Applied Microbiology and Biotechnology

, Volume 101, Issue 23–24, pp 8331–8344 | Cite as

Bovine single chain Fv antibody inhibits bovine herpesvirus-1 infectivity by targeting viral glycoprotein D

  • Jian Xu
  • Jing Wu
  • Bo Jiang
  • Houjun He
  • Xixi Zhang
  • Xiaoyang Li
  • Dawei Yang
  • Xiufen Huang
  • Joshua E. Sealy
  • Munir Iqbal
  • Yongqing LiEmail author
Biotechnological products and process engineering


Glycoprotein D (gD) of bovine herpesvirus-1 (BoHV-1) is essential for attachment and penetration of cells during infection and is a major target for neutralizing antibodies during an adaptive immune response. Currently there are no recombinant antibodies capable of binding gD epitopes for use in treating BoHV-1 infection. In this study, a bovine scFv gene derived from a hybridoma secreting monoclonal antibodies (McAbs) against the amino acid motif MEESKGYEPP of gD was expressed in E. coli. Molecular modeling, western blot and ELISA analysis showed that this scFv had a high affinity for BoHV-1 gD, with a Kd of 161.2 ± 37.58 nM and for whole BoHV-1 virus, with a Kd of 67.44 ± 16.99 nM. In addition, this scFv displayed a high affinity for BoHV-1 antigen in an ELISA and competed with BoHV-1 anti-serum in a competitive ELISA. Immunofluorescence assay (IFA) and laser confocal microscopy showed that this scFv could efficiently bind to and be internalized by BoHV-1 infected Madin-Darby bovine kidney (MDBK) cells. Importantly, this scFv was shown to inhibit BoHV-1 infectivity and to reduce the number of viral plaques by blocking viral attachment to MDBK cells. Our study suggests that this bovine single-chain antibody could be developed for use as a diagnostic and therapeutic agent against BoHV-1 infection in cattle.


Bovine herpesvirus-1 Single chain Fv antibody Glycoprotein D Neutralizing epitope Binding affinity Blocking viral attachment Virus neutralization 



This work was supported by a grant from The National Key Project of Research and Development Program of China (Grant No. 2016YFD0500900), funding from Beijing Innovation Team of Technology System in Dairy Industry (award no. bjcystx-ny-3) and the Special Program on Science and Technology Innovation Capacity Building of BAAFS (award no. KJCX20170406).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All animal research was approved by the Beijing Association for Science and Technology (approval ID SYXK (Beijing) 2007-0023) and was in compliance with Beijing Laboratory Animal Welfare and Ethics guidelines as issued by the Beijing Administration Committee of Laboratory Animals. All animal studies were also performed in accordance with the Beijing Academy of Agricultural and Forestry Sciences Institutional Animal Care and Use Committee guidelines. The protocol was approved by the Committee on Experimental Animal Management of Beijing Academy of Agricultural and Forestry Sciences.

Supplementary material

253_2017_8566_MOESM1_ESM.pdf (187 kb)
ESM 1 (PDF 186 kb)


