Seroepidemiology for MERS coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in Egypt, June 2013

1www.eurosurveillance.org
Research articles
Seroepidemiology for MERS coronavirus using
microneutralisation and pseudoparticle virus
neutralisation assays reveal a high prevalence of
antibody in dromedary camels in Egypt, June 2013
R A Perera1,2
, P Wang2,3,4
, M R Gomaa5
, R El-Shesheny5
, A Kandeil5
, O Bagato5
, L Y Siu3
, M M Shehata5
, A S Kayed5
, Y Moatasim5
,
M Li3
, L L Poon1
, Y Guan1
, R J Webby6
, M A Ali5
, J S Peiris (malik@hku.hk)1
, G Kayali (ghazi.kayali@stjude.org)6
1. Centre of Influenza Research, School of Public Health, The University of Hong Kong, Hong Kong, China
2. These authors contributed equally to the work and are joint first authors
3. Hong Kong University-Pasteur Research Pole, The University of Hong Kong, Hong Kong, China
4. Key Laboratory of Protein and Peptide Pharmaceuticals, Chinese Academy of Sciences - University of Tokyo Joint Laboratory of
Structural Virology and Immunology, Beijing, China
5. Division of Environmental Research, National Research Centre, Giza, Egypt
6. Division of Virology, Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis, United States
Citation style for this article:
Perera RA, Wang P, Gomaa MR, El-Shesheny R, Kandeil A, Bagato O, Siu LY, Shehata MM, Kayed AS, Moatasim Y, Li M, Poon LL, Guan Y, Webby RJ, Ali MA, Peiris JS,
Kayali G. Seroepidemiology for MERS coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in
dromedary camels in Egypt, June 2013.
Euro Surveill. 2013;18(36):pii=20574. Available online: http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=20574
Article submitted on 26 August 2013 / published on 5 September 2013
We describe a novel spike pseudoparticle neutralisa-
tion assay (ppNT) for seroepidemiological studies on
Middle East respiratory syndrome coronavirus (MERS-
CoV) and apply this assay together with conventional
microneutralisation (MN) tests to investigate 1,343
human and 625 animal sera. The sera were collected
in Egypt as a region adjacent to areas where MERS has
been described, and in Hong Kong, China as a control
region. Sera from dromedary camels had a high preva-
lence of antibody reactive to MERS-CoV by MERS NT
(93.6%) and MERS ppNT (98.2%) assay. The antibody
titres ranged up to 1,280 and higher in MN assays
and 10,240 and higher in ppNT assays. No other
investigated species had any antibody reactivity to
MERS-CoV. While seropositivity does not exclude the
possibility of infection with a closely related virus, our
data highlight the need to attempt detection of MERS-
CoV or related coronaviruses in dromedary camels. The
data show excellent correlation between the conven-
tional MN assay and the novel ppNT assay. The newly
developed ppNT assay does not require Biosafety Level
3 containment and is thus a relatively high-throughput
assay, well suited for large-scale seroepidemiology
studies which are needed to better understand the
ecology and epidemiology of MERS-CoV.
Introduction
A novel lineage C beta-coroanvirus was isolated from
a patient with fatal viral pneumonia in Saudi Arabia in
2012 and termed Middle East respiratory syndrome cor-
onavirus (MERS-CoV) [1]. As of 3 September 2013, 108
human cases have been confirmed, 50 of which were
fatal [2]. Locally acquired cases have been reported
from Jordan, Qatar, Saudi Arabia and the United Arab
Emirates, and imported index cases, sometimes with
secondary local transmission, have been reported in
France, Germany, Italy, Tunisia and the United Kingdom
[2-4]. Clusters of cases suggestive of limited human-
to-human transmission have been reported; the larg-
est cluster of cases to date occurred at a healthcare
facility in Al-Hasa, Saudi Arabia [5]. The epidemiology
of the disease so far is suggestive of multiple zoonotic
transmissions from an animal reservoir leading to
human infection, sometimes with secondary transmis-
sion events in humans.
Phylogenetically closely related, although not identi-
cal, viruses have been found in insectivorous bats in
Africa and Europe [6,7]. More recently, a very short
fragment (181 bp) of the RNA-dependent RNA polymer-
ase gene that was genetically identical to MERS-CoV
has been detected in a Taphozous perforatus bat cap-
tured in the vicinity of the residence of a human case
with MERS [8]. These findings remain to be confirmed
with more definitive sequence data. Even if MERS-CoV
is found in bats, the possibility of an intermediate peri-
domestic host remains important to explore.
Since antibody responses following coronavirus infec-
tion remain detectable for many years [9], seroepidemi-
ology of potential animal species for MERS-CoV-specific
antibody is a logical approach to identify candidate
species for further investigation. A recent report sug-
gests that MERS-CoV antibody was detected in drom-
edary camels in the Arabian peninsula [10]. While a
number of serological tests, including ELISA assays,
immunoflourescence assays and immunoassays using
recombinant viral proteins have been used for detect-
ing serological responses in infected humans [11,12],
virus neutralisation is the most specific serological

