Rescue of a duck circovirus from an infectious DNA clone in ducklings

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. 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. 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. 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.


Introduction
The duck circovirus (DuCV) is a member of the genus Circovirus within the family Circoviridae. The DuCV virion is icosahedral, non-enveloped, and 15 to 16 nm in diameter [1]. DuCV was originally reported in two female 6-weekold Mulard ducks from a German farm; both ducks had a feathering disorder and poor body condition [1,2]. The virus has since been reported in Hungary [3], Taiwan [4], the US [5] and Mainland China [6][7][8][9]. DuCV has been detected in Muscovy, Mule and Pekin ducks, where it causes stunting and feather abnormalities. Although controversial, lymphoid depletion predisposes the host to immunosuppression, and disease progression is further complicated by co-infections with other bacterial and viral pathogens [2,10].
DuCV consists of a single-stranded, circular DNA genome that contains approximately 1988-1996 nucleotides (nts) and two major open reading frames (ORFs) [1]. ORF1, denoted the rep gene, encodes the replication-associated protein, which is required for viral replication initiation.
Meanwhile, ORF2, denoted the cap gene, encodes a viral structural and virulence-associated protein that stimulates the host immune response. The intergenic regions of these ORFs contain a stem loop, which is considered the site of viral DNA replication initiation [1,5].
At present, DuCV is not considered to be directly associated with a particular disease, although recent studies have suggested that DuCV partially contributes to lymphoid depletion [2], may predispose the host to immunosuppression and may serve as an immunological trigger for further complicated disease progression [3][4][5][6][7]. Indeed, DuCV-affected ducks exhibited a higher prevalence and greater loads of other bacterial and viral pathogens than non-DuCV-affected ducks [1,11]. However, the results from the above-mentioned studies do not support a direct association of DuCV with another pathogen or with host damage.
Due to the lack of a cell culture system for propagating DuCV, little is known regarding the molecular biology and pathogenesis of DuCV. To definitively characterize diseases associated with DuCV infection, an appropriate animal model is needed [12]. In addition, reverse genetics is a powerful tool for addressing these questions [13][14][15]. Because infections with multiple different genotypes or Fig. 1 Construction strategy of pIC-2DuCV. a Two full-length genomes of DuCV strain GH01, denoted IC1 and IC2, were amplified. b IC1 and IC2 were ligated into the pUC19 vector to yield pIC-1 and pIC-2, respectively. c IC-2 was ligated head-to-tail to pIC-1 to produce a tandem-dimerized DuCV DNA clone, which was denoted pIC-2DuCV. d pIC-2DuCV subtypes of DuCV are common events, a biologically pure and isolated form of a specific DuCV that is generated from a full-length infectious DNA clone is also required to study the pathology due to a single phenotype [15]. To date, no infectious DNA clones of DuCV in cultured cells or animals have been reported; therefore, it is important to construct an infectious DuCV DNA clone that can be used as a model for studying the replication and transcription mechanisms of DuCV as well as for dissecting the structural and functional relationships between host and DuCV genes. Here, we describe the construction and initial in vivo characterization of full-length DNA clones of DuCV. Furthermore, the rescue of a DuCV containing the introduced genetic markers was confirmed by sequencing of viral DNA obtained from ducks experimentally inoculated with circular DuCV genomic DNA.

Ethics statement
The experimental procedures were performed in strict accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals and were approved by the National Institute of Animal Health Animal Care and Use Committee of Sichuan Agricultural University (Approval Number 2012-032).

Viruses and animals
Duck circovirus strain GH01 (GenBank No. JX499186) was isolated and maintained at the Institute of Preventive Veterinary Medicine of Sichuan Agricultural University. A cloned strain with a genetic marker, termed RMDV, was obtained and used as the animalchallenge strain in this study to avoid contamination Fig. 2 Construction strategy of pIC-Mu2DuCV. a Two full-length genomes of DuCV strain GH01, denoted IC-Mu1 and IC-Mu2, were amplified by overlapping PCR. b IC-Mu1 and IC-Mu2 were ligated into the pUC19 vector to yield pIC-Mu1 and pIC-Mu2, respectively. c IC-Mu2 was ligated head-to-tail to pIC-Mu1 to produce a tandem-dimerized DuCV DNA clone, which was denoted pIC-Mu2DuCV. d pIC-Mu2DuCV by the parental virus and other unknown viruses. Ninety-six healthy, 10-day-old commercial ducklings were obtained from a duck farm that was negative for DuCV, as detected by PCR.

