Novel paramyxoviruses in free-ranging European bats.
ABSTRACT The zoonotic potential of paramyxoviruses is particularly demonstrated by their broad host range like the highly pathogenic Hendra and Nipah viruses originating from bats. But while so far all bat-borne paramyxoviruses have been identified in fruit bats across Africa, Australia, South America, and Asia, we describe the detection and characterization of the first paramyxoviruses in free-ranging European bats. Moreover, we examined the possible impact of paramyxovirus infection on individual animals by comparing histo-pathological findings and virological results. Organs from deceased insectivorous bats of various species were sampled in Germany and tested for paramyxovirus RNA in parallel to a histo-pathological examination. Nucleic acids of three novel paramyxoviruses were detected, two viruses in phylogenetic relationship to the recently proposed genus Jeilongvirus and one closely related to the genus Rubulavirus. Two infected animals revealed subclinical pathological changes within their kidneys, suggestive of a similar pathogenesis as the one described in fruit bats experimentally infected with Hendra virus.Our findings indicate the presence of bat-born paramyxoviruses in geographic areas free of fruit bat species and therefore emphasize a possible virus-host co-evolution in European bats. Since these novel viruses are related to the very distinct genera Rubulavirus and Jeilongvirus, a similarly broad genetic diversity among paramyxoviruses in other Microchiroptera compared to Megachiroptera can be assumed. Given that the infected bats were either found in close proximity to heavily populated human habitation or areas of intensive agricultural use, a potential risk of the emergence of zoonotic paramyxoviruses in Europe needs to be considered.
- SourceAvailable from: Claudia Kohl[Show abstract] [Hide abstract]
ABSTRACT: Bats are being increasingly recognized as reservoir hosts of highly pathogenic and zoonotic emerging viruses (Marburg virus, Nipah virus, Hendra virus, Rabies virus, and coronaviruses). While numerous studies have focused on the mentioned highly human-pathogenic bat viruses in tropical regions, little is known on similar human-pathogenic viruses that may be present in European bats. Although novel viruses are being detected, their zoonotic potential remains unclear unless further studies are conducted. At present, it is assumed that the risk posed by bats to the general public is rather low. In this review, selected viruses detected and isolated in Europe are discussed from our point of view in regard to their human-pathogenic potential. All European bat species and their roosts are legally protected and some European species are even endangered. Nevertheless, the increasing public fear of bats and their viruses is an obstacle to their protection. Educating the public regarding bat lyssaviruses might result in reduced threats to both the public and the bats.Viruses 08/2014; 63390:3110-3128. · 3.28 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Bat-borne viruses can pose a serious threat to human health, with examples including Nipah virus (NiV) in Bangladesh and Malaysia, and Marburg virus (MARV) in Africa. To date, significant human outbreaks of such viruses have not been reported in the European Union (EU). However, EU countries have strong historical links with many of the countries where NiV and MARV are present and a corresponding high volume of commercial trade and human travel, which poses a potential risk of introduction of these viruses into the EU. In assessing the risks of introduction of these bat-borne zoonotic viruses to the EU, it is important to consider the location and range of bat species known to be susceptible to infection, together with the virus prevalence, seasonality of viral pulses, duration of infection and titre of virus in different bat tissues. In this paper, we review the current scientific knowledge of all these factors, in relation to the introduction of NiV and MARV into the EU.Viruses 05/2014; 6(5):2084-121. · 3.28 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The Paramyxoviridae form an increasingly diverse viral family, infecting a wide variety of different hosts and have been, in recent years, linked to disease emergence in many different animal populations and in man. Bats and rodents have been identified as major animal populations capable of harboring paramyxoviruses, and host shifting between these animals is likely to be important driving factor in the underlying evolutionary processes that eventually lead to disease emergence. Here, we have studied paramyxovirus circulation within populations of endemic and introduced wild small mammals of the southwestern Indian Ocean region and belonging to four taxonomic orders: Rodentia, Afrosoricida, Soricomorpha and Chiroptera. We report elevated infection levels, as well as widespread paramyxovirus dispersal and frequent host exchange of a newly emerging genus of the Paramyxoviridae, currently referred to as the Unclassified Morbilli-Related Viruses (UMRVs). In contrast with other genera of the Paramyxoviridae, where bats have been shown to be key host species, we show that rodents (and in particular Rattus rattus) are significant spreaders of UMRVs. We predict that the ecological particularities of the southwestern Indian Ocean, where small mammal species often live in densely packed, multi-species communities, in combination with the increasing invasion of R. rattus and perturbations of endemic animal communities by active anthropological development will have a major influence on the dynamics of UMRV infection. Identification of the infectious agents that circulate within wild animal reservoirs is essential for several reasons: i) Infectious disease outbreaks often originate from wild fauna; ii) Anthropological expansion increases the risk of contact between human and animal populations and hence of disease emergence; iii) Evaluation of pathogen reservoirs helps elaborating preventive measures to limit the risk of disease emergence. Many paramyxoviruses for which bats and rodents serve as major reservoirs, have demonstrated their potential to cause disease in humans and animals. In the context of the biodiversity hot spot of southwestern Indian Ocean islands and their rich endemic fauna, we show that highly diverse Unclassified Morbilli-related viruses exchange between various endemic animal species and their dissemination is likely facilitated by the introduced Rattus rattus. Hence, many members of the Paramyxoviridae appear well adapted for the study of the viral phylodynamics that may potentially be associated with disease emergence.Journal of Virology 05/2014; · 4.65 Impact Factor
