DOI QR코드

DOI QR Code

Development of monoclonal antibodies against viral hemorrhagic septicemia virus (VHSV, genotype IVa), the causative agent of VHS

VHS (viral hemorrhagic septicemia)의 원인병원체인 VHSV (genotype IVa)에 대한 단클론 항체 개발

  • Kong, Kyoung-Hui (Department of Aqualife Medicine, College of Fisheries and Ocean Science, Chonnam National University) ;
  • Oh, Myung-Joo (Department of Aqualife Medicine, College of Fisheries and Ocean Science, Chonnam National University) ;
  • Jang, Min-Seok (South Sea Fisheries Research Institute, National Institute of Fisheries Science (NIFS)) ;
  • Kim, Choon-Sup (Enbiogene) ;
  • Kim, Wi-Sik (Department of Aqualife Medicine, College of Fisheries and Ocean Science, Chonnam National University)
  • 공경희 (전남대학교 수산생명의학과) ;
  • 오명주 (전남대학교 수산생명의학과) ;
  • 장민석 (국립수산과학원 남해수산연구소) ;
  • 김춘섭 ((주)엔바이로젠) ;
  • 김위식 (전남대학교 수산생명의학과)
  • Received : 2019.12.04
  • Accepted : 2019.12.11
  • Published : 2019.12.31

Abstract

We developed and subsequently characterized mouse antibodies (MAbs) against viral hemorrhagic septicemia virus (VHSV, genotype IVa), the causative agent of VHS. Five hybridoma clones secreting MAbs against VHSV were established. The MAbs recognized the glycoprotein (MAbs 2C10, 18H4, 23H6, and 30B7) and nucleocapsid protein (15E10) of VHSV by western blot analysis. All five MAbs reacted with VHSV-infected cells and tissue homogenates of VHSV-infected olive flounder (Paralichthys olivaceus) by western blot analysis. Whereas, no reactivity was observed in normal cells and tissue homogenates of normal olive flounder. Moreover, these MAbs reacted with VHSV, but did not react with other fish viruses (infectious hematopoietic necrosis virus, hirame rhabdovirus, spring viraemia of carp virus, infectious pancreatic necrosis virus, marine birnavirus, and nervous necrosis virus) by enzyme linked immunosorbent assay (ELISA). These results indicate that the MAbs are specific to VHSV and can be of value in VHSV detection.

본 연구에서는 넙치(Paralichthys olivaceus)로부터 분리한 바이러스성출혈성패혈증바이러스(viral hemorrhagic septicemia virus, VHSV, genotype IVa)에 대한 단클론 항체(monoclonal antibody, MAb)를 개발하였다. VHSV에 대한 항체를 생산하는 총 5개의 hybridoma clone을 생산하였다. 4개의 MAbs (2C10, 18H4, 23H6, 30B7)는 glycoprotein을 인식하였고, MAb 15E10은 nucleocapsid protein을 인식하였다. 5개의 MAbs는 western blot 상에서 VHSV에 감염된 세포와 넙치시료에 반응하였으나, 정상 세포와 넙치시료에는 반응하지 않았다. 또한 ELISA상에서 VHSV에만 반응하였고 6종의 어류바이러스(IHNV, HIRRV, SVCV, IPNV, MABV, NNV)에는 반응하지 않았다. 이상의 결과, 본 연구에서 제작된 MAbs는 VHSV에만 특이적으로 반응하는 것이 확인되어 VHSV를 검사하는데 사용될 수 있을 것으로 사료된다.

