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Transboundary and Emerging Diseases

ORIGINAL ARTICLE

Phylogenetic Analysis of Canine Parvovirus VP2 Gene in


China
L. Yi1, M. Tong1, Y. Cheng1, W. Song2 and S. Cheng1
1
Institute of Special Wild Economic Animal and Plant Science, Chinese Academy of Agricultural Sciences, Changchun, Jilin province, China
2
Wuhan ZhenAi Pet Clinic, Jiang’an District Wuhan, Hubei province, China

Keywords: Summary
canine parvovirus; VP2 gene; phylogenetic
analysis In this study, a total of 37 samples (58.0%) were found through PCR assay to be
positive for canine parvovirus (CPV) of 66 suspected faecal samples of dogs col-
Correspondence lected from various cities throughout China. Eight CPV isolates could be obtained
S. Cheng. Institute of Special Wild Economic in the CRFK cell line. The sequencing of the VP2 gene of CPV identified the pre-
Animal and Plant Science, Chinese Academy dominant CPV strain as CPV-2a (Ser297Ala), with two CPV-2b (Ser297Ala).
of Agricultural Sciences, 4899 Juye Street,
Sequence comparison revealed homologies of 99.3–99.9%, 99.9% and 99.3–99.7%
Changchun, Jilin province, China 130122.
Tel.: +86-431-81919840;
within the CPV 2a isolates, within the CPV 2b isolates and between the CPV 2a
Fax: +86-431-81919840; and 2b isolates, respectively. In addition, several non-synonymous and synony-
E-mail: tcscsp@126.com mous mutations were also recorded. The phylogenetic tree revealed that most of
the CPV strains from different areas in China were located in the formation of a
Received for publication April 29, 2014 large branch, which were grouped together along with the KU143-09 strain from
Thailand and followed the same evolution. In this study, we provide an updated
doi:10.1111/tbed.12268
molecular characterization of CPV 2 circulation in China.

or named New CPV-2a/2b, have also been reported in


Introduction
recent times by scholars in various countries, including
Canine parvovirus type 2 (CPV-2) causes acute haemor- China (Xu et al., 2015; Zhao et al., 2013). And another
rhagic enteritis in dogs. It emerged in 1978 in the USA and report (Ikeda et al., 2000) show the variants of CPV 2a/2b
rapidly spread among domestic dog populations through- have a natural mutation of VP2 residue G300D.
out the world with high morbidity and frequent mortality Although the CPV vaccines available in China are CPV-2
(Meunier et al., 1985). type, there are evidences to suggest that complete immunity
The virus has some persistent genetic variations, and at may not be provided to pups (Ohshima et al., 2008). The
present, CPV-2a, CPV-2b and CPV-2c are the three major variants of CPV 2a/2b emerged in China, considering that
antigenic variants of CPV-2 (Buonavoglia et al., 2000; the CPV2 vaccine appears to provide a comparatively lower
Martella et al., 2004; Decaro et al., 2006). and shorter immunity against heterologous CPVs (Zhang
Nowadays, CPV 2a or CPV 2b are the predominant in et al., 2010).
Asian countries including Korea, China, Thailand, Japan, CPV-2 is a small non-enveloped, single-stranded DNA
Taiwan, India and Australia, although a few CPV 2c strains virus (5.2 kb) and is a member of the genus Parvovirus of
have been isolated in India. Outside of Asia, CPV 2a and 2b the family Parvoviridae. The VP2 capsid protein is a major
isolates are common in the United States, whereas CPV 2c capsid protein and plays an important role in the determi-
is more widespread in Uruguay, Brazil and Argentina. nation of antigenicity and host range of CPVs.
European epidemiological surveys show that CPV 2c is The amplification of VP2 gene (capsid protein) and sub-
now predominant in Italy, Germany and Spain and is also sequent sequencing of the PCR products covering the
widely co-distributed with CPV 2a or CPV 2b in Portugal, informative amino acids would certainly aid in detecting
France and Belgium (Decaro and Buonavoglia, 2012). genetic variation existing between CPV-2 and its variants
The variants of CPV 2a/2b, undergoing mutation at resi- (Chinchkar et al., 2006; Jeoung et al., 2008; Phromnoi
due Ser297Ala and designated as CPV-2a (Ser297Ala)/2b, et al., 2010).

