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.

Diversity and dynamic changes of anelloviruses in plasma following allogeneic hematopoietic stem cell transplantation.

Monitoring of alphatorquevirus (torque teno virus [TTV]) DNA in plasma may prove to be useful to assess the net state of immune competence following allogeneic hematopoietic stem cell transplantation (allo-HSCT). There are scarce data published on the prevalence of beta (torque teno mini virus [TTMV]) and gammatorqueviruses (torque teno midi virus [TTMDV]) and, in particular, on the dynamics of anelloviruses in allo-HSCT patients. Twenty-five allo-HSCT recipients with available plasma specimens obtained before conditioning and after engraftment were included. Degenerated primers targeting a highly conserved genomic sequence across all anelloviruses were designed for genomic amplification and high-throughput sequencing. Co-detection of TTV, TTMV, and TTMDV both in pre-transplant and post-engraftment plasma specimens was documented in more than two-thirds of patients. The use of quantitative real-time polymerase chain reaction (PCR) assays targeting TTMV and TTMDV in addition to TTV may add value to TTV-specific PCR assays in the inference of the net state of immunosuppresion or immune competence in this clinical setting.

A novel anellovirus from hospitalized neonates.

Here, using viral metagenomics combined with conventional PCR, the complete genome sequence of a novel anellovirus (named anel-ch-zj and GenBank no. MT157223) from nasopharynx secretion specimens from hospitalized neonates was determined, and the deduced amino acid sequence of its ODF1 protein was found to be only 33.19%-39.33% identical to those of related anelloviruses with sequences available in the GenBank database, suggesting that it represents a putative new genus within the family Anelloviridae. PCR screening of 135 samples (including 45 nasopharynx secretion, 45 blood, and 45 fecal specimens collected from 45 individual hospitalized neonates) indicated that two nasopharynx secretion, two blood, and four fecal samples were positive for anel-ch-zj. Further PCR screening of 50 blood samples, 115 fecal samples, and 396 nasopharynx secretions collected from hospitalized children 1-5 years old did not yield any positive results. Whether this novel anellovirus detected in neonates is associated with specific diseases needs future investigation.

In-depth serum virome analysis in patients with acute liver failure with indeterminate etiology.

In clinical virome research, whole-genome/transcriptome amplification is required when starting material is limited. An improved method, named "template-dependent multiple displacement amplification" (tdMDA), has recently been developed in our lab (Wang et al. in BioTechniques 63:21-25. https://doi.org/10.2144/000114566, 2017). In combination with Illumina sequencing and bioinformatics pipelines, its application in virome sequencing was explored using a serum sample from a patient with chronic hepatitis C virus (HCV) infection. In comparison to an amplification-free procedure, virome sequencing via tdMDA showed a 9.47-fold enrichment for HCV-mapped reads and, accordingly, an increase in HCV genome coverage from 28.5% to 70.1%. Eight serum samples from acute patients liver failure (ALF) with or without known etiology were then used for virome sequencing with an average depth at 94,913x. Both similarity-based (mapping, NCBI BLASTn, BLASTp, and profile hidden Markov model analysis) and similarity-independent methods (machine-learning algorithms) identified viruses from multiple families, including Herpesviridae, Picornaviridae, Myoviridae, and Anelloviridae. However, their commensal nature and cross-detection ruled out an etiological interpretation. Together with a lack of detection of novel viruses in a comprehensive analysis at a resolution of single reads, these data indicate that viral agents might be rare in ALF cases with indeterminate etiology.

High-Throughput Sequencing-Based Investigation of Viruses in Human Cancers by Multienrichment Approach.

BACKGROUND: Viruses and other infectious agents cause more than 15% of human cancer cases. High-throughput sequencing-based studies of virus-cancer associations have mainly focused on cancer transcriptome data. METHODS: In this study, we applied a diverse selection of presequencing enrichment methods targeting all major viral groups, to characterize the viruses present in 197 samples from 18 sample types of cancerous origin. Using high-throughput sequencing, we generated 710 datasets constituting 57 billion sequencing reads. RESULTS: Detailed in silico investigation of the viral content, including exclusion of viral artefacts, from de novo assembled contigs and individual sequencing reads yielded a map of the viruses detected. Our data reveal a virome dominated by papillomaviruses, anelloviruses, herpesviruses, and parvoviruses. More than half of the included samples contained 1 or more viruses; however, no link between specific viruses and cancer types were found. CONCLUSIONS: Our study sheds light on viral presence in cancers and provides highly relevant virome data for future reference.

