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. 2015 Sep 8;10(9):e0137288. doi: 10.1371/journal.pone.0137288

Co-Circulation of the Rare CPV-2c with Unique Gln370Arg Substitution, New CPV-2b with Unique Thr440Ala Substitution, and New CPV-2a with High Prevalence and Variation in Heilongjiang Province, Northeast China

Yufei Geng 1, Donghua Guo 1, Chunqiu Li 1, Enyu Wang 1, Shan Wei 1, Zhihui Wang 1, Shuang Yao 1, Xiwen Zhao 1, Mingjun Su 1, Xinyu Wang 1, Jianfa Wang 1, Rui Wu 1, Li Feng 2, Dongbo Sun 1,*
Editor: Zhefeng Meng3
PMCID: PMC4562665  PMID: 26348721

Abstract

To trace evolution of canine parvovirus-2 (CPV-2), a total of 201 stool samples were collected from dogs with diarrhea in Heilongjiang province of northeast China from May 2014 to April 2015. The presence of CPV-2 in the samples was determined by PCR amplification of the VP2 gene (568 bp) of CPV-2. The results revealed that 95 samples (47.26%) were positive for CPV-2, and they showed 98.8%–100% nucleotide identity and 97.6%–100% amino acid identity. Of 95 CPV-2-positive samples, types new2a (Ser297Ala), new2b (Ser297Ala), and 2c accounted for 64.21%, 21.05%, and 14.74%, respectively. The positive rate of CPV-2 and the distribution of the new2a, new2b and 2c types exhibited differences among regions, seasons, and ages. Immunized dogs accounted for 48.42% of 95 CPV-2-positive samples. Coinfections with canine coronavirus, canine kobuvirus, and canine bocavirus were identified. Phylogenetic analysis revealed that the identified new2a, new2b, and CPV-2c strains in our study exhibited a close relationship with most of the CPV-2 strains from China; type new2a strains exhibited high variability, forming three subgroups; type new2b and CPV-2c strains formed one group with reference strains from China. Of 95 CPV-2 strains, Tyr324Ile and Thr440Ala substitutions accounted for 100% and 64.21%, respectively; all type new2b strains exhibited the Thr440Ala substitution, while the unique Gln370Arg substitution was found in all type 2c strains. Recombination analysis using entire VP2 gene indicated possible recombination events between the identified CPV-2 strains and reference strains from China. Our data revealed the co-circulation of new CPV-2a, new CPV-2b, and rare CPV-2c, as well as potential recombination events among Chinese CPV-2 strains.

Introduction

Canine parvovirus-2 (CPV-2) causes a highly contagious and often fatal disease characterized by vomiting and hemorrhagic gastroenteritis in dogs of all ages, and by myocarditis in pups of less than three months of age [1]. The vaccine could provide protective immunity against the classic CPV-2 infection in canine populations, reducing the mortality of animals and spreading of the virus. However, the long-term interaction of the virus and its host may have led to the emergence of CPV-2 antigenic variants. During the past decades, new antigenic types of the CPV-2a, CPV-2b, CPV-2c, new CPV-2a, and new CPV-2b were frequently reported in the dog population, and they replaced the original CPV-2, which has caused widespread concern [29]. Therefore, molecular epidemiological investigation of CPV-2 is an undergoing hotspot issue worldwide.

The VP2 protein of CPV-2 is the major capsid protein, and it is highly antigenic, playing important roles in determining viral host ranges and tissue tropisms [10, 11]. At present, VP2 is widely used for the identification and monitoring of the types of CPV-2 circulating in the canine population based on the amino acid residues of the VP2 protein at positions 426Asp/Glu/Asn and 297Ser/Ala [10, 1215]. In the current study, the molecular epidemiology of the CPV-2 strains circulating in northeast China was investigated by polymerase chain reaction (PCR) targeting the partial VP2 gene. Moreover, potential recombination events of the identified CPV-2 strains were analyzed using the selected entire VP2 gene. Our aim was to provide insights into the epidemiology and genetic diversity of the CPV-2 strains circulating in northeast China, which can be useful for preventing and controlling CPV-2 infections.

Materials and Methods

Ethics statement

All animal experiments were conducted according to the Guide for the Care and Use of Laboratory Animals of Harbin Veterinary Research Institute (HVRI), Chinese Academy of Agricultural Sciences, China. Sampling and data publication also were approved by animal’s owners. The field study did not involve endangered or protected species. No specific permissions were required for locations of samples because the samples were collected from public areas or non-protection areas.

Sampling

Commercial virus sampling tubes (YOCON Biological Technology Co. Ltd., Beijing, China) containing 3.5 mL of Hank’s balanced salt solution, streptomycin, and penicillin, were used to collect fecal samples. A total of 201 fecal samples were collected in the form of rectal swabs from animal hospitals of the Harbin, Daqing, and Mudanjiang districts of Heilongjiang province in northeast China from May 2014 to April 2015. These animals, which were aged between 1 month and 1 year, included animals with symptoms of different degrees of diarrhea, animals with or without a vaccination history, and animals from different breeds and both genders. Of the 201 samples, 141 were collected in Harbin, 20 were collected in Daqing, and 40 were collected in Mudanjiang. All rectal swab samples were stored at −80°C, and they were also used for etiological investigations in our other studies.

DNA extraction

After 1 mL of fecal samples was centrifuged at 1,500 × g for 10 min at 4°C, the supernatant of each sample was transferred to a 1.5 mL EP tube. Genomic DNA was extracted from the supernatant using a commercial TIANamp Stool DNA Kit (Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer’s instructions. The extracted genomic DNAs were stored at −40°C. A commercial vaccine was used as a positive control, and distilled water was used as a negative control.

Sequencing of the VP2 fragment

For all samples, 568 bp of the VP2 gene was amplified using the primers CPV-FP (5’−TGATTGTAAACCATGTAGACTAAC−3’) and CPV-RP (5’−TAATGCAGTTAAAGGACCATAAG−3’) as described by Mittal et al. (2014). PCR amplifications were conducted in a 50 μL reaction volume containing 0.1 μM of forward primer, 0.1 μM of reverse primer, 4 μL of genomic DNA, 25μL of EmeraldAmp PCR Master Mix (2 × Premix) (TaKaRa Biotechnology Co. Ltd., Dalian, China), and an appropriate volume of double-distilled (dd) H2O. The mixtures were amplified by 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min, with a final extension at 72°C for 10 min using an automated Applied Biosystems GeneAmp PCR System 9700 thermal cycler (Thermo Fisher Scientific, Waltham, MA, USA). After the amplification, products were purified using the AxyPrep DNA Gel Extraction kit (A Corning Brand, Suzhou, China), and they were directly subjected to Sanger sequencing. Each sample was sequenced three times. Searches for nucleotide similarities were conducted using the BLAST algorithm at the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/blast). Sequence analysis was performed using the EditSeq program in the Lasergene DNASTAR version 5.06 software (DNASTAR Inc., Madison, WI, USA). Multiple sequence alignments were performed using the Multiple Sequence Alignment program of the DNAMAN version 6.0 software (Lynnon BioSoft, Point-Claire, Quebec, Canada). A total of 95 sequences (507 bp in length) were submitted to the NCBI Genbank (http://www.ncbi.nlm.nih.gov) under the accession numbers KT074258–KT074352.

