Composition Vendor Newgen KnowledgeWorks (P) Ltd. PLoS Pathog plos plospath PLOS Pathogens 1553-7366 1553-7374 Public Library of Science San Francisco, CA USA 10.1371/journal.ppat.1013235 PPATHOGENS-D-25-00320 Research Article Biology and life sciencesOrganismsEukaryotaAnimalsVertebratesAmniotesMammalsBats Biology and life sciencesZoologyAnimalsVertebratesAmniotesMammalsBats Biology and life sciencesAnatomyRenal systemKidneys Medicine and health sciencesAnatomyRenal systemKidneys Biology and life sciencesOrganismsVirusesRNA virusesParamyxovirusesHenipavirus Biology and life sciencesMicrobiologyMedical microbiologyMicrobial pathogensViral pathogensParamyxovirusesHenipavirus Medicine and health sciencesPathology and laboratory medicinePathogensMicrobial pathogensViral pathogensParamyxovirusesHenipavirus Biology and life sciencesOrganismsVirusesViral pathogensParamyxovirusesHenipavirus Biology and life sciencesMicrobiologyMedical microbiologyMicrobial pathogensViral pathogens Medicine and health sciencesPathology and laboratory medicinePathogensMicrobial pathogensViral pathogens Biology and life sciencesOrganismsVirusesViral pathogens Medicine and health sciencesMedical conditionsParasitic diseasesProtozoan infections Biology and life sciencesEvolutionary biologyEvolutionary systematicsPhylogeneticsPhylogenetic analysis Biology and life sciencesTaxonomyEvolutionary systematicsPhylogeneticsPhylogenetic analysis Computer and information sciencesData managementTaxonomyEvolutionary systematicsPhylogeneticsPhylogenetic analysis Biology and life sciencesOrganismsVirusesRNA virusesParamyxovirusesHenipavirusHendra virus Biology and life sciencesMicrobiologyMedical microbiologyMicrobial pathogensViral pathogensParamyxovirusesHenipavirusHendra virus Medicine and health sciencesPathology and laboratory medicinePathogensMicrobial pathogensViral pathogensParamyxovirusesHenipavirusHendra virus Biology and life sciencesOrganismsVirusesViral pathogensParamyxovirusesHenipavirusHendra virus Biology and life sciencesOrganismsVirusesRNA virusesParamyxovirusesHendra virus Biology and life sciencesMicrobiologyMedical microbiologyMicrobial pathogensViral pathogensParamyxovirusesHendra virus Medicine and health sciencesPathology and laboratory medicinePathogensMicrobial pathogensViral pathogensParamyxovirusesHendra virus Biology and life sciencesOrganismsVirusesViral pathogensParamyxovirusesHendra virus Biology and life sciencesOrganismsEukaryotaProtozoansParasitic protozoans Infectome analysis of bat kidneys from Yunnan province, China, reveals novel henipaviruses related to Hendra and Nipah viruses and prevalent bacterial and eukaryotic microbes Meta-transcriptomic analysis of bat kidney infectome in Yunnan province, China Kuang Guopeng Conceptualization Data curation Formal analysis Investigation Methodology Project administration Resources Software Validation Visualization Writing – original draft Writing – review & editing 1 ☯ Yang Tian Conceptualization Data curation Formal analysis Investigation Methodology Project administration Resources Software Validation Visualization Writing – original draft Writing – review & editing 1 2 ☯ Yang Weihong Conceptualization Data curation Formal analysis Funding acquisition Investigation Methodology Project administration Resources Writing – review & editing 1 Wang Jing Investigation Methodology Project administration Resources Software Writing – review & editing 3 4 5 Pan Hong Conceptualization Data curation Formal analysis Investigation Methodology Writing – review & editing 1 Pan Yuanfei Methodology Software Supervision Validation Visualization Writing – review & editing 6 Gou Qin-yu Methodology Software Supervision Validation Visualization Writing – review & editing 3 4 5 Wu Wei-chen Investigation Methodology Resources Software Supervision Writing – review & editing 3 4 5 Wang Juan Investigation Methodology Resources Writing – review & editing 1 Yang Lifeng Investigation Methodology Resources Writing – review & editing 1 Han Xi Investigation Methodology Resources Writing – review & editing 1 Chen Yao-qing Conceptualization Investigation Methodology Writing – review & editing 7 Eden John-Sebastian Investigation Supervision Writing – review & editing 8 Holmes Edward C. Conceptualization Writing – original draft Writing – review & editing 8 9 https://orcid.org/0000-0002-6154-4437 Shi Mang Conceptualization Data curation Funding acquisition Investigation Methodology Project administration Software Supervision Validation Visualization Writing – original draft Writing – review & editing 3 4 5 * https://orcid.org/0000-0002-7354-0513 Feng Yun Conceptualization Data curation Formal analysis Funding acquisition Investigation Methodology Project administration Resources Supervision Validation Writing – original draft Writing – review & editing 1 2 10 * Yunnan Provincial Key Laboratory for Zoonosis Control and Prevention, Yunnan Institute of Endemic Disease Control and Prevention, Kunming, China School of Public Health, Dali University, Dali, China National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University, Shenzhen, China State Key Laboratory for Biocontrol, Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University, Shenzhen, China Shenzhen Key Laboratory for Systems Medicine in Inflammatory Diseases, School of Medicine, Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University, Shenzhen, China Ministry of Education Key Laboratory of Biodiversity Science and Ecological Engineering, School of Life Sciences, Fudan University, Shanghai, China School of Public Health (Shenzhen), Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University, Shenzhen, China School of Medical Sciences, The University of Sydney, Sydney, New South Wales, Australia Laboratory of Data Discovery for Health Limited, Hong Kong SAR, China State Key Laboratory of Remote Sensing Science, Center for Global Change and Public Health, Faculty of Geographical Science, Beijing Normal University, Beijing, China Obbard Darren J. Editor University of Edinburgh, UNITED KINGDOM OF GREAT BRITAIN AND NORTHERN IRELAND

The authors have declared that no competing interests exist.

These authors contributed equally to this work.

 * E-mail: ynfy428@163.com (YF); shim23@mail.sysu.edu.cn (MS) 2462025 62025 21 6 e1013235 622025 2652025 2025 Kuang et al This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Bats are natural reservoirs for a wide range of microorganisms, including many notable zoonotic pathogens. However, the composition of the infectome (i.e., the collection of viral, bacterial and eukaryotic microorganisms) within bat kidneys remains poorly understood. To address this gap, we performed meta-transcriptomic sequencing on kidney tissues from 142 bats, spanning ten species sampled at five locations in Yunnan province, China. This analysis identified 22 viral species, including 20 novel viruses, two of which represented newly discovered henipaviruses closely related to the highly pathogenic Hendra and Nipah viruses. These henipaviruses were found in the kidneys of bats inhabiting an orchard near villages, raising concerns about potential fruit contamination via bat urine and transmission risks to livestock or humans. Additionally, we identified a novel protozoan parasite, tentatively named Klossiella yunnanensis, along with two highly abundant bacterial species, one of which is a newly discovered species—Flavobacterium yunnanensis. These findings broaden our understanding of the bat kidney infectome, underscore critical zoonotic threats, and highlight the need for comprehensive, full-spectrum microbial analyses of previously understudied organs to better assess spillover risks from bat populations.

 Author summary

Although extensive investigations have been conducted on the bat virome, most studies have focused on fecal samples, leaving other tissues, such as the kidney, largely unexplored. However, the kidney can harbor important zoonotic pathogens, including the highly pathogenic Hendra and Nipah viruses, and genomic evidence of henipaviruses in bats from China has remained undocumented. In this study, we report the first detection of two novel henipavirus genomes from bat kidneys in China, one of which is the closest known relative of pathogenic henipaviruses identified to date. Beyond virome analysis, our study also examined highly prevalent bacteria and eukaryotic microbes, identifying those potentially relevant to bat infections. Overall, these findings provide valuable insights into the infectome of the bat kidney, highlighting the need for broader microbial surveillance beyond the gastrointestinal tract.

 the National Key R&D Program of China 2024YFC2607501 https://orcid.org/0000-0002-6154-4437 ShiMang the National Key R&D Program of China 2024YFC2607502 https://orcid.org/0000-0002-6154-4437 ShiMang Yunnan Revitalization Talent Support Program Top Physician Project XDYC-MY-2022-0074 https://orcid.org/0000-0002-7354-0513 FengYun the National Natural Science Foundation of China 82341118 https://orcid.org/0000-0002-6154-4437 ShiMang Natural Science Foundation of Guangdong Province of China 2022A1515011854 https://orcid.org/0000-0002-6154-4437 ShiMang Shenzhen Science and Technology Program KQTD20200820145822023 https://orcid.org/0000-0002-6154-4437 ShiMang Major Project of Guangzhou National Laboratory GZNL2023A01001 https://orcid.org/0000-0002-6154-4437 ShiMang Guangdong Province “Pearl River Talent Plan” Innovation, Entrepreneurship Team Project 2019ZT08Y464 https://orcid.org/0000-0002-6154-4437 ShiMang the Fund of Shenzhen Key Laboratory ZDSYS20220606100803007 https://orcid.org/0000-0002-6154-4437 ShiMang National Health & Medical Research Council (NHMRC) Investigator grant GNT2017197 HolmesEdward C. AIR@InnoHK administered by the Innovation and Technology Commission, Hong Kong Special Administrative Region, China. HolmesEdward C. This study was funded by grants from the National Key R&D Program of China (2024YFC2607501 & 2024YFC2607502 to M.S.), Yunnan Revitalization Talent Support Program Top Physician Project (XDYC-MY-2022-0074 to Y.F.), the National Natural Science Foundation of China (82341118 to M.S.), Natural Science Foundation of Guangdong Province of China (2022A1515011854 to M.S.), Shenzhen Science and Technology Program (KQTD20200820145822023 to M.S.), Major Project of Guangzhou National Laboratory (GZNL2023A01001 to M.S.), Guangdong Province “Pearl River Talent Plan” Innovation, Entrepreneurship Team Project (2019ZT08Y464 to M.S.), and the Fund of Shenzhen Key Laboratory (ZDSYS20220606100803007 to M.S.), National Health & Medical Research Council (NHMRC) Investigator grant (GNT2017197 to E.C.H.) and AIR@InnoHK administered by the Innovation and Technology Commission, Hong Kong Special Administrative Region, China (to E.C.H.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data Availability The meta-transcriptomic sequencing reads generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) database under BioProject accession PRJNA1184956. The whole genome sequences of viruses generated in this study are available at NCBI/GenBank under accession numbers PQ621837 to PQ621860, PQ815815, and PQ824231. Introduction

