Viral metagenomics characterizes known and identifies unknown viruses
based on sequence similarities to any previously sequenced viral genomes. A
metagenomics approach was used to identify virus sequences in Australian
mosquitoes causing cytopathic effects in inoculated mammalian cell cultures.
Sequence comparisons revealed strains of Liao Ning virus
(Reovirus, Seadornavirus), previously
detected only in China, livestock-infecting Stretch Lagoon virus
(Reovirus, Orbivirus), two novel
dimarhabdoviruses, named Beaumont and North Creek viruses, and two novel
orthobunyaviruses, named Murrumbidgee and Salt Ash viruses. The novel virus
proteomes diverged by ≥50% relative to their closest previously
genetically characterized viral relatives. Deep sequencing also generated
genomes of Warrego and Wallal viruses, orbiviruses linked to kangaroo blindness,
whose genomes had not been fully characterized. This study highlights viral
metagenomics in concert with traditional arbovirus surveillance to characterize
known and new arboviruses in field-collected mosquitoes. Follow-up
epidemiological studies are required to determine whether the novel viruses
virus discovery; deep sequencing; arbovirus surveillance; bunyavirus; rhabdovirus; reovirus; novel virus; mosquito; Australia
Members of the family Picornaviridae consist of small positive-sense single-stranded RNA (+ssRNA) viruses capable of infecting various vertebrate species, including birds. One of the recently identified avian picornaviruses, with a remarkably long (>9,040-nucleotide) but still incompletely sequenced genome, is turkey hepatitis virus 1 (THV-1; species Melegrivirus A, genus Megrivirus), a virus associated with liver necrosis and enteritis in commercial turkeys (Meleagris gallopavo). This report presents the results of the genetic analysis of three complete genomes of megriviruses from fecal samples of chickens (chicken/B21-CHV/2012/HUN, GenBank accession no. KF961186, and chicken/CHK-IV-CHV/2013/HUN, GenBank accession no. KF961187) (Gallus gallus domesticus) and turkey (turkey/B407-THV/2011/HUN, GenBank accession no. KF961188) (Meleagris gallopavo) with the largest picornavirus genome (up to 9,739 nucleotides) so far described. The close phylogenetic relationship to THV-1 in the nonstructural protein-coding genome region and possession of the same internal ribosomal entry site type (IVB-like) suggest that the study strains belong to the genus Megrivirus. However, the genome comparisons revealed numerous unique variations (e.g., different numbers of potential 2A peptides, unusually long 3′ genome parts with various lengths of a potential second open reading frame, and multiple repeating sequence motifs in the 3′ untranslated region) and heterogeneous sequence relationships between the structural and nonstructural genome regions. These differences suggest the classification of chicken megrivirus-like viruses into a candidate novel species in the genus Megrivirus. Based on the different phylogenetic positions of chicken megrivirus-like viruses at the structural and nonstructural genome regions, the recombinant nature of these viruses is plausible.
IMPORTANCE The comparative genome analysis of turkey and novel chicken megriviruses revealed numerous unique genome features, e.g., up to four potential 2A peptides, unusually long 3′ genome parts with various lengths containing a potential second open reading frame, multiple repeating sequence motifs, and heterogeneous sequence relationships (possibly due to a recombination event) between the structural and nonstructural genome regions. Our results could help us to better understand the evolution and diversity (in terms of sequence and genome layout) of picornaviruses.
We describe a 8,724-nucleotide-long picornavirus genome encoding a single 2,470-aa polyprotein obtained from the feces of a wild mouse. Rosavirus is genetically closest to the double ORF Dicipivirus found in canine feces that is currently the only picornavirus with a second internal ribosome entry site (IRES). Of note, a section of rosavirus’ 5′UTR showed strong sequence and structural conservation with the type II IRES from the Parechovirus and Hungarovirus genera possibly reflecting exchange of genetic modules between genera. Based on genetic distance criteria rosavirus qualifies as prototype of a new genus of the Picornaviridae family.
