Scientia Agricultura Sinica ›› 2025, Vol. 58 ›› Issue (17): 3561-3570.doi: 10.3864/j.issn.0578-1752.2025.17.015

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles    

Construction of Infectious Clones for Canine Parvovirus CPV-2c SX-LC Strain and Virus Rescue

LIU ChenXi(), ZHAO BingBing, SHI ZhiBin, WANG ShiDa, WANG JingFei()   

  1. State Key Laboratory of Animal Disease Control and Prevention/Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069
  • Received:2025-03-04 Accepted:2025-06-12 Online:2025-09-02 Published:2025-09-02
  • Contact: WANG JingFei

RichHTML

2

PDF

11

Abstract:

【Background】Canine parvovirus type 2 (CPV-2), a member of the genus Parvovirus within the family Parvoviridae, is a non-enveloped, single-stranded DNA virus that mainly infects canines and poses a great threat to puppies in particular. The virus has become one of the most significant epidemic threats to the global canine breeding industry. Currently, vaccination is the main strategy for preventing CPV-2 infection; however, cases of disease have still been reported in vaccinated dogs. Therefore, the fundamental research on the pathogenic mechanisms and immunological characteristics of circulating CPV-2 strains is essential for optimizing prevention and control strategies.【Objective】This study aimed to establish a reverse genetics system for the currently prevalent CPV-2c subtype, providing an essential experimental tool for future investigations into viral pathogenicity, immune escape mechanisms, and host adaptive evolution.【Method】Based on the published genome sequence of the CPV-Y1 strain (GenBank ID: D26079.1), specific primers were designed to amplify the near full-length genome of the CPV-2c SX-LC strain, which was isolated in our laboratory using segmented PCR. The inverted terminal repeats (ITRs) of CPV-2 were synthesized and combined with the PCR products to clone the complete CPV-2 genome into the pBluescript SK (+) vector. An XhoI restriction site was introduced into the genome as a genetic marker, facilitating the construction of a stable and operable reverse genetics plasmid. The purified recombinant plasmid was transfected into canine-derived F81 cells to rescue the virus, followed by serial passaging. Successfully rescue of the virus was confirmed through the observation of cytopathic effects (CPE), detection of the VP2 major capsid protein using indirect immunofluorescence assay (IFA), and morphological identification of viral particles by negative staining under transmission electron microscopy (TEM). Additionally, the proliferation kinetics of the rescued virus were assessed via a one-step growth curve assay, and hemagglutination activity (HA) was evaluated too. These characteristics were compared to those of the parental wild-type strain to verify the stability and utility of the constructed reverse genetics system.【Result】Restriction enzyme digestion and sequencing confirmed the successful construction of the full-length CPV-2c SX-LC genome plasmid. After transfection of the recombinant plasmid into F81 cells and five serial passages, the significant cytopathic effects were observed. IFA results confirmed stable expression of the VP2 antigen in infected cells. TEM analysis revealed virus particles with typical CPV-2 morphological characteristics, further validating the successful rescue of the recombinant virus rCPV-2c SX-LC. Growth curve analysis and hemagglutination assays demonstrated that the rescued virus exhibited replication kinetics and hemagglutination activity comparable to those of the parental strain, indicating similar biological characteristics.【Conclusion】By optimizing the cloning of ITRs, a plasmid containing the complete CPV-2 ITR structure was successfully constructed, which improved rescue efficiency and ensured genome integrity compared to traditional construction strategies. The established reverse genetics system for the CPV-2c SX-LC strain provided a robust technical platform for further basic and applied research on canine parvovirus.

Key words: canine parvovirus, infectious clone, reverse genetics system, virus rescue, viral genome

Fig. 1

Strategy for constructing the full-length infectious clone of canine parvovirus A: Schematic diagram of the CPV genome segments; B: Schematic diagram of the plasmid p-AE, containing the 3′ half (fragment A) and 5′ half (fragment E) of the ITRs; C: Schematic diagram of the plasmid p-ABE, containing the complete 3′ ITR (fragments A and B) and the 5′ half of ITR (fragment E); D: Schematic diagram of the plasmid p-ABDE, containing the complete 3′ (fragments A and B) and 5′ (fragments D and E) ITRs; E: Schematic diagram of the plasmid p-M, containing the viral intermediate fragment (M); F: Schematic diagram of the plasmid p-CPV, containing the full-length viral genome sequence"

Table 1

Primers used for PCR and synthesized sequences"

