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.