==== Front PLoS One PLoS One plos PLOS ONE 1932-6203 Public Library of Science San Francisco, CA USA 10.1371/journal.pone.0288007 PONE-D-23-08923 Research Article Biology and Life Sciences Organisms Eukaryota Animals Vertebrates Amniotes Mammals Primates Apes Chimpanzees Biology and Life Sciences Zoology Animals Vertebrates Amniotes Mammals Primates Apes Chimpanzees Biology and Life Sciences Anatomy Body Fluids Saliva Medicine and Health Sciences Anatomy Body Fluids Saliva Biology and Life Sciences Physiology Body Fluids Saliva Biology and Life Sciences Microbiology Medical Microbiology Microbial Pathogens Viral Pathogens Medicine and Health Sciences Pathology and Laboratory Medicine Pathogens Microbial Pathogens Viral Pathogens Biology and Life Sciences Organisms Viruses Viral Pathogens Biology and life sciences Organisms Viruses DNA viruses Herpesviruses Biology and Life Sciences Microbiology Medical Microbiology Microbial Pathogens Viral Pathogens Herpesviruses Medicine and Health Sciences Pathology and Laboratory Medicine Pathogens Microbial Pathogens Viral Pathogens Herpesviruses Biology and Life Sciences Organisms Viruses Viral Pathogens Herpesviruses Biology and life sciences Organisms Viruses DNA viruses Herpesviruses Epstein-Barr virus Biology and life sciences Microbiology Medical microbiology Microbial pathogens Viral pathogens Herpesviruses Epstein-Barr virus Medicine and health sciences Pathology and laboratory medicine Pathogens Microbial pathogens Viral pathogens Herpesviruses Epstein-Barr virus Biology and life sciences Organisms Viruses Viral pathogens Herpesviruses Epstein-Barr virus Biology and life sciences Organisms Viruses DNA viruses Papillomaviruses Human Papillomavirus Biology and Life Sciences Microbiology Medical Microbiology Microbial Pathogens Viral Pathogens Papillomaviruses Human Papillomavirus Medicine and Health Sciences Pathology and Laboratory Medicine Pathogens Microbial Pathogens Viral Pathogens Papillomaviruses Human Papillomavirus Biology and Life Sciences Organisms Viruses Viral Pathogens Papillomaviruses Human Papillomavirus Biology and Life Sciences Microbiology Virology Viral Transmission and Infection Biology and life sciences Organisms Viruses DNA viruses Papillomaviruses Biology and Life Sciences Microbiology Medical Microbiology Microbial Pathogens Viral Pathogens Papillomaviruses Medicine and Health Sciences Pathology and Laboratory Medicine Pathogens Microbial Pathogens Viral Pathogens Papillomaviruses Biology and Life Sciences Organisms Viruses Viral Pathogens Papillomaviruses Viruses in saliva from sanctuary chimpanzees (Pan troglodytes) in Republic of Congo and Uganda Viruses in sanctuary chimpanzee saliva https://orcid.org/0000-0002-5689-8118 Dunay Emily Conceptualization Formal analysis Funding acquisition Investigation Methodology Writing – original draft Writing – review & editing 1 Rukundo Joshua Investigation Methodology Resources Writing – review & editing 2 Atencia Rebeca Investigation Methodology Resources Writing – review & editing 3 Cole Megan F. Investigation Writing – review & editing 4 Cantwell Averill Investigation Writing – review & editing 5 Emery Thompson Melissa Conceptualization Funding acquisition Investigation Methodology Resources Supervision Writing – review & editing 4 https://orcid.org/0000-0002-6906-7807 Rosati Alexandra G. Conceptualization Funding acquisition Investigation Methodology Resources Supervision Writing – review & editing 5 6 Goldberg Tony L. Conceptualization Funding acquisition Investigation Methodology Resources Supervision Writing – original draft Writing – review & editing 1 * 1 Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, United States of America 2 Ngamba Island Chimpanzee Sanctuary / Chimpanzee Trust, Entebbe, Uganda 3 Jane Goodall Institute Congo, Pointe-Noire, Republic of Congo 4 Department of Anthropology, University of New Mexico, Albuquerque, New Mexico, United States of America 5 Department of Psychology, University of Michigan, Ann Arbor, Michigan, United States of America 6 Department of Anthropology, University of Michigan, Ann Arbor, Michigan, United States of America Berber Engin Editor Lerner Research Institute - Cleveland Clinic, UNITED STATES Competing Interests: The authors have declared that no competing interests exist. * E-mail: tony.goldberg@wisc.edu 29 6 2023 2023 18 6 e028800727 3 2023 16 6 2023 © 2023 Dunay et al 2023 Dunay et al https://creativecommons.org/licenses/by/4.0/ 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. Pathogen surveillance for great ape health monitoring has typically been performed on non-invasive samples, primarily feces, in wild apes and blood in sanctuary-housed apes. However, many important primate pathogens, including known zoonoses, are shed in saliva and transmitted via oral fluids. Using metagenomic methods, we identified viruses in saliva samples from 46 wild-born, sanctuary-housed chimpanzees at two African sanctuaries in Republic of Congo and Uganda. In total, we identified 20 viruses. All but one, an unclassified CRESS DNA virus, are classified in five families: Circoviridae, Herpesviridae, Papillomaviridae, Picobirnaviridae, and Retroviridae. Overall, viral prevalence ranged from 4.2% to 87.5%. Many of these viruses are ubiquitous in primates and known to replicate in the oral cavity (simian foamy viruses, Retroviridae; a cytomegalovirus and lymphocryptovirus; Herpesviridae; and alpha and gamma papillomaviruses, Papillomaviridae). None of the viruses identified have been shown to cause disease in chimpanzees or, to our knowledge, in humans. These data suggest that the risk of zoonotic viral disease from chimpanzee oral fluids in sanctuaries may be lower than commonly assumed. http://dx.doi.org/10.13039/100000049 National Institute on Aging R37AG049395 Thompson Melissa Emery http://dx.doi.org/10.13039/100000049 National Institute on Aging R01AG049395 Thompson Melissa Emery National Institutes of Health Office of the Director T32OD010423 https://orcid.org/0000-0002-5689-8118 Dunay Emily University of Wisconsin-Madison Global Health Institute Graduate Student Research Award https://orcid.org/0000-0002-5689-8118 Dunay Emily "This research was supported by National Institutes of Health awards R37AG049395 and R01AG049395 through the National Institute for Aging (https://nia.nih.gov) and the Office of Research on Women's Health (https://orwh.od.nih.gov) to MET, AGR, and TLG and a University of Wisconsin-Madison Global Health Institute (https://ghi.wisc.edu) Graduate Student Research Award to ED for research materials. ED was supported in the form of salary by the University of Wisconsin-Madison Comparative Biomedical Sciences Training Grant T32OD010423 from the National Institutes of Health Office of the Director (https://nih.gov). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." Data AvailabilityViral nucleotide sequences are available in GenBank under accession numbers OP934203-OP934225. Metadata is available under BioProject PRJNA888083 and BioSamples SAMN31207558 - SAMN31207565. All other relevant data are within the paper and supporting information files. Data Availability Viral nucleotide sequences are available in GenBank under accession numbers OP934203-OP934225. Metadata is available under BioProject PRJNA888083 and BioSamples SAMN31207558 - SAMN31207565. All other relevant data are within the paper and supporting information files. ==== Body pmcIntroduction Sanctuaries throughout Africa house and care for thousands of wild-born non-human primates (NHPs) that have been rescued from the illegal bushmeat and pet trades [1, 2]. In this setting, human caretakers typically have frequent, direct contact with NHPs with different geographic origins and histories which provides great opportunity for zoonotic pathogen transmission [3, 4]. One risk in such settings is NHP bites [5, 6]. NHP bites can cause severe physical injury, can become infected, and can transmit infectious agents, some of which are severely pathogenic in people [5, 7–11]. For example, herpes B virus, which infects macaques (Macaca spp.) often asymptomatically, is frequently lethal in humans [5]. Human infections with apathogenic simian foamy viruses (SFVs) and with oncogenic primate T-lymphotropic viruses have resulted from NHP bites in hunters and occupational workers (e.g., people employed in research and zoo settings) [12–15]. Some viruses, such as Epstein-Barr virus of humans, are thought to be transmitted in saliva without an inciting bite [16, 17]. Furthermore, many viruses that can be detected in saliva can be transmitted through other routes (e.g., SARS-CoV-2: respiratory droplets [18]; herpes B virus: urine, feces, scratches [19]). In addition to concern for virus transmission between sanctuary NHPs and sanctuary workers, the possible introduction and spread of viruses among and between NHP social groups and species warrants screening for pathogens that could impact relocation or reintroduction efforts [5, 20–23]. For example, human herpes simplex virus type 1, which has yet to be documented in wild mountain gorilla (Gorilla beringei beringei) populations, was detected in oral lesions of a sanctuary-housed juvenile eastern lowland gorilla (G. b. graueri) in Democratic Republic of Congo [20, 23]. The most commonly housed NHP at Pan African Sanctuary Alliance member sanctuaries is the chimpanzee (Pan troglodytes) [24, 25]. Thus, the majority of prior studies regarding sanctuary NHP health have been focused on chimpanzees [27]. Historically, research addressing viral infections in sanctuary-housed chimpanzees has been limited to using fecal and blood samples to determine what viruses are present in these populations [26, 27]. Thus far, these studies have shown that sanctuary chimpanzees are infected with a number of viruses that have rarely been associated with disease [28–30]. Despite the recognized risk of pathogen transmission, studies focused on viral infections in saliva from captive chimpanzees are few. Experimental infection of chimpanzees in research settings has suggested that saliva is a transmitting agent for certain human hepatitis viruses [31, 32]. In zoo- and laboratory-housed chimpanzees, there are reports of focal epithelial hyperplasia [33–35], a benign oral condition that has been associated with a papillomavirus, Pan paniscus papillomavirus type 1, first identified in zoo-housed bonobos (Pan paniscus) [36–38]. In wild chimpanzee populations, non-invasive saliva sampling has been recognized as a potentially valuable tool for pathogen detection, however, to our knowledge, only once has it been used to screen for a certain virus of interest (i.e., monkeypox virus) [39, 40]. When possible, the use of diverse and alternative sample types, such as saliva, may contribute to great ape health assessments by improving pathogen detection capabilities [16, 39–41]. There are limitations innate to all types of clinical samples regarding their suitability for the detection of pathogens due to differences in microbial properties, cellular and tissue tropism, and transmission routes [16, 42–44]. For example, rhinovirus C, a human respiratory pathogen that was associated with a lethal respiratory outbreak in wild chimpanzees in Uganda in 2013, is unlikely to be reliably detected in fecal samples due to virion characteristics that are not compatible with survival in the gastrointestinal tract [45]. Furthermore, certain enteric viruses (i.e., viruses of the gastrointestinal tract), which are known to be transmitted by the fecal-oral route, have been shown to replicate in the salivary glands and transmit through saliva in a mouse model [46]. Yet the results of viral diagnostics using saliva are likely to overlap with some of the more commonly utilized sample types. For example, certain viruses that are known to replicate in the oral cavity (e.g., SFV) can also be detected in peripheral blood mononuclear cells (an approach that has been employed in the chimpanzee sanctuary setting [6, 47]) and feces [48]. In this study, using metagenomic methods, we characterized viruses in saliva collected from two African sanctuary chimpanzee populations: A) Tchimpounga Chimpanzee Rehabilitation Centre (TCRC) in Republic of Congo and B) Ngamba Island Chimpanzee Sanctuary (NICS) in Uganda. Previously, we analyzed plasma samples from these same populations and found no evidence of pathogenic viruses and that sanctuary chimpanzees are infected with many of the same types of viruses as their wild counterparts [26]. To our knowledge, this study is the first to identify viruses in saliva samples from sanctuary-housed chimpanzees which addresses the possibility that pathogenic viruses are being shed in saliva and could be transmitted to sanctuary personnel. Together with our prior surveillance using plasma, results from the current study could have implications for occupational health and safety of NHP sanctuary personnel, sanctuary management, and wild chimpanzee conservation efforts. Methods Ethics statement Research at the sanctuaries was approved by the Institutional Animal Care and Use Committees at the University of Michigan (#8102) and Harvard University (#14-07-206-1). Additionally, research was approved by the Republic of Congo Ministry of Scientific Research and Technological Innovation and Jane Goodall Institute Congo for TCRC and the Uganda Wildlife Authority, the Uganda National Council for Science and Technology, and Chimpanzee Sanctuary and Wildlife Conservation Trust for NICS. Samples were shipped to the USA under Convention on International Trade in Endangered Species of Wild Fauna and Flora permits: Republic of Congo permit CG1126038 and USA permit 20US56953D/9 (TCRC); Uganda permit 004877 and USA permit 20US09881D/9 (NICS). Research complied with the standards outlined by the Pan African Sanctuary Alliance and adhered to the American Society of Primatologists Principles for the Ethical Treatment of Non-Human Primates. Study sites, study populations, and sample collection The study sites were two Pan African Sanctuary Alliance member chimpanzee sanctuaries: A) TCRC and B) NICS (Fig 1). TCRC is located approximately 30km north of Pointe-Noire, Republic of Congo, in the Tchimpounga Nature Reserve, and cares for approximately 150 chimpanzees. NICS is located on Ngamba Island in Lake Victoria, Uganda, and cares for approximately 52 chimpanzees. After rescue, arrival at the sanctuaries, and rehabilitation, chimpanzees are integrated into social groups that semi-free range in forested enclosures during the day and stay in enclosed dens at night. At both sites, chimpanzees are provisioned with a variety of species-appropriate fruits, vegetables, and other foods multiple times per day, but the sanctuary chimpanzees are also able to forage within their forest enclosures. 