Canine parvovirus (CPV;
The genomic DNA of CPV-2 contains two major open reading frames (ORFs): ORF1 and OFR2 (16). ORF1 encodes nonstructural proteins (NS1 and NS2) that regulate gene expression and are the first translated in the early stage of viral infection; ORF2 encodes capsid proteins (VP1 and VP2) that regulate viral tendency and antigenicity (7). VP2 constitutes the main antigenic determinant, which is strongly immunogenic and can stimulate the body to produce neutralising antibodies as well as be used to prepare subunits or DNA vaccines. In addition, VP2 can bind to transferrin receptors on the host cell membrane; this mediates the extent of infection of the virus particles, and is related to the haemagglutination of the virus (4). Therefore, amino acid substitution in the protein sequence of VP2 may lead to changes in host range, tissue tropism, and the genetic and antigenic properties of the virus.
In the early 1980s, the two new antigenic variants CPV-2a and CPV-2b acquired high virulence and pathogenicity, and they gradually replaced CPV-2 worldwide. As CPV-2a strains, the virus has regained its ability to infect cats and other canines (1). Compared with the
All 56 strains (length- 1,755 bp) were designated as follows: CN/HN1601–CN/HN1723, CN/HB1601–CN/ HB1715, CN/AH1601–CN/AH1710, and CN/JS1601– CN/HNJS1708. All complete genome sequences of the
Epidemiological data of the 56 strains in the study and the reference strains for genotyping
Strains | Accession nos. | Origin | Vaccination record | Age | CPV type | Substitution of amino acid residues in VP2 | ||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
5 | 267 | 297 | 324 | 370 | 426 | 440 | ||||||
CPV-6.us.80 | EU659117 | the USA, 1980 | N.I. no information |
N.I. | CPV-2 | A | F | S | Y | Q | N | T |
CPV-13.us.81 | EU659118 | the USA, 1981 | N.I. | N.I. | CPV-2a | A | F | S | Y | Q | N | A |
CPV-411b.us.98 | EU659121 | the USA, 1998 | N.I. | N.I. | CPV-2b | A | F | A | Y | Q | N | A |
CPV-HN1506 | MF467240 | Henan, 2017 | N.I. | N.I. | New-2a | A | Y | A | I | Q | N | A |
RVC57 KNA | KY399053 | SNA, 2016 | N.I. | N.I. | New-2a | A | F | A | I | Q | N | A |
BJ15-15 | KT162046 | Beijing, 2015 | N.I. | N.I. | New-2a | A | Y | A | I | Q | N | A |
BJ15-11 | KT162026 | Beijing, 2015 | N.I. | N.I. | New-2b | A | Y | A | I | Q | D | A |
VP2 | KR058183 | China, 2013 | N.I. | N.I. | New-2b | A | Y | A | I | Q | D | A |
CPV-HN1506 | MF467239 | Henan, 2015 | N.I. | N.I. | New-2b | A | Y | A | I | Q | D | A |
CPV dog HCM | LC216910 | Indonesia,2013 | N.I. | N.I. | CPV-2c | G | Y | A | I | R | E | T |
CPV-HN1617 | MF467229 | Henan, 2016 | N.I. | N.I. | CPV-2c | G | Y | A | I | R | E | T |
CPV-SH1516 | MG013488 | Shanghai, 2017 | N.I. | N.I. | CPV-2c | G | Y | A | I | R | E | T |
CN/HN1601 | MK517966 | Henan, 2016 | N.I. | 3 m | CPV-2c | A | Y | A | I | R | E | T |
CN/HN1602 | MK517967 | Henan, 2016 | 1 dose | 2 m | CPV-2c | A | Y | A | I | R | E | T |
CN/HN1603 | MK517968 | Henan, 2016 | 2 doses | 5 m | CPV-2c | G | Y | A | I | R | E | T |
CN/HN1604 | MK517969 | Henan, 2016 | 1 dose | 2 m | CPV-2c | A | Y | A | I | R | E | T |
CN/HN1605 | MK517970 | Henan, 2016 | 1 dose | 2 m | CPV-2c | G | Y | A | I | R | E | T |
