Archives of Virology

, Volume 161, Issue 10, pp 2653–2665 | Cite as

The amino-terminal domain of ORF149 of koi herpesvirus is preferentially targeted by IgM from carp populations surviving infection

  • F. Torrent
  • A. Villena
  • P. A. Lee
  • W. Fuchs
  • S. M. Bergmann
  • J. M. CollEmail author
Original Article


Recombinantly expressed fragments of the protein encoded by ORF149 (pORF149), a structural protein from the common- and koi-carp-infecting cyprinid herpesvirus-3 (CyHV-3) that was previously shown to be antigenic, were used to obtain evidence that its amino-terminal part contains immunodominant epitopes in fish populations that survived the infection. To obtain such evidence, nonspecific binding of carp serum tetrameric IgM had to be overcome by a novel ELISA protocol (rec2-ELISA). Rec2-ELISA involved pre-adsorption of carp sera with a heterologous recombinant fragment before incubation with pORF149 fragments and detection with anti-carp IgM monoclonal antibodies. Only in this way was it possible to distinguish between sera from uninfected and survivor carp populations. Although IgM from survivors recognised pORF149 fragments to a lesser degree than whole virus, specificity was confirmed by correlation of rec2- and CyHV-3-ELISAs, inhibition of rec2-ELISA by an excess of frgIIORF149, ELISA using IgM-capture, Western blotting, and reduction of reactivity in CyHV-3-ELISA by pre-adsorption of sera with frgIIORF149. The similarity of IgM-binding profiles between frgIORF149 (amino acid residues 42-629) and frgIIORF149 (42-159) and their reactivities with previously described anti-CyHV-3 monoclonal antibodies confirmed that most pORF149 epitopes were localised in its amino-terminal part.


Recombinant Fragment ELISA Protocol Carp Population Carp Serum Infectious Haematopoietic Necrosis Virus 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



Thanks go to Paula Perez Gonzalez, who helped with the experimental work. Thanks are due to Dra. D. Chee (Animal & Plant Health Laboratories, Aquatic Animal health Section Laboratories Group, Singapore) for providing sera from carp surviving CyHV-3, to Dra. K. Thompson (AquaTic Diagnostics, Ltd) for providing some of the anti-CyHV-3 monoclonal antibodies, and Dra. P. Fernandez (MAGRAMA, Algete, Madrid, Spain) for her help with CyHV-3-U. We also thank J.L. Gonzalez and his team (CBC, SL) for obtaining common carp sera samples from all over Spain and from imported koi, and P. Encinas, B. Chinchilla and Dr. E. Gomez-Casado, who helped with Spanish koi sera at UPM (Madrid). This work was supported by RTA2013-00008-00-00 of INIA, and AGL2014-51773-C3 and AGL2014-53190 REDC of the Ministerio de Economía y Competitividad of Spain.

Compliance with ethical standards

This study was funded by RTA2013-00008-00-00 of the Instituto Nacional de Investigaciones y Tecnología Agraria y Alimentaria, INIA, and AGL2014-51773-C3 and AGL2014-53190 REDC of the Ministerio de Economía y Competitividad of Spain. The authors declare that they have no conflict of interest. All applicable international, national and/or institutional guidelines for the care and use of animals were followed. This article does not contain any studies with human participants by any of the authors.

Supplementary material

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Supplementary material 1 (DOC 129 kb)
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Supplementary material 2 (DOCX 15 kb)
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Supplementary material 3 (TIFF 1383 kb)
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Supplementary material 4 (TIFF 108 kb)


