Arch Virol (2011) 156:1883–1890
DOI 10.1007/s00705-011-1057-1
BRIEF REPORT
Detection of alpha and betacoronaviruses in multiple
Iberian bat species
Ana Falcón • Sonia Vázquez-Morón • Inmaculada Casas • Carolina Aznar
Guillermo Ruiz • Francisco Pozo • Pilar Perez-Breña • Javier Juste •
Carlos Ibáñez • Inazio Garin • Joxerra Aihartza • Juan E. Echevarrı́a
•
Received: 2 February 2011 / Accepted: 20 June 2011 / Published online: 16 July 2011
Ó The Author(s) 2011. This article is published with open access at Springerlink.com
Abstract Bat coronaviruses (CoV) are putative precursors of the severe acute respiratory syndrome (SARS) CoV
and other CoV that crossed the species barrier from zoonotic reservoirs into the human population. To determine
the presence and distribution of CoV in Iberian bats, 576
individuals of 26 different bat species were captured in 13
locations in Spain. We report for the first time the presence
of 14 coronaviruses in 9 Iberian bat species. Phylogenetic
analysis of a conserved CoV genome region (RdRp gene)
shows a wide diversity and distribution of alpha and
betacoronavirus in Spain. Interestingly, although some of
these viruses are related to other European BatCoV, or to
Asian CoV, some of the viruses found in Spain cluster in
new groups of a and b CoV.
GenBank accession numbers: Partial sequences of RdRp gene of bat
coronaviruses obtained in this study were deposited in GenBank
under the accession numbers HQ184049 to HQ184062.
A. Falcón S. Vázquez-Morón I. Casas C. Aznar G. Ruiz
F. Pozo P. Perez-Breña J. E. Echevarrı́a
Centro Nacional de Microbiologı́a, Instituto de Salud Carlos III,
Madrid, Spain
A. Falcón (&)
Centro Nacional de Biotecnologı́a, CSIC, Darwin 3,
Campus Cantoblanco, 28049 Madrid, Spain
e-mail: afalcon@cnb.csic.es
J. Juste C. Ibáñez
Estación Biológica de Doñana, CSIC, Seville, Andalusia, Spain
I. Garin J. Aihartza
Department of Zoology and Animal Cell Biology,
University of the Basque Country (UPV/EHU),
Leioa, The Basque Country, Spain
The emergence of infectious diseases is a major threat to
global public health in this century (WHO, World Health
http://www.who.int/whr/previous/en/index.html)
Report
and many of these new infectious human diseases are
caused by viruses emerging from wildlife. In the last
50 years, more than 30 new infectious human diseases
have been identified (WHO, World Health Report
http://www.who.int/whr/previous/en/index.html) including
the Severe Acute Respiratory Syndrome (SARS). The
aetiological agent of this disease was identified as a
previously unknown coronavirus (SARS-CoV) [1] and
BatCoV are putative precursors of SARS-CoV [2]. The
outbreak of SARS-CoV and subsequent identification of
two additional human coronaviruses (HCoV-NL63[3] and
HCoV-HKU1 [4]) has drawn human and animal health
attention to Coronavirinae subfamily, that includes three
genera, Alphacoronavirus (a CoV), Betacoronavirus
(b CoV) and Gammacoronavirus (c CoV), replacing the
classical groups 1, 2 and 3 [5] (http://talk.ictvonline.org/
media/g/vertebrate-2008/default.aspx).
The relevance and possible re-emergence of the pandemic SARS-CoV and other emerging viruses of zoonotic
origin have activated surveillance systems of hazard agents
in wild animals, including bats. As a result of these studies,
bats have been described as putative reservoirs for some
emerging viruses affecting humans [6]. BatCoV are putative precursors of CoV affecting humans and mammals [7,
8], including SARS-CoV [2] and other CoV that crossed
the species barrier from zoonotic reservoirs into the human
population [9]. In fact, association of some of these CoV to
certain bat species has been suggested [10, 11], reinforcing
the notion that there may be a relationship between some
BatCoV and their hosts. Nowadays the presence of CoV
has been shown in bats in China [12, 13], North and South
America [14–17], Africa [18] and a number of regions in
123
1884
Europe [11, 19–21] but not the Iberian Peninsula (Spain
and Portugal), which is a bridge for European and African
bat populations [22]. Thus, surveillance of wildlife reservoirs of putative zoonotic CoV is necessary, not only to
unveil the ecology of these viruses, but also to permit early
detection of viruses that might pose a threat to human
health.