CN/HN1606 | MK517971 | Henan, 2016 | 2 doses | 7 m | New-2a | A | Y | A | I | Q | N | A |
CN/HN1607 | MK517972 | Henan, 2016 | 1 dose | 2 m | CPV-2c | G | Y | A | I | R | E | T |
CN/HN1708 | MK517973 | Henan, 2017 | N.I. | 3 m | CPV-2c | A | Y | A | I | R | E | T |
CN/HN1709 | MK517974 | Henan, 2017 | N.I. | 4 m | New-2a | A | Y | A | I | Q | N | A |
CN/HN1710 | MK517975 | Henan, 2017 | 1 dose | 3 m | New -2a | A | Y | A | I | Q | N | A |
CN/HN1711 | MK517976 | Henan, 2017 | 1 dose | 2 m | CPV-2c | A | Y | A | I | R | E | T |
CN/HN1712 | MK517977 | Henan, 2017 | N.I. | 5 m | New-2a | A | Y | A | I | Q | N | A |
CN/HN1713 | MK517978 | Henan, 2017 | 1 dose | 42 d | CPV-2c | G | Y | A | L | R | E | T |
CN/HN1714 | MK517979 | Henan, 2017 | 1 dose | 2 m | CPV-2c | A | Y | A | Y | R | E | T |
CN/HN1715 | MK517980 | Henan, 2017 | 1 dose | 5 m | New-2a | A | Y | A | I | Q | N | A |
CN/HN1716 | MK517981 | Henan, 2017 | 2 doses | 4 m | CPV-2c | G | Y | A | I | R | E | T |
CN/HN1717 | MK517982 | Henan, 2017 | N.I. | 2 m | New-2a | G | Y | A | I | Q | N | A |
CN/HN1718 | MK517983 | Henan, 2017 | 1 dose | 37 d | New-2b | A | Y | A | I | Q | D | A |
CN/HN1719 | MK517984 | Henan, 2017 | 1 dose | 2 m | New-2b | A | Y | A | I | Q | D | A |
CN/HN1720 | MK517985 | Henan, 2017 | 2 doses | 4 m | New-2a | G | Y | A | I | Q | N | A |
CN/HN1721 | MK517986 | Henan, 2017 | 1 dose | 2 m | CPV-2c | G | Y | A | I | R | E | T |
CN/HN1722 | MK517987 | Henan, 2017 | N.I. | 6 m | CPV-2c | A | Y | A | I | R | E | T |
CN/HN1723 | MK517988 | Henan, 2017 | 1 dose | 5 m | CPV-2c | A | Y | A | I | R | E | T |
CN/HB1601 | MK517989 | Hubei, 2016 | N.I. | 40 d | CPV-2c | G | Y | A | I | R | E | T |
CN/HB1602 | MK517990 | Hubei, 2016 | 1 dose | 5 m | CPV-2c | G | Y | A | I | R | E | T |
CN/HB1603 | MK517991 | Hubei, 2016 | 1 dose | 3 m | CPV-2c | G | Y | A | I | R | E | T |
CN/HB1704 | MK517992 | Hubei, 2017 | 1 dose | 3 m | CPV-2c | G | Y | A | I | R | E | A |
CN/HB1705 | MK517993 | Hubei, 2017 | 2 doses | 4 m | CPV-2c | A | Y | A | I | R | E | T |
CN/HB1706 | MK517994 | Hubei, 2017 | 1 dose | 2 m | CPV-2c | A | Y | A | I | R | E | T |
CN/HB1707 | MK517995 | Hubei, 2017 | N.I. | 3 m | New-2a | A | Y | A | I | Q | N | A |
CN/HB1708 | MK517996 | Hubei, 2017 | 1 dose | 33 d | CPV-2c | A | Y | A | I | R | E | T |
CN/HB1709 | MK517997 | Hubei, 2017 | 1 dose | 2 m | CPV-2c | G | Y | A | I | R | E | T |
CN/HB1710 | MK517998 | Hubei, 2017 | 2 doses | 4 m | CPV-2c | G | Y | A | I | R | E | T |
CN/HB1711 | MK517999 | Hubei, 2017 | N.I. | 6 m | CPV-2c | A | Y | A | I | R | E | T |
CN/HB1712 | MK518000 | Hubei, 2017 | 1 dose | 39 d | New-2a | A | Y | A | I | Q | N | A |
CN/HB1713 | MK518001 | Hubei, 2017 | 1 dose | 3 m | CPV-2c | G | Y | A | L | R | E | T |
CN/HB1714 | MK518002 | Hubei, 2017 | N.I. | 5 m | CPV-2a | A | Y | S | Y | Q | N | T |
CN/HB1715 | MK518003 | Hubei, 2017 | 1 dose | 2 m | New-2a | G | Y | A | I | R | E | T |
CN/AH1601 | MK518004 | Anhui, 2016 | 2 doses | 6 m | CPV-2c | G | Y | A | I | R | E | T |
CN/AH1602 | MK518005 | Anhui, 2016 | N.I. | 4 m | CPV-2c | G | Y | A | I | R | E | T |
CN/AH1603 | MK518006 | Anhui, 2016 | 1 dose | 3 m | CPV-2c | G | Y | A | I | R | E | T |
