Background
Canine parvovirus type 2c (CPV-2c) was first detected in Italy in 2000 [
1]. Antigenic differences among CPV-2a, −2b, and -2c are observed only in residue 426 (Asn in 2a, Asp in 2b, and Glu in 2c) [
2], which is located in the major VP2 antigenic site of the parvovirus [
3]. The functions of capsid protein VP2 include facilitating receptor binding, controlling host range [
4], and eliciting neutralizing antibodies [
3]. Although CPV-2c infection results in almost the same clinical signs as for CPV-2a and CPV-2b, including anorexia, vomiting, acute gastroenteritis, and hemorrhagic diarrhea, infection by CPV-2c has been reported to be indicative of a more severe disease [
5,
6].
A retrospective analysis revealed that the oldest CPV-2c strain was identified in Germany in 1996 [
7]. Another retrospective analysis revealed that the frequency of CPV-2 variants underwent rapid fluctuation in Italy between 1995 and 2005, with CPV-2c very rapidly replacing CPV-2b [
8]. CPV-2c infection has not only been observed in Italy, but it is also widely distributed in other European countries [
7,
9,
10], including Germany, Portugal [
11], Spain [
12], Belgium, France, Greece [
13], Bulgaria [
14], Sweden [
15], Turkey [
16], and the United Kingdom. In recent years, CPV-2c has also been found to be widespread in Tunisia [
17], the USA [
18], Uruguay [
19], Brazil [
20], Argentina [
21], Ecuador [
5], Mexico [
22], and Morocco [
23]. Surprisingly, since the first reported finding in Vietnam in 2004, the CPV-2c variant has not been prevalent in Asia [
24]. Indeed, only a few CPV-2c strains have been isolated in India [
25] and China [
26‐
28], with either CPV-2a or -2b being prevalent in Asian countries thus far [
25‐
27,
29‐
42].
In Taiwan, as in other Asian countries, both the CPV-2a and -2b genotypes constitute the prevalent CPV-2 field strains circulating in the last two decades [
30,
31,
39,
41]. Before the present study, no report indicated the occurrence of a CPV-2c variant in Taiwan [
30,
31,
39,
41]. However, in January 2015, the first report of CPV-2c in a puppy in Taiwan occurred, and there is to date limited information about the CPV-2c variant in Taiwan. In the present study, we examined this CPV-2c variant and investigated the distribution of CPV-2 variants in Taiwan.
Methods
Specimen collection
Clinical specimens (feces and/or rectal swab) were collected from 99 dogs with suspected CPV-2 infection from northern, central, southern, and eastern Taiwan from January 2014 to April 2016. These samples were mainly acquired from dogs with diarrhea and/or bloody diarrhea. The year of sampling and the age, clinical history, and CPV-2 types of the sampled dogs are summarized in Additional file
1.
CPV-2 screening and partial VP2 gene amplification
Viral DNA was extracted from the clinical samples (either feces or rectal swab) and screened for CPV-2 by real-time PCR, as described by Lin et al. [
43]. Samples showing positive results for either type of specimen were included in this study. The partial VP2 gene of CPV-2 was amplified by PCR, as described by Buonavoglia et al. [
1], and the DNA fragments were purified and sequenced as described by Lin et al. [
41].
Sequence and phylogenetic analyses
The VP2 DNA sequences of our samples were compared to those of reference FPV (M38246), CPV-2 (M38245), CPV-2a (M24003), CPV-2b (M74849), new CPV-2a (JX048605), new CPV-2b (JX048607), and CPV-2c (JF414818, JF414820, JF414822, FJ005247, GU380303, GU380305, KR611522, KT074339, KT162005, KF149962, FJ005196, GQ865518, FJ222821, FJ005213, FJ005238, FJ005214, KC196099, KM457119, AB120727, KP071956, KR559893, FJ005235). Multiple alignments of the nucleic acid and amino acid sequences were performed using the Clustal W method and the MegAlign program (DNASTAR, Madison, WI, USA). Phylogenetic analyses were performed with the maximum likelihood method using MEGA 6, version 6.06.
Discussion
This is the first study to demonstrate the CPV-2c variant in Taiwan. Previous studies have shown that both the CPV-2a and -2b genotypes constitute the prevalent CPV-2 field strains circulating in Taiwan, and no CPV-2c cases were reported in the last two decades (Table
3). In our continuous surveillance and sequence analysis, Taiwan was considered to be free of CPV-2c. However, since January 2015, one case of CPV-2c occurred in a puppy in Taiwan. The CPV-2c variant appears thus far to have the highest detection rate in the dog population of Taiwan. Similar results have shown that CPV-2c replaced the previous circulation of CPV-2 strains in Italy [
8], Uruguay [
19], Argentina [
21], Brazil [
20], and the United States [
44]. Interestingly, comparative VP2 genome analysis of the isolated CPV-2c reference revealed that the partial VP2 genome sequence of the Taiwanese strain is similar (97.7–100 %) to that of Chinese CPV-2c strains. Our results indicate that the recent CPV-2c isolate from Taiwan shares a common evolutionary origin with the Chinese CPV-2c strains and should be classified as novel Asian CPV-2c isolates. According to genotype surveillance between Taiwan [
30,
31,
39,
41] and China [
26‐
28,
37,
45‐
48], CPV-2c was first detected in China in 2009 [
28]. In contrast, no CPV-2c variant was observed in studies conducted over the last two decades in Taiwan (Table
3). In addition, the Taiwanese CPV-2c variant is more closely related to the Chinese CPV-2c strains than the recent Taiwanese CPV-2a (JX048605) and -2b (JX048607) isolates (Fig.
