Identification of novel Chaphamaparvovirus in breeding Muscovy ducks

Identification of novel Chaphamaparvovirus in breeding Muscovy ducks | 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 Short Report Identification of novel Chaphamaparvovirus in breeding Muscovy ducks Yadong Gao, Jiawen Dong, Zhuanqiang Yan, Meiting Chen, Yingshan Yin, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7451845/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 Since 2022, outbreaks of hepatitis-like disease have been frequently reported in Muscovy duck farms across South China, leading to a significant decrease of egg production and hatchability. With the help of metagenomic sequencing, a novel Chaphamaparvovirus (ChPV) was identified from livers of the diseased ducks. Phylogenetic analysis revealed that the isolated strain shares 61.8-77.4% genome identities with duck-origin Chaphamaparvoviruses, and 44.3-77.4% with avian-origin Chaphamaparvoviruses. In addition, specific probes and primers were designed for quantitative PCR detection. The methods’ specificity, sensitivity, and repeatability were validated, suggesting the suitability for field detection of MuChPV. Chaphamaparvovirus breeding Muscovy duck metagenomic sequencing Real-time fluorescent quantitative PCR Figures Figure 1 Figure 2 Introduction, methods, and results Parvovirus are non-enveloped viruses with linear, single-stranded DNA genomes of approximately 4-6 kb in length[1]. The family comprises three subfamilies: Parvovirinae , Densovirinae , and Hamaparvovirinae . Since 2022, infectious disease outbreaks have occurred frequently among breeding Muscovy ducks on several commercial farms in southern China. The diseased ducks exhibited a 10-15% reduction in egg production, a 10-30% decrease of hatching rate, and green feces. Necropsy revealed hepatitis symptoms, including hepatic swelling, hemorrhage and yellow color (Fig. 1A). Histopathology analysis confirmed mild liver damage characterized by hepatocyte granular degeneration, steatosis, and infiltration of inflammatory cells. In addition, significant inflammatory cell infiltration in the portal area and around the central veins, along with degeneration of renal tubular epithelia cells, was also observed in kidneys (Fig. 1B). Ducks are significant reservoirs for diverse viruses[2]. However, no pathogenic bacteria or viruses, including MDPV, MDRV, DAstV, GAstV, DTMUV, DEV, NDPV, DCV, AIV, DAdV, and NDV, were detected. To identify the pathogen, metagenomic sequencing was applied via the Illumina Novaseq 6000 platform. Clean data was generated via SOAPnuke (v1.5.6) and aligned with the ribosome database (Silva 132), the virus reference database (Virus-NT), and host database via BWA (v0.7.17). Of 33,205,372 clean reads, 9,677 corresponded to virus. Megahit (v1.1.2) assembled these clean reads into 890 contigs, which were compared with virus database via BLAST. Six contigs were identified as virus: two matched Chestnut teal chaphamaparvovirus , and one shared 77.4% nucleotide sequence identity with Pacific black duck chaphamaparvovirus 1 (GenBank accession number: MT247730). This contig was designated as MuChPV-GD2022 (GenBank accession number: OR854229). in situ hybridization (ISH) using a digoxigenin-labelled probe targeting the NS1 gene (Table 1) detected positive signals in livers of the diseased ducks (Fig. 1C). Transmission electron microscopy (TEM) revealed non-enveloped, spherical virions with a diameter of about 25 nm (Fig. 1D). The complete MuChPV-GD2022 genome (4407 bp, G+C content: 39.1%) comprises two open reading frames (ORFs). ORF1 (nt 86-739, 224 aa) locates at 5’ end. ORF2 encodes a nonstructural protein NS1 (nt 640–2703, 687 aa) and two structural proteins: VP1 (nt 2696–4282, 528 aa) and NP (nt 1844-2545, 234 aa). Alignment using BioEdit (v7.2.5) and MEGA 7.0 indicated that the NS1 and VP1 protein share < 71.5% and <69.4% amino acid identity, respectively, with other Parvoviridae members. The NP protein shares 76.0% identity with that of Chestnut teal chaphamaparvovirus (GenBank accession number: QMI57906). Notable, the VP1 is overlapped with the