Discovery of three cycloviruses in fecal samples from silver-haired bats (Lasionycteris noctivagans) in Arizona (USA).

Bats harbour a diverse array of viruses, some of which are zoonotic, and are one of the most speciose groups of mammals on earth. As part of an ongoing bat-associated viral diversity research project, we identified three cycloviruses (family Circoviridae) in fecal samples of silver-haired bats (Lasionycteris noctivagans) caught in Cave Creek Canyon of Arizona (USA). Two of the three identified genomes represent two new species in the genus Cyclovirus. Cycloviruses have been found in a wide range of environments and hosts; however, little is known about their biology. These new genomes of cycloviruses are the first from silver-haired bats, adding to the broader knowledge of cyclovirus diversity. With continuing studies, it is likely that additional viruses of the family Circoviridae will be identified in Arizona bat populations.

Cyclovirus detection in Chilean adults with and without community-acquired pneumonia.

Cycloviruses (CyV) (genus Cyclovirus, family Circoviridae) are nonenveloped DNA viruses. The first report in humans was in 2010 and research has focused only on disease-associated human sample detection. The only HuACyV (CyCV-ChileNPA1, HuACyV10) reported in the Chilean population was in children (3.3%) with an acute respiratory infection. Its detection in respiratory samples from adults, with/without respiratory disease remains unknown. The aim of this study was to detect HuACyV10 in adults with and without respiratory disease. HuACyV10 was studied in nasopharyngeal swabs from 105 hospitalized adults with community-acquired pneumonia (CAP) and 104 adults without respiratory symptoms. Total nucleic acids were extracted, and viral rep and cp gene fragments were amplified by real-time polymerase chain reaction. HuACyV10 was detected in 19.05% adults with CAP and in 0.96% asymptomatic adults, being significantly higher in adult CAP than asymptomatic (n = 1) ones (p = 0.0001). C t values were between 26.7 and 39.6, and the median was 34.1 for rep and 33.8 for the CAP in adults CAP (p = 0.68), and 35.7 and 36.0, respectively, in the asymptomatic case. HuACyV10 detection in CAP adults concentrated in the Autumn-Winter season of the Southern hemisphere. The only asymptomatic adult with HuACyV10 was detected in the Spring-Summer period. In this first report of HuACyV10 in respiratory samples from adults, detection was significantly higher in CAP than in asymptomatic adults. As the sensitivity of both rep and cp genes was similar, both can be applied for detecting HuACyV10. It would be advisable to investigate the pathogenic role of HuACyV10 in adult respiratory infections. ​.

Insights into Circovirus Host Range from the Genomic Fossil Record.

A diverse range of DNA sequences derived from circoviruses (family Circoviridae) has been identified in samples obtained from humans and domestic animals, often in association with pathological conditions. In the majority of cases, however, little is known about the natural biology of the viruses from which these sequences are derived. Endogenous circoviral elements (CVe) are DNA sequences derived from circoviruses that occur in animal genomes and provide a useful source of information about circovirus-host relationships. In this study, we screened genome assemblies of 675 animal species and identified numerous circovirus-related sequences, including the first examples of CVe derived from cycloviruses. We confirmed the presence of these CVe in the germ line of the elongate twig ant (Pseudomyrmex gracilis), thereby establishing that cycloviruses infect insects. We examined the evolutionary relationships between CVe and contemporary circoviruses, showing that CVe from ants and mites group relatively closely with cycloviruses in phylogenies. Furthermore, the relatively random interspersion of CVe from insect genomes with cyclovirus sequences recovered from vertebrate samples suggested that contamination might be an important consideration in studies reporting these viruses. Our study demonstrates how endogenous viral sequences can inform metagenomics-based virus discovery. In addition, it raises doubts about the role of cycloviruses as pathogens of humans and other vertebrates.IMPORTANCE Advances in DNA sequencing have dramatically increased the rate at which new viruses are being identified. However, the host species associations of most virus sequences identified in metagenomic samples are difficult to determine. Our analysis indicates that viruses proposed to infect vertebrates (in some cases being linked to human disease) may in fact be restricted to arthropod hosts. The detection of these sequences in vertebrate samples may reflect their widespread presence in the environment as viruses of parasitic arthropods.