To determine the presence and distribution of putative
zoonotic CoV in Iberian bats, 576 individuals from 26 bat
species were captured and sampled in 13 different locations
throughout Spain during 2004-2007 (Fig. 1). These samples were collected in the context of bat rhabdoviruses and
lyssaviruses Surveillance Program in Spain. Most of the
sampled bat species are also distributed across Europe, but
Eptesicus isabellinus is a meridional serotine bat restricted
to North Africa and the Iberian Peninsula [23], and Myotis
escalerai is endemic in the Iberian Peninsula. Bats were
caught with mist nets mainly as they left diurnal roost and
by hand with polyethylene butterfly nets within roosts.
Oro-pharyngeal swabs (n=390) between 2004 and 2007 as
well as faecal samples from individual bats (n=216) in
2007 were taken before bats were released (Table 1).
Oro-pharyngeal swabs collected between 2004 and 2006
were preserved in 1 ml of lysis buffer (4 M GuSCN (Sigma),
OS% N-lauroyl Sarcosine (Sigma), 1 mM dithiothreitol
(DTT, Sigma), 25 mM Sodium Citrate and 20 pg/tube
Glycogen (Boehringer Mannheim). Oro-pharyngeal swabs
and faeces collected in 2007 were preserved in both 1 ml of
lysis buffer and 1 ml of viral transport medium (VTM)
(Eagle’s minimal essential medium (EMEM) supplemented
with 10 UI/ml of penicillin, 10 lg/ml of streptomycin,
160 lg/ml of gentamicin, 50 UI/ml of mycostatin and 1% of
Fig. 1 Geographical location of bat capture sites in Spain. 1: A
Coruña, 2: Lugo, 3: La Rioja, 4: Gerona, 5: Castellón, 6: Valencia, 7:
Alicante, 8: Málaga, 9: Cádiz, 10: Sevilla, 11: Huelva, 12: Cáceres,
13: Menorca. The stars show the locations where positive samples
were found
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A. Falcón et al.
bovine serum albumin). All samples were frozen at -80°C
before sending them to the Rabies Reference Laboratory at
the Centro Nacional de Microbiologı́a, ISCIII in Madrid.
Faecal samples were clarified by centrifugation. Total
nucleic acid was extracted from a 200 ll aliquot of each
specimen for PCR assays and the rest were stored to -80°C
in two different aliquots. Final pellets were always resuspended to 55 ll of water.
A pan-coronavirus nested PCR was designed in the
RdRp gene. A total of 5 ll of extracted RNA was added to
45 ll of reaction mixture of OneStep RT-PCR kit
(QIAGEN, Valencia, CA, USA) containing 200 lM
dNTPs and 60 pmol of generic CoV-specific degenerated
primers (forward 50 -CARATGAATYTIAARTAYGC-30
and reverse 50 -TGYTGWGARCAAAAYTCRTG-30 ) and
following manufacturer indications. Amplifications were
carried out into thin-walled reaction tubes (Sorenson,
BioScience, UT) in a PTC-200 (Peltier Thermal Cycler, MJ
Research, Watertown, MA). Nested PCR amplifications
were performed using 2 ll of first amplification product
and 48 ll of reaction mixture containing 60 mM Tris-HCl
(pH 8.5), 15 mM (NH4)2SO4, 200 lM dNTPs (Amersham
Pharmacia Biotech, Piscataway, NJ), 3 mM MgCl2,
35 pmol of generic CoV-specific degenerated primers
(forward 50 -ATGGGWTGGGAYTAYCCIAARTG-30 and
reverse 50 -ACRTTRTTYTGRWARTA-30 ) and 1.25 U
AmpliTaq DNA Polymerase (Perkin-Elmer Cetus, Norwalk). Amplification product size of 512 nt was visualized
by agarose gel electrophoresis and sequenced directly in
both directions using an automated ABI PRISM 377 model
sequencer. For phylogeny reconstruction, consensus
sequences were aligned together with others obtained from
public genomic databases using the program CLUSTAL X
(version 1.83) (Table 2). A Bayesian phylogenetic inference was obtained using Mr Bayes version 3.1 [24] with
random starting trees without constraints. For the analyses
GTR substitution model, gamma estimation and two
simultaneous runs of 107 generations were done, each with
four Markov chains, and the trees were sampled every 100
generations. Amino acid identity was calculated with
MEGA 4 using the pairwise deletion option. The alignment
comprised the same 396 bp of the RdRp gene used for the
phylogenetic reconstruction.