Novel Paramyxoviruses in Free-Ranging European Bats
Andreas Kurth1*., Claudia Kohl1., Annika Brinkmann1, Arnt Ebinger1, Jennifer A. Harper2, Lin-Fa Wang2,
Kristin Mu ¨hldorfer3, Gudrun Wibbelt3
1Robert Koch Institute, Centre for Biological Security, Berlin, Germany, 2CSIRO Livestock Industries, Australian Animal Health Laboratory, Victoria, Australia, 3Leibniz
Institute for Zoo and Wildlife Research, Berlin, Germany
The zoonotic potential of paramyxoviruses is particularly demonstrated by their broad host range like the highly pathogenic
Hendra and Nipah viruses originating from bats. But while so far all bat-borne paramyxoviruses have been identified in fruit
bats across Africa, Australia, South America, and Asia, we describe the detection and characterization of the first
paramyxoviruses in free-ranging European bats. Moreover, we examined the possible impact of paramyxovirus infection on
individual animals by comparing histo-pathological findings and virological results. Organs from deceased insectivorous
bats of various species were sampled in Germany and tested for paramyxovirus RNA in parallel to a histo-pathological
examination. Nucleic acids of three novel paramyxoviruses were detected, two viruses in phylogenetic relationship to the
recently proposed genus Jeilongvirus and one closely related to the genus Rubulavirus. Two infected animals revealed
subclinical pathological changes within their kidneys, suggestive of a similar pathogenesis as the one described in fruit bats
experimentally infected with Hendra virus.Our findings indicate the presence of bat-born paramyxoviruses in geographic
areas free of fruit bat species and therefore emphasize a possible virus–host co-evolution in European bats. Since these
novel viruses are related to the very distinct genera Rubulavirus and Jeilongvirus, a similarly broad genetic diversity among
paramyxoviruses in other Microchiroptera compared to Megachiroptera can be assumed. Given that the infected bats were
either found in close proximity to heavily populated human habitation or areas of intensive agricultural use, a potential risk
of the emergence of zoonotic paramyxoviruses in Europe needs to be considered.
Citation: Kurth A, Kohl C, Brinkmann A, Ebinger A, Harper JA, et al. (2012) Novel Paramyxoviruses in Free-Ranging European Bats. PLoS ONE 7(6): e38688.
Editor: Jean-Pierre Vartanian, Institut Pasteur, France
Received July 7, 2011; Accepted May 8, 2012; Published June 21, 2012
Copyright: ? 2012 Kurth et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Funding was provided by the Konrad Adenauer Foundation (fellowship to CK) and by the Isler-Stiftung, Clara-Samariter-Stiftung and FAZIT-Stiftung
(KM). 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.
* E-mail: KurthA@rki.de
. These authors contributed equally to this work.
Members of the virus family Paramyxoviridae are divided into two
subfamilies, Paramyxovirinae and Pneumovirinae, comprising a vast
variety of animal- and human-pathogenic viruses . Within the
subfamily Paramyxovirinae, five genera have been classified, Respiro-
, Morbilli-, Rubula-, Avula-, and Henipavirus, as well as a fast-
growing group of unclassified viruses. The increased molecular
characterization of recently isolated paramyxoviruses indicates
a much greater genetic diversity within the subfamily Paramyxovir-
inae than previously assumed. Furthermore, the detection of highly
human-pathogenic paramyxoviruses has also influenced the
attention drawn to paramyxovirus research and to the isolation
of further novel paramyxoviruses from hosts that are suggested as
likely species to transmit newly emerging viruses. Bats are among
this highly suspected group of animals . They belong to the
most successful and diverse mammals on earth and comprise
approximately 1,200 chiropteran species distributed worldwide. In
the last two decades important zoonotic viruses including Ebola,
Marburg, and SARS virus, but also paramyxoviruses such as
Hendra and Nipah virus have been identified in various Pteropus
spp. (flying foxes) fruit bats [3–11]. For Menangle virus, another
paramyxovirus isolated from fruit bats, less pathogenic courses of
disease in humans have been described . For other bat
paramyxoviruses isolated, infections in humans have yet to be
associated, e.g. Tioman virus from flying fox , bat parain-
fluenza virus from flying fox , Tuhoko virus from flying fox
, Mapuera virus from non-pteropid fruit bat , and Henipa-
like viruses also from non-pteropid fruit bat . All viruses of the
family Paramyxoviridae so far detected in bat species have been
identified in fruit bats across Africa, Australia, South America,
Asia, and Madagascar [3–6,10]. Only a few studies attempting the
isolation of paramyxoviruses in bats concerned insectivorous bat
species, and all of them turned out with negative results [16,17].
The only indication of paramyxoviruses in this group of bat species
was the detection of Nipah virus antibodies in lesser Asiatic yellow
bats (Scotophilus kuhlii) .