Construction of a DuCV molecular DNA clone
The full-length genome of DuCV strain GH01 was amplified by PCR using two pairs of primers, namely IC-1F/IC-1R and IC-2 F/IC-2R (Fig. 1a), and the amplification products were named IC1 and IC2, respectively. The products were subsequently inserted into a pUC19 vector (TaKaRa, Dalian, China) that had been previously digested with Hind III/BamH I or BamH I/EcoR I, respectively. The resulting constructs were termed monomeric DuCV DNA pIC-1 or pIC-2, respectively, and then transformed into Escherichia coli DH5α competent cells (Fig. 1b). The recombinant plasmids were verified by PCR, restriction enzyme digestion and DNA sequencing. The full-length IC-2 was excised from pIC-2 by digestion with the BamH I and EcoR I restriction enzymes, gel-purified and ligated head-to-tail with pIC-1 to construct a tandem-dimerized DuCV DNA clone, which was denoted pIC-2DuCV ( Fig. 1c and 1d). pIC-2DuCV was also confirmed by PCR, restriction enzyme digestion and DNA sequencing.

Introduction of genetic markers into the tandem-dimerized DuCV DNA clone
An Xho I restriction enzyme site was engineered into the DuCV genome within the pIC-2DuCV clone to introduce a genetic marker that would allow discrimination between the cloned virus and the potential indigenous viruses in the subsequent animal study. To create the unique Xho I site (C′TCGAG; mutation is underlined), an A-to-G point mutation at nucleotide position 482 of the IC-2 genome was generated by a fusion PCR technique using two pairs of primers (IC-MuF/IC-R1 and IC-F1/IC-MuR) containing the desired mutations (Fig. 2a). The corresponding region in GH01 was replaced by the fusion PCR product using the Xho I sites at both ends. The mutation did not change the putative ORFV1 or its complementary amino acid sequence. The resulting full-length DNA clones were named IC-Mu1 and IC-Mu2, respectively (Fig. 2b). All mutations were confirmed by restriction enzyme digestion and DNA sequencing. Using the same strategy, IC-Mu1 and IC-Mu2 were ligated to the pUC19 vector to produce a tandemdimerized DuCV DNA clone, denoted pIC-Mu2DuCV ( Fig. 2c and 2d).

In vivo transfection of ducklings with the DuCV molecular DNA clone
To determine whether the plasmid DNA of the dimerized DuCV or muDuCV clone was infectious when directly injected intramuscularly into ducklings, fifteen 10-day-old ducklings were randomly assigned into three rooms of five animals each, and prior to inoculation, the absence of DuCV in the ducklings was verified by PCR. The ducklings were then inoculated intramuscularly with approximately 100 μg/kg recombinant plasmid DNA pIC-2DuCV, pIC-Mu2DuCV or pUC19 vector combined with 200 μg/kg Lipofectamine 2000 (Invitrogen, Shanghai) ( Table 1). Blood samples were then collected at 0 (before inoculation), 1, 3, 7, 10, 15, 21 and 28 DPC and submitted to DuCV detection.
Eighty-one 10-day-old ducklings were divided randomly into two challenge groups (groups 1 and 2) and a control group (group 3; 27 ducklings in each group) and were raised separately in different isolation rooms with individual ventilation systems. The animals received food and water ad libitum. The group-1 and group-2 ducklings were inoculated intramuscularly with a defined dose (0.5 ml × 105 ID50/ml) of pIC-Mu2DuCV, and the ducklings in the control group were mock-inoculated with the same volume of PBS using the above-mentioned inoculation route and procedure ( Table 2). On the day of challenge and at the end of the experiment, all of the ducklings were weighed, and the average daily weight gain (ADWG) (kg/day) was determined and expressed as (body weight at 28 DPCbody weight at 0 DPC)/28. Blood samples were obtained at 0, 1, 3, 5, 7, 10, 15, 21 and 28 DPC for virological and serological examination. After challenge, all of the ducklings were examined clinically, and their rectal temperatures were taken daily during the experimental period. At each examination point, three randomly selected ducklings were killed and subjected to postmortem examinations, determination of any gross lesions, and pathological examinations.

Clinical examination
The ducklings of the above-mentioned DuCV-challenged groups and of the control group were examined clinically at 0, 1, 3, 5, 7, 10, 15, 21 and 28 DPC and were each given a physical condition score. The evaluated clinical signs included the presence of wasting, feathering disorder and depression. The clinical parameters were scored using a numeric value ranging from 0 to 2 (0 = normal, 1 = mild, 2 = severe).

