伪狂犬病病毒荧光标签毒株构建及其在抗病毒药物筛选中的初步应用

doi:10.11843/j.issn.0366-6964.2024.08.031

摘要/Abstract

摘要：

为筛选针对伪狂犬病病毒(pseudorabies virus，PRV)的有效抗病毒药物，本研究以PRV人源分离毒株hSD-1/2019和经典猪源流行毒株Ea为亲本病毒，采用同源重组技术将mCherry报告基因插入到PRV的UL35基因终止子之前位点，构建了表达红色荧光蛋白mCherry的荧光标签毒株PRV-mCherry。基于构建的荧光标签病毒，以荧光强度为指标，建立高通量药物筛选平台，对天然产物库中1 621种化合物进行抗病毒药物筛选，并初步探索药物特性。结果表明，mCherry标签插入位置正确且稳定遗传，荧光标签毒株PRV-mCherry与亲本毒株在PK-15细胞上的增殖曲线趋势一致，且荧光强度可反映病毒增殖情况。使用PRV-mCherry从天然产物库中成功筛选出3种可在体外有效抑制PRV增殖的药物，根据测定的50%细胞毒性浓度和50%抑制浓度，计算出3种药物的选择指数范围为203.63~564.58 μmol·L^-1，表明其具有良好的成药性。本研究为临床防治PRV感染的抗病毒药物发掘提供了新的策略和思路。

关键词: 伪狂犬病病毒, 人源分离株, 荧光标签毒株, 抗病毒药物

Abstract:

In order to screen effective antiviral drugs against pseudorabies virus (PRV), we here constructed the recombinant strains PRV-mCherry expressing red fluorescent protein mCherry based on the human-originated isolate hSD-1/2019 and classical strain Ea. Specifically, mCherry reporter gene was inserted into the site before the terminator of PRV UL35 through homologous recombination technique. Based on the constructed recombinant fluorescent viruses, a high-throughput drug screening platform indicated by fluorescence intensity was established and applied for testing 1 621 drugs in the natural product library for screening effective antiviral drugs. Properties of the drugs were preliminarily explored. The results showed that mCherry was inserted in the correct position and was stably inherited. The fluorescence intensity of the recombinant strain PRV-mCherry was representative of the viral proliferation. The strains PRV-mCherry showed similar proliferation curves as the parental strains, supporting the usability of the PRV-mCherry strains in drug screening. Three drugs that could effectively inhibit PRV proliferation in vitro were screened out from the natural product library by using PRV-mCherry. Based on the determined 50% cytotoxic concentration and 50% inhibitory concentration, the selection indices of the three drugs were calculated to be 203.63-564.58 μmol·L^-1, indicating that they had good drug-forming properties. The research provided new ideas in antiviral drugs for the treatment of PRV infection.

Key words: pseudorabies virus, human-originated isolate, fluorescently labeled strains, antiviral drugs

中图分类号:

S852.659.1

呙会会, 张浩, 杨丹, 旷燕, 李亚菲, 刘绍蒙, 刘青芸, 王湘如. 伪狂犬病病毒荧光标签毒株构建及其在抗病毒药物筛选中的初步应用[J]. 畜牧兽医学报, 2024, 55(8): 3600-3611.

Huihui GUO, Hao ZHAGN, Dan YANG, Yan KUANG, Yafei LI, Shaomeng LIU, Qingyun LIU, Xiangru WANG. Construction of Fluorescently Labelled Pseudorabies Viruses and Their Preliminary Application in Antiviral Drug Screening[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(8): 3600-3611.