Picornavirus; Feces; Mice; Genus
A divergent parvovirus genome was the only eukaryotic viral sequence detected in feces of a Tunisian child with unexplained diarrhea. Tusavirus 1 shared 44% and 39% identity with the nonstructural protein 1 and viral protein 1, respectively, of the closest genome, Kilham rat parvovirus, indicating presence of a new human viral species in the Protoparvovirus genus.
parvovirus; diarrhea; feces; child; viruses; Tunisia; Tusavirus
The Gyrovirus genus consists of the immunosuppressive Chicken Anemia Virus (CAV) prototype and since 2011 three other viral species found in sera/tissues of chickens, human feces, and on human skin. Here the genomes of two other gyrovirus species were characterized in diarrhea samples from Tunisian children whose main ORFs shared amino acid identity of 46–59% with those of the previously characterized gyroviruses and were provisionally named GyV5 and GyV6. All known gyroviruses grouped into two clades with distinct genomic features including replacement of the VP2 overlapping Apoptin gene with a distinct ORF of unknown function. Previous reports of gyrovirus DNA in human blood and on human skins warrant studies of possible human tropisms for these newly characterized gyroviruses.
Gyrovirus; diarrhea; feces; children
Using metagenomics and molecular cloning methods, we characterized five novel small, circular viral genomes from pig feces that are distantly related to chimpanzee and porcine stool-associated circular viruses, (ChiSCV and PoSCV1). Phylogenetic analysis placed these viruses into a highly divergent clade of this rapidly growing new viral family. This new clade of viruses, provisionally named porcine stool-associated circular virus 2 and 3 (PoSCV2 and PoSCV3), encodes a stem–loop structure (presumably the origin of DNA replication) in the small intergenic region and a replication initiator protein commonly found in other biological systems that replicate their genomes via the rolling–circle mechanism. Furthermore, these viruses also exhibit three additional overlapping open reading frames in the large intergenic region between the capsid and replication initiator protein genes.
Deep sequencing; Porcine stool-associated circular virus
Despite the continuously growing number of known avian picornaviruses (family Picornaviridae), knowledge of their genetic diversity in wild birds, especially in long-distance migrant species is very limited. In this study, we report the presence of a novel picornavirus identified from one of 18 analysed faecal samples of an Afro-Palearctic migrant bird, the European roller (Coracias garrulus L., 1758), which is distantly related to the marine-mammal-infecting seal aquamavirus A1 (genus Aquamavirus). The phylogenetic analyses and the low sequence identity (P1 26.3 %, P2 25.8 % and P3 28.4 %) suggest that this picornavirus could be the founding member of a novel picornavirus genus that we have provisionally named ‘Kunsagivirus’, with ‘Greplavirus A’ (strain roller/SZAL6-KuV/2011/HUN, GenBank accession no. KC935379) as the candidate type species.
This study reports the metagenomic detection and complete genome characterization of a novel turkey picornavirus from faecal samples of healthy (1/3) and affected (6/8) commercial turkeys with enteric and/or stunting syndrome in Hungary. The virus was detected at seven of the eight farms examined. The turkey/M176-TuASV/2011/HUN genome (KC465954) was genetically different from the currently known picornaviruses of turkey origin (megriviruses and galliviruses), and showed distant phylogenetic relationship and common genomic features (e.g. uncleaved VP0 and three predicted and unrelated 2A polypeptides) to duck hepatitis A virus (DHAV) of the genus Avihepatovirus. The complete genome analysis revealed multiple distinct genome features like the presence of two in-tandem aphthovirus 2A-like sequence repeats with DxExNPG/P ‘ribosome-skipping’ sites (76 %, 23/30 amino acids identical), with the first aphthovirus 2A-like sequence being located at the end of the VP1 capsid protein (VP1/2A1 ‘ribosome-skipping’ site). The phylogenetic analyses, low sequence identity (33, 32 and 36 % amino acid identity in P1, P2 and P3 regions) to DHAV, and the type II-like internal ribosome entry site suggests that this turkey picornavirus is related to, but distinct from the genus Avihepatovirus and it could be the founding member of a novel Avihepatovirus sister-clade genus. This is the third, taxonomically highly distinct picornavirus clade identified from turkeys exhibiting varied symptoms.
Parvovirus; bufavirus; bocavirus; viruses; Primate protoparvovirus; diarrhea; quantitative PCR; gastroenteritis; Burkina Faso; Finland
The near-complete genomes of two picobirnaviruses (PBVs) in diarrheal stool samples, human picobirnaviruses D and E (HuPBV-D and -E), were genetically characterized. Their RNA-dependent RNA polymerase (RdRp) protein sequences had <66% identities to known PBVs. Due to a single nucleotide insertion, the open reading frame 2 (ORF2) in segment 1 of HuPBV-D was interrupted by a stop codon. A small stem-loop structure overlying the stop codon may result in translational readthrough into the rest of ORF2.