Keywords

References

  1. Banerjee, R. and Jaiswal, A.: Recent advances in nanoparticle-based lateral flow immunoassay as a pointof-care diagnostic tool for infectious agents and diseases. Analyst., 143(9): 1970-1996, 2018. https://doi.org/10.1039/c8an00307f
  2. Baron, J., Fishbourne, E., Couacy-Hyman, E., Abubakar, M., Jones, B.A., Frost, L., Herbert, R., Chibssa, T.R., Kliister, G. van't, Afzal, M., Ayebazibwe, C., Toye, P., Bashiruddin, J. and Baron, D.: Development and testing of a field diagnostic assay for peste des petits ruminants virus. Transbound. Emerg. Dis., 61: 390-396, 2014. https://doi.org/10.1111/tbed.12266
  3. Egusa, S., Wakabayashi, H. and Muroga, K.: Infectious and parasitic diseases of fish and shellfish. Life Science Publishing Co. Seoul, pp. 54-58, 2006.
  4. Huang, X., Aguilar, Z.P., Xu, H., Lai, W. and Xiong, Y.: Membrane-based lateral flow immunochromatographic strip with nanoparticles as reporters for detection: a review. Biosens. Bioelectron., 75: 166-180, 2016. https://doi.org/10.1016/j.bios.2015.08.032
  5. Isshiki, T., Nishizawa, T., Kobayashi, T., Nagano, T. and Miyazaki, T.: An outbreak of VHSV (viral hemorrhagic septicemia virus) infection in farmed Japanese flounder Paralichthys olivaceus in Japan. Dis. Aquat. Org., 47: 87-99, 2001. https://doi.org/10.3354/dao047087
  6. Ito, T., Olesen, N.J., Skall, H.F., Sano, M., Kurita, J., Nakajima, K. and Iida, T.: Development of a monoclonal antibody against viral haemorrhagic septicaemia virus (VHSV) genotype IVa. Dis. Aquat. Organ., 89: 17-27, 2010. https://doi.org/10.3354/dao02183
  7. Ito, T., Kurita, J., Sano, M., Skall, H.F., Lorenzen, N., Einer-Jensen, K. and Olesen N.J.: Typing of viral hemorrhagic septicemia virus by monoclonal antibodies. J. Gen. Virol., 93: 2546-2557, 2012. https://doi.org/10.1099/vir.0.043091-0
  8. Jang, M.S., Kim, J.O., Oh, M.J. and Kim, W.S.: Study on tendency of viral hemorrhagic septicemia virus (VHSV) detection in Korea from 2001 to 2016 based on reported cases. J. Fish Pathol., 31(1): 49-55, 2018. https://doi.org/10.7847/JFP.2018.31.1.049
  9. Jang, M.S.: The status of viral hemorrhagic septicemia (VHS) occurrence in olive flounder (Paralichthys olivaceus) farms of Korea. Ph.D. Thesis, Chonnam national university, Korea, 2019.
  10. Jeong, H.N., Jang, M.S., Oh, M.J. and Kim, W.S.: Production of monoclonal antibodies against viral hemorrhagic septicemia virus (VHSV, genotype IVa) from olive flounder. J. Fish Pathol., 30(2): 149-154, 2017. https://doi.org/10.7847/JFP.2017.30.2.149
  11. Kim, J.W., Cho, M.Y., Lee, H.N., Han, H.J., Oh, Y.K., Lee, S.J., Jee, B.Y., Myeong, J.I., and Won, K.M.: Diagnosis case of viral hemorrhagic septicemia (VHS) in adult olive flounder Paralichthys olivaceus. Kor J. Fish Aquat. Sci., 45(6): 666-674, 2012. https://doi.org/10.5657/KFAS.2012.0666
  12. Kim, S.M., Lee, J.I., Hong., M.J., Park, H.S. and Park, S.I.: Genetic relationship of the VHSV (viral hemorrhagic septicemia virus) isolated from cultured olive flounder, Paralichthys olivaceus in Korea. J. Fish Pathol., 16(1): 1-12, 2003.
  13. Kim, W.S., Kim, S.R., Kim, D.W., Kim, J.O., Park, M.A., Kitamura, S.I., Kim, H.Y., Kim, D.H., Han, H.J., Jung, S.J. and Oh, M.J.: An outbreak of VHSV (viral hemorrhagic septicemia virus) infection in farmed olive flounder Paralichthys olivaceus in Korea. Aquaculture, 296: 165-168, 2009. https://doi.org/10.1016/j.aquaculture.2009.07.019
