The virome investigation of the globally endangered Eld's deer (Rucervus eldii) on Hainan Island, China

The virome investigation of the globally endangered Eld's deer (Rucervus eldii) on Hainan Island, China | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The virome investigation of the globally endangered Eld's deer (Rucervus eldii) on Hainan Island, China Haoxiang Su, Dandan Zheng, Zihan Li, Xuming Qi, Yunxing Chang, and 21 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5371247/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Mar, 2025 Read the published version in Scientific Reports → Version 1 posted 13 You are reading this latest preprint version Abstract More than 60% of emerging (re-emerging) infectious diseases worldwide are caused by animal-derived pathogens, of which 71% are of wild animal origin. It is important to obtain insight into the viral pathogens present in the wildlife reservoir from a public health perspective. Based on next-generation sequencing (NGS), we report a metagenomic viral survey of Eld's deer ( Rucervus eldii ) (n = 33) in Bangxi Provincial Nature Reserve in Hainan Province. We collected 33 nasal swabs, 33 anal swabs, and 9 tick bite wound swabs and combined them into 5 pools according to the type of swabs. The metagenomic analysis showed that there were differences in the 5pools of viral reads, but the overall viral reads were closely related to mammals. We amplified and obtained the complete genomes of novel papillomaviruses (PV) and circoviruses (CV) in Eld's deer, PsPV-HMU-1 and PsaCV-HMU-1. The novel PV and CV genomic structure revealed that it was consistent with that of the known mammalian PV and CV. The L1 of PsPV-HMU-1 and the rep of PsaCV-HMU-1 showed less than 77.20% and 45.43% amino acid homology with the closest viruses, respectively. The positive rate of PV carried by Eld's deer is relatively high. Multiple Eld's deer with positive nase or anal swabs, accompanied by positive swabs from tick bites and wounds, indicate that insect bites may be closely related viral infection. This study helps us monitor Eld's deer viral diseases and understand the genetic diversity and evolutionary history of FV and CV carried by Eld's deer. Biological sciences/Microbiology/Pathogens Biological sciences/Microbiology/Virology Biological sciences/Molecular biology Eld's deer Papillomaviridae Circoviridae Genetic diversity Phylogenetic analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Eld's deer ( Rucervus eldii ) is a rare and globally endangered tropical Southeast Asian deer species, belonging to Artiodactyla, Family Cervidae and Subfamily Cervinae. It has been listed in Appendix I of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) and classified as endangered on the Red List of Threatened Species by the International Union for Conservation of Nature (IUCN) and the class I national key protected wildlife in China 1 . Eld's deer was once widely distributed on Hainan Island in China, but due to factors like habitat loss and destruction, blind hunting, and illegal trade, their population has sharply declined, and they are in danger of going extinct 2 . There were only 26 Eld's deer remaining in Hainan in the 1970s 3 . At present, Eld's deer are mainly distributed on the Hainan Datian National Nature Reserve and Hainan Bangxi Provincial Nature Reserve in China. Despite fact that the Eld's deer population has recovered and grown after over 40 years of development and preservation, it continues to be extremely vulnerable to extinction because of things like inbreeding, poor genetic diversity, and the diminishing evolutionary capacity of tiny populations. According to reports, there are near 300 deer in Hainan Bangxi Provincial Nature Reserve which clearly exceeded the reserve's carrying capacity and increased the risk of virus transmission. In other countries and regions of Southeast Asia, Eld's deer has been facing huge threats. Over 360 Eld's deer were surviving in India in 2003 4 . This species saw a 90% decline in Cambodia between 1998 and 2008 and a 50% decline in the ensuing 10 to 15 years. The number of Eld's deer in Laos and Myanmar is also declining annually, and the wild population of Eld's deer are thought to be regional extinct in Thailand and Vietnam 4 . Animal pathogen monitoring is usually limited to pathogens with known economic impacts on livestock and is extremely rare in wild animal species. However, most re-emerging viral infections originate from animals, so routine testing cannot provide sufficient vigilance 5 . Pathogenic organisms can cause outbreaks and even lead to population collapse in wild animals. The severe acute respiratory syndrome coronavirus (SARS-CoV) 6, 7 that broke out in China in 2003, the influenza A virus (H1N1) 8 that prevailed in the global influenza pandemic in 2009, the Middle East respiratory syndrome coronavirus (MERS-CoV) 9 in the Middle East in 2012, the Corona Virus Disease 2019 (SARS-CoV-2) that caused extremely high death in the world in 2019, and the animal-derived hantavirus 10 suggest that growing global virus diversity and potential spillover effects require attention. The cross-species transmission and clinical pathogenic mechanisms of viruses carried by Artiodactyla deserve attention. The serum samples of captive dromedary camels 11 and wild Eld's deer in Thailand 12 both contain pandemic (H1N1) and 2009 influenza (H1N1pdm09) virus antibodies, indicating the susceptibility of those animals to natural infection. Circular Rep-encoding (replication-associated protein encoding) single-stranded DNA (CRESS DNA) form a highly diverse group of small viruses that have been detected worldwide in prokaryotes and eukaryotes, and environmental samples 13 . However, the persistence of CRESS DNA viruses in herds of domestic animals has 13 . PVs are a diverse group of small, non-enveloped viruses belonging to the family Papillomaviridae, with double-stranded DNA genomes 14 . Papillomaviridae The Papillomaviridae has two subfamilies Firstpapillomavirinae and Secondpapillomavirinae , with more than 50 virus genera. PVs can cause hyperplasia in the skin and mucous membranes of humans, mammals, and non-mammalian animals, and are a significant risk factor for cervical and genital cancers 14 . The development of viral metagenomics platforms has made it feasible to compile a more comprehensive list of the viruses that are present in animals 5 . Using viral metagenomic techniques, a considerable number of new tiny circular DNA viruses have been discovered and described from fecal sources during the last five years 15-19 . Several mammalian viruses, including papillomavirus, parvovirus, poliovirus, and coronavirus, were detected in Arctic reindeer in western Alaska, USA, and the complete genome of papillomavirus was obtained from clinically manifested Roe deer in Slovenia through virus NGS 20 . Using NGS technology, we have obtained two complete virus sequences of papillomavirus and circovirus from Elder's deer and subsequently identified and described as PsPV-HMU-1 and PsaCV-HMU-1. We particularly focus on the origin of PV oncogenes, assessing whether they are monophyletic or result from processes like mutations or recombination based on the conserved sections of PV, and evaluate the potential for novel PV genes to cause cancer. It is crucial to perform viral metagenomics research on Hainan Eld's deer to comprehend the diversity, evolution, and variation patterns of virus genotypes carried by Eld's deer. Results Viral Metagenome of Eld's deer Efforts were made to eliminate bacteria, microbial eukaryotes, archaea, and organisms that showed no discernible relationship to any amino acid sequences in the viral non-redundant (NR) protein database. Five pools yielded 59.50 GB of nucleotide data (58,402,837 valid reads, 150 bp in length). The sequences of less than 50bp, archaea, bacteria, microbial eukaryotes (such as fungi), and those that had no significant similarity with any amino acid sequences in the viral NR protein database were eliminated (Supplementary Table 2). Figure 2 shows the virus composition of 5 pools. Unfortunately, the viruses of the Picornavirdae in pool 1 and Parvoviridae in pool 2 have not been identified, possibly due to prolonged degradation of samples (Fig. 2 A and 2 B). In pools 4 and 5, the proportion of virus-associated reads was 2,039,836 (16.97%) and 3,128,644 (28.15%), respectively. Retrotranscribing viruses, ssRNA, double-stranded (ds) DNA, single-stranded (ss) DNA, and ssRNA made up the viromes. The viral composition of swabs taken from tick bite wounds, throats, and anuses differs significantly. Both pool 4 and pool 5 included virus-related reads from the families Ackermannviridae , Myoviridae , Podoviridae , and Schitoviridae (Fig. 2 ). Most sequence reads showed low nucleotide and amino acid sequence identities with known viruses. Unclassified reads and a sizable number of unclassified viruses are present in the sample pools. In pool 4 and pool 5, the percentage of reads that corresponded to circovirus and papillomavirus were 0.13% and 0.12% of all the viruses, respectively. Papillomaviridae Genomic characterization of novel PV We obtained a complete genome Eld's deer Papillomavirus (PsPV-HMU-1) from