表达增强型绿色荧光蛋白的报告猪瘟病毒C株的构建及在家兔中生物学特性评价

doi:10.11843/j.issn.0366-6964.2020.07.022

Abstract

Abstract: C-strain (also known as HCLV strain) of classical swine fever virus (CSFV) is a lapinized live attenuated vaccine against classical swine fever (CSF). However, C-strain lacks the capacity for the serological differentiation between infected and vaccinated animals (DIVA). The purpose of this study is to insert the gene encoding the enhanced green fluorescent protein (EGFP) into CSFV C-strain to construct a reporter virus, and to provide a virus tracing tool for basic research of C-strain, and also to provide a method for constructing C-strain marker vaccine. Here, we constructed and characterized two reporter viruses based on C-strain. One is the reporter virus rHCLV-EGFP, which expressing the enhanced green fluorescent protein (EGFP) fused in frame with the N^pro protein. The other one is the chimeric reporter virus rHCLV-N^pro(SM)-EGFP in which the N^pro gene is replaced with the counterpart of the highly virulent CSFV Shimen (SM) strain in the context of C-strain. The researchers identified the rescued reporter viruses by ELISA kit for CSFV antigen, observing the fluorescence directly and detecting the EGFP protein by Western blot, as well as evaluated the biological characteristics of these two reporter viruses in cells and rabbits. The antigens of the two reporter viruses were positive by ELISA. The reporter virus rHCLV-EGFP remained viable but not fluorescent, containing an intact EGFP gene but expressed as an N^pro-EGFP fusion protein with unexpected size. Interestingly, the EGFP fluorescence was restored in the chimeric reporter virus rHCLV-N^pro(SM)-EGFP. The experiment in cells showed that the two reporter viruses had similar growth characteristics to the parental virus and retained genetic stability. Furthermore, the animal experiment in rabbits revealed that rHCLV-N^pro(SM)-EGFP exhibited similar biological properties to the parental virus C-strain, while rHCLV-EGFP lost the ability to induce the fever response of C-strain in rabbits. In this study, the chimeric reporter virus rHCLV-N^pro (SM)-EGFP is able to be used as the reporter virus of C-strain. It can be used for virus tracing to study the replication process of the C-strain and the interaction between the virus and cells. The chimeric reporter virus also has the potential for a marker vaccine, which has the DIVA capability by detecting the anti-EGFP antibody.

Key words: classical swine fever virus, C-strain, N^pro protein, enhanced green fluorescent protein, characterization

CLC Number:

S852.651

HAN Yuying, XIE Libao, LI Yongfeng, QIU Huaji. Generation and Characterization in Rabbits of a Reporter Classical Swine Fever Virus Vaccine C-strain Expressing the Enhanced Green Fluorescent Protein[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(7): 1699-1709.