2 www.eurosurveillance.org
test and currently considered the gold-standard.
However, virus neutralisation requires the handling of
live virus and requires Biosafety Level 3 containment.
We have therefore developed a pseudoparticle neutral-
isation (ppNT) assay where the spike protein of MERS-
CoV is expressed by a replication-incompetent human
immunodeficiency (HIV) virus that contains a luciferase
reporter gene. Similar pseudotype viruses have been
used successfully in serological tests for severe acute
respiratory syndrome coronavirus (SARS-CoV) and
influenza viruses such as the highly pathogenic avian
influenza A(H5N1) virus [13]. Pseudotyped MERS-CoV
has been used to study the mechanisms of virus entry,
and it has been shown that cell transduction by such
particles is blocked by neutralising antibodies specific
for MERS-CoV [14].
The geographical distribution of MERS-CoV in its ani-
mal reservoir is not defined. Being a Middle Eastern
country with an ecology and domestic livestock prac-
tices fairly similar to some countries where human
MERS infections have occurred, we reasoned that
Egypt would be a relevant geographical location for
seroepidemiological studies. We have used both the
ppNT assay and conventional microneutralisation (MN)
tests to carry out seroepidemiological surveillance in
humans and livestock in Egypt. Human and animal sera
collected in Hong Kong were used as controls.
Methods
Sera from dromedary camels (n=110), water buffaloes
(n=8) and cows (n=25) were collected from two abat-
toirs, one located in Cairo and the second located in
the Qalyubia governorate in the Nile Delta region. The
dromedary camels were mostly imported from Sudan
for slaughter and were five to seven years-old. Upon
import, they were held on Egyptian farms for four to
five months before transport to the abattoirs in open
trucks. Sera from sheep (n=5) and goats (n=13) were
collected from backyard animals from a village in the
Nile Delta. All sera were collected in June 2013.
Human sera (n=815) were collected in 2012–13 as part
of an ongoing community-based seroepidemiological
study on influenza virus among healthy subjects in
Cairo and the Nile Delta region. The age range of the
subjects was between two and 79 years-old (median:
29 years). Fifty-eight per cent of the study subjects
were female.
Sera collected in Hong Kong served as un-exposed
controls. These included archived age-stratified
human sera (n=528) collected in Hong Kong in 2011 and
2012, with more than 50 sera from each decade of age
(range: <10 to 80 years-old). Swine sera (n=260) were
collected from an abattoir in Hong Kong in 2011 and
2012. Sera (n=204) from wild northern pintails (Anas
acuta) and Eurasian widgeons (Anas penelope) were
collected in December 2010 from the Mai Po wetlands
nature reserve in Hong Kong.
As positive controls, we used a convalescent serum
from a human patient with MERS, kindly provided by
Dr C Drosten (Institute of Virology, University of Bonn
Medical Centre, Bonn, Germany), and sera from two
experimentally infected macaques and a non-infected
control macaque kindly provided by Bart Haagmans
(Erasmus University Medical Center, Rotterdam, the
Netherlands).
An acute and convalescent serum from a patient with
SARS was used as a further negative control. The MN
antibody titre was <10 to SARS-CoV in the acute serum,
and 160 in the convalescent serum.
The study was approved by the institutional review
boards of the University of Hong Kong and St Jude
Children’s Research Hospital and the Ethics Committee
of the National Research Centre, Egypt.
Viruses and virus titration
MERS-CoV (strain EMC) virus was obtained from
Dr R Fouchier (Erasmus University Medical Center,
Rotterdam, the Netherlands). SARS-CoV (strain HKU-
39849) was taken from the virus repository at Hong
Kong University. Virus stock for MERS-CoV was pre-
pared in Vero cell culture (ATCC CCL-81) in minimal
essential medium containing 2% fetal bovine serum,
100 units/mL penicillin and 100 μg/mL streptomycin.
Virus aliquots were stored at -80 °C. Virus was titrated
in serial half-log10
dilutions (from 0.5 log to 7 log) to
obtain 50% tissue culture infectious dose (TCID50
) on
96-well tissue culture plates of Vero cells. The plates
were observed in a phase contrast microscope for cyto-
pathic effect (CPE) daily for three days. The endpoint of
viral dilution leading to CPE in 50% of inoculated wells
was estimated by using the Reed Muench method and
designated as one TCID50
. SARS-CoV was grown and
titrated in the same manner with the exception that
Vero E6 cells (ATCC CRL-1586) were used.
Microneutralisation tests
Serial two-fold dilutions of heat-inactivated sera (56 °C
for 30 minutes) were made, starting with a dilution of
1:10. The serum dilutions were mixed with equal vol-
umes of 200 TCID50
of MERS-CoV or SARS-CoV as indi-
cated. After 1 h of incubation at 37 °C, 35 µL of the
virus–serum mixture was added in quadruplicate to
Vero or Vero-E6 cell monolayers for MERS-CoV and
SARS-CoV, respectively, in 96-well microtiter plates.
After 1 h of adsorption, an additional 150 µL of cul-
ture medium were added to each well and the plates
incubated for three more days at 37 °C in 5% CO2
in a
humidified incubator. A virus back-titration was per-
formed without immune serum to assess input virus
dose. CPE was read at three days post infection. The
highest serum dilution that completely protected the
cells from CPE in half of the wells was taken as the
neutralising antibody titre and was estimated using
the Reed-Muench method. Positive and negative con-
trol sera were included to validate the assay.