pool 5, with genbank accession PQ037580. A core collection of early (E6, E7, E1, and E2) and late (L2 and L1) open-reading frames (ORFs) was predicted using Pfam software (Fig. 3 A). PsPV-HMU-1 also exhibits conserved LCR organizations among PV types belonging to the same genus. The non-coding LCR region of PsPV-HMU-1 (Fig. 3 B), located between the late and early genes, contained a single polyadenylation (PolyA) signal, TATA box, nuclear factor (Nf1) (TTGGC), and four E2 binding sites, following the ‘traditional’ four E2-binding site (ACCN2-11GGT) pattern found in most mammalian papillomaviruses. The E7 and E6 protein upregulate DNA replication, inhibit cell checkpoints, and block apoptosis in mammalian hosts. The novel PV has a prototypical mammalian E6 protein consisting of two highly conserved zinc finger motifs (C-X2-C-X29-C-X2-C). The C-terminal zinc-binding domain suggests that the E7 protein conforms to the classical E7 protein observed in mammalian papillomaviruses (Fig. 3 C). An ATP/GTP binding site (G-X4-GKS) was present in the E1 helicase domain. Phylogenetic Classification of the Genomes of novel PV Our selection of mammalian PVs as references was primarily based on BLAST results, and the early evolutionary analysis of the ICTV novel PVs. A small number of sauropsid and fish PV clades were selected as outgroups. The phylogenetic tree is consistent with the evolutionary relationships among three distinct monophyletic branches: avian papillomavirus, sauropsid, and mammalian, as proposed by the International Committee on Taxonomy of Viruses (ICTV). The papillomaviruses most closely related to PsPV-HMU-1 were those from the genus Dyokappapapillomavirus (Supplementary Table 3), particularly bovine papillomavirus 22 (BVP 22) (NC035208) (Fig. 4 ). Circroviridae Genomic characterization of novel CV We obtained a complete Eld's deer-associated circular single-stranded (ssDNA) virus genome (PsaCV-HMU-1) from pool 4, with GenBank accession: PQ119789. PsaCV-HMU-1 had genome length of 2,722 bp and C + G content of 40.56%. The genome contains two major open reading frames (ORFs), encoding the replication-associated protein (Rep) and the capsid protein (Cp) on different strands of a double-stranded DNA (dsDNA) replicative form. The rolling circle replication (RCR) mechanism of circular CRESS DNA viruses is associated with conserved Rep motifs, including N-terminal RCR motifs (I, II, and III), C-terminal superfamily 3 helicase motifs (Walker-A, -B, and Motif C), and an arginine finger, that together have a role in the initiation, elongation, and termination of the RCR of CRESS DNA viruses of eukaryote origin 21. Similar to other CRESS DNA viruses, PsaCV-HMU-1 contains a conserved sequence (CAGTGTTAC) located in a putative stem loop structure at the origin of replication. Phylogenetic Classification of the Genomes of novel CV Due to the diversity of circovirus species and genomes, effectively classifying circoviruses is challenging. Therefore, we selected representative virus sequences from the Circoviridae , Smacoviridae , and CRESS DNA viruses to jointly establish a phylogenetic tree. The phylogenetic trees of Rep and Cp are almost consistent with each other; however, the Rep of PsaCV-HMU-1 was segregated from the known CVs at the deep root of the phylogenetic tree. PsaCV-HMU-1 was only distantly related to the Circoviridae and Smacoviridae but was grouped together in a separate branch with unclassified CRESS virus 1 (CRESSV1) genomes, particularly the camel-associated drosmacovirus (KM573766) (Fig. 5 A-B). The Rep and Cp genome sequences of PsaCV-HMU-1 showed lower amino acid identities of 45.43% and 31.73%, respectively, compared to other known viruses (Supplementary Table 4). Additionally, these sequences were difficult to compare with those of other known circular viruses at the nucleic acid level in the NCBI nucleotide database. Prevalence of novel virus infection in Eld's deer Quick sampling within two months is necessary to track the percentage of deer that are viral carriers within the same season. Five pools were examined for positivity of PsPV-HMU-1 and PsaCV-HMU-1. For each individual sample in the positive-result pool, conduct a second nucleic acid extraction and a positive rate screening. PsPV-HMU-1 and PsaCV-HMU-1 positive samples yielded amplification products of 1529 bp and 1169 bp, respectively. Both nasal and anal swabs from Eld’s deer displayed a high PsPV-HMU-1 positivity rate (Table 1 ), but only one animal’s anal swab showed PsaCV-HMU-1 positivity (Table 2 ). The results showed that PsPV-HMU-1 was detected in 15% (5/33) of the nasal swabs, 9% (3/33) of the anal swabs, and 55% (5/9) of the wound swabs (Table 1 ). Only one anal swabs (1/33) detected PsaCV-HMU-1, while nasal swabs and wound swabs were negative (Table 2 ). Table 1 Detection rate of PsPV-HMU-1 among Eld's deer from Baisha No. nose swab anal swabs wound swabs (bitten by ticks) Sampling date Sampling place 1 - - - 2023/12/29 Fence No.2 2 - - Null 2023/12/29 Fence No.2 3 - - Null 2023/12/29 Fence No.2 4 - - Null 2023/12/29 Fence No.2 5 - - Null 2023/12/29 Fence No.2 6 - - Null 2024/1/9 Fence No.8 7 - - Null 2024/1/9 Fence No.8 8 - - Null 2024/1/9 Fence No.8 9 - - Null 2024/1/9 Fence No.8 10 - - Null 2024/1/9 Fence No.8 11 - - Null 2024/1/9 Fence No.8 12 - - Null 2024/1/9 Fence No.8 13 - - - 2024/1/9 Fence No.8 14 - - Null 2024/1/9 Fence No.8 15 - - Null 2024/1/9 Fence No.8 16 - - Null 2024/1/9 Fence No.8 17 - - Null 2024/1/9 Fence No.8 18 - - Null 2024/1/9 Fence No.8 19 - - Null 2024/1/9 Fence No.8 20 - - Null 2024/1/10 Fence No.1 21 - - - 2024/1/10 Fence No.1 22 + - + 2024/1/10 Fence No.1 23 - + + 2024/1/10 Fence No.1 24 - + + 2024/1/10 Fence No.1 25 + - - 2024/1/10 Fence No.1 26 + - + 2024/1/10 Fence No.1 27 - - Null 2024/1/10 Fence No.1 28 - - Null 2024/1/10 Fence No.1 29 + - Null 2024/1/10 Fence No.2 30 + - + 2024/1/10 Fence No.2 31 - + Null 2024/1/10 Fence No.2 32 - - Null 2024/1/10 Fence No.2 33 - - Null 2024/1/10 Fence No.2 Table 1 Detection rate of PsPV-HMU-1 among Eld's deer from Baisha. The nasal and anal swab samples from fence 2 form pools 1 and 3, respectively. The nasal and anal swab samples from fence 1 and fence 8 form pools 2 and 4, respectively. Table 2 Detection rate of PsaCV-HMU-1 among Eld's deer from Baisha No. nose swab anal swabs wound swabs (bitten by ticks) Sampling date Sampling place 1 - - - 2023/12/29 Fence No.2 2 - - Null 2023/12/29 Fence No.2 3 - - Null 2023/12/29 Fence No.2 4 - - Null 2023/12/29 Fence No.2 5 - - Null 2023/12/29 Fence No.2 6 - - Null 2024/1/9 Fence No.8 7 - - Null 2024/1/9 Fence No.8 8 - - Null 2024/1/9 Fence No.8 9 - - Null 2024/1/9 Fence No.8 10 - - Null 2024/1/9 Fence No.8 11 - - Null 2024/1/9 Fence No.8 12 - - Null 2024/1/9 Fence No.8 13 - - - 2024/1/9 Fence No.8 14 - - Null 2024/1/9 Fence No.8 15 - - Null 2024/1/9 Fence No.8 16 - - Null 2024/1/9 Fence No.8 17 - - Null 2024/1/9 Fence No.8 18 - - Null 2024/1/9 Fence No.8 19 - - Null 2024/1/9 Fence No.8 20 - - Null 2024/1/10 Fence No.1 21 - - - 2024/1/10 Fence No.1 22 - - - 2024/1/10 Fence No.1 23 - - - 2024/1/10 Fence No.1 24 - - - 2024/1/10 Fence No.1 25 - - - 2024/1/10 Fence No.1 26 - - - 2024/1/10 Fence No.1 27 - + Null 2024/1/10 Fence No.1 28 - - Null 2024/1/10 Fence No.1 29 - - Null 2024/1/10 Fence No.2 30 - - - 2024/1/10 Fence No.2 31 - - Null 2024/1/10 Fence No.2 32 - - Null 2024/1/10 Fence No.2 33 - - Null 2024/1/10 Fence No.2 Discussion Artiodactyls have a higher rate of carriage papillomavirus in mammals. Based on PsPV-HMU-1 and other recent studies, all mammalian papillomaviruses in the Dyokap genus contain seven typical sequences (LCR E6, E7, E1, E2, L2, and L1). The pairwise alignment of the L1 ORF showed a maximum nucleotide sequence homology of 76.11% with the most similar sequence of the BvPV 22. The amino acid identities of L1 between PsPV-HMU-1 and species within the Dyokap genus range from 56.47–77.20% (Supplementary Table 3). PsPV is distantly related to human-associated papillomaviruses. Based on sequence identity criteria and the distance of PsPV-HMU-1 to other viruses in the Dyokap genus, PsPV-HMU-1 is likely to be classified as a new papillomavirus species within the Dyokap genus (Fig. 4 ). PsPV-HMU-1 shares many features with other viruses in the Dyokap genus, providing evidence for host-virus co-speciation. However, utilizing "molecular morphology" information can more accurately determine the phylogenetic placement of Fulmarus glacialis papillomavirus 1 (FgPV1) 22 , emphasizing the importance of molecular-level analysis, especially early protein analysis. Animals with positive PsPV-HMU-1 in wound swabs must also have tested positives in throat or anal swabs, indicating that the infection was persistent. The high PsPV-HMU-1 positivity rate in swabs from tick bites on Eld's deer highlights the potential threat of insect vectors to animal populations (Table 1 ). The evolutionary history of this group of viruses extends back at least 400 million years ago (MYA) as evidenced by the recent discovery of fish papillomaviruses 23 . Osteichthyes and Amniota shared a common ancestor around 400 MYA 24 . The host range and virological categorization have been broadened by the isolation and characterization of the novel