Bats (order Chiroptera) are one of the most diverse and abundant groups of mammals, comprising nearly 1,500 species with a near global distribution [1]. Bats are also well-known natural reservoirs for a wide variety of microbial pathogens, a characteristic often attributed to their unique immune systems which maintain a delicate balance between host defenses and immune tolerance to viral infections [2–4]. Importantly, bats have been implicated in a number of major emerging disease outbreaks, including Hendra [5], Nipah [6], Marburg and Ebola [7] virus disease, severe acute respiratory syndrome (SARS) [8], Middle East respiratory syndrome (MERS) [8], and coronavirus disease 2019 (COVID-19) [9]. Indeed, comparative studies indicate that bats harbor a greater diversity of viruses than many other mammalian groups, underscoring their significance for zoonotic disease surveillance [10].

Metagenomic approaches have greatly advanced the characterization of bat viromes, deepening our understanding of the diversity of bat-borne pathogens and their potential role in disease emergence and transmission [11–14]. As of October 2024, viral sequences from at least 31 families have been identified in 340 bat species across 111 countries [15]. Bat-borne viruses are transmitted to humans either through direct contact with bats or via so-called “intermediate” hosts, often linked to the ingestion of food or water contaminated with bat saliva, feces, or urine [16]. Although most research has concentrated on the bat gut virome, viruses residing in other tissues, including the kidneys where they may be excreted via urine, also present potential transmission risks. Indeed, zoonotic viruses have been detected in bat kidneys and urine, including henipaviruses [17–20], pararubulaviruses [20–25], and betacoronaviruses [25]. As these kidney-associated pathogens can be excreted through urine they are at heightening risk of human exposure.

Beyond viruses, bats harbor a diverse array of bacteria, fungi, and protozoan parasites that infect bats or even humans [26,27]. A notable example is the psychrophilic fungus Pseudogymnoascus destructans, which has caused a devastating disease in bats and led to the deaths of millions of animals across eastern North America [27]. Although this fungus is not known to pose a direct threat to humans, disturbances during bat hibernation—such as flying during the day and gathering near cave and mine entrances in winter—may increase human-bat encounters. Additionally, zoonotic bacteria and protozoan parasites, such as members of Leptospira [28,29] and Toxoplasma [30], have been identified in bat kidneys. However, as with viruses, research on the bacteria and eukaryotic pathogens in bat kidneys remain sparse, highlighting a critical gap in our understanding of the diversity of bat pathogens.

Yunnan province, located in southwestern China and bordering a number of Southeast Asian countries, is renowned as a hotspot for bat diversity and bat-borne viral pathogens, including close relatives of Marburg virus [31], SARS-CoV [32,33], and SARS-CoV-2 [9,13,34]. Herein, we utilized a meta-transcriptomics approach to investigate the total infectome—comprising viruses, bacteria, and eukaryotic microbes—in bat kidneys collected from this geographic region. We further identified and characterized potential human pathogens of notable zoonotic risk and explored interactions between viruses and their protozoan parasite hosts, offering valuable insights into the complexity of the bat kidney infectome.

 Results Bat species identification

Between 2017 and 2021, kidney tissues were sampled from 142 individual bats across five cities/counties in Yunnan province, China (Fig 1A and S1 Table). Species identification was initially performed by recovering partial cytochrome c oxidase I (cox1) gene sequences using targeted PCR assay and Sanger sequencing. Phylogenetic analysis of full-length cox1 sequences, generated from meta-transcriptomic sequencing, confirmed the presence of ten bat species spanning five genera and three families (Fig 1B and S1 Table). Based on mitochondrial sequences and sampling locations, the samples were pooled into 20 groups for sequencing library constructions, with each group containing 2–8 individuals (S1 Table). Meta-transcriptomic sequencing of total RNA extracted from these pools yielded an average of 56.33 million clean non-rRNA reads, totaling approximately 1.13 billion clean non-rRNA reads.

10.1371/journal.ppat.1013235.g001Bat kidney sampling and species identification.

(A) Map showing the five sampling locations in Yunnan province, China, with nearby countries (Myanmar, Laos, Vietnam, Thailand, Cambodia, and Malaysia) shown for reference. Pie charts indicate the species composition of the bats sampled at each site. The basemap shapefile used in ArcGIS was obtained from the publicly available GADM data set (https://gadm.org/download_country.html). (B) Unrooted phylogenetic tree inferred from full-length COX1 gene sequences of bat kidney samples analyzed in this study. Colors correspond to different bat species, matching the color scheme used in the pie charts. Branch lengths are scaled to the number of nucleotide substitutions per site.

 Overview of the Yunnan bat kidney infectome

Meta-transcriptomic analysis of the bat kidneys identified a diverse microbial community (Fig 2 and S2 Table). Based on our detection criteria (see Methods), microbes were detected in 18 of the 20 libraries analyzed, comprising 0.06% to 1.28% of total clean non-rRNA reads per library (Fig 2A). Two libraries—one from Hipposideros armiger (8 individuals, LS) and another from Rhinolophus stheno (2 individuals, SB)—showed no microbial presence. RNA viruses dominated the microbial community, with 20 species from 12 families identified, as well as one DNA virus, one reverse transcribing virus, two bacterial species, and one eukaryotic species (Fig 2B). Among these, the eukaryote genus Phyllobacterium was the most frequently detected, present in 13 (65%) libraries. Notably, one library contains a total of 12 microbial species, all of which were viruses (Fig 2C).

10.1371/journal.ppat.1013235.g002Overview of the bat kidney infectome.

(A) Numbers of total reads (light blue) and microbial reads (orange) for each library. (B) Number of viral, bacterial, and eukaryotic microbial species detected, with color schemes corresponding to those used in panel C. (C) Heatmap illustrating the distribution and relative abundance of viral, bacterial, and eukaryotic microbes, represented as RNA abundance (RPM: reads per million non-rRNA reads) in each library. Host species and orders are labeled at the top and color-coded according to their respective categories.

 Virome of bat kidneys

We identified 22 viral species across 12 families in bat kidneys (Figs 2 and 3). These included six RNA viruses from the Paramyxoviridae, three from the Totiviridae, two each from the Partitiviridae, Solemoviridae, and Narnaviridae, and one each from the Phenuiviridae, Chuviridae, Nodaviridae, Picornaviridae, and Tombusviridae. Additionally, we discovered one DNA virus from the Parvoviridae and one reverse transcribing virus from the Hepadnaviridae (Figs 2 and 3). Of these, 20 species (90.91%) spanning 10 families were newly identified per ICTV (International Committee on Virus Taxonomy) species demarcation criteria (S2 Table).

10.1371/journal.ppat.1013235.g003Phylogenetic diversity of viruses identified in this study.

Phylogenetic trees of viruses from 12 virus families estimated using the maximum likelihood method based on conserved protein sequences (RdRp for RNA viruses, NS1 for Parvoviridae, and DNA polymerase for Hepadnaviridae). Colored dots on the trees, corresponding to host genera as indicated in the legend, represent viral species identified in this study. Red stars mark members of known mammal-associated viral lineages. All trees are mid-pointed rooted for clarity only with horizontal branch lengths depicting the number of amino acid substitutions per site.

Phylogenetic analyses revealed that nine species (40.91%) were related to known mammal-associated viruses, representing one reverse transcribing virus, one DNA virus and seven RNA viruses (Fig 3). The Paramyxoviridae exhibited the highest diversity, with two species from the genus Henipavirus and four from the genus Jeilongvirus identified. Notably, the two newly identified henipaviruses had a relatively close evolutionary relationship two human pathogens—Hendra virus (HeV, 52.23–56.94% amino acid identity in the L protein) and Nipah virus (NiV, 52.17–57.03% amino acid identity) (Fig 3). In addition, we identified a hepatotropic virus (Hepadnaviridae, genus Orthohepadnavirus), denoted Bat hepatitis virus variant YNBS16, in bat kidneys. Phylogenetic analysis revealed that this sequence was closely related to an hepadnavirus sequence, ZYPR16 (Clade BtHBV 7), previously identified in bat livers (S1 Fig).

