Nanobodies against African swine fever virus p72 and CD2v proteins as reagents for developing two cELISAs to detect viral antibodies

Jiahong Zhu; Qingyuan Liu; Liuya Li; Runyu Zhang; Yueting Chang; Jiakai Zhao; Siyu Liu; Xinyu Zhao; Xu Chen; Yani Sun; Qin Zhao

doi:10.1016/j.virs.2024.04.002

June 2024

Jiahong Zhu, Qingyuan Liu, Liuya Li, Runyu Zhang, Yueting Chang, Jiakai Zhao, Siyu Liu, Xinyu Zhao, Xu Chen, Yani Sun and Qin Zhao. Nanobodies against African swine fever virus p72 and CD2v proteins as reagents for developing two cELISAs to detect viral antibodies[J]. Virologica Sinica, 2024, 39(3): 478-489. doi: 10.1016/j.virs.2024.04.002

Citation: Jiahong Zhu, Qingyuan Liu, Liuya Li, Runyu Zhang, Yueting Chang, Jiakai Zhao, Siyu Liu, Xinyu Zhao, Xu Chen, Yani Sun, Qin Zhao. Nanobodies against African swine fever virus p72 and CD2v proteins as reagents for developing two cELISAs to detect viral antibodies .VIROLOGICA SINICA, 2024, 39(3) : 478-489. http://dx.doi.org/10.1016/j.virs.2024.04.002

基于抗p72和CD2v蛋白纳米抗体建立2种检测猪血清中抗非洲猪瘟病毒抗体的cELISAs

cstr: 32224.14.j.virs.2024.04.002

西北农林科技大学动物医学院

通讯作者： 孙亚妮, sunyani@nwsuaf.edu.cn; 赵钦, qinzhao_2004@nwsuaf.edu.cn
收稿日期： 2023-09-11
录用日期： 2024-04-01

摘要

非洲猪瘟病毒（ASFV）对全球养猪业构成巨大威胁。目前，没有有效的疫苗或治疗药物对抗ASFV感染。快速诊断并消灭感染猪仍是控制病毒传播的主要手段。最近，中国报道了EP402R基因缺失（CD2v-deleted）的低毒力ASFV分离株，进一步增加了猪场ASFV感染的复杂性。此外，EP402R基因缺失ASFV株已被用作潜在的减毒活疫苗候选毒株。因此，开发能够区分野生型和EP402R基因缺失型ASFV感染的检测方法至关重要。本研究利用原核系统表达了重组ASFV p72和CD2v蛋白，并从免疫的双峰驼中，分别筛选出8株抗ASFV-p72和10株抗ASFV-CD2v纳米抗体。将这些纳米抗体与辣根过氧化物酶（HRP）融合表达，选择ASFV-p72-Nb2-HRP和ASFV-CD2v-Nb22-HRP融合蛋白为竞争探针，建立了两种检测猪血清中抗ASFV抗体的竞争ELISA (cELISA)。两种cELISA具有高灵敏度、良好的特异性、重复性和稳定性。与商用ELISA试剂盒的符合率分别为98.6%和97.6%。总之，这两种cELISA方法易于操作，成本低，生产过程简单。两种cELISA联合使用可区分野生型或CD2v缺失型ASFV感染，可在猪场ASFV感染的监测中发挥重要作用。
- 非洲猪瘟病毒
- , ASFV p72和CD2v
- , 纳米抗体
- , 竞争ELISA

Nanobodies against African swine fever virus p72 and CD2v proteins as reagents for developing two cELISAs to detect viral antibodies

Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling Observing and Experimental Station of National Data Center of Animal Health, Ministry of Agriculture, Yangling, 712100, China

Corresponding author: Yani Sun, sunyani@nwsuaf.edu.cn Qin Zhao, qinzhao_2004@nwsuaf.edu.cn
Received Date: 11 September 2023
Accepted Date: 01 April 2024

