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Duck circovirus (DuCV) is widely recognized as a prominent virus in China's duck farming industry, known for its ability to cause persistent infections and significant immunosuppression, which can lead to an increased susceptibility to secondary infections, posing a significant threat to the duck industry. Moreover, clinical evidence also indicates the potential vertical transmission of the virus through duck embryos to subsequent generations of ducklings. However, the limited availability of suitable cell lines for in vitro cultivation of DuCV has hindered further investigation into the molecular mechanisms underlying its infection and pathogenicity. In this study, we observed that oral DuCV infection in female breeding ducks can lead to oviduct, ovarian, and follicular infections. Subsequently, the infection can be transmitted to the fertilized eggs, resulting in the emergence of virus-carrying ducklings upon hatching. In contrast, the reproductive organs of male breeding ducks were unaffected by the virus, thus confirming that vertical transmission of DuCV primarily occurs through infection in female breeding ducks. By analyzing transcriptome sequencing data from the oviduct, we focused on claudin-2, a gene encoding the tight junction protein CLDN2 located on the cell membrane, which showed significantly increased expression in DuCV-infected oviducts of female breeding ducks. Notably, CLDN2 was confirmed to interact with the unique structural protein of DuCV, namely capsid protein (Cap), through a series of experimental approaches including co-immunoprecipitation (co-IP), GST pull-down, immunofluorescence, and adhesion-blocking assays. Furthermore, we demonstrated that the Cap protein binds to the extracellular loop structural domains EL1 and EL2 of CLDN2. Subsequently, by constructing a series of truncated bodies of the CLDN2 promoter region, we identified the transcription factor SP5 for CLDN2. Moreover, we found that DuCV infection triggers the activation of the MAPK-ERK signaling pathway in DEF cells and ducks, leading to an upregulation of SP5 and CLDN2 expression. This process ultimately leads to the transportation of mature CLDN2 to the cell surface, thereby facilitating increased virus adherence to the target organs. In conclusion, we discovered that DuCV utilizes host CLDN2 proteins to enhance adhesion and infection in oviducts and other target organs. Furthermore, we elucidated the signaling pathways involved in the interaction between DuCV Cap proteins and CLDN2, which provides valuable insights into the molecular mechanism underlying DuCV's infection and vertical transmission. [ABSTRACT FROM AUTHOR]

Background and Aim: Ducks worldwide are infected with duck circovirus (DuCV), which causes feather abnormality, emaciation, and poor growth performance. DuCV is similar to other circoviruses that induce immunosuppression due to the occurrence of the bursae of Fabricius (BF) and spleen atrophies. In Thailand, retarded ducks with feather losses were submitted for disease investigation. The ducks presented low body weight gain, had small BF and spleens, and were consistent with duck-infected DuCV. Our study investigated the possibility of DuCV infection in duck flocks in Thailand. We also analyzed the genetic characteristics of the virus. Materials and Methods: BF and spleen samples were collected from affected meat and layer ducks from six farms thought to have been infected with DuCV. These tissues were then subjected to histopathological examination and molecular identification using conventional polymerase chain reaction and nucleotide sequencing. To identify DuCV, phylogenetic trees were generated using MEGA version X software. Samples of tissues or swabs were collected to determine whether coinfections with bacteria and viruses existed. Results: Phylogenetic analysis using the entire genome (1995–1996 bp) and cap gene (762 bp) revealed that the DuCV isolates circulating in Thailand belonged to DuCV genotype I, which was further subdivided into two sub-genotypes: subgenotype I b and an unclassified sub-genotype based on reference sub-genotypes. Thai isolates have variations in 10 amino acid residues in the capsid protein. Ducks infected with Thai DuCV were also coinfected with Riemerella anatipestifer, Escherichia coli, Pasteurella multocida, duck viral enteritis, and duck Tembusu virus, which is consistent with previous DuCV infection studies. Conclusion: Six DuCVs from ducks who were previously found to have feather loss, were underweight, had growth retardation, and had poor body condition were identified in this study as belonging to genotype Ⅰ and constituting at least two sub-genotypes. Due to the immunosuppressive effects of DuCV, coinfection of bacterial and viral pathogens was typically observed in Thai DuCV-infected ducks. [ABSTRACT FROM AUTHOR]

Background and Aim: Ducks worldwide are infected with duck circovirus (DuCV), which causes feather abnormality, emaciation, and poor growth performance. DuCV is similar to other circoviruses that induce immunosuppression due to the occurrence of the bursae of Fabricius (BF) and spleen atrophies. In Thailand, retarded ducks with feather losses were submitted for disease investigation. The ducks presented low body weight gain, had small BF and spleens, and were consistent with duck-infected DuCV. Our study investigated the possibility of DuCV infection in duck flocks in Thailand. We also analyzed the genetic characteristics of the virus. Materials and Methods: BF and spleen samples were collected from affected meat and layer ducks from six farms thought to have been infected with DuCV. These tissues were then subjected to histopathological examination and molecular identification using conventional polymerase chain reaction and nucleotide sequencing. To identify DuCV, phylogenetic trees were generated using MEGA version X software. Samples of tissues or swabs were collected to determine whether coinfections with bacteria and viruses existed. Results: Phylogenetic analysis using the entire genome (1995–1996 bp) and cap gene (762 bp) revealed that the DuCV isolates circulating in Thailand belonged to DuCV genotype I, which was further subdivided into two sub-genotypes: sub-genotype I b and an unclassified sub-genotype based on reference sub-genotypes. Thai isolates have variations in 10 amino acid residues in the capsid protein. Ducks infected with Thai DuCV were also coinfected with Riemerella anatipestifer, Escherichia coli, Pasteurella multocida, duck viral enteritis, and duck Tembusu virus, which is consistent with previous DuCV infection studies. Conclusion: Six DuCVs from ducks who were previously found to have feather loss, were underweight, had growth retardation, and had poor body condition were identified in this study as belonging to genotype Ⅰ and constituting at least two sub-genotypes. Due to the immunosuppressive effects of DuCV, coinfection of bacterial and viral pathogens was typically observed in Thai DuCV-infected ducks.

Duck circovirus (DuCV) is one of the most prevalent infectious viruses in the duck industry in China. Although the clinical signs vary, it often causes immunosuppression in the host and leads to secondary infection with other pathogens. Novel goose parvovirus (NGPV) mainly infects ducks and causes short beak and dwarfism syndrome in ducks. However, the incidence of infection in ducks has increased in recent years, and the phenomenon of mixed infection with DuCV is common, resulting in more severe clinical morbidity. However, there are no systematic studies evaluating the presence of mixed infections. In order to investigate the synergistic pathogenicity of DuCV and NGPV co-infection in SPF ducks, a comparative experiment using DuCV and NGPV co-infection and mono-infection bird models was established. The results showed that the clinical signs of short beak, dwarfism and immunosuppression were more obvious in DuCV and NGPV co-infected ducks; the tissue damage of target organs was more serious, and the viral titre in organs and cloacal swabs were more significant compared with those of SPF ducks infected with only one virus. The results indicated that co-infection with DuCV and NGPV could promote viral replication and cause more severe tissue damage and immunosuppression than single virus infection. The present study reveals that the co-infection of NGPV and DuCV has a synergistic pathogenic effect from the aspect of pathogenicity, and the conclusions drawn not only clarify the direction of the subsequent research on the mechanism of co-infection of NGPV and DuCV, but also provide a scientific basis for the research on the co-infection of immunosuppressive pathogens and other pathogens. [ABSTRACT FROM AUTHOR]

