Identification and Characterization of Novel CRESS-DNA viruses in the Human Respiratory Tract

Identification and Characterization of Novel CRESS-DNA viruses in the Human Respiratory Tract | 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 Research Article Identification and Characterization of Novel CRESS-DNA viruses in the Human Respiratory Tract Huanyan Zhang, Yutong Fu, Cheng Cao, Heping Jiang, Ran Tang, Ziyuan Dai, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6208723/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Jun, 2025 Read the published version in Virology Journal → Version 1 posted 10 You are reading this latest preprint version Abstract CRESS-DNA viruses are small, circular, single-stranded DNA viruses that have been identified in diverse environments and hosts, including vertebrates, invertebrates, and environmental samples. However, their diversity and role in the human respiratory tract remain poorly understood. In this study, we employed viral metagenomics to analyze 140 nasopharyngeal swab samples from asymptomatic individuals. High-throughput sequencing and bioinformatics analyses were used to identify and characterize novel CRESS-DNA viruses. Phylogenetic relationships were inferred based on Rep protein sequences using maximum likelihood analysis. We identified and characterized eight novel CRESS-DNA viruses, which were classified into the families Endolinaviridae and Naryaviridae , with one potentially representing a novel viral family. These viruses exhibited typical circular genomic structures encoding Rep and Cap proteins, with conserved motifs associated with rolling circle replication. Phylogenetic analysis showed that some viruses were closely related to sequences from vertebrate hosts or environmental samples, suggesting a diverse ecological distribution. Our findings expand the known diversity of CRESS-DNA viruses in the human respiratory tract and highlight their potential ecological and evolutionary significance. Further studies are needed to explore their host specificity, replication mechanisms, and potential roles in human health and disease. Metagenomics Cressdnaviricota Endolinaviridae Naryaviridae Phylogenetics Respiratory tract Figures Figure 1 Figure 2 Figure 3 Figure 4 1 Introduction Circular Replication-associated protein (Rep)-Encoding Single-Stranded (CRESS) DNA viruses constitute a highly diverse group and are the smallest known autonomously replicating and capsid-encoding animal pathogens( 1 ). As of March 8, 2025, according to the classification by the International Committee on Taxonomy of Viruses (ICTV), CRESS-DNA viruses (phylum Cressdnaviricota ) include two classes, 13 orders, 24 families, and 269 genera, with these numbers continuing to grow. They have been identified worldwide in both prokaryotic and eukaryotic hosts, as well as in environmental samples( 2 ). CRESS-DNA viruses possess compact genomes, typically encoding at least two open reading frames (ORFs), which encode a conserved replication-associated protein and a capsid protein (Cap)( 3 ). Several cressdnavirus families have been experimentally verified to infect eukaryotic hosts, including Bacilladnaviridae , Circoviridae , Geminiviridae , and Genomoviridae , which are known to infect diatoms, vertebrates, plants, and fungi, respectively( 4 ). Moreover, numerous unclassified viruses with typical Cressdnaviricota features are frequently detected in human and non-human primate stool samples, sparking interest in their host specificity and potential health implications( 5 – 7 ). Due to their compact genomes, high sequence variability, and limited representation in reference databases, CRESS-DNA viruses are often underrepresented in conventional virome analyses. Their identification poses unique challenges, particularly in distinguishing them from other circular DNA molecules or host-derived sequences. To overcome these limitations, recent studies have increasingly relied on viral metagenomics combined with de novo assembly and specialized bioinformatic approaches designed to identify viral sequences, including highly divergent CRESS-DNA viruses( 1 , 8 ). Nevertheless, a significant portion of CRESS-DNA-related sequences remain unclassified, representing a reservoir of viral “dark matter” and emphasizing the need for continued methodological development and expansive surveillance to uncover their full diversity. The human respiratory tract harbors a variety of viruses, some of which may contribute to infections or diseases under certain conditions, such as immunosuppression or co-infection. For instance, certain coronaviruses (e.g., HCoV-229E and HCoV-OC43)( 9 ), human cytomegalovirus( 10 ), Epstein-Barr virus( 11 ), and human papillomavirus( 12 ) can persist in the respiratory tract for prolonged periods without immediately causing acute symptoms. Previous studies have shown that anelloviruses and herpesviruses are the predominant viruses in human respiratory tract samples( 13 – 20 ). Subsequently, Abbas et al. reported the presence of Redondoviridae, a group of CRESS-DNA viruses, in human respiratory and oral samples. They identified Redondoviridae as the second most prevalent viral family in human respiratory samples after anelloviruses and revealed their potential association with disease( 21 ). Furthermore, microbe-infecting viruses play a significant role in human health by influencing microbial abundance, diversity, and evolution within the body [5,9]. Therefore, understanding the diversity and evolutionary history of both vertebrate-infecting and microbe-infecting viruses in the respiratory tract is crucial for elucidating their broader implications for human health and disease. Although CRESS-DNA viruses have been widely detected across various hosts and environments, their diversity and evolutionary relationships within the human respiratory tract remain largely unexplored. In this study, we employed viral metagenomics to analyze the viral community in nasopharyngeal swab samples from healthy individuals, aiming to identify novel and previously uncharacterized viruses. Through phylogenetic analysis, we examined their evolutionary relationships with known viruses and explored their potential ecological niches and host ranges. Our findings further expand our understanding of the respiratory virome and provide new insights into the biological roles of CRESS-DNA viruses in the respiratory tract, as well as their interactions with the host. 2 Materials and Methods 2.1 Sample collection and preparation In order to study the composition of the respiratory virome in healthy individuals, a total of 140 nasopharyngeal swab samples from 140 healthy persons (age range is 8–60 years old) were collected from Jiangsu province of China in 2024. The sample collection protocol was approved by the Ethics Committee of Jiangsu University (Approval No. JSDX2023100702). The posterior pharyngeal wall and both palatine tonsils of each participant were gently swabbed by trained personnel using a sterile throat swab, with the swab rotated 3 to 5 times to ensure sufficient sample collection. The swab was immediately placed into a sterile collection tube and stored at 4 ℃, with initial sample processing completed within 6 hours to minimize viral nucleic acid degradation. Before viral metagenomic analysis, the swab tip was immersed in 0.5 mL of Dulbecco’s phosphate-buffered saline (DPBS) and vortexed vigorously for 5 minutes. The sample was then incubated at 4 ℃ for 30 minutes. The sample was centrifuged at 15,000×ɡ for 10 minutes, and the supernatant was collected and stored at -80 ℃ for further use. 2.2 Viral metagenomic sample processing and analysis Approximately 50 µL of supernatant from each nasopharyngeal swab specimen was pooled together. This specimen pool was selected for viral metagenomics analysis. Briefly, 500 µL of specimen was passed through a 0.45-µm syringe filter (Millipore) and centrifuged at 13,000×ɡ for 5 min. By this step, 166.5 µL of filtrate enriched in viral particles was collected. The filtrate was then treated with a cocktail of DNase, RNase, benzonase and Baseline-ZERO to digest unprotected nucleic acid at 37℃ for 60 min ( 22 ). Total nucleic acids were then extracted using QIAamp MinElute Virus Spin Kit (Qiagen) according to the manufacturer’s protocol. Fourteen library was then constructed using a Nextera XT DNA Sample Preparation Kit (Illumina) and sequenced using the Illumina NovaSeq platform with 150 bases paired ends with dual barcoding. For bioinformatics analysis, pair-end reads of 150 bp generated by NovaSeq were debarcoded using vendor software from Illumina. An in-house analysis pipeline running on a 32-node Linux cluster was used to process the data. Clonal reads were removed, and low-sequencing-quality tails were trimmed using a Phred quality score of ten as the threshold. Adaptors were trimmed using the default parameters of VecScreen which is NCBI BLASTn with specialized parameters designed for adapter removal. The cleaned reads were de novo assembled in the same barcode using the ENSEMBLE assembler [16]. Contigs and unassembled reads are then matched against a customized viral proteome database using BLASTx with an E-value cutoff of < 10 − 5 , where the virus BLASTx database was compiled using NCBI virus reference proteome ( ftp://ftp.ncbi.nih.gov/refseq/release/viral/ ) to which was added viral proteins sequences from NCBI nr fasta file (based on annotation taxonomy in Virus Kingdom). Candidate viral hits are then compared to an in-house non-virus non-redundant (NVNR) protein database to remove false positive viral hits, where the NVNR database was compiled using non-viral protein sequences extracted from NCBI nr fasta file (based on annotation taxonomy excluding Virus Kingdom). Contigs without significant BLASTx similarity to viral proteome database are searched against viral protein families in vFam database ( 23 ) using HMMER3 ( 24 – 26 ) to detect remote viral protein similarities. 2.3 Characterization of novel viral genome sequences Taxonomic annotation of contigs was performed using DIAMOND BLASTx (v2.1.8) against a custom viral protein database derived from the NCBI RefSeq viral protein sequences (updated March 2024), with an e-value threshold of 1e-5. Non-viral sequences were removed by comparison to the non-redundant non-viral protein database (NCBI NR-NVNR) using the same parameters. Contigs assigned as viruses were further screened using the vFam database (HMMER v3.3.2) to identify conserved viral protein domains. Contigs identified as CRESS-DNA viruses were selected based on significant sequence similarity to known Rep or Cap proteins and were further evaluated for genome completeness. Manual analysis in this context refers to the systematic curation and characterization of viral genomes using consistent and objective criteria, including the presence of a circular genome structure or terminal redundancy (as evidence of circularity), at least two major ORFs encoding Rep and Cap proteins, the presence of conserved Rep domains such as RCR motifs I-III and SF3 helicase motifs (Walker A, B, and C), a genome length greater than 1.5 kb, and genomic architecture, including gene orientation and intergenic regions, consistent with representative CRESS-DNA viral taxa. ORFs were predicted using Geneious Prime (v2023.2.1), and protein domains were annotated with the Conserved Domain Database (CDD, NCBI)( 27 ). Sequence similarity among viral genomes was calculated using the Sequence Demarcation Tool (SDT v1.2) with MUSCLE alignment and default settings. 