Phylogenetic analysis

For the phylogenetic analysis, the VP2 gene sequences of CPV-2, CPV-2a, CPV-2b, CPV-2c, new CPV-2a, and new CPV-2b strains from different geographical locations within China and the rest of the world were retrieved from the NCBI nucleotide database. A nucleotide phylogenetic tree of the VP2 fragments (507 bp) of CPV-2 was generated from the ClustalX-generated alignments by MEGA6.06 software using the neighbor-joining method [16]. Neighbor-joining phylogenetic tree was built with the p-distance model and 1000 replicates of interior-branch test; otherwise, the default parameters in MEGA 6 were used. The support of reliability (above 50%) was shown in the obtained phylogenetic tree.

Sequencing of the entire selected VP2 genes

A total of 13 entire VP2 genes identified in our study, representing different genetic diversity in the phylogenetic tree of the 507 bp VP2 fragments of CPV-2, were chosen for recombination analysis. The entire VP2 gene of CPV-2 was amplified using the primers CPVvp2-F (5'-AACTAAAAGAAGTAAACCAC-3') and CPVvp2-R (5'-TGTAATAAACATAAAAACAT-3'), which resulted in a 1882 bp fragment covering the open reading frame of the VP2 gene. PCR amplifications were conducted in a 50 μL reaction volume containing 0.1 μM of forward primer, 0.1 μM of reverse primer, 4 μL of genomic DNA, 5 μL of 10 × buffer, 0.5 U of ExTaq DNA polymerase (TaKaRa), and an appropriate volume of ddH2O. The mixtures were amplified by 35 cycles of 94°C for 1 min, 50°C for 1 min, and 72°C for 2 min, with a final extension at 72°C for 10 min using an automated Applied Biosystems GeneAmp PCR System 9700 thermal cycler. The VP2 PCR products were purified using the AxyPrep DNA Gel Extraction kit (Corning), and then purified products were directly subjected to Sanger sequencing. Each sample was sequenced three times. All sequences were submitted to the NCBI Genbank (http://www.ncbi.nlm.nih.gov) under the accession numbers KT156825–KT156837.

Recombination analysis

Thirteen entire VP2 sequences in this study and 25 VP2 gene reference sequences from different areas in China were used for the recombination analysis (Table 1). The 38 VP2 genes were aligned in the ClustalX (1.83) program, and then were screened for possible recombination by using the Recombination Detection Program (RDP) [17], GENECONV [18], BOOTSCAN [19], MaxChi [20], CHIMAERA [21], and SISCAN [22] methods embedded in RDP4. Only potential recombination events detected by two or more of the programs, coupled with phylogenetic evidence of recombination, were considered significant using the highest acceptable P-value cutoff of 0.05.

Table 1. List of the entire VP2 gene for recombination analysis.

No. Strains Accession no. Genotyping Location in China Year
1 MDJ-1 KT156825 New2a Northeast (Mudanjiang) 2014
2 MDJ-7 KT156826 New2a Northeast (Mudanjiang) 2014
3 MDJ-9 KT156827 New2a Northeast (Mudanjiang) 2014
4 MDJ-15 KT156828 New2a Northeast (Mudanjiang) 2014
5 MDJ-20 KT156829 New2a Northeast (Mudanjiang) 2015
6 MDJ-22 KT156830 New2a Northeast (Mudanjiang) 2015
7 HRB-A4 KT156831 New2b Northeast (Harbin) 2014
8 HRB-A6 KT156832 New2c Northeast (Harbin) 2014
9 HRB-b2 KT156833 New2b Northeast (Harbin) 2014
10 HRB-e2 KT156834 New2a Northeast (Harbin) 2015
11 HRB-ee7 KT156835 New2a Northeast (Harbin) 2014
12 HRB-F8 KT156836 New2a Northeast (Harbin) 2014
13 HRB-J8 KT156837 New2b Northeast (Harbin) 2014
14 bj-5 GQ169549 New2a North (Beijing) 2010
15 Wu KJ674820 New2a South-central (Wuhan) 2013
16 CPV/WH02/06 EU377537 New2a South-central (Wuhan) 2006
17 06–11-NJ FJ432716 New2a East (Nanjing) 2006
18 08–5-WH FJ432717 New2a South-central (Wuhan) 2008
19 Wh-3 GQ169539 New2a South-central (Wuhan) 2009
20 1-nj GQ169550 New2a East (Nanjing) 2009
21 09/09 GU452713 New2a NA 2009
22 11/09 GU452715 New2a NA 2009
23 CPV-6(10) JQ743904 New2a NA 2010
24 CPVSH-3/2011 JX121627 New2a East (Shanghai) 2011
25 GZ0202 GU569937 New2b Southwest (Guizhou) 2002
26 YN0201 GU569938 New2b Southwest (Yunnan) 2002
27 YN0203 GU569940 New2b Southwest (Yunnan) 2002
28 GZ0201 GU569944 New2b Southwest (Guizhou) 2002
29 CPV-4(10) JQ743890 New2b NA 2010
30 CPV-10(10) JQ743891 New2b NA 2010
31 CPV-BG(11) JQ743892 New2b NA 2011
32 CPV-HS(11) JQ743893 New2b NA 2011
33 CPV-BM(11) JQ743894 New2b NA 2011
34 CPV-2b-wuhan2 KC881278 New2b South-central (Wuhan) 2010
35 06/09 GU380303 2c NA 2009
36 08/09 GU380305 2c NA 2009
37 G1 KF482468 2c South 2009
38 G15 KF482471 2c South 2009

Screening for canine enteric pathogens

Fecal samples that tested positive for CPV-2 were also screened for canine bocavirus (CBoV), canine coronavirus (CCoV), canine astrovirus (CaAstV), canine norovirus (CNoV), canine kobuvirus (CaKV), and group A-rotavirus (RV-A), by either PCR or reverse transcription-PCR (RT-PCR) and sequencing, as previously described [2328].