  1. 1.
    Adamek M, Rakus KL, Chyb J, Brogden G, Huebner A, Irnazarow I, Steinhagen D (2012) Interferon type I responses to virus infections in carp cells: In vitro studies on Cyprinid herpesvirus 3 and Rhabdovirus carpio infections. Fish Shellfish Immunol 33:482–493CrossRefPubMedGoogle Scholar
  2. 2.
    Adamek M, Steinhagen D, Irnazarow I, Hikima J, Jung TS, Aoki T (2014) Biology and host response to Cyprinid herpesvirus 3 infection in common carp. Dev Compar Immunol 43:151–159CrossRefGoogle Scholar
  3. 3.
    Adkison MA, Gilad O, Hedrick RP (2005) An enzyme linked immunosorbent assay (ELISA) for detection of antibodies to the koi herpesvirus (KHV) in the serum of koi Cyprinus carpio. Fish Pathol 40:53–62CrossRefGoogle Scholar
  4. 4.
    Aoki T, Hirono I, Kurokawa K, Fukuda H, Nahary R, Eldar A, Davison AJ, Waltzek TB, Bercovier H, Hedrick RP (2007) Genome sequences of three koi herpesvirus isolates representing the expanding distribution of an emerging disease threatening koi and common carp worldwide. J Virol 81:5058–5065CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Bergmann SM, Schutze H, Fischer U, Fichtner D, Riechardt M, Meyer K, Schrudde D, Kempter J (2009) Detection of koi herpes-virus (KHV) genome in apparently healthy fish. Bull Euro Assoc Fish Pathol 29:145–152Google Scholar
  6. 6.
    Bergmann SM, Kempter J (2011) Detection of koi herpesvirus (KHV) after re-activation in persistently infected common carp (Cyprinus carpio L.) using non-lethal sampling methods. Bull Euro Assoc Fish Pathol 31:92–100Google Scholar
  7. 7.
    Cho MY, Won KM, Kim JW, Jee BY, Park MA, Hong S (2014) Detection of koi herpesvirus (KHV) in healthy cyprinid seed stock. Dis Aquat Organ 112:29–36CrossRefPubMedGoogle Scholar
  8. 8.
    Del Rio-Rodriguez RE, Inglis V, Millar SD (1997) Survival of Escherichia coli in the intestine of fish. Aquacult Res 28:257–264CrossRefGoogle Scholar
  9. 9.
    Dong CF, Li XZ, Weng SP, Xie SX, He JG (2013) Emergence of fatal European genotype CyHV-3/KHV in mainland China. Veter Microbiol 162:239–244CrossRefGoogle Scholar
  10. 10.
    Encinas P, Gomez-Casado E, Estepa A, Coll JM (2011) An ELISA for detection of trout antibodies to viral haemorrhagic septicemia virus using recombinant fragments of their viral G protein. J Virol Methods 176:14–23CrossRefPubMedGoogle Scholar
  11. 11.
    Encinas P, Gomez-Casado E, Fregeneda G, Olesen NJ, Lorenzen N, Estepa A, Coll JM (2011) Rainbow trout surviving infections of viral haemorrhagic septicemia virus (VHSV) show lasting antibodies to recombinant G protein fragments. Fish Shellfish Immunol 30:929–935CrossRefPubMedGoogle Scholar
  12. 12.
    Encinas P, Gomez-Sebastian S, Nuñez MC, Gomez-Casado E, Escribano JM, Estepa A, Coll JM (2011) Antibody recognition of the protein G of viral haemorrhagic septicemia virus purified in large amount from insect larvae. BMC reports 4:210–217Google Scholar
  13. 13.
    Fernandez-Alonso M, Lorenzo G, Perez L, Bullido R, Estepa A, Lorenzen N, Coll JM (1998) Mapping of the lineal antibody epitopes of the glycoprotein of VHSV, a salmonid rhabdovirus. Dis Aqu Organ 34:167–176CrossRefGoogle Scholar
  14. 14.
    Fuchs W, Granzow H, Dauber M, Fichtner D, Mettenleiter TC (2014) Identification of structural proteins of koi herpesvirus. Arch Virol 159:3257–3268CrossRefPubMedGoogle Scholar