The present study aimed to detect and isolate novel paramyx-
oviruses in free-ranging European insectivorous bats and to
estimate a possible impact of paramyxovirus infection on infected
individual animals by comparing histo-pathological findings and
Materials and Methods
As part of a study to investigate diseases in free-ranging bats in
Germany , 120 deceased bats from 2009 of 15 different
European vespertilionid species (Eptesicus nilssoni, E. serotinus, Myotis
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bechsteini, M. daubentonii, M. mystacinus, M. nattereri, Nyctalus leisleri,
N. noctula, Pipistrellus kuhli, P. nathusii, P. pipistrellus, P. pygmaeus,
Plecotus auritas, P. austriacus, Vespertilio murinus) were examined. The
bat carcasses originated from 4 different geographic regions in
Germany, i.e. Berlin greater metropolitan area (n=83), Bavaria
(n=30), Brandenburg (n=5), and Baden-Wuerttemberg (n=2).
Bat carcasses were stored at –20uC for transportation before
performing a full necropsy. For histo-pathological examination,
a small piece of tissue from all organs was fixed in buffered 4%
formalin, processed routinely and embedded in liquid paraffin.
Paraffin blocks were cut at 2–5 mm thickness and stained with
hematoxylin-eosin . Immuno-histochemistry was performed
on all organs of PCR positive bats using rabbit immune sera
against Beilong virus, J-virus, Menangle virus, Tioman virus and
Nipah virus as described previously . Samples of lung, liver,
heart, and kidney, and conspicuous tissues (e.g. enlarged spleen)
from each bat were homogenized in buffer and transferred to
Pooled organ tissue from each bat was used for RNA/DNA
extraction (PureLinkTMViral RNA/DNA Mini Kit, Invitrogen,
Germany) and further cDNA synthesis according to the manu-
facturer’s instructions (TaqManH Reverse Transcription Reagents,
Applied Biosystems, Germany). Broadly reactive paramyxovirus-
specific RT-PCR assays were applied , yielding amplicons of
538 base pairs (PAR primers) and 486 base pairs (RES-MOR-
HEN primers) located across domains I and II of the RNA
polymerase (L)-coding sequence, a region of the genome suitable
for phylogenetic analyses . Since cDNA was readily available,
PCR conditions were modified using the optimization method by
Taguchi . For this, based on the use of orthogonal arrays
representing individual reactions with components at different
concentration levels, a minimal number of experiments is allowed.
To increase PCR sensitivity, the product yield for each reaction is
used to calculate the optimal concentration of each reaction
component. By using this method, a novel PCR reaction mixture
was determined and henceforth used. For first-round PCR in the
seminested assay, PCR mixtures contained 3 pmol each of
forward and reverse primers, 16PlatinumH Taq buffer (Invitro-
gen), 250 nmol MgCl2 (Invitrogen), 2.5 pmol desoxynucleoside
triphosphates (Invitrogen), 2 ml of cDNA, and 1.25 U of
PlatinumH Taq polymerase (Invitrogen). Water was then added
to a final volume of 25 ml. The PCR mixture was sequentially
incubated at 94uC for 2 min for denaturation, and then 40 cycles
at 94uC for 15 s, 50uC for 30 s, 72uC for 30 s, and a final
extension at 72uC for 7 min. For the second amplification in the
seminested PCR assay, 16PlatinumH Taq buffer, 25 nmol MgCl2,
2.5 pmol desoxynucleoside triphosphates, 3 pmol each of forward
and reverse primers, 1.25 U PlatinumH Taq, 1 ml PCR product
from the first reaction, adding water to a final volume of 25 ml.
The cycling conditions were identical to the ones of the first round.
PCR products were run on an 1.5% agarose gel containing
ethidium bromide. Images were captured on E.A.S.Y. RH-3 gel
documentation system (Herolab, Germany). Amplicons from the
PCR reaction were purified using the MSBH Spin PCRapace kit
(Invitek, Germany). Both strands of the amplicons were sequenced
with a BigDye Terminator v 3.1 Cycle Sequencing kit on an ABI
Genetic Analyzer 3500 6l D6 automated sequencer (Applied
Biosystems, Germany) using the corresponding PCR primers.
Remaining reaction conditions were performed in accordance to
the manufacturer’s protocol.
On the basis of newly acquired sequence information, specific
qPCR assays were designed (Table 1) to screen pooled organ
tissues of all 120 bats. Cycler conditions for all qPCR assays were
as follows: predenaturation (95uC for 10 min), 45 amplification
cycles (95uC for 30 s, 60uC for 30 s, 72uC for 30 s), and final
extension (72uC for 10 min).
