Viruses 2014, 6, 2138-2154; doi:10.3390/v6052138
viruses
ISSN 1999-4915
www.mdpi.com/journal/viruses
Article
Evidence for Retrovirus and Paramyxovirus Infection of
Multiple Bat Species in China
Lihong Yuan 1, Min Li
1, Linmiao Li
1, Corina Monagin
2, Aleksei A. Chmura
3,
Bradley S. Schneider 2, Jonathan H. Epstein
3, Xiaolin Mei
1, Zhengli Shi
4,
Peter Daszak 3 and Jinping Chen
1,*
1 Guangdong Entomological Institute, South China Institute of Endangered Animals,
Guangzhou 510260, China; E-Mails: [email protected] (L.Y.); [email protected] (M.L.);
[email protected] (L.L.); [email protected] (X.M.); [email protected] (J.C.) 2 Metabiota, San Francisco, CA 94104, USA; E-Mails: [email protected] (C.M.);
[email protected] (B.S.S.) 3 EcoHealth Alliance, New York, NY 10001, USA; E-Mails: [email protected] (A.A.C.);
[email protected] (J.H.E.); [email protected] (P.D.) 4 Wuhan Institute of Virology of Chinese Academy of Sciences, Wuhan 430071, China;
E-Mail: [email protected]
* Author to whom correspondence should be addressed; E-Mail: [email protected];
Tel.: +86-20-8910-0920; Fax: +86-20-8418-3704.
Received: 19 February 2014; in revised form: 27 April 2014 / Accepted: 6 May 2014 /
Published: 16 May 2014
Abstract: Bats are recognized reservoirs for many emerging zoonotic viruses of public
health importance. Identifying and cataloguing the viruses of bats is a logical approach to
evaluate the range of potential zoonoses of bat origin. We characterized the fecal pathogen
microbiome of both insectivorous and frugivorous bats, incorporating 281 individual bats
comprising 20 common species, which were sampled in three locations of Yunnan province,
by combining reverse transcription polymerase chain reaction (RT-PCR) assays and
next-generation sequencing. Seven individual bats were paramyxovirus-positive by RT-PCR
using degenerate primers, and these paramyxoviruses were mainly classified into three
genera (Rubulavirus, Henipavirus and Jeilongvirus). Various additional novel pathogens
were detected in the paramyxovirus-positive bats using Illumina sequencing. A total of
7066 assembled contigs (≥200 bp) were constructed, and 105 contigs matched eukaryotic
viruses (of them 103 belong to 2 vertebrate virus families, 1 insect virus, and 1 mycovirus),
17 were parasites, and 4913 were homologous to prokaryotic microorganisms. Among the
OPEN ACCESS
Viruses 2014, 6 2139
103 vertebrate viral contigs, 79 displayed low identity (<70%) to known viruses including
human viruses at the amino acid level, suggesting that these belong to novel and
genetically divergent viruses. Overall, the most frequently identified viruses, particularly
in bats from the family Hipposideridae, were retroviruses. The present study expands our
understanding of the bat virome in species commonly found in Yunnan, China, and provides
insight into the overall diversity of viruses that may be capable of directly or indirectly
crossing over into humans.
Keywords: bat; metagenomics; hipposideridae; pathogens; viral emergence; virome; zoonoses
1. Introduction
Bats (order Chiroptera) comprise the second largest mammalian group in the world and are widely
distributed across six continents [1]. Numerous bat species have been implicated as natural reservoirs for
significant zoonotic viruses, including Ebola and, Marburg viruses; Nipah and Hendra viruses; SARS
coronavirus; and most recently [2–5]. Collectively, bats harbor a broad diversity of viruses, and it has
been proposed that entire viral families and genera, such as pararmyxoviruses, hepaciviruses, and
pegiviruses, may have originated from bats [6,7]. More than 170 viruses have been detected in bats, and
many of these viruses are highly pathogenic to humans [8], including, Nipah and Hendra viruses [9],
rabies virus [10], Australian Bat Lyssavirus [11], Marburg virus [12], and the SARS-like coronavirus [13].
Notably, the outbreak of SARS coronavirus in 2002, which originated in horseshoe bats in China and
infected more than 8000 people globally, has spurred increased efforts from the international research
community to characterize the diversity and distribution of bat viruses around the world [4,14].
Over 120 bat species have been identified in China, and many are widely distributed throughout the
southern provinces of Yunnan, Guangdong, Guangxi, and Fujian [15]. These more ubiquitous bat
species, which are in the genera of Rousettus, Myotis, Miniopterus and Hipposideros, naturally reside in
close proximity to humans, thus increasing the potential of transmission of zoonotic pathogens to
humans. Highly pathogenic paramyxoviruses have been identified in a variety of vertebrates, including
humans, and phylogenetic reconstruction of host associations suggests a predominance of host switches
from bats to other mammals and birds [6], and often cause serious outbreaks of diseases [16,17]. Thus,
assessing the variety of paramyxoviruses circulating in bats in China could provide important guidance
for the control and prevention of future epidemics.
Recently, high-throughput sequencing (e.g., Illumina, Solexa, etc.)—a rapid and efficient technique
using sequence-independent amplification of nucleic acids followed by shotgun sequencing—has been
employed with great success to discover an enormous diversity of viruses in a range of samples,
including marine and fresh water [18,19], animals tissues [20,21], human feces, and bat fecal, urine and
oral samples [22–29]. In this study, we used reverse transcription polymerase chain reaction (RT-PCR)
to survey the prevalence of paramyxoviruses in bats from Yunnan China, and then conducted Illumina
sequencing to characterize paramyxoviruses from bat samples. To date, this is the first reported
metagenomic analysis in fecal samples of both insectivorous and frugivorous bats in China.
Viruses 2014, 6 2140
2. Experimental
2.1. Ethics Statement
This study was approved by the Guangdong Entomological Institute Administrative Panel on Laboratory
Animal Care (Guangzhou, China). All bats were released unharmed immediately after sampling.
2.2. Sample Collection and Viral Nucleic Acids Preparation
Between November 2011 and March 2012, a total of 562 oral and rectal swab samples from
281 individual bats of 20 species were collected at multiple sites in the Yunnan Province of China.
Of the samples collected, 252 samples (44.84%) were from 126 insectivorous bats and 310 samples
(55.16%) from 155 frugivorous bats. The sampling locations include Yuanjiang Gulong hole (N23°35.180',
E101°57.540'; H: 405 m), Xishuangbanna Botanical Garden (N: 21°55.368', E: 101°15.235', H: 535 m),
and the Natural Arch (N: 21°59.235', E: 101°21.418', H: 919 m). Samples were suspended in a
phosphate buffer solution (PBS) with antibiotics and stored at −80 °C until nucleic acid extraction. For
the sampling details, such as numbers, locations and specific bat species, see Table S1.
To extract viral nucleic acid, the swab suspensions were centrifuged at 5000× g for 10 min. Then, the
supernatant was transferred to fresh tubes and centrifuged at 12,000× g for 20 min. The viral DNA
and RNA were simultaneously extracted from a 140 μL sample with the QIAamp Viral RNA Mini
Kit (QIAgen, Düsseldorf, Germany) according to the manufacturer’s protocol and eluted into 60 μL
AVE buffer.