NS1 by 8 nucleotides (Fig. 1E). Phylogenetic analysis based on NS1 sequences suggests that the identified MuChPV-GD2022 strain belongs to waterfowl/duck-associated Chaphamaparvoviruses , which is a new genus in the subfamily Hamaparvovirinae (Fig. 1F). Given that NS1 exhibits < 85% amino acid identity to known Chaphamaparvoviruses, this novel strain likely represents a novel species according to ICTV criteria for Chaphamaparvovirus. To enable early intervention, we developed a rapid, sensitive diagnostic assay targeting the NS1 gene of MuChPV. A 335 bp fragment was amplified using MuChPv-F1/R1 primers (Fig. 2A). The PCR product was purified, cloned into the pMD™18-T vector (Takara Biomedical Technology, China), and transformed into E. coli DH5α competent cells (Tsingke Biotechnology Co., Ltd., China). The amplification conditions were as follows: 95°C for 5 minutes, 45 cycles at 95°C for 20 seconds, annealing for 20 seconds, then extension at 72°C for 20 seconds. A standard curve was generated using 10-fold serial dilutions (2.11×10 9 to 2.11×10 0 copies/mL) of the recombinant plasmid. After optimization (probe: 0.2 mmol/L; primers: 0.3 mmol/L; annealing: 51 °C), the standard curve equation was y=-3.4937x+40.4, with a correlation coefficient (R2) of 0.9986 and an amplification efficiency (E) of 93% (Fig. 2B), confirming a robust linear relationship between DNA concentration and the Ct value. The qPCR assay showed no cross-reactivity with MDPV, TMUV, DuCV, MDRV, NDRV, or DAstV (Fig. 2C), demonstrating specificity for MuChPV. Its limit of detection (LOD) was 2.11×10 1 copies/mL (Fig. 2D), compared to 2.11×10 3 copies/mL for conventional PCR assay (using primers MuChPV-F2/R2, Fig. 2E), suggesting a 100-fold greater sensitivity. Furthermore, across plasmid concentrations from 2.11×10 5 to 2.11×10 7 , intra-assay and inter-assay variability yielded a coefficient of variation (CV) of 0.32-0.44% and 0.25-0.12%, respectively, confirming high reproducibility (CV < 0.5%). The CV was calculated using the following formula: CV = (Standard Deviation (SD) / Mean) × 100%. Since there is no golden standard for detection of the virus, the qPCR method was applied to 90 field samples. 45 out of 90 samples were tested positive for the virus with a positivity rate of 50.00% using the established qPCR assay, while 34 samples (37.78%) were detected as positive using conventional PCR method. All conventional PCR-positive samples were qPCR-positive, while 11 qPCR-positive samples were undetected by conventional PCR method, underscoring the superior sensitivity of qPCR. Discussion While Parvovirinae and Densovirina subfamilies infect vertebrates and invertebrates respectively, the recently identified Hamaparvovirinae subfamily infects both[3] . First identified in birds in 2019[4], Chaphamaparvoviru s, a genus within Hamaparvovirinae , infects diverse vertebrate hosts including mammals, reptiles, avian species, and fish[5-8]. Avian infections are usually associated with reduced egg production and poor growth performance[5, 9]. However, pathogenicity and genetic diversity of parvoviruses in domestic ducks remain poorly characterized. This study identifies a novel Chaphamaparvoviru s strain, MuChPV-GD2022, presenting a distinct specie s . The sequence homology of the NS1, VP1 and NP proteins with other Chaphamaparvoviruses suggest broad avian host tropism and potential for cross-species transmission[10]. To facilitate epidemiological studies in Muscovy ducks, we established a highly sensitive, specific, and reproducible TaqMan qPCR assay targeting the NS1 gene. This assay could provide a reliable tool for early diagnosis and surveillance of MuChPV infections. Declarations Fundings This study was supported by Guangdong Province Key Laboratory of Livestock Disease Prevention (2023B1212060040), National Key R&D Program of China (2022YFD1801000), Special Fund for Scientific Innovation Strategy-Construction of High Level Academy of Agriculture Science (202110TD, R2020PY-JX014, R2020QD-049, R2020PY-JC001). Competing interests: The authors declare that they have no competing interests. Consent to Publish