e262 © 2014 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 63 (2016) e262–e269
L. Yi et al. Phylogenetic Analysis of Canine Parvovirus VP2 Gene

Similarly, the origin and source of the CPV, as well as its The PCR products were analysed on 1.0% agarose gel
transmission routes, may be identified via sequence analysis containing ethidium bromide, at a final concentration of
and by constructing a phylogenetic tree using the amplified 0.5 lg ml1. The gel was visualized under a UV transillu-
VP2 gene (Mukhopadhyay et al., 2014). minator, and the images were documented in a gel
In China, as in other Asian countries, CPV 2a and 2b documentation system.
variants have predominated since the first outbreak (Xu
et al., 2015; Zhu et al., 2014). Therefore, constant surveil-
Isolation of CPV
lance and monitoring of CPV variants which may poten-
tially escape the host immune system and detection The CRFK cell line, maintained in an RPMI-1640 med-
methods are always necessary. ium (Hyclone, Beijing, China), was used for isolation of
Therefore, the aim of this study is to clarify the evolution CPV from eight PCR-positive samples according to the
of CPV 2 isolated from northern, central and eastern China procedure recommended as reported previously. (Parthi-
during the period of 2009–2013. ban et al., 2011). The virus was harvested the third day
after inoculation, following three alternative freeze–thaw
cycles and clarified at 6000 g for 15 min in a refrigerated
Materials and Methods
centrifuge. The supernatants were stored at 40°C for
Clinical samples and laboratory processing further use. The presence of the virus in the cell culture
A total of 66 rectal swabs were collected from CPV-sus- fluids at the third passage level was confirmed through
pected dogs, regardless of their vaccination status. The PCR assay.
samples were collected from the south-central Chinese
city of Wuhan, eastern city of Shanghai and northern city
Genotyping of CPV samples/isolates
of Beijing during a period of 4 years, from 2009 to 2013.
The collected samples were emulsified in 1 ml of 0.1 M The amplified PCR products (2023 bp) of the VP2 gene of
PBS of pH 7.4 and centrifuged at 2656 g for 15 min at CPV from 18 randomly selected clinical samples and four
4°C. The supernatant was collected and used for PCR cell culture isolates (for a total of 22), from diverse locations
amplification. throughout China, were sent for sequence analysis using the
automated sequencer Applied Biosystem 3100. The specific-
ity of the sequences was determined using BLAST (Basic
Polymerase chain reaction
Local Alignment Search Tool, http://blast.st-va.ncbi.nlm.
DNA was prepared by boiling at 96°C for 10 min and nih.gov/Blast.cgi), and the sequences were aligned using a
chilling immediately in crushed ice (Schuncka et al., 1995). biological sequence alignment editor (BioEdit, Micro
The supernatants were diluted to a ratio of 1 : 10 in Focus, England), revealing important informative amino
distilled water to reduce the residual inhibitors of DNA acid residues. The nucleotide sequences obtained were
polymerase activity. aligned with corresponding sequences available in GenBank
The polymerase chain reaction was conducted utilizing using Clustal W of the MEGA 5.0 program (http://www.
the primers VPfor (50 -GCCGGTGCAGGACAAGTAAAAA megasoftware.net). The sequences were also submitted to
GAG-30 , located at nucleotide position 2747–2771)/VPrev GenBank for allotment of accession numbers.
(50 -CAACCACCCACACCATAACAACATAC-30 , located at
nucleotide position 4919–4944) and yielded a 2023-bp
Phylogenetic analyses
product, designed according to the DNA sequence of
CPV-15 in GenBank (M24003) and by PRIMER PREMIER A phylogenetic and molecular evolution tree was constructed
5.0 software. from the entire VP2 gene nucleotide sequences of the CPV
The polymerase chain reaction was conducted by prepar- strains obtained in this study with MEGA version 5.0, using
ing the reaction mixture (50 ll) at a final concentration of the neighbour-joining (NJ) method. The reliability of the
19 Taq buffer, 200 lM of each dNTP, 1 lM of each VPfor/ phylogenetic tree obtained for the VP2 region was then
VPrev primer, 2 U of Taq DNA polymerase and 10 ll of evaluated by running 500 replicates in the bootstrap test.
the prepared template DNA. All of the reagents were
obtained from Beijing CoWin Biotech, China.
Results
The PCR amplification was carried out in an Eppendorf
Master Cycler (Hamburger, Germany), followed by initial Polymerase chain reaction and isolation
denaturation at 95°C for 5 min, 35 cycles at 94°C for 30 s, Among the 66 samples screened by PCR assay using
at 50°C for 1 min, at 72°C for 1 min and a final extension VPfor/VPrev primers, 37 samples yielded a specific ampli-
at 72°C for 10 min. con of 2023 bp. Among the eight samples subjected to