图/表 6

表 1

图 1

图 2

图 3

图 4

表 2

参考文献 36

1	姜平, 董永毅, 许家荣, 等.华东部分省市PRRS和猪伪狂犬病血清流行病学调查[J].畜牧与兽医,1999,31(6):22-23.
	JIANG P , DONG Y Y , XU J R , et al.Seroepidemiologic survey of PRRS and porcine pseudorabies in selected provinces and cities of East China[J].Animal Husbandry and Veterinary Medicine,1999,31(6):22-23.
2	ITOH S , KOBAYASHI K , YONEDA N , et al.Clinical study of late potentials: comparison of late potentials in myocardial infarction, cardiomyopathy and idiopathic ventricular tachycardia[J].Jpn Circ J,1988,52(1):21-29. doi: 10.1253/jcj.52.21
3	WOŹNIAKOWSKI G , SAMOREK-SALAMONOWICZ E .Animal herpesviruses and their zoonotic potential for cross-species infection[J].Ann Agric Environ Med,2015,22(2):191-194. doi: 10.5604/12321966.1152063
4	THOMFORD N E , SENTHEBANE D A , ROWE A , et al.Natural products for drug discovery in the 21st century: innovations for novel drug discovery[J].Int J Mol Sci,2018,19(6):1578. doi: 10.3390/ijms19061578
5	VAN DE SAND L , BORMANN M , SCHMITZ Y , et al.Antiviral active compounds derived from natural sources against herpes simplex viruses[J].Viruses,2021,13(7):1386. doi: 10.3390/v13071386
6	HOGUE I B , JEAN J , ESTEVES A D , et al.Functional carboxy-terminal fluorescent protein fusion to pseudorabies virus small capsid protein VP26[J].J Virol,2017,92(1):e01193-17.
7	PEDERSEN K , TURNAGE C T , GASTON W D , et al.Pseudorabies detected in hunting dogs in Alabama and Arkansas after close contact with feral swine (Sus scrofa)[J].BMC Vet Res,2018,14(1):388. doi: 10.1186/s12917-018-1718-3
8	CHIARI M , FERRARI N , BERTOLETTI M , et al.Long-term surveillance of Aujeszky's disease in the alpine wild boar (Sus scrofa)[J].EcoHealth,2015,12(4):563-570. doi: 10.1007/s10393-015-1064-x
9	曹明珠, 吕林, 李玉峰, 等.2018年山东省猪伪狂犬病野毒抗体流行病学调查[J].畜牧与兽医,2019,51(5):128-130.
	CAO M Z , LÜ L , LI Y F , et al.Epidemiological investigation of swine serum antibody against gE of pseudorabies in Shandong Province in 2018[J].Animal Husbandry & Veterinary Medicine,2019,51(5):128-130.
10	SEHL J , TEIFKE J P .Comparative pathology of pseudorabies in different naturally and experimentally infected species-a review[J].Pathogens,2020,9(8):633. doi: 10.3390/pathogens9080633
11	METTENLEITER T C .Pathogenesis of neurotropic herpesviruses: role of viral glycoproteins in neuroinvasion and transneuronal spread[J].Virus Res,2003,92(2):197-206. doi: 10.1016/S0168-1702(02)00352-0
12	SKINNER G R B , AHMAD A , DAVIES J A .The infrequency of transmission of herpesviruses between humans and animals; postulation of an unrecognised protective host mechanism[J].Comp Immunol Microbiol Infect Dis,2001,24(4):255-269. doi: 10.1016/S0147-9571(01)00014-5
13	MRAVAK S , BIENZLE U , FELDMEIER H , et al.Pseudorabies in man[J].Lancet,1987,1(8531):501-502.
14	LIU J B , CHEN C H , LI X D .Novel Chinese pseudorabies virus variants undergo extensive recombination and rapid interspecies transmission[J].Transbound Emerg Dis,2020,67(6):2274-2276. doi: 10.1111/tbed.13784
15	LIU Q Y , WANG X J , XIE C H , et al.A novel human acute encephalitis caused by pseudorabies virus variant strain[J].Clin Infect Dis,2021,73(11):e3690-e3700. doi: 10.1093/cid/ciaa987
16	FAN S Y , YUAN H X , LIU L , et al.Pseudorabies virus encephalitis in humans: a case series study[J].J Neurovirol,2020,26(4):556-564. doi: 10.1007/s13365-020-00855-y
17	赵伟丽, 乌依罕, 李红芳, 等.伪狂犬病毒脑炎临床观察与脑脊液二代测序鉴定[J].中华医学杂志,2018,98(15):1152-1157.
	ZHAO W L , WU Y H , LI H F , et al.Clinical experience and next-generation sequencing analysis of encephalitis caused by pseudorabies virus[J].National Medical Journal of China,2018,98(15):1152-1157.
18	WANG D , TAO X G , FEI M M , et al.Human encephalitis caused by pseudorabies virus infection: a case report[J].J Neurovirol,2020,26(3):442-448. doi: 10.1007/s13365-019-00822-2
19	WANG Y W , NIAN H , LI Z W , et al.Human encephalitis complicated with bilateral acute retinal necrosis associated with pseudorabies virus infection: A case report[J].Int J Infect Dis,2019,89,51-54. doi: 10.1016/j.ijid.2019.09.019
20	YANG X , GUAN H Z , LI C , et al.Characteristics of human encephalitis caused by pseudorabies virus: A case series study[J].Int J Infect Dis,2019,87,92-99. doi: 10.1016/j.ijid.2019.08.007
21	ZHENG L H , LIU X J , YUAN D Q , et al.Dynamic cerebrospinal fluid analyses of severe pseudorabies encephalitis[J].Transbound Emerg Dis,2019,66(6):2562-2565. doi: 10.1111/tbed.13297
22	YANG H N , HAN H , WANG H , et al.A case of human viral encephalitis caused by pseudorabies virus infection in China[J].Front Neurol,2019,10,534. doi: 10.3389/fneur.2019.00534
23	HU F , WANG J W , PENG X Y .Bilateral necrotizing retinitis following encephalitis caused by the pseudorabies virus confirmed by next-generation sequencing[J].Ocul Immunol Inflamm,2021,29(5):922-925.
24	AI J W , WENG S S , CHENG Q , et al.Human endophthalmitis caused by pseudorabies virus infection, China, 2017[J].Emerg Infect Dis,2018,24(6):1087-1090.
25	VERPOEST S , CAY B , FAVOREEL H , et al.Age-dependent differences in pseudorabies virus neuropathogenesis and associated cytokine expression[J].J Virol,2017,91(2):e02058-16.
26	STANGHERLIN L M , DE PAULA F N , ICIMOTO M Y , et al.Positively selected sites at HCMV gB furin processing region and their effects in cleavage efficiency[J].Front Microbiol,2017,8,934.
27	WONG G , LU J H , ZHANG W H , et al.Pseudorabies virus: a neglected zoonotic pathogen in humans?[J].Emerg Microbes Infect,2019,8(1):150-154.
28	LAVAL K , ENQUIST L W .The neuropathic itch caused by pseudorabies virus[J].Pathogens,2020,9(4):254.
29	WHITLEY R J , ROIZMAN B .Herpes simplex virus infections[J].Lancet,2001,357(9267):1513-1518.
30	JIANG Y C , FENG H , LIN Y C , et al.New strategies against drug resistance to herpes simplex virus[J].Int J Oral Sci,2016,8(1):1-6.
31	THOMAS E , STEWART L E , DARLEY B A , et al.Plant-based natural products and extracts: Potential source to develop new antiviral drug candidates[J].Molecules,2021,26(20):6197.
32	YANG Y , HAN J , MA Y L , et al.Demethylzeylasteral inhibits cell proliferation and enhances cell chemosensitivity to 5-fluorouracil in Colorectal Cancer cells[J].J Cancer,2020,11(20):6059-6069.
33	LV H W , JIANG L P , ZHU M D , et al.The genus Tripterygium: A phytochemistry and pharmacological review[J].Fitoterapia,2019,137,104190.
34	ZHU Z L , QIU X D , WU S , et al.Blocking effect of demethylzeylasteral on the interaction between human ACE2 protein and SARS-CoV-2 RBD protein discovered using SPR technology[J].Molecules,2021,26(1):57.
35	HARTWELL D , JONES J , LOVEMAN E , et al.Topotecan for relapsed small cell lung cancer: A systematic review and economic evaluation[J].Cancer Treat Rev,2011,37(3):242-249.
36	ZHAI H Y , HU S Y , LIU T X , et al.Nitidine chloride inhibits proliferation and induces apoptosis in colorectal cancer cells by suppressing the ERK signaling pathway[J].Mol Med Rep,2016,13(3):2536-2542.