Torquetenovirus detection in exosomes enriched vesicles circulating in human plasma samples.

BACKGROUND: Torquetenovirus (TTV) belongs to Anelloviridae family, infects nearly all people indefinitely without causing overt disease establishing a fine and successful interaction with the host. Increasing evidence have shown some human viruses exploit extracellular vesicles thereby helping viral persistence in the host. Here, the presence of TTV in extracellular vesicles circulating in human plasma was investigated. METHODS: TTV DNA was quantified in plasma-derived exosomes from 122 samples collected from 97 diseased patients and 25 healthy donors. Exosomes enriched vesicles (EEVs) were extracted from plasma and characterized by Nanoparticle tracking analysis, by western blot for presence of tetraspanin CD63, CD81 and annexin II protein and, finally, by electron microscopy (EM). Presence and quantitation of TTV DNA were assessed with an universal single step real-time TaqMan PCR assay. RESULTS: Preliminary investigation showed that the human plasma extracted extracellular vesicles exhibited a main size of 70 nm, had concentration of 2.5 × 109/ml, and scored positive for tetraspanin CD63, CD81 and annexin II, typical characteristic of the exosomes vesicles. EEVs extracted from pooled plasma with TTV DNA viremia of 9.7 × 104 copies/ml showed to contain 6.3 × 102 TTV copies/ml, corresponding to 0.65% of total viral load. Important, TTV yield changed significantly following freezing/thawing, detergents and DNAse treatment of plasma before EEVs extraction. EEVs purified by sucrose-density gradient centrifugation and analysis of gradient fraction positive for exosomes marker CD63 harbored 102 TTV copies/ml. Moreover, EM evidenced the presence of TTV-like particles in EEVs. Successive investigation of plasma EEVs from 122 subjects (37 HIV-positive, 20 HCV infected, 20 HBV infected, 20 kidney transplant recipients, and 25 healthy) reported TTV DNA detection in 42 (34%) of the viremic samples (37 were from diseased patients and 5 from healthy people) at a mean level of 4.8 × 103 copies/ml. The examination of EEVs selected samples reported the presence of TTV genogroup 1, 3, 4 and 5, with genogroup 3 highly observed. CONCLUSIONS: Collectively, although these observations should be confirmed by further studies, circulation of TTV particles in EEVs opens new avenues and mechanistic insights on the molecular strategies adopted by anelloviruses to persist in the host.

Novel anelloviruses identified in buccal swabs of Antarctic fur seals.

Viral diversity associated with Antarctic wildlife remains poorly studied. Nonetheless, over the past 5 years, there has been a concerted effort using viral metagenomics approaches to identify and characterize viruses associated with Antarctic pinniped and avian species. Here we used a viral metagenomics approach to identify circular DNA viruses in buccal swab samples from Antarctic fur seals (Arctocephalus gazella) breeding on Livingston Island, Antarctica during the 2016/2017 field season. We identified two new lineages of anelloviruses, torque teno Arctocephalus gazella virus (TTAgV)-1 (2083 nts) and -2 (2127-2143 nts), which are most closely related to and cluster with a previously identified anellovirus associated with California sea lions (Zalophus californianus) sharing ~ 60% genome-wide pairwise identity. The ORF1 of TTAgVs share 26-41% amino acid similarity to the ORF1 of other previously identified pinniped-associated anelloviruses.

Prevalence of human anelloviruses in Romanian healthy subjects and patients with common pathologies.

