非洲猪瘟病毒抗体间接免疫荧光检测方法的建立

doi:10.1016/j.jia.2023.05.021

Journal of Integrative Agriculture ›› 2024, Vol. 23 ›› Issue (1): 228-238.DOI: 10.1016/j.jia.2023.05.021

非洲猪瘟病毒抗体间接免疫荧光检测方法的建立

收稿日期:2023-03-15 接受日期:2023-04-19 出版日期:2024-01-20 发布日期:2024-01-06

Establishment of an indirect immunofluorescence assay for the detection of African swine fever virus antibodies

Wan Wang^1*, Zhenjiang Zhang^{1, 2*}, Weldu Tesfagaber¹, Jiwen Zhang¹, Fang Li¹, Encheng Sun¹, Lijie Tang², Zhigao Bu¹, Yuanmao Zhu^1#, Dongming Zhao^1# #br#

¹State Key Laboratory for Animal Disease Control and Prevention, National African Swine Fever Para-reference Laboratory, National High Containment Facilities for Animal Diseases Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China
²College of Veterinary Medicine, Northeast Agricultural University, Harbin 150069, China

Received:2023-03-15 Accepted:2023-04-19 Online:2024-01-20 Published:2024-01-06
About author:Wan Wang, E-mail: 82101205510@caas.cn; Zhenjiang Zhang, E-mail: 82101199503@caas.cn; #Correspondence Dongming Zhao, E-mail: zhaodongming@caas.cn; Yuanmao Zhu, E-mail: zhuyuanmao@caas.cn * These authors contributed equally to this study.
Supported by:
This work was supported by the National Key R&D Program of China (2019YFE0107300 and 2021YFD1800101), the Applied Technology Research and Development Project of Heilongjiang Province, China (GA19B301), and the Central Public-interest Scientific Institution Basal Research Fund, China (1610302022003).

摘要/Abstract

摘要：

非洲猪瘟（African Swine Fever，ASF）给我国养猪业造成巨大的经济损失。由于没有安全有效的疫苗，所以快速准确的诊断对ASF有效防控至关重要。间接免疫荧光法（Indirect Immunofluorescence Assay，IFA）是世界动物卫生组织（World Organization for Animal Health，WOAH）推荐的一种ASF血清学检测的金标准方法。

在本研究中，我们制备了野猪肾细胞（BK2258），该细胞能够支持非洲猪瘟病毒（African Swine Fever Virus，ASFV）SD/DY-I/21株高效复制，并表现出明显的细胞病变效应。利用BK2258建立了一种用于ASFV抗体检测的IFA方法。我们使用接种低毒力基因I型SD/DY-I/21株、基因II型HLJ/HRB1/20株和候选疫苗HLJ/18-7GD株免疫猪只的血清样品，同时也使用现地血清样品和阴性血清样品对该方法的特性进行了评估。利用IFA检测ASFV阳性血清，显示出明亮且特异的绿色荧光灶，没有因细胞衰老或其他细胞损伤因素引起的非特异性绿色荧光。与商业化的间接酶联免疫吸附方法（indirect Enzyme-Linked Immunosorbent Assay，iELISA）相比，检测病毒不同感染时间的血清发现，IFA可提前1-4天检测到ASFV抗体。IFA和iELISA对同一份ASFV抗体阳性血清样本的检出限分别为1:25600和1:6400，表明IFA比iELISA更敏感。新建立的IFA检测方法具有高度特异性，与其他6种重要猪病原体，包括经典猪瘟病毒（Classical Swine Fever Virus，CSFV）、猪繁殖与呼吸综合征病毒（Porcine Reproductive and Respiratory Syndrome Virus，PRRSV）、猪圆环病毒2型（Porcme Circovirus Type 2，PCV2）、伪狂犬病病毒（Pseudorabies Virus，PRV）、口蹄疫病毒O型（Foot-and-Mouth Disease Virus Type O，FMDV/O）和猪流行性腹泻病毒（Porcine Epidemic Diarrhea Virus，PEDV）的阳性血清无交叉反应。该研究提供了一种灵敏、特异且可靠的ASF血清学检测的金标准方法。

Abstract:

African swine fever (ASF) continues to cause enormous economic loss to the global pig industry. Since there is no safe and effective vaccine, accurate and timely diagnosis of ASF is essential to implement control measures. Indirect immunofluorescence assay (IFA) is a gold standard serological method recommended by the World Organization for Animal Health (WOAH). In this study, we used primary fetal kidney cells to establish a wild boar cell line (BK2258) that supported the efficient replication of ASF virus (ASFV) SD/DY-I/21 and showed visible cytopathic effect (CPE). Moreover, using BK2258, we established a sensitive and specific IFA for ASFV antibody detection. To standardize and evaluate the performance of this assay, we used serum samples from pigs infected with the low virulent genotype I SD/DY-I/21 and genotype II HLJ/HRB1/20, and immunized with the vaccine candidate HLJ/18-7GD, field samples, and negative serum samples. The IFA reacted with the ASFV-positive sera and displayed bright fluorescence foci. There was no non-specific green fluorescence due to cellular senescence or other cell damage-causing factors. Compared to a commercial indirect enzyme-linked immunosorbent assay (iELISA), ASFV antibodies were detected 1–4 days earlier using our IFA. The detection limits of the IFA and iELISA for the same ASFV-antibody positive serum samples were 1:25,600 and 1:6,400, respectively, indicating that the IFA is more sensitive than iELISA. The newly established IFA was highly specific and did not cross-react with sera positive for six other important porcine pathogens (i.e., Classical swine fever virus (CSFV), Porcine reproductive and respiratory syndrome virus (PRRSV), Porcme circovirus type 2 (PCV2), Pseudorabies virus (PRV), Foot-and-Mouth disease virus type O (FMDV/O), and Porcine epidemic diarrhea virus (PEDV)). This study thus provides a sensitive, specific, and reliable detection method that is suitable for the serological diagnosis of ASF.

Key words: African swine fever , antibody , IFA , serological method

. 非洲猪瘟病毒抗体间接免疫荧光检测方法的建立[J]. Journal of Integrative Agriculture, 2024, 23(1): 228-238.

Wan Wang, Zhenjiang Zhang, Weldu Tesfagaber, Jiwen Zhang, Fang Li, Encheng Sun, Lijie Tang, Zhigao Bu, Yuanmao Zhu, Dongming Zhao. Establishment of an indirect immunofluorescence assay for the detection of African swine fever virus antibodies[J]. Journal of Integrative Agriculture, 2024, 23(1): 228-238.

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非洲猪瘟病毒抗体间接免疫荧光检测方法的建立

Establishment of an indirect immunofluorescence assay for the detection of African swine fever virus antibodies

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