A total of 26 out of the 30 known bat species known for
Iberian Peninsula were screened for CoV and 14 samples
taken from 9 bat species, all included in the family
Vespertilionidae, were positive for CoV RNA (Tables 1
and 2). Twelve of them were found within faecal samples
(5.5%) of 7 different bat species in 6 locations and two
were obtained in oral samples (0.5%) of 2 other different
species in the same location (Table 1). In view of these
results, it is not surprising that the presence of CoV RNA is
significantly more frequent in faeces than in oral cavity
Alpha and Betacoronaviruses in Iberian bats
1885
Table 1 Results of detection of CoV RNA in faecal or oral samples of bats collected in Spain
Bat Species
Faecal samples
positive/no. tested
Oral samples
positive/no. tested
Location
Genus
Barbastella barbastellus
0/4
0/2
3, 4
Eptesicus isabellinus
1/8
NA
10a
Eptesicus serotinus
0/7
NA
1, 12
Hypsugo savii
2/26
0/10
2, 4a, 12a,b
a,b
Miniopterus schreibersii
0/2
1/71
2, 5, 6a, 7, 8, 9, 12, 13
a
Myotis alcathoe
0/1
NA
3
Myotis bechsteinii
Myotis blythii
0/2
NA
0/3
1/11
4, 8
6a, 7, 12
Myotis capaccinii
NA
0/14
5, 6, 13
Myotis daubentonii
1/39
0/52
2, 3, 8a, 11, 12
Myotis emarginatus
NA
0/2
8, 12
Myotis escalerai
NA
0/15
11, 7
Myotis myotis
1/1
0/17
6, 7, 8a, 12
Myotis mystacinus
0/5
NA
2, 3
Myotis nattereri
0/3
0/3
4
Nyctalus lasiopterus
5/37
0/137
3, 8a,b, 9, 10, 11
Nyctalus leisleri
0/23
0/11
2, 3, 4, 8
Pipistrellus kuhlii
1/4
0/6
8, 12a
Pipistrellus pipistrellus
0/3
0/1
3, 12
Pipistrellus pygmaeus
NA
0/1
12
Pipistrellus sp.
1/29
0/5
1, 2, 4, 8, 12a,b
Plecotus auritus
Plecotus austriacus
0/7
0/7
NA
0/10
1, 3, 4
4, 11, 12
Rhinolophus euryale
NA
0/13
6, 7, 12
Rhinolophus ferrumequinum
0/3
0/5
4, 8, 12
Rhinolophus hipposideros
0/4
NA
4, 12
Rhinolophus mehelyi
NA
0/1
12
Total
12/216
2/390
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13
b
a
a
a
a
a
a
a,b
NA no samples available
a
Locations where positives samples were found
b
These samples were collected in different localities than other positive samples with the same number location
(p\0.0001, FISHER EXACT TEST). It is of interest that
none of the viruses has been found in oropharingeal and
faecal samples of the same individual, when both samples
were available. This fact may indicate either that the
infection was at different stage in the different individuals
at the time of sampling or that replication of virus may take
place independently in the intestinal and respiratory tracts
[25]. Most of the CoV RNA sequences found in faecal
samples (83%) correspond to a CoV, the remaining two
belonging to b CoV. All CoV RNAs from oral samples
(100%) were found to contain viral RNA sequences corresponding to a CoV. In agreement with all previous
studies [7], none of the coronavirus detected in Spanish
bats belong to group c.
The phylogenetic analysis of Spanish BatCoV was
performed using 396nt out of the 512 nt RT-PCR amplified
fragments. 116nt fragment information was lost to allowed
us include more sequences from other European countries
and other continents deposited in GenBanK to perform a
meaningful analysis. This small part of the RdRp gene has
been previously used, and sufficiently represents the full
gene information, for phylogenetic analysis of BatCoV [11,
14, 20]
The phylogenetic reconstruction showed 6 different
lineages of Spanish BatCoV (Fig. 2). BatCoV A and B
were closely related to other a BatCoV found in China
[12], although they appeared to display certain genetic
differentiation (Fig. 2). Myotis daubentonii-associated
CoV H, and Pipistrellus-associated CoV K, clustered
respectively with lineages 4 and 3 of a CoV previously
described in Germany and are hosted by the same bat
species or genera [11] (Fig. 2). BatCoV L was closely
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1886
A. Falcón et al.
Table 2 Data of interest related to the 91 coronavirus sequences used for the generation of the phylogenetic tree
Access no
Host species
Country
Genus
Cluster
DQ249221
Bat
China
b
HKU5
DQ249219
Bat
China
b
HKU5
DQ249218
Pipistrellus sp.
