Archives of Virology

, Volume 161, Issue 12, pp 3391–3404 | Cite as

Production of monoclonal antibodies against the ORF3 protein of rat hepatitis E virus (HEV) and demonstration of the incorporation of the ORF3 protein into enveloped rat HEV particles

  • Masaharu Takahashi
  • Tominari Kobayashi
  • Tanggis
  • Suljid Jirintai
  • Mulyanto
  • Shigeo Nagashima
  • Tsutomu Nishizawa
  • Satoshi Kunita
  • Hiroaki Okamoto
Original Article
First Online:
Received:
Accepted:

Abstract

Eight murine monoclonal antibodies (MAbs) against a synthetic peptide corresponding to the C-terminal 15-amino-acid portion of the ORF3 protein of rat hepatitis E virus (ratHEV) were produced and characterized. Immunofluorescence assays using the anti-ratHEV ORF3 MAbs revealed the accumulation of ORF3 protein in the cytoplasm of PLC/PRF/5 cells transfected with ORF3-expressing plasmids or inoculated with cell-culture-generated ratHEV strains. Anti-ORF3 MAbs could capture ratHEV particles in culture supernatant and serum following treatment with 0.5 % deoxycholate, but not those without prior detergent treatment or fecal ratHEV particles. Following treatment with 0.5 % deoxycholate and 0.5 % trypsin, the buoyant density of ratHEV particles in culture supernatant with ORF3 protein on the surface shifted from 1.15 g/cm3 to 1.26 g/cm3 in a sucrose gradient; the resulting particles were capturable by an anti-ORF2 MAb but not by an anti-ORF3 MAb. This indicates that the ORF3 protein (at least its C-terminal portion) is incorporated into the enveloped ratHEV virions released from infected cells but that it is not found in the virions in the feces, supporting the hypothesis that the ratHEV ORF3 protein is associated with the egress of virions from infected cells, similar to human HEV, despite the fact that the ratHEV ORF3 protein lacks a PSAP amino acid motif.

Keywords

ORF3 Protein Efficient Cell Culture System ORF3 Peptide 
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.
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Notes

Acknowledgments

This study was supported in part by the Research Program on Hepatitis from the Japan Agency for Medical Research and Development, AMED (15fk0210030h0001 and 16fk0210201h0002) and a MEXT-Supported Program for the Strategic Research Foundation at Private Universities, 2013-2017 (S1311030).

Compliance with ethical standards

Funding

This study was supported in part by the Research Program on Hepatitis from the Japan Agency for Medical Research and Development, AMED (15fk0210030h0001 and 16fk0210201h0002) and a MEXT-Supported Program for the Strategic Research Foundation at Private Universities, 2013-2017 (S1311030).