Results

Investigation and genotyping of CPV-2

A total of 201 samples from the Harbin, Daqing, and Mudanjiang districts of Heilongjiang province in northeast China were detected via PCR amplification of 568 bp of the VP2 gene of CPV-2. Nucleotide sequences of the partial VP2 of CPV-2 strains identified in our study were shown in Supporting Information (S1 Fig). The characteristics of the CPV-2-positive dogs, the genotypes of CPV-2 strains, and the amino acid substitutions of the VP2 protein are shown in Table 2, and a further analysis of the CPV-2-positive samples is shown in Table 3. Of the 201 samples, 47.26% (95/201) were positive for CPV-2, and three types, new2a (Ser297Ala), new2b (Ser297Ala), and 2c, were found, accounting for 64.21%, 21.05%, and 14.74%, respectively. The new2a type was identified in Harbin, Daqing, and Mudanjiang; the new2b type was identified in Harbin and Daqing; and the 2c type was identified in Harbin and Mudanjiang.

Table 2. Characteristics of the CPV-2 positive dogs, the genotypes of CPV-2 strains, and amino acid substitution of the VP2 protein in northeast China.

No. Strain Accession No. Collection date Location Breed Gender Age Vaccine Other enteric viruses (Accession No.) CPV type Substitution of amino acid residues in VP2 protein of CPV
297 324 370 419 426 440
Reference strains 1 CPV-b M38245 1996 USA NA NA NA NA NA 2 Ser Tyr Gln Asn Asn Thr
2 YB8301 GU569943 1983 China NA NA NA NA NA 2 Ser Try Gln Asn Asn Thr
3 CPV-15 M24003 1984 USA NA NA NA NA NA 2a Ser Tyr Gln Asn Asn Thr
4 CC8601 GU569948 1986 China NA NA NA NA NA 2a Ser Tyr Gln Asn Asn Thr
5 CPV-39 M74849 1984 USA NA NA NA NA NA 2b Ser Tyr Gln Asn Asp Thr
6 56/00 FJ222821 2009 Italy NA NA NA NA NA 2c Ala Tyr Gln Asn Glu Thr
7 06/09 GU380303 2009 China NA NA NA NA NA 2c Ala Ile Gln Asn Glu Thr
8 G1 KF482468 2009 China NA NA NA NA NA 2c Ala Tyr Gln Asn Glu Thr
9 CPV-435 AY742953 2005 USA NA NA NA NA NA New2a Ala Tyr Gln Asn Asn Thr
10 Wh-6 GQ169542 2009 China NA NA NA NA NA New2a Ala Ile Gln Asn Asn Thr
11 CPVSH-2/2011 JX121626 2011 China NA NA NA NA NA New2a Ala Ile Gln Asn Asn Ala
12 CPV-436 AY742955 2005 USA NA NA NA NA NA New2b Ala Tyr Gln Asn Asp Thr
13 GZ0201 GU569944 2002 China NA NA NA NA NA New2b Ala Tyr Gln Asn Asp Thr
14 CPV-2b-wuhan2 KC881278 2010 China NA NA NA NA NA New2b Ala Ile Gln Asn Asp Ala
Strains in this study 1 MDJ-1 KT074258 Dec-2014 MDJ SH F 2M Yes CCoV (KT192642) New2a Ala Ile Gln Asn Asn Ala
2 MDJ-22 KT074265 Oct-2014 MDJ GM F 6M Yes New2a Ala Ile Gln Asn Asn Ala
3 MDJ-28 KT074267 Dec-2014 MDJ JS M 3M Yes New2a Ala Ile Gln Asn Asn Ala
4 MDJ-29 KT074268 May-2015 MDJ MX M 5M No New2a Ala Ile Gln Asn Asn Ala
5 MDJ-32 KT074269 Jan-2015 MDJ NA NA NA Yes New2a Ala Ile Gln Asn Asn Ala
6 MDJ-40 KT074272 May-2015 MDJ PD F NA Yes New2a Ala Ile Gln Asn Asn Ala
7 DQ-alpha1 KT074273 Dec-2014 DQ NA F 3M No CCoV (KT192660) New2a Ala Ile Gln Asn Asn Ala
8 DQ-beta1 KT074274 May-2015 DQ PD NA 3M Yes New2a Ala Ile Gln Asn Asn Ala
9 HRB-A5 KT074278 Sep-2014 HRB MX M NA NA New2a Ala Ile Gln Asn Asn Ala
10 HRB-A8 KT074280 Oct-2014 HRB BF M 4M NA CCoV (KT192655), CaKV (KT210398) New2a Ala Ile Gln Asn Asn Ala
11 HRB-B0 KT074279 Oct-2014 HRB PM F 3M Yes CCoV (KT192665), CaKV (KT210404) New2a Ala Ile Gln Asn Asn Ala
12 HRB-B7 KT074283 Oct-2014 HRB MI F 3M Yes CCoV (KT192668) New2a Ala Ile Gln Asn Asn Ala
13 HRB-b9 KT074318 Sep-2014 HRB NA M 3M Yes New2a Ala Ile Gln Asn Asn Ala
14 HRB-C2 KT074285 Oct-2014 HRB TM F NA No CCoV (KT192678), CaKV (KT210410) New2a Ala Ile Gln Asn Asn Ala
15 HRB-C5 KT074286 Oct-2014 HRB YT M NA No New2a Ala Ile Gln Asn Asn Ala
16 HRB-C6 KT074287 Oct-2014 HRB BF F 4M Yes New2a Ala Ile Gln Asn Asn Ala
17 HRB-C9 KT074289 Oct-2014 HRB MX F 3M Yes New2a Ala Ile Gln Asn Asn Ala
18 HRB-b5 KT074290 Oct-2014 HRB NA NA 3M No New2a Ala Ile Gln Asn Asn Ala
19 HRB-bb7 KT074281 Oct-2014 HRB GM NA 2M NA CCoV (KT192671), New2a Ala Ile Gln Asn Asn Ala
20 HRB-bb8 KT074284 Oct-2014 HRB SM F 2M No CCoV (KT192672), CaKV (KT210406) New2a Ala Ile Gln Asn Asn Ala
21 HRB-dd2 KT074291 Nov-2014 HRB BD M 5M NA New2a Ala Ile Gln Asn Asn Ala
22 HRB-D4 KT074294 Oct-2014 HRB YT F 4M NA CCoV (KT192683) New2a Ala Ile Gln Asn Asn Ala
23 HRB-D8 KT074295 Nov-2014 HRB YT M 3.5M No New2a Ala Ile Gln Asn Asn Ala
24 HRB-D9 KT074296 Nov-2014 HRB NA NA 3.5M Yes CaKV (KT210414) New2a Ala Ile Gln Asn Asn Ala
25 HRB-E0 KT074297 Nov-2014 HRB NA F 3.5M No New2a Ala Ile Gln Asn Asn Ala
26 HRB-E1 KT074298 Nov-2014 HRB NA M 3.5M Yes CCoV (KT192686) New2a Ala Ile Gln Asn Asn Ala
27 HRB-e2 KT074299 Apr-2015 HRB NA M 3M No New2a Ala Ile Gln Asn Asn Ala
28 HRB-E8 KT074300 Nov-2014 HRB GM M 3M Yes CCoV (KT192687), CaKV (KT210415) New2a Ala Ile Gln Asn Asn Ala
29 HRB-F0 KT074301 Feb-2015 HRB SM F 4M Yes New2a Ala Ile Gln Asn Asn Ala
30 HRB-F4 KT074302 Nov-2014 HRB NA M NA No New2a Ala Ile Gln Asn Asn Ala
31 HRB-G4 KT074305 Mar-2015 HRB YT F 4M Yes New2a Ala Ile Gln Asn Asn Ala
32 HRB-H2 KT074307 Dec-2014 HRB PD F 3M NA CaKV (KT210423) New2a Ala Ile Gln Asn Asn Ala
