Presence of Antibodies against Genogroup VI
Norovirus in Humans
João Rodrigo Mesquita1,2, Verónica P Costantini3, Jennifer L Cannon4, Seh-ching Lin3,
Maria São José Nascimento1and Jan Vinjé3*
Background: Noroviruses are important enteric pathogens in humans and animals. Recently, we reported a novel
canine norovirus (CaNoV) in dogs with diarrhea belonging to a new genogroup (GVI). No data are available on
exposure of humans to this virus.
Methods: Sera from 373 small animal veterinarians and 120 age-matched population controls were tested for IgG
antibodies to CaNoV by a recombinant virus like particle based enzyme-linked immunosorbent assay.
Results: Antibodies to CaNoV were found in 22.3% of the veterinarians and 5.8% of the control group (p <0.001).
Mean corrected OD450values for CaNoV antibodies were significantly higher in small animal veterinarians compared
to the control group.
Conclusions: These findings suggest that CaNoV may infect humans and small animal veterinarians are at an
increased risk for exposure to this virus. Additional studies are needed to assess if this virus is able to cause disease
Noroviruses (NoVs) are the leading cause of epidemic and
sporadic acute gastroenteritis in humans with worldwide an
estimated 1 million hospitalizations and up to 200,000
deaths in children<5 years of age each year [1,2]. Outbreaks
occur in various settings including long-term care facilities,
hospitals, schools, restaurants and cruise ships. The main
modes of transmission of NoV are person-to-person and
through the consumption of contaminated food or water.
During outbreaks, however, multiple transmission routes
may play a role . In recent years NoVs have been detected
in a number of mammalian species and several studies have
suggested that zoonotic transmission from animal to
humans may occur [4-6] and that an animal reservoir might
be the source of the introduction of new strains in the hu-
man population. Although no zoonotic events have been
reported, there are several indications that NoVs may be
able to cross the species barrier. Gnotobiotic pigs have been
experimentally infected with a human NoV strain , and
viruses closely related to human NoVs have been detected
in swine . Moreover, NoV sequences have been detected
in livestock and in retail meat samples highlighting a pos-
sible route for indirect zoonotic transmission of NoVs
through the food chain and the risk for emergence of
animal/human recombinants .
Noroviruses are a group of non-enveloped, single-
stranded, RNA viruses with an icosahedral capsid sym-
metry classified into the genus Norovirus of the family
Caliciviridae. They can be grouped in at least 5 different
genogroups (designated GI-GV) [1,9]. Strains infecting
humans are found in GI, GII and GIV. Porcine NoVs are
classified in distinct genotypes within GII, bovine and
ovine viruses belong uniquely to GIII, and murine NoVs
are grouped in GV. Recently, several research groups
have reported NoVs in domestic carnivores with diar-
rhea [10,11]. Canine NoVs (CaNoVs) genetically related
to GIV have been reported in Italy, Greece and Japan
[10-13], whereas viruses belonging to a proposed new
genogroup (GVI) were found in fecal samples from dogs
with diarrhea in Portugal and Italy [9,14-16].
The zoonotic potential of an infectious disease agent
has been inferred by comparing pathogen-specific anti-
body levels between individuals that are in close contact
* Correspondence: email@example.com
3Division of Viral Diseases, National Center for Immunization and Respiratory
Diseases, Centers for Disease Control and Prevention, Mail Stop G-04, 1600
Clifton Rd, Atlanta, GA 30333, USA
Full list of author information is available at the end of the article
© 2013 Mesquita et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Mesquita et al. Virology Journal 2013, 10:176
with a particular animal and a matched control population
with no professional exposure to animals [17,18]. For ex-
ample, a higher serum antibody level against bovine NoV
was detected in large animal veterinarians compared to
the general population, indicating that bovine NoV strains
could infect the human population . Additionally,
antibodies to human NoVs have been detected in pigs
highlighting the possibility of human-to-animal transmis-
sion of NoV .
In most industrialized countries, pets are an integral
part of the household leading to well-documented health
risks associated with owning a pet. Bites, scratches and
allergies are more frequent; however, infections includ-
ing parasitic, bacterial, fungal and viral diseases can be
transmitted to humans . In a recent report, human
NoV sequences were detected in fecal samples from pet
dogs which had been in direct contact with humans with
NoV gastroenteritis, suggesting that human NoVs can at
least survive in the gastrointestinal tract of dogs .
To investigate if CaNoV may infect humans, sera from
pet veterinarians and age-matched population controls
were tested for IgG antibodies to recombinant virus-like
particles of CaNoV. This Study Protocol was published
Of the 373 veterinarians, 83 (22.3%) had IgG antibodies
against CaNoV compared to 7 (5.8%) of the 120 matched
population controls (p<0.001). Moreover, the mean
corrected OD450values for CaNoV antibodies was signifi-
cantly higher in veterinarians than in controls (p<0.001)
(Figure 1). CaNoV antibodies were detected in veterinar-
ians from all four countries.