Additional primers were designed using conserved regions
between Jeilongviruses and Henipaviruses to extend the sequence
obtained by PAR primers (primers and protocol are available on
Bayesian reconstruction of phylogenetic trees was performed in
concordance with the current proposals of Paramyxoviridae taxon-
omy using MrBayes, version 3.1.2 [24,25]. The underlying
alignment by ClustalW was based on a 529 base pair fragment
(PAR Primer) and 1,593 base pairs amplicon (long fragment) from
PCR reactions. The evolutionary history was inferred using the
bayesian MCMC method. First, a model selection for these
calculations was performed with jModelTest  and model
GTR+I+G (invariable sites, gamma distribution) was selected for
the PAR and the RES-MOR-HEN fragment, and GTR+G to
study the long fragment alignment. The calculation parameters
were as follows: number of runs: four, number of generations:
1,000,000, sample frequency: 100, burn in: 25%. The results were
finally visualized by the FigTree v1.2.1 program, a graphical
viewer of phylogenetic trees. Based on the GTR substitution
model the estimated transversion ratio, proportion of invariable
sites and gamma distribution parameters were estimated auto-
For confirmation of virus isolation and determination of the
infected organs, RNA/DNA extraction and PCR analysis in-
cluding sequencing was performed on all individual organs from
infected bats. For two isolates, a second RT-PCR with primers
Table 1. Primer sequences.
Virus PrimerSequence 59 to 39
BatPV/Myo.mys/E20/09 E20/09 FTgACAgATgATTTATgTgTTCgTTACT55.6
E20/09 RgAATCCCACTCTgATTTCAACg 56.1
BatPV/Pip.pip/E95/09E95/09 F ggTgCTTggCCACCTCT 57.3
E95/09 R gCgATgAAgTTTgTCTTggA56.4
E95/09 MGB CACTgCTTTATgCCTTTAA70
BatPV/Nyc.noc/E155/09 E155/09 F2 ggAgATTgCACTCAgTCTTCCTgT57.4
E155/09 R2 gTCCCCCTACTTgAgATggCA56.3
E155/09 MGB TCCgAgCTAAAATgTCA68
Novel Paramyxoviruses in European Bats
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RES-MOR-HEN  and the same PCR conditions as described
above was conducted to acquire fragments comparable to
previously isolated novel Henipa-like viruses from African fruit
bats . Bat species confirmation was achieved by sequencing and
analyzing the mitochondrial DNA as described .
The modified PCR protocol (PAR primers) resulted in a 10-
fold increase of sensitivity compared to the published protocol
which was applied as a two-step PCR (Figure 1). With this
optimization, three out of 120 pooled samples were PCR positive
for paramyxoviruses using PAR primers (Table 2). The identified
viruses were termed after the infected bat species: BatPV/
Myo.mys/E20/09 (Accession number JN086950), BatPV/Pip.-
pip/E95/09 (Accession number JN086951), and BatPV/Nyc.-
noc/E155/09 (Accession number JN086952). Fragments of
529 bp length were aligned with homologous fragments of the
partial polymerase gene of other members of the family
Paramyxoviridae from GenBank (Figure 2). Phylogenetic analysis
confirmed three distinct isolates within the subfamily Paramyx-
ovirinae. BatPV/Nyc.noc/E155/09 was in basal association to
other members of the genus Rubulavirus. For both BatPV/
Myo.mys/E20/09 and BatPV/Pip.pip/E95/09, the closest asso-
ciation was observed to J-virus and Beilong virus (unclassified
viruses) [28,29]. A longer sequence of 1,593 bp was generated for
BatPV/Pip.pip/E95/09 and used for an extended phylogenetic
analysis (Figure 3). The highest similarity was revealed for
BatPV/Myo.mys/E20/09 to J-Virus with 66.4%, for BatPV/
Pip.pip/E95/09 also to J-Virus with 64.1%, and for BatPV/
Nyc.noc/E155/09 to Rubulavirus with 62.1% (Table 3). Within
the subfamily Paramyxovirinae the extent of minimal nucleotide
homology for the partial polymerase gene between different
viruses in the same genus ranges from 64.1% (Rubulavirus) to
76.8% (Henipavirus), whereas the extent of nucleotide similarity
between viruses from different genera is between 40.3%
(Morbillivirus) and 58.1% (Henipavirus). Analysing the nucleo-
tide homology of the partial polymerase gene of the new
insectivorous bat paramyxoviruses, no definite correlation to one
of the other paramyxovirus genera could be obtained. The
comparison of sequences of BatPV/Myo.mys/E20/09 (Accession
number JN086954), obtained from the PCR assay with RES-
MOR-HEN primers (Table 2), confirmed the results of the
above-mentioned phylogenetic analysis (data not shown).
The first paramyxovirus termed BatPV/Myo.mys/E20/09 was
detected in pooled organs and was subsequently confirmed in the
kidney only of one adult male whiskered bat (Myotis mystacinus)
found in Bavaria. Histological examination of the internal organs
revealed multifocal mild interstitial nephritis with lymphoplasma-
cytic infiltrates and occasional neutrophiles. Lungs had mild non-
suppurative interstitial pneumonia and marked leucocytostasis in
most blood vessels. Additionally, there was distinct activation of
the lymphoreticular tissue of the spleen with moderate follicular
hyperplasia and sparse irregularly distributed small foci of
lymphocytes and plasma cell aggregations within the liver.
The second virus (BatPV/Pip.pip/E95/09) was detected in the
pooled organs of an adult female common pipistrelle bat
(Pipistrellus pipistrellus) also found in Bavaria. No specific infected
organ could subsequently be determined due to sample size
limitations. Histologically the animal had multifocal moderate
interstitial nephritis with segmental infiltrates of lymphocytes,
plasma cells, and occasional single neutrophiles (Figure 4). There
was mild generalized interstitial pneumonia and moderate
follicular hyperplasia of the spleen. There were mild intrasinusoi-
dal infiltrates of neutrophiles, lymphocytes, and plasma cells within
The third virus (BatPV/Nyc.noc/E155/09) was detected in
pooled organs and was subsequently confirmed in the lung of only
one adult female noctule bat (Nyctalus noctula) found in Berlin.
