Serological Studies Confirm the Novel Astrovirus HMOAstV-C as a Highly Prevalent Human Infectious Agent
Molecular identification of a microbe is the first step in determining its prevalence of infection and pathogenic potential. Detection of specific adaptive immune responses can provide insights into whether a microbe is a human infectious agent and its epidemiology. Here we characterized human anti-IgG antibody responses by luciferase immunoprecipitation systems (LIPS) against two protein fragments derived from the capsid protein of the novel HMOAstV-C astrovirus. While antibodies to the N-terminal fragment were not informative, the C-terminal capsid fragment of HMOAstV-C showed a high frequency of immunoreactivity with serum from healthy blood donors. In contrast, a similar C-terminal capsid fragment from the related HMOAstV-A astrovirus failed to show immunoreactivity. Detailed analysis of adult serum from the United Sates using a standardized threshold demonstrated HMOAstV-C seropositivity in approximately 65% of the samples. Evaluation of serum samples from different pediatric age groups revealed that the prevalence of antibodies in 6-12 month, 1-2 year, 2-5 year and 5-10 year olds was 20%, 23%, 32% and 36%, respectively, indicating rising seroprevalence with age. Additionally, 50% (11/22) of the 0-6 month old children showed anti-HMOAstV-C antibody responses, likely reflecting maternal antibodies. Together these results document human humoral responses to HMOAstV-C and validate LIPS as a facile and effective approach for identifying humoral responses to novel infectious agents.
Serological Studies Confirm the Novel Astrov irus
HMOAstV-C as a Highly Prevalent Human Infectious
Peter D. Burbelo
*, Kathryn H. Ching
, Frank Esper
, Michael J. Iadarola
, Eric Delwart
, W. Ian Lipkin
1 Neurobiology and Pain Therapeutics Section, Laboratory of Sensory Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health,
Bethesda, Maryland, United States of America, 2 Department of Pediatrics, University Hospitals Case Medical Center, Cleveland, Ohio, United States of America, 3 Blood
Systems Research Institute and Department of Laboratory Medicine, University of California San Francisco, San Francisco, California, United States of America, 4 Center for
Infection and Immunity, Columbia University, New York, New York, United States of America
Molecular identification of a microbe is the first step in determining its prevalence of infection and pathogenic potential.
Detection of specific adaptive immune responses can provide insights into whether a microbe is a human infectious agent
and its epidemiology. Here we characterized human anti-IgG antibody responses by luciferase immunoprecipitation
systems (LIPS) against two protein fragments derived from the capsid protein of the novel HMOAstV-C astrovirus. While
antibodies to the N-terminal fragment were not informative, the C-terminal capsid fragment of HMOAstV-C showed a high
frequency of immunoreactivity with serum from healthy blood donors. In contrast, a similar C-terminal capsid fragment
from the related HMOAstV-A astrovirus failed to show immunoreactivity. Detailed analysis of adult serum from the United
Sates using a standardized threshold demonstrated HMOAstV-C seropositivity in approximately 65% of the samples.
Evaluation of serum samples from different pediatric age groups revealed that the prevalence of antibodies in 6–12 month,
1–2 year, 2–5 year and 5–10 year olds was 20%, 23%, 32% and 36%, respectively, indicating rising seroprevalence with age.
Additionally, 50% (11/22) of the 0–6 month old children showed anti-HMOAstV-C antibody responses, likely reflecting
maternal antibodies. Together these results document human humoral responses to HMOAstV-C and validate LIPS as a
facile and effective approach for identifying humoral responses to novel infectious agents.