declaration ： The work described in this manuscript is entirely original and has not been published previously. All the listed authors have reviewed and approved this manuscript. Ethics and Consent to Participate declarations: The authors confirm that the ethical policies of the journal, as noted on the journal’s author guidelines page, have been adhered to. All animal experiments in this study were approved by the Committee on the Ethics of Animal Experiments of Institute of Animal Health, Guangdong Academy of Agricultural Sciences Experimental Animal Welfare Ethics Committee (Approve ID: 2018-007). Author Contribution LL,YZ, and MS contributed to the design of the study. YG, JD performed the virological study and comparative analysis. ZY contributed to preparation of the samples. MC, YY, and JZ performed the ISH. YX and ZQ performed TEM. YH, JG and LQ performed the sequencing of the MuChPV isolate. YG prepared figures 1-2. YZ and LL wrote and revised the main manuscript text. All authors reviewed and approved the final manuscript. References López-Astacio RA, A.O., Lee H, Hafenstein SL, Parrish CR, The Structures and Functions of Parvovirus Capsids and Missing Pieces: the Viral DNA and Its Packaging, Asymmetrical Features, Nonprotein Components, and Receptor or Antibody Binding and Interactions. J Virol, 2023. 97 (7): p. e0016123. Dong, H.V., et al., Epidemiological Analysis and Genetic Characterization of Parvovirus in Ducks in Northern Vietnam Reveal Evidence of Recombination. Animals (Basel), 2022. 12 (20). Pénzes JJ, S.-V.M., Canuti M, Eis-Hübinger AM, Hughes J, Cotmore SF, Harrach B, Reorganizing the family Parvoviridae: a revised taxonomy independent of the canonical approach based on host association. Arch Virol, 2020. 165 (9): p. 2133-2146. Wang, Y., et al., The fecal virome of red-crowned cranes. Arch Virol, 2019. 164 (1): p. 3-16. Canuti M, V.J., Munro HJ, Roul S, Ojkic D, Robertson GJ, Whitney HG, Dufour SC, Lang AS, Investigating the Diversity and Host Range of Novel Parvoviruses from North American Ducks Using Epidemiology, Phylogenetics, Genome Structure, and Codon Usage Analysis. Viruses, 2021. 13 (2): p. 193. Di Profio, F., et al., Exploring the Enteric Virome of Cats with Acute Gastroenteritis. Vet Sci, 2023. 10 (5). Matos, M., et al., A novel Chaphamaparvovirus is the etiological agent of hepatitis outbreaks in pheasants (Phasianus colchicus) characterized by high mortality. Transbound Emerg Dis, 2022. 69 (5): p. e2093-e2104. Cui, H., et al., Molecular characteristics of novel chaphamaparvovirus identified in chickens. Poult Sci, 2023. 102 (3): p. 102449. Vibin, J., et al., Metagenomic characterisation of avian parvoviruses and picornaviruses from Australian wild ducks. Sci Rep, 2020. 10 (1): p. 12800. Wille, M. and E.C. Holmes, Wild birds as reservoirs for diverse and abundant gamma- and deltacoronaviruses. FEMS Microbiol Rev, 2020. 44 (5): p. 631-644. Table Table 1. Primer list Primer and probe Sequence Length（bp） MD chap-1F GTGGATGTGGATGTGAGGGTA 1236 MD chap-1R TCCTGGGTCATTGGATTGC MD chap-2F CAGTTAGTGTGGATGAGGGCA 1281 MD chap-2R CCAATCGCTGTTGTTCGG MD chap-3F ACGAGACGCTTGATTCTTTGG 1025 MD chap-3R GCTTCATATGGTCCTGTTGGTG MD chap-4F ACAGCAGATGCAGATAAATGGG 982 MD chap-4R CGGCAGAGTACGGTTCAATAGT qMD chap -F TTCCTACCGGATCTGGATCAAC 137 qMD chap -R GGTCCTACTGCTGGCCATAC MD chap-ISH AGAGGATCAAGCGTCTCGTAACACCA MuChPv-F1 5′-TGGGACAAACATGGTCTCTT-3′ 335 MuChPv-R1 5′-CATGGTTCTCCTCCTGCTAA-3′ MuChPv-F2 5′-CTGACCTCTTTGACTCTAC-3′ 154 MuChPv-R2 5′-GCTCCATTACATACCTCTC-3′ MuChPv-P FAM-5′-TGCTGGACATAATGCTCTGAACC-3′-BHQ-1 Additional Declarations No competing interests reported. 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14:21:20","extension":"html","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":50783,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7451845/v1/6bad2f7a9a475b427219221a.html"},{"id":92269174,"identity":"2f02e3dc-c1f6-4650-aeee-e5217e64b291","added_by":"auto","created_at":"2025-09-26 14:13:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2427123,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of duck MuChPV.