Despite the vaccination procedures adopted in China in the last decades, CDV is still a serious threat to breeding raccoon dogs as well as to domestic dogs and it is responsible for relevant economic losses in Chinese fur industry . In order to determine the frequency of this infection, we analyzed the samples taken from raccoon dogs with suspect of CDV infection. Our result revealed a relatively high (51.8%) prevalence of CDV infection animals sampled with clinical signs consistent with CDV infection. This indicated that CDV still represents a high risk to the canine population in China. Furthermore, all samples were tested twice and the results were reproducible. One possible explanation for the 52 animals thought to be infected with CDV were found negative by PCR screening is the ability of detecting low copy numbers of RNA. Another possible explanation for the negative is the presence of Taq polymerase inhibitors on blood samples, since it is known that blood material contains nucleases that degrades RNA, and substances, such as heparin and hemoglobin were found to inhibit the activity of Taq DNA polymerase .
The molecular analysis of the amplifications obtained by the use of primer pair “B” helped to clarify the relationship of the China strains with other CDV strains reported in other parts of the world. The analysis has shown that the strains of giant panda and raccoon dogs in this study are placed on one branches of the phylogenetic tree, were characterized as Asia-1 genotype. The H gene-based neighbor-joining phylogenetic tree analysis using selected sequences retrieved from GenBank revealed geographic-related patterns of segregation. Our findings are consistent with previous studies demonstrating the occurrence of strains belonging to genetically distinct CDV lineages in Asia [20, 28, 29]. The Chinese Asia-1 strains displayed a high genetic conservation with other Asia-1 CDVs, which detected from raccoon dogs or other carnivores in China over nearly two decades (Figure 1). The strain Sichuan08 was obtained from a mild fever giant panda in the Mar of 2012, and it showed the identity between Sichuan02-06 were 94.5-95.5%. Because there were few reports of giant panda clinical onset of CDV infection up to now, so we couldn't determine whether or not the fever is the clinical manifestations of the giant panda.
The observed diversity between the vaccine strains and recent wild-type CDVs may be accounted for by several mechanisms, such as adaptation to new host species [30, 31], antigenic escape [32–35] and/or genetic recombination between wild-type strains , variously driving the evolution of the virus. It has been described that changes in N-glycosylation of the H protein may affect neutralization by antibodies and replication in vitro [15, 37, 38]. So, the connected aspartic amide N glycosylation site potentially is a spotlight in H proteins between vaccine and wild strains of CDV. Usually, there are four (Onderstepoort strain) or seven (CDV3, Lederle and Convac strain) potential sites in the vaccine strains. However, seven or nine sites have been detected in all wild CDV strains, of which 309–311 N-connected amide asparagine glycosylation sites are specific to CDV field strains. Some studies believed that the variants from H protein glycosylation played a crucial role in the antigenic differences and increase in N glycosylation may eventually result in vaccine failure [15, 39]. So, we conjectured that the giant panda and raccoon dogs infected CDV maybe due to this reason in this time.
Based on phylogenetic analyses of complete sequences data from CDV strains retrieved from domestic dog and non-dog species it was hypothesized that positive selection drives residues 530 and 549 of the HA gene, and that the presence of specific residues at these positions resulted in the emergence of CDV as a disease of novel host species . Recently, it has been suggested that there no evidence that amino acid 530 was strongly affected by host species, while wild canids there was a nearly significant trend towards a 549Y bias, non-canid strains showed no significant bias towards either H or Y at site 549 . In this study, We found that amino acid 549Y was conserved within CDV lineages, regardless of host species. CDV transmission in wild carnivore and non-canids species may also most often occur between individuals within a species, and will be influenced by a range of factors such as population size and ranging patterns [40, 41].