Histologically the bat revealed marked follicular hyperplasia of the
spleen without further inflammatory organ lesions. The lung was
severely congested, and oedematous fluid was present in the lung
Using immune sera against Beilong virus, J-virus, Menangle
virus, Tioman virus and Nipah virus in immuno-histochemistry,
no stained antigens were visualized in any of the paramyxovirus-
positive bats although all immune sera worked well against their
homologous virus in corresponding positive controls.
After screening all 120 bats of 15 species with three virus-
specific qPCR assays, an identical paramyxovirus to BatPV/
Myo.mys/E20/09 was detected in the spleen of one additional
Myotis mystacinus (E120/09).
During the past decade, bats have increasingly been recognized
as members of the animal group with the highest relative risk to
harbour novel emerging zoonotic pathogens . The emergence
of Hendra and Nipah virus provided the first evidence of
a zoonotic paramyxovirus originating from bats with a broad host
range including humans. Interestingly, despite the enormously
diverse chiropteran animal order, so far, with the exception of
rabies, only fruit bats have been implicated as a reservoir of
a number of new and emerging zoonotic viruses [3–10].
With this study, we were able to describe the detection and
characterization of the first three paramyxoviruses in insectivorous
bats. The genetic distance between these three novel paramyx-
oviruses and the closest related member known is higher than that
of members within other paramyxovirus genera, suggesting that all
three viruses might be considered as unassigned paramyxoviruses.
Thus the two viruses BatPV/Myo.mys/E20/09 and BatPV/
Figure 1. Improved detection sensitivity after Taguchi optimi-
zation of the Paramyxovirinae subfamily-specific PCR . Gel
electrophoresis of amplification products of the second round
seminested PCR using 10-fold serial dilutions (100to 1024) of a cDNA-
sample (kidney of sample E20/09). (A) PCR protocol adopted for two-
step PCR as previously published  using the pan-PAR-F1/PAR-R
primer pair (1st run) and the pan-PAR-F2/PAR-R primer pair (2nd run).
(B) Optimized protocol using the pan-PAR-F1/PAR-R primer pair (1st
run) and the pan-PAR-F2/PAR-R primer pair (2nd run).
Novel Paramyxoviruses in European Bats
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Figure 2. Phylogenetic analysis of the partial L-gene sequence obtained from PCR fragments after Pan-Paramyxovirinae-PCR with
PAR primers (529 bp) . The revealed gap-free alignment was used to generate a phylogenetic tree of the novel bat paramyxoviruses (red)
concordant with representatives from all known genera of paramyxoviruses with MrBayes. Posterior probability rates are given next to the tree nodes.
Novel Paramyxoviruses in European Bats
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Pip.pip/E95/09 might even be considered as members of a new
putative genus, as they contain an amino acid identity of 79.5% of
the partial L-gene, the highest conserved region of the para-
myxovirus genome. Further precise genetic analyses will have to
prove whether they ought to be integrated into the proposed new
genus Jeilongvirus , comprising J-virus and Beilong virus, with
amino acid identities between 69.9% and 74%, respectively,
although their viral antigens in PCR positive organs are not
immunologically cross-reactive. The third novel paramyxovirus,
BatPV/Nyc.noc/E155/09, has a basal association with the genus
Rubulavirus and could therefore become a member of this genus,
although nucleic acid identity was slightly lower than that between
already classified members within this genus and no cross-
reactivity of viral antigens with immune sera of closely related
rubulaviruses was obtained.
Besides virus detection, our study allowed a direct correlation of
virology and histo-pathology results. In previous studies in which
bats were examined for paramyxovirus infections, no overt clinical
disease was noted [5,30], despite occasional high prevalences of
antibodies against various paramyxoviruses (e.g. Hendra and
Nipah virus) and the detection of paramyxovirus RNA in different
bat organs [5,30–32]. For Hendra virus infections of pteropid bats,
the subclinical course has been confirmed by experimental
infection. Kidneys are the only site of pathological lesions after
Hendra virus infection of pteropid bats, with mild interstitial
perivascular infiltrates by mononuclear inflammatory cells, while
virus nucleic acids were also detected in lung, spleen, gastrointes-
tinal tract, and urine . In contrast, Nipah virus was only
detected in kidneys (male) and uteri (female) of pteropid bats after
experimental infection . In all Hendra virus and Nipah virus
experimental infection studies the amount of virus recovered
reached the limit of detection level, a similar situation encountered
in our study. In infected insectivorous bats, low band intensities of
PCR products of organs tested positive for paramyxovirus
infection indicated a low virus load. Likewise, the kidneys were
the only organ infected in the male whiskered bat (BatPV/
Myo.mys/E20/09) and presumably in the female common
pipistrelle. Interestingly, both animals had mononuclear inflam-
matory interstitial infiltrates similar to the reported experimental
Hendra virus infections. Unfortunately, attempts to prove the
evidence by specific immunohistochemistry with antibodies di-
rected against Beilong virus, J-virus, Menangle virus, Tioman virus
and Nipah virus were not successful. A number of reasons could
account for this result. Either the noted inflammatory changes
GenBank Accession numbers of novel paramyxoviruses PAR fragment: JN086950 (BatPV/Myo.mys/E20/09), JN086951 (BatPV/Pip.pip/E95/09),
JN086952 (BatPV/Nyc.noc/E155/09). RSV = respiratory syncytial virus.
