Bat Airway Epithelial Cells: A Novel Tool for the Study of Zoonotic Viruses
Citation: Eckerle I, Ehlen L, Kallies R, Wollny R, Corman VM, et al. (
Bat Airway Epithelial Cells: A Novel Tool for the Study of Zoonotic Viruses
Isabella Eckerle 0
Lukas Ehlen 0
Rene Kallies 0
Robert Wollny 0
Victor M. Corman 0
Veronika M. Cottontail 0
Marco Tschapka 0
Samuel Oppong 0
Christian Drosten 0
Marcel A. Mu ller 0
Vincent Jacobus Munster, NIH, United States of America
0 1 Institute of Virology, University of Bonn Medical Centre , Bonn, Germany , 2 Institute of Experimental Ecology, University of Ulm , Ulm, Germany , 3 Smithsonian Tropical Research Institute, Balboa, Panama, 4 Kwame Nkrumah University of Science and Technology , Kumasi , Ghana
Bats have been increasingly recognized as reservoir of important zoonotic viruses. However, until now many attempts to isolate bat-borne viruses in cell culture have been unsuccessful. Further, experimental studies on reservoir host species have been limited by the difficulty of rearing these species. The epithelium of the respiratory tract plays a central role during airborne transmission, as it is the first tissue encountered by viral particles. Although several cell lines from bats were established recently, no well-characterized, selectively cultured airway epithelial cells were available so far. Here, primary cells and immortalized cell lines from bats of the two important suborders Yangochiroptera and Yinpterochiroptera, Carollia perspicillata (Seba's short-tailed bat) and Eidolon helvum (Straw-colored fruit bat), were successfully cultured under standardized conditions from both fresh and frozen organ specimens by cell outgrowth of organ explants and by the use of serum-free primary cell culture medium. Cells were immortalized to generate permanent cell lines. Cells were characterized for their epithelial properties such as expression of cytokeratin and tight junctions proteins and permissiveness for viral infection with Rift-Valley fever virus and vesicular stomatitis virus Indiana. These cells can serve as suitable models for the study of bat-borne viruses and complement cell culture models for virus infection in human airway epithelial cells.
Funding: The study was supported by the German Research Foundation (DFG grant DR 772/3- 1). The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Emerging infectious diseases are perceived as a significant threat
to human health. Among emerging infectious diseases,
approximately 2/3 are of zoonotic origin . While bats fulfill many
ecologically important functions, several species were recognized
as potential reservoirs of zoonotic viruses. Emerging human
diseases caused by bat-borne viruses include the severe acute
respiratory syndrome, Hendra and Nipah encephalitides, as well
as Ebola- and Marburg hemorrhagic fever . A recent
outbreak of severe respiratory infection in the Middle East region
caused by the novel human coronavirus MERS-CoV with close
identity to bat coronaviruses emphasizes the relevance of
batborne zoonotic diseases . The conditions and mechanisms
behind animal-to-human spillover are unknown.
Research on bats has led to the discovery of a plethora of novel
bat virus sequences, including viruses that are presumably
candidates for switching their host species due to a close identity
with human viruses . To assess the risk and potential for
human pathogenicity of novel bat viruses, it is crucial to establish
valid research tools for comparative in vitro infection modeling.
The two main obstacles for studying novel bat viruses are: 1)
isolation attempts by conventional methods in cell culture or
animal models have been unsuccessful for many viruses; and 2)
suitable model systems to study virus isolates in their natural host
are missing. In most cases it is not possible to study virus
replication in bats under experimental conditions, because species
of interest cannot be kept or bred in captivity. Also, due to
conservation reasons and ethical concerns, it is not justifiable to
sample an arbitrary number of these animals from their natural
habitat for infection studies in the laboratory.
To isolate and study zoonotic viruses with airborne
transmission, cell culture models representing the respiratory tract are of
special interest. During infection, the airway epithelium plays a
crucial role: i) By forming a physical barrier between a host and its
environment, these are often the first cells that encounter
pathogens ii) they represent a barrier that must be crossed by
the virus for successful entry into the host. The airway epithelium
is not only a pure physical barrier consisting of a tightly packed
layer of cells covered with mucus. Recent studies also show that
airway epithelial cells provide a complex contribution to host
defense, innate immunity, and immune regulation (for a review see
So far, only one cell line from the respiratory tract of a bat is
widely available. This cell line is derived from the lung of Tadarida
brasiliensis (Tb 1 Lu, American Type Culture Collection, ATCC
Nr. CCL-88), but no information on the cell type of origin exists.