© 2014 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 63 (2016) e262–e269 e263
Phylogenetic Analysis of Canine Parvovirus VP2 Gene L. Yi et al.

isolation, four showed a mild CPE in the form of rounding Table 2. Details of samples sequenced, place of collection, year of
of cells, increased granularity, decreased refractility and isolation and their GenBank accession numbers
detached cells during the third passage. The cryolysates Sample no./year Place of GenBank accession
were also confirmed as positive for CPV by PCR. The virus S. No. of collection collection numbers
in the remaining four samples failed to grow in the CRFK
1 CPV 1-1/2013 Wuhana KJ674806
cell line.
2 CPV 1-10/2013 Wuhana KJ674807
3 CPV 1-11/2013 Wuhana KJ674808
Sequencing and CPV Genotyping analysis 4 CPV 1-12/2013 Wuhana KJ674809
5 CPV 1-7/2013 Wuhana KJ674815
Sequencing was performed on the amplified VP2 gene of 6 CPV 1-8/2013 Wuhana KJ674816
the representative isolates (total of 22) selected from diverse 7 CPV si/2013 Wuhana KJ674819
geographical locations throughout China. The sequencing 8 CPV wu/2013 Wuhana KJ674820
9 CPV SH-3/2011 Shanghaic JX121627
results revealed 20 sequences as the CPV-2a (Ser297Ala)
10 CPV SH-2/2011 Shanghaic JX121626
strain and two sequences, namely CPV 1-8 and CPV 1-10 11 CPV SH-1/2011 Shanghaic JX121625
(Wuhan, south-central China), as the CPV-2b (Ser297Ala) 12 CPV BJ-2/2011 Beijingb JX121624
strain. 13 CPV BJ-1/2011 Beijingb JX121623
The VP2 nucleotide sequences were analysed using 14 CPV 1-nj/2009 Nanjingc GQ169550
DNASTAR software, revealing homologies of 99.3–99.9%, 15 CPV bj-5/2010 Beijingb GQ169549
99.9% and 99.3–99.7% within the local CPV 2a isolates, 16 CPV bj-3/2010 Beijingb GQ169547
17 CPV bj-2/2010 Beijingb GQ169546
within the local CPV 2b isolates and between the local CPV
18 CPV bj-1/2010 Beijingb GQ169545
2a and 2b isolates, respectively (Table 1). In comparison 19 CPV wh-8/2009 Wuhana GQ169544
with the low nucleotide sequence, similarity between the 20 CPV wh-6/2009 Wuhana GQ169542
reference CPV 2a Chinese western strains (CPV-04/08/ 21 CPV wh-3/2009 Wuhana GQ169539
CN-2 and CPV-04/08/CN-4) and our CPV 2a strains 22 CPV wh-1/2009 Wuhana GQ169537
(99.5–99.7% and 99.3–99.6%, respectively) and the homol- a
South-central China.
ogy levels between our analysed CPV 2a isolates and refer- b
Northern China.
ence CPV Chinese 2a strains (99.3–99.9%) appeared to be c
Eastern China.
much higher.