viruses from the Cervidae family. These include a virus isolated from a European elk fibropapilloma 25 , two related papillomaviruses isolated from the fibromas of American mule deer 25 , and a papillomavirus isolated from a red deer fibropapilloma 25 . Papillomaviruses are often host-specific. However these cross-species transmission occurrences within papillomavirus-affected species have also been reported in mallards and gulls 26 , suggesting that host specificity limitations may loosen in cases of genetically closely related hosts. It is important to assess the possibility of cross-species transmission of PV carried by wild Eld's deer. Prokaryotes, eukaryotes, and environmental samples have all been found to contain CRESS DNA viruses, a very varied collection of tiny viruses 27 , 28 . The porcine circovirus 2 is associated with multisystemic diseases in swine, posing a threat to swine herds and losses for breeders. In this study, the occurrence of CRESS DNA viruses was investigated in anal swab samples of healthy Eld's deer collected from the Provincial Nature Reserve. The rep of PsaCV-HMU-1 is difficult to compare with other CVs at the nucleotide level, and its amino acid homology with the closest Dromedary stool-associated circular ssDNA virus is 45.43% (Supplementary Table 4). In the phylogenetic tree, PsaCV-HMU-1 was grouped with CRESSV1, but it belonged to separate groups of the Cirlivirales order (Fig. 5 ). Although species demarcation criteria were not defined for CRESSV1 and CRESSV2 viruses, the distance between PsaCV-HMU-1 on the phylogenetic tree and known sequence suggests that the new virus may belong to a different virus species. Therefore,it is difficult to assign PsaCV-HMU-1 to the appropriate viral families. However, these findings provide some insight into the ecology of tiny circular DNA virus genomes and aid in the construction of data sets related to them. The CRESS DNA viruses associated with eukaryotic hosts have been classified by the ICTV, namely Circoviridae , Genomoviridae , Geminiviridae , Nanoviridae , Bacilladnaviridae , and Smacoviridae 29. Compared to wild animals, there are more studies on the impact of virus diversity in pig manure on economic benefits. CRESS DNA virus sequences are frequently detected in the fecal virome of Hungarian pigs, indicating a correlation between semi-enclosed animal husbandry and environmental pollution 30. These findings provides a reference for the protection of wild deer. Closely related CRESS DNA viruses of the Cirlivirales and Cremevirales orders, such as those in the Circoviridae and Smacoviridae families, may be present in the intestines of swine and other mammals (such as bovines, fur seals, or primates), indicating that multiple host species can be infected despite differences in genomic sequences 13 . Gaining more insight into the evolutionary processes of the virus carried by Eld's deer will be possible through extensive genomic and phylogenetic analysis of novel PVs and CVs, as well as comparisons with other non-mammalian viruses. Further research is necessary to determine the pathogenicity and potential effects of novel mammal-borne PVs and CVs on wild deer, as these viruses have expanded both the viral genomic information and host range. Identification susceptible wild animals and understanding transmission within animal populations are important for the assessment of zoonotic potential. Materials and methods Swab sample collection Thirty-three Eld's deer ( Rucervus eldii ) were captured from three isolated fenced areas (n = 33, 20 females, 13 males), with nine having tick bites on their ears in December 2023 and January 2024 (Fig. 1 ). We used tweezers to pick up the tick and collected the bite wound specimen using a swab. No specific clinical disease signs were recorded in any of the animals. We collect 33 nasal swabs, 33 anal swabs, and 9 tick bite wound swabs. Thoese wild animals live in the Bangxi Provincial Nature Reserve in Hainan Province. To ensure sample quality, the collected samples were promptly submerged in virus-sampling tubes that will not inactivate the virus and stored at -20°C at the sampling site (Yocon Biology, Beijing, China). They were then sent to the laboratory in an ice-cold dry chain within 24 hours 31 . After that, the samples were kept at -80°C. Samples from each individual were combined by adding 1 mL into one fresh sample tube (10–23 samples in each pool). We combined the same type of swabs and obtained 2 throat pools, 2 anal pools, and 1 tick bite wound pool. All animals are protected in accordance with the biosafety commitment letter signed before sampling. The sampling procedures were approved by the Ethics Committee of the Hainan Medical University (approval no. HMUEC20180059). Viral nucleic acid library construction and NGS The combined sample was ultracentrifuged for three hours at 100,000×g and 4°C after being passed through a 0.45 µm filter (Millipore Sigma, Burlington, MA, USA). The precipitates from the samples were resuspended in 100 µL of Hank's balanced salt solution and digested with a cocktail of DNase and RNase enzymes to decompose and remove unprotected nucleic acids, as previously reported 14 . Viral DNA and RNA were extracted using QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions (Qiagen, Hilden, Germany). The first strand of cDNA was synthesized using Superscript III Reverse Transcriptase (Invitrogen, Thermo Fisher Scientific), and the double-stranded DNA was synthesized in compliance with previous guidelines 14 , 32 . The nucleic acid library was then constructed using the DNA Library Prep Kit. (Invitrogen Collibri, USA). The amplified viral nucleic acid libraries were sequenced on an Illumina HiSeq 2500 using the 150 bp paired-end method (Illumina Inc., San Diego, CA, USA). The valid sequence data that have been cleaned and filtered were deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive under the accession number PRJNA1138552. Taxonomic assignment The methods and parameters for extracting effective sequences after cleaning and screening the original sequences were applied using the previously described criteria 14 , followed by sequence similarity-based taxonomic assignments 14 , 33 . First, we used Trinity (version 2.5.1) to assemble the randomly distributed reads across the entire viral genome into contigs, and compared them with the National Center for Biotechnology Information (NCBI) nonredundant protein (NR) database using basic local alignment search tool (BLAST) (E-value < 10 − 5 , -F: filter query sequence, default = T), and then with our in-house database ( ftp://ftp.ncbi.nih.gov/pub/taxonomy/accession2taxid/ ) for classification annotation and other information obtained from the NCBI Entrez server. Sequences that were difficult to align with the NR database were extracted and reanalyzed to avoid missing important or novel data. Genome sequencing and annotation of novel virus Based on the results of the virus categorization annotation, extract pertinent circovirus and papillomavirus reads, create nested primers for polymerase chain reaction (PCR) amplification, and follow the conditions as described previously. Sequence the amplified products and then splice them using SeqMan with the default settings (Version 7.1.0.44). A list of primers used to amplify the sequences found during this inquiry is provided in Supplementary Table 1. Use BLAST software on NCBI to compare the new sequence with a known virus library to obtain nucleotide and amino acid information. Use EditSeq (Version 7.1.0.44) to predict the position of the open reading frame. Conserved protein families and domains were predicted using Pfam ( http://www.ebi.ac.uk/services/proteins ), Blastp ( https://blast.ncbi.nlm.nih.gov ), and InterProScan 5 ( http://www.ebi.ac.uk/services/proteins ). The IBS 1.0.3 program was used to create the genomic structure diagram. Phylogenetic and data analyses The ICTV was used to obtain the representative reference sequences required to construct the phylogenetic tree ( https://ictv.global/report/genome ). The reference sequences with close genetic relationships were obtained from GenBank. Alignments were prepared with MEGAX using the MUSCLE package with default parameters, and the best substitution model was evaluated using the Model Selection tool. To determine the origins and evolutionary history of novel PV, we randomly select the complete E1-E2-L2-L1 nucleotide sequences of 42 representative viruses for phylogenetic analysis. The phylogenetic tree was constructed using the maximum likelihood method (GTR + G) with 1,000 bootstrap replicates. The classification and phylogeny of novel circular virus are based on the available replicase (Rep) and capsid (Cp) sequences using the maximum likelihood method (LG + F + G + I) with 1,000 bootstrap replicates. Calculation of prevalence RNA was extracted from all individual samples using the QIAamp Virus RNA Mini Kit (Qiagen, Hilden, Germany). Specific PCR primers were designed (Supplementary Table 1) to amplify the conserved regions of L1 in PV and Rep in CV. Sequencing results revealed > 99% similarity between the amplified products and the novel viruses, which can be used for positive rate detection. PCR was performed