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.

Circoviruses and cycloviruses identified in Weddell seal fecal samples from McMurdo Sound, Antarctica.

Circoviridae is a family of circular single-stranded DNA viruses whose members infect a wide variety of hosts. While well characterized in avian and mammalian hosts, little is known about circoviruses associated with Antarctic animals. From 48 Weddell seal (Leptonychotes weddellii) fecal samples collected on the sea ice in McMurdo between Nov 2014 and Dec 2014, we identified and determined the genomes of novel viruses that fall within two genera of the family Circoviridae, i.e. Circovirus (n = 7) and Cyclovirus (n = 45). We named these viruses as werosea circovirus (WerCV) and werosea cyclovirus (WerCyV). The genomes of WerCV and WerCyV share ~63-64% genome-wide pairwise identity with classified circoviruses and cycloviruses, respectively. Based on the species demarcation threshold of 80% for members of the Circoviridae, the genomes of WerCV and WerCyV represent new species in their respective genera. Evidence indicated recombination in five of the 45 WerCyV genomes identified in this study. These are the first circoviruses found associated with Antarctic pinnipeds, adding to those recently identified associated with Adélie (Pygoscelis adeliae) and chinstrap penguins (P. antarcticus).

Metatranscriptomic Analysis of Virus Diversity in Urban Wild Birds with Paretic Disease.

Wild birds are major natural reservoirs and potential dispersers of a variety of infectious diseases. As such, it is important to determine the diversity of viruses they carry and use this information to help understand the potential risks of spillover to humans, domestic animals, and other wildlife. We investigated the potential viral causes of paresis in long-standing, but undiagnosed, disease syndromes in wild Australian birds. RNA from diseased birds was extracted and pooled based on tissue type, host species, and clinical manifestation for metagenomic sequencing. Using a bulk and unbiased metatranscriptomic approach, combined with clinical investigation and histopathology, we identified a number of novel viruses from the families Astroviridae, Adenoviridae, Picornaviridae, Polyomaviridae, Paramyxoviridae, Parvoviridae, and Circoviridae in common urban wild birds, including Australian magpies, magpie larks, pied currawongs, Australian ravens, and rainbow lorikeets. In each case, the presence of the virus was confirmed by reverse transcription (RT)-PCR. These data revealed a number of candidate viral pathogens that may contribute to coronary, skeletal muscle, vascular, and neuropathology in birds of the Corvidae and Artamidae families and neuropathology in members of the Psittaculidae The existence of such a diverse virome in urban avian species highlights the importance and challenges in elucidating the etiology and ecology of wildlife pathogens in urban environments. This information will be increasingly important for managing disease risks and conducting surveillance for potential viral threats to wildlife, livestock, and human health.IMPORTANCE Wildlife naturally harbor a diverse array of infectious microorganisms and can be a source of novel diseases in domestic animals and human populations. Using unbiased RNA sequencing, we identified highly diverse viruses in native birds from Australian urban environments presenting with paresis. This research included the clinical investigation and description of poorly understood recurring syndromes of unknown etiology: clenched claw syndrome and black and white bird disease. As well as identifying a range of potentially disease-causing viral pathogens, this study describes methods that can effectively and efficiently characterize emergent disease syndromes in free-ranging wildlife and promotes further surveillance for specific pathogens of potential conservation and zoonotic concern.

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.

Complete genome sequences of three novel cycloviruses identified in a dragonfly (Odonata: Anisoptera) from China.

Three cycloviruses (genus Cyclovirus, family Circoviridae) were recovered from a dragonfly (Odonata: Anisoptera) captured in Fuzhou, China. The three cycloviruses, named dragonfly associated cyclovirus 9, 10 and 11 (DfCyV-9, -10, -11), respectively, show 56.1-79.6% genome-wide identity to known cycloviruses and 61.6-65.1% among themselves. Thus, according to the current species demarcation criteria, they represent three novel cycloviruses. Notably, DfCyV-10 has a predicted replication-associated protein (Rep) that is most similar to that of bat associated cyclovirus 2 (BatACyV-2), a cyclovirus discovered in China, with 79.4% amino acid sequence identity, but a putative capsid protein (Cp) most similar to that of BatACyV-10, a cyclovirus discovered in Brazil, with 71.7% amino acid sequence identity. These data are useful for understanding the diversity and evolution of cycloviruses, especially those found in insects.

Diverse replication-associated protein encoding circular DNA viruses in guano samples of Central-Eastern European bats.

