The Potential of O’nyong-nyong Virus Strain SG650 Murine Monoclonal Antibodies for Detection of O’nyong-nyong and Chikungunya Viruses

  • Albina Makio Jomo Kenyatta University
  • Lillian Musila, PhD United States Army Medical Research Directorate-Africa
  • Eddy Okoth Odari, PhD Jomo Kenyatta University
  • Juliette Rose Ongus, PhD Jomo Kenyatta University
  • Rosemary Sang, PhD Kenya Medical Research Institute
Keywords: O’nyong-Nyong Virus, Chikungunya Virus, Monoclonal Antibodies, Diagnostic-Potential
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O’nyong-nyong virus (ONNV) and Chikungunya virus (CHIKV) are antigenically related alphaviruses responsible for febrile illnesses common to the tropics and associated with relatively high morbidity and mortality. Murine monoclonal antibodies (mAbs) targeting alphaviruses like Chikungunya have been developed and used to make commercially available kits. However, few studies have been conducted to develop antibodies specific to ONNV and no commercial kits are available for use in endemic regions where outbreak potential is high. We demonstrate the potential of in-house generated monoclonal antibodies against ONNV to detect both ONNV and CHIKV. The objective of this study was to generate mAbs using hybridoma technology, characterize the developed mAbs, determine their specificity against selected alphaviruses and check their diagnostic potential using an indirect IgG enzyme-linked immunosorbent assay (ELISA) and focus neutralization assay (FRNT50). BALB/c mice were immunized with ONNV purified proteins from ONNV infectious culture fluid. After four rounds of booster injections, the mice were sacrificed, spleen cells harvested and fused with parental myeloma cells then cultured in selective media and the successful hybrid clones with antibody-producing ability purified to yield the desired mAbs. Five monoclonal antibodies targeting the ONNV E1 protein of isotypes IgG2a/kappa, IgG2b/kappa and IgM/kappa (P1B12, P1E9, P1G11, P1B4 and P1G6) demonstrated a potential to detect both ONNV and CHIKV isolates by indirect IgG ELISA but no potential for neutralization of the viruses by FRNT50. This study demonstrates the potential efficacy of in-house serological tools as an alternative in the absence of commercial assays in screening and diagnosis of ONN and CHIK viruses which are often co-circulating. It is our recommendation that this work may be pursued further to design and optimize ELISA assays, using the developed mAbs, for the detection of both ONN and CHIK viruses in the research laboratory set-up


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Adungo, F., Yu, F., Kamau, D., Inoue, S., Hayasaka, D., Posadas-Herrera, G., et al. (2016). Development and Characterization of Monoclonal Antibodies to Yellow Fever Virus and Application in Antigen Detection and IgM Capture Enzyme-Linked Immunosorbent Assay. Clin Vaccine Immunol, 23(8), 689-697.

Alexander C. Outhread, J. K., Dominic, E. & Dwyer. (2011). Viral Arthritides. [Review]. Expert Review Anti-infective Therapy, 9(5), 545- 554.

Athmaram, T. N., Saraswat, S., Misra, P., Das, T. K., & Srinivasan, A. (2013). A two-step purification strategy for chikungunya virions purification using sucrose buoyant density gradient seperation. Unpublished manuscript.

Basore, K., Earnest, J. T., Diamond, M. S., & Fremont, D. H. (2018). Structural characterization of broadly neutralizing alphavirus antibodies. The Journal of Immunology, 200(1 Supplement), 126.133-126.133.

Blackburn, N., Besselaar, T., & Gibson, G. (1995). Antigenic relationship between chikungunya virus strains and o'nyong nyong virus using monoclonal antibodies. Res Virol, 146, 69 - 73.

Bréhin, A.-C., Rubrecht, L., Navarro-Sanchez, M. E., Maréchal, V., Frenkiel, M.-P., Lapalud, P., et al. (2008). Production and characterization of mouse monoclonal antibodies reactive to Chikungunya envelope E2 glycoprotein. Virology, 371(1), 185-195.

Chanas, A., Hubalek, Z., Johnson, B., & Simpson, D. (1979). A comparative study of O'nyong nyong virus with Chikungunya virus and plaque variants. Arch Virol, 59, 231 - 238.

Clements, T. L., Rossi, C. A., Irish, A. K., Kibuuka, H., Eller, L. A., Robb, M. L., et al. (2019). Chikungunya and O’nyong-nyong Viruses in Uganda: Implications for Diagnostics. Open Forum Infectious Diseases, 6(3).

Corrêa, A. L., Senna, J. P. M., & de Sousa, Á. P. B. (2016). Effects of passage number on growth and productivity of hybridoma secreting MRSA anti-PBP2a monoclonal antibodies. Cytotechnology, 68(3), 419-427.

de St. Groth, S. F., & Scheidegger, D. (1980). Production of monoclonal antibodies: Strategy and tactics. Journal of Immunological Methods, 35(1), 1-21.