33 HRB-I1 KT074308 Sep-2014 HRB NA F 4M NA CCoV (KT192697) New2a Ala Ile Gln Asn Asn Ala
34 HRB-I3 KT074309 Dec-2014 HRB NA F 3M Yes New2a Ala Ile Gln Asn Asn Ala
35 HRB-I4 KT074310 Sep-2014 HRB SN M 3.5M NA New2a Ala Ile Gln Asn Asn Ala
36 HRB-I8 KT074312 Feb-2015 HRB MI M 3M No New2a Ala Ile Gln Asn Asn Ala
37 HRB-J1 KT074313 Sep-2014 HRB NA M 3M Yes New2a Ala Ile Gln Asn Asn Ala
38 HRB-K1 KT074316 Mar-2015 HRB NA M 3.5M Yes New2a Ala Ile Gln Asn Asn Ala
39 HRB-K3 KT074317 Jan-2015 HRB NA F 3M Yes CaKV (KT210425) New2a Ala Ile Gln Ser Asn Ala
40 HRB-G0 KT074304 Dec-2014 HRB PK M 4M Yes New2a Ala Ile Gln Ser Asn Ala
41 HRB-F8 KT074303 Nov-2014 HRB PD M 3M No CCoV (KT192691), CaKV (KT210418) New2a Ala Ile Gln Ser Asn Ala
42 MDJ-7 KT074259 Nov-2014 MDJ NA M 6M Yes New2a Ala Ile Gln Asn Asn Thr
43 MDJ-9 KT074260 Dec-2014 MDJ RC M 3M No New2a Ala Ile Gln Asn Asn Thr
44 MDJ-15 KT074261 Nov-2014 MDJ RC F 1.5M Yes CBoV (KR998482) New2a Ala Ile Gln Asn Asn Thr
45 MDJ-18 KT074262 Mar-2015 MDJ MX M 3M Yes New2a Ala Ile Gln Asn Asn Thr
46 MDJ-20 KT074263 Mar-2015 MDJ NA F 3M Yes New2a Ala Ile Gln Asn Asn Thr
47 MDJ-21 KT074264 Dec-2014 MDJ RC M 3M No CBoV (KR998484) New2a Ala Ile Gln Asn Asn Thr
48 MDJ-27 KT074266 Apr-2015 MDJ NA F NA NA CBoV (KR998487), CaKV (KT210392) New2a Ala Ile Gln Asn Asn Thr
49 MDJ-33 KT074270 Jan-2015 MDJ MX NA 3M No New2a Ala Ile Gln Asn Asn Thr
50 MDJ-37 KT074271 Apr-2015 MDJ NA M NA Yes New2a Ala Ile Gln Asn Asn Thr
51 DQ-beta7 KT074275 Feb-2015 DQ NA M 1M No CBoV (KR998491) New2a Ala Ile Gln Asn Asn Thr
52 HRB-A2 KT074277 Sep-2014 HRB CH M 4M No New2a Ala Ile Gln Asn Asn Thr
53 HRB-B8 KT074282 Oct-2014 HRB GM NA NA NA New2a Ala Ile Gln Asn Asn Thr
54 HRB-C7 KT074288 Oct-2014 HRB NA M 2M NA CBoV (KR998493) New2a Ala Ile Gln Asn Asn Thr
55 HRB-a1 KT074276 Sep-2014 HRB NA F 3.5M Yes CCoV (KT192656), CBoV (KR998488), CaKV (KT210394) New2a Ala Ile Gln Asn Asn Thr
56 HRB-ee7 KT074292 Dec-2014 HRB NA M 3.5M Yes New2a Ala Ile Gln Asn Asn Thr
57 HRB-D2 KT074293 Oct-2014 HRB PD F 2M No New2a Ala Ile Gln Asn Asn Thr
58 HRB-G6 KT074306 Dec-2014 HRB GM F 3.5M Yes New2a Ala Ile Gln Asn Asn Thr
59 HRB-I7 KT074311 Nov-2014 HRB NA F NA Yes New2a Ala Ile Gln Asn Asn Thr
60 HRB-J4 KT074314 Oct-2014 HRB NA M 4M No New2a Ala Ile Gln Asn Asn Thr
61 HRB-J7 KT074315 Nov-2014 HRB NA F 3.5M Yes New2a Ala Ile Gln Asn Asn Thr
62 DQ-beta0 KT074336 Dec-2014 DQ PM M 1.5M No CCoV (KT192673) New2b Ala Ile Gln Asn Asp Ala
63 DQ-beta4 KT074337 Dec-2014 DQ SN F 5M Yes New2b Ala Ile Gln Asn Asp Ala
64 DQ-beta9 KT074338 Mar-2015 DQ CO M NA Yes New2b Ala Ile Gln Asn Asp Ala
65 HRB-A4 KT074319 Sep-2014 HRB NA F 3M NA CCoV (KT192652), CaKV (KT210396) New2b Ala Ile Gln Asn Asp Ala
66 HRB-A9 KT074320 Sep-2014 HRB PM NA 3M NA New2b Ala Ile Gln Asn Asp Ala
67 HRB-B6 KT074321 Oct-2014 HRB CH F 2M No New2b Ala Ile Gln Asn Asp Ala
68 HRB-aa8 KT074322 Oct-2014 HRB BF M 4M NA New2b Ala Ile Gln Asn Asp Ala
69 HRB-b2 KT074323 Oct-2014 HRB GM NA 3M NA CBoV (KR998490) New2b Ala Ile Gln Asn Asp Ala
70 HRB-d1 KT074324 Nov-2014 HRB LR F 7M Yes New2b Ala Ile Gln Asn Asp Ala
71 HRB-d3 KT074325 Oct-2014 HRB RC NA NA NA New2b Ala Ile Gln Asn Asp Ala
72 HRB-d5 KT074326 Sep-2014 HRB NA M 3M No New2b Ala Ile Gln Asn Asp Ala
73 HRB-E7 KT074327 Nov-2014 HRB SM F 3M Yes New2b Ala Ile Gln Asn Asp Ala
74 HRB-F5 KT074328 Nov-2014 HRB YT F 4M Yes CCoV (KT192690) New2b Ala Ile Gln Asn Asp Ala
75 HRB-F6 KT074329 Nov-2014 HRB NA M 3M No New2b Ala Ile Gln Asn Asp Ala
76 HRB-F7 KT074330 Nov-2014 HRB MI F 3M Yes New2b Ala Ile Gln Asn Asp Ala
77 HRB-H3 KT074331 Jan-2015 HRB SM M 3M No CCoV (KT192693) New2b Ala Ile Gln Asn Asp Ala
78 HRB-H4 KT074332 Dec-2014 HRB NA F 3.5M No New2b Ala Ile Gln Asn Asp Ala
79 HRB-H8 KT074333 Dec-2014 HRB BF M 3.5M No New2b Ala Ile Gln Asn Asp Ala
80 HRB-I5 KT074334 Oct-2014 HRB BD M 3.5M Yes New2b Ala Ile Gln Asn Asp Ala
81 HRB-J8 KT074335 Dec-2014 HRB SM NA 3.5M Yes New2b Ala Ile Gln Asn Asp Ala
82 MDJ-4 KT074348 Apr-2015 MDJ NA M 4M No 2c Ala Ile Arg Asn Glu Thr
83 MDJ-6 KT074349 Dec-2014 MDJ NA F NA Yes 2c Ala Ile Arg Asn Glu Thr
84 MDJ-11 KT074350 Oct-2014 MDJ PD F 4M No CCoV (KT192644) 2c Ala Ile Arg Asn Glu Thr
85 MDJ-24 KT074351 Nov-2014 MDJ RC F 1.5M Yes 2c Ala Ile Arg Asn Glu Thr
86 MDJ-39 KT074352 Mar-2015 MDJ NA F 3M No 2c Ala Ile Arg Asn Glu Thr
87 HRB-A6 KT074339 Sep-2014 HRB NA F 3M Yes CCoV (KT192653) 2c Ala Ile Arg Asn Glu Thr
88 HRB-aa9 KT074340 Oct-2014 HRB CH M 3.5M No 2c Ala Ile Arg Asn Glu Thr
89 HRB-b3 KT074341 Oct-2014 HRB PM NA 2M No CCoV (KT192670), CaKV (KT210409) 2c Ala Ile Arg Asn Glu Thr
90 HRB-E5 KT074342 Nov-2014 HRB NA M 3M Yes 2c Ala Ile Arg Asn Glu Thr
91 HRB-e6 KT074343 Mar-2015 HRB PD F 3.5M No 2c Ala Ile Arg Asn Glu Thr
92 HRB-E9 KT074344 Nov-2014 HRB CH NA NA Yes 2c Ala Ile Arg Asn Glu Thr
93 HRB-F9 KT074345 Feb-2015 HRB NA M NA NA 2c Ala Ile Arg Asn Glu Thr
94 HRB-G8 KT074346 Nov-2014 HRB PM NA 4M Yes CaKV (KT210421) 2c Ala Ile Arg Asn Glu Thr
95 HRB-J6 KT074347 Nov-2014 HRB PD NA NA Yes 2c Ala Ile Arg Asn Glu Thr