  14. Kim, W.S., Jung, S.J., Kim, J.O., Kim, D.W., Kim, J.H. and Oh, M.J.: Genetic positioning of Korean viral hemorrhagic septicemia virus (VHSV) from cultured and wild marine fishes. J. Fish Pathol., 24(1): 1-9, 2011. https://doi.org/10.7847/jfp.2011.24.1.001
  15. Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685. 1970. https://doi.org/10.1038/227680a0
  16. Liddell, J.E. and Cryer, A.: A practical guide to monoclonal antibodies, pp. 90-104, John Wiley and Sons, Inc., New York, 1991.
  17. Lorenzen, N., Olesen, N.J. and Jorgensen, P.E.V.: Production and characterization of monoclonal antibodies to four Egtved virus structural proteins. Dis. Aquat. Organ., 4: 35-42, 1988. https://doi.org/10.3354/dao004035
  18. Nishizawa, T., Iida, H., Takano, T., Nakajima, K. and Muroga, K.: Genetic relatedness among Japanese, American and European isolates of viral hemorrhagic septicemia virus (VHSV) based on partial G and P genes. Dis. Aquat. Org., 48: 143-148, 2002. https://doi.org/10.3354/dao048143
  19. OIE. Manual of Diagnostic tests for Aquatic Animals. Viral hemorrhagic septicemia virus (VHSV); Office International des Epizooties: Paris, France, 2019.
  20. Skall, H., Olesen, N.J. and Mellergaard, S.: Viral haemorrhagic septicemia virus in marine fish and its implications for fish farming-a review. J. Fish Dis., 28: 509-529, 2005. https://doi.org/10.1111/j.1365-2761.2005.00654.x
  21. Snow, M., Cunningham, C.O., Melvin, W.T. and Kurath, G.: Analysis of the nucleoprotein gene identifies distinct lineages of viral haemorrhagic septicaemia virus within the European marine environment. Virus Res., 63: 35-44, 1999. https://doi.org/10.1016/S0168-1702(99)00056-8
  22. Song, D., Ha, G., Serhan, W., Eltahir, Y., Yusof, M., Hashem, F., Elsayed, E., Marzoug, B., Abdelazim, A. and Al Muhairi, S.: Development and validation of a rapid immunochromatographic assat for detection of middle east respiratory syndrome coronavirus antigen in dremedary camels. J. Clin. Microbiol., 53: 1178-1182, 2015. https://doi.org/10.1128/JCM.03096-14
  23. Stone, D.M., Way, K. and Dixon, P.F.: Nucleotide sequence of the glycoprotein gene of viral haemorrhagic septicaemia (VHS) viruses from different geographic areas: a link between VHS in farmed fish species and viruses isolated from North Sea cod (Gadus morha L.). J. Gen. Viol., 78: 1319-1326, 1997. https://doi.org/10.1099/0022-1317-78-6-1319
  24. Tordo, N., Benmansour, A., Calisher, C., Dietgen, R., Fang, R.-X., Jackson, A.O., Kurath, G., Nadin-Davis, S., Tesh, R.B. and Walker, P.J.: Family Rhabdoviridae. In Fauquet, C.M., Mayo, M.A., Maniloff, J., Desselberger, U and Ball, L. A., editors. Virus taxonomy: Eighth report of the international committee on taxonomy of viruses. Elsevier/Academic Press, London, United Kingdom. pp. 623-644, 2005.
  25. Towbin, H., Staehelin, T. and Gordon, J.: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets; procedure and some applications. Proc. Natl. Acad. Sci., 76: 4350-4354, 1979. https://doi.org/10.1073/pnas.76.9.4350
  26. Wolf, K.: Viral hemorrhagic septicemia. In Wolf, K., editor. Fish viruses and fish viral diseases. Cornell University Press, Ithaca, New York, USA. pp. 217-249, 1988.
  27. Wong, R. and Tse, H.: Lateral flow immunoassay. Humana Press, New York. Springer, 2008.