There was also considerable variation in diversity among viral families (Figs 2B, C and 3). While the Paramyxoviridae dominated the samples obtained, in many cases the highest abundance members of this family were not associated with the infection of vertebrates, including Yunnan narnavirus 1 and 2 (Narnaviridae) and Yunnan totivirus 1–4 (Totiviridae). Yunnan totivirus 1 was especially abundant in pools YNBS16 (RPM = 7393.98) and YNBS17 (RPM = 4171.90) (Fig 2C), indicating that its presence was unlikely due to environmental contamination or dietary origin.

 Characterization of newly identified henipaviruses

Among the viruses identified, we focused on those with potential emergence risks based on their phylogenetic relationship to known high-impact human pathogens, specifically Yunnan bat henipavirus 1 and 2. Of the 20 pooled libraries, one (YNBS03) was positive for Yunnan bat henipavirus 1, while two (YNBS02 and YNBS04) contained reads corresponding to Yunnan bat henipavirus 2. These positive pools were all derived from the kidneys of Rousettus leschenaultii bats inhabiting an orchard near villages in RL (WD) (Fig 2C).

Using henipavirus genome sequences assembled from these libraries, primers were designed to further examine individual kidneys through qRT-PCR. The results revealed that one kidney from pool YNBS03 (sample WDBS1745), one from YNBS02 (sample WDBS1733), and two from pool YNBS04 (samples WDBS1762 and WDBS1769) tested positive for henipavirus. Further testing of other organs (heart, liver, lung, gut, and brain) from the same individuals (WDBS1733 and WDBS1745) using qRT-PCR and meta-transcriptomic sequencing confirmed the multi-organ presence of henipaviruses within these bats, with the exception of brain tissues (Table 1). Notably, the kidneys exhibited significantly higher viral abundance compared to other organs, suggesting that they are the primary site of henipavirus replication within the host.

10.1371/journal.ppat.1013235.t001Detection of henipavirus in various organs within individual bats.
Library Organ Virus query Length (bp) Number of reads qRT-PCR (Ct) Nested- PCR
WDBN1745 brain Yunnan bat henipavirus 1 19755 0 NoCt –
WDBX1745 heart Yunnan bat henipavirus 1 19755 0 NoCt –
WDBG1745 liver Yunnan bat henipavirus 1 19755 0 NoCt –
WDBF1745 lung Yunnan bat henipavirus 1 19755 16 33.35 –
WDBC1745 gut Yunnan bat henipavirus 1 19755 0 NoCt –
WDBS1745 kidney Yunnan bat henipavirus 1 19755 3953 23.82 +
WDBN1733 brain Yunnan bat henipavirus 2 17723 N/A NoCt –
WDBX1733 heart Yunnan bat henipavirus 2 17723 16 31.37 +
WDBG1733 liver Yunnan bat henipavirus 2 17723 15 26.04 +
WDBF1733 lung Yunnan bat henipavirus 2 17723 20 30.34 +
WDBC1733 gut Yunnan bat henipavirus 2 17723 29 30.74 +
WDBS1733 kidney Yunnan bat henipavirus 2 17723 161657 16.91 +

The complete genomes of Yunnan bat henipavirus 1 and 2 were successfully assembled from individual kidney samples WDBS1745 and WDBS1733, achieving mean sequencing depths of 27.99X and 1,274.77X, respectively (Fig 4A). These two sequences were designated as Yunnan bat henipavirus 1 variant WDBS1745 and Yunnan bat henipavirus 2 variant WDBS1733. The open reading frames (ORFs) and gene arrangements of both viruses were consistent with other members of the genus Henipavirus, with each encoding six proteins (Fig 4A).

10.1371/journal.ppat.1013235.g004Characterization of the novel henipavirus species examined in this study.

(A) Genome organization and sequencing coverage of two novel henipavirus species. Coverage across the full-length genome is displayed, with open reading frames (ORFs) depicted as colored arrows below the coverage plots. Regions confirmed by Sanger sequencing for Yunnan bat henipavirus 1 are marked with a red bar beneath the coverage graph. (B) Maximum likelihood phylogenetic trees estimated using amino acid sequences of each gene within the genus Henipavirus, rooted with J-virus. Color blocks indicate different species groups, and newly identified viruses are marked with solid red circles. All trees are mid-pointed rooted for clarity only with horizontal branch lengths depicting the number of amino acid substitutions per site.

Phylogenetic analysis of all six genes revealed a clear separation between the predominantly rodent-associated and bat/human-associated clades of the genus Henipavirus (Fig 4B). Notably, the newly identified viruses formed distinct lineage, generally grouping with other bat-hosted henipaviruses, including the zoonotic pathogens HeV and NiV, both known for their high mortality rates in humans [35] Yunnan bat henipavirus 1 was most closely related to HeV and NiV in the N (70.33–71.33% amino acid identity) and L proteins (56.94–57.03% amino acid identity), which underscores its potential risk as an emerging pathogen (Fig 4B). However, the phylogenetic positions of Yunnan bat henipavirus 1 and 2 showed marked variability. In particular, Yunnan bat henipavirus 1 was most closely related to HeV and NiV in the N and L proteins trees, but occupied variable positions in the other trees. Although the bootstrap support for these groupings was generally weak, the topological movement of Yunnan bat henipavirus 1 among the henipaviruses likely reflects the action of recombination. Alternatively, the topological inconsistency may result from high sequence divergence, leading to substitution saturation, increased phylogenetic noise, and hence fewer informative sites for reliable inference. Conversely, the phylogenetic positions of Yunnan bat henipavirus 2 was more consistent across gene trees and also exhibited a greater divergence from other bat henipaviruses.

 Identification and characterization of abundant bacteria in bat kidneys

Our meta-transcriptomic analysis revealed the presence of two relatively abundant bacterial taxa, Flavobacterium and Phyllobacterium, with conserved marker genes (rpoB, groEL, recA, and gyrB) identified via the BLASTx analysis of assembled contigs (S3 Table). Both taxa were represented by assembled contigs with high sequence coverage and depth, enabling the confident reconstruction of marker genes for phylogenetic analysis. Specifically, the representative rpoB gene of Flavobacterium was assembled from group YNBS16, with a mean coverage of 76.8% and an average depth of 3.30X, while the representative groEL gene of Phyllobacterium was recovered from group YNBS01, with 91.9% coverage and a mean depth of 5.29X. Phylogenetic analysis revealed that the Flavobacterium species forms a distinct branch closely related to Flavobacterium ammonificans (94.12% nucleic acid identity in the ropB gene) (Fig 5A). This bacterium was tentatively classified as a novel species and named Flavobacterium yunnanensis. Similarly, the Phyllobacterium species was confirmed as Phyllobacterium calauticae based on 97.30% nucleic acid identity and phylogenetic placement of the groEL gene (Fig 5B). Further transcriptomic profiling across all 20 pools demonstrated diverse gene expression patterns for these bacteria (Fig 5C), indicating that they are metabolically active within the bat hosts.

10.1371/journal.ppat.1013235.g005Gene expression profiles, prevalence and identification of the two bacterial microbes.

(A) Maximum likelihood phylogenetic tree of the genus Flavobacterium, constructed using the rpoB gene. (B) Maximum likelihood phylogenetic tree of the genus Phyllobacterium, constructed using the groEL gene. (C) Top 25 expressed genes (measured in RPM) for Flavobacterium yunnanensis and Phyllobacterium calauticae in pools YNBS16 and YNBS01, respectively (left panel), compared with their expression in other positive pools (right panel).

 Eukaryotic microbe identified in bat kidneys

Analysis of the cox1 and cytochrome b (cytB) genes identified a protozoan microbe closely related to the Klossiella equi of the family Klossiellidae (phylum Apicomplexa), known to infect the kidney of horses [36]. Phylogenetic and sequence divergence analyses revealed 87.7% nucleotide identity to K. equi (MH203050.1) in the COX1 gene and 91.4% identity in the cytB gene. Based on these findings, the newly identified protozoan was proposed as a novel species, tentatively named Klossiella yunnanensis (Fig 6A, B). Klossiella mitochondrial reads were detected in six pools, exhibiting uneven gene expression levels across different libraries (Fig 6C).

10.1371/journal.ppat.1013235.g006Identification and characterization of a eukaryotic microbe.

(A, B) Phylogenetic trees of Klossiella, estimated using nucleotide sequences of the cox1 gene (a) and cytb gene (b). Colored dots indicate newly identified eukaryotic species, with colors corresponding to host genera. (C) Transcriptomic profiles of the Klossiella mitochondrion, represented as RPM, across positive pools. (D) Spearman’s correlation analysis showing the relationship between the total relative abundance (RPM) of totiviruses, narnaviruses, and Klossiella yunnanensis.

Interestingly, members of the viral families Totiviridae and Narnaviridae, known to naturally infect protozoa or fungi, showed co-occurrence with K. yunnanensis (Fig 2C). To explore this relationship, we analyzed the correlation between the relative abundances (in RPM) of these viruses and Klossiella across the six positive pools. Strong positive correlations were observed, with Spearman’s ρ ranging from 0.83 to 0.94 (p < 0.05) (Fig 6D), suggesting that the viruses in these families are likely hosted by K. yunnanensis rather than by bats.