Nucleotide sequence accession numbers Phylogenetic analysis


The GenBank accession numbers (Table 2) were obtained. The phylogenetic tree based on nucleotide sequences relat-
Additionally, nine non-synonymous (Table 3) and 11 ing to the place of origin of the samples is depicted in
synonymous (Table 4) mutations were also observed in the Fig. 1. The sequences obtained from Wuhan (south-central
CPV sequences under study. China; 1-11, wu, si, 1-12, 1-1, 1-7 and wh-3), Shanghai

Table 1. Sequence homology of local Chinese CPV 2a and 2b isolates and reference strains

Genotypes

CPV-2a China CPV-2b China

CPV-04/08/CN-2 CPV-04/08/CN-4 Chinese GZ0201 Chinese


(FJ435343) Western China (FJ435345) Western China 2a strains (GU569944) 2b strains

CPV-04/08/CN-2 (FJ435343) 100.0 99.5 99.5–99.7 99.3 99.5


Western China
CPV-04/08/CN-4 (FJ435345) 100.0 99.3–99.6 99.0 99.3
Western China
Chinese 2a strainsa 99.3–99.9 99.4–99.5 99.6–99.7
GZ0201 (GU569944) 100.0 99.5
Chinese 2b strainsb 99.9
a
Accession numbers: KJ674806, KJ674808, KJ674809, KJ674815, KJ674819, KJ674820, JX121627, JX121626, JX121625, JX121624, JX121623,
GQ169550, GQ169549, GQ169547, GQ169546, GQ169545, GQ169544, GQ169542, GQ169539, GQ169537.
b
Accession numbers: KJ674816, KJ674807.

e264 © 2014 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 63 (2016) e262–e269
L. Yi et al. Phylogenetic Analysis of Canine Parvovirus VP2 Gene

Table 3. Non-synonymous mutations in the VP2 gene sequences analysed in this study

Positions indicating Nucleotide/Amino acid residue

3370 & 3757 &


3201 (139) 3582 (167) 3371 (195) 3586 (267) 3758 (324) 3964 (393) 4030 (415) 4062 (426) 4104 (440)

Nucleotide change and change in the amino acid residue

G?A A?G TG ?AT T?A TA?AT A?G T?C A?G A?G


S. no. Sample No. (Val ?Ile) (Asn ? Asp) (Leu ?Tyr) (Phe ? Tyr) (Tyr ? Ile) (Glu?Gly) (Ile?Thr) (Asn ?Asp) (Thr ? Ala)

1. CPV 1-1 – – – A – – – – G
2. CPV 1-10 – – – A – – – T –
3. CPV 1-11 – – – A – – – – G
4. CPV 1-12 – – – A – – – – G
5. CPV 1-7 – – – A – – – – G
6. CPV 1-8 – – – A – – – T –
7. CPV si – – – A – – – – G
8. CPV wu – – – A – – – – G
9. CPV SH-3/2011 – – – A – – – – G
10. CPV SH-2/2011 – – – A – G – – G
11. CPV SH-1/2011 – – – – – – – – –
12. CPV BJ-2/2011 – – – – – – C – –
13. CPV BJ-1/2011 – – – A – – – – G
14. CPV 1-nj – – – – – – – – –
15. CPV bj-5 – – – – – – – – –
16. CPV bj-3 – – AT A – – – – G
17. CPV bj-2 – – – – – G – – –
18. CPV bj-1 A G – – – – – – –
19 CPV wh-8 A – – A AT – – – –
20 CPV wh-6 – – – – – – – – –
21 CPV wh-3 – – – A – – – – G
22 CPV wh-1 – – – – – – – – –