基因 Genes	引物 Primer	序列(5′→3′) Sequences
上游同源臂	PRV-HindⅢ-F	CCCAAGCTTAGGCCGCGTACCCTCCG
Upstream homologous arm	PRV-KpnⅠ-R	CGGGGTACCGGGCGAGGGGCGAGGG
mCherry	mCherry-KpnⅠ-F	CGGGGTACCGGTGGAGGCGGTTCAGGCGGAG
		GTGGCTCTATGGTGAGCAAGGGCGAGGA
下游同源臂	mCherry-BamHⅠ-R	CGCGGATCCTAGCCCCGCGCGATCAATAAAG
Downstream homologous arm	PRV-BamHⅠ-F	CGCGGATCCTAGCCCCGCGCGATCAATAAAG
	PRV-EcoRⅠ-R	CCGGAATTCCCGCGCGTGGTGGAGTCG

编号 Number	药物 Drug	半数细胞毒性浓度/(μmol·L^-1) CC₅₀	半数抑制浓度/(μmol·L^-1) IC₅₀	选择指数 (CC₅₀/IC₅₀)
1	去甲泽拉木醛Demethylzeylasteral	135.6	0.618 2	219.346 50
2	盐酸石蒜碱Lycorine hydrochloride	34.0	2.750 0	12.363 64
3	石蒜碱Lycorine	31.9	1.480 0	21.554 05
4	托泊替康Topotecan	219.0	0.387 9	564.578 50
5	氯化两面针碱Nitidine Chloride	448.2	2.201 0	203.634 70

