JOURNAL OF VIROLOGY, July 2010, p. 6955–6965
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 14
Bat Guano Virome: Predominance of Dietary Viruses from Insects
and Plants plus Novel Mammalian Viruses?
Linlin Li,1,2Joseph G. Victoria,1,2Chunlin Wang,3Morris Jones,4Gary M. Fellers,5
Thomas H. Kunz,6and Eric Delwart1,2*
Blood Systems Research Institute, San Francisco, California1; Department of Laboratory Medicine, University of California,
San Francisco, California2; Stanford Genome Technology Center, Stanford, California3; Clinical Investigation Facility,
David Grant USAF Medical Center, Travis Air Force Base, California4; U.S. Geological Survey,
Western Ecological Research Center, Point Reyes, California5; and Center for Ecology and
Conservation Biology, Department of Biology, Boston University, Boston, Massachusetts6
Received 5 March 2010/Accepted 30 April 2010
Bats are hosts to a variety of viruses capable of zoonotic transmissions. Because of increased contact
between bats, humans, and other animal species, the possibility exists for further cross-species transmis-
sions and ensuing disease outbreaks. We describe here full and partial viral genomes identified using
metagenomics in the guano of bats from California and Texas. A total of 34% and 58% of 390,000 sequence
reads from bat guano in California and Texas, respectively, were related to eukaryotic viruses, and the
largest proportion of those infect insects, reflecting the diet of these insectivorous bats, including members
of the viral families Dicistroviridae, Iflaviridae, Tetraviridae, and Nodaviridae and the subfamily Densoviri-
nae. The second largest proportion of virus-related sequences infects plants and fungi, likely reflecting the
diet of ingested insects, including members of the viral families Luteoviridae, Secoviridae, Tymoviridae, and
Partitiviridae and the genus Sobemovirus. Bat guano viruses related to those infecting mammals comprised
the third largest group, including members of the viral families Parvoviridae, Circoviridae, Picornaviridae,
Adenoviridae, Poxviridae, Astroviridae, and Coronaviridae. No close relative of known human viral pathogens
was identified in these bat populations. Phylogenetic analysis was used to clarify the relationship to known
viral taxa of novel sequences detected in bat guano samples, showing that some guano viral sequences fall
outside existing taxonomic groups. This initial characterization of the bat guano virome, the first meta-
genomic analysis of viruses in wild mammals using second-generation sequencing, therefore showed the
presence of previously unidentified viral species, genera, and possibly families. Viral metagenomics is a
useful tool for genetically characterizing viruses present in animals with the known capability of direct or
indirect viral zoonosis to humans.
Bats belong to one of the most diverse, abundant, and widely
distributed group of mammals. More than 1,100 bat species
belong to the order of Chiroptera, representing approximately
20% of all mammalian species (54). Most bat species feed on
insects and other arthropods, while others feed on fruit nectar,
bird or mammal blood, and small vertebrates such as fish,
frogs, mice, and birds (30). Of the 47 species of bats reported
in the United States, most of them are insectivorous (http:
Bats are considered the natural reservoir of a large variety of
zoonotic viruses causing serious human diseases such as lyssa-
viruses, henipaviruses, severe acute respiratory syndrome coro-
navirus, and Ebola virus (6, 38, 46, 59, 63, 65). Characteristics
of bats, including their genetic diversity, broad geological dis-
tribution, gregarious habits, high population density, migratory
habits, and long life span (30, 58), likely endow them with the
ability to host diverse viruses, some of which are also able to
infect humans and other mammals (41, 63).
More than 80 virus species have been isolated or detected in
bats using nucleic acid-based methods (6, 38, 59, 65). Viruses
that have been recently discovered in bats include astroviruses,
adeno-associated viruses (AAVs), adenoviruses, herpesviruses,
and polyomavirus (8, 9, 13, 31, 32, 35, 37, 39, 40, 42, 61, 62, 68).
For example, it was recently reported that a newly identified
adenovirus isolated from bat guano was capable of infecting
various vertebrate cell lines, including those of humans, mon-
keys, dogs, and pigs (35). With increasing human populations
in previously wild areas, contact of bats with humans and with
wild and domestic animals has increased, providing greater
opportunities for cross-species transmissions of potentially
pathogenic bat viruses. To better understand the range of
viruses carried by bats, we undertook an initial character-
ization of the guano viromes of several common bat species
in the United States.
