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Isolation of single-chain variable fragment (scFv) antibodies for detection of Chickpea chlorotic dwarf virus (CpCDV) by phage display

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Abstract

Chickpea chlorotic dwarf virus (CpCDV, genus Mastrevirus), has a wide host range and geographic distribution in many parts of the world, and it is one of the most important legume-infecting viruses. Detection of CpCDV-infected plants in the field and evaluation of viral resistance of plant cultivars are possible by conducting serological assays. Here, development and characterization of a specific recombinant monoclonal antibody for CpCDV as a diagnostic tool are described. For this purpose, the coat protein of CpCDV was expressed in Escherichia coli strain Rosetta (DE3) and used to screen a Tomlinson phage display antibody library to select a specific single-chain variable fragment (scFv). In each round of biopanning, the affinity of the phage for CpCDV-CP was tested by enzyme-linked immunosorbent assay (ELISA). The results showed that the specificity of the eluted phages increased after each round of panning. Testing of individual clones by ELISA showed that five clones of the monoclonal phage were more strongly reactive against CpCDV than the other clones. All selected positive clones contained the same sequence. The phage-displayed scFv antibody, which was named CpCDV-scFvB9, did not bind to other tested plant pathogens and showed high sensitivity in the detection of CpCDV. A Western blot assay demonstrated that CpCDV-scFvB9 reacted with the recombinant coat protein of CpCDV. Finally, the interaction CpCDV-scFvB9 and CpCDV-CP was analyzed in a molecular docking experiment. This is the first report on production of an scFv antibody against CpCDV, which could be useful for immunological detection of the virus.

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References

  1. Zerbini FM, Briddon RW, Idris A (2017) ICTV virus taxonomy profile: Geminiviridae. J Gen Virol 98:131–133. https://doi.org/10.1099/jgv.0.000738

    Article  CAS  PubMed  Google Scholar 

  2. Zaagueri T, Miozzi L, Mnari-Hattab M, Noris E, Accotto GP, Vaira AM (2017) Deep sequencing data and infectivity assays indicate that chickpea chlorotic dwarf virus is the etiological agent of the "hard fruit syndrome" of watermelon. Viruses 9(11):311. https://doi.org/10.3390/v9110311

    Article  CAS  Google Scholar 

  3. Horn NM, Reddy SV, Reddy DVR (1995) Assessment of yield losses caused by chickpea chlorotic dwarf geminivires in chickpea (Cicer arientinum) in India. Eur J Plant Pathol 101:221–224. https://doi.org/10.1007/BF01874768

    Article  Google Scholar 

  4. Makkouk KM, Kumari SG, Shahraeen N (2002) Identification and seasonal variation of viral diseases of chickpea and lentil in Iran. J Plant Dis Prot 110(2):157–169

    Google Scholar 

  5. Kumari SG, Makkouk KM, Attar N, Ghulam W (2004) First report of Chickpea chlorotic dwarf virus infecting spring chickpea in Syria. Plant Dis 88:424. https://doi.org/10.1094/PDIS.2004.88.4.424C

    Article  CAS  PubMed  Google Scholar 

  6. Nahid N, Amin I, Mansoor S, Rybicki EP, Van der Walt E, Briddon RW (2008) Two dicot-infecting mastreviruses (family Geminiviridae ) occur in Pakistan. Arch Virol 153:1441–1451. https://doi.org/10.1007/s00705-008-0133-7

    Article  CAS  PubMed  Google Scholar 

  7. Mumtaz H, Kumari SG, Mansoor S, Martin DP, Briddon RW (2011) Analysis of the sequence of a dicot-infecting mastrevirus (Family Geminiviridae) originating from Syria. Virus Genes 42:422–428. https://doi.org/10.1007/s11262-011-0586-8

    Article  CAS  PubMed  Google Scholar 

  8. Kraberger S, Harkins GW, Kumari SG (2013) Evidence that dicot-infecting mastreviruses are particularly prone to inter-species recombination and have likely been circulating in Australia for longer than in Africa and the Middle East. Virology 444:282–291. https://doi.org/10.1016/j.virol.2013.06.024