using GoTaq Colorless Master Mix (Promega). Nested PCR used two microliters of the first-round PCR product as the template for the second round of PCR. The thermal cycling conditions for both PCRs were as follows: 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 57°C for 35 s, 72°C for 30 s, and a final elongation step at 72°C for 10 min. If the product is consistent with expectations 31 , it is determined to be a positive sample. Declarations Acknowledgements We would like to extend our gratitude to all the institution and individuals who participate and provided their kind assistance, especially generous permission and collaboration in the sample collection process from the Hainan Bangxi Provincial Nature Reserve Administration and the Hainan Tropical Infectious Diseases Biobank. Author contributions Y.Z., G.L. and Y.L. (Youyou Li) designed the study. Y.Z., X.Q., Y.C., Q.L., G.R., R.P., G.W., X.H. and Y.H. collected the specimens. H.S., Z.L., J.D., M.Z. and X.T., performed the experiments. D.Z., J.D., Y.L. (Youyou Li), C.T. (Chuanning Tang), X.C., L.N., J.Y., F.Y., G.L., J.L., Y.L. (Yu Li) and C.T. (Chuan Tian) analysed the data. Y.L. (Youyou Li) wrote the draft manuscript. D.Z., F.Y. and Y.Z. edited the manuscript. All authors read and approved the final manuscript. Funding This work was supported by the Key Research and Development Plan of Hainan Province (No. ZDYF2022SHFZ085), Hainan Provincial Natural Science Foundation of China (No. 822RC695), National Natural Science Foundation of China (Nos. 32060015 and 82060378), Hainan Medical University Talent Development Project (No. XRC2021002), Science and Technology Planning Project of Guizhou Province (No. qkhjc-zk2022-yb435), Gradutae student innovation grant of Hainan Medical University (No. HYYS2020-33), Research Project of the Hainan Academician Innovation Platform (No. YSPTZX202004), and Hainan Talent Development Project (No. SRC200003). Data availability statement The datasets presented in this study can be found in online repositories of the National Center for Biotechnology Information (NCBI). The accession number(s) was PRJNA1138552. Ethics Statement The sampling strategy in this study did not involve hunting or euthanized method. During the translocation process in the nature reserves where Eld's deer live, we collected throat swabs, anal swabs, and swab samples from tick bite sites. After sample collecting, all Eld's deer were released on-site as soon as possible. We have no experiments on live vertebrates. All methods were carried out in accordance with relevant guidelines and regulations of the Ethics Committee of the Hainan Medical University (approval no. HMUEC20180059), and reported in accordance with ARRIVE guidelines (https://arriveguidelines.org). Competing interests The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Supplementary Files SupplementaryTable.xlsx Cite Share Download PDF Status: Published Journal Publication published 11 Mar, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 30 Dec, 2024 Reviews received at journal 28 Dec, 2024 Reviewers agreed at journal 05 Dec, 2024 Reviewers agreed at journal 04 Dec, 2024 Reviews received at journal 03 Dec, 2024 Reviews received at journal 03 Dec, 2024 Reviewers agreed at journal 29 Nov, 2024 Reviewers agreed at journal 26 Nov, 2024 Reviewers invited by journal 25 Nov, 2024 Editor assigned by journal 25 Nov, 2024 Editor invited by journal 21 Nov, 2024 Submission checks completed at journal 20 Nov, 2024 First submitted to journal 01 Nov, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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07:08:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5371247/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5371247/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-92781-4","type":"published","date":"2025-03-11T15:57:10+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":69967524,"identity":"88f78ad3-ce42-4216-af1f-6bb748e716f0","added_by":"auto","created_at":"2024-11-27 05:39:27","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":291340,"visible":true,"origin":"","legend":"\u003cp\u003eSample collection from Eld's deer in Hainan Bangxi Provincial Nature Reserve. The specific sampling points have been marked on the map.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5371247/v1/d48d9ca5267bf37e310f2670.jpg"},{"id":69967526,"identity":"9d42552e-72d4-4e62-9126-fa3a13884983","added_by":"auto","created_at":"2024-11-27 05:39:27","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":218893,"visible":true,"origin":"","legend":"\u003cp\u003eNext-generation sequencing (NGS) sequencing data of the virome profile of 5 pools (A) Viral content in pool 1, which is composed of nasal swab samples. (B) Viral content in pool 2, which is composed of nasal swab samples. (C) Viral content in pool 3, which is composed of anal swab samples. (D) Viral content in pool 4, which is composed of anal swab samples. (E) Viral content in pool 5, which is composed of tick bite wound swab samples.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5371247/v1/8cdb518b03caed51bb9d72b5.jpg"},{"id":69967529,"identity":"595b23dd-5c38-4891-9513-5e00c68a844b","added_by":"auto","created_at":"2024-11-27 05:39:28","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":663629,"visible":true,"origin":"","legend":"\u003cp\u003eGenomic structure and motif annotation of PsPV-HMU-1. (A) Using the ORF predictor, putative and confirmed early and late ORFs were found in the genomes of PsPV-HMU-1. LTR, long-terminal repeat; early (E6, E7, E1, E2); and late (L2, L1). (B) The motif structure diagram of LTR in PsPV-HMU-1. (C) The CR1, CR2, and N-terminal structure diagram of E7.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5371247/v1/cd88afa22648016697160d38.jpg"},{"id":69968997,"identity":"ead4f2d5-9928-484e-a14d-1377139b2c21","added_by":"auto","created_at":"2024-11-27 05:56:04","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":700746,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree of PsPV-HMU-1, concatenated early (E1, E2) and late (L2, L1) papillomavirus nucleotide sequences. The maximum likelihood approach and a 1000 bootstrap replication were used to construct the tree. The scale bar indicates the number of amino acid changes per site, and bootstrap values \u0026lt;50 percent are not shown.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5371247/v1/b8adb163474f436fdf972f9b.jpg"},{"id":69967527,"identity":"25e84a2c-844a-4444-8a50-70fb62ded989","added_by":"auto","created_at":"2024-11-27 05:39:27","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":724344,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree based on the amino acid sequences of the complete Rep and Cp amino acids of PsaCV-HMU-1. Phylogenetic tree constructed by the maximum likelihood method using the best-fit models (LG+G+I). The scale bar indicates the number of amino acid changes per site, and bootstrap values \u0026lt;50 percent are not shown. (A) Phylogenetic tree based on Rep amino acid sequence. (B) Phylogenetic tree based on the Cp amino acid sequence.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5371247/v1/e80ee86befa0b6743057e509.jpg"},{"id":78688915,"identity":"b9749c1f-f8aa-4b25-8020-fd667b70c748","added_by":"auto","created_at":"2025-03-17 16:07:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3842262,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5371247/v1/49082e4e-6a86-407e-804c-a1daf9637f8c.pdf"},{"id":69969755,"identity":"2eca7079-ea46-437c-93f7-51da34f52994","added_by":"auto","created_at":"2024-11-27 06:03:54","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17432,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5371247/v1/707ec273377e1fe816b47339.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The virome investigation of the globally endangered Eld's deer (Rucervus eldii) on Hainan Island, China","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEld\u0026apos;s deer\u0026nbsp;(\u003cem\u003eRucervus eldii\u003c/em\u003e)\u0026nbsp;is a rare and\u0026nbsp;globally\u0026nbsp;endangered\u0026nbsp;tropical Southeast Asian\u0026nbsp;deer species, belonging to Artiodactyla, Family Cervidae and Subfamily Cervinae.\u0026nbsp;It has been listed in Appendix I\u0026nbsp;of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) and classified as endangered on the Red List of Threatened Species by the International Union for Conservation of Nature (IUCN) and\u0026nbsp;the class I national key protected wildlife in China\u0026nbsp;\u003csup\u003e1\u003c/sup\u003e. Eld\u0026apos;s deer was\u0026nbsp;once widely distributed on Hainan Island in China, but due to factors like habitat loss and destruction, blind hunting, and illegal trade, their population has sharply declined, and they are in danger of going extinct\u0026nbsp;\u003csup\u003e2\u003c/sup\u003e. There were only 26 Eld\u0026apos;s deer remaining in Hainan in the 1970s\u0026nbsp;\u003csup\u003e3\u003c/sup\u003e. At present, Eld\u0026apos;s deer are mainly distributed on the Hainan Datian National Nature Reserve and Hainan Bangxi Provincial Nature Reserve in China. Despite\u0026nbsp;fact that the Eld\u0026apos;s deer population has recovered and grown after over 40 years of development and preservation, it continues to be extremely vulnerable to extinction because of things like inbreeding, poor genetic diversity, and the diminishing evolutionary capacity of tiny populations. According to reports, there are near 300 deer in Hainan Bangxi Provincial Nature Reserve which clearly exceeded the reserve\u0026apos;s carrying capacity and increased the risk of virus transmission. In other countries and regions of Southeast Asia,\u0026nbsp;Eld\u0026apos;s deer\u0026nbsp;has been facing huge threats.