Canine parvovirus (CPV) emerged in the late 1970s as a host-range variant of feline panleukopenia virus (FPV) (21). CPV belongs to the
Currently, only CPV-2 has been thoroughly investigated. The VP2 capsid protein, accounting for 90% of the CPV nucleocapsid and 426 amino acids in size, plays a crucial role in the structure of this virus and serves as the main protective antigen - two characteristics which are the classification criteria for CPV viral typing. Owing to variations in the antigenicity of VP2 across strains, multiple genotypes have emerged, including CPV-2a, CPV-2b, and CPV-2c. CPV-2a, CPV-2b, and CPV-2c differ at the 426th amino acid (Asn in 2a, Asp in 2b, and Glu in 2c) of the parvovirus VP2 protein (11, 17, 18). In general, a novel CPV-2 variant replaces the old variants rapidly (11, 15). In recent decades, CPV-2c has become widespread in European countries (6, 11), the United States (11, 12), South America (2, 11, 19, 20), and Africa (1, 11, 23). In Asia, CPV-2c was first reported from Vietnam in 2004 (11, 16); however, since then, this strain has not been prevalent in Asia (4, 11). Surprisingly, in the past few years, novel CPV-2c isolates from Asia have been identified in mainland China (8, 11, 25, 28), Taiwan (4, 11, 14), Laos (11, 24), and Thailand (3, 11). Therefore, in the present study, VP2 sequence analysis of strains collected from central and eastern China was performed to determine the mutation tendency of CPV-2 in infected dogs sampled from October 2018 to April 2019.
Reference strains used in the study
Strain | Accession number | Genotype | Place of isolation | Year of isolation |
---|---|---|---|---|
nn171025 | MK332005 | CPV-2a | Guangxi | 2017 |
nn17101 | MK332003 | CPV-2a | Guangxi | 2017 |
nn1693 | MK332002 | CPV-2b | Guangxi | 2016 |
nn1681 | MK331996 | CPV-2b | Guangxi | 2016 |
nn171105 | MK332007 | CPV-2c | Guangxi | 2017 |
nn171024 | MK332004 | CPV-2c | Guangxi | 2017 |
CPV-411b.us.9 | EU659121 | CPV-2b | The USA | 1998 |
CPV-13.us.81 | EU659118 | CPV-2a | The USA | 1981 |
CPV-6.us.80 | EU659117 | CPV-2 | The USA | 1980 |
Raccoon/ WI/ 37/ 10 | JN867618 | CPV-2a | The USA | 2010 |
110/ 07-27 | FJ005236 | CPV-2c | The USA | 2007 |
08-B | GU362934 | CPV-2a | Italy | 2008 |
260-00 | MF177231 | CPV-2a | Italy | 2000 |
140/ 05 | FJ005265 | CPV-2b | Italy | 2005 |
CPV /IZSSI /25835/ 09 | KU508407 | CPV-2c | Italy | 2009 |
56/00 | FJ222821 | CPV-2c | Italy | 2000 |
CPV/dog/HCM/20/2013 | LC216910 | CPV-2c | Indonesia | 2013 |
Pome | EF599098 | CPV-2c(a) | South Korea | 2007 |