    Article  CAS  PubMed  Google Scholar 

  9. Ouattara A, Tiendrebeogo F, Lefeuvre P (2017) New strains of Chickpea chlorotic dwarf virus discovered on diseased papaya and tomato plants in Burkina Faso. Arch Virol 162:1791–1794. https://doi.org/10.1007/s00705-017-3262-z

    Article  CAS  PubMed  Google Scholar 

  10. Fiallo-olive E, Mohammed IU, Turaki AA, Muhammad A, Navas-Castillo JA (2017) Novel Strain of the mastrevirus Chickpea chlorotic dwarf virus infecting papaya in Nigeria. Plant Dis 101:1684. https://doi.org/10.1094/PDIS-03-17-0430-PDN

    Article  Google Scholar 

  11. Horn NM, Reddy SV, RobertS IM, Reddy DVR (1993) Chickpea chlorotic dwarf virus, a new leafhopper-transmitted geminivirus of chickpea in India. Ann Appl Biol 122:467–479. https://doi.org/10.1111/j.1744-7348.1993.tb04050.x

    Article  Google Scholar 

  12. Akhtar KP, Ahmad M, Shah TM, Atta BM (2011) Transmission of chickpea chlorotic dwarf virus in chickpea by the leafhopper Orosius albicinctus (Distant) in Pakistan. Plant Prot Sci 47:1–4. https://doi.org/10.17221/45/2009-PPS

    Article  Google Scholar 

  13. Manzoor M, Ii Yas M, Shafiq M, Haider M, Shahid A, Briddon R (2014) A distinct strain of chickpea chlorotic dwarf virus (genus Mastrevirus, family Geminiviridae) identified in cotton plants affected by leaf curl disease. Arch Virol 159(5):1217–1221. https://doi.org/10.1007/s00705-013-1911-4

    Article  CAS  PubMed  Google Scholar 

  14. Kanakala S, Sakhare A, Verma HN, Malathi VG (2012) Infectivity and the phylogenetic relationship of a mastrevirus causing chickpea stunt disease in India. Eur J Plant Pathol 135:429–438. https://doi.org/10.1007/s10658-012-0100-8

    Article  Google Scholar 

  15. Abd El-Aziz MH (2019) Three modern serological methods to detect plant viruses. J Plant Sci Phytopathol. https://doi.org/10.29328/journal.jpsp.1001039

    Article  Google Scholar 

  16. Fang Y, Ramasamy RP (2015) Current and prospective methods for plant disease detection. Biosensors 5(3):537–561. https://doi.org/10.3390/bios5030537

    Article  CAS  PubMed  Google Scholar 

  17. Makkouk KM, Kumari SG (2002) Low cost paper can be used in tissue blot immunoassay for detection of cereal and legume viruses. Phytopathol Mediterr 41:275–278. https://doi.org/10.14601/Phytopathol_Mediterr-1683

    Article  Google Scholar 

  18. Seepiban C, Charoenvilaisiri S, Warin N (2017) Development and application of triple antibody sandwich enzyme-linked immunosorbent assays for begomovirus detection using monoclonal antibodies against Tomato yellow leaf curl Thailand virus. Virol J 14(99):2–14. https://doi.org/10.1186/s12985-017-0763-z

    Article  CAS  Google Scholar 

  19. Lima JAA, Nascimento AKQ, Radaelli P, Purcifull DE (2012) Serology applied to plant virology. Serological diagnosis of certain human, animal and plant diseases. Rijeka Croacia. InTech. https://doi.org/10.5772/38038

    Article  Google Scholar 

  20. Khatabi B, He B, Hajimorad MR (2012) Diagnostic potential of polyclonal antibodies against bacterially expressed recombinant coat protein of Alfalfa mosaic virus. Plant Dis 96:1352–1357. https://doi.org/10.1094/PDIS-08-11-0683-RE

    Article  CAS  PubMed  Google Scholar 

  21. Lipman NS, Jackson LR, Trudel LJ, Weis-Garcia F (2005) Monoclonal versus polyclonal antibodies: distinguishing characteristics, applications, and information resources. ILAR J 46(3):258–268. https://doi.org/10.1093/ilar.46.3.258

    Article  CAS  PubMed  Google Scholar 

  22. Ahmad ZA, Yeap SK, Ali AM, Ho WY, Alitheen NBM, Hamid M (2012) scFv antibody: principles and clinical application. Clin Dev Immunol 2012:980250. https://doi.org/10.1155/2012/980250