Abstract

African swine fever virus (ASFV) poses a significant threat to the global swine industry. Currently, there are no effective vaccines or treatments available to combat ASFV infection in pigs. The primary means of controlling the spread of the disease is through rapid detection and subsequent elimination of infected pig. Recently, a lower virulent ASFV isolate with a deleted EP402R gene (CD2v-deleted) has been reported in China, which further complicates the control of ASFV infection in pig farms. Furthermore, an EP402R-deleted ASFV variant has been developed as a potential live attenuated vaccine candidate strain. Therefore, it is crucial to develop detection methods that can distinguish wild-type and EP402R-deleted ASFV infections. In this study, two recombinant ASFV-p72 and -CD2v proteins were expressed using a prokaryotic system and used to immunize Bactrian camels. Subsequently, eight nanobodies against ASFV-p72 and ten nanobodies against ASFV-CD2v were screened. Following the production of these nanobodies with horse radish peroxidase (HRP) fusion proteins, the ASFV-p72-Nb2-HRP and ASFV-CD2v-Nb22-HRP fusions were selected for the development of two competitive ELISAs (cELISAs) to detect anti-ASFV antibodies. The two cELISAs exhibited high sensitivity, good specificity, repeatability, and stability. The coincidence rate between the two cELISAs and commercial ELISA kits was 98.6% and 97.6%, respectively. Collectively, the two cELISA for detecting antibodies against ASFV demonstrated ease of operation, a low cost, and a simple production process. The two cELISAs could determine whether pigs were infected with wild-type or CD2v-deleted ASFV, and could play an important role in monitoring ASFV infections in pig farms.
- African swine fever virus(ASFV)
- , ASFV-p72
- , ASFV-CD2v
- , Nanobody-HRP
- , Competitive ELISA