Note. For breed, GM = Golden Malinois, LR = Labrador Retriever, RC = Rough Collie, CO = Caucasian Owtcharka, TM = Tibetan Mastiff, PD = Poodle, CH = Chihuahua, and JS = Japanese Spitz; For gender, F = female, and M = male; for age, M = month; for location, MDJ = Mudanjiang, HRB = Harbin, DQ = Daqing.

Table 3. Further analysis of the CPV-2 positive samples.

Harbin Daqing Mudanjiang Total
Numbers of sample 141 20 40 201
Positive rate for CPV-2 48.94% (69/141) 30% (6/20) 50% (20/40) 47.26% (95/201)
Genotyping of CPV-2 New2a 62.32% (43/69) 50% (3/6) 75% (15/20) 64.21% (61/95)
New2b 24.64% (17/69) 50% (3/6) 21.05% (20/95)
2c 13.04% (9/69) 25% (5/20) 14.74% (14/95)
Substitution of amino acid residues in VP2 protein of CPV-2 297Ser/Ala Ser
Ala 100% (69/69) 100% (6/6) 100% (20/20) 100% (95/95)
324Tyr/Ile Tyr
Ile 100% (69/69) 100% (6/6) 100% (20/20) 100% (95/95)
370Gln/Arg Gln 86.96% (60/69) 100% (6/6) 75% (15/20) 85.26% (81/95)
Arg 13.04% (9/69) 25% (5/20) 14.74% (14/95)
419Asn/Ser Asn 95.65% (66/69) 100% (6/6) 100% (20/20) 96.84% (92/95)
Ser 4.35% (3/69) 3.16% (3/95)
440Thr/Ala Thr 27.54% (19/69) 16.67% (1/6) 70% (14/20) 35.79% (34/95)
Ala 72.46% (50/69) 83.33% (5/6) 30% (6/20) 64.21% (61/95)
Vaccined Yes 44.93% (31/69) 50% (3/6) 60% (12/20) 48.42% (46/95)
No 30.43% (21/69) 50% (3/6) 35% (7/20) 32.63% (31/95)
Collection data Jan. to Mar. 11.59% (8/69) 33.33% (2/6) 25% (5/20) 15.78% (15/95)
Apr. to Jun. 1.45% (1/69) 16.67% (1/6) 25% (5/20) 7.37% (7/95)
Jul. to Sep. 15.94% (11/69) 11.58% (11/95)
Oct. to Dec. 71.01% (49/69) 50% (3/6) 50% (10/20) 65.26% (62/95)
Age 0< Age ≤2M 8.70% (6/69) 33.33% (2/6) 15% (3/20) 11.58% (11/95)
2M< Age ≤4M 73.91% (51/69) 33.33% (2/6) 45% (9/20) 65.26% (62/95)
4M< Age ≤6M 1.45% (1/69) 16.67% (1/6) 15% (3/20) 5.26% (5/95)
>6M 1.45% (1/69) 1.05% (1/95)
Other enteric viruses in the CPV positive samples CCoV 24.64% (17/69) 33.33% (2/6) 10% (2/20) 22.11% (21/95)
CaKV 18.84% (13/69) 5% (1/20) 14.74% (14/95)
CBoV 4.35% (3/69) 16.67% (1/6) 15% (3/20) 7.37% (7/95)
CCoV+CaKV 13.04% (9/69) 5% (1/20) 10.53% (10/95)
CBoV+CaKV 1.45% (1/69) 5% (1/20) 2.11% (2/95)
CCoV+CBoV+CaKV 1.45% (1/69) 1.05% (1/95)
Identity Nucleotides New2a 99.2%–100% 99.2%–100% 99.2%–100% 99.0%–100%
New2b 99.6%–100% 100% 99.8%–100%
2c 100% 100% 100%
New2a+ New2b+2c 98.8%–100% 99.0%–100% 98.6%–100% 98.8%–100%
Amino acids New2a 98.8%–100% 99.4%–100% 99.4%–100% 98.8%–100%
New2b 100% 100% 100%
2c 100% 100% 100%
New2a+ New2b+2c 97.6%–100% 98.8%–100% 98.2%–100% 97.6%–100%