To evaluate possible cross-reactivity between these
antibodies and human NoV, two serum samples from
veterinarians with high (HAT) and low antibody titers
(LAT) to CaNoV were pre-incubated with 2-fold serial
dilutions of CaNoV VLPs. The corrected OD450values
for CaNoV antibodies in both serum samples decreased
significantly with increasing concentration of CaNoV
VLPs (β=−0.150±0.043, 95%CI −0.287 to −0.013, p<0.05
and β=−0.073±0.018, 95%CI −0.132 to −0.014, p <0.05
for serum HATand LAT, respectively) (Figure 2A). By con-
trast, no significant change in the corrected OD450
values was observed when the pre-incubated sera were
tested for the presence of GII.4 New Orleans anti-
bodies (β = −0.011 ± 0.031; 95%CI −0.11 to 0.089 and
β = 0.0219 ±0.056; 95%CI 0.158 to 0.202 for samples
68 and 25, respectively) (Figure 2B).
We detected IgG antibodies against CaNoV in 22.3% of
the small animal veterinarians and in 5.8% of the age-
matched population controls. These findings suggest
that CaNoV may infect humans and that small animal
veterinarians are at an increased risk for exposure and
possibly infection with this virus. The presence of anti-
bodies in the population control samples may be
explained by household contact as dogs are popular pet
An increased exposure risk to bovine NoV has been
reported for large animal veterinarians in the Netherlands
who had a higher seroprevalence of bovine NoV anti-
bodies than the general population . Conversely, a
high prevalence of antibodies against human NoV (Nor-
walk strain) was detected in pigs and captive juvenile ma-
caques [5,22]. These data suggest that NoV may be able to
cross the species barrier. Exposure to zoonotic agents is a
widely recognized risk in veterinary medicine. In an
Australian survey, 4% of veterinarians reported having
acquired at least one zoonotic disease from animal-
related exposure . In another study, IgG antibodies
against Brucella spp and Coxiella burnetii were higher
among veterinarians working in an endemic region
. The zoonotic risk of veterinarians and pig han-
dlers for hepatitis E virus (HEV) infections has been
Figure 1 Corrected optical densities [OD] at 450 nm for canine
norovirus antibodies in sera from veterinarians and controls.
Sera were tested for the presence of CaNoV antibodies in a VLP-
based ELISA at 1:1,500 dilution. Values are the corrected optical
densities (OD) at 450 nm [OD450(VLP coated) - OD450(non-coated)]
for each serum sample and the mean corrected OD450value of each
group (horizontal bars). Groups were compared and analyzed by
Mann–Whitney U-test. Differences were considered significant (*)
Mesquita et al. Virology Journal 2013, 10:176
Page 2 of 5
demonstrated in studies in Taiwan and the US [25,26]. In
the Taiwan study, 27% of pig handlers tested positive for
anti-HEV antibodies compared to 8% of the control sub-
jects , while in the US study 26% of veterinarians
working with swine and 17% of blood donors were
seropositive for HEV, suggesting that veterinarians may
be at a higher risk of HEV infection through animal
contact, compared to normal blood donors .
A limitation of our study was that the antibodies
detected by the CaNoV VLPs may be cross-reactive
against human NoVs. However, the blocking assay data
showed that binding of CaNoV antibodies but not hu-
man NoV antibodies, could be blocked by CaNoV VLPs,
demonstrating the VLP-based ELISA used in this study
measured CaNoV-specific antibodies.
In conclusion, our data suggest that CaNoV may infect
humans and that small animal veterinarians are at an in-
creased risk. Studies that test human stool samples in
households with dogs with CaNoV diarrhea are needed
to confirm that this virus is able to cause diarrheal dis-
ease in humans.
Materials and methods
A total of 373 pet veterinarians from four different
countries (Portugal, Spain, Brazil, and United Kingdom)
who attended the Annual Veterinary Meeting in January
2012 in Santa Maria da Feira, Portugal, were enrolled in
the study after giving informed consent (Table 1). Blood
was obtained by venipuncture from all enrolled veteri-
narians. In addition, 120 sera matched by age (in 5-year
age groups) and sex were collected from anonymous vol-
unteers from the University of Porto. This study was ap-
proved by the institutional review board at the University
of Porto (Parecer nº18/CEUP/2011).
Figure 2 Evaluation of potential cross-reactivity between human GII.4 NoV IgG antibodies and canine NoV VLPs. Two sera with low
(LAT) and high (HAT) CaNoV antibody titer were pre-incubated with a 2-fold serial dilution of CaNoV VLPs (5, 2.5, 1.25, 0.625, 0.3125 μg/ml) and
tested for antibodies against CaNoV VLPs (A) and human GII.4 NoV VLPs (B). Values are the corrected optical densities (OD) at 450 nm [OD450
(VLP coated) - OD450(non-coated)] for each serum sample. Dotted lines represent the logistic regression of HAT values, solid lines represent the
logistic regression of LAT values.
Mesquita et al. Virology Journal 2013, 10:176
Page 3 of 5
Canine norovirus VLP-based antibody ELISA
Recombinant virus-like particles (VLPs) of CaNoV
[dog/C33/Viseu/2007/PRT, GenBank accession num-
ber: GQ443611.1] were produced by cloning full-
length VP1/VP2 (ORF2 and ORF3 of the genome) in a
baculovirus-insect cell expression system. Recombin-
ant VLPs were recovered from the culture media and
purified through sucrose and CsCl gradients .