ABSTRACT: Duck circovirus (DuCV) can infect domestic and wild ducks, retarding growth and suppressing immunity, thereby increasing the possibility of secondary infection by other pathogens. In this study, for the first time, 2 DuCV strains (G221116 and G210917) were identified in geese from China. To study the genetic characteristics of the 2 goose-originated DuCVs, multiple sequence alignment and phylogenetic analyses were perforemed according to genome sequences of 2 DuCV strains g and reference waterfowl circoviruses retrieved from the GenBank database. Pairwise analysis showed that the genome sequence identities between the 2 DuCVs with reference DuCV-1 and DuCV-2 strains were 80.95% to 98.24%, and 58.04% to 59.55% with Goose circovirus (GoCV). Phylogenetic analysis showed that the 2 DuCVs belonged to DuCV-1 and DuCV-2 genotypes. These results broaden our understanding of the genetic heterogeneity and evolution of DuCV and suggest trans-host transmission of DuCV between ducks and geese.

Simple Summary: Vertically transmitted duck viruses are viruses that are transmitted from a female duck to its offspring when it is an egg, which seriously threatens production in the duck breeding industry. In this study, we evaluated the distribution and genetic variation in four vertically transmitted duck pathogens, including DHBV, DuCV, DHAV-3, and ARV. This study found that DHBV was the most prevalent virus, followed by DuCV, and then ARV and DHAV-3. The genetic analysis results showed that all the identified duck viruses here had complex qualities. These findings will improve our knowledge of the evolution of DuCV, DHAV-3, and ARV and help choose suitable strains for vaccination. To investigate the distribution and genetic variation in four vertically transmitted duck pathogens, including duck hepatitis B virus (DHBV), duck circovirus (DuCV), duck hepatitis A virus 3 (DHAV-3), and avian reoviruses (ARV), we conducted an epidemiology study using PCR and RT-PCR assays on a duck population. We found that DHBV was the most prevalent virus (69.74%), followed by DuCV (39.48%), and then ARV (19.92%) and DHAV-3 (8.49%). Among the 271 duck samples, two, three or four viruses were detected in the same samples, indicating that the coinfection of vertical transmission agents is common in ducks. The genetic analysis results showed that all four identified DuCV strains belonged to genotype 1, the DHAV-3 strain was closely clustered with previously identified strains from China, and the ARV stain was clustered under genotype 1. These indicate that different viral strains are circulating among the ducks. Our findings will improve the knowledge of the evolution of DuCV, DHAV-3, and ARV, and help choose suitable strains for vaccination. [ABSTRACT FROM AUTHOR]

ABSTRACT: Birds infected with duck circovirus (DuCV) can potentially cause immunosuppression by damaging lymphoid tissues, causing great losses in the duck breeding industry. Duck circovirus can be divided into two genotypes (DuCV-1 and DuCV-2), but simultaneous detection and differentiation of DuCV-1 and DuCV-2 by high-resolution melting (HRM) analysis is still lacking. Here, we designed specific primers according to the sequence characteristics of the newly identified ORF3 gene and then established a PCR-HRM method for the simultaneous detection and differentiation of DuCV-1 and DuCV-2 via high-resolution melting analysis. Our data showed that the established PCR-HRM assay had the advantages of specificity, with the lowest detection limits of 61.9 copies/μL (for DuCV-1) and 60.6 copies/μL (for DuCV-2). The melting curve of the PCR-HRM results indicated that the amplification product was specific, with no cross-reaction with common waterfowl origin pathogens and a low coefficient of variation less than 1.50% in both intra-batch and inter-batch repetitions, indicating the advantages of repeatability. We found that the percentage of DuCV-2-positive ducks was higher than that of DuCV-1-positive ducks, with 8.62% rate of DuCV-1 and DuCV-2 coinfection. In addition, we found DuCV-2-positive in geese firstly. In conclusion, this study provides a candidate PCR-HRM assay for the detection and accurate differentiation of DuCV-1 and DuCV-2 infection, which will help us for further epidemiological surveillance of DuCVs.

ABSTRACT: In recent years, with the expansion of duck breeding industry in China, the infection rate of duck circovirus (DuCV) in duck and the mixed infection rate of DuCV with other diseases increased significantly, which seriously endanger the development of duck breeding industry. To study the epidemic status of duck circovirus in China, analyze the virus's genetics and evolution, and establish a foundation for scientific prevention and control of duck circovirus, our laboratory collected 4 disease materials preliminarily diagnosed as duck circovirus infections. Conventional PCR was used to amplify 4 strains of duck circovirus with a full length of 1993bp, and their sequences were compared and analyzed. The analysis showed that the 4 DuCVs had typical circovirus characteristics, including 3 major ORFs: ORFV1 (Rep protein), ORFC1 (Cap protein), ORFC2 (apoptosis-related protein), and a stem ring structure. The 4 strains were compared with 22 other reference strains, and the results revealed that all 4 strains belonged to the DuCV-I type represented by the German strain AY228555. Furthermore, the homology between the 4 DuCVs and the reference strains was up to 98.6%, which help us to understand the genotype and genetic variation of DuCV in these regions and provide a reference for the prevention and control of DuCV.

Duck Tembusu virus (DTMUV), duck circovirus (DuCV), and new duck reovirus (NDRV) have seriously hindered the development of the poultry industry in China. To detect the three pathogens simultaneously, a multiplex digital PCR (dPCR) was developed and compared with multiplex qPCR in this study. The multiplex dPCR was able to specifically detect DTMUV, DuCV, and NDRV but not amplify Muscovy duck reovirus (MDRV), Muscovy duck parvovirus (MDPV), goose parvovirus (GPV), H4 avian influenza virus (H4 AIV), H6 avian influenza virus (H6 AIV), and Newcastle disease virus (NDV). The standard curves showed excellent linearity in multiplex dPCR and qPCR and were positively correlated. The sensitivity results showed that the lowest detection limit of multiplex dPCR was 1.3 copies/μL, which was 10 times higher than that of multiplex qPCR. The reproducibility results showed that the intra- and interassay coefficients of variation were 0.06–1.94%. A total of 173 clinical samples were tested to assess the usefulness of the method; the positive detection rates for DTMUV, DuCV, and NDRV were 18.5, 29.5, and 14.5%, respectively, which were approximately 4% higher than those of multiplex qPCR, and the kappa values for the clinical detection results of multiplex dPCR and qPCR were 0.85, 0.89, and 0.86, indicating that the two methods were in excellent agreement.

