Novel adenoviruses and herpesviruses detected in bats

Abstract

Samples from native Hungarian or captive bats were tested by PCR for the presence of adenoviruses and herpesviruses. Two novel adenoviruses from a common noctule (Nyctalus noctula) and a greater horseshoe (Rhinolophus ferrum-equinum) bat were detected. In captive Egyptian fruit bats (Rousettus aegyptiacus), DNA from two novel herpesviruses was demonstrated. Phylogenetic analysis facilitated provisional taxonomic placement of the newly detected viruses. Such analysis and the existence of unique, shared early proteins (E3 and E4) suggest that canine adenoviruses may have originated in vespertilinoid bats.

Full text

Short Communication
Novel adenoviruses and herpesviruses detected in bats
Máté Jánoska
a,1
, Márton Vidovszky
a
, Viktor Molnár
b
, Mátyás Liptovszky
b
, Balázs Harrach
a
, Mária Benk}
o
a,
⇑
a
Veterinary Medical Research Institute, Hungarian Academy of Sciences, 1581 Budapest, P.O. Box 18, Hungary
b
Budapest Zoological and Botanical Garden, 1371 Budapest, P.O. Box 469, Hungary
article info
Article history:
Accepted 26 June 2010
Available online xxxx
Keywords:
Adenoviruses
Herpesviruses
Bats
Chiroptera
Nested PCR
abstract
Samples from native Hungarian or captive bats were tested by PCR for the presence of adenoviruses and
herpesviruses. Two novel adenoviruses from a common noctule (Nyctalus noctula) and a greater horse-
shoe (Rhinolophus ferrum-equinum) bat were detected. In captive Egyptian fruit bats (Rousettus aegyptia-
cus), DNA from two novel herpesviruses was demonstrated. Phylogenetic analysis facilitated provisional
taxonomic placement of the newly detected viruses. Such analysis and the existence of unique, shared
early proteins (E3 and E4) suggest that canine adenoviruses may have originated in vespertilinoid bats.
Ó2010 Elsevier Ltd. All rights reserved.
Relative to the very many bat species and our increasing aware-
ness of their role as virus reservoirs (Calisher et al., 2006) or as ‘do-
nors’ of novel adapting viruses, the number of adenovirus (AdV)
and herpesvirus (HV) types identified in bats is small. The isolation
of the first recognised bat AdV was published recently by Maeda
et al. (2008) and over the past 3 years a dozen or so HV types have
been identified in bats of different megachiropteran and microchir-
opteran species (Wibbelt et al., 2007; Molnár et al., 2008; Razafin-
dratsimandresy et al., 2009). However, this probably represents
only the ‘tip of the iceberg’ in terms of the actual number of AdVs
and HVs that infect bats. In this study we carried out PCR-based
assessment of organ and faecal samples collected from bats and
identified two novel AdVs and two novel HVs, respectively.
A total of 57 samples originated from native species of bat,
including three common noctules (Nyctalus noctula), two serotine
bats (Eptesicus serotinus) and one parti-coloured bat (Vespertilio
murinus). The animals had been found moribund or dead in the
vicinity of Budapest during the previous 3 years and were submit-
ted to Budapest Zoo which provides a nature conservation service
for such threatened species. The moribund animals were euthan-
ased by a veterinarian with expertise in handling bats and were
processed lege artis. In addition, 43 Egyptian fruit bats (Rousettus
aegyptiacus) and five Lyle’s flying foxes (Pteropus lylei) captive-bred
within the zoo, were examined. These bats had died from a range
of different causes, but a viral aetiology was not suspected and
pathognomonic lesions had not been evident on post-mortem
examination. DNA extraction was carried out on an organ pool of
liver, lungs and small intestines. Three faecal samples from greater
horseshoe bats (Rhinolophus ferrum-equinum) randomly collected
at the Aggtelek National Park in North-eastern Hungary in Novem-
ber 2009 were also examined.
Nested PCR was used in the viral detection process with degen-
erate, consensus primers targeting the DNA-dependent DNA poly-
merase gene of AdVs (Wellehan et al., 2004) or HVs (VanDevanter
et al., 1996). The PCR products of 318 and 321 bp from the AdVs
and of 219 and 228 bp from the HVs, respectively, were purified
and sequenced directly using the corresponding inner PCR primers.
Of the 57 samples, three were found to contain adenoviral DNA.
In two of the faecal samples from the greater horseshoe bats, iden-
tical sequences were demonstrated implying the presence of a
hitherto unknown AdV (GenBank Accession number GU289918).
A further novel AdV sequence was detected in the pooled organs
of a common noctule (Accession number GU198877). Phylogenetic
tree reconstruction indicated that these novel viruses were masta-
denoviruses (Fig. 1).
Two novel HV sequences were detected in two samples from
Egyptian fruit bats (Accession numbers. FJ797654 and FJ597655).
On phylogenetic analysis, these HVs were found to belong to the
subfamilies Betaherpesvirinae and Gammaherpesvirinae, respec-
tively (Fig. 2).
The first AdV (designated FBV1) identified in a bat was isolated
during attempts to establish a specific cell line from the megachir-
opteran Ryukyu flying fox (Pteropus dasymallus yaeyamae)(Maeda
et al., 2008). Isolation of the first AdV (designated PPV1) from a
microchiropteran bat, the common pipistrelle (Pipistrellus pipistrel-
lus) was reported in Germany (Sonntag et al., 2009). More recently,
the isolation and genomic analysis of an additional microchiropter-
an AdV (BtAdV-TJM) originating from a Rickett’s big-footed bat
1090-0233/$ - see front matter Ó2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tvjl.2010.06.020
⇑
Corresponding author. Tel.: +36 1 4674081; fax: +36 1 4674076.
E-mail address: benko@vmri.hu (M. Benk}
o).
1
This article is dedicated to the memory of the late Máté Jánoska.
The Veterinary Journal xxx (2010) xxx–xxx
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Please cite this article in press as: Jánoska, M., et al. Novel adenoviruses and herpesviruses detected in bats. The Veterinary Journal (2010), doi:10.1016/
j.tvjl.2010.06.020

