The FASEB Journal article fj.201601168R. Published online February 8, 2017.

THE

JOURNAL • RESEARCH • www.fasebj.org

Viruses comprise an extensive pool of mobile genetic

elements in eukaryote cell cultures and human
clinical samples
Jakob Thannesberger,* Hans-Joerg Hellinger,† Ingeborg Klymiuk,‡ Marie-Theres Kastner,* Franz J. J. Rieder,*
Martina Schneider,* Susanne Fister,§ Thomas Lion,{ Karin Kosulin,{ Johannes Laengle,k Michael Bergmann,k
Thomas Rattei,† and Christoph Steininger*,1
*Division of Infectious Diseases, Department of Medicine 1, and kDepartment of General Surgery, Medical University of Vienna, Vienna,
Austria; †CUBE–Division of Computational Systems Biology, Department of Microbiology and Ecosystem Science, University of Vienna,
Vienna, Austria; ‡Center for Medical Research, Core Facility Molecular Biology, Medical University of Graz, Graz, Austria; §Christian Doppler
Laboratory for Monitoring of Microbial Contaminants, University of Veterinary Medicine, Vienna, Austria; and {Children’s Cancer Research
Institute, Vienna, Austria

ABSTRACT: Viruses shape a diversity of ecosystems by modulating their microbial, eukaryotic, or plant host me-
tabolism. The complexity of virus–host interaction networks is progressively fathomed by novel metagenomic
approaches. By using a novel metagenomic method, we explored the virome in mammalian cell cultures and clinical
samples to identify an extensive pool of mobile genetic elements in all of these ecosystems. Despite aseptic treat-
ment, cell cultures harbored extensive and diverse phage populations with a high abundance of as yet unknown and
uncharacterized viruses (viral dark matter). Unknown phages also predominated in the oropharynx and urine of
healthy individuals and patients infected with cytomegalovirus despite demonstration of active cytomegalovirus
replication. The novelty of viral sequences correlated primarily with the individual evaluated, whereas relative
abundance of encoded protein functions was associated with the ecologic niches probed. Together, these obser-
vations demonstrate the extensive presence of viral dark matter in human and artificial ecosystems.—
Thannesberger, J., Hellinger, H.-J., Klymiuk, I., Kastner, M.-T., Rieder, F. J. J., Schneider, M., Fister, S., Lion, T.,
Kosulin, K., Laengle, J., Bergmann, M., Rattei, T., Steininger, C. Viruses comprise an extensive pool of mobile
genetic elements in eukaryote cell cultures and human clinical samples. FASEB J. 31, 000–000 (2017). www.fasebj.org
KEY WORDS: viral microbiome • next-generation sequencing • viral dark matter • bacteriophages

The viral microbiome, or virome (i.e., the collective ge-
nomes of all viral habitants), is large, medically relevant,
and, thus far, grossly neglected (1). Humans are in per-
sistent and immediate contact with an immense diversity
of prokaryotic, eukaryotic, and plant viruses and provide
ecologic niches for a large fraction of these viruses. The
viral community further provides an enormous pool of
mobile genetic information that may confer novel func-
tional properties to its hosts. Microbial viruses are thought
to comprise the most abundant entities in the biosphere—it
ABBREVIATIONS: AdV, adenovirus A; CMV, human cytomegalovirus;
COG, Clusters of Orthologous Group; ds, double-stranded; FCS, fetal calf
serum; InfluA, influenza A virus; MDA, multistrand displacement am-
plification; MOI, multiplicity of infection; OMV, outer membrane vesicle;
ss, single-stranded; VIPEP, viral isolation, purification, and enrichment
procedure
1
Correspondence: Medical University Vienna, Department of Medicine I,
Währinger Gürtel 18-20, 1090 Vienna, Austria. E-mail: christoph.steininger@
meduniwien.ac.at
doi: 10.1096/fj.201601168R
This article includes supplemental data. Please visit http://www.fasebj.org to
obtain this information.
was estimated that ;1025 phages initiate an infection every
second on the global scale. In the course of those infections,
phages encounter nucleic acids of microbial or proviral
origin with which they can potentially recombine to gen-
erate novel genomic arrangements, thereby rapidly ex-
ploring the sequence space (2). The diversity of the genetic
pool is constrained by the permissiveness of cells for viral
infection, as most viruses are highly host specific and may
infect only a limited number of eukaryotic cell types or
microbial strains.
Functional roles of viruses for their hosts are in-
creasingly elucidated in the context of modulation of mi-
crobial interaction networks and long-term coevolution of
virus and host, with feedback loops on associated ecosys-
tems (3–7). Phages influence the microbiome by trans-
mitting genes from one microbial host to another, thereby
conferring accessory capabilities, such as increased patho-
genicity and antibiotic resistance as well as other metabolic
functions that are associated with evolutionary advantages
(8–10). Phages are known to contribute to human health by
infecting and lysing invading microbial pathogens or by