China
b
HKU5
DQ648809
Bat
China
b
DQ648807
Bat
China
b
DQ249217
Pipistrellus sp.
China
b
DQ648819
Bat
China
b
HKU5
DQ249215
Tylonycteris
China
b
HKU4
DQ249214
Tylonycteris
China
b
HKU4
DQ074652
Tylonycteris
China
b
HKU4
DQ249216
DQ648803
Tylonycteris
Bat
China
China
b
b
HKU4
HQ184059
Hypsugo savii
Spain
b
HQ184062
Eptesicus isabellinus
Spain
b
b
GQ404795
Rhinolophus hipposideros
Slovenia
GQ404796
Rhinolophus hipposideros
Slovenia
b
GQ404797
Rhinolophus hipposideros
Slovenia
b
DQ022305
Rhinolophus sinicus
China
b
NC_009696
Rhinolophus macrotis
b
b
NC_004718
Human
NC_009021
Rousettus leschenaulti
NC_006577
Human
b
NC_006852
Mouse
b
NC_007732
Pig
b
NC_005147
Human
b
EF544563
Myotis occultus
USA
a
EF544565
HQ184049
Myotis occultus
Miniopterus schreibersii
USA
Spain
a
a
HQ184050
Myotis blythii
Spain
a
DQ648838
Bat
China
a
China
b
DQ648855
Rhinolophus ferrumequinum
China
a
DQ648854
Rhinolophus ferrumequinum
China
a
a
NC_003436
Pig
EU375862
Myotis dasycneme
Germany
a
EU375859
Myotis dasycneme
Germany
a
a
EU375858
Myotis dasycneme
Germany
EU375855
Myotis dasycneme
Germany
a
EU375863
Myotis dasycneme
Germany
a
EU375861
Myotis dasycneme
Germany
a
EU375856
Myotis dasycneme
Germany
a
EU375854
Myotis dasycneme
Germany
a
EU375857
EU375865
Myotis dasycneme
Myotis bechsteinii
Germany
Germany
a
a
EU375853
Myotis bechsteinii
Germany
a
EU375860
Myotis bechsteinii
Germany
a
EU375869
Pipistrellus nathusii
Germany
a
EU375864
Pipistrellus nathusii
Germany
a
EU375870
Pipistrellus pygmaeus
Germany
a
123
HKU9
Alpha and Betacoronaviruses in Iberian bats
1887
Table 2 continued
Access no
Host species
Country
Genus
EU375868
Pipistrellus pygmaeus
Germany
a
EU375867
Pipistrellus pygmaeus
Germany
a
HQ184060
Pipistrellus sp.
Spain
a
DQ648822
Bat
China
a
DQ648821
Bat
China
a
DQ648824
Bat
China
a
DQ648823
Bat
China
a
EU375875
Myotis daubentonii
Germany
a
EU375873
Myotis daubentonii
Germany
a
EU375874
Myotis daubentonii
Germany
a
a
EU375872
Myotis daubentonii
Germany
EU375866
Myotis daubentonii
Germany
a
HQ184056
EU375871
Myotis daubentonii
Myotis daubentonii
Spain
Germany
a
a
DQ648833
Myotis ricketti
China
a
DQ249224
Myotis ricketti
China
a
DQ648837
Myotis ricketti
China
a
Cluster
HKU6
DQ249235
Rhinolophus sinicus
China
a
HKU2
DQ249213
Rhinolophus sinicus
China
a
HKU2
DQ648840
Bat
China
a
DQ249228
Miniopterus sp.
China
a
EU834954
Miniopterus australis
Australia
a
EU834952
Miniopterus australis
Australia
a
EU834955
Miniopterus schreibersii
Australia
a
EU834953
Rhinolophus megaphyllus
Australia
a
DQ648835
Miniopterus schreibersii
China
a
DQ648796
Bat
China
a
DQ648797
DQ249226
Bat
Miniopterus magnater
China
China
a
a
HQ184061
Hypsugo savii
Spain
a
HQ184051
Nyctalus lasiopterus
Spain
a
HQ184054
Nyctalus lasiopterus
Spain
a
HQ184053
Nyctalus lasiopterus
Spain
a
HQ184052
Nyctalus lasiopterus
Spain
a
HQ184055
Nyctalus lasiopterus
Spain
a
HQ184057
Myotis myotis
Spain
a
HQ184058
Pipistrellus kuhlii
Spain
a
Australia
a
EU834951
Myotis macropus
NC_002645
Human
a
NC_005831
Human
a
AY994055
Cat
a
NC_002306
Pig
a
NC_001451
Chicken
c
HKU8
HKU7
123
1888
Fig. 2 CoV phylogenetic reconstruction based on 396 bp of the RdRp
gene including 14 Spanish CoV from different bat species and 77 alpha,
beta and gammacoronaviruses obtained from GenBank. Accession
numbers are shown in brackets. BatCoV detected in Spain are
highlighted in italics. For the analyses GTR substitution model, gamma
estimation and two simultaneous runs of 107 generations were done,
each with four Markov chains, and the trees were sampled every 100
generations. First 25% trees were excluded as burn-in from the analysis.
