Viruses of free-roaming and hunting dogs in Uganda show elevated prevalence, richness and abundance across a gradient of contact with wildlife.

Domestic dogs (Canis lupus familiaris) live with humans, frequently contact other animals and may serve as intermediary hosts for the transmission of viruses. Free-roaming dogs, which account for over 70% of the world's domestic dog population, may pose a particularly high risk in this regard. We conducted an epidemiological study of dog viromes in three locations in Uganda, representing low, medium and high rates of contact with wildlife, ranging from dogs owned specifically for traditional hunting in a biodiversity and disease 'hotspot' to pets in an affluent suburb. We quantified rates of contact between dogs and wildlife through owner interviews and conducted canine veterinary health assessments. We then applied broad-spectrum viral metagenomics to blood plasma samples, from which we identified 46 viruses, 44 of which were previously undescribed, in three viral families, Sedoreoviridae, Parvoviridae and Anelloviridae. All 46 viruses (100 %) occurred in the high-contact population of dogs compared to 63 % and 39 % in the medium- and low-contact populations, respectively. Viral prevalence ranged from 2.1 % to 92.0 % among viruses and was highest, on average, in the high-contact population (22.3 %), followed by the medium-contact (12.3 %) and low-contact (4.8 %) populations. Viral richness (number of viruses per dog) ranged from 0 to 27 and was markedly higher, on average, in the high-contact population (10.2) than in the medium-contact (5.7) or low-contact (2.3) populations. Viral richness was strongly positively correlated with the number of times per year that a dog was fed wildlife and negatively correlated with the body condition score, body temperature and packed cell volume. Viral abundance (cumulative normalized metagenomic read density) varied 124-fold among dogs and was, on average, 4.1-fold higher and 2.4-fold higher in the high-contact population of dogs than in the low-contact or medium-contact populations, respectively. Viral abundance was also strongly positively correlated with the number of times per year that a dog was fed wildlife, negatively correlated with packed cell volume and positively correlated with white blood cell count. These trends were driven by nine viruses in the family Anelloviridae, genus Thetatorquevirus, and by one novel virus in the family Sedoreoviridae, genus Orbivirus. The genus Orbivirus contains zoonotic viruses and viruses that dogs can acquire through ingestion of infected meat. Overall, our findings show that viral prevalence, richness and abundance increased across a gradient of contact between dogs and wildlife and that the health status of the dog modified viral infection. Other ecological, geographic and social factors may also have contributed to these trends. Our finding of a novel orbivirus in dogs with high wildlife contact supports the idea that free-roaming dogs may serve as intermediary hosts for viruses of medical importance to humans and other animals.

The Emerging Tool of the Human Anellovirome.

The blood virome is dominated by the Anelloviridae family, which emerges early in life; the anellome, which represents the variety of anelloviruses within an individual, stabilizes by adulthood [...].

Long-Read Nanopore-Based Sequencing of Anelloviruses.

Routinely used metagenomic next-generation sequencing (mNGS) techniques often fail to detect low-level viremia (<104 copies/mL) and appear biased towards viruses with linear genomes. These limitations hinder the capacity to comprehensively characterize viral infections, such as those attributed to the Anelloviridae family. These near ubiquitous non-pathogenic components of the human virome have circular single-stranded DNA genomes that vary in size from 2.0 to 3.9 kb and exhibit high genetic diversity. Hence, species identification using short reads can be challenging. Here, we introduce a rolling circle amplification (RCA)-based metagenomic sequencing protocol tailored for circular single-stranded DNA genomes, utilizing the long-read Oxford Nanopore platform. The approach was assessed by sequencing anelloviruses in plasma drawn from people who inject drugs (PWID) in two geographically distinct cohorts. We detail the methodological adjustments implemented to overcome difficulties inherent in sequencing circular genomes and describe a computational pipeline focused on anellovirus detection. We assessed our protocol across various sample dilutions and successfully differentiated anellovirus sequences in conditions simulating mixed infections. This method provides a robust framework for the comprehensive characterization of circular viruses within the human virome using the Oxford Nanopore.