DH326 | EF599097 | CPV-2b | South Korea | 2007 |
DH426 | EF599096 | CPV-2a | South Korea | 2007 |
16M130 | MH643886 | CPV-2 | South Korea | 2016 |
2670/CPV-2c/2010/Ind | KX425920 | CPV-2c | India | 2010 |
CU267 | MH711901 | CPV-2c | Thailand | 2017 |
TH011401 | KT364589 | CPV-2c | Thailand | 2014 |
T37 | CPU72698 | CPV-2a | Taiwan | 1996 |
T10 | CPU72696 | CPV-2b | Taiwan | 1996 |
2017090801 | MH127909 | CPV-2c | Taiwan | 2017 |
Protein (VP2) | KU244254 | CPV-2c | Taiwan | 2015 |
PV/PL/HeN02/08 | EU441280 | CPV-2a | Henan | 2008 |
Henan42 | KJ438805 | CPV-2a | Henan | 2013 |
CPV-HN1617 | MF467229 | CPV-2c | Henan | 2016 |
CPV-zj18 | KM386948 | CPV-2b | Zhejiang | 2014 |
CPV-zj7 | KM386937 | CPV-2a | Zhejiang | 2014 |
Beijing | HQ883267 | CPV-2a | Beijing | 2010 |
BJ-1 | MN101726 | CPV-2a | Beijing | 2018 |
2011-BJ-B43 | KF803527 | CPV-2b | Beijing | 2011 |
2011-BJ-B6 | KF803606 | CPV-2b | Beijing | 2011 |
CPV-SH1516 | MG013488 | CPV-2c | Shanghai | 2017 |
Shanghai/04g/2016 | KY937646 | CPV-2a | Shanghai | 2016 |
ShangHai/3-2/2016 | KY937640 | CPV-2a | Shanghai | 2016 |
Shanghai/03g/2016 | KY937637 | CPV-2c | Shanghai | 2016 |
CPVpf/2007(vaccine) | FJ197847 | CPV-2 | South Korea | 2007 |
29/97(vaccine) | FJ222823 | CPV-2b | N.I. no information | 2008 |
CPV-GX1581 | MF467242 | CPV-2c | Guangxi | 2015 |
Clinical information on sources of Chinese canine parvovirus type 2 (CPV-2) obtained in this study
Strain | Genotype | Site | Date of sampling | Age | Breed | Sex | Vaccinated status | Accession number |
---|---|---|---|---|---|---|---|---|
CH-AH-D1 | CPV-2b | Hefei | Oct. 7, 2018 | 5 months | Poodle | Female | Unvaccinated | MN119560 |
CH-AH-D2 | CPV-2c | Anqing | Oct. 7, 2018 | 2 years | Poodle | Male | 1 dose | MN119561 |
CH-AH-D3 | CPV-2c | Suzhou | Oct. 8, 2018 | 1 year | Mixed | Female | 2 doses | MN119562 |
CH-AH-D4 | CPV-2c | Suzhou | Oct. 11, 2018 | 3 months | Poodle | Male | Unvaccinated | MN119563 |
CH-AH-D5 | CPV-2b | Wuhu | Nov. 9, 2018 | 1 year | Mixed | Female | 2 doses | MN119564 |
CH-AH-D6 | CPV-2c | Anqing | Nov. 11, 2018 | 4 months | Poodle | Female | 1 dose | MN119565 |
CH-AH-D7 | CPV-2c | Suzhou | Nov. 13, 2018 | 45 days | Schnauzer | Male | Unvaccinated | MN119566 |
CH-AH-D8 | CPV-2c | Hefei | Nov. 30, 2018 | 7 months | Mixed | Male | 1 dose | MN119567 |
CH-AH-D9 | CPV-2c | Wuhu | Dec. 1, 2018 | 6 months | Retriever | Male | Unvaccinated | MN119568 |
CH-AH-D10 | CPV-2c | Suzhou | Dec. 3, 2018 | 3 months | Schnauzer | Female | Unvaccinated | MN119569 |
CH-AH-D11 | CPV-2c | Anqing | Jan. 7, 2019 | 8 months | Mixed | Male | 2 doses | MN119570 |