10.1371/journal.pone.0288007.g001 Fig 1 Map of study sites. (A) Tchimpounga Chimpanzee Rehabilitation Centre (TCRC) in Republic of Congo (purple) and (B) Ngamba Island Chimpanzee Sanctuary (NICS) in Uganda (blue). Map created using R v. 4.2.0 with Natural Earth (R Core Team, 2022). At TCRC, we analyzed saliva from 1 sanctuary-born and 22 wild-born chimpanzees (11 females and 12 males, ages 7–31 years old) that was collected between July 4th and July 24th, 2019. At NICS, we analyzed saliva from 24 wild-born chimpanzees (13 females and 11 males, ages 7–30 years old) that was collected between July 29th and August 14th, 2016. Saliva samples were collected by researchers from chimpanzees who voluntarily allowed their mouth to be swabbed as in previous studies, following the same protocols [49–51]. To do so, the researchers first thoroughly washed their hands and then poured ground SweeTARTS (Ferrera Candy Company, Chicago, IL, USA) powder onto a cotton round. While holding the chimpanzee’s bottom lip through the wire mesh enclosure, the cotton round was placed between the bottom lip and gums to absorb saliva. To minimize sample contamination, saliva was not collected from chimpanzees during provisioning periods (e.g., in the morning before breakfast at NICS, and either before breakfast or at least a half hour after feeding at TCRC), from chimpanzees who were observed eating anything remaining from prior feeding periods, or from chimpanzees who had visible cuts or other potential contamination sources in their mouths. After becoming saturated with saliva, a process that typically took 2–3 minutes and no more than 5 minutes, the cotton round was placed into a 10-mL syringe and the plunger was used to expel the saliva into a 1.2 mL cryogenic vial (Fisher Scientific, Waltham, MA, USA). TCRC saliva samples were stored in the field in a cooler with ice packs and moved to a -20°C freezer within two hours. NICS saliva samples were frozen immediately in liquid nitrogen. All samples were kept frozen during shipment to the USA and were then stored at −80°C until processing. Sample preparation and sequencing Following previously described methods optimized for virus detection in chimpanzee blood, feces, and respiratory swabs [4, 26, 45, 52–54], saliva samples were subject to metagenomic next-generation sequencing. First, we added 125ul of saliva to 125ul of Hank’s Balanced Salt Solution (MilliporeSigma, Burlington, MA, USA), and the mixture was homogenized by bead beating and clarified by centrifugation. After nuclease digestion [55], we extracted nucleic acids using the QIAmp MinElute Virus Spin Kit (Qiagen, Hilden, Germany). We synthesized double-stranded cDNA using the SuperScript double-stranded cDNA Synthesis Kit (Invitrogen, Carlsbad, CA, USA). We purified the cDNA using AmpureXP beads (Beckman Coulter, Brea, CA, USA) and prepared DNA libraries using the Nextera XT DNA sample preparation kit (Illumina, San Diego, CA, USA). DNA libraries were sequenced on a MiSeq instrument (MiSeq Reagent Kit, V2 chemistry, 300 cycle kit; Illumina). Virus identification We trimmed and prepared sequencing data using CLC Genomics Workbench v. 20.0.4 (Qiagen) as previously described [26]. First, we trimmed reads of low quality (Phred quality score <30) and <50 bases in length. We then filtered out reads mapping to known contaminants and to a chimpanzee (Pan troglodytes) reference genome (GenBank accession number GCF_002880755.1). We performed de novo assembly of the remaining reads using SPAdes v. 3.15.2 with the metaSPAdes pipeline [56, 57]. We compared resulting contiguous sequences to the GenBank database using both the BLASTn (nucleotide) and BLASTx (protein) algorithms to identify viruses [58, 59]. We retained contigs representing viruses associated with mammalian hosts for further analyses (i.e., we excluded confirmed bacteriophage and viruses associated with invertebrates per ICTV reports). We used NOVOplasty to build out circular virus genomes [60]. To quantify viral abundances, we mapped reads from each individual to the sequence of a target gene (see Table 1 footnote) for each virus at a length fraction of 1.0 and similarity of 0.9 and classified an individual as positive for a virus if it had a normalized read count of ≥ 1 read per million at a length of ≥ 50 bases matching that virus. To estimate viral relative abundance, we determined the proportion of reads mapping to each virus and the proportion of reads mapping to any virus in the population (total viral abundance). We then normalized this measure to one million reads and to the length of the target sequence for each virus and applied a log transformation to calculate a metagenomic measure of viral abundance (log10 viral reads per million per kilobase of target sequence, or log10vRPM/kb) which is correlated with results from quantitative PCR assays [61] and has proven informative in our prior studies of chimpanzee viruses [26, 45, 52–54]. Complete papillomavirus genomes were annotated using PuMA [62]. 10.1371/journal.pone.0288007.t001 Table 1 Viruses in saliva from sanctuary chimpanzees at TCRC and NICS. ID Virus Name Sanctuary Abbreviation Genome Familya Genusa Closest match (source, location, year, accession)b E-Valueb % ID (NT)b Accessionc 1 simian foamy virus Pan troglodytes schweinfurthii NICS SFVpsc ssRNA-RT Retroviridae Simiispumavirus Human foamy virus (human, Germany, EU381420) 0 97.95 OP934216 2 simian foamy virus Pan troglodytes troglodytes TCRC SFVptr ssRNA-RT Retroviridae Simiispumavirus Human spumaretrovirus (chimpanzee, AF232918) 0 96.53 OP934220 3 nabpantry virus 12 NICS NAPTV-12 dsRNA (segmented) Picobirnaviridae Orthopicobirnavirus Picobirnavirus sp. (human, Netherlands, 2016, OK562155) 2.00E-87 66.54 OP934217 4 nabpantry virus 13 NICS NAPTV-13 dsRNA (segmented) Picobirnaviridae Orthopicobirnavirus Marmot picobirnavirus (marmot, China, 2013, KY928684) 0 71.41 OP934218 5 nabpantry virus 14 NICS NAPTV-14 dsRNA (segmented) Picobirnaviridae Orthopicobirnavirus Porcine picobirnavirus (pig, USA, 2018, MW977424) 0 75.72 OP934219 6 ticpantry virus 7 TCRC TCPTV-7 dsRNA (segmented) Picobirnaviridae Orthopicobirnavirus Chimpanzee picobirnavirus (chimpanzee, Sierra Leone, 2013–2016, MT350351) 0 97.96 OP934221 7 ticpantry virus 8 TCRC TCPTV-8 dsRNA (segmented) Picobirnaviridae Orthopicobirnavirus Picobirnavirus sp. (cow, 2017–2019, China, MZ556513) 0 73.46 OP934222 8 ticpantry virus 9 TCRC TCPTV-9 dsRNA (segmented) Picobirnaviridae Orthopicobirnavirus Porcine picobirnavirus (pig, USA, 2018, MW977506) 0 87.26 OP934223 9 ticpantry virus 10 TCRC TCPTV-10 dsRNA (segmented) Picobirnaviridae Orthopicobirnavirus Picobirnavirus sp. (human, Cameroon, 2014, MH933806) 0 81.72 OP934224 10 ticpantry virus 11 TCRC TCPTV-11 dsRNA (segmented) Picobirnaviridae Orthopicobirnavirus Human picobirnavirus (human, USA, 2018, OL875327) 0 89.13 OP934225 11 nabpantry virus 15 NICS NAPTV-15 ssDNA (circular) Circoviridae Cyclovirus Cyclovirus Chimp53 (chimpanzee, Cameroon, 2003, GQ404881) 0 95.15 OP934203 12 chimpanzee associated cyclovirus 2 NICS ChimpACyV2 ssDNA (circular) Circoviridae Cyclovirus Cyclovirus ZM36a (shrew, Zambia, 2012, AB937982) 0 90.41 OP934204 13 nabpantry virus 17 NICS NAPTV-17 ssDNA (circular) Circoviridae Cyclovirus Mongoose-associated cyclovirus (mongoose, Saint Kitts and Nevis, 2017, MZ382573) 0 93.17 OP934205 14 ticpantry virus 12 TCRC TCPTV-12 ssDNA (circular) Circoviridae Cyclovirus Swine cyclovirus (pig, Cameroon, 2012, KM392285) 0 97.10 OP934208 15 ticpantry virus 13 TCRC TCPTV-13 ssDNA (circular) Unclassified Unclassified Circovirus sp. (pig, China, 2017, MK377638) 2.00E-92 70.64 OP934209 16 Pan paniscus papillomavirus type 1 NICS PpPV1 dsDNA (circular) Papillomaviridae Alphapapillomavirus Common chimpanzee papillomavirus 1 (chimpanzee, AF020905) 0 95.94 OP934206 17 Pan troglodytes papillomavirus type 1 NICS PtroPV1 dsDNA (circular) Papillomaviridae Alphapapillomavirus Human papillomavirus type 177 (human, KR816168) 0 84.16 OP934207 18 Pan paniscus papillomavirus type 1 TCRC PpPV1 dsDNA (circular) Papillomaviridae Alphapapillomavirus Common chimpanzee papillomavirus 1 (chimpanzee, AF020905) 0 95.39 OP934210 19 Pan troglodytes papillomavirus type 1 TCRC PtroPV1 dsDNA (circular) Papillomaviridae Alphapapillomavirus Human papillomavirus type 177 (human, KR816168) 0 84.16 OP934211 20 ticpantry virus 16 TCRC TCPTV-16 dsDNA (circular) Papillomaviridae Gammapapillomavirus Human papillomavirus (human, USA, 2015, MH777284) 7.00E-152 78.55 OP934212 21 chimpanzee cytomegalovirus NICS CCMV dsDNA (linear) Herpesviridae Cytomegalovirus Panine betaherpesvirus 2 (chimpanzee, Germany, 2020, MZ151943) 0 94.12 OP934213 22 Pan troglodytes lymphocryptovirus 1 NICS PtroLCV-1 dsDNA (linear) Herpesviridae Lymphocryptovirus Macaca arctoides gammaherpesvirus 1 (macaque, Georgia, 1985, MG471437) 0 93.47 OP934214 23 Pan troglodytes lymphocryptovirus 1 TCRC PtroLCV-1 dsDNA (linear) Herpesviridae Lymphocryptovirus Pan paniscus lymphocryptovirus 