Anelloviruses versus human immunity: how do we control these viruses?

One continuous companion and one of the major players in the human blood virome are members of the Anelloviridae family. Anelloviruses are probably found in all humans, infection occurs early in life and the composition (anellome) is thought to remain stable and personal during adulthood. The stable anellome implies a great balance between the host immune system and the virus. However, the lack of a robust culturing system hampers direct investigation of interactions between virus and host cells. Other techniques, however, including next generation sequencing, AnelloScan-antibody tests, evolution selection pressure analysis, and virus protein structures, do provide new insights into the interactions between anelloviruses and the host immune system. This review aims at providing an overview of the current knowledge on the immune mechanisms acting on anelloviruses and the countering viral mechanisms allowing immune evasion.

The Impact of First-Time SARS-CoV-2 Infection on Human Anelloviruses.

Members of the Anelloviridae family dominate the blood virome, emerging early in life. The anellome, representing the variety of anelloviruses within an individual, stabilizes by adulthood. Despite their supposedly commensal nature, elevated anellovirus concentrations under immunosuppressive treatment indicate an equilibrium controlled by immunity. Here, we investigated whether anelloviruses are sensitive to the immune activation that accompanies a secondary infection. As a model, we investigated 19 health care workers (HCWs) with initial SARS-CoV-2 infection, with blood sampling performed pre and post infection every 4 weeks in a 3-month-follow-up during the early 2020 COVID-19 pandemic. A concurrently followed control group (n = 27) remained SARS-CoV-2-negative. Serum anellovirus loads were measured using qPCR. A significant decrease in anellovirus load was found in the first weeks after SARS-CoV-2 infection, whereas anellovirus concentrations remained stable in the uninfected control group. A restored anellovirus load was seen approximately 10 weeks after SARS-CoV-2 infection. For five subjects, an in-time anellome analysis via Illumina sequencing could be performed. In three of the five HCWs, the anellome visibly changed during SARS-CoV-2 infection and returned to baseline in two of these cases. In conclusion, anellovirus loads in blood can temporarily decrease upon an acute secondary infection.

Torque teno sus virus k2a (TTSuVk2a) in wild boars from northeastern Patagonia, Argentina.

Torque teno sus virus k2a (TTSuVk2a) is a member of the family Anelloviridae that can establish persistent infections in both domestic pigs and wild boars. Its association with diseases has not been precisely elucidated, and it is often considered only as a commensal virus. This infectious agent has been reported in herds throughout the world. In this study, we investigated the detection rate and diversity of TTSuVk2a in free-living wild boars from northeastern Patagonia, Argentina. Total DNA was extracted from tonsil samples of 50 animals, nested PCR assays were carried out, and infection was verified in 60% of the cases. Sequence analysis of the viral non-coding region revealed distinct phylogenetic groups. These clusters showed contrasting patterns of spatial distribution, which presented statistically significant differences when evaluating spatial aggregation. In turn, the sequences were compared with those available in the database to find that the clusters were distinguished by having similarity with TTSuVk2a variants of different geographic origin. The results suggested that Patagonian wild boar populations are bearers of diverse viral strains of Asian, European, and South American provenance.

The mysterious anelloviruses: investigating its role in human diseases.

Anelloviruses (AVs) that infect the human population are members of the Anelloviridae family. They are widely distributed in human populations worldwide. Torque teno virus (TTV) was the first virus of this family to be identified and is estimated to be found in the serum of 80-90% of the human population. Sometime after the identification of TTV, Torque teno mini virus (TTMV) and Torque teno midi virus (TTMDV) were also identified and classified in this family. Since identifying these viruses, have been detected in various types of biological fluids of the human body, including blood and urine, as well as vital organs such as the liver and kidney. They can be transmitted from person to person through blood transfusions, fecal-oral contact, and possibly sexual intercourse. Recent studies on these newly introduced viruses show that although they are not directly related to human disease, they may be indirectly involved in initiating or exacerbating some human population-related diseases and viral infections. Among these diseases, we can mention various types of cancers, immune system diseases, viral infections, hepatitis, and AIDS. Also, they likely use the microRNAs (miRNAs) they encode to fulfill this cooperative role. Also, in recent years, the role of proliferation and their viral load, especially TTV, has been highlighted to indicate the immune system status of immunocompromised people or people who undergo organ transplants. Here, we review the possible role of these viruses in diseases that target humans and highlight them as important viruses that require further study. This review can provide new insights to researchers.