Porcine stool-associated circular virus 5 (PoSCV5) was detected in the feces of a pig with diarrhea. The complete 3,062-nucleotide genome contains two bidirectionally transcribed open reading frames (ORFs). Phylogenetic analysis of the deduced replication initiator protein (Rep) places PoSCV5 alone on a deep branch among the small circular Rep-encoding single-stranded DNA viruses.
Using a metagenomic approach and molecular cloning methods, we identified, cloned, and sequenced the complete genome of a novel circular DNA virus, porcine stool-associated virus (PoSCV4), from pig feces. Phylogenetic analysis of the deduced replication initiator protein showed that PoSCV4 is most related to a fur seal feces-associated circular DNA virus.
We compared the plasma viromes of HIV-infected subjects with low versus high CD4+ T cell counts from the United States and Uganda by using deep sequencing and detected HIV, hepatitis C virus, hepatitis B virus, GB virus C, anellovirus, and human endogenous retrovirus (HERV) reads. An increase in the proportion of reads for anelloviruses, a family of highly prevalent and genetically diverse human viruses, was seen in subjects with AIDS from both countries. The proportion of endogenous human retrovirus reads was increased in AIDS subjects from Uganda but not the United States. Progression to AIDS is therefore associated with changes in the plasma concentration of commensal viruses.
During both regulatory and routine surveillance sampling of baitfish from the states of Illinois, Minnesota, Montana, and Wisconsin, USA, isolates (n = 20) of a previously unknown picornavirus were obtained from kidney/spleen or entire viscera of fathead minnows (Pimephales promelas) and brassy minnows (Hybognathus hankinsoni). Following the appearance of a diffuse cytopathic effect, examination of cell culture supernatant by negative contrast electron microscopy revealed the presence of small, round virus particles (∼30–32 nm), with picornavirus-like morphology. Amplification and sequence analysis of viral RNA identified the agent as a novel member of the Picornaviridae family, tentatively named fathead minnow picornavirus (FHMPV). The full FHMPV genome consisted of 7834 nucleotides. Phylogenetic analysis based on 491 amino acid residues of the 3D gene showed 98.6% to 100% identity among the 20 isolates of FHMPV compared in this study while only 49.5% identity with its nearest neighbor, the bluegill picornavirus (BGPV) isolated from bluegill (Lepomis macrochirus). Based on complete polyprotein analysis, the FHMPV shared 58% (P1), 33% (P2) and 43% (P3) amino acid identities with BGPV and shared less than 40% amino acid identity with all other picornaviruses. Hence, we propose the creation of a new genus (Piscevirus) within the Picornaviridae family. The impact of FHMPV on the health of fish populations is unknown at present.
Defining clear hepatitis C virus (HCV) infection outcomes, including reinfection and viral intercalation after clearance of infection, requires ongoing, frequent follow-up, most importantly with longitudinal viral sequencing. Patients who have cleared HCV infection may demonstrate sustained viral clearance despite ongoing HCV exposure.
Background. Detection of hepatitis C virus (HCV) reinfection and intercalation (ie, intermittent recurrent bouts of viremia with homologous virus interspersed with aviremic periods) requires extensive and frequent evaluation and viral sequencing.
Methods. HCV infection outcomes were studied prospectively in active injection drug users with recurrent HCV RNA–positive tests after serial negative results. HCV viremia and viral sequences (Core/E1) were assessed from monthly blood samples.
Results. Viral clearance, reinfection, and intercalating infection were all detected. Among 44 participants with apparently resolved HCV (26 incident HCV clearers and 18 enrolled with already resolved infection), 36 (82%) remained persistently HCV RNA negative, but 8 demonstrated intermittent recurrent viremia. Four of these (50%) had confirmed reinfection with a heterologous virus; 3 demonstrated viral intercalation, and 1 was not classifiable as either. Estimated incidence of first reinfection was 5.4 per 100 person-years (95% confidence interval, 2.0–14.5). Six (75%) participants, including 3 of 4 with reinfection, demonstrated sustained viral clearance for a median of 26 months since last HCV RNA test.