2.3. Screening of Paramyxoviruses and Phylogenetic Analysis
Sensitive and broadly reactive RT-PCR assays were performed at Wuhan Institute of Virology,
Chinese Academy of Sciecnces. In this study, the primers, which were designed according to the
conserved motifs of the RNA polymerase (L)-coding sequence, were from the reference Tong et al. [30],
and could be used for identification of novel paramyxoviruses. The 560 bp fragments of the RNA
polymerase (L)-coding sequence conserved in the Paramyxovirinae subfamily were amplified,
according to the reference Tong et al. [30] with no modification. The PCR productions were sequenced
using Big Dye Terminator kits (Life Technologies, Carlsbad, CA, USA) on an ABI 3730 automated
sequencer with primer PAR-R.
To assess the phylogenetic relationship of paramyxoviruses identified in this study, 79 partial and full
L-gene nucleotide sequences were downloaded from NCBI with the GenBank accession numbers listed
in Table S2. Nucleotide sequences were then aligned and translated by using ClustalX 1.83 [31]. The
best-fit model (GTR + I + G) of the phylogenetic relationship was determined by Modeltest 3.7 [32] and
the phylogenetic trees of L-gene were constructed by MrBayes 3.1.1 [33], with the sequences of
Pneumovirinae subfamily as an outgroup.
Viruses 2014, 6 2141
2.4. High-throughput Sequencing and Pathogen Analysis
To further characterize the co-infected viruses in bat paramyxovirus-positive samples, Illumina
high-throughput sequencing was conducted. Total nucleic acids were extracted as described above.
Viral nucleic acid samples were then pooled, and a 3 μg pooled sample was used for sequencing library
preparation. The synthesis of first and second-strand cDNA from the viral RNA was performed using
random oligonucleotides/SuperScript II and DNA Polymerase I/RNase H, respectively. After the 3'-end
adenylation, DNA fragments were ligated with Illumina PE adapters on both sides to purify and
selectively enrich the 200 bp fragments using the AMPure XP system (Beckman Coulter, Fullerton, CA,
USA) and Illumina PCR Primer Cocktail in a 10 cycle PCR reaction. Finally, a sequencing library was
generated by Illumina TruSeqTM
RNA Sample Preparation Kit (Illumina, San Diego, CA, USA)
following the manufacturer’s recommendations and four index codes were added to attribute sequences.
The clustering of the index-coded samples was performed on a cBot Cluster Generation System using
TruSeq PE Cluster Kit v3-cBot-HS (Illumina, Inc.) according to the manufacturer’s instructions. After
cluster generation, the library preparations were sequenced on an Illumina Hiseq 2000 platform and 100
bp paired-end reads were generated. Reads that were contaminated with adapter or of low quality, as
well as poly-N reads, were removed from raw data and clean reads were obtained.
2.5. Identification of Viral Homologous Sequences
Clean reads from Illumina were compiled with de novo assembly, using de Bruijn graphs assembly
algorithms [34], and contigs <200 bp in length were not analyzed further. Remaining sequences
underwent sequential BLASTx searching against GenBank database to eliminate the bacteria and
eukaryotes contigs and identify suspect-viral sequences, which were longer than 200 bp, and taxonomic
classification was queried from the NCBI taxonomy web service. The reference sequence of the source
organism with the best contig alignment (i.e., the alignment with the lowest e-value ≤ 0.0001) was
retrieved. Contigs of bacteria and eukaryotes were eliminated. If the reference sequence taxonomy was
viral and aligned with the contig with an e-value of ≤0.0001, the sequence was flagged as suspect-viral
and retained for further analysis.
2.6. Phylogenetic Analysis of Viral Sequences
Suspect viral sequences related to invertebrate virus families were excluded. Reference sequences
were downloaded from NCBI. Global alignments with contigs were generated using ClustalX 1.83 [31],
translated in MEGA 5 [35] and edited with BIOEDIT v7.0 [36]. Gap-stripped alignments were then used
to construct the phylogenetic trees by MrBayes 3.1.1 [33].
2.7. Nucleotide Sequence Accession Numbers
The Illumina sequence data obtained in this study have been deposited in the Sequence Read
Archive (SRA) database (Accession No. SRX368740). The trimmed and binned vertebrate viral contigs
(≥200 bp) used for phylogenetic analysis in this study were deposited in GenBank (Accession No.
KF547868-KF547871).
Viruses 2014, 6 2142
3. Results
3.1. Detection of Paramyxoviruses
Among the samples, seven fecal samples of seven individual bats were identified as
paramyxovirus-positive, and in these bats, two individuals belong to Hipposideros cineraceus (sampled
in the Yuanjiang Gulong hole), one of Rousettus leschenaultii, one of Eonycteris spelaea, one of
Hipposideros armiger (sampled from the Xishuangbanna Botanical Garden, Mengla, China), and three
Taphozous melanopogon (sampled in Natural Arch, Mengla, China) (Table S1). The ~560-bp L-gene
sequences were submitted to GenBank (Accession No. KC599255, KC599257- KC599261, KC599263).
The phylogenetic tree of the L-gene, based on a 529 bp alignment is shown in Figure 1. The
phylogenetic analysis indicates that the paramyxovirus sequences identified in this study are separated
into three distinct genera. Of them, KC599259 identified in R. leschenaultii was clustered with
Rubulavirus. KC599257 detected in E. spelaea showed a close relationship with paramyxoviruses from
Eidolon helvum in urban Africa, which formed an unclassified sister clade to the genus Henipavirus. The
third cluster is that of five novel paramyxoviruses sharing a common ancestor and forming a
phylogenetically diverse subgroup, closely related to genus Jeilongvirus, comprising Jeilongvirus
(J-virus) and Beilong virus [37]. Moreover, this cluster can be further divided into two clades,
KC599255 and KC599258 from Hipposideridae; and KC599260, KC599261, and KC599263 from
T. melanopogon (family Emballonuridae). The deduced amino acid identity analysis supported the
phylogenetic relationship observed using the nucleotide sequences (Table S3). KC599259 had the
lowest homology (37.58%–40.61%) with other paramyxovirus sequences identified in this study and
had a higher identity (61.49%–78.74%) with known Rubulavirus and Rubulavirus-related virus. The
novel paramyxoviruses (KC599255, KC599258, KC599260, KC599261 and KC599263) from
insectivorous bats shared the highest amino acid identities (74.55%–100%), and had a relatively high
homology with Jeilongvirus (74.25%–78.44%) and KC599257 (59.39%–64.85%). On the other hand,
KC599257 was most closely related to Henipaviruses (92.94%).
3.2. In-Depth Analysis of Pathogens by Illumina High-throughput Sequencing
Individual sequence reads with base quality scores were produced by Illumina. After removing the
contaminant reads (0.01% the adapter reads, 2.43% low quality reads and 0.38% containing N reads), a
total of 7,963,701 clean reads (97.15%) were obtained in this study (Figure 2A).
3.2.1. Assembled Contigs and BLASTx Analysis
A total of 7066 assembled contigs (≥200 bp) were constructed and 6880 contigs with e-value ≤ 0.0001
were obtained. Data showed that the longest contig was 6481 bp and 310 contigs were longer
than 1000 bp. A total of 73.1% (5029/6880) of contigs associated with microorganisms. 118 contigs that
were homologous to eukaryotic viruses and phage. Moreover, we found 17 contigs that were homologs
of parasites (Table 1, Figure 2, Table S4 and Table S5).