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.

Characterization of Diverse Anelloviruses, Cressdnaviruses, and Bacteriophages in the Human Oral DNA Virome from North Carolina (USA).

The diversity of viruses identified from the various niches of the human oral cavity-from saliva to dental plaques to the surface of the tongue-has accelerated in the age of metagenomics. This rapid expansion demonstrates that our understanding of oral viral diversity is incomplete, with only a few studies utilizing passive drool collection in conjunction with metagenomic sequencing methods. For this pilot study, we obtained 14 samples from healthy staff members working at the Duke Lemur Center (Durham, NC, USA) to determine the viral diversity that can be identified in passive drool samples from humans. The complete genomes of 3 anelloviruses, 9 cressdnaviruses, 4 Caudoviricetes large bacteriophages, 29 microviruses, and 19 inoviruses were identified in this study using high-throughput sequencing and viral metagenomic workflows. The results presented here expand our understanding of the vertebrate-infecting and microbe-infecting viral diversity of the human oral virome in North Carolina (USA).

Monitoring of plasma Torque teno virus, total Anelloviridae and Human Pegivirus 1 viral load for the prediction of infectious events and acute graft versus host disease in the allogeneic hematopoietic stem cell transplantation setting.

Anelloviridae and Human Pegivirus 1 (HPgV-1) blood burden have been postulated to behave as surrogate markers for immunosuppression in transplant recipients. Here, we assessed the potential utility plasma Torque teno virus (TTV), total Anelloviridae (TAV), and HPgV-1 load monitoring for the identification of allogeneic hematopoietic stem cell transplantation recipients (allo-HSCT) at increased risk of infectious events or acute graft versus host disease (aGvHD). In this single-center, observational study, plasma TTV DNA, TAV DNA, and HPgV-1 RNA loads were monitored in 75 nonconsecutive allo-HSCT recipients (median age, 54 years). Monitoring was conducted before at baseline or by days +30, +60, +90, +120, and +180 after transplantation. Pneumonia due to different viruses or Pneumocystis jirovecii, BK polyomavirus-associated haemorrhagic cystitis (BKPyV-HC), and Cytomegalovirus DNAemia were the infectious events considered in the current study. Kinetics of plasma TTV, TAV DNA, and HPgV-1 RNA load was comparable, with though and peak levels measured by days +30 and day +90 (+120 for HPgV-1). Forty patients (53%) developed one or more infectious events during the first 180 days after allo-HSCT, whereas 29 patients (39%) had aGvHD (grade II-IV in 18). Neither, TTV, TAV, nor HPgV-1 loads were predictive of overall infection or CMV DNAemia. A TTV DNA load cut-off ≥4.40 log10 (pretransplant) and ≥4.58 log10 (baseline) copies/mL predicted the occurrence of BKPyV-HC (sensitivity ≥89%, negative predictive value, ≥96%). TTV DNA loads ≥3.38 log10 by day +30 anticipated the occurrence of aGvHD (sensitivity, 90%; negative predictive value, 97%). Pretransplant HPgV-1 loads were significantly lower (p = 0.03) in patients who had aGvHD than in those who did not. Monitoring of TTV DNA or HPgV-1 RNA plasma levels either before or early after transplantation may be ancillary to identify allo-HSCT recipients at increased risk of BKPyV-HC or aGvHD.

Blood virome of patients with traumatic sepsis.