MERS-CoV spike pseudoparticle
neutralisation assay
A codon-optimised spike gene was designed according
to published MERS-CoV genome sequence (GenBank
accession number: JX869059.1), synthesised by
GeneCust (Luxembourg) and subcloned into pcDNA3.1+
vector to generate pcDNA-S. To produce HIV/MERS
spike pseudoparticles, 10 µg pNL Luc E-
R-
and 10 µg
pcDNA-S were co-transfected into 4x106
293T cells [13].
Supernatants of transfected cells were harvested 48 h
later and quantified for HIV p24 viral protein using a
p24 ELISA Kit (Cell Biolabs, San Diego, United States).
For the ppNT assay, HIV/MERS pseudoparticles con-
taining 5 ng p24 were used to infect Vero E6 cells
(ATCC CRL-1586) in a single well (96-well plate format;
1x104
cells/well). Infected cells were lysed in 20 µl
lysis buffer and 100 µl of luciferase substrate at two
days postinfection (Promega Corporation, Madison,
United States). Luciferase activity was measured in a
Microbeta luminometer (PerkinElmer, Waltham, United
States).
For the ppNT, HIV/MERS pseudoparticles (5 ng of p24)
were pre-incubated with serially diluted sera for 30 min
at 4 °C and then added to cells in triplicate. Residual
virus replication was assayed at two days post infec-
tion, as described above. The highest serum dilu-
tion giving a 90% reduction of luciferase activity was
regarded as the ppNT antibody titre.
Results
Overall, 976 human and animal sera from Egypt and 992
human and animal sera from Hong Kong were tested by
MN at a screening dilution of 1:10 and 1:20 (Table 1).
None of the age-stratified human sera (n=528), swine
sera (n=260) or wild bird sera (n=204) collected in
Hong Kong had any neutralising activity for MERS-
CoV in the MN tests. Similarly, none of the sera from
humans (n=815), water buffaloes (n=8), cows (n=25),
sheep (n=5) and goats (n=13) collected in Egypt were
positive in the screening MN tests. In contrast, 103 of
110 sera collected in Egypt from dromedary camels
neutralised MERS-CoV at the screening dilution of 1:20
or higher.
Entry of MERS pseudoparticles was shown to be inhib-
ited by increasing concentrations of 0–20 mM NH4
Cl
(data not shown), demonstrating pH dependent entry of
the MERS pseudoparticles as previously reported [14].
The MERS ppNT assay was evaluated using two sera
from experimentally infected macaques, one negative
control serum from an uninfected macaque, a human
convalescent serum from a MERS patient and five neg-
ative human control sera from Hong Kong (Figure 1).
The MERS ppNT assay was then used to screen 115
human sera from Hong Kong and 100 randomly selected
human sera from Egypt which were all serologically
negative for MERS-CoV. Sixteen dromedary camel sera
that were positive in the MN screening assay were all
found to have a high neutralising activity in the ppNT
assay. In addition, five of six sera that were negative
in the MN assay had a weak, but detectable, activity
in the ppNT test (Table 1, Table 2, Figure 2). The camel
sera that were found to be positive at a screening dilu-
tion of 1:20 in the MN test had antibody titres in the
MERS NT screen ranging from 40 to 1,280 and higher,
and MERS ppNT titres ranging from 640 to 10,240 and
higher. One of the five MERS MN-negative sera was
negative in the MERS ppNT assay, while the other four
had low MERS ppNT titres ranging from 40 to 160.
Table 1
Screening results for MERS-CoV microneutralisation and MERS-CoV spike protein pseudoparticle neutralisation, human
and animal samples from Egypt and Hong Kong, 2012–2013 (n=1,968)
Sera Source of sera
MERS-CoV micro-neutralisation titre ≥1:20 MERS-CoV spike pseudotype antibody titre ≥1:20
Total tested % Positive (n) Total tested % Positive (n)
Humana
Egypt
815 0 (0/815) 100 0 (0/100)
Goatb
13 0 (0/13) ND ND
Sheepb
5 0 (0/5) ND ND
Water buffalob
8 0 (0/8) ND ND
Cowb
25 0 (0/25) ND ND
Camelb
110 93.6 (103/110) 110 98.2 (108/110)
Human
Hong Kong
528 0 (0/528) 115 0 (0/115)
Swine 260 0 (0/260) ND ND
Wild bird 204 0 (0/204) ND ND
MERS-CoV: Middle East respiratory syndrome coronavirus; ND: not done.
a
Collected in 2012–13.
b
Collected in June 2013.
Details of sera collected in Hong Kong as given in Methods.

