Members of the Circoviridae family are small, non-enveloped, spherical viruses which contain circular, single-stranded DNA genomes. This family comprises two genera, Gyrovirus and Circovirus [27, 28]. Chicken anemia virus (CAV) is the only member of the Gyrovirus , while the Circovirus genus has many members including Porcine circovirus type Ⅰ (PCV-1) and Porcine circovirus type Ⅱ (PCV-2) [8, 18], psittacine beak and feather disease virus (BFDV) , Gull circovirus (GuCV) , pigeon circovirus (PiCV) or columbid circovirus (CoCV) [17, 21, 33], canary circovirus (CaCV) [22, 29], goose circovirus (GoCV) , Duck circovirus (DuCV) , Finch circovirus (FiCV) , and so on.
DuCV was detected from a mulard duck for the first time in German and the complete genome was sequenced [9, 25]. Later, DuCV were successively identified from some sick Muscovy ducks in Hungary , Taiwan of China  and China , Pekin ducks in America , and Cherry Valley ducks in China . The birds infected by DuCV were in a poor body condition and showed marked feather dystrophy with haemorrhagic shafts to many feathers, especially in the dorsal part. Histopathological examination of the dorsal skin revealed a mainly heterophilic inflamma tory infiltration of the follicular and perifollicular tissue. The gross pathological appearance of the internal organs was attributable to a concurrent infection by Riemerella anatipestifer. Histopathological examination of bursal tissue demonstrated lymphocytic depletion, necrosis and histiocytosis . Since there is no in vitro culture method for DuCV detection, the study of DuCV mostly depends on molecular approaches, such as PCR, real time PCR or nucleotide sequencing [5-7, 9, 11, 25, 31, 35].
In this study, we sequenced the complete genome of six DuCV strains which were found in Cherry Valley ducks in China between 2007 and 2008. Sequence and phylogenetic analysis were carried out to compare the six strains with other DuCVs, including strains from Mulard duck, Muscovy duck, Pekin ducks and Mule duck. The results from this study will aid a better understanding of DuCV in Cherry Valley ducks.
The bursa of Fabricius (BF) of Cherry Valley ducks were collected from six commercial farms in Shandong Province of China between 2007 and 2008. These ducks, aged between 4 and 8 weeks, showed feathering disorders, poor body condition and low weight under field conditions.
BF (0.1g) was homogenized with 500 µL sterile 0.9% NaCl. DNA was extracted using DNAzol reagent (Invitrogen, USA) according to the manufacturer's instructions. Briefly, homogenate (500 µL) was combined with 2× volume DNAzol reagent and incubated at room temperature for 10 min followed by centrifugation at 10, 000 rmp for 10 min.
Based on the published sequence of DuCV, four pairs of primers were designed to amplify four overlapping fragments that covered the complete genome of DuCV . Sequences of the four pairs primers were:
P3f5'-CCAATAAACTACTGAGAC-3'/P3r5'-ATC GGCGTGCATATCGTG-3' and
A Taq PCR Master Mix Kit (QIAgen, Germany) was used to amplify four segments of DuCV following the manufacturer's instructions. Briefly, 2 μL of extracted DNA was added to the reaction mixture with final concentrations of 2.5 mmol/L MgCl2, 1×PCR buffer, 0.3 mmol/L dNTPs, 0.5 µL of each primer (100 ng/µL) and 0.2 µL of Taq DNA polymerase (5 U/µL) per 25 μL. DNA was amplified for 33 cycles with the following parameters: a first denaturation at 94 ℃ for 5 min, followed by denaturation at 95 ℃ for 50 s, annealing at 55 ℃ for 40 s and elongation at 72 ℃ for 1 min; and a final extension at 72 ℃ for 10 min. PCR products were visualized in a 1.0% agarose gel and stained with ethidium bromide.
PCR products were purified using a Gel Purification Kit (Invitrogen, USA), and the purified PCR-products were TA-cloned into pMD18-T vector (TaKaRa, China) and transformed into a DH5a competent cell according to the manufacturer's protocol. To avoid base mismatching resulting from PCR, three clones were selected to sequence for each fragment. The nucleotide (nt) sequences of the positive clones were determined using T7 and SP6 sequence primers from a commercial service (Shanghai Sangon Biological Engineering Technology & Service Co., Ltd).
The complete genome sequences of the six DuCV strains examined in this study have been deposited into GenBank under accession numbers EU022374, EU022375 and GU131340 to GU131343. The acces sion numbers of all sequences used in this study are given in Table 1. Nucleotide and amino acid sequence alignments were performed by the Clustal W method with Megalin 5.08 (DNAStar Inc., USA). Phylogenetic trees were constructed from the aligned 33 strains' nucleotide sequences and polyprotein amino acid sequences of DuCV and by the Clustal W method with 1000 bootstrap replicates.