Serology for DuCV antibody detection
The presence of specific antibodies against DuCV in serum samples obtained throughout the study at 0, 1, 3, 5, 7, 10, 15, 21 and 28 DPC was determined by ELISA using a protocol established by our laboratory (data not published). ELISA was also used to detect changes in the DuCV-specific antibodies over time in vivo post DuCV challenge. Ninety-six-well ELISA plates (Corning, USA) were coated with optimized prepared Cap peptide solution (data not published) and incubated at 4°C overnight. After washing three times with PBS containing 0.05 % Tween-20 (PBST), the plates were blocked with 1 % bovine serum albumin (BSA) in PBS for 1 h at 37°C. Following this incubation, the wells were washed three times with PBST, duck serum diluted with 1 % BSA-PBST (1:50) was added, and the plates were incubated at 37°C for 1 h. After three washes, as described above, HRPlabeled goat anti-duck IgG (1:5000) was added, and the plates were incubated at 37°C for 1 h. After three additional washes, 100 μL of TMB was added to each well, and the plates were incubated at 37°C for 20 min for color development. The color-development reaction was terminated by adding 100 μL of 2 M H 2 SO 4 to each well, and the absorbance of each well at a wavelength of 450 nm (OD 450 nm ) was determined using a microplate reader (Bio-Rad).   Table 4 The results of transfection experiment

Statistical analysis
Comparisons of a single treatment among the different challenge groups (using the ADWG, ELISA and qPCR results) were all performed using nonparametric oneway ANOVA followed by LSD multiple comparisons. The statistical analysis of the data was performed using SPSS for Windows (version 16.0), and P < 0.05 was considered statistically significant in all cases.

Construction of an infectious viral clone
We first generated two monomeric, full-length DuCV DNA clones, denoted IC-1 and IC-2, that were derived from the prototype Sichuan isolate GH01 ( Fig. 1a and  1b). Each full-length DuCV genome was inserted into a pUC19 vector containing a eukaryotic promoter. Hind III/BamH I and BamH I/EcoR I, which were unique restriction sites in the pIC-1 and pIC-2 genomes, respectively, were incorporated at the ends of the genomic DNA to facilitate the generation of concatemers and thus mimic the DuCV circular DNA genome. Double digestion of the plasmid DNA of each clone with BamH I or EcoR I resulted in 2.6-kb, 2-kb, and 4.6-kb fragments. The 2.6-kb fragment represented the backbone vector, whereas the 2-kb fragment represented the inserted monomeric DuCV genomic DNA. The 4.6-kb fragment consisted of the linearized backbone vector with the  The clinical parameters were scored using a numeric value ranging from 0 to 2 (0 = normal, 1 = mild, 2 = severe); number of elements × scores b Pairs of treatments with (*) were significantly different with control group (p < 0.05) inserted monomeric DuCV genomic DNA. Subsequently, the 4.6-kb and 2-kb fragments were ligated in tandem to generate the pIC-2DuCV clone, and the recombinant plasmids were verified by PCR, restriction enzyme digestion and DNA sequencing (Fig. 3). A fusion PCR technique using two pairs of primers (IC-MuF/IC-R1 and IC-F1/IC-MuR) containing the desired mutations was utilized to create the unique Xho I site (Fig. 2a). The resulting fusion PCR products were named IC-Mu1 and IC-Mu2, respectively ( Fig. 2a and 2b), and processing was continued using the above-described technique to produce a tandem-dimerized DuCV DNA clone, which was denoted pIC-Mu2DuCV ( Fig. 2c and  2d). All mutagenesis was confirmed by restriction enzyme digestion and DNA sequencing (Fig. 4).
The viral nucleic acids in the serum in the challenged groups were detected at 15 DPC using PCR (Table 4), and restriction enzyme digestion and DNA sequencing were performed to differentiate the rescued virus from the parental virus (Fig. 5). The results from these experiments indicated that the constructed full-length cDNA clones were infectious and that viable viruses could be recovered; the rescued virus containing the genetic marker was named RMDV.
Because the genetic marker that was introduced into the full-length DNA clones can be used to distinguish between infections caused by the cloned virus or by a potential indigenous contaminating virus, we performed a study to further verify the in vivo infectivity of the RMDV rescue mutant virus in ducklings. Eighty-one ducklings were assigned to three groups of 27 ducklings each, and the ducklings in each group were inoculated with RMDV, WT-DuCV or PBS.