The correlation of the MERS MN and MERS ppNT titres
are shown in Figure 3 (Pearson’s correlation coef-
ficient: R=0.88). The MERS ppNT test appears to be
more sensitive than the MERS MN test, and thus some
of the apparently MN-negative camel sera give low
titre-positive results in the MERS ppNT assay. However,
none of the human sera from Egypt (n=100) or Hong
Kong (n=115) had any detectable antibody in the MERS
ppNT test. None of the camel sera with high antibody
titres to MERS-CoV had any cross-neutralising activity
to SARS-CoV (Table 2).
Discussion
Of 1,968 human and animal sera tested by MERS-CoV
MN and 325 human and animal sera tested by MERS-
CoV ppNT assays, only sera from dromedary camels
had any neutralising antibody activity to the MERS-
CoV. Of the 110 camel sera, 93.6% were seropositive
by MERS-CoV MN test and 98.2% were seropositive
by MERS-CoV ppNT test. The antibody titres were very
high in MN as well as ppNT, suggesting that the virus
infecting these camels was MERS-CoV virus itself or a
very closely related virus.
It is known that dromedary camels host bovine corona-
viruses (BCoV) which are lineage A beta-coronaviruses.
However cross-neutralisation between MERS-CoV (lin-
eage C beta-coronavirus) and BCoV was excluded by
Reusken and colleagues in their study of sera from
dromedary camels [10]. Furthermore, BCoV is antigeni-
cally closely related to the human coronavirus OC43.
Human beta-coronavirus lineage A viruses OC43 and
Figure 1
MERS-CoV spike protein pseudoparticle neutralisation, human and animal samples from Egypt and Hong Kong, 2012–13
(n=9)
CPS: counts per second; MERS-CoV: Middle East respiratory syndrome coronavirus;
As positive controls, we used a convalescent human serum (CHS) from a patient with MERS, kindly provided by Dr C Drosten (Institute of
Virology, University of Bonn Medical Centre, Bonn, Germany) and sera from two experimentally infected macaques (MAC1, MAC2), kindly
provided by Bart Haagmans (Erasmus University Medical Center, Rotterdam, the Netherlands). As negative controls we used serum from a
non-infected control macaque (NMS) and five human sera (NHS 1–5) from Hong Kong. The horizontal dotted line represents the 90% reduction
in luciferase activity which represents the cut-off for positivity in the assay. Each batch of assays had the cut-off determined with reference to
a serum-free negative control, and the data represented here are a compilation of two experiments. Thus the cut-off line is a representative
indication based on an average of cut-offs used in seperate experiments.
MAC 1
MAC 2
CHS
100
1,000
10,000
100,000
1,000,000
1:20 1:40 1:80 1:160 1:320 1:640 1:1,280 1:2,560 1:5,120
Luciferaseactivity(CPS)
Serum dilution
NMS
MAC 1
MAC 2
NHS 1
NHS 2
NHS 3
NHS 4
NHS 5
CHS
Cut-oﬀ