Sepsis is one of the possible outcomes of severe trauma, and it poses a dire threat to human life, particularly in immunocompromised people. The most prevalent pathogens are bacteria and fungi, but viruses should not be overlooked. For viral metagenomic analysis, we collected blood samples from eight patients with post-traumatic sepsis before and seven days after treatment. The results demonstrated that Anellovirus predominated the viral community, followed by Siphoviridae and Myoviridae, and that the variations in viral community and viral load before and after treatment were not statistically significant. This study allows us to investigate methods for establishing NGS-based viral diagnostic instruments for detecting viral infections in the blood of sepsis patients so that antiviral therapy can be administered quickly.

Expansion of Betatorquevirus and/or Gammatorquevirus in Patients with Severe Clinical Outcomes of the Liver Diseases.

Anellovirus (AV) is a ubiquitous virus in the human population. Individuals can be infected with multiple AV genera and species to form a heterogeneous repertoire, termed the anellome. Using advanced methods, we examined the anellomes from 12 paired serum and liver samples, as well as 2701 subjects with different clinical diagnoses. Overall, anellomes are remarkably individualized, with significant among-group differences (Kruskal-Wallis test p = 6.6 × 10-162 for richness and p = 7.48 × 10-162 for Shannon entropy). High dissimilarity scores (beta diversity) were observed between patient groups, except for paired serum and liver samples. At the population level, the relative abundance of combinational AV genus Betatorquevirus (torque teno mini viruses, TTMV), and Gammatorquevirus (torque teno midi viruses, TTMDV) exhibited an exponential distribution with a low bound point at 32%. Defined by this value, the AV TTMV/TTMDV-expanded anellome was significantly enriched among patients with acute liver failure (31.7%) and liver transplantation (40.7%), compared with other patient groups (χ2 test: p = 4.1 × 10-8-3.2 × 10-3). Therefore, anellome heterogeneity may be predictive of clinical outcomes in certain diseases, such as liver disease. The consistency of anellome between paired serum and liver samples indicates that a liquid biopsy approach would be suitable for longitudinal studies to clarify the causality of the AV TTMV/TTMDV-expanded anellome in the outcomes of liver disease.

Detection of porcine circovirus 2, porcine parvovirus 1, and torque teno sus virus k2a in wild boars from northeastern Patagonia, Argentina.

Wild boars can act as a reservoir of pathogenic viruses that affect the pig industry. Here, we assessed the presence of porcine circovirus 2, porcine parvovirus 1, and torque teno sus virus k2a in wild boars in northeastern Patagonia (Argentina). Total DNA was extracted from the tonsils of 27 animals (collected between early 2016 and mid-2019) and used to prepare sample pools, which were subjected to viral detection through two-round PCR assays. Sequencing of the amplification products and phylogenetic analysis confirmed the occurrence of all of the aforementioned infectious agents.

Characterization of the Human Blood Virome in Iranian Multiple Transfused Patients.

Blood transfusion safety is an essential element of public health. Current blood screening strategies rely on targeted techniques that could miss unknown or unexpected pathogens. Recent studies have demonstrated the presence of a viral community (virobiota/virome) in the blood of healthy individuals. Here, we characterized the blood virome in patients frequently exposed to blood transfusion by using Illumina metagenomic sequencing. The virome of these patients was compared to viruses present in healthy blood donors. A total number of 155 beta-thalassemia, 149 hemodialysis, and 100 healthy blood donors were pooled with five samples per pool. Members of the Anelloviridae and Flaviviridae family were most frequently observed. Interestingly, samples of healthy blood donors harbored traces of potentially pathogenic viruses, including adeno-, rota-, and Merkel cell polyomavirus. Viruses of the Anelloviridae family were most abundant in the blood of hemodialysis patients and displayed a higher anellovirus richness. Pegiviruses (Flaviviridae) were only observed in patient populations. An overall trend of higher eukaryotic read abundance in both patient groups was observed. This might be associated with increased exposure through blood transfusion. Overall, the findings in this study demonstrated the presence of various viruses in the blood of Iranian multiple-transfused patients and healthy blood donors.

SCANellome: Analysis of the Genomic Diversity of Human and Non-Human Primate Anelloviruses from Metagenomics Data.