    Article  CAS  PubMed  Google Scholar 

  23. Hoogenboom HR (2005) Selecting and screening recombinant antibody libraries. Nat Biotechnol 23(9):1105–1116. https://doi.org/10.1038/nbt1126

    Article  CAS  PubMed  Google Scholar 

  24. Liu HL, Lin WF, Hu WC, Lee YA, Chang YC (2015) A strategy for generating a broad-spectrum monoclonal antibody and soluble single-chain variable fragments against plant potyviruses. Appl Environ Microbiol 81:6839–6849. https://doi.org/10.1128/AEM.01198-15

    Article  CAS  PubMed  Google Scholar 

  25. Villani ME, Roggero P, Bitti O, Benvenuto E, Franconi R (2005) Immunomodulation of cucumber mosaic virus infection by intrabodies selected in vitro from a stable single frame work phage display library. Plant Mol Biol 58:305–316. https://doi.org/10.1007/s11103-005-4091-0

    Article  CAS  PubMed  Google Scholar 

  26. De Haard HJ, van Neer N, Reurs A (1999) A large non-immunized human Fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies. J Biol Chem 274:18218–18230. https://doi.org/10.1074/jbc.274.26.18218

    Article  PubMed  Google Scholar 

  27. Holliger P, Hudson PJ (2005) Engineered antibody fragments and the rise of single domains. Nat Biotechnol 23(9):1126–1136. https://doi.org/10.1038/nbt1142

    Article  CAS  PubMed  Google Scholar 

  28. Kunik V, Peters B, Ofran Y (2012) Structural consensus among antibodies defines the antigen binding site. PLOS Comput Biol 8(2):e1002388. https://doi.org/10.1371/journal.pcbi.1002388

    Article  CAS  PubMed  Google Scholar 

  29. Griep RA, Van Twisk C, Van Beckhoven JRCM, Van Der Wolf JM, Schots A (1998) Development of specific recombinant monoclonal antibodies against the lipopolysaccharide of Ralstonia solanacearum Race 3. Phytopathology 98:795–803. https://doi.org/10.1094/PHYTO.1998.88.8.795

    Article  Google Scholar 

  30. Toth RL, Harper K, Mayo MA, Torrance L (1999) Fusion proteins of single-chain variable fragments derived from phage display libraries are effective reagents for routine diagnosis of potato Leaf roll Virus infection in potato. Phytopathology 89:1015–1021. https://doi.org/10.1094/PHYTO.1999.89.11.1015

    Article  CAS  PubMed  Google Scholar 

  31. Yang ZY, Liu H, Zheng Z, Wang R, Wang S, Zhuang Z (2013) Preparation of scFv against HrpA of Pseudomonas syringae pv. tomato DC3000. Afr J Microbiol Res 7(44):5090–5096. https://doi.org/10.5897/AJMR12.1661

    Article  CAS  Google Scholar 

  32. Yuan Q, Jordan R, Brlansky RH, Istomina O, Hartung J (2015) Development of single chain variable fragment (scFv) antibodies against Xylella fastidiosa subsp. pauca by phage display. J Microbiol Methods 117:148–154. https://doi.org/10.1016/j.mimet.2015.07.020

    Article  CAS  PubMed  Google Scholar 

  33. Raeisi H, Safarnejad MR, Alavi SM, Elahinia SA, Farrokhi N (2019) Applying of pthA effector protein of Xanthomonas citri subsp. citri for production of specific antibodies and its application for detection of infected plants. J Plant Pathol. https://doi.org/10.1007/s42161-019-00385-5

    Article  Google Scholar 

  34. Safarnejad MR, Bananej K, Sokhansanj Y (2019) Developing of specific antibody against chickpea chlorotic dwarf virus (CpCDV) through recombinant coat protein. J Crop Protect 8(2):179–190

    Google Scholar 

  35. Raeisi H, Safarnejad MR, Alavi SM, Farrokhi N, Elahinia SA, Safarpour H, Sharifian F (2019) Development and molecular analyses of Xanthomonas pthA specific scFv recombinant monoclonal antibodies. J Crop Protect 8(4):417–429