  15. 15.
    Gotesman M, Kattlun J, Bergmann SM, El-Matbouli M (2013) CyHV-3: the third cyprinid herpesvirus. Dis Aqu Organ 105:163–174CrossRefGoogle Scholar
  16. 16.
    Gotesman M, Soliman H, El-Matbouli M (2013) Antibody screening identifies 78 putative host proteins involved in Cyprinid herpesvirus 3 infection or propagation in common carp, Cyprinus carpio L. J Fish Dis 36:721–733CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Gotesman M, Abd-Elfattah A, Kattlun J, Soliman H, El-Matbouli M (2014) Investigating the interactions of Cyprinid herpesvirus-3 with host proteins in goldfish Carassius auratus. J Fish Dis 37:835–841CrossRefPubMedGoogle Scholar
  18. 18.
    Kattlun J, Gotesman M, Bergmann SM, El-Matbouli M (2013) Infection of carp (Cyprinus carpio) with Cyprinid Herpesvirus 3 (CyHV-3) - an update. Wiener Tierarztliche Monatsschrift 100:72–84Google Scholar
  19. 19.
    Khan F, He M, Taussig MJ (2006) Double-hexahistidine tag with high-affinity binding for protein immobilization, purification, and detection on ni-nitrilotriacetic acid surfaces. Anal Chem 78:3072–3079CrossRefPubMedGoogle Scholar
  20. 20.
    Kim HJ, Park JS, Kwon SR (2015) Development of a stringent ELISA protocol to evaluate anti-viral hemorrhagic septicemia virus-specific antibodies in olive flounder Paralichthys olivaceus with improved specificity. J Microbiol 53:481–485CrossRefGoogle Scholar
  21. 21.
    Kim WS, Nishizawa T, Yoshimizu M (2007) Non-specific adsorption of fish immunoglobulin M (IgM) to blocking reagents on ELISA plate wells. Dis Aqu Organ 78:55–59CrossRefGoogle Scholar
  22. 22.
    Kim WS, Mochizuki M, Nishizawa T, Yoshimizu M (2008) Detection of specific antibodies against infectious hematopoietic necrosis virus from rainbow trout sera by ELISA using two novirhabdoviruses. Fish Pathol 43:112–116CrossRefGoogle Scholar
  23. 23.
    Lang D, Vornhagen R, Rothe M, Hinderer W, Sonneborn HH, Plachter B (2001) Cross-reactivity of Epstein-Barr virus-specific immunoglobulin M antibodies with cytomegalovirus antigens containing glycine homopolymers. Clin Diagn Lab Immunol 8:747–756PubMedPubMedCentralGoogle Scholar
  24. 24.
    Lorenzen N, Olesen NJ, Jorgensen PEV (1990) Neutralization of Egtved virus pathogenicity to cell cultures and fish by monoclonal antibodies to the viral G protein. J General Virol 71:561–567CrossRefGoogle Scholar
  25. 25.
    Lorenzen N, Olesen NJ, Jorgensen PEV (1993) Antibody response in rainbow trout to VHS virus proteins. Fish Shellfish Immunol 3:461–473CrossRefGoogle Scholar
  26. 26.
    Lorenzen N, Olesen NJ (1997) Immunization with viral antigens: viral haemorrhagic septicemia. Dev Biol Stand 90:201–209PubMedGoogle Scholar
  27. 27.
    Lorenzen N, Olesen NJ, Koch C (1999) Inmunity to VHS virus in rainbow trout. Aquaculture 172:41–61CrossRefGoogle Scholar
  28. 28.
    Magadan S, Sunyer OJ, Boudinot P (2015) Unique features of fish immune repertoires: particularities of adaptive immunity within the largest group of vertebrates. Results Probl Cell Differ 57:235–264CrossRefPubMedGoogle Scholar
  29. 29.
    Matras M, Antychowicz J, Castric J, Bergmann SM (2012) Cyhv-3 infection dynamics in common carp (Cyprinus carpio)—evaluation of diagnostic methods. Bull Veterin Instit Pulawy 56:127–132Google Scholar
