ESPID Reports and Reviews

CONTENTS
Viruses in Human Gastroenteritis

EDITORIAL BOARD
Co-Editors: Delane Shingadia and Nicole Ritz
Board Members
David Burgner (Melbourne, Cristiana Nascimento-Carvalho George Syrogiannopoulos
Australia) (Bahia, Brazil) (Larissa, Greece)
Kow-Tong Chen (Tainan,Taiwan) Ville Peltola (Turku, Finland) Tobias Tenenbaum (Mannhein, Germany)
Luisa Galli (Florence, Italy) Emmanuel Roilides (Thessaloniki, Marc Tebruegge (Southampton, UK)
Steve Graham (Melbourne, Greece) Marceline van Furth (Amsterdam,
Australia) Ira Shah (Mumbai, India) The Netherlands)

Newly Identified Viruses in Human Gastroenteritis

Pathogens or Not?
Saskia L. Smits, PhD,* Albert D.M.E. Osterhaus, PhD, and Marion P. Koopmans, PhD*

Key Words: human, gastroenteritis, diarrhea,
virus, pathogen, age, viral metagenomics, disease,
picornavirus, polyomavirus, parvovirus, picobirna-
virus, astrovirus, case-control
I nfectious diarrheal diseases remain a com-
mon cause of morbidity and mortality
especially in children younger than 5 years in
low-income and middle-income countries.1
The most common viral pathogens known
to cause acute gastroenteritis in humans are
From the *Department of Viroscience, Erasmus Medi-
cal Center, Rotterdam, The Netherlands; Viroclin-
ics Biosciences BV, Rotterdam, The Netherlands;
Research Center for Emerging Infections and
Zoonoses, University of Veterinary Medicine, Han-
nover, Germany; Artemis One Health, Utrecht,
The Netherlands; and Virology Division, Centre
for Infectious Diseases Research, Diagnostics and
Screening, National Institute for Public Health and
the Environment, Bilthoven, The Netherlands.
This work was partially funded by the European Com-
mission's COMPARE H2020 project under grant
agreement No 643476, the Dutch government pro-
ject number FES0908, by Netherlands Genomics
Initiative (NGI) project number 050-060-452, and
ZonMW TOP project 91213058.
Address for correspondence: Saskia Smits, Depart-
ment of Viroscience, Erasmus MC, P.O. Box 2040,
3000 Rotterdam, Netherlands, E-mail: s.smits@
erasmusmc.nl.
Supplemental digital content is available for this arti-
cle. Direct URL citations appear in the printed
text and are provided in the HTML and PDF
versions of this article on the journals website
(www.pidj.com).
Copyright 2015 Wolters Kluwer Health, Inc. All
rights reserved.
ISSN: 0891-3668/16/3501-0104
DOI: 10.1097/INF.0000000000000950
rotavirus A, norovirus, sapovirus, enteric ade-
noviruses and astrovirus. In addition to these
well-established etiologies, a large number of
moderate to severe diarrhea cases go without
etiological diagnosis and many unanswered
questions surrounding the burden and etiol-
ogy of diarrhea, and the factors determining
clinical expression of infection remain.
Sequence-independent amplification of
nucleic acids combined with next-generation
sequencing technology and bioinformatics
analyses or viral metagenomics is a relatively
new strategy for rapid identification of viruses
in clinical and public health settings. In contrast
to classical molecular detection techniques,
even those detecting multiple viruses, viral
metagenomics theoretically allows the char-
acterization of the entire virome in a clinical
sample, consisting of known pathogens, novel
pathogens that elude conventional testing, bac-
teriophages, plant viruses and unannotated
sequences that may be of viral origin. Since the
first applications of metagenomics to the field
of gastroenteritis,2,3 it has been increasingly
applied for virus discovery purposes resulting
in the identification of a plethora of previously
unknown viruses in human and animal enteric
specimens.4 Many previously unknown viruses
have been characterized in human stool alone
in recent years, such as salivirus, cosavirus,
bufavirus, picobirnavirus, recovirus, anellovi-
rus, astrovirus, circovirus and polyomavirus
(see Table, Supplemental Digital Content 1,
http://links.lww.com/INF/C297).5
The increasing application of such
unbiased or catch all detection methods raises
questions for the clinician, trying to understand
which information is relevant for patient man-
agement, and for the public health professional
focusing on population health decisions. The
expanding universe of microbes, discovered
through virome and microbiome studies, has
opened an entirely new field of research, seek-
ing to understand the interactions between hosts
and the healthy and disease causing microor-
ganisms that inhabit the human body. This is
particularly the case for the gut microbiome
and virome, which not only consists of human-
associated microbes but also reflect the host
environment. Viruses identified in human stool
samples may originate from a dietary source
or may replicate in the gut microbiota, such as
bacteria, parasites, protozoan or nematodes.
Even in the case of viruses with human tropism,
not only many factors, such as immune status,
age and nutritional status, but also geographic
location and even seasonal differences, play an
important role in the exposure to and clinical
outcome of virus infection.6 Viruses may only
become pathogenic in the context of certain
host backgrounds (ie, immunodeficiency, coin-
fections, genetic factors), exemplified by sim-
ian immunodeficiency virus infections in dif-
ferent primate host species.7 This underlines the
importance of studying both host and pathogen
parameters in an integrated way in pathogenic-
ity studies.
NEWLY IDENTIFIED VIRUSES
IN HUMAN GASTROENTERITIS:
PATHOGENS OR NOT?
Establishing disease causation in the
molecular era has been debated since the start