\u0026nbsp;Over 360 Eld\u0026apos;s deer were surviving in India in 2003\u003csup\u003e4\u003c/sup\u003e. This species saw a 90% decline in Cambodia between 1998 and 2008 and a 50% decline in the ensuing 10 to 15 years. The number of Eld\u0026apos;s deer in Laos and Myanmar is also declining annually, and the\u0026nbsp;wild population\u0026nbsp;of\u0026nbsp;Eld\u0026apos;s deer are thought to be regional extinct in Thailand and Vietnam\u0026nbsp;\u003csup\u003e4\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnimal pathogen monitoring is usually limited to pathogens with known economic impacts on livestock\u0026nbsp;and\u0026nbsp;is extremely rare in wild animal species. However, most re-emerging viral infections originate from animals, so routine testing cannot provide sufficient vigilance\u0026nbsp;\u003csup\u003e5\u003c/sup\u003e.\u0026nbsp;Pathogenic organisms can cause outbreaks and even lead to population collapse in wild animals. The severe acute respiratory syndrome coronavirus (SARS-CoV)\u0026nbsp;\u003csup\u003e6, 7\u003c/sup\u003e that broke out in China in 2003, the\u0026nbsp;influenza A virus\u0026nbsp;(H1N1)\u0026nbsp;\u003csup\u003e8\u003c/sup\u003e that prevailed in the global influenza pandemic in 2009, the Middle East respiratory syndrome coronavirus (MERS-CoV)\u0026nbsp;\u003csup\u003e9\u003c/sup\u003e in the Middle East in 2012, the Corona Virus Disease 2019 (SARS-CoV-2) that caused extremely high death in the world in 2019, and the animal-derived hantavirus\u0026nbsp;\u003csup\u003e10\u003c/sup\u003e suggest that growing global virus diversity and potential spillover effects require attention.\u0026nbsp;The cross-species transmission and clinical pathogenic mechanisms of viruses carried by Artiodactyla deserve attention. The serum samples of captive dromedary camels\u0026nbsp;\u003csup\u003e11\u003c/sup\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003eand wild Eld\u0026apos;s deer in Thailand\u0026nbsp;\u003csup\u003e12\u003c/sup\u003e both contain pandemic (H1N1) and 2009 influenza (H1N1pdm09) virus antibodies, indicating the susceptibility of those animals to natural infection.\u0026nbsp;Circular Rep-encoding (replication-associated protein encoding) single-stranded DNA (CRESS DNA) form a highly diverse group of small viruses that have been detected worldwide in prokaryotes and eukaryotes, and environmental samples\u0026nbsp;\u003csup\u003e13\u003c/sup\u003e.\u0026nbsp;However, the persistence of CRESS DNA viruses in herds of domestic animals has\u0026nbsp;\u003csup\u003e13\u003c/sup\u003e. PVs are a diverse group of small, non-enveloped viruses belonging to the family Papillomaviridae, with double-stranded DNA genomes\u0026nbsp;\u003csup\u003e14\u003c/sup\u003e. \u003cem\u003ePapillomaviridae\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eThe \u003cem\u003ePapillomaviridae\u003c/em\u003e has two subfamilies \u003cem\u003eFirstpapillomavirinae\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Secondpapillomavirinae\u003c/em\u003e, with more than 50 virus genera. PVs can cause hyperplasia in the skin and mucous membranes of humans, mammals, and non-mammalian animals, and are a significant risk factor for cervical and genital cancers\u0026nbsp;\u003csup\u003e14\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe development of viral metagenomics platforms has made it feasible to compile a more comprehensive list of the viruses that are present in animals\u0026nbsp;\u003csup\u003e5\u003c/sup\u003e.\u0026nbsp;Using viral metagenomic techniques, a considerable number of new tiny circular DNA viruses have been discovered and described from fecal sources during the last five years\u0026nbsp;\u003csup\u003e15-19\u003c/sup\u003e. Several mammalian viruses, including papillomavirus, parvovirus, poliovirus, and coronavirus, were detected in Arctic reindeer in western Alaska, USA, and the complete genome of papillomavirus was obtained from clinically manifested Roe deer in Slovenia through virus NGS\u0026nbsp;\u003csup\u003e20\u003c/sup\u003e. Using NGS technology, we have obtained two complete virus sequences of papillomavirus and circovirus from Elder\u0026apos;s deer and subsequently identified and described as PsPV-HMU-1 and PsaCV-HMU-1. We particularly focus on the origin of PV oncogenes, assessing whether they are monophyletic or result from processes like mutations or recombination based on the conserved sections of PV, and evaluate the potential for novel PV genes to cause cancer. It is crucial to perform viral metagenomics research on Hainan Eld\u0026apos;s deer to comprehend the diversity, evolution, and variation patterns of virus genotypes carried by Eld\u0026apos;s deer.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e\n \u003ch2\u003eViral Metagenome of Eld\u0026apos;s deer\u003c/h2\u003e\n \u003cp\u003eEfforts were made to eliminate bacteria, microbial eukaryotes, archaea, and organisms that showed no discernible relationship to any amino acid sequences in the viral non-redundant (NR) protein database. Five pools yielded 59.50 GB of nucleotide data (58,402,837 valid reads, 150 bp in length). The sequences of less than 50bp, archaea, bacteria, microbial eukaryotes (such as fungi), and those that had no significant similarity with any amino acid sequences in the viral NR protein database were eliminated (Supplementary Table 2). Figure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e shows the virus composition of 5 pools. Unfortunately, the viruses of the \u003cem\u003ePicornavirdae\u003c/em\u003e in pool 1 and \u003cem\u003eParvoviridae\u003c/em\u003e in pool 2 have not been identified, possibly due to prolonged degradation of samples (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). In pools 4 and 5, the proportion of virus-associated reads was 2,039,836 (16.97%) and 3,128,644 (28.15%), respectively. Retrotranscribing viruses, ssRNA, double-stranded (ds) DNA, single-stranded (ss) DNA, and ssRNA made up the viromes. The viral composition of swabs taken from tick bite wounds, throats, and anuses differs significantly. Both pool 4 and pool 5 included virus-related reads from the families \u003cem\u003eAckermannviridae\u003c/em\u003e, \u003cem\u003eMyoviridae\u003c/em\u003e, \u003cem\u003ePodoviridae\u003c/em\u003e, and \u003cem\u003eSchitoviridae\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Most sequence reads showed low nucleotide and amino acid sequence identities with known viruses. Unclassified reads and a sizable number of unclassified viruses are present in the sample pools. In pool 4 and pool 5, the percentage of reads that corresponded to circovirus and papillomavirus were 0.13% and 0.12% of all the viruses, respectively.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003ePapillomaviridae\u003c/h2\u003e\n \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e\n \u003ch2\u003eGenomic characterization of novel PV\u003c/h2\u003e\n \u003cp\u003eWe obtained a complete genome Eld\u0026apos;s deer Papillomavirus (PsPV-HMU-1) from pool 5, with genbank accession PQ037580. A core collection of early (E6, E7, E1, and E2) and late (L2 and L1) open-reading frames (ORFs) was predicted using Pfam software (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). PsPV-HMU-1 also exhibits conserved LCR organizations among PV types belonging to the same genus. The non-coding LCR region of PsPV-HMU-1 (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB), located between the late and early genes, contained a single polyadenylation (PolyA) signal, TATA box, nuclear factor (Nf1) (TTGGC), and four E2 binding sites, following the \u0026lsquo;traditional\u0026rsquo; four E2-binding site (ACCN2-11GGT) pattern found in most mammalian papillomaviruses. The E7 and E6 protein upregulate DNA replication, inhibit cell checkpoints, and block apoptosis in mammalian hosts. The novel PV has a prototypical mammalian E6 protein consisting of two highly conserved zinc finger motifs (C-X2-C-X29-C-X2-C). The C-terminal zinc-binding domain suggests that the E7 protein conforms to the classical E7 protein observed in mammalian papillomaviruses (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC). An ATP/GTP binding site (G-X4-GKS) was present in the E1 helicase domain.