Table 1. General information and accession number of DuCV isolates used in this study
Polymerase chain reaction (PCR)
Cloning and sequencing of the genomic sequences
Sequence analysis of the genome sequence
The complete genome sequences of the six DuCV strains from Cherry Valley ducks in this study contained 1988, 1991 and 1995 nucleotides (Table 1) respectively. Like other members of the DuCV family, the genomes of the six strains contained two major ORFs encoding replicase protein (Rep) and capsid protein (Cap). Two minor ORFs were identified in the complementary strand. The ORFC2 was observed in three DuCV strains (WF0701, WF0801 and WF0803), whereas the ORF3 was observed in all the six DuCV strains. The other two minor ORFs were identified only in the viral strand of WF0701 (Table 2). The initiation codons of the two major ORFs varied in the six DuCV strains. In the intergenic region between Rep and Cap genes, there existed a loop-stem structure with the nonanucleotide motif TATTATTAC. In the up and down streams of the nucleotide sequence, there were two inverted repeat sequences (10 bp in length). After the stem-loop structure, two tandem arranged direct repeats of the hexamer GTACTC were found from nt 11 to 17 and 18 to 23. As in all the other DuCV strains, the genomes of the six strains contained four copies of repeat sequences (CACTTGGGCAG or CACTTGGCAG).
Table 2. Genomic organization analysis of the six DuCV isolates in this study
As shown in Fig. 1A, all 33 available complete genome sequences were clustered into two clear genetic groups, strongly supported by 100% bootstrap values. The nucleotide identities in DuCV complete genomes within the same group were at least 91.9%, whereas those between members of the two groups did not exceed 86.8% (Table 3). We labelled the genetic groups DuCV genotypes A and B. Genotype A, represented by nineteen DuCV strains with genomes of 1988 and 1989 nucleotides, included three DuCV strains (LY0701, WF0802 and WF0804) examined in this study and another sixteen viruses from Taiwan and China [5, 11, 31]. Genotype B contained fourteen DuCV strains whose genome were 1991, 1992, 1995 and 1996 nucleotides and the other three DuCV strains (WF0701, WF0801 and WF0803) determined in this study and another sixteen viruses from Germany, America and China [3, 7, 9] (http://www.ncbi.nlm.nih.gov/Genbank/index.html).
Figure 1. The phylogenetic trees of DuCV isolates based on the nucleotide sequences of complete genome (A), the complete nucleotide sequences of Cap gene (B), the complete amino acid sequences of Rep gene (C) and the complete amino acid sequences of ORF3 gene(D). Numbers at nodes indicate bootstrap percentages obtained after 1 000 replicates. Genotypes are indicated on the right side of the trees. The bar indicates genetic distance and *, the strains sequenced in this study.
Table 3. Percentage nucleotide (nt) and amino acid (aa) identity in the complete genome and Cap gene within and between DuCV genotypes, Rep gene and ORF3 gene
The Caps of all the 33 DuCVs consisted of 794 nucleotides and encoded 257 amino acid residues. The phylogenetic trees constructed by the Cap (Fig. 1B) nucleotide sequences of 33 DuCVs further confirmed the existence of the same two genotypes defined by the complete genome analysis, which contained the same DuCVs as the complete sequences. The nucleotide and amino acid identities in Cap region within the same genotype were from 88.8% to 99.7%, whereas those between members of the two genotypes were from 80.2% to 89.5%, respectively (Table 3). Comparisons of deduced amino acid sequences of the 33 available Cap revealed there were 22 genotype-associated alterations in capsid protein between the two genotypes of DuCVs (Fig. 2).
In the six DuCV strains, the Reps of four DuCVs consisted of 879 nucleotides and encoded 292 amino acid residues, whereas the Reps of the other two DuCVs (LY0701 and WF0802) consisted of 894 nucleotides and encoded 297 amino acid residues (Table 2). The phylogenetic trees constructed by the Rep (Fig. 1C) amino acid sequences of 33 DuCVs showed the existence of two genotypes, but these differed from the two genotypes defined by the complete genome and Cap gene analysis. The nucleotide and amino acid identities in Rep region within the same genotype were from 91.9% to 100% and from 96.6% to 100%, whereas those between members of any of the two genotypes were from 87.1% to 93.7% and from 92.6% to 96.6%, respectively (Table 4). The conserved amino acid motifs associated with rolling circle replication (Ⅰ, Ⅱ, and Ⅲ) were indicated by shadowed boxes, and the dNTP binding domain (A) was similarily indicated (Fig. 3).
Figure 3. Deduced amino acid sequences of the Rep proteins from the six DuCVs. Conserved amino acid motifs associated with rolling circle replication (Ⅰ, Ⅱ, and Ⅲ) are indicated by shadowed boxes, and the the dNTP binding domain (A) is similarily indicated. A dot indicates a position where the amino acid is identical to that of the majority. Alignments were prepared using Megalin Clustal W Method (Dnastar Inc.).
Table 4. Percentage nucleotide (nt) and amino acid (aa) identity in the complete Rep gene and ORF3 gene within and between DuCV genotypes
The ORF3 was identified in all the 33 DuCV strains. Based on the complete amino acid sequences, the 33 ORF3 genes were clustered into the same two genotypes defined by the analysis of the complete amino acid sequence of Rep gene (Fig. 1D). The nucleotide and amino acid identities in ORF3 gene within the same genotype were higher than 97% and 91.1%, whereas those between members of any of the two genotypes were only between 88.6% to 91.1% and 73.4% to 81%, respectively (Table 4). In this study, the ORF3 genes of three DuCVs (WF0701, WF0801 and WF0803) belonging to genotype 1 consisted of 237 nucleotides and encoded 78 amino acid residues, whereas the ORF3s of the other three DuCVs (LY0701, WF0802 and WF0804) belonging to genotype 2 consisted of 297 nucleotides and encoded 98 amino acid residues (Fig. 4).