(Myotis ricketti) has been reported in China (Li et al., 2010). These
authors, using the same PCR (Wellehan et al., 2004) found 19 pos-
itives in >300 samples from bats. These positive samples repre-
sented six putative novel AdVs (Fig. 1) and were from Rickett’s
big-footed bats (Myotis ricketti), lesser Asiatic yellow house bats
(Scotophilus kuhlii), Horsfield’s bats (Myotis horsfieldii), and a great
roundleaf bat (Hipposideros armiger). It is noteworthy that 102 fae-
cal samples from Rhinolophus species in China were found to be
negative, whereas two out of three similar samples from bats of
a different species of the Rhinolophus genus found in Hungary
were positive.
It is possible to make direct comparisons between the putative
AdVs found in the bats in Hungary with those detected in Japan,
Germany, and China (Fig. 1). In general, the divergence among
the different bat AdVs was very large, considerably exceeding that
observed among the AdVs of primates. The order Chiroptera is di-
vided into suborders Megachiroptera and Microchiroptera. The
Ryukyu flying fox is currently the only megachiropteran bat known
to be infected with an AdV (strain FBV1), and the AdVs detected in
microchiropterans are quite distinct from the strain FBV1. With
one exception, the AdVs found in microchiropteran bats from the
family Vespertilinoidae form a monophyletic clade (Fig. 1). This
lineage is close to branches of the AdVs found in the two bats of
the Rhinolophidae family, Hipposideros armiger and Rhinolophus
ferrum-equinum.
Phylogenetic tree topology suggests there has been a long co-
evolution of most AdVs found in bats with their hosts. A striking
exception to this is the AdV detected in a sample (No. 1252) of
Horsfield’s bats (Li et al., 2010), which occupies a branch distant
from the cluster of AdVs found in the vespertilinoid bats. The fact
that the latter clade also includes the two canine AdV types (CAdV-
1 and 2), leads us to speculate on the origin of CAdVs that have an
unusually broad host range. The early regions of CAdVs show strik-
ing similarity with those of BtAdV-TJM (Li et al., 2010), including
0.1
white sturgeon
blue-tongued skink
tokay gecko
duck-1
bovine-4
ovine-7
Meyer parrot
fowl-1
fowl-9
fowl-5
turkey-3
great tit
raptor-1
Travancore tortoise
frog-1
murine-3
murine-1
M. horsfieldii 1252
bat FBV1 Japan
porcine-3
bovine-3
tree shrew
greater horseshoe bat
Hipposideros armi
g
er
M. horsfieldii 1213
common noctule
M. ricketti 1069
M. ricketti 1282, 1285
8 Scotophilus kuhlii
canine-2
canine-1
4 M. rickettii/2 M. sp.
bat PPV1 German
y
porcine-5
bovine-2
bovine-1
simian-1
human-40
simian-3
simian-25
human-12
human-9
human-2
Atadenovirus
Siadenovirus
Aviadenovirus
Mastadenovirus
92
60
100
97
98
79
67
100
99
98
62
24
25
66
100
82
80
93
68
37
84
63
31
65
28
44
85
Bat hosts from
family
Vespertilinoidae
Ichtadenovirus
Fig. 1. Phylogenetic tree reconstruction of adenoviruses (AdVs) based on distance matrix analysis of a 91 amino acid-long alignment from partial sequences of the viral DNA-
dependent DNA polymerase (ProtDist categories model, Fitch global rearrangements). AdVs associated with bats are printed in bold. Unrooted tree: the white sturgeon AdV
was selected as an ‘outgroup’. Bootstrap values are shown at branch nodes when they confirm the calculated topology. Latin names designate AdVs detected in bats in China.
The branch marked ‘4 M. rickettii/2 M. Sp.’ includes the sequence from the BtAdV-TJM strain (Li et al., 2010). The virus demonstrated from a megachiropteran fruit bat
(Pteropus dasymallus yaeyamae) is highlighted by a grey background. The virus cluster that contains the canine AdVs and the AdVs from bats of the family Vespertilinoidae, is
circled with a discontinuous line. M. ricketti,Myotis ricketti; bat FBV1, fruit bat AdV (Maeda et al., 2008); bat PPV1, AdV from Pipistrellus pipistrellus (Sonntag et al., 2009).
Numbers in front of the Latin name of the host indicate the number times identical AdV sequences were detected.
2M. Jánoska et al. / The Veterinary Journal xxx (2010) xxx–xxx
Please cite this article in press as: Jánoska, M., et al. Novel adenoviruses and herpesviruses detected in bats. The Veterinary Journal (2010), doi:10.1016/
j.tvjl.2010.06.020