Citation: Burbelo PD, Ching KH, Esper F, Iadarola MJ, Delwart E, et al. (2011) Serological Studies Confirm the Novel Astrovirus HMOAstV-C as a Highly Prevalent
Human Infectious Agent. PLoS ONE 6(8): e22576. doi:10.1371/journal.pone.0022576
Editor: Man-Seong Park, College of Medicine, Hallym University, Korea
Received April 5, 2011; Accepted June 24, 2011; Published August 4, 2011
Copyright: ß 2011 Burbelo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Division of Intramural Research, National Institute of Dental and Craniofacial Research. This study is supported in part
by the National Institute of Allergy and Infectious Diseases K23 AI065829 (F.E.), AI090196 (A.K.) and AI57158 (Northeast Biodefense Center - A.K., W.I.L), and the
Department of Defense (A.K., W.I.L.) and the Blood Systems Research Institute and NIH R01220 HL083254 (E.D.) . The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
The family Astroviridae consists of small (28–30 nm in diameter),
non-lipid enveloped, single-stranded positive-sense RNA viruses
with genomes ranging in size from 6.4 to 7.3 kb. The genome
includes three open reading frames (ORFs) designated ORF1a,
ORF1b and ORF2. ORF1a encodes the non-structural poly-
protein 1a, while the longer ORF1b encodes polyprotein 1ab
including the RNA dependent RNA polymerase (RdRp) expressed
through a ribosomal frameshift at the ORF1a/1b junction. ORF2
encodes the viral capsid structural polyprotein [1,2]. To date the
Astroviridae family consists of two genera, Astrovirus and Mamas-
trovirus, which infect avian and mammalian hosts, respectively.
These astroviruses, transmitted through the fecal-oral route can
cause gastroenteritis in numerous avian and mammalian species,
including humans [3,4]. All eight known human astrovirus
serotypes belonging to the first identified human Astrovirus species
(HAstV) have been associated with gastroenteritis [5,6,7,8,9].
Clinical symptoms of HAstV infection in humans usually lasts
between two and four days  and consists of watery diarrhea
and less commonly, vomiting, headache, fever, abdominal pains
and anorexia. HAstV can also cause significant disease in the
elderly and immunocompromised patients .
Several metagenomics studies have recently used random
amplification and mass sequencing of nucleic acid extracted from
human stool to systematically catalogue viruses, phage and
bacteria present in patients with diarrhea [12,13,14,15]. For
example, a novel astrovirus species, AstV-MLB1, was identified in
stool from children and adults, including some with diarrhea
[13,16,17,18]. In addition, a new group of astroviruses, provi-
sionally named HMOAstV/AstV-VA, were discovered by con-
sensus PCR using stool samples from different continents [13,18].
Phylogenic analysis of the HMOAstV viruses revealed that they
consisted of three subgroups, HMOAstV-A, HMOAstV-B, and
HMOAstV-C. HMOAstV-C RNA was also identified in individ-
uals from a gastroenteritis outbreak in a daycare center in Virginia
and tentatively named AstV-VA1 . Despite the identification
of novel astroviruses in human stool, it is unclear if these new
viruses are pathogenic or whether they are passengers associated
with ingested food.
PLoS ONE | www.plosone.org 1 August 2011 | Volume 6 | Issue 8 | e22576
Our objective was to determine the seroprevalence of anti-
HMOAstV-C antibodies in children and adults in the US.
Traditionally, ELISA assays are used for serological testing and
require purified virus or recombinant viral proteins. Unfortunate-
ly, ELISA tests often show cross-reactivity, have inherent high
background signals and require extensive optimization and
standardization. Luciferase Immunoprecipitation System (LIPS)
is a new technology that employs luciferase-tagged antigens in a
liquid phase immunoprecipitation assay . LIPS offers several
advantages over ELISA including low backgrounds, highly
quantitative data and the ability to generate diagnostically useful
serodeterminations without pre-determined cut-off values [20,21].
To determine if the novel HMOAstV-C astrovirus is a prevalent
human virus, LIPS was used to screen for antibodies against
conserved regions of the HMOAstV-C viral capsid.
Materials and Methods
Serum samples from adult, healthy blood donors (n = 106) were
collected without any clinical information under IRB approved
protocols at the National Institutes of Health, Bethesda, Maryland.
Children serum samples were from Rainbow Babies and
Children’s Hospital, Cleveland, Ohio and obtained under IRB
approval from University Hospitals – Case Medical Center. A
total of 103 serum samples from children from different ages were
analyzed as follows: 0-6 months (n = 22), 6–12 months (n = 15), 1-2
years (n = 22), 2-5 years (n = 22) and 5-10 years old (n = 22).
