 2], Sophoyesatellite [n = 4] in the family Alphasatellitidae. Identification of PSSaV and other nanoviruses in wild and cultivated plants in Iran reveals that nanoviruses could be causing yield reduction in crops plants in this country.

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.