Identification of a novel parvovirus in the Arctic wolf (Canis lupus)

Identification of a novel parvovirus in the Arctic wolf (Canis lupus) | 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 of a novel parvovirus in the Arctic wolf (Canis lupus) Ziyuan Dai, Rong Zhu, Hongmei Chen, Mingzhong Sun This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3337233/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Through the utilization of a viral metagenomic approach, a novel virus has been found in a pharyngeal metagenomic library derived from an Arctic wolf ( Canis lupus ). This virus has been temporarily designated as AWPV and assigned a GenBank accession number BK063423. The genome of AWPV is comprised of 4,920 base pairs, and its nucleotide composition is composed of 36.4% A, 23.4% T, 18.2% G, and 22.0% C, with a GC content of 40.2%. The viral genome demonstrates a typical pattern of parvovirus organization, with two predicted ORFs: ORF1, which encodes non-structural proteins NS1 and NS2, and ORF2, which encodes VP1 and VP2. By performing a pairwise sequence comparison and a phylogenetic analysis based on the NS1 and VP1 protein sequences, it has been suggested that AWPV may represent a novel species within the genus Protoparvovirus . This discovery of a novel parvovirus has enhanced our comprehension of the mammalian virus ecology and has facilitated an improved understanding of potential future infectious diseases. Parvovirus Virome Metagenomics Arctic wolf Novel Figures Figure 1 Figure 2 Introduction The family Parvoviridae comprises round, non-enveloped viruses that have linear, single-stranded DNA genomes ranging from 4–6 kb( 1 ), and they have been detected in nearly all major groups of vertebrates, as well as in both proto- and deuterostome invertebrates( 2 ). In 1975, the family Parvoviridae was established, and in 1993 it was divided into two subfamilies, Parvovirinae and Densovirinae , to classify viruses that infect either vertebrate or invertebrate hosts( 1 ). As of now, according to the classification principles of the International Committee for the Taxonomy of Viruses (ICTV), the family Parvoviridae comprises 3 subfamilies, 28 genera, and 175 species. Reports of parvoviruses have surfaced in numerous countries and have affected a wide range of hosts, including mammals such as humans( 3 ), mice( 4 ), canines( 5 ), and chimpanzees( 6 ), as well as arthropods such as crickets( 7 ), and birds such as ducks( 8 ), red-crowned cranes( 9 ), and pigeons( 10 ). Parvoviral genomes are characterized by long inverted terminal repeats (LTRs) located at both the 5' and 3' ends, which can adopt hairpin-like structures and play a role in the viruses' expression and transcription strategies( 11 ). They encode two open reading frames (ORFs), where ORF1 encodes non-structural proteins NS1 and NS2, and ORF2 encodes structural proteins VP1 and VP2( 12 ). Parvoviruses can be classified as belonging to the same species if their NS1 proteins have a shared amino acid sequence identity of over 85%. Similarly, a genus can be defined as a cluster of species that form a single branch and have a minimum of 35–40% amino acid sequence identity with a coverage of over 80% between any two members( 2 ). At present, there are 18 species within the genus Protoparvovirus , consisting of 15 officially recognized species and three that are currently proposed( 13 ). Protoparvovirus carnivoran2 (CPV-2), belonging to the genus Protoparvovirus of the family Parvoviridae , is a highly contagious viral pathogen that primarily affects canids, particularly dogs. It is characterized by causing severe gastroente ric disease in its hosts, including symptoms such as vomiting, diarrhea, loss of appetite, and dehydration( 5 , 14 ). CPV-2 has a wide range of hosts, including dogs, foxes, and wolves, and is mainly transmitted through the fecal-oral route( 15 ). The CPV-2 first invades the pharynx and then enters the bloodstream within a few days of infection, reaching the intestines and bone marrow, causing severe leukopenia and may also cause viremia, which may subsequently lead to myocarditis( 15 , 16 ). Here, we present the genome characterization of a novel Arctic wolf parvovirus (named AWPV) identified from an Arctic wolf pharyngeal metagenomic library. The discovery of this new genome expands our understanding of the diversity of parvoviruses. Our analysis suggests that AWPV could potentially be classified as a new species in the genus Protoparvovirus . Materials and Methods Metagenome assembly While studying potential pathogenic viruses in mammals, an available library - SRR12366691 - was downloaded from the SRA database. This library was uploaded by Du et al. from Hainan Medical University and corresponds to the host Arctic wolf, collected from an aquarium in Xi'an, China. The method for processing the samples has been described in the previous( 17 ). The SRA file format was transformed to fastq format utilizing Pfastq-dump v0.1.6 ( https://github.com/inutano/pfastq-dump ), and the elimination of host sequences was executed using Bowtie2 v2.4.5( 18 , 19 ). The potential primer sequences present in the raw reads were removed by applying Trim Galore v0.6.5 ( https://www.bioinformatics.babraham.ac.uk/projects/trim_galore ). Afterwards, the resultant files were subjected to quality control using the options ‘--phred33 --length 35 --stringency 3 --fastqc’. PRINSEQ-lite v0.20.4 (-derep 1)( 20 ) was employed to mark duplicated reads. An in-house pipeline was utilized to assemble this library. The assembly of single-end reads was carried out using MEGAHIT v1.2.9( 21 ) with default parameters. Contigs with a sequence length greater than 1,500 bp were kept after the assembly process. Following the aforementioned steps, the outcomes were imported into Geneious Prime v2022.0.1( 22 ) to be sorted and confirmed manually. Search for novel vertebrate-associated viruses The contigs were aligned with the non-redundant protein (nr) database (downloaded in February 2023) utilizing the BLASTx program built in DIAMOND v2.0.15( 23 ), with a cut-off E-value of < 10 − 5 . The taxonomic identification was carried out using the built-in rma2info program in MEGAN6( 24 ), and the viruses of interest were filtered out from the results. Geneious Prime was used to predict putative open reading frames (ORFs) using built-in parameters (Minimum size: 400) ( 22 ). These predictions were subsequently validated by comparing them to ORFs found in related viruses. Comparisons to the Conserved Domain Database (CDD) were used to annotate these ORFs. Finally, we obtained a putative novel parvovirus with complete genome organization structure. Phylogenetic analysis To infer phylogenetic relationships, reference protein sequences related to parvovirus were downloaded from the NCBI GenBank database. The protein sequences were aligned using the alignment program in Geneious Prime. Subsequently, the resulting alignment was optimized further by utilizing MUSCLE in MEGA v7.0( 25 ) and MAFFT v7.3.1, which employed the E-INS-I algorithm( 26 ). MrBayes v3.2( 27 ) was utilized to construct Bayesian inference trees. A Markov chain was executed for a maximum of 1 million generations, with sampling occurring every 50 generations. The first 25% of Markov chain Monte Carlo (mcmc) samples were discarded as burn-in. In addition, Maximum Likelihood trees were constructed using MEGA v7.0( 25 ) software to verify all Bayesian inference trees. The Sequence Demarcation Tool v1.2( 28 ) was used to conduct color-coded pairwise identity matrix analysis comparing the novel parvovirus to other members of Parvoviridae . The identity score for each pair of sequences is computed as 1-M/N, where M is the number of mismatched nucleotides and N is the total number of columns along the alignment where neither sequence has a gap character( 28 ). Prediction of potential genome recombination events The Recombination Detection Program v4.39 (RDP4) software was used to analyze genomic alignments of both reference strains and the AWPV strain. This was done through various algorithms including RDP, GENECONV, Chimaera, MaxChi, BootScan, and SiScan, in order to detect possible recombination events( 29 ). Prediction of spatial structure The three-dimensional structure of the viral structural