HKU1, and alpha-coronaviruses (229E and NL63) are
ubiquitous respiratory viruses infecting humans and
the panel of human sera of different ages tested can
be expected to have varying levels of antibody to these
viruses. The lack of any MERS-neutralising activity
in the human sera we studied also indicates that the
MN and ppNT assays are specific for MERS-CoV. The
lack of cross-reactivity with convalescent serum from
patients with SARS provides additional evidence of
the lack of cross-reactivity in the MERS-CoV serology
assays. Furthermore, it is notable that the camel sera
with high antibody titres to MERS-CoV did not cross-
react with SARS-CoV, a beta-coronavirus of lineage B.
Taken together these data indicate that a MERS-CoV or
a highly related virus is endemic in dromedary camels
imported for slaughter in Egypt. These findings pro-
vide independent confirmation of the results recently
reported by Reusken et al. who found very high anti-
body titres to MERS-CoV in dromedary camels [10].
The dromedary camels sampled in our study were
those brought to abattoirs for slaughter in Cairo and
in the Qalyubia governorate in the Nile Delta region.
These animals were sourced from other East African
countries such as Sudan and held in Egypt for some
time prior to slaughter. Thus it is unclear where the ani-
mals originally acquired the infection. Considering the
similar data from dromedary camels in Oman and the
Canary Islands [10], it is likely that this coronavirus is
widespread in North and East Africa and the Arabian
peninsula.
There is substantial movement of people between
Egypt and Saudi Arabia and other states on the Arabian
peninsula, and thus it is possible that people may get
infected, either as part of their travel to endemic areas
or through zoonotic transmission within the coun-
try. There is also much movement of livestock across
these Middle Eastern countries. The lack of antibody to
MERS-CoV in sera of people resident in Egypt indicates
Figure 2
MERS-CoV spike protein pseudoparticle neutralisation on selected sera from dromedary camels, Egypt, June, 2013 (n=21)
CPS: counts per second; MERS-CoV: Middle East respiratory syndrome coronavirus; MN: microneutralisation; ppNT: pseudoparticle
neutralisation.
Sixteen sera found to be positive and five sera found to be negative in the MERS-CoV MN screening assay were titrated in the MERS-CoV ppNT
assay. The sera used are shown in Table 2. The horizontal dotted line represents the 90% reduction in luciferase activity which represents the
cut-off for positivity in the assay.Each batch of assays had the cut-off determined with reference to a serum-free negative control and the data
represented here are a compilation of two experiments. Thus the cut-off line is a representative indication based on an average of cut-offs
used in seperate experiments.
10
100
1,000
10,000
100,000
1,000,000
1:20 1:40 1:80 1:160 1:320 1:640 1:1,280 1:2,560 1:5,120 1:10,240
Luciferaseactivity(CPS)
Serum dilution
C29
C101
C107
C108
C109
C110
C111
C112
C113
C115
C116
C117
C118
C119
C120
C121
C127
C132
C144
C147
C585
Cut-oﬀ