CN/AH1604 | MK518007 | Anhui, 2016 | 1 dose | 5 m | New-2b | A | Y | A | I | R | D | T |
CN/AH1705 | MK518008 | Anhui, 2017 | N.I. | 8 m | CPV-2c | G | Y | A | I | R | E | T |
CN/AH1706 | MK518009 | Anhui, 2017 | 1 dose | 3 m | New-2b | A | Y | A | I | Q | D | A |
CN/AH1707 | MK518010 | Anhui, 2017 | N.I. | 2 m | CPV-2c | A | Y | A | I | R | E | T |
CN/AH1708 | MK518011 | Anhui, 2017 | N.I. | 3 m | New-2b | G | Y | A | I | Q | D | T |
CN/AH1709 | MK518012 | Anhui, 2017 | 2 doses | 4 m | CPV-2c | A | Y | A | I | R | E | T |
CN/AH1710 | MK518013 | Anhui, 2017 | 1 dose | 5 m | New -2a | G | Y | A | I | Q | N | T |
CN/JS1601 | MK518014 | Jiangsu, 2016 | N.I. | 3 m | CPV-2c | A | Y | A | I | R | E | T |
CN/JS1602 | MK518015 | Jiangsu, 2016 | 1 dose | 2 m | CPV-2c | G | Y | A | I | R | E | T |
CN/JS1603 | MK518016 | Jiangsu, 2016 | N.I. | 41 d | New-2a | A | Y | A | I | Q | N | A |
CN/JS1704 | MK518017 | Jiangsu, 2017 | N.I. | 4 m | New-2a | G | Y | A | I | Q | N | A |
CN/JS1705 | MK518018 | Jiangsu, 2017 | 1 dose | 4 m | CPV-2c | A | Y | A | I | R | E | T |
CN/JS1706 | MK518019 | Jiangsu, 2017 | N.I. | 2 m | CPV-2c | A | Y | A | I | R | E | T |
CN/JS1707 | MK518020 | Jiangsu, 2017 | 2 doses | 5 m | CPV-2c | G | Y | A | I | R | E | T |
CN/JS1708 | MK518021 | Jiangsu, 2017 | 1 dose | 3 m | CPV-2c | A | Y | A | I | R | E | T |
Information about the reference strains in our study
Strain | Accession no. | Genotype | Place of isolation | Submission date |
---|---|---|---|---|
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 | CPV2c(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 | CPV2a | 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 |
CPV-HN1506 | MF467240 | New-2a | Henan | 2017 |
RVC57 KNA 2016 | KY399053 | New-2a | Saint Kitts and Nevis | 2016 |
SY40 | KY625992 | New-2a | China | 2016 |
PU4 | KC429669 | New-2a | India | 2011 |
BJ15-15 | KT162046 | New-2a | Beijing | 2015 |
SY38 | KY625998 | New-2b | China | 2016 |
BJ15-11 | KT162026 | New-2b | Beijing | 2015 |
VP2 | KR058183 | New-2b | China | 2013 |
CPV-HN1506 | MF467239 | New-2b | Henan | 2015 |
The corresponding ranges elsewhere were 97.4%– 99.9% nucleotide homology and 96.1%–99.8% amino acid homology among the 15 strains isolated from Hubei Province, 98.8%–99.8% nucleotide homology and 98.3%–99.8% amino acid homology among the 8 strains isolated from Jiangsu Province, and 98.9%–99.8% nucleotide homology and 98.8%–99.7% amino acid homology among the 10 strains isolated from Anhui Province.
The genotype of the CPV-2 strain and the amino acid mutation in the VP2 sequence are shown in Table 1. Among the four provinces in Central China, the genotypes of 23 strains in Henan were new CPV-2a (30.43%), new CPV-2b (8.70%), and CPV-2c (60.87%). Among the 15 strains in Hubei, the genotypes of CPV-2a comprised 6.67%, those of new CPV-2a 13.33%, and those of CPV-2c 80.00%. There were only two genotypes of new CPV-2a (25.00%) and CPV-2c (75.00%) in Jiangxi. The three genotypes in Anhui were new CPV-2a (10.00%), new CPV-2b (30.00%), and CPV-2c (60.00%). The geographical distribution and epidemiological survey results are presented in Fig. 2.