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 1A. \u003c/strong\u003ePathological lesions of clinical samples with hepatitisobserved in the diseased ducks. \u003cstrong\u003eFig. 1B. \u003c/strong\u003eHistopathological lesions of liver and kidney of the diseased ducks. Black arrows indicate eosinophilic granular bodies, yellow arrows indicate inflammatory cell infiltration, and blue arrows indicate Cytoplasmic porosity and vacuolar degeneration of the cells. \u003cstrong\u003eFig. 1C\u003c/strong\u003e. Demonstration of MuChPV DNA in liver histologic section of the diseased duck by ISH. Bar = 25 µm. \u003cstrong\u003eFig. 1D.\u003c/strong\u003eTEM images of MuChPV virion. Bar = 200 nm. \u003cstrong\u003eFig. 1E. \u003c/strong\u003eIllustration of genomic organization of the complete genome sequences of MuChPV- GD2022strain. \u003cstrong\u003eFig. 1F\u003c/strong\u003e. Phylogenetic analysis of the complete genome sequences of the MuChPV-GD2022 strain using the neighbor-joining method, with 1,000 bootstrap replications. The black triangle represents the MuChPV-GD2022strain.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7451845/v1/615ed209cafa0b9b2af077d2.png"},{"id":92269178,"identity":"0f7e9851-ec35-484b-b511-73c79fb15f46","added_by":"auto","created_at":"2025-09-26 14:13:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5940533,"visible":true,"origin":"","legend":"\u003cp\u003eThe establishment of a quantitative PCR method for duck MuChPV detection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 2A.\u003c/strong\u003e PCR amplification results of MuChPV NS1 gene. The band in strip 1 indicates the size of the PCR product of the NS1 gene is 335 bp; Strip M shows the DNA marker. \u003cstrong\u003eFig. 2B.\u003c/strong\u003e The standard curve of the qPCR method. \u003cstrong\u003eFig. 2C. \u003c/strong\u003eThe specific test of the established TaqMan qPCR method. The red line indicates the amplification curve of duck MuChPV, while lines of other colors at the bottom indicate the amplification curves of other viruses. \u003cstrong\u003eFig. 2D. \u003c/strong\u003eThe sensitivity test of the established TaqMan qPCR method. The numbered lines with different colors are amplification curves of a series of plasmid dilutions ranging from 2.11×10\u003csup\u003e9\u003c/sup\u003e to 2.11×10\u003csup\u003e0\u003c/sup\u003e copies/mL. \u003cstrong\u003eFig. 2E. \u003c/strong\u003eThe sensitivity test of the traditional PCR method. The numbered stripes show the PCR products of a series of plasmid dilutions ranging from 2.11×10\u003csup\u003e9\u003c/sup\u003e to 2.11×10\u003csup\u003e0\u003c/sup\u003e copies/mL. Stripe M shows the DNA marker.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7451845/v1/f1ba32dbad7158b9c994b2d1.png"},{"id":98775580,"identity":"67f3a0cb-e612-4e44-8e8b-80fa35fd5c6f","added_by":"auto","created_at":"2025-12-22 12:20:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8222519,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7451845/v1/fbaa9a33-77a0-4da0-b3cf-a32b01def980.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Identification of novel Chaphamaparvovirus in breeding Muscovy ducks","fulltext":[{"header":"Introduction, methods, and results","content":"\u003cp\u003eParvovirus are non-enveloped viruses with linear, single-stranded DNA genomes of approximately 4-6 kb in length[1]. The family comprises three subfamilies: \u003cem\u003eParvovirinae\u003c/em\u003e, \u003cem\u003eDensovirinae\u003c/em\u003e, and \u003cem\u003eHamaparvovirinae\u003c/em\u003e. Since 2022, infectious disease outbreaks have occurred frequently among breeding Muscovy ducks on several commercial farms in southern China. The diseased ducks exhibited a 10-15% reduction in egg production, a 10-30% decrease of hatching rate, and green feces. Necropsy revealed hepatitis symptoms, including hepatic swelling, hemorrhage and yellow color (Fig. 1A). Histopathology analysis confirmed mild liver damage characterized by hepatocyte granular degeneration, steatosis, and infiltration of inflammatory cells. In addition, significant inflammatory cell infiltration in the portal area and around the central veins, along with degeneration of renal tubular epithelia cells, was also observed in kidneys (Fig. 1B). Ducks are significant reservoirs for diverse viruses[2]. However, no pathogenic bacteria or viruses, including MDPV, MDRV, DAstV, GAstV, DTMUV, DEV, NDPV, DCV, AIV, DAdV, and NDV, were detected.