1 (bonobo, AF534220) 0 99.56 OP934215 a Determined by phylogenetic analyses. See Figs 3–5 and S1, S2 Figs. b Closest match, E-value, and percent identity (nucleotide) were identified by querying the target gene (Bet (Retroviridae), polymerase (Picobirnaviridae, Herpesviridae), Rep (Circoviridae), and L1 (Papillomaviridae)) nucleotide sequences against the NCBI’s nonredundant nucleotide database using the discontiguous megablast homology searching algorithm. Length of target gene obtained for each virus and genome information is reported in S11 Table. c GenBank accession number of viral sequence from this study. Phylogenetics and viral sequence comparisons To infer phylogenetic relationships, we generated multiple sequence alignments of nucleotide sequences from the viruses identified in this study and known, related viruses in GenBank using the T-Coffee algorithm implemented by EMBL-EBI and hand edited the alignments as needed [63, 64]. We then inferred maximum-likelihood phylogenetic trees using PhyML with Smart Model Selection and 1000 bootstrap replicates [65, 66]. We displayed resulting phylogenetic trees in FigTree v. 1.4.4 [67]. We used MEGA X to calculate genetic distances (nucleotide p-distance ± standard error, 1000 bootstrap replicates) of viruses within and between the two sanctuary populations [68]. To compare the viruses identified in this study to each other and known relatives, we used Sequence Demarcation Tool v. 1.2 to generate pairwise alignments using the MUSCLE algorithm and calculate pairwise identities for nucleotide and amino acid sequences [69, 70]. Statistical analyses We conducted statistical analyses using R v. 4.2.0 implemented in R Studio [71, 72]. We calculated the prevalence of each virus (percentage of positive individuals) with modified Wald 95% confidence intervals (Agresti & Coull, 1998). We calculated odds ratios with 95% confidence intervals and performed two-tailed Fisher’s exact tests (‘fisher.test’ in R) to assess the association between sex (male or female) and viral infection. We used Mann-Whitney U tests (‘wilcox.test’ in R) to assess the association between sex and viral abundance in infected individuals. We compared viral richness (number of viruses per individual) and total viral abundance between the two sanctuaries using Mann-Whitney U tests. Results Virus characterization Next-generation sequencing produced an average of 1,533,717 reads per sample (SD ± 621,460) for TCRC and 1,589,629 (SD ± 203,431) for NICS after filtering for quality and length. From these data, we identified 20 viruses of four genome types (ssRNA-RT, dsRNA, ssDNA, and dsDNA) (Table 1 and Fig 2). We identified 12 of these viruses at TCRC and 11 at NICS which are 66.5–99.6% similar to their closest match in GenBank based on BLASTn percent identity. All but one of these viruses, an unclassified CRESS (circular rep-encoding single-stranded) DNA virus identified at TCRC (TCPTV-13), were classified into five viral families: Circoviridae, Herpesviridae, Papillomaviridae, Picobirnaviridae, and Retroviridae. At least one virus from each of these families was identified at both sanctuaries. For the families Herpesviridae and Papillomaviridae, viruses from two genera (Cytomegalovirus and Lymphocryptovirus; Alphapapillomavirus and Gammapapillomavirus) were identified. Detailed pairwise sequence comparisons between viruses identified in this study and known relatives (S2–S9 Tables) as well as within- and between-population genetic distances (S10 Table) are provided in the supplementary information. 10.1371/journal.pone.0288007.g002 Fig 2 Prevalence of viruses identified in saliva from chimpanzees at TCRC and NICS. (A) Total number of viruses in each family and genus identified at TCRC and NICS. Family and genus were determined using phylogenetic analyses (Figs 3–5 and S1, S2 Figs). (B) Barplot displays the proportion (%) of individuals at each sanctuary who were positive for at least one virus in the family. (C) Barplot displays the proportion of individuals at each sanctuary who were positive for at least one virus in each identified Papillomaviridae genus. D) Barplot displays the proportion of individuals at each sanctuary who were positive for a virus in each identified Herpesviridae genus. Simian foamy virus Pan troglodytes troglodytes (Retroviridae: Simiispumavirus; SFVptr) was identified at TCRC, and simian foamy virus Pan troglodytes schweinfurthii (SFVpsc) was identified at NICS (Fig 3). The TCRC SFVptr variant shares 96% Bet nucleotide sequence (NT) identity to known SFVptr variants (JQ867463, JQ867462, AF232917, AF232918) (S2 Table). The NICS SFVpsc variant shares 98% Bet NT identity with known SFVpsc variants (KX087159, U21247, EU381240). 10.1371/journal.pone.0288007.g003 Fig 3 Maximum-likelihood phylogenetic tree of simian foamy virus Bet gene nucleotide sequences. Viruses identified in this study are marked with a colored star to indicate the sanctuary of origin (purple = TCRC, blue = NICS). Virus names are followed by (host, location, year, GenBank accession number). Bootstrap values ≥ 50% are represented by numbers beside branches (1000 replicates). Scale bar is equal to nucleotide substitutions per site. We identified eight picobirnaviruses (Picobirnaviridae: Orthopicobirnavirus), all of which clustered phylogenetically in genogroup 1 except TCPTV-7 which clustered in genogroup 2 (S1 Fig). Picobirnavirus diversity within and between the two sanctuaries was similar (S3 and S10 Tables). Of the cycloviruses (Circoviridae: Cyclovirus) identified in this study (TCPTV-12, NAPTV-15, ChimpACyV2, NAPTV-17), two contain introns in their replicase genes (TCPTV-12 and ChimpACyV2), which has been reported for other cycloviruses [73] (S2 Fig and S4 Table). We identified the complete genome for ChimpACyV2 and NAPTV-17. ChimpACyV2 is most closely related to a cyclovirus identified in shrew feces (AB937982; [74]) to which it shares 75.4% genome-wide NT identity. Based on ICTV guidelines for cyclovirus species demarcation (<80% genome-wide NT identity; [73]), ChimpACyV2 represents a novel species. NAPTV-17 shares 91% genome-wide NT identity with a mongoose associated cyclovirus (MZ382573; [75]), a novel species that has not yet been formally classified [76], making NAPTV-17 a member of this putative species [73]. A single unclassified CRESS DNA virus (TCPTV-13) was identified at TCRC (S2 Fig and S2 and S5 Tables). Pan paniscus papillomavirus type 1 (Papillomavirus: Alphapapillomavirus; PpPV1) was identified at each sanctuary (Fig 4A). The TCRC and NICS PpPV1 variants share 98.6% L1 NT identity (S6 Table). We also identified a novel alphapapillomavirus, Pan troglodytes papillomavirus type 1 (PtroPV1), at both sanctuaries. These variants share 99.9% L1 NT identity. PtroPV1 is most closely related to human papillomavirus type 177 (KR816168; 85% L1 NT identity) and represents a novel papillomavirus type based on the papillomavirus type demarcation threshold (<90% complete L1 NT identity) [77–79]. A single gammapapillomavirus (TCPTV-16; Papillomaviridae: Gammapapillomavirus) was identified at TCRC (Fig 4B and S7 Table). 10.1371/journal.pone.0288007.g004 Fig 4 Maximum likelihood phylogenetic trees of papillomavirus L1 gene nucleotide sequences. (A) alphapapillomaviruses and (B) gammapapillomaviruses. Viruses identified in this study are marked with a colored star to indicate the sanctuary of origin (purple = TCRC, blue = NICS). Virus names are followed by (host, location, year, GenBank accession number). Bootstrap values ≥ 50% are represented by numbers beside branches (1000 replicates). Scale bar is equal to nucleotide substitutions per site. We identified chimpanzee cytomegalovirus (CCMV) (Herpesviridae: Cytomegalovirus) at NICS which shares 95% DNA polymerase NT identity to the original CCMV (AF480884) and 81% to human cytomegalovirus (AY446894) (Fig 5A and S8 Table). Lastly, we identified Pan troglodytes lymphocryptovirus 1 (PtroLCV-1) (Herpesviridae: Lymphocryptovirus) at each sanctuary (Fig 5B). These variants share 99.8% DNA polymerase NT identity to each other, 99.0% to the original PtroLCV-1 (AF534226), and 92% to Epstein-Barr virus (AJ507799) (S9 Table). 