Figure 3. Phylogenetic analysis of the partial L-gene sequence obtained from a longer PCR fragment after Pan-Paramyxovirinae-
PCR with novel primers (1,593 bp). The revealed gap-free alignment was used to generate a phylogenetic tree of the novel bat paramyxovirus
(red) concordant with closely related representatives from other paramyxoviruses with MrBayes. Posterior probability rates are given next to the tree
nodes. GenBank Accession number of the long fragment of BatPV/Pip.pip/E95/09: JN086951.
Novel Paramyxoviruses in European Bats
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were indeed unrelated to paramyxovirus infection or the
immunogenic epitopes of paramyxoviruses in European insectiv-
orous bats differ significantly from their australo-asian relatives,
hence prohibiting bonding between the reagents or, taking into
account that molecular investigations indicated a low virus load,
the number of infectious particles was too low to be picked up by
immunohistochemistry. Although unequivocal evidence of the
causative association between paramyxovirus infection and renal
inflammation remains open it is important to note, that nephritis is
a rare finding in insectivorous bats. Out of 500 examined deceased
bats only 3% had inflammatory changes within their kidneys,
while 20% of these respective cases were clearly associated to
bacterial disease . Considering this background information
together with findings from experimental paramyovirus infections
in fruit bats a possible link between the renal mononuclear
infiltrates and the detected novel paramyxovirus nucleic acids
seems feasible. With this, a possible transmission route via urine
like in Hendra virus infection could also be assumed for these
viruses. In contrast, the detection of BatPV/Nyc.noc/E155/09
limited to the lungs of the female noctule bat is suggestive of an
oronasal and/or salivary route of transmission. Our findings
confirm a promising approach for the ultra-sensitive detection of
paramyxoviruses by applying a modified PCR protocol as
a powerful tool, including non-invasive sampling (oral swabs,
urine, faeces). However, it should be emphasized that substantial
insights regarding the estimation of possible spill-over events can
only be achived by a combination of virology, histo-pathology, and
bat ecology investigations.
Emerging paramyxoviruses from fruit bats in spill-over hosts
have regularly been associated with ecosystem and land-use
changes resulting in an increased overlap of bats, domestic
animals, and human ecologies and thereby increased opportunities
for bat-borne zoonotic diseases to emerge . As demonstrated
in this study, paramyxoviruses basally related to Henipaviruses
also exist in geographic areas distant to the distribution range of
fruit bats, the suspected natural hosts for Henipaviruses, indicating
a possible virus–host co-evolution beyond this animal group. Since
infected bats were found in close proximity to heavily populated
human habitations as well as intensive agricultural use, a potential
risk for the emergence of zoonotic paramyxoviruses in Europe
should be further elucidated. Although the three novel paramyx-
oviruses detected in three distinct European bat species cannot be
readily assigned to any previously described paramyxovirus genus,
they are associated to two very distinct genera (Rubulavirus and
Jeilongvirus) , indicating a similarly broad genetic diversity
among paramyxoviruses in insectivorous bats compared to fruit
bats. Given the much larger diversity amongst insectivorous bats
with over 1,000 bat species in contrast to 186 fruit bat species ,
Table 2. Animal properties of paramyxovirus-infected adult insectivorous bats.
Positive PCR/sequencing (RES-
Organ poolOrgan Organ poolOrgan
BatPV/Myo.mys/E20/09 Myotis mystacinus MaleBavaria
BatPV/Pip.pip/E95/09Pipistrellus pipistrellus Female Bavarian.d.*n.d.*
BatPV/Nyc.noc/E155/09 Nyctalus noctulaFemaleBerlin Lung -–
*not determined due to sample volume limitations.
Table 3. Relatedness of the novel paramyxoviruses to other members of currently established genera of the family
Genus / Species (1)(2)(3) (4) (5) (6)(7)(8) (9)(10) (11)
(1) Pneumovirus n.d.
(2) Metapneumovirus 63.8n.d.
(4) Rubulavirus 33.8 34.2 48.8
(5) Respirovirus 33.432.9 44.246.0
(6) Morbillivirus34.433.5 42.745.952.5
(7) Henipavirus126.96.36.199.544.0 45.7
(8) J-virus34.735.342.5 48.054.058.848.6n.d.
Table lists percentage of nucleotide homology within the partial L-gene (529 base pairs) obtained by pan-Paramyxovirinae-specific PCR (PAR primers). Bold numbers
present the highest identities of the novel bat paramyxoviruses with the following established species: Human respiratory syncytial virus M74568 (Pneumovirus),
Human metapneumovirus AY297749 (Metapneumovirus), Newcastle disease virus AY505072 (Avulavirus), Mumps virus HQ416907 (Rubulavirus), Sendai virus EF679198
(Respirovirus), Measles virus AF266290 (Morbillivirus), Hendra virus HM044317 (Henipavirus), J-virus (AY900001), BatPV/Myo.mys/E20/09 (JN086950), BatPV/Pip.pip/E95/
09 (JN086951), BatPV/Nyc.noc/E155/09 (JN086952). n.d.: not determined.