We and others have recently established bat cell lines from a larger
number of bat species and different organ types, which have
already provided important insight into immunology and
virushost interaction of different zoonotic viruses . To
specifically address the role of the airway epithelium in virus-host
interaction in the reservoir host, we present a further specification
of bat cell culture models.
We chose two bat species which reflect the two bat suborders
Yangochiroptera and Yinpterochiroptera, that both include
species presenting properties relevant for zoonotic transmission:
broad distribution range, high population densities, and in some
species frequent interactions with humans due to a high
adaptability to environments altered by humans. Sebas
shorttailed fruit bat, Carollia perspicillata, a member of the highly diverse
family Phyllostomidae (the largest and most diverse bat family in
the Neotropics), is stable in its population trend, according to the
International Union for the Conservation of Nature and Natural
Resources (IUCN, www.iucnredlist.org). It is highly abundant in
many localities of the Neotropics. The straw-colored fruit bat,
Eidolon helvum, a member of the Pteropodidae, is found in very large
colonies across a wide area in both East- and West Africa, and
lives close to human settlements and in urban areas. In contrast to
C. perspicillata, the population trend of E. helvum is suspected to be
decreasing due to intensive hunting for meat (IUCN: near
threatened), but the sometimes very large colonies and the
adaptability to urban habitats pose potential risks for zoonotic
transmission. Furthermore, E. helvum is capable of travelling
thousands of kilometers across Africa within a seasonal migration
Of note, these characteristics are not limited to the two bat
species named above but are true for many bat species around the
world. Among them are typical characteristics of bats (i.e. large
population size) that distinguish bats from other mammals as
reservoirs for potentially zoonotic viruses.
To generate a tool for isolation of bat-borne viruses and to
facilitate studies on virus transmission in the natural reservoir host,
we aimed at establishing airway epithelial cell lines derived from
Capturing and sampling of E. helvum was done with permission
from the Wildlife Division, Forestry Commission, Accra, Ghana.
Geographic co-ordinates of the sampling site in Kumasi/Ghana
were N06u42902.00 W001u37929.90. Under the auspices of Ghana
authorities bats were caught with mist nets, anaesthetized with a
Ketamine/Xylazine mixture and euthanized by cervical
dislocation (permit no. CHRPE49/09; A04957) as described previously
. Veterinary skilled staff performed all procedures on E. helvum.
Additional export permission was obtained from the Veterinary
Services of the Ghana Ministry of Food and Agriculture (permit
no. CHRPE49/09; A04957). For the generation of
Yangochiroptera cell lines, the Institute of Zoology, University of Veterinary
Medicine Hannover, Germany, kindly provided a post-mortem
trachea sample of C. perspicillata from a breeding colony established
for research purposes on Neurophysiology and Neurobiology of
bats (Landeshauptstadt Hannover, Fachbereich Recht und
Ordnung, Gewerbe und Veterinarangelegenheiten, permit
no. 42500/1H). Animals were euthanized (permit No. 11/0435)
by cervical dislocation while under halothane anesthesia.
After the animals were euthanized as described above, tracheas
were removed in toto. Trachea specimens were immediately placed
in ice-cold medium for transport or, if longer transport was
necessary (as in the case of samples of E. helvum from Ghana),
frozen at 280uC in cell culture freezing medium (PAA, Pasching,
Austria). The following steps were done under sterile conditions
using a laminar flow hood. Upon arrival at the laboratory, frozen
specimens were thawed and washed in 37uC warm, sterile
phosphate buffered saline (PBS). The trachea and large bronchi
were roughly cleaned from the attached surrounding tissue, and
then longitudinally chopped into pieces with a sterile blade. Tissue
fragments were placed in a 6-well cell culture plate and submerged
in 37uC warm medium. The medium consisted of primary airway
epithelial cell medium basal mix supplemented with bovine
pituitary extract 0.004 ml/ml, epidermal growth factor
(recombinant human) 10 ng/ml, insulin (recombinant human) 5 mg/ml,
hydrocortisone 0.5 mg/ml, epinephrine 0.5 mg/ml,
triiodo-Lthyronine 6.7 ng/ml, holo-transferrin (human) 10 mg/ml, and
retinoic acid 0.1 ng/ml (Promocell, Heidelberg, Germany). The
primary cell medium was additionally supplemented with
penicillin/streptomycin (Life Technologies GmbH, Darmstadt,
Germany), ofloxacin (TarividH, Sanofi-Aventis), and amphotericin B
(PAA, Pasching, Austria) for the initial culturing of the tissue to
avoid bacterial and fungal contamination. Any movement of the
cell culture plates was avoided during the first 3 days, then cells
were observed daily for quality of beating of ciliated cells and
outgrowth of primary cells. After an outgrowth of primary cells
was observed, the medium was changed every 2 days. When
nearly confluent, cells were immortalized by lentiviral transduction
of the large T antigen of SV40 as described previously [14,15]. All
cell cultures were genotyped by amplification of mitochondrial
cytochrome c oxidase I .