To investigate the distribution and genetic variation in four vertically transmitted duck pathogens, including duck hepatitis B virus (DHBV), duck circovirus (DuCV), duck hepatitis A virus 3 (DHAV-3), and avian reoviruses (ARV), we conducted an epidemiology study using PCR and RT-PCR assays on a duck population. We found that DHBV was the most prevalent virus (69.74%), followed by DuCV (39.48%), and then ARV (19.92%) and DHAV-3 (8.49%). Among the 271 duck samples, two, three or four viruses were detected in the same samples, indicating that the coinfection of vertical transmission agents is common in ducks. The genetic analysis results showed that all four identified DuCV strains belonged to genotype 1, the DHAV-3 strain was closely clustered with previously identified strains from China, and the ARV stain was clustered under genotype 1. These indicate that different viral strains are circulating among the ducks. Our findings will improve the knowledge of the evolution of DuCV, DHAV-3, and ARV, and help choose suitable strains for vaccination.

A multiplex qPCR assay was developed to simultaneously detect duck circovirus (DuCV), duck Tembusu virus (DTMUV), Muscovy duck reovirus (MDRV), and novel duck reovirus (NDRV), but it did not amplify other viruses, including duck virus enteritis (DVE), infectious bursal disease virus (IBDV), avian reovirus (ARV), H5 avian influenza virus (H5 AIV), H7 avian influenza virus (H7 AIV), H9 avian influenza virus (H9 AIV), Newcastle disease virus (NDV), and Muscovy duck parvovirus (MDPV), and the detection limit for DuCV, DTMUV, MDRV, and NDRV was 1.51 × 101 copies/μL. The intra- and interassay coefficients of variation were less than 1.54% in the repeatability test with standard plasmid concentrations of 1.51 × 107, 1.51 × 105, and 1.51 × 103 copies/μL. The developed multiple qPCR assay was used to examine 404 clinical samples to verify its practicability. The positivity rates for DuCV, DTMUV, MDRV, and NDRV were 26.0%, 9.9%, 4.0%, and 4.7%, respectively, and the mixed infection rates for DuCV + DTMUV, DuCV + MDRV, DuCV + NDRV, MDRV + NDRV, DTMUV + MDRV, and DTMUV + NDRV were 2.7%, 1.2%, 1.2%, 1.0%, 0.5%, and 0.7%, respectively. [ABSTRACT FROM AUTHOR]

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Duck circovirus (DuCV) is widely recognized as a prominent virus in China's duck farming industry, known for its ability to cause persistent infections and significant immunosuppression, which can lead to an increased susceptibility to secondary infections, posing a significant threat to the duck industry. Moreover, clinical evidence also indicates the potential vertical transmission of the virus through duck embryos to subsequent generations of ducklings. However, the limited availability of suitable cell lines for in vitro cultivation of DuCV has hindered further investigation into the molecular mechanisms underlying its infection and pathogenicity. In this study, we observed that oral DuCV infection in female breeding ducks can lead to oviduct, ovarian, and follicular infections. Subsequently, the infection can be transmitted to the fertilized eggs, resulting in the emergence of virus-carrying ducklings upon hatching. In contrast, the reproductive organs of male breeding ducks were unaffected by the virus, thus confirming that vertical transmission of DuCV primarily occurs through infection in female breeding ducks. By analyzing transcriptome sequencing data from the oviduct, we focused on claudin-2 , a gene encoding the tight junction protein CLDN2 located on the cell membrane, which showed significantly increased expression in DuCV-infected oviducts of female breeding ducks. Notably, CLDN2 was confirmed to interact with the unique structural protein of DuCV, namely capsid protein (Cap), through a series of experimental approaches including co-immunoprecipitation (co-IP), GST pull-down, immunofluorescence, and adhesion-blocking assays. Furthermore, we demonstrated that the Cap protein binds to the extracellular loop structural domains EL1 and EL2 of CLDN2. Subsequently, by constructing a series of truncated bodies of the CLDN2 promoter region, we identified the transcription factor SP5 for CLDN2. Moreover, we found that DuCV infection triggers the activation of the MAPK-ERK signaling pathway in DEF cells and ducks, leading to an upregulation of SP5 and CLDN2 expression. This process ultimately leads to the transportation of mature CLDN2 to the cell surface, thereby facilitating increased virus adherence to the target organs. In conclusion, we discovered that DuCV utilizes host CLDN2 proteins to enhance adhesion and infection in oviducts and other target organs. Furthermore, we elucidated the signaling pathways involved in the interaction between DuCV Cap proteins and CLDN2, which provides valuable insights into the molecular mechanism underlying DuCV's infection and vertical transmission., Importance: Although duck circovirus (DuCV) poses a widespread infection and a serious hazard to the duck industry, the molecular mechanisms underlying DuCV infection and transmission remain elusive. We initially demonstrated vertical transmission of DuCV through female breeding ducks by simulating natural infection. Furthermore, a differentially expressed membrane protein CLDN2 was identified on the DuCV-infected oviduct of female ducks, and its extracellular loop structural domains EL1 and EL2 were identified as the interaction sites of DuCV Cap proteins. Moreover, the binding of DuCV Cap to CLDN2 triggered the intracellular MAPK-ERK pathway and activated the downstream transcription factor SP5. Importantly, we demonstrated that intracellular Cap also interacts with SP5, leading to upregulation of CLDN2 transcription and facilitating enhanced adherence of DuCV to target tissue, thereby promoting viral infection and transmission. Our study sheds light on the molecular mechanisms underlying vertical transmission of DuCV, highlighting CLDN2 as a promising target for drug development against DuCV infection., Competing Interests: The authors declare no conflict of interest.

Duck circovirus (DuCV) can infect domestic and wild ducks, retarding growth and suppressing immunity, thereby increasing the possibility of secondary infection by other pathogens. In this study, for the first time, 2 DuCV strains (G221116 and G210917) were identified in geese from China. To study the genetic characteristics of the 2 goose-originated DuCVs, multiple sequence alignment and phylogenetic analyses were perforemed according to genome sequences of 2 DuCV strains g and reference waterfowl circoviruses retrieved from the GenBank database. Pairwise analysis showed that the genome sequence identities between the 2 DuCVs with reference DuCV-1 and DuCV-2 strains were 80.95% to 98.24%, and 58.04% to 59.55% with Goose circovirus (GoCV). Phylogenetic analysis showed that the 2 DuCVs belonged to DuCV-1 and DuCV-2 genotypes. These results broaden our understanding of the genetic heterogeneity and evolution of DuCV and suggest trans-host transmission of DuCV between ducks and geese., Competing Interests: DISCLOSURES The authors have no competing interests to declare., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

ABSTRACT: Duck circovirus (DuCV) is the smallest known virus in waterfowl that infects both domestic and wild duck. Infected ducks often show stunted growth and immunosuppression, which increases the rate of secondary infection with other pathogens. In this study, 270 liver tissue samples were collected to screen the presence of DuCV in Guangdong province, China, and the complete genome sequences were recovered and systematically analyzed. Genetic analyses revealed that sequences determined in this study shared 81.6% to 100.0% genome-wide pairwise identity with previously identified DuCV genomes. Phylogenetic analyses showed that 2 DuCV genotypes with a high infection rate were co-circulating in duck population in Guangdong province, and extensive recombination events have occurred during the evolution of DuCV. Our results expand upon the knowledge regarding the genetic diversity and evolution of DuCV, and also indicate that extensive genetically divergent DuCV are co-circulating in the duck populations in Guangdong, southern China.

