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

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

  • 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.

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  • 10.1016j.virs.2024.04.002-ESM.docx
    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.

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

      Corresponding author: Yani Sun, sunyani@nwsuaf.edu.cn
      Corresponding author: Qin Zhao, qinzhao_2004@nwsuaf.edu.cn
    • 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

    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.

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