one ORF from the E3, and four ORFs from the E4 regions, respec-
tively. Homologues of these ORFs have not been identified in other
mastadenoviruses to date, indicating a close common ancestry be-
tween canine and vespertilinoid bat AdVs.
In the present survey, a betaherpesvirus (BHV) and a gamma-
herpesvirus (GHV) were demonstrated in Egyptian fruit bats. Previ-
ously, >16 HV types have been found in bats of different species. As
seen on the phylogenetic tree reconstruction in Fig. 2, several HVs
found in bats have been assigned to each of the three sub-families
of Herpesviridae. It has been proposed that five HVs of the sub-fam-
ily Alphaherpesvirinae be classified in the genus Simplexvirus (Raza-
findratsimandresy et al., 2009). The two bat HVs belonging to the
subfamily Betaherpesvirinae are bat BHV-1 from a microchiropter-
an host, the Natterer’s bat (Myotis nattereri)(Wibbelt et al., 2007),
and a BHV (Accession number AB125970) detected in a lesser
dawn or cave nectar bat (Eonycteris spelaea), which, despite its rel-
atively small body size, is a species of the sub-order Megachirop-
tera. In accordance with host taxonomic relationships, the BHV
Percavirus
Rhadinovirus
Lymphocryptovirus
Cytomegalovirus
Muromegalovirus
Roseolovirus
Simplexvirus
Varicellovirus
Mardivirus
Iltovirus
Iguanid-2
Passerid-1
Gallid-1
Meleagrid-1
Gallid-3
Gallid-2
Human-3
Cercopithecine-9
Bat simplexvirus 1
Lonchophylla thomasi 1
Pteropus lylei 1
Eidolon helvum 1
Eidolon dupraenum 1
Cercopithecine-2
Human-2
Human-1
Human-6
Human-7
Murid-2
Murid-1
Bat BHV-1
Egyptian fruit bat
Eonycteris spelaea
Cercopithecine-5
Panine-2
Human-5
Bat GHV-3
Ovine-2
Alcelaphine-1
Alcelaphine-2
Hipposideros diadema
Callithrix jacchus 1
Callithrix penicillata 1
Human-4
Papiine-1
Macacine-4
Bovine-4
Egyptian fruit bat
Saimirine-2
Ateline-2
Rhinolophus ferrum-equinum
Mustelid-1
Equid-2
Equid-5
Bat GHV-2
Bat GHV-7
Bat GHV-1
Bat GHV-5
Bat GHV-6
Bat GHV-4
0.5
α
β
Macavirus
37
51
65
41
62
72
59
73
100
97
98
89
69
89
80
36
99
94
28
19
29
85
100
55
98
32
95
55
42
14
94
66
33
43
57
61
70
100
100
95
47
14 81
99
30
γ
Fig. 2. Distance matrix analysis of a 53 amino acid-long alignment from partial sequences of herpesvirus DNA-dependent DNA polymerase. The three sub-families are
labelled
a
,band
c
and the sub-family Alphaherpesvirinae was used as an ‘outgroup’. Bootstrap values are shown when they support the tree topology. Bat viruses are marked
according to the designation of their original description (see text). The herpesviruses detected in megachiropteran fruit bats are highlighted with a grey background, and
those identified in the present study are labelled using the name of the host species.
M. Jánoska et al. / The Veterinary Journal xxx (2010) xxx–xxx 3
Please cite this article in press as: Jánoska, M., et al. Novel adenoviruses and herpesviruses detected in bats. The Veterinary Journal (2010), doi:10.1016/
j.tvjl.2010.06.020