2.4 Phylogenetic analysis Representative Rep amino acid sequences from the identified CRESS-DNA viruses were aligned with those of reference viruses using MAFFT (v7.526) with the L-INS-i algorithm( 28 ). The resulting alignments were manually inspected and trimmed using trimAl (v2.rev0) with the “automated1” mode to remove poorly aligned regions( 29 ). Context sequences for the phylogenetic analyses of potentially novel CRESS-DNA viruses were selected based on the most recent taxonomic updates ( https://ictv.global/vmr ) and retrieved from GenBank. In addition, the three most similar RefSeq sequences identified by BLASTn but not included among the primary ICTV reference sequences were incorporated into the context dataset. Maximum likelihood phylogenetic trees were constructed using IQ-TREE (v2.1.4)( 30 ), with the best-fitting substitution model determined by ModelFinder( 31 ). Branch support was evaluated using 1,000 ultrafast bootstrap replicates (UFBoot)( 32 ). Final trees were visualized and annotated using Interactive Tree of Life (iTOL, https://itol.embl.de/ ). 2.5 Pairwise sequence identity analysis Pairwise nucleotide identity comparisons among all identified CRESS-DNA viruses were calculated using the Sequence Demarcation Tool (SDT v1.2) with the MAFFT alignment option( 33 ). Each sequence set was aligned within SDT, and identity matrices were generated to assist in species-level classification and similarity assessment. 3 Results 3.1 Viral metagenomic overview Metagenomic sequencing was performed on 14 libraries, generating a total of 283,320,898 raw reads with an average GC content of 39.81%. Low-quality reads, along with those of mammalian and bacterial/archaeal origin, were removed. The assembled sequences were screened against the GenBank nonredundant (nr) viral protein database using BLASTx (E value of < 10 − 5 ), identifying 61,425 viral contigs. Viral species richness was assessed by rarefaction curves (Fig. 1 A). As the number of sampled contigs increased, the curves gradually approached a plateau, indicating that the sequencing depth was sufficient, and additional sequencing would likely reveal only a limited number of new species. Species accumulation curves based on a random sampling strategy supported this conclusion and showed that these 14 pools contained approximately 450 different viral species (Fig. 1 B). A stacked bar plot illustrates the composition and relative abundance of viral phyla in each library, showing that Uroviricota was the predominant phylum, consistently accounting for more than 80% of viral sequences across all libraries. Furthermore, Cressdnaviricota was detected in all libraries, with relatively high abundance in libraries 137J3 and 152L2, accounting for 33.85% and 43.10% of the total viral contigs in these libraries, respectively (Fig. 1 C). Additionally, approximately 5% of the contigs were predicted to have viral signature but could not be assigned to any known viral phylum, suggesting the potential existence of numerous unidentified viruses in the human virome. Metagenomic sequencing identified viral contigs spanning 59 viral families, including 21 families of double-stranded DNA (dsDNA) viruses, 12 single-stranded DNA (ssDNA) families, 5 double-stranded RNA (dsRNA) viral families, and 21 single-stranded RNA (ssRNA) viral families (Fig. 1 D). Notably, the number of viral contigs classified within the Circoviridae , Microviridae , and Schitoviridae families was significantly higher than that of other known viral families. 3.2 Identification of novel CRESS-DNA viruses from human respiratory tract A total of six complete and two nearly complete viral genomes were identified and their features are summarized in Table 1 . The genome lengths of the identified human-associated CRESS-DNA viruses ranged from 1,881 to 2,969 bp, with G + C content varying from 27.30–44.00%. These viruses exhibited a typical circular genomic organization, encoding two major genes: the replication-associated protein ( rep ) and capsid protein ( cap ). Notably, two isolates (GZ2_44556 and JX2_20455) exhibited a monosense genomic organization, with both genes oriented in the same direction, whereas the remaining six genomes displayed the classical ambisense arrangement, with genes oriented in opposite directions (Fig. 2 A). The putative Rep proteins ranged from 236 to 405 amino acids (aa), whereas the Cap proteins ranged from 247 and 364 aa. BLASTp analysis showed that the Rep proteins shared sequence identities ranging from 47.62% and 82.52% with known viruses, whereas the Cap proteins exhibited lower sequence identities, ranging from 27.37–57.26%. Additionally, all identified genomes contained a highly conserved nonamer sequence in the intergenic region, a hallmark of CRESS DNA viruses (Table 1 ). These viruses also retained three conserved motifs (motif-I, -II, and -III) within the HUH endonuclease domain, which is associated with rolling circle replication (RCR), as well as three conserved motifs (Walker-A, Walker-B, and Walker-C) within the superfamily 3 helicase (SF3H) domain, which is characteristic feature of CRESS DNA viruses( 3 , 34 ) (Fig. 2 B). Figure 2 C presented a heatmap illustrating the distribution of mapped reads for the eight identified viral genomes across 14 metagenomic libraries. The results revealed a highly heterogeneous distribution pattern. For example, JX2_20455, GZ2_44556, and TJ2_186 were mapped with higher read counts in libraries 153L3 and 134D3, whereas their presence was relatively lower in other libraries. This suggested that these viruses might have been more dominant or had a more localized distribution in those samples. The variation in mapped read abundance indicated potential differences in host specificity, viral prevalence, or environmental distribution among the identified CRESS-DNA viruses. Table 1 Genomic features of genomes of CRESS-DNA viruses sequenced in this study Sample ID Accession Size (nt) GC (%) Classification Putative Rep (aa) Putative Cap (aa) Blastp hits on Rep protein Blastp hits on Cap protein conserved nonamer sequence (5' to 3') Accession Identity (%) Accession Identity (%) JX2_20455 PV246545 1881 34.90 Cressdnaviricota sp. 236 300 AUM62033 49.57 BDF97698 45.13 TAGTATTAC SH3_22592 PV246547 2244 27.30 Naryaviridae sp. 329 247 XNT20971 54.4 WDW25918 27.37 TAGTATTAC HB2_18678 PV246542 2101 35.30 Naryaviridae sp. 284 337 WCD56378 82.52 QXF14467 57.26 TAGTATTAC GZ2_44556 PV246541 2010 41.30 Naryaviridae sp. 331 249 QTE03437 47.62 BDF97692 38.72 TAGTATTAC TJ2_186 PV246548 2900 44.00 Endolinaviridae sp. 352 364 WQA29659 63.22 ASU55894 34.64 TAGTATTAC SH3_18839 PV246546 2969 43.30 Endolinaviridae sp. 405 320 WCR62272 52.85 QCH00636 53.57 TACATATGA JX2_13661 PV246544 2900 39.30 Endolinaviridae sp. 381 351 WCR62234 56.19 AXQ03915 32.38 TAGTATTAC HB2_24497 PV246543 2871 42.90 Endolinaviridae sp. 350 333 WQA29659 64.84 WCR62088 41.4 TAGTATTAC 3.3 Phylogenetic Analysis The maximum likelihood phylogenetic tree (Fig. 3 ) was constructed based on Rep protein sequences of members of the phylum Cressdnaviricota, incorporating all known viral families within this phylum. Phylogenetic analysis revealed that the eight novel viruses identified in this study clustered into distinct groups within the phylum Cressdnaviricota . Specifically, SH3_22592, HB2_18678, and GZ2_44556 were assigned to the Naryaviridae family, while TJ2_186, SH3_18839, JX2_13661, and HB2_24497 clustered within the Endolinaviridae clade. Notably, JX2_20455 did not cluster with any known families, suggesting that it may represent a novel viral family or higher taxonomic rank. Phylogenetic analysis showed that HB2_18678 and SH3_22592, belonging to the family Naryaviridae , clustered with viruses isolated from Bos taurus (OQ198243) and Bos grunniens (PQ792334), respectively, with Rep protein sequence identities of 82.52% and 54.40%. In contrast, GZ2_44556 shared less than 50% sequence identity in the Rep protein with all known viruses and formed a distinct branch. Within Endolinaviridae family, TJ2_186, HB2_24497, and SH3_18839 clustered with a virus isolated from Sus scrofa (MK377714), forming a small clade with a bootstrap support of 87%. Additionally, JX2_13661 and a virus identified in Equus quagga (OM892383) formed sister lineages. Whole-genome distance matrix analysis showed that the pairwise identity between the newly discovered viruses and known viruses ranged from 55.07–69.14% (Fig. 4 ). According to the ICTV species demarcation criteria for Naryaviridae and Endolinaviridae , which define new species based on whole-genome nucleotide sequence identity below 78%( 35 ), all seven isolates identified in this study were classified as novel species. Furthermore, JX2_20455 clustered with viruses detected in wastewater, soil, and rainbow trout tissue (KY487983, OM154321, MH617056), forming a distinct phylogenetic branch. This branch was positioned as a sister lineage to the clade comprising members of Gredzevirales and Cremevirales . Notably, JX2_20455 exhibited the highest whole-genome identity (61.76%) with Miresoil virus 515 (OM154321), which was isolated from soil. As this viral cluster could not be assigned to any known viral taxonomy, it likely represents a novel family or higher taxonomic rank. 4 Discussion In this study, we identified and characterized eight novel CRESS-DNA viruses from human respiratory secretions, expanding the known diversity of this viral group. These newly identified viruses were phylogenetically classified into the families Endolinaviridae and Naryaviridae , with one virus potentially representing a novel viral family. Their genomic characteristics, including the presence of conserved Rep protein motifs, suggest a shared evolutionary origin with previously identified CRESS-DNA viruses. However, their host specificity, evolutionary history, and potential implications for human health remain largely unexplored. The detection of CRESS-DNA viruses in human respiratory samples aligns with previous reports identifying members of Redondoviridae