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Figure 1. Phylogenetic analysis of paramyxovirus L-gene identified by RT-PCR. Phylogenetic
tree of paramyxoviruse L-gene was constructed based on a 529 bp sequence alignment with
Pneumovirinae subfamily as an outgroup. The accession numbers of sequences identified in
this study are red and posterior probability values are shown next to the tree nodes. Posterior
probability values are shown at each node (>70%) and the bar represents the expected
number of amino acid substitutions per site.
Viruses 2014, 6 2144
Table 1. Details of assembled contigs related to eukaryotic viruses and phages as determined
with Blastx and the GenBank database. Detailed information is shown in Table S6.
Clade Family Genus Virus Name Contigs
Vertebrate virus Retroviridae Gammaretrovirus Human endogenous retrovirus 24
Murine leukemia virus 5
Spleen necrosis virus 4
Moloney murine leukemia virus 3
Porcine endogenous retrovirus 2
Woolly monkey sarcoma virus 2
Friend murine leukemia virus 1
Feline leukemia virus 1
Xenotropic Murine Leukemia Virus 1
Chick syncytial virus 1
Gammaretrovirus RfRV/China/2011 1
Unknown
gammaretrovirus Reticuloendotheliosis virus 40
Bat gammaretrovirus 1
Baboon endogenous virus strain M7 1
Rousettus leschenaultii retrovirus 1
Betaretrovirus Ovine enzootic nasal tumor virus
(ENTV) 2
Jaagsiekte sheep retrovirus 2
Squirrel monkey retrovirus 2
Simian endogenous retrovirus 1
Unclassified Simian retrovirus 4
Multiple sclerosis associated retrovirus 1
Polyomaviridae Polyomavirus STL polyomavirus 1
Hamster polyomavirus 1
Merkel cell polyomavirus 1
Insect virus Solenopsis invicta virus 3 1
Mycovirus Grapevine partitivirus 1
Phage Myoviridae T4-like virus Acinetobacter phage Ac42 1
Myoviridae T4-like virus Acinetobacter phage Acj61 1
Myoviridae T4-like virus Acinetobacter phage Acj9 2
Myoviridae T4-like virus Aeromonas phage 44RR2.8t 1
Myoviridae T4-like virus Aeromonas phage Aeh1 1
Myoviridae T4-like virus Aeromonas phage Aes508 1
Myoviridae T4-like virus Enterobacteria phage Bp7 1
Myoviridae T4-like virus Enterobacteria phage JSE 1
Myoviridae T4-like virus Enterobacteria phage Phi1 1
Myoviridae T4-like virus Enterobacteria phage RB69 1
Myoviridae unclassified Myoviridae Salmonella phage STML-198 1
Myoviridae unclassified Myoviridae Yersinia phage phiR1-RT 1
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Figure 2. Schematic summary of the number of Illumina high-throughput sequencing reads.
(A) Classification of raw reads, and contigs (≥200 bp) compared with Genbank using
BLASTx searches (e-value < 0.0001); (B) Reads related to eukaryotic viruses and phages.
(A)
(B)
3.2.2. Identification of Bacteriophage Sequences
There were 13 contigs related to the sequences of known phages with a similarity of 61%–89% and an
e-value of 6e−71
–2.12e−5
. All of them were members of Myoviridae and 11 contigs were related to
T4-like entero-bacterial phages (Table 1, Figure 2B and Table S5).
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3.2.3. Analysis of Vertebrate Viral Sequences by Family
Thirteen contigs of phage, 1 insect-virus, and 1 mycovirus contig were removed, and 103 contigs
belonging to two mammalian-infecting viral families were left. Of them, 97% (100/103) were related to
retroviruses and three contigs were related to a polyomavirus of double-stranded DNA (dsDNA).
For the 103 contigs of vertebrate viruses, the amino acid sequences were 35% to 90% homologous with
viral protein sequences (e-value ≤ 0.0001). In these, 79 cotigs displayed low identity (<70%) to known
viruses at the amino acid level, suggesting that these belong to novel and genetically divergent viruses.
The nucleotide BLASTx searches against the GenBank database are listed in Table S6.
3.2.4. Retroviridae
We identified 100 contigs, primarily related to three genera of retroviruses (Gamma, beta and
unclassified retroviruses), and with e-values of 5e−117
–1e−4
(Figure 3A, also see Table S6). These
retroviral sequences related to all three canonical genes of retrovirus, including 28 Gag proteins,
66 protease/polymerase (Pol) and 6 envelope glycoproteins (Env), and some of them are highly closed to
known bat tretroviruses [38–40]. Three retrovirual contigs, which code Pol, Gag and Env of
gammaretrovirus respectively and have the longest CDS, were used for phylogenetic analysis. Due to
the amplification of the short retroviruses sequences, we have adjusted the tree according to the
published bat retrovirus trees [39,40] by removing some of the reference sequences. The phylogenetic
trees indicated that Pol (KF547868) and Gag (KF547870) showed high homologous with bat
gammaretroviruses, whereas Env (KF547869) couldn’t clade with those of bat gammaretroviruses
indicating the possibility of new viruses (Figure 3B–D). Moreover, sequence analysis indicated that the
translations of 55 of the 100 retrovirus contigs contained stop codons within the region of BLASTx
alignments, suggesting that they originated from non-functional, endogenous/exogeneous retroviruses.
In addition, BLASTx searching indicated that many contigs (such as comp6291_c0_seq1,
comp48905_c0_seq1, comp8098_c0_seq1, comp6984_c0_seq1, comp92776_c0_seq1, comp71626_c0_seq1
and comp36376_c0_seq1) shared equivalent or relatively higher amino acid identity (52%–85%,
e-values of 7e−12
–4e−35
) with poxvirus rather than retroviruses, with Fowlpox virus (FPV) being the
closest relative.
3.2.5. Polyomaviridae
We assembled two sequences related to the VP1 capsid protein and a sequence related to the large T
antigen protein of polyomaviruses (Table S6). They are phylogenetically clustered with hamster
polyomavirus, merker cell polyomavirus and STL polyomavirus respectively, and with high confidence
of e-values of 2e−15
–2e−53
(Table S6 and Figure 4).
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Figure 3. Classification and phylogenetic analysis of retroviral contigs. (A) The chart shows
proportions of retroviral sequences related to different retroviral genera. The number of
sequences related to each genus is shown in parentheses; (B)–(D) The midpoint-rooted
phylogenetic tree based on alignments of 83 aa Pol (starred), 142 aa Gag (starred) and 150 aa
Env (starred) of bat retroviruses, respectively. Posterior probability values are shown at each
node and the bar represents the expected number of amino acid substitutions per site.
Sequence information is shown in Table S7.
(A)
(B)
(C)
(D)
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Figure 4. Phylogenetic analysis of polyomaviral contig. The midpoint-rooted phylogenetic
tree based on 111 aa VP1 protein of polyomavirus (starred). Posterior probability values are
shown at each node and the bar represents the expected number of amino acid substitutions
per site.