BACKGROUND: Human anelloviruses (TTV, TTMDV and TTMV) are at high prevalence all across the globe, having also a controversial disease-inducing potential. This study aimed to estimate the prevalence of anelloviral DNA in the Romanian human population and to investigate the association of infections with common pathologies in Romanian population. METHODS: After informed consent, blood samples were collected from 2000 subjects represented by: clinically healthy individuals (n = 701) and a group of patients with pathologies linked to low grade inflammation or alteration of carbohydrate metabolism (n = 1299). All samples were analysed for the presence of TTV, TTMDV and TTMV DNA by hemi-nested PCR. RESULTS: The prevalence of TTV, TTMDV and TTMV in the studied population was 68.2, 54.4%, respectively 40.1%, lower than the recent reports from other geographic regions. The three viral species were significantly more frequent in the group of patients compared to the healthy subjects and were associated with type 2 diabetes mellitus. The presence of anelloviral DNA was also associated with medical procedures (e.g. haemodialysis/transfusions, surgical procedures) and previous hepatitis A virus infection. Lifestyle choices related to alcohol consumption, smoking, physical activity and living environment were not associated with differences in distribution of the three viruses. CONCLUSION: Further evidence is needed to establish a correlation between infection with human anelloviruses and a pathology or group of pathologies.

Anellovirus loads are associated with outcomes in pediatric lung transplantation.

Anelloviruses are DNA viruses ubiquitously present in human blood. Due to their elevated levels in immunosuppressed patients, anellovirus levels have been proposed as a marker of immune status. We hypothesized that low anellovirus levels, reflecting relative immunocompetence, would be associated with adverse outcomes in pediatric lung transplantation. We assayed blood samples from 57 patients in a multicenter study for alpha- and betatorquevirus, two anellovirus genera. The primary short-term outcome of interest was acute rejection, and longer-term outcomes were analyzed individually and as "composite" (death, chronic rejection, or retransplant within 2 years). Patients with low alphatorquevirus levels at 2 weeks post-transplantation were more likely to develop acute rejection within 3 months after transplant (P = .013). Low betatorquevirus levels at 6 weeks and 6 months after transplant were associated with death (P = .047) and the composite outcome (P = .017), respectively. There was an association between low anellovirus levels and adverse outcomes in pediatric lung transplantation. Alphatorquevirus levels were associated with short-term outcomes (ie, acute rejection), while betatorquevirus levels were associated with longer-term outcomes (ie, death, or composite outcome within 2 years). These observations suggest that anelloviruses may serve as useful biomarkers of immune status and predictors of adverse outcomes.

Viral nucleic acids in human plasma pools.

BACKGROUND: The identification of viruses in human blood is required for epidemiologic surveillance and to detect potentially emerging threats to blood transfusion safety. STUDY DESIGN AND METHODS: Viral nucleic acids in plasma fractionation pools assembled from blood donors in the United States and Europe were analyzed by viral metagenomics. RESULTS: Anelloviruses were detected in each of the 10 plasma pools. Human pegivirus A (HPgV; GB virus type C) sequences were identified in eight of the 10 pools, more than 90% of which belong to Genotype 2. The recently described human HPgV2 in Flaviviridae was not detected. A small number of sequence reads of human papillomavirus were also detected in three pools. In one pool, two different gemycircularvirus genomes were identified and fully sequenced. The capsid protein of one gemycircularvirus shared 83% to 84% identity to those of genomes from human serum and sewage. The presence of the gemycircularvirus genomes in the plasma pool was independently confirmed and the viral concentration estimated by digital PCR at more than 10(6) copies/mL assuming their origin from single donors. CONCLUSION: Further research is required to elucidate whether gemycircularviruses can infect humans or are indicative of contamination occurring during phlebotomy, plasma pool processing, or ongoing donor fungal infections.

Identification and genomic characterization of a novel species of feline anellovirus.

Here, a novel feline anellovirus strain (named FelineAV621 and GenBank no. KX262893) was detected in two cats with diarrhea. The complete genome of FelineAV621 is 2409 nt long with a G+C content of 56.67 %, including three open reading frames (ORFs). Phylogenetic analysis based on the amino acid sequence of the putative capsid protein (ORF1) indicated that FelineAV621 belonged to a novel anellovirus species inside a clade containing the seal anellovirus, canine TTVs, and porcine TTVs, but was distant from all the previous feline anelloviruses.

Prevalence of anelloviruses (TTV, TTMDV, and TTMV) in healthy blood donors and in patients infected with HBV or HCV in Qatar.