and Anelloviridae in the human respiratory tract( 36 , 37 ). Notably, metagenomic studies have increasingly uncovered diverse CRESS-DNA viruses in various environments, including human fecal samples, wastewater, and animal tissues. While some CRESS-DNA viruses have been experimentally confirmed to infect vertebrate hosts( 38 , 39 ), the biological significance of many newly identified members, including those in this study, remains uncertain. The presence of these viruses in the respiratory tract could result from transient exposure, asymptomatic colonization, or integration into the broader human virome. Phylogenetic analysis revealed that some of the Naryaviridae and Endolinaviridae members identified in this study clustered with viruses detected in diverse vertebrate hosts, including Bos taurus , Sus scrofa , and Equus quagga , suggesting potential cross-host transmission events. This observation raises the possibility that these viruses have undergone host-switching events or horizontal gene transfer, contributing to their genetic diversity. Notably, one of the newly identified viruses, JX2_20455, formed a distinct phylogenetic lineage, clustering with viruses detected in environmental sources such as wastewater and soil. This suggests a broader ecological distribution of CRESS-DNA viruses beyond host-associated niches. Despite these findings, the potential pathogenicity and replication mechanisms of these viruses remain unclear. The relatively low sequence similarity of their Cap proteins to known viral species suggests that these viruses may employ distinct host interactions or structural adaptations. Future research should focus on experimentally validating their replication competence in human or microbial cell lines and investigating their prevalence in larger human cohorts. Moreover, metatranscriptomic approaches could elucidate their transcriptional activity in the respiratory tract. Given the increasing recognition of CRESS-DNA viruses as a diverse and ubiquitous viral group, their potential role in shaping microbial communities and human health warrants further exploration. Understanding their interactions with host immunity, co-infections with other respiratory viruses, and potential transmission dynamics will be crucial for defining their biological relevance. Our study adds to the growing body of evidence indicating that human respiratory secretions harbor previously unrecognized viral diversity, underscoring the need for ongoing metagenomic surveillance to fully characterize the human virome. Declarations 5 Funding This research was financially supported by National Natural Science Foundation of China (No. 82341106), National Key Research and Development Programs of China (No. 2023YFD1801301 and 2022YFC2603801) and Clinical Medicine Science and Technology Development Foundation of Jiangsu University (No. JLY2021151) 6.1 Conflict of interest The 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. 6.2 Human Rights Statement The study was approved by the Jiangsu University Ethics Committee and complied with Chinese ethics laws and regulations. Informed consent to participate was obtained from all human participants. 7 Authors' contributions WZ and ZD contributed to the conception of the study. HZ and YF contributed significantly to analysis and manuscript preparation. CC performed the data analysis and wrote the manuscript. HJ and RT helped perform the analysis with constructive discussions. All authors reviewed the manuscript. 8 Data availability The novel CRESS-DNA viruses reported in this study are publicly available in the GenBank database under the accession numbers PV246541–PV246548. Additionally, the raw sequencing data supporting this study's findings have been deposited in the NCBI Sequence Read Archive (SRA) under the BioProject accession number PRJNA1230567. These datasets are openly accessible through the NCBI SRA database. References Capozza P, Lanave G, Diakoudi G, Pellegrini F, Cardone R, Vasinioti VI, et al. Diversity of CRESS DNA Viruses in Squamates Recapitulates Hosts Dietary and Environmental Sources of Exposure. Microbiol Spectr. 2022;10(3):e0078022. Feher E, Mihalov-Kovacs E, Kaszab E, Malik YS, Marton S, Banyai K. Genomic Diversity of CRESS DNA Viruses in the Eukaryotic Virome of Swine Feces. Microorganisms. 2021;9(7). Krupovic M, Varsani A, Kazlauskas D, Breitbart M, Delwart E, Rosario K et al. Cressdnaviricota: a Virus Phylum Unifying Seven Families of Rep-Encoding Viruses with Single-Stranded, Circular DNA Genomes. J Virol. 2020;94(12). Kinsella CM, Bart A, Deijs M, Broekhuizen P, Kaczorowska J, Jebbink MF, et al. Entamoeba and Giardia parasites implicated as hosts of CRESS viruses. Nat Commun. 2020;11(1):4620. Phan TG, Mori D, Deng X, Rajindrajith S, Ranawaka U, Fan Ng TF, et al. Small circular single stranded DNA viral genomes in unexplained cases of human encephalitis, diarrhea, and in untreated sewage. Virology. 2015;482:98–104. Phan TG, da Costa AC, Del Valle Mendoza J, Bucardo-Rivera F, Nordgren J, O'Ryan M, et al. The fecal virome of South and Central American children with diarrhea includes small circular DNA viral genomes of unknown origin. Arch Virol. 2016;161(4):959–66. Altan E, Del Valle Mendoza J, Deng X, Phan TG, Sadeghi M, Delwart EL. Small Circular Rep-Encoding Single-Stranded DNA Genomes in Peruvian Diarrhea Virome. Genome Announc. 2017;5(38). Dai Z, Wang H, Xu J, Lu X, Ni P, Yang S et al. Unveiling the Virome of Wild Birds: Exploring CRESS-DNA Viral Dark Matter. Genome Biol Evol. 2024;16(10). Pene F, Merlat A, Vabret A, Rozenberg F, Buzyn A, Dreyfus F, et al. Coronavirus 229E-related pneumonia in immunocompromised patients. Clin Infect Dis. 2003;37(7):929–32. Landolfo S, Gariglio M, Gribaudo G, Lembo D. The human cytomegalovirus. Pharmacol Ther. 2003;98(3):269–97. Damania B, Kenney SC, Raab-Traub N. Epstein-Barr virus: Biology and clinical disease. Cell. 2022;185(20):3652–70. Szydlowski J, Jonczyk-Potoczna K, Pucher B, Buraczynska-Andrzejewska B, Prauzinska M, Kolasinska-Lipinska J, et al. Prevalence of human papillomavirus (HPV) in upper respiratory tract mucosa in a group of pre-school children. Ann Agric Environ Med. 2014;21(4):822–4. Willner D, Furlan M, Haynes M, Schmieder R, Angly FE, Silva J, et al. Metagenomic analysis of respiratory tract DNA viral communities in cystic fibrosis and non-cystic fibrosis individuals. PLoS ONE. 2009;4(10):e7370. Abeles SR, Ly M, Santiago-Rodriguez TM, Pride DT. Effects of Long Term Antibiotic Therapy on Human Oral and Fecal Viromes. PLoS ONE. 2015;10(8):e0134941. Wylie KM, Mihindukulasuriya KA, Sodergren E, Weinstock GM, Storch GA. Sequence analysis of the human virome in febrile and afebrile children. PLoS ONE. 2012;7(6):e27735. Perez-Brocal V, Moya A. The analysis of the oral DNA virome reveals which viruses are widespread and rare among healthy young adults in Valencia (Spain). PLoS ONE. 2018;13(2):e0191867. Young JC, Chehoud C, Bittinger K, Bailey A, Diamond JM, Cantu E, et al. Viral metagenomics reveal blooms of anelloviruses in the respiratory tract of lung transplant recipients. Am J Transpl. 2015;15(1):200–9. Clarke EL, Lauder AP, Hofstaedter CE, Hwang Y, Fitzgerald AS, Imai I, et al. Microbial Lineages in Sarcoidosis. A Metagenomic Analysis Tailored for Low-Microbial Content Samples. Am J Respir Crit Care Med. 2018;197(2):225–34. Abbas AA, Diamond JM, Chehoud C, Chang B, Kotzin JJ, Young JC, et al. The Perioperative Lung Transplant Virome: Torque Teno Viruses Are Elevated in Donor Lungs and Show Divergent Dynamics in Primary Graft Dysfunction. Am J Transpl. 2017;17(5):1313–24. Abbas AA, Young JC, Clarke EL, Diamond JM, Imai I, Haas AR, et al. Bidirectional transfer of Anelloviridae lineages between graft and host during lung transplantation. Am J Transpl. 2019;19(4):1086–97. Abbas AA, Taylor LJ, Dothard MI, Leiby JS, Fitzgerald AS, Khatib LA, et al. Redondoviridae, a Family of Small, Circular DNA Viruses of the Human Oro-Respiratory Tract Associated with Periodontitis and Critical Illness. Cell Host Microbe. 2019;25(5):719–29. e4. Zhang W, Li L, Deng X, Kapusinszky B, Pesavento PA, Delwart E. Faecal virome of cats in an animal shelter. J Gen Virol. 2014;95(Pt 11):2553–64. Skewes-Cox P, Sharpton TJ, Pollard KS, DeRisi JL. Profile hidden Markov models for the detection of viruses within metagenomic sequence data. PLoS ONE. 2014;9(8):e105067. Johnson LS, Eddy SR, Portugaly E. Hidden Markov model speed heuristic and iterative HMM search procedure. BMC Bioinformatics. 2010;11:431. Finn RD, Clements J, Eddy SR. HMMER web server: interactive sequence similarity searching. Nucleic Acids Res. 2011;39(Web Server issue):W29–37. Eddy SR. A new generation of homology search tools based on probabilistic inference. Genome Inf. 2009;23(1):205–11. Wang J, Chitsaz F, Derbyshire MK, Gonzales NR, Gwadz M, Lu S, et al. The conserved domain database in 2023. Nucleic Acids Res. 2023;51(D1):D384–8. Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013;30(4):772–80. Capella-Gutierrez S, Silla-Martinez JM, Gabaldon T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics. 2009;25(15):1972–3. Minh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD, von Haeseler A, et al. IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era. Mol Biol Evol. 2020;37(5):1530–4. Kalyaanamoorthy S, Minh BQ, Wong TKF, von Haeseler A, Jermiin LS. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat Methods. 2017;14(6):587–9. Hoang DT, Chernomor O, von Haeseler A, Minh BQ, Vinh LS. UFBoot2: Improving the Ultrafast Bootstrap Approximation. Mol Biol Evol. 2018;35(2):518–22. Muhire BM, Varsani A, Martin DP. SDT: a virus classification tool based on pairwise sequence alignment and identity calculation. PLoS ONE. 2014;9(9):e108277. Zhao L, Rosario K, Breitbart M, Duffy S. Eukaryotic Circular Rep-Encoding Single-Stranded DNA (CRESS DNA) Viruses: Ubiquitous Viruses With Small Genomes and a Diverse Host Range. Adv Virus Res. 2019;103:71–133. Varsani A, Hopkins A, Lund MC, Krupovic M. 2024 taxonomic update for the families Naryaviridae, Nenyaviridae, and Vilyaviridae. Arch Virol. 2024;170(1):18. Abbas AA, Taylor LJ, Dothard MI, Leiby JS, Fitzgerald AS, Khatib LA, et al. Redondoviridae, a Family of Small, Circular DNA Viruses of the Human Oro-Respiratory Tract Associated with Periodontitis and Critical Illness. Cell Host Microbe. 2019;26(2):297. Taylor LJ, Keeler EL, Bushman FD, Collman RG. The enigmatic roles of Anelloviridae and Redondoviridae in humans. Curr Opin Virol. 2022;55:101248. Hamelin B, Perot P, Pichler I, Haslbauer JD, Hardy D, Hing D, et al. Circovirus Hepatitis in Immunocompromised Patient, Switzerland. Emerg Infect Dis. 2024;30(10):2140–4. Meng XJ. Porcine circovirus type 2 (PCV2): pathogenesis and interaction with the immune system. Annu Rev Anim Biosci. 2013;1:43–64. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 30 Jun, 2025 Read the published version in Virology Journal → Version 1 posted Editorial decision: Accepted 14 Apr, 2025 Reviews received at journal 14 Apr, 2025 Reviews received at journal 10 Apr, 2025 Reviews received at journal 03 Apr, 2025 Reviewers agreed at journal 03 Apr, 2025 Reviewers agreed at journal 02 Apr, 2025 Reviewers agreed at journal 02 Apr, 2025 Reviewers invited by journal 02 Apr, 2025 Submission checks completed at journal 02 Apr, 2025 First submitted to journal 01 Apr, 2025 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6208723","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":437597779,"identity":"d40a3a30-0008-475b-a0a7-9ea5aa67266f","order_by":0,"name":"Huanyan Zhang","email":"","orcid":"","institution":"Jintan Affiliated Hospital of Jiangsu University","correspondingAuthor":false,"prefix":"","firstName":"Huanyan","middleName":"","lastName":"Zhang","suffix":""},{"id":437597780,"identity":"4f9e7244-3150-41c2-826a-a6841a1b5729","order_by":1,"name":"Yutong Fu","email":"","orcid":"","institution":"Jiangsu University","correspondingAuthor":false,"prefix":"","firstName":"Yutong","middleName":"","lastName":"Fu","suffix":""},{"id":437597781,"identity":"043f67db-1b38-4668-8baf-ea4699d72132","order_by":2,"name":"Cheng Cao","email":"","orcid":"","institution":"Jintan Affiliated Hospital of Jiangsu University","correspondingAuthor":false,"prefix":"","firstName":"Cheng","middleName":"","lastName":"Cao","suffix":""},{"id":437597782,"identity":"44929a11-5453-473d-97ee-93ed67b78bd7","order_by":3,"name":"Heping Jiang","email":"","orcid":"","institution":"Jintan First People's Hospital in Changzhou City","correspondingAuthor":false,"prefix":"","firstName":"Heping","middleName":"","lastName":"Jiang","suffix":""},{"id":437597783,"identity":"b1979c78-16d7-4e38-ab5e-14d161b3f606","order_by":4,"name":"Ran Tang","email":"","orcid":"","institution":"Jintan First People's Hospital in Changzhou City","correspondingAuthor":false,"prefix":"","firstName":"Ran","middleName":"","lastName":"Tang","suffix":""},{"id":437597784,"identity":"9bbeb503-601f-4df5-945d-177e2f37d763","order_by":5,"name":"Ziyuan Dai","email":"","orcid":"","institution":"Yancheng Third People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ziyuan","middleName":"","lastName":"Dai","suffix":""},{"id":437597785,"identity":"4f103cf5-b70a-4aa1-8039-adbf6619af43","order_by":6,"name":"Wen Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA40lEQVRIiWNgGAWjYDACCSjNz8CQAKSYSdAi2UCyFoMDYIoILfKzm589/Npmk2d8I+HxB4YK68QG9rMH8GphnHPM3Fi2La3Y7EZCmgTDmfTEBp68BLxamCUSzKQltx1O3AbUwsDYdjixQYLHAK8WNon0b0At/xM3z0hI/sD4jwgtPBI5ZpIftx1I3CCRkCDB2ECEFgmJnDJpxn/JiTPOPEiTSDiWbtzGk4Nfi/yM9G2SP87YJfa35yR/+FBjLdvPfga/FhBg5oG4MQEcmWwE1QMB4w8wxX6AGMWjYBSMglEwAgEATstFJNON6WoAAAAASUVORK5CYII=","orcid":"","institution":"Jintan Affiliated Hospital of Jiangsu University","correspondingAuthor":true,"prefix":"","firstName":"Wen","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-03-12 05:23:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6208723/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6208723/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12985-025-02742-6","type":"published","date":"2025-06-30T15:57:46+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79827191,"identity":"e3bb0f86-c77a-443c-94fa-22058b1f514e","added_by":"auto","created_at":"2025-04-03 09:44:29","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1617133,"visible":true,"origin":"","legend":"\u003cp\u003eViral metagenomic overview of the 14 libraries. (A) Species rarefaction curve annotated by MEGAN v.6.22.2 after log-scale transformation. Legends are displayed to the side of the panel. (B) Accumulation curve of viral contigs in this study. Error bars represent the range, and the blue area in the background represents the 95% confidence interval. (C) Bar graphs showing the relative proportion and taxonomy based on viral Phyla. (D) Heat map representing the viral contigs of each viral family of each library on a log\u003csub\u003e10\u003c/sub\u003e scale. Viral genome types and viral families are annotated with corresponding colors (see color legend).\u003c/p\u003e","description":"","filename":"Figture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6208723/v1/b2c9309f9da9d2aee753398e.jpg"},{"id":79827189,"identity":"38e9e201-b41f-4704-80a4-f5909783aef6","added_by":"auto","created_at":"2025-04-03 09:44:28","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":782494,"visible":true,"origin":"","legend":"\u003cp\u003eGenomic characterization and distribution of identified novel CRESS-DNA viruses. (A) Genome schematic organizations of the human-associated CRESS-DNA viruses sequenced in this study. (B) Identification of the rolling circle replication domain and superfamily 3 helicase domain in the Rep protein. (C) Heatmap showing the normalized read counts mapped to eight novel human-associated CRESS-DNA viruses across 14 metagenomic libraries. Column-wise normalization was applied to highlight relative abundance variations. The hierarchical clustering on the left groups viruses with similar mapping patterns.\u003c/p\u003e","description":"","filename":"Figture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6208723/v1/c5130c98ed62fa332cac4784.jpg"},{"id":79827668,"identity":"7855cc81-86e3-4176-98dd-f618181be035","added_by":"auto","created_at":"2025-04-03 09:52:29","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":864183,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum likelihood phylogenetic tree inferred from Rep proteins of members of the phylum \u003cem\u003eCressdnaviricota\u003c/em\u003e. The maximum likelihood phylogenetic tree was constructed using IQtree with automatic selection of the best-fit substitution model for a given alignment, which was Q.pfam+F+R8. Numbers at the nodes represent ultrafast bootstrap support values. The scale bar represents the number of substitutions per site.\u003c/p\u003e","description":"","filename":"Figture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6208723/v1/fffbe3312a91b4e2d76b4803.jpg"},{"id":79827192,"identity":"ee665a3f-8b0d-404d-afcf-2a6730eeb077","added_by":"auto","created_at":"2025-04-03 09:44:29","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":338696,"visible":true,"origin":"","legend":"\u003cp\u003ePairwise identity matrix of members of the families \u003cem\u003eEndolinaviridae \u003c/em\u003eand \u003cem\u003eNaryaviridae\u003c/em\u003e, with a 78% species demarcation threshold inferred using SDT v1.3.\u003c/p\u003e","description":"","filename":"Figture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6208723/v1/e9f50b3a69ff69ffddb30151.jpg"},{"id":86180230,"identity":"a05d91a4-e041-4fc8-95de-f400bd9795c5","added_by":"auto","created_at":"2025-07-07 16:21:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5234739,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6208723/v1/2ec7981e-f7f5-4689-9148-18353d2db35c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eIdentification and Characterization of Novel CRESS-DNA viruses in the Human Respiratory Tract\u003c/p\u003e","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eCircular Replication-associated protein (Rep)-Encoding Single-Stranded (CRESS) DNA viruses constitute a highly diverse group and are the smallest known autonomously replicating and capsid-encoding animal pathogens(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). As of March 8, 2025, according to the classification by the International Committee on Taxonomy of Viruses (ICTV), CRESS-DNA viruses (phylum \u003cem\u003eCressdnaviricota\u003c/em\u003e) include two classes, 13 orders, 24 families, and 269 genera, with these numbers continuing to grow. They have been identified worldwide in both prokaryotic and eukaryotic hosts, as well as in environmental samples(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). CRESS-DNA viruses possess compact genomes, typically encoding at least two open reading frames (ORFs), which encode a conserved replication-associated protein and a capsid protein (Cap)(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSeveral cressdnavirus families have been experimentally verified to infect eukaryotic hosts, including \u003cem\u003eBacilladnaviridae\u003c/em\u003e, \u003cem\u003eCircoviridae\u003c/em\u003e, \u003cem\u003eGeminiviridae\u003c/em\u003e, and \u003cem\u003eGenomoviridae\u003c/em\u003e, which are known to infect diatoms, vertebrates, plants, and fungi, respectively(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Moreover, numerous unclassified viruses with typical \u003cem\u003eCressdnaviricota\u003c/em\u003e features are frequently detected in human and non-human primate stool samples, sparking interest in their host specificity and potential health implications(\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Due to their compact genomes, high sequence variability, and limited representation in reference databases, CRESS-DNA viruses are often underrepresented in conventional virome analyses. Their identification poses unique challenges, particularly in distinguishing them from other circular DNA molecules or host-derived sequences. To overcome these limitations, recent studies have increasingly relied on viral metagenomics combined with de novo assembly and specialized bioinformatic approaches designed to identify viral sequences, including highly divergent CRESS-DNA viruses(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Nevertheless, a significant portion of CRESS-DNA-related sequences remain unclassified, representing a reservoir of viral \u0026ldquo;dark matter\u0026rdquo; and emphasizing the need for continued methodological development and expansive surveillance to uncover their full diversity. The human respiratory tract harbors a variety of viruses, some of which may contribute to infections or diseases under certain conditions, such as immunosuppression or co-infection. For instance, certain coronaviruses (e.g., HCoV-229E and HCoV-OC43)(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e), human cytomegalovirus(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), Epstein-Barr virus(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), and human papillomavirus(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e) can persist in the respiratory tract for prolonged periods without immediately causing acute symptoms. Previous studies have shown that anelloviruses and herpesviruses are the predominant viruses in human respiratory tract samples(\u003cspan additionalcitationids=\"CR14 CR15 CR16 CR17 CR18 CR19\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Subsequently, Abbas et al. reported the presence of Redondoviridae, a group of CRESS-DNA viruses, in human respiratory and oral samples. They identified Redondoviridae as the second most prevalent viral family in human respiratory samples after anelloviruses and revealed their potential association with disease(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Furthermore, microbe-infecting viruses play a significant role in human health by influencing microbial abundance, diversity, and evolution within the body [5,9]. Therefore, understanding the diversity and evolutionary history of both vertebrate-infecting and microbe-infecting viruses in the respiratory tract is crucial for elucidating their broader implications for human health and disease. Although CRESS-DNA viruses have been widely detected across various hosts and environments, their diversity and evolutionary relationships within the human respiratory tract remain largely unexplored. In this study, we employed viral metagenomics to analyze the viral community in nasopharyngeal swab samples from healthy individuals, aiming to identify novel and previously uncharacterized viruses. Through phylogenetic analysis, we examined their evolutionary relationships with known viruses and explored their potential ecological niches and host ranges. Our findings further expand our understanding of the respiratory virome and provide new insights into the biological roles of CRESS-DNA viruses in the respiratory tract, as well as their interactions with the host.