Sequence comparisons revealed 99.0%–100%, 99.8%–100%, and 100% nucleotide identities within the new2a, new2b, and 2c strains, respectively, and 98.8%–100% nucleotide identities between the new2a, new2b, and 2c types, respectively. At the amino acid level, 98.8%–100%, 100%, 100%, 97.6%–100% identities were revealed within the new2a, new2b, and 2c strains, and between the new2a, new2b, and 2c types, respectively. A total of five substitutions of amino acid residues occurred in all 95 CPV-2-positive samples based on an analysis of partial VP2 sequences. Residue 324Ile was present in all 95 CPV-2-positive samples when compared with the reference strains, and residue 370Arg was specific for type CPV-2c when compared with CPV-2c reference strains. Only three new CPV-2a strains, HRB-K3, HRB-G0, and HRB-F8, showed amino acids substitutions (Asn→Ser) at position 419. At position 440, all new2b strains and most of the new2a strains identified in our study showed amino acids substitutions (Thr→Ala) when compared with the reference strains.

For the CPV-2-positive animals, 65.26% (62/95) were aged from 2–4 months, 48.42% (46/95) had a vaccination history, and 32.63% (31/95) had no vaccination history, while 65.26% (62/95) were collected from October to December. Coinfections with CCoV, CaKV, and CBoV were found in the 95 CPV-2-positive samples, of which 22.11% (21/95) were positive for CCoV, 14.74% (14/95) were positive for CaKV, 7.37% (7/95) were positive for CBoV, 10.53% (10/95) were positive for CCoV and CaKV, 2.11% (2/95) were positive for CBoV and CaKV, and 1.05% (1/95) were positive for CCoV, CBoV, and CaKV.

Phylogenetic analysis

To analyze the genetic diversity of the new2a, new2b, and 2c strains identified in northeast China, partial VP2 gene sequences of CPV-2, CPV-2a, CPV-2b, CPV-2c, new CPV-2a, and new CPV-2b strains from different geographical locations within China and the rest of the world were used to construct a phylogenetic tree. Nucleotide sequences of the partial VP2 used for construction of phylogenetic tree was shown in Supporting Information (S2 Fig). The generated phylogenetic tree was composed of four groups: GI, GII, GIII, and GIV. The 95 CPV-2 strains from northeast China were divided into two different groups: GI and GII (Fig 1). All 14 CPV-2c strains and 20 new CPV-2b strains identified in our study were divided into the GI group. Meanwhile, the GI group included the new2a, new2b and 2c reference strains from China. The GII group, consisting of four subgroups, GIIa, GIIb, GIIc, and GIId, was specific for the new CPV-2a strains, except for one new CPV-2b strain (HRB-J8). The new CPV-2a strains identified in northeast China were divided into the three subgroups GIIa, GIIb, and GIIc, and the new CPV-2a reference strains from other countries were divided into the subgroup GIId. The GIV group consisted of different genotypes of CPV-2 reference strains from China and other countries.

Fig 1. Phylogenetic analysis of CPV-2 strains on the basis of the partial VP2 gene (507 bp).

Fig 1

The red spot diagram represents CPV-2 strains identified in this study. The values in parentheses indicate the number of strains with identical sequences to that included in the tree. (1): KT074275 from Daqing; (2): KT074317 and KT074304 from Harbin; (6): KT074266, KT074270 from Mudanjiang; KT074276, KT074288, KT074293, and KT074306 from Harbin; (8): KT074262, KT074264, and KT074271 from Mudanjiang; KT074277, KT074282, KT074311, KT074314, and KT074315 from Harbin. (13): KT074340-KT074347 from Harbin; KT074348-KT074352 from Mudanjiang. (17): KT074320-KT074322 and KT074324-KT074334 from Harbin; KT074336-KT074338 from Daqing; (35): KT074267-KT074269 and KT074272 from Mudanjiang; KT074273 and KT074274 from Daqing; KT074278, KT074279, KT074280-KT074287, KT074289- KT074291, KT074294- KT074298, KT074300- KT074302, KT074305, KT074307- KT074310, KT074312, KT074313, KT074316, and KT074318 from Harbin.

Recombination analysis

The recombination analysis based on the VP2 gene revealed that two potential recombination events occurred between the selected 13 strains in this study and the 25 reference strains from China, but no potential recombination events were found within the selected 13 strains in this study. Nucleotide sequences of the entire VP2 of CPV-2 strains identified in our study were shown in Supporting Information (S3 Fig), and the breakpoint positions of recombination events were shown in Supporting Information (S4 Fig). Fig 2 shows the recombination event that occurred between the HRB-F8 strain (accession no. KT156836) and a G1 strain (accession no. KF482468), which led to the recombinant MDJ-20 strain (accession no. KT156829). The bootscan plot of this recombination event is shown in Fig 2A, which used HRB-F8 and G1 as minor parent strain, and major parent strain, respectively. The recombination event was confirmed by fast neighbor-joining trees that were constructed using the regions derived from minor parent strain (1–133 and 558–1755) (Fig 2B), the recombinant region (134–557) (Fig 2C), and the non-recombinant region (Fig 2D). Fig 3 shows the recombination event that occurred between MDJ-20 strain (KT156829) and a G1 strain (KF482468), which led to the recombinant 08/09 strain (GU380305). The bootscan plot of this recombination event is shown in Fig 3A, which used MDJ-20 and G1 as major parent strain, and minor parent strain, respectively. The recombination event was confirmed by fast neighbor-joining trees that were constructed using the regions derived from minor parent strain (1–809 and 1708–1755) (Fig 3B), the recombinant region (810–1707) (Fig 3C), and the non-recombinant region (Fig 3D). The two recombinant strains MDJ-20 and 80/09 were divided into different clusters in the recombination region-based phylogenetic trees when compared with the non-recombination region-based phylogenetic trees.