 Discussion

There have been many studies examining the presence of viruses, bacteria, and eukaryotic microbes (i.e., fungi and protozoan parasites) in various bat tissues, including the brain, lung, liver, rectum, feces, urine, throat, and fecal swabs [9,13,31–34,37,38]. In contrast, the infectome composition of kidneys has received comparatively little attention. Our meta-transcriptomic sequencing of bat kidneys revealed a diverse array of microorganisms, shedding light on the broader bat infectome. Although viruses were the predominant microbial group identified, only 9 of the 22 detected viral species were categorized as mammalian viruses. Notably, the mammal-associated viruses identified in the kidneys differed from those identified in the rectal tissues of the same individual bats [13]. These findings align with previous research showing that viruses from different families exhibit marked variation in their organ-specific distribution in bats [14]. As a consequence, these results underscore the importance of adopting a multi-organ approach to comprehensively understand the microbial diversity harbored by bats, particularly for identifying host-microbe interactions. Furthermore, considering that kidney-associated pathogens, such as henipaviruses, may be shed through urine [39,40], future research should incorporate both kidney and urine sampling to comprehensively evaluate pathogen shedding and the associated transmission risk.

Of particular note, our study identified two novel henipaviruses that cluster within the bat-associated clade of this genus, including the lineage containing the Hendra and Nipah viruses. Nipah virus (NiV) are lethal pathogens that cause severe diseases in humans, including acute respiratory distress and encephalitis, with a mortality rate of 35–75% [35,41]. Similarly, Hendra virus (HeV) has caused multiple fatal outbreaks in humans and horses, including the death of veterinarians [35]. These viruses are naturally hosted by fruit bats (Pteropus species) and are typically transmitted to humans through bat urine or saliva, often via contamination of food sources [39,40]. HeV and NiV were first identified in Australia and Malaysia, respectively, and associated with Pteropus and other bat species [5,6]. In this study, we identified two related henipaviruses in Rousettus leschenaultii bats, marking the first detection of full-length henipavirus genomes in bats from China. This finding is particularly significant as Yunnan province is a recognized hotspot for bat diversity [31–34] and is located in southwestern China, representing the region of China geographically closest to Malaysia, where NiV first emerged (Fig 1A). Previously, antibodies to Nipah or Nipah-like viruses have been reported in bats from multiple regions in China, including Yunnan, Guangdong, Hainan and Hubei provinces, suggesting potential exposure to such viruses [42]. A recent large-scale study of bat RNA viral metagenomes detected genomic fragments of Nipah virus in bats from Southwest China [43], highlighting the potential circulation risk of Nipah-like viruses. However, these findings were not confirmed by RT-PCR, and the absence of full-length genomes further underscores the importance of our study, which provides genome-scale evidence for the diversity of henipaviruses in China and their zoonotic risk. Notably, more distantly related viruses have been discovered in rodents and shrews, including Mojiang virus [44] and Langya virus [45], with the latter confirmed to infect humans. These findings highlight the significance of the continued surveillance and genomic characterization of henipaviruses in bats, which are critical for understanding their potential spillover risk.

We also identified at least one bacterial species prevalent in bat kidneys. While the gut microbiota of bats has been extensively studied, less attention has been given to those of other organs, including the kidneys [16]. Previous research identified Leptospira spp. in bat kidney, supporting the hypothesis that bat kidneys may serve as a reservoir for zoonotic Leptospira [28,29,46,47], and we previously detected pathogenic Leptospira in bat kidneys using nested PCR in individual tissue samples [48]. However, no Leptospira-associated reads were detected in the meta-transcriptomic sequencing of this study, possibly due to sample pooling which might obscure the detection of low-abundance microbes. Instead, we identified Flavobacterium and Phyllobacterium, of which Phyllobacterium calauticae exhibited relatively high abundance and prevalence (Figs 2 and 5). Phyllobacterium calauticae is an aerobic, motile bacterium isolated from microaerophilic freshwater sediments, adapted to efficiently utilize oxygen in low-oxygen environments [49]. This is not unprecedented, as Listeria monocytogenes, an environmentally ubiquitous bacterium, has previously been isolated from various wild animals, including bat kidneys [50,51]. Although these bacteria were relatively abundant, their biological significance in bats remains unclear. On one hand, their high abundance in tissue may suggest a pathogenic role. Alternatively, they may represent commensals or opportunistic colonizers, although their presence in the kidney is less probable.

Previous studies have shown that bats harbor a diverse range of protozoan parasites, some of which are capable of infecting humans [52]. However, there is only limited research on protozoan parasites present in bat kidneys. Toxoplasma gondii, a zoonotic protozoan parasite, has been detected in bats collected in Yunnan [53], and herein we identified a protozoan parasite, tentatively named Klossiella yunnanensis, in six (30%) of the libraries. Phylogenetic analysis suggests that K. yunnanensis is closely related to species known to infect horses, K. equi, which is generally considered non-pathogenic but can cause kidney alterations in cases of heavy infection [36]. The pathogenicity of this eukaryotic parasite to humans or even bats remains unclear. Moreover, the absence of a complete reference genome and the low sequence similarity to available references prevented the recovery of the full nuclear genome, preventing a more comprehensive genomic characterization of this parasite.

In addition, viruses from the families Totiviridae and Narnaviridae [54,55], which are known to infect a wide range of non-vertebrate host types including protozoan parasites, were detected in high abundance in bat kidneys. Spearman’s correlation analysis of relative abundances indicated that these viruses were associated with K. yunnanensis rather than the bat hosts themselves (Fig 6D). This highlights the importance of conducting studies of the total infectome to better elucidate the interactions between viruses within an animal and their potential relationship with the primary host. Furthermore, we detected near-complete genomes of chuviruses in bat kidney tissues, which questions their usual assignment as viruses that do not infect vertebrates. While it is possible that these viruses infect eukaryotic microbes within the host, recent studies have increasingly identified chuviruses in mammals [14], suggesting that their host range may be broader than their previous designation as insect-specific viruses.

Our study has several limitations. Uneven sampling across locations and bat species—in which each species was sampled at only one or two sites—complicated our ability to assess virus distribution, compare viral compositions between species, and identify transmission networks. Although practical for broad surveys, pooling precludes resolving whether microbes originated from co-infections in single bats or from distinct individuals. Pooling may have also reduced sensitivity for detecting low-abundance microbes and potentially compromised the accuracy of microbial quantification. In addition, the lack of a complete genome assembly for the newly discovered eukaryotic parasite, coupled with the absence of a reference genome in existing databases, limited our ability to accurately quantify its abundance. The reliance on reference genomes from closely related species for abundance estimation may also introduce inaccuracies, particularly for divergent or poorly characterized taxa. These limitations collectively underscore the value of sequencing individual samples and integrating DNA-based approaches to obtain a more comprehensive view of the total infectome. Despite these limitations, our study offers the first comprehensive characterization of the bat kidney infectome, providing a foundation for more effective discovery and characterization of potential bat-borne pathogens.

 Materials and methods Ethics statement

This research, including the specimen collection and processing procedures, was reviewed and approved by the Ethics Committee of the Yunnan Institute of Endemic Disease Control and Prevention (File No. 20160002). All experiments were conducted with the approval of the Biosafety Committee of the same institute.

 Sample collection

Five sampling sites in Yunnan province were selected, denoted RL, ML, SB, LS, and JP (Fig 1A). From 2017 to 2021, bats were captured using mist nets (12 m × 2.5 m, mesh size 38 mm) deployed near caves and orchards during evening hours. To minimize stress, nets were inspected every 15–20 minutes by trained personnel. Only bats displaying signs of weakened vital functions were selected for dissection. They were first humanely euthanized via intracardiac injection of sodium pentobarbitone and subsequently dissected for internal organ collection, following approved ethical protocols. Initial identification of bat species was performed by experienced field biologists based on morphological characteristics. Captured bats were then transported to the laboratory, euthanized by intracardiac delivery of sodium pentobarbitone, and dissected. Kidney tissues were collected and stored at −80 °C until further analysis. Preliminary species identification was confirmed by sequencing the cytochrome c oxidase I (cox1) gene for each specimen [56]. Mammalian species confirmation was achieved using de novo assembled cox1 gene contigs. The final clean cox1 contigs were compared against the database within the BARCODE OF LIFE DATA SYSTEM (BOLDSYSTEMS) [57], and phylogenetic analyses were conducted using PHYML 3.0 [58] for species identification.

 Meta‑transcriptomic sequencing

Individual tissues were initially organized into sample groups based on species identification and collection location. Specifically, 142 kidney tissues were grouped into 20 libraries, each comprising 2–8 individuals (S1 Table). Total RNA was extracted and purified from each pool using the RNeasy Plus Universal Mini Kit (Qiagen, Germany). RNA libraries were constructed using the Zymo-Seq RiboFree Total RNA Library Kit (No. R3003) (Zymo Research, USA), following the manufacturer’s instructions. These libraries were sequenced using paired-end 150 bp reads on the Illumina NovaSeq 6000 sequencing platform.

 Characterization of total infectomes

Adapter sequences were removed from the sequencing reads, and initial quality control was performed using the pipeline implemented in bbduk.sh (https://sourceforge.net/projects/bbmap/). Duplicate reads were filtered out using cd-hit-dup with default settings [59]. rRNA reads were removed by mapping the processed reads against the SILVA rRNA database (Release 138.1) using Bowtie2 (version 2.3.5.1) in ‘--local’ mode [60]. The remaining high-quality, non-rRNA reads were either (i) directly compared against the non-redundant protein (nr) database using DIAMOND BLASTx [61], or (ii) assembled into contigs using MEGAHIT (version 1.2.8) [62] before comparison against the National Center for Biotechnology Information (NCBI) non-redundant protein (nr) database. An e-value threshold of 1 × 10−5 was set to maintain high sensitivity and minimize false positives.