Dutton, R. L., Scharer, J. M., & Moo-Young, M. (1999). Hybridoma growth and productivity: effects of conditioned medium and of inoculum size. Cytotechnology, 29(1), 1-10.

Fischer, W. B. (Ed.). (2005). ProteinReviews (Vol. Volume 1). NewYork: Kluwer Academic/ Plenum Publishers.

Fong, R. H., Banik, S. S., Mattia, K., Barnes, T., Tucker, D., Liss, N., et al. (2014). Exposure of epitope residues on the outer face of the chikungunya virus envelope trimer determines antibody neutralizing efficacy. J Virol, 88(24), 14364-14379.

Fox, J. M., Long, F., Edeling, M. A., Lin, H., van Duijl-Richter, M. K. S., Fong, R. H., et al. (2015). Broadly Neutralizing Alphavirus Antibodies Bind an Epitope on E2 and Inhibit Entry and Egress. Cell, 163(5), 1095-1107.

Hanly, W. C., Artwohl, J. E., & Bennett, B. T. (1995). Review of Polyclonal Antibody Production Procedures in Mammals and Poultry. ILAR Journal, 37(3), 93-118.

Hunt, A. R., Frederickson, S., Maruyama, T., Roehrig, J. T., & Blair, C. D. (2010). The first human epitope map of the alphaviral E1 and E2 proteins reveals a new E2 epitope with significant virus neutralizing activity. PLoS Negl Trop Dis, 4(7), 0000739.

Inoue, S., Alonzo, M. T. G., Kurosawa, Y., Mapua, C. A., Reyes, J. D., Dimaano, E. M., et al. (2010). Evaluation of a dengue IgG indirect enzyme-linked immunosorbent assay and a Japanese encephalitis IgG indirect enzyme-linked immunosorbent assay for diagnosis of secondary dengue virus infection. [Report]. Vector-Borne and Zoonotic Diseases, 10(2), 143+.

Johnson, B. K., Gichogo, A., Gitau, G., Patel, N., Ademba, G., Kirui, R., et al. (1981). Recovery of o'nyong-nyong virus from Anopheles funestus in Western Kenya. Transactions of The Royal Society of Tropical Medicine and Hygiene, 75(2), 239-241.

Karabatsos, N. (1975). Antigenic relationships of group A arboviruses by plaque reduction neutralization testing. Am J Trop Med Hyg, 24(3), 527-532.

Kiwanuka, N., Sanders, E. J., Rwaguma, E. B., Kawamata, J., Ssengooba, F. P., Najjemba, R., et al. (1999). O'nyong-nyong fever in south-central Uganda, 1996-1997: clinical features and validation of a clinical case definition for surveillance purposes. Clin Infect Dis, 29(5), 1243-1250.

Konishi, E., & Hotta, S. (1980). Studies on structural proteins of Chikungunya Virus. I. Separation of three species of proteins and their preliminary characterization. Microbiol Immunol, 24(5), 419-428.

LaBeaud, A. D., Banda, T., Brichard, J., Muchiri, E. M., Mungai, P. L., Mutuku, F. M., et al. (2015). High rates of o'nyong nyong and Chikungunya virus transmission in coastal Kenya. PLoS Negl Trop Dis, 9(2).

Lanciotti, R. S., Ludwig, M. L., Rwaguma, E. B., Lutwama, J. J., Kram, T. M., Karabatsos, N., et al. (1998). Emergence of epidemic O'nyong-nyong fever in Uganda after a 35-year absence: genetic characterization of the virus. Virology, 252(1), 258-268.

Leenaars, M., & Hendriksen, C. F. M. (2005). Critical Steps in the Production of Polyclonal and Monoclonal Antibodies: Evaluation and Recommendations. ILAR Journal, 46(3), 269-279.

Levinson, R. S., Strauss, J. H., & Strauss, E. G. (1990). Complete sequence of the genomic RNA of O'nyong-nyong virus and its use in the construction of alphavirus phylogenetic trees. Virology, 175(1), 110-123.

Li, Z., Yi, Y., Luo, X., Xiong, N., Liu, Y., Li, S., et al. (2020). Development and Clinical Application of A Rapid IgM-IgG Combined Antibody Test for SARS-CoV-2 Infection Diagnosis. J Med Virol, 27(10), 25727.

Mbiguino, A., & Menezes, J. (1991). Purification of human respiratory syncytial virus: superiority of sucrose gradient over percoll, renografin, and metrizamide gradients. J Virol Methods, 31(2-3), 161-170.

Mease, L., Coldren, R., Musila, L., Prosser, T., Ogolla, F., Ofula, V., et al. (2011). Seroprevalence and distribution of arboviral infections among rural Kenyan adults: A cross-sectional study. Virology Journal, 8(1), 371.

