牛结节性皮肤病病毒荧光重组毒株的构建及其在抗病毒药物研究中的应用

Construction of fluorescent gene recombinant strain of lumpy skin disease virus and its application in antiviral drug research

摘要

[目的]本文旨在构建 1 株牛结节性皮肤病病毒(lumpy skin disease virus, LSDV)荧光重组毒株，并应用于抗病毒药物研究。[方法]基于 CRISPR/Cas9 技术构建 LSDV XJ 株重组毒株(rLSDV)，并通过 TCID₅₀试验测定病毒的生长曲线，再以纯化后的病毒样品作为免疫原制备兔多克隆抗体；用 Western blot(WB)试验评估 rLSDV 荧光信号强度与病毒蛋白表达量的相关性；用间接免疫荧光(IFA)试验评估 rLSDV 自发荧光信号的特异性；采用 CCK8 试验评价不同浓度 3′-甲氧基鸟苷对 MDBK 细胞的毒性，利用 rLSDV 荧光成像、WB、qPCR 和 TCID₅₀试验检测抗病毒药物 3′-甲氧基鸟苷对 LSDV 在 MDBK 细胞中增殖的影响。[结果]通过 CRISPR/Cas9 技术成功构建了 rLSDV，TCID₅₀试验测定的 rLSDV 与亲本病毒株的生长曲线基本一致；WB 试验结果显示，rLSDV 产生的荧光信号强度与病毒蛋白的表达量呈极显著正相关(P<0.01)；IFA 结果显示，抗体标记与 rLSDV 自身的荧光区域高度重合，且荧光信号强度趋势一致；在进一步的抗病毒药物研究中，CCK8、rLSDV 荧光成像、WB、qPCR 和 TCID₅₀试验结果均表明 3′-甲氧基鸟苷在安全浓度范围内对 LSDV 在 MDBK 细胞中的增殖具有极显著的抑制作用(P<0.01)。[结论]基于 CRISPR/Cas9 技术成功制备了 rLSDV，rLSDV 能够准确反映病毒的复制水平，3′-甲氧基鸟苷在体外能有效抑制 rLSDV 的增殖，可作为抗 LSDV 的一个潜在候选药物。

Abstract

[Objectives] This study aimed to construct a fluorescent gene recombinant strain of lumpy skin disease virus (LSDV) and apply it to antiviral drug research, laying the foundation for further elucidating the infection and defense mechanisms of LSDV. [Methods] Construction of a fluorescent gene recombinant strain (rLSDV) of LSDV XJ strain based on CRISPR/Cas9 technology and the growth curve of the virus was determined by TCID₅₀ assay. Simultaneously, rabbit polyclonal antibody was prepared using purified virus samples as immunogens. The correlation between the fluorescence signal intensity of rLSDV and viral protein expression was evaluated through Western blot (WB) assays. The autofluorescence signal of rLSDV was validated through indirect immunofluorescence assays (IFA). The cytotoxicity of 3′-O-methylguanosine on MDBK cell lines was examined by CCK8 assay. Furthermore, the inhibitory effects of 3′-O-methylguanosine on LSDV proliferation in MDBK were evaluated through rLSDV, WB, qPCR, and TCID₅₀ assays. [Results] The rLSDV was successfully constructed using CRISPR/Cas9 technology, and the growth curve of rLSDV, as determined by the TCID₅₀ assay, was consistent with that of the parental strain. WB results showed a significant positive correlation (P < 0.01) between the fluorescence signal intensity produced by rLSDV and the expression level of viral proteins. IFA results indicated that the antibody labeling highly overlapped with the fluorescent regions of rLSDV, and the fluorescence signal intensity trends were consistent. In further antiviral drug research, CCK8, IFA, qPCR, and TCID₅₀ assays collectively demonstrated that 3′-O-methylguanosine significantly (P < 0.01) inhibited the proliferation of LSDV in MDBK cells within a non-toxic concentration range. [Conclusions] The rLSDV was successfully constructed using CRISPR/Cas9 technology, and it accurately reflected the replication level of the virus. Furthermore, 3′-O-methylguanosine was identified as a potent inhibitor of LSDV proliferation in vitro, providing a promising drug candidate for further antiviral drug development.

