Circovirus are small, non-enveloped viruses composed of icosahedra particles with a diameter of 17-26.5 nm and a circular single-stranded DNA genome, approximately 2kb in size [9, 28]. Currently, the family Circoviridae comprises two genera Gyrovirus and Circovirus. To date, the genus Gyrovirus contains only one member, the chicken anaemia virus (CAV) . Within the genus Circovirus there are several members, including porcine circovirus types 1 and 2 (PCV 1 and PCV 2) , psittactine beak and feather disease virus (BFDV) , pigeon circovirus (PiCV, also known as columbid circorus, CoCV) [13, 27], canary circovirus (CaCV) [14, 26], goose circovirus (GoCV) [21, 27], duck circovirus (DuCV) , raven circovirus (RaCV) , starling circovirus (StCV) , finch circovirus (FiCV), gull circovirus (GuCV) , ostrich (Struthio camelus) (OCV) and the recently identified circoviruses infecting mute swan (Cygnus olor) (SwCV) .
Duck circovirus (DuCV) was first reported in Germany in 2003 [8, 20]. Since then, DuCV has subsequently been reported in Hungary , Taiwan  and the USA . We reported the first detection of DuCV in the mainland China in Fujian Province , since then DuCV has been reported in Shandong (SD-LY0701), Guangdong , Zhejiang (this study) and Jiangsu (JS-AQ0901) provinces. To better understand the characteristics of DuCV in Southern China, we report here the detection of DuCV at different farms in Southern China, and the complete nucleotide sequences of 10 strains. Comparative analysis was conducted with DuCVs reported from Taiwan, Shangdong, Guangdong, USA and Germany.
A total of 138 ducks were collected from 18 different duck farms located in different areas of Fujian, Zhejiang and Guangdong Province in Southern China from March 2006 to December 2009 (Table 1). In general, there was no obvious clinical abnormities among the sampled duck flocks, although sporadic deaths and growth retardation in a small number of ducks were often observed.
Table 1. Detection of DuCV from different regions in Southern China*
DNA was isolated from a mixture of the lung, liver, spleen and fabricius bursa of each sample using a DNeasy Blood & Tissue kit (Qiagen, Germany) according to the manufacturer's instructions. Total DNA was precipitated and used for DuCV examination, and each DNA sample was defined as a virus strain.
A primer pair was designed according to Chen , with the following sequences for forward: 5'-ATATT ATTACCGGCGC(C/T) TGTA-3' and reverse: 5'-TC AGGAATCCCTG(A/C)AGGTGA-3'. The targeted amplificon is a 228-bp segment of DuCV genome.
A total of 138 clinic samples were examined using a GoTaq Green Master Mix (Promega, USA) according to the manufacturer's instructions. The PCR mixture (50μL) contained 25μL GoTaq Green Master Mix, 22μL distilled water, 1μL primers (20 pmol each) and 1 μL of template DNA. PCR condition was 1 cycle of 94 ℃ for 5 min; 35 cycles of 94 ℃ 30 s (melting), 54 ℃ 30 s (annealing), and 72 ℃ 30 s (extension) and a final extension period of 72 ℃ for 5 min. The PCR product was separated by electrophoresis in a 1.5% agarose gel. The specific PCR products were purified using an agarose gel DNA purification kit (Takara, Dalian) according to the manufacture's instructions. Purified DNA samples were cloned using a pMD18-T kit (TaKaRa, Dalian) and then trans-formed into Escherichia coli DH5α cells. The positive recombinant clones were sequenced to confirm the sequences.
The embryonated eggs of Ducks, Muscovy ducks and Mule ducks (20 for each), non-embryonated budgerigar eggs (15 each) were collected in Fujian Province, together with a mixture of the lung, liver and spleen of newly hatched animals (15 each). These samples were analyzed for the presence of DuCV DNA using the same method as described above.