References 36

[1]	LUO Y Z, LI S, SUN Y, et al. Classical swine fever in China:a minireview[J]. Vet Microbiol, 2014, 172(1-3):1-6.
[2]	ZHANG H L, LENG C L, FENG L P, et al. A new subgenotype 2. 1d isolates of classical swine fever virus in China, 2014[J]. Infect Genet Evol, 2015, 34:94-105.
[3]	WANG M M, LINIGER M, MUÑOZ-GONZÁLEZ S, et al. A polyuridine insertion in the 3' untranslated region of classical swine fever virus activates immunity and reduces viral virulence in piglets[J]. J Virol, 2020, 94(2):e01214-19.
[4]	MA S M, MAO Q, CHEN W X, et al. Serum lipidomics analysis of classical swine fever virus infection in piglets and emerging role of free fatty acids in virus replication in vitro[J]. Front Cell Infect Microbiol, 2019, 9:410.
[5]	BECHER P, AVALOS RAMIREZ R, ORLICH M, et al. Genetic and antigenic characterization of novel pestivirus genotypes:implications for classification[J]. Virology, 2003, 311(1):96-104.
[6]	BORCA M V, HOLINKA L G, RAMIREZ-MEDINA E, et al. Identification of structural glycoprotein E2 domain critical to mediate replication of classical swine fever virus in SK6 cells[J]. Virology, 2019, 526:38-44.
[7]	WU R, LI L, ZHAO Y, et al. Identification of two amino acids within E2 important for the pathogenicity of chimeric classical swine fever virus[J]. Virus Res, 2016, 211:79-85.
[8]	BLOME S, MOß C, REIMANN I, et al. Classical swine fever vaccines-State-of-the-art[J]. Vet Microbiol, 2017, 206:10-20.
[9]	BLOME S, STAUBACH C, HENKE J, et al. Classical swine fever-An updated review[J]. Viruses, 2017, 9(4):86.
[10]	LUO Y Z, JI S W, LEI J L, et al. Efficacy evaluation of the C-strain-based vaccines against the subgenotype 2. 1d classical swine fever virus emerging in China[J]. Vet Microbiol, 2017, 201:154-161.
[11]	HUANG Y L, DENG M C, WANG F I, et al. The challenges of classical swine fever control:modified live and E2 subunit vaccines[J]. Virus Res, 2014, 179:1-11.
[12]	JI W, GUO Z, DING N Z, et al. Studying classical swine fever virus:making the best of a bad virus[J]. Virus Res, 2015, 197:35-47.
[13]	TONG C, CHEN N, LIAO X, et al. Continuous passaging of a recombinant C-Strain virus in PK-15 cells selects culture-adapted variants that showed enhanced replication but failed to induce fever in rabbits[J]. J Microbiol Biotechnol, 2017, 27(9):1701-1710.
[14]	ZHANG L K, LI Y F, XIE L B, et al. Secreted expression of the Cap gene of porcine circovirus type 2 in classical swine fever virus C-strain:potential of C-strain used as a vaccine vector[J]. Viruses, 2017, 9(10):298.
[15]	LI Y F, XIE L B, ZHANG L K, et al. The E2 glycoprotein is necessary but not sufficient for the adaptation of classical swine fever virus lapinized vaccine C-strain to the rabbit[J]. Virology, 2018, 519:197-206.
[16]	QIU H J, SHEN R X, TONG G Z. The lapinized Chinese strain vaccine against classical swine fever virus:a retrospective review spanning half a century[J]. Agric Sci China, 2006, 5(1):1-14.
[17]	XIA H Y, WAHLBERG N, QIU H J et al. Lack of phylogenetic evidence that the Shimen strain is the parental strain of the lapinized Chinese strain (C-strain) vaccine against classical swine fever[J]. Arch Virol, 2011, 156(6):1041-1044.
[18]	FAN Z C, DENNIS J C, BIRD R C. Bovine viral diarrhea virus is a suitable viral vector for stable expression of heterologous gene when inserted in between Npro and C genes[J]. Virus Res, 2008, 138(1-2):97-104.
[19]	HU Q Q, CHEN W Y, HUANG K H, et al. Rescue of recombinant peste des petits ruminants virus:creation of a GFP-expressing virus and application in rapid virus neutralization test[J]. Vet Res, 2012, 43:48.
[20]	李超. 猪瘟兔化弱毒疫苗株适应家兔的分子基础[D]. 北京:中国农业科学院, 2013.LI C. Genetic determinants of adaption to rabbits of C-strain, a lapinized attenuated vaccine against classical swine fever[D]. Beijing:Chinese Academy of Agricultural Sciences, 2013. (in Chinese)
