3.2 | Phylogenetic analysis
A phylogenetic tree was constructed using the neighbor-joining method with MEGA 6 based on the full-length VP2 nucleotide sequences obtained in this study, along with sequences retrieved from GenBank. As shown in Figure 1, all of the CPV-2c isolates were roughly divided into two clusters. The first cluster includes European and American isolates of CPV-2c. The second cluster consists of all 31 CPV-2c strains isolated in this study and other CPV-2c strains isolated from China and other countries previously. This shows that the Chinese CPV-2c isolates are obviously distinct from the prototypical CPV-2c strains from European countries such as Germany, Greece, Italy, and so on. Meanwhile, the 31 CPV-2c isolates in Jilin Province in this study were far from the earliest CPV-2c strains (GU380303, GU380305) isolated in Jilin Province in 2009. These newly identified CPV-2c strains are closely related to local CPV-2c isolates in cluster Ⅱ, such as Anhui (MK518005), Henan (MF467229), Sichuan (MH476587), Shandong (MK268682), and so on. Sequence comparisons also revealed 98.7%–100% nucleotide similarity in VP2 between these CPV-2c subtype strains and local CPV-2c isolates. However, the similarities of the nucleotides among the CPV-2c strains identified in this study and reported abroad or in the original Jilin isolates (GU380303, GU380305) were 98.8%–99.4% and 98.8%–99.3%, respectively. These results indicate that the CPV-2c isolates from China were likely to be derived from the local adaption of the original CPV-2c isolates rather than introduction from other countries. Of even more concern, all strains from recent epidemics clustered in a separate group that is far from the vaccine strains.