Virologica Sinica

, Volume 31, Issue 4, pp 324–330 | Cite as

Monoclonal antibody-based serological methods for detecting Citrus tristeza virus in citrus groves

  • Zhen Liu
  • Zhe Chen
  • Jian Hong
  • Xuefeng Wang
  • Changyong Zhou
  • Xueping Zhou
  • Jianxiang Wu
Research Article
  • 93 Downloads

Abstract

Citrus tristeza virus (CTV) is one of the most economically important citrus viruses and harms the citrus industry worldwide. To develop reliable and effective serological detection assays of CTV, the major capsid protein (CP) gene of CTV was expressed in Escherichia coli BL21 (DE3) using the expression vector pET-28a and purified through Ni+-NTA affinity chromatography. The recombinant protein was used to immunize BALB/c mice. Four hybridoma cell lines (14B10, 14H11, 20D5, and 20G12) secreting monoclonal antibodies (MAbs) against CTV were obtained through conventional hybridoma technology. The titers of MAb-containing ascitic fluids secreted by the four hybridoma lines ranged from 10-6 to 10-7 in indirect enzyme-linked immunosorbent assay (ELISA). Western blots showed that all four MAbs could specifically react with CTV CP. Using the prepared MAbs, dot-ELISA, Tissue print-ELISA, and triple antibody sandwich (TAS)-ELISA were developed to detect CTV in tree nurseries and epidemiological studies. The developed dot-ELISA and TAS-ELISA methods could detect CTV in crude extracts of infected citrus leaves with dilutions of 1:2560 and 1:10, 240 (w/v, g/mL), respectively. Tissue print-ELISA was particularly useful for large-scale field sample detection, mainly owing to its simplicity and lack of sample preparation requirements. The field survey revealed that CTV is prevalent on citrus trees in the Chongqing Municipality, Jiangxi Province, and Zhejiang Province of China. The coincidence rate of serological and RT-PCR test results reached more than 99.5%. The prepared MAbs against CTV and established sensitive and specific serological assays have a significant role in the detection and prevention and control of CTV in our country.