\u003c/p\u003e\n\u003cp\u003eTo identify the pathogen, metagenomic sequencing was applied via the Illumina Novaseq 6000 platform. Clean data was generated via SOAPnuke (v1.5.6) and aligned with the ribosome database (Silva 132), the virus reference database (Virus-NT), and host database via BWA (v0.7.17). Of 33,205,372 clean reads, 9,677 corresponded to virus. Megahit (v1.1.2) assembled these clean reads into 890 contigs, which were compared with virus database via BLAST. Six contigs were identified as virus: two matched \u003cem\u003eChestnut teal chaphamaparvovirus\u003c/em\u003e, and one shared 77.4% nucleotide sequence identity with \u003cem\u003ePacific black duck chaphamaparvovirus 1\u003c/em\u003e (GenBank accession number: MT247730). This contig was designated as MuChPV-GD2022 (GenBank accession number: OR854229). \u003cem\u003ein situ\u003c/em\u003e hybridization (ISH) using a digoxigenin-labelled probe targeting the NS1 gene (Table 1) detected positive signals in livers of the diseased ducks (Fig. 1C). Transmission electron microscopy (TEM) revealed non-enveloped, spherical virions with a diameter of about 25 nm (Fig. 1D).\u003c/p\u003e\n\u003cp\u003eThe complete MuChPV-GD2022 genome (4407 bp, G+C content: 39.1%) comprises two open reading frames (ORFs). ORF1 (nt 86-739, 224 aa) locates at 5’ end. ORF2 encodes a nonstructural protein NS1 (nt 640–2703, 687 aa) and two structural proteins: VP1 (nt 2696–4282, 528 aa) and NP (nt 1844-2545, 234 aa). Alignment using BioEdit (v7.2.5) and MEGA 7.0 indicated that the NS1 and VP1 protein share \u0026lt; 71.5% and \u0026lt;69.4% amino acid identity, respectively, with other \u003cem\u003eParvoviridae\u003c/em\u003e members. The NP protein shares 76.0% identity with that of \u003cem\u003eChestnut teal chaphamaparvovirus\u003c/em\u003e (GenBank accession number: QMI57906). Notable, the VP1 is overlapped with the NS1 by 8 nucleotides (Fig. 1E). Phylogenetic analysis based on NS1 sequences suggests that the identified MuChPV-GD2022 strain belongs to waterfowl/duck-associated \u003cem\u003eChaphamaparvoviruses\u003c/em\u003e, which is a new genus in the subfamily \u003cem\u003eHamaparvovirinae\u003c/em\u003e (Fig. 1F). Given that NS1 exhibits \u0026lt; 85% amino acid identity to known Chaphamaparvoviruses, this novel strain likely represents a novel species according to ICTV criteria for Chaphamaparvovirus.\u003c/p\u003e\n\u003cp\u003eTo enable early intervention, we developed a rapid, sensitive diagnostic assay targeting the NS1 gene of MuChPV. A 335 bp fragment was amplified using MuChPv-F1/R1 primers (Fig. 2A). The PCR product was purified, cloned into the pMD™18-T vector (Takara Biomedical Technology, China), and transformed into \u003cem\u003eE. coli\u003c/em\u003e DH5α competent cells (Tsingke Biotechnology Co., Ltd., China). The amplification conditions were as follows: 95°C for 5 minutes, 45 cycles at 95°C for 20 seconds, annealing for 20 seconds, then extension at 72°C for 20 seconds. A standard curve was generated using 10-fold serial dilutions (2.11×10\u003csup\u003e9\u003c/sup\u003e to 2.11×10\u003csup\u003e0\u003c/sup\u003e copies/mL) of the recombinant plasmid. After optimization (probe: 0.2 mmol/L; primers: 0.3 mmol/L; annealing: 51 °C), the standard curve equation was y=-3.4937x+40.4, with a correlation coefficient (R2) of 0.9986 and an amplification efficiency (E) of 93% (Fig. 2B), confirming a robust linear relationship between DNA concentration and the Ct value.