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003ch3\u003ePhylogenetic Classification of the Genomes of novel PV\u003c/h3\u003e\n\u003cp\u003eOur selection of mammalian PVs as references was primarily based on BLAST results, and the early evolutionary analysis of the ICTV novel PVs. A small number of sauropsid and fish PV clades were selected as outgroups. The phylogenetic tree is consistent with the evolutionary relationships among three distinct monophyletic branches: avian papillomavirus, sauropsid, and mammalian, as proposed by the International Committee on Taxonomy of Viruses (ICTV). The papillomaviruses most closely related to PsPV-HMU-1 were those from the genus \u003cem\u003eDyokappapapillomavirus\u003c/em\u003e (Supplementary Table 3), particularly bovine papillomavirus 22 (BVP 22) (NC035208) (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eCircroviridae\u003c/h3\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eGenomic characterization of novel CV\u003c/h2\u003e\n \u003cp\u003eWe obtained a complete Eld\u0026apos;s deer-associated circular single-stranded (ssDNA) virus genome (PsaCV-HMU-1) from pool 4, with GenBank accession: PQ119789. PsaCV-HMU-1 had genome length of 2,722 bp and C\u0026thinsp;+\u0026thinsp;G content of 40.56%. The genome contains two major open reading frames (ORFs), encoding the replication-associated protein (Rep) and the capsid protein (Cp) on different strands of a double-stranded DNA (dsDNA) replicative form. The rolling circle replication (RCR) mechanism of circular CRESS DNA viruses is associated with conserved Rep motifs, including N-terminal RCR motifs (I, II, and III), C-terminal superfamily 3 helicase motifs (Walker-A, -B, and Motif C), and an arginine finger, that together have a role in the initiation, elongation, and termination of the RCR of CRESS DNA viruses of eukaryote origin 21. Similar to other CRESS DNA viruses, PsaCV-HMU-1 contains a conserved sequence (CAGTGTTAC) located in a putative stem loop structure at the origin of replication.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003ePhylogenetic Classification of the Genomes of novel CV\u003c/h2\u003e\n \u003cp\u003eDue to the diversity of circovirus species and genomes, effectively classifying circoviruses is challenging. Therefore, we selected representative virus sequences from the \u003cem\u003eCircoviridae\u003c/em\u003e, \u003cem\u003eSmacoviridae\u003c/em\u003e, and CRESS DNA viruses to jointly establish a phylogenetic tree. The phylogenetic trees of Rep and Cp are almost consistent with each other; however, the Rep of PsaCV-HMU-1 was segregated from the known CVs at the deep root of the phylogenetic tree. PsaCV-HMU-1 was only distantly related to the \u003cem\u003eCircoviridae\u003c/em\u003e and \u003cem\u003eSmacoviridae\u003c/em\u003e but was grouped together in a separate branch with unclassified CRESS virus 1 (CRESSV1) genomes, particularly the camel-associated drosmacovirus (KM573766) (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA-B). The Rep and Cp genome sequences of PsaCV-HMU-1 showed lower amino acid identities of 45.43% and 31.73%, respectively, compared to other known viruses (Supplementary Table 4). Additionally, these sequences were difficult to compare with those of other known circular viruses at the nucleic acid level in the NCBI nucleotide database.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003ePrevalence of novel virus infection in Eld\u0026apos;s deer\u003c/h3\u003e\n\u003cp\u003eQuick sampling within two months is necessary to track the percentage of deer that are viral carriers within the same season. Five pools were examined for positivity of PsPV-HMU-1 and PsaCV-HMU-1. For each individual sample in the positive-result pool, conduct a second nucleic acid extraction and a positive rate screening. PsPV-HMU-1 and PsaCV-HMU-1 positive samples yielded amplification products of 1529 bp and 1169 bp, respectively. Both nasal and anal swabs from Eld\u0026rsquo;s deer displayed a high PsPV-HMU-1 positivity rate (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), but only one animal\u0026rsquo;s anal swab showed PsaCV-HMU-1 positivity (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). The results showed that PsPV-HMU-1 was detected in 15% (5/33) of the nasal swabs, 9% (3/33) of the anal swabs, and 55% (5/9) of the wound swabs (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Only one anal swabs (1/33) detected PsaCV-HMU-1, while nasal swabs and wound swabs were negative (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eDetection rate of PsPV-HMU-1 among Eld\u0026apos;s deer from Baisha\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNo.\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003enose swab\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eanal swabs\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ewound swabs\u003c/p\u003e\n \u003cp\u003e(bitten by ticks)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSampling date\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSampling place\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2023/12/29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2023/12/29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2023/12/29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2023/12/29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2023/12/29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u0026nbsp;\u003c/strong\u003eDetection rate of PsPV-HMU-1 among Eld\u0026apos;s deer from Baisha. The nasal and anal swab samples from fence 2 form pools 1 and 3, respectively. The nasal and anal swab samples from fence 1 and fence 8 form pools 2 and 4, respectively.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eDetection rate of PsaCV-HMU-1 among Eld\u0026apos;s deer from Baisha\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNo.\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003enose swab\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eanal swabs\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ewound swabs\u003c/p\u003e\n \u003cp\u003e(bitten by ticks)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSampling date\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSampling place\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2023/12/29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2023/12/29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2023/12/29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2023/12/29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2023/12/29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2024/1/10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFence No.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eArtiodactyls have a higher rate of carriage papillomavirus in mammals. Based on PsPV-HMU-1 and other recent studies, all mammalian papillomaviruses in the Dyokap genus contain seven typical sequences (LCR E6, E7, E1, E2, L2, and L1). The pairwise alignment of the L1 ORF showed a maximum nucleotide sequence homology of 76.11% with the most similar sequence of the BvPV 22. The amino acid identities of L1 between PsPV-HMU-1 and species within the Dyokap genus range from 56.47\u0026ndash;77.20% (Supplementary Table\u0026nbsp;3). PsPV is distantly related to human-associated papillomaviruses. Based on sequence identity criteria and the distance of PsPV-HMU-1 to other viruses in the Dyokap genus, PsPV-HMU-1 is likely to be classified as a new papillomavirus species within the Dyokap genus (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). PsPV-HMU-1 shares many features with other viruses in the Dyokap genus, providing evidence for host-virus co-speciation. However, utilizing \"molecular morphology\" information can more accurately determine the phylogenetic placement of Fulmarus glacialis papillomavirus 1 (FgPV1) \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, emphasizing the importance of molecular-level analysis, especially early protein analysis. Animals with positive PsPV-HMU-1 in wound swabs must also have tested positives in throat or anal swabs, indicating that the infection was persistent. The high PsPV-HMU-1 positivity rate in swabs from tick bites on Eld's deer highlights the potential threat of insect vectors to animal populations (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe evolutionary history of this group of viruses extends back at least 400\u0026nbsp;million years ago (MYA) as evidenced by the recent discovery of fish papillomaviruses \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Osteichthyes and Amniota shared a common ancestor around 400 MYA \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The host range and virological categorization have been broadened by the isolation and characterization of the novel viruses from the Cervidae family. These include a virus isolated from a European elk fibropapilloma \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, two related papillomaviruses isolated from the fibromas of American mule deer \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, and a papillomavirus isolated from a red deer fibropapilloma \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Papillomaviruses are often host-specific. However these cross-species transmission occurrences within papillomavirus-affected species have also been reported in mallards and gulls \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, suggesting that host specificity limitations may loosen in cases of genetically closely related hosts. It is important to assess the possibility of cross-species transmission of PV carried by wild Eld's deer.