The development of massively parallel sequencing technol-
ogy makes is possible to reveal uncultured viral assemblages
within biological or environmental samples (11, 28). To date,
this approach has been used to characterize viruses in equine
feces (7), human blood (5), tissue (14), human feces (3, 4, 15,
45, 60, 67), and human respiratory secretions (64), which in
turn has facilitated the discovery of many novel viruses (18, 20,
25, 33, 47, 50). In the present study, we analyzed the viruses
present in guano from several bat species in California and
Texas, using sequence-independent PCR amplification, pyro-
sequencing, and sequence similarity searches.
* Corresponding author. Mailing address: Blood Systems Research
Institute. 270 Masonic Ave., San Francisco, CA 94118. Phone: (415)
923-5763 Fax: (415) 567-5899. E-mail: firstname.lastname@example.org.
?Published ahead of print on 12 May 2010.
MATERIALS AND METHODS
Collection of bat guano. All of the bat species sampled are insectivorous.
Plastic sheets were laid down on flat surfaces beneath bat roosts. Freshly pro-
duced bat guano was then collected 1 day later and stored at ?80°C. Samples
were collected from a bat roost near San Saba, TX, on two occasions that were
3 days apart during the summer of 2008. The roost (TM) was occupied mostly by
Tadarida brasiliensis (Brazilian free-tailed bat). Three other species present in
smaller numbers, Myotis velifer (Cave myotis), Nycticeus humeralis (evening bat),
and Perimyotis subflavus (tricolored bat), are also known to share the roost and
may have also been sampled.
Guano samples from northern California were collected from five different
roosts at Point Reyes National Seashore. One roost (GF-4) was occupied by
Antrozous pallidus (pallid bat), and the other four roosts (GF-3, -5, -6, and -7)
were occupied by Myotis spp. and/or Tadarida brasiliensis (Table 1).
Sample preparation and viral nucleic acid extraction. Bat guano was pro-
cessed as previously described (60). Briefly, groups of 12 bat guano pellets from
the same roosts were resuspended by vigorous vortexing in Hank’s buffered
saline solution (Gibco BRL) and cleared of debris by low-speed centrifugation
(5 min at 11,000 ? g). A total of 500 ?l of guano supernatant was filtered through
a 0.45-?m filter (Millipore) to remove bacterium-sized particles. The viral par-
ticles containing filtrate were digested with a mixture of DNases and RNase to
remove unprotected nucleic acids (i.e., those not in viral capsids) (1). Viral
nucleic acids were then extracted using the QIAamp viral RNA minikit (Qiagen).
DNA and RNA library construction and pyrosequencing. Viral nucleic acid
libraries were constructed by random PCR amplification as previously described
(60). Both a RNA virus-only and DNA plus RNA virus sequence-independent
amplifications were performed and then pooled prior to sequencing. For RNA
virus-only amplification, an aliquot of the extracted viral nucleic acid collected
from each pool of 12 guano pellets was treated with DNase (Ambion) to remove
viral DNA. A total of 100 pmol of primer, consisting of an arbitrarily designed
20-base oligonucleotide followed by a randomized octamer sequence at the 3?
end, was then used in a reverse transcription (RT) reaction (Moloney murine
leukemia virus reverse transcriptase; Promega). For the RNA plus DNA virus
amplification, the DNase step prior to RT was excluded. A single round of DNA
synthesis was then performed using Klenow fragment polymerase (New England
Biolabs), followed by PCR amplification of double-stranded DNA using a primer
consisting of only the 20-base fixed portion of the random primer.