ABSTRACT: Duck circovirus (DuCV) infection occurs frequently in ducks in China and is generally believed to lead to immunosuppression and secondary infection, though there has been a lack of detailed research and direct evidence. In this study, one-day-old Cherry Valley ducklings were artificially infected with DuCV alone and co-infected with DuCV and Avian Pathogenic Escherichia coli (APEC). The immune indexes at 32 d old were systematically monitored, including immune organ weight, lymphocyte transformation rate, IL-10, IL-12, soluble CD4 (sCD4), soluble CD8 (sCD8), IFN-γ, viral loads in each organ, APEC colonization, and so on. The results showed the development of immune organs in ducklings was affected, resulting in a decrease in the lymphocyte transformation rate (LTR), IL-12, sCD4, sCD8, IFN-γ and an increase in IL-10 content at 8 to 32 d postinfection (dpi). In the detection of virus loads in some organs, it was found that 8 dpi, DuCV existed stably in various organs, suggesting the importance of preventing and controlling the virus in the early stage of culture. The results of exploring the DuCV infection that shows some influence on secondary infection by APEC. The results showed that DuCV infection could significantly enhance the pathogenicity of APEC and the colonization ability of APEC in vivo. DuCV can induce more serious APEC infection in 24 dpi than in 14 dpi. Based on the above results, it can be concluded that DuCV infection will affect the immune system, cause immunosuppression, and lead to more serious secondary infection.

ABSTRACT: Duck circovirus disease (DuCVD), as an immunosuppressive disease, is a threat to the poultry industry. In order to diagnose this disease quickly and accurately, a real-time fluorescence-based recombinase-aided amplification (RF-RAA) method was established to detect duck circovirus (DuCV). The results showed that the quantity of amplification products was positively correlated with the value of fluorescence signal. Obvious detection results can be observed at 41°C after 15 min reaction. This method has good specificity and has no cross reaction with Muscovy duck parvovirus (MDPV), duck enteritis virus (DEV), fowl adenovirus (FAdV), porcine circovirus (PCV), and duck hepatitis A virus (DHAV). The sensitivity test showed that the minimum concentration of template detected by RF-RAA for DuCV was 10° copies/μL, and its sensitivity was 10 times higher than that of real-time fluorescence-based quantitative PCR (RFQ-PCR) and 10,000 times higher than that of polymerase chain reaction (PCR). Fifty-two clinical samples were detected by RF-RAA and RFQ-PCR, and the coincidence rate of the two methods was 98.08%. This method has the advantages of simple operation, good specificity and high sensitivity, and can be used for laboratory detection and clinical diagnosis of DuCV.

Coinfection of goose parvovirus (GPV) and duck circovirus (DuCV) occurs commonly in field cases of short beak and dwarfism syndrome (SBDS). However, whether there is synergism between the two viruses in replication and pathogenicity remains undetermined.

Abstract Background Duck circovirus (DuCV) is a potential immunosuppressive virus that causes feather disorders in young ducks. In this study, DuCV obtained from various species of ducks was investigated by polymerase chain reaction (PCR) in southern and southwestern China (Guangdong, Guangxi and Yunnan provinces) from 2018 to 2019. Results A total of 848 bursa samples were collected from dead Mulard, Cherry Valley Pekin, Muscovy and Mallard ducks from duck farms. The positivity rate of DuCV in the total sample was approximately 36.91%. We found that the prevalence of DuCV in Yunnan (43.09%) was higher than those in Guangxi (34.38%) and Guangdong (34.4%). However, the positivity rates of DuCV in the four duck species were not significantly different (P > 0.05). Nineteen randomly selected complete viral genomes were sequenced. The complete genomes of the DuCV were 1987 to 1995 nt in length, and were 81.7–99.3% homologous to the other 57 sequences in GenBank. Phylogenetic analyses based on the complete genomes of 76 DuCVs showed that the 19 novel DuCV sequences from Guangdong and Guangxi provinces mainly belonged to the DuCV-1 and DuCV-2 genetic groups, respectively. However, the two genotype groups coexisted in Yunnan Province. In addition, recombination analysis showed putative recombination sites in 3 strains in Yunnan that originated from strains Guangdong and Guangxi. Interestingly, the epidemiological investigation showed that Mulard ducks, Cherry Valley Pekin ducks and Muscovy ducks more than 4 weeks old were more susceptible to infection with the novel DuCV than ducks less than 4 weeks old. Conclusions These data provide insight into the molecular epidemiology and genetic diversity of DuCVs circulating in southern and southwestern China for the first time.

Duck circovirus (DuCV) is widespread across the world and causes feather disorders in young ducks. It was identified as the causative pathogen of duck beak atrophy and dwarfism syndrome and primary sclerosing cholangitis. In this study, we aimed to establish a TaqMan-based real-time PCR assay to detect DuCV. The primers and probe were designed based on the conserved region of the DuCV Rep gene. After optimizing the reaction conditions, the minimum virus detection limit of the designed PCR technique was 39.4 copies/μL, 100 times that of conventional PCR (cPCR). No cross-reaction with six other common duck viruses was observed. The intra- and inter-assay variations were less than 1%. The detection rate of DuCV-positive clinical samples using TaqMan-based real-time PCR was higher than that using SYBR Green-based real-time PCR and cPCR. Collectively, these results showed that the established TaqMan-based real-time PCR detected DuCV with high sensitivity and specificity, and significant repeatability, making it suitable for clinical use. Hence, it may be used as a novel tool for the diagnosis and epidemiological investigation of DuCV. [ABSTRACT FROM AUTHOR]

In this study, we developed a real-time quantitative polymerase chain reaction (qPCR) assay based on a dual-labeled hydrolysis probe to simultaneously detect both duck circovirus (DuCV) 1 and DuCV-2. The reproducibility, sensitivity and specificity of the primer set and probe were evaluated using other duck pathogens. The detection limit was 20 copies per µL. The intra-assay coefficients of variation (CVs) were ≤ 0.73% and the inter-assay CVs were ≤ 1.89%. No cross-reaction occurred with other duck pathogens. In addition, the qPCR assay was successfully applied to the simultaneous detection of DuCV-1 and DuCV-2 in clinical field samples. Therefore, this assay will be useful for laboratory diagnosis and epidemiological field studies of DuCV. [ABSTRACT FROM AUTHOR]

ABSTRACT: Duck circovirus disease (DuCVD) caused by duck circovirus (DuCV) continues to spread in recent years, which brings serious harm to the poultry industry, so early diagnosis of DuCVD is of great significance for the prevention and control of this disease. Specific primers and probes for DuCV were designed in this study. Reverse primers and probes were modified at the 5′ ends with biotin and fluorescein, respectively, and they were combined with dipsticks labeled with biotin antibodies and fluorescein antibodies to establish a recombinase-aided amplification-lateral flow dipstick (RAA-LFD) assay for detection of duck circovirus. By using this method, the reaction products reached detectable levels in about 20 min as a result of rapid amplification at a constant temperature of 37℃. The detection results could be observed by dripping the reaction products onto the dipstick within 2 to 3 min. The RAA-LFD method has good specificity and high sensitivity (102 copies/μL). Compared with conventional polymerase chain reaction (PCR), RAA-LFD has no power limit on the testing instrument, and is easy to use, saving more time and manpower, so it is more suitable for clinical detection.