\u003c/p\u003e\n\u003cp\u003eThe qPCR assay showed no cross-reactivity with MDPV, TMUV, DuCV, MDRV, NDRV, or DAstV (Fig. 2C), demonstrating specificity for MuChPV. Its limit of detection (LOD) was 2.11×10\u003csup\u003e1\u003c/sup\u003e copies/mL (Fig. 2D), compared to 2.11×10\u003csup\u003e3\u003c/sup\u003e copies/mL for conventional PCR assay (using primers MuChPV-F2/R2, Fig. 2E), suggesting a 100-fold greater sensitivity. Furthermore, across plasmid concentrations from 2.11×10\u003csup\u003e5\u003c/sup\u003e to 2.11×10\u003csup\u003e7\u003c/sup\u003e, intra-assay and inter-assay variability yielded a coefficient of variation (CV) of 0.32-0.44% and 0.25-0.12%, respectively, confirming high reproducibility (CV \u0026lt; 0.5%). The CV was calculated using the following formula: CV = (Standard Deviation (SD) / Mean) × 100%.\u003c/p\u003e\n\u003cp\u003eSince there is no golden standard for detection of the virus, the qPCR method was applied to 90 field samples. 45 out of 90 samples were tested positive for the virus with a positivity rate of 50.00% using the established qPCR assay, while 34 samples (37.78%) were detected as positive using conventional PCR method. All conventional PCR-positive samples were qPCR-positive, while 11 qPCR-positive samples were undetected by conventional PCR method, underscoring the superior sensitivity of qPCR.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWhile \u003cem\u003eParvovirinae\u003c/em\u003e and \u003cem\u003eDensovirina\u0026nbsp;\u003c/em\u003esubfamilies infect vertebrates and invertebrates respectively, the recently identified \u003cem\u003eHamaparvovirinae\u003c/em\u003e subfamily infects both[3] . First identified in birds in 2019[4], \u003cem\u003eChaphamaparvoviru\u003c/em\u003es, a genus within \u003cem\u003eHamaparvovirinae\u003c/em\u003e, infects diverse vertebrate hosts including mammals, reptiles, avian species, and fish[5-8]. Avian infections are usually associated with reduced egg production and poor growth performance[5, 9]. However, pathogenicity and genetic diversity of parvoviruses in domestic ducks remain poorly characterized.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study identifies a novel \u003cem\u003eChaphamaparvoviru\u003c/em\u003es strain, MuChPV-GD2022, presenting a distinct specie\u003cem\u003es\u003c/em\u003e. The sequence homology of the NS1, VP1 and NP proteins with other Chaphamaparvoviruses suggest broad avian host tropism and potential for cross-species transmission[10]. To facilitate epidemiological studies in Muscovy ducks, we established a highly sensitive, specific, and reproducible TaqMan qPCR assay targeting the NS1 gene. This assay could provide a reliable tool for early diagnosis and surveillance of MuChPV infections.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFundings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by Guangdong Province Key Laboratory of Livestock Disease Prevention (2023B1212060040), National Key R\u0026amp;D Program of China (2022YFD1801000), Special Fund for Scientific Innovation Strategy-Construction of High Level Academy of Agriculture Science (202110TD, R2020PY-JX014, R2020QD-049, R2020PY-JC001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish declaration\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003eThe work described in this manuscript is entirely original and has not been published previously. All the listed authors have reviewed and approved this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics and Consent to Participate declarations:\u003c/strong\u003e The authors confirm that the ethical policies of the journal, as noted on the journal’s author guidelines page, have been adhered to. All animal experiments in this study were approved by the Committee on the Ethics of Animal Experiments of Institute of Animal Health, Guangdong Academy of Agricultural Sciences Experimental Animal Welfare Ethics Committee (Approve ID: 2018-007).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eLL,YZ, and MS contributed to the design of the study. YG, JD performed the virological study and comparative analysis. ZY contributed to preparation of the samples. MC, YY, and JZ performed the ISH. YX and ZQ performed TEM. YH, JG and LQ performed the sequencing of the MuChPV isolate. YG prepared figures 1-2. YZ and LL wrote and revised the main manuscript text. All authors reviewed and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eL\u0026oacute;pez-Astacio RA, A.O., Lee H, Hafenstein SL, Parrish CR, \u003cem\u003eThe Structures and Functions of Parvovirus Capsids and Missing Pieces: the Viral DNA and Its Packaging, Asymmetrical Features, Nonprotein Components, and Receptor or Antibody Binding and Interactions.