Anelloviruses are extremely prevalent in the human population and are considered to be commensal parts of the human virome. The best-known member in humans is the Torque teno virus. Recent metagenomic next-generation sequencing investigations have helped reveal the considerable number of species and genotypes from the same genus that can be co-detected within a single individual and that this diversity increases as a function of age during the first months/years of life. As a result, to date, the bioinformatics analysis of this genetic diversity remains complex and constraining for researchers. Here, we present SCANellome, a user-friendly tool to investigate the anellome composition at the genus, species, and genotype levels of samples from metagenomics data generated by the Illumina and Nanopore platforms. SCANellome is based on an in-house up-to-date database that includes all human and non-human primate anellovirus reference sequences available on GenBank and meets the latest classification criteria established by the International Committee on Taxonomy of Viruses.

Multiple genotypes infection and molecular characterization of Torque teno neovison virus: A novel Anelloviridae of mink in China.

A novel Torque teno neovison virus (TTVs) was identified in specimens collected from dead mink during an outbreak of the Aleutian mink disease virus. Eighteen complete genomic sequences were obtained, ranging from 2109 to 2158 nucleotides in length and consisting of an untranslated region and three open reading frames. The genomic organization of mink TTVs is similar to previously reported anelloviruses. However, the deduced amino acid sequence of its ORF1 protein shows genetic diversity compared to related anelloviruses, suggesting that it represents a putative new species within the Anelloviridae family. This study provides a detailed molecular characterization of the novel mink anelloviruses, including its codon usage pattern, origin, and evolution. Analysis of the viral genomic sequences reveals the existence of multiple genotypes of co-infection. Principal component analysis and phylogenetic trees confirm the coexistence of multiple genotypes. Furthermore, the codon usage analyses indicate that mink TTVs have a genotype-specific codon usage pattern and show a low codon usage bias. Host-specific adaptation analysis suggests that TTVs are less adapted to mink. The possible origin and evolutionary history of mink TTVs were elucidated. Mink TTVs was genetically closely related to giant panda anellovirus, representing a new species. The observed incongruence between the phylogenetic history of TTVs and that of their hosts suggests that the evolution of anellovirus is largely determined by cross-species transmission. The study provides insights into the co-infection and genetic evolution of anellovirus in China.

Respiratory eukaryotic virome expansion and bacteriophage deficiency characterize childhood asthma.

Asthma development and exacerbation is linked to respiratory virus infections. There is limited information regarding the presence of viruses during non-exacerbation/infection periods. We investigated the nasopharyngeal/nasal virome during a period of asymptomatic state, in a subset of 21 healthy and 35 asthmatic preschool children from the Predicta cohort. Using metagenomics, we described the virome ecology and the cross-species interactions within the microbiome. The virome was dominated by eukaryotic viruses, while prokaryotic viruses (bacteriophages) were independently observed with low abundance. Rhinovirus B species consistently dominated the virome in asthma. Anelloviridae were the most abundant and rich family in both health and asthma. However, their richness and alpha diversity were increased in asthma, along with the co-occurrence of different Anellovirus genera. Bacteriophages were richer and more diverse in healthy individuals. Unsupervised clustering identified three virome profiles that were correlated to asthma severity and control and were independent of treatment, suggesting a link between the respiratory virome and asthma. Finally, we observed different cross-species ecological associations in the healthy versus the asthmatic virus-bacterial interactome, and an expanded interactome of eukaryotic viruses in asthma. Upper respiratory virome "dysbiosis" appears to be a novel feature of pre-school asthma during asymptomatic/non-infectious states and merits further investigation.

Human Anelloviruses: Influence of Demographic Factors, Recombination, and Worldwide Diversity.