Birds infected with duck circovirus (DuCV) can potentially cause immunosuppression by damaging lymphoid tissues, causing great losses in the duck breeding industry. Duck circovirus can be divided into two genotypes (DuCV-1 and DuCV-2), but simultaneous detection and differentiation of DuCV-1 and DuCV-2 by high-resolution melting (HRM) analysis is still lacking. Here, we designed specific primers according to the sequence characteristics of the newly identified ORF3 gene and then established a PCR-HRM method for the simultaneous detection and differentiation of DuCV-1 and DuCV-2 via high-resolution melting analysis. Our data showed that the established PCR-HRM assay had the advantages of specificity, with the lowest detection limits of 61.9 copies/μL (for DuCV-1) and 60.6 copies/μL (for DuCV-2). The melting curve of the PCR-HRM results indicated that the amplification product was specific, with no cross-reaction with common waterfowl origin pathogens and a low coefficient of variation less than 1.50% in both intra-batch and inter-batch repetitions, indicating the advantages of repeatability. We found that the percentage of DuCV-2-positive ducks was higher than that of DuCV-1-positive ducks, with 8.62% rate of DuCV-1 and DuCV-2 coinfection. In addition, we found DuCV-2-positive in geese firstly. In conclusion, this study provides a candidate PCR-HRM assay for the detection and accurate differentiation of DuCV-1 and DuCV-2 infection, which will help us for further epidemiological surveillance of DuCVs. [ABSTRACT FROM AUTHOR]

To explore genetic variations in duck circovirus (DuCV) and the molecular epidemiology of its infection, tissue samples were collected from 219 dead ducks from 20 farms in the central and eastern regions of China. All farms tested positive for DuCV, with duck‐origin goose parvovirus, reovirus and Tembusu virus having co‐infection rates of 100%, 0% and 0%, respectively. A total of 20 strains from the DuCV‐positive flock were sequenced. The total sequence length was 1987–1996 nt, and the sequences shared 82% (JX499186, DuCV2 from Sichuan province, China) to 99.7% (KY328304, DuCV1 from Shandong Province, China) sequence identity with DuCV sequences available in GenBank. Hyper‐variable regions were mainly located in open reading frame (ORF)2, ORF3 and intergenic regions. The tertiary structure of ORF2 from four provinces (Henan, Anhui, Zhejiang and Fujian) in China showed a canonical viral jelly roll and the antigenic epitope of ORF2 located in the bulge of the protein surface. Overall, 15 of the 20 DuCV strains are possibly derived through inter‐genotypic and intragenotypic recombination. Based on sequence and phylogenetic analyses, six strains from Fujian Province clustered into a novel genotype—DuCV‐1d. These findings may enrich our understanding of DuCV evolution and circulation and lay the foundation for vaccine strain selection. [ABSTRACT FROM AUTHOR]

Background: Duck circovirus (DuCV) is a potential immunosuppressive virus that causes feather disorders in young ducks. In this study, DuCV obtained from various species of ducks was investigated by polymerase chain reaction (PCR) in southern and southwestern China (Guangdong, Guangxi and Yunnan provinces) from 2018 to 2019. Results: A total of 848 bursa samples were collected from dead Mulard, Cherry Valley Pekin, Muscovy and Mallard ducks from duck farms. The positivity rate of DuCV in the total sample was approximately 36.91%. We found that the prevalence of DuCV in Yunnan (43.09%) was higher than those in Guangxi (34.38%) and Guangdong (34.4%). However, the positivity rates of DuCV in the four duck species were not significantly different (P > 0.05). Nineteen randomly selected complete viral genomes were sequenced. The complete genomes of the DuCV were 1987 to 1995 nt in length, and were 81.7–99.3% homologous to the other 57 sequences in GenBank. Phylogenetic analyses based on the complete genomes of 76 DuCVs showed that the 19 novel DuCV sequences from Guangdong and Guangxi provinces mainly belonged to the DuCV-1 and DuCV-2 genetic groups, respectively. However, the two genotype groups coexisted in Yunnan Province. In addition, recombination analysis showed putative recombination sites in 3 strains in Yunnan that originated from strains Guangdong and Guangxi. Interestingly, the epidemiological investigation showed that Mulard ducks, Cherry Valley Pekin ducks and Muscovy ducks more than 4 weeks old were more susceptible to infection with the novel DuCV than ducks less than 4 weeks old. Conclusions: These data provide insight into the molecular epidemiology and genetic diversity of DuCVs circulating in southern and southwestern China for the first time. [ABSTRACT FROM AUTHOR]

The capsid (Cap) protein, which is the only structural protein of duck circovirus (DuCV), is the most important antigen for the development of vaccines against DuCV and the virus's serological diagnostic methods. In order to use yeast expression system to produce a large quantities of DuCV Cap protein which is close to its natural form to display the antigen peptides perfectly, the Cap gene was optimized into the codon-optimized capsid (Opt-Cap) gene towards the preference of yeast firstly. Then, the genes of Cap and Opt-Cap were separately cloned into pPIC9K plasmid and transformed into Picha pastoris GS115. The strains that displayed the phenotype of Mut+ and contained multiple inserts of expression cassette were selected from those colonies. After the induction expression, the secretory type of Cap protein, which was about 43 kDa, was best expressed under 0.5% (v/v) methanol and sorbitol induction. Compared with the Cap gene, the expression level of Opt-Cap gene was much higher. What's more, the purified Cap protein had a good reactivity to its specific polyclone antibody and DuCV-positive serum, and it was able to self-assemble into virus-like particles (VLPs). These VLPs, with a diameter of 15–20 nm and without a nucleic acid structure, showed a high level of similarity to DuCV particles in size and shape. All of the results demonstrated that, based on the codon-optimization, it is suitable to use the P. pastoris expression system to produce DuCV VLPs on a large scale. It is the first time that a large amounts of DuCV VLPs were produced successfully in P. pastoris, which might be particularly useful for the further studies of serological diagnosis and vaccines of DuCV.

To investigate the distribution and genetic variation in four vertically transmitted duck pathogens, including duck hepatitis B virus (DHBV), duck circovirus (DuCV), duck hepatitis A virus 3 (DHAV-3), and avian reoviruses (ARV), we conducted an epidemiology study using PCR and RT-PCR assays on a duck population. We found that DHBV was the most prevalent virus (69.74%), followed by DuCV (39.48%), and then ARV (19.92%) and DHAV-3 (8.49%). Among the 271 duck samples, two, three or four viruses were detected in the same samples, indicating that the coinfection of vertical transmission agents is common in ducks. The genetic analysis results showed that all four identified DuCV strains belonged to genotype 1, the DHAV-3 strain was closely clustered with previously identified strains from China, and the ARV stain was clustered under genotype 1. These indicate that different viral strains are circulating among the ducks. Our findings will improve the knowledge of the evolution of DuCV, DHAV-3, and ARV, and help choose suitable strains for vaccination.