  1. Alves Dummer L, Pereira Leivas Leite F, van DrunenLittel–van den Hurk S (2014) Bovine herpesvirus glycoprotein D: a review of its structural characteristics and applications in vaccinology. Vet Res 45:111. CrossRefPubMedPubMedCentralGoogle Scholar
  2. Barnwal B, Mok CK, Wu J, Diwakar MK, Gupta G, Zeng Q, Chow VT, Song J, Yuan YA, Tan YJ (2015) A monoclonal antibody binds to threonine 49 in the non-structural 1protein of influenza A virus and interferes with its ability to modulate viral replication. Antivir Res 116:55–61. CrossRefPubMedGoogle Scholar
  3. Batra SA, Shanthalingam S, Donofrio G, Haldorson GJ, Chowdhury S, White SN, Srikumaran S (2017) Immunization of bighorn sheep against Mannheimiahaemolytica with a bovine herpesvirus 1-vectored vaccine. Vaccine 35:1630–1636. CrossRefPubMedGoogle Scholar
  4. Bird RE, Hardman KD, Jacobson JW, Johnson S, Kaufman BM, Lee SM, Lee T, Pope SH, Riordan GS, Whitlow M (1988) Single-chain antigen-binding proteins. Science 242:423–426. CrossRefPubMedGoogle Scholar
  5. Biswas S, Bandyopadhyay S, Dimri U, Patra PH (2013) Bovine herpesvirus-1 (BoHV-1) - a re-emerging concern in livestock: a revisit to its biology, epidemiology, diagnosis, and prophylaxis. Vet Q 33:68–81. CrossRefPubMedGoogle Scholar
  6. Bork P, Holm L, Sander C (1994) The immunoglobulin fold. Structural classification, sequence patterns and common core. J Mol Biol 242:309–320. PubMedGoogle Scholar
  7. Chatterjee D, Chandran B, Berger EA (2012) Selective killing of Kaposi's sarcoma-associated herpesvirus lytically infected cells with a recombinant immunotoxin targeting the viral gpK8.1A envelope glycoprotein. MAbs 4:233–242. CrossRefPubMedPubMedCentralGoogle Scholar
  8. Chothia C, Gelfand I, Kister A (1998) Structural determinants in the sequences of immunoglobulin variable domain. J Mol Biol 278:457–479. CrossRefPubMedGoogle Scholar
  9. Chowdhury SI (2016) Identification of an epitope within the bovine herpesvirus 1 glycoprotein E cytoplasmic tail and use of a monoclonal antibody directed against the epitope for the differentiation between vaccinated and infected animals. J Virol Methods 233:97–104. CrossRefPubMedGoogle Scholar
  10. de Witte WE, Danhof M, van der Graaf PH, de Lange EC (2016) In vivo target residence time and kinetic selectivity: the association rate constant as determinant. Trends Pharmacol Sci 37:831–842. CrossRefPubMedGoogle Scholar
  11. Del Medico Zajac MP, Zanetti FA, Esusy MS, Federico CR, Zabal O, Valera AR, Calamante G (2017) Induction of both local immune response in mice and protection in a rabbit model by intranasal immunization with modified vaccinia Ankara virus expressing a secreted form of bovine herpesvirus 1 glycoprotein D. Viral Immunol 30:70–76. CrossRefPubMedGoogle Scholar
  12. Della Cristina P, Castagna M, Lombardi A, Barison E, Tagliabue G, Ceriotti A, Koutris I, Di Leandro L, Giansanti F, Vago R, Ippoliti R, Flavell SU, Flavell DJ, Colombatti M, Fabbrini MS (2015) Systematic comparison of single-chain Fv antibody-fusion toxin constructs containing Pseudomonas exotoxin A or saporin produced in different microbial expression systems. Microb Cell Factories 14:19. CrossRefGoogle Scholar
  13. Denton PW, Long JM, Wietgrefe SW, Sykes C, Spagnuolo RA, Snyder OD, Perkey K, Archin NM, Choudhary SK, Yang K, Hudgens MG, Pastan I, Haase AT, Kashuba AD, Berger EA, Margolis DM, Garcia JV (2014) Targeted cytotoxic therapy kills persisting HIV infected cells during ART. PLoS Pathog 10:e1003872. CrossRefPubMedPubMedCentralGoogle Scholar
  14. Eldridge GM, Weiss GA (2015) Identifying reactive peptides from phage-displayed libraries. Methods Mol Biol 1248:189–199. CrossRefPubMedPubMedCentralGoogle Scholar
  15. Fan Y, Garcia-Rodriguez C, Lou J, Wen W, Conrad F, Zhai W, Smith TJ, Smith LA, Marks JD (2017) A three monoclonal antibody combination potently neutralizes multiple botulinum neurotoxin serotype F subtypes. PLoS One 12:e0174187. CrossRefPubMedPubMedCentralGoogle Scholar