DuCV was examined using a GoTaq Green Master Mix (Promega, USA) according to the manufacturer's instructions using the following primer pair 5'-CAAT GGCGAAGAGCGGCAACTACT-3' (forward) and 5'-AGCTGCCCAAGTGTTTAATCCCT-3' (reverse). The reaction mixtures contained 1 μL of template DNA. PCR thermal cycles were denaturation at 95 ℃ for 5 min, 35 cycles each comprising 94 ℃ for 50 s (melting), 55 ℃ for 30 s (annealing), and 72 ℃ extension 90 s (elongation) of amplification and a final extension period of 72 ℃ for 10 min, although small variations in the annealing temperature were used according to the manufacturer's directions. The DuCV-sepecfic PCR product was cloned into pMD18-T and sequenced. Based on the determined sequence, another primer pair 5'-CCCAATAAAC TACTGAGACGAA-3' (forward) and 5'-GAA CATG AACATAATCTTCAAAGG -3' (reverse) was used to amply the remaining genomic sequences by inverse-PCR. Also, the DuCV-sepecfic PCR product was cloned into pMD18-T and sequenced.
The complete genome sequences of DuCV were obtained by assembling sequences of the continuous overlapping PCR products using the Lasergene software package (DNASTAR Version 7.0 Madison, WI, USA). Nucleotide sequence alignments were performed using the ClustalW method with the MegAlign program in the Lasergene software package.
A total of 23 sequences were used for homology analysis and phylogeny construction (Table 2). Sequence alignment and phylogenetic analysis were performed using MEGA 4 and the Neighbor-Joining method . The numbers on the branches represented bootstrap support for 1 000 replicates. The sequence of goose circovirus (GenBank accession number GU320569) was used as the out-group.
Table 2. List of all DuCVs used in this study
The age ranges of the 138 samples are detailed in Table 1 and Table 3. 11 out of 46 samples tested positive for ages of 0 to 4 weeks, 26 of 65 samples for 4 to 8 weeks, and 12 of 27 samples for those more than 8 weeks. Overall the DuCV infection rate was 35.5%. The results suggested that ducks at the age of more than 4 weeks were more susceptible to the DuCV infection(P < 0.05). It is interesting to note that ducks in most PCR-positive farms exhibited no apparent clinical abnormality, indicating that the infection of DuCV may be subclinical. Meanwhile, no DuCV-specific amplicon was obtained from all the vertical transmission samples.
Table 3. Age distribution of DuCV positive ducks isolated in this study
The 10 DuCVs sequenced in this study had genome length varying from 1988 to 1996 nt (Table 1). The sequences were deposited in GenBank (see table 2 for accession numbers). The overall genome nt identity of the strains ranged between 83.2% to 99.8 % (data not shown).
Sequence analysis indicated that there were two major open reading frames (ORFs) in the genome of DuCVs: one was Rep, coding for the replicating proteins, and the other was Cap, coding for the capsid protein. The Rep genes of SD-LY0701, FJ0601 , FQ312 and PT60 (this study) encode a 297-aa protein and start at 33 nt, and these four strains all have 1988-nt complete genome, while Reps from other strains were 292-aa. The Cap proteins of all strains were 257-aa in length. The 3' intergenic region, which is defined as being located between the termination codons of the major V1 and C1 ORFs, were about 110-111 nt for most DuCVs, but 96 nt for SD-LY0701, FJ0601, FQ312 and PT60.
Based on the complete nucleotide sequences of 10 isolates in this study and 3 Taiwan strains, 1 German strain, 1 USA strain and others representative DuCVs (Table 2), a phylogenetic tree was constructed by the MEGA 4 Neighbor-Joining method. The sequence of GoCV was used as the out-group. The results showed that the DuCVs could be divided into two distinct genetic groups, the Euro-USA lineage (detected in Germany mullard duck and the USA Pekin duck) and the Taiwan lineage (detected in Taiwan Muscovy duck) (Fig. 1), with about 10.0% sequence identity difference between the two subgroups (data not shown).
Figure 1. Phylogenetic analysis of 23 DuCV genome nucleotide sequences. The tree is based on the DuCVs (Table 2) was constructed with the MEGA 4.0 Neighbor-Joining method. The GoCV was used as the out-group. The taxon Separation is 24 pixels, the tree width is 400 pixels, and the SBL is 0.46806491.