Circular replication-associated protein encoding single-stranded DNA (CRESS DNA) viruses are increasingly recognized worldwide in a variety of samples. Representative members include well-described veterinary pathogens with worldwide distribution, such as porcine circoviruses or beak and feather disease virus. In addition, numerous novel viruses belonging to the family Circoviridae with unverified pathogenic roles have been discovered in different human samples. Viruses of the family Genomoviridae have also been described as being highly abundant in different faecal and environmental samples, with case reports showing them to be suspected pathogens in human infections. In order to investigate the genetic diversity of these viruses in European bat populations, we tested guano samples from Georgia, Hungary, Romania, Serbia and Ukraine. This resulted in the detection of six novel members of the family Circoviridae and two novel members of the family Genomoviridae. Interestingly, a gemini-like virus, namely niminivirus, which was originally found in raw sewage samples in Nigeria, was also detected in our samples. We analyzed the nucleotide composition of members of the family Circoviridae to determine the possible host origins of these viruses. This study provides the first dataset on CRESS DNA viruses of European bats, and members of several novel viral species were discovered.

Genome sequence of a mallard duck origin cyclovirus, DuACyV-1.

The genome sequence of a novel avian cyclovirus is described in this study. The genome size and orientation of predicted genes was similar to those described in other vertebrate and insect origin cycloviruses. The greatest genome sequence identity was shared with a dragonfly cyclovirus (nt, 60.6%). Phylogenetic analysis showed marginal relatedness with another avian cyclovirus, the chicken associated cyclovirus 1. In contrast, along a short fragment of the replication-associated protein coding gene (rep) (spanning nt 1240-1710) the duck origin cyclovirus was very similar to human origin and honey bee origin rep sequences (human - TN4, 98%; honey bee - hb10, 100%). Related cyclovirus strains existing amongst various animal species living in diverse ecosystems and separated by large geographic distances show the need for additional studies to better understand the ecology and epidemiology of cycloviruses.

ICTV Virus Taxonomy Profile: Circoviridae.

The family Circoviridae comprises viruses with small, circular, single-stranded DNA (ssDNA) genomes, including the smallest known animal viruses. Members of this family are classified into two genera, Circovirus and Cyclovirus, which are distinguished by the position of the origin of replication relative to the coding regions and the length of the intergenic regions. Within each genus, the species demarcation threshold is 80 % genome-wide nucleotide sequence identity. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Circoviridae, which is available at www.ictv.global/report/circoviridae.

Highly divergent cyclo-like virus in a great roundleaf bat (Hipposideros armiger) in Vietnam.

Members of the viral family Circoviridae are increasingly recognized worldwide. Bats seem to be natural reservoirs or dietary-related dispensers of these viruses. Here, we report a distantly related member of the genus Cyclovirus detected in the faeces of a great roundleaf bat (Hipposideros armiger). Interestingly, the novel virus lacks a Circoviridae-specific stem-loop structure, although a Geminiviridae-like nonamer sequence was detected in the large intergenic region. Based on these differences and its phylogenetic position, we propose that our new virus represents a distant and highly divergent member of the genus Cyclovirus. However it is lacking several characteristics of members of the genus, which raises a challenge in its taxonomic classification.

Novel cyclovirus detected in the intestinal contents of Taiwan squirrels (Callosciurus erythraeus thaiwanensis).

A novel cyclovirus was identified in the intestinal contents of Taiwan squirrels (Callosciurus erythraeus thaiwanensis) collected in Kanagawa prefecture, Japan, by metagenomic analysis, and was named Taiwan squirrel cyclovirus-1 (TsCyV-1). Phylogenetic analysis showed that TsCyV-1 formed a branch separate from other representative cyclovirus strains. TsCyV-1 is considered to be a new species in the genus Cyclovirus because the criteria for demarcation of cyclovirus species is proposed as nucleotide identities <80 %.

Distinct circular single-stranded DNA viruses exist in different soil types.

The potential dependence of virus populations on soil types was examined by electron microscopy, and the total abundance of virus particles in four soil types was similar to that previously observed in soil samples. The four soil types examined differed in the relative abundances of four morphological groups of viruses. Machair, a unique type of coastal soil in western Scotland and Ireland, differed from the others tested in having a higher proportion of tailed bacteriophages. The other soils examined contained predominantly spherical and thin filamentous virus particles, but the Machair soil had a more even distribution of the virus types. As the first step in looking at differences in populations in detail, virus sequences from Machair and brown earth (agricultural pasture) soils were examined by metagenomic sequencing after enriching for circular Rep-encoding single-stranded DNA (ssDNA) (CRESS-DNA) virus genomes. Sequences from the family Microviridae (icosahedral viruses mainly infecting bacteria) of CRESS-DNA viruses were predominant in both soils. Phylogenetic analysis of Microviridae major coat protein sequences from the Machair viruses showed that they spanned most of the diversity of the subfamily Gokushovirinae, whose members mainly infect obligate intracellular parasites. The brown earth soil had a higher proportion of sequences that matched the morphologically similar family Circoviridae in BLAST searches. However, analysis of putative replicase proteins that were similar to those of viruses in the Circoviridae showed that they are a novel clade of Circoviridae-related CRESS-DNA viruses distinct from known Circoviridae genera. Different soils have substantially different taxonomic biodiversities even within ssDNA viruses, which may be driven by physicochemical factors.