References
1. Andres, G., Charro, D., Matamoros, T., Dillard, R.S.,Abrescia, N.G.A., 2020. The cryo-EM structure of African swine fever virus unravels a unique architecture comprising two icosahedral protein capsids and two lipoprotein membranes. J. Biol. Chem. 295, 1-12.
2. Biagetti, M., Cuccioloni, M., Bonfili, L., Cecarini, V., Sebastiani, C., Curcio, L., Giammarioli, M., De Mia, G.M., Eleuteri, A.M.,Angeletti, M., 2018. Chimeric DNA/LNA-based biosensor for the rapid detection of African swine fever virus. Talanta. 184, 35-41.
3. Boinas, F.S., Hutchings, G.H., Dixon, L.K.,Wilkinson, P.J., 2004. Characterization of pathogenic and non-pathogenic African swine fever virus isolates from Ornithodoros erraticus inhabiting pig premises in Portugal. J. Gen. Virol. 85, 2177-2187.
4. Burmakina, G., Malogolovkin, A., Tulman, E.R., Zsak, L., Delhon, G., Diel, D.G., Shobogorov, N.M., Morgunov, Y.P., Morgunov, S.Y., Kutish, G.F., Kolbasov, D.,Rock, D.L., 2016. African swine fever virus serotype-specific proteins are significant protective antigens for African swine fever. J. Gen. Virol. 97, 1670-1675.
5. Caixia, W., Songyin, Q., Ying, X., Haoyang, Y., Haoxuan, L., Shaoqiang, W., Chunyan, F.,Xiangmei, L., 2022. Development of a blocking ELISA kit for detection of ASFV antibody based on a monoclonal antibody against full-length p72. J. AOAC Int. 105, 1428-1436.
6. Cao, Y., Han, D., Zhang, Y., Zhang, K., Du, N., Tong, W., Li, G., Zheng, H., Liu, C., Gao, F.,Tong, G., 2021. Identification of one novel epitope targeting p54 protein of African swine fever virus using monoclonal antibody and development of a capable ELISA. Res. Vet. Sci. 141, 19-25.
7. Chen, D., Wang, D., Wang, C., Wei, F., Zhao, H., Lin, X.,Wu, S., 2021. Application of an AlphaLISA method for rapid sensitive detection of African swine fever virus in porcine serum. Appl. Microbiol. Biotechnol. 105, 4751-4759.
8. Chen, W., Zhao, D., He, X., Liu, R., Wang, Z., Zhang, X., Li, F., Shan, D., Chen, H., Zhang, J., Wang, L., Wen, Z., Wang, X., Guan, Y., Liu, J.,Bu, Z., 2020. A seven-gene-deleted African swine fever virus is safe and effective as a live attenuated vaccine in pigs. Sci. China Life Sci. 63, 623-634.
9. Dixon, L.K., Chapman, D.A., Netherton, C.L.,Upton, C., 2013. African swine fever virus replication and genomics. Virus Res. 173, 3-14.
10. Du, T., Zhu, G., Wu, X., Fang, J.,Zhou, E.M., 2019. Biotinylated single-domain antibody-based blocking ELISA for detection of antibodies against swine influenza virus. Int J Nanomedicine. 14, 9337-9349.
11. Freije, J.M., Munoz, M., Vinuela, E.,Lopez-Otin, C., 1993. High-level expression in Escherichia coli of the gene coding for the major structural protein (p72) of African swine fever virus. Gene. 123, 259-262.
12. Gallardo, C., Soler, A., Rodze, I., Nieto, R., Cano-Gomez, C., Fernandez-Pinero, J.,Arias, M., 2019. Attenuated and non-haemadsorbing (non-HAD) genotype II African swine fever virus (ASFV) isolated in Europe, Latvia 2017. Transbound. Emerg. Dis. 66, 1399-1404.
13. Ge, S., Li, J., Fan, X., Liu, F., Li, L., Wang, Q., Ren, W., Bao, J., Liu, C., Wang, H., Liu, Y., Zhang, Y., Xu, T., Wu, X.,Wang, Z., 2018. Molecular characterization of African swine fever virus, China, 2018. Emerg. Infect. Dis. 24, 2131-2133.
14. Geng, R., Sun, Y., Li, R., Yang, J., Ma, H., Qiao, Z., Lu, Q., Qiao, S.,Zhang, G., 2022. Development of a p72 trimer-based colloidal gold strip for detection of antibodies against African swine fever virus. Appl. Microbiol. Biotechnol. 106, 2703-2714.
15. Goatley, L.C.,Dixon, L.K., 2011. Processing and localization of the african swine fever virus CD2v transmembrane protein. J. Virol. 85, 3294-3305.