After immortalization, cells were passaged frequently, at least
once a week, until a sudden increase in cell growth was observed,
at which point they were partly stock frozen for further use. After
three to four passages, cells were characterized for expression of
epithelial marker proteins cytokeratin and zonula occludens
protein 1 (ZO-1) by immunofluorescence staining. Cells were
subcloned by end-point-limiting dilution and those cell
populations that were initially derived from a single cell and presented an
epithelial morphology most closely resembling the primary cell
morphology were selected. Characterization was repeated for all
subclones. After subcloning, cells were adapted to a supplemented
Dulbeccos modified Eagles medium (DMEM) (PAA, Co lbe,
Germany) with 10% heat-inactivated FBS as described previously
. The cell line Tb 1 Lu (American Type Culture Collection,
ATCC Nr. CCL-88) was cultured under the same conditions.
Cells were incubated at 37uC and 5% CO2.
Nucleic acid preparation and real-time PCR
Viral RNA was extracted from the cell culture supernatant
according to the manufacturers instructions (NucleospinH RNA
virus, Machery Nagel, Du ren, Germany). PCR was performed
using the SuperScriptH III One-Step RT-PCR System with
PlatinumH Taq DNA Polymerase (Invitrogen, Karlsruhe,
Germany). Cycling conditions for the vesicular stomatitis virus Indiana
(VSV) and Rift Valley fever virus (RVFV) real-time RT-PCR
included a reverse transcription step for 15 min at 55uC, initial
denaturation for 2 min at 95uC, and 45 cycles consisting of
denaturation for 15 seconds at 95uC and primer annealing/
elongation for 30 seconds at 58uC. Real-time RT-PCR was
carried out using the LightCycler 480 Real-Time PCR System
(Roche, Basel, Switzerland).
Immunofluorescence assay (IFA)
Cells were seeded on glass slides and washed with PBS on the
following day, then fixed with acetone-methanol. Slides were
incubated with primary mouse monoclonal antibodies against
pancytokeratin (mouse-monoclonal, Abcam, C-11, ab7753) and rabbit
polyclonal antibodies against zonula occludens protein (ZO-1 Mid)
(rabbit polyclonal, Invitrogen 40-2200) diluted 1:400 in PBS
overnight at 4uC. The secondary antibodies were cyanine
3labeled donkey-anti-mouse serum and cyanine 2-labeled
donkeyanti-rabbit serum (Dianova). Nuclei were counterstained with
DAPI. All photographs were taken with a 207 Motic Axiovision
microscope (Zeiss). Antibodies with reactivity to a broad variety of
species were chosen. To compare bat and rodent staining to the
staining in cells with confirmed reactivity, all antibodies were
additionally tested in porcine airway epithelial cells. A comparable
staining pattern was seen in all cells (data not shown). Cells were
then subcloned by end-point-limiting dilution and multiple clones
were selected for generation of homogeneous cell lines. After
selection of clones, characterization by IFA was repeated in the
Although the species from which cells were obtained were
identified by experienced field workers (E. helvum) or from an
established breeding colony (C. perspicillata), species confirmation of
the cell lines was performed by sequencing subunit I of the
cytochrome c oxidase gene (CO1) and compared to available
databases (GenBank and BOLD System, www.barcodinglife.com).
As the cells were derived from feral animals or from breeding
populations without special pathogen free (SPF) housing
conditions, the immortalized cells were investigated for putative
contaminants which might be cultured along with the cells, posing
a risk for laboratory workers as well as a source of bias when
assessing virus-host interaction. Cells were detached and
dissociated with Accutase, and washed twice in PBS. Then, viral DNA/
RNA was extracted as described above. Cells were tested for viral
nucleic acids using generic (RT)-PCR assays specific for different
virus families and genera. (RT)-PCR amplification products were
pooled and analyzed by agarose gel electrophoresis. Amplicons
ranging in size from 150 bp to 700 bp were cut from the gel and
purified using the QIAEX II Gel Extraction Kit (Qiagen, Hilden,
Germany). Fragment ends were repaired, 454 sequencing adaptors
were ligated, and emulsion PCR was performed according to
standard 454 sequencing protocols (Roche, Mannheim,
Germany). Deep sequencing of (RT)-PCR products was done on a 454
Genome Sequencer Junior (Roche). The resulting reads were
aligned against the virus database using the BLASTn (wordsize 7)
and tBLASTx (wordsize 2) algorithms  with an e-value cut-off
of 1023. All cell lines were controlled for mycoplasma  and SV
5 (in-house assay, see also ). Additionally, cell lines were
screened for lyssaviruses .