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modifying the commensal microbiome (e.g., gut and skin)
in human favor (11). Eukaryotic viruses may cause clini-
cally apparent diseases, such as infectious mononucleosis
in patients with primary infection by human cytomegalo-
virus (CMV). CMV may increase susceptibility to oppor-
tunistic viral, fungal, or bacterial pathogens by modulation
of the immunity of the host (12); however, the same virus,
in turn, may also protect from bacterial pathogens, such as
Listeria monocytogenes or Yersinia pestis (13–15). Further-
more, humans ingest by nutrition large quantities of plant
and eukaryotic animal viruses that passage the gut with
preservation of their infectivity, but do not infect human
cells (16). The role of this large, transient fraction of the viral
microbiome is largely unknown, but these viruses may
occasionally trigger an immune response (16).
The human virome is only marginally characterized;
therefore, it is not surprising that gut virome samples
taken from different human individuals yield mostly
novel viruses (8, 9, 17, 18) and that only a small minority of
viral open reading frames resemble previously studied
genes (17). Metagenomic data sets are dominated by
uncharacterized sequences (60–95%) that very likely orig-
inate from viral genomes (7). Moreover, the recent mining
of publicly available microbial genomic data for the viral
signal revealed a considerable number of novel viral se-
quences, increasing the number of known viruses by .10-
fold (19). Hence, viral dark matter (i.e., the uncharacterized
fraction of the viral world) is thought to be far more ex-
tensive than has been thus far fathomed (7, 18, 19).
A major limitation that is responsible for this gap in
knowledge is lack of sensitive metagenomic methods for
characterization of viral communities. The low concentra-
tion of viruses in clinical samples relative to contaminant
genomes requires preanalytic enrichment for efficient
metagenomic analysis (20). In contrast to the conserved
genomic regions that are available for preanalytic targeted
amplification of genes of interest, such as the gene that codes
for the 16S fragment of the small rRNA subunit in bacteria,
the genomes of viruses are too diverse (21). Recently, several
novel methods have been developed for preanalytic prep-
aration of samples before next-generation sequencing anal-
ysis. Nevertheless, only a few methods were evaluated for
isolation of virions from clinical samples and most require
large volumes of samples, which limits the clinical useful-
ness of these methods (21, 22). These technological short-
comings greatly limit our understanding of ecological and
evolutionary interference between viruses and their hosts.
This study aimed for exploration of the diversity of
mobile genetic information carried by the entirety of
viruses that are present in clinical samples, using a novel
method for their sensitive study. In this work, we discov-
ered compelling evidence that the viral dark matter has
been massively underestimated in eukaryotic cell cultures
and distinct human ecologic niches.
MATERIALS AND METHODS
Clinical samples
Oropharyngeal lavages and urine and fecal samples were col-
lected from healthy individuals for evaluation of sensitivity and
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reproducibility of the preanalytic procedure of virus isolation
and purification in different clinical specimens. Bile fluid
samples were collected and pooled from swine cadavers as
these samples most closely mimic human bile fluid and are
available in sufficient quantity for experimental procedures.
In addition, oropharyngeal lavages and urine samples were
collected from 4 patients with primary CMV infection. Di-
agnosis of primary CMV infection was established by the
presence of CMV-specific IgM and CMV DNA that was de-
tectable in plasma and by observation of clinical signs and
symptoms that were compatible with infectious mono-
nucleosis. Clinical samples were collected throughout a me-
dian period of 41 d after onset of symptoms, and the range of
plasma viremia at the time of sample collection was 7.9 3 102
to 2.6 3 103 copies/ml (median, 2.1 3 103 copies/ml). One of
these patients (N) also presented flu-like symptoms that
started 2 d before initial evaluation and an influenza A virus
(InfluA) infection was confirmed by detection of virus-
specific DNA in a respiratory sample. All patients were
without evident underlying diseases and none had received
antibiotics in the 6-mo period before sampling. Clinical study
was performed in accordance with the Declaration of Helsinki
good clinical practice guidelines and was approved by the
Medical University of Vienna Ethics Committee (EK26/2012).
Viruses, bacteria, and cells
Viruses selected in the present study represented extensive di-
versity of structure and genome: CMV (AD169 strain) is an
enveloped, double-stranded (ds) DNA virus; adenovirus A
(AdV; A12 strain VR-863) is a nonenveloped, dsDNA virus;
InfluA [A/PuertoRico/8/34 virus (H1N1)] is an enveloped,
single stranded (ss)RNA virus; Listeria phage P100 is a non-
enveloped dsDNA phage; and bacteriophage MS2 is a non-
enveloped ssRNA phage.
For propagation of CMV, human foreskin fibroblasts were
cultured in DMEM (GlutaMax; Thermo Fisher Scientific,
Waltham, MA, USA) that was supplemented with 10% fetal calf
serum (FCS; Linaris, Dossenheim, Germany) and Antibiotic-
Antimycotic mix (Thermo Fisher Scientific). Human foreskin
fibroblasts were then trypsinized, seeded to approximately
80% confluence, and subsequently infected with CMV (AD160
strain) at a multiplicity of infection (MOI) of 0.01. Viral sus-
pension was replaced with culture medium after 90 min. Virus
culture was harvested after a total cytopathic effect was visible.
Cell debris was removed by centrifugation at 3000 g for 20 min,
and virus supernatant was stored in DMEM that was supple-
mented with 20% FCS at 280°C. MOI was measured by using a
plaque assay as described previously (23). Human adenovirus
type 2 (American Type Culture Collection, Manassas, VA,
USA) was propagated in the human alveolar adenocarcinoma
epithelial cell line A549 that was grown in DMEM culture
medium supplemented with 5% FCS and 1% penicillin/
streptomycin mix (Thermo Fisher Scientific). Upon observa-
tion of a cytopathic effect, virus was harvested by performing 3
freezing–thawing cycles followed by centrifugation at 4000 g
for 10 min. Aliquots of supernatant were used for subsequent
experiments. For propagation of InfluA strain A/PuertoRico/
8/34 (H1N1), A549 cells were maintained in DMEM/nutrient
mixture F-12 (Thermo Fisher Scientific) that was supplemented
with 10% heat-inactivated FCS (Linaris, Dossenheim, Ger-
many). African green monkey kidney Vero cells (European
Collection of Authenticated Cell Cultures; 88020401) were
cultivated in OptiPro medium (Thermo Fisher Scientific).
InfluA propagation was done in Vero cells in the presence of
5 mg/ml trypsin (Sigma-Aldrich, St. Louis, MO, USA) after
infection with an MOI of 0.1. Virus titer concentrations were
assessed by plaque assays performed on Vero cells. All mam-
malian cell cultures were tested for bacterial contaminations
THANNESBERGER ET AL.
with Mycoplasma spp. infection with use of Venor GeM Myco-
plasma detection kit (Minerva Biolabs, Berlin, Germany).
Phage P100 was used as the Listex P100 commercial prepa-
ration (batch 12G26,lLot 308; EBI Food Safety, Wageningen, The
Netherlands). Phage MS2 was kindly provided by Prof. Regina
Sommer (Medical University of Vienna, Vienna, Austria). For
production of MS2 stocks, phage dilutions were used for Double
Agar Overlay Plaque Assay (24). Plates with confluent lysis were
overlaid with 5 ml SM buffer (5.8 g NaCl, 2.4 g Tris HCl, 1.0 g
CaCl2, 0.1 g gelatin, add 1 L H2O) and shaken overnight at 4°C.
Thereafter, supernatant was centrifuged at 6000 g for 2 min. Su-
pernatant was filtered (0.02 mm), aliquoted, and stored at 220°C.
For propagation and enumeration of the phage MS2, Escherichia
coli Top 10F9 (Thermo Fisher Scientific) was used. For propaga-
tion and enumeration of the phage P100 L. monocytogenes EGDe
(American Type Culture Collection, Manassas, VA, USA; BAA-
679) was used. E. coli and L. monocytogenes strains were grown
overnight in tryptone soya broth with 0.6% (w/v) yeast extract
(Oxoid, Waltham, MA, USA). Overnight bacteria cultures were
diluted 10-fold in fresh medium and incubated at 37°C for 3–4 h
to obtain a maximum number of viable cells in their logarithmic
growth phase (log phase). HEK293 cells were grown in DMEM
with 10% FCS (Linaris, Dossenheim, Germany) and Antibiotic-
Antimycotic mix (Thermo Fisher Scientific). Cells were harvested
at an approximately 90% confluence. All bacteria and cell lines
were grown in a humidified 5% CO2 atmosphere at 37°C.
Detection and quantification of viral, bacterial, and
cellular nucleic acids
Contaminant 18S rDNA and 16S rDNA was detected with use of
specific PCR assays as previously described (25). In short, PCR
mixture contained 13 iTaq PCR buffer (Bio-Rad, Hercules, CA,
USA), 200 mM of each deoxynucleotide triphosphate (dNTP)-
Mix, 2 mM MgCl2, 5 mM of each forward and reverse primer, and
0.4 U iTaq DNA Polymerase (Bio-Rad) in 20 ml reaction volume. L.
monocytogenes genomic DNA (5 ng; for 16S rDNA) or HEK193 cell
genomic DNA (5 ng; for 18S rDNA) were used as positive control
and nucleic acid–free water as negative control. PCR reactions
included initial denaturation at 94°C for 15 min, followed by 35
cycles of denaturation at 94°C for 60 s, annealing at 55°C (16S) or
56°C (18S) for 60 s and extension at 72°C for 2 min, followed by a
final extension at 72°C for 10 min in a thermal cycler. Amplicons
were visualized by electrophoresis in 1% agarose gel.
Real time PCR assays were performed for quantification of
CMV, AdV, and InfluA in different samples as previously de-
scribed (26–28). For CMV, AdV, and InfluA quantitative PCR, all
reactions were done in a total volume of 20 ml that contained 10 ml
iTaq Universal Supermix (Bio-Rad), 300 nM concentration of
primers, 200 nM TaqMan probe, and 2 ml of template DNA.
Mixtures were prepared in 96-well optical microtiter plates
(Thermo Fisher Scientific) and amplified on StepOnePlus Real
Time PCR System (Thermo Fisher Scientific) by using the fol-
lowing cycling parameters: denaturation for 90 s at 95°C, fol-
lowed by 50 cycles of denaturation for 15 s at 95°C, and annealing
and extension for 60 s on 57°C (CMV) or 60°C (InfluA, AdV).
Standards for viral quantification were produced by cloning the
CMV UL17 gene (3077497), the AdV Hexon-encoding gene
(1460858), and the InfluA M-protein–encoding gene (21693175)
into expression vector pcDNA 3.1. (Thermo Fisher Scientific).
P100 was quantified by quantitative PCR—carried out as pre-
viously described (29)—that amplified a fragment of the gp104
gene. MS2 was quantified by quantitative PCR as described
previously using a FAM-labeled probe (30). For quantification of
L. monocytogenes, a quantitative PCR that targeted the prfA locus
of L. monocytogenes was carried out according to the study by
Rossmanith et al. (31). Reaction mixtures for PCR assays for L.
monocytogenes, P100, and MS2 included 0.5 mM primer, 0.25 mM
VIRAL MICROBIOME
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probe, 3.5 mM MgCl2, 0.8 mM dNTP, 1.5 U Platinum Taq DNA
Polymerase (Thermo Fisher Scientific), 13 PCR buffer, and 5 ml
DNA or cDNA per 25 ml PCR reaction. Reactions were carried
out by using the Mx3000P Real-Time cycler (Stratagene, La Jolla,
CA, USA). All quantitative PCRs included serial dilution of
plasmids or genomic DNA within the range of 30 to 3 3 105
copies/well for calculation of a standard curve and quantifica-
tion of target. Each DNA sample was analyzed in triplicate and
multiple negative controls were included in each assay.
Viral isolation, purification, and
enrichment procedure
The work flow of viral isolation, purification, and enrichment
procedure (VIPEP) is depicted in Fig. 1. A prototype version of
the QiaAmp Virome RNA/DNA kit (Qiagen, Venlo, The Neth-
erlands) was used to enrich virions and isolate viral nucleic acids.
In brief, clinical samples were diluted 1:5 in PBS buffer pH7
(Dulbecco’s PBS, no calcium, no magnesium; Thermo Fisher
Scientific) before further purification steps to conserve intact vi-
rions; 2 steps of low-speed centrifugation were performed with
centrifugation for 5 min at 2500 g and 21°C, followed by transfer
of supernatant to another tube and centrifugation for 15 min at
4800 g and 21°C; to separate bacteria and other large particulate
matter, such as cell organelles from virions, supernatants were
passed through a 0.45-mM syringe filter; flow throughs were
further enriched for virions by an ultrafiltration step; after ul-
trafiltration, retentate was treated with DNase (Qiagen) to
remove any unprotected DNA; protected nucleic acids that were
contained inside the intact virions in the sample after DNase
digestion were isolated by using a standard silica spin column
procedure (QiaAmp MinElute Virus Spin Kit; Qiagen, Venlo, The
Netherlands)—immediately after nucleic acid elution, RNase
inhibitor (Qiagen) was added for protection of viral RNA; Elon-
gation of rRNA was blocked from nonspecific amplification by
using a set of 5 specific oligonucleotides that contain a 39-dideoxy
C6 amino modification as previously described (32). After 10 min
of incubation at 37°C, reverse transcription was performed by
using the Omniscript kit (Qiagen) and a set of 96 nonribosomal
hexanucleotides, as previously described (33). Finally, multi-
strand displacement amplification (MDA) was performed to
amplify the remaining genomic sequences by using the Repli-g
kit (Qiagen).
Library preparation and
next-generation sequencing
MDA-amplified dsDNA was quantified with QuantiFluor ONE
dsDNA Dye on a Quantus Fluorometer (Promega, Mannheim,
Germany) according to manufacturer instructions. Shotgun li-
brary for sequencing on an Illumina MiSeq platform (Illumina,
San Diego, CA, USA) was prepared with NebNext Ultra DNA
Library Prep Kit for Illumina in combination with the Index
Primers Set 1 (New England BioLabs, Frankfurt, Germany)
according to manufacturer instructions. In brief, 600 ng dsDNA
was randomly fragmented by ultrasonication in a microTUBE on
a M220 focused ultrasonicator (Covaris, Woburn, MA, USA) in a
total volume of 130 ml 13 TE for 1 min and 21 s. Of shared DNA,
55 ml was used for end repair and adapter ligation reactions
according to manufacturer instructions. Size selection and puri-
fication were performed according to instructions for 300 to 400
bp insert size. Subsequent PCR amplification was performed
with 4 cycles, and libraries were eluted after successful amplifi-
cation and purification in 33 ml 13 TE buffer (pH 8.0). For quality
control, libraries were analyzed with a DNA High Sensitivity Kit
on a 2100 Bioanalyzer system (Agilent Technologies, Santa Clara,
CA, USA) and were again quantified on a Quantus Fluorometer.
3
Figure 1. Workflow of VIPEP. Step 1 stabilzes viral particles by providing favorable environmental conditions. Physical separation
steps (2–4) reduce cellular and bacterial contamination, followed by digestion of cell-free nucleic acids (5). After extraction of nucleic
acids from cells (6), rRNA blocking, reverse transcription, and MDA (7), sample may be subjected to next-generation sequencing.