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Sample collection and preparation\u003c/h2\u003e \u003cp\u003eIn order to study the composition of the respiratory virome in healthy individuals, a total of 140 nasopharyngeal swab samples from 140 healthy persons (age range is 8\u0026ndash;60 years old) were collected from Jiangsu province of China in 2024. The sample collection protocol was approved by the Ethics Committee of Jiangsu University (Approval No. JSDX2023100702). The posterior pharyngeal wall and both palatine tonsils of each participant were gently swabbed by trained personnel using a sterile throat swab, with the swab rotated 3 to 5 times to ensure sufficient sample collection. The swab was immediately placed into a sterile collection tube and stored at 4 ℃, with initial sample processing completed within 6 hours to minimize viral nucleic acid degradation. Before viral metagenomic analysis, the swab tip was immersed in 0.5 mL of Dulbecco\u0026rsquo;s phosphate-buffered saline (DPBS) and vortexed vigorously for 5 minutes. The sample was then incubated at 4 ℃ for 30 minutes. The sample was centrifuged at 15,000\u0026times;ɡ for 10 minutes, and the supernatant was collected and stored at -80 ℃ for further use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Viral metagenomic sample processing and analysis\u003c/h2\u003e \u003cp\u003eApproximately 50 \u0026micro;L of supernatant from each nasopharyngeal swab specimen was pooled together. This specimen pool was selected for viral metagenomics analysis. Briefly, 500 \u0026micro;L of specimen was passed through a 0.45-\u0026micro;m syringe filter (Millipore) and centrifuged at 13,000\u0026times;ɡ for 5 min. By this step, 166.5 \u0026micro;L of filtrate enriched in viral particles was collected. The filtrate was then treated with a cocktail of DNase, RNase, benzonase and Baseline-ZERO to digest unprotected nucleic acid at 37℃ for 60 min (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Total nucleic acids were then extracted using QIAamp MinElute Virus Spin Kit (Qiagen) according to the manufacturer\u0026rsquo;s protocol. Fourteen library was then constructed using a Nextera XT DNA Sample Preparation Kit (Illumina) and sequenced using the Illumina NovaSeq platform with 150 bases paired ends with dual barcoding. For bioinformatics analysis, pair-end reads of 150 bp generated by NovaSeq were debarcoded using vendor software from Illumina. An in-house analysis pipeline running on a 32-node Linux cluster was used to process the data. Clonal reads were removed, and low-sequencing-quality tails were trimmed using a Phred quality score of ten as the threshold. Adaptors were trimmed using the default parameters of VecScreen which is NCBI BLASTn with specialized parameters designed for adapter removal. The cleaned reads were de novo assembled in the same barcode using the ENSEMBLE assembler [16]. Contigs and unassembled reads are then matched against a customized viral proteome database using BLASTx with an E-value cutoff of \u0026lt;\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e, where the virus BLASTx database was compiled using NCBI virus reference proteome (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003eftp://ftp.ncbi.nih.gov/refseq/release/viral/\u003c/span\u003e\u003cspan address=\"http://ftp://ftp.ncbi.nih.gov/refseq/release/viral/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to which was added viral proteins sequences from NCBI nr fasta file (based on annotation taxonomy in Virus Kingdom). Candidate viral hits are then compared to an in-house non-virus non-redundant (NVNR) protein database to remove false positive viral hits, where the NVNR database was compiled using non-viral protein sequences extracted from NCBI nr fasta file (based on annotation taxonomy excluding Virus Kingdom). Contigs without significant BLASTx similarity to viral proteome database are searched against viral protein families in vFam database (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) using HMMER3 (\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) to detect remote viral protein similarities.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Characterization of novel viral genome sequences\u003c/h2\u003e \u003cp\u003eTaxonomic annotation of contigs was performed using DIAMOND BLASTx (v2.1.8) against a custom viral protein database derived from the NCBI RefSeq viral protein sequences (updated March 2024), with an e-value threshold of 1e-5. Non-viral sequences were removed by comparison to the non-redundant non-viral protein database (NCBI NR-NVNR) using the same parameters. Contigs assigned as viruses were further screened using the vFam database (HMMER v3.3.2) to identify conserved viral protein domains.\u003c/p\u003e \u003cp\u003eContigs identified as CRESS-DNA viruses were selected based on significant sequence similarity to known Rep or Cap proteins and were further evaluated for genome completeness. Manual analysis in this context refers to the systematic curation and characterization of viral genomes using consistent and objective criteria, including the presence of a circular genome structure or terminal redundancy (as evidence of circularity), at least two major ORFs encoding Rep and Cap proteins, the presence of conserved Rep domains such as RCR motifs I-III and SF3 helicase motifs (Walker A, B, and C), a genome length greater than 1.5 kb, and genomic architecture, including gene orientation and intergenic regions, consistent with representative CRESS-DNA viral taxa. ORFs were predicted using Geneious Prime (v2023.2.1), and protein domains were annotated with the Conserved Domain Database (CDD, NCBI)(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Sequence similarity among viral genomes was calculated using the Sequence Demarcation Tool (SDT v1.2) with MUSCLE alignment and default settings.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Phylogenetic analysis\u003c/h2\u003e \u003cp\u003eRepresentative Rep amino acid sequences from the identified CRESS-DNA viruses were aligned with those of reference viruses using MAFFT (v7.526) with the L-INS-i algorithm(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). The resulting alignments were manually inspected and trimmed using trimAl (v2.rev0) with the \u0026ldquo;automated1\u0026rdquo; mode to remove poorly aligned regions(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Context sequences for the phylogenetic analyses of potentially novel CRESS-DNA viruses were selected based on the most recent taxonomic updates (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ictv.global/vmr\u003c/span\u003e\u003cspan address=\"https://ictv.global/vmr\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and retrieved from GenBank. In addition, the three most similar RefSeq sequences identified by BLASTn but not included among the primary ICTV reference sequences were incorporated into the context dataset. Maximum likelihood phylogenetic trees were constructed using IQ-TREE (v2.1.4)(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), with the best-fitting substitution model determined by ModelFinder(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Branch support was evaluated using 1,000 ultrafast bootstrap replicates (UFBoot)(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Final trees were visualized and annotated using Interactive Tree of Life (iTOL, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://itol.embl.de/\u003c/span\u003e\u003cspan address=\"https://itol.embl.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Pairwise sequence identity analysis\u003c/h2\u003e \u003cp\u003ePairwise nucleotide identity comparisons among all identified CRESS-DNA viruses were calculated using the Sequence Demarcation Tool (SDT v1.2) with the MAFFT alignment option(\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Each sequence set was aligned within SDT, and identity matrices were generated to assist in species-level classification and similarity assessment.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Viral metagenomic overview\u003c/h2\u003e \u003cp\u003eMetagenomic sequencing was performed on 14 libraries, generating a total of 283,320,898 raw reads with an average GC content of 39.81%. Low-quality reads, along with those of mammalian and bacterial/archaeal origin, were removed. The assembled sequences were screened against the GenBank nonredundant (nr) viral protein database using BLASTx (E value of \u0026lt;\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e), identifying 61,425 viral contigs. Viral species richness was assessed by rarefaction curves (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). As the number of sampled contigs increased, the curves gradually approached a plateau, indicating that the sequencing depth was sufficient, and additional sequencing would likely reveal only a limited number of new species. Species accumulation curves based on a random sampling strategy supported this conclusion and showed that these 14 pools contained approximately 450 different viral species (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). A stacked bar plot illustrates the composition and relative abundance of viral phyla in each library, showing that \u003cem\u003eUroviricota\u003c/em\u003e was the predominant phylum, consistently accounting for more than 80% of viral sequences across all libraries. Furthermore, \u003cem\u003eCressdnaviricota\u003c/em\u003e was detected in all libraries, with relatively high abundance in libraries 137J3 and 152L2, accounting for 33.85% and 43.10% of the total viral contigs in these libraries, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Additionally, approximately 5% of the contigs were predicted to have viral signature but could not be assigned to any known viral phylum, suggesting the potential existence of numerous unidentified viruses in the human virome. Metagenomic sequencing identified viral contigs spanning 59 viral families, including 21 families of double-stranded DNA (dsDNA) viruses, 12 single-stranded DNA (ssDNA) families, 5 double-stranded RNA (dsRNA) viral families, and 21 single-stranded RNA (ssRNA) viral families (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Notably, the number of viral contigs classified within the \u003cem\u003eCircoviridae\u003c/em\u003e, \u003cem\u003eMicroviridae\u003c/em\u003e, and \u003cem\u003eSchitoviridae\u003c/em\u003e families was significantly higher than that of other known viral families.