\u003c/em\u003e J Virol, 2023. \u003cstrong\u003e97\u003c/strong\u003e(7): p. e0016123.\u003c/li\u003e\n\u003cli\u003eDong, H.V., et al., \u003cem\u003eEpidemiological Analysis and Genetic Characterization of Parvovirus in Ducks in Northern Vietnam Reveal Evidence of Recombination.\u003c/em\u003e Animals (Basel), 2022. \u003cstrong\u003e12\u003c/strong\u003e(20).\u003c/li\u003e\n\u003cli\u003eP\u0026eacute;nzes JJ, S.-V.M., Canuti M, Eis-H\u0026uuml;binger AM, Hughes J, Cotmore SF, Harrach B, \u003cem\u003eReorganizing the family Parvoviridae: a revised taxonomy independent of the canonical approach based on host association.\u003c/em\u003e Arch Virol, 2020. \u003cstrong\u003e165\u003c/strong\u003e(9): p. 2133-2146.\u003c/li\u003e\n\u003cli\u003eWang, Y., et al., \u003cem\u003eThe fecal virome of red-crowned cranes.\u003c/em\u003e Arch Virol, 2019. \u003cstrong\u003e164\u003c/strong\u003e(1): p. 3-16.\u003c/li\u003e\n\u003cli\u003eCanuti M, V.J., Munro HJ, Roul S, Ojkic D, Robertson GJ, Whitney HG, Dufour SC, Lang AS, \u003cem\u003eInvestigating the Diversity and Host Range of Novel Parvoviruses from North American Ducks Using Epidemiology, Phylogenetics, Genome Structure, and Codon Usage Analysis.\u003c/em\u003e Viruses, 2021. \u003cstrong\u003e13\u003c/strong\u003e(2): p. 193.\u003c/li\u003e\n\u003cli\u003eDi Profio, F., et al., \u003cem\u003eExploring the Enteric Virome of Cats with Acute Gastroenteritis.\u003c/em\u003e Vet Sci, 2023. \u003cstrong\u003e10\u003c/strong\u003e(5).\u003c/li\u003e\n\u003cli\u003eMatos, M., et al., \u003cem\u003eA novel Chaphamaparvovirus is the etiological agent of hepatitis outbreaks in pheasants (Phasianus colchicus) characterized by high mortality.\u003c/em\u003e Transbound Emerg Dis, 2022. \u003cstrong\u003e69\u003c/strong\u003e(5): p. e2093-e2104.\u003c/li\u003e\n\u003cli\u003eCui, H., et al., \u003cem\u003eMolecular characteristics of novel chaphamaparvovirus identified in chickens.\u003c/em\u003e Poult Sci, 2023. \u003cstrong\u003e102\u003c/strong\u003e(3): p. 102449.\u003c/li\u003e\n\u003cli\u003eVibin, J., et al., \u003cem\u003eMetagenomic characterisation of avian parvoviruses and picornaviruses from Australian wild ducks.\u003c/em\u003e Sci Rep, 2020. \u003cstrong\u003e10\u003c/strong\u003e(1): p. 12800.\u003c/li\u003e\n\u003cli\u003eWille, M. and E.C. Holmes, \u003cem\u003eWild birds as reservoirs for diverse and abundant gamma- and deltacoronaviruses.\u003c/em\u003e FEMS Microbiol Rev, 2020. \u003cstrong\u003e44\u003c/strong\u003e(5): p. 631-644.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003ePrimer list\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003ePrimer and probe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 340px;\"\u003e\n \u003cp\u003eSequence\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003eLength（bp）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eMD chap-1F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 340px;\"\u003e\n \u003cp\u003eGTGGATGTGGATGTGAGGGTA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 99px;\"\u003e\n \u003cp\u003e1236\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eMD chap-1R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 340px;\"\u003e\n \u003cp\u003eTCCTGGGTCATTGGATTGC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eMD chap-2F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 340px;\"\u003e\n \u003cp\u003eCAGTTAGTGTGGATGAGGGCA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 99px;\"\u003e\n \u003cp\u003e1281\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eMD chap-2R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 340px;\"\u003e\n \u003cp\u003eCCAATCGCTGTTGTTCGG\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eMD chap-3F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 340px;\"\u003e\n \u003cp\u003eACGAGACGCTTGATTCTTTGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 