An equimolar pool was sequenced on an Illumina MiSeq desktop
sequencer (Illumina). Libraries were diluted to 8 pM and run with
5% PhiX control (Illumina) and v3 600 cycles chemistry according
to manufacturer instructions.
Identification of known viruses and nonviral reads
All sequence reads were mapped by using bwa (34) to the version
GRCh38 of the human genome retrieved from the Genome Ref-
erence Consortium (35). Read pairs, of which one read was
identified as rRNA by using SortMeRNA (36), were considered
rRNA fragments. All nonhuman and non-rRNA read pairs were
mapped by using bwa (34) to all genome sequences from the
National Center for Biotechnology Information Viral Genome
Resource (37). The fraction of each virome, which was covered by
proper pairs, was determined, and genomes with .50% genome
coverage were selected for further manual inspection in Tablet
(38). If homogeneous coverage of the entire genome by read pairs
was observed, the virus was considered to be present in the
sample. All remaining reads were considered unassigned se-
quence reads (Table 1) in subsequent analyses.
Phage Orthologous Groups (39). Nonphage viral genomes were
retrieved from the National Center for Biotechnology In-
formation Viral Genome Resource (37). Protein sequences of one
representative genome per species were clustered into Clusters of
Orthologous Groups (COGs) of nonphage viruses by using Na-
tional Center for Biotechnology COGsoft (39). Sequence simi-
larities between all COG members and proteins from eggNOG
4.0 (40) were determined by BLASTP (41) with an e-Value cutoff
of 1e25. Only COGs in which ,10% of members had sequence
similarities with eggNOG proteins (from not more than 2 species)
were considered to be virus specific. The common lineage of all
members from each COG was determined by a custom python
script. Phage and nonphage virus-specific marker genes were
used as a joint database for searching hits of all unassigned se-
quencing reads by BLASTX (41) with an e-Value cutoff of 1e24. A
custom python script was used to add lineage information to
each hit, and the resulting files were visualized in KRONA
(Supplemental File SI1) (42). The number of different taxonomic
lineages present in a sample was taken as a measure of diversity.
Assembly and annotation of unknown viruses