Conclusions. These results show that frequent monitoring and viral sequencing are required to correctly assess HCV outcomes and estimate incidence of reinfection (which was previously overestimated). Sustained clearance may take many months and occur after episodes of reinfection and viral intercalation. Three of 4 subjects who had confirmed reinfection showed evidence of long-term clearance. Viral intercalation occurs with significant frequency. Further studies of these events, especially immunological, are needed to inform HCV clinical care and vaccine development.
hepatitis C virus; viral sequencing; reinfection; intercalation; young IDU
Fecal samples (N=10) from 6- to 8-week-old wild boar piglets (Sus scrofa), collected from an animal park in Hungary in April 2011, were analyzed using viral metagenomics and complete genome sequencing. Kobuvirus (genus Kobuvirus, family Picornaviridae) was detected in all (100%) specimens, with the closest nucleotide (89%) and amino acid (94%) sequence identity of the strain wild boar/WB1-HUN/2011/HUN (JX177612) to the prototype porcine kobuvirus S-1-HUN (EU787450). This study suggests that genetically highly similar (practically the same geno-/serotype) porcine kobuvirus circulate in wild boars, the wildlife counterparts of domestic pigs. Wild boars could be an important host and reservoir for kobuvirus.
We identified a novel rhabdovirus, American bat vesiculovirus, from postmortem tissue samples from 120 rabies-negative big brown bats with a history of human contact. Five percent of the tested bats were infected with this virus. The extent of zoonotic exposure and possible health effects in humans from this virus are unknown.
Rhabdoviridae; vesiculovirus; viral metagenomic; bats; virus discovery; big brown bats; viruses; North America; Maryland; United States; rabies; Eptesicus fuscus
Next-generation sequencing was used for discovery and de novo assembly of a novel, highly divergent DNA virus at the interface between the Parvoviridae and Circoviridae. The virus, provisionally named parvovirus-like hybrid virus (PHV), is nearly identical by sequence to another DNA virus, NIH-CQV, previously detected in Chinese patients with seronegative (non-A-E) hepatitis. Although we initially detected PHV in a wide range of clinical samples, with all strains sharing ∼99% nucleotide and amino acid identity with each other and with NIH-CQV, the exact origin of the virus was eventually traced to contaminated silica-binding spin columns used for nucleic acid extraction. Definitive confirmation of the origin of PHV, and presumably NIH-CQV, was obtained by in-depth analyses of water eluted through contaminated spin columns. Analysis of environmental metagenome libraries detected PHV sequences in coastal marine waters of North America, suggesting that a potential association between PHV and diatoms (algae) that generate the silica matrix used in the spin columns may have resulted in inadvertent viral contamination during manufacture. The confirmation of PHV/NIH-CQV as laboratory reagent contaminants and not bona fide infectious agents of humans underscores the rigorous approach needed to establish the validity of new viral genomes discovered by next-generation sequencing.
Members of the family Picornaviridae are important pathogens of humans and animals, although compared with the thousands of known bird species (>10 000), only a few (n = 11) picornaviruses have been identified from avian sources. This study reports the metagenomic detection and complete genome characterization of a novel turkey picornavirus from faecal samples collected from eight turkey farms in Hungary. Using RT-PCR, both healthy (two of three) and affected (seven of eight) commercial turkeys with enteric and/or stunting syndrome were shown to be shedding viruses in seven (88 %) of the eight farms. The viral genome sequence (turkey/M176/2011/HUN; GenBank accession no. JQ691613) shows a high degree of amino acid sequence identity (96 %) to the partial P3 genome region of a picornavirus reported recently in turkey and chickens from the USA and probably belongs to the same species. In the P1 and P2 regions, turkey/M176/2011/HUN is related most closely to, but distinct from, the kobuviruses and turdivirus 1. Complete genome analysis revealed the presence of characteristic picornaviral amino acid motifs, a potential type II-like 5′ UTR internal ribosome entry site (first identified among avian-origin picornaviruses) and a conserved, 48 nt long ‘barbell-like’ structure found at the 3′ UTR of turkey/M176/2011/HUN and members of the picornavirus genera Avihepatovirus and Kobuvirus. The general presence of turkey picornavirus – a novel picornavirus species – in faecal samples from healthy and affected turkeys in Hungary and in the USA suggests the worldwide occurrence and endemic circulation of this virus in turkey farms. Further studies are needed to investigate the aetiological role and pathogenic potential of this picornavirus in food animals.