Virus infection studies
Cells were seeded in 24-well plates at a density of 46105 cells
per ml. The following day, cells were infected with VSV Indiana
or RVFV clone 13 at a multiplicity of infection (MOI) of 0.1 or
0.001 as described previously [14,21]. Briefly, the medium was
removed and cells were infected with virus diluted in Optipro
serum-free medium (Life Technologies GmBH, Darmstadt,
Germany) for 1 h. Cells were inoculated for 1 h at 37uC and
washed twice with PBS after infection. Samples were taken at time
points 0, 4, 8, 12, 24, and 48 hours post infection (hpi) for VSV
and 0, 8, 12, 24, 48, and 72 hpi for RVFV.
Sebas short-tailed bat (C. perspicillata)
Straw-colored fruit bat (E. helvum)
Middle- and South America
Understory resident of rain forests and secondary forests,
roosting in caves and hollow trees. Widespread and highly
abundant in many localities of the Neotropics.
Africa, Arabian Peninsula
Tropical rain forest, dry savanna, modified habitats and
urban areas. Animals may move during their seasonal
migrations over thousands of km.
Sebas short-tailed bat
Viruses were detected either as virus isolates, on the basis of genetic sequence, or indirectly by detection of antibodies. Table adapted from ; modified and
supplemented. Viruses that have been reported to infect humans are marked with *.
A protocol for isolation of primary airway epithelial cells was
established and immortalized cell lines were generated from the
two bat species, C. perspicillata and E. helvum, of the suborders
Yangochiroptera and Yinpterochiroptera (Table 1, Figure 1).
An extensive review of the literature identified a high diversity of
viruses in both species, among them zoonotic viruses or viruses
related to zoonotic viruses (Table 2).
Establishment of primary cells from outgrowths
Growth of primary cells from the tissue was observed between
35 days after tissue fragments were placed in the dish (see
Figure 2 A, B). Directly after placing the tissue fragments in the
dish, beating of ciliated cells was observed on the luminal side of
the tracheal rings from both fresh and frozen trachea specimens
(see Video S1). Outgrowing cells displayed a homogeneous,
cobblestone-like morphology typical of epithelial cells (see
Figure 2 C). To increase the number of primary cells, tracheal
rings were carefully removed once the dish was fully coated with
cells (usually after 23 weeks) and placed in a new dish, where
repeated outgrowth of cells was observed. This procedure could be
repeated five to six times; however, a decrease in the number of
outgrowing cells as well as loss of active ciliated cells within the
tracheal ring over time was observed. In general, outgrowth of
cells from the tissue specimens was observed for more than 3
months when frequently placed in new dishes and supplemented
with fresh medium every 2 days.
Immortalization and epithelial characterization
Primary cells of both species were successfully immortalized by
lentiviral transduction of the large T antigen of SV40. After
immortalization, a decline in the cell population was observed.
One to two weeks afterwards, surviving cells started to proliferate
rapidly. Cells were expanded and characterized for epithelial cell
markers by IFA with broadly reactive antibodies against
cytokeratins (anti-pan-cytokeratin) as well as zonula occludens protein
(ZO-1). The cells in both of the cell lines generated were positive
for both of these epithelial markers, in contrast to the
commercially available cell line Tb 1 Lu (see Figure 3).
The species of origin for both cell lines were confirmed by their
CO1 sequences in the BOLD system.
Screening of cells for viral contamination
Apart from testing all cells for lyssaviruses, 454 sequencing was
used for broad pathogen screening. After lysis of cell pellets and
purification of RNA and DNA, nucleic acid amplification was
performed using degenerate primers targeting conserved regions of
major virus families or genera (Table S1). All sequences obtained
were identified as host sequences; no sequences from viral or
bacterial organisms were found.