  16. Geoghegan EM, Zhang H, Desai PJ, Biragyn A, Markham RB (2015) Antiviral activity of a single-domain antibody immunotoxin binding to glycoprotein D of herpes simplex virus 2. Antimicrob Agents Chemother 59:527–535. CrossRefPubMedGoogle Scholar
  17. Geraghty RJ, Krummenacher C, Cohen GH, Eisenberg RJ, Spear PG (1998) Entry of alpha herpesviruses mediated by poliovirus receptor-related protein 1 and poliovirus receptor. Science 280:1618–1620. CrossRefPubMedGoogle Scholar
  18. Gupta SK, Shukla P (2017) Microbial platform technology for recombinant antibody fragment production: a review. Crit Rev Microbiol 43:31–42. CrossRefPubMedGoogle Scholar
  19. Hanke L, Knockenhauer KE, Brewer RC, van Diest E, Schmidt FI, Schwartz TU, Ploegh HL (2016) The antiviral mechanism of an influenza A virus nucleoprotein-specific single-domain antibody fragment. MBio 7: pii: e01569–16. doi:
  20. Huston JS, Levinson D, Mudgett-Hunter M, Tai MS, Novotný J, Margolies MN, Ridge RJ, Bruccoleri RE, Haber E, Crea R (1988) Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc Natl Acad Sci U S A 85:5879–5883. CrossRefPubMedPubMedCentralGoogle Scholar
  21. Iwasa S, Sato N, Wang CW, Cheng YH, Irokawa H, Hwang GW, Naganuma A, Kuge S (2016) The phospholipid: diacylglycerolacyl transferaselro1 is responsible for hepatitis C virus core-induced lipid droplet formation in a yeast model system. PLoS One 11:e0159324. CrossRefPubMedPubMedCentralGoogle Scholar
  22. Keuser V, Schynts F, Detry B, Collard A, Robert B, Vanderplasschen A, Pastoret PP, Thiry E (2004) Improved antigenic methods for differential diagnosis of bovine, caprine, and cervine alpha herpesviruses related to bovine herpesvirus 1. J Clin Microbiol 42:1228–1235.
  23. Krah S, Schröter C, Zielonka S, Empting M, Valldorf B, Kolmar H (2016) Single-domain antibodies for biomedical applications. Immunopharmacol Immunotoxicol 38:21–28. CrossRefPubMedGoogle Scholar
  24. Lee G, Yu J, Cho S, Byun SJ, Kim DH, Lee TK, Kwon MH, Lee S (2014) A nucleic-acid hydrolyzing single chain antibody confers resistance to DNA virus infection in hela cells and C57BL/6 mice. PLoS Pathog 10:e1004208. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Levings RL, Roth JA (2013a) Immunity to bovine herpesvirus 1: II. Adaptive immunity and vaccinology. Anim Health Res Rev 14:103–123. CrossRefPubMedGoogle Scholar
  26. Levings RL, Roth JA (2013b) Immunity to bovine herpesvirus 1: I. Viral lifecycle and innate immunity. Anim Health Res Rev 14:88–102. CrossRefPubMedGoogle Scholar
  27. Levings RL, Collins JK, Patterson PA, Roth JA (2015) Virus, strain, and epitope specificities of neutralizing bovine monoclonal antibodies to bovine herpesvirus 1glycoproteins gB, gC, and gD, with sequence and molecular model analysis. Vet Immunol Immunopathol 164:179–193. CrossRefPubMedGoogle Scholar
  28. Li B, Ye J, Lin Y, Wang M, Zhu J (2014) Preparation and identification of a single-chain variable fragment antibody against Newcastle diseases virus F48E9. Vet Immunol Immunopathol 161:258–264. CrossRefPubMedGoogle Scholar
  29. Liu Y, Jones C (2016) Regulation of notch-mediated transcription by a bovine herpesvirus 1 encoded protein (ORF2) that is expressed in latently infected sensory neurons. J Neuro-Oncol 22:518–528. Google Scholar
  30. Ma H, O'Kennedy R (2015) The structure of natural and recombinant antibodies. Methods Mol Biol 1348:7–11. CrossRefPubMedGoogle Scholar
  31. Margolis DM, Garcia JV, Hazuda DJ, Haynes BF (2016) Latency reversal and viral clearance to cure HIV-1. Science 353:f6517. CrossRefGoogle Scholar
  32. Muylkens B, Thiry J, Kirten P, Schynts F, Thiry E (2007) Bovine herpesvirus 1 infection and infectious bovine rhinotracheitis. Vet Res 38:181–209. CrossRefPubMedGoogle Scholar