For virus identification, contigs identified from the kingdom ‘Viruses’ were extracted, and those shorter than 600 bp were excluded to ensure the quality of virus genomes. The remaining overlapping contigs were merged into extended viral sequences using the SeqMan program implemented in the Lasergene software package version 7.1 (DNAstar, USA) [63]. To assign species-level classifications, all viral contigs were clustered using CD-HIT (v4.8.1) [59], applying identity thresholds based on ICTV species demarcation criteria for the corresponding viral genera [55]. Representative contigs from each cluster were then compared to known viral species to determine whether they matched previously recognized or potentially novel viruses. For genera lacking explicit species demarcation criteria, a 90% amino acid identity threshold for the RdRP or replicase protein was applied (S2 Table). The abundance of these viral contigs was estimated by mapping reads back to the assembled genomes using Bowtie2 version 2.5.2 with ‘--end-to-end’ and ‘--very-fast’ settings. Reads mapped to all contigs assigned to the same viral species were aggregated to calculate the final abundance of that species in each library. Alignments were sorted and indexed with SAMtools version 1.18 and visualized with Geneious Prime version 2020.2.4 [64,65].

For bacteria and eukaryotic microbes, we initially utilized MetaPhlAn version 4 to identify potential microbial taxonomy [66]. We then performed a de novo assembly of the reads using MEGAHIT (version 1.2.8) as described above [62]. Assembled contigs were compared against conserved bacterial marker genes (e.g., rpoB, groEL, recA, and gyrB) and eukaryotic microbial genes (e.g., EF1-alpha) using DIAMOND BLASTx [61]. Complete reference genome sequences of the corresponding bacterial and protozoan genera were subsequently downloaded from GenBank and used as templates for read mapping and gene abundance estimation with Bowtie2 (version 2.5.2) [60]. Highly conserved regions, such as rRNA genes, were excluded from the reference genome sequences before conducting mapping analyses. From the aligned reads, we generated consensus sequences for well-covered protein-coding regions, with a focus on phylogenetically informative loci such as rpoB and groEL, which provide high species-level resolution in bacterial systematics. Finally, these consensus sequences were subsequently subjected to BLASTn comparisons against the NCBI nucleotide (nt) database to determine microbial taxonomy at the species level.

 Evolutionary analyses

To determine the evolutionary relationships of the newly identified microbes, reference nucleotide/amino acid sequences for microbial taxa in question were downloaded from the NCBI GenBank Database. In all cases, sequences were then aligned using MAFFT [67], with the 5’ and 3’ unaligned regions (when present) removed manually and ambiguously aligned sequences excluded using TrimAl version 1.5.0 [68]. Phylogenetic trees on these data were then estimated using the maximum likelihood method implemented in PHYML 3.0, employing the GTR model of nucleotide substitution and SPR branch swapping [58]. Node support was estimated using an approximate likelihood ratio test using Shimodaira–Hasegawa-like procedures.

 Characterization of henipaviruses

To assess the prevalence of novel henipaviruses in bats and in different organs, real-time quantitative reverse transcription PCR (qRT-PCR) and nested RT-PCR were performed on all individual kidney samples. Specific primers were designed using the virus genome sequences obtained from libraries YNBS03 (Yunnan bat henipavirus 1) and YNBS02 and YNBS04 (Yunnan bat henipavirus 2). To investigate viral distributions across various bat organs, PCR detection and individual meta-transcriptomics assays were performed on the brain, heart, liver, kidney, and gut sample of the positive bats (WD1733 and WD1745). However, the library construction for the brain sample from WDBN1733 failed.

As the full-length sequence of Yunnan bat henipaviruses 1 was not initially obtained, PCR assays and Sanger sequencing were employed to complete it. The final genome consensus sequences were confirmed by mapping the reads against draft genome sequences, and viral abundance was estimated based on the number of reads mapped to genome [60]. For each complete genomes, potential open reading frames (ORFs) and coding arrangements were predicted using ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/) and annotated by blastp program (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic trees for each gene were estimated following the standard protocol described above.

 Supporting information Maximum likelihood phylogenetic tree estimated using amino acid sequences of the DNA polymerase within the genus <italic>Orthohepadnavirus</italic> (<italic>Hepadnaviridae</italic>).

The newly identified virus in this study is marked with a solid red circle. Bat-derived viruses and their corresponding clades—determined according to divergence levels used by ICTV as species demarcation criteria—are labeled on the right.

(TIF)

 Information of sample group and RNA library in this study.

(XLSX)

 Viruses in bat kidneys identified in this study.

(XLSX)

 Summary of contigs with BLASTx hits to conserved bacterial marker genes.

(XLSX)

We wish to thank the local Centers for Disease Control and Prevention in five trapping sites for their assistance in specimen collection.

