Abstract
Infecting ducks with duck hepatitis B virus (DHBV) is widely accepted as a relevant model for studying aspects of human HBV infection. However, efficient and sensitive diagnostic methods for the various infection models are limited. In order to provide a more simple and convenient method for serologic diagnosis, we improved the production of recombinant DHBV viral capsid protein (core protein) and then used it to develop an indirect enzyme-linked immunosorbent assay (ELISA) for detecting anti-DHBc antibodies (DHBcAg ELISA) in DHBV-infected ducks. Given the positive/negative cut-off value, the maximum dilution of duck sera in which anti-DHBc antibodies could be detected was 1:12,800. In addition, the DHBcAg ELISA displayed no cross reactivity with duck antisera against duck circovirus (DuCV), duck plague virus (DPV), duck hepatitis virus (DHV), duck swollen head septicemia virus (DSHSV), avian influenza virus (AIV), Riemerella anatipestifer, Salmonella anatum, or Escherichia coli. Furthermore, the coefficients of variation (CVs) of inter-assay and intra-assay experiments were both below than 10 %. When compared to PCR for accuracy on clinical samples from cases of suspected DHBV infection, the DHBcAg showed 95.45 % coincidence with PCR. In conclusion, recombinant DHBc was readily produced and used to establish a simple DHBcAg ELISA that provided a highly specific and sensitive method for analysis of clinical samples.
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References
Alonso A, Gomes M, Martins M, Sondahl M (1990) Detection of foot-and-mouth disease virus infection-associated antigen antibodies: comparison of the enzyme-linked immunosorbent assay and agar gel immunodiffusion tests. Prev Vet Med 9:223–240
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Cai MS, Cheng AC, Wang MS, Zhao LC, Zhu DK, Luo QH, Liu F, Chen XY (2009) His6-tagged UL35 protein of duck plague virus: expression, purification, and production of polyclonal antibody. Intervirology 52:141–151
Chen YX, Huang AL, Qi ZY, Guo SH (2004) Establishment and assessment of two methods for quantitative detection of serum duck hepatitis B virus DNA. World J Gastroenterol 10:2666–2669
Dubendorff JW, Studier FW (1991) Creation of a T7 autogene. Cloning and expression of the gene for bacteriophage T7 RNA polymerase under control of its cognate promoter. J Mol Biol 219:61–68
Guo H, Mao R, Block TM, Guo JT (2010) Production and function of the cytoplasmic deproteinized relaxed circular DNA of hepadnaviruses. J Virol 84:387–396
Jia R, Cheng A, Wang M, Qi X, Zhu D, Ge H, Luo Q, Liu F, Guo Y, Chen X (2009) Development and evaluation of an antigen-capture ELISA for detection of the UL24 antigen of the duck enteritis virus, based on a polyclonal antibody against the UL24 expression protein. J Virol Methods 161:38–43
Jia R, Cheng A, Wang M, Zhu D, Ge H, Xin H, Liu F, Luo Q, Guo Y, Qi X, Yin Z, Chen X (2009) Cloning, expression, purification and characterization of UL24 partial protein of duck enteritis virus. Intervirology 52:326–334
Jilbert AR, Wu TT, England JM, Hall PM, Carp NZ, O’Connell AP, Mason WS (1992) Rapid resolution of duck hepatitis B virus infections occurs after massive hepatocellular involvement. J Virol 66:1377–1388
Jilbert AR, Botten JA, Miller DS, Bertram EM, Hall PM, Kotlarski J, Burrell CJ (1998) Characterization of age- and dose-related outcomes of duck hepatitis B virus infection. Virology 244:273–282
Jilbert AR, Kotlarski I (2000) Immune responses to duck hepatitis B virus infection. Dev Comp Immunol 24:285–302
Lambert V, Fernholz D, Sprengel R, Fourel I, Deleage G, Wildner G, Peyret C, Trepo C, Cova L, Will H (1990) Virus-neutralizing monoclonal antibody to a conserved epitope on the duck hepatitis B virus pre-S protein. J Virol 64:1290–1297
Li C, Cheng A, Wang M, Zhang N, Shen C, Yang J, Zhu D, Jia R, Luo Q, Chen X (2010) Development and validation of an indirect enzyme-linked immunosorbent assay for the detection of antibodies against duck swollen head hemorrhagic disease virus. Avian Dis 54:1270–1274
Mason WS, Seal G, Summers J (1980) Virus of Pekin ducks with structural and biological relatedness to human hepatitis B virus. J Virol 36:829–836
Middelberg AP (2002) Preparative protein refolding. Trends Biotechnol 20:437–443
Miller DS, Bertram EM, Scougall CA, Kotlarski I, Jilbert AR (2004) Studying host immune responses against duck hepatitis B virus infection. Methods Mol Med 96:3–26
Miller DS, Boyle D, Feng F, Reaiche GY, Kotlarski I, Colonno R, Jilbert AR (2008) Antiviral therapy with entecavir combined with post-exposure “prime-boost” vaccination eliminates duck hepatitis B virus-infected hepatocytes and prevents the development of persistent infection. Virology 373:329–341
Oneda H, Inouye K (1999) Refolding and recovery of recombinant human matrix metalloproteinase 7 (matrilysin) from inclusion bodies expressed by Escherichia coli. J Biochem 126:905–911
Pinto PS, Vaz AJ, Germano PM, Nakamura PM (2000) ELISA test for the diagnosis of cysticercosis in pigs using antigens of Taenia solium and Taenia crassiceps cysticerci. Rev Inst Med Trop Sao Paulo 42:71–79
Schein CH (1990) Solubility as a function of protein structure and solvent components. Biotechnology (NY) 8:308–317
Schmid B, Rösler C, Nassal M (2011) A high level of mutation tolerance in the multifunctional sequence encoding the RNA encapsidation signal of an avian hepatitis B virus and slow evolution rate revealed by in vivo infection. J Virol 85:9300–9313
Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185:60–89
Tohidi-Esfahani R, Vickery K, Cossart Y (2010) The early host innate immune response to duck hepatitis B virus infection. J Gen Virol 91:509–520
Walters KA, Joyce MA, Addison WR, Fischer KP, Tyrrell DL (2004) Superinfection exclusion in duck hepatitis B virus infection is mediated by the large surface antigen. J Virol 78:7925–7937
Wen Y, Cheng A, Wang M, Ge H, Shen C, Liu S, Xiang J, Jia R, Zhu D, Chen X, Lian B, Chang H, Zhou Y (2010) A Thymidine Kinase recombinant protein-based ELISA for detecting antibodies to Duck Plague Virus. Virol J 7:77–85
Wu Y, Cheng A, Wang M, Zhang S, Zhu D, Jia R, Luo Q, Chen Z, Chen X (2011) Serologic detection of duck enteritis virus using an indirect ELISA based on recombinant UL55 protein. Avian Dis 55:626–632
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This research was supported by China Agricultural Research System (CARS-43-8), the Ministry of Education Program (20125103110013), Sichuan Province Research Programs (2013HH0042/ 2013TD0015/ 11ZA084/ 12TD005/ 2011ZO0034/ 2011JO0040), and China 973 program (2011CB111606).
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Q. Liu, R. Jia, M. Wang, and J. Huang contributed equal to this work.
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Liu, Q., Jia, R., Wang, M. et al. Cloning, expression and purification of duck hepatitis B virus (DHBV) core protein and its use in the development of an indirect ELISA for serologic detection of DHBV infection. Arch Virol 159, 897–904 (2014). https://doi.org/10.1007/s00705-013-1897-y
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DOI: https://doi.org/10.1007/s00705-013-1897-y