  30. 30.
    Michel B, Leroy B, Raj VS, Lieffrig F, Mast J, Wattiez R, Vanderplasschen AF, Costes B (2010) The genome of cyprinid herpesvirus 3 encodes 40 proteins incorporated in mature virions. J General Virol 91:452–462CrossRefGoogle Scholar
  31. 31.
    Monaghan SJ (2013) Approaches to DIVA vaccination for fish using infectious salmon anaemia and koi herpesvirus disease as models. pHD Thesis. University of Stirling. p 462Google Scholar
  32. 32.
    Monaghan SJ, Thompson KD, Adams A, Bergmann SM (2015) Sensitivity of seven PCRs for early detection of koi herpesvirus in experimentally infected carp, Cyprinus carpio L., by lethal and non-lethal sampling methods. J Fish Dis 38:303–319CrossRefPubMedGoogle Scholar
  33. 33.
    Monaghan SJ, Thompson KD, Bron JE, Bergmann SM, Jung TS, Aoki T, Muir KF, Dauber M, Reiche S, Chee D, Chong SM, Chen J, Adams A (2016) Expression of immunogenic structural proteins of cyprinid herpesvirus 3 in vitro assessed using immunofluorescence. Vet Res 47:8CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Olesen NJ, Lorenzen N, Jorgensen PEV (1991) Detection of rainbow trout antibody to Egtved virus by enzyme- linked immunosorbent assay (ELISA), immunofluorescence (IF), and plaque neutralization tests (50 % PNT. Dis Aqu Organ 10:31–38CrossRefGoogle Scholar
  35. 35.
    Perelberg A, Ronen A, Hutoran M, Smith Y, Kotler M (2005) Protection of cultured Cyprinus carpio against a lethal viral disease by an attenuated virus vaccine. Vaccine 23:3396–3403CrossRefPubMedGoogle Scholar
  36. 36.
    Perelberg A, Ilouze M, Kotler M, Steinitz M (2008) Antibody response and resistance of Cyprinus carpio immunized with cyprinid herpes virus 3 (CyHV-3). Vaccine 26:3750–3756CrossRefPubMedGoogle Scholar
  37. 37.
    Pionnier N, Adamek M, Miest JJ, Harris SJ, Matras M, Rakus KL, Irnazarow I, Hoole D (2014) C-reactive protein and complement as acute phase reactants in common carp Cyprinus carpio during CyHV-3 infection. Diseases of Aquatic Organisms 109:187–199CrossRefPubMedGoogle Scholar
  38. 38.
    Pokorova D, Vesely T, Piackova V, Reschova S, Hulova J (2005) Current knowledge on koi herpesvirus (KHV): a review. Veterinarni Medicina 50:139–147Google Scholar
  39. 39.
    Rakus KL, Wiegertjes GF, Adamek M, Siwicki AK, Lepa A, Irnazarow I (2009) Resistance of common carp (Cyprinus carpio L.) to Cyprinid herpesvirus-3 is influenced by major histocompatibility (MH) class II B gene polymorphism. Fish Shellfish Immunol 26:737–743CrossRefPubMedGoogle Scholar
  40. 40.
    Rakus KL, Irnazarow I, Adamek M, Palmeira L, Kawana Y, Hirono I, Kondo H, Matras M, Steinhagen D, Flasz B, Brogden G, Vanderplasschen A, Aoki T (2012) Gene expression analysis of common carp (Cyprinus carpio L.) lines during Cyprinid herpesvirus 3 infection yields insights into differential immune responses. Dev Comp Immunol 37:65–76CrossRefPubMedGoogle Scholar
  41. 41.
    Reed AN, Izume S, Dolan BP, LaPatra S, Kent M, Dong J, Jin L (2014) Identification of B Cells as a Major Site for Cyprinid Herpesvirus 3 Latency. J Virol 88:9297–9309CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Rocha A, Fernandez-Alonso M, Mas V, Perez L, Estepa A, Coll JM (2002) Antibody response to a linear epitope of the protein G of VHS rhabdovirus in immunized trout. Veter Immunol Immunopathol 86:89–99CrossRefGoogle Scholar