Birds are frequent sources of emerging human infectious diseases. Viral particles were enriched from the feces of 51 wild urban pigeons (Columba livia) from Hong Kong and Hungary, their nucleic acids randomly amplified and then sequenced. We identified sequences from known and novel species from the viral families Circoviridae, Parvoviridae, Picornaviridae, Reoviridae, Adenovirus, Astroviridae, and Caliciviridae (listed in decreasing number of reads), as well as plant and insect viruses likely originating from consumed food. The near full genome of a new species of a proposed parvovirus genus provisionally called Aviparvovirus contained an unusually long middle ORF showing weak similarity to an ORF of unknown function from a fowl adenovirus. Picornaviruses found in both Asia and Europe that are distantly related to the turkey megrivirus and contained a highly divergent 2A1 region were named mesiviruses. All eleven segments of a novel rotavirus subgroup related to a chicken rotavirus in group G were sequenced and phylogenetically analyzed. This study provides an initial assessment of the enteric virome in the droppings of pigeons, a feral urban species with frequent human contact.
Molecular detection of viruses has been aided by high-throughput sequencing, permitting the genomic characterization of emerging strains. In this study, we comprehensively screened 500 respiratory secretions from children with upper and/or lower respiratory tract infections for viral pathogens. The viruses detected are described, including a divergent human parainfluenza virus type 4 from GS FLX pyrosequencing of 92 specimens. Complete full-genome characterization of the virus followed, using Single Molecule, Real-Time (SMRT) sequencing. Subsequent “primer walking” combined with Sanger sequencing validated the RS platform's utility in viral sequencing from complex clinical samples. Comparative genomics reveals the divergent strain clusters with the only completely sequenced HPIV4a subtype. However, it also exhibits various structural features present in one of the HPIV4b reference strains, opening questions regarding their lifecycle and evolutionary relationships among these viruses. Clinical data from patients infected with the strain, as well as viral prevalence estimates using real-time PCR, is also described.
Using viral metagenomics of brain tissue from a young adult crossbreed steer with acute onset of neurologic disease, we sequenced the complete genome of a novel astrovirus (BoAstV-NeuroS1) that was phylogenetically related to an ovine astrovirus. In a retrospective analysis of 32 cases of bovine encephalitides of unknown etiology, 3 other infected animals were detected by using PCR and in situ hybridization for viral RNA. Viral RNA was restricted to the nervous system and detected in the cytoplasm of affected neurons within the spinal cord, brainstem, and cerebellum. Microscopically, the lesions were of widespread neuronal necrosis, microgliosis, and perivascular cuffing preferentially distributed in gray matter and most severe in the cerebellum and brainstem, with increasing intensity caudally down the spinal cord. These results suggest that infection with BoAstV-NeuroS1 is a potential cause of neurologic disease in cattle.
Astrovirus; brain; neurologic disease; bovine; cattle; next-generation sequencing; in situ hybridization; viruses
The genus Teschovirus, family Picornaviridae, currently includes 12 serotypes (PTV 1 to 12) isolated from swine. PTVs have been well studied in domestic pigs, but knowledge about PTVs in wild boars is deficient. Here, we report the first complete PTV genome sequence from 7 (70%) of 10 fecal samples of wild boar piglets (Sus scrofa) by RT-PCR and pyrosequencing. Analysis of the wild boar PTV strain WB2C-TV/2011/HUN (JQ429405) showed considerable difference, especially in VP1 (66–74% amino acid identity) compared to the available PTVs. PTV is present in wild boars, and WB2C-TV/2011/HUN represents a novel PTV genotype, provisionally named PTV-13.
Mice (Mus musculus) are the most commonly used laboratory animals. Viral metagenomics on tissues of immunodeficient mice revealed sequences of a novel mammalian astrovirus. Using PCR, we screened mice from 4 breeders, 4 pharmaceutical companies, 14 research institutes and 30 universities in the US and Japan. Mice from one US breeder tested positive while none from Japanese breeders were positive for MuAstV. Mice in over half of the universities (19/30), institutes (7/14) and pharmaceutical animal facilities (2/4) investigated revealed the presence of MuAstV. Nine mice strains tested positive including both immunodeficient strains (NSG, NOD-SCID, NSG-3GS, C57BL6-Timp-3−/−, and uPA-NOG) and immunocompetent strains (B6J, ICR, Bash2, BALB/c). Our data indicates that MuAstV has a wide geographical, institutional and host strain distribution. Comparison of the MuAstV RdRp sequences showed numerous mutations indicating ongoing viral divergence in different facilities. This study demonstrates the need for metagenomic screening of laboratory animals to identify adventitious infections that may affect experimental outcomes.