Detection of Circovirus in Foxes with Meningoencephalitis, United Kingdom, 2009-2013.

A fox circovirus was identified in serum samples from foxes with unexplained neurologic signs by using viral metagenomics. Fox circovirus nucleic acid was localized in histological lesions of the cerebrum by in situ hybridization. Viruses from the family Circoviridae may have neurologic tropism more commonly than previously anticipated.

Circoviridae Survey in Captive Non-Human Primates, Italy.

Circoviruses (CVs) and cycloviruses (CyVs), members of the family Circoviridae, have been identified only occasionally in non-human primates (NHPs). In this study, we investigated the presence and genetic features of these viruses in 48 NHPs housed in the Bioparco-Rome Zoological Garden (Italy) and in the Anima Natura Wild Sanctuary Semproniano (Grosseto, Italy), testing fecal, saliva, and serum samples with a broadly reactive consensus nested PCR able of amplifying a partial region of the replicase (Rep) gene of members of the family Circoviridae. Viral DNA was detected in a total of 10 samples, including a saliva swab and 9 fecal samples collected, respectively from five Japanese macaques (Macaca fuscata) and four mandrills (Mandrillus sphinx), with an overall prevalence of 18.7% (9/48). On genome sequencing, five strains revealed the highest nucleotide identity (98.3-98.6%) to a CyV strain (RI196/ITA) detected in the intestinal content of a Maltese wall lizard (Podarcis filfolensis) in Italy. Although the origin of the Italian NHP strains, genetically distant from previously detected NHP CyVs, is uncertain, our results also highlight that the virome of captive animals is modulated by the different dietary and environmental sources of exposure.

Novel cyclovirus in human cerebrospinal fluid, Malawi, 2010-2011.

To identify unknown human viruses, we analyzed serum and cerebrospinal fluid samples from patients with unexplained paraplegia from Malawi by using viral metagenomics. A novel cyclovirus species was identified and subsequently found in 15% and 10% of serum and cerebrospinal fluid samples, respectively. These data expand our knowledge of cyclovirus diversity and tropism.

Metagenomic Detection of Divergent Insect- and Bat-Associated Viruses in Plasma from Two African Individuals Enrolled in Blood-Borne Surveillance.

Metagenomic next-generation sequencing (mNGS) has enabled the high-throughput multiplexed identification of sequences from microbes of potential medical relevance. This approach has become indispensable for viral pathogen discovery and broad-based surveillance of emerging or re-emerging pathogens. From 2015 to 2019, plasma was collected from 9586 individuals in Cameroon and the Democratic Republic of the Congo enrolled in a combined hepatitis virus and retrovirus surveillance program. A subset (n = 726) of the patient specimens was analyzed by mNGS to identify viral co-infections. While co-infections from known blood-borne viruses were detected, divergent sequences from nine poorly characterized or previously uncharacterized viruses were also identified in two individuals. These were assigned to the following groups by genomic and phylogenetic analyses: densovirus, nodavirus, jingmenvirus, bastrovirus, dicistrovirus, picornavirus, and cyclovirus. Although of unclear pathogenicity, these viruses were found circulating at high enough concentrations in plasma for genomes to be assembled and were most closely related to those previously associated with bird or bat excrement. Phylogenetic analyses and in silico host predictions suggested that these are invertebrate viruses likely transmitted through feces containing consumed insects or through contaminated shellfish. This study highlights the power of metagenomics and in silico host prediction in characterizing novel viral infections in susceptible individuals, including those who are immunocompromised from hepatitis viruses and retroviruses, or potentially exposed to zoonotic viruses from animal reservoir species.