Keywords

Citrus tristeza virus (CTV) monoclonal antibody dot-ELISA Tissue print-ELISA TAS-ELISA 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Ananthakrishna G, Venkataprasanna T, Roy A, Brlansky RH. 2010. Characterization of the mixture of genotypes of a Citrus tristeza virus isolate by reverse transcription-quantitative realtime PCR. J Virol Methods, 164: 75–82.CrossRefGoogle Scholar
  2. Bar-Joseph M, Marcus R, Lee RF. 1989. The continuous challenge of Citrus tristeza viruscontrol. Ann Rev Phytopathology, 27: 291–316.CrossRefGoogle Scholar
  3. Bertolini E, Moreno A, Capote N, Olmos A, Luis A, Eduardo VE, Jordi PP, Cambra M. 2008. Quantitative detection of Citrus tristeza virus in plant tissues and single aphids by real-time RTPCR. Eur J Plant Pathol, 120: 177–188.CrossRefGoogle Scholar
  4. Cambra M, Garnsey SM, Permar TA, Henderson CT, Gumpf DJ, Vela C. 1990. Detection of Citrus tristeza virus (CTV) with a mixture of monoclonal antibodies. Phytopathology, 80: 1034.Google Scholar
  5. Cambra M, Camarasa E, Gorris MT, Garnsey SM, Carbonell E. 1991. Comparison of different immunosorbent assays for citrus tristeza virus (CTV) using CTV-specific monoclonal and polyclonal antibodies. In: Proc 11th Conf IOCV. Riverside, CA. pp. 38–45.Google Scholar
  6. Cambra M, Gorris MT, Roman MP, Terrada E, Garnsey SM, Camarasa E, Olmos A, Colomer M. 2000. Routine detection of Citrus tristeza virus by direct Immunoprinting-ELISA method using specific monoclonal and recombinant antibodies. In: Proc 14th Conf IOCV. Riverside, CA. pp. 34–41.Google Scholar
  7. Cambra M, Gorris MT, Olmos A, Martinez MC, Romian MP, Bertolini E, Lopez A, Carbonell EA. 2002. European diagnostic protocol (DIAGPRO) for Citrus tristeza virus in adult trees. In: Duran-Vila, N., Milne, R.G., da Graca (Eds.), Proc 15th Conf IOCV. Riverside, CA, pp. 69–77.Google Scholar
  8. Davino S, Davino M, Sambade A, Guardo M, Caruso A. 2003. The first Citrus tristeza virus outbreak found in a relevant citrus producing area of Sicily, Italy. Plant Dis, 87: 285–289.CrossRefGoogle Scholar
  9. Febres VJ, Ashoulin L, Mawassi M, Frank A, Bar-Joseph M, Manjunath KL, Lee F, Niblett CL. 1997. The p27 protein is present at one end of Citrus tristeza virus particles. Phytopathology, 86: 1331–1335.Google Scholar
  10. Garnsey SM, Cambra M. 1993. Enzyme-linked immunosorbent assay (ELISA). G.P. Martelli (Ed.), In: Graft transmissible diseases of grapevines. pp. 169–192.Google Scholar
  11. Garnsey SM, Gottwald TR, Hilf ME, Matos L, Borbón J. 2000. Emergence and spread of severe strains of Citrus tristeza virus isolates in the Dominican Republic. In: Proc14th Conf IOCV. Riverside, CA. pp. 57–68.Google Scholar
  12. Garnsey SM, Permar TA, Cambra M, Henderson CT. 1993. Direct tissue blot immunoassay (DTBIA) for detection of citrus tristeza virus (CTV). In: Proc 12th Conf IOCV. CA, pp. 39–50.Google Scholar
  13. Gottwald TR, Polek M, Riley KM. 2002. History, present incidence, and spatial distribution of Citrus tristeza virus in the California Central Valley. In: Proc 15th Conf IOCV, Riverside, CA. pp. 83–94.Google Scholar
  14. Huang Z, Rundell AP, Guan X, Powell AC. 2004. Detection and isolate differentiation of Citrus tristeza virus in infected field trees based on reverse transcription-polymerase chain reaction. Plant Dis, 88: 625–629.CrossRefGoogle Scholar
  15. Hung TH, Wu ML, Su HJ. 2000. A rapid method based on the one step reverse transcriptase-polymerase chain reaction (RT-PCR) technique for detection of different strains of Citrus tristeza virus. J Phytopathol, 148: 469–475.CrossRefGoogle Scholar
  16. Karasev OV, Karasev AV, Gumpf DJ, Lee RF, Garnsey, SM. 1995. Production of polyclonal antisera to the coat protein of Citrus tristeza virus expressed in Escherichia coli: application for immunodiagnosis. Phytopathology, 85: 691–694.CrossRefGoogle Scholar
  17. Mehta P, Brlansky RH, Gowda S, Yokomi RK. 1997. Reverse transcription polymerase chain reaction detection of Citrus tristeza virus in aphids. Plant Dis, 81: 1066–1069.CrossRefGoogle Scholar
  18. Nikolaeva OV, Karasev AV, Powell CA, Gumpf DJ, Garnsey SM Lee RF. 1996. Mapping of epitopes for Citrus tristeza virusspecific monoclonal antibodies using bacterially expressed coat protein fragments. Phytopathology, 86: 974–979.CrossRefGoogle Scholar