关键词

牛结节性皮肤病病毒 / CRISPR/Cas9 / 荧光重组毒株 / 抗病毒药物 / 3′-甲氧基鸟苷

Key words

lumpy skin disease virus (LSDV) / CRISPR/Cas9 / fluorescent gene recombinant strain / antiviral drugs / 3′-O-methylguanosine

参考文献

[1] Davies F G. Lumpy skin disease, an African capripoxvirus disease of cattle[J]. The British Veterinary Journal, 1991, 147(6): 489-503.

[2] Ma J, Yuan Y, Shao J, et al. Genomic characterization of lumpy skin disease virus in southern China[J]. Transboundary and Emerging Diseases, 2022, 69(5): 2788-2799.

[3] Tulman E R, Afonso C L, Lu Z, et al. Genome of lumpy skin disease virus[J]. Journal of Virology, 2001, 75(15): 7122-7130.

[4] Babuik S, Bowden T R, Boyle D B, et al. Capripoxviruses: an emerging worldwide threat to sheep, goats and cattle[J]. Transboundary and Emerging Diseases, 2008, 55(7): 263-272.

[5] Sprygin A, Pestova Y, Wallace D B, et al. Transmission of lumpy skin disease virus: a short review[J]. Virus Research, 2019, 269: 197637.

[6] 孙映雪, 宋建德, 郑雪光, 等. 2020-2022 年全球牛结节性皮肤病流行状况[J]. 中国动物检疫, 2023, 40(5): 1-4.

[7] Sun Y X, Song J D, Zheng X G, et al. Global prevalence of lumpy skin disease from 2020 to 2022[J]. China Animal Health Inspection, 2023, 40(5): 1-4(in Chinese).

[8] Kononov A, Byadovskaya O, Kononova S, et al. Detection of vaccine-like strains of lumpy skin disease virus in outbreaks in Russia in 2017[J]. Archives of Virology, 2019, 164(6): 1575-1585.

[9] Mathijs E, Vandebussche F, Nguyen L, et al. Coding-complete sequences of recombinant lumpy skin disease viruses collected in 2020 from four outbreaks in northern Vietnam[J]. Microbiology Resource Announcements, 2021, 10(48): e0089721.

[10] Sprygin A, Sainnokhoi T, Gombo-Ochir D, et al. Genetic characterization and epidemiological analysis of the first lumpy skin disease virus outbreak in Mongolia, 2021[J]. Transboundary and Emerging Diseases, 2022, 69(6): 3664-3672.

[11] Lu G, Xie J, Luo J, et al. Lumpy skin disease outbreaks in China, since 3 August 2019[J]. Transboundary and Emerging Diseases, 2021, 68(2): 216-219.

[12] Mcfadden G. Poxvirus tropism[J]. Nature Reviews: Microbiology, 2005, 3(3): 201-213.

[13] Moss B. Smallpox vaccines: targets of protective immunity[J]. Immunological Reviews, 2011, 239(1): 8-26.

[14] Zhao Y, Lu Y, Richardson S, et al. TRIM5α restricts poxviruses and is antagonized by CypA and the viral protein C6[J]. Nature, 2023, 620(7975): 873-880.

[15] Ren S, Zhang Y, Gao X, et al. 2024 Platform establishment of the Cre-loxP recombination system for genetic manipulation of the lumpy skin disease virus[J]. Veterinary Microbiology, 2024, 294: 110122.

[16] Wang J, Ji J, Zhong Y, et al. Construction of recombinant fluorescent LSDV for high-throughput screening of antiviral drugs[J]. Veterinary Research, 2024, 55(1): 33.

[17] van der Oost J, Jore M M, Westra E R, et al. CRISPR-based adaptive and heritable immunity in prokaryotes[J]. Trends in Biochemical Sciences, 2009, 34(8): 401-407.

[18] Jansen R, van Embden J D A, Gaastr W, et al. Identification of genes that are associated with DNA repeats in prokaryotes[J]. Molecular Microbiology, 2002, 43(6): 1565-1575.

[19] Brouns S J J, Jore M M, Lundgren M, et al. Small CRISPR RNAs guide antiviral defense in prokaryotes[J]. Science, 2008, 321(5891): 960-964.