Norovirus morphology and size of the purified VLPs
was confirmed by electron microscopy (Figure 3).
Canine NoV VLPs (0.25 μg per well) were coated into
96-well microtiter plates (NUNC, Milford, USA) in car-
bonate–bicarbonate buffer (0.01 M, pH 9.6), and incu-
bated overnight at 4°C. Coated plates were washed with
PBS/0.5% Tween-20 and blocked with PBS/0.5% Tween
20/ 5% non-fat dry milk (blocking buffer) for 2 h at 37°C.
Serum samples were diluted 1:1,500 in blocking buffer
and tested in duplicate in VLP-coated and non-coated
wells, to correct for sample background. After 1 hour
incubation at 37°C, bound IgG was detected by
peroxidase-labeled goat anti-dog IgG (H+L) (1:12,800)
and TMB substrate (Kirkegaard & Perry Laboratories, Gai-
thersburg, USA). Background signal [OD450 (non-coated wells)]
was subtracted from each sample to obtain a corrected
OD450[OD450 (VLP coated)- OD450 (non-coated)]. Cut off
value of the test was established as the mean of the
OD450 (non-coated wells)plus 3 standard deviations (3SD).
A serum sample was considered positive when the
corrected OD450was higher than the cut off.
Two serum samples with high (HAT) and low antibodies
levels (LAT) against CaNoV and high levels of anti GII.4
New Orleans antibodies were pre-incubated with 2-fold
serial dilutions of CaNoV VLPs (5, 2.5, 1.25, 0.625,
0.3125 μg/ml) for 1 h at 37°C. After incubation, 50 μL of
pre-incubated sera were tested in duplicate for the pres-
ence of CaNoV antibodies as described above, and for
the presence of human NoV antibodies using GII.4 New
Orleans VLPs [Hu/GII.4/New Orleans1805/2009/USA,
GenBank accession number: GU445325.2] with slight
modifications (coating with 0.0625 μg per well and de-
tection of bound IgG by peroxidase-labeled goat anti-
human IgG (H+L) (1:12,800)).
A χ2 test for unequal odds with Yates’ continuity correc-
tion was used to determine significant differences in
CaNoV prevalence between study groups. Mann–Whitney
U-test was used to assess differences in CaNoV antibody
magnitude between study groups. P values less than 0.05
were considered statistically significant. Statistical analyses
were performed with R software .
The authors declare that they have no competing interests.
JM conceived the study, collected the sera, carried out the immunoassays
and drafted the manuscript. VC helped design the immunoassays, and
helped draft the manuscript. JC cloned and sequenced the canine norovirus
strain. SL produced the virus-like particles. MSJN conceived the study and
participated in the design of the study. JV participated in the design and
Table 1 Descriptive epidemiology of 373 veterinarians
participating in the study
% (N1) 95%CI2
19-2951.7 (193) 46.7-56.8
30-39 38.8 (145)33.9-43.8
40-497.8 (29) 5.1-10.5
≥501.7 (6) 0.3-2.9
Female 70.5 (263)65.9-75.1
Male 29.5 (110)24.9-34.1
Years in practice
1Number of individuals in each category;2Confidence Interval;3Foreign:
residents of Brazil, Spain or United Kingdom.
Figure 3 Electron microscopy of CaNoV VLPs confirming a
norovirus-like morphology and size. Grids were stained with 2%
Mesquita et al. Virology Journal 2013, 10:176
Page 4 of 5
coordination of the study and drafted the manuscript. All authors read and Download full-text
approved the final manuscript.
The authors would like to thank Dr. Charles Humphrey (CDC) for performing
electron microscopy analyses on the VLPs. The study received financial
support from FEDER funds through Programa Operacional Factores de
Competividade – COMPETE and FCT - Fundação para a Ciência e a
Tecnologia (project PTDC/CVT/113218/2009), and by grant SFRH/BD/45407/
2008 to J.R.M. from Fundação para a Ciência e a Tecnologia. The funding
sources did have no influence on the design, collection, analysis, and
interpretation of data; writing of the manuscript; or the decision to submit
this manuscript for publication.
The findings and conclusions in this article are those of the authors and do
not necessarily represent the views of the Centers for Disease Control and
Prevention. This article did receive clearance through the appropriate
channels at the CDC prior to submission.
1Department of Biological Sciences, Faculty of Pharmacy, University of Porto,
Porto, Portugal.2Agrarian Superior School, Polytechnic Institute of Viseu,
Viseu, Portugal.3Division of Viral Diseases, National Center for Immunization
and Respiratory Diseases, Centers for Disease Control and Prevention, Mail
Stop G-04, 1600 Clifton Rd, Atlanta, GA 30333, USA.4Center for Food Safety,
University of Georgia, Griffin, GA, USA.
Received: 19 December 2012 Accepted: 31 May 2013
Published: 4 June 2013
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Cite this article as: Mesquita et al.: Presence of Antibodies against
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