CH-AH-D12 | CPV-2c | Suzhou | Feb. 7, 2019 | 4 months | Mixed | Female | Unvaccinated | MN119571 |
CH-AH-D13 | CPV-2c | Wuhu | Mar. 11, 2019 | 2 months | Poodle | Female | 1 dose | MN119572 |
CH-AH-D14 | CPV-2a | Hefei | Mar. 16, 2019 | 52 days | Mixed | Female | Unvaccinated | MN119573 |
CH-AH-D15 | CPV-2a | Anqing | Apr. 2, 2019 | 4 months | Retriever | Male | 1 dose | MN119574 |
CH-AH-D16 | CPV-2b | Suzhou | Apr. 3, 2019 | 3 months | Poodle | Female | Unvaccinated | MN119575 |
CH-HN-D1 | CPV-2c | Zhengzhou | Oct. 3, 2018 | 40 days | Mixed | Male | Unvaccinated | MN119576 |
CH-HN-D2 | CPV-2c | Hebi | Oct. 4, 2018 | 4 months | Mixed | Female | 1 dose | MN119577 |
CH-HN-D3 | CPV-2c | Nanyang | Oct. 8, 2018 | 10 months | Mixed | Male | 1 dose | MN119578 |
CH-HN-D4 | CPV-2c | Nanyang | Nov. 6, 2018 | 5 months | Retriever | Female | 1 dose | MN119579 |
CH-HN-D5 | CPV-2b | Luoyang | Nov. 7, 2018 | 4 months | Mixed | Male | Unvaccinated | MN119580 |
CH-HN-D6 | CPV-2c | Xinxiang | Nov. 12, 2018 | 5 months | Schnauzer | Male | Unvaccinated | MN119581 |
CH-HN-D7 | CPV-2a | Xinyang | Nov. 14, 2018 | 4 months | Mixed | Female | 1 dose | MN119582 |
CH-HN-D8 | CPV-2c | Luoyang | Nov. 16, 2018 | 2 months | Mixed | Female | 1 dose | MN119583 |
CH-HN-D9 | CPV-2c | Zhengzhou | Nov. 28, 2018 | 2 months | Schnauzer | Male | Unvaccinated | MN119584 |
CH-HN-D10 | CPV-2c | Anyang | Nov. 29, 2018 | 4 months | Mixed | Female | 1 dose | MN119585 |
CH-HN-D11 | CPV-2c | Xinyang | Dec. 3, 2018 | 50 days | Mixed | Female | 1 dose | MN119586 |
CH-HN-D12 | CPV-2c | Nanyang | Jan. 1, 2019 | 3 months | Retriever | Male | Unvaccinated | MN119587 |
CH-HN-D13 | CPV-2c | Shangqiu | Jan. 3, 2019 | 3 months | Mixed | Female | 1 dose | MN119588 |
CH-HN-D14 | CPV-2a | Anyang | Jan. 4, 2019 | 4 months | Mixed | Female | 1 dose | MN119589 |
CH-HN-D15 | CPV-2a | Zhengzhou | Jan. 5, 2019 | 1 month | Mixed | Male | Unvaccinated | MN119590 |
CH-HN-D16 | CPV-2b | Anyang | Jan. 5, 2019 | 2 months | Poodle | Male | Unvaccinated | MN119591 |
CH-HN-D17 | CPV-2c | Zhengzhou | Feb. 3, 2019 | 1 year | Mixed | Female | 2 doses | MN119592 |
CH-HN-D18 | CPV-2b | Nanyang | Feb. 4, 2019 | 4 months | Poodle | Male | Unvaccinated | MN119593 |
CH-HN-D19 | CPV-2c | Xinxiang | Feb. 5, 2019 | 5 months | Mixed | Female | 1 dose | MN119594 |
CH-HN-D20 | CPV-2b | Hebi | Mar. 8, 2019 | 2 months | Poodle | Female | Unvaccinated | MN119595 |
CH-HN-D21 | CPV-2c | Xinxiang | Mar. 9, 2019 | 5 months | Mixed | Male | 1 dose | MN119596 |
CH-HN-D22 | CPV-2c | Nanyang | Mar. 10, 2019 | 8 months | Retriever | Male | 2 doses | MN119597 |