(eastern China; SH-2; Sh-3) and Beijing (northern China; of the PCR assay were in concordance with the results
BJ-1 and bj-3) formed a separate lineage and were also obtained by Xu (46.6%) in western China (Xu et al.,
placed close to each other along with the CPV sequence 2015).
from the Thailand KU143-09 strain. The sequences CPV-2b This study revealed that CPV-2a (Ser297Ala) was the
1-10 and 1-8 (Wuhan) were shown to be closely related to predominating strain circulating in China, along with the
each other and were supported by a bootstrap value of CPV-2b (Ser297Ala) strain co-circulating in certain areas
99%. The sequences wh-8, wh-1 (Wuhan), 1-nj and SH-1 (Wuhan, south-central China). Similar observations were
(Shanghai) from south-central and eastern China formed a made by Zhang and Zhao in their studies involving limited
separate lineage and were closely related with the CPV-04/ geographical areas of China (Zhang et al., 2010; Zhao et al.,
08/CN-2 strain (western China). The sequence bj-2 2013). A similar epidemiological pattern has also been
(Beijing, northern China) formed a clade and was closely reported in Brazil, where all circulating strains were charac-
related with KU5-08 from Thailand. The sequences bj-5, terized as CPV-2a (Castro et al., 2011). In addition, some
bj-1, BJ-2 (Beijing) and wh-6 (Wuhan) formed a separate reports have shown that CPV-2a is the predominant variant
clade and were closely related with the G13 strain (southern in Asia (Jeoung et al., 2008; Phromnoi et al., 2010; Soma
China), K026 strain (South Korea) and CPV-42 strain et al., 2013; Mukhopadhyay et al., 2014) and Australia
(Taiwan). (Meers et al., 2007). It has been reported that mutation
Ala297 at this position may be responsible for changes in
the antigenicity of CPV variants (Truyen, 2006). Further-
Discussion
more, the emergence and spread of this variant indicates
In this study, 66 samples were collected from CPV-sus- that the Ala297 mutation may have had a remarkable
pected dogs, and 37 CPV-positive samples, accounting influence on the process of host adaptation (Pereira et al.,
for 56.0% of positive rate, were identified. The results 2007).

© 2014 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 63 (2016) e262–e269 e265
Phylogenetic Analysis of Canine Parvovirus VP2 Gene L. Yi et al.

Table 4. Synonymous mutations in the VP2 gene sequences analysed in this study

Positions indicating Nucleotide/Amino acid residue

3032 3518 3605 3617 3878 3944 4270 4444 4481 4508 4538
(82) (244) (273) (277) (364) (386) (495) (553) (565) (574) (584)

Amino acid residue and Nucleotide change

S. No. Sample No. Val G-A Tyr C-T Cys T-C His C-T Ala G-A Gln A-G Pro T-C Ile T-C Asp T-C Gln A-G Thr T-C

1. CPV 1-1 – – – – – – – – – – –
2. CPV 1-10 A – – T – – – C – – –
3. CPV 1-11 – – – – A – – – – – –
4. CPV 1-12 – – – – – – – – – – –
5. CPV 1-7 – – – – – – – – – – –
6. CPV 1-8 A – – T – – – C – – –
7. CPV si – – – – – – – – – – –
8. CPV wu – – – – A – – – – – –
9. CPV SH-3/2011 – – – – – – – – – – –
10. CPV SH-2/2011 – – – – – – – – – – C
11. CPV SH-1/2011 – T C T – G C – C G –
12. CPV BJ-2/2011 – T – T – – – – – – –
13. CPV BJ-1/2011 – – – – – – – – – – C
14. CPV 1-nj – T C T – G C – C G –
15. CPV bj-5 A T – T – – – – – – –
16. CPV bj-3 – – – – – – – – – – –
17. CPV bj-2 – T – T – – – – – – –
18. CPV bj-1 – T – T – – – – – – –
19 CPV wh-8 – T C T – G C – C G –
20 CPV wh-6 A T – T – – – – – – –
21 CPV wh-3 – – – – – – – – – – C
22 CPV wh-1 – T C T – G C – C G –