4. Discussion
With the increase of human activities, habitat and survival of bats are severely reduced, thus causing
habitat shifts in bat populations and increased opportunity for contact with humans. The close contact
between humans and bats expands the opportunity for cross-species transmission of new zoonotic
viruses to humans [41]. In the current study, we identified seven bats [including three insectivorous bats
(Hipposideros cineraceus, H. armiger and Taphozous melanopogon) and two frugivorous bats
(Eonycteris spelaea and Rousettus leschenaultii)] positive for paramyxovirus by RT-PCR. Phylogenetic
analysis indicated that paramyxoviruses carried by insectivorous bats were distinct from those found in
frugivorous bats (Figure 1). Virus sequences from insectivorous bats could not be definitely assigned to
any existing paramyxovirus genus, however, they appeared to cluster within the relatively new genus of
the Jeilongvirus [37]. Moreover, we found that KC599257 (E. spelaea) and KC599259 (R. leschenaulti)
from fruit bats belonged to henipavirus-related paramyxovirus and Rubulavirus genus, respectively. The
finding of a henipavirus-related agent in E. spelaea provides further evidence that there is a large
spectrum of henipa-like viruses among bats, including insectivorous species. Li et al. [42] previously
found serological evidence of henipa-like viruses in bats, including Rousettus spp., Myotis spp.,
Hipposideros spp., and Miniopterus spp. in China. Henipaviruses have thus far overwhelmingly been
identified in Pteropus and Eidolon species, though this may, in part, be due to a lack of surveillance in
non-pteropodid bats [2,17,43]. The phylogenetic distance of species may constrain both cross-species
Viruses 2014, 6 2149
transmission and host shifts and thus result in the separation of viruses [44]. Previous studies demonstrate
that humans can be infected by paramyxoviruses through a secondary host species, such as horses and
pigs, or directly from bats [9,45–47]. Although it is currently unknown whether paramyxoviruses, such
as Jeilongvirus, are capable of infecting people, the potential risks of bat borne paramyxoviruses to
human health should be considered and surveys of human populations highly exposed to bats for
evidence of spillover of bat paramyxoviruses would help establish the public health risk.
High-throughput sequencing-based viral metagenomics is a powerful tool to explore known and
unknown viruses existing in host animals. Recently, it has been successfully used to detect viruses in
bats in North America, Africa and Asia [22,23,26–28], where the viromes of one frugivorous bat
species, E. helvum in Africa, and several insectivorous bats were described. From our metagenomic
study of paramyxovirus-positive bat samples, full-length viral genomic sequences were unable to be
assembled from the clean reads. This result is similar with that of previous studies and may be
due to the low quantity of the virus within samples, the short reads, and the lack of known reference
sequences [22,23,26]. Previous studies demonstrated that protein-based comparisons are more effective
than those based on nucleotides, and thus BLASTx usually identifies more suspect-viral sequences than
BLASTn [28,48]. Among the viral contigs, most of the bacterioviruses detected in this study are
enterobacterial phages, suggesting the similarity between the intestinal bacterial population of bats and
that of humans. Five contigs are highly similar to Moloney Murine Leukemia Virus (M-MLV), which
may have resulted from contamination of RT reagents and also have been detected in previous virome
analysis of insectivorous bats in China [26]. Many contigs are closed to bat retrovirues, which were
identified by previous studies [38–40]. Furthermore, we found that many viral contigs showed
equivalent or higher identity with Fowlpox virus (FPV) more than Reticuloendotheliosis virus (REV).
As a prototypical member of Avipoxvirus, FPV only infects non-mammalian hosts [49], and there is no
previous data available to indicate that bats are natural reservoirs or vectors of FPV. Previous studies
show that REVs derive directly from mammalian retroviruses and integrate into FPV genomes, which
become endogenous in the genome of larger and more complex DNA viruses [50–52]. Thus, these
FPV-like contigs were deemed as REVs and classified into unknown gammaretroviruses temporarily,
and the source of FPV-like contigs in this study should be explored further.
Our sequence analysis indicates that majority of the vertebrate viral sequences were distinct from
those previously identified in bats and were often diverse within the viral family. The novel viruses
identified were classified into two viral families, Retroviridae and Polyomaviridae, which were also
detected in previous studies [26–28]. Retroviruses have both endogenous and exogenous forms in
nature, comparing the results from this study with previous studies, metagenomic virome analysis
indicated that retroviruses are the most common viral sequences found in bats, especially in
Hipposideridae [26,29]. This finding may be due to the frequent integration of retroviral genomic
material into host genomes, rather than an indication of viral infection. Interestingly, the Illumina
screening did not detect paramyxovirus nucleic acid, which had been detected by RT-PCR, suggesting
that these viruses may be present in samples in relatively small amounts and below the sensitivity of
Illumina detection. These findings highlight an important limitation of high throughput sequencing as a
tool for virome characterization. Furthermore, the limited size or number of contigs generated by
Illumina sequencing may necessitate follow-up with conventional PCR assays in order to obtain enough
sequence to perform meaningful phylogenetic analyses [6,28]. The sensitivity of high-throughput
Viruses 2014, 6 2150
sequencing to detect viral nucleic acid is also limited if samples are not properly filtered and treated to
remove host genome and other non-target nucleic acid and enrich for viral sequences. Host nucleic acid
are typically present in significantly higher abundance relative to viral nucleic acid and can create
background noise in the results [26]. As the cost of high-throughput sequencing decreases and methods
of sample preparation improve to optimize results, Illumina sequencing can be an effective tool for
characterizing virus diversity in host organisms. Bats represent a massively diverse group of mammals,
and as a group they have been increasingly identified as natural reservoirs for virus families such as
paramyxoviruses, coronaviruses, and retroviruses, that contain zoonotic members. Emerging zoonotic
bat-borne viruses, such as SARS coronavirus; Nipah and Hendra virus; and Marburg and Ebola virus;
illustrate that bat viruses are capable of infecting people through various routes of transmission
and in various contexts such as through domestic animals, food-borne routes, or the wildlife trade.
Understanding the underlying diversity of viruses within common bat species and the types of
interactions they have with people, wildlife and livestock in China may provide further insight into the
risk of viral spillover, and may ultimately allow for interventions that reduce the risk of outbreaks.
5. Conclusions
We, for the first time, carry out the metagenomic analysis in fecal samples of both insectivorous
and frugivorous bats in China. The present study contributes to the understanding of the bat
virome, and sheds light on the overall diversity of zoonotic viruses.
Acknowledgment
This work was supported by CAS Cross-disciplinary Collaborative Teams Program for Science,
Technology and Innovationand was made possible by the generous support of the American people
through the United States Agency for International Development (USAID) Emerging Pandemic Threats
PREDICT. The contents are the responsibility of the authors and do not necessarily reflect the views of
USAID or the United States Government. LH Yuan is supported by the NSFC (31301012), the
Guangzhou Pearl River Scientific and Technological New Star Project (2012J2200003), and the
Outstanding Youth Science and Technology Talent Fund of Guangdong Academy of Sciences (rcjj201402).