16. Heimerman, M.E., Murgia, M.V., Wu, P., Lowe, A.D., Jia, W.,Rowland, R.R., 2018. Linear epitopes in African swine fever virus p72 recognized by monoclonal antibodies prepared against baculovirus-expressed antigen. J. Vet. Diagn. Invest. 30, 406-412.
17. Hemmink, J.D., Khazalwa, E.M., Abkallo, H.M., Oduor, B., Khayumbi, J., Svitek, N., Henson, S.P., Blome, S., Keil, G., Bishop, R.P.,Steinaa, L., 2022. Deletion of the CD2v gene from the genome of ASFV-Kenya-IX-1033 partially reduces virulence and induces protection in pigs. Viruses. 14, 1917.
18. Jia, N., Ou, Y., Pejsak, Z., Zhang, Y.,Zhang, J., 2017. Roles of African swine fever virus structural proteins in viral infection. J. Vet. Res. 61, 135-143.
19. Jia, R., Zhang, G., Bai, Y., Liu, H., Chen, Y., Ding, P., Zhou, J., Feng, H., Li, M., Tian, Y.,Wang, A., 2022. Identification of linear B cell epitopes on CD2V protein of African swine fever virus by monoclonal antibodies. Microbiol. Spectr. 10, e0105221.
20. Jiang, W., Jiang, D., Li, L., Wan, B., Wang, J., Wang, P., Shi, X., Zhao, Q., Song, J., Zhu, Z., Ji, P.,Zhang, G., 2022. Development of an indirect ELISA for the identification of African swine fever virus wild-type strains and CD2v-deleted strains. Front. Vet. Sci. 9, 1006895.
21. Jolaoluwa, A.E., Oluseyi, O.B., Ido, G.F., Yusoff, S.M.,Adetunji, O.G., 2021. Detection of African swine fever virus in pigs in Southwest Nigeria. J. Vet. World. 14, 1840-1845.
22. King, D.P., Reid, S.M., Hutchings, G.H., Grierson, S.S., Wilkinson, P.J., Dixon, L.K., Bastos, A.D.,Drew, T.W., 2003. Development of a TaqMan PCR assay with internal amplification control for the detection of African swine fever virus. J. Virol. Methods. 107, 53-61.
23. Liu, Q., Ma, B., Qian, N., Zhang, F., Tan, X., Lei, J.,Xiang, Y., 2019. Structure of the African swine fever virus major capsid protein p72. Cell Res. 29, 953-955.
24. Liu, Y., Xie, Z., Li, Y., Song, Y., Di, D., Liu, J., Gong, L., Chen, Z., Wu, J., Ye, Z., Liu, J., Yu, W., Lv, L., Zhong, Q., Tian, C., Song, Q., Wang, H.,Chen, H., 2023. Evaluation of an I177L gene-based five-gene-deleted African swine fever virus as a live attenuated vaccine in pigs. Emerg. Microbes Infect. 12, 2148560.
25. Lv, C., Zhao, Y., Jiang, L., Zhao, L., Wu, C., Hui, X., Hu, X., Shao, Z., Xia, X., Sun, X., Zhang, Q., Jin, M., 2021. Development of a dual ELISA for the detection of CD2v-unexpressed lower-virulence mutational ASFV. Life (Basel). 11, 1214.
26. Muyldermans, S., 2021. Applications of nanobodies. Annu. Rev. Anim. Biosci. 9, 401-421.
27. Nah, J.J., Kwon, O.G., Choi, J.D., Jang, S.H., Lee, H.J., Ahn, D.G., Lee, K., Kang, B., Hae-Eun, K.,Shin, Y.K., 2022. Development of an indirect ELISA against African swine fever virus using two recombinant antigens, partial p22 and p30. J. Virol. Methods. 309, 114611.
28. Oura, C.A., Edwards, L.,Batten, C.A., 2013. Virological diagnosis of African swine fever--comparative study of available tests. Virus Res. 173, 150-158.
29. Perez-Nunez, D., Sunwoo, S.Y., Garcia-Belmonte, R., Kim, C., Vigara-Astillero, G., Riera, E., Kim, D.M., Jeong, J., Tark, D., Ko, Y.S., You, Y.K.,Revilla, Y., 2022. Recombinant African swine fever virus Arm/07/CBM/c2 lacking CD2v and A238L is attenuated and protects pigs against virulent Korean Paju strain. Vaccines (Basel). 10, 1992.
30. Pillay, T.S.,Muyldermans, S., 2021. Application of single-domain antibodies (“nanobodies”) to laboratory diagnosis. Ann. Lab. Med. 41, 549-558.
31. Revilla, Y., Perez-Nunez, D.,Richt, J.A., 2018. African swine fever virus biology and vaccine approaches. Adv. Virus Res. 100, 41-74.