Novel Paramyxoviruses in European Bats
PLoS ONE | www.plosone.org6 June 2012 | Volume 7 | Issue 6 | e38688
we predict a far higher diversity of paramyxoviruses in in-
sectivorous bats. Extensive phylogenetic studies of insectivorous
bat-born paramyxoviruses will provide further insight into the
suggested co-evolution of paramyxoviruses and bats . In
addition to Africa, Australia, South America, and Asia, the
detection of novel paramyxoviruses in European bats extends the
possible geographic overlap with other susceptible spill-over hosts.
Is there a possibility for paramyxoviruses of insectivorous bats to
emerge as zoonotic pathogens? Before any answer to this question
can be attempted, further research on paramyxovirus diversity and
distribution combined with the understanding of dynamics of
pathogen cycles within bat populations will be needed as well as
investigations into pathogenicity factors of these viruses, like
receptors for host invasion. Particularly as so far transmission of
bat related paramyxoviruses did not occur directly between bats
and humans but depended on a secondary host species like horses
The authors are grateful to Berliner Artenschutz Team–BAT-e.V., F.
Brandes, I. Frey-Mann, H. Geiger, J. Haensel, J. Harder, L. Ittermann, M.
Kistler, M. Kredler, S. Morgenroth, E. Mu ¨hlbach, K. Mu ¨ller, R. Pfeiffer,
W. Rietschel, S. Rosenau, R. Straub, G. Strauss, and W. and H. Zoels for
providing the bat carcasses, to D. Stern for Taguchi methodical support, to
A. Nitsche for designing PCR primers and probes, G. Crameri for helpful
scientific discussion, U. Erikli for copy-editing and to D. Krumnow for her
excellent technical assistance.
Conceived and designed the experiments: AK CK GW. Performed the
experiments: CK AB AE KM JAH. Analyzed the data: AK CK LW GW.
Contributed reagents/materials/analysis tools: AK CK AB AE JAH LW
KM GW. Wrote the paper: AK GW.
1. Lamb RA, Collins PL, Kolakowsky D, Melero JA, Nagai Y (2005) Family
Paramyxoviridae. In: Fauquet CM, Mayo J, Maniloff J, Desselberger U, Ball
LA, editors. Virus taxonomy: 8th report of the International Committee on
Taxonomy of Viruses. San Diego: Elsevier. 655–668.
2. Cleaveland S, Haydon DT, Taylor L (2007) Overview of Pathogen Emergence:
Which Pathogens Emerge, When and Why? In: Childs JE, Mackenzie JSRJA,
editors. Wildlife and Emerging Zoonotic Diseases: The Biology, Circumstances
and Consequences of Cross-Species Transmission. Berlin: Springer. 85–111.
3. Chua KB, Koh CL, Hooi PS, Wee KF, Khong JH, et al. (2002) Isolation of
Nipah virus from Malaysian Island flying-foxes. MicrobesInfect 4: 145–151.
4. Drexler JF, Corman VM, Gloza-Rausch F, Seebens A, Annan A, et al. (2009)
Henipavirus RNA in African bats. PLoSOne 4: e6367.
5. Halpin K, Young PL, Field HE, Mackenzie JS (2000) Isolation of Hendra virus
from pteropid bats: a natural reservoir of Hendra virus. JGenVirol 81: 1927–
6. Lau SK, Woo PC, Wong BH, Wong AY, Tsoi HW, et al. (2010) Identification
and complete genome analysis of three novel paramyxoviruses, Tuhoko virus 1,
2 and 3, in fruit bats from China. Virology 404: 106–116.
7. Leroy EM, Kumulungui B, Pourrut X, Rouquet P, Hassanin A, et al. (2005)
Fruit bats as reservoirs of Ebola virus. Nature 438: 575–576.
8. Li W, Shi Z, Yu M, Ren W, Smith C, et al. (2005) Bats are natural reservoirs of
SARS-like coronaviruses. Science 310: 676–679.
9. Towner JS, Amman BR, Sealy TK, Carroll SA, Comer JA, et al. (2009) Isolation
of genetically diverse Marburg viruses from Egyptian fruit bats. PLoSPathog 5:
10. Wong S, Lau S, Woo P, Yuen KY (2007) Bats as a continuing source of
emerging infections in humans. RevMedVirol 17: 67–91.
11. Wibbelt G, Speck S, Field H (2009) Methods for Assessing Diseases in bats. In:
Kunz TH, Parsons S, editors. Ecological and Behavioral Methods for the Study
of Bats: The Johns Hopkins University Press. 775–794.
12. Halpin K, Young PL, Field H, Mackenzie JS (1999) Newly discovered viruses of
flying foxes. VetMicrobiol 68: 83–87.