\u003c/p\u003e \u003cp\u003eProkaryotes, eukaryotes, and environmental samples have all been found to contain CRESS DNA viruses, a very varied collection of tiny viruses \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. The porcine circovirus 2 is associated with multisystemic diseases in swine, posing a threat to swine herds and losses for breeders. In this study, the occurrence of CRESS DNA viruses was investigated in anal swab samples of healthy Eld's deer collected from the Provincial Nature Reserve. The rep of PsaCV-HMU-1 is difficult to compare with other CVs at the nucleotide level, and its amino acid homology with the closest Dromedary stool-associated circular ssDNA virus is 45.43% (Supplementary Table\u0026nbsp;4). In the phylogenetic tree, PsaCV-HMU-1 was grouped with CRESSV1, but it belonged to separate groups of the \u003cem\u003eCirlivirales\u003c/em\u003e order (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Although species demarcation criteria were not defined for CRESSV1 and CRESSV2 viruses, the distance between PsaCV-HMU-1 on the phylogenetic tree and known sequence suggests that the new virus may belong to a different virus species. Therefore,it is difficult to assign PsaCV-HMU-1 to the appropriate viral families. However, these findings provide some insight into the ecology of tiny circular DNA virus genomes and aid in the construction of data sets related to them.\u003c/p\u003e \u003cp\u003eThe CRESS DNA viruses associated with eukaryotic hosts have been classified by the ICTV, namely \u003cem\u003eCircoviridae\u003c/em\u003e, \u003cem\u003eGenomoviridae\u003c/em\u003e, \u003cem\u003eGeminiviridae\u003c/em\u003e, \u003cem\u003eNanoviridae\u003c/em\u003e, \u003cem\u003eBacilladnaviridae\u003c/em\u003e, and \u003cem\u003eSmacoviridae\u003c/em\u003e 29. Compared to wild animals, there are more studies on the impact of virus diversity in pig manure on economic benefits. CRESS DNA virus sequences are frequently detected in the fecal virome of Hungarian pigs, indicating a correlation between semi-enclosed animal husbandry and environmental pollution 30. These findings provides a reference for the protection of wild deer. Closely related CRESS DNA viruses of the \u003cem\u003eCirlivirales\u003c/em\u003e and \u003cem\u003eCremevirales\u003c/em\u003e orders, such as those in the \u003cem\u003eCircoviridae\u003c/em\u003e and \u003cem\u003eSmacoviridae\u003c/em\u003e families, may be present in the intestines of swine and other mammals (such as bovines, fur seals, or primates), indicating that multiple host species can be infected despite differences in genomic sequences \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGaining more insight into the evolutionary processes of the virus carried by Eld's deer will be possible through extensive genomic and phylogenetic analysis of novel PVs and CVs, as well as comparisons with other non-mammalian viruses. Further research is necessary to determine the pathogenicity and potential effects of novel mammal-borne PVs and CVs on wild deer, as these viruses have expanded both the viral genomic information and host range. Identification susceptible wild animals and understanding transmission within animal populations are important for the assessment of zoonotic potential.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSwab sample collection\u003c/h2\u003e \u003cp\u003eThirty-three Eld's deer (\u003cem\u003eRucervus eldii\u003c/em\u003e) were captured from three isolated fenced areas (n\u0026thinsp;=\u0026thinsp;33, 20 females, 13 males), with nine having tick bites on their ears in December 2023 and January 2024 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We used tweezers to pick up the tick and collected the bite wound specimen using a swab. No specific clinical disease signs were recorded in any of the animals. We collect 33 nasal swabs, 33 anal swabs, and 9 tick bite wound swabs. Thoese wild animals live in the Bangxi Provincial Nature Reserve in Hainan Province. To ensure sample quality, the collected samples were promptly submerged in virus-sampling tubes that will not inactivate the virus and stored at -20\u0026deg;C at the sampling site (Yocon Biology, Beijing, China). They were then sent to the laboratory in an ice-cold dry chain within 24 hours \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. After that, the samples were kept at -80\u0026deg;C. Samples from each individual were combined by adding 1 mL into one fresh sample tube (10\u0026ndash;23 samples in each pool). We combined the same type of swabs and obtained 2 throat pools, 2 anal pools, and 1 tick bite wound pool. All animals are protected in accordance with the biosafety commitment letter signed before sampling. The sampling procedures were approved by the Ethics Committee of the Hainan Medical University (approval no. HMUEC20180059).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eViral nucleic acid library construction and NGS\u003c/h2\u003e \u003cp\u003eThe combined sample was ultracentrifuged for three hours at 100,000\u0026times;g and 4\u0026deg;C after being passed through a 0.45 \u0026micro;m filter (Millipore Sigma, Burlington, MA, USA). The precipitates from the samples were resuspended in 100 \u0026micro;L of Hank's balanced salt solution and digested with a cocktail of DNase and RNase enzymes to decompose and remove unprotected nucleic acids, as previously reported \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Viral DNA and RNA were extracted using QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer\u0026rsquo;s instructions (Qiagen, Hilden, Germany). The first strand of cDNA was synthesized using Superscript III Reverse Transcriptase (Invitrogen, Thermo Fisher Scientific), and the double-stranded DNA was synthesized in compliance with previous guidelines \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The nucleic acid library was then constructed using the DNA Library Prep Kit. (Invitrogen Collibri, USA). The amplified viral nucleic acid libraries were sequenced on an Illumina HiSeq 2500 using the 150 bp paired-end method (Illumina Inc., San Diego, CA, USA). The valid sequence data that have been cleaned and filtered were deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive under the accession number PRJNA1138552.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eTaxonomic assignment\u003c/h2\u003e \u003cp\u003eThe methods and parameters for extracting effective sequences after cleaning and screening the original sequences were applied using the previously described criteria \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, followed by sequence similarity-based taxonomic assignments \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. First, we used Trinity (version 2.5.1) to assemble the randomly distributed reads across the entire viral genome into contigs, and compared them with the National Center for Biotechnology Information (NCBI) nonredundant protein (NR) database using basic local alignment search tool (BLAST) (E-value\u0026thinsp;\u0026lt;\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e, -F: filter query sequence, default\u0026thinsp;=\u0026thinsp;T), and then with our in-house database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003eftp://ftp.ncbi.nih.gov/pub/taxonomy/accession2taxid/\u003c/span\u003e\u003cspan address=\"http://ftp://ftp.ncbi.nih.gov/pub/taxonomy/accession2taxid/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for classification annotation and other information obtained from the NCBI Entrez server. Sequences that were difficult to align with the NR database were extracted and reanalyzed to avoid missing important or novel data.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eGenome sequencing and annotation of novel virus\u003c/h2\u003e \u003cp\u003eBased on the results of the virus categorization annotation, extract pertinent circovirus and papillomavirus reads, create nested primers for polymerase chain reaction (PCR) amplification, and follow the conditions as described previously. Sequence the amplified products and then splice them using SeqMan with the default settings (Version 7.1.0.44). A list of primers used to amplify the sequences found during this inquiry is provided in Supplementary Table\u0026nbsp;1. Use BLAST software on NCBI to compare the new sequence with a known virus library to obtain nucleotide and amino acid information. Use EditSeq (Version 7.1.0.44) to predict the position of the open reading frame. Conserved protein families and domains were predicted using Pfam (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ebi.ac.uk/services/proteins\u003c/span\u003e\u003cspan address=\"http://www.ebi.ac.uk/services/proteins\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), Blastp (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov\u003c/span\u003e\u003cspan address=\"https://blast.ncbi.nlm.nih.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and InterProScan 5 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ebi.ac.uk/services/proteins\u003c/span\u003e\u003cspan address=\"http://www.ebi.ac.uk/services/proteins\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The IBS 1.0.3 program was used to create the genomic structure diagram.