名称 Name 序列Sequence (5′-3′) 扩增位置Position (nt)
MF1 GGAAAAGGTGGCGGGCT 158—174
MR1 GTCATAATTACTGGAGTTG 1711—1729
MF2 TTGGATTGAAGAAGCTGGT 1598—1616
MR2 GGAGTTGGTATGGTTGGTT 3391—3409
MF3 TTAAAGACTGTTTCAGAATC 3237—3256
MR3 CACACCATAACAACATACATTA 4754—4775
M-XhoI F GGAAATCACAGCAAACTCGAGCAGACTTGT 2966—2995
M- XhoI R AGTCTGCTCGAGTTTGCTGTGATTTCCACCCA 2961—2992
TF ATGAGTGATGGAGGAGTTCA 2790—2809
TR TGTTCCTGTAGCAAATTCATCACC 3561—3584
A GGTACCATTCTTTAGAACCAACTGACCAAGTTCACGTACGTATGACGTGATGACGCGCGCTGCG
CGCGCTGCCTACGGCAGTAAGCTT		 1—82
B GCGCGCTGCCTACGGCAGTCACACGTCATACGTACGCTCCTTGGTCAGTTGGTTCTAAAGAATGA
TAGGCGGTTTGTGTGTTTAAACTTGGGCG GGAAAAGGTGGCGGGCTCTGGAT		 83—174
D CTGGAT TAATGTATGTTGTTATGGTGTGGGTGGTTGGTTGGTTTGCCCTTAGAATATGTTAAGGA
CCAAAAAAAATCAATAAAAGACATTTAAAACTAAATGGCCTCGTATACTGTCTATAAGGTGAACT
AACCTTACCATAAGTATCAATCTGTCTTTAAGGGGGGGGTGGGTGGGAGATGCACAACATCAGTA
GACTGACTGGCCTGGTTGGTTGCGCTTAATCAACCAGACCGCTACAAGCTT		 4748—4993
E AAGCTTGGTCTGGTTGATTAAGCAGAGCAACCAACCAGGCCAGTCAGTCTACTGATGTTGTGCAT
CTCCCACCCACCCCCCCCTTAAAGACAGATTGAGGATCC		 4988—5093

Fig. 2

Identification of the full-length p-CPV plasmid A: PCR results of viral intermediate fragments: M: DNA marker; 1: PCR result of viral intermediate fragment M1; 2: PCR result of viral intermediate fragment M2; 3: PCR result of viral intermediate fragment M3; 4: PCR result of viral intermediate fragment M. B: PCR identification of plasmids containing ITRs: 1: PCR identification of plasmid p-AE; 2: PCR identification of plasmid p-ABDE. C: XhoI single enzyme digestion identification of the plasmid containing the full-length viral sequence; 1: PCR identification of plasmid p-CPV"

Fig. 3

Cytopathic effects, IFA identification, and electron microscopy observation of the rescued CPV A: Cytopathic effects of CPV on cells; B: Identification of the rescued CPV by IFA; C: Identification of the rescued CPV by electron microscope"

Fig. 4

Identification of genetic marker of the rescued virus A: XhoI digestion analysis. M: DNA Marker; 1: PCR product of parental virus; 2: PCR product of rescued virus; 3: PCR product of parental virus digested with XhoI; 4: PCR product of rescued virus digested with XhoI. B: Sequence analysis"

Fig. 5

One-step growth curve and hemagglutination assay of the rescued virus"

[1]
DECARO N, BUONAVOGLIA C, BARRS V R. Canine parvovirus vaccination and immunisation failures: Are we far from disease eradication. Veterinary Microbiology, 2020, 247: 108760.
[2]
TUTEJA D, BANU K, MONDAL B. Canine parvovirology - A brief updated review on structural biology, occurrence, pathogenesis, clinical diagnosis, treatment and prevention. Comparative Immunology, Microbiology and Infectious Diseases, 2022, 82: 101765.
[3]
高艳, 张峣, 都兴洋, 蒋烈戈, 涂亚斌, 张兴山, 韩雪, 高宏雷. 犬细小病毒黑龙江流行株的分离鉴定及其VP2基因的遗传进化分析. 中国预防兽医学报, 2022, 44(2): 146-151.
GAO Y, ZHANG Y, DU X Y, JIANG L G, TU Y B, ZHANG X S, HAN X, GAO H L. Isolation and identification of canine parvovirus strain in Heilongjiang province and genetic evolution analysis of VP2 gene. Chinese Journal of Preventive Veterinary Medicine, 2022, 44(2): 146-151. (in Chinese)
[4]
PARRISH C R. Host range relationships and the evolution of canine parvovirus. Veterinary Microbiology, 1999, 69(1/2): 29-40.
[5]
LI J H, CHENG B Y, LI Z H, CUI Y L, YANG H Y, LIU W Q, ZHANG C M, YU Y L. Detection and molecular epidemiology of canine parvovirus and identification of highly pathogenic CPV-2c isolates from Shandong, China. Virus Genes, 2025, 61(1): 97-109.