Diversity of CRESS DNA Viruses in Squamates Recapitulates Hosts Dietary and Environmental Sources of Exposure.

Replication-associated protein (Rep)-encoding single-stranded (CRESS) DNA viruses comprise viruses with covalently closed, circular, single-stranded DNA (ssDNA) genomes, and are considered the smallest known autonomously replicating, capsid-encoding animal pathogens. CRESS DNA viruses (phylum Cressdnaviricota) encompass several viral families including Circoviridae. Circoviruses are classified into two genera, Circovirus and Cyclovirus, and they are known to cause fatal diseases in birds and pigs. Circoviruses have also been identified in human stools, blood, and cerebrospinal fluid (CSF), as well as in various wild and domestic vertebrates, including reptiles. The synanthropic presence of Squamata reptiles has increased in the last century due to the anthropic pressure, which has shifted forested animal behavior to an urban and peri-urban adaptation. In this paper, we explored the diversity of CRESS DNA viruses in Squamata reptiles from different Italian areas representative of the Mediterranean basin. CRESS DNA viruses were detected in 31.7% (33/104) of sampled lizards and geckoes. Different CRESS DNA viruses likely reflected dietary composition or environmental contamination and included avian-like (n = 3), dog (n = 4), bat-like (n = 1), goat-like (n = 1), rodent-like (n = 4), and insect-like (n = 2) viruses. Rep sequences of at least two types of human-associated cycloviruses (CyV) were identified consistently, regardless of geographic location, namely, TN9-like (n = 11) and TN12-like (n = 6). A third human-associated CyV, TN25-like, was detected in a single sample. The complete genome of human-like CyVs, of a rodent-like, insect-like, and of a bat-like virus were generated. Collectively, the results recapitulate hosts dietary and environmental sources of exposure and may suggest unexpected ecological niches for some CRESS DNA viruses. IMPORTANCE CRESS DNA viruses are significant pathogens of birds and pigs and have been detected repeatedly in human samples (stools, serum, and cerebrospinal fluid), both from healthy individuals and from patients with neurological disease, eliciting in 2013 a risk assessment by the European Centre for Disease Prevention and Control (ECDC). Sequences of CRESS DNA viruses previously reported in humans (TN9, TN12, and TN25), and detected in different animal species (e.g., birds, dogs, and bats) were herein detected in fecal samples of synanthropic squamates (geckos and lizards). The complete genome sequence of six viruses was generated. This study extends the information on the genetic diversity and ecology of CRESS DNA viruses. Because geckos and lizards are synanthropic animals, a role in sustaining CRESS DNA virus circulation and increasing viral pressure in the environment is postulated.

Novel Cyclovirus Species in Dogs with Hemorrhagic Gastroenteritis.

Nested PCRs with circovirus/cyclovirus pan-rep (replicase gene) primers detected eukaryotic circular Rep-encoding single-stranded DNA (CRESS DNA) viruses in three (samples CN9E, CN16E and CN34) of 18 canine parvovirus-2-positive fecal samples from household dogs with hemorrhagic gastroenteritis on the Caribbean island of Nevis. The complete genomes of CRESS DNA virus CN9E, CN16E and CN34 were determined by inverse nested PCRs. Based on (i) genome organization, (ii) location of the putative origin of replication, (iii) pairwise genome-wide sequence identities, (iv) the presence of conserved motifs in the putative replication-associated protein (Rep) and the arginine-rich region in the amino terminus of the putative capsid protein (Cp) and (v) a phylogenetic analysis, CN9E, CN16E and CN34 were classified as cycloviruses. Canine-associated cycloviruses CN16E and CN34 were closely related to each other and shared low genome-wide nucleotide (59.642-59.704%), deduced Rep (35.018-35.379%) and Cp (26.601%) amino acid sequence identities with CN9E. All the three canine-associated cycloviruses shared < 80% genome-wide pairwise nucleotide sequence identities with cycloviruses from other animals/environmental samples, constituting two novel species (CN9E and CN16E/34) within the genus Cyclovirus. Considering the feeding habits of dogs, we could not determine whether the cycloviruses were of dietary origin or infected the host. Interestingly, the CN9E putative Rep-encoding open reading frame was found to use the invertebrate mitochondrial genetic code with an alternative initiation codon (ATA) for translation, corroborating the hypothesis that cycloviruses are actually arthropod-infecting viruses. To our knowledge, this is the first report on the detection and complete genome analysis of cycloviruses from domestic dogs.