  43. 43.
    Ronen A, Perelberg A, Abramowitz J, Hutoran M, Tinman S, Bejerano I, Steinitz M, Kotler M (2003) Efficient vaccine against the virus causing a lethal disease in cultured Cyprinus carpio. Vaccine 21:4677–4684CrossRefPubMedGoogle Scholar
  44. 44.
    Rosenkranz D, Klupp BG, Teifke JP, Granzow H, Fichtner D, Mettenleiter TC, Fuchs W (2008) Identification of envelope protein pORF81 of koi herpesvirus. J Gen Virol 89:896–900CrossRefPubMedGoogle Scholar
  45. 45.
    Sanz FA, Coll JM (1992) Detection of hemorrhagic septicemia virus of salmonid fishes by use of an enzyme-linked immunosorbent assay containing high sodium chloride concentration and two noncompetitive monoclonal antibodies against early viral nucleoproteins. Am J Veter Res 53:897–903Google Scholar
  46. 46.
    St-Hilaire S, Beevers N, Way K, Le Deuff RM, Martin P, Joiner C (2005) Reactivation of koi herpesvirus infections in common carp Cyprinus carpio. Dis Aqu Organ 67:15–23CrossRefGoogle Scholar
  47. 47.
    St-Hilaire S, Beevers N, Joiner C, Hedrick RP, Way K (2009) Antibody response of two populations of common carp, Cyprinus carpio L., exposed to koi herpesvirus. J Fish Dis 32:311–320CrossRefPubMedGoogle Scholar
  48. 48.
    Sunyer JO (2013) Fishing for mammalian paradigms in the teleost immune system. Nat Immunol 14:320–326CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Tu C, Lu YP, Hsieh CY, Huang SM, Chang SK, Chen MM (2014) Production of monoclonal antibody against ORF72 of koi herpesvirus isolated in Taiwan. Folia Microbiol (Praha) 59:159–165CrossRefGoogle Scholar
  50. 50.
    Zhang YA, Salinas I, Oriol Sunyer J (2011) Recent findings on the structure and function of teleost IgT. Fish Shellfish Immunol 31:627–634CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Zhou JX, Wang H, Li XW, Zhu X, Lu WL, Zhang DM (2014) Construction of KHV-CJ ORF25 DNA vaccine and immune challenge test. J Fish Dis 37:319–325CrossRefPubMedGoogle Scholar
  52. 52.
    Zhou JX, Xue JD, Wang QJ, Zhu X, Li XW, Lv WL, Zhang DM (2014) Vaccination of plasmid DNA encoding ORF81 gene of CJ strains of KHV provides protection to immunized carp. In Vitro Cell Dev Biol-Animal 50:489–495CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Wien 2016

Authors and Affiliations

  • F. Torrent
    • 2
  • A. Villena
    • 7
  • P. A. Lee
    • 3
    • 4
  • W. Fuchs
    • 5
  • S. M. Bergmann
    • 6
  • J. M. Coll
    • 1
    Email author
  1. 1.Departamento BiotecnologíaInstituto Nacional Investigaciones y Tecnologías Agrarias y Alimentarias, INIAMadridSpain
  2. 2.Escuela Superior de Ingenieros de Montes, Universidad Politécnica de Madrid (UPM), PiscifactoríaMadridSpain
  3. 3.Graduate Institute of BiotechnologyCentral Taiwan University of Science and TechnologyTaichungTaiwan
  4. 4.GeneReach BiotechnologyTaichungTaiwan
  5. 5.Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal HealthGreifswald-Insel RiemsGermany
  6. 6.Institute of Infectology, German Reference Laboratory for KHVD, Friedrich-Loeffler-Institut, Federal Research Institute for Animal HealthGreifswald-Insel RiemsGermany
  7. 7.Departamento de Biología MolecularUniversidad de LeónLeonSpain

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