A total of 37 distinct random primers (containing different 20-base fixed
sequences) were applied to guano collected from the 6 bat roosts (Table 1). The
number of primers assigned per roost was based on the number of guano pellets
collected. Viral nucleic acids were therefore amplified from 37 pools of 12 guano
pellets per pool, with each pellet presumed to be from a different animal. Guano
obtained from up to 96 bats in the Texas roost (8 primers) and up to 348 bats in
the 5 California roosts (29 primers) was analyzed (Table 1). To further improve
viral nucleic acid sampling within each pool, the random PCR amplifications
were performed in duplicate, starting with the Klenow-treated products, result-
ing in four PCRs per original pool (2 viral RNA-only inputs and 2 viral RNA plus
DNA inputs). The DNA obtained from these four PCRs was mixed and purified,
and the DNA concentration was measured. Equal amounts of DNA from each
of the 37 different pools were then mixed together and run on a 2% agarose gel,
and DNA fragments from the 500- to 1,000-bp region were excised and purified.
The DNA was then sequenced on a single pyrosequencing gasket using GS FLX
Titanium reagents (Roche).
A subset of random primer sequences used was previously published (60). The
other random primers were designed by generating random sequences using
Primo (http://www.changbioscience.com/primo/primor.html) that were then an-
alyzed by BLASTn to remove those primers likely to bind to human and bacterial
Bioinformatics. The pyrosequencing reads were grouped in 37 bins, according
to their unique sequence tags (the 20 fixed bases of the random PCR primer).
The fixed primer sequences plus eight additional downstream nucleotides (en-
coded by the 3? NNNNNNNN part of the random primers), were then trimmed
from each read. Trimmed reads within each sequence bin were then assembled
by Sequencher software (Gene Codes), with a criterion of 95% identity or
greater over at least 35 bp. Contigs were therefore assembled using sequences
from at most 12 animals. When overlapping sequences in contigs contained
mutations (due to pyrosequencing error or because multiple viral variants from
different animals in the same pool were sequenced), the consensus sequence was
used. The assembled sequence contigs and singlets greater than 100 bp were then
compared to the GenBank nonredundant nucleotide and protein databases using
BLASTn and BLASTx, respectively. Using BLAST searches, sequences were
classified as likely originating from a eukaryotic virus, bacteria, phage, or
eukaryote or deemed unclassifiable based on the taxonomic origin of the best-hit
sequence. An E value of 0.001 was used as the cutoff value for significant hits.
Phylogenetic analysis. Reference viral sequences from different viral families
were obtained from GenBank. Amino acid sequence alignments were generated
using ClustalW and implemented in MEGA 4.1 with the default settings (29).
Aligned sequences were trimmed to match the genomic regions of the viral
sequences obtained in our study and phylogenetic trees generated by MEGA4,
using neighbor-joining with amino acid p distances and 1,000 bootstrap repli-
cates. The GenBank accession numbers of the viral sequences used in the
phylogenetic analyses are shown in the trees.
Nucleotide sequence accession numbers. Trimmed and binned sequence reads
and contigs of metagenomes from bat guano in California and Texas have been
deposited in the GenBank sequence reads archive under accession number
SRA012669. Sequences from the genomes described in more detail can be found
under GenBank accession numbers HM228873 to HM228895 and HM234168 to
Sequence data overview. Approximately 390,000 raw se-
quences (average length, 296 bases) were generated from viral
particle-enriched nucleic acids from bat guano. Sequence con-
tigs were then formed and, together with singlets longer
than 100 bases, were classified based on best BLAST scores
(E value ? 0.001) to taxonomically assigned sequences in the
GenBank nonredundant database. Summaries of the classifi-
cations of viral nucleic acid in bat guano from California and
Texas are shown in Fig. 1A. Approximately 51% and 39% of all
the sequence reads from California and Texas, respectively,
had no significant similarity to any sequences in GenBank
(E value ? 0.001), similar to the percentages of unclassified
sequences in a previous metagenomic study of human stool
(60). The most abundant matches of viral sequences from bat
guano in California and Texas were with eukaryotic viruses,
with 34% and 58% of total reads, respectively. Sequences from
both California and Texas bat guano yielded approximately 1%
TABLE 1. Summary of bat guano sample information
No. of roostsMajor bat species
No. of random primers used at roost:
TMGF-3 GF-4 GF-5 GF-6 GF-7
GF-3 to -7)
Myotis spp. or/and Tadarida
brasiliensis (roosts GF-3,
-5, -6, and -7); Antrozous
pallidus (roost GF4)
3 12446384 DNA and
TM San Saba,
1Tadarida brasiliensis4 at both
6956LI ET AL.J. VIROL.