protein identified in this study was predicted using ColabFold( 30 ), and SWISS-MODEL( 30 ) was employed to compare and screen models that possess comparable spatial structures from the PDB database. To visualize the results, PyMOL v2.0 ( www.pymol.org ) was utilized. Data availability The novel parvovirus sequence obtained in this study have been deposited in GenBank database under accession number BK063423. Results Overview of the pharyngeal metagenomic library of the Arctic Wolf This library was sequenced using the Illumina HiSeq 2500 platform, generating 28,946,890 raw reads. Following quality control, 28,945,811 clean reads were obtained, which were then assembled into 736 contigs greater than 1,500 bp in length. Upon searching these contigs against the nr database, 260 contigs exhibited the highest identity with viral proteins (Supplementary Table 1). Eleven viral families were identified, with the family Myoviridae being the most prevalent, accounting for 28.85% of all viral contigs, followed by Siphoviridae (18.08%), Podoviridae (12.31%), Ackermannviridae (11.54%), Demerecviridae (6.54%), Autographiviridae (6.15%), Microviridae (4.23%), and Herelleviridae (3.08%) (Supplementary Table 1). However, the vast majority of them are already known. Moreover, 20 contigs were identified, which could not be confidently assigned to any viral taxonomic group (E-value > 10 − 5 ). This finding suggests the potential existence of novel viruses within these contigs or the prevalence of non-coding regions in the assembled sequences. In order to determine whether the host of this library is the Arctic wolf, we performed a BLASTx search by comparing the contigs assembled from the NGS data of this library (specifically those containing potential mammalian sequence reads) against the complete mitochondrial proteome database downloaded from GenBank. The search results revealed that the majority of sequences among the mammalian matches corresponded to the Arctic wolf (Supplementary Table 2). Identification of a novel parvovirus In this research, a virus named AWPV, belonging to the family Parvoviridae and possessing a complete genome organization structure, was obtained using the sequence assembly tool of Geneious Prime. AWPV has a genome size of 4,920 bp, characterized by a GC content of 40.2% and a nucleotide composition consisting of 36.4% A, 23.4% T, 18.2% G, and 22.0% C. The typical organizational pattern of this viral genome is shown in Fig. 1 A. Specifically, AWPV possesses a partial 5' non-translated region (68 bp), a complete NS1 open reading frame (620 aa), a complete virus protein (VP) 1 open reading frame (739 aa), a complete virus protein (VP) 2 open reading frame (581 aa), and a partial 3' non-translated region (214 bp). The NS1 protein contains several conserved motifs that are related to ATP- or GTP-binding, including a Walker A loop 408 GPASTGKS 415 [GXXXXGK(T/S)], a Walker B loop at 448 IIWVEE 453 (xxxxEE), and a Walker B' loop at 465 KAICSGQSIRIDQK 478 (KxxxxGxxxxxxxK). In addition, we identified a conserved replication initiator motif 132 KLHIHVLLHH 141 (xxHxHxxxxx) in the NS1 protein. By aligning with other protoparvovirus sequences, potential splicing signals for expressing VP1 were identified. In the N-terminal of VP1, the phospholipase A 2 (PLA2) motif was identified, which contains the expected calcium-binding (YLGPG) site and catalytic residues (Fig. 1 A). Phylogenetic analysis Representative sequences with high identity to the NS1 and VP1 proteins of AWPV, as well as other parvoviruses, were included in subsequent analysis. Based on the phylogenetic analysis of NS1 and VP1 protein sequences, AWPV was positioned between the parvoviruses identified in California sea lions( 31 ) and foxes( 13 , 32 ), forming a distinct branch (Fig. 1 B). Based on the distance matrix analysis of NS1 and VP1 proteins, it was determined that AWPV shares less than 77% identity with the protein sequences of other Parvovirus members. This suggests that AWPV should be classified as a new species of the genus Protoparvovirus (Fig. 2 and Supplementary Table 3). In addition, the RDP4 software did not indicate any significant evidence of recombination signals. Predicting the spatial structure of the AWPV's VP The structure and biophysical properties of Protoparvovirus capsids are essential for its survival in the natural environment and entry into host cells( 33 ). To predict and contrast the similarity of the spatial structure of AWPV's VP with the structure dictated by present known sequences, the sequence exhibiting the greatest degree of similarity to AWPV's VP, which is the Newlavirus strain ITA/2016/51.20–153 (UZZ82241), was retrieved from the GenBank database. The spatial structure of sequences encoding VP was predicted using ColabFold. Furthermore, SWISS-MODEL was utilized to search and obtain the virus model (6x2k.1) of Tusavirus( 34 ), which exhibited the highest similarity to AWPV. The PyMOL software was used to import all PDB files and conduct pairwise comparisons. The Root Mean Square Deviation (RMSD) is an indicator of the overall similarity between two spatial structures. Typically, an RMSD of less than 2 Å suggests a significant degree of similarity. The AWPV identified in this study did not show significant similarity in the VP spatial structure with the Newlavirus strain ITA/2016/51.20–153 and Tusavirus (RMSD = 2.888 and 4.458), indicating that AWPV and other related viruses may have different characteristics in mediating cell attachment during infection (Supplementary Fig. 1). Discussion Metagenomics has revolutionized our understanding of viral diversity, greatly expanding the number of identified viral families, genera, and species beyond traditional methods. This is measured through sequence diversity and evolutionary distances( 35 ). Additionally, metagenomics enables the detection of novel viruses and the characterization of viromes in diverse samples, which enhances our knowledge of virus-host interactions and their potential public health implications, particularly for emerging and re-emerging infectious diseases. The Arctic wolf, also known as the white wolf or polar wolf, is a subspecies of the gray wolf in the family Canidae and is distributed in the northern regions of Eurasia, northern Canada, and northern Greenland. In recent years, there have been reports about Arctic wolves being infected with protoparvovirus, but most of them are related to CPV-2. For example, Justin M. Stilwell and others identified CPV-2 in a female Arctic wolf that died at the age of six weeks due to systemic canine distemper virus (CDV) infection( 36 ). In this study, we recovered a novel virus belonging to the genus Protoparvovirus from the Arctic wolf pharyngeal metagenomic library. The virus, tentatively named AWPV, has a genome length of 4,920 bp, and its best match in the GenBank database shows a nucleotide sequence identity of 75.76% and coverage of 70%. The Bayesian phylogenetic trees constructed based on NS1 and VP1 amino acid sequences exhibited overall similar topology, with AWPV forming a unique branch located between the recently identified parvoviruses from California sea lions (California sea lion parvovirus, MN982959) and foxes (Newlaviruses, ON959793-96), and AWPV having a relatively distant relationship with CPVs. The California sea lion parvovirus was derived from a 2–3-year-old subadult emaciated female California sea lion that was found stranded and deceased in 2010( 31 ). The Newlaviruses were detected and identified in the carcasses of Italian foxes( 32 ). Due to the possibility of this Arctic wolf being captive, we cautiously speculate that AWPV may also have originated from interactions with other captive animals. Further research was deemed necessary to investigate the epidemiology and transmission dynamics of AWPV in both captive and wild Arctic wolf populations, in order to gain a comprehensive understanding of this virus and its potential impact on the health of these animals. The vast temporal and geographic range of both wild and captive hosts raises questions as to whether other, as-yet-undiscovered intermediate hosts may be carrying these viruses. While the role of these newly identified viruses in host morbidity and mortality remains uncertain, it is clear that members of the family Parvoviridae have achieved a worldwide distribution. In summary, the identification of AWPV increases our understanding of the diversity of canine parvoviruses and provides limited assistance in improving virus taxonomy. While there is no direct evidence to suggest whether AWPV will have significant negative effects on its host, further dynamic monitoring of the virus is needed in the future. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Written informed consent for publication was obtained from all participants. Availability of data and materials The novel parvovirus sequence obtained in this study have been deposited in GenBank database under accession number BK063423. Competing interests The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding This work was supported by The Special Funds for Science Development of the Clinical Teaching Hospitals of Jiangsu Vocational College of Medicine no. 20229152 to ZD. Authors' contributions ZD and MS collected data and wrote this article, HC, RZ and ZD assisted in data processing and proofreading. All authors reviewed the manuscript. Acknowledgments Not applicable. References Cotmore SF, Agbandje-McKenna M, Canuti M, Chiorini JA, Eis-Hubinger AM, Hughes J et al (2019) ICTV Virus Taxonomy Profile: Parvoviridae. J Gen Virol 100(3):367–368 Epub 2019/01/24. 10.1099/jgv.0.001212 Penzes JJ, Soderlund-Venermo M, Canuti M, Eis-Hubinger AM, Hughes J, Cotmore SF et al (2020) Reorganizing the family Parvoviridae: a revised taxonomy independent of the canonical approach based on host association. Arch Virol 165(9):2133–2146 Epub 2020/06/14. 10.1007/s00705-020-04632-4 Phan TG, Vo NP, Bonkoungou IJ, Kapoor A, Barro N, O'Ryan M et al (2012) Acute diarrhea in West African children: diverse enteric viruses and a novel parvovirus genus. J Virol 86(20):11024–11030 Epub 2012/08/03. 10.1128/JVI.01427-12 Janus LM, Bleich A (2012) Coping with parvovirus infections in mice: health surveillance and control. Lab Anim 46(1):14–23 Epub 2011/11/01. 10.1258/la.2011.011025 Decaro N, Buonavoglia C (2012) Canine parvovirus–a review of epidemiological and diagnostic aspects, with emphasis on type 2c. Vet Microbiol 155(1):1–12 Epub 2011/10/04. 10.1016/j.vetmic.2011.09.007 Adlhoch C, Kaiser M, Loewa A, Ulrich M, Forbrig C, Adjogoua EV et al (2012) Diversity of parvovirus 4-like viruses in humans, chimpanzees, and monkeys in hunter-prey relationships. Emerg Infect Dis 18(5):859–862 Epub 2012/04/21. 10.3201/eid1805.111849 Meng G, Zhang X, Plevka P, Yu Q, Tijssen P, Rossmann MG (2013) The structure and host entry of an invertebrate parvovirus. J Virol 87(23):12523–12530 Epub 2013/09/13. 10.1128/JVI.01822-13 Liu J, Yang X, Hao X, Feng Y, Zhang Y, Cheng Z (2020) Effect of Goose Parvovirus and Duck Circovirus Coinfection in Ducks. J Vet Res 64(3):355–361 Epub 2020/09/29. 10.2478/jvetres-2020-0048 Wang Y, Yang S, Liu D, Zhou C, Li W, Lin Y et al (2019) The fecal virome of red-crowned cranes. Arch Virol 164(1):3–16 Epub 2018/09/19. 10.1007/s00705-018-4037-x Bergmann V, Kiupel H (1982) [Inclusion body enteritis in pigeons caused by adenovirus and parvovirus]. Arch Exp Veterinarmed 36(3):445–453 Epub 1982/01/01. PubMed PMID: 6291478 Brown KE (2010) The expanding range of parvoviruses which infect humans. Rev Med Virol 20(4):231–244 Epub 2010/06/30. 10.1002/rmv.648 de Oliveira Santana W, Silveira VP, Wolf JM, Kipper D, Echeverrigaray S, Canal CW et al (2022) Molecular phylogenetic assessment of the canine parvovirus 2 worldwide and analysis of the genetic diversity and temporal spreading in Brazil. Infect Genet Evol 98:105225 Epub 2022/02/02. 10.1016/j.meegid.2022.105225 Canuti M, Bouchard E, Rodrigues B, Whitney HG, Hopson M, Gilroy C et al (2021) Newlavirus, a Novel, Highly Prevalent, and Highly Diverse Protoparvovirus of Foxes (Vulpes spp). Viruses 13(10). 10.3390/v13101969 PubMed PMID: 34696399; PubMed Central PMCID: PMCPMC8537079 Epub 2021/10/27 Decaro N, Buonavoglia C, Barrs VR (2020) Canine parvovirus vaccination and immunisation failures: Are we far from disease eradication? Vet Microbiol 247:108760 Epub 2020/08/10. 10.1016/j.vetmic.2020.108760 Kurucay HN, Tamer C, Muftuoglu B, Elhag AE, Gozel S, Cicek-Yildiz Y et al (2023) First isolation and molecular characterization of canine parvovirus-type 2b (CPV-2b) from red foxes (Vulpes vulpes) living in the wild habitat of Turkey. Virol J 20(1):27 Epub 2023/02/12. 10.1186/s12985-023-01988-2 Tuteja D, Banu K, Mondal B (2022) Canine parvovirology - A brief updated review on structural biology, occurrence, pathogenesis, clinical diagnosis, treatment and prevention. Comp Immunol Microbiol Infect Dis 82:101765 Epub 2022/02/20. 10.1016/j.cimid.2022.101765 Zhang P, Su H, Peng R, Chan JF, Bai S, Wang G et al (2022) Identification of a Novel Astrovirus in Pinnipeds. Front Microbiol 13:845601 Epub 2022/05/24. 10.3389/fmicb.2022.845601 Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9(4):357–359 Epub 2012/03/06. doi: 10.1038/nmeth.1923. PubMed PMID: 22388286; PubMed Central PMCID: PMCPMC3322381 Langmead B, Wilks C, Antonescu V, Charles R (2019) Scaling read aligners to hundreds of threads on general-purpose processors. Bioinformatics 35(3):421–432 Epub 2018/07/19. 10.1093/bioinformatics/bty648 Schmieder R, Edwards R (2011) Quality control and preprocessing of metagenomic datasets. Bioinformatics 27(6):863–864 Epub 2011/02/01. 10.1093/bioinformatics/btr026 Li D, Luo R, Liu CM, Leung CM, Ting HF, Sadakane K et al (2016) MEGAHIT v1.0: A fast and scalable metagenome assembler driven by advanced methodologies and community practices. Methods 102:3–11 Epub 2016/03/26. 10.1016/j.ymeth.2016.02.020 Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S et al (2012) Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28(12):1647–1649 Epub 2012/05/01. 10.1093/bioinformatics/bts199 Buchfink B, Reuter K, Drost HG (2021) Sensitive protein alignments at tree-of-life scale using DIAMOND. Nat Methods 18(4):366–368 Epub 2021/04/09. 10.1038/s41592-021-01101-x Gautam A, Felderhoff H, Bagci C, Huson DH (2022) Using AnnoTree to Get More Assignments, Faster, in DIAMOND + MEGAN Microbiome Analysis. mSystems 7(1):e0140821. Epub 2022/02/23. 10.1128/msystems.01408-21 . PubMed PMID: 35191776; PubMed Central PMCID: PMCPMC8862659 Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol 33(7):1870–1874 Epub 2016/03/24. 10.1093/molbev/msw054 Kuraku S, Zmasek CM, Nishimura O, Katoh K (2013) aLeaves facilitates on-demand exploration of metazoan gene family trees on MAFFT sequence alignment server with enhanced interactivity. Nucleic Acids Res 41(Web Server issue):W22-8. Epub 2013/05/17. 10.1093/nar/gkt389 . PubMed PMID: 23677614; PubMed Central PMCID: PMCPMC3692103 Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Hohna S et al (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61(3):539–542 Epub 2012/02/24. 10.1093/sysbio/sys029 Muhire BM, Varsani A, Martin DP (2014) SDT: a virus classification tool based on pairwise sequence alignment and identity calculation. PLoS ONE 9(9):e108277 Epub 2014/09/27. 10.1371/journal.pone.0108277 Martin DP, Murrell B, Golden M, Khoosal A, Muhire B (2015) RDP4: Detection and analysis of recombination patterns in virus genomes. Virus Evol 1(1):vev003 Epub 2015/05/26. 10.1093/ve/vev003 Mirdita M, Schutze K, Moriwaki Y, Heo L, Ovchinnikov S, Steinegger M (2022) ColabFold: making protein folding accessible to all. Nat Methods 19(6):679–682 Epub 2022/06/01. 10.1038/s41592-022-01488-1 Altan E, Delaney MA, Colegrove KM, Spraker TR, Wheeler EA, Deng X et al (2020) Complex Virome in a Mesenteric Lymph Node from a Californian Sea Lion (Zalophus Californianus) with Polyserositis and Steatitis. Viruses 12(8). Epub 2020/07/29. 10.3390/v12080793 . PubMed PMID: 32718049; PubMed Central PMCID: PMCPMC7472147 Lanave G, Ndiana LA, Pellegrini F, Diakoudi G, Di Martino B, Sgroi G et al (2022) Detection at high prevalence of newlavirus (protoparvovirus) in the carcasses of red foxes. Virus Res 323:198971 Epub 2022/10/19. 10.1016/j.virusres.2022.198971 Ros C, Bayat N, Wolfisberg R, Almendral JM (2017) Protoparvovirus Cell Entry. Viruses 9(11). Epub 2017/10/27. 10.3390/v9110313 . PubMed PMID: 29072600; PubMed Central PMCID: PMCPMC5707520 Mietzsch M, McKenna R, Vaisanen E, Yu JC, Ilyas M, Hull JA et al (2020) Structural Characterization of Cuta- and Tusavirus: Insight into Protoparvoviruses Capsid Morphology. Viruses 12(6). Epub 2020/06/21. 