[21]	LI C, HUANG J H, LI Y F, et al. Efficient and stable rescue of classical swine fever virus from cloned cDNA using an RNA polymerase Ⅱ system[J]. Arch Virol, 2013, 158(4):901-907.
[22]	LI C, LI Y F, SHEN L, et al. The role of noncoding regions of classical swine fever virus C-strain in its adaptation to the rabbit[J]. Virus Res, 2014, 183:117-122.
[23]	LI Y F, SHEN L, SUN Y, et al. Simplified serum neutralization test based on enhanced green fluorescent protein-tagged classical swine fever virus[J]. J Clin Microbiol, 2013, 51(8):2710-2712.
[24]	PENG W P, HOU Q, XIA Z H, et al. Identification of a conserved linear B-cell epitope at the N-terminus of the E2 glycoprotein of classical swine fever virus by phage-displayed random peptide library[J]. Virus Res, 2008, 135(2):267-272.
[25]	ZHANG X J, HAN Q Y, SUN Y, et al. Development of a triplex TaqMan real-time RT-PCR assay for differential detection of wild-type and HCLV vaccine strains of classical swine fever virus and bovine viral diarrhea virus 1[J]. Res Vet Sci, 2012, 92(3):512-518.
[26]	LI Y F, WANG X, SUN Y, et al. Generation and evaluation of a chimeric classical swine fever virus expressing a visible marker gene[J]. Arch Virol, 2016, 161(3):563-571.
[27]	KIM D Y, CALVERT J G, CHANG K O, et al. Expression and stability of foreign tags inserted into nsp2 of porcine reproductive and respiratorysyndrome virus (PRRSV)[J]. Virus Res, 2007, 128(1-2):106-114.
[28]	TUMKOSIT U, MAEDA Y, KISHISHITA N, et al. The use of green fluorescent protein-tagged virus-like particles as a tracer in the early phase of chikungunya infection[J]. Virus Res, 2019, 272:197732.
[29]	张玲楷, 李永锋, 谢利豹, 等. 表达猪圆环病毒2型Cap蛋白的重组猪瘟兔化弱毒疫苗株的构建与鉴定[J]. 生物工程学报, 2018, 34(2):216-223.ZHANG L K, LI Y F, XIE L B, et al. Generation and characterization of recombinant classical swine fever virus C-strain expressing the cap protein of porcine circovirus type 2[J]. Chinese Journal of Biotechnology, 2018, 34(2):216-223. (in Chinese)
[30]	HOLINKA L G, FERNANDEZ-SAINZ I, O'DON-NELL V, et al. Development of a live attenuated antigenic marker classical swine fever vaccine[J]. Virology, 2009, 384(1):106-113.
[31]	HOLINKA L G, FERNANDEZ-SAINZ I, SANFORD B, et al. Development of an improved live attenuated antigenic marker CSF vaccine strain candidate with an increased genetic stability[J]. Virology, 2014, 471-473:13-18.
[32]	KORTEKAAS J, VLOET R P M, WEERDMEESTER K, et al. Rational design of a classical swine fever C-strain vaccine virus that enables the differentiation between infected and vaccinated animals[J]. J Virol Methods, 2010, 163(2):175-185.
[33]	KORTEKAAS J, KETELAAR J, VLOET R P M, et al. Protective efficacy of a classical swine fever virus C-strain deletion mutant and ability to differentiate infected from vaccinated animals[J]. Vet Microbiol, 2011, 147(1-2):11-18.
[34]	EBLÉ P L, GEURTS Y, QUAK S, et al. Efficacy of chimeric Pestivirus vaccine candidates against classical swine fever:protection and DIVA characteristics[J]. Vet Microbiol, 2013, 162(2-4):437-446.
[35]	韩玉莹, 李永锋, 谢利豹, 等. 猪瘟兔化弱毒疫苗(C株)——10年回眸[J]. 中国预防兽医学报, 2019, 41(6):654-659.HAN Y Y, LI Y F, XIE L B, et al. C-strain:an update review during the last decade[J]. Chinese Journal of Preventive Veterinary Medicine, 2019, 41(6):654-659. (in Chinese)
[36]	刘元杰. 猪瘟疫苗C株WH303表位突变毒株的构建及在细胞和兔体中特性研究[D]. 北京:中国兽医药品监察所, 2018.LIU Y J. Construction and research of characteristics in cells and rabbits of a WH303 epitope mutant based on C-strain[D]. Beijing:China Institute of Veterinary Drug Control, 2018. (in Chinese)

Generation and Characterization in Rabbits of a Reporter Classical Swine Fever Virus Vaccine C-strain Expressing the Enhanced Green Fluorescent Protein

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