FIG. 1. Sequence classification for California and Texas bat guano-derived sequences based on BLASTx (E value ? 0.001). (A) Percentages
of sequences with similarity to those of eukaryotes, bacteria, phages, and eukaryotic virus in GenBank and to unclassifiable sequences. (B) Per-
centages of most abundant eukaryotic viral matches classified by viral families. Plant viruses are highlighted in green, insect viruses are highlighted
in red, and mammalian viruses are not highlighted. (C) Eukaryotic viral families in California roost GF-3 to -7 and in one Texas roost at two
collection time points (TM1-4 and TM5-8).
of eukaryote sequences, indicating the DNase and RNase
treatment was largely effective in removing non-capsid-pro-
tected bat host nucleic acids.
Phages in bat guano. Based on prior studies, phages com-
posed a significant fraction of human and equine fecal viral
populations (3, 4, 7, 60). The levels of phage sequences in the
feces of South Asian children with nonpolio acute flaccid pa-
ralysis and health contacts processed in the same manner were
approximately 16% and 12% of total reads, respectively (60).
In our study, the sequences with similarities to phages made up
4% and 0.1% of sequences in bat guano from California and
Texas, respectively. Among the phages in bat guano samples
from California, the majority belonged to the families Sipho-
viridae (67%) and Microviridae (28%), consistent with earlier
viral metagenomic studies of which siphophages were the most
abundant phages in human and equine feces (3, 4, 7). The most
abundant sequence matches were to c2-like Lactococcus
phages, T1-like enterobacterium phages, Chlamydia phage 3,
and Spiroplasma phage 4 (data not shown).
Eukaryotic virus population in bat guano. Many previously
characterized and highly divergent eukaryotic viral sequences
were detected in bat guano. The families of eukaryotic viruses
that were found, based on their most significant BLASTx
matches, are shown in Fig. 1B. Sequences of DNA viruses
infecting eukaryotes made up a smaller fraction (approxi-
mately 10%) than eukaryotic RNA viral sequences in both
California and Texas bat metagenomes. The DNA viruses were
dominated by single-stranded DNA (ssDNA) viruses, includ-
ing animal viruses from the families Parvoviridae and Circoviri-
dae and plant viruses from the family Geminiviridae. Most of
the proteins encoded by ssDNA eukaryotic virus-like se-
quences showed less than 60% amino acid identities to known
viral protein, suggesting the presence of numerous novel viral
species in bats. Sequences related to the newly discovered
Cyclovirus genus in the family Circoviridae, commonly found in
the tissues of hoofed farm animals and chickens as well as in
human and wild chimpanzee feces, were also detected (33),
showing that these viruses also exist in wild bats. Single-
stranded RNA viruses belonged largely to the families Dicis-
troviridae, Nodaviridae, and Picornaviridae. Double-stranded
RNA viral sequences in the family Partitiviridae (18%) were
also detected in the bat guano from California.
Guano collected from bats in California had a more diverse
viral composition than guano collected from bats in Texas,
which may reflect the multiple roosts and bats species sampled
in California. The most common eukaryotic viral families var-
ied greatly between each of the five California roosts sampled
(Fig. 1C). The guano viromes of the GF-3, -5, and -6 roosts
were dominated by plant viruses, whereas the GF-4 roost, the
only one with pallid bats, was richest in insect dicistroviruses,
and the GF-7 roost had a more diversified virus profile. The
viral compositions of the two guano samplings from the same
Texas roost were highly distinct, with the earlier collection
dominated by dicistroviruses and the later one by plant virus
leutoviruses and tymoviruses (Fig. 1C, TM1-4 and TM5-8).
Insect viruses. The largest fraction of the bat guano virome
was related to insect viruses from the family Dicistroviridae,
consisting of 29% of California viral sequences and 61% of
Texas viral sequences, likely reflecting the insect-based diet of
the bat species analyzed. Viral sequences related to viruses
from Iflaviridae, Tetraviridae, Alphanodavirus, and Densovirinae
were also detected. Most of the viruses were novel, sharing less
than 60% amino acid (aa) similarity to known viral proteins,
while some shared high (?90%) amino acid similarity with
known insect viruses.