Morita, K., & Igarashi, A. (1989). Suspension culture ofAedes albopictus cells for flavivirus mass production. [journal article]. Journal of tissue culture methods, 12(1), 35-37.

Ngwe Tun, M. M., Thant, K. Z., Inoue, S., Kurosawa, Y., Lwin, Y. Y., Lin, S., et al. (2013). Serological characterization of dengue virus infections observed among dengue hemorrhagic fever/dengue shock syndrome cases in upper Myanmar. J Med Virol, 85(7), 1258-1266.

Okabayashi, T., Sasaki, T., Masrinoul, P., Chantawat, N., Yoksan, S., Nitatpattana, N., et al. (2015). Detection of Chikungunya Virus Antigen by a Novel Rapid Immunochromatographic Test. Journal of Clinical Microbiology, 53(2), 382-388.

Pezzi, L., LaBeaud, A. D., Reusken, C. B., Drexler, J. F., Vasilakis, N., Diallo, M., et al. (2019). GloPID-R report on chikungunya, o'nyong-nyong and Mayaro virus, part 2: Epidemiological distribution of o'nyong-nyong virus. Antiviral Res, 172(104611), 20.

Pezzi, L., Reusken, C. B., Weaver, S. C., Drexler, J. F., Busch, M., LaBeaud, A. D., et al. (2019). GloPID-R report on Chikungunya, O'nyong-nyong and Mayaro virus, part I: Biological diagnostics. Antiviral Res, 166, 66-81.

Posey, D. L., O'Rourke, T., Roehrig, J. T., Lanciotti, R. S., Weinberg, M., & Maloney, S. (2005). O'Nyong-nyong fever in West Africa. Am J Trop Med Hyg, 73(1), 32.

Powers, A. M., Brault, A. C., Shirako, Y., Strauss, E. G., Kang, W., Strauss, J. H., et al. (2001). Evolutionary relationships and systematics of the alphaviruses. Journal of virology, 75(21), 10118-10131.

Powers, A. M., Brault, A. C., Tesh, R. B., & Weaver, S. C. (2000). Re-emergence of chikungunya and o’nyong-nyong viruses: evidence for distinct geographical lineages and distant evolutionary relationships. Journal of General Virology, 81(2), 471-479.

Schmid, G., Blanch, H. W., & Wilke, C. R. (1991). Hybridoma gowth, metabolism, and product formation in hepes-buffered medium: effect of passage number and pH. In R. E. Spier, J. B. Griffiths & B. Meignier (Eds.), Production of Biologicals from Animal Cells in Culture (pp. 73-75): Butterworth-Heinemann.

Shore, H. (1961). O'nyong-nyong fever: An epidemic virus disease in East Africa: III. Some clinical and epidemiological observations in the Northern Province of Uganda. Transactions of The Royal Society of Tropical Medicine and Hygiene, 55(4), 361-373.

Shukla, J., Khan, M., Tiwari, M., Sannarangaiah, S., Sharma, S., Rao, P. V., et al. (2009). Development and evaluation of antigen capture ELISA for early clinical diagnosis of chikungunya. Diagn Microbiol Infect Dis, 65(2), 142-149.

Strauss, J. H., & Strauss, E. G. (1994). The alphaviruses: gene expression, replication, and evolution. Microbiol Rev, 58(3), 491-562.

Uchime, O., Fields, W., & Kielian, M. (2013). The role of E3 in pH protection during alphavirus assembly and exit. Journal of virology, 87(18), 10255-10262.

Wasonga, C., Inoue, S., Kimotho, J., Morita, K., Ongus, J., Sang, R., et al. (2015). Development and Evaluation of an in-house IgM-capture ELISA for the Detection of Chikungunya and Application to a Dengue Outbreak Situation in Kenya in 2013. Japanese Journal of Infectious Diseases, advpub.

Williams, M. C., & Woodall, J. P. (1961). O'nyong-nyong fever: an epidemic virus disease in East Africa. II. Isolation and some properties of the virus. Trans R Soc Trop Med Hyg, 55, 135-141.

Wu, S.-C., Liu, C.-C., & Lian, W.-C. (2004). Optimization of microcarrier cell culture process for the inactivated enterovirus type 71 vaccine development. Vaccine, 22(29-30), 3858-3864.

Yao, J. S., Strauss, E. G., & Strauss, J. H. (1996). Interactions between PE2, E1, and 6K required for assembly of alphaviruses studied with chimeric viruses. J Virol, 70(11), 7910-7920.

5 August, 2021
How to Cite
Makio, A., Musila, L., Odari, E., Ongus, J., & Sang, R. (2021). The Potential of O’nyong-nyong Virus Strain SG650 Murine Monoclonal Antibodies for Detection of O’nyong-nyong and Chikungunya Viruses. East African Journal of Health and Science, 3(1), 97-114.