that this infection is not common in Egypt, either as an
infection acquired through travel or as an occasional
zoonotic infection.
The MERS-CoV ppNT assay described here is a safe
and specific assay for large scale seroepidemiological
studies in a range of animal species, and such studies
are urgently needed in regions where MERS-CoV cases
have been detected as well as other regions. The HIV
backbone used for pseudoparticle production is not
replication-competent and the MERS-CoV pseudoparti-
cles can therefore be produced and used in Biosafety
Level 2 containment; in contrast, MN assays involve
handling of the live MERS-CoV and require Biosafety
Level 3 containment which is not always available in
affected regions. Unlike immunoassays, there is no
requirement for finding and optimising an enzyme-
labelled anti-Ig conjugate for each species to be
investigated. Furthermore, the MERS-CoV ppNT assay
appears around 10 times more sensitive than the con-
ventional MN assay (Figure 3, Table 2). The MN assay
is a neutralisation assay based on TCID50
rather than
a plaque reduction assay, which perhaps makes it less
sensitive than a plaque neutralisation assay. In any
event, experience with influenza virus serology using
pseudoparticle assays has shown that they are more
sensitive than conventional MN assays for detecting
neutralising antibodies. Thus MERS-CoV ppNT can be
used as a screening assay, and positive sera can be
retested for confirmation in a MERS MN tests.
Serological data does not provide proof that the virus
infecting dromedary camels is the MERS CoV, and
infection by a closely related coronavirus or a chimeric
virus with a MERS-CoV-like spike protein cannot be
ruled out until the dromedary camel virus is detected
and genetically sequenced. However, it provides a
strong impetus to attempt to seek the virus in speci-
mens from these animals and to identify the MERS-
related virus that appears to be infecting them. These
serological studies also need to be extended to other
domestic animals species to define the circulation of
MERS-CoV or related viruses in animals in close con-
tact with humans. Such studies should also include
humans exposed to dromedary camels. It is important
to note that waning antibody levels may result in false-
negative serology results, and this is particularly rel-
evant in mild or asymptomatic episodes of infection
where the peak antibody titre may be lower and drop
more quickly.
Table 2
Antibody titres of selected sera from dromedary camels
tested by microneutralisation for MERS-CoV and
SARS-CoV and by MERS spike protein pseudoparticle
neutralisation, Egypt, June, 2013 (n=21)
Camel sera
Antibody titres
MERS-CoV MN
test
SARS-CoV MN
test
MERS-CoV
ppNT test
C101 <10 Negative <10 Negative 40
C585 <10 Negative <10 Negative <20 Negative
C29 320 <10 Negative 2,560
C107 160 <10 Negative 5,120
C108 160 <10 Negative 5,120
C109 640 <10 Negative ≥10,240
C110 ≥1,280 <10 Negative ≥10,240
C111 320 <10 Negative 5,120
C112 320 <10 Negative 5,120
C113 320 <10 Negative 2,560
C115 160 <10 Negative 1,280
C116 320 <10 Negative 5,120
C117 640 <10 Negative 5,120
C118 640 <10 Negative 5,120
C119 80 <10 Negative 640
C120 40 <10 Negative 1,280
C121 160 <10 Negative 2,560
C147 ≥1,280 <10 Negative ≥10,240
MERS-CoV: Middle East respiratory syndrome coronavirus; MN:
microneutralisation; ppNT: pseudoparticle neutralisation; SARS-
CoV: severe acute respiratory syndrome coronavirus.
Figure 3
Correlation of MERS-CoV antibody titres determined by
MERS-CoV microneutralisation and MERS-CoV spike
protein pseudoparticle neutralisation in selected sera from
dromedary camels, Egypt, June, 2013 (n=21)
MERS-CoV: Middle East respiratory syndrome coronavirus; MN:
microneutralisation; ppNT: pseudoparticle neutralisation.
The data used as those shown in Table 2. In the event of
overlapping dots, their MN titre (X axis) was increased or
decreased by 0.05% to slightly offset the overlap for ease of
observation. The limit of detection in the MN and ppNT assays
were titres of 10 and 20 respectively; and thus these values on the
Y and X axis correspond to <10 and <20, respectively.
100
1,000
10,000
100 1,000 10,000 100,000<20
<10
Neutralising titre by MERS-CoV ppNT
NeutralisingtitrebyMERS-CoVMN