Virus infection studies
To investigate whether the cells generated were permissive for
virus infection, we performed virus infection studies with two
zoonotic viruses, VSV and RVFV clone 13 (Figure 4). Cells were
infected with two different MOIs, 0.1 or 0.001, and viral copies in
the supernatant were detected. Upon infection, cells of both
species showed cytopathic effects. Both cell lines showed high
replication of VSV with maximum copy numbers in the range of
109 viral copies per ml and lower maximum copy numbers for
The present report describes the establishment of airway
epithelial cell lines from two bat species pertaining to the
suborders Yangochiroptera and Yinpterochiroptera for the study
of bat-borne, including several zoonotic viruses. The detection of
an increasing number of bat-borne viruses necessitates suitable
model systems for the isolation and study of these viruses. Several
new cell lines from bats have been established in recent years, but
so far there has been no focus on targeted generation of epithelial
cells [13,14]. Successful cultures of primary and immortalized cells
of airway epithelial origin are already well established for humans
and a few animal species. These include mainly equine, porcine,
bovine, and mouse cell lines , but to the best of our
knowledge, no characterized airway epithelial cell lines from bats
So far, only two bat cell lines are available from the American
Type Culture Collection (ATCC): Tb 1 Lu, derived from the lung
of the neotropical bat species Tadarida brasiliensis (Mexican
freetailed Bat), which was established in 1965; and Myi/It, derived
from a skin tumor of the North American bat species Myotis velifer
incautus (cave bat). However, given the high phylogenetic diversity
of bats, these cell lines may not always be appropriate to study
batrelated zoonotic viruses in detail. Cell lines are needed that allow
the study of virus-host interaction in a wide range of host species;
species that may differ in many aspects, including not only their
geographic distribution, ecology, and biology, but also most likely
in their immunological and receptor-associated characteristics as
Successful selective culturing of epithelial cells is dependent on
the use of serum-free medium supplemented with hormones and
growth factors . Widely used formulations of cell culture
Figure 3. Immunofluorescence staining for markers of epithelial origin of Tb 1 Lu and airway epithelial cells from C. perspicillata
(CarperAEC.B) and E. helvum (EidheAEC.B) prior to subcloning. The markers used to confirm epithelial origin were cytokeratin (CK, red) and
zonula occludens-1 (ZO-1, green); nuclei are counterstained with DAPI (blue). Expression of both markers is present in all cell lines generated by the
described methods, indicating an epithelial origin. By contrast, the commercially available Tb 1 Lu does not show expression of the respective
medium supplemented with fetal bovine serum (FBS) are less
suitable for epithelial cell growth, as, during the manufacturing
process, serum platelet derived growth factor (PDGF) is released.
In cell culture, PDGF has a stimulatory effect on fibroblasts, while
it inhibits epithelial cell growth and quickly leads to terminal
squamous differentiation in the primary cell culture .
Furthermore, even if epithelial cells are present in the primary
cell culture, overgrowth of the epithelial proportion by fibroblasts
after several passages can occur if medium containing FBS are
used (unpublished observation). Most bat-derived cell lines
published so far, however, were obtained and cultured using
conventional medium containing FBS. It is therefore suggested
that most of them contain at least a mix of several different cell
types, including fibroblasts, or consist of fibroblasts only, as they
normally overgrow other cell types after several passages. To
prevent this, we used serum-free medium only for growth of
primary cells, and found this approach to be efficacious. By
contrast, Crameri et al. established primary and immortalized bat
cell lines from Pteropus alecto and found serum-free medium the
least successful approach in their culture compared to
conventional medium formulations, however they do not comment on the
cell type (i.e. epithelial vs. fibroblast) . Their study primarily
evaluated overall isolation success of primary cells but not targeted
isolation of a certain cell type such as epithelial cells as presented
here. Furthermore, there are various formulations of serum-free
medium, which differ in their composition and are optimized for
different primary cell types; it is therefore difficult to compare
In our approach, adaptation of a standard medium containing
FBS was only performed after subcloning of immortalized cell
cultures and repeated confirmation of the selected clones for
epithelial cell markers. By using the method of endpoint limiting
dilution, we ensured that each subclone originated from a single
cell, so that contamination with fibroblasts or other non-epithelial
cells could be excluded. Characterization of epithelial origin was
additionally repeated in all subclones after adaptation to standard
medium with FBS. Culture of the original immortalized cell lines
was done with serum-free medium only until subcloning was
In contrast to many cell lines obtained previously by us and
other groups, we were able to culture and establish indefinite cell
lines from an adult animal without the use of embryonic tissue.