Viral sequences similar to those of Kashmir bee virus (12)
and acute bee paralysis virus (19) were very abundant in bat
guano from California and Texas. Sequences covered ?70% of
the complete genomes of these viruses (GenBank accession
numbers HM228885 to HM228895). The translated full-length
structural proteins shared 98% similarity with Kashmir bee
virus and 97% aa similarity with acute bee paralysis virus,
indicating that the viruses found in bat guano were variants of
Kashmir bee virus and acute bee paralysis virus rather than
new viral species. Because the sampled bats are nocturnal, it is
unlikely that they feed on diurnal bees. Kashmir bee virus and
acute bee paralysis virus may therefore also infect nocturnal
bees or other insect hosts, or the bat species studied may have
previously unknown dietary activities.
Viral sequences related to those of betanodaviruses known
to infect fishes (36, 55) were also found in bat guano from
California and Texas. We generated the almost full-length
RNA1 segment (?90% coverage) of a nodavirus (bat guano-
associated nodavirus GF-4), with a best match (E value of
3e?108) to Epinephelus tauvina nervous necrosis virus, with aa
similarity of 34% (GenBank accession number HM228873).
Phylogenetically, this nodavirus sequence in guano fell be-
tween the alphanodaviruses and betanodaviruses (Fig. 2).
Given that none of the bat species sampled in our study are
known to eat fish, this virus may represent a highly divergent
Plant viruses. The second largest proportion of the bat
guano viromes detected, with 46% of the viral sequences from
Californian bats and 27% of those from Texan bats, was re-
lated to plant and fungal viral families, including Luteoviridae,
Secoviridae, Tymoviridae, and Partitiviridae, and the Sobemovi-
rus genus. Both previously characterized and newly identified
plant viruses were detected. Plant viruses previously identified
in human feces were mostly dominated by the tobamovirus
pepper mild mottle virus (67).
Mammalian viruses in bat guano. Sequences related to
mammalian and bird viruses made up less than 10% of the
viromes from bat guano in California and Texas. Viral
sequences related to viruses from the families Parvoviridae,
Circoviridae, Adenoviridae, Poxviridae, Picornaviridae, Astroviri-
dae, and Coronaviridae were found in bat guano. Most of the
sequence reads showed limited amino acid identity (?60%)
with known viruses. Phylogenetic analyses were used to assess
the relationship of novel viral sequences to known viruses.
Bat cyclovirus and circovirus-like virus. Cyclovirus, a new
genus in the family Circoviridae, was recently described in stool
samples from humans as well as in muscle tissue samples from
hoofed farm animals and chickens from Pakistan and Nigeria
(33). Stools from wild chimpanzees also contained cycloviruses
(33). In California bat stool samples from roost GF-4, we
identified a virus that is closely related to cycloviruses. The
complete circular genome was 1,844 bp (GenBank accession
number HM228874). The genome organization of bat cyclovi-
rus GF-4 had characteristic features of cycloviruses, including
two major inversely arranged open reading frames (ORFs)
6958LI ET AL.J. VIROL.
encoding the putative replication-associated protein (Rep; 281
aa) and capsid protein (Cap; 227 aa). A characteristic potential
stem-loop structure with a conserved nonanucleotide motif
(5?-TAATACTAT-3?) was also found in the 5? intergenic re-
gion (between the start codons of the two major ORFs) (Fig.
3A). The putative Rep proteins of the bat cyclovirus GF-4 had
45% to 68% aa similarity to cycloviruses found in human and
chimpanzee feces and 39% to 43% similarity to the Rep pro-
teins of porcine and avian circoviruses (data not shown).
In Texas bat stool sample TM6, we found a small, circular
DNA virus with a full-genome size of 1,696 bp (bat circovirus-
like virus TM6) (GenBank accession number HM228876). The
virus had two major ORFs arranged in opposite directions,
with Rep at 264 aa and Cap at 226 aa, and two noncoding
intergenic regions (Fig. 3B). The stem-loop structure also had
the cyclovirus-conserved nonanucleotide motif (5?-TAATACT
AT-3?) but was instead located at the 3? intergenic region
(between the stop codons of the two major ORFs).