 References Simmons NB, Cirranello AL. Bat Species of the World: A taxonomic and geographic database. Version 1.6. 2024. https://zenodo.org/records/12802826 Calisher CH, Childs JE, Field HE, Holmes KV, Schountz T. Bats: important reservoir hosts of emerging viruses. Clin Microbiol Rev. 2006;19(3):531–45. doi: 10.1128/CMR.00017-06 16847084 Hayman DTS. Bats as Viral Reservoirs. Annu Rev Virol. 2016;3(1):77–99. doi: 10.1146/annurev-virology-110615-042203 27578437 Irving AT, Ahn M, Goh G, Anderson DE, Wang L-F. Lessons from the host defences of bats, a unique viral reservoir. Nature. 2021;589(7842):363–70. doi: 10.1038/s41586-020-03128-0 Halpin K, Young PL, Field HE, Mackenzie JS. Isolation of Hendra virus from pteropid bats: a natural reservoir of Hendra virus. J Gen Virol. 2000;81(Pt 8):1927–32. doi: 10.1099/0022-1317-81-8-1927 10900029 Chua KB, Koh CL, Hooi PS, Wee KF, Khong JH, Chua BH, et al. Isolation of Nipah virus from Malaysian Island flying-foxes. Microbes Infect. 2002;4(2):145–51. doi: 10.1016/s1286-4579(01)01522-2 11880045 Brainard J, Pond K, Hooper L, Edmunds K, Hunter P. Presence and Persistence of Ebola or Marburg Virus in Patients and Survivors: A Rapid Systematic Review. PLoS Negl Trop Dis. 2016;10(2):e0004475. doi: 10.1371/journal.pntd.0004475 26927697 Cui J, Li F, Shi Z-L. Origin and evolution of pathogenic coronaviruses. Nat Rev Microbiol. 2019;17(3):181–92. doi: 10.1038/s41579-018-0118-9 30531947 Zhou P, Yang X-L, Wang X-G, Hu B, Zhang L, Zhang W, et al. Addendum: A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020;588(7836):E6. doi: 10.1038/s41586-020-2951-z 33199918 Olival KJ, Hosseini PR, Zambrana-Torrelio C, Ross N, Bogich TL, Daszak P. Erratum: Host and viral traits predict zoonotic spillover from mammals. Nature. 2017;548(7669):612. doi: 10.1038/nature23660 29411779 Van Brussel K, Holmes EC. Zoonotic disease and virome diversity in bats. Curr Opin Virol. 2022;52:192–202. doi: 10.1016/j.coviro.2021.12.008 34954661 Wu Z, Yang L, Ren X, He G, Zhang J, Yang J, et al. Deciphering the bat virome catalog to better understand the ecological diversity of bat viruses and the bat origin of emerging infectious diseases. ISME J. 2016;10(3):609–20. doi: 10.1038/ismej.2015.138 26262818 Wang J, Pan Y-F, Yang L-F, Yang W-H, Lv K, Luo C-M, et al. Individual bat virome analysis reveals co-infection and spillover among bats and virus zoonotic potential. Nat Commun. 2023;14(1):4079. doi: 10.1038/s41467-023-39835-1 37429936 Chen Y-M, Hu S-J, Lin X-D, Tian J-H, Lv J-X, Wang M-R, et al. Host traits shape virome composition and virus transmission in wild small mammals. Cell. 2023;186(21):4662–4675.e12. doi: 10.1016/j.cell.2023.08.029 37734372 Zhou S, Liu B, Han Y, Wang Y, Chen L, Wu Z, et al. ZOVER: the database of zoonotic and vector-borne viruses. Nucleic Acids Res. 2022;50(D1):D943–9. doi: 10.1093/nar/gkab862 34634795 Han H-J, Wen H, Zhou C-M, Chen F-F, Luo L-M, Liu J, et al. Bats as reservoirs of severe emerging infectious diseases. Virus Res. 2015;205:1–6. doi: 10.1016/j.virusres.2015.05.006 25997928 Halpin K, Hyatt AD, Fogarty R, Middleton D, Bingham J, Epstein JH, et al. Pteropid bats are confirmed as the reservoir hosts of henipaviruses: a comprehensive experimental study of virus transmission. Am J Trop Med Hyg. 2011;85(5):946–51. doi: 10.4269/ajtmh.2011.10-0567 22049055 Marsh GA, de Jong C, Barr JA, Tachedjian M, Smith C, Middleton D, et al. Cedar virus: a novel Henipavirus isolated from Australian bats. PLoS Pathog. 2012;8(8):e1002836. doi: 10.1371/journal.ppat.1002836 22879820 Barr J, Smith C, Smith I, de Jong C, Todd S, Melville D, et al. Isolation of multiple novel paramyxoviruses from pteropid bat urine. J Gen Virol. 2015;96(Pt 1):24–9. doi: 10.1099/vir.0.068106-0 25228492 Johnson RI, Tachedjian M, Rowe B, Clayton BA, Layton R, Bergfeld J, et al. Erratum: Johnson et al. Alston Virus, a Novel Paramyxovirus Isolated from Bats Causes Upper Respiratory Tract Infection in Experimentally Challenged Ferrets. Viruses 2018, 10, 675. Viruses. 2021;13(7):1204. doi: 10.3390/v13071204 34201906 Barr JA, Smith C, Marsh GA, Field H, Wang L-F. Evidence of bat origin for Menangle virus, a zoonotic paramyxovirus first isolated from diseased pigs. J Gen Virol. 2012;93(Pt 12):2590–4. doi: 10.1099/vir.0.045385-0 22915696 Baker KS, Todd S, Marsh GA, Crameri G, Barr J, Kamins AO, et al. Novel, potentially zoonotic paramyxoviruses from the African straw-colored fruit bat Eidolon helvum. J Virol. 2013;87(3):1348–58. doi: 10.1128/JVI.01202-12 23152534 Baker KS, Tachedjian M, Barr J, Marsh GA, Todd S, Crameri G, et al. Achimota Pararubulavirus 3: A New Bat-Derived Paramyxovirus of the Genus Pararubulavirus. Viruses. 2020;12(11):1236. doi: 10.3390/v12111236 33143230 Federici L, Masulli M, De Laurenzi V, Allocati N. An overview of bats microbiota and its implication in transmissible diseases. Front Microbiol. 2022;13:1012189. doi: 10.3389/fmicb.2022.1012189 36338090 Rizzo F, Edenborough KM, Toffoli R, Culasso P, Zoppi S, Dondo A, et al. Coronavirus and paramyxovirus in bats from Northwest Italy. BMC Vet Res. 2017;13(1):396. doi: 10.1186/s12917-017-1307-x 29273042 Dhivahar J, Parthasarathy A, Krishnan K, Kovi BS, Pandian GN. Bat-associated microbes: Opportunities and perils, an overview. Heliyon. 2023;9(12):e22351. doi: 10.1016/j.heliyon.2023.e22351 38125540 US Fish and Wildlife Service. North American bat death toll exceeds 5.5 million from white-nose syndrome. US Fish & Wildlife Service news release; 17 January 2012. http://www.fws.gov Bevans AI, Fitzpatrick DM, Stone DM, Butler BP, Smith MP, Cheetham S. Phylogenetic relationships and diversity of bat-associated Leptospira and the histopathological evaluation of these infections in bats from Grenada, West Indies. PLoS Negl Trop Dis. 2020;14(1):e0007940. doi: 10.1371/journal.pntd.0007940 31961893 Ramos-Nino ME, Fitzpatrick DM, Eckstrom KM, Tighe S, Dragon JA, Cheetham S. The Kidney-Associated Microbiome of Wild-Caught Artibeus spp. in Grenada, West Indies. Animals (Basel). 2021;11(6):1571. doi: 10.3390/ani11061571 34072244 Zamora-Vélez A, Cuadrado-Ríos S, Hernández-Pinsón A, Mantilla-Meluk H, Gómez-Marín JE. First Detection of Toxoplasma gondii DNA in a Wild Bat from Colombia. Acta Parasitol. 2020;65(4):969–73. doi: 10.2478/s11686-020-00222-1 32436053 He B, Hu T, Yan X, Pa Y, Liu Y, Liu Y, et al. Isolation, characterization, and circulation sphere of a filovirus in fruit bats. Proc Natl Acad Sci U S A. 2024;121(7):e2313789121. doi: 10.1073/pnas.2313789121 38335257 Ge X-Y, Li J-L, Yang X-L, Chmura AA, Zhu G, Epstein JH, et al. Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor. Nature. 2013;503(7477):535–8. doi: 10.1038/nature12711 24172901 Hu B, Zeng L-P, Yang X-L, Ge X-Y, Zhang W, Li B, et al. Discovery of a rich gene pool of bat SARS-related coronaviruses provides new insights into the origin of SARS coronavirus. PLoS Pathog. 2017;13(11):e1006698. doi: 10.1371/journal.ppat.1006698 29190287 Zhou H, Ji J, Chen X, Bi Y, Li J, Wang Q, et al. Identification of novel bat coronaviruses sheds light on the evolutionary origins of SARS-CoV-2 and related viruses. Cell. 2021;184(17):4380-4391.e14. doi: 10.1016/j.cell.2021.06.008 34147139 Aljofan M. Hendra and Nipah infection: emerging paramyxoviruses. Virus Res. 2013;177(2):119–26. doi: 10.1016/j.virusres.2013.08.002 23954578 Léveillé AN, Bland SK, Carlton K, Larouche CB, Kenney DG, Brouwer ER, et al. Klossiella equi infecting kidneys of Ontario horses: life cycle features and multilocus sequence-based genotyping confirm the genus Klossiella belongs in the Adeleorina (Apicomplexa: Coccidia). J Parasitol. 2019. Karunarathna SC, Dong Y, Karasaki S, Tibpromma S, Hyde KD, Lumyong S, et al. Discovery of novel fungal species and pathogens on bat carcasses in a cave in Yunnan Province, China. Emerg Microbes Infect. 2020;9(1):1554–66. doi: 10.1080/22221751.2020.1785333 32573334 Cai Y, Wang X, Zhang N, Li J, Gong P, He B, et al. First report of the prevalence and genotype of Trypanosoma spp. in bats in Yunnan Province, Southwestern China. Acta Trop. 2019;198:105105. doi: 10.1016/j.actatropica.2019.105105 31348896 Luby SP, Rahman M, Hossain MJ, Blum LS, Husain MM, Gurley E, et al. Foodborne transmission of Nipah virus, Bangladesh. Emerg Infect Dis. 2006;12(12):1888–94. doi: 10.3201/eid1212.060732 17326940 Luby SP, Hossain MJ, Gurley ES, Ahmed BN, Banu S, Khan SU, et al. Recurrent zoonotic transmission of Nipah virus into humans, Bangladesh, 2001-2007. Emerg Infect Dis. 2009;15(8):1229–35. doi: 10.3201/eid1508.081237 19751584 Sawatsky B, Grolla A, Kuzenko N, Weingartl H, Czub M. Inhibition of henipavirus infection by Nipah virus attachment glycoprotein occurs without cell-surface downregulation of ephrin-B2 or ephrin-B3. J Gen Virol. 2007;88(Pt 2):582–91. doi: 10.1099/vir.0.82427-0 17251577 Li Y, Wang J, Hickey AC, Zhang Y, Li Y, Wu Y, et al. Antibodies to Nipah or Nipah-like viruses in bats, China. Emerg Infect Dis. 2008;14(12):1974–6. doi: 10.3201/eid1412.080359 19046545 Yan X, Liu Y, Hu T, Huang Z, Li C, Guo L, et al. A compendium of 8,176 bat RNA viral metagenomes reveals ecological drivers and circulation dynamics. Nat Microbiol. 2025;10(2):554–68. doi: 10.1038/s41564-024-01884-7 39833544 Wu Z, Yang L, Yang F, Ren X, Jiang J, Dong J, et al. Novel Henipa-like virus, Mojiang Paramyxovirus, in rats, China, 2012. Emerg Infect Dis. 2014;20(6):1064–6. doi: 10.3201/eid2006.131022 24865545 Zhang X-A, Li H, Jiang F-C, Zhu F, Zhang Y-F, Chen J-J, et al. A Zoonotic Henipavirus in Febrile Patients in China. N Engl J Med. 2022;387(5):470–2. doi: 10.1056/NEJMc2202705 35921459 Vashi NA, Reddy P, Wayne DB, Sabin B. Bat-associated leptospirosis. J Gen Intern Med. 2010;25(2):162–4. doi: 10.1007/s11606-009-1210-7 20012224 Ballados-González GG, Sánchez-Montes S, Romero-Salas D, Colunga Salas P, Gutiérrez-Molina R, León-Paniagua L, et al. Detection of pathogenic Leptospira species associated with phyllostomid bats (Mammalia: Chiroptera) from Veracruz, Mexico. Transbound Emerg Dis. 2018;65(3):773–81. doi: 10.1111/tbed.12802 29318786 Yang T, Yang W, Kuang G, Pan H, Han X, Yang L, et al. Prevalence and Characteristics of Novel Pathogenic Leptospira Species in Bats in Yunnan Province, China. Microorganisms. 2023;11(6):1619. doi: 10.3390/microorganisms11061619 37375121 Lustermans JJM, Bjerg JJ, Schramm A, Marshall IPG. Phyllobacterium calauticae sp. nov. isolated from a microaerophilic veil transversed by cable bacteria in freshwater sediment. Antonie Van Leeuwenhoek. 2021;114(11):1877–87. doi: 10.1007/s10482-021-01647-y 34491484 Höhne K, Loose B, Seeliger HP. Isolation of Listeria monocytogenes in slaughter animals and bats of Togo (West Africa). Ann Microbiol (Paris). 1975;126A(4):501–7. 812407 Povolyaeva O, Chalenko Y, Kalinin E, Kolbasova O, Pivova E, Kolbasov D, et al. Listeria monocytogenes Infection of Bat Pipistrellus nathusii Epithelial cells Depends on the Invasion Factors InlA and InlB. Pathogens. 2020;9(11):867. doi: 10.3390/pathogens9110867 33105852 Santana Lima VF, Rocha PA, Dias Silva MA, Beltrão-Mendes R, Ramos RAN, Giannelli A, et al. Survey on helminths and protozoa of free-living Neotropical bats from Northeastern Brazil. Acta Trop. 2018;185:267–72. doi: 10.1016/j.actatropica.2018.06.002 29883575 Jiang HH, Qin SY, Wang W, He B, Hu TS, Wu JM, et al. Prevalence and genetic characterization of Toxoplasma gondii infection in bats in southern China. Vet Parasitol. 2014;203(3–4):318–21. doi: 10.1016/j.vetpar.2014.04.016 24813744 Goodman RP, Ghabrial SA, Fichorova RN, Nibert ML. Trichomonasvirus: a new genus of protozoan viruses in the family Totiviridae. Arch Virol. 2011;156(1):171–9. doi: 10.1007/s00705-010-0832-8 20976609 ICTV. The ICTV report on virus classification and taxon nomenclature. 2024. https://ictv.global/report IVANOVA NV, DEWAARD JR, HEBERT PDN. An inexpensive, automation‐friendly protocol for recovering high‐quality DNA. Molecular Ecology Notes. 2006;6(4):998–1002. doi: 10.1111/j.1471-8286.2006.01428.x Ratnasingham S, Hebert PDN. bold: The Barcode of Life Data System (http://www.barcodinglife.org). Mol Ecol Notes. 2007;7(3):355–64. doi: 10.1111/j.1471-8286.2007.01678.x 18784790 Guindon S, Dufayard J-F, Lefort V, Anisimova M, Hordijk W, Gascuel O. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol. 2010;59(3):307–21. doi: 10.1093/sysbio/syq010 20525638 Fu L, Niu B, Zhu Z, Wu S, Li W. CD-HIT: accelerated for clustering the next-generation sequencing data. Bioinformatics. 2012;28(23):3150–2. doi: 10.1093/bioinformatics/bts565 23060610 Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9(4):357–9. doi: 10.1038/nmeth.1923 22388286 Buchfink B, Xie C, Huson DH. Fast and sensitive protein alignment using DIAMOND. Nat Methods. 2015;12(1):59–60. doi: 10.1038/nmeth.3176 25402007 Li D, Liu C-M, Luo R, Sadakane K, Lam T-W. MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph. Bioinformatics. 2015;31(10):1674–6. doi: 10.1093/bioinformatics/btv033 25609793 Clewley JP. Macintosh sequence analysis software. DNAStar’s LaserGene. Mol Biotechnol. 1995;3(3):221–4. doi: 10.1007/BF02789332 7552691 Li H. A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data. Bioinformatics. 2011;27(21):2987–93. doi: 10.1093/bioinformatics/btr509 21903627 Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, et al. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012;28(12):1647–9. doi: 10.1093/bioinformatics/bts199 22543367 Segata N, Waldron L, Ballarini A, Narasimhan V, Jousson O, Huttenhower C. Metagenomic microbial community profiling using unique clade-specific marker genes. Nat Methods. 2012;9(8):811–4. doi: 10.1038/nmeth.2066 22688413 Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013;30(4):772–80. doi: 10.1093/molbev/mst010 23329690 Capella-Gutiérrez S, Silla-Martínez JM, Gabaldón T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics. 2009;25(15):1972–3. doi: 10.1093/bioinformatics/btp348 19505945 10.1371/journal.ppat.1013235.r001 Author response to Decision Letter 0 Submission Version 0

