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: yuanlh@gdei.gd.cn (L.Y.); limm0620@126.com (M.L.);

lilinmiao-8245524@163.com (L.L.); twenty-five@live.cn (X.M.); Chenjp@gdei.gd.cn (J.C.) 2 Metabiota, San Francisco, CA 94104, USA; E-Mails: cmonagin@metabiota.com (C.M.);

bschneider@metabiota.com (B.S.S.) 3 EcoHealth Alliance, New York, NY 10001, USA; E-Mails: chmura@ecohealthalliance.org (A.A.C.);

epstein@ecohealthalliance.org (J.H.E.); daszak@conservationmedicine.org (P.D.) 4 Wuhan Institute of Virology of Chinese Academy of Sciences, Wuhan 430071, China;

E-Mail: zlshi@wh.iov.cn

* Author to whom correspondence should be addressed; E-Mail: chenjp@gdei.gd.cn;

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).

Viruses 2014, 6 2143

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

Viruses 2014, 6 2145

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).

Viruses 2014, 6 2147

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)

Viruses 2014, 6 2148

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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