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Identification of novel CRESS-DNA viruses from human respiratory tract\u003c/h2\u003e \u003cp\u003eA total of six complete and two nearly complete viral genomes were identified and their features are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The genome lengths of the identified human-associated CRESS-DNA viruses ranged from 1,881 to 2,969 bp, with G\u0026thinsp;+\u0026thinsp;C content varying from 27.30\u0026ndash;44.00%. These viruses exhibited a typical circular genomic organization, encoding two major genes: the replication-associated protein (\u003cem\u003erep\u003c/em\u003e) and capsid protein (\u003cem\u003ecap\u003c/em\u003e). Notably, two isolates (GZ2_44556 and JX2_20455) exhibited a monosense genomic organization, with both genes oriented in the same direction, whereas the remaining six genomes displayed the classical ambisense arrangement, with genes oriented in opposite directions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The putative Rep proteins ranged from 236 to 405 amino acids (aa), whereas the Cap proteins ranged from 247 and 364 aa. BLASTp analysis showed that the Rep proteins shared sequence identities ranging from 47.62% and 82.52% with known viruses, whereas the Cap proteins exhibited lower sequence identities, ranging from 27.37\u0026ndash;57.26%. Additionally, all identified genomes contained a highly conserved nonamer sequence in the intergenic region, a hallmark of CRESS DNA viruses (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These viruses also retained three conserved motifs (motif-I, -II, and -III) within the HUH endonuclease domain, which is associated with rolling circle replication (RCR), as well as three conserved motifs (Walker-A, Walker-B, and Walker-C) within the superfamily 3 helicase (SF3H) domain, which is characteristic feature of CRESS DNA viruses(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC presented a heatmap illustrating the distribution of mapped reads for the eight identified viral genomes across 14 metagenomic libraries. The results revealed a highly heterogeneous distribution pattern. For example, JX2_20455, GZ2_44556, and TJ2_186 were mapped with higher read counts in libraries 153L3 and 134D3, whereas their presence was relatively lower in other libraries. This suggested that these viruses might have been more dominant or had a more localized distribution in those samples. The variation in mapped read abundance indicated potential differences in host specificity, viral prevalence, or environmental distribution among the identified CRESS-DNA viruses.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGenomic features of genomes of CRESS-DNA viruses sequenced in this study\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"12\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSample ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAccession\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSize\u003c/p\u003e \u003cp\u003e(nt)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGC\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eClassification\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePutative Rep\u003c/p\u003e \u003cp\u003e(aa)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePutative Cap\u003c/p\u003e \u003cp\u003e(aa)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eBlastp hits on Rep protein\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003eBlastp hits on Cap protein\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003econserved nonamer sequence\u003c/p\u003e \u003cp\u003e(5' to 3')\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eAccession\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eIdentity\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eAccession\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eIdentity\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eJX2_20455\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePV246545\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1881\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCressdnaviricota sp.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e236\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eAUM62033\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e49.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eBDF97698\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e45.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eTAGTATTAC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSH3_22592\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePV246547\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2244\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e27.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eNaryaviridae sp.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e329\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e247\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eXNT20971\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e54.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eWDW25918\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e27.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eTAGTATTAC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHB2_18678\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePV246542\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e35.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eNaryaviridae sp.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e284\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e337\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eWCD56378\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e82.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eQXF14467\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e57.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eTAGTATTAC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGZ2_44556\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePV246541\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e41.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eNaryaviridae sp.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e331\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e249\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eQTE03437\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e47.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eBDF97692\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e38.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eTAGTATTAC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTJ2_186\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePV246548\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2900\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e44.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eEndolinaviridae sp.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e352\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e364\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eWQA29659\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e63.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eASU55894\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e34.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eTAGTATTAC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSH3_18839\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePV246546\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2969\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e43.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eEndolinaviridae sp.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e405\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e320\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eWCR62272\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e52.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eQCH00636\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e53.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eTACATATGA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eJX2_13661\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePV246544\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2900\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e39.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eEndolinaviridae sp.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e381\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e351\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eWCR62234\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e56.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eAXQ03915\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e32.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eTAGTATTAC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHB2_24497\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePV246543\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2871\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e42.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eEndolinaviridae sp.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e350\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e333\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eWQA29659\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e64.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eWCR62088\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e41.