The ESPID Reports and Reviews of Pediatric Infectious Diseases series topics, authors and contents are chosen and approved
independently by the Editorial Board of ESPID.

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The Pediatric Infectious Disease Journal Volume 35, Number 1, January 2016 ESPID Reports and Reviews
of the use of molecular detection techniques
in biomedical research laboratories.8 Fulfill-
ing Koch postulates for all newly identified
viruses remains unlikely. Comparing viral
incidence or prevalence in diseased versus
matched controls, seroconversion and/or
detection of antigen/nucleic acid in affected
tissue may aid in studying viral pathogenic-
ity. As nicely reviewed previously,9 major
hurdles exist in performing these types of
studies. The comparison of virus preva-
lence in disease cases with healthy controls
requires careful subject matching to preclude
misleading results explained by host differ-
ences instead of viral pathogenicity. Healthy
control samples are often a limiting factor, as
these are not ordinarily collected in medical
settings. Measurement of antibody responses
requires (often difficult) cell culture systems
and/or proper antigen synthesis and positive
control sera, and biopsy results of patients for
antigen detection are often not available.9 In
addition, the extent of viral genetic and phe-
notypic diversity is complicating develop-
ment of specific and sensitive assays. Thus,
finding a new virus does not automatically
mean unveiling its clinical significance as is
highlighted below by conveying what is cur-
rently known of a subset of the most studied
viruses, focusing on virus families known to
infect vertebrates and for which case-control
studies were conducted (see Table, Supple-
mental Digital Content 1, http://links.lww.
com/INF/C297).
Picornaviridae
Many new viruses belonging to the
family Picornaviridae have been identified
in recent years in stool samples. The Picor-
naviridae family contains human and animal
viruses of considerable clinical and socioeco-
nomic importance, such as enterovirus, pare-
chovirus and hepatitis A virus. In addition, 4
new genera are recognized: cardiovirus (saf-
fold virus), cosavirus, kobuvirus (Aichivirus)
and salivirus (klassevirus), viruses which
were identified in human stool but for which
the clinical significance in gastroenteritis or
other disease syndromes remains unclear.10,11
Cardiovirus
Phylogenetic analysis suggests the
existence of 8 distinct genetic saffold virus
lineages. Screening for cardiovirus infec-
tions has occurred in many countries, includ-
ing Afghanistan, Canada, China, Germany,
Pakistan and the US, showing that this virus
has spread worldwide with polymerase chain
reaction (PCR) prevalence rates in enteric
specimens ranging from 0.5% to 12%.1015
Cardioviruses are mostly found in infants
and children <6 years of age.1015 Seropreva-
lence studies of saffold viruses 2 and 3 using
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virus neutralization assays in Africa, Asia
and Europe revealed that generally 75% of
children <24 months are seropositive versus
>90% in older children and adults.13,15 This
epidemiologic pattern suggests that cardiovi-
rus infection is acquired early in life, similar
to other viruses in the family Picornaviridae.
So far, epidemiological studies and 2 case-
control studies in young children have failed
to provide a clear picture of the relationship
between cardiovirus infection and actual dis-
ease in humans.10,12 Rather, available data
suggest that saffold viruses rarely cause dis-
ease and most likely go unnoticed in a high
proportion of infections.11,16 However, this is
also the case for other picornaviruses, that
nonetheless cause serious disease: for polio,
an estimated 1:100 to 1:1000 infected indi-
viduals develop neurological illness. Such
disease associations, however, cannot be
detected through the studies that have been
conducted so far.
Cosavirus
Cosaviruses are classified into 5
different species11 and were originally
identified in 2008 in the feces of South
Asian children with non-polio acute flac-
cid paralysis17 and shown to be present at
high prevalence in feces of both healthy
(44% and 25.4%) and paralyzed (49%
and 42.8%) children from Pakistan and
Tunisia, respectively.17,18 In China, cosavi-
rus was detected in children with (3.2%)
and without (1.6%) diarrhea.19 Cosavirus
prevalence was also reported in the feces
of healthy Brazilian children from a com-
munity child-care center (49% in 2008 and
6.5% in 2011) and with gastroenteritis in
a pediatrics department (3.6%).20 Strong
associations of cosavirus infection with
gastroenteritis were not obtained.
Kobuvirus
The Kobuvirus genus contains 3 spe-
cies, Aichivirus AC, of which only Aichi-
virus 1 from species A infects humans.21
