4. Discussion
CPV-2c strains have been reported at a high prevalence in dogs in many geographic regions in Europe, America, and Asia, and specifically in Vietnam (Geng et al., 2015; Hoang et al., 2019; Kang et al., 2008; Ohshima et al., 2008). In this study, we identified CPV-2c infections at a high incidence (98.3%), which is concordant with a previous study of CPV in Vietnam (Hoang et al., 2019). Previous genetic characterization and analysis has been based on the individual VP genes, whereas this study attempted to characterize the whole genome of CPV-2c isolates obtained from 59 Vietnamese dogs.
In this study, Vietnamese CPV-2c presented the unique non-synonymous Thr112Ile and Ile447Met mutations in the VP1 and VP2 genes, respectively. These two novel mutations are seemingly currently restricted to Vietnam. Thus, further studies are needed to monitor these mutations and elucidate their impact on the pathogenicity and virulence of CPV-2c. In addition, the common mutations that have been widely reported in other Asian CPV-2 strains, such as Phe267Tyr, Tyr324Ile, and Gln370Arg, and the recent Ala5Gly mutation, were observed in the VP2 sequence of all Vietnamese CPV-2c strains. Interestingly, these mutations were not observed in the previous CPV-2c HNI-4-1strain (AB120727), which was firstly isolated in Vietnam in 2002 (Nakamura et al., 2004). Moreover, these CPV-2c isolates exhibit the Ile60Val, Tyr544Phe, Glu545Val, and Leu630Pro mutations in the NS1 gene and the Arg116Lys, Leu125Ile, and Ala131Thr in the VP1 genes, which are similar to those described in Chinese and Taiwanese CPV-2 isolates (Chiang et al., 2016; J. Wang et al., 2016; Wu, Li, Wang, Liu, & Tian, 2018). These findings indicated that Vietnamese CPV-2c strains likely shared a common evolutionary pattern in both their nonstructural and structural proteins with other CVP-2 variants, including the Italian CPV-2c (MF510157) that was isolated from a puppy imported from Thailand to Italy in 2017.
The phylogenetic analysis showed that the mutation of 324Ile in the VP2 gene, which has been frequently observed in the most recent CPV-2 isolates in Asia (Geng et al., 2015; Kang et al., 2008; Mukhopadhyay et al., 2014; Phromnoi, Sirinarumitr, & Sirinarumitr, 2010; Soma, Taharaguchi, Ohinata, Ishii, & Hara, 2013; H. Zhao et al., 2017; Y. Zhao, Lin, Zeng, Lu, & Hou, 2013), plays a role as the hallmark amino acid to separate the Asian CPV-2 clade from the Western counterpart. In addition, the prevalence of CPV-2 strains carrying 267Tyr is increasing in Asia (Chiang et al., 2016) as the next step of the evolution process in the CPV-2 subclade. Thus, the 267Tyr and 324Ile mutations of VP2 may serve at present as genetic markers for the Asian CPV-2 strains.
Apart from the amino acid changes in VP1/VP2, mutations in NS1/NS2 might represent the emergence of a subclade in the phylogeny by sharing common characterizations. In the WT-II group, all strains had the Tyr544Phe genotype in NS1, similar to that in a previous study (Grecco et al., 2018). In addition, the Tyr544Phe and Glu545Val mutations in the NS1/NS2 were present in the Asia-III and -IV subclades, and residues Ile60Val and Leu630Pro were in the Asia-IV subclade. These findings suggest that these mutations in NS1/NS2 might play a role in the emergence of new variants. Future studies on genomic analysis, including the NS1/NS2 genes, should be conducted to be better understanding the viral evolution.
Although CPV-2 is a DNA virus, it has been reported (and observed in this study) to have a high nucleotide substitution rate, perhaps as rapid as that found in RNA viruses (Shackelton, Parrish, Truyen, & Holmes, 2005). More retrieved sequences could lead to a greater precision on the substitution rate estimation. The substitution analysis in this study suggested that the CPV-2 genome had a high background mutation rate of 2.49 x 10-4 nt/s/y. Notably, the individual NS and VP gene analysis showed a similar substitution rate, suggesting that viral evolution of CPV-2 may not only be observed in the structural proteins, which are associated with immune escape and cellular tropism, but also in the nonstructural proteins. In addition, we found that the evolutionary rate of the Asian clade was higher than that for the Western clade. Further studies are needed to verify this observation and search for the factors that alter the evolutionary rate in the Asian group. Interestingly, the selective pressure analysis in this study revealed that even though most of the CPV-2 has undergone negative selection, there were potential positive selection sites located in both the NS and VP genes. Thus, the function of mutation(s) in the nonstructural protein needs to be verified and might be interesting for further study of CPV evolution.