10.1371/journal.pone.0288007.g005 Fig 5 Maximum likelihood phylogenetic trees of herpesvirus DNA polymerase catalytic subunit gene nucleotide sequences. (A) cytomegaloviruses and (B) lymphocryptoviruses. Viruses identified in this study are marked with a colored star to indicate the sanctuary of origin (purple = TCRC, blue = NICS). Virus names are followed by (host, location, year, GenBank accession number). Bootstrap values ≥ 50% are represented by numbers beside branches (1000 replicates). Scale bar is equal to nucleotide substitutions per site. Viral prevalence Overall, viral prevalence ranged from 4.2% to 87.5%. At TCRC, a cyclovirus and PtroPV1 were the most prevalent (53.3%; 95% confidence interval (CI): 33%, 70.8%). At NICS, PtroLCV-1 was the most prevalent virus (87.5% CI: 68.2%, 96.5%). The least prevalent viruses at TCRC were the gammapapillomavirus (TCPTV-16) and PpPV1, each infecting one individual (4.3% CI: <0.01%, 22.7%). At NICS, a cyclovirus (NAPTV-15) was the least prevalent (4.2% CI: <0.01%, 21.9%). Viral prevalence by family and genus is shown in Fig 2. The prevalence of individual viruses (overall and by sex) is listed in S12 Table. There were no statistically significant associations between viral presence and sex. Viral richness and abundance Viral richness ranged from 0 to 7 at both sanctuaries (Fig 6A and S13, S14 Tables). The average viral richness was 2.8 (SD ± 1.8) at TCRC and 3.5 (SD ± 1.5) at NICS. Viral abundance (log10vRPM/kb) ranged among viruses and individuals from 0 to 4.22 at TCRC and 0 to 3.51 at NICS (Fig 6B, S3 Fig, and S13, S14 Tables). Among infected individuals, the virus with the highest abundance was NAPTV-15 (1.65), and the virus with the lowest abundance was NAPTV-17 (0.62), both cycloviruses. Total viral abundance (all viruses combined) was on average 0.63 (SD ± 0.7) at TCRC and 0.97 (SD ± 0.8), reaching a maximum of 3.23 at TCRC and 2.8 at NICS. We compared the distribution viral richness and total viral abundance at TCRC and NICS and did not observe a statistically significant difference (p-values: viral richness 0.19 and total viral abundance 0.07). For each sample, the number of sequencing reads mapping to each virus is reported in S15, S16 Tables. 10.1371/journal.pone.0288007.g006 Fig 6 Boxplots of (A) viral richness and (B) total viral abundance for TCRC and NICS chimpanzees. Mann-Whitney U test comparisons between TCRC and NICS populations were not statistically significant (ns). Discussion We identified a total of 20 viruses in saliva samples from two populations of wild-born, sanctuary-housed chimpanzees in Africa. Of the viruses identified, none are known to cause disease in chimpanzees or humans. Chimpanzees at these two sanctuaries were infected with viruses from the same families and genera including highly similar variants of two papillomaviruses and a herpesvirus (i.e., PpPV1, PtroPV1, and PtroLCV-1). With the exception of papillomaviruses, all types of viruses identified in this study have been documented previously in fecal samples from wild chimpanzees [48, 52, 53, 80–82]. Additionally, herpesviruses and SFV have been detected in tissues from deceased wild chimpanzees [83–87]. NHPs of many species are persistently infected with SFVs, which replicate in the superficial epithelial cells of the oral mucosa but do not cause known pathology [44, 88, 89]. Herpesviruses, also ubiquitous, naturally establish persistent, latent infections in primates and are intermittently shed in saliva [90, 91]. Several human herpesviruses (e.g., Epstein-Barr virus, human cytomegalovirus) are opportunistic pathogens, particularly in immunocompromised individuals [92, 93]. Mountain gorilla lymphocryptovirus has been associated with Epstein-Barr virus-like (human lymphocryptovirus) pathology in infant mountain gorillas [91]. Epstein-Barr virus-like pathology has not been documented for chimpanzees infected with PtroLCV-1. The true hosts of CRESS DNA viruses, including cycloviruses [73, 75, 76, 94], and picobirnaviruses [95, 96] are unknown, although arthropods and bacteria or fungi, respectively, are suspected. Thus, these viruses, neither of which are considered pathogenic, may actually represent dietary or environmental contaminants and not viral infection in chimpanzees [76, 95, 97, 98]. Papillomavirus infections can be persistent or transient and are usually asymptomatic or cause mild disease. Papillomaviruses have been detected in healthy skin swabs from zoo-housed chimpanzees and in oral cavity samples from laboratory-housed chimpanzees exhibiting focal epithelial hyperplasia [35, 99]. Previously, vaginal swabs from chimpanzees at NICS were tested for papillomaviruses using PCR, which yielded negative results [100]. Some papillomavirus types do cause disease (e.g., cervical cancer in humans and macaques) [101–103]. However, PpPV1 and PtroPV1 are distantly related to the carcinogenic human papillomavirus types, 16 and 18 (K02718 and X05015; 69–75% L1 NT identity; S6 Table) [104, 105]. In fact, PpPV1 clusters phylogenetically with members of the species Alphapapillomavirus 10 which are associated with benign oral conditions such as focal epithelial hyperplasia in humans, chimpanzees, and bonobos [34–36, 77]. PtroPV1, the novel alphapapillomavirus identified in this study, is most closely related to human papillomaviruses of species Alphapapillomavirus 11 including human papillomavirus type 73 which is classified as possibly carcinogenic to humans [105, 106]. Zoonotic transmission of SFVs has been documented both in the wild and in captivity [11, 14, 15, 47, 107, 108], but infections have not been shown to be pathogenic in the human host [88]. The SFVs detected in this study cluster phylogenetically with known SFV variants from central chimpanzees (P. t. troglodytes, SFVptr) and eastern chimpanzees (P. t. schweinfurthii, SFVpsc), consistent with virus-host co-speciation [109]. Herpesviruses are generally considered host-specific; however, the ability for cross-species transmission of herpesviruses between humans and NHPs is known for members of the subfamily Alphaherpesvirinae (i.e., herpes B virus, herpes simplex virus) [20, 110]. In contrast, transmission of cytomegaloviruses and lymphocryptoviruses between primates of different species (including humans) has not been documented, even where exposure would be high, such as in a chimpanzee and colobus monkey predator-prey system in the wild [81, 110–113]. Like most herpesviruses, papillomaviruses also demonstrate host-specificity [79]. Zoonotic transmission of papillomaviruses between chimpanzees and humans has not been reported. Overall, our findings show that the sanctuary chimpanzees we analyzed are shedding viruses in their saliva that are known to replicate in the oral cavity but, to our knowledge, do not pose a risk to chimpanzee or human health. This conclusion is important because frequent contact between sanctuary apes and human staff has been an ongoing concern for sanctuary management [6]. Surveys of NHP workers in field, laboratory, zoo, or sanctuary settings have reported NHP bites in ~40% of workers [114, 115]. Contact with chimpanzee oral fluids could also occur during feeding, cleaning of enclosures, and especially during intensive rehabilitation of newly rescued individuals. Fortunately, our results suggest that the likelihood of sanctuary personnel acquiring pathogenic viral infections from chimpanzee saliva is low, at least in the sanctuaries we studied. Nevertheless, measures to minimize this risk should be taken particularly when viral infection status of sanctuary chimpanzees is unknown, which is often the case, and to avoid other sequelae (e.g., bacterial