A phylogenetic analysis of the complete Rep protein of bat
cyclovirus GF-4 and bat circovirus-like virus TM6, including
cycloviruses, circoviruses, chicken anemia virus (CAV), and
non-Circoviridae Rep proteins from the plant Nanovirus milk
vetch dwarf virus, Geminivirus pepper golden mosaic virus,
canarypox virus, Bifidobacterium pseudocatenulatum plasmid
pM4, Giardia intestinalis, and Entamoeba histolytica was per-
formed (Fig. 3C). Examination of the phylogenetic tree
showed that bat cyclovirus GF-4 grouped with known cyclovi-
ruses, forming a distinct species of cyclovirus. Bat circovirus-
like virus TM6 fell outside the Circovirus and Cyclovirus clades,
grouping with canarypox virus. While most closely related to
the replicase sequence of canarypox virus, these two proteins
showed only 40% aa similarity.
Bat kobuvirus. Kobuvirus, a genus in the family Picornaviri-
dae, currently contains three species: Aichi virus, bovine kobu-
virus, and porcine kobuvirus. Kobuvirus-related viruses named
salivirus and klassevirus have been recently described in hu-
man stool samples (20, 21, 34). Aichi virus and salivirus have
been associated with human gastroenteritis, while bovine
kobuvirus and porcine kobuvirus are associated with bovine
and porcine diarrhea, respectively (26, 27, 48, 66). In one of the
Texan sets of guano samples (TM7), we found approximately
400 reads which assembled into 5 contigs covering more than
60% of the viral genome of a virus closely related to kobuvi-
HM228884). BLASTx searches showed that these contigs
shared 39% to 59% aa similarity to kobuviruses. We tentatively
named this virus bat kobuvirus. According to the International
Committee on Taxonomy of Viruses (ICTV) (http://www
members of a picornavirus genus should share ?40%, ?40%,
and ?50% aa similarity in their P1, P2, and P3 regions, re-
spectively. The largest contig (1,998 bp) covered about 70% of
the P1 region and shared 46% aa similarity with the closest
match, human Aichi virus. Bat kobuvirus therefore appears to be
a new viral species within the genus Kobuvirus (Fig. 4). Phyloge-
netic analysis using the contig (1,335 bp) covering more than 80%
of the 3-D region produces a similar tree topology (data not
Bat astrovirus. The family Astroviridae includes positive sin-
gle-stranded RNA viruses, with genomes of 6.4 to 7.3 kb,
encoding nonstructural proteins with ORF1a and ORF1b and
structural protein with ORF2 (43). Astroviruses (AstV) have
been identified in a variety of mammals and birds, including
humans, cattle, pigs, sheep, mink, dogs, cats, mice, bats, chick-
ens, and turkeys. In the California bat roost GF-7, we detected
a highly divergent astrovirus-like sequence (677 bp) (GenBank
accession number HM228876). The translated amino acid se-
quence most closely matched the serine protease region of the
newly characterized human HMOAstV-A viral genome (34%
similarity) (16, 23). Phylogenetic analysis based on this region
yielded a tree topology that was congruent with those of anal-
yses using other genome regions (8, 49, 68). The tree showed
that the bat astrovirus GF-7 sequence fell in a basal position
relative to other mamastroviruses (infecting mammals) be-
FIG. 2. Phylogenetic analysis of bat guano-associated nodavirus GF-4 based on its 950-aa RNA-dependent RNA polymerase (RdRp) region.
RNA1 of nodaviruses encodes the RdRp protein of ?1,000 aa.
VOL. 84, 2010 DIVERSE VIRUSES FOUND IN BAT GUANO6959
tween genera Mamastrovirus and Avastrovirus (infecting birds)
(Fig. 5). Because the same genome region was not sequenced
for recently reported bat astroviruses from China (8, 68), the
relationship between bat astrovirus GF-7 and other bat astro-
viruses is currently unknown.