Since the first report on CPV-2 infection, the epidemic characteristics of CPV-2 have substantially varied among different regions (13). CPV isolates from the United States and some European countries mainly belong to the CPV-2b and CPV-2c subtypes, and the CPV-2a and CPV-2c subtypes are the most prevalent in Asian countries (5). In China, CPV-2 was the first isolated in 1982 and has developed into one of the most harmful canine pathogens (6). At present, vaccine-based immunisation is the major strategy to prevent and control CPV-2 in China; thus, understanding the latest epidemic situation and genetic variation of the circulating CPV-2 strains is necessary.
Recently, epidemiological investigation in Central China has only been reported in Henan Province. In the present study, dogs suspected of CPV-2 infections in Henan, Hubei, Anhui, and Jiangsu provinces in Central China were investigated, and a total of 56 dogs were identified to be infected with it. The
In recent years, new CPV-2a has been emerging as the predominant epidemic strain of CPV-2 in some Chinese provinces (7, 19). To the best of our knowledge, a wide distribution of CPV-2c and new CPV-2a variants has not been reported in Central China before this study.
To summarise the current reports on residue analysis, the seven reported mutation residues related to virulence strength and host range were analysed in this study and compared with mutation residues in the reference strains. The amino acid mutation Thr440Ala in VP2 is associated with the evolution of antigenic variants. All new CPV-2a strains described in this study harboured a mutation of residue 440 (Thr→Ala). The mutation of residue 426 as Asn→Asp serves as a marker for distinguishing CPV-2b, whereas that of residue 426 as Asn→Glu helps determine CPV-2c genotype. Regarding the identified CPV-2c, the mutation rate for residue 370 (Gln→Arg) reached 69.64%, which is suspected to be caused by long-term antibody pressure. The mutation of residue 324 (Tyr→Ile) displayed in the 56 strains and all reference strains has spread worldwide since its first report, which indicated that the change at residue 324 has been a common mutation in CPV-2c in Asian countries. Furthermore, the amino acids at position 324 are present on the outer surface of the virions, and mutation at this position may cause changes in the tertiary structural model structure of the protein, leading to the enhancement of the binding ability of the viral receptor and a change in the host range (10, 14). In this study, mutations of Ser297Ala (98.21%) and Phe267Tyr (100%) were found in CPV-2c and new CPV-2a/2b; a change at these points causes a change in the antigenicity of CPV-2 and is, therefore, used as a marker to distinguish new CPV-2a/2b strains (14). It can be clearly seen from the tertiary structural model of the protein that change at an amino acid mutation site will affect the protein structure, which may change the pathogenicity of the virus. During virus transmission, the risk of enhanced virulence or increased clinical severity is extremely high. All these mutation sites may be affected by changes in the fine structure of the virus, thereby weakening the binding ability of specific antibodies to viral antigens. Therefore, the immune response of immunised animals should be strengthened, and protective measures should be taken for the clinical treatment and prevention of CPV disease. The biological function of all site mutations identified in the present study needs to be confirmed with animal regression studies, and extensive epidemiological investigations are needed. According to the currently reported information on site mutations, timely monitoring of mutations at each site as well as adjusting CPV-2 treatment and prevention measures are necessary.
In the present study, the type and geographical distribution of the reference strains in the phylogenetic tree were analysed, and the results were found to be highly comprehensive and complete. Evolutionary analysis of the classical strains isolated from the United States and Italy compared with those isolated from four provinces showed the degree of difference. Since the discovery of CPV-2, its evolution has been rapid. New CPV-2a and new CPV-2b were highly similar to CPV-2a and CPV-2b, respectively, which indicated that these two new genotypes mutated from their respective ancestors. The trend of the VP2 phylogenetic tree was consistent with that reported in a previous study (7). Meanwhile, the CN/HN/1714 strain belonged to CPV-2a, which was closely related to the Japanese reference strains.
In conclusion, the present study revealed new epidemiological data of CPV-2 in Central China, including the co-circulation of new CPV-2a and new CPV-2c with high variation. In addition, the molecular characterisation of the strains isolated from Central China increased the understanding of the epidemic characteristics of CPV-2 strains worldwide. This study may be a scientific reference for CPV vaccine-related research and development.