Author Contributions
Lihong Yuan and Jinping Chen conceived and designed the experiments. Lihong Yuan, Min Li,
Linmiao Li and Xiaolin Mei carried out the lab work. Lihong Yuan and Min Li participated in data
analysis. Lihong Yuan, Jinping Chen, Corina Monagin, Aleksei Chmura, Bradley S. Schneider,
Jonathan H. Epstein, Zhengli Shi and Peter Daszak wrote the paper.
Viruses 2014, 6 2151
Conflicts of Interest
The authors declare no conflict of interest.
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Supplementary for Evidence for Retrovirus and Paramyxovirus Infection of Multiple Bat Species
in China
Table S1. Sample information of 20 bat species tested for paramyxoviruses.
Bat species Bat family Roosting
sites Sampling site Total tested Total positive (n/r)
Rhinolophus pusillus
Rhinolophidae
Cave B/C 1
9
Rhinolophus affinis Cave B 1 0/0
Rhinolophus huananus Cave B 1
Rhinolophus yunanesis Cave B/C 6
Taphozous melanopogon Emballonuridae
Cave B/C 34 36
3/8.33%
Taphozous perforatus Cave C 2
Eonycteris spelaea
Pteropodidae
Tree C 23
155
1/4.35%
Macroglossus sobrinus Cave A 3
Cynopterus sphinx Tree B 78
Rousettus leschenaulti Cave B 51 1/1.96%
Hipposideros cineraceus
Hipposideridae
Cave B 17
27
1/5.88%
Hipposideros larvatus Cave A/B 7
Hipposideros armiger Cave B 2 1/50%
Hipposideros pomona Cave B/C 1
Chaerephon plicata Molossidae Cave A 7 7 0/0
Pipistrellus abramus
Vespertilionidae
House C 2
47
Pipistrellus pipistrellus House B 1
Tylonycteris pachypus Tree B 6 0/0
Scotophilus heathi Tree B 28
Miniopterus schreibersii Cave B 10
281 7/2.49%
Cave, Tree and House are all near to humans’ habitation and there are close relationships with human life.
A: Yuanjiang cologne hole; B: Xishuangbanna botanic garden; C: Natural arch; n: the number of positive bat
individuals; r: rate of positive bat individuals.
Table S2. Paramyxovirus available in Genebank used for phylogenetic analyses.
Virus abbreviation Genus Accession Number host Country
Nipah virus AF212302NiV Henipavirus AF212302 Pteropus hypomelanus Malaysia
Hendra virus AF017149HeV Henipavirus AF017149 Pteropus poliocephalus Australia
Nipah virus FJ513078NiV Henipavirus FJ513078 Homo sapiens India
Nipah virus AJ627196NiV Henipavirus AJ627196 Sus scrofa Malaysia
Nipah virus AY988601NiV Henipavirus AY988601 Homo sapiens Bangladesh
Hendra virus NC_001906HeV Henipavirus NC_001906 Horse Australia
Eidolon helvum
Henipavirus
HQ660129Eid
HenipaV Henipavirus HQ660129 Eidolon helvum Ghana
Eidolon helvum PV JN648075U5B Henipavirus-related JN648075 Eidolon helvum Ghana
Eidolon helvum PV JN648086U6B Henipavirus-related JN648086 Eidolon helvum Ghana
Eidolon helvum PV JN648061U46B Henipavirus-related JN648061 Eidolon helvum Ghana
Eidolon helvum PV JN648063U47C Henipavirus-related JN648063 Eidolon helvum Ghana
Eidolon helvum PV JN648079U67J Henipavirus-related JN648079 Eidolon helvum Ghana
Human parainfluenza
virus 4b
EU627591HPIV4
b Rubulavirus EU627591 Homo sapiens Canada
Simian virus 5 AF052755SV5 Rubulavirus AF052755 Chlorocebus sabaeus USA
S2
Table S2. Cont.
Virus abbreviation Genus Accession Number host Country
Simian virus 41 NC_006428SV41 Rubulavirus NC_006428 Chlorocebus sabaeus Japan
Menangle virus AF326114MenPV Rubulavirus AF326114 Pteropus alecto Australia
Mapuera virus EF095490MprPV Rubulavirus EF095490 Sturnira lilium Brazil
Mumps virus EU370207MuV Rubulavirus EU370207 Homo sapiens Croatia
Mumps virus GU980052MuV Rubulavirus GU980052 Homo sapiens United
Kingdom
Mumps virus AY669145MuV Rubulavirus AY669145 Homo sapiens Russia
Porcine rubulavirus BK005918PorPV Rubulavirus BK005918 Sus scrofa America
Tioman virus AF298895TioPV Rubulavirus AF298895 Pteropus hypomelanus Malaysia
Human
parainfluenza virus 2 AF533012HPIV2 Rubulavirus AF533012 Homo sapiens USA
Tuhoko viruses 1 GU128080ThkPV1 Rubulavirus GU128080 Rousettus leschenaulti China
Tuhoko viruses 2 GU128081ThkPV2 Rubulavirus GU128081 Rousettus leschenaulti China
Tuhoko viruses 3 GU128082ThkPV3 Rubulavirus GU128082 Rousettus leschenaulti China
Achimota viruses 1 JX051319AchPV1 Rubulavirus JX051319 Eidolon helvum Ghana
Achimota viruses 2 JX051320AchPV2 Rubulavirus JX051320 Eidolon helvum Ghana
Epomophorus
species Rubulavirus
HQ660095Epo
RublaV Rubulavirus HQ660095 Epomophorus species Congo
Eidolon helvum PV JN648076U5D Rubulavirus-related JN648076 Eidolon helvum Ghana
Eidolon helvum PV JN648088U9B Rubulavirus-related JN648088 Eidolon helvum Ghana
Eidolon helvum PV JN648062U46G Rubulavirus-related JN648062 Eidolon helvum Ghana
Eidolon helvum PV JN648058U47E Rubulavirus-related JN648058 Eidolon helvum Ghana
Eidolon helvum PV JN648070U53A Rubulavirus-related JN648070 Eidolon helvum Ghana
Eidolon helvum PV JN648080U67N Rubulavirus-related JN648080 Eidolon helvum Ghana
Peste-des-petits-rumi
nants virus AJ849636PPRV Morbillivirus AJ849636 Ovis aries Turkey
Cetacean
morbillivirus AJ608288CeMV Morbillivirus AJ608288 Delphinus delphis
United
Kingdom
Measles virus EF565859MeV Morbillivirus EF565859 Homo sapiens France
Measles virus AB254456MeV Morbillivirus AB254456 Homo sapiens Japan
Rinderpest virus NC_006296RPV Morbillivirus NC_006296 Bos auben Kenya
Phocine distemper
virus Y09630PDV Morbillivirus Y09630 Phoca vitulina
United
Kingdom
Canine distemper
virus AY443350CDV Morbillivirus AY443350 Procyon lotor USA
Sendai virus AB039658SeV Respirovirus AB039658 Mus musculus Japan
Tianjin Sendai virus EF679198Tianjin
SeV Respirovirus EF679198 Callithrix jacchus China
Bovine parainfluenza
virus 3 AF178654BPIV3 Respirovirus AF178654 Bovine USA
S3
Table S2. Cont.