Detection and Complete Genome Analysis of Circoviruses and Cycloviruses in the Small Indian Mongoose (Urva auropunctata): Identification of Novel Species.

Fecal samples from 76 of 83 apparently healthy small Indian mongooses (Urva auropunctata) were PCR positive with circovirus/cyclovirus pan-rep (replicase gene) primers. In this case, 30 samples yielded high quality partial rep sequences (~400 bp), of which 26 sequences shared maximum homology with cycloviruses from an arthropod, bats, humans or a sheep. Three sequences exhibited maximum identities with a bat circovirus, whilst a single sequence could not be assigned to either genus. Using inverse nested PCRs, the complete genomes of mongoose associated circoviruses (Mon-1, -29 and -66) and cycloviruses (Mon-20, -24, -32, -58, -60 and -62) were determined. Mon-1, -20, -24, -29, -32 and -66 shared <80% maximum genome-wide pairwise nucleotide sequence identities with circoviruses/cycloviruses from other animals/sources, and were assigned to novel circovirus, or cyclovirus species. Mon-58, -60 and -62 shared maximum pairwise identities of 79.90-80.20% with human and bat cycloviruses, which were borderline to the cut-off identity value for assigning novel cycloviral species. Despite high genetic diversity, the mongoose associated circoviruses/cycloviruses retained the various features that are conserved among members of the family Circoviridae, such as presence of the putative origin of replication (ori) in the 5'-intergenic region, conserved motifs in the putative replication-associated protein and an arginine rich region in the amino terminus of the putative capsid protein. Since only fecal samples were tested, and mongooses are polyphagous predators, we could not determine whether the mongoose associated circoviruses/cycloviruses were of dietary origin, or actually infected the host. To our knowledge, this is the first report on detection and complete genome analysis of circoviruses/cycloviruses in the small Indian mongoose, warranting further studies in other species of mongooses.

Novel Cyclovirus Identified in Broiler Chickens With Transmissible Viral Proventriculitis in China.

In October 2018, an outbreak of transmissible viral proventriculitis (TVP) occurred in 30-day-old commercial broiler chickens on a farm in Weifang, China. TVP, an infectious viral disease characterized by runting and stunting, is associated with many viruses, and has a significant economic impact on the global poultry industry. TVP is diagnosed according to clinical symptoms, gross and histological lesions, and negative PCR results for pathogenic bacteria, avian leukosis virus subgroup J, Marek's disease virus, reticuloendotheliosis virus, infectious bursa disease virus, avian reovirus, chicken anemia virus, infectious bronchitis virus, chicken proventricular necrosis virus, gyrovirus 3 and chicken circovirus. To further detect the possible causative pathogens of TVP, we used PacBio third-generation sequencing to examine proventricular samples. A dominant abundance of the novel cyclovirus (CyCV), chCyCV-SDAU-1, was identified in broilers with TVP. The complete chCyCV-SDAU-1 genome was verified via inverse PCR, was 1936 bp long, and consisted of Rep, Cp, and two intergenic regions. Phylogenetic tree analysis showed that chCyCV-SDAU-1 formed an independent branch with other cycloviruses. The homology of chCyCV-SDAU-1 with 20 others known cycloviruses was < 40%. Retrospective investigation showed that the CyCV infection rate in the broilers with TVP was 80% (16/20), while no CyCV was found in healthy chickens. In conclusion, a novel CyCV was identified in chickens with TVP, though its role in this disease is unclear.