Primers used for Epidemiological investigation
Epidemiological investigation for field samples
Vertical transmission investigation
Primers used for amplification the complete genomes
Analysis of the DuCV complete genome sequences
Genomes organizations and overall similarity
Phylogenetic analysis of the complete genome of DuCVs
Because no in vivo culture system is available for the propagation of members in the genus Circovirus except for PCV1 and PCV2, polymerase chain reaction (PCR) based methods, including both real time PCR and nested PCR [2, 5, 7, 11], represent important tools for DuCV epidemiological investigation, and was the technology platform used for our current study.
The epidemiological investigation results showed the ducks at an age of more than 4 weeks were more susceptible to DuCV. Our results suggest that that infection of DuCV was mostly subclinical, which is different from previously published reports claiming obvious association of DuCV infection with clinical characteristics, e.g. a feathering disorder, poor body condition and low weight, which may induce damage to lymphoid tissue and immunosuppression [20, 28, 29]. There were also reports indicating that ducks positive for DuCV by PCR were more susceptible to co-infection with RA (Riemerella anatipestifer), E. coli (Escherichia coli), Duck Cholera (Pasteurella multocida), DHV-I (Duck hepatitis virus-I), AIV (Avian influenza virus), DPV (Duck parvovirus), Avian paramyxovirus type-1(APMV-1) and/or DRV (Duck reovirus) . Due to the lack of a commercial vaccine at present, there is no effective measure to control the infection of DuCV and the associated co-infection of other pathogens. Previous studies found that BFDV DNA was detected in both embryonated eggs and non-embryonated budgerigar eggs, suggesting that the BFDV can be transmitted horizontally and vertically . However, no DuCV-specific amplicon was obtained from the vertical transmission investigations.
Data obtained from this study and previous studies indicate that positive detection was observed in different species, including the Muscovy duck, the Mule duck, the Cheery Duck, the Mullard Duck and also the Pekin Duck in different regions and countries, suggesting that DuCV infection were ubiquitous, showing no species dependency.
In general, genetic variation of virus genome may lead to the change of their pathogenicity . This study revealed an obvious difference in the Rep protein, with those of SD-LY0701, FJ0601, FQ312 and PT60 coding 297-aa whiles other strains coding for a 292-aa Rep protein due to a different translational initiation site. Further investigation is warranted to examine whether this difference will result in any biological changes, such as changes in replication or virulence.
However, there are several differences among the DuCV genomes. There are some similar regions among the genus Circovirus. A stem-loop structure (Table 4) with a highly conserved sequence of nine nucleotides nonamer "TAT TATT①AC"(①position 1) was found in the untranslated region between the two DuCV ORFs (5' intergenic regions) when all DuCV sequences in GenBank were compared (regardless of geographical origin and species). While the PCV1, PCV2, GuCV, PiCV and FiCV had nine nucleotides "NANTATT①AC" (①position 1).When designing a diagnostic PCR test for the genus Circovirus, it is important to examine all available sequences of known isolates to avoid false negative results [9, 24], these genomic characteristics may be used to develop a broad-spectrum PCR method for identifying circovirus in the genus Circovirus from other avian species.
Table 4. Homology of the nonamer sequence amongst circoviruses
Analysis of the complete genomes sequences showed that there were two distinct groups located in two genotype branches of the phylogenetic tree: the Euro-USA lineage containing DuCV detected in Germany, USA, and the mainland China, which the genomes length were longer than 1988nt; while the other is the Taiwan lineage, consisting with the strains found in the Taiwan area and the mainland China, which the genomes length were all 1988nt, including the coding 297-aa Rep protein strains SD-LY0701, FJ0601, FQ312 and PT60. The two lineages displayed an overall sequence difference of approximately 10.0% across the genome. The phylogenetic analysis demonstrated that there maybe two distinguish origin and evolution of DuCV, while these two lineage were co-circulating in mainland China. Earlier clinic samples and more species need to be examined to determine the exact origin.