Molecular surveillance of canine distemper virus in diarrhoetic puppies in northeast China from May 2014 to April 2015

LI, Chunqiu; GUO, Donghua; WU, Rui; KONG, Fanzhi; ZHAI, Junjun; YUAN, Dongwei; SUN, Dongbo

To trace evolution of the CDV strains identified in our study, we attempted to perform the sequencing of the complete H gene of the 50 identified CDV strains according to the protocol described by Budaszewski Rda et al. [3] . The first round of amplification was conducted by using primer pair RH3-F and RH4-R, and the nested PCR was performed by using inner primer pairs H1F/H1R, H2F/H2RB, CDVF10B/CDVR10 and H3FB/H3R generating overlapping fragments of 789, 700, 870 and 542 bp, respectively. The amplified PCR products for the complete H genes were directly subjected to Sanger sequencing. Sequence analysis was carried out by using the EditSeq program in the Lasergene DNASTARâ„¢ version 5.06 software). All nucleotide sequences generated in our study were submitted to GenBank. Furthermore, phylogenetic tree of the complete H genes was generated from the ClustalXgenerated alignments by MEGA6.06 software using the neighbor-joining method [15] . Result indicated that a total of six complete H genes were successfully amplified and sequenced in our study. The sequence comparison revealed that the six complete H genes showed nucleotide homologies of 96.7-99% and amino acids homologies of 96.1-99% (Table 1 ). The six complete H genes identified in our study exhibited a high nucleotide similarity (97.5-97.9%) with the CDV Asia 1 reference strain (Table 1) . Furthermore, the phylogenetic analysis revealed that the six CDV strains were divided into the CDV Asia 1 group, forming three clades (Fig. 1) . At present, CDV strains include ten distinct lineages known as America-1, America-2, Arctic-like, Rockborn-like, Asia-1, Asia-2, Africa-1, European Wildlife, Europe/South America-1 and South America-2 [6] . In our study, only CDV Asia-1 strains were identified, which is in line with most reports from China [2, 12] . Although the type Asia-1 is the most predominant CDV group in China, the CDV Asia-1 strains identified in our study also exhibited genetic diversity (Fig. 1) . The genetic diversity of CDV Asia-1 strains may be attributed to the long-term immune pressure which induces the adaptive mutation of CDV strains to varying degrees.