Fig 2. Identification of the recombination event between the minor parent strain HRB-F8 (KT156836) (blue) and the major parent strain G1 (KF482468) (green), which led to the recombinant MDJ-20 strain (KT156829) (red).

Fig 2

(A) Bootscan evidence for the recombination origin on the basis of pairwise distance, modelled with a window size 200, step size 20 and 100 bootstrap replicates. (B, C, and D) Fast neighbour-joining (NJ) tree (1000 replicates, Kimura two-parameter distance) constructed using the regions derived from minor parent strain (1–133, 558–1755) (B), the recombination region (134–557) (C), and non-recombinant region (D). Note. The potential recombination event was detected in Maxchi (P = 3.74E-02) and SiSscan (P = 1.42E-04) methods.

Fig 3. Identification of the recombination event between the major parent strain MDJ-20 (KT156829) (green) and the minor parent strain G1 (KF482468) (blue), which led to the recombinant 08/09 strain (GU380305) (red).

Fig 3

(A) Bootscan evidence for the recombination origin on the basis of pairwise distance, modelled with a window size 200, step size 20 and 100 bootstrap replicates. (B, C, and D) Fast neighbour-joining (NJ) tree (1000 replicates, Kimura two-parameter distance) constructed using the regions derived from minor parent strain (1–809, 1708–1755) (B), the recombination region (810–1707) (C), and non-recombinant region (D). Note. The potential recombination event was detected in Maxchi (P = 8.51E-04), SiSscan (P = 1.05E-02), and Chimaera (P = 7.25E-03) methods.

Discussion

It has been reported that a high proportion (29.2%–84%) of fecal samples from diarrhea-affected dogs tested positive for CPV-2 [11, 29]. In our study, 201 samples were collected from diarrhea-affected dogs in three districts in Heilongjiang province in northeast China, and 95 CPV-2-positive samples were identified, accounting for 47.26% of all samples. The results were in line with recent reports in China [15, 30]. The CPV-2-positive rate differed among the three districts (Harbin, 48.94%; Daqing, 30%; Mudanjiang, 50%). In our study, mixed infections of CCoV (22.11%), CaKV (14.74%), and CBoV (7.37%) were found in the CPV-2-positive samples. A coinfection of CCoV, CBoV, and CaKV with CPV-2 was also found in one sample. Cavalli et al. (2014) reported that mixed infections of CPV-2 and CCoV reached 49.12% in diarrheic dogs in Albania [31]. CaKV and CBoV had been reported to be associated with severe enteritis in a litter of puppies [28, 32]. The high prevalence of co-infections of CPV-2 with three enteric viruses may be associated with viral diarrhea in dogs in northeast China.

In our study, CPV-2-positive rates showed clear differences among seasons and ages. 65.26% of CPV-2-positive samples were collected from October to December. The high prevalence of CPV-2 between October and December may be indicative of a seasonal variation in northeast China. Additionally, a high CPV-2-positive rate was found in animals aged from 2–4 months, which is similar to the report described by Cavalli et al. (2014) [31]. At present, most dogs in China are vaccinated for CPV-2. However, in the CPV-2-positive samples identified in this study, 48.42% came from dogs with a vaccination history. This data demonstrated that the CPV-2 vaccines used in northeast China may fail to generate protective antibody titers against heterogeneous CPV antigenic types.

The Ser297Ala mutation may have had a remarkable influence on the process of host adaptation [33]. This mutation was used as a marker of the new CPV-2a/2b variant. This study indicated that the new CPV-2a (Ser297Ala) variant, accounting for 64.21% of CPV-2 infections, was the predominating strain circulating in the three districts of Heilongjiang province in northeast China. The new CPV-2b (Ser297Ala) variant, accounting for 21.05% of CPV-2 infections, was the second most predominant strain circulating in the Harbin and Daqing districts of Heilongjiang province. In our study, the prevalence of the new CPV-2a is in agreement with most of the reports that were recently conducted in China [8, 15, 30, 34, 35], African countries [36], other Asian countries, including Korea, Thailand, Taiwan, and Turkey, and Australia [6, 3739]. However, the prevalence of the new CPV-2b variant reported here in CPV-2-positive samples was higher than those in other reports in China. Interestingly, the presence of the rare CPV-2c, accounting for 14.74% of the CPV-2-positive samples, was confirmed in the Harbin and Mudanjiang of Heilongjiang province. The co-circulation of the CPV-2c, new CPV-2a, and new CPV-2b has previously not been reported in China. It has been reported that the CPV-2c variant is widely distributed and co-exists with other CPV types in Europe and North and South America [4045]. Compared with an earlier report that detected the CPV-2, new CPV-2a, and CPV-2b variants in China [46], the emergence of the CPV-2c in northeast China suggests that CPV-2 strains circulating in China exhibit genetic variations. Additionally, our results are similar to those reported for the genotypes of CPV-2 strains circulating in India, where the new CPV-2a and new CPV-2b were the predominant strains [14], and the co-circulation of the CPV-2c was also found [47].

In the VP2 protein of CPV-2, the Tyr324Ile mutation is adjacent to residue 323, which affects binding to canine transferrin receptors, resulting in changes in the host range of canine parvovirus [13]. Presumably, this mutation may result in stronger receptor binding. In our study, all 95 identified CPV-2 strains, including the new2a, new2b, and 2c types, contained the unique Tyr324Ile mutation. The Tyr324Ile mutation in CPV-2a strains has been reported in China [8, 15, 30, 46], South Korea [38], Thailand [48], Uruguay [49], Japan [50], Taiwan [6], and India [51]. These data support the view that the Try324Ile substitution in CPV-2 strains may be a common amino acid alteration in Asia countries, especially in China. This conclusion needs to be validated by an extensive epidemiological survey in a future study. In addition, further studies should be conducted to understand the relationship between the Try324Ile substitution and the severity of clinical symptoms.