This has several advantages, as the use of embryonic tissue is more
laborious and involves sampling of multiple individuals to obtain a
gravid animal in the field. Further, separation of specific cell types
is more difficult in embryos and contamination by multiple cell
types is more likely.
Limitations of our method include the use of medium adapted
to humans and human growth factors, which might not be optimal
for other mammalian species such as bats. It is therefore likely that
the cells may not retain all of the epithelial cell properties that are
present in vivo. While it is unlikely that cell culture medium
optimized for individual non-human species such as bats will ever
become available, adaptation of protocols already successfully used
for human and livestock cell cultures is a feasible approach. There
is a general pitfall in all cell cultures, especially if further
modifications such as immortalization or subcloning are
performed. Thorough characterization of our cells showed that
growth of the desired cell type was successful.
Recently, several findings supported the approach of isolation of
bat-borne viruses in cell lines developed from the natural host
species. Examples include the isolation of a henipa-related
paramyxovirus in primary kidney cells from Pteropus alecto  as
well as isolation of Menangle virus from the urine of the same
species . Zhang et al. showed isolation and growth of a bat
herpesvirus from the bat Miniopterus schreibersii in primary bat cells
after failure in 14 other mammalian cell lines . Therefore bat
cell lines could provide an opportunity for isolation and
characterization of bat-borne viruses that otherwise fail to grow
in mammalian cell lines.
On the other hand, bat cell lines can provide a hint for zoonotic
origin of human viruses, for example Huynh and coworkers
showed replication of HCoV-NL63 in immortalized lung cells
from the North American tri-colored bat (Perimyotis subflavus) for
multiple passages and therefore conclude a bat origin of the virus
Due to the large number of species that build the order
Chiroptera, it will not be possible to find a perfect match between
virus and natural host in all instances however; there are
examples of virus receptors that are conserved between different
members of the Chiropteran order, and therefore bat cell lines that
are representatives of the order can serve as valuable
representatives. The most recent example is the conserved receptor
Dipeptidyl-peptidase-4 for MERS-coronavirus that allows
infection of bat cell lines, not only originating from the presumed
reservoir host, bats of the family Vespertilionidae, but across
several other bat families .
To study airborne transmission of viruses in a system that
resembles the in vivo situation as closely as possible, we aimed at
establishing a protocol for generating primary cell lines as well as
indefinite cell cultures. Even though generation of primary cells
from repeated outgrowth of trachea specimens was successful, only
a small number of cells were obtained in total from a single animal,
limiting complex experiments with primary cells. This problem
could be overcome by culturing organ specimens from multiple
animals; but for almost all relevant wild zoonotic species it is not
possible to harvest material of the required quality from a large
number of individuals. The generation of immortalized cell lines
that are not limited in their passage number can overcome this
problem but the immortalization process comes at the cost of
interfering with at least some primary cell properties. As shown,
immortalized cell lines and cell lines from a subcloned single cell
still maintain many of their original characteristics, such as
confluent growth as a monolayer and expression of tight junction
proteins, an important feature of epithelial cells. Furthermore,
immortalized cell lines are valuable and easy to maintain. They
can therefore be shared with other groups, facilitating zoonotic
research beyond the lab of origin. Finally, adapting single cell
clones of confirmed epithelial origin to standard medium will allow
for less costly propagation and use compared with cells adapted to
serum-free primary cell medium.
With these issues in mind, we believe the protocols and cell
cultures developed in the present study will provide a useful model
for the study of bat-borne viruses and virus-host interaction in
Table S1 Virus families and genera for which screening
by nucleic acid amplification was performed. Details on
assays available upon request.
We thank Stephan Kallies for excellent technical help and Andrea Rasche
for help with organ specimens. We thank Karl-Heinz Esser and Arne
Liebau (Institute of Zoology, University of Veterinary Medicine,
Hannover) for organ specimens and Florian Gloza-Rausch (Noctalis, Bad
Segeberg) for photographs of E. helvum. We thank Friedemann Weber,
Institute of Virology, University of Marburg for Rift Valley Fever virus
clone 13 and Alexander Pfeifer and Katrin Zimmermann, Institute of
Pharmacology and Toxicology, University of Bonn, for providing large T
Conceived and designed the experiments: IE LE RK RW V. Corman CD
MAM. Performed the experiments: IE LE RK RW V. Corman. Analyzed
the data: IE LE RK RW V. Corman V. Cottontail MT SO CD MAM.
Contributed reagents/materials/analysis tools: IE LE RK RW MAM.
Wrote the paper: IE CD MAM.
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