Samples were collected from September 2009 through March
2010. Other than the age of the individual from whom the serum
was obtained, no other clinical information was available.
Based on the similarity of HMOAstV-C astrovirus with animal
astroviruses , serum samples from different domesticated
animals, including horses, pigs and rabbits, were tested. Pig and
horse serum samples were the kind gift of Yanjin Zhang and Utpal
Pal (VA-MD Regional College of Veterinary Medicine, Univ. of
Maryland). Rabbit serum samples were obtained from NIH
laboratories and commercial vendors. All serum samples were
stored at 280uC, thawed, and then left at 4uC prior to processing
for LIPS analysis.
Generation of Ruc-Astrovirus antigen fusion constructs
Templates for capsid coding sequences of HMOAstV-C and
HMOAstV-A were generated by RT-PCR amplification using
human stool as described . Due to the possibility of antibody
cross-reactivity to different regions of the HMOAstV-C capsid,
two different fragments encompassing the N-terminal (amino acids
1-393) and C-terminal fragment (amino acids 402-704) were
generated by PCR. The primer adapter sequences used to clone
each protein fragment are as follows: N-terminal capsid fragment
of HMOAstV-C, 59-GAGGGATC CATGGCTGGTAAA-
CAGCCC-39 and 59-GAGCTCGAGTCAAGGGCCTGTGT-
TAGGTGC-39 and C-terminal capsid fragment of HMOAstV-
C, 59- GAGGGATCCAACACCACTACAGGGTCA-39 and 59-
GAGCTCGAGTCAATCCAGTGGGGTCAATCT-39. A third
C-terminal capsid fragment from the HMOAstV-A astrovirus
(amino acids 396-700), spanning a similar region to HMOAstV-C
was constructed with the primers: 59-GAGGGATCCAGCA-
CAGCCTCAGCGG TT -39 and 59-GAGCTCGAGT-
CAATCCTTAGGCTTCTTTCT-39. The three capsid frag-
ments were subcloned downstream of Renilla luciferase (Ruc)
using the pREN2 vector . DNA sequencing was used to
confirm the integrity of the three DNA constructs. The sequence
for the C-terminal capsid fragment of HMOAstV-C has been
deposited in GenBank with accession (JF313458). Plasmid DNA
was then prepared from these two different pREN2 expression
vectors using a Qiagen Midi preparation kit. Following transfec-
tion of mammalian expression vectors, crude protein extracts were
obtained as described for use as antigen .
Briefly, human and animal sera were processed in a 96-well
format at room temperature as previously described . Serum
samples were first diluted 1:10 in assay buffer A (50 mM Tris,
pH 7.5, 100 mM NaCl, 5 mM MgCl
, 1% Triton X-100) using a
96-well polypropylene microtiter plate. Antibody titers were
measured by adding 40
ml of buffer A, 10 ml of diluted sera (1 ml
equivalent), and 1610
light units (LU) of each of the Ruc-
HMOAstV antigen fragments containing crude Cos1 cell extract
to wells of a polypropylene plate and incubated for 60 minutes at
room temperature on a rotary shaker. Next, 5
ml of a 30%
suspension of Ultralink protein A/G beads (Pierce Biotechnology,
Rockford, IL) in PBS were added to the bottom of each well of a
96-well filter HTS plate (Millipore, Bedford, MA). To this filter
plate, the 100
ml antigen-antibody reaction mixture was trans-
ferred and incubated for 60 minutes at room temperature on a
rotary shaker. The washing steps of the retained protein A/G
beads were performed on a Biomek Workstation or Tecan plate
washer with a vacuum manifold. After the final wash, LU were
measured in a Berthold LB 960 Centro microplate luminometer
(Berthold Technologies, Bad Wilbad, Germany) using coelenter-
azine substrate mix (Promega, Madison, WI). All LU data were
obtained from the average of at least two separate experiments.
Using the C-terminal capsid fragment of HMOAstV-C as the
query sequence, a BLAST search was performed against the non-
redundant NCBI protein databases. From this analysis, the highest
homology was with HMOAstV-B and HMOAstV-A astroviruses.