infection from bites [10]). We emphasize that our results are specific to viruses in saliva, which may not reflect viruses in other body compartments. For example, we previously described viruses in plasma from TCRC and NICS chimpanzees (from primarily different individuals at TCRC but the same individuals at NICS) [27], but no viruses identified in this previous study of plasma were found in the present study of saliva (distinct picobirnaviruses were identified in both sample types) (S17 Table). We did, however, detect PtroLCV-1 at NICS, which has been previously detected in the blood buffy coat of these chimpanzees, but we did not detect herpesviruses in the genus Rhadinovirus which have also been previously detected at NICS [116]. Given that we detected other herpesviruses in this study, we suspect that the latter difference is due to intermittent shedding of herpesviruses in saliva [117, 118]. Likewise, SFV has also been detected in chimpanzees at NICS using buffy coat from blood [6]. Our results share little overlap with prior work using fecal samples from sanctuary chimpanzees to detect viruses which have primarily utilized virus-specific diagnostics (i.e., PCR). Notably, PtroLCV-1 was detected at lower prevalence in feces (3/21; 14.3%) [119] from NICS chimpanzees than in buffy coat (14/40; 35%) [116] or here in saliva (21/24; 87.5%). Lastly, we recognize that we are unable to comment on the infectivity and transmissibility of the viruses identified in this study as our methods do not distinguish between viral nucleic acids and infectious viruses [61]. Non-invasive saliva sampling has been utilized, albeit infrequently in comparison to fecal samples, to assess the health of wild chimpanzee populations. In wild western chimpanzees (P. t. verus), saliva collected non-invasively from food wadges has been used to detect Staphylococcus and Streptococcus spp. and monkeypox virus [40, 120–122]. Chewed ropes and vegetation have been used to detect herpesviruses and SFVs in saliva from wild mountain gorillas, olive baboons (Papio anubis), rhesus macaques (M. mulatta), and golden monkeys (Cercopithecus kandti) [23, 41, 91, 123]. Flavored chew swabs have been utilized to detect Mycobacterium tuberculosis in free-ranging macaques [124]. Our results suggest that metagenomic methods might be beneficial if combined with these or similar collection methods. NHP sanctuary managers are faced with the need to develop long-term solutions to balance ongoing demand for care and capacity limits [125, 126]. In light of this, our findings add to prior studies that have shown that wild-born chimpanzees housed at sanctuaries appear broadly healthy with respect to viral infections [27], cardiovascular [127], endocrine [49, 51], and psychological health [51], in some ways similar to wild populations. We envision that our methods could be used to screen newly rescued individuals for viral pathogens upon arrival at a sanctuary, to prevent the introduction of orally transmitted pathogens, and to make decisions about husbandry and management of infected individuals, including decisions about relocation and reintroduction [24, 128]. Supporting information S1 Fig Maximum-likelihood phylogenetic tree of picobirnavirus RNA dependent RNA polymerase gene nucleotide sequences. Viruses identified in this study are marked with a colored star to indicate the sanctuary of origin (purple = TCRC, blue = NICS). Virus names are followed by (host, location, year, GenBank accession number). Bootstrap values (%) ≥ 50 are represented by numbers beside branches (1000 replicates). Scale bar is equal to nucleotide substitutions per site. (TIF) Click here for additional data file. S2 Fig Maximum-likelihood phylogenetic trees of cyclovirus and unclassified CRESS DNA virus replicase gene nucleotide sequences. Viruses identified in this study are marked with a colored star to indicate the sanctuary of origin (purple = TCRC, blue = NICS). Virus names are followed by (host, location, year, GenBank accession number). Bootstrap values (%) ≥ 50 are represented by numbers beside branches (1000 replicates). Scale bar is equal to nucleotide substitutions per site. (TIF) Click here for additional data file. S3 Fig Heatmap of saliva viral abundance of sanctuary chimpanzees at TCRC and NICS. Displays viral abundance data (log10vRPM/kb) for each genus and total viral abundance data (log10vRPM/kb for all viruses) for each individual at each sanctuary. Values range from 0 (lightest) to 4.22 (darkest). a Genus refers to Table 1 and Fig 2. b For individuals infected with more than one virus from a genus, the average viral abundance is shown. (TIF) Click here for additional data file. S1 Table Detailed sample inventory. (XLSX) Click here for additional data file. S2 Table Pairwise sequence comparisons of simian foamy viruses. (XLSX) Click here for additional data file. S3 Table Pairwise sequence comparisons of picobirnaviruses. (XLSX) Click here for additional data file. S4 Table Pairwise sequence comparisons of cycloviruses. (XLSX) Click here for additional data file. S5 Table Pairwise sequence comparisons of CRESS DNA viruses. (XLSX) Click here for additional data file. S6 Table Pairwise sequence comparisons of alphapapillomaviruses. (XLSX) Click here for additional data file. S7 Table Pairwise sequence comparisons of gammapapillomaviruses. (XLSX) Click here for additional data file. S8 Table Pairwise sequence comparisons of cytomegaloviruses. (XLSX) Click here for additional data file. S9 Table Pairwise sequence comparisons of lymphocryptoviruses. (XLSX) Click here for additional data file. S10 Table Within- and between-population (sanctuary) genetic distances of viruses identified in this study. (XLSX) Click here for additional data file. S11 Table Sequence information for viruses identified in this study. (XLSX) Click here for additional data file. S12 Table Prevalence and univariate statistical associations between sex (male or female) and prevalence and abundance of viruses in saliva from chimpanzees at TCRC and NICS. (XLSX) Click here for additional data file. S13 Table Viral richness and viral abundance for TCRC chimpanzees. (XLSX) Click here for additional data file. S14 Table Viral richness and viral abundance for NICS chimpanzees. (XLSX) Click here for additional data file. S15 Table Sequencing reads mapping to viruses identified in saliva from TCRC chimpanzees. (XLSX) Click here for additional data file. S16 Table Sequencing reads mapping to viruses identified in saliva from NICS chimpanzees. (XLSX) Click here for additional data file. S17 Table Reported viral diversity by ante-mortem sample type for sanctuary chimpanzees in Africa. (XLSX) Click here for additional data file. S1 File Reference list for S17 Table. (DOCX) Click here for additional data file. We are grateful to the Republic of Congo Ministry of Scientific Research and Technological Innovation and Jane Goodall Institute Congo for research approval at TCRC as well as the Uganda Wildlife Authority, the Uganda National Council for Science and Technology, and Chimpanzee Sanctuary and Wildlife Conservation Trust for research approval at NICS. We thank Sofia Fernandez-Navarro, Rosemary Bettle, Alex Tumukunde, Titus Mukungu, and the animal caretakers and staff at both sites for assistance with data collection, and Emily Otali and Audrey Salvy for assistance with research permissions. We also thank Koenraad Van Doorslaer for assistance with papillomavirus genome annotation and virus naming and Christopher Dunn for assistance in the lab. 10.1371/journal.pone.0288007.r001 Decision Letter 0 Berber Engin Academic Editor © 2023 Engin Berber 2023 Engin Berber https://creativecommons.org/licenses/by/4.0/ 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 Version0 3 May 2023 PONE-D-23-08923Viruses in saliva from sanctuary chimpanzees (Pan troglodytes) in Republic of Congo and UgandaPLOS ONE Dear Dr. Dunay, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. 