doi: 10.1007/s11262-024-02125-z pmid: 39625586
[6]
PAN S S, MAN Y Z, XU X, JI J, ZHANG S Y, HUANG H H, LI Y, BI Y Z, YAO L G. Genetic diversity and recombination analysis of canine parvoviruses prevalent in central and Eastern China, from 2020 to 2023. Microorganisms, 2024, 12(11): 2173.
[7]
LI Z S, CAI J X, FENG C C, WANG Y, FANG S R, XUE X H. Two novel sites determine genetic relationships between CPV-2 and FPV: An epidemiological survey of canine and feline parvoviruses in Changchun, China (2020). Frontiers in Veterinary Science, 2024, 11: 1444984.
[8]
YU Z H, WANG W J, YU C, HE L, DING K, SHANG K, CHEN S B. Molecular characterization of feline parvovirus from domestic cats in Henan Province, China from 2020 to 2022. Veterinary Sciences, 2024, 11(7): 292.
[9]
PU J Y, ZHANG Y, ZHONG D K, CHEN Q S. Detection and genetic characterization of circulating canine parvovirus from stray dogs in Shanghai, China. Virology, 2024, 595: 110041.
[10]
CHEN S B, SHANG K, CHEN J, YU Z H, WEI Y, HE L, DING K. Global distribution, cross-species transmission, and receptor binding of canine parvovirus-2: Risks and implications for humans. Science of the Total Environment, 2024, 930: 172307.
[11]
GROPPETTI D, PECILE A, FILIPE J, RIVA F, INGLESI A, KUHN P A, GIUSSANI E, DALL’ARA P. Canine amniotic fluid at birth holds information about neonatal antibody titres against core vaccine viruses. Veterinary Sciences, 2024, 11(6): 234.
[12]
GARCIN P, COHEN S, TERPSTRA S, KELLY I, FOSTER L J, PANTÉ N. Proteomic analysis identifies a novel function for galectin-3 in the cell entry of parvovirus. Journal of Proteomics, 2013, 79: 123-132.

doi: 10.1016/j.jprot.2012.12.010 pmid: 23268121
[13]
LÓPEZ-ASTACIO R A, ADU O F, LEE H, HAFENSTEIN S L, PARRISH C R. The structures and functions of parvovirus capsids and missing pieces: The viral DNA and its packaging, asymmetrical features, nonprotein components, and receptor or antibody binding and interactions. Journal of Virology, 2023, 97(7): e0016123.
[14]
XU P, GANAIE S S, WANG X M, WANG Z K, KLEIBOEKER S, HORTON N C, HEIER R F, MEYERS M J, TAVIS J E, QIU J M. Endonuclease activity inhibition of the NS1 protein of parvovirus B19 as a novel target for antiviral drug development. Antimicrobial Agents and Chemotherapy, 2019, 63(3): e01879-18.
[15]
WANG X R, HAO X Q, ZHAO Y N, XIAO X Y, LI S J, ZHOU P. Canine parvovirus NS1 induces host translation shutoff by reducing mTOR phosphorylation. Journal of Virology, 2025, 99(1): e0146324.
[16]
MIETZSCH M, PÉNZES J J, AGBANDJE-MCKENNA M. Twenty-five years of structural parvovirology. Viruses, 2019, 11(4): 362.
[17]
孙岩, 仲飞, 李秀锦, 王幸兴, 王璐, 贾启恒, 韩冬梅, 李振, 张峰, 潘红丽. 犬白细胞介素-7基因对犬细小病毒DNA疫苗的免疫增强作用. 中国农业科学, 2012, 45(10): 2058-2066. doi: 10.3864/j.issn.0578-1752.2012.10.019.
SUN Y, ZHONG F, LI X J, WANG X X, WANG L, JIA Q H, HAN D M, LI Z, ZHANG F, PAN H L. Immune enhancing effects of canine interleukin-7 gene on canine parvovirus DNA vaccine. Scientia Agricultura Sinica, 2012, 45(10): 2058-2066. doi: 10.3864/j.issn.0578-1752.2012.10.019. (in Chinese)
[18]
CHIANG S R, LIN C Y, CHEN D Y, TSAI H F, LIN X C, HSU T C, TZANG B S. The effects of human parvovirus VP1 unique region in a mouse model of allergic asthma. PLoS ONE, 2019, 14(5): e0216799.
[19]
KARIATSUMARI T, HORIUCHI M, HAMA E, YAGUCHI K, ISHIGURIO N, GOTO H, SHINAGAWA M. Construction and nucleotide sequence analysis of an infectious DNA clone of the autonomous parvovirus, mink enteritis virus. The Journal of General Virology, 1991, 72 (Pt 4): 867-875.
[20]
YU Y L, SU J, WANG J G, XI J, MAO Y P, HOU Q, ZHANG X M, LIU W Q. A rapid method for establishment of a reverse genetics system for canine parvovirus. Virus Genes, 2017, 53(6): 876-882.