This is the first study to investigate the genotype preva- residues 265 and 267 are not exposed on the capsid
lence of CPV 2 in northern, central and eastern China in surface (Xie and Chapman, 1996), substitutions of these
recent years. Our results indicate that CPV-2a (Tyr324Ile) residues may not affect the antigenicity of the viruses.
is a prevalent CPV 2 field strain currently circulating in However, interestingly, the frequency of the strains car-
China. This residue is adjacent to residue 323, which affects rying this residue has increased and has reached a high
binding to canine transferrin receptors, resulting in the prevalence among Chinese CPVs. In this study, 14 of
changes of canine parvovirus’ host range (Hueffer et al., 22 (26.7%) CPV isolates showed Phe267Tyr mutation,
2003). Presumably, this Tyr324Ile alteration may result in indicating that this mutation probably arose quite
stronger receptor binding. quickly.
Surprisingly, all of the CPV 2a/2b isolates, except one, Most of the sequences under study carried another
contained a unique amino acid substitution (Tyr324Ile). amino acid change, namely Thr440Ala, which was located
Similar reports have shown that this Ile324 variant of CPV at the top of the threefold spike (within the GH loop), and
2a is also found in South Korea (Jeoung et al., 2008), China was considered to be the main antigenic site of the virus.
(Xu et al., 2015), Thailand (Phromnoi et al., 2010), This finding is in accordance with the previously study
Uruguay (Maya et al., 2013), Japan (Soma et al., 2013), (Battilani et al., 2002).
Taiwan (Lin et al., 2014) and India (Mukhopadhyay et al., In addition, two sequences under study carried another
2014). amino acid change, namely Val139 Ile, which has also been
Among the non-synonymous mutations, some Chi- found in Hungary (Csagola et al., 2014). This variant
nese CPV isolates covered in this study were shown to requires further observation and confirmation.
have a common substitution of Phe-267 to Tyr, which Four non-synonymous mutations in the present study,
was also recorded in CPV-2a strains from Thailand resulting in amino acid changes Asn167Asp, Leu195Tyr,
(Phromnoi et al., 2010), India (Mukhopadhyay et al., Glu393Gly and Ile415Thr, had not been reported previ-
2014) and China (Xu et al., 2013). Due to the fact that ously.

e266 © 2014 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 63 (2016) e262–e269
L. Yi et al. Phylogenetic Analysis of Canine Parvovirus VP2 Gene

Fig. 1. Neighbor Joining tree constructed from the nucleotide sequences of the VP2 gene of CPV strains under study and the reference
sequences. Phylogenetic analysis of CPV VP2 sequences with the lines connecting the sequences to the place of origin on the map of China,
The number of each node represents the bootstrap value resulting from 1000 replicates. The tree is drawn to scale, with branch lengths
measured in the number of substitutions per site. Each isolate obtained in this study is indicated by the CPV sample number followed by the
year of isolation and strain identity. The GenBank accession numbers of the sequences under study are provided in Table 1. The CPV isolates
of the current study in the present study are indicated (●). The GenBank accession numbers of the reference sequences are FPV CU-4
(M38246), CPV KU53-03 (FJ869138), CPV TWN1 (EF592511), CPV-42 (JX048605), CPV K001 (EU009200), CPV K026 (EU009204), CPV K031
(EU009206), CPV KU5_08 (FJ869126), CPV KU143_09 (KU143_09), CPV-04/08/CN-2(FJ435343), CPV-04/08/CN-4 (FJ435345), CPV-G13
(KF785790), CPV Sho-nan (AB128923), and GZ0201 (GU569944).