BACKGROUND: Anelloviruses (TTV, TTMV, and TTMDV) have been associated with non A-G hepatitis. The goal of the current study was to estimate the prevalence of these anelloviruses in Qatar. METHODS: A total of 607 blood samples (500 healthy donors, and 53 HBV-and 54 HCV-positive patients) representing different nationalities were tested for the presence of TTV, TTMV, and TTMDV DNA by nested PCR. RESULTS: Prevalence rates for the three viruses were high in all studied groups, and exceeding 95% in the HBV group (for TTV and TTMDV). Infection with more than one type of viruses was common and significant in most of the positive patients (p < 0.05) and ranging from 55.4% for TTV/TTMV and TTMV/TTMDV co-infections in the healthy group, to 96.3% for TTV/TTMV co-infections in the HBV group. Further, and as with most previous studies, no significant association was found between anelloviruses infections and age, nationality, or gender (p > 0.05) albeit the detection of higher infection rates among females and Qatari subjects. CONCLUSION: This was the first published study to look at prevalence of Anellowviruses in the Middle East. High prevalence rates of the three viruses in all studied groups was noted. Further studies are needed to explore and compare the different genotypes of these viruses in the region.

Viral metagenomics reveal blooms of anelloviruses in the respiratory tract of lung transplant recipients.

Few studies have examined the lung virome in health and disease. Outcomes of lung transplantation are known to be influenced by several recognized respiratory viruses, but global understanding of the virome of the transplanted lung is incomplete. To define the DNA virome within the respiratory tract following lung transplantation we carried out metagenomic analysis of allograft bronchoalveolar lavage (BAL), and compared with healthy and HIV+ subjects. Viral concentrates were purified from BAL and analyzed by shotgun DNA sequencing. All of the BAL samples contained reads mapping to anelloviruses, with high proportions in lung transplant samples. Anellovirus populations in transplant recipients were complex, with multiple concurrent variants. Quantitative polymerase chain reaction quantification revealed that anellovirus sequences were 56-fold more abundant in BAL from lung transplant recipients compared with healthy controls or HIV+ subjects (p < 0.0001). Anellovirus sequences were also more abundant in upper respiratory tract specimens from lung transplant recipients than controls (p = 0.006). Comparison to metagenomic data on bacterial populations showed that high anellovirus loads correlated with dysbiotic bacterial communities in allograft BAL (p = 0.008). Thus the respiratory tracts of lung transplant recipients contain high levels and complex populations of anelloviruses, warranting studies of anellovirus lung infection and transplant outcome.

Metagenomic profiling of the viromes of plasma collected from blood donors with elevated serum alanine aminotransferase levels.

BACKGROUND: In Japanese Red Cross (JRC) blood centers, blood collected from donors with serum alanine aminotransferase (ALT) levels of more than 60 U/L are disqualified even if serologically negative for transfusion-transmitted infections (TTIs). To assess potential risks of TTIs in plasma with elevated serum ALT levels in the current donor screening program of the JRC, we conducted a metagenomic analysis (MGA) of virome profiles in the plasma of blood donors with or without elevated serum ALT levels. STUDY DESIGN AND METHODS: Based on serum ALT levels, donors were classified into three groups: "high," more than 79 U/L; "middle," 61 to 79 U/L; and "low," less than 61 U/L. We individually analyzed 100 plasma samples from each group by MGA, employing shotgun sequencing. Viral sequences detected using MGA were partly confirmed using real-time polymerase chain reaction (PCR). RESULTS: Donors with high and middle ALT levels were significantly younger than those with low ALT levels, and more than 90% were males. Herpesviridae, Anelloviridae, Picornaviridae, and Flaviviridae sequences were identified in plasma samples, and their distribution and frequency were not significantly different among the three groups. CONCLUSION: The serum ALT test may be unsuitable for monitoring for additional risks of TTIs in blood donors who were negative for typical TTIs using serologic and nucleic acid tests. Although MGA is less sensitive than PCR, it remains the best technology to detect known viruses in these donors.

Human anelloviruses: an update of molecular, epidemiological and clinical aspects.