CH-HN-D23 | CPV-2c | Hebi | Mar. 12, 2019 | 3 months | Mixed | Male | 1 dose | MN119598 |
CH-HN-D24 | CPV-2c | Luoyang | Mar. 15, 2019 | 2 months | Mixed | Female | 1 dose | MN119599 |
CH-HN-D25 | CPV-2c | Anyang | Mar. 29, 2019 | 2 months | Schnauzer | Female | Unvaccinated | MN119600 |
CH-HN-D26 | CPV-2c | Zhengzhou | Mar. 31, 2019 | 4 months | Mixed | Male | Unvaccinated | MN119601 |
CH-HN-D27 | CPV-2c | Luoyang | Apr. 1, 2019 | 1 year | Mixed | Female | 1 dose | MN119602 |
CH-HN-D28 | CPV-2c | Nanyang | Apr. 4, 2019 | 9 months | Mixed | Female | 1 dose | MN119603 |
CH-HN-D29 | CPV-2c | Zhengzhou | Apr. 5, 2019 | 4 months | Poodle | Male | Unvaccinated | MN119604 |
CH-ZJ-D1 | CPV-2c | Huzhou | Oct. 2, 2018 | 1 month | Mixed | Female | 1 dose | MN119605 |
CH-ZJ-D2 | CPV-2c | Hangzhou | Oct. 3, 2018 | 3 months | Mixed | Male | Unvaccinated | MN119606 |
CH-ZJ-D3 | CPV-2c | Jinhua | Oct. 4, 2018 | 2 months | Mixed | Female | 1 dose | MN119607 |
CH-ZJ-D4 | CPV-2c | Ningbo | Oct. 8, 2018 | 4 months | Retriever | Male | 1 dose | MN119608 |
CH-ZJ-D5 | CPV-2c | Hangzhou | Nov. 10, 2018 | 3 months | Mixed | Male | 1 dose | MN119609 |
CH-ZJ-D6 | CPV-2c | Ningbo | Nov. 11, 2018 | 6 months | Mixed | Female | 2 doses | MN119610 |
CH-ZJ-D7 | CPV-2c | Hangzhou | Nov. 17, 2018 | 2 months | Mixed | Female | Unvaccinated | MN119611 |
CH-ZJ-D8 | CPV-2c | Jinhua | Dec. 1, 2018 | 5 months | Mixed | Male | 1 dose | MN119612 |
CH-ZJ-D9 | CPV-2c | Huzhou | Dec. 1, 2018 | 3 months | Mixed | Male | 1 dose | MN119613 |
CH-ZJ-D10 | CPV-2c | Shaoxing | Dec. 3, 2018 | 4 months | Retriever | Female | 1 dose | MN119614 |
CH-ZJ-D11 | CPV-2c | Jinhua | Jan. 4, 2019 | 7 months | Mixed | Male | 2 doses | MN119615 |
CH-ZJ-D12 | CPV-2c | Hangzhou | Feb. 6, 2019 | 2 months | Mixed | Male | 1 dose | MN119616 |
CH-ZJ-D13 | CPV-2c | Jinhua | Mar. 14, 2019 | 3 months | Mixed | Male | 1 dose | MN119617 |
CH-ZJ-D14 | CPV-2c | Shaoxing | Mar. 14, 2019 | 1 year | Mixed | Female | 1 dose | MN119618 |
CH-ZJ-D15 | CPV-2c | Hangzhou | Apr. 4, 2019 | 8 months | Mixed | Male | 2 doses | MN119619 |
CH-ZJ-D16 | CPV-2c | Jinhua | Apr. 4, 2019 | 9 months | Retriever | Female | 2 doses | MN119620 |
Statistics of the main amino acid mutation sites in the VP2 capsid protein of canine parvovirus type 2 in Chinese and reference strains
Strains/GenBank Accession number | Mutation sites: amino acid residue | |||||
---|---|---|---|---|---|---|
5 | 30 | 130 | 370 | 426 | 440 | |
EU659117-2/the USA/1980 | A | G | V | Q | N | T |