If the detection of MERS-CoV in insectivorous bats is
confirmed [8] and if indeed the coronavirus we and oth-
ers demonstrated to be common in dromedary camels
is confirmed to be MERS-CoV, we will have a scenario
of a virus reservoir in bats with a peridomestic animal
such as the camel as intermediate host, which may in
fact be the immediate source of human infection. It is
notable that a number of index cases with MERS-CoV
had a history of exposure to camels, although this is by
no means universally the case. Given that the MERS-
like coronavirus in camels appears to be ubiquitous, it
remains to be explained why MERS in humans appears
relatively rare. Coronaviruses are well known to mutate
to markedly change virulence or host range. Examples
are the emergence of the less pathogenic porcine res-
piratory coronavirus from virulent transmissible gas-
troenteritis virus of pigs, or virulent feline infectious
peritonitis viruses emerging from low pathogenic feline
coronaviruses [15]. Furthermore, the SARS-like virus
detected in civets and other small mammals in live
animal markets in southern China in 2002–03 initially
appeared to infect humans, who appear to have sero-
converted, but with minimal disease and onward trans-
mission [16], while a few amino acid changes in the
SARS-CoV spike protein allowed that virus to acquire
efficient transmissibility and virulence in humans [17].
Thus, previous experience with animal and human
coronaviruses highlights the public health urgency of
investigations of MERS-CoV and MERS-CoV-like viruses
in domestic and wild animals.
Acknowledgements
We thank Dr CYH Leung for providing wild bird sera from
Hong Kong. This study was supported in part by research
contract from the National Institute of Allergy and Infectious
Diseases (NIAID) contract HHSN266200700005C and a
grant from the European Community Seventh Framework
Programme (FP7/2007-2013) under project European man-
agement Platform for Emerging and Re-emerging Disease
entities (Grant agreement No. 223498) (EMPERIE).
Conflict of interest
None declared.
Authors’ contributions
Pei-gang Wang developed the MERS-CoV pseudotype assay
and carried out the tests. Ranawaka AMP Perera developed
the MERS-CoV microneutralisation test and carried out the
tests in BSL3 containment. Leo LLM Poon and Yi Guan pro-
vided advice on laboratory methods. Lewis YL Siu and Ming-
yuan Li carried out the MERS-CoV pseudoparticle assays.
Mokhtar R. Gomaa, Rabeh El-Shesheny, Ahmed Kandeil, Ola
Bagato, Mahmoud M. Shehata, Ahmed S. Kayed and Yassmin
Moatasim collected human and animal sera in Egypt. Richard
J. Webby and Mohamed A. Ali provided advice on field study
design. Joseph SM Peiris and Ghazi Kayali designed and co-
ordinated the study and wrote the manuscript. All authors
reviewed and commented on the manuscript.
References
1. Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus AD,
Fouchier RA. Isolation of a novel coronavirus from a man with
pneumonia in Saudi Arabia. N Engl J Med. 2012;367(19):1814-
20. http://dx.doi.org/10.1056/NEJMoa1211721. PMid:23075143.
2. World Healh Organization (WHO). Middle East respiratory
syndrome coronavirus (MERS-CoV) – update. Geneva: WHO;
29 Aug 2013. Available from: http://www.who.int/csr/
don/2013_08_29/en/index.html
3. World Healh Organization (WHO). Middle East respiratory
syndrome coronavirus (MERS-CoV) summary and literature
update – as of 9 July 2013. Geneva: WHO. [Accessed 20 Aug
2013]. Available from: http://www.who.int/csr/disease/
coronavirus_infections/update_20130709/en/index.html
4. Bermingham A, Chand MA, Brown CS, Aarons E, Tong C,
Langrish C, et al. Severe respiratory illness caused by a
novel coronavirus, in a patient transferred to the United
Kingdom from the Middle East, September 2012. Euro Surveill.
2012;17(40):pii=20290. http://www.eurosurveillance.org/
ViewArticle.aspx?ArticleId=20290
5. Assiri A, McGeer A, Perl TM, Price CS, Al Rabeeah