Viral capsid sequences were aligned using the global alignment
program COBALT (www.ncbi.nlm.nih.gov/guide/sequence-
analysis/) with default parameters.
GraphPad Prism software (San Diego, CA) was used for
statistical analysis. For the calculation of sensitivity and specificity,
a cut-off limit was used, which was derived from the combined
value of the mean value plus 3 standard deviations (SD) of the
replica samples containing only buffer, Ruc-extract and protein
A/G beads. Human blood donor samples highly positive for anti-
HMOAstV-C antibodies were used as internal positive controls to
standardize the LIPS parameters for testing of serum samples.
Identification of human antibody responses to the capsid
While most bona fide antigenic targets used in LIPS assays show
high sensitivity and specificity , the exact antigens useful for
diagnosis of HMOAstV-C are not known. As a screening
approach and to potentially eliminate cross-reactivity spanning
the full-length capsid regions of these viruses, we chose to first test
two different protein fragments encompassing conserved N- and
C-terminal capsid fragments of HMOAstV-C. From LIPS
screening of 45 adult blood donor samples, the HMOAstV-C N-
terminal capsid fragment showed higher background binding to
the mock protein A/G beads than to clinical samples and was
judged not to be immunoreactive (Fig. 1). However, the C-
HMOAstV-C Is a Highly Prevalent Human Infection
PLoS ONE | www.plosone.org 2 August 2011 | Volume 6 | Issue 8 | e22576
terminal capsid fragment of HMOAstV-C protein demonstrated a
robust response up to 125,900 LU above background (Fig. 1). The
HMOAstV-C immunoreactivity in these human seropositive
samples was highly reproducible and could be completely
eliminated by pre-adsorption with protein A/G beads (data not
shown). A third protein fragment derived from a similar C-
terminal capsid region of the related HMOAstV-A virus failed to
show immunoreactivity with any of the human samples tested
(Fig.1). These results suggest that detectable human antibody
responses are specific for HMOAstV-C and not for HMOAstV-A
Sequence analysis of the C-terminal fragment of HMOAstV-C
revealed 68% and 30% amino acid identity with corresponding
capsid region from the novel Astrovirus, HMOAstV-B and
HMOAstV-A astroviruses, respectively (Fig. 2). Importantly, no
significant homology of the HMOAstV-C protein fragment was
detected with the capsids derived from the eight known serotypes
of HAstV strains (data not shown).
High prevalence of anti-HMOAstV-C antibodies in adult
To determine if HMOAstV-C infection is unique to humans,
additional LIPS analysis was performed to compare immunore-
activity in animal and human serum samples. For these studies,
immunoreactivity against the C-terminal capsid fragment of
HMOAstV-C was examined in 106 healthy adult US blood
donors side-by-side serum samples from different domesticated
animals. Buffer blanks in the LIPS format were used as negative
controls to evaluate seropositivity  and a cut-off value was
calculated from the mean plus 3 SD of 19 replica samples
containing only buffer, Ruc-extract and protein A/G beads. Using
a threshold of .9,525 LU, 65% (69 of 106) of healthy adult US
blood donors were seropositive (Fig. 3). In contrast, no
immunoreactivity was detected in any of the rabbit (n = 6), or
pig (n = 16) serum samples (Fig. 3). Of 16 horse serum samples,
one sample (6.25%) was immunoreactive to HMOAstV-C antigen
(Fig. 3). As a control, several antigens were tested and revealed
highly robust antibody titers in these same animal serum samples
against known animal pathogens (data not shown). Collectively,
these results suggest that antibodies against HMOAstV-C are
relatively common in human adults, but are rare or non-existent in
pigs, rabbits and horses.