Therefore, the existence of all these non-synonymous strain KU143-09 (FJ 869126) and followed the same evo-
mutations in the present study indicated that the CPV lution. However, interestingly, the sequences obtained
strains were under constant selection pressure and were from Wuhan and Shanghai (wh-8, 1-nj, wh-1 and SH-1)
constantly mutating, leading to the evolution of newer obtained during 2009–2011 formed a distinctly separate
CPV types/variants. lineage from the samples obtained from others selected in
The phylogenetic tree shows that most of the CPV this study, indicating an additional separation but shared
strains in China are located in a large branch, although ancestral origins.
there are some differences among them. The distribution The VP2 sequence of CPV isolates in this research was,
of CPV in China did not show geographical correlation. respectively, located in northern China, south-
Phylogenetic studies revealed that most of the CPV central China and eastern China and showed a high homol-
sequences obtained from Wuhan (south-central China) ogy between 99.3 and 99.7% in comparison with the Sichu-
and four sequences obtained from Beijng and Shanghai an CPV isolates (western China). The degree of CPV
during 2009–2013 were grouped together with the Thai variation is almost the same, showing that there are no

© 2014 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 63 (2016) e262–e269 e267
Phylogenetic Analysis of Canine Parvovirus VP2 Gene L. Yi et al.

geographical features in the Chinese CPV isolates. Similar enteritis in Spain. J. Vet. Med. B Infect. Dis. Vet. Public Health
results demonstrated verification by the phylogenetic tree 53, 468–472.
based on the VP2 gene. The CPV isolates in this research Hueffer, K., J. S. Parker, W. S. Weichert, R. E. Geisel, J. Y. Sgro,
and the reference isolate CPV-G13 from southern China and C. R. Parrish, 2003: The natural host range shift and
(KF 785790) are in a large branch and may share the same subsequent evolution of canine parvovirus resulted from
ancestral origins. This result may be due to the increasing virus-specific binding to the canine transferrin receptor. J.
amounts of extensive trade, exchange and circulation of Virol. 77, 1718–1726.
goods among large cities in China, which indicates that Ikeda, Y., M. Mochizuki, R. Naito, K. Nakamura, T. Miyazawa,
T. Mikami, and E. Takahashi, 2000: Predominance of canine
CPV transmission and epidemics tend to be consistent,
parvovirus (CPV) in unvaccinated cat populations and emer-
with no geographical features.
gence of new antigenic types of CPVs in cats. Virology 278,
It is thus certain that the results of this study add valu-
13–19.
able information to the evolution of CPV strains in China.
Jeoung, S. Y., S. J. Ahn, and D. Kim, 2008: Genetic analysis of
Further monitoring and surveillance of larger areas, includ-
VP2 gene of canine parvovirus isolates in Korea. J. Vet. Med.
ing all cities, will be useful for identifying and analysing Sci. 70, 719–722.
newer CPV strains/variants. Lin, C. N., C. H. Chien, M. T. Chiou, L. L. Chueh, M. Y. Hung,
and H. S. Hsu, 2014: Genetic characterization of type 2a
Acknowledgements canine parvoviruses from Taiwan reveals the emergence of an
Ile324 mutation in VP2. Virol. J. 11, 39.
The present study was partly financially supported by the Ji Martella, V., A. Cavalli, A. Pratelli, G. Bozzo, M. Camero, D. Bu-
Lin Province Major Scientific and Technological Achieve- onavoglia, D. Narcisi, M. Tempesta, and C. Buonavoglia,
ments Transformation Program (no. 10ZDZH010) and Ji 2004: A canine parvovirus mutant is spreading in Italy. J. Clin.
Lin Province Scientific and Technological Program Microbiol. 42, 1333–1336.
(20140204066NY). Maya, L., L. Calleros, L. Francia, M. Hernandez, G. Iraola, Y.
Panzera, K. Sosa, and R. Perez, 2013: Phylodynamics analy-
sis of canine parvovirus in Uruguay: evidence of two suc-
Competing interests cessive invasions by different variants. Arch. Virol. 158,
The authors declare that they have no competing interests. 1133–1141.
Meers, J., M. Kyaw-Tanner, Z. Bensink, and R. Zwijnenberg,
2007: Genetic analysis of canine parvovirus from dogs in Aus-
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