Human torque teno viruses (TTVs) are new, emerging infectious agents, recently assigned to the family Anelloviridae. The first representative of the genus, torque teno virus (TTV), was discovered in 1997, followed by torque teno mini virus (TTMV) in 2000, and torque teno midi virus (TTMDV) in 2007. These viruses are characterized by an extremely high prevalence, with relatively uniform distribution worldwide and a high level of genomic heterogeneity, as well as an apparent pan-tropism at the host level. Although these viruses have a very high prevalence in the general population across the globe, neither their interaction with their hosts nor their direct involvement in the etiology of specific diseases are fully understood. Since their discovery, human anelloviruses, and especially TTV, have been suggested to be associated with various diseases, such as hepatitis, respiratory diseases, cancer, hematological and autoimmune disorders, with few arguments for their direct involvement. Recent studies have started to reveal interactions between TTVs and the host's immune system, leading to new hypotheses for potential pathological mechanisms of these viruses. In this review article, we discuss the most important aspects and current status of human TTVs in order to guide future studies.

Identification of novel anelloviruses with broad diversity in UK rodents.

Anelloviruses are a family of small circular ssDNA viruses with a vast genetic diversity. Human infections with the prototype anellovirus, torque teno virus (TTV), are ubiquitous and related viruses have been described in a number of other mammalian hosts. Despite over 15 years of investigation, there is still little known about the pathogenesis and possible disease associations of anellovirus infections, arising in part due to the lack of a robust cell culture system for viral replication or tractable small-animal model. We report the identification of diverse anelloviruses in several species of wild rodents. The viruses are highly prevalent in wood mice (Apodemus sylvaticus) and field voles (Microtus agrestis), detectable at a low frequency in bank voles (Myodes glareolus), but absent from house mice (Mus musculus). The viruses identified have a genomic organization consistent with other anelloviruses, but form two clear phylogenetic groups that are as distinct from each other as from defined genera.

Faecal virome of cats in an animal shelter.

We describe the metagenomics-derived feline enteric virome in the faeces of 25 cats from a single shelter in California. More than 90 % of the recognizable viral reads were related to mammalian viruses and the rest to bacterial viruses. Eight viral families were detected: Astroviridae, Coronaviridae, Parvoviridae, Circoviridae, Herpesviridae, Anelloviridae, Caliciviridae and Picobirnaviridae. Six previously known viruses were also identified: feline coronavirus type 1, felid herpes 1, feline calicivirus, feline norovirus, feline panleukopenia virus and picobirnavirus. Novel species of astroviruses and bocaviruses, and the first genome of a cyclovirus in a feline were characterized. The RNA-dependent RNA polymerase region from four highly divergent partial viral genomes in the order Picornavirales were sequenced. The detection of such a diverse collection of viruses shed within a single shelter suggested that such animals experience robust viral exposures. This study increases our understanding of the viral diversity in cats, facilitating future evaluation of their pathogenic and zoonotic potentials.

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.

Rescue of a porcine anellovirus (torque teno sus virus 2) from cloned genomic DNA in pigs.

Anelloviruses are a group of single-stranded circular DNA viruses infecting humans and other animal species. Animal models combined with reverse genetic systems of anellovirus have not been developed. We report here the construction and initial characterization of full-length DNA clones of a porcine anellovirus, torque teno sus virus 2 (TTSuV2), in vitro and in vivo. We first demonstrated that five cell lines, including PK-15 cells, are free of TTSuV1 or TTSuV2 contamination, as determined by a real-time PCR and an immunofluorescence assay (IFA) using anti-TTSuV antibodies. Recombinant plasmids harboring monomeric or tandem-dimerized genomic DNA of TTSuV2 from the United States and Germany were constructed. Circular TTSuV2 genomic DNA with or without introduced genetic markers and tandem-dimerized TTSuV2 plasmids were transfected into PK-15 cells, respectively. Splicing of viral mRNAs was identified in transfected cells. Expression of TTSuV2-specific open reading frame 1 (ORF1) in cell nuclei, especially in nucleoli, was detected by IFA. However, evidence of productive TTSuV2 infection was not observed in 12 different cell lines transfected with the TTSuV2 DNA clones. Transfection with circular DNA from a TTSuV2 deletion mutant did not produce ORF1 protein, suggesting that the observed ORF1 expression is driven by TTSuV2 DNA replication in cells. Pigs inoculated with either the tandem-dimerized clones or circular genomic DNA of U.S. TTSuV2 developed viremia, and the introduced genetic markers were retained in viral DNA recovered from the sera of infected pigs. The availability of an infectious DNA clone of TTSuV2 will facilitate future study of porcine anellovirus pathogenesis and biology.