FJ197847-2/South Korea/2007/Vaccine | \ | \ | \ | \ | \ | \ |
MH643886-2/South Korea/2016 | \ | \ | \ | \ | \ | \ |
EU659118-2a/the USA/1981 | \ | \ | \ | \ | \ | \ |
MF177231-2a/Italy/2000 | \ | \ | \ | \ | \ | \ |
GU362934-2a/Italy/2008 | \ | \ | \ | \ | \ | \ |
EU441280-2a/Henan/2008 | \ | \ | \ | \ | \ | \ |
HQ883267-2a/Beijing/2010 | \ | \ | \ | \ | \ | A |
KJ438805-2a/Henan/2013 | \ | \ | \ | \ | \ | A |
KY937646-2a/Shanghai/2016 | \ | \ | \ | \ | \ | A |
MK332005-2a/Guangxi/2017 | \ | \ | \ | \ | \ | A |
MN101726-2a/Beijing/2018 | \ | \ | \ | \ | \ | A |
CPU72696-2b/Taiwan/1996 | \ | \ | \ | \ | D | \ |
EU659121-2b/the USA/1998 | \ | \ | \ | \ | D | \ |
FJ005265-2b/Italy/2005 | \ | \ | \ | \ | D | \ |
KF803606-2b/Beijing/2011 | \ | \ | \ | \ | D | \ |
MK332002-2b/Guangxi/2016 | \ | \ | \ | \ | D | A |
MK331996-2b/Guangxi/2016 | \ | \ | \ | \ | D | A |
KU508407-2c/Italy/2009 | \ | \ | \ | \ | E | \ |
KX425920-2c/India/2010 | \ | \ | \ | \ | E | \ |
KU244254-2c/Taiwan/2015 | \ | \ | \ | \ | E | \ |
LC216910-2c/Indonesia/2013 | G | \ | \ | R | E | \ |
MF467242-2c/Vaccine/2015 | G | \ | \ | R | E | \ |
MF467229-2c/Henan/2016 | G | \ | \ | R | E | \ |
MGo13488-2c/Shanghai/2017 | G | \ | \ | R | E | \ |
CH-AH-D1 | G | \ | \ | \ | D | A |
CH-AH-D2 | \ | \ | \ | R | E | \ |
CH-AH-D3 | \ | W | \ | R | E | \ |
CH-AH-D4 | G | \ | \ | R | E | \ |
CH-AH-D5 | \ | \ | \ | \ | D | A |
CH-AH-D6 | G | \ | \ | R | E | \ |
CH-AH-D7 | \ | \ | \ | R | E | \ |
CH-AH-D8 | \ | \ | \ | R | E | \ |
CH-AH-D9 | G | \ | \ | R | E | \ |
CH-AH-D10 | G | \ | \ | R | E | \ |
CH-AH-D11 | G | \ | \ | R | E | \ |
CH-AH-D12 | G | \ | \ | R | E | \ |
CH-AH-D13 | G | \ | \ | R | E | \ |
CH-AH-D14 | \ | \ | \ | \ | \ | A |
CH-AH-D15 | \ | \ | \ | \ | \ | A |
CH-AH-D16 | \ | \ | \ | \ | D | A |
CH-HN-D1 | G | \ | \ | R | E | \ |
CH-HN-D2 | G | \ | \ | R | E | \ |
CH-HN-D3 | G | \ | \ | R | E | \ |
CH-HN-D4 | G | \ | \ | R | E | \ |
CH-HN-D5 | \ | \ | \ | \ | D | A |
CH-HN-D6 | G | \ | \ | R | E | \ |
CH-HN-D7 | \ | \ | A | \ | \ | A |
CH-HN-D8 | \ | \ | \ | R | E | \ |
CH-HN-D9 | G | \ | \ | R | E | \ |
CH-HN-D10 | G | \ | \ | R | E | \ |
CH-HN-D11 | G | \ | \ | R | E | \ |
CH-HN-D12 | G | \ | \ | R | E | \ |
CH-HN-D13 | G | \ | \ | R | E | \ |
CH-HN-D14 | \ | \ | \ | \ | \ | A |
CH-HN-D15 | \ | \ | \ | \ | \ | A |
CH-HN-D16 | \ | \ | \ | \ | D | A |
CH-HN-D17 | G | \ | \ | R | E | \ |
CH-HN-D18 | \ | \ | \ | \ | D | A |
CH-HN-D19 | G | \ | \ | R | E | \ |
CH-HN-D20 | \ | \ | \ | \ | D | A |
CH-HN-D21 | G | \ | \ | R | E | \ |
CH-HN-D22 | G | \ | \ | R | E | \ |
CH-HN-D23 | G | \ | \ | R | E | \ |