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

References

  1. 1.
    Chandra V, Kalia M, Hajela K, Jameel S (2010) The ORF3 protein of hepatitis E virus delays degradation of activated growth factor receptors by interacting with CIN85 and blocking formation of the Cbl-CIN85 complex. J Virol 84:3857–3867CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Debing Y, Moradpour D, Neyts J, Gouttenoire J (2016) Update on hepatitis E virology: implications for clinical practice. J Hepatol 65(1):200–212Google Scholar
  3. 3.
    Emerson SU, Nguyen H, Torian U, Purcell RH (2006) ORF3 protein of hepatitis E virus is not required for replication, virion assembly, or infection of hepatoma cells in vitro. J Virol 80:10457–10464CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Emerson SU, Nguyen HT, Torian U, Burke D, Engle R, Purcell RH (2010) Release of genotype 1 hepatitis E virus from cultured hepatoma and polarized intestinal cells depends on open reading frame 3 protein and requires an intact PXXP motif. J Virol 84:9059–9069CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Freed EO (2002) Viral late domains. J Virol 76:4679–4687CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Hurley JH (2010) The ESCRT complexes. Crit Rev Biochem Mol Biol 45:463–487CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Imai M, Nomura M, Gotanda T, Sano T, Tachibana K, Miyamoto H, Takahashi K, Toyama S, Miyakawa Y, Mayumi M (1982) Demonstration of two distinct antigenic determinants on hepatitis B e antigen by monoclonal antibodies. J Immunol 128:69–72PubMedGoogle Scholar
  8. 8.
    Jirintai S, Tanggis, Mulyanto, Suparyatmo JB, Takahashi M, Kobayashi T, Nagashima S, Nishizawa T, Okamoto H (2014) Rat hepatitis E virus derived from wild rats (Rattus rattus) propagates efficiently in human hepatoma cell lines. Virus Res 185:92–102CrossRefPubMedGoogle Scholar
  9. 9.
    Johne R, Heckel G, Plenge-Bönig A, Kindler E, Maresch C, Reetz J, Schielke A, Ulrich RG (2010) Novel hepatitis E virus genotype in Norway rats, Germany. Emerg Infect Dis 16:1452–1455CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Johne R, Plenge-Bonig A, Hess M, Ulrich RG, Reetz J, Schielke A (2010) Detection of a novel hepatitis E-like virus in faeces of wild rats using a nested broad-spectrum RT-PCR. J Gen Virol 91:750–758CrossRefPubMedGoogle Scholar
  11. 11.
    Johne R, Dremsek P, Kindler E, Schielke A, Plenge-Bonig A, Gregersen H, Wessels U, Schmidt K, Rietschel W, Groschup MH, Guenther S, Heckel G, Ulrich RG (2012) Rat hepatitis E virus: geographical clustering within Germany and serological detection in wild Norway rats (Rattus norvegicus). Infect Genet Evol 12:947–956CrossRefPubMedGoogle Scholar
  12. 12.
    Johne R, Dremsek P, Reetz J, Heckel G, Hess M, Ulrich RG (2014) Hepeviridae: an expanding family of vertebrate viruses. Infect Genet Evol 27:212–229CrossRefPubMedGoogle Scholar
  13. 13.
    Kamar N, Selves J, Mansuy JM, Ouezzani L, Peron JM, Guitard J, Cointault O, Esposito L, Abravanel F, Danjoux M, Durand D, Vinel JP, Izopet J, Rostaing L (2008) Hepatitis E virus and chronic hepatitis in organ-transplant recipients. N Engl J Med 358:811–817CrossRefPubMedGoogle Scholar
  14. 14.
    Kamar N, Bendall R, Legrand-Abravanel F, Xia NS, Ijaz S, Izopet J, Dalton HR (2012) Hepatitis E. Lancet 379:2477–2488CrossRefPubMedGoogle Scholar
  15. 15.
    Kannan H, Fan S, Patel D, Bossis I, Zhang YJ (2009) The hepatitis E virus open reading frame 3 product interacts with microtubules and interferes with their dynamics. J Virol 83:6375–6382CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Kar-Roy A, Korkaya H, Oberoi R, Lal SK, Jameel S (2004) The hepatitis E virus open reading frame 3 protein activates ERK through binding and inhibition of the MAPK phosphatase. J Biol Chem 279:28345–28357CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Kenney SP, Pudupakam RS, Huang YW, Pierson FW, LeRoith T, Meng XJ (2012) The PSAP motif within the ORF3 protein of an avian strain of the hepatitis E virus is not critical for viral infectivity in vivo but plays a role in virus release. J Virol 86:5637–5646CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Kenney SP, Wentworth JL, Heffron CL, Meng XJ (2015) Replacement of the hepatitis E virus ORF3 protein PxxP motif with heterologous late domain motifs affects virus release via interaction with TSG101. Virology 486:198–208CrossRefPubMedGoogle Scholar