6 Feb 2025

 10.1371/journal.ppat.1013235.r002 Decision Letter 0 Obbard Darren Guest Editor 2025 Darren Obbard This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 0

19 Apr 2025

PPATHOGENS-D-25-00320

Infectome analysis of bat kidneys from Yunnan province, China, reveals close relatives of Hendra-Nipah viruses and prevalent bacterial and eukaryotic pathogens

PLOS Pathogens

Dear Dr. Feng,

Thank you for submitting your manuscript to PLOS Pathogens. After careful consideration, we feel that it has merit but does not fully meet PLOS Pathogens's publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript within 30 days Jun 18 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plospathogens@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/ppathogens/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

* A rebuttal letter that responds to each point raised by the editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. This file does not need to include responses to any formatting updates and technical items listed in the 'Journal Requirements' section below.

* A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

* An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, competing interests statement, or data availability statement, please make these updates within the submission form at the time of resubmission. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

Darren J. Obbard

Guest Editor

PLOS Pathogens

Ronald Swanstrom

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Additional Editor Comments:

Thank you for your submission. As you will see, all three reviewers agree on the value in this work, and feel that - overall - it is very well done. The manuscript is interesting, and relatively easy to read, and the novel henipavirus give the paper quite a broad level of interest.

In any revision, I ask that you take on board all of the reviewers' comments. I suspect the vast majority of comments should be relatively easy to address through textual changes (e.g. more stringent use of the word 'pathogen') or through a change in framing. While I would welcome more detailed analysis of non-viral microbiota and any further validation that might be possible, given the scale of the manuscript as it stands I recognise that further work along these lines might be beyond the scope of what is feasible.

Nevertheless, in addition to the scientific / analytical suggestions, I particularly want to draw your attention to the absolute need for

(1) A clear statement on the methodology and nets used for trapping (mist nets, sticky nets?) and how the approach used conforms to the relevant ethical standards,

and (2) Full availability of all raw and processed sequences under PRJNA1184956 - which is not yet publicly available.

Journal Requirements:

1) Please upload all main figures as separate Figure files in .tif or .eps format. For more information about how to convert and format your figure files please see our guidelines: 

https://journals.plos.org/plospathogens/s/figures

2) Please ensure that the funders and grant numbers match between the Financial Disclosure field and the Funding Information tab in your submission form. Note that the funders must be provided in the same order in both places as well.

- State the initials, alongside each funding source, of each author to receive each grant. For example: "This work was supported by the National Institutes of Health (####### to AM; ###### to CJ) and the National Science Foundation (###### to AM)."

- State what role the funders took in the study. If the funders had no role in your study, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.".

If you did not receive any funding for this study, please simply state: u201cThe authors received no specific funding for this work.u201d

Reviewers' Comments:

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: The manuscript by Kuang et al. investigates the total infectome present in bat kidneys collected from Yunnan, identifying close relatives of highly significant pathogens, including henipaviruses—BSL-4 level viruses—alongside abundant bacteria and parasites. While the study is limited in scale in terms of the number of animals and geographic coverage, its findings are highly valuable and of great interest to the virology community. Notably, the discovery of bat-associated henipaviruses is both novel and significant. Previous studies were either based solely on serological evidence or lacked reliability due to the absence of PCR confirmation. Another key strength of this work is its novel approach in characterizing the total infectome, rather than focusing solely on virome or specific viral families like coronaviruses, providing a more comprehensive perspective.

Reviewer #2: This article presents interesting data on the infectome of the kidneys of 10 different bat species in five regions of Yunnan Province in China (Fig1). New viruses, including some with zoonotic potential (henipaviruses), as well as a novel protozoan and 2 bacteria, were identified using a meta-transcriptomic approach (Fig2 and 3). The two henipaviruses were subsequently confirmed by qRT-PCR and nested RT-PCR testing of different tissues to determine virus distribution within 2 bats, one testing positive in the lung and kidney and the other testing positive in all tissues (heart, liver, kidney, gut) except the brain. The newly identified henipavirus 1 was present in 1 of the 20 libraries, and the henipavirus 2 was present in 2 of the 20 libraries, both corresponding to Rousettus leschenaultii and collected near areas with fruit orchards. Individual bat testing identified 4 bats carrying these henipaviruses, and within these bats, several tissues tested positive for the virus with the kidney showing the highest sequence yield (supplemental material), suggesting this is the target tissue tropism for these viruses. Characterization of the ORFs, identified the sequences for the 6 proteins found in henipaviruses, and phylogenetic analysis grouped these new viruses with other bat henipaviruses, including the zoonotic Hendra and Nipah. Observation of variable clustering depending on which of the 6 genes was being evaluated suggests recombination events (Fig4). The 2 novel bacterial species described include a Flavobacterium (94% nucleic acid identity in the ropB gene) and a Phyllobacterium (97% nucleic acid identity in the groEL gene) (Fig 5). The rationale for the selection of these genes was unclear, and the relevance of these findings was only briefly mentioned. It was noted that bacteria such as Leptospira were expected to be found in the kidneys of these bats, but weren’t. The protozoan from the Klossiellidae (Fig6) was present in 6 pools, and analysis of the COX1 and the cytB genes resulted in high similarity to Klosiella equi, an organism not usually considered pathogenic. The bat species identification was done by morphological characteristics and confirmed by genetic testing of COX1. Bats were pooled into 20 groups with variable numbers (2-8 individuals) for sequencing library construction. The authors acknowledge the drawback of sample pooling (lines 264 and 290) but also demonstrate the value of this approach. Two of the 20 libraries rendered no microbial presence. Library preparation, sequence curation, and phylogenetic analysis protocols described in the materials and methods seemed appropriate. The results of this study provide valuable information: The results indicated a preponderance of RNA viruses (20 species within 12 families), one DNA virus, one reverse-transcribing virus, one protozoan, and two abundant bacterial species (one of them present in 65% of the libraries). Nine of the virus species were phylogenetically related to known mammalian viruses. The methodology is consistent with other studies, the manuscript is well-organized and written, and the figures are clear.