Bat parvovirus. Classified within the Parvoviridae family Par-
vovirinae are a subfamily of linear, nonsegmented single-
stranded DNA viruses largely infecting mammals but also
some birds, with an average genome size of 4 to 6 kbp. Viruses
in this subfamily possess two major ORFs, encoding the non-
structural protein (NS) and structural protein (VP) (22, 24,
57). In one of the guano samples from Texas bats (TM2), we
generated a parvovirus-like sequence of 1,346 bases, including
the C terminus of the nonstructural (NS) protein and the N
terminus of the viral protein (VP) (GenBank accession num-
ber HM228877). Phylogenetic analysis based on this partial NS
region demonstrated that this virus fell between sequences
belonging to the subfamily Parvovirinae, infecting mammals/
birds, and those belonging to the subfamily Densovirinae, in-
fecting insects (Fig. 6).
Bat adeno-associated virus and adenovirus. Adeno-associ-
ated virus (AAV) belongs to the genus Dependovirus in the
subfamily Parvovirinae. AAV usually requires coinfection with
a helper adenovirus for its replication. AAV has been found in
FIG. 3. (A) Genome organization of bat cyclovirus GF-4; (B) genome organization of bat circovirus-like virus TM6; (C) phylogenetic analysis
of bat cyclovirus GF-4 and circovirus-like virus TM6 based on the complete amino acid sequence of the Rep protein (?280 aa).
6960LI ET AL. J. VIROL.
many vertebrate species, but no bat AAV has previously been
reported (17, 44, 52, 53). Members of the family Adenoviridae
are double-stranded DNA viruses, with relatively large ge-
nomes ranging from 26 to 45 kb. Adenovirus infection was
identified in at least 40 vertebrate species, including mammals,
birds, amphibians, reptiles, and fishes (35, 51, 52, 56). In the
present study, we found both AAV and adenovirus sequences in
AAV sequence encoded a partial capsid protein VP1 (160 aa)
(GenBank accession number HM228878), which exhibited 74%
or less aa similarity over that region with known AAVs. Phyloge-
netically, bat AAV was related to known AAV as a deep-rooted
lineage (Fig. 7). The bat adenovirus-related sequence was short
(60 aa), showing 82% similarity over that region with that of
polypeptide VIII of the capsid protein of tree shrew adenovirus
(227 aa) (GenBank accession number HM234169). It also
showed 80% aa similarity with the sequence of the recently char-
acterized bat adenovirus strain TJM (polypeptide VIII; 222 aa)
(35). The same genome region was not described for the other
available bat adenovirus strains FBV1 and PPV1 (56).
Bat coronavirus. The family Coronaviridae includes positive
single-stranded RNA viruses, with genomes of 16 to 31 kb. Bat
coronaviruses have received much attention since the global
outbreak caused by severe acute respiratory syndrome corona-
virus (SARS-CoV), and numerous bat coronaviruses (CoVs)
have been identified from different bat species (32). In our
study, a CoV sequence (116 aa) (GenBank accession number
HM234168) was detected in one of the guano samples from
Texas (TM5), showing a best match to bat genus Scotophilus
CoV 512 (73% aa identity). Phylogenetic analysis based on this
partial replicase region confirmed that this virus is most closely
related to Scotophilus bat CoV 512 (nonstructural proteins 8
and 9; 303 aa) but forms a distinct genetic lineage (Fig. 8).
Another guano virus-like sequence had more ambiguous
viral origins. A sequence of 200 aa in length showed a best
BLASTx match to a simian hepatitis A virus in the family
FIG. 4. Phylogenetic analysis of bat kobuvirus based on its 660-aa partial P1 region. The P1 region of kobuviruses encodes structural proteins
of ?870 aa.
FIG. 5. Phylogenetic analysis of bat astrovirus based on its 225-aa partial ORF1a region. ORF1a of astroviruses encodes nonstructural protein
proteases of ?900 aa.
VOL. 84, 2010 DIVERSE VIRUSES FOUND IN BAT GUANO6961
Picornaviridae (E value of 4e?17) (GenBank accession num-
ber HM228879) but phylogenetically fell between the families
Picornaviridae and Dicistroviridae, reflecting the possible pres-
ence of a novel viral family (data not shown).