All unassigned sequence reads were trimmed and filtered

Identification of viral marker proteins by their per-base quality values by using prinseq-lite and pa-
rameters -min_qual_mean 30 -min_len 100 -trim_qual_right 30
Orthologous groups of phage-specific marker genes (viral quo- -trim_qual_type min -trim_qual_window 5 (43). Pairs in which
tient $ 0.9) were retrieved from a recently compiled database of both members have passed were de novo assembled by using Trinity

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 2.0.6 (44) and parameters –full_cleanup–normalize_max_read_cov

2.0E+06 (84.2)
9.0E+06 (98.8)
1.1E+07 (90.2)

(66.5)

(66.0)
(63.2)

(97.9)
(99.4)
(99.3)
(84.8)
(8.8)
Unassigned
50–min_contig_length 1000. Coverage was estimated by mapping
all read pairs with bwa (34) to the contigs. Unmapped reads were

9.1E+05
1.2E+05
1.0E+06
9.7E+05

1.6E+06
1.6E+06
1.8E+06
1.3E+06
extracted by using a custom python script. Genes on contigs were
predicted by using MetaGeneMark (45). For each predicted gene,
the best matches to phage and nonphage specific marker genes (see
above) in Uniprot/SwissProt (46) were obtained from BLASTP (41)
searches (max e-Value 1e210; for nonredundant (nr) database max

(86.2)
JC polvomavirus

2.1E+01 (0.0)
3.3E+01 (0.0)
4.7E+05 (3.9)

(0.2)

(0.0)
(0.0)

(0.0)
(0.0)
(0.0)
(0.0)
e-Value 1e24). Enrichment of marker genes was calculated by
the ratio of abundance in target sample vs. reference sample or

0
3.3E+03
1.2E+06
2.0E+01
1.7E+01

1.5E+01
1.3E+01
4.4E+01
SwissProt Database entries, and each protein was manually clas-
sified into 17 categories: antimicrobial, cofactor binding domain,
eukaryotic host, genome alteration, host structure–bacteria, host
structure–eukaryotic, host structure–unspecific, ion binding do-
main, ion transport, metabolic function, oncogene, replication,
5.0E+00 (0.0)
0 (0.0)
0 (0.0)

(0.0)
(0.0)
(0.0)
(0.0)

(0.0)
(0.0)
(0.0)
(0.0)
resistance, signal transduction, transcription related, unspecific
InfluA

molecular function, virus nonstructure, or virus structure (Supple-

0
0
0
0

0
0
0
1.0E+00
mental Table SI2).
Reads that were not assembled were dereplicated by using cd-
hit-est (47) at 98% identity. For each representative sequence, best
Pairs, n (% of total)

matches to an in-house database of universal cellular COGs from

4.3E+02 (0.0)
1.1E+01 (0.0)
9.0E+03 (0.1)

(0.6)
(0.0)
(0.0)
(0.8)

(0.0)
(0.0)
(0.0)
(0.0)
eggNOG 4.0 (40), Uniprot/SwissProt (46), and National Center
for Biotechnology Information nr (48) were obtained from
CMV

8.6E+03
3.0E+00
8.6E+01
1.3E+04

9.6E+01
3.9E+01
8.0E+00
0

BLASTX (41) searches (max e-Value 0.01).

RESULTS
1.5E+05 (6.2)
7.3E+03 (0.1)
2.2E+04 (0.2)

(0.0)
(0.0)
(0.0)
(0.0)

(0.0)
(0.0)
(0.0)
(0.0)

Novel VIPEP yields highly purified

AdV

1.0E+00

1.0E+00
3.0E+00
2.2E+01
0
0
0

viral samples

The most significant limitation to high-throughput vi-

rus discovery is currently the lack of a preanalytic
6.1E+01 (0.0)
1.8E+01 (0.0)
4.2E+03 (0.0)

(8.6)
(0.1)
(1.8)
(9.7)

(0.0)
(0.1)
(0.1)
(0.3)

method that allows efficient purification of viral parti-

Human

cles from low volumes of a diversity of clinical speci-

1.2E+05
1.8E+03
2.7E+04
1.5E+05

4.0E+01
1.8E+03
9.5E+02
5.2E+03

mens (21). To overcome this shortcoming, we developed

a novel protocol for purification and enrichment of vi-
rions from a maximum of 3 ml of clinical sample (VIPEP;
Fig. 1) on basis of previously described methods. Vali-
(20.9)

(16.4)
(18.7)
1.1E+05 (4.5)
4.0E+04 (0.4)
2.2E+05 (1.9)

(2.2)

(1.0)
(0.4)
(0.3)
(7.3)

dation of each step of this protocol, with use of virus-

All rRNA

specific quantitative PCR assays and laboratory reference

2.9E+05
3.1E+04
2.5E+05
2.9E+05

1.7E+04
5.7E+03
6.0E+03
1.1E+05

samples, demonstrated a highly efficient elimination of

nonviral genomes and enrichment of viral genomes (Fig.
2). Elimination of contaminating nonviral genomes was
achieved by using centrifugation, filtration, digestion of
1183
3970
4813

673
678
738
749

814
787
878
748
Mbp

free DNA, and rDNA blocking, whereas viral enrich-

Total (n)

ment was achieved by using ultrafiltration and MDA

(Fig. 2). Contaminating prokaryotic and eukaryotic ge-
2.41E+06
9.10E+06
1.20E+07

1.37E+06
1.40E+06
1.51E+06
1.54E+06

1.67E+06
1.60E+06
1.80E+06
1.53E+06

nomes could be reduced clearly below ,1% of original

Pairs

samples. Evaluation of VIPEP with 3 different eukaryotic

and 2 prokaryotic reference viruses revealed consider-
TABLE 1. Separation of known sequences

able differences in enrichment factors, which ranged

Oropharyngeal lavage, patient ID

from 1235% for P100 phage and 130,958% for AdV,

respectively.
To evaluate whether these differences in viral re-
Cell culture supernatant

covery may be related to environmental conditions and

Oropharyngeal lavage

the environmental resistance of different viruses, we

Urine, patient ID

spiked cell-free CMV and AdV cell culture supernatants

Reference sample

into clinical samples with significantly different genomic

Clinical sample

background, osmolarity, pH, or immune factors and sub-

jected these samples to VIPEP (Fig. 3). Viral recovery was
Urine

found to be clearly associated with the clinical specimen

Sample

N
K

K
P

P
J

tested (Fig. 3). CMV recovery from feces was more efficient
than from bile or oropharyngeal lavage and undetectable