In the VP2 protein of CPV-2, the amino acid residue at position 440 is located in the GH loop. It has been reported that the high levels of the Thr440Ala substitution in VP2 are associated with the evolution of antigenic variants in circulating parvovirus types [52, 53]. In our study, the Thr440Ala substitution was found in 64.21% of the 95 CPV-2-positive samples, of which 100% of the new2b strains had the Thr440Ala substitution, 67.22% of the new2a strains exhibited the Thr440Ala substitution, while the type 2c strains did not have the Thr440Ala substitution. At present, the Thr440Ala substitution has been frequently reported in China and other countries [8, 14, 15, 36]. Therefore, we suggest further studies to better understand the relationship between this mutation and the severity of clinical symptoms.

In this study, both new CPV-2b strains and CPV-2c strains exhibited 100% amino acid identity on the basis of the deduced sequence of the partial VP2 protein among different districts. These data demonstrated that type new2b and 2c strains of CPV-2 circulating in northeast China exhibited genetic stability. However, compared with CPV-2 reference strains, the type new2b strains showed a unique Thr440Ala substitution (100%), and the type 2c strains exhibited a unique Gln370Arg substitution (100%). The amino acid substitutions resulted in phylogenetic changes in the new2b and 2c strains identified in our study. The new2b and 2c strains (except one strain) differed genetically from the reference strains of other countries, forming one group (GI) with Chinese reference strains of the new2b and 2c types. In our study, the close relationship between the new 2b and 2c types suggests that these two types share a similar evolutionary history in China. For the identified type 2c strains, the unique Gln370Arg substitution, which was previously not described, was highlighted as evidence of a potential CPV-2c variant or new CPV-2c. Further studies regarding potential variant CPV-2c strains should be conducted to understand the relationship between the Gln370Arg substitution and viral pathogenicity.

The phylogenetic analysis revealed that the type new2a strains identified in our study exhibited high variability, forming three subgroups. Of the 61 new2a strains, a total of two amino acid substitution sites were revealed, Asn419Ser and Thr440Ala. The two amino acid substitutions, as well as some non-synonymous substitutions, accounted for the genetic diversity of the new2a strains in the phylogenetic tree. In our study, the Asn419Ser substitution was detected in only three of the 61 new2a strains. We suggest that the Asn419Ser substitution, first described here, should be the focus of further epidemiological investigations of CPV-2. Accumulating reports have indicated that the new CPV-2a strains, accounting for high positive rates among CPV-2 types, are the predominating strains circulating in China. The high variability of the new CPV-2a strains in our study may be attributed to long-term interactions between the new CPV-2a strains and their hosts. It is suggested that vaccinations against CPV-2 should be updated on the basis of these new CPV-2a strains.

Although point mutations are considered the main mechanism for generating genomic diversity in CPV, co-infection and recombination have also been explored as variability-inducing mechanisms [54]. Pérez et al. (2014) revealed that a recombinant strain arose from inter-genotypic recombination between CPV-2c and CPV-2a strains within the VP1/VP2 gene boundary [55]. In our study, two inter-genotypic recombination events between the 13 CPV-2 strains in this study and the Chinese reference strains were identified in the VP2 gene. The two recombination events occurred between type new2a and 2c strains, resulting in two potential recombinant type new2a strains. Our finding is in line with the occurrence of a possible recombination event in the CPV-2 strain 364-rec, as described by Pérez et al. (2014) [55]. Although the evidence of naturally occurring recombination events was not obtained in our study, possible recombination events among Chinese CPV-2 strains suggest that co-infections with CPV-2 strains with different genotypes, especially types 2a and 2c, as well as potential recombinant strains of CPV-2, should be monitored in China by extensive molecular epidemiology investigations in the future.

Since 1983, the epidemiological information of CPV-2 strains circulating in China has been frequently reported [8, 15, 30, 34, 35, 46]. Accumulating reports indicated that circulation of CPV-2, CPV-2a, CPV-2b, new CPV-2a, and new CPV-2b strains was identified in different districts of China, of which new CPV-2a was predominant strains in China, followed by new CPV-2b. Compared with the previous epidemiological investigation of CPV-2 in China, the detailed information of samples used in our study, including collection date, location, animal breed, animal gender, animal age, animal immunization, and coinfection with other enteric viruses, resulted in the generation of more precise and valuable epidemiological data which not only enriches basic epidemiological data of Chinese CPV-2 strains, but also increases the understanding of epidemic characteristics of CPV-2 strains circulating in northeast China. In addition, our findings revealed novel epidemiological information of CPV-2 in China, involving in the co-circulation of new CPV-2a with high variation, new CPV-2b, and rare CPV-2c in Heilongjiang province of northeast China, the unique amino acids substitutions including Gln370Arg substitution in identified CPV-2c strains, and Thr440Ala substitution in identified new CPV-2b strains, and the potential recombination events which could happen among type new2a and 2c CPV-2 field strains circulating in China.

Conclusions

Taken together, in the current study, the resulting data increase our understanding of the genetic evolution of CVP-2 strains in China, and they also provide valuable information for further studies of CPV-2 in other countries. However, some speculations resulting from our data still need to be validated by further and extensive epidemiological investigation in the future.

Supporting Information

S1 Fig. Nucleotide sequences of the partial VP2 gene of CPV-2 strains identified in our study.

(TXT)

S2 Fig. Nucleotide sequences of the partial VP2 gene were used for construction of phylogenetic tree.

(TXT)

S3 Fig. Nucleotide sequences of the entire VP2 gene of CPV-2 strains identified in our study were used for analysis of recombination events.

(TXT)

S4 Fig. The breakpoint positions of recombination events in our study.

(XLS)

Data Availability

All sequence files are available from the GenBank database (accession numbers KT074258–KT074352 and KT156825–KT156837).

Funding Statement

This work was supported by the Program for New Century Excellent Talents in Heilongjiang Provincial University (grant no. 1252-NCET-016).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Nucleotide sequences of the partial VP2 gene of CPV-2 strains identified in our study.

(TXT)

S2 Fig. Nucleotide sequences of the partial VP2 gene were used for construction of phylogenetic tree.

(TXT)

S3 Fig. Nucleotide sequences of the entire VP2 gene of CPV-2 strains identified in our study were used for analysis of recombination events.

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S4 Fig. The breakpoint positions of recombination events in our study.

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Data Availability Statement

All sequence files are available from the GenBank database (accession numbers KT074258–KT074352 and KT156825–KT156837).


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