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic and data analyses\u003c/h2\u003e \u003cp\u003eThe ICTV was used to obtain the representative reference sequences required to construct the phylogenetic tree (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ictv.global/report/genome\u003c/span\u003e\u003cspan address=\"https://ictv.global/report/genome\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The reference sequences with close genetic relationships were obtained from GenBank. Alignments were prepared with MEGAX using the MUSCLE package with default parameters, and the best substitution model was evaluated using the Model Selection tool. To determine the origins and evolutionary history of novel PV, we randomly select the complete E1-E2-L2-L1 nucleotide sequences of 42 representative viruses for phylogenetic analysis. The phylogenetic tree was constructed using the maximum likelihood method (GTR\u0026thinsp;+\u0026thinsp;G) with 1,000 bootstrap replicates. The classification and phylogeny of novel circular virus are based on the available replicase (Rep) and capsid (Cp) sequences using the maximum likelihood method (LG\u0026thinsp;+\u0026thinsp;F\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;I) with 1,000 bootstrap replicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCalculation of prevalence\u003c/h2\u003e \u003cp\u003eRNA was extracted from all individual samples using the QIAamp Virus RNA Mini Kit (Qiagen, Hilden, Germany). Specific PCR primers were designed (Supplementary Table\u0026nbsp;1) to amplify the conserved regions of L1 in PV and Rep in CV. Sequencing results revealed\u0026thinsp;\u0026gt;\u0026thinsp;99% similarity between the amplified products and the novel viruses, which can be used for positive rate detection. PCR was performed using GoTaq Colorless Master Mix (Promega). Nested PCR used two microliters of the first-round PCR product as the template for the second round of PCR. The thermal cycling conditions for both PCRs were as follows: 94\u0026deg;C for 5 min, followed by 35 cycles of 94\u0026deg;C for 30 s, 57\u0026deg;C for 35 s, 72\u0026deg;C for 30 s, and a final elongation step at 72\u0026deg;C for 10 min. If the product is consistent with expectations \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, it is determined to be a positive sample.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to extend our gratitude to all the institution and individuals who participate and provided their kind assistance, especially generous permission and collaboration in the sample collection process from the Hainan Bangxi Provincial Nature Reserve Administration and the Hainan Tropical Infectious Diseases Biobank.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.Z., G.L. and Y.L. (Youyou Li) designed the study. Y.Z., X.Q., Y.C., Q.L., G.R., R.P., G.W., X.H. and Y.H. collected the specimens. H.S., Z.L., J.D., M.Z. and X.T., performed the experiments. D.Z., J.D., Y.L. (Youyou Li), C.T. (Chuanning Tang), X.C., L.N., J.Y., F.Y., G.L., J.L., Y.L. (Yu Li) and C.T. (Chuan Tian) analysed the data. Y.L. (Youyou Li) \u0026nbsp;wrote the draft manuscript. D.Z., F.Y. and Y.Z. edited the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Key Research and Development Plan of Hainan Province (No. ZDYF2022SHFZ085), Hainan Provincial Natural Science Foundation of China (No. 822RC695), National Natural Science Foundation of China (Nos.\u0026nbsp;32060015 and 82060378), Hainan Medical University Talent Development Project (No. XRC2021002),\u0026nbsp;Science and Technology Planning Project of Guizhou Province (No.\u0026nbsp;qkhjc-zk2022-yb435),\u0026nbsp;Gradutae student innovation grant of Hainan Medical University (No.\u0026nbsp;HYYS2020-33),\u0026nbsp;Research Project of the Hainan Academician Innovation Platform (No. YSPTZX202004), and Hainan Talent Development Project (No. SRC200003).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets presented in this study can be found in online repositories of the National Center for Biotechnology Information (NCBI). The accession number(s) was PRJNA1138552.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sampling strategy in this study did not involve hunting or euthanized method.\u0026nbsp;During the translocation process in the nature reserves where Eld's deer live, we collected throat swabs, anal swabs, and swab samples from tick bite sites. After sample collecting, all Eld's deer were released on-site as soon as possible. We have no experiments on live vertebrates. All methods were carried out in accordance with relevant guidelines and regulations of the Ethics Committee of the Hainan Medical University (approval no. HMUEC20180059), and reported in accordance with ARRIVE guidelines (https://arriveguidelines.org). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePublisher’s note\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eGhazi, M.G. et al. 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Unbiased parallel detection of viral pathogens in clinical samples by use of a metagenomic approach. \u003cem\u003eJ Clin Microbiol\u003c/em\u003e. \u003cstrong\u003e49\u003c/strong\u003e, 3463-3469 (2011).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Eld's deer, Papillomaviridae, Circoviridae, Genetic diversity, Phylogenetic analysis","lastPublishedDoi":"10.21203/rs.3.rs-5371247/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5371247/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMore than 60% of emerging (re-emerging) infectious diseases worldwide are caused by animal-derived pathogens, of which 71% are of wild animal origin. It is important to obtain insight into the viral pathogens present in the wildlife reservoir from a public health perspective. Based on next-generation sequencing (NGS), we report a metagenomic viral survey of Eld's deer (\u003cem\u003eRucervus eldii\u003c/em\u003e) (n = 33) in Bangxi Provincial Nature Reserve in Hainan Province. We collected 33 nasal swabs, 33 anal swabs, and 9 tick bite wound swabs and combined them into 5 pools according to the type of swabs. The metagenomic analysis showed that there were differences in the 5pools of viral reads, but the overall viral reads were closely related to mammals. We amplified and obtained the complete genomes of novel papillomaviruses (PV) and circoviruses (CV) in Eld's deer, PsPV-HMU-1 and PsaCV-HMU-1. The novel PV and CV genomic structure revealed that it was consistent with that of the known mammalian PV and CV. The L1 of PsPV-HMU-1 and the rep of PsaCV-HMU-1 showed less than 77.20% and 45.43% amino acid homology with the closest viruses, respectively. The positive rate of PV carried by Eld's deer is relatively high. Multiple Eld's deer with positive nase or anal swabs, accompanied by positive swabs from tick bites and wounds, indicate that insect bites may be closely related viral infection. This study helps us monitor Eld's deer viral diseases and understand the genetic diversity and evolutionary history of FV and CV carried by Eld's deer.\u003c/p\u003e","manuscriptTitle":"The virome investigation of the globally endangered Eld's deer (Rucervus eldii) on Hainan Island, China","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-27 05:39:22","doi":"10.21203/rs.3.rs-5371247/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-12-30T10:16:19+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-28T09:27:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"275328033720623988749831263102839944697","date":"2024-12-05T09:42:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"1277933426277029560106734681457873211","date":"2024-12-04T15:32:09+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-03T15:22:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-03T09:07:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"234929456749214667740066023065944795972","date":"2024-11-29T07:52:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"146243848689920933031060624599923996667","date":"2024-11-26T13:29:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-26T03:51:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-26T02:23:02+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-11-21T12:45:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-20T05:44:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-11-01T06:58:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"86d40377-be4b-4c28-849f-60490b87f6bf","owner":[],"postedDate":"November 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":40796466,"name":"Biological sciences/Microbiology/Pathogens"},{"id":40796467,"name":"Biological sciences/Microbiology/Virology"},{"id":40796468,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2025-03-17T16:00:01+00:00","versionOfRecord":{"articleIdentity":"rs-5371247","link":"https://doi.org/10.1038/s41598-025-92781-4","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-03-11 15:57:10","publishedOnDateReadable":"March 11th, 2025"},"versionCreatedAt":"2024-11-27 05:39:22","video":"","vorDoi":"10.1038/s41598-025-92781-4","vorDoiUrl":"https://doi.org/10.1038/s41598-025-92781-4","workflowStages":[]},"version":"v1","identity":"rs-5371247","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5371247","identity":"rs-5371247","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}