detected in the Egyptian fruit bat in the present study is more clo-
sely related to the HV of the lesser dawn bat than to that of the
Natterer’s bat.
The picture is more complex in the sub-family Gammaherpesvir-
inae. Seven gammaherpesviruses (GHV) infecting bats of the family
Vespertilionidae (Microchiroptera), have been reported (Wibbelt
et al., 2007). On the phylogenetic tree, six of these viruses (bat
GHV-1, -2, -4, -5, -6, and -7) form a separate clade, whereas the
bat GHV-3 appears closer to a branch of the genus Macavirus.In
the Philippines, Watanabe et al. (2009) detected the sequence of
an additional GHV that clusters close to the genus Lymphocryptovi-
rus in a diadem roundleaf bat (Hipposideros diadema). In primary
cell cultures prepared from a greater horseshoe bat (Rhinolophus
ferrum-equinum), Maeda et al. (2008) isolated a further GHV
(Accession number AB298558), which occupies the branch closest
to that of the genus Percavirus (Fig. 2).
The GHV detected in the second Egyptian fruit bat in the pres-
ent study, is the first gammaherpesvirus found in a megachiropter-
an host and it clustered with the genus Rhadinovirus.
In conclusion, our results corroborate that bats are hosts to a di-
verse range of viruses, including AdVs and HVs. It must be remem-
bered that the amplified DNA polymerase gene fragment we used
in the phylogenetic analyses is short, allowing for preliminary virus
classification only. PCR amplification and sequencing of additional
genome regions will be required to confirm taxonomic
categorisation.
Conflict of interest statement
None of the authors of this paper has a financial or personal
relationship with other people or organisations that could inappro-
priately influence or bias the content of the paper.
Acknowledgements
This work was supported by Grants OTKA K61317 and NKTH-
OTKA K67781 provided by the Hungarian Scientific Research Fund.
Máté Jánoska, a promising young scientist, passed away tragically
during the submission process.
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Please cite this article in press as: Jánoska, M., et al. Novel adenoviruses and herpesviruses detected in bats. The Veterinary Journal (2010), doi:10.1016/
j.tvjl.2010.06.020