  19. 19.
    Kobayashi T, Takahashi M, Tanggis, Mulyanto, Jirintai S, Nagashima S, Nishizawa T, Okamoto H (2016) Characterization and epitope mapping of monoclonal antibodies raised against rat hepatitis E virus capsid protein: an evaluation of their neutralizing activity in a cell culture system. J Virol Methods 233:78–88CrossRefPubMedGoogle Scholar
  20. 20.
    Lee GH, Tan BH, Chi-Yuan Teo E, Lim SG, Dan YY, Wee A, Kim Aw PP, Zhu Y, Hibberd ML, Tan CK, Purdy MA, Teo CG (2016) Chronic infection with camelid hepatitis E virus in a liver transplant recipient who regularly consumes camel meat and milk. Gastroenterology 150(355–357):e353Google Scholar
  21. 21.
    Li TC, Ami Y, Suzaki Y, Yasuda SP, Yoshimatsu K, Arikawa J, Takeda N, Takaji W (2013) Characterization of full genome of rat hepatitis E virus strain from Vietnam. Emerg Infect Dis 19:115–118CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Li TC, Yoshizaki S, Yang T, Kataoka M, Nakamura T, Ami Y, Yuriko S, Takeda N, Wakita T (2016) Production of infectious ferret hepatitis E virus in a human hepatocarcinoma cell line PLC/PRF/5. Virus Res 213:283–288CrossRefPubMedGoogle Scholar
  23. 23.
    Li W, Guan D, Su J, Takeda N, Wakita T, Li TC, Ke CW (2013) High prevalence of rat hepatitis E virus in wild rats in China. Vet Microbiol 165:275–280CrossRefPubMedGoogle Scholar
  24. 24.
    Moin SM, Panteva M, Jameel S (2007) The hepatitis E virus Orf3 protein protects cells from mitochondrial depolarization and death. J Biol Chem 282:21124–21133CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Mulyanto, Depamede SN, Sriasih M, Takahashi M, Nagashima S, Jirintai S, Nishizawa T, Okamoto H (2013) Frequent detection and characterization of hepatitis E virus variants in wild rats (Rattus rattus) in Indonesia. Arch Virol 158:87–96CrossRefPubMedGoogle Scholar
  26. 26.
    Mulyanto, Suparyatmo JB, Andayani IGAS, Khalid, Takahashi M, Ohnishi H, Jirintai S, Nagashima S, Nishizawa T, Okamoto H (2014) Marked genomic heterogeneity of rat hepatitis E virus strains in Indonesia demonstrated on a full-length genome analysis. Virus Res 179:102–112CrossRefPubMedGoogle Scholar
  27. 27.
    Nagashima S, Takahashi M, Jirintai, Tanaka T, Yamada K, Nishizawa T, Okamoto H (2011) A PSAP motif in the ORF3 protein of hepatitis E virus is necessary for virion release from infected cells. J Gen Virol 92:269–278CrossRefPubMedGoogle Scholar
  28. 28.
    Nagashima S, Takahashi M, Jirintai S, Tanaka T, Nishizawa T, Yasuda J, Okamoto H (2011) Tumour susceptibility gene 101 and the vacuolar protein sorting pathway are required for the release of hepatitis E virions. J Gen Virol 92:2838–2848CrossRefPubMedGoogle Scholar
  29. 29.
    Nagashima S, Jirintai S, Takahashi M, Kobayashi T, Tanggis, Nishizawa T, Kouki T, Yashiro T, Okamoto H (2014) Hepatitis E virus egress depends on the exosomal pathway, with secretory exosomes derived from multivesicular bodies. J Gen Virol 95:2166–2175CrossRefPubMedGoogle Scholar
  30. 30.
    Pavio N, Meng XJ, Doceul V (2015) Zoonotic origin of hepatitis E. Curr Opin Virol 10:34–41CrossRefPubMedGoogle Scholar
  31. 31.
    Purcell RH, Emerson SU (2008) Hepatitis E: an emerging awareness of an old disease. J Hepatol 48:494–503CrossRefPubMedGoogle Scholar
  32. 32.
    Purcell RH, Engle RE, Rood MP, Kabrane-Lazizi Y, Nguyen HT, Govindarajan S, St Claire M, Emerson SU (2011) Hepatitis E virus in rats, Los Angeles, California, USA. Emerg Infect Dis 17:2216–2222CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Ratra R, Kar-Roy A, Lal SK (2009) ORF3 protein of hepatitis E virus interacts with the Bbeta chain of fibrinogen resulting in decreased fibrinogen secretion from HuH-7 cells. J Gen Virol 90:1359–1370CrossRefPubMedGoogle Scholar