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Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ********** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The paper for Dunay and collegues reports on viruses that are detected in saliva from chimpanzees housed in two sanctuaries in Africa, one in the Republic of Congo and the other in Uganda. In both sanctuaries saliva was collected from 22 and 24 chimpanzees and subsequently analyzed by next generation metagenomic sequencing to document viral diversity in saliva, especially for the presence of viral families that include viruses that can be pathogenic for humans and and be transmitted to humans, especially animal keepers, via bites. The methods are correct and are standard for this kind of studies. Moreover this is one of the first studies reporting on saliva. The authors identified around 20 viruses from 5 viral families, but none of them is currently associated with pathogenicity in chimpanzees or humans. I have a few points that need to be clarified: The authors mention also results on plasma or blood samples rom the same chimpanzee communities, can they specify whether this is also from the same animals for whom saliva samples are studies in this report? Table 1 seems to contain important information, but the table is truncated and many information is not displayed. This should be rectified!! The authors should provide more details on the number of reads for the different viruses they reported and the different gene fragments that are obtained. For some viruses , they obtained whole genomes, and for others they did phylogenetic analysis on genes fragments, but how much of the different virus genomes were recovered in the different samples? Can the authors also provide information on what percentage of the total reads are represented by the viruses reported here, what is percentage of other viruses that were excluded and what was the overall diversity of viruses in saliva. This can be useful information on whether saliva has a specific virome or whether some viruses are also observed in fecal samples from apes, which are more frequently studied, because these samples are easier to collect. Reviewer #2: The authors present a manuscript based on the presence of viruses in saliva from african chimpanzees. This manuscript explain that viruses found in saliva do not cause disease in chimpanzee and human. The authors used phylogenetic analysis for viruses identification and characterisation. 1- The authors have detected viruses in saliva but did not tell about the proviral load for all these viruses. This information is important to best understand the infectiosity of each virus and to best evaluate the risk for transmission to other non human primates and to human. 2- Some bibliographic references number are missed or used in duplicate or triplicate. The authors are requested to use a good logiciel for formating bibliography at the end of the manuscript. 3- Line 267, page 13: What does the sentence means:" Accesssion...this study." 4- Page 16, line 344: What does 70.8% represents? a CI? 5- At the line 419, the authors explain that "primates" include also humans. Is the word "primates" used several time in the manuscript include also "humans" or only animal? If so, the authors are requested to write NHPS and not only "primates". 6- Page 20, line 428: "...has been..." is written twice Reviewer #3: The present study looks to use metagenomic methods to identify viruses in saliva samples from chimpanzees in two wildlife sanctuaries. I believe the study is interesting, well presented and valuable for practitioners in the field that work in wildlife rehabilitation facilities. The abstract is succinct, and identifies the uniqueness of the study, as well as clearly summarizes the results and greater implications. The authors do a great job introducing the subject material and highlighting the importance of the study. The figures and tables visualize and support the findings well. There is a great number of both as supplementary material. The methods are well detailed and comprehensively written so that others could repeat the methodology. The discussion includes limitations and relevant comparisons with previous research. It presents relevant findings, and I believe the conclusions are within the realm of the results. Minor revision: Please have a look over the reference list as some numbers are missing, repeated or out of order (5 and 6 missing, 21 repeated, 125 comes between 46 and 50). ********** 6. 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.] 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. 10.1371/journal.pone.0288007.r002 Author response to Decision Letter 0 Submission Version1 22 May 2023 Response to Reviewers **All line numbers reference refer to Revised Manuscript with Track Changes** Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ________________________________________ 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: I Don't Know ________________________________________ 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ________________________________________ 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ________________________________________ 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The paper for Dunay and collegues reports on viruses that are detected in saliva from chimpanzees housed in two sanctuaries in Africa, one in the Republic of Congo and the other in Uganda. In both sanctuaries saliva was collected from 22 and 24 chimpanzees and subsequently analyzed by next generation metagenomic sequencing to document viral diversity in saliva, especially for the presence of viral families that include viruses that can be pathogenic for humans and and be transmitted to humans, especially animal keepers, via bites. The methods are correct and are standard for this kind of studies. Moreover this is one of the first studies reporting on saliva. The authors identified around 20 viruses from 5 viral families, but none of them is currently associated with pathogenicity in chimpanzees or humans. I have a few points that need to be clarified: 1 - The authors mention also results on plasma or blood samples rom the same chimpanzee communities, can they specify whether this is also from the same animals for whom saliva samples are studies in this report? Response to Comment 1: We are grateful to the reviewer for making this suggestion. In order to address the reviewer's critique, we have added information to the Discussion stating whether the same individuals were sampled in the previous plasma manuscript and the current saliva manuscript (lines 444-445). 2 - Table 1 seems to contain important information, but the table is truncated and many information is not displayed. This should be rectified!! Response to Comment 2: We have followed the PLOS submission guidelines for Tables as listed on the website (https://journals.plos.org/plosone/s/tables), specifically, “Do not split your table or otherwise try to make the table appear within the manuscript margins if it does not fit on one page. In Word, tables that run off of the manuscript page can be seen using Draft View.” If you would like us to submit the table in a different format for better viewing, please advise what is best. 3 - The authors should provide more details on the number of reads for the different viruses they reported and the different gene fragments that are obtained. For some viruses , they obtained whole genomes, and for others they did phylogenetic analysis on genes fragments, but how much of the different virus genomes were recovered in the different samples? 