VIRAL MICROBIOME 5
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Figure 2. Efficacy of VIPEP. A) Change in relative concentration of viral or bacterial nucleic acids for different steps of VIPEP.
Cell-free supernatant of 3 eukaryotic virus cultures (CMV, AdV, and InfluA H1N1) were spiked at mean concentrations of 4.65 3
105 copies/ml into 3 ml PBS buffer. In addition, 2 bacteriophages (P100 and MS2), cell-free Listeria monocytogenes DNA, and
Listeria monocytogenes cells were spiked into 3 ml PBS buffer. All samples were also contaminated with intact and lysed HEK293
cells. All experiments were performed in triplicate. Bars show change in copy number in relation to feed as determined by
specific quantitative PCR and error bars indicate SEM. B) PCR products of a 16S and 18S rDNA-specific PCR assay from aliquots of
samples collected at each purification step and from feed (far left).

from urine. AdV recovery was similarly efficient in the
different clinical specimen, although to a lesser extent,
which was compatible with the well-documented envi-
ronmental resistance of AdV. Dilution of samples with an
iso-osmolar, neutral buffer before viral recovery signifi-
cantly increased the efficiency of the procedure for both
viruses and all clinical specimens evaluated (Fig. 3).
Mammalian cell culture supernatants contain
a high diversity of viruses
Eukaryotic viruses are propagated under aseptic condi-
tions in mammalian cell cultures. In addition to those
intended viruses, a wide range of other, putatively present
viruses that resist inactivation by standard procedures
were introduced to the sample by diverse biological
components (49). To evaluate the presence of unintended
viruses in cell cultures, 3 different eukaryotic viruses—
CMV, InfluA, and AdV—were propagated in 3 different
cell lines under aseptic conditions, respectively. Cell-free
cell culture supernatants were harvested after release of
viruses from cells, pooled, and tested by VIPEP. Meta-
genomic analysis of pooled supernatants yielded a total of
2.4 Mio read pairs, of which only 61 pairs were of human
origin and only 4.5% originated from rRNA genes, which
was indicative of the high efficacy of VIPEP purification
(Table 1). Contrary to expectation, the screen for whole-
genome coverage against a library of publicly available
virus genome sequences indicated the presence solely of
AdV in this sample—only 6.2% of read pairs had simi-
larities with AdV (Table 1). Coverage of CMV (425 pairs)
and InfluA (5 pairs) was negligible, despite high viral loads
in supernatants as confirmed by quantitative PCR (Fig. 2
and Table 1).
We searched the large fraction of unassigned reads (2 3
106 read pairs) for similarities with the previously estab-
lished phage and nonphage virus-specific marker data-
base to identify sequences of putative viral origin among
those unassigned reads and to further gain taxonomic in-
formation for sequences that were hit by a viral marker.
The majority of matches (90%) could be taxonomically
classified as phages, inferred by the known lineage of the
markers, which elevated a distinction between pro-
karyotic and eukaryotic viruses (Fig. 4A and Supplemen-
tal File SI1, KRONA chart). The largest fraction of those
phage sequences (57%) are assigned to Microviridae, and of
these, 99% were assigned to enterobacteria phages of the
genus Microvirus. Matches that were assigned to other
taxonomic phage families accounted for only 4% of the
total. A large fraction of matches (40%) were assigned to
the taxonomic order of tailed phages, but not to specific
subranks of this order, which indicated a high abundance

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of as yet unknown and uncharacterized phages (i.e., viral
dark matter). In contrast, 98% of matched reads by
nonphage viral markers could be assigned at the family
level (Fig. 4A and Supplemental File SI1, KRONA
chart). Matches were most commonly assigned to Ade-
noviridae (57%), followed by Poxviridae (17%), Herpes-
viridae (7%), and Phycodnaviridae (7%). Identified
adenovirus and herpesvirus matches differed from
those viruses that were intentionally propagated, as
99% of Adenoviridae matches were unassigned and 66%
of Herpesviridae matches were assigned to herpesvi-
ruses other than b-herpesvirus CMV.
To estimate the novelty of viral sequences that were
discovered by analysis of their sequence similarity to
viral markers, we used the degree of sequence identity
between marker and read as a measure (Fig. 4B).
Under the assumption that a known virus—already
present in public databases—would show a 100% se-
quence identity to the respective marker, we observed
that most detected phage, as well as eukaryotic, viral
sequences showed low identity to known sequences,
which indicated that, congruent with our expectations,
the majority is indeed novel. This observation might be
indicative of a likely diverse and largely unknown
viral community in cell culture supernatants. We also
assessed the coding potential of this discovered viral
dark matter by de novo assembling the metagenome
sequence reads (Table 2), predicting genes on the
resulting contigs, obtaining best matches to proteins
in Uniprot/SwissProt, and finally grouping them
into significant keyword categories (Fig. 4C and Sup-
plemental Table SI2). Compared with the entirety
of Uniprot/SwissProt entries, proteins that confer
survival advantages, such as antibiotic or environ-
mental resistance, signal transduction, or bacterial
structure, to the bacterial host of phages were
enriched. In contrast, genes that code for oncogens,
replication, or eukaryotic structure were found to be
under-represented.
VIRAL MICROBIOME
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Figure 3. Environmental resistance of free
virions in different clinical samples. Cell-free
CMV or AdV virions were spiked at mean
concentrations of 4.35 3 104 copies/ml into 3 ml
of PBS buffer or into 1 of 4 different clinical
specimens. Samples were either subjected di-
rectly to VIPEP or prediluted 1:5 in PBS buffer.
Change in relative viral DNA concentration was
determined by virus-specific quantitative PCR
before and after VIPEP. All experiments were
performed in triplicate. Bars show change in
copy number in relation to feed and error bars
indicate SEM.
Viral communities differ in healthy
individuals between the ecologic
niche probed
To study the viral microbiome in healthy individuals, we
next evaluated the viral microbiome in oropharyngeal and
urinary samples, respectively. As a reference, samples
were spiked with CMV, AdV, and InfluA at mean con-
centrations of 1.5 3 107 copies/ml. Nevertheless, read
pairs that were assigned to these 3 viruses represented
a minute fraction of the total number (Table 1). John
Cunningham (JC) polyomavirus sequences at a frequency
of 4% of total read pairs were detected in the urinary
sample; however, .90% of read pairs were unassigned,
and, of these, sequences with matches to phage markers
predominated in both samples. The majority of these pu-
tative phage sequences were assigned in the oropharyn-
geal and urinary samples (97 and 81%, respectively) to
chlamydiamicrovirus that infects Chlamydia spp. (Supple-
mental File S1). The majority of nonphage sequences were
assigned in both samples to Anelloviridae (65 and 68%, re-
spectively). Nevertheless, the oropharyngeal sample con-
tained a lower diversity of nonphage matches than the
urinary and included Papillomaviridae, which represented
the second largest fraction (33%).
Sequence similarities with phage and nonphage marker
genes differed between urine and oropharyngeal lavage
samples (Fig. 5A). Similarities ranged for nonphage
matches in both samples from 20–100% with a peak of
identity of 50–60% in urine and 40–50% in oropharyngeal
lavage; however, the range of similarity was wider for
phage matches in urine sample than in the oropharyngeal
sample and indicated a predominance of unknown
phages in the oropharynx. Functional keywords, assessed
from the assembly (Table 2), were differently abundant
among proteins in the 2 samples (Fig. 5B). Compared with
the oropharyngeal sample, genes that code for proteins
related to ion transport were clearly enriched in the urine
sample (76-fold), which was compatible with a potential
7
Figure 4. Metagenomic analysis of unassigned sequence reads from mammalian cell culture supernatant. Virus-rich supernatants
of 3 infected cell lines [CMV in human foreskin fibroblasts (HFF), AdV in human alveolar adenocarcinoma cells (A549), and
InfluA in Vero cells] were spiked into PBS buffer before being subjected to VIPEP. Next-generation sequencing–derived
sequence reads were mapped by using bwa (34) to human genome (version hg19), to rRNA genes (SortMe RNA), and to all
genome sequences from the National Center for Biotechnology Information Viral Genomes Database. Remaining unassigned
sequence reads were aligned to a set of marker genes inferred from orthologous groups, which indicated their taxonomic lineage
when hit by such a marker. A) Distribution of reads in percentage of total number of unassigned reads (middle), reads matched
to phage markers (left), and matched to nonphage (right) marker genes. The complete data set is presented in Supplemental
KRONA File SI1. The inconsistency of taxonomic hierarchy between panel A and the Supplemental KRONA File SI1 chart
occurred because of differences in reference databases used. The sum of relative frequency of the different phage virus families
do not add up to 100% because of settling rounding differences. B) Sequence identity between unassigned reads and set of
marker genes. Bars indicate absolute number of reads at identity rates from 0–100%. A sequence identity of 100% indicates a
sequence that is identical to a known virus. C ) Gene ontology categories with significant enrichment (P , 0.001). After de novo
assembly of unassigned sequence reads, predicted genes were searched against SwissProt and revealed keywords were grouped
into gene categories (Supplemental Table SI2). Frequency of categories in cell culture supernatant was compared with frequency
of SwissProt entries. Bars indicate enrichment (.1) or depletion (,1) for each category.