10.3390/v12060653 . PubMed PMID: 32560452; PubMed Central PMCID: PMCPMC7354515 Greninger AL (2018) A decade of RNA virus metagenomics is (not) enough. Virus Res 244:218 – 29. Epub 2017/10/23. 10.1016/j.virusres.2017.10.014 . PubMed PMID: 29055712; PubMed Central PMCID: PMCPMC7114529 Stilwell JM, Anis E, Wilkes RP, Rissi DR (2019) Dual infection with an emergent strain of canine distemper virus and canine parvovirus in an Arctic wolf under managed care. J Vet Diagn Invest 31(4):594–597 PubMed PMID: 31113289; PubMed Central PMCID: PMCPMC6857037 Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigure1.jpg Supplementary Figure 1. The structural model visualization of the VP protein was done through PyMOL v2.0 software, using the PDB files and pairwise alignment. SupplementaryTable1.xlsx Supplementary Table 1. A summary of information regarding an Arctic wolf pharyngeal metagenomic library. SupplementaryTable2.xlsx Supplementary Table 2. BLASTx results for potential host species. SupplementaryTable3.xlsx Supplementary Table 3. Additional information related to the distance matrix analysis of AWPV. Cite Share Download PDF Status: Posted Version 1 posted 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. 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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-3337233","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":231759070,"identity":"ea30dd1c-91ef-4830-a6d6-29515093b74b","order_by":0,"name":"Ziyuan Dai","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYDACZgY2BoYCCyArgeHAhwobHn72BmK0GEiAtRyccSZNRrLnAEF7EFqYedsO2xjccMCvXred+dmDDwYScgzsyQ8P87ad52G4wcD44WMObi1mh9nMDWcYSBgz8DwzODjn3G0extkNzJIzt+HTwsMmzWMgkdggkWBw4E3ZbR5mmQNszLyEtPwxkKhvkEj/cICH7RwPm0QCEVqA3k9gkMgxOMjTdoCHh7AWNjPJHgMJwwaeNwXAQE7mkeA52IzfL+cPP5P4UWEjz8CevvnDhwo7e/vjzQc/fMSjBQ7sD8CZjA1EqB8Fo2AUjIJRgA8AABt8TgcEIMRWAAAAAElFTkSuQmCC","orcid":"","institution":"Affiliated Hospital 6 of Nantong University, Yancheng Third People's Hospital","correspondingAuthor":true,"prefix":"","firstName":"Ziyuan","middleName":"","lastName":"Dai","suffix":""},{"id":231759071,"identity":"d834d533-d638-48c2-8303-ebce8a581d74","order_by":1,"name":"Rong Zhu","email":"","orcid":"","institution":"Affiliated Hospital 6 of Nantong University, Yancheng Third People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Rong","middleName":"","lastName":"Zhu","suffix":""},{"id":231759072,"identity":"7e948b00-a4a1-4533-a6ec-22003eac3ad1","order_by":2,"name":"Hongmei Chen","email":"","orcid":"","institution":"Affiliated Hospital 6 of Nantong University, Yancheng Third People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hongmei","middleName":"","lastName":"Chen","suffix":""},{"id":231759073,"identity":"27bf6c7e-ed56-4714-b5c6-edba5908a267","order_by":3,"name":"Mingzhong Sun","email":"","orcid":"","institution":"Affiliated Hospital 6 of Nantong University, Yancheng Third People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Mingzhong","middleName":"","lastName":"Sun","suffix":""}],"badges":[],"createdAt":"2023-09-08 10:29:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3337233/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3337233/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":43069451,"identity":"a482a69f-afef-4694-bb6b-90c91ba957a5","added_by":"auto","created_at":"2023-09-13 14:04:39","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":883385,"visible":true,"origin":"","legend":"\u003cp\u003eGenome and Phylogenetic Analysis of a novel parvovirus. \u003cstrong\u003e(A)\u003c/strong\u003e Organization of the AWPV genome.\u003cstrong\u003e (B)\u003c/strong\u003e Phylogenetic analysis of AWPV. The Bayesian inference tree has been constructed based on protein sequences obtained from the NS1 and VP1 regions of AWPV. The virus discovered in this study is denoted in red within the tree.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3337233/v1/3e792695d58c7e98281b2f6f.jpg"},{"id":43070749,"identity":"9f9e5d84-2e90-4e5d-93d5-faa596db9657","added_by":"auto","created_at":"2023-09-13 14:12:39","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1047675,"visible":true,"origin":"","legend":"\u003cp\u003ePairwise identity matrix analysis of AWPV. Pairwise sequence comparison produced with VP protein sequences within Bayesian consensus tree.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3337233/v1/83e691d12826630a5db05147.jpg"},{"id":43252327,"identity":"28bda2e7-84fb-4faf-ab70-ec41d34e24aa","added_by":"auto","created_at":"2023-09-17 10:22:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":648209,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3337233/v1/fcd8abe1-2b2a-4123-aa82-e994991b3dae.pdf"},{"id":43069452,"identity":"8c500dc4-7f31-4d88-acc4-7d91c9307648","added_by":"auto","created_at":"2023-09-13 14:04:39","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":461347,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 1. The structural model visualization of the VP protein was done through PyMOL v2.0 software, using the PDB files and pairwise alignment.\u003c/p\u003e","description":"","filename":"SupplementaryFigure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3337233/v1/d6e82d678dc423d45a8e5212.jpg"},{"id":43069447,"identity":"8765a573-e472-4b38-b0e5-ead1f27f8fc2","added_by":"auto","created_at":"2023-09-13 14:04:39","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11799,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 1. A summary of information regarding an Arctic wolf pharyngeal metagenomic library.\u003c/p\u003e","description":"","filename":"SupplementaryTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3337233/v1/eee325d97df80c8f2a378a05.xlsx"},{"id":43069446,"identity":"72c5343b-5014-477f-b5de-d56645d44e88","added_by":"auto","created_at":"2023-09-13 14:04:39","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":11432,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 2. BLASTx results for potential host species.\u003c/p\u003e","description":"","filename":"SupplementaryTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3337233/v1/e779819e35cf5ce27059abb8.xlsx"},{"id":43069449,"identity":"a409d614-79a2-4095-9e33-d4c2d20dd421","added_by":"auto","created_at":"2023-09-13 14:04:39","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":16080,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 3. Additional information related to the distance matrix analysis of AWPV.\u003c/p\u003e","description":"","filename":"SupplementaryTable3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3337233/v1/15d45c4c3cbf404b0f284730.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Identification of a novel parvovirus in the Arctic wolf (Canis lupus)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe family \u003cem\u003eParvoviridae\u003c/em\u003e comprises round, non-enveloped viruses that have linear, single-stranded DNA genomes ranging from 4\u0026ndash;6 kb(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e), and they have been detected in nearly all major groups of vertebrates, as well as in both proto- and deuterostome invertebrates(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). In 1975, the family \u003cem\u003eParvoviridae\u003c/em\u003e was established, and in 1993 it was divided into two subfamilies, \u003cem\u003eParvovirinae\u003c/em\u003e and \u003cem\u003eDensovirinae\u003c/em\u003e, to classify viruses that infect either vertebrate or invertebrate hosts(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). As of now, according to the classification principles of the International Committee for the Taxonomy of Viruses (ICTV), the family \u003cem\u003eParvoviridae\u003c/em\u003e comprises 3 subfamilies, 28 genera, and 175 species. Reports of parvoviruses have surfaced in numerous countries and have affected a wide range of hosts, including mammals such as humans(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), mice(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e), canines(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e), and chimpanzees(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e), as well as arthropods such as crickets(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), and