Bat guano fertilizer as a source of Betacoronavirus: First molecular evidence linking Rhinolophus coelophyllus to viral reservoirs in Thailand

Figures Abstract Commercial bat guano fertilizers distributed in Thailand were screened for the presence of coronavirus genomes to assess potential public health risks. A total of 41 samples were analyzed for Alphacoronavirus (AlphaCoV) and Betacoronavirus (BetaCoV) using partial Spike (S) gene sequences. Two samples (4.88%) tested positive for AlphaCoV, showing 97.08–99.27% sequence similarity, while one sample (2.44%) was positive for BetaCoV with 98.91% similarity. Due to the greater relevance of BetaCoVs to human health, the BetaCoV-positive sample underwent host identification via partial cytochrome oxidase I (COI) gene analysis and next-generation sequencing. The results revealed Rhinolophus coelophyllus as the likely natural reservoir. This study provides the first evidence of bat-derived BetaCoV genome in a commercial fertilizer in Thailand and highlights the importance of monitoring wildlife-derived products for emerging zoonotic viruses. Citation: Suwannasing R, Kimprasit T (2026) Bat guano fertilizer as a source of Betacoronavirus: First molecular evidence linking Rhinolophus coelophyllus to viral reservoirs in Thailand. PLoS One 21(3): e0344265. https://doi.org/10.1371/journal.pone.0344265 Editor: Daniel Oladimeji Oluwayelu, University of Ibadan Faculty of Veterinary Medicine, NIGERIA Received: September 3, 2025; Accepted: February 17, 2026; Published: March 11, 2026 Copyright: © 2026 Suwannasing, Kimprasit. 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. Data Availability: All relevant data are within the manuscript and its Supporting Information files. Funding: This research project is supported by Science Research and Innovation Fund. Agreement No. FF66-P1-059. Rajamangala University of Technology Isan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Introduction Bats (Order: Chiroptera) represent one of the most diverse groups of mammals, comprising over 1,400 species capable of thriving across a wide range of ecological niches and habitats [1,2]. As volant mammals, bats play a significant role in the ecology of infectious diseases, serving as natural reservoirs for numerous zoonotic viruses. These include lyssaviruses (agents of rabies), henipaviruses, severe acute respiratory syndrome coronavirus (SARS-CoV), and Ebola virus [3,4]. Their ability to travel long distances enhances the potential for widespread pathogen dissemination [5], underscoring their importance in veterinary public health and disease surveillance. The animals contribute significantly to ecosystem services, including natural pest control, pollination, and seed dispersal. Their excreta, particularly guano, is rich in nutrients and widely utilized as an organic fertilizer [6]. In Southeast Asia, including Thailand, bat guano is commercially harvested and sold as fertilizer [7]. However, the precise origin of guano contained in packaged fertilizers often remains unknown, as it is collected from multiple bat roosts and distributed across regions and neighboring countries. While bats play a beneficial role in agriculture, their potential to harbor and transmit zoonotic pathogens has raised public health concerns [8,9]. Notably, several bat-borne viruses, including coronaviruses, have been detected in guano samples [5,7,10,11], underscoring the need for biosafety measures in guano handling and trade. Coronaviruses are lipid-enveloped, positive-sense single-stranded RNA viruses with genome sizes ranging from 27 to 32 kilobases—among the largest of all RNA viruses [12]. Veterinary and zoonotic surveillance studies have identified bats as key reservoirs for several coronaviruses that have crossed species barriers to infect humans, including SARS-CoV and MERS-CoV. Notably, coronavirus strains have been isolated from guano samples of the Pteropus medius, a common flying fox in Sri Lanka [13]. These viral sequences exhibited over 97% nucleotide similarity with coronaviruses previously detected in Cynopterus sphinx, fruit bats, and insectivorous bats (Scotophilus heathii and Scotophilus kuhlii) in Thailand. Similar viral strains were also identified in guano and rectal swab samples collected from bat populations in Myanmar [14]. These findings underscore the importance of wildlife monitoring in veterinary public health to mitigate the risk of emerging zoonotic diseases. This suggests the viruses were circulated in different bat species and posed serious health risk to mine operators, guano packagers, sellers, and/or purchasers of the guano without the use of preventive measures. Due to the limited existing knowledge, this study aimed to investigate the presence of coronaviruses in commercial bat guano fertilizers distributed throughout Thailand, with particular emphasis on products sold in border areas shared with