Increased exposure to HMOAstV-C with age
We next explored differences in HMOAstV-C seroprevalence
amongst different aged children in the United States. LIPS
analysis revealed that the prevalence of infection in 6–12 month,
1–2 year, 2–5 year and 5–10 year olds was 20%, 23%, 32% and
36%, respectively. These results suggest a trend of increased
antibody prevalence with increasing age, although no statistical
difference was observed between age groups (Fig. 4). Additionally,
50% (11/22) of the 0-6 month old children showed anti-
HMOAstV-C antibody responses, which likely reflects the
presence of maternal antibodies. No statistical difference in
antibody titers was observed between these different groups of
children. All of the different children age groups showed a
significantly lower prevalence of HMOAstV-C antibodies than
adults (Fischer Exact T test; p,0.005). These results are consistent
with increased infection by HMOAstV-C over time.
Although several novel viruses have been identified in human
stool [12,13,14,15], few studies have examined humoral responses
to them. We have established a robust, sensitive serology platform
that is ideally suited for pathogen discovery applications. LIPS
holds several advantages over ELISA for studying humoral
responses against potentially new infectious agents stemming from
the low background binding, high sensitivity innate to this assay
and the ability to rapidly test different antigens/antigen fragments
using a standard format with little or no assay optimization .
Here we used LIPS assays to demonstrate humoral responses to
the HMOAstV-C Astrovirus capsid protein and provide experi-
mental data supporting human infection.
Several different lines of evidence indicate that the HMOAstV-
C capsid fragment is a target of humoral responses. First, only the
C-terminal capsid region of HMOAstV-C was immunoreactive
with human serum samples. Although RNA from HMOAstV-A
was previously identified in human stool samples, we were unable
to detect human immunoreactivity to this analogous C-terminal
capsid region of HMOAstV-A. The exact reason for the lack of
immunoreactivity is not clear, but it is possible that this capsid
region of HMOAstV-A is less antigenic or its conformational
folding is not recapitulated in the current Renilla luciferase fusion
protein using the LIPS system. Second, the HMOAstV-C
astrovirus capsid region used in LIPS was relatively unique and
had no significant homology with the eight known serotypes of
HAstV strains. Based on its homology, it is possible that some of
the observed HMOAstV-C capsid immunoreactivity is against the
related HMOAstV-B capsid. However, our previous LIPS studies
Figure 1. LIPS detection of antibodies to a C-terminal capsid
fragment of HMOAstV-C. Antibodies to the N- and C-terminal capsid
protein of HMOAstV-C and the C-terminal capsid protein of HMOAstV-
CA capsid protein fragments were analyzed in 45 adult serum samples.
Each symbol represents individual serum samples tested with each
protein fragment s and LU values were adjusted by subtracting
background binding to protein A/G beads. The short solid line
represents the mean antibody titer for each group.
HMOAstV-C Is a Highly Prevalent Human Infection
PLoS ONE | www.plosone.org 3 August 2011 | Volume 6 | Issue 8 | e22576
demonstrate markedly different serologic responses to similarly
homologous antigens in subjects infected with related pathogens
[25,26]. In these studies, variations in both linear and conforma-
tional epitopes are more clearly differentiated using the LIPS
liquid assay than using ELISAs . Further studies examining
the humoral responses against the HMOAstV-B capsid, as well as
additional proteins from the HMOAstV viruses should resolve this
issue. Third, the anti-HMOAstV-C capsid antibody titers detected
by LIPS assay in human samples was substantially higher than the
background binding associated with mock protein A/G beads
alone. This magnitude is comparable to those seen by LIPS
against other infection agents such as Borrelia burgdorferi  and
Kaposi Sarcoma associated virus . Our strategy of using buffer
blanks instead of seronegative uninfected samples has been used in
other seroepidemiologic investigations and provide comparable
diagnostic thresholds as seronegative samples [24,29]. Fourth, the
relatively low immunoreactivity against HMOAstV-C in young
children (age 6-12 months) and a corresponding increase in
seroprevalence with childhood age is consistent with human
infection. Lastly, additional studies evaluating rabbit, pig and
horse samples showed that anti-HMOAstV-C antibodies were rare
or absent in animals. The lack of immunoreactivity against
HMOAstV-C in animals, despite the greater homology of
HMOAstV-C to animal Astroviruses , supports the notion
that humans are a host for these viruses. The approach of
analyzing humoral responses to a panel of different animal serums
side-by-side human samples by LIPS is useful for understanding
the host range for this and other potential pathogens.