Virus abbreviation Genus Accession Number host Country
Human parainfluenza
virus 1 NC_003461HPIV1 Respirovirus NC_003461 Homo sapiens USA
Sus scrofa
parainfluenza virus 3 EU439428SwPIV3 Respirovirus EU439428 Sus scrofa USA
Newcastle disease
virus AF077761NDV Avulavirus AF077761 Gallus gallus Netherlands
Avian paramyxovirus 2 EU338414APMV-2 Avulavirus EU338414 Gallus gallus USA
Avian paramyxovirus 3 EU403085APMV-3 Avulavirus EU403085 Meleagris gallopavo Netherland
Avian paramyxovirus 4 EU877976APMV-4 Avulavirus EU877976 Anas platyrhynchos South Korea
Avian paramyxovirus 5 GU206351APMV-5 Avulavirus GU206351 Melopsittacus
aubenton Japan
Avian paramyxovirus 6 EF569970APMV-6 Avulavirus EF569970 Anas domesticus Russia
Avian paramyxovirus 7 FJ231524APMV-7 Avulavirus FJ231524 Columbidae species USA
Avian paramyxovirus 8 FJ215863APMV-8 Avulavirus FJ215863 Branta aubentoni USA
Avian paramyxovirus 9 EU910942APMV-9 Avulavirus EU910942 Anas domesticus USA
Atlantic salmon
paramyxovirus EU156171AsaPV Aquaparamyxovirus EU156171 Salmo salar Norway
Fer-de-Lance
paramyxovirus AY141760FdlPV Ferlavirus AY141760 Fer-de-Lance Viper USA
Mossman virus AY286409MosPV Undefined AY286409 wild rat Australia
Salem virus JQ697837SalPV Undefined JQ697837 horse USA
Tupaia paramyxovius AF079780TupPV Undefined AF079780 Tupaia belangeri Germany
J-virus AY900001JPV Undefined AY900001 Mus musculus Australia
Beilong virus DQ100461BeiPV Undefined DQ100461 Rattus norvegicus China
Eidolon helvum PV JN648085U6A Undefined JN648085 Eidolon helvum Ghana
Eidolon helvum PV JN648089U9D Undefined JN648089 Eidolon helvum Ghana
Eidolon helvum PV JN648081U68E Undefined JN648081 Eidolon helvum Ghana
Human
metapneumovirus AF371337HMPV Metapneumovirus AF371337 Homo sapiens Netherlands
Avian
metapneumovirus C DQ009484AMPV-C Metapneumovirus DQ009484 Branta aubentoni USA
Avian
metapneumovirus B AB548428AMPV-B Metapneumovirus AB548428 Meleagris gallopavo France
Murine pneumonia
virus AY743910MPV Pneumovirus AY743910 Mus musculus USA
Bovine respiratory
syncytial virus AF092942BRSV Pneumovirus AF092942 Bovine kidney cell Germany
Human respiratory
syncytial virus S2 U39662HRSV Pneumovirus U39662 Homo sapiens UK
Abbreviation represents virus sequences in the phylogenic tree.
S4
Table S3. Comparision of amino acid identities.
YN12137 YN12069 YN12162 YN12193 YN12167 YN12003 YN12103
YN12137
YN12069
YN12162
YN12193
YN12167
YN12003
YN12103
100
39.39 100
38.18 63.64 100
38.18 63.64 100 100
37.58 61.82 96.36 96.36 100
40.61 59.39 75.76 75.76 74.55 100
38.18 64.85 78.79 78.79 79.39 83.03 100
MenPV
R
u
b
u
l
a
v
i
r
u
s
67.24 33.53 32.92 33.72 33.94 36.88 36.36
TioPV 63.22 32.94 32.3 33.72 33.94 33.12 34.66
ThkPV1 65.52 31.76 34.78 36.05 35.15 35 34.66
ThkPV2 78.74 35.88 32.3 33.14 33.33 35.62 34.09
ThkPV3 72.41 35.88 32.92 34.88 33.94 35.62 35.8
AchPV1 72.41 32.94 35.23 33.14 35.15 36.25 35.23
AchPV2 74.71 35.88 32.3 33.72 35.15 36.88 38.07
HPIV4b 62.64 37.06 37.27 35.47 35.76 36.88 38.07
SV5 64.94 35.88 32.92 38.37 40.61 38.12 38.07
SV41 58.05 34.71 34.78 34.3 32.73 34.38 33.52
MuV 63.22 37.06 36.65 37.21 38.79 38.12 37.5
MprPV 63.79 35.88 34.78 36.05 36.97 35 35.23
PorPV 62.07 35.29 32.92 34.3 34.55 33.12 34.09
U5D 71.84 32.94 32.3 33.14 35.15 36.88 35.23
U9B 72.41 32.94 32.3 33.14 35.15 36.25 35.23
U46G 70.11 32.35 32.3 33.14 35.15 36.25 35.23
U47E 68.39 32.94 33.54 34.3 34.55 34.38 34.09
U53A 61.49 34.71 34.78 35.47 36.97 36.25 35.8
U67N 68.39 32.94 32.92 33.72 33.94 33.75 33.52
EpoRublaV 62.07 34.71 36.65 37.21 38.79 37.5 37.5
NC_001906HeV
H
e
n
i
p
a
v
i
r
u
s
37.06 65.88 61.49 61.49 59.63 58.12 60.62
AF017149HeV 37.06 65.88 61.49 61.49 59.63 58.12 60.62
FJ513078NiV 38.24 66.47 63.35 63.35 62.73 59.38 62.5
AY988601NiV 38.24 66.47 63.35 63.35 62.73 59.38 62.5
AJ627196NiV 37.65 65.88 63.98 63.98 63.35 58.75 61.87
AF212302NiV 37.65 65.88 63.98 63.98 63.35 58.75 61.87
U5B 38.24 67.65 60.87 60.87 60.87 58.75 61.87
U6A 35.88 92.94 55.9 55.9 56.52 57.5 58.75
U6B 41.76 68.24 57.76 57.76 57.14 56.25 58.75
U9D 38.82 70 60.25 60.25 60.87 60 62.5
U46B 40 65.88 62.11 62.11 60.25 60 60
U47C 40 65.88 62.11 62.11 60.25 60 60
U67J 41.76 70.59 60.87 60.87 59.63 59.38 60.62
U68E 41.18 70.59 62.73 62.73 59.63 65.62 66.25
EidHenipaV 37.06 62.94 57.14 57.14 57.14 56.25 58.12
JPV 40.61 61.08 76.65 77.25 76.05 74.25 78.44
BeiPV 40.61 59.28 74.85 75.45 76.05 76.05 75.45
Table lists percentage of identity between deduced amino acid sequences from partial L gene fragments
identified in the present study and homologous sequences from selected known paramyxoviruses.
S5
Table S4.
Total contigs (≥200 bp) 7066
e-value(p ≤ 0.0001) 6880
Microorganism 5029
Prokaryotic microorganism 4913
Eukaryotic microorganism 2
Eukaryotic viruses and phages 118
Animal 1748
Protozoa 3
Metazoa 1745
Parasite 17
Insects and Vertebrates 1728
Plant (Alga) 17
Unknown 81
Table S5. Contigs related to prokaryotic microorganism as determined with Blastx and the
GenBank database.