[1]	张颖, 宋春莲, 张莹, 沈鸿, 舒相华, 杨洪贵. 伪狂犬病病毒感染小鼠基质金属蛋白酶-9介导紧密连接蛋白损伤血脑屏障的研究[J]. 畜牧兽医学报, 2024, 55(5): 2186-2194.
[2]	李艺璇, 牛静轶, 李港, 万超, 方仁东, 叶超. 伪狂犬病病毒编码的内膜蛋白生物学功能研究进展[J]. 畜牧兽医学报, 2024, 55(3): 957-970.
[3]	王姿月, 张子卉, 吴文学, 彭辰. 猴痘的诊断、预防和治疗[J]. 畜牧兽医学报, 2023, 54(8): 3195-3205.
[4]	陈宏建, 曹艳, 樊杰, 甘荣萱, 宋文博, 喻盛炜, 杨婷, 赵艳霞, 魏春燕, 谢锐, 华琳, 彭忠, 吴斌. 2020—2022年湖北省生猪屠宰场伪狂犬病病毒的分离鉴定及遗传进化分析[J]. 畜牧兽医学报, 2023, 54(7): 2972-2981.
[5]	袁生, 李安琪, 吕文珂, 羊露露, 周峰, 黄良宗, 白挨泉, 温峰, 黄淑坚, 郭锦玥. 一株猪伪狂犬病病毒的主要毒力相关基因的变异分析及其对家兔的致病性[J]. 畜牧兽医学报, 2023, 54(5): 2195-2199.
[6]	倪征, 叶伟成, 陈柳, 云涛, 华炯钢, 朱寅初, 张存. 1株伪狂犬病病毒的基因变异及其对小鼠的致病性分析[J]. 畜牧兽医学报, 2023, 54(4): 1766-1770.
[7]	何文峰, 李琛, 常洪涛, 李隆熙, 陈静, 杨国庆, 刘慧敏. 抑制伪狂犬病病毒复制的宿主蛋白的筛选与鉴定[J]. 畜牧兽医学报, 2023, 54(3): 1177-1186.
[8]	吴学敏, 陈如敬, 陈秋勇, 车勇良, 严山, 刘玉涛, 周伦江, 王隆柏. 伪狂犬病病毒变异株gC抗体间接ELISA检测方法的建立及初步应用[J]. 畜牧兽医学报, 2022, 53(6): 2029-2034.
[9]	李兆龙, 张惠芳, 丰志华, 方舟. 携带CRISPR/Cas9的重组腺联病毒对伪狂犬病病毒感染小鼠的治疗效应[J]. 畜牧兽医学报, 2022, 53(3): 834-846.
[10]	郭振华, 李翔, 翁茂洋, 金前跃, 郭军庆, 邢广旭, 张改平. 宿主膜联蛋白A2与伪狂犬病病毒US3蛋白互作及其对细胞凋亡的影响[J]. 畜牧兽医学报, 2022, 53(11): 3927-3935.
[11]	王家敏, 李自良, 马芳芳, 康碧静, 田玲, 李倬, 马忠仁, 乔自林. 生物反应器培养BHK-21悬浮细胞增殖伪狂犬病病毒工艺优化[J]. 畜牧兽医学报, 2022, 53(11): 3936-3947.
[12]	李琛, 何文峰, 赵丽娜, 凡启, 杨国庆, 刘慧敏. PK-15细胞的ISG15基因敲除促进PRV的复制[J]. 畜牧兽医学报, 2022, 53(10): 3621-3630.
[13]	翟云云, 李佳佳, 张爽, 任子昱, 金前跃, 杜永坤, 万博. 凋亡相关斑点样蛋白敲除PK-15细胞系的建立及对PRV感染的影响[J]. 畜牧兽医学报, 2021, 52(2): 478-487.
[14]	张晓战, 杨磊, 邓同炜, 赵攀登, 彭志锋, 陈露露, 郭懿文, 夏艳勋, 乔宏兴, 边传周, 王增. 表达绿色荧光蛋白重组猪塞内卡病毒的构建及初步应用[J]. 畜牧兽医学报, 2021, 52(10): 2978-2985.
[15]	赵硕, 王若木, 党佳佳, 许力士, 秦树英, 薛辉, 韦祖樟, 陈樱, 欧阳康, 黄伟坚. 2013—2018年广西地区猪伪狂犬病病毒gB、gE与TK基因遗传变异分析[J]. 畜牧兽医学报, 2020, 51(4): 810-819.