  34. 34.
    Smith DB, Simmonds P, Jameel S, Emerson SU, Harrison TJ, Meng XJ, Okamoto H, Van der Poel WH, Purdy MA (2014) Consensus proposals for classification of the family Hepeviridae. J Gen Virol 95:2223–2232CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Takahashi M, Yamada K, Hoshino Y, Takahashi H, Ichiyama K, Tanaka T, Okamoto H (2008) Monoclonal antibodies raised against the ORF3 protein of hepatitis E virus (HEV) can capture HEV particles in culture supernatant and serum but not those in feces. Arch Virol 153:1703–1713CrossRefPubMedGoogle Scholar
  36. 36.
    Takahashi M, Tanaka T, Takahashi H, Hoshino Y, Nagashima S, Jirintai, Mizuo H, Yazaki Y, Takagi T, Azuma M, Kusano E, Isoda N, Sugano K, Okamoto H (2010) Hepatitis E virus (HEV) strains in serum samples can replicate efficiently in cultured cells despite the coexistence of HEV antibodies: characterization of HEV virions in blood circulation. J Clin Microbiol 48:1112–1125CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Tanaka T, Takahashi M, Kusano E, Okamoto H (2007) Development and evaluation of an efficient cell-culture system for hepatitis E virus. J Gen Virol 88:903–911CrossRefPubMedGoogle Scholar
  39. 39.
    Thiry D, Mauroy A, Pavio N, Purdy MA, Rose N, Thiry E, de Oliveira-Filho EF (2015) Hepatitis E virus and related viruses in animals. Transbound Emerg Dis. doi: 10.1111/tbed.12351
  40. 40.
    Tyagi S, Surjit M, Roy AK, Jameel S, Lal SK (2004) The ORF3 protein of hepatitis E virus interacts with liver-specific alpha1-microglobulin and its precursor alpha1-microglobulin/bikunin precursor (AMBP) and expedites their export from the hepatocyte. J Biol Chem 279:29308–29319CrossRefPubMedGoogle Scholar
  41. 41.
    Usuda S, Tsuda F, Gotanda T, Tachibana K, Nomura M, Okamoto H, Imai M, Nakamura T, Miyakawa Y, Mayumi M (1986) A solid-phase enzyme immunoassay for the common and subtypic determinants of hepatitis B surface antigen with monoclonal antibodies. J Immunol Methods 87:203–210CrossRefPubMedGoogle Scholar
  42. 42.
    Welsch S, Muller B, Krausslich HG (2007) More than one door—budding of enveloped viruses through cellular membranes. FEBS Lett 581:2089–2097CrossRefPubMedGoogle Scholar
  43. 43.
    Widen F, Ayral F, Artois M, Olofson AS, Lin J (2014) PCR detection and analysis of potentially zoonotic hepatitis E virus in French rats. Virol J 11:90CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Wolf S, Reetz J, Johne R, Heiberg AC, Petri S, Kanig H, Ulrich RG (2013) The simultaneous occurrence of human norovirus and hepatitis E virus in a Norway rat (Rattus norvegicus). Arch Virol 158:1575–1578CrossRefPubMedGoogle Scholar
  45. 45.
    Yamada K, Takahashi M, Hoshino Y, Takahashi H, Ichiyama K, Nagashima S, Tanaka T, Okamoto H (2009) ORF3 protein of hepatitis E virus is essential for virion release from infected cells. J Gen Virol 90:1880–1891CrossRefPubMedGoogle Scholar
  46. 46.
    Yin X, Ambardekar C, Lu Y, Feng Z (2016) Distinct entry mechanisms for nonenveloped and quasi-enveloped hepatitis E viruses. J Virol 90:4232–4242CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Zhou X, de Man RA, de Knegt RJ, Metselaar HJ, Peppelenbosch MP, Pan Q (2013) Epidemiology and management of chronic hepatitis E infection in solid organ transplantation: a comprehensive literature review. Rev Med Virol 23:295–304CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Wien 2016

Authors and Affiliations

  1. 1.Division of Virology, Department of Infection and ImmunityJichi Medical University School of MedicineShimotsukeJapan
  2. 2.West Nusa Tenggara Hepatitis LaboratoryMataramIndonesia
  3. 3.Immunobiology Laboratory, Faculty of MedicineUniversity of MataramMataramIndonesia
  4. 4.Center for Experimental MedicineJichi Medical University School of MedicineShimotsukeJapan

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