birds such as ducks(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), red-crowned cranes(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e), and pigeons(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eParvoviral genomes are characterized by long inverted terminal repeats (LTRs) located at both the 5' and 3' ends, which can adopt hairpin-like structures and play a role in the viruses' expression and transcription strategies(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). They encode two open reading frames (ORFs), where ORF1 encodes non-structural proteins NS1 and NS2, and ORF2 encodes structural proteins VP1 and VP2(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Parvoviruses can be classified as belonging to the same species if their NS1 proteins have a shared amino acid sequence identity of over 85%. Similarly, a genus can be defined as a cluster of species that form a single branch and have a minimum of 35\u0026ndash;40% amino acid sequence identity with a coverage of over 80% between any two members(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAt present, there are 18 species within the genus \u003cem\u003eProtoparvovirus\u003c/em\u003e, consisting of 15 officially recognized species and three that are currently proposed(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Protoparvovirus carnivoran2 (CPV-2), belonging to the genus \u003cem\u003eProtoparvovirus\u003c/em\u003e of the family \u003cem\u003eParvoviridae\u003c/em\u003e, is a highly contagious viral pathogen that primarily affects canids, particularly dogs. It is characterized by causing severe gastroente ric disease in its hosts, including symptoms such as vomiting, diarrhea, loss of appetite, and dehydration(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). CPV-2 has a wide range of hosts, including dogs, foxes, and wolves, and is mainly transmitted through the fecal-oral route(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). The CPV-2 first invades the pharynx and then enters the bloodstream within a few days of infection, reaching the intestines and bone marrow, causing severe leukopenia and may also cause viremia, which may subsequently lead to myocarditis(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHere, we present the genome characterization of a novel Arctic wolf parvovirus (named AWPV) identified from an Arctic wolf pharyngeal metagenomic library. The discovery of this new genome expands our understanding of the diversity of parvoviruses. Our analysis suggests that AWPV could potentially be classified as a new species in the genus \u003cem\u003eProtoparvovirus\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and Methods","content":" \u003ch2\u003eMetagenome assembly\u003c/h2\u003e \u003cp\u003eWhile studying potential pathogenic viruses in mammals, an available library - SRR12366691 - was downloaded from the SRA database. This library was uploaded by Du et al. from Hainan Medical University and corresponds to the host Arctic wolf, collected from an aquarium in Xi'an, China. The method for processing the samples has been described in the previous(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). The SRA file format was transformed to fastq format utilizing Pfastq-dump v0.1.6 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/inutano/pfastq-dump\u003c/span\u003e\u003cspan address=\"https://github.com/inutano/pfastq-dump\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and the elimination of host sequences was executed using Bowtie2 v2.4.5(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). The potential primer sequences present in the raw reads were removed by applying Trim Galore v0.6.5 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.babraham.ac.uk/projects/trim_galore\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.babraham.ac.uk/projects/trim_galore\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Afterwards, the resultant files were subjected to quality control using the options \u0026lsquo;--phred33 --length 35 --stringency 3 --fastqc\u0026rsquo;. PRINSEQ-lite v0.20.4 (-derep 1)(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e) was employed to mark duplicated reads. An in-house pipeline was utilized to assemble this library. The assembly of single-end reads was carried out using MEGAHIT v1.2.9(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) with default parameters. Contigs with a sequence length greater than 1,500 bp were kept after the assembly process. Following the aforementioned steps, the outcomes were imported into Geneious Prime v2022.0.1(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) to be sorted and confirmed manually.\u003c/p\u003e \u003ch2\u003eSearch for novel vertebrate-associated viruses\u003c/h2\u003e \u003cp\u003eThe contigs were aligned with the non-redundant protein (nr) database (downloaded in February 2023) utilizing the BLASTx program built in DIAMOND v2.0.15(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), with a cut-off E-value of \u0026lt;\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e. The taxonomic identification was carried out using the built-in rma2info program in MEGAN6(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), and the viruses of interest were filtered out from the results. Geneious Prime was used to predict putative open reading frames (ORFs) using built-in parameters (Minimum size: 400) (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). These predictions were subsequently validated by comparing them to ORFs found in related viruses. Comparisons to the Conserved Domain Database (CDD) were used to annotate these ORFs. Finally, we obtained a putative novel parvovirus with complete genome organization structure.\u003c/p\u003e \u003ch2\u003ePhylogenetic analysis\u003c/h2\u003e \u003cp\u003eTo infer phylogenetic relationships, reference protein sequences related to parvovirus were downloaded from the NCBI GenBank database. The protein sequences were aligned using the alignment program in Geneious Prime. Subsequently, the resulting alignment was optimized further by utilizing MUSCLE in MEGA v7.0(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e) and MAFFT v7.3.1, which employed the E-INS-I algorithm(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). MrBayes v3.2(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e) was utilized to construct Bayesian inference trees. A Markov chain was executed for a maximum of 1 million generations, with sampling occurring every 50 generations. The first 25% of Markov chain Monte Carlo (mcmc) samples were discarded as burn-in. In addition, Maximum Likelihood trees were constructed using MEGA v7.0(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e) software to verify all Bayesian inference trees. The Sequence Demarcation Tool v1.2(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) was used to conduct color-coded pairwise identity matrix analysis comparing the novel parvovirus to other members of \u003cem\u003eParvoviridae\u003c/em\u003e. The identity score for each pair of sequences is computed as 1-M/N, where M is the number of mismatched nucleotides and N is the total number of columns along the alignment where neither sequence has a gap character(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e).\u003c/p\u003e \u003ch2\u003ePrediction of potential genome recombination events\u003c/h2\u003e \u003cp\u003eThe Recombination Detection Program v4.39 (RDP4) software was used to analyze genomic alignments of both reference strains and the AWPV strain. This was done through various algorithms including RDP, GENECONV, Chimaera, MaxChi, BootScan, and SiScan, in order to detect possible recombination events(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e).\u003c/p\u003e \u003ch2\u003ePrediction of spatial structure\u003c/h2\u003e \u003cp\u003eThe three-dimensional structure of the viral structural protein identified in this study was predicted using ColabFold(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), and SWISS-MODEL(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e) was employed to compare and screen models that possess comparable spatial structures from the PDB database. To visualize the results, PyMOL v2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.pymol.org\u003c/span\u003e\u003cspan address=\"http://www.pymol.