Anelloviruses represent the major and most diverse component of the healthy human virome, referred to as the anellome. In this study, we determined the anellome of 50 blood donors, forming two sex- and age-matched groups. Anelloviruses were detected in 86% of the donors. The number of detected anelloviruses increased with age and was approximately twice as high in men as in women. A total of 349 complete or nearly complete genomes were classified as belonging to torque teno virus (TTV), torque teno mini virus (TTMV), and torque teno midi virus (TTMDV) anellovirus genera (197, 88, and 64 sequences, respectively). Most donors had intergenus (69.8%) or intragenus (72.1%) coinfections. Despite the limited number of sequences, intradonor recombination analysis showed 6 intragenus recombination events in ORF1. As thousands of anellovirus sequences have been described recently, we finally analyzed the global diversity of human anelloviruses. Species richness and diversity were close to saturation in each anellovirus genus. Recombination was found to be the main factor promoting diversity, although its effect was significantly lower in TTV than in TTMV and TTMDV. Overall, our results suggest that differences in diversity between genera may be caused by variations in the relative contribution of recombination. IMPORTANCE Anelloviruses are the most common human infectious viruses and are considered essentially harmless. Compared to other human viruses, they are characterized by enormous diversity, and recombination is suggested to play an important role in their diversification and evolution. Here, by analyzing the composition of the plasma anellome of 50 blood donors, we find that recombination is also a determinant of viral evolution at the intradonor level. On a larger scale, analysis of anellovirus sequences currently available in databases shows that their diversity is close to saturation and differs among the three human anellovirus genera and that recombination is the main factor explaining this intergenus variability. Global characterization of anellovirus diversity could provide clues about possible associations between certain virus variants and pathologies, as well as facilitate the implementation of unbiased PCR-based detection protocols, which may be relevant for using anelloviruses as endogenous markers of immune status.

Herpesviruses, polyomaviruses, parvoviruses, papillomaviruses, and anelloviruses in vestibular schwannoma.

Etiology of vestibular schwannoma (VS) is unknown. Viruses can infect and reside in neural tissues for decades, and new viruses with unknown tumorigenic potential have been discovered. The presence of herpesvirus, polyomavirus, parvovirus, and anellovirus DNA was analyzed by quantitative PCR in 46 formalin-fixed paraffin-embedded VS samples. Five samples were analyzed by targeted next-generation sequencing. Viral DNA was detected altogether in 24/46 (52%) tumor samples, mostly representing anelloviruses (46%). Our findings show frequent persistence of anelloviruses, considered normal virome, in VS. None of the other viruses showed an extensive presence, thereby suggesting insignificant role in VS.

Discovery and comparative genomic analysis of a novel equine anellovirus, representing the first complete Mutorquevirus genome.

The complete genome of a novel torque teno virus species (Torque teno equus virus 2 (TTEqV2) isolate Alberta/2018) was obtained by high-throughput sequencing (HTS) of nucleic acid extracted from the lung and liver tissue of a Quarter Horse gelding that died of nonsuppurative encephalitis in Alberta, Canada. The 2805 nucleotide circular genome is the first complete genome from the Mutorquevirus genus and has been approved as a new species by the International Committee on Taxonomy of Viruses. The genome contains several characteristic features of torque teno virus (TTV) genomes, including an ORF1 encoding a putative 631 aa capsid protein with an arginine-rich N-terminus, several rolling circle replication associated amino acid motifs, and a downstream polyadenylation signal. A smaller overlapping ORF2 encodes a protein with an amino acid motif (WX7HX3CXCX5H) which, in general, is highly conserved in TTVs and anelloviruses. The UTR contains two GC-rich tracts, two highly conserved 15 nucleotide sequences, and what appears to be an atypical TATA-box sequence also observed in two other TTV genera. Codon usage analysis of TTEqV2 and 11 other selected anelloviruses from five host species revealed a bias toward adenine ending (A3) codons in the anelloviruses, while in contrast, A3 codons were observed at a low frequency in horse and the four other associated host species examined. Phylogenetic analysis of TTV ORF1 sequences available to date shows TTEqV2 clusters with the only other currently reported member of the Mutorquevirus genus, Torque teno equus virus 1 (TTEqV1, KR902501). Genome-wide pairwise alignment of TTEqV2 and TTEqV1 shows the absence of several highly conserved TTV features within the UTR of TTEqV1, suggesting it is incomplete and TTEqV2 is the first complete genome within the genus Mutorquevirus.