AA, Cummings DA, et al. Hospital outbreak of Middle
East respiratory syndrome coronavirus. N Engl J
Med. 2013;369(5):407-16. http://dx.doi.org/10.1056/
NEJMoa1306742. PMid:23782161.
6. Annan A, Baldwin HJ, Corman VM, Klose SM, Owusu M,
Nkrumah EE, et al. Human betacoronavirus 2c EMC/2012-
related viruses in bats, Ghana and Europe. Emerg Infect Dis.
2013;19(3):456-9. http://dx.doi.org/10.3201/eid1903.121503.
PMid:23622767. PMCid:PMC3647674.
7. Ithete NL, Stoffberg S, Corman VM, Cottontail VM, Richards
LR, Schoeman MC, et al. Close relative of human Middle
East respiratory syndrome coronavirus in bat, South Africa.
Emerg Infect Dis. 2013. EPub ahead of print. DOI: 10.3201/
eid1910.130946. http://dx.doi.org/10.3201/eid1910.130946
8. Memish ZA, Mishra N, Olival KJ, Fagbo SF, Kapoor V, Epstein
JH, et al. Middle East respiratory syndrome coronavirus in
bats, Saudi Arabia. Emerg Infect Dis. 2013. EPub ahead of
print. DOI: 10.3201/eid1911.131172. http://dx.doi.org/10.3201/
eid1911.131172
9. Cao WC, Liu W, Zhang PH, Zhang F, Richardus JH.
Disappearance of antibodies to SARS-associated coronavirus
after recovery. N Engl J Med. 2007;357(11):1162-3. http://dx.doi.
org/10.1056/NEJMc070348. PMid:17855683.
10. Reusken CB, Haagmans BL, Müller MA, Gutierrez C, Godeke GJ,
Meyer B, et al. Middle East respiratory syndrome coronavirus
neutralising serum antibodies in dromedary camels: a
comparative serological study. Lancet Infect Dis. 2013; pii:
S1473-3099(13)70164-6. DOI: 10.1016/S1473-3099(13)70164-6.
http://dx.doi.org/10.1016/S1473-3099(13)70164-6
11. Corman VM, Muller MA, Costabel U, Timm J, Binger T,
Meyer B, et al. Assays for laboratory confirmation of novel
human coronavirus (hCoV-EMC) infections. Euro Surveill.
2012;17(49):pii=20334. Available from: http://www.
eurosurveillance.org/ViewArticle.aspx?ArticleId=20334
12. Reusken C, Mou H, Godeke GJ, van der Hoek L, Meyer B, Muller
MA, et al. Specific serology for emerging human coronaviruses
by protein microarray. Euro Surveill. 2013;18(14):20441.
Available from: http://www.eurosurveillance.org/ViewArticle.
aspx?ArticleId=20441.PMid:23594517
13. Garcia JM, Lagarde N, Ma ES, de Jong MD, Peiris JS.
Optimization and evaluation of an influenza A (H5)
pseudotyped lentiviral particle-based serological assay.
J Clin Virol. 2010;47(1):29-33. http://dx.doi.org/10.1016/j.
jcv.2009.10.009. PMid:19897409.
14. Gierer S, Bertram S, Kaup F, Wrensch F, Heurich A, Krämer-Kühl
A, et al. The spike protein of the emerging betacoronavirus
EMC uses a novel coronavirus receptor for entry, can be
activated by TMPRSS2, and is targeted by neutralizing
antibodies. J Virol. 2013;87(10):5502-11. http://dx.doi.
org/10.1128/JVI.00128-13. PMid:23468491.
15. Saif LJ. Animal coronaviruses: what can they teach us
about the severe acute respiratory syndrome? Rev Sci Tech.
2004;23(2):643-60. PMid:15702725
16. Guan Y, Zheng BJ, He YQ, Liu XL, Zhuang ZX, Cheung CL,
et al. Isolation and characterization of viruses related to
the SARS coronavirus from animals in southern China.
Science. 2003;302(5643):276-8. http://dx.doi.org/10.1126/
science.1087139. PMid:12958366.
17. Li F. Structural analysis of major species barriers between
humans and palm civets for severe acute respiratory
syndrome coronavirus infections. J Virol. 2008;82(14):6984-
91. http://dx.doi.org/10.1128/JVI.00442-08. PMid:18448527.
PMCid:PMC2446986.

Seroepidemiology for MERS coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in Egypt, June 2013

Recommended

Recommended

More Related Content

What's hot

What's hot (19)

Viewers also liked

Viewers also liked (18)

Similar to Seroepidemiology for MERS coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in Egypt, June 2013

Similar to Seroepidemiology for MERS coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in Egypt, June 2013 (20)

Recently uploaded

Recently uploaded (20)

Seroepidemiology for MERS coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in Egypt, June 2013