CH-HN-D24 | G | \ | \ | R | E | \ |
CH-HN-D25 | G | \ | \ | R | E | \ |
CH-HN-D26 | G | \ | \ | R | E | \ |
CH-HN-D27 | G | \ | \ | R | E | \ |
CH-HN-D28 | G | \ | \ | R | E | \ |
CH-HN-D29 | G | \ | \ | R | E | \ |
CH-ZJ-D1 | G | \ | \ | R | E | \ |
CH-ZJ-D2 | G | \ | \ | R | E | \ |
CH-ZJ-D3 | G | \ | \ | R | E | \ |
CH-ZJ-D4 | G | \ | \ | R | E | \ |
CH-ZJ-D5 | G | \ | \ | R | E | \ |
CH-ZJ-D6 | G | \ | \ | R | E | \ |
CH-ZJ-D7 | G | \ | \ | R | E | \ |
CH-ZJ-D8 | G | \ | \ | R | E | \ |
CH-ZJ-D9 | G | \ | \ | R | E | \ |
CH-ZJ-D10 | G | \ | \ | R | E | \ |
CH-ZJ-D11 | G | \ | \ | R | E | \ |
CH-ZJ-D12 | G | \ | \ | R | E | \ |
CH-ZJ-D13 | G | \ | \ | R | E | \ |
CH-ZJ-D14 | G | \ | \ | R | E | \ |
CH-ZJ-D15 | G | \ | \ | R | E | \ |
CH-ZJ-D16 | G | \ | \ | R | E | \ |
Strains with designations starting with “CH” are Chinese strains obtained in this study. (A:Ala, D:Asp, E: Glu, G: Gly, N:Asn, Q:Gln, R:Arg, T:Thr, V: Val, W:Try)
A CPV is widely distributed in various regions of China, and the virus undergoes rapid mutations. Since its identification, three important variations in the CPV genotype have occurred, resulting in the emergence of subtypes CPV-2a, CPV-2b, and CPV-2c. During 2009– 2012, CPV-2a was the dominant genotype in the southern region of Nanjing (29). In this period and continuing until 2014, CPV-2c was not widespread in Henan Province (30). By 2015–2016, the novel CPV-2a strain had become the prevalent subtype in Henan, Guangxi, and Jiangsu Provinces and in these years Aln5Gly and Gln370Arg mutations in genotype 2c were reported for the first time (26). In this study, the epidemiological trend of CPV-2 was observed from 2018 to 2019 through sequencing the CPV-2 strains collected from three provinces (Henan, Anhui and Zhejiang). Variations in the CPV-2 genotype were strongly affected by vaccination and tended to be due to 2c mutations. In the present study, trends of CPV infections in Anhui, Henan, and Zhejiang Provinces revealed CPV-2c as the most widespread genotype. Three genotypes coexisted in Anhui and Henan Provinces, which are not perfectly covered by the vaccination program. In Zhejiang Province, in contrast, the vaccination rate was high and CPV-2c was the only strain detected. Based on genotypic and immunisation history, CPV-2c shows high transmissibility and strong adaptability. It is therefore the genotype most likely to cause vaccination failure when the program uses classical CPV-2, and this genotype’s dominance may be explained by its having evolved under pressure from immunisation.