3). Taken together, our results suggest that the Taiwanese CPV-2c may be present due to import from China at some time between the end of 2014 to early 2015 rather than to evolution of existing CPV-2a or -2b genotypes. An ongoing investigation and complete VP2 genome sequence analysis is needed to trace the genetic evolution of this novel CPV-2c variant.
Table 3
Review of CPV-2 genotyping in Taiwan
1994–1995 | North | 10 | 1 | 0 | |
2003–2004 | Central | 2 | 34 | 0 | |
2011 | South | 35 | 19 | 0 | |
2008–2012 | North, Central, and South | 15 | 13 | 0 | |
2014–2016 | North, Central, South, and East | 17 | 23 | 48 | This study |
The recent Taiwanese CPV-2a is composed of two divergent lineages that have different ancestors. Most Taiwanese CPV-2a strains belong to the recent Taiwanese lineage of CPV-2a, sharing a common amino acid substitution (Tyr324Ile). The second lineage of the Taiwanese CPV-2a variant is more closely related to recent Uruguayan and Chinese CPV-2a strains and has distinctive amino acid substitutions of Phe267Tyr, Tyr324Ile, and Thr440Ala. This new CPV-2a variant was discovered in China and Uruguay between 2006 and 2009 and in 2010 [
49], respectively. This is the first detection of this lineage of CPV-2a in eastern Taiwan in 2015. However, this new CPV-2a variant recently emerged in Uruguay and underwent clonal expansion [
49]. An ongoing investigation is aimed at determining whether this new CPV-2a variant will replaced the CPV-2c variant in the Taiwanese dog population.
Amino acid substitution of Tyr324Ile has been observed in Korea [
32,
34], China [
26,
27,
37,
45‐
48], Thailand [
36], Uruguay [
49,
50], Japan [
38], Taiwan [
39,
41], and India [
40,
42]. Interestingly, the frequency of the Ile324 variant has reached a high prevalence among Taiwanese CPV-2 isolates (94.5 %), and our results revealed that this variant is not only present among Taiwanese CPV-2a strains but also CPV-2b and -2c strains. In addition, this study reports for the first time the amino acid substitution of Phe267Tyr in Taiwan. Surprisingly, all of the Tyr267 variants among Taiwanese CPV-2a, −2b, and −2c strains contain the amino acid substitution of Tyr324Ile. Our review of sequence analysis in the literature indicated that this phenomenon is also found in Uruguay [
51]. The functions of CPV-2 residues 267 and 324 are still unknown and remain to be elucidated.
The substitution of Gln370Arg is unique to the Taiwanese CPV-2c strains, and this mutation is also observed in Chinese panda parvovirus [
52] and Chinese CPV-2c strains [
26,
27]. Residue 359 and 375 constitute a flexible surface loop of the capsid protein that is adjacent to a double Ca
2+-binding site; this region is essential for virus infectivity, and changes are correlated with the ability of the virus to cause erythrocyte hemagglutination [
53]. Therefore, it remains to be investigated whether Gln370Arg substitution causes antigenic alterations.
Mutation of residue 420 had been reported in Brazilian reference CPV-2c strains (Phe420Leu) [
54]. In the present study, we detected 10 CPV-2c strains with a unique change at the same position, yet Phe420Ser was unique in these 10 Taiwanese CPV-2c strains. Therefore, further studies focusing on potential variants of the CPV-2c strains should be conducted to elucidate the relationship between Phe420Ser substitution and viral pathogenicity.
Although several studies have demonstrated the efficacy of the current CPV-2 vaccine against CPV-2c infection [
55,
56], some evidence suggests that dogs with the complete vaccination program still suffer from CPV-2c [
6]. In the present study, four of 22 CPV-2c-diseased dogs died despite vaccination (C104-030, C104-031, C104-042, C104-216) (Additional file
1). Among those that died, three were under 6 months of age. Surprisingly, despite having undergone the complete vaccination program, one adult dog (strain no. C104-216) was infected by this novel CPV-2c variant. Therefore, co-infection with other diseases needs to examined, and the efficacy of the current vaccine against this novel CPV-2c variant remains to be evaluated, especially in regard to the amino acid substitutions observed in this novel CPV-2c variant compared to the CPV-2c prototype.
Conclusions
This is the first report to identify a novel CPV-2c variant in Taiwan. The novel CPV-2c variant was found to be distributed throughout Taiwan, revealing that this novel CPV-2c variant is currently circulating on the island. Phylogenetic analysis demonstrated that the recent CPV-2c isolate from Taiwan shares a common evolutionary origin with Chinese strains of CPV-2c, as classified into novel Asian CPV-2c isolates (Phe267Tyr, Tyr324Ile, Gln370Arg). Continuous and intensive surveillance of this novel CPV-2c is needed, especially in previously disease-free countries.
Acknowledgements
The authors would like to thank Prof. Ling-Ling Chueh and Prof. Hsin-Fu Liu for comments that greatly improved the manuscript.