doi: 10.1007/s11262-017-1497-0 pmid: 28808849
[21]
OKWEE-ACAI J, AGWAI B, MAWADRI P, KESIIME C, TUBIHEMUKAMA M, KUNGU J, ODUR B. Prevalence of common conditions and associated mortalities of dogs treated at the small animal clinic, Makerere University, Kampala, Uganda. BMC Veterinary Research, 2024, 20(1): 590.
[22]
ULAS N, OZKANLAR Y, OZKANLAR S, TIMURKAN M O, AYDIN H. Clinical and inflammatory response to antiviral treatments in dogs with parvoviral enteritis. Journal of Veterinary Science, 2024, 25(1): e11.

doi: 10.4142/jvs.23139 pmid: 38311324
[23]
段树丽. 一例犬细小病毒病的诊断及治疗. 甘肃畜牧兽医, 2021, 51(6): 36-39.
DUAN S L. Diagnosis and treatment of a canine parvovirus disease. Gansu Animal Husbandry and Veterinary, 2021, 51(6): 36-39. (in Chinese)
[24]
GALLINELLA G. New insights into parvovirus research. Viruses, 2019, 11(11): 1053.
[25]
ZHOU H Z, ZHANG H H, SU X, XU F Z, XIAO B, ZHANG J, QI Q, LIN L L, CUI K D, LI Q Q, LI S P, YANG B. Identification of host-protein interaction network of canine parvovirus capsid protein VP2 in F81 cells. Microorganisms, 2025, 13(1): 88.
[26]
LIU K, XU P, LI Y C, QIN J L, ZHU J P, LI Y. Inhibition of canine parvovirus 2 (CPV-2) replication by TAT-scFv through targeting of the viral structural protein VP2 of CPV-2. The New Microbiologica, 2024, 46(4): 381-389.
[27]
YUAN D L, WANG J G, LI Z L, MAO Y P, SUN J Z, XI J, WANG S, HOU Q, YI B, LIU W Q. Establishment of a rescue system for an autonomous Parvovirus mink enteritis virus. Virus Research, 2014, 183: 1-5.

doi: 10.1016/j.virusres.2014.01.012 pmid: 24463297
[28]
CHENG N, ZHAO Y K, HAN Q X, ZHANG W J, XI J, YU Y L, WANG H L, LI G H, GAO Y W, YANG S T, LIU W Q, XIA X Z. Development of a reverse genetics system for a feline panleukopenia virus. Virus Genes, 2019, 55(1): 95-103.