survival benefit of bacteria thriving in this high osmolar,
low pH environment. Genome alteration genes, onco-
genes, and genes for ion binding domains were also
enriched in urine, whereas genes that code for resistance
proteins were enriched in the oropharyngeal sample.
Viral diversity during acute eukaryotic
virus infection
In patients with primary CMV infection, the virus is shed
in urine and the oropharynx for prolonged periods and
was proposed to modify the susceptibility to a range of
other infectious pathogens (12). Therefore, we character-
ized the oropharyngeal and urinary viral microbiome of 4
patients with acute, primary CMV infection and signifi-
cant CMV load in plasma. CMV DNA was undetectable in
the oropharyngeal and urinary samples, despite use of a
highly sensitive, virus-specific quantitative PCR assay
(Fig. 6A). Viral enrichment by VIPEP yielded positive
quantitative PCR results in 2 oropharyngeal and 3 urinary
samples. Metagenomic analysis yielded a mean 1.6 3 106
read pairs (Table 1). Again, CMV-specific reads comprised
a negligible fraction of the total reads not exceeding 0.8% in
any of the 8 samples tested, and JC polyomavirus–specific

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 reads were detected in one of the urine samples. Re-

Unassembled unique

5.3E105 (13.1)

(10.1)
(29.5)
(13.2)
(14.3)

(11.9)

(14.9)
markably, significant numbers of CMV read pairs were

6.2E105 (3.5)
5.7E105 (2.6)

(9.0)
(9.5)
associated with higher human read pairs (P = 0.002),

1.9E105
7.3E104
2.6E105
2.8E105

3.9E105
2.9E105
3.4E105
3.9E105
which may indicate copurification of virions with sub-
Pairs, n (% of total)
cellular particles (Table 1).
The abundance of hits to phage markers was highly
variable among the different samples and ranged from
29 to 99% in urinary samples and from 47 to 99% in
1.6E106 (79.7)
8.0E106 (89.3)
9.3E106 (85.4)

(50.4)
(76.2)
(73.3)
(69.3)

(93.7)
(59.7)
(72.5)
(88.4)
oropharyngeal samples (Supplemental File S1). In
Unassembled

addition to the known JC polyomavirus that was de-

tected in one urine sample, a large fraction of non-

4.6E105
9.4E104
7.3E105
6.7E105

1.5E106
9.5E105
1.3E106
1.1E106
phage matches were assigned to other Polyomaviridae
(58%). In urine samples, overall sequence similarity of
unassigned reads with known phage markers was
high (80–90%), which was indicative of a population of
5583

5325
7591

9100

8544
7965
8908
9747
28487
19141

11448
N50

phage species that was closely related to known species

(Fig. 6B). In contrast, sequence similarity with known
phage markers was lower overall in oropharyngeal
Longest

samples, which was indicative of an abundance of novel

67061
21033
15276

17348
36955
54746
49942

34764
16088
51188
37348
phages. Sequence identity of unassigned reads with
Assembly results

nonphage markers was more variable than with phage

markers and covered in all samples 30–70%, which was
Mbp, n

indicative of a high abundance of novel nonphage

0.9
1.0

0.2
0.2
1.3
0.8

1.0
0.4
0.7
2.8
11.4

viruses. Our overall observation is that the novelty

of the viruses correlated rather with the patient than
with the ecological niche probed (Fig. 6B). The rela-
3.17E103
2.13E102
2.63E102

4.80E101
4.50E101
2.64E102
1.84E102

2.35E102
1.05E102
1.57E102
5.51E102
Contig, n

tive abundance of encoded protein functions differed

clearly between the 2 ecological niches probed (Fig. 6C).
Genes that code for proteins related to oncogenesis,
host structure, and ion transport were found to be
enriched in urine samples compared with oropha-
Assembled reads, n (% of total)

ryngeal samples. In contrast, genes that code for pro-

teins involved in antimicrobial mechanisms or cellular
4.1E105 (20.3)
9.6E105 (10.7)
1.6E106 (14.6)

(49.6)
(23.8)
(26.7)
(30.7)

(40.3)
(27.5)
(11.6)
(6.3)

replication, in turn, were enriched in oropharyngeal

samples.
4.5E105
2.9E104
2.7E105
3.0E105

1.0E105
6.4E105
4.9E105
1.5E105

DISCUSSION

In this study, we provide evidence for an enormous viral

diversity of known and novel viruses in mammalian cell
997

447

486
472

797
782
872
634
3922
4346

60
Mbp

cultures as well as in diverse human specimens. Our

findings underline the abundance and genetic diversity of
Total, n

viruses and contribute to the overall consensus and body

2.03E106
8.99E106
1.08E107

9.13E105
1.23E105
9.95E105
9.70E105

1.63E106
1.59E106
1.79E106
1.29E106

of evidence of viral impact on multiple ecosystems. The

Pairs
TABLE 2. De novo assembly and dereplication

present observations underline the enormous abundance,

diversity, and functional properties of genetic information
that is packaged into highly mobile viral particles, which
makes viruses a flexible genetic reservoir with far reach
Oropharyngeal lavage, patient ID