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was utilized.\u003c/p\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe novel parvovirus sequence obtained in this study have been deposited in GenBank database under accession number BK063423.\u003c/p\u003e "},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eOverview of the pharyngeal metagenomic library of the Arctic Wolf\u003c/h2\u003e \u003cp\u003eThis library was sequenced using the Illumina HiSeq 2500 platform, generating 28,946,890 raw reads. Following quality control, 28,945,811 clean reads were obtained, which were then assembled into 736 contigs greater than 1,500 bp in length. Upon searching these contigs against the nr database, 260 contigs exhibited the highest identity with viral proteins (Supplementary Table\u0026nbsp;1). Eleven viral families were identified, with the family \u003cem\u003eMyoviridae\u003c/em\u003e being the most prevalent, accounting for 28.85% of all viral contigs, followed by \u003cem\u003eSiphoviridae\u003c/em\u003e (18.08%), \u003cem\u003ePodoviridae\u003c/em\u003e (12.31%), \u003cem\u003eAckermannviridae\u003c/em\u003e (11.54%), \u003cem\u003eDemerecviridae\u003c/em\u003e (6.54%), \u003cem\u003eAutographiviridae\u003c/em\u003e (6.15%), \u003cem\u003eMicroviridae\u003c/em\u003e (4.23%), and \u003cem\u003eHerelleviridae\u003c/em\u003e (3.08%) (Supplementary Table\u0026nbsp;1). However, the vast majority of them are already known. Moreover, 20 contigs were identified, which could not be confidently assigned to any viral taxonomic group (E-value\u0026thinsp;\u0026gt;\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e). This finding suggests the potential existence of novel viruses within these contigs or the prevalence of non-coding regions in the assembled sequences. In order to determine whether the host of this library is the Arctic wolf, we performed a BLASTx search by comparing the contigs assembled from the NGS data of this library (specifically those containing potential mammalian sequence reads) against the complete mitochondrial proteome database downloaded from GenBank. The search results revealed that the majority of sequences among the mammalian matches corresponded to the Arctic wolf (Supplementary Table\u0026nbsp;2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of a novel parvovirus\u003c/h2\u003e \u003cp\u003eIn this research, a virus named AWPV, belonging to the family \u003cem\u003eParvoviridae\u003c/em\u003e and possessing a complete genome organization structure, was obtained using the sequence assembly tool of Geneious Prime. AWPV has a genome size of 4,920 bp, characterized by a GC content of 40.2% and a nucleotide composition consisting of 36.4% A, 23.4% T, 18.2% G, and 22.0% C. The typical organizational pattern of this viral genome is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. Specifically, AWPV possesses a partial 5' non-translated region (68 bp), a complete NS1 open reading frame (620 aa), a complete virus protein (VP) 1 open reading frame (739 aa), a complete virus protein (VP) 2 open reading frame (581 aa), and a partial 3' non-translated region (214 bp). The NS1 protein contains several conserved motifs that are related to ATP- or GTP-binding, including a Walker A loop \u003csup\u003e408\u003c/sup\u003eGPASTGKS\u003csup\u003e415\u003c/sup\u003e [GXXXXGK(T/S)], a Walker B loop at \u003csup\u003e448\u003c/sup\u003eIIWVEE\u003csup\u003e453\u003c/sup\u003e (xxxxEE), and a Walker B' loop at \u003csup\u003e465\u003c/sup\u003eKAICSGQSIRIDQK\u003csup\u003e478\u003c/sup\u003e (KxxxxGxxxxxxxK). In addition, we identified a conserved replication initiator motif \u003csup\u003e132\u003c/sup\u003eKLHIHVLLHH\u003csup\u003e141\u003c/sup\u003e (xxHxHxxxxx) in the NS1 protein. By aligning with other protoparvovirus sequences, potential splicing signals for expressing VP1 were identified. In the N-terminal of VP1, the phospholipase A\u003csub\u003e2\u003c/sub\u003e (PLA2) motif was identified, which contains the expected calcium-binding (YLGPG) site and catalytic residues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analysis\u003c/h2\u003e \u003cp\u003eRepresentative sequences with high identity to the NS1 and VP1 proteins of AWPV, as well as other parvoviruses, were included in subsequent analysis. Based on the phylogenetic analysis of NS1 and VP1 protein sequences, AWPV was positioned between the parvoviruses identified in California sea lions(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e) and foxes(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e), forming a distinct branch (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Based on the distance matrix analysis of NS1 and VP1 proteins, it was determined that AWPV shares less than 77% identity with the protein sequences of other Parvovirus members. This suggests that AWPV should be classified as a new species of the genus \u003cem\u003eProtoparvovirus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Supplementary Table\u0026nbsp;3). In addition, the RDP4 software did not indicate any significant evidence of recombination signals.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePredicting the spatial structure of the AWPV's VP\u003c/h2\u003e \u003cp\u003eThe structure and biophysical properties of Protoparvovirus capsids are essential for its survival in the natural environment and entry into host cells(\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). To predict and contrast the similarity of the spatial structure of AWPV's VP with the structure dictated by present known sequences, the sequence exhibiting the greatest degree of similarity to AWPV's VP, which is the Newlavirus strain ITA/2016/51.20\u0026ndash;153 (UZZ82241), was retrieved from the GenBank database. The spatial structure of sequences encoding VP was predicted using ColabFold. Furthermore, SWISS-MODEL was utilized to search and obtain the virus model (6x2k.1) of Tusavirus(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e), which exhibited the highest similarity to AWPV. The PyMOL software was used to import all PDB files and conduct pairwise comparisons. The Root Mean Square Deviation (RMSD) is an indicator of the overall similarity between two spatial structures. Typically, an RMSD of less than 2 \u0026Aring; suggests a significant degree of similarity. The AWPV identified in this study did not show significant similarity in the VP spatial structure with the Newlavirus strain ITA/2016/51.20\u0026ndash;153 and Tusavirus (RMSD\u0026thinsp;=\u0026thinsp;2.888 and 4.458), indicating that AWPV and other related viruses may have different characteristics in mediating cell attachment during infection (Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eMetagenomics has revolutionized our understanding of viral diversity, greatly expanding the number of identified viral families, genera, and species beyond traditional methods. This is measured through sequence diversity and evolutionary distances(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Additionally, metagenomics enables the detection of novel viruses and the characterization of viromes in diverse samples, which enhances our knowledge of virus-host interactions and their potential public health implications, particularly for emerging and re-emerging infectious diseases.