Highlights • DuCV induces primary sclerosing cholangitis (PSC) in natural and reproductive duck cases. • DuCV shows high tropism to epithelial cell of bile duct. • The pathology model of PSC that induced by DuCV sheds light on mechanism of PSC study. Abstract Primary sclerosing cholangitis (PSC) is a chronic, cholestatic liver disease of unknown cause. In the study, we found that duck circovirus (DuCV) induces PSC in natural and reproductive cases. PSC in DuCV naturally infected ducks was investigated by PCR and histopathology. A model of PSC was developed in one-day old duck by infection of DuCV. Effects on serum levels of liver enzymes and histology were evaluated, and DuCV tropism for bile duct in liver was analyzed by immuohistochemistry. Pathology observation of natural or reproductive DuCV infected ducks showed that the lesion of liver were characterized by cholangiocytic injuries and progressive fibrous obliteration of the biliary tree associated with lymphocytes infiltration. ALT, AST, ALP, GGT, ALB, TBIL and TP were significantly increased in serum of DuCV infected ducks. DuCV showed higher tropism for epithelial cells of bile duct than other cells in PSC. [ABSTRACT FROM AUTHOR]

Short beak and dwarfism syndrome (SBDS), which was previously identified only in mule ducks, is now an emerging disease of Pekin ducks in China and Egypt. The disease is caused by the infection of ducks with a genetic variant of goose parvovirus—novel goose parvovirus (nGPV). In 2019, SBDS was observed for the first time in Poland in eight farms of Pekin ducks. Birds in the affected flock were found to show growth retardation and beak atrophy with tongue protrusions. Morbidity ranged between 15% and 40% (in one flock), while the mortality rate was 4–6%. Co-infection with duck circovirus, a known immunosuppressive agent, was observed in 85.7% of ducks. The complete coding regions of four isolates were sequenced and submitted to GenBank. The phylogenetic analysis revealed a close relationship of Polish viral sequences with the Chinese nGPV. Genomic sequence alignments showed 98.57–99.28% identity with the nGPV sequences obtained in China, and 96.42% identity with the classical GPV (cGPV; Derzsy’s disease). The rate of amino acid mutations in comparison to cGPV and Chinese nGPV was higher in the Rep protein than in the Vp1 protein. To our knowledge, this is the first report of nGPV infection in Pekin ducks in Poland and Europe. It should be emphasized that monitoring and sequencing of waterfowl parvoviruses is important for tracking the viral genetic changes that enable adaptation to new species of waterbirds.

In recent years, with the expansion of duck breeding industry in China, the infection rate of duck circovirus (DuCV) in duck and the mixed infection rate of DuCV with other diseases increased significantly, which seriously endanger the development of duck breeding industry. To study the epidemic status of duck circovirus in China, analyze the virus's genetics and evolution, and establish a foundation for scientific prevention and control of duck circovirus, our laboratory collected 4 disease materials preliminarily diagnosed as duck circovirus infections. Conventional PCR was used to amplify 4 strains of duck circovirus with a full length of 1993bp, and their sequences were compared and analyzed. The analysis showed that the 4 DuCVs had typical circovirus characteristics, including 3 major ORFs: ORFV1 (Rep protein), ORFC1 (Cap protein), ORFC2 (apoptosis-related protein), and a stem ring structure. The 4 strains were compared with 22 other reference strains, and the results revealed that all 4 strains belonged to the DuCV-I type represented by the German strain AY228555. Furthermore, the homology between the 4 DuCVs and the reference strains was up to 98.6%, which help us to understand the genotype and genetic variation of DuCV in these regions and provide a reference for the prevention and control of DuCV. [ABSTRACT FROM AUTHOR]

Duck circovirus (DuCV) is one of the most prevalent viruses in the duck breeding industry, and causes persistent infection and severe immunosuppression. Currently, there is a serious lack of prevention and control measures and no commercial vaccine against DuCV. Therefore, effective antiviral drugs are important for treating DuCV infection. Interferon (IFN) is an important component of antiviral innate immunity, but it remains unclear whether duck IFN-α has a clinical effect against DuCV. Antibody therapy is an important way to treat viral infections. The DuCV structural protein (cap) is immunogenic, and it remains to be determined whether an anti-cap protein antibody can effectively block DuCV infection. In this study, the duck IFN-α gene and the DuCV structural protein cap gene were cloned, expressed and purified in Escherichia coli to prepare duck recombinant IFN-α and the cap protein. Then, rabbits were immunized with the recombinant cap protein to prepare a rabbit polyclonal antibody. This study investigated the antiviral effect of duck recombinant IFN-α and the anti-cap protein antibody and their combined effect on Cherry Valley ducks infected with DuCV. The results showed that the treatment significantly alleviated the clinical symptoms of immune organ atrophy and immunosuppression compared with the control. The histopathological damage of the target organs was alleviated, and replication of DuCV in the immune organs was significantly inhibited. The treatment also reduced the damage caused by DuCV to the liver and immune function, and increased the level of the DuCV antibody in the blood, thereby improving antiviral activity. Notably, the combination of duck IFN-α and the polyclonal antibody completely blocked DuCV infection after 13 days under the experimental conditions, showing a better inhibitory effect on DuCV infection than single treatments. These results showed that duck recombinant IFN-α and the anti-cap protein antibody can be used as antiviral drugs to clinically treat and control DuCV infection, particularly the vertical transmission of the virus in breeding ducks. • Duck IFN-α and the anti-cap protein polyclonal antibody exhibited antiviral effects on Cherry Valley ducks infected with DuCV. • The combination of duck IFN-α and the anti-cap protein polyclonal antibody completely blocked DuCV infection under the experimental conditions. • Duck IFN-α and the anti-cap protein polyclonal antibody can be used as antiviral drugs to treat and control DuCV infection. [ABSTRACT FROM AUTHOR]

Highlights • Three DNA vaccines were developed based on the Capsid (Cap) protein of DuCV. • DNA vaccines encoding Cap protein-related antigens induced efficient systemic immune responses. • DNA vaccine-expressing secretory Cap protein enhanced the protective immune responses against DuCV infection. Abstract Duck circovirus (DuCV) is an immunosuppressive pathogen that causes a huge economic loss in the avian industry. Efficient vaccination has become a necessary strategy for preventing DuCV infection in the breeding industry. Three DNA vaccines encoding the Capsid (Cap) protein of DuCV were developed in this study, which were based on the eukaryotic vector pcDNA3.1 containing (i) the full length of Cap gene, pcDNA3.1-Cap, (ii) the Cap gene with a deletion of its nuclear localization signal (NLS) peptide encoding sequence, pcDNA3.1-CapΔNLS, and (iii) the Cap gene without NLS but harboring a fragment encoding the secretory signal peptide of tissue plasminogen activator (tPA), pcDNA3.1-tPA-CapΔNLS. Production of Cap protein-derived antigens from these three DNA vaccines was confirmed in vitro. The deletion of the NLS coding sequence of the Cap gene changed the subcellular location of the Capsid protein from the nucleus to the cytoplasm. Secretion of the Cap protein was observed in pcDNA3.1-tPA-CapΔNLS-transfected cells. The immunogenicity of these three DNA vaccines was assessed in vivo by measuring Cap-specific antibody and related cytokine levels. The results demonstrated that all these vaccines could induce a significant, specific immune response to protect ducks from DuCV challenge. Notably, higher titers of Cap-specific antibody were produced in ducks vaccinated with pcDNA3.1-tPA-CapΔNLS, which provided the highest protective efficacy at a rate of 90% in the challenge experiment. Taken together, DNA vaccines expressing the DuCV Cap protein show promising immunogenicity, which can be enhanced by replacing the NLS of the Cap protein with a secretory signal peptide of tPA. [ABSTRACT FROM AUTHOR]