Genomic characterization of novel circular ssDNA viruses from insectivorous bats in Southern Brazil.

Circoviruses are highly prevalent porcine and avian pathogens. In recent years, novel circular ssDNA genomes have recently been detected in a variety of fecal and environmental samples using deep sequencing approaches. In this study the identification of genomes of novel circoviruses and cycloviruses in feces of insectivorous bats is reported. Pan-reactive primers were used targeting the conserved rep region of circoviruses and cycloviruses to screen DNA bat fecal samples. Using this approach, partial rep sequences were detected which formed five phylogenetic groups distributed among the Circovirus and the recently proposed Cyclovirus genera of the Circoviridae. Further analysis using inverse PCR and Sanger sequencing led to the characterization of four new putative members of the family Circoviridae with genome size ranging from 1,608 to 1,790 nt, two inversely arranged ORFs, and canonical nonamer sequences atop a stem loop.

Limited geographic distribution of the novel cyclovirus CyCV-VN.

A novel cyclovirus, CyCV-VN, was recently identified in cerebrospinal fluid (CSF) from patients with central nervous system (CNS) infections in central and southern Vietnam. To explore the geographic distribution of this novel virus, more than 600 CSF specimens from patients with suspected CNS infections in northern Vietnam, Cambodia, Nepal and The Netherlands were screened for the presence of CyCV-VN but all were negative. Sequence comparison and phylogenetic analysis between CyCV-VN and another novel cyclovirus recently identified in CSF from Malawian patients indicated that these represent distinct cycloviral species, albeit phylogenetically closely related. The data suggest that CyCV-VN has a limited geographic distribution within southern and central Vietnam. Further research is needed to determine the global distribution and diversity of cycloviruses and importantly their possible association with human disease.

Two novel circo-like viruses detected in human feces: complete genome sequencing and electron microscopy analysis.

The application of viral metagenomic techniques and a series of PCRs in a human fecal sample enabled the detection of two novel circular unisense DNA viral genomes with 92% nucleotide similarity. The viruses were tentatively named circo-like virus-Brazil (CLV-BR) strains hs1 and hs2 and have genome lengths of 2526 and 2533 nucleotides, respectively. Four major open reading frames (ORFs) were identified in each of the genomes, and differences between the two genomes were primarily observed in ORF 2. Only ORF 3 showed significant amino acid similarities to a putative rolling circle replication initiator protein (Rep), although with low identity (36%). Our phylogenetic analysis, based on the Rep protein, demonstrated that the CLV-BRs do not cluster with members of the Circoviridae, Nanoviridae or Geminiviridae families and are more closely related to circo-like genomes previously identified in reclaimed water and feces of a wild rodent and of a bat. The CLV-BRs are members of a putative new family of circular Rep-encoding ssDNA viruses. Electron microscopy revealed icosahedral (~23 nm) structures, likely reflecting the novel viruses, and rod-shaped viral particles (~65-460 × 21 × 10 nm in length, diameter, and axial canal, respectively). Circo-like viruses have been detected in stool samples from humans and other mammals (bats, rodents, chimpanzees and bovines), cerebrospinal fluid and sera from humans, as well as samples from many other sources, e.g., insects, meat and the environment. Further studies are needed to classify all novel circular DNA viruses and elucidate their hosts, pathogenicity and evolutionary history.

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.

Identification of a new cyclovirus in cerebrospinal fluid of patients with acute central nervous system infections.

Acute central nervous system (CNS) infections cause substantial morbidity and mortality, but the etiology remains unknown in a large proportion of cases. We identified and characterized the full genome of a novel cyclovirus (tentatively named cyclovirus-Vietnam [CyCV-VN]) in cerebrospinal fluid (CSF) specimens of two Vietnamese patients with CNS infections of unknown etiology. CyCV-VN was subsequently detected in 4% of 642 CSF specimens from Vietnamese patients with suspected CNS infections and none of 122 CSFs from patients with noninfectious neurological disorders. Detection rates were similar in patients with CNS infections of unknown etiology and those in whom other pathogens were detected. A similar detection rate in feces from healthy children suggested food-borne or orofecal transmission routes, while high detection rates in feces from pigs and poultry (average, 58%) suggested the existence of animal reservoirs for such transmission. Further research is needed to address the epidemiology and pathogenicity of this novel, potentially zoonotic virus.

