MATERIALS AND METHODS

Total RNA preparation: Total RNA was extracted from the virus-infected Vero cells using QIAamp viral RNA mini kit (Qiagen) according to the manufacturer’s instruction.

Primer pair and PCR amplification: To understand the genetic characteristics of the newly isolated CDV strains, nine pairs of primers covering the whole genome were designed according to reference CDV sequences from GenBank (Fig. 1). RT-PCR was carried out using mRNA as template.

The PCR reactions contained 2 μL DNA Template, 2 μL primer pairs, 2 μL dNTPs (TaKaRa, Dalian, China), 2.5 μL 10xEx Taq buffer and 0.5 U Ex Taq polymerase (TaKaRa, Dalian, China) in a total volume of 25 μL. The PCR amplification was initiated by a pre-denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 94°C for 30 sec, annealing at temperatures 55°C for 30 sec and an extension at 72°C for 30 or 90 sec (depending on the amplified fragment length). Subsequently, the target fragment of PCR products were purified and cloned into pMD18-T vector (TaKaRa, Japan) and then transfected into E. coli DH5α cells. The positive clones were selected for sequencing by Shanghai Invitrogen Biotechnology Co., Ltd. The sequence assembly was carried out using the SeqMan program of the DNASTAR Software (Madison, WI).

Sequences alignment analysis: To investigate the differences between the published main sequences of the CDV wild strain and vaccine strain. Multiple sequence alignment was carried out using the software package MegAlign (DNASTAR).

Amino acid analysis: The potential N-linked glycosylation sites of the H protein were determined using the software NetNGlyc1.0. Phylogenetic trees were constructed based on the H, F, N and whole genome by the neighbor-joining method and a bootstrap analysis with 1000 replicates was done to assess the confidence level of the branch pattern. Bootstrap values >70% were considered to be significant.

RESULTS AND DISCUSSION

In the present study, 9 cDNA fragments covering genome and whole length genome sequence were obtained by using RT-PCR. The full-length genome sequence was obtained by cloning, sequencing and splicing of the 9 overlapping gene fragments. The sequence named CDV-RD-JL strains was submitted to GenBank and the accession number is KJ848781.

Sequence analysis results showed that the complete genome of CDV-RD-JL was 15690 nt and the 3’ end leader sequence and the 5’ end tailer sequence were composed of 52 and 38 nucleotides, respectively. The N, P, M, F, H and L gene sizes were 1683, 1655, 1447, 2209, 1946, 6642 nt.

Fig. 1:	Schema graph of nine pairs of primers covering the whole genome

M and F genes were spaced by a length of about 400 bp non coding region. The recently research demonstrated that the spacer region played an important role in the regulation of F protein expression and control of virus virulence (Avila et al., 2014; Imhoff et al., 2007).

The sequence alignment analysis of the 15 different sources of CDV whole genomes deposited in the GenBank showed that the sequence homology of the genome sequences of the CDV-RD-JL strain and Asia-1 type CDV strains was as high as 98.1-98.8%. The genome homology of the CDV genomes in the present study with the other Genotype CDV strains was 92.9-97%. The genome homology of the CDV with Onderstepoort isolate was the lowest and genome homology of the CDV with HLJ1-06 and CDV SY in China were the highest. The identity values with CDV other reference sequences ranged from 93.2-98.9% for N gene, 93.8-99.0% for P gene, 90.6-98.4% for H gene and 90.6-98.1% for F gene, respectively (Table 1).

The CDV H protein N-linked glycosylation site prediction showed that there were 9 potential N-linked glycosylation sites in the CDV-RD-JL strain located in the amino acids 19-21, 149-151, 309-311, 391-393, 422-424, 456-458, 584-586, 587-589 and 603-605 (Table 2). Among them, six glycosylation sites (positions 19-21, 149-151, 391-393, 422-424, 456-458 and 587-590) were conserved for all of the CDV strains. The potential N-linked glycosylation sites number is the same as CDV Asian-1 strains in China such as strain HLJ1-06 and strain Hebei.

Phylogenetic trees were constructed based on the H, F, N and whole genome (Fig. 2). Results showed that the CDV strain CDV-RD-JL having highest identity with the strain HeBei (99.0%) originated from China in 2008, belonging to CDV Asia-1 type. The phylogenetic tree for the H, F and N regions had a topology similar to that of whole genome which clustered with CDV Asia-1 type.

With the rapid development of Fur-animal farm, the number of Chinese raccoon dog reached 8-15 million. Emergence of the CDV infection was more complex and need more attention to highlight the infection of CDV.

Table 1:	Nucleotide identity of CDV-RD-JL with reference sequences
ND: Not determined

Fig. 2(a-d):	Phylogenetic trees of (a) H, (b) F, (c) N and (d) Whole genome

Table 2:	CDV H protein N- linked glycosylation site prediction
*Potential N-link glycosylation sites (N-X-S/T), Among them, the 309-311 N-glycosylation site, which is specific for the wild-type strain, is boxed in red and the 584-586 N-glycosylation site, acquired in the Asian-1 strains

This new finding give us a support to pay more attention to the CDV in China. It should take appropriate measures to control the disease and take a surveillance program in a larger Chinese raccoon dog population in vast geographical areas.

ACKNOWLEDGMENT

The present study was partly financially supported by the Ji Lin Province Major Scientific and Technological Achievements Transformation Program (No. 10ZDZH010) and (No. 20140204066NY).