Ducks infected with duck circovirus (DuCV) exhibit feathering disorder, growth retardation, and low body weight. The virus can induce immunosuppression and increase rates of infection caused by other pathogens. The purpose of the present study was to investigate the pathogenesis of DuCV in experimentally infected Pekin ducks. At postmortem examination, gross lesions were observed in the immune organs including bursa of Fabricius (BF), thymus, and spleen. Hemorrhage, lymphocytic depletion, necrosis, and degeneration were observed in the bursal tissues by histological examination. The TUNEL assay was performed with bursal tissue. There was a significant difference of the apoptosis rate between the negative and DuCV-infected ducks. The earliest time point for detection of DuCV DNA in sera, cloacal swabs, and organs was 1 wk post-infection (WPI). Viral shedding was persistent and detectable at the end of the experiment (10 WPI). The findings provide evidence that horizontal transmission and persistent infection are the characteristics of DuCV. The organ with the highest mean viral load was the spleen, followed by BF, cecal tonsil, lung, thymus, liver, and kidney. We successfully established an experimental DuCV genotype 1 (DuCV-1) infection in Pekin ducks and demonstrated the pathogenicity and persistence of DuCV-1. In conclusion, DuCV-1 caused extensive damage to the immune organs that may have resulted in immunosuppression. Pathobiological characteristics of DuCV-1 include systemic infection, persistent infection, and horizontal transmission. These features allow DuCV-1 to circulate more easily in farms and increase the susceptibility of ducks to other diseases. [ABSTRACT FROM AUTHOR]

Summary: As a newly emerged infectious disease, duck “beak atrophy and dwarfism syndrome (BADS)” disease has caused huge economic losses to waterfowl industry in China since 2015. Novel goose parvovirus‐related virus (NGPV) is believed the main pathogen of BADS disease; however, BADS is rarely reproduced by infecting ducks with NGPV alone. As avian circovirus infection causes clinical symptoms similar to BADS, duck circovirus (DuCV) is suspected the minor pathogen of BADS disease. In this study, an investigation was carried out to determine the coinfection of NGPV and DuCV in duck embryos and in ducks with BADS disease. According to our study, the coinfection of emerging NGPV and DuCV was prevalent in East China (Shandong, Jiangsu and Anhui province) and could be vertical transmitted, indicating their cooperative roles in duck BADS disease. [ABSTRACT FROM AUTHOR]

To probe the epidemiology of duck circovirus (DuCV) in migrating wild ducks in China, 189 samples collected from 11 species of wild ducks from 2013 to 2016 were analyzed by PCR. Four positive samples were obtained from Mallard (2), Green-winged Teal (1), and Falcated Duck (1), and the positive rate was 2.12%. The homologous alignment of the complete genome shows that the homology of the wild duck strains is 81.6 to 95.1% to each other and 81.4 to 100% to the 32 strains from other origins. Amino acids alignment revealed that the mutable ORF2 gene has six major variable regions and many random mutation points. Phylogenetic analysis showed that the four sequences belong to two genotypes: wd2013017, wd2013046 and AY228555 (representative strain of genotype 1) belong to genotype 1, while wd2014012, wd2015028 and AY394721 (representative strain of genotype 2) belong to genotype 2. The results indicate that both genotypes of DuCV are distributed and common in migrating wild ducks. Further analysis shows that duck circovirus infection is mainly concentrated in the eastern coastal cities of China, which are part of the East Asian-Australian flyway. This suggests that wild ducks with circovirus may be an important factor in the epidemic and spread of DuCV. [ABSTRACT FROM AUTHOR]

Duck circovirus (DuCV) is divided into genotypes 1 and 2. The DuCV ORF3 protein is a newly identified viral protein with apoptotic activity. In this study, the differences in the gene sequences, subcellular localization, and apoptotic activities of the ORF3 proteins of DuCV genotypes 1 and 2 were analyzed. A T-to-A point mutation at nucleotide 236 (T236A) in the ORF3 gene sequence of DuCV genotype 1 was observed, which generates a premature stop codon (TAG) and resulted in a truncated ORF3 protein. The ORF3 protein of DuCV genotype 2 is 20 amino acids longer at its C-terminus than the truncated ORF3 protein of genotype 1. A variant monopartite-type nuclear localization signal (RRLRTCNCRACRTLK) was identified within the C-terminal region of the ORF3 protein of DuCV genotype 2, which is essential for the nuclear localization of the protein. The 20 C-terminal residues of the DuCV genotype 2 ORF3 protein also inhibits the apoptotic activity of the protein. Our findings provide insight into the biological and functional characteristics of the DuCV ORF3 protein. [ABSTRACT FROM AUTHOR]

Duck circovirus (DuCV), as a causative agent of long-term immunosuppressive disease, has caused heavy damage to waterfowl breeding worldwide. In this study, the full-length Cap (capsid) gene of DuCV was expressed in Escherichia coli ( E. coli ) for the first time by optimizing the codons in its nuclear localization signal (NLS) regions. The recombinant protein was expressed as inclusion bodies, and the quantification of purified Cap protein could reach 0.29 mg mL −1 . Moreover, an indirect ELISA method (Cap-ELISA) was established based on the recombinant Cap protein. The results of the optimization for Cap-ELISA revealed that the optimum concentration of the coating antigen and serum dilution ratio were 19.5 ng per well and 1:1280, respectively. The cut-off value of the Cap-ELISA for positive sample detection was 0.145, the sensitivity of that reached 1:25600, and it was specific for the detection of DuCV anti-sera. In comparative experiments using 56 clinically suspected DuCV-infection samples, the Cap-ELISA showed a 94.64% coincidence rate with the PCR test. These results indicated that codon optimization is a reasonable strategy to obtain an intact Cap protein, and this Cap-ELISA is suitable for extensive applications in DuCV serological diagnosis. [ABSTRACT FROM AUTHOR]