(50–53).
Mammalian cell cultures are widely used for prop-
Cell culture supernatant

agation of a diversity of viral pathogens. Efficient virus

Oropharyngeal lavage

propagation requires careful selection of cells, culture

Urine, patient ID

media, and supplements from human and animal

Reference sample

sources, because of the strict species specificity of

Clinical sample

viruses (e.g., propagation of CMV, AdV, and InfluA;

Materials and Methods). Quality control of culture
Urine

conditions, cells, and media must be very strict to

Sample

N
K

K
P

P
J

avoid microbial contaminations, which would be

detrimental to viral propagation. The complex virome

VIRAL MICROBIOME 9
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Figure 5. Metagenomic analysis of unassigned sequence reads in urine and oropharyngeal lavage samples from healthy individuals.
Urine or oropharyngeal lavage samples of 2 healthy individuals were subjected to VIPEP. As a reference, all samples were spiked with
cell-free CMV, AdV, and InfluA viruses at concentrations of 1.5 3 107 copies/ml. Sequence reads were mapped by bwa (34) to human
genome (version hg19), to rRNA genes (SortMe RNA), and to all genome sequences from the National Center for Biotechnology
Information Viral Genome Database. A) Similarity of unassigned sequence reads to a set of phage and nonphage viral marker genes.
Bars indicate absolute numbers of reads at identity rates from 0–100% to known virus sequences. A sequence identity of 100% indicates
a sequence identical to a known virus. B) Gene ontology categories with significant enrichment (P , 0.001). After de novo assembly of
unassigned sequence reads, possible genes were aligned to SwissProt and revealed keywords were grouped into gene categories
(Supplemental Table SI2). Frequency of categories in cell culture supernatant was compared with frequency of SwissProt entries. Bars
indicate enrichment (.1) or depletion (,1) for each category.

that we discovered in mammalian cell culture, there-
fore, is astonishing.
Adenoviridae accounted for the largest fraction of
eukaryotic viruses detected in cell culture supernatant
and infects a broad range of vertebrate hosts. Para-
poxvirus accounted for 46% of Poxviridae matches and
infects a diversity of mammals, including sheep, goat,
cattle, and humans. Betaentomopoxvirus accounted for
49% of Poxviridae matches and infects Lepidoptera (moths
and butterflies) (54). Chlorovirus is a member of Phy-
codnaviridae and infects eukaryotic green algae, which are
common in freshwater lakes. Phycodnaviridae, Poxviridae,
Iridoviridae, and Mimiviridae belong to the Megavirales,
with genomes exceeding 500 kb and particles .150 nm in
diameter. Natural hosts for Iridoviridae are amphibia, fish,
invertebrates, and insects for Mimiviridae amoeba, and
for Poxviridae, humans, vertebrates, and arthropods. Se-
quences that match these 4 Megavirales comprised 27% of
all nonphage matches. Alpha-, Beta-, and Gammaherpesvir-
idae accounted for 7% of nonphage matches and are ca-
pable of infecting a range of mammals and birds. Hence,
detection of highly host-specific viruses in cell culture
suggests contamination from a diversity of sources, in-
cluding materials from surface water, mammals, birds,
fish, and arthropods. The large number of unassigned
sequences represents very likely novel viruses and im-
pedes assignment of sequences to specific hosts for fur-
ther identification of viral sources.
Observation of a vast diversity of viruses found in cell
culture may have far-reaching consequences for many
areas of biotechnology. Cell cultures are widely used for
propagation of viruses, generation of recombinant pro-
teins, or propagation of cells for transplantation. Products
are used as antigens for vaccines or immunologic assays or
for replacement of human hormones, such as insulin or
blood clotting factors. Products are virus inactivated, often
with a combination of methods to ensure safety, and
proteins are purified to eliminate contamination of cellular
components. However, not all products would resist harsh
antiviral treatments. Industrial fermentation, for example,
rests upon the use of beneficial microbes (55). Bacterio-
phage infections of starter lactic acid bacteria pose a seri-
ous risk to the dairy industry, in particular because
pasteurization processes are not adequate to deactivate viral
particles (49). Moreover, we show here that viral sequences
detected in mammalian cell culture were packaged into
virus-like particles and, hence, were associated with a
significant protein mass that may interfere with diagnostic
assays or immunization. Our findings may warrant evalu-
ation of the significance of unintended viral contamination
of cell cultures for the performance of immunologic assays
or immune responses to medical products.
We observed a high abundance of Microviridae, which is
likely not derived from sequencing control contaminations
of PhiX174 for several reasons: the relative abundance of
Microviridae was highly variable between, and clearly
lower in, clinical samples. Moreover, the observed genetic
diversity among Microvirus contigs and quality controls
throughout the sequencing process indicate that the
Microviridae family markers that were detected align with
wild-type viruses. In addition, we exclusively analyzed
Microviridae reads from indexed sample reads and did not
analyze the undetermined reads fraction that contained
PhiX control reads. Contaminations by remaining nucleic
acids in reagents can be excluded as we used negative
controls for all steps and as all patient Ki samples were
processed in one batch, which resulted in clearly dis-
criminative viral patterns.

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Figure 6. Metagenomic analysis of urine and oropharyngeal lavage samples from patients with primary CMV infection. Urine and
oropharyngeal lavage samples were collected from 4 different patients (J, K, P, N) and subjected to VIPEP. A) CMV-specific DNA
concentrations in different clinical samples were determined before and after VIPEP by using CMV-specific quantitative PCR. Error
bars indicate SEM. All experiments were performed in triplicate. B) Similarity of unassigned sequence reads to a set of phage and
nonphage viral marker genes. Bars indicate absolute numbers of reads at identity rates from 0–100% to known virus sequences. A
sequence identity of 100% indicates a sequence identical to a known virus. C) Gene ontology categories with significant enrichment
(P , 0.001). After de novo assembly of unassigned sequence reads, possible genes were aligned to SwissProt and revealed keywords were
grouped into gene categories (Supplemental Table SI2). Frequency of categories in cell culture supernatant was compared with
frequency of SwissProt entries. Bars indicate enrichment (.1) or depletion (,1) for each category.