\u003c/p\u003e \u003cp\u003eThe Arctic wolf, also known as the white wolf or polar wolf, is a subspecies of the gray wolf in the family Canidae and is distributed in the northern regions of Eurasia, northern Canada, and northern Greenland. In recent years, there have been reports about Arctic wolves being infected with protoparvovirus, but most of them are related to CPV-2. For example, Justin M. Stilwell and others identified CPV-2 in a female Arctic wolf that died at the age of six weeks due to systemic canine distemper virus (CDV) infection(\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). In this study, we recovered a novel virus belonging to the genus \u003cem\u003eProtoparvovirus\u003c/em\u003e from the Arctic wolf pharyngeal metagenomic library. The virus, tentatively named AWPV, has a genome length of 4,920 bp, and its best match in the GenBank database shows a nucleotide sequence identity of 75.76% and coverage of 70%. The Bayesian phylogenetic trees constructed based on NS1 and VP1 amino acid sequences exhibited overall similar topology, with AWPV forming a unique branch located between the recently identified parvoviruses from California sea lions (California sea lion parvovirus, MN982959) and foxes (Newlaviruses, ON959793-96), and AWPV having a relatively distant relationship with CPVs. The California sea lion parvovirus was derived from a 2\u0026ndash;3-year-old subadult emaciated female California sea lion that was found stranded and deceased in 2010(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). The Newlaviruses were detected and identified in the carcasses of Italian foxes(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Due to the possibility of this Arctic wolf being captive, we cautiously speculate that AWPV may also have originated from interactions with other captive animals. Further research was deemed necessary to investigate the epidemiology and transmission dynamics of AWPV in both captive and wild Arctic wolf populations, in order to gain a comprehensive understanding of this virus and its potential impact on the health of these animals. The vast temporal and geographic range of both wild and captive hosts raises questions as to whether other, as-yet-undiscovered intermediate hosts may be carrying these viruses. While the role of these newly identified viruses in host morbidity and mortality remains uncertain, it is clear that members of the family \u003cem\u003eParvoviridae\u003c/em\u003e have achieved a worldwide distribution.\u003c/p\u003e \u003cp\u003eIn summary, the identification of AWPV increases our understanding of the diversity of canine parvoviruses and provides limited assistance in improving virus taxonomy. While there is no direct evidence to suggest whether AWPV will have significant negative effects on its host, further dynamic monitoring of the virus is needed in the future.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent for publication was obtained from all participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe novel parvovirus sequence obtained in this study have been deposited in GenBank database under accession number BK063423.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe\u0026nbsp;authors\u0026nbsp;declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by The Special Funds for Science Development of the Clinical Teaching Hospitals of Jiangsu Vocational College of Medicine no. 20229152 to ZD.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZD and MS collected data and wrote this article, HC, RZ and ZD assisted in data processing and proofreading. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eCotmore SF, Agbandje-McKenna M, Canuti M, Chiorini JA, Eis-Hubinger AM, Hughes J et al (2019) ICTV Virus Taxonomy Profile: Parvoviridae. J Gen Virol 100(3):367\u0026ndash;368 Epub 2019/01/24. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1099/jgv.0.001212\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003ePenzes JJ, Soderlund-Venermo M, Canuti M, Eis-Hubinger AM, Hughes J, Cotmore SF et al (2020) Reorganizing the family Parvoviridae: a revised taxonomy independent of the canonical approach based on host association. Arch Virol 165(9):2133\u0026ndash;2146 Epub 2020/06/14. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00705-020-04632-4\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003ePhan TG, Vo NP, Bonkoungou IJ, Kapoor A, Barro N, O\u0026apos;Ryan M et al (2012) Acute diarrhea in West African children: diverse enteric viruses and a novel parvovirus genus. J Virol 86(20):11024\u0026ndash;11030 Epub 2012/08/03. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/JVI.01427-12\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eJanus LM, Bleich A (2012) Coping with parvovirus infections in mice: health surveillance and control. Lab Anim 46(1):14\u0026ndash;23 Epub 2011/11/01. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1258/la.2011.011025\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eDecaro N, Buonavoglia C (2012) Canine parvovirus\u0026ndash;a review of epidemiological and diagnostic aspects, with emphasis on type 2c. Vet Microbiol 155(1):1\u0026ndash;12 Epub 2011/10/04. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.vetmic.2011.09.007\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eAdlhoch C, Kaiser M, Loewa A, Ulrich M, Forbrig C, Adjogoua EV et al (2012) Diversity of parvovirus 4-like viruses in humans, chimpanzees, and monkeys in hunter-prey relationships. Emerg Infect Dis 18(5):859\u0026ndash;862 Epub 2012/04/21. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3201/eid1805.111849\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMeng G, Zhang X, Plevka P, Yu Q, Tijssen P, Rossmann MG (2013) The structure and host entry of an invertebrate parvovirus. J Virol 87(23):12523\u0026ndash;12530 Epub 2013/09/13. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/JVI.01822-13\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLiu J, Yang X, Hao X, Feng Y, Zhang Y, Cheng Z (2020) Effect of Goose Parvovirus and Duck Circovirus Coinfection in Ducks. J Vet Res 64(3):355\u0026ndash;361 Epub 2020/09/29. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2478/jvetres-2020-0048\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eWang Y, Yang S, Liu D, Zhou C, Li W, Lin Y et al (2019) The fecal virome of red-crowned cranes. Arch Virol 164(1):3\u0026ndash;16 Epub 2018/09/19. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00705-018-4037-x\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBergmann V, Kiupel H (1982) [Inclusion body enteritis in pigeons caused by adenovirus and parvovirus]. Arch Exp Veterinarmed 36(3):445\u0026ndash;453 Epub 1982/01/01. PubMed PMID: 6291478\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBrown KE (2010) The expanding range of parvoviruses which infect humans. Rev Med Virol 20(4):231\u0026ndash;244 Epub 2010/06/30. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/rmv.648\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003ede Oliveira Santana W, Silveira VP, Wolf JM, Kipper D, Echeverrigaray S, Canal CW et al (2022) Molecular phylogenetic assessment of the canine parvovirus 2 worldwide and analysis of the genetic diversity and temporal spreading in Brazil. Infect Genet Evol 98:105225 Epub 2022/02/02. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.meegid.2022.105225\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eCanuti M, Bouchard E, Rodrigues B, Whitney HG, Hopson M, Gilroy C et al (2021) Newlavirus, a Novel, Highly Prevalent, and Highly Diverse Protoparvovirus of Foxes (Vulpes spp). Viruses 13(10). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/v13101969\u003c/span\u003e\u003c/span\u003ePubMed PMID: 34696399; PubMed Central PMCID: PMCPMC8537079 Epub 2021/10/27\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eDecaro N, Buonavoglia C, Barrs VR (2020) Canine parvovirus vaccination and immunisation failures: Are we far from disease eradication? Vet Microbiol 247:108760 Epub 2020/08/10. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.vetmic.2020.108760\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eKurucay HN, Tamer C, Muftuoglu B, Elhag AE, Gozel S, Cicek-Yildiz Y et al (2023) First isolation and molecular characterization of canine parvovirus-type 2b (CPV-2b) from red foxes (Vulpes vulpes) living in the wild habitat of Turkey. Virol J 20(1):27 Epub 2023/02/12. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12985-023-01988-2\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eTuteja D, Banu K, Mondal B (2022) Canine parvovirology - A brief updated review on structural biology, occurrence, pathogenesis, clinical diagnosis, treatment and prevention. Comp Immunol Microbiol Infect Dis 82:101765 Epub 2022/02/20. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cimid.2022.101765\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eZhang P, Su H, Peng R, Chan JF, Bai S, Wang G et al (2022) Identification of a Novel Astrovirus in Pinnipeds. Front Microbiol 13:845601 Epub 2022/05/24. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fmicb.2022.845601\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLangmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9(4):357\u0026ndash;359 Epub 2012/03/06. doi: 10.1038/nmeth.1923. PubMed PMID: 22388286; PubMed Central PMCID: PMCPMC3322381\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLangmead B, Wilks C, Antonescu V, Charles R (2019) Scaling read aligners to hundreds of threads on general-purpose processors. Bioinformatics 35(3):421\u0026ndash;432 Epub 2018/07/19. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/bioinformatics/bty648\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eSchmieder R, Edwards R (2011) Quality control and preprocessing of metagenomic datasets. Bioinformatics 27(6):863\u0026ndash;864 Epub 2011/02/01. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/bioinformatics/btr026\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLi D, Luo R, Liu CM, Leung CM, Ting HF, Sadakane K et al (2016) MEGAHIT v1.0: A fast and scalable metagenome assembler driven by advanced methodologies and community practices. Methods 102:3\u0026ndash;11 Epub 2016/03/26. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ymeth.2016.02.020\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eKearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S et al (2012) Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28(12):1647\u0026ndash;1649 Epub 2012/05/01. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/bioinformatics/bts199\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBuchfink B, Reuter K, Drost HG (2021) Sensitive protein alignments at tree-of-life scale using DIAMOND. Nat Methods 18(4):366\u0026ndash;368 Epub 2021/04/09. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41592-021-01101-x\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eGautam A, Felderhoff H, Bagci C, Huson DH (2022) Using AnnoTree to Get More Assignments, Faster, in DIAMOND + MEGAN Microbiome Analysis. \u003cem\u003emSystems\u003c/em\u003e 7(1):e0140821. Epub 2022/02/23. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/msystems.01408-21\u003c/span\u003e\u003c/span\u003e. PubMed PMID: 35191776; PubMed Central PMCID: PMCPMC8862659\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eKumar S, Stecher G, Tamura K (2016) MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol 33(7):1870\u0026ndash;1874 Epub 2016/03/24. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/molbev/msw054\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eKuraku S, Zmasek CM, Nishimura O, Katoh K (2013) aLeaves facilitates on-demand exploration of metazoan gene family trees on MAFFT sequence alignment server with enhanced interactivity. \u003cem\u003eNucleic Acids Res\u003c/em\u003e 41(Web Server issue):W22-8. Epub 2013/05/17. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/nar/gkt389\u003c/span\u003e\u003c/span\u003e. PubMed PMID: 23677614; PubMed Central PMCID: PMCPMC3692103\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eRonquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Hohna S et al (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61(3):539\u0026ndash;542 Epub 2012/02/24. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/sysbio/sys029\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMuhire BM, Varsani A, Martin DP (2014) SDT: a virus classification tool based on pairwise sequence alignment and identity calculation. PLoS ONE 9(9):e108277 Epub 2014/09/27. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pone.0108277\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMartin DP, Murrell B, Golden M, Khoosal A, Muhire B (2015) RDP4: Detection and analysis of recombination patterns in virus genomes. Virus Evol 1(1):vev003 Epub 2015/05/26. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/ve/vev003\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMirdita M, Schutze K, Moriwaki Y, Heo L, Ovchinnikov S, Steinegger M (2022) ColabFold: making protein folding accessible to all. Nat Methods 19(6):679\u0026ndash;682 Epub 2022/06/01. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41592-022-01488-1\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eAltan E, Delaney MA, Colegrove KM, Spraker TR, Wheeler EA, Deng X et al (2020) Complex Virome in a Mesenteric Lymph Node from a Californian Sea Lion (Zalophus Californianus) with Polyserositis and Steatitis. \u003cem\u003eViruses\u003c/em\u003e 12(8). Epub 2020/07/29. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/v12080793\u003c/span\u003e\u003c/span\u003e. PubMed PMID: 32718049; PubMed Central PMCID: PMCPMC7472147\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLanave G, Ndiana LA, Pellegrini F, Diakoudi G, Di Martino B, Sgroi G et al (2022) Detection at high prevalence of newlavirus (protoparvovirus) in the carcasses of red foxes. Virus Res 323:198971 Epub 2022/10/19. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.virusres.2022.198971\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eRos C, Bayat N, Wolfisberg R, Almendral JM (2017) Protoparvovirus Cell Entry. \u003cem\u003eViruses\u003c/em\u003e 9(11). Epub 2017/10/27. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/v9110313\u003c/span\u003e\u003c/span\u003e. PubMed PMID: 29072600; PubMed Central PMCID: PMCPMC5707520\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMietzsch M, McKenna R, Vaisanen E, Yu JC, Ilyas M, Hull JA et al (2020) Structural Characterization of Cuta- and Tusavirus: Insight into Protoparvoviruses Capsid Morphology. \u003cem\u003eViruses\u003c/em\u003e 12(6). Epub 2020/06/21. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/v12060653\u003c/span\u003e\u003c/span\u003e. PubMed PMID: 32560452; PubMed Central PMCID: PMCPMC7354515\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eGreninger AL (2018) A decade of RNA virus metagenomics is (not) enough. \u003cem\u003eVirus Res\u003c/em\u003e 244:218 \u0026ndash; 29. Epub 2017/10/23. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.virusres.2017.10.014\u003c/span\u003e\u003c/span\u003e. PubMed PMID: 29055712; PubMed Central PMCID: PMCPMC7114529\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eStilwell JM, Anis E, Wilkes RP, Rissi DR (2019) Dual infection with an emergent strain of canine distemper virus and canine parvovirus in an Arctic wolf under managed care. J Vet Diagn Invest 31(4):594\u0026ndash;597 PubMed PMID: 31113289; PubMed Central PMCID: PMCPMC6857037\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Parvovirus, Virome, Metagenomics, Arctic wolf, Novel","lastPublishedDoi":"10.21203/rs.3.rs-3337233/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3337233/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThrough the utilization of a viral metagenomic approach, a novel virus has been found in a pharyngeal metagenomic library derived from an Arctic wolf (\u003cem\u003eCanis lupus\u003c/em\u003e). This virus has been temporarily designated as AWPV and assigned a GenBank accession number BK063423. The genome of AWPV is comprised of 4,920 base pairs, and its nucleotide composition is composed of 36.4% A, 23.4% T, 18.2% G, and 22.0% C, with a GC content of 40.2%. The viral genome demonstrates a typical pattern of parvovirus organization, with two predicted ORFs: ORF1, which encodes non-structural proteins NS1 and NS2, and ORF2, which encodes VP1 and VP2. By performing a pairwise sequence comparison and a phylogenetic analysis based on the NS1 and VP1 protein sequences, it has been suggested that AWPV may represent a novel species within the genus \u003cem\u003eProtoparvovirus\u003c/em\u003e. This discovery of a novel parvovirus has enhanced our comprehension of the mammalian virus ecology and has facilitated an improved understanding of potential future infectious diseases.\u003c/p\u003e","manuscriptTitle":"Identification of a novel parvovirus in the Arctic wolf (Canis lupus)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-09-13 14:04:34","doi":"10.21203/rs.3.rs-3337233/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1c4f3366-4880-4c3f-934b-cbcf7ea37ecc","owner":[],"postedDate":"September 13th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-09-17T10:14:23+00:00","versionOfRecord":[],"versionCreatedAt":"2023-09-13 14:04:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3337233","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3337233","identity":"rs-3337233","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}