Seroprevalence studies performed in Asia,
Europe and North Africa demonstrated high
levels of Aichivirus 1 antibodies (8099%)
in adults, suggesting a worldwide distribu-
tion of the virus.21 In contrast, much lower
reverse-transcriptionPCR prevalence data
were obtained, with Aichivirus 1 being
detected in 0.5%3% of human gastroen-
teritis cases in Asia, Europe, South-Amer-
ica and Africa, similar to observations with
saffold viruses.10,11,20,21 The epidemiological
data suggest that the virus might circulate
without causing any symptoms or with
mild symptoms that do not require medical
attention.
Salivirus
Since the identification of saliviruses,
they have been detected in stool samples
of children with gastroenteritis worldwide,
with prevalence rates ranging from 2.1% to
8.6%.10,11,22 A possible association was reported
between salivirus detection and gastroenteritis
in case-control studies.10,23,24 However, the virus
has also been detected in healthy children and
in association with other known enteric patho-
genic viruses, such as norovirus and adenovi-
rus, and thus, indicating that the potential asso-
ciation of salivirus with enteric disease needs
to be studied in more detail, similar to other
members of the family Picornaviridae.22,23
Polyomaviridae
Virus discovery programs using clini-
cal samples from a range of conditions have
yielded multiple newly identified viruses
in the family Polyomaviridae, including KI
polyomavirus (KIPyV), WU polyomavi-
rus (WUPyV), Merkel cell polyomavirus
(MCPyV), human polyomavirus 6 (HPyV6),
HPyV7, trichodysplasia spinulosa-associated
polyomavirus, HPyV9, MW polyomavirus
(MWPyV), HPyV10, MX polyomavirus, STL
polyomavirus and HPyV12. A case-control
study to explore the prevalence of 10 human
polyomaviruses (BKPyV, JCPyV, KIPyV,
WUPyV, MCPyV, HPyV6, HPyV7, trichod-
ysplasia spinulosa-associated polyomavirus,
HPyV9 and MWPyV) was conducted in
China by testing fecal specimens from 211
hospitalized children with diarrhea and from
208 asymptomatic control subjects collected
between April 2011 and January 2012.25
Only KIPyV (0.5 and 0%), WUPyV (4.3%
and 1.9%), MCPyV (30.3% and 27.9%) and
MWPyV (1.4% and 2.9%) were detected in
healthy controls and children with diarrhea,
respectively.25 STL polyomavirus was present
in 2.2% of hospitalized Chinese children with
gastroenteritis and 3.0% of healthy children.26
Despite the relatively frequent detection of
(some of the) human polyomaviruses in fecal
samples, a causative role of polyomaviruses in
gastroenteritis was not supported.25
Parvoviridae
New human bocaviruses (HBoVs)
and bufaviruses were identified in the family
Parvoviridae. HBoV subtypes 14 have been
detected in respiratory and gastrointestinal
infections worldwide.27 In contrast to HBoV1,
HBoV2, HBoV3 and HBoV4 occur mainly in
stool.28 HBoV2 is the most common with PCR
prevalence up to 26%, followed by HBoV3
(5%) and HBoV4 (2%). The detection rates in
adults may be lower than in children.28 Case-
control studies to investigate a link between
bocavirus 1 and gastroenteritis in China
between 2006 and 2008 showed no statisti-
cally significant differences in the prevalence
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Smits et al The Pediatric Infectious Disease Journal Volume 35, Number 1, January 2016
(3.5%4.3%), clinical disease presentation
and virus loads between children with and
without gastroenteritis.29,30 Human bocavirus
2 was detected at much higher prevalences of
20.4% and 12.3% in children with diarrhea and
healthy controls, respectively.30 An association
of human bocavirus 2 infection and acute gas-
troenteritis was shown in a case-control study
in Australian children, but not in adults or chil-
dren in the United Kingdom.31,32 Bufaviruses
were first detected in West African children
with diarrhea33 and have subsequently been
shown in diarrhea specimens from all over the
world with PCR prevalence rates between 0.8%
and 4% in patients with gastroenteritis.34,35
Astroviridae
The classic human astroviruses sero-
types 18 have been associated with acute
gastroenteritis in humans. Many more differ-
ent human astroviruses have been identified
in diarrhea specimens in recent years, such
as MLB1-3 and VA1-4, which seem to have a
worldwide distribution.36 A case-control study
on MLB1 and classic astrovirus infections in
India showed that classic astroviruses were sig-
nificantly associated with diarrhea, whereas the
MLB1 strain was not. In addition, MLB1 loads
were not different between symptomatic and
asymptomatic children.37 However, MLB1 was
significantly associated with diarrhea in Kenya
but not in The Gambia.38 A seroprevalence
study in North America indicated that MLB1
seropositivity was high in children <6 months
old, decreased to a nadir at 1223 months old