The Virome of Acute Respiratory Diseases in Individuals at Risk of Zoonotic Infections.

The ongoing coronavirus disease 2019 (COVID-19) pandemic emphasizes the need to actively study the virome of unexplained respiratory diseases. We performed viral metagenomic next-generation sequencing (mNGS) analysis of 91 nasal-throat swabs from individuals working with animals and with acute respiratory diseases. Fifteen virus RT-PCR-positive samples were included as controls, while the other 76 samples were RT-PCR negative for a wide panel of respiratory pathogens. Eukaryotic viruses detected by mNGS were then screened by PCR (using primers based on mNGS-derived contigs) in all samples to compare viral detection by mNGS versus PCR and assess the utility of mNGS in routine diagnostics. mNGS identified expected human rhinoviruses, enteroviruses, influenza A virus, coronavirus OC43, and respiratory syncytial virus (RSV) A in 13 of 15 (86.7%) positive control samples. Additionally, rotavirus, torque teno virus, human papillomavirus, human betaherpesvirus 7, cyclovirus, vientovirus, gemycircularvirus, and statovirus were identified through mNGS. Notably, complete genomes of novel cyclovirus, gemycircularvirus, and statovirus were genetically characterized. Using PCR screening, the novel cyclovirus was additionally detected in 5 and the novel gemycircularvirus in 12 of the remaining samples included for mNGS analysis. Our studies therefore provide pioneering data of the virome of acute-respiratory diseases from individuals at risk of zoonotic infections. The mNGS protocol/pipeline applied here is sensitive for the detection of a variety of viruses, including novel ones. More frequent detections of the novel viruses by PCR than by mNGS on the same samples suggests that PCR remains the most sensitive diagnostic test for viruses whose genomes are known. The detection of novel viruses expands our understanding of the respiratory virome of animal-exposed humans and warrant further studies.

High prevalence of cyclovirus Vietnam (CyCV-VN) in plasma samples from Madagascan healthy blood donors.

Cycloviruses, small ssDNA viruses belonging to the Circoviridae family, have been suggested as possible causes of enteric, respiratory and neurological disorders in human patients. One of these species, cyclovirus-Vietnam (CyCV-VN), initially isolated from cerebrospinal fluid samples of patients with unexplained neurological disorders, has since been reported in serum samples from chronically patients infected with HBV, HCV or HIV, in Italy. On the other hand, CyCV-VN was not detected in serum samples from healthy individuals. Here, we report on a high prevalence of 43.4% (40/92) of CyCV-VN in plasma samples from asymptomatic blood donors from Madagascar. Interestingly, this virus was not detected by metagenomics and PCR in six other African countries, suggesting regional differences in CyCV-VN prevalence across Africa. Phylogenetic analysis based on the complete genomes showed that CyCV-VN sequences isolated from blood were most closely related to sequences previously reported from human stool in Madagascar. Further investigations using larger cohorts are required to determine the global epidemiology, the natural history and the pathological significance, if any, of CyCV-VN infection in humans.

Cyclovirus Vietnam DNA in immunodeficient patients.

BACKGROUND: Cyclovirus Vietnam (CyCV-VN) is a CyCV detected in 2013 from cerebrospinal fluid (CSF) samples of patients with neurological disorders. Information on prevalence, pathogenesis and disease association of CyCV-VN is still very patchy. OBJECTIVES AND STUDY DESIGN: In this study, we have used a PCR assay targeting the Rep gene to investigate the prevalence of CyCV-VN infection in blood and CSF samples of 346 Italian subjects. RESULTS: Overall, 7% of blood samples were positive for CyCV-VN while the virus was not detected in any of the CSF samples. The prevalence of CyCV-VN was relatively high in HIV positive patients (21%), modest in patients with HBV or HCV infection (6%), and low in transplant recipient patients (2%). Positive patients showed low levels of CyCV-VN viremia. The virus was not detected in serum samples from healthy individuals. Longitudinal analysis of serum samples obtained from selected patients showed a stable or transient presence of circulating CyCV-VN. CONCLUSIONS: The present study is the first to demonstrate CyCV-VN DNA circulation in Italy and to cast light on some biological aspects of this novel virus of men.