    Google Scholar 

  36. Lopez MM, Bertolini E, Olmos A, Caruso P, Gorris MT (2003) Innovative tools for detection of plant pathogenic viruses and bacteria. Int Microbiol 6:233–243. https://doi.org/10.1007/s10123-003-0143-y

    Article  CAS  PubMed  Google Scholar 

  37. Emanuel P (2000) Recombinant antibodies: a new reagent for biological agent detection. Biosens Bioelectron 14(10–11):751–759. https://doi.org/10.1016/s0956-5663(99)00058-5

    Article  CAS  PubMed  Google Scholar 

  38. McCafferty J, Griffiths AD, Winter G, Chiswell DJ (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348(6301):552–554. https://doi.org/10.1038/348552a0

    Article  CAS  PubMed  Google Scholar 

  39. Schillberg S, Zimmermann S, Zhang MY, Fischer R (2001) Antibody-based resistance to plant pathogens. Transgenic Res 10(1):1–12. https://doi.org/10.1023/A:1008945126359

    Article  CAS  PubMed  Google Scholar 

  40. Krishnaswamy S, Kabir ME, Rahman MM, Miyamoto M, Furuichi Y, Komiyama T (2011) Isolation and characterization of recombinant single chain fragment variable anti-idiotypic antibody specific to Aspergillus fumigates membrane protein. J Immunol Methods 366(1):60–68. https://doi.org/10.1016/j.jim.2011.01.006

    Article  CAS  PubMed  Google Scholar 

  41. Yuan Q, Jordan R, Brlansky RH, Minenkova O, Hartung J (2016) Development of single chain variable fragment (scFv) antibodies against surface proteins of ‘Ca Liberibacter asiaticus’. J Microbiol Methods 122:1–7. https://doi.org/10.1016/j.mimet.2015.12.015

    Article  CAS  PubMed  Google Scholar 

  42. Raeisi H, Safarnejad MR, Alavi SM, Elahinia SA, Farrokhi N (2018) Production of polyclonal phages harbouring antibody fragment genes against Xanthomonas citri subsp. citri using phage display technology. JAEP 85(2):265–276. https://doi.org/10.22092/jaep.2017.115980.1194

    Article  Google Scholar 

  43. Cerovska N, Moravec T, Rosecka P, Dedic P, Filigaro-va M (2003) Production of polyclonal antibodies to a recombinant coat protein of Potato mop-top virus. J Phytopathol 151:195–200. https://doi.org/10.1046/j.1439-0434.2003.00705.x

    Article  CAS  Google Scholar 

  44. Halk EL, De Boer SH (1985) Monoclonal antibodies in plant-disease research. Annu Rev Phytopathol 23(1):321–350. https://doi.org/10.1146/annurev.py.23.090185.001541

    Article  Google Scholar 

  45. Zhang Y (2008) I-TASSER server for protein 3D structure prediction. BMC Bioinf 9:40. https://doi.org/10.1186/1471-2105-9-40

    Article  CAS  Google Scholar 

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Acknowledgements

The authors would like to thank the Iranian Research Institute of Plant Protection for providing research facilities.

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Authors and Affiliations

  1. Department of Plant Protection, College of Agricultural Sciences, Guilan University, Rasht, Iran

    Hamideh Raeisi

  2. Department of Plant Viruses, Iranian Research Institute of Plant Protection, Agricultural Research Education and Extension Organization of Iran, Tehran, Iran

    Mohammad Reza Safarnejad

  3. Plant Virology Research Center, College of Agriculture, Shiraz University, Shiraz, Iran

    Pedram Moeini

  4. Cellular and Molecular Research Center, Birjand University of Medical Sciences, Birjand, Iran

    Hossein Safarpour

  5. Department of Plant Pathology, Faculty of Agriculture and Natural Resources, Tehran Science and Research Branch, Islamic Azad University, Tehran, Iran

    Yalda Sokhansanj

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  1. Hamideh Raeisi
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Correspondence to Hamideh Raeisi.

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Raeisi, H., Safarnejad, M.R., Moeini, P. et al. Isolation of single-chain variable fragment (scFv) antibodies for detection of Chickpea chlorotic dwarf virus (CpCDV) by phage display. Arch Virol 165, 2789–2798 (2020). https://doi.org/10.1007/s00705-020-04813-1

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