99px;\"\u003e\n \u003cp\u003e1025\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eMD chap-3R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 340px;\"\u003e\n \u003cp\u003eGCTTCATATGGTCCTGTTGGTG\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eMD chap-4F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 340px;\"\u003e\n \u003cp\u003eACAGCAGATGCAGATAAATGGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 99px;\"\u003e\n \u003cp\u003e982\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eMD chap-4R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 340px;\"\u003e\n \u003cp\u003eCGGCAGAGTACGGTTCAATAGT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eqMD chap -F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 340px;\"\u003e\n \u003cp\u003eTTCCTACCGGATCTGGATCAAC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e137\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eqMD chap -R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 340px;\"\u003e\n \u003cp\u003eGGTCCTACTGCTGGCCATAC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eMD chap-ISH\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 340px;\"\u003e\n \u003cp\u003eAGAGGATCAAGCGTCTCGTAACACCA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eMuChPv-F1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 340px;\"\u003e\n \u003cp\u003e5\u0026prime;-TGGGACAAACATGGTCTCTT-3\u0026prime;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 99px;\"\u003e\n \u003cp\u003e335\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eMuChPv-R1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 340px;\"\u003e\n \u003cp\u003e5\u0026prime;-CATGGTTCTCCTCCTGCTAA-3\u0026prime;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eMuChPv-F2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 340px;\"\u003e\n \u003cp\u003e5\u0026prime;-CTGACCTCTTTGACTCTAC-3\u0026prime;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 99px;\"\u003e\n \u003cp\u003e154\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eMuChPv-R2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 340px;\"\u003e\n \u003cp\u003e5\u0026prime;-GCTCCATTACATACCTCTC-3\u0026prime;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eMuChPv-P\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 340px;\"\u003e\n \u003cp\u003eFAM-5\u0026prime;-TGCTGGACATAATGCTCTGAACC-3\u0026prime;-BHQ-1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\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":"Chaphamaparvovirus, breeding Muscovy duck, metagenomic sequencing, Real-time fluorescent quantitative PCR","lastPublishedDoi":"10.21203/rs.3.rs-7451845/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7451845/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSince 2022, outbreaks of hepatitis-like disease have been frequently reported in Muscovy duck farms across South China, leading to a significant decrease of egg production and hatchability. With the help of metagenomic sequencing, a novel Chaphamaparvovirus (ChPV) was identified from livers of the diseased ducks. Phylogenetic analysis revealed that the isolated strain shares 61.8-77.4% genome identities with duck-origin Chaphamaparvoviruses, and 44.3-77.4% with avian-origin Chaphamaparvoviruses. In addition, specific probes and primers were designed for quantitative PCR detection. The methods’ specificity, sensitivity, and repeatability were validated, suggesting the suitability for field detection of MuChPV.\u003c/p\u003e","manuscriptTitle":"Identification of novel Chaphamaparvovirus in breeding Muscovy ducks","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-26 14:13:15","doi":"10.21203/rs.3.rs-7451845/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":"c92b6f4e-2201-4f2c-b058-8dabce105783","owner":[],"postedDate":"September 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-19T09:24:39+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-26 14:13:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7451845","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7451845","identity":"rs-7451845","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}