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eTAGTATTAC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Phylogenetic Analysis\u003c/h2\u003e \u003cp\u003eThe maximum likelihood phylogenetic tree (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) was constructed based on Rep protein sequences of members of the phylum Cressdnaviricota, incorporating all known viral families within this phylum. Phylogenetic analysis revealed that the eight novel viruses identified in this study clustered into distinct groups within the phylum \u003cem\u003eCressdnaviricota\u003c/em\u003e. Specifically, SH3_22592, HB2_18678, and GZ2_44556 were assigned to the \u003cem\u003eNaryaviridae\u003c/em\u003e family, while TJ2_186, SH3_18839, JX2_13661, and HB2_24497 clustered within the \u003cem\u003eEndolinaviridae\u003c/em\u003e clade. Notably, JX2_20455 did not cluster with any known families, suggesting that it may represent a novel viral family or higher taxonomic rank.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePhylogenetic analysis showed that HB2_18678 and SH3_22592, belonging to the family \u003cem\u003eNaryaviridae\u003c/em\u003e, clustered with viruses isolated from \u003cem\u003eBos taurus\u003c/em\u003e (OQ198243) and \u003cem\u003eBos grunniens\u003c/em\u003e (PQ792334), respectively, with Rep protein sequence identities of 82.52% and 54.40%. In contrast, GZ2_44556 shared less than 50% sequence identity in the Rep protein with all known viruses and formed a distinct branch. Within \u003cem\u003eEndolinaviridae\u003c/em\u003e family, TJ2_186, HB2_24497, and SH3_18839 clustered with a virus isolated from \u003cem\u003eSus scrofa\u003c/em\u003e (MK377714), forming a small clade with a bootstrap support of 87%. Additionally, JX2_13661 and a virus identified in \u003cem\u003eEquus quagga\u003c/em\u003e (OM892383) formed sister lineages. Whole-genome distance matrix analysis showed that the pairwise identity between the newly discovered viruses and known viruses ranged from 55.07\u0026ndash;69.14% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). According to the ICTV species demarcation criteria for \u003cem\u003eNaryaviridae\u003c/em\u003e and \u003cem\u003eEndolinaviridae\u003c/em\u003e, which define new species based on whole-genome nucleotide sequence identity below 78%(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e), all seven isolates identified in this study were classified as novel species. Furthermore, JX2_20455 clustered with viruses detected in wastewater, soil, and rainbow trout tissue (KY487983, OM154321, MH617056), forming a distinct phylogenetic branch. This branch was positioned as a sister lineage to the clade comprising members of \u003cem\u003eGredzevirales\u003c/em\u003e and \u003cem\u003eCremevirales\u003c/em\u003e. Notably, JX2_20455 exhibited the highest whole-genome identity (61.76%) with Miresoil virus 515 (OM154321), which was isolated from soil. As this viral cluster could not be assigned to any known viral taxonomy, it likely represents a novel family or higher taxonomic rank.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eIn this study, we identified and characterized eight novel CRESS-DNA viruses from human respiratory secretions, expanding the known diversity of this viral group. These newly identified viruses were phylogenetically classified into the families \u003cem\u003eEndolinaviridae\u003c/em\u003e and \u003cem\u003eNaryaviridae\u003c/em\u003e, with one virus potentially representing a novel viral family. Their genomic characteristics, including the presence of conserved Rep protein motifs, suggest a shared evolutionary origin with previously identified CRESS-DNA viruses. However, their host specificity, evolutionary history, and potential implications for human health remain largely unexplored.\u003c/p\u003e \u003cp\u003eThe detection of CRESS-DNA viruses in human respiratory samples aligns with previous reports identifying members of \u003cem\u003eRedondoviridae\u003c/em\u003e and \u003cem\u003eAnelloviridae\u003c/em\u003e in the human respiratory tract(\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Notably, metagenomic studies have increasingly uncovered diverse CRESS-DNA viruses in various environments, including human fecal samples, wastewater, and animal tissues. While some CRESS-DNA viruses have been experimentally confirmed to infect vertebrate hosts(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e), the biological significance of many newly identified members, including those in this study, remains uncertain. The presence of these viruses in the respiratory tract could result from transient exposure, asymptomatic colonization, or integration into the broader human virome.\u003c/p\u003e \u003cp\u003ePhylogenetic analysis revealed that some of the \u003cem\u003eNaryaviridae\u003c/em\u003e and \u003cem\u003eEndolinaviridae\u003c/em\u003e members identified in this study clustered with viruses detected in diverse vertebrate hosts, including \u003cem\u003eBos taurus\u003c/em\u003e, \u003cem\u003eSus scrofa\u003c/em\u003e, and \u003cem\u003eEquus quagga\u003c/em\u003e, suggesting potential cross-host transmission events. This observation raises the possibility that these viruses have undergone host-switching events or horizontal gene transfer, contributing to their genetic diversity. Notably, one of the newly identified viruses, JX2_20455, formed a distinct phylogenetic lineage, clustering with viruses detected in environmental sources such as wastewater and soil. This suggests a broader ecological distribution of CRESS-DNA viruses beyond host-associated niches. Despite these findings, the potential pathogenicity and replication mechanisms of these viruses remain unclear. The relatively low sequence similarity of their Cap proteins to known viral species suggests that these viruses may employ distinct host interactions or structural adaptations. Future research should focus on experimentally validating their replication competence in human or microbial cell lines and investigating their prevalence in larger human cohorts. Moreover, metatranscriptomic approaches could elucidate their transcriptional activity in the respiratory tract.\u003c/p\u003e \u003cp\u003eGiven the increasing recognition of CRESS-DNA viruses as a diverse and ubiquitous viral group, their potential role in shaping microbial communities and human health warrants further exploration. Understanding their interactions with host immunity, co-infections with other respiratory viruses, and potential transmission dynamics will be crucial for defining their biological relevance. Our study adds to the growing body of evidence indicating that human respiratory secretions harbor previously unrecognized viral diversity, underscoring the need for ongoing metagenomic surveillance to fully characterize the human virome.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003e5 Funding\u003c/h2\u003e\n\u003cp\u003eThis research was financially supported by National Natural Science Foundation of China (No. 82341106), National Key Research and Development Programs of China (No. 2023YFD1801301 and 2022YFC2603801) and Clinical Medicine Science and Technology Development Foundation of Jiangsu University (No. JLY2021151)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.1 Conflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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\u003e6.2 Human Rights Statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Jiangsu University Ethics Committee and complied with Chinese ethics laws and regulations. Informed consent to participate was obtained from all human participants.\u003c/p\u003e\n\u003ch2\u003e7 Authors\u0026apos; contributions\u003c/h2\u003e\n\u003cp\u003eWZ and ZD contributed to the conception of the study. HZ and YF contributed significantly to analysis and manuscript preparation. CC performed the data analysis and wrote the manuscript. HJ and RT helped perform the analysis with constructive discussions. All authors reviewed the manuscript.\u003c/p\u003e\n\u003ch2\u003e8 Data availability\u003c/h2\u003e\n\u003cp\u003eThe novel CRESS-DNA viruses reported in this study are publicly available in the GenBank database under the accession numbers PV246541\u0026ndash;PV246548. Additionally, the raw sequencing data supporting this study\u0026apos;s findings have been deposited in the NCBI Sequence Read Archive (SRA) under the BioProject accession number PRJNA1230567. These datasets are openly accessible through the NCBI SRA database.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCapozza P, Lanave G, Diakoudi G, Pellegrini F, Cardone R, Vasinioti VI, et al. Diversity of CRESS DNA Viruses in Squamates Recapitulates Hosts Dietary and Environmental Sources of Exposure. Microbiol Spectr. 2022;10(3):e0078022.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeher E, Mihalov-Kovacs E, Kaszab E, Malik YS, Marton S, Banyai K. Genomic Diversity of CRESS DNA Viruses in the Eukaryotic Virome of Swine Feces. Microorganisms. 2021;9(7).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKrupovic M, Varsani A, Kazlauskas D, Breitbart M, Delwart E, Rosario K et al. Cressdnaviricota: a Virus Phylum Unifying Seven Families of Rep-Encoding Viruses with Single-Stranded, Circular DNA Genomes. J Virol. 2020;94(12).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKinsella CM, Bart A, Deijs M, Broekhuizen P, Kaczorowska J, Jebbink MF, et al. Entamoeba and Giardia parasites implicated as hosts of CRESS viruses. Nat Commun. 2020;11(1):4620.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePhan TG, Mori D, Deng X, Rajindrajith S, Ranawaka U, Fan Ng TF, et al. Small circular single stranded DNA viral genomes in unexplained cases of human encephalitis, diarrhea, and in untreated sewage. Virology. 2015;482:98\u0026ndash;104.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePhan TG, da Costa AC, Del Valle Mendoza J, Bucardo-Rivera F, Nordgren J, O'Ryan M, et al. The fecal virome of South and Central American children with diarrhea includes small circular DNA viral genomes of unknown origin. Arch Virol. 2016;161(4):959\u0026ndash;66.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAltan E, Del Valle Mendoza J, Deng X, Phan TG, Sadeghi M, Delwart EL. Small Circular Rep-Encoding Single-Stranded DNA Genomes in Peruvian Diarrhea Virome. Genome Announc. 2017;5(38).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDai Z, Wang H, Xu J, Lu X, Ni P, Yang S et al. Unveiling the Virome of Wild Birds: Exploring CRESS-DNA Viral Dark Matter. Genome Biol Evol. 2024;16(10).