Significant posterior probabilities are indicated. Complementary
123
A. Falcón et al.
information about sequences used in this phylogenetic reconstruction
are shown in Table 2. Positive samples described in this work are shown
in shaded rectangles and ovals. The new alpha and betacoronavirus
groups described in this work are shown in shaded ovals. Amino acid
identity was calculated with MEGA 4 using the pairwise deletion
option. The alignment comprised the same 396 bp of the RdRp gene
used for the phylogenetic reconstruction. Amino acid identities across
132 amino acids are indicated next to the brackets that links every
Spanish BatCoV to the phylogenetic neighbours
Alpha and Betacoronaviruses in Iberian bats
related to cluster HKU7 described in China [10]. BatCoV
C, D, E, F, G and I belonged to the same lineage and
showed an intriguing new independent cluster (significant
posterior probability = 0.95) including BatCoV I0 (Fig. 2).
In addition, BatCoV J and M were genetically related to b
CoV, although they did not really associate with any of
their previously described linages. Sequences corresponding to two different groups of CoV were found in the same
bat species (Hypsugo savii) as it was also found for other
bat species elsewhere [12]. Spanish BatCoV described here
were also classified calculating amino acid distances of
these viruses from phylogenetic neighbours and related
reference species [21]. Amino acid distance criteria
recently described for separating RdRp grouping units
(RGU) were adopted [21]. Interestingly, the amino acid
distance criteria ([4.8% for alphacoronavirus and [6.3%
for betacoronavirus) reinforced the presence of the new
group of alphacoronaviruses mentioned above including
Spanish BatCoV C, D, E, F, G and I; and additionally
showed four new tentative groups (Fig. 2). BatCoV A and
B were included in a new alphacoronavirus group and
BatCoV I0 constituted another independent alphacoronavirus group. BatCoV J, and M represented two new betacoronavirus groups and BatCoV H, K and L remained as
part of several established species (Fig. 2).
It has been previously suggested that some CoV associate to certain bat species [10, 11]. However, we found
that different bat species from the same colony or location
harbour CoV of the same genetic lineage (BatCoV A and
B; G and I), indicating a greater diversity and higher
complexity than previously described for the ecology of
BatCoV. Similar exceptions were found in China and
South America [12, 16, 26] and could also be observed
with Australian BatCoV (Fig. 2 and Table 2).
In conclusion, previous studies showed the presence of
BatCoV in Europe. However, to our knowledge, this is the
first report describing the presence of CoV RNA in Iberian
bat species. Phylogenetic data indicate high diversity, wide
distribution and complex ecology of CoV in bats captured
in diverse Spanish locations. The sequences reported herein
provide new insights into the diversity of coronaviruses and
describe new phylogenetic linages that appear to diverge
from all genotypes previously detected in other European
locations. Future studies should clarify whether such
apparently high diversity reflects the bio-geographical
peculiarities of the Iberian Peninsula or not. This study
contributes with a new dataset to the global surveillance of
emerging BatCoV with pathogenic potential in humans.
Our data reinforce the notion that the ecology and transmission of CoV in bat reservoirs is far from being completely understood and that more studies will be necessary
to evaluate the magnitude of the potential threat that these
viruses pose to human health.
1889
Acknowledgments We gratefully thank S. Perlman and P. Woo for
providing MHV RNA and HCoV-HKU1 RT-PCR products respectively, as controls for pancoronavirus PCR development. We also
thank L. Enjuanes for scientific discussions and P. Gastaminza for
critical comments on the manuscript. We are grateful to David Garcı́a,
Ana Popa, Grupo Drosera, Museu de Granollers and everyone who
participated in the collection of field samples and the Genomic Unit of
the National Center of Microbiology for analyzing the genomic
sequences. This work was supported by a Spanish Ministry of Science
and Innovation grant (Projects SAF2006-12784-C02-01 and
SAF2006-12784-C02-02) and a grant from the Basque Government
(ref. IT301-10).
Conflict of interest
The authors declare no conflict of interest.
Open Access This article is distributed under the terms of the
Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and source are credited.
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