Figure 2. Comparison of the C-terminal capsid fragment of HMOAstV-C with related viruses. From BLASTP analysis, the highest
homology of the HMOAstV-C capsid fragment was with HMOAstV-B (68% identity) and HMOAstV-A (30% identity) astroviruses. Identical amino acid
residues with HMOAstV-C are shaded.
Figure 3. LIPS detection of antibodies to the C-terminal capsid
fragment of HMOAstV-C in human and animal serum samples.
Immunoreactivity to the HMOAstV-C was determined in 106 healthy
adult US blood donors, 6 rabbits, 16 horses, 16 pigs and 14 buffer only
controls. Raw LU values are shown without subtracting background
binding to protein A/G beads. The short solid line represents the mean
titer for each group. The dashed line represents the diagnostic cut-off,
derived from the mean plus 3 SD of 19 replica buffer blank samples.
Figure 4. Prevalence of HMOAstV-C antibodies with childhood
age. A total of 103 child serum samples were analyzed by LIPS
including from the following age brackets: 0–6 months (n = 22), 6–12
month (n = 15), 1–2 year (n = 22), 2–5 year (n = 22), and 5–10 year olds
(n = 22). Raw LU values are shown without subtracting background
binding to protein A/G beads. The dashed line represents the
diagnostic cut-off value derived from the mean plus 3SD of replica
buffer blank sample s. The fraction and percent seropositive for
HMOAstV-C are shown above each group.
HMOAstV-C Is a Highly Prevalent Human Infection
PLoS ONE | www.plosone.org 4 August 2011 | Volume 6 | Issue 8 | e22576
Overall the results suggest that HMOAstV-C is a common
infectious agent circulating in human populations. It is also
possible that the studies described here underestimate the true
seroprevalence of HMOAstV-C that may result from transient
antibody titers from older infections and the genetic diversity of
this newly recognized agent. The higher prevalence of antibodies
in adults (65%) suggests that most adults are seropositive; however,
it is unknown whether the presence of antibody is protective
against infection. Nevertheless, compared to children, adults show
a markedly higher incidence of HMOAstV-C infection. Further-
more, the detection of high prevalence of antibodies in the 0-6
month old children is consistent with the presence of maternal
antibodies. In infants, waning maternal antibodies for many
infectious agents is often observed [30,31].
Additional investigation including prospective analysis of
antibody responses by LIPS from acute infection would be
worthwhile. It is interesting to note that HMOAstV-C/AstV-VA1
was identified as an Astrovirus associated with an outbreak of
gastroenteritis in a child daycare center in Virginia . RT-PCR
and sequencing of affected individuals showed identical
HMOAstV-C sequences. It is highly likely that there are diverse
HMOAstV-C strains causing a spectrum of clinical symptoms.
Along these lines, an Astrovirus, phylogenetically related to
HMOAstV-C (95% identical capsids) was recently detected in
the brain of a child with encephalitis who had a genetic form of
agammaglobulinemia . Further studies are clearly needed to
establish the clinical spectrum of disease caused by HMOAstV-C.
Although a single antigenic capsid target was diagnostically useful
here with HMOAstV-C, the ability of detecting antibody
responses to multiple independent protein fragments can increase
assay sensitivity and has the potential to stratify different
conditions caused by the same infectious agent . The
development of Renilla luciferase antigen fusions for additional
HMOAstV-C Astrovirus proteins and other Astrovirus in the LIPS
system will likely be useful for identifying and understanding their
role in diarrheal and other illnesses.
Conceived and designed the experiments: PDB ED WIL AK. Performed
the experiments: PDB KHC AK. Analyzed the data: PDB KHC.
Contributed reagents/materials/analysis tools: FE MJI. Wrote the paper:
PDB FE ED WIL AK.
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HMOAstV-C Is a Highly Prevalent Human Infection
PLoS ONE | www.plosone.org 5 August 2011 | Volume 6 | Issue 8 | e22576