and increased to 100% by adulthood.39 Similar
high prevalence rates were seen for astrovirus
VA1 in healthy U.S. blood donors and evalua-
tion of serum samples from different pediatric
age groups revealed that the prevalence ofanti-
bodies in 612 months, 12 years, 25 years
and 510 years old was 20%, 23%, 32% and
36%, respectively, indicating rising seropreva-
lence with age.40 The seroprevalence studies
indicate that infection with these new astrovi-
ruses is common, but a causative role in gas-
troenteritis remains to be shown.
Overall, the development of next-gen-
eration sequence platforms in combination
with metagenomics approaches has yielded
a growing list of viruses detected in human
gastroenteritis cases, for which the (possible)
clinical disease association remains to be elu-
cidated, highlighting the fact that identifying
new viruses is at present easier than show-
ing association with disease.
FUTURE ROLE OF VIRAL
METAGENOMICS IN DISEASE
ASSOCIATION STUDIES?
It is clear that virome profiling at
present does not add much to the needs
of the clinician or public health specialist.
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Nevertheless, viral metagenomics can be
used as a potentially cost-saving research
tool to study viral incidence, level of viral
coinfections and their correlation to clini-
cal diarrheic disease in well-designed cohort
studies in the years to come and to resolve
some of the outstanding questions simultane-
ously for a wide range of viruses. Provided
samples from large enough matched cohorts
are analyzed from different human popula-
tions with and without disease, the obtained
data may not only provide basic insight into
the viral burden of diarrhea in general instead
of focusing on one or a few viral pathogens
as is classically done, but it may allow (i) elu-
cidation of viral pathogens or combinations
of pathogens involved in disease burden,
(ii) surveillance for as yet unidentified enteric
viruses and zoonotic events, (iii) the study of
effects of vaccination on viral incidence lev-
els (eg, rotavirus) and whether other viruses
fill the niche that vaccination leaves behind
and (iv) an appraisal of host differences in
viral disease burden. Theoretically, it would
be possible to decipher the entire spectrum of
viruses present in the human enteric tract at
a certain point in time. This gut virome is not
a given: it will vary over time, with diet, with
genetic profile and health status of the host,
among others.6 Finding clinically relevant
information on viruses and their evolutionary
changes becomes theoretically possible but
requires in depth bioinformatics analyses of
systematically sampled stool with sufficient
metadata for meaningful analysis. Currently,
such analyses are time-consuming, expensive
and complex precluding routine use of next-
generation sequencing in clinical and public
health settings. The fast developments in the
metagenomics field, however, undoubtedly
will lead to advances in this respect.
CONCLUSIONS
When talking about newly identi-
fied viral pathogens of human gastroenteri-
tis, one can conclude that breakthroughs
in the field of metagenomics have had far-
reaching effects on the identification and
characterization of new viruses. It also led
to the recognition that a growing number of
enteric gastroenteritis cases that were once
attributed to unknown causes may be caused
or triggered by infectious viral agents.
Solid evidence, however, that these new
viruses actually cause disease is difficult to
obtain. Still, the technology is fast evolv-
ing, and viral metagenomics when applied
judiciously could address some important
outstanding questions, relevant both for
virus diagnostics and surveillance. Future
metagenomics will potentially enable gen-
erating the complete genomic information
from clinical samples independent of the
sector (public health, veterinary health and
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food safety), the type of pathogen (viruses,
bacteria and parasites) and the sample type.
The primary sequence output and derived
data, such as assemblies and functional
annotation data, in combination with associ-
ated clinical, microbiological, and epidemi-
ological data would lead to a more in depth
view of the human virome in correlation to
disease. This does require engagement of
clinicians, bioinformaticians, epidemiolo-
gists and laboratory scientists, to fully cap-
ture the potential of these novel technologies
for infectious disease research.
ACKNOWLEDGMENTS
This work was partially funded by the
European Commission COMPARE H2020
project under grant agreement No 643476,
the Dutch government project number
FES0908, by Netherlands Genomics Initia-
tive (NGI) project number 050-060-452 and
ZonMW TOP project 91213058.
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