Of note, whole-genome amplification by MDA intro-
duces a bias to taxonomic analysis. MDA favors short,
circular ssDNA, such as the genome of Microviridae, be-
cause of the rolling cycle method. This bias can be mini-
mized, but not completely eliminated, by including an
initial denaturation step (56). On the one hand, DNA
amplification by MDA thus limits, in general, accurate
estimation of taxa frequency and may also have affected
our observations (57). On the other hand, high sensitivity
of MDA also allows detection of minor species; therefore,
our study presents a nonquantitative and diversity-wise
representative picture of viral species composition.
The present study required adaptation of bioinformatic
methods to characterize the large fraction of unassigned
sequences that were obtained. In previous studies, the
presence of a large number of novel viruses was suspected
(58–61). A major advantage of our novel method for viral
purification and enrichment is the high efficacy in elimi-
nation of bacterial and human contaminating genomes
and the enrichment of virions from small and clinically
attainable samples prior to metagenomic analysis (62, 63).
Viruses constitute only a minor fraction (2–5%) of the total
amount of nucleic acids that are present in clinical samples
as a result of their relatively small genome size, which may
lead to an underestimation of the viral abundance clinical
samples (22, 64, 65); however, the high efficacy of VIPEP
allowed assessment of the viral diversity by using a spe-
cific bioinformatics workflow, which was established for
this study. We automatically assessed the homogeneity of
virus genome coverage that compared reads with all
public genomes, thereby preventing false positives, which
would have arisen from partial conservation between
different genomes. To clearly distinguish between viruses
and potential cellular contamination (62, 66), we estab-
lished a database of characteristic markers of phages and
nonphages, grouped to orthologous groups even when
sequence similarity was low between different viruses.
For functional profiling, we developed a tool on the basis
of keywords annotated in Uniprot/Swissprot.
A potential shortcoming of VIPEP may be use of
physical enrichment procedures, which may favor iso-
lation of environmentally more stable viruses. The effect
could be clearly mitigated in the VIPEP procedure but has
to be considered when comparing relative abundance of

VIRAL MICROBIOME 11
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viruses by using metagenomic virome data. Still, we ob-
served clearly different viral community profiles for dif-
ferent ecological niches probed, which were biologically
plausible, such as preferential detection of Polyomaviridae
or genes that encode proteins related to ion transport in
urine, and in concordance with previous metagenomic
studies (67).
Oropharynx and urinary tract differ significantly with
regard to temperature, osmolarity, pH, oxygenation, or
secreted immune factors. Phage diversity differs signifi-
cantly between ecological niches (68, 69). Nevertheless,
relative abundances of bacteria thriving under different
environmental conditions do not necessarily predict the
relative abundances of their phages (68, 69). In contrast,
replication of eukaryotic viruses in specialized cells, for
example, explains the detection of influenza viruses in the
oropharynx but not in the urinary tract. We found, how-
ever, that conditions in the different ecological niches may
also have direct, detrimental effects on eukaryotic viruses.
Recovery of eukaryotic virus spikes was clearly associated
with the clinical sample tested and could be increased by
neutralizing buffers. In both ecological niches that were
sampled during active CMV infection, phages were
clearly more abundant than eukaryotic viruses, including
CMV. Even in the single patient with influenza virus
coinfection, neither CMV nor influenza virus sequences
were detectable in significant quantities.
Moreover, the different compartments facilitated by
phages and eukaryotic viruses for replication should be
considered when studying the viral microbiome. CMV
replicates in the epithelial mucosa of the oropharynx,
whereas free phages adhere to the mucosa to wait for
bacterial bait (70). Similarly, urine holds large bacterial
loads—the normal range is up to 105 cfu/ml—and few
host cells that harbor eukaryotic viruses. Hence, a possible
selection bias toward phages has to be considered when
collecting fluids from extracellular compartments.
The high abundance of eukaryotic viruses in a few urine
samples is remarkable as well. Purification and enrich-
ment of virions by physical methods may also enrich other
particles with physical properties that are similar to vi-
rions. In patients with CMV infection, we noticed consid-
erable CMV-specific signals only in urinary samples with
comparably high loads of human DNA and rRNA. The
generally high efficiency of VIPEP in the elimination of
cellular genomes may indicate that CMV particles were
associated and copurified with small, subcellular particles
in these cases. CMV, polyomaviruses, and, occasionally,
adenoviruses form inclusion bodies in desquamated uri-
nary tract epithelial cells (so-called decoy cells) (71). Decoy
cells would have been eliminated by VIPEP, but inclusion
bodies contain viral DNA, cellular chromatin, and rRNA
(72, 73) and may have been copurified with cellular DNA
and rRNA from cell lysate.
Moreover, outer membrane vesicles (OMVs) that were
generated by gram-negative bacteria may have also been
copurified with virions. OMVs are 50–200 nm in diameter
and composed of LPS, phospholipids, outer membrane
proteins, and soluble periplasmic proteins, as well as RNA
or DNA (74). Nucleic acids, however, are associated with
the exterior of the vesicle and were very likely eliminated
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by the VIPEP procedure as indicated by the uniformly
negative 16S rRNA PCR results. Phages may erroneously
inject their viral DNA into OMVs instead of a bacterial cell
(75), and subsequent fusion of OMVs with sensitive re-
cipient cells may transfer genetic information (74, 76, 77).
Hence, we may not entirely exclude that some of the phage
sequences detected were associated with OMVs instead of
virions.
Phages in humans can act as both commensals and
pathogens (78, 79). We found a high diversity of phage
sequences that encode functional properties of potential
significance for their bacterial host, including resistance to
environment or antimicrobials. The diversity of genes re-
flects a high content of mobile genetic information present
in human ecological niches and underlines their significant
roles in the ecology of the human microbiome. Horizontal
gene transfer facilitates in prokaryotes the exchange of
alleles within a population, and phages play an important
role in this mechanism (80, 81). Phage genes have been
integrated into almost all sequenced pathogenic and
nonpathogenic bacterial genomes (prophages) and may
constitute up to 20% of bacterial DNA (82). Prophages may
become trapped in the host’s chromosome as a result of
genome rearrangements (cryptic prophages) (83). Cryptic
prophage genes, however, facilitate bacterial survival by
encoding gene products that confer resistance to antibi-
otics, osmotic, oxidative, and acid stress or that favor
biofilm formation (84). Thus, the vast diversity of the
phage gene pool that is present in the human oropharynx
and urinary tract forms a bazaar where bacteria may
acquire new, beneficial gene functions.
In summary, previous studies have shown that viral
sequence space remains widely undersampled through-
out multiple ecosystems. A significant part of viral ge-
nomes have not yet been characterized or annotated, or
remain hidden in microbial genomes. Our present obser-
vations represent further evidence for the extensive pres-
ence of viral dark matter in human and eukaryotic
specimen. The abundance and diversity of viruses de-
tected warrants further evaluation with respect to genetic
and immunologic consequences.
ACKNOWLEDGMENTS
This work was supported by a research grant from the
Austrian Science Fund P25353-B21. Financial support by the
Austrian Federal Ministry of Science, Research, and Economy
and the National Foundation of Research, Technology, and
Development is gratefully acknowledged. The authors declare
no conflicts of interest.
AUTHOR CONTRIBUTIONS
C. Steininger designed research; J. Thannesberger,
I. Klymiuk, and S. Fister performed research; H.-J.
Hellinger and T. Rattei analyzed data; M.-T. Kastner,
F. J. J. Rieder, M. Schneider, T. Lion, K. Kosulin,
J. Laenngle, and M. Bergmann contributed new reagents
or analytic tools; and J. Thannesberger, T. Rattei, and
C. Steininger wrote the paper.
THANNESBERGER ET AL.
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Received for publication October 25, 2016.
Accepted for publication January 9, 2017.
THANNESBERGER ET AL.
Viruses comprise an extensive pool of mobile genetic elements
in eukaryote cell cultures and human clinical samples
Jakob Thannesberger, Hans-Joerg Hellinger, Ingeborg Klymiuk, et al.

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