Family Name Contig number
Acinetobacter Acinetobacter sp. 2
Clostridium Clostridium difficile 544
Clostridium sp. 159
Clostridium sticklandii 28
Coprococcus comes 1
Coprococcus eutactus 1
Corynebacterium Corynebacterium efficiens 1
Enterococcus Enterococcus faecalis 3
Enterococcus saccharolyticus 1
Flavobacteria Flavobacteria bacterium 2
Francisella Francisella philomiragia subsp. philomiragia 1
Fusobacterium Fusobacterium necrophorum 1
Gemella Gemella haemolysans 3
Gemella morbillorum 2
Gemella sanguinis 1
Haemophilus Haemophilus parainfluenzae 57
Haemophilus influenzae 17
Klebsiella Klebsiella pneumoniae 12
Micrococcus Micrococcus luteus 19
Mycoplasma Mycoplasma fermentans 1
Paenibacillus Paenibacillus sp. 3
Paenibacillus popilliae 1
Streptococcus Streptococcus infantis 18
Streptococcus mutans 15
Streptococcus equinus 12
Streptococcus gallolyticus 8
Streptococcus bovis 3
Streptococcus equi ssp equi 2
Staphylococcus chromogenes 1
Streptococcus alactolyticus 1
S6
Table S5. Cont.
Family Name Contig number
Helicobacter Heliobacterium modesticaldum 9
Helicobacter mustelae 2
Aggregatibacter Aggregatibacter segnis 3
Aspergillus Aspergillus niger 2
Aspergillus nidulans 1
Aspergillus oryzae 1
Bacillus Bacillus 6
Bacillus stratosphericus 3
Bacillus thuringiensis 1
Bacteroides Bacteroides uniformis 2
Bacteroides caccae 1
Bacteroides fragilis 1
Bacteroides ovatus 1
Bacteroides vulgatus 1
Beggiatoa Beggiatoa 2
Bergeyella Bergeyella zoohelcum 2
Bifidobacterium Bifidobacterium adolescentis 1
Brevibacillus Brevibacillus laterosporus 5
Citrobacter Citrobacter koseri 5
Citrobacter rodentium 3
Citrobacter 2
Citrobacter youngae 1
Clostridium Clostridium butyricum 187
Clostridium bartlettii 100
Clostridium celatum 75
Clostridium beijerinckii 35
Clostridium sp. Maddingley 29
Clostridium saccharoperbutylacetonicum 23
Clostridium carboxidivorans 7
Clostridium sporogenes 7
Clostridium cellulovorans 6
Clostridium ultunense Esp 6
Clostridium acetobutylicum 5
Clostridium clostridioforme 5
Clostridium leptum 4
Clostridium kluyveri 3
Clostridium arbusti 2
Clostridium ljungdahlii 2
Clostridium polysaccharolyticum 2
Clostridium acidurici 1
Clostridium bolteae 1
Clostridium botulinum 1
Clostridium clariflavum 1
Clostridium pasteurianum 1
Clostridium roseum 1
Clostridium sp. 1
Clostridium symbiosum 1
Clostridium thermocellum 1
S7
Table S5. Cont.
Family Name Contig number
Collinsella Collinsella 2
Deinococcus Deinococcus radiodurans 2
Escherichia Escherichia Coli 873
Elizabethkingia Elizabethkingia meningoseptica 1
Enterobacter Enterobacter cloacae 6
Enterobacter hormaechei 1
Enterobacteriaceae bacterium 1
Erysipelotrichaceae Erysipelotrichaceae 3
Eubacteriaceae Eubacteriaceae bacterium 3
Eubacterium dolichum 2
Eubacterium limosum 1
Eubacterium siraeum 1
Eubacterium ventriosum 1
Finegoldia Finegoldia magna 1
Fusobacterium Fusobacterium gonidiaformans 1
Fusobacterium nucleatum subsp. vincentii 1
Fusobacterium varium 1
Gordonia Gordonia amarae 1
Granulicatella Granulicatella elegans 11
Haemophilus somnus 1
Hafnia Hafnia alvei 1
Halobacillus Halobacillus sp. 1
Halomanas Halomonas titanicae 1
Helicobacter Helicobacter cinaedi 2
Helicobacter hepaticus 1
Helicobacter pullorum 1
Lachnospiraceae Lachnospiraceae bacterium 6
Lachnospiraceae oral taxon 1
Lactobacillu Lactobacillus jensenii 6
Lactobacillus rhamnosus 3
Lactobacillus fermentum 2
Lactobacillus brevis 1
Lactobacillus casei 1
Lactobacillus curvatus 1
Lactobacillus pentosus 1
Lactobacillus saerimneri 1
Lactobacillus salivarius 1
Leptotrichia Leptotrichia goodfellowii 2
Leptotrichia hofstadii 1
Listeria Listeria grayi 1
Magnetospirillum Magnetospirillum gryphiswaldense 5
Methanobrevibacter Methanobrevibacter smithii 1
Neisseria Neisseria polysaccharea 30
Peptoniphilus Peptoniphilus indolicus 2
Peptostreptococcus Peptostreptococcus stomatis 5
S8
Table S5. Cont.
Family Name Contig number
Photorhabdus Photorhabdus asymbiotica 2
Photorhabdus asymbitotica 1
Planctomyces Planctomyces 1
Propionibacterium Propionibacterium acidipropionici 3
Propionibacterium acidipropionici 1
Ruminococcus Ruminococcus obeum 6
Ruminococcus gnavus 2
Scheffersomyces Scheffersomyces stipitis 2
Selenomonas Selenomonas noxia 1
Staphylococcus Staphylococcus epidermidis 1
Stigmatella Stigmatella aurantiaca 1
Streptococcus Streptococcus mitis 11
Streptococcus oralis 10
Streptococcus tigurinus 9
Streptococcus sanguinis 7
Streptococcus thermophilus 7
Streptococcus macacae 5
Streptococcus australis 4
Streptococcus ictaluri 4
Streptococcus macedonicus 4
Streptococcus macedonicus 4
Streptococcus parasanguinis 4
Streptococcus pasteurianus 4
Streptococcus ratti 4
Streptococcus sobrinus 4
Streptococcus criceti 2
Streptococcus criceti 2
Streptococcus constellatus ssp constellatus 1
Streptomyces Streptomyces sp. 36
Thermoanaerobacter Thermoanaerobacter ethanolicus 1
Thermoanaerobacter mathranii 1
Thermoanaerobacter tengcongensis 1
Thermoanaerobacterium thermosaccharolyticum 1
Thermoanaerobacterium xylanolyticum 1
Veillonella Veillonella spp. 2
Vivrio Vivrio mimicus 1
Yersinia Yersinia frederiksenii 1
Yokenella Yokenella regensburgei 1
Ajellomyces Ajellomyces capsulatus 2
Ureaplasma Ureaplasma parvum serovar 2
Thioflavicoccus Thioflavicoccus mobilis 1
Kurthia Kurthia sp. 1
Cyanothece Cyanothece sp. 1
Chlorobaculum Chlorobaculum tepidum 1
Deinococcus Deinococcus radiodurans 2
S9
Table S5. Cont.