Cycloviruses, gemycircularviruses and other novel replication-associated protein encoding circular viruses in Pacific flying fox (Pteropus tonganus) faeces.

Viral metagenomic studies have demonstrated that animal faeces can be a good sampling source for exploring viral diversity associated with the host and its environment. As part of an continuing effort to identify novel circular replication-associated protein encoding single-stranded (CRESS) DNA viruses circulating in the Tongan archipelago, coupled with the fact that bats are a reservoir species of a large number of viruses, we used a metagenomic approach to investigate the CRESS DNA virus diversity in Pacific flying fox (Pteropus tonganus) faeces. Faecal matter from four roosting sites located in Ha'avakatolo, Kolovai, Ha'ateiho and Lapaha on Tongatapu Island was collected in April 2014 and January 2015. From these samples we identified five novel cycloviruses representing three putative species, 25 gemycircularviruses representing at least 14 putative species, 17 other CRESS DNA viruses (15 putative species), two circular DNA molecules and a putative novel multi-component virus for which we have identified three cognate molecules. This study demonstrates that there exists a large diversity of CRESS DNA viruses in Pacific flying fox faeces.

Small circular single stranded DNA viral genomes in unexplained cases of human encephalitis, diarrhea, and in untreated sewage.

Viruses with small circular ssDNA genomes encoding a replication initiator protein can infect a wide range of eukaryotic organisms ranging from mammals to fungi. The genomes of two such viruses, a cyclovirus (CyCV-SL) and gemycircularvirus (GemyCV-SL) were detected by deep sequencing of the cerebrospinal fluids of Sri Lankan patients with unexplained encephalitis. One and three out of 201 CSF samples (1.5%) from unexplained encephalitis patients tested by PCR were CyCV-SL and GemyCV-SL DNA positive respectively. Nucleotide similarity searches of pre-existing metagenomics datasets revealed closely related genomes in feces from unexplained cases of diarrhea from Nicaragua and Brazil and in untreated sewage from Nepal. Whether the tropism of the cyclovirus and gemycircularvirus reported here include humans or other cellular sources in or on the human body remains to be determined.

False-positive PCR detection of cyclovirus Malawi strain VS5700009 in human cerebrospinal fluid.

BACKGROUND: Cyclovirus (CyCV) Malawi strain VS5700009 has recently been discovered and reported in clinical cerebrospinal fluid (CSF) samples. Further epidemiological and case-control studies are warranted. The availability of a highly sensitive and specific detection assay for this new virus is thus crucial. OBJECTIVES: To evaluate the performance of the first and the only available PCR assay for CyCV-VS5700009. STUDY DESIGN: A total of 100 CSF samples collected during January-December 2010 were selected for PCR detection of CyCV-VS5700009. Positive PCR amplicons were subjected to sequencing confirmation and BLAST analysis. RESULTS: Initial PCR screening for CyCV-VS5700009 identified one sample, showing a PCR band of expected size (380 bp). Sequencing and BLAST analysis, however, indicated that the band was 364 bp in length and showed >99% nucleotide homology to a human gene known as nuclear receptor coactivator 6 (NCOA6). Pairwise sequence alignment confirmed that both the forward and reverse PCR primers used had significant homology (>70%) to NCOA6. None of the CSF samples tested were positive for CyCV-VS5700009. CONCLUSIONS: The original PCR assay for CyCV-VS5700009 detection may have potential cross-reactivity with contaminating human genomic DNA. The assay may be of little diagnostic use on clinical specimens that are rich in host DNA such as biopsy tissues.