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePene F, Merlat A, Vabret A, Rozenberg F, Buzyn A, Dreyfus F, et al. Coronavirus 229E-related pneumonia in immunocompromised patients. Clin Infect Dis. 2003;37(7):929\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLandolfo S, Gariglio M, Gribaudo G, Lembo D. The human cytomegalovirus. Pharmacol Ther. 2003;98(3):269\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDamania B, Kenney SC, Raab-Traub N. Epstein-Barr virus: Biology and clinical disease. Cell. 2022;185(20):3652\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSzydlowski J, Jonczyk-Potoczna K, Pucher B, Buraczynska-Andrzejewska B, Prauzinska M, Kolasinska-Lipinska J, et al. Prevalence of human papillomavirus (HPV) in upper respiratory tract mucosa in a group of pre-school children. Ann Agric Environ Med. 2014;21(4):822\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWillner D, Furlan M, Haynes M, Schmieder R, Angly FE, Silva J, et al. Metagenomic analysis of respiratory tract DNA viral communities in cystic fibrosis and non-cystic fibrosis individuals. PLoS ONE. 2009;4(10):e7370.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbeles SR, Ly M, Santiago-Rodriguez TM, Pride DT. Effects of Long Term Antibiotic Therapy on Human Oral and Fecal Viromes. PLoS ONE. 2015;10(8):e0134941.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWylie KM, Mihindukulasuriya KA, Sodergren E, Weinstock GM, Storch GA. Sequence analysis of the human virome in febrile and afebrile children. PLoS ONE. 2012;7(6):e27735.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePerez-Brocal V, Moya A. The analysis of the oral DNA virome reveals which viruses are widespread and rare among healthy young adults in Valencia (Spain). PLoS ONE. 2018;13(2):e0191867.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoung JC, Chehoud C, Bittinger K, Bailey A, Diamond JM, Cantu E, et al. Viral metagenomics reveal blooms of anelloviruses in the respiratory tract of lung transplant recipients. Am J Transpl. 2015;15(1):200\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClarke EL, Lauder AP, Hofstaedter CE, Hwang Y, Fitzgerald AS, Imai I, et al. Microbial Lineages in Sarcoidosis. A Metagenomic Analysis Tailored for Low-Microbial Content Samples. Am J Respir Crit Care Med. 2018;197(2):225\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbbas AA, Diamond JM, Chehoud C, Chang B, Kotzin JJ, Young JC, et al. The Perioperative Lung Transplant Virome: Torque Teno Viruses Are Elevated in Donor Lungs and Show Divergent Dynamics in Primary Graft Dysfunction. Am J Transpl. 2017;17(5):1313\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbbas AA, Young JC, Clarke EL, Diamond JM, Imai I, Haas AR, et al. Bidirectional transfer of Anelloviridae lineages between graft and host during lung transplantation. Am J Transpl. 2019;19(4):1086\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbbas AA, Taylor LJ, Dothard MI, Leiby JS, Fitzgerald AS, Khatib LA, et al. Redondoviridae, a Family of Small, Circular DNA Viruses of the Human Oro-Respiratory Tract Associated with Periodontitis and Critical Illness. Cell Host Microbe. 2019;25(5):719\u0026ndash;29. e4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang W, Li L, Deng X, Kapusinszky B, Pesavento PA, Delwart E. Faecal virome of cats in an animal shelter. J Gen Virol. 2014;95(Pt 11):2553\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSkewes-Cox P, Sharpton TJ, Pollard KS, DeRisi JL. Profile hidden Markov models for the detection of viruses within metagenomic sequence data. PLoS ONE. 2014;9(8):e105067.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJohnson LS, Eddy SR, Portugaly E. Hidden Markov model speed heuristic and iterative HMM search procedure. BMC Bioinformatics. 2010;11:431.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFinn RD, Clements J, Eddy SR. HMMER web server: interactive sequence similarity searching. Nucleic Acids Res. 2011;39(Web Server issue):W29\u0026ndash;37.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEddy SR. A new generation of homology search tools based on probabilistic inference. Genome Inf. 2009;23(1):205\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang J, Chitsaz F, Derbyshire MK, Gonzales NR, Gwadz M, Lu S, et al. The conserved domain database in 2023. Nucleic Acids Res. 2023;51(D1):D384\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKatoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013;30(4):772\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCapella-Gutierrez S, Silla-Martinez JM, Gabaldon T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics. 2009;25(15):1972\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMinh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD, von Haeseler A, et al. IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era. Mol Biol Evol. 2020;37(5):1530\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKalyaanamoorthy S, Minh BQ, Wong TKF, von Haeseler A, Jermiin LS. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat Methods. 2017;14(6):587\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoang DT, Chernomor O, von Haeseler A, Minh BQ, Vinh LS. UFBoot2: Improving the Ultrafast Bootstrap Approximation. Mol Biol Evol. 2018;35(2):518\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMuhire BM, Varsani A, Martin DP. SDT: a virus classification tool based on pairwise sequence alignment and identity calculation. PLoS ONE. 2014;9(9):e108277.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao L, Rosario K, Breitbart M, Duffy S. Eukaryotic Circular Rep-Encoding Single-Stranded DNA (CRESS DNA) Viruses: Ubiquitous Viruses With Small Genomes and a Diverse Host Range. Adv Virus Res. 2019;103:71\u0026ndash;133.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVarsani A, Hopkins A, Lund MC, Krupovic M. 2024 taxonomic update for the families Naryaviridae, Nenyaviridae, and Vilyaviridae. Arch Virol. 2024;170(1):18.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbbas AA, Taylor LJ, Dothard MI, Leiby JS, Fitzgerald AS, Khatib LA, et al. Redondoviridae, a Family of Small, Circular DNA Viruses of the Human Oro-Respiratory Tract Associated with Periodontitis and Critical Illness. Cell Host Microbe. 2019;26(2):297.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaylor LJ, Keeler EL, Bushman FD, Collman RG. The enigmatic roles of Anelloviridae and Redondoviridae in humans. Curr Opin Virol. 2022;55:101248.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHamelin B, Perot P, Pichler I, Haslbauer JD, Hardy D, Hing D, et al. Circovirus Hepatitis in Immunocompromised Patient, Switzerland. Emerg Infect Dis. 2024;30(10):2140\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeng XJ. Porcine circovirus type 2 (PCV2): pathogenesis and interaction with the immune system. Annu Rev Anim Biosci. 2013;1:43\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"virology-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"virj","sideBox":"Learn more about [Virology Journal](http://virologyj.biomedcentral.com/)","snPcode":"12985","submissionUrl":"https://submission.nature.com/new-submission/12985/3","title":"Virology Journal","twitterHandle":"@VirologyJ","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Metagenomics, Cressdnaviricota, Endolinaviridae, Naryaviridae, Phylogenetics, Respiratory tract","lastPublishedDoi":"10.21203/rs.3.rs-6208723/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6208723/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCRESS-DNA viruses are small, circular, single-stranded DNA viruses that have been identified in diverse environments and hosts, including vertebrates, invertebrates, and environmental samples. However, their diversity and role in the human respiratory tract remain poorly understood. In this study, we employed viral metagenomics to analyze 140 nasopharyngeal swab samples from asymptomatic individuals. High-throughput sequencing and bioinformatics analyses were used to identify and characterize novel CRESS-DNA viruses. Phylogenetic relationships were inferred based on Rep protein sequences using maximum likelihood analysis. We identified and characterized eight novel CRESS-DNA viruses, which were classified into the families \u003cem\u003eEndolinaviridae\u003c/em\u003e and \u003cem\u003eNaryaviridae\u003c/em\u003e, with one potentially representing a novel viral family. These viruses exhibited typical circular genomic structures encoding Rep and Cap proteins, with conserved motifs associated with rolling circle replication. Phylogenetic analysis showed that some viruses were closely related to sequences from vertebrate hosts or environmental samples, suggesting a diverse ecological distribution. Our findings expand the known diversity of CRESS-DNA viruses in the human respiratory tract and highlight their potential ecological and evolutionary significance. Further studies are needed to explore their host specificity, replication mechanisms, and potential roles in human health and disease.\u003c/p\u003e","manuscriptTitle":"Identification and Characterization of Novel CRESS-DNA viruses in the Human Respiratory Tract","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-03 09:44:24","doi":"10.21203/rs.3.rs-6208723/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accepted","date":"2025-04-14T12:43:18+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-14T12:22:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-10T17:42:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-03T17:46:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"148174332744058331886437759844776276659","date":"2025-04-03T12:24:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"94904283100100640545865744224397551133","date":"2025-04-02T18:45:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"304439487408693248571133262682184980003","date":"2025-04-02T12:09:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-02T11:16:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-02T06:14:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Virology Journal","date":"2025-04-01T17:44:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"virology-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"virj","sideBox":"Learn more about [Virology Journal](http://virologyj.biomedcentral.com/)","snPcode":"12985","submissionUrl":"https://submission.nature.com/new-submission/12985/3","title":"Virology Journal","twitterHandle":"@VirologyJ","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b51607c3-3b44-41c9-9b90-bf11771688d6","owner":[],"postedDate":"April 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-07T16:14:51+00:00","versionOfRecord":{"articleIdentity":"rs-6208723","link":"https://doi.org/10.1186/s12985-025-02742-6","journal":{"identity":"virology-journal","isVorOnly":false,"title":"Virology Journal"},"publishedOn":"2025-06-30 15:57:46","publishedOnDateReadable":"June 30th, 2025"},"versionCreatedAt":"2025-04-03 09:44:24","video":"","vorDoi":"10.1186/s12985-025-02742-6","vorDoiUrl":"https://doi.org/10.1186/s12985-025-02742-6","workflowStages":[]},"version":"v1","identity":"rs-6208723","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6208723","identity":"rs-6208723","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}