doi: 10.1007/s11262-018-1621-9 pmid: 30519855
[29]
王诗研, 印春生, 曹众达, 张石豪, 张嘉雯. 犬细小病毒病防控技术研究进展. 中国兽药杂志, 2024, 58(6): 86-94.
WANG S Y, YIN C S, CAO Z D, ZHANG S H, ZHANG J W. Advances in prevention and control techniques for canine parvovirus disease. Chinese Journal of Veterinary Drug, 2024, 58(6): 86-94. (in Chinese)
[1] ZHANG XiaoZhan, DONG XuanZhi, LÜ NanNan, LIU YiWen, MA XinTian, WANG LinQing, XIA YanXun, JIANG ZengHai, GUO YunZe, ZHAO PanDeng, SONG YuZhen, YANG DeCheng, BIAN Chuanzhou. Effect of 12 Nucleotides Natural Insertion within the Internal Ribosome Entry Site Core Region on the Replication and Cellular Tropism of Porcine Senecavirus A [J]. Scientia Agricultura Sinica, 2024, 57(7): 1407-1416.
[2] WANG Yuan, DU MengDan, LI ZhengGang, SHE XiaoMan, YU Lin, LAN GuoBing, DING ShanWen, HE ZiFu, TANG YaFei. Identification of Pathogen Causing Tomato White Tip and Curl Leaf Disease and Its Pathogenicity in Guangdong Province [J]. Scientia Agricultura Sinica, 2024, 57(12): 2350-2363.
[3] GAO XiaoXiao, TU LiQin, YANG Liu, LIU YaNan, GAO DanNa, SUN Feng, LI Shuo, ZHANG SongBai, JI YingHua. Construction of an Infectious Clone of Tobacco Mild Green Mosaic Virus Isolate Infecting Pepper from Jiangsu Based on Genomic Clone [J]. Scientia Agricultura Sinica, 2023, 56(8): 1494-1502.
[4] DU BingChen, WANG Ming, LIU ChunGuo, WANG ShiDa, WEI XinYu, LU YaMan, SUN ZhenZhao, LIU ZaiSi, WEI LiLi, WANG JingFei, YANG DeCheng. Construction of Infectious cDNA Clone of GETV SC483 Strain [J]. Scientia Agricultura Sinica, 2023, 56(17): 3479-3486.
[5] JIANG QiQi,XU JianJian,SU Yue,ZHANG Qi,CAO Peng,SONG ChenHu,LI ZhongAn,SONG Zhen. Construction and Application of Infectious Clone of Citrus Yellow Mosaic Virus [J]. Scientia Agricultura Sinica, 2022, 55(24): 4840-4850.
[6] LI ZhengGang,TANG YaFei,SHE XiaoMan,YU Lin,LAN GuoBing,HE ZiFu. Molecular Characteristics and Pathogenicity Analysis of Youcai Mosaic Virus Guangdong Isolate Infecting Radish [J]. Scientia Agricultura Sinica, 2022, 55(14): 2752-2761.
[7] ZHANG Li,TANG YaFei,LI ZhengGang,YU Lin,LAN GuoBing,SHE XiaoMan,HE ZiFu. Molecular Characteristic of Squash Leaf Curl China Virus (SLCCNV) Infecting Cucurbitaceae Crops in Guangdong Province [J]. Scientia Agricultura Sinica, 2021, 54(19): 4097-4109.
[8] XU JianJian,WANG YanJiao,DUAN Yu,MA ZhiMin,BIN Yu,ZHOU ChangYong,SONG Zhen. Construction of Genome-Length cDNA of Citrus Vein Enation Virus and Identification of Its Infectivity [J]. Scientia Agricultura Sinica, 2020, 53(18): 3707-3715.
[9] CUI TianTian, YAN JianHong, BIN Yu, LI ZhongAn, ZHOU ChangYong, SONG Zhen. Construction of Citrus leaf blotch virus Infectious cDNA Clone by Yeast Homologous Recombination System [J]. Scientia Agricultura Sinica, 2018, 51(9): 1695-1705.
[10] TANG Ya-fei, HE Zi-fu, DU Zhen-guo, SHE Xiao-man, LAN Guo-bing. The Complex of Cotton leaf curl Multan virus and Its Associated Betasatellite Molecule Causing Cotton Leaf Curl Disease in Guangdong Province [J]. Scientia Agricultura Sinica, 2015, 48(16): 3166-3175.
[11] SUN Yan, ZHONG Fei, LI Xiu-Jin, WANG Xing-Xing, WANG Lu, JIA Qi-Heng, HAN Dong-Mei, LI Zhen, ZHANG Feng, PAN Hong-Li. Immune Enhancing Effects of Canine Interleukin-7 Gene on Canine Parvovirus DNA Vaccine [J]. Scientia Agricultura Sinica, 2012, 45(10): 2058-2066.
[12] ZOU Xing-qi,ZHAO Qi-zu,FAN Yun-feng,ZHU Yuan-yuan,WANG Qin,XU Lu,FAN Xue-zheng,NING Yi-bao
. Construction of the Full Length Infectious cDNA Clones of CSFV C Strain and Virus Rescue
 [J]. Scientia Agricultura Sinica, 2011, 44(2): 409-416 .
[13]
YAO Min; ZHANG Tian-qi; TIAN Zhi-chao; WANG Yuan-chao; TAO Xiao-rong
.

Construction of Agrobacterium-mediated Cucumber mosaic virus Infectious cDNA Clones and 2b Deletion Viral Vector

[J]. Scientia Agricultura Sinica, 2011, 44(14): 3060-3068 .
[14] . Rescue of Infectious Foot-and-Mouth Disease Virus in vivo from Full-Length cDNA Clone of Asia1 Type
[J]. Scientia Agricultura Sinica, 2009, 42(2): 688-693 .
[15] . Development of Infectious Molecular Clones of Infectious Bursal Disease Virus Attenuated strain [J]. Scientia Agricultura Sinica, 2007, 40(10): 2343-2349 .
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd