3.4 Distinctive amino acid mutations in Vietnamese CVP-2c
creating a new Asia subclade
Phylogenetic analysis of the 199 full-length CPV-2 genome sequences,
including the 11 sequences from this study, revealed that the CPV-2
lineages separated into two main clades. The first clade consisted of
sequences from Europe and America (Western clade), while the other clade
was comprised of most of the strains from Asian countries (Asian clade),
where the VP2 residue 324 is 324Ile in the Asian clade and 324Tyr in the
Western clade (Table 3).
The most recent common ancestor (MRCA) of the Western clade was
estimated to be in 1982, while the MRCA of the Asian clade was estimated
to be in 1994 (Figure 1). Later, in 1986, the Western clade was further
divided into the Western-I (WT-I; including the CPV-2a/b strains from
Brazil, Argentina, Uruguay, and Germany) and Western-II (WT-II;
including the CPV-2a/b/c strains from Italy, Germany, Ecuador, Brazil,
USA, Canada, Japan, and New Zealand), based on the mutation of NS1
residue Tyr544Phe. By 1996, the Western-III (WT-III) sub-clade, which
was composed of only CPV-2c strains from Italy, Argentina, Uruguay,
Paraguay, Brazil, Australia, Ecuador, France, and Albania, emerged and
was well-defined by the change in the VP2 residue to either Asn426Glu or
Asp426Glu.
In the Asian clade, the majority of the CPV-2 isolates expressed
Phe267Tyr in VP2, forming an Asia-II subclade in 1998, which included
CPV-2a strains from Vietnam (HCM16 in this study), China, and Uruguay,
and a few strains that carried Phe 267 but clustered in the Asia-I
subclade (CPV-2a isolated in China, India, and Canada). Thereafter,
CPV-2 sequences further separated into Asia-III (CPV-2a/b strains from
China and Uruguay) based on the Tyr544Phe and Glu545Val mutations in the
NS1 gene in 2002. Interestingly, the most recent mutations of NS1
residues at Ile60Val, Leu630Pro, and Ala5Gly in VP2 in the Vietnamese
CPV-2c (10 strains from this study), Chinese CPV-2a (KR002800), and
Italian CPV-2c (MF510157) established a new Asian subclade since 2005,
named here as Asia-IV (Tables 2 and 3, Figure 1, Supplementary Table
S4).