The main amino acid mutations in the VP2 protein of the 61 strains occurred at sites 5, 370, 426, and 440. In Anhui, Henan and Zhejiang Provinces, 45 (73.77%) strains harboured a similar Ala5Gly mutation to the reference strain CPV-2c. This finding indicates that the amino acid mutation at site 5 may determine the CPV subtype (CPV-2c), with such mutations gradually becoming increasingly common in China and overseas. The nearly three quarters proportion of strains collected from Anhui, Henan and Zhejiang Provinces with the Ala5Gly mutation in the CPV-VP2 protein suggests that it has become prevalent. A total of 49 (80.33%) strains in the three provinces displayed a similar Gln370Arg mutation to the reference strain CPV-2c. This site may alter the spatial structure of the VP2 protein to some extent, thereby affecting the pathogenicity of the virus (8). Mutations at the amino acid site 426 markedly affected the classification of CPV-2a, CPV-2b, and CPV-2c (30). The reference strain CPV-2a showed 426Asn, and mutation at this site occurred in 5 of the 61 strains (8.20%) tested in this study. Seven (11.48%) strains collected from Anhui and Henan Provinces were Asn426Asp-mutated in a similar way to the reference strain CPV-2b, although no mutation was detected in the strains collected from Zhejiang Province. A total of 49 (80.33%) strains revealed a similar Asn426Gln mutation to the reference strain CPV-2c. These findings indicate that the Asn426Gln mutation was highly prevalent in the three provinces studied. Furthermore, the strains collected from Henan and Anhui Provinces (19.67%) were like the reference strains CPV-2a and CPV-2b in having the Thr440Ala mutation. This result demonstrates that CPV strains carrying the Thr440Ala mutation are prevalent in Anhui and Henan Provinces. In terms of mutation rates, CPV-2c was the dominant mutant genotype in Anhui, Henan, and Zhejiang Provinces from October 2018 to April 2019.
Sequence identities and phylogenetic relationships of the 61 strains collected from the three provinces revealed in this study will contribute to a better understanding of the frequency and mutation tendencies of CPV strains in these provinces. The VP2 sequences of the CPV strains collected from Anhui, Henan, and Zhejiang Provinces shared high identity without any outstanding variations; however, these strains showed relatively distant relationships with the classical vaccine strains. Moreover, there were no obvious regional differences in CPV genotype distribution according to the evolutionary tree. This result indicates that CPV genotypes are characterised by mutations at individual amino acid sites which do not affect the overall evolutionary characteristics of CPV.
In this study, mutations at four major amino acid sites led to the emergence of different genotypes, which further evolved into the highly immune-resistant subtype CPV-2c under pressure from immunisation as previously described (4, 8). The epitope is the antigenic sequence that stimulates B cells to produce antibodies, leading to antigen–antibody binding. The VP2 protein of CPV-2c presents multiple epitopes; therefore, multiple amino acid mutations might be triggered via adaptive selection for survival under pressure from immunisation. The present study provides a theoretical and technical basis for evolutionary analysis and vaccine strain selection against CPV-2c. Selection pressure analysis revealed that the genomic regions carrying mutations were complex, diverse, and highly prone to mutations, which further explains the differentiation of CPV-2 into additional genotypes in recent years (4). Two regions (0–25 and 350–450) in epitope prediction (Fig. 3A) and two regions (0–51 and 251–451) in selection pressure analysis (Fig. 3B) showed a high degree of coincidence in their entropy values. Moreover, the mutations at the sites 5, 370, 426, and 440 in this study conform to the epidemic trends of the virus, further confirming the authenticity of the mutated sites in this study. Unfortunately, owing to the unavailability of specific sera for genotypic variants in our laboratory, we were unable to provide data for serological analysis in this study. Nonetheless, based on the results of epitope prediction and selection pressure analyses combined with vaccination history, the genotypic variations in strains collected from Anhui, Henan and Zhejiang Provinces in China may be attributed to pressure from immunisation. Animal inoculation experiments are needed to determine whether immunisation failure is a possible consequence of antibody pressure on evolution of CPV-2a, CPV-2b, and CPV-2c.
In conclusion, amino acid sequence analysis of the VP2 protein of 61 CPV strains collected from central and eastern China indicated that CPV-2c is the predominant genotype of CPV in the study regions, particularly in Zhejiang Province. In addition, strains with CPV-2a and CPV-2b genotypes harbouring novel mutations were also detected. These results highlight the need for further research targeting different CPV genotypes to develop vaccines and establish more effective vaccination programs that increase the scope of immunisation.