Family Name Contig number
Wolinella Wolinella succinogenes 1
Exophiala Exophiala dermatitidis 2
Prevotella Prevotella sp. 1
Penicillium Penicillium chrysogenum Wisconsin 2
Desulfotomaculum Desulfotomaculum gibsoniae 3
Desulfovibrio Desulfovibrio magneticus 1
Dichomitus squalens Dichomitus squalens 1
mixed culture bacterium mixed culture bacterium 2
uncultured uncultured Acidobacteria bacterium 4
uncultured uncultured alpha proteobacterium 1
uncultured uncultured bacterium 1
uncultured uncultured beta proteobacterium 2
uncultured uncultured Chromatiales bacterium 3
uncultured uncultured Desulfobacterales bacterium 1
uncultured uncultured Desulfobacterium sp. 1
uncultured uncultured gamma proteobacterium 3
uncultured uncultured Oceanospirillales bacterium 1
uncultured uncultured Rhodobacterales bacterium 1
Caenorhabditis Caenorhabditis remanei 1
Bacillus Bacillus macauensis 1
Geobacillus Geobacillus thermoleovorans 1
Sporolactobacillus Sporolactobacillus vineae 1
Acidaminococcus fermentans Acidaminococcus fermentans 1
Actinobacillus Actinobacillus pleuropneumoniae 1
Actinomyces viscosus 2
Aggregatibacter Aggregatibacter actinomycetemcomitans 4
Arthrobacter Arthrobacter citreus 1
Aspergillus Aspergillus flavus 1
Bacillus Bacillus cereus 5
Bacillus licheniformis 2
Bacillus bataviensis 1
Bacillus halodurans 1
Bacillus mycoides 1
Brachyspira Brachyspira hyodysenteriae 2
Campylobacter Campylobacter jejuni ssp jejuni 3
Chryseobacterium Chryseobacterium gleum 2
Ciona Ciona intestinalis 1
Clostridium Clostridium perfringens 1526
Clostridium hiranonis 70
Clostridium botulinum 52
Clostridium lentocellum 13
Clostridium novyi 3
Clostridium tetani 3
Clostridium septicum 2
Clostridium termitidis 2
S10
Table S5. Cont.
Family Name Contig number
Edwardsiella Edwardsiella tard 3
Enterobacter Enterobacter hormaechei 1
Enterococcus Enterococcus casseliflavus 1
Erwinia Erwinia amylovory 4
Escherichia Escherichia fergusonii 10
Gallibacterium Gallibacterium anatis 2
Haemophilus Haemophilus haemolyticus 7
Helicobacter Helicobacter bilis 4
Helicobacter pylori 1
higella higella flexneri 92
Klebsiella Klebsiella oxytoca 5
Lactococcus Lactococcus garvieae 2
Lawsonia Lawsonia intracellularis 4
Legionella Legionella pneumophila subsp. Pneumophila 2
Listeria Listeria monocytogenes 2
Mycobacterium Mycobacterium tuberculosis 1
Magnaporthe Magnaporthe oryzae 5
Mannheimia Mannheimia haemolytica 4
Mannheimia succiniciproducens 1
Marinobacter Marinobacter hydrocarbonoclasticus 3
Melissococcus Melissococcus plutonius 1
Metarhizium Metarhizium anisopliae 1
Methanococcus Methanococcus maripaludis 2
Methanococcus voltae 1
Mycoplasma Mycoplasma gallisepticum 1
Myxococcus Myxococcus sp. 1
Neisseria Neisseria meningitidis 3
Neisseria subflava 1
Paenibacillus Paenibacillus alvei 2
Paracoccidioides Paracoccidioides brasiliensis 2
Pasteurella Pasteurella multocida 6
Pediococcus Pediococcus acidilactici 2
Peptostreptococcus Peptostreptococcus anaerobius 3
Providencia Providencia stuartii 1
Psychrobacter Psychrobacter cryohalolentis 1
Rhodococcus Rhodococcus equi 1
Rhodospirillum Rhodospirillum photometricum 6
Riemerella Riemerella anatipestifer 1
Saccharomonospora Saccharomonospora paurometabolica 1
Salmonella Salmonella enterica ssp enterica 128
Salmonella enterica ssp arizonae 2
Salmonella bongori 1
Salmonella enterica subsp. houtenae str 1
Salmonella paratyphi 1
Salmonella typhimurium 1
S11
Table S5. Cont.
Family Name Contig number
Shigella Shigella dysenteriae 26
Shigella sonnei 16
Shigella boydii 13
Shigella spp. 2
Staphylococcus Staphylococcus aureus 5
Streptococcus Streptococcus agalactiae 30
Streptococcus pneumoniae 17
Streptococcus anginosus 14
Streptococcus suis 14
Streptococcus dysgalactiae ssp dysgalactiae 11
Streptococcus pyogenes 10
Streptococcus iniae 6
Streptococcus uberis 6
Streptococcus equi ssp zooepidemicus 5
Streptococcus porcinus 5
Streptococcus salivarius 5
Streptococcus vestibularis 5
Streptococcus intermedius 3
Streptococcus canis 1
Streptomyces Streptomyces ghanaensis 4
Streptomyces coelicoflavus 3
Streptomyces albus 2
Streptomyces roseosporus 1
Talaromyces Talaromyces stipitatus 4
Trichoderma Trichoderma harzianum 1
Vibrio Vibrio cholerae 3
Weissella Weissella 1
Yersinia Yersinia pestis 2
Marssonina Marssonina brunnea f. sp 1
Serratia Serratia plymuthica 1
Total 4913
Table S7. Gammaretroviruses used in the phylogenetic analyses.
Virus Abbreviation ID Host Reference
Rhinolophus pusillus retrovirus RpuRV JQ292909 bat 1
R. pearsoni retrovirus RpeRV JQ292914 bat 1
R. megaphyllus retrovirus RmRV JQ292911 bat 1
R. affinis retrovirus RaRV JQ292913 bat 1
Myotis ricketti retrovirus MrRV JQ292912 bat 2
Pteropusalecto retrovirus PaRV JQ292910 bat 1
Megaderma lyra retrovirus MlRV JQ951956 bat 2
Rousettus leschenaultia retrovirus RIRV JQ951957 bat 2
S12
Table S7. Cont.
Virus Abbreviation ID Host Reference
R. ferrumequinum retrovirus RfRV JQ303225 bat 1
Reticuloendotheliosis virus REV NC_006934 bird 3
Friend murine leukemia virus F-MuLV NC_001362 mouse 4
Moloney murine leukemia virus M-MuLV NC_001501 mouse 5
Murinetype C retrovirus M-CRV NC_001702 mouse
Porcineendogenous type C retrovirus class A PERV-A AJ293656 pig 6
Porcineendogenous type C retrovirus class B PERV-B AY099324 pig 7
Porcineendogenous type C retrovirus class C PERV-C EF133960 pig 8
Mus dunni endogenous virus MDEV AF053745 mouse 9
RD114 retrovirus RD114 NC_009889 cat
Koala retrovirus KoRV AF151794 koala 10
Baboon endogenous virus BaEV D10032 nonhuman
primates 11
Xenotropic murine leukemia virus X-MuLV AEI59728 mouse 12
Porcineendogenous retrovirus PERV CAA76582.1 pig 13
Orcinus orca endogenous retrovirus OOEV GQ222416 whale 14
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