Duck circovirus (DuCV) is one of the most prevalent infectious viruses in the duck industry in China. Although the clinical symptoms vary, it often causes immunosuppression in the host and leads to secondary infection with other pathogens. Fowl adenovirus serotype 4 (FAdV-4) mainly infects chickens and causes hydropericardium hepatitis syndrome. However, the incidence of infection in ducks has increased in recent years, and the phenomenon of mixed infection with DuCV is very common, resulting in more severe clinical morbidity. However, there is no systematic study evaluating the presence of mixed infection. To explore the synergistic pathogenicity of DuCV and FAdV-4 co-infection in Cherry Valley ducks, a comparative experiment was established between DuCV and FAdV-4 co-infection and single infection animal models. It was found that DuCV and FAdV-4 co-infected ducks showed more pronounced clinical signs of pericardial effusion, hepatitis and immunosuppression; more severe tissue damage in target organs; and more significant levels of viral load, biochemical indicators and immune indicators in various organs compared with Cherry Valley ducks infected with just one virus. The results showed that co-infection with DuCV and FAdV-4 may promote greater viral replication, causing more severe tissue damage and immunosuppression than infection with just one virus. Therefore, the monitoring and prevention of the two viruses should be strengthened clinically, with a particular focus on the potential harm of DuCV as it carries the highest infection rate. • A model building method was used to simulate the pathogenicity of DuCV and FAdV-4 coinfection. • DuCV and FAdV-4 co-infection caused more severe pathogenicity than the presence of just one infection. • DuCV and FAdV-4 co-infection synergistically promotes the replication of both viruses. • DuCV and FAdV-4 co-infection causes more severe lymphocyte damage in the duck population than in the groups infected by either virus alone. [ABSTRACT FROM AUTHOR]

Duck Tembusu virus (DTMUV), duck circovirus (DuCV), and new duck reovirus (NDRV) have seriously hindered the development of the poultry industry in China. To detect the three pathogens simultaneously, a multiplex digital PCR (dPCR) was developed and compared with multiplex qPCR in this study. The multiplex dPCR was able to specifically detect DTMUV, DuCV, and NDRV but not amplify Muscovy duck reovirus (MDRV), Muscovy duck parvovirus (MDPV), goose parvovirus (GPV), H4 avian influenza virus (H4 AIV), H6 avian influenza virus (H6 AIV), and Newcastle disease virus (NDV). The standard curves showed excellent linearity in multiplex dPCR and qPCR and were positively correlated. The sensitivity results showed that the lowest detection limit of multiplex dPCR was 1.3 copies/μL, which was 10 times higher than that of multiplex qPCR. The reproducibility results showed that the intra- and interassay coefficients of variation were 0.06-1.94%. A total of 173 clinical samples were tested to assess the usefulness of the method; the positive detection rates for DTMUV, DuCV, and NDRV were 18.5, 29.5, and 14.5%, respectively, which were approximately 4% higher than those of multiplex qPCR, and the kappa values for the clinical detection results of multiplex dPCR and qPCR were 0.85, 0.89, and 0.86, indicating that the two methods were in excellent agreement., Competing Interests: CL is employed by Guangxi State Farms Yongxin Animal Husbandry Group Xijiang Co. Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Yin, Xiong, Shi, Long, Feng, Qu, Lu, Huang, Lin, Sun and Li.)

Duck circovirus (DuCV) is an immunosuppressive virus and can induce immunosuppression increasing rates of infection caused by other pathogens, which has resulted in gross economic losses in poultry industries. The genome sizes of DuCVs varied from 1987 to 1996 nucleotides (nt). Up to date two DuCV genotypes/lineages, DuCV1 and DuCV2, have been defined, with genome identities of around 83% between each other. In this study, a novel duck circovirus having a genome size of 1755 nt was identified from laying ducks with a disease of egg production declining or abrogation from Hunan province, China. Two major open reading frames (ORFs) were identified, with ORF1 (882 nt) and ORF2 (738 nt) encoding replicase (Rep) and capsid protein (Cap), respectively. Its genome showed the highest identities of 62.3–63.7% and 66.3%− 67.8% to the known genomes of DuCVs and goose circoviruses (GoCVs) available in GenBank, respectively, while it showed less than 50% identities to the genomes of other circoviruses. The amino acid sequence of capsid of this virus showed the highest identities of 45.4%− 47.3% and 42.9%− 44.5% to capsids of the known DuCVs and GoCVs, respectively, while it showed less than 27% identity to the capsid proteins of other circoviruses available in GenBank. Further phylogenetic analyses based on genomes, amino acid sequences of Rep and Cap proteins demonstrated that the present duck circovirus was clustered in a separate clade, distinct from other DuCVs and GoCVs, corroborating it is a distinct novel circovirus species in ducks, tentatively designated as duck cirovirus 3 (DuCV3). The clinical significance and pathogenesis of this virus needs further investigation. • This study firstly identified a novel circovirus in diseased ducks, designated as duck circovirus 3 (DuCV3). • DuCV3 genome has unique features different from other known duck circoviruses. • Phylogeneticaly, DuCV3 is a distinct species located between the known duck circoviruses and goose circoviruses. [ABSTRACT FROM AUTHOR]

Duck circovirus (DuCV) is the smallest known virus in waterfowl that infects both domestic and wild duck. Infected ducks often show stunted growth and immunosuppression, which increases the rate of secondary infection with other pathogens. In this study, 270 liver tissue samples were collected to screen the presence of DuCV in Guangdong province, China, and the complete genome sequences were recovered and systematically analyzed. Genetic analyses revealed that sequences determined in this study shared 81.6% to 100.0% genome-wide pairwise identity with previously identified DuCV genomes. Phylogenetic analyses showed that 2 DuCV genotypes with a high infection rate were co-circulating in duck population in Guangdong province, and extensive recombination events have occurred during the evolution of DuCV. Our results expand upon the knowledge regarding the genetic diversity and evolution of DuCV, and also indicate that extensive genetically divergent DuCV are co-circulating in the duck populations in Guangdong, southern China. [ABSTRACT FROM AUTHOR]

Background: Duck circovirus may predispose the host to immunosuppression and may serve as an immunological trigger for further complicated disease progression. Due to the lack of a cell culture system for propagating DuCV, little is known regarding the molecular biology and pathogenesis of DuCV. The aim of this study was to describe the construction and initial in vivo characterization of full-length DNA clones of DuCV (pIC-Mu2DuCV) and its infectivity under in vivo conditions. Method: The constructed pIC-Mu2DuCV contained two copies of the whole DuCV genome and an introduced Xho I restriction enzyme site. Eighty-one 10-day-old conventional ducklings that were free of DuCV were randomly divided equally into three groups (1, 2 and 3). The ducklings in groups 1, 2 and 3 were inoculated intramuscularly with pIC-Mu2DuCV, wild-type virus GH01 and PBS, respectively. Subsequently, all of the ducklings were examined clinically, which were each given a physical condition score, and their rectal temperatures were taken daily during the experimental period. DuCV genomes in serum samples and in various tissues from all of the ducklings at 0, 1, 3, 5, 7, 10, 15, 21 and 28 DPC were detected by PCR and real-time quantitative PCR, respectively. Results: The average daily weight gain (ADWG) of group 3 was significantly higher than those of groups 1 and 2, and the temperature of all ducklings was stable between 41.7 °C and 42.2 °C. The clinical values (physical condition scores) of groups 1, 2 and 3 were 12.5, 15.6 and 0, respectively. In addition, viremia occurred at 15 and 10 days post-challenge (DPC) in groups 1 and 2, and antibodies could be detected in these ducklings at 21 and 15 DPC. Proliferation ability analysis showed that the viral titers of group 1 were lower than those of their parental viruses in group 2. Conclusion: This study shows that the rescued viruses are not significantly different but exhibit lower pathogenicity and proliferation ability compared with the parental virus. The results will facilitate future studies on DuCV pathogenesis and biology. [ABSTRACT FROM AUTHOR]