Abstract
Ten murine monoclonal antibodies (MAbs) against a synthetic peptide corresponding to the well-conserved, C-terminal 24-amino acid portion of ORF3 protein of hepatitis E virus (HEV) were produced and characterized. Immunofluorescent assays using the anti-ORF3 MAbs revealed accumulation of ORF3 protein in the cytoplasm of PLC/PRF/5 cells transfected with ORF3-expressing plasmid or inoculated with cell-culture-generated HEV. The anti-ORF3 MAbs could capture HEV particles in culture medium and serum at variable efficiency of up to 61 and