High global diversity of cycloviruses amongst dragonflies.

Members of the family Circoviridae, specifically the genus Circovirus, were thought to infect only vertebrates; however, members of a sister group under the same family, the proposed genus Cyclovirus, have been detected recently in insects. In an effort to explore the diversity of cycloviruses and better understand the evolution of these novel ssDNA viruses, here we present five cycloviruses isolated from three dragonfly species (Orthetrum sabina, Xanthocnemis zealandica and Rhionaeschna multicolor) collected in Australia, New Zealand and the USA, respectively. The genomes of these five viruses share similar genome structure to other cycloviruses, with a circular ~1.7 kb genome and two major bidirectionally transcribed ORFs. The genomic sequence data gathered during this study were combined with all cyclovirus genomes available in public databases to identify conserved motifs and regulatory elements in the intergenic regions, as well as determine diversity and recombinant regions within their genomes. The genomes reported here represent four different cyclovirus species, three of which are novel. Our results confirm that cycloviruses circulate widely in winged-insect populations; in eight different cyclovirus species identified in dragonflies to date, some of these exhibit a broad geographical distribution. Recombination analysis revealed both intra- and inter-species recombination events amongst cycloviruses, including genomes recovered from disparate sources (e.g. goat meat and human faeces). Similar to other well-characterized circular ssDNA viruses, recombination may play an important role in cyclovirus evolution.

Molecular and microscopic evidence of viruses in marine copepods.

As dominant members of marine mesozooplankton communities, copepods play critical roles in oceanic food webs and biogeochemical cycling. Despite the ecological significance of copepods, little is known regarding the causes of copepod mortality, and up to 35% of total copepod mortality cannot be accounted for by predation alone. Viruses have been established as ecologically important infectious agents in the oceans; however, viral infection has not been investigated in mesozooplankton communities. Here we used molecular and microscopic techniques to document viral infection in natural populations of the calanoid copepods Acartia tonsa (Dana) and Labidocera aestiva (Wheeler) in Tampa Bay, FL. Viral metagenomics revealed previously undocumented viruses in each species, named Acartia tonsa copepod circo-like virus (AtCopCV) and Labidocera aestiva copepod circo-like virus (LaCopCV). LaCopCV was found to be extremely prevalent and abundant in L. aestiva populations, with up to 100% prevalence in some samples and average viral loads of 1.13 × 10(5) copies per individual. LaCopCV transcription was also detected in the majority of L. aestiva individuals, indicating viral activity. AtCopCV was sporadically detected in A. tonsa populations year-round, suggesting temporal variability in viral infection dynamics. Finally, virus-like particles of unknown identity were observed in the connective tissues of A. tonsa and L. aestiva by transmission electron microscopy, demonstrating that viruses were actively proliferating in copepod connective tissue as opposed to infecting gut contents, parasites, or symbionts. Taken together, these results provide strong independent lines of evidence for active viral infection in dominant copepod species, indicating that viruses may significantly influence mesozooplankton ecology.

Diverse circular ssDNA viruses discovered in dragonflies (Odonata: Epiprocta).

Viruses with circular ssDNA genomes that encode a replication initiator protein (Rep) are among the smallest viruses known to infect both eukaryotic and prokaryotic organisms. In the past few years an overwhelming diversity of novel circular Rep-encoding ssDNA (CRESS-DNA) viruses has been unearthed from various hosts and environmental sources. Since there is limited information regarding CRESS-DNA viruses in invertebrates, this study explored the diversity of CRESS-DNA viruses circulating among insect populations by targeting dragonflies (Epiprocta), top insect predators that accumulate viruses from their insect prey over space and time. Using degenerate PCR and rolling circle amplification coupled with restriction digestion, 17 CRESS-DNA viral genomes were recovered from eight different dragonfly species collected in tropical and temperate regions. Nine of the genomes are similar to cycloviruses and represent five species within this genus, suggesting that cycloviruses are commonly associated with insects. Three of the CRESS-DNA viruses share conserved genomic features with recently described viruses similar to the mycovirus Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1, leading to the proposal of the genus Gemycircularvirus. The remaining viruses are divergent species representing four novel CRESS-DNA viral genera, including a gokushovirus-like prokaryotic virus (microphage) and three eukaryotic viruses with Reps similar to circoviruses. The novelty of CRESS-DNA viruses identified in dragonflies using simple molecular techniques indicates that there is an unprecedented diversity of ssDNA viruses among insect populations.