4 - Can the authors also provide information on what percentage of the total reads are represented by the viruses reported here, what is percentage of other viruses that were excluded and what was the overall diversity of viruses in saliva. This can be useful information on whether saliva has a specific virome or whether some viruses are also observed in fecal samples from apes, which are more frequently studied, because these samples are easier to collect. Response to Comments 3 and 4: We are grateful to the reviewer for making these suggestions. This is indeed useful information. To address these points, we have created additional supporting information tables (S11, S15, and S16). S11 Table (now referenced in line 268) lists for each virus what gene was utilized for phylogenetic analyses and viral abundance calculations, its length, whether the complete or partial gene was obtained, and for which viruses we obtained the complete genome. Additionally, full sequence data (partial or complete genome) for each virus is annotated and available on GenBank (see corresponding accession numbers in Table 1). S15 Table (TCRC) and S16 Table (NICS), both now referenced in lines 366-367, provide the number of reads from each sample that mapped to each virus and what percentage of total reads after quality trim and removal of contaminants/host are viral for each sample. Lastly, as mentioned in the discussion section all types of viruses identified in this study with the exception of papillomaviruses have been documented in the feces of wild chimpanzees (lines 379-382) and one, PtroLCV-1, has been documented in feces from sanctuary chimpanzees at NICS (lines 454-457). This information is further detailed in the S17 Table. Reported viral diversity by ante-mortem sample type for sanctuary chimpanzees in Africa. Reviewer #2: The authors present a manuscript based on the presence of viruses in saliva from african chimpanzees. This manuscript explain that viruses found in saliva do not cause disease in chimpanzee and human. The authors used phylogenetic analysis for viruses identification and characterisation. 1- The authors have detected viruses in saliva but did not tell about the proviral load for all these viruses. This information is important to best understand the infectiosity of each virus and to best evaluate the risk for transmission to other non human primates and to human Response to Comment 1: We thank the reviewer for raising this important point. We agree that it would be useful to understand the infectivity and risk of transmission of these viruses. We have therefore added a statement to the Discussion explaining this idea and how it applies to our study (lines 458-461), and we have provided supporting citations. 2- Some bibliographic references number are missed or used in duplicate or triplicate. The authors are requested to use a good logiciel for formating bibliography at the end of the manuscript. Response to Comment 2: The reviewer is indeed correct. We have gone through the manuscript with a fine-tooth comb to ensure that all references are properly formatted. 3- Line 267, page 13: What does the sentence means:" Accesssion...this study." Response to Comment 3: This is an excellent point. This sentence means that the accession number listed in Table 1 is the corresponding GenBank accession number corresponding to the nucleotide sequence of each virus identified in this study. We have changed the wording of this sentence to clarify this point (line 269). 4- Page 16, line 344: What does 70.8% represents? a CI? Response to Comment: Yes, this represents the upper limit of the 95% confidence interval as defined and indicated throughout the manuscript: (53.3%; 95% confidence interval (CI): 33%, 70.8%). 5- At the line 419, the authors explain that "primates" include also humans. Is the word "primates" used several time in the manuscript include also "humans" or only animal? If so, the authors are requested to write NHPS and not only "primates". Response to Comment 5: This is indeed a valid point, many thanks. Based on the reviewer's excellent suggestion, we have reviewed the entire manuscript and made changes to reflect appropriate use of “NHP” and “primate” throughout (lines 48 and 433). 6- Page 20, line 428: "...has been..." is written twice Response to Comment 6: Good catch! We have fixed the typo by removing the duplication (line 432). Reviewer #3: The present study looks to use metagenomic methods to identify viruses in saliva samples from chimpanzees in two wildlife sanctuaries. I believe the study is interesting, well presented and valuable for practitioners in the field that work in wildlife rehabilitation facilities. The abstract is succinct, and identifies the uniqueness of the study, as well as clearly summarizes the results and greater implications. The authors do a great job introducing the subject material and highlighting the importance of the study. The figures and tables visualize and support the findings well. There is a great number of both as supplementary material. The methods are well detailed and comprehensively written so that others could repeat the methodology. The discussion includes limitations and relevant comparisons with previous research. It presents relevant findings, and I believe the conclusions are within the realm of the results. Response: Many thanks for the positive comments! Minor revision: Please have a look over the reference list as some numbers are missing, repeated or out of order (5 and 6 missing, 21 repeated, 125 comes between 46 and 50). Response: We thank the reviewer for raising this important point. To address this point, we have carefully looked over the entire reference list and in-text citations and ensured that numbering is now correct throughout. ________________________________________ 6. 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 ________________________________________ Attachment Submitted filename: Response to Reviewers.docx Click here for additional data file. 10.1371/journal.pone.0288007.r003 Decision Letter 1 Berber Engin Academic Editor © 2023 Engin Berber 2023 Engin Berber https://creativecommons.org/licenses/by/4.0/ 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 Version1 19 Jun 2023 Viruses in saliva from sanctuary chimpanzees (Pan troglodytes) in Republic of Congo and Uganda PONE-D-23-08923R1 Dear Dr. Dunay, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Engin Berber Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #2: All comments have been addressed Reviewer #3: All comments have been addressed ********** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #2: Yes Reviewer #3: Yes ********** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: Yes Reviewer #3: Yes ********** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #2: Yes Reviewer #3: Yes ********** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #2: Yes Reviewer #3: Yes ********** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #2: (No Response) Reviewer #3: (No Response) ********** 7. 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 #2: No Reviewer #3: No ********** 10.1371/journal.pone.0288007.r004 Acceptance letter Berber Engin Academic Editor © 2023 Engin Berber 2023 Engin Berber https://creativecommons.org/licenses/by/4.0/ 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. 22 Jun 2023 PONE-D-23-08923R1 Viruses in saliva from sanctuary chimpanzees (Pan troglodytes) in Republic of Congo and Uganda Dear Dr. Dunay: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Engin Berber Academic Editor PLOS ONE ==== Refs References 1 Ferrie GM , Farmer KH , Kuhar CW , Grand AP , Sherman J , Bettinger TL . The social, economic, and environmental contributions of Pan African Sanctuary Alliance primate sanctuaries in Africa. Biodivers Conserv. 2014 Jan 1;23 (1 ):187–201. 2 Stokes R , Tully G , Rosati AG . Pan African Sanctuary Alliance: securing a future for the African great apes. Int Zoo Yearb. 2018;52 (1 ):173–81. 3 Dunay E , Apakupakul K , Leard S , Palmer JL , Deem SL . Pathogen Transmission from Humans to Great Apes is a Growing Threat to Primate Conservation. 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