32. Sastre, P., Perez, T., Costa, S., Yang, X., Raber, A., Blome, S., Goller, K.V., Gallardo, C., Tapia, I., Garcia, J., Sanz, A.,Rueda, P., 2016. Development of a duplex lateral flow assay for simultaneous detection of antibodies against African and Classical swine fever viruses. J. Vet. Diagn. Invest. 28, 543-549.
33. Sheng, Y., Wang, K., Lu, Q., Ji, P., Liu, B., Zhu, J., Liu, Q., Sun, Y., Zhang, J., Zhou, E.M.,Zhao, Q., 2019. Nanobody-horseradish peroxidase fusion protein as an ultrasensitive probe to detect antibodies against Newcastle disease virus in the immunoassay. J. Nanobiotechnology. 17, 35.
34. Sun, E., Zhang, Z., Wang, Z., He, X., Zhang, X., Wang, L., Wang, W., Huang, L., Xi, F., Huangfu, H., Tsegay, G., Huo, H., Sun, J., Tian, Z., Xia, W., Yu, X., Li, F., Liu, R., Guan, Y., Zhao, D.,Bu, Z., 2021. Emergence and prevalence of naturally occurring lower virulent African swine fever viruses in domestic pigs in China in 2020. Sci. China Life Sci. 64, 752-765.
35. Vincke, C., Gutierrez, C., Wernery, U., Devoogdt, N., Hassanzadeh-Ghassabeh, G.,Muyldermans, S., 2012. Generation of single domain antibody fragments derived from camelids and generation of manifold constructs. Methods Mol. Biol. 907, 145-176.
36. Wang, A., Jia, R., Liu, Y., Zhou, J., Qi, Y., Chen, Y., Liu, D., Zhao, J., Shi, H., Zhang, J.,Zhang, G., 2020a. Development of a novel quantitative real-time PCR assay with lyophilized powder reagent to detect African swine fever virus in blood samples of domestic pigs in China. Transbound. Emerg. Dis. 67, 284-297.
37. Wang, D., Yu, J., Wang, Y., Zhang, M., Li, P., Liu, M.,Liu, Y., 2020b. Development of a real-time loop-mediated isothermal amplification (LAMP) assay and visual LAMP assay for detection of African swine fever virus (ASFV). J. Virol. Methods. 276, 113775.
38. Wang, L., Fu, D., Tesfagaber, W., Li, F., Chen, W., Zhu, Y., Sun, E., Wang, W., He, X., Guo, Y., Bu, Z.,Zhao, D., 2022. Development of an ELISA method to differentiate animals infected with wild-type African swine fever viruses and attenuated HLJ/18-7GD vaccine candidate. Viruses. 14, 1731.
39. Wang, Y., Xu, L., Noll, L., Stoy, C., Porter, E., Fu, J., Feng, Y., Peddireddi, L., Liu, X., Dodd, K.A., Jia, W.,Bai, J., 2020c. Development of a real-time PCR assay for detection of African swine fever virus with an endogenous internal control. Transbound. Emerg. Dis. 67, 2446-2454.
40. Wilkinson, P.J., Pegram, R.G., Perry, B.D., Lemche, J.,Schels, H.F., 1988. The distribution of African swine fever virus isolated from Ornithodoros moubata in Zambia. Epidemiol. Infect. 101, 547-564.
41. Yang, H., Peng, Z., Song, W., Zhang, C., Fan, J., Chen, H., Hua, L., Pei, J., Tang, X., Chen, H.,Wu, B., 2022. A triplex real-time PCR method to detect African swine fever virus gene-deleted and wild type strains. Front. Vet. Sci. 9, 943099.
42. Zhao, H., Ren, J., Wu, S., Guo, H., Du, Y., Wan, B., Ji, P., Wu, Y., Zhuang, G., Zhang, A.,Zhang, G., 2022. HRP-conjugated-nanobody-based cELISA for rapid and sensitive clinical detection of ASFV antibodies. Appl. Microbiol. Biotechnol. 106, 4269-4285.
43. Zhao, J., Zhu, J., Wang, Y., Yang, M., Zhang, Q., Zhang, C., Nan, Y., Zhou, E.M., Sun, Y.,Zhao, Q., 2022. A simple nanobody-based competitive ELISA to detect antibodies against African swine fever virus. Virol. Sin. 37, 922-933.
44. Zhu, Z., Xiao, C.T., Fan, Y., Cai, Z., Lu, C., Zhang, G., Jiang, T., Tan, Y.,Peng, Y., 2019. Homologous recombination shapes the genetic diversity of African swine fever viruses. Vet. Microbiol. 236, 108380.
Proportional views
Electronic Supplementary Material

10.1016j.virs.2024.04.002-ESM.docx