Our study examined the viral assemblages in the guano of
bats from California and Texas. The viral metagenomic ap-
FIG. 6. Phylogenetic analysis of bat parvovirus based on its 210-aa partial nonstructural (NS) ORF. The NS ORF of parvoviruses encodes
replication-associated protein REP of ?700 aa.
FIG. 7. Phylogenetic analysis of bat AAV based on its 160-aa partial capsid protein VP1 region. The VP1 protein of AAVs is ?700 aa.
6962LI ET AL.J. VIROL.
proach used here, involving the partial purification of viral
nucleic acids, random PCR amplification, and pyrosequencing,
detected viral sequences very closely related to known viruses
as well as novel viruses.
The proportion of phage sequences was relatively low, com-
pared with that of the viromes reported for human and equine
feces (3, 4, 6, 60), consisting mostly of siphophages. The eukary-
otic viruses included species from multiple DNA and RNA viral
families. The majority of the eukaryotic viruses in bat guano from
California and Texas were related to viruses infecting insects and
plants. The presence of insect viruses in guano was not unex-
are insectivorous. The high frequency of plant viral sequences
might reflect the plant diet of the eaten insects.
Outbreaks of white-nose syndrome have been associated
with infection with the Geomyces destructans fungus in the
family Helotiaceae of the Ascomycota phylum (2). No out-
breaks of the white-nose syndrome have been reported in
Texas or California. Some of the Partitivirus-like sequences
detected in guano from bats in California and Texas were
related to those of viruses known to infect members of the
phyla Ascomycota (which accounts for 75% of all fungal spe-
cies) and Basidiomycota, indicating the likely presence of fun-
gal viruses in the guts of bats.
The present study revealed numerous new mammalian viruses,
including a highly divergent kobuvirus, astrovirus, parvovirus,
AAV, adenovirus, and coronavirus. No close homologue of a
known human viral pathogen was detected in our study. A large
fraction of sequences was unclassifiable using BLAST methods. It
is conceivable that viral sequences that are too divergent from
those of known viruses to be recognized by BLAST methods were
included in this large group of unclassifiable sequences.
Viral metagenomics, while providing sequence data on the
most prevalent viruses present in a sample, is not currently as
sensitive as PCR. For example, in a previous study, cycloviruses
were found in approximately 9% of Nigerian stool samples
(33), while these cycloviruses were not detected in the same
cohort using viral metagenomics (data not shown). The viral
survey reported here is therefore likely to underestimate the
diversity of low-concentrations viruses in bat guano. Large
differences in virome composition between guano samples
taken only 3 days apart from the same bat roost in Texas were
also observed. This result indicates that representative sam-
pling of the enteric viruses in this population was not achieved
and/or that the bat guano virome in this roost changed within
a few days. Analyzing fecal pellets from a greater number of
bats using deeper sequencing methods will result in improved
sampling of the viral populations.
The viral metagenomic data obtained from the present study
provide a preliminary view of the viromes in bat guano. Future
study involving a wider sampling of bat species in different
locations will doubtlessly increase our understanding of the
diversity of viruses present in these mammals. Except for the
recognition of viruses very closely related to known human
pathogens, it is not possible to predict, based on genetic infor-
mation alone, which bat viruses already are or may evolve into
human pathogens, a rare occurrence also influenced by the
extent of contact with bats. Further studies, such as in vitro
replication using cell lines from different species in frequent con-
tact with bats, may help define which viruses have zoonotic po-
tential (10, 35, 63). The further characterization of the bat virome
will therefore increase our understanding of mammalian virus
diversity but may also be useful for the detection of potential
zoonotic viruses. Identifying bat pathogens will also help preserve
these mammals and their beneficial effects on the environment
largely due to their voracious appetite for insects and role as
We acknowledge NHLBI grant R01HL083254 and BSRI for sus-
tained support to E.L.D. and support from Boston University’s Center
for Ecology and Conservation Biology to T.H.K.
FIG. 8. Phylogenetic analysis of bat CoV based on its 110-aa partial replicase region. Nonstructural proteins 8 and 9 of CoV are ?300 aa.
VOL. 84, 2010DIVERSE VIRUSES FOUND IN BAT GUANO 6963
The use of trade, product, or firm names is for descriptive purposes
alone and does not imply endorsement by the U.S. Government.
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