  19. Olmos A, Cambra M, Esteban O, Gorris MT, Terrada E. 1999. New device and method for capture, reverse transcription and nested PCR in a single closed tube. Nucleic Acids Res, 27: 1564–1565.CrossRefPubMedPubMedCentralGoogle Scholar
  20. Ozturk S, Cirakoglu B. 2003. Production of a monoclonal antibody specific for Citrus tristeza virus. Food Agr Immunol, 15: 65–73.CrossRefGoogle Scholar
  21. Permar TA, Garnsey SM, Gumpf DJ, Lee R. 1990. A monoclonal antibody that discriminates strains of Citrus tristeza virus. Phytopathology, 80: 224–228.CrossRefGoogle Scholar
  22. Roistacher CN. 1991. Graft-transmissible diseases of citrus: Handbook for detection and diagnosis. Rome: IOCV and FAO, pp. 286.Google Scholar
  23. Sekiya ME, Lawrence SD, Mccaffery M, Cline K. 1991. Molecular cloning and nucleotide sequencing of the coat protein gene of Citrus tristeza virus. J Gen Virol, 72: 1013–1020.CrossRefPubMedGoogle Scholar
  24. Sagheer A, Zhou CY, Zhou Y, Cao MJ, Wang XF. 2012. Distribution and research advances of Citrus tristeza virus. J Integr Agr, 11: 346–358.CrossRefGoogle Scholar
  25. Shang HL, Xie Y, Zhou XP, Qian YJ, Wu JX. 2011. Monoclonal antibody-based serological methods for detection of Cucumber green mottle mosaic virus. Virol J, 8: 228–236.CrossRefPubMedPubMedCentralGoogle Scholar
  26. Saponari M, Manjunath K, Yokomi RK. 2008. Quantitative detection of Citrus tristeza virus in citrus and aphids by real-time reverse transcription-PCR (TaqMan). J Virol Methods, 147: 43–53.CrossRefPubMedGoogle Scholar
  27. Moreno P, Ambros S, Albiachmarti MR, Guerri J, Pena L. 2008. Citrus tristeza virus: a pathogen that changed the course of the citrus industry. Mol Plant Pathol, 9: 51–268.CrossRefGoogle Scholar
  28. Vela C, Cambra M, Cortes E, Moreno P, Miguet JG, Perez De San Roman C, Sanz A. 1986. Production and characterization of monoclonal antibodies specific for Citrus tristeza virus and their use in diagnosis. J Gen Virol, 67: 91–96.CrossRefGoogle Scholar
  29. Wu GW, Tang M, Wang GP, Wang CX, Liu Y, Yang F, Hong N. 2014. The epitope structure of Citrus tristeza virus coat protein mapped by recombinant proteins and monoclonal antibodies. Virology, 448: 238–246.CrossRefPubMedGoogle Scholar
  30. Wu JX, Meng CM, Shang HL, Rong S, Zhang C, Hong J, Zhou XP. 2011. Monoclonal antibody-based tripleantibody sandwichenzyme- linked immunosorbentassay and immunocapture reverse transcription polymerase chain reaction for odontoglossum ringspot virus detection. J Virol Methods, 171: 40–45.CrossRefPubMedGoogle Scholar
  31. Wu JX, Wang Q, Liu H, Qian YJ, Xie Y, Zhou XP. 2013. Monoclonal antibody-based serological methods for maize chlorotic mottle virus detection in China. J Zhejiang Univ Sci B, 14: 555–562.CrossRefPubMedPubMedCentralGoogle Scholar
  32. Wu JX, Yu C, Yang CY, Deng FL, Zhou XP. 2009. Monoclonal antibodies against the recombinant nucleocapsid protein of tomato spotted wilt virus and its application in the virus detection. J Phytopathol, 157: 344–349.CrossRefGoogle Scholar
  33. Wu JX, Yu L, Li L, Hu JQ, Zhou JY, Zhou XP. 2007. Oral immunization with transgenic rice seeds expressing VP2 protein of infectious bursal disease virus induces protective immune responses in chickens. Plant Biotechnol J, 5: 570–578.CrossRefPubMedGoogle Scholar
  34. Wu JX, Ni YQ, Liu H, Ding M, Zhou XP. 2014. Monoclonal antibody- based serological assays and immunocapture-RT-PCR for detecting Rice dwarf virus in field rice plants and leafhopper vectors. J Virol Methods, 195: 134–140.CrossRefPubMedGoogle Scholar
  35. Zhou CY. 1997. Occurrence guidelines and outlook of Citrus tristeza virus in China. In: Proceedings of 1st Chinese Symposium on Plant Virus and Viral Diseases Control Researches. Beijing: China Agr Sci Pre. pp. 182–187. (In Chinese)Google Scholar
  36. Zhou Y, Zhou CY, Song Z, Liu KH, Yang FY. 2007. Characterization of Citrus tristeza virus isolates by indicators and molecular biology methods. Agr Sci China, 6: 573–579.CrossRefGoogle Scholar

Copyright information

© Wuhan Institute of Virology, CAS and Springer Science+Business Media Singapore 2016

Authors and Affiliations

  • Zhen Liu
    • 1
  • Zhe Chen
    • 1
  • Jian Hong
    • 1
  • Xuefeng Wang
    • 2
  • Changyong Zhou
    • 2
  • Xueping Zhou
    • 1
  • Jianxiang Wu
    • 1
  1. 1.State Key Laboratory of Rice Biology, Institute of BiotechnologyZhejiang UniversityHangzhouChina
  2. 2.China Citrus Research Institute of Chinese Academy of Agricultural SciencesSouthwest UniversityChongqingChina

Personalised recommendations