Clinical examination
No apparent gross lesions were observed in the control group, but clinical signs characterized by wasting, feathering disorder and depression were observed mainly from 10 to 28 DPC. The mean clinical score (CS) was Fig. 6 Changes in the rectal temperatures of the ducklings from the different experimental groups. The data are presented as the means ± SD Fig. 7 Comparison of the average daily weight gains for the different experimental groups. *indicates a statistically significant difference (P < 0.05) significantly higher (P < 0.05) in the RMDV-and WT-DuCV-challenged groups compared with the PBSchallenged group throughout the study period, but no significant differences were observed between the RMDV-and WT-DuCV-challenged groups (P > 0.05). The detailed CS values are shown in Table 5.
The rectal temperatures of the ducklings in both of the challenge groups and in the control group did not exceed 42.2°C up to the termination of the experiment (Fig. 6).
As shown in Fig. 7, the ADWG was higher in the control group than in the challenge groups, and this difference was statistically significant (P < 0.05). Moreover, no significant difference in ADWG was observed between the RMDV-challenged group and the WT-DuCV-challenged group (P > 0.05), which could represent persuasive proof of the similar virulence of RMDV and WT-DuCV. The results confirmed similar virulence of RMDC as WT-DuCV in ducklings, but further demonstrations are required.

Viremia
To detect viremia, the viral DNA from serum samples was collected at 0, 1, 3, 5, 7, 10, 15, 21 and 28 DPC and investigated using routine PCR. Moreover, restriction enzyme digestion and DNA sequencing were performed to differentiate the rescued virus from the parental virus (data not shown). No viremia was detected in the ducklings of the control group, whereas ducklings challenged with RMDV showed viremia from 15 to 28 DPC. In addition, earlier and more severe viremia was observed in the WT-DuCV-challenged group compared with the RMDV-challenged group ( Table 6).

Detection of DuCV-specific antibody
ELISAs were used to investigate the level of DuCVspecific antibodies in the serum of ducklings from the DuCV-challenged and control groups. As shown in Fig. 8, DuCV-specific antibodies appeared at approximately 21 DPC in RMDV-challenged ducklings and at approximately 15 DPC in WT-DuCV-challenged ducklings, and the antibody titers increased slightly. In contrast, no DuCVspecific antibodies were detected in the control-group ducklings.

Virus distribution and quantification in different tissues, as shown by real-time quantitative PCR (qPCR)
DuCV was localized mainly in the bursa of Fabricius (BF), spleen, liver, kidney, thymus, and Harderian gland of the ducklings. The earliest time point at which virus was observed in the serum of RMDV-challenged ducklings was 15 DPC, which was 5 days later than the earliest time point at which virus was observed in WT-DuCVchallenged ducklings; however, the viral loads of RMDV-challenged ducklings at 15 DPC were lower than 10 4 copies/mg. Meanwhile, the viral loads in the BF of all of the challenged groups were significantly greater than those of the other tissues (P < 0.05) and exceeded 10 4 copies/mg or 10 5 copies/mg, reaching  the highest load at 21 DPC (4.72 × 10 4 -8.34 × 10 4 copies/ mg in the RMDV-challenged group and 3.25 × 10 55 .47 × 10 5 copies/mg in the WT-DuCV-challenged group). In addition, no virus was detected in the control group. The tissue sample qPCR results are summarized in Table 7.

Discussion
Due to the lack of a cell culture system for propagating DuCV, many features of the genomic structure, function and molecular biology of DuCV remain unknown, even though DuCV was identified more than ten years ago. In particular, the molecular basis of the pathogenesis of this virus is unclear.
Reverse genetics is a powerful tool for addressing these questions, and in this study, we demonstrated the first generation of an infectious DNA clone of Sichuan isolate GH01. The main aim of our work was to produce a tandem-dimerized DuCV DNA clone. We further inserted an Xho I restriction enzyme site into the DuCV genome of the pIC-2DuCV clone to introduce a genetic marker that could be used to discriminate between the cloned virus and the potential indigenous viruses in the subsequent animal study. The recombinant pIC-2DuCV and pIC-Mu2DuCV plasmids and the pUC19 vector were inoculated intramuscularly into five ducklings. It appeared that both the pIC-2DuCV and pIC-Mu2DuCV DNA concatemers were replicationcompetent when transfected in vivo because they mimicked the natural DuCV circular genome. The rescue of pIC-2DuCV and pIC-Mu2DuCV was then demonstrated through in vivo animal experiments.

Conclusion
In conclusion, we constructed a genetically stable and similarly pathogenic infectious clone of DuCV, which will facilitate studies of the pathogenesis, host tropism, replication, and transcription of this virus. This study shows that the rescued viruses are not significantly different but exhibit lower pathogenicity and proliferation ability compared with the parental virus. Further study is needed to explore the replication, host-DuCV interactions, and clinical significance of RMDV and WT-DuCV. In summary, we report the first demonstration that cloned DuCV genomic DNA with a genetic marker is infectious when directly injected into ducklings. In addition, we confirmed that the clinical signs of the ducklings injected with RMDV are similar to those of ducklings injected with WT-DuCV.