13. Chua KB, Wang LF, Lam SK, Crameri G, Yu M, et al. (2001) Tioman virus,
a novel paramyxovirus isolated from fruit bats in Malaysia. Virology 283: 215–
Figure 4. Kidney of a common pipistrelle bat (E95/09) with interstitial nephritis comprised by segmental infiltrates of lymphocytes,
plasma cells, and occasional single neutrophilic granulocytes.
Novel Paramyxoviruses in European Bats
PLoS ONE | www.plosone.org7 June 2012 | Volume 7 | Issue 6 | e38688
14. Pavri KM, Singh KR, Hollinger FB (1971) Isolation of a new parainfluenza virus
from a frugivorous bat, Rousettus leschenaulti, collected at Poona, India.
AmJTropMedHyg 20: 125–130.
15. Henderson GW, Laird C, Dermott E, Rima BK (1995) Characterization of
Mapuera virus: structure, proteins and nucleotide sequence of the gene encoding
the nucleocapsid protein. JGenVirol 76 ( Pt 10): 2509–2518.
16. Yob JM, Field H, Rashdi AM, Morrissy C, van der Heide B, et al. (2001) Nipah
virus infection in bats (order Chiroptera) in peninsular Malaysia. Emerg Infect
Dis 7: 439–441.
17. Young PL, Halpin K, Selleck PW, Field H, Gravel JL, et al. (1996) Serologic
evidence for the presence in Pteropus bats of a paramyxovirus related to equine
morbillivirus. EmergInfectDis 2: 239–240.
18. Muhldorfer K, Speck S, Kurth A, Lesnik R, Freuling C, et al. (2011) Diseases
and causes of death in European bats: dynamics in disease susceptibility and
infection rates. PLoS One 6: e29773.
19. Muhldorfer K, Speck S, Wibbelt G (2011) Diseases in free-ranging bats from
Germany. BMC Vet Res 7: 61.
20. Bowden TR, Bingham J, Harper JA, Boyle DB (2012) Menangle virus, a pteropid
bat paramyxovirus infectious for pigs and humans, exhibits tropism for
secondary lymphoid organs and intestinal epithelium in weaned pigs. J Gen
Virol 93: 1007–1016.
21. Tong S, Chern SW, Li Y, Pallansch MA, Anderson LJ (2008) Sensitive and
broadly reactive reverse transcription-PCR assays to detect novel paramyx-
oviruses. JClinMicrobiol 46: 2652–2658.
22. McCarthy AJ, Goodman SJ (2010) Reassessing conflicting evolutionary histories
of the Paramyxoviridae and the origins of respiroviruses with Bayesian multigene
phylogenies. InfectGenetEvol 10: 97–107.
23. Cobb BD, Clarkson JM (1994) A simple procedure for optimising the
polymerase chain reaction (PCR) using modified Taguchi methods. Nucleic
Acids Res 22: 3801–3805.
24. Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of
phylogenetic trees. Bioinformatics 17: 754–755.
25. Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference
under mixed models. Bioinformatics 19: 1572–1574.
26. Posada D (2008) jModelTest: phylogenetic model averaging. Mol Biol Evol 25:
27. Sonntag M, Muhldorfer K, Speck S, Wibbelt G, Kurth A (2009) New
adenovirus in bats, Germany. Emerg Infect Dis 15: 2052–2055.
28. Jack PJ, Boyle DB, Eaton BT, Wang LF (2005) The complete genome sequence
of J virus reveals a unique genome structure in the family Paramyxoviridae.
JVirol 79: 10690–10700.
29. Li Z, Yu M, Zhang H, Magoffin DE, Jack PJ, et al. (2006) Beilong virus, a novel
paramyxovirus with the largest genome of non-segmented negative-stranded
RNA viruses. Virology 346: 219–228.
30. Chua KB, Bellini WJ, Rota PA, Harcourt BH, Tamin A, et al. (2000) Nipah
virus: a recently emergent deadly paramyxovirus. Science 288: 1432–1435.
31. Eaton BT, Broder CC, Middleton D, Wang LF (2006) Hendra and Nipah
viruses: different and dangerous. NatRevMicrobiol 4: 23–35.
32. Hayman DT, Suu-Ire R, Breed AC, McEachern JA, Wang L, et al. (2008)
Evidence of henipavirus infection in West African fruit bats. PLoSOne 3: e2739.
33. Williamson MM, Hooper PT, Selleck PW, Westbury HA, Slocombe RF (2000)
Experimental hendra virus infectionin pregnant guinea-pigs and fruit Bats
(Pteropus poliocephalus). JComp Pathol 122: 201–207.
34. Middleton DJ, Morrissy CJ, van der Heide BM, Russell GM, Braun MA, et al.
(2007) Experimental Nipah virus infection in pteropid bats (Pteropus
poliocephalus). JComp Pathol 136: 266–272.
35. Halpin K, Hyatt AD, Plowright RK, Epstein JH, Daszak P, et al. (2007)
Emerging viruses: coming in on a wrinkled wing and a prayer. ClinInfectDis 44:
36. Almeida FC, Giannini NP, DeSalle R, Simmons NB (2009) The phylogenetic
relationships of cynopterine fruit bats (Chiroptera: Pteropodidae: Cynopterinae).
MolPhylogenetEvol 53: 772–783.
Novel Paramyxoviruses in European Bats
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