[20] Niu Y, Shen B, Cui Y, et al. Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos[J]. Cell, 2014, 156(4): 836-843.

[21] Cong L, Ran F A, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems[J]. Science, 2013, 339(6121): 819-823.

[22] Bi Y, Sun L, Gao D, et al. High-efficiency targeted editing of large viral genomes by RNA-guided nucleases[J]. PLoS Pathogens, 2014, 10(5): e1004090.

[23] Yuan M, Zhang W, Wang J, et al. Efficiently editing the vaccinia virus genome by using the CRISPR-Cas9 system[J]. Journal of Virology, 2015, 89(9): 5176-5179.

[24] Yuan M, Wang P, Chard L S, et al. A simple and efficient approach to construct mutant vaccinia virus vectors[J]. Journal of Visualized Experiments, 2016(116): 54171.

[25] van Diepen M, Chapman R, Douglass N, et al. Advancements in the growth and construction of recombinant lumpy skin disease virus (LSDV) for use as a vaccine vector[J]. Vaccines, 2021, 9(10): 1131.

[26] Goswami B B, Sharma O K. Inhibition of vaccinia virus growth and virus-specific RNA synthesis by 3′-O-methyladenosine and 3′-O-methylguanosine[J]. Journal of Virology, 1983, 45(3): 1164-1167.

[27] Schlosser-Perrin L, Holzmuller P, Fernandez B, et al. Constitutive proteins of lumpy skin disease virion assessed by next-generation proteomics[J]. Journal of Virology, 2023, 97(10): e00723-23.

[28] Cyrklaff M, Risco C, Fernández J J, et al. Cryo-electron tomography of vaccinia virus[J]. Proc Natl Acad Sci USA, 2005, 102(8): 2772-2777.

[29] Tan L, Shu X, Xu K, et al. Homologous recombination technology generated recombinant pseudorabies virus expressing EGFP facilitates to evaluate its susceptibility to different cells and screen antiviral compounds[J]. Research in Veterinary Science, 2022, 145: 125-134.

[30] Fu P F, Cheng X, Su B Q, et al. CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivo[J]. Veterinary Research, 2021, 52(1): 95.

[31] 刘铭, 邢晓旭, 孙东波. 紫铆因对牛冠状病毒的体外抗病毒作用[J]. 东北农业大学学报, 2025, 56(6): 126-134.

[32] Liu M, Xing X X, Sun D B. Antiviral activity of butein on bovine coronavirus in vitro[J]. Journal of Northeast Agricultural University, 2025, 56(6): 126-134(in Chinese with English abstract).

[33] Frumence E, Viranaicken W, Gadea G, et al. A GFP reporter MR766-based flow cytometry neutralization test for rapid detection of Zika virus-neutralizing antibodies in serum specimens[J]. Vaccines, 2019, 7(3): 66.

[34] 王浩, 刘聪, 贾笑冉, 等. 牛结节性皮肤病病毒 ORF005 基因缺失重组病毒的构建及生物学特性分析[J]. 畜牧与兽医, 2025, 57(12): 83-91.

[35] Wang H, Liu C, Jia X R, et al. Construction and biological characteristics of an ORF005 gene deleted lumpy skin disease virus strain[J]. Animal Husbandry & Veterinary Medicine, 2025, 57(12): 83-91(in Chinese with English abstract).

[36] Hubert M, Guivel-Benhassine F, Bruel T, et al. Complement-dependent mpox-virus-neutralizing antibodies in infected and vaccinated individuals[J]. Cell Host & Microbe, 2023, 31(6): 937-948.e4.

[37] Lu C, Li H, Chen W, et al. Immunological characteristics of a recombinant alphaherpes virus with an envelope-embedded Cap protein of circovirus[J]. Frontiers in Immunology, 2024, 15: 1438371.

[38] Liu Y, Shen Z, Xie Z, et al. African swine fever virus I73R is a critical virulence-related gene: a potential target for attenuation[J]. Proc Natl Acad Sci USA, 2023, 120(15): e2210808120.

基金资助

国家重点研发计划项目(2022YFD1800500)