neighboring Southeast Asian countries. Materials and methods Bat guano fertilizers sampling From October 2022 to September 2023, a total of 41 bat guano fertilizers were collected from 10 provinces located in the north, northeast, west, east, and south of Thailand. The sampling sites were chosen based on their bounding area to neighboring countries (Fig 1). The fertilizers were collected from both agricultural markets and stand-alone shops in urban and rural areas and then put on ice during transport to our laboratory. Detection of bat coronaviruses Following homogenization, 25 grams of each fertilizer sample were aseptically collected using sterilized spoons to ensure sample integrity. Total viral nucleic acids were subsequently extracted using the High Pure Viral Nucleic Acid Extraction Kit (Roche, Germany), in accordance with the manufacturer’s protocol. This procedure was conducted to facilitate downstream molecular detection of potential zoonotic viral agents present in the organic fertilizer matrix. Coronavirus RNA extracted from the samples was reverse transcribed into complementary DNA (cDNA) using SuperScript IV Reverse Transcriptase (Invitrogen™, USA) with random hexamer primers to ensure broad coverage of viral genomes. The resulting cDNA was subjected to nested PCR targeting the Spike (S) gene, a conserved region among coronaviruses. The primer sets developed by the authors and presented in Table 1 were employed for amplification in this study. For Alphacoronaviruses (AlphaCoV), the primer sets included: AlphaCoV-F and AlphaCoV-R1 for the first round. AlphaCoV-F and AlphaCoV-R2 for the second round. For Betacoronaviruses (BetaCoV), First round: BetaCoV-F1 and BetaCoV-R. Second round: BetaCoV-F1 and BetaCoV-R. PCR amplification was conducted using GoTaq® Green Master Mix (Promega, USA) under standardized thermocycling conditions: initial denaturation at 95°C for 2 minutes, followed by 35 cycles of denaturation at 95°C for 30 seconds, annealing at 50°C for 30 seconds, and extension at 72°C for 30 seconds. A final extension step was performed at 72°C for 5 minutes. This protocol facilitates sensitive detection of coronavirus RNA in environmental and biological samples, contributing to veterinary epidemiological surveillance and zoonotic risk assessment. Post-amplification, PCR products were resolved via agarose gel electrophoresis, visualized, excised from the gel, and purified using the QIAquick® Gel Extraction Kit (Qiagen), ensuring high-quality DNA for downstream sequencing and analysis. PCR products that tested positive for CoV were subjected to direct Sanger sequencing to obtain nucleotide sequences. The resulting sequences were analyzed using the BLAST algorithm against the NCBI nucleotide database to confirm viral identity and assess sequence similarity. For phylogenetic analysis, sequences were aligned with reference BetaCoV genomes retrieved from the NCBI database, including those reported by Hassanin et al., 2024 [15]. A phylogenetic tree was inferred using the Maximum Likelihood approach implemented in the IQ-TREE web server, employing appropriate substitution models selected by ModelFinder. Branch support was evaluated using ultrafast bootstrap approximation to ensure robustness of the inferred topology. Detection of host species To identify the animal host species associated with bat BetaCoV-positive guano fertilizer samples, DNA barcoding was performed targeting the mitochondrial cytochrome c oxidase I (COI) gene. The amplification protocol followed the method described by Francis et al., 2010 [16], utilizing specific primers for COI. PCR reactions were carried out using GoTaq® Green Master Mix (Promega, USA) under the following conditions: initial denaturation at 95°C for 2 minutes, followed by 35 cycles of 30 seconds at 95°C, 1 minute at 50°C, and 45 seconds at 72°C, with a final extension at 72°C for 7 minutes. PCR amplicons were separated using agarose gel electrophoresis to verify product size and integrity. Bands corresponding to the expected size of approximately 650 base pairs were visualized and carefully excised from the gel. These target fragments were then purified using the QIAquick® Gel Extraction Kit (Qiagen), yielding high-quality DNA suitable for downstream applications such as next-generation sequencing and host species identification through COI barcoding. Animalia sequences of COI that had been recorded worldwide were obtained from the NCBI and used as references (S1 File). Next generation sequencing and data analyses The purified COI PCR products were further processed using the Agencourt AMPure XP reagent (Beckman Coulter, Brea, CA, USA) in accordance with the instructions provided by the manufacturer. Sequencing libraries were prepared from purified COI amplicons using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (New England Biolabs, Massachusetts, USA) with unique dual indices, following the manufacturer’s instructions. Library construction comprised end repair, dA-tailing, adapter ligation, and limited-cycle PCR enrichment. Indexed libraries were sequenced on an Illumina® MiSeq with the MiSeq Reagent Kit v2 (500 cycles; 2 × 250 bp). Base calling and demultiplexing were performed with MiSeq Reporter, and raw reads were exported in FASTQ format. Using CLC Genomics Workbench v10.1.1 (Filgen, Nagoya, Japan), all analyses were carried out. Read trimming and quality control include adapter trimming and the removal of low-quality bases using CLC parameters Reads with undecipherable bases (N), reads outside the predicted range of COI lengths, and reads that were obviously identical were not included. The bat species reference sequences were culled from the National Center for Biotechnology Information (NCBI). Sequences that were unusually lengthy were removed and duplicate copies were eliminated. To assign and map the merged readings to the curated COI reference set, penalties that were compatible with the merging settings were applied (CLC “Map Reads to Reference”). Coverage per base, average depth, and mapping % were all included for each reference point. Segmental BLASTn was used to check against the COI database for any PCR or merger artifacts, and coverage-break algorithms were used to identify probable chimeras. Sequences showing 89.00%–100.00% similarity were grouped as a single haplotype of host mitochondrial DNA, following the criteria established by Yassin et al., 2010 [17]. This analytical pipeline enabled precise identification of bat host species contributing to the guano samples. Results A total of 41 bat guano fertilizer samples were obtained from 10 provinces across regions of Thailand, especially in the provinces located near the border of Thailand-Myanmar, Thailand-Laos, Thailand-Cambodia, and Thailand-Malaysia, except for Sakon Nakhon (Fig 1). All the samples were subjected to screening for bat AlphaCoV and bat BetaCoV partial genome sequencess using the specific primers for the Spike (S) gene. The results showed that 2 samples (2/41; 4.88%) obtained from different local agriculture markets – MH1 and NK3 were positive for the bat AlphaCoV, while the bat BetaCoV partial genome sequences was detected only from a sample obtained from the local agriculture market in Loei province (1/41; 2.44%) – LY3 by the reverse transcriptase PCR (RT-PCR). The sample MH1 and NK3 were homologous to Bat alphacoronavirus isolate BACV/CH0008 (Acesssion No. MT708749) and Bat alphacoronavirus isolate BACV/CH002 (Acesssion No. MT708749) with 97.08% and 99.27% similarity, respectively. While the sample LY3 was identified as positive for the bat BetaCoV partial genome sequence with 98.91% identities to BetaCoV strain BtRf-BetaCoV/HeN2013 (Accession no. KJ473817). Given the greater public health relevance of BetaCoV compared to AlphaCoV, the nucleotide sequence of LY3 was deposited in the GenBank database through the NCBI website and designated as THLY3 strain with the GenBank Accession No. PV730171. Then, the strain was further analyzed for the viral genomic evolution and host species identification. The viral THLY3 strain identified in this study demonstrated a close genetic relationship to the sarbecoviruses previously detected from the Rhinolophus bats, in China and Korea. Moreover, the genetics of the strain was closer to the strains detected in East Asian countries compared to the Southeast Asian countries. Therefore, the strain was assumed to originate in China and Korea then dispersed to Thailand. This phylogenetic proximity is illustrated in Fig 2, highlighting the potential zoonotic linkage and evolutionary origin of the viral THLY3 strain. This Maximum Likelihood tree, generated via IQ-TREE, is based on partial Spike gene sequences. Each entry is identified by its GenBank accession number, strain, country, and host. Branch support values are displayed at the respective nodes. Considering the reported maximum intraspecific divergence of COI sequences ranging from 0.00% to 11.0% [17], a similarity threshold of 89.00%–100.00% was applied to determine which animals were the possible hosts for the BetaCoV THLY3 strain. Reads with less than 89.00% similarity to reference COI sequences were excluded to ensure taxonomic accuracy. After filtering, a total of 88 high-confidence reads were classified within the Order Chiroptera. These sequences corresponded to multiple bat species as shown in Table 2. This diversity highlights the presence of multiple rhinolophid bat species contributing to the guano fertilizer samples. BLAST analysis confirmed that one of the consensus sequences exhibited 100% nucleotide identity with Rhinolophus coelophyllus voucher EBD 23517 (GenBank Accession No. HM541556), previously reported in Southeast Asia [16]. The sequence has been deposited in the NCBI GenBank database under the designation Rhinolophus coelophyllus