Reviewer #3: Kuang and colleagues have investigated and reported the infectome of bat kidneys sampled in Yunnan Province, China. This manuscript is both timely and relevant, as it provides further evidence of novel microorganisms that infect wild animals, demonstrating specific tissue tropism and tissue specific viromes. The findings are particularly significant due to the identification of a divergent paramyxovirus that clusters within the same clade as lethal human pathogens such as Nipah and Hendra viruses. However, while the results are robust, the authors tend to overstate the pathogenic potential of the microorganisms identified. Throughout the manuscript, the microorganisms are frequently referred to as pathogens, despite the absence of experimental or indirect evidence—such as observed disease signs in animals—to support this classification. Follow additional comments below.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: 1. The authors should reference the recent publication in Nature Microbiology by He et al., which also reports the discovery of Nipavirus-like viruses in China. Although that study only identified viral fragments, and the authors of this manuscript failed to obtain longer genomic sequences using PCR, some discussion comparing the findings would be valuable.

2. The authors have done a thorough job in characterizing henipaviruses, making full use of the available data. However, the characterization of bacteria is relatively limited. While I understand that these may be of lesser priority, it would be valuable to perform single-library RNA and DNA sequencing on the samples with highest abundance of bacteria to obtain draft genomes. Any novel pathogens would merit revealing full genomes.

3. Similarly, the transcriptomic characterization of parasites is currently limited to mitochondrial genes—what about nuclear genes? Expanding this analysis would provide a more comprehensive understanding of the parasite component in the infectome.

Reviewer #2: Some clarification on the nets used for trapping is necessary. Can the type of net and source be provided?

Reviewer #3: Page 4 line 57 - The authors stated “broader pathogen surveillance beyond the gastrointestinal tract” A pathogen per definition is a microorganisms that induce disease. The authors have no data to infer pathogenicity even for the new henipaviruses. I recommend the authors to moderate their claims throughout the manuscript.

Data availability:

PRJNA1184956 - I could not find the raw dataset at NCBI SRA. Neither the consensus genomic data with the accession numbers provided.

Page 16 line 352-360 - Does the authors consider that mapping reads to reference bacterial and fungi genomes can severely bias the results? For the taxa that are closely related to reference genomes it should be ok once reads will map against with high confidence, but for more diverging taxa reads will not even map since Bowtie 2 has a limited number of snps allowed to map a read against a reference sequence. As pointed out by the authors, there is no study investigating bat kidneys using metagenomics/metatranscriptomics. Therefore, most of the bacterial and fungi species likely infecting these bats may be substantially divergent from the reference genomes available in the databases. I recommend to the authors to take a more holistic view of bacteria and fungi performing Diamond search on the assembled contigs from Megahit and extracting the taxonomic identification from it.

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: 4. Terms like “infectome” should be defined in the abstract as well.

5. The geographic locations and ecological factors (in any) of the newly found viruses should be discussed.

Reviewer #2: The references are adequate, but there are at least 3 references that are repeated (ref 21 is a repeat of 19, 49 is a repeat of 29, and 50 is a repeat of 30).

Reviewer #3: Abstract

How the authors can ascertain if the protozoan and bacteria found are relevant for infection/pathogenicity, maybe they are just highly prevalent non deleterious symbionts

Page 5 line 79-80 - The authors stated “While much of this research has focused on the bat gut virome, viruses present in other body sites—such as the kidneys—also pose transmission risks.” Those viruses present in the kidney are not supposed to be excreted through urine? That is the way Nipah is excreted and gets in contact with humans through a natural beverage contaminated with urine carrying infectious Nipah viral particles, for instance. One thing that may impact the chance of detecting the virus in the urine is that the viral particle is perhaps labile and degrades quickly and viral loads may not be that high compared with kidney tissue. An interesting strategy to sample wild bats and test this hypothesis which also can provide critical data to evaluate if the virus is being excreted is to sample bat urine and only after euthanasiate the bat to collect the kidney. But very good sampling procedures are required to avoid cross contamination of samples of the same bat in the field.

Figure 1 A - I think it would be worthwhile for the readers to see the Yunnan province contextualized within China and bordering countries. Besides, if the authors can also depict it including the geography and biome of the region it is interesting for the readers to understand bats species and populations residing in these environments and why they are more likely related to bat species and population of neighbouring countries and not to other Chinese provinces. Providing the context of neighbouring countries is particularly important because Nipah outbreaks have been reported in neighbouring countries.

Page 7 line 112 - “Based on mitochondrial sequences and sampling locations, the samples were pooled into 20 groups for” - Does it mean that samples from the same species and caves/sites were pooled together? What about samples of the same population from different time points or there were no resampling of the same population in different time points?

Figure 2 B - This is a very difficult graph to interpret. I suggest the authors look for an alternative way to show these results. In fact, these results are already shown in much more detail in section C. It is not the same metric, but the presence of viruses per taxonomic level distributed per host species is already there in much more detail.

Figure 2 B- How the authors determined that, for instance, Yannan bat henipavirus 2 what was present in different pools of H. armiger? Did these henipaviruses show high sequence identity? I have not seen a methodological description of how the authors approached the detection of the same virus in multiple samples in the material and methods. The only section about this regards the use of ICTV parameters, but it would be important to provide more detail stating for instance, that “we considered the same virus present in multiple samples when ???? clustered together in the phylogenetic tree and showed aa ou nucl identity of ??? in the entire genome??”

It is interesting that the authors found Chuvirus and other viruses in the kidney tissue of several different bat species and they did not comment on the possibility of these viruses to infect mammals, even though the current knowledge suggests that these viruses have other animals taxa (invertebrates etc) as main hosts. Finding a full genome of a virus within the kidney suggests that the virus may be infecting that tissue. On the other hand, it is hard to associate the presence of such viruses as of dietary sources as pointed out in page 8 line 154-155. Except for the Toti and Narna comments by the authors on Page 11 line 217.223.

Page 8 line 142-145 - amino acid ou nucleotide identity? L protein I assume amino acid. This is a substantial difference.

Page 10 line 193 - “likely reflects the action of recombination.“ or due to the high divergence it may represent the accumulation of several mutations leading to the phylogenetic signal saturation leaving few informative sites that allows more consistent phylogenetic grouping inferences. Please, add alternative explanations even more considering that the data is not suitable to evaluate recombination (divergent viruses).

Page 10 line 197 - “bacterial pathogens” How the authors know that these are pathogenic bacteria, this same question also applies to viruses and fungi found? Were there any signs of disease in the sampled bats? Although there is some indirect evidence that some may be pathogens (belonging to a family/taxa that include several known pathogens and are phylogenetically related to known pathogens) there is no other supporting evidence to suggest pathogenicity in this manuscript.

Page 12 line 239 - “our study identified close relatives”. Close relatives is a bit strong and arbitrary adjective based on the amino acid divergence and instability of the phylogenetic positioning depending on the protein used for phylogenetic reconstruction. I suggest the authors moderate it. The results are important and interesting, but those are not close relatives of Hendra and Nipah.

Page 12 line 246 - italicize Pteropus

Page 13 line 278-9 - “The pathogenicity of these eukaryotic parasites to humans or even bats remains unclear.” Here the authors emphasized that more studies are necessary to ascertain if the identified microbes are pathogenic. But I think the authors must review more throughout the wording and tone throughout the manuscript as pointed out multiple times above including in the title.

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

Figure resubmission:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. If there are other versions of figure files still present in your submission file inventory at resubmission, please replace them with the PACE-processed versions.

Reproducibility:

To enhance the reproducibility of your results, we recommend that authors of applicable studies deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

 10.1371/journal.ppat.1013235.r003 Author response to Decision Letter 1 Submission Version 1

22 May 2025

Submitted filename: Response to Reviewers.docx

 10.1371/journal.ppat.1013235.r004 Decision Letter 1 Obbard Darren Guest Editor 2025 Darren Obbard This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 1

26 May 2025

Dear Dr. Feng,

We are pleased to inform you that your manuscript 'Infectome analysis of bat kidneys from Yunnan province, China, reveals novel henipaviruses related to Hendra and Nipah viruses and prevalent bacterial and eukaryotic microbes' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Darren J. Obbard

Guest Editor

PLOS Pathogens

Ronald Swanstrom

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

Thank you for your positive and wholehearted engagement with my and the reviewers' comments and questions. I do not see any further scientific or presentation issues in need of response.

Reviewer Comments (if any, and for reference):

 10.1371/journal.ppat.1013235.r005 Acceptance letter Obbard Darren Guest Editor 2025 Darren Obbard This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Dear Dr. Feng,

We are delighted to inform you that your manuscript, "Infectome analysis of bat kidneys from Yunnan province, China, reveals novel henipaviruses related to Hendra and Nipah viruses and prevalent bacterial and eukaryotic microbes," has been formally accepted for publication in PLOS Pathogens.

We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Pearls, Reviews, Opinions, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064