  33. Organtini LJ, Lee H, Iketani S, Huang K, Ashley RE, Makhov AM, Conway JF, Parrish CR, Hafenstein S (2016) Near-atomic resolution structure of a highly neutralizing fab bound to canine parvovirus. J Virol 90:9733–9742. CrossRefPubMedPubMedCentralGoogle Scholar
  34. Patrusheva I, Perelygina L, Torshin I, LeCher J, Hilliard J (2016) B virus (Macacine Herpesvirus 1) divergence: variations in glycoprotein D from clinical and laboratory isolates diversify virus entry strategies. J Virol 90:9420–9432. CrossRefPubMedPubMedCentralGoogle Scholar
  35. Pendzialek J, Roose K, Smet A, Schepens B, Kufer P, Raum T, Baeuerle PA, Muenz M, Saelens X, Fiers W (2017) Bispecific T cell engaging antibody constructs targeting a universally conserved part of the viral M2 ectodomain cure and prevent influenza A virus infection. Antivir Res 141:155–164. 2017.02.016 CrossRefPubMedGoogle Scholar
  36. Peralta A, Molinari P, Conte-Grand D, Calamante G, Taboga O (2007) A chimeric baculovirus displaying bovine herpesvirus-1 (BoHV-1) glycoprotein D on its surface and their immunological properties. Appl Microbiol Biotechnol 75:407–414. CrossRefPubMedGoogle Scholar
  37. Rader C, Barbas CF (1997) Phage display of combinatorial antibody libraries. Curr Opin Biotechnol 8:503–508. CrossRefPubMedGoogle Scholar
  38. Saeed AF, Wang R, Ling S, Wang S (2017) Antibody engineering for pursuing a healthier future. Front Microbiol 8:495. PubMedPubMedCentralGoogle Scholar
  39. Safdari Y, Ahmadzadeh V, Khalili M, Jaliani HZ, Zarei V, Erfani-Moghadam V (2016) Use of single chain antibody derivatives for targeted drug delivery. Mol Med 22:258–270. CrossRefPubMedCentralGoogle Scholar
  40. Spiess K, Jakobsen MH, Kledal TN, Rosenkilde MM (2016) The future of antiviral immunotoxins. J Leukoc Biol 99:911–925. CrossRefPubMedGoogle Scholar
  41. Weisshaar M, Cox R, Morehouse Z, Kumar Kyasa S, Yan D, Oberacker P, Mao S, Golden JE, Lowen AC, Natchus MG, Plemper RK (2016) Identification and characterization of influenza virus entry inhibitors through dual Myxovirus high-throughput screening. J Virol 90:7368–7387. CrossRefPubMedPubMedCentralGoogle Scholar
  42. Yang J, Chen R, Wei J, Zhang F, Zhang Y, Jia L, Yan Y, Luo W, Cao Y, Yao L, Sun J, Xu Z, Yang A (2010) Production and characterization of a recombinant single-chain antibody against Hantaan virus envelop glycoprotein. Appl Microbiol Biotechnol 86:1067–1075. CrossRefPubMedGoogle Scholar
  43. Yodsheewan R, Maneewatch S, Srimanote P, Thueng-In K, Songserm T, Dong-Din-On F, Bangphoomi K, Sookrung N, Choowongkomon K, Chaicumpa W (2013) Human monoclonal scFv specific to NS1 protein inhibits replication of influenza viruses across types and subtypes. Antivir Res 100:226–237. CrossRefPubMedGoogle Scholar
  44. Zhang X, Qi X, Zhang Q, Zeng X, Shi Z, Jin Q, Zhan F, Xu Y, Liu Z, Feng Z, Jiao Y (2013) Human 4F5 single-chain Fv antibody recognizing a conserved HA1 epitope has broad neutralizing potency against H5N1 influenza a viruses of different clades. Antivir Res 99:91–99. CrossRefPubMedGoogle Scholar
  45. Zhang L, Li Q, Ding X, Zhang B, Zhang Q, Qu X, Huo Y, Yang J, Wang S (2017) Antisense oligonucleotides targeting Raf-1 block Japanese encephalitis virus in vitro and in vivo. Nucleic Acid Ther 27:78–86. CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • Jian Xu
    • 1
  • Jing Wu
    • 1
    • 2
  • Bo Jiang
    • 1
  • Houjun He
    • 2
  • Xixi Zhang
    • 1
    • 3
  • Xiaoyang Li
    • 1
    • 2
  • Dawei Yang
    • 1
  • Xiufen Huang
    • 1
  • Joshua E. Sealy
    • 4
  • Munir Iqbal
    • 4
  • Yongqing Li
    • 1
    Email author
  1. 1.Institute of Animal Husbandry and Veterinary MedicineBeijing Academy of Agricultural and Forestry SciencesBeijingPeople’s Republic of China
  2. 2.College of Animal Science and TechnologyJiangxi Agricultural UniversityNanchangPeople’s Republic of China
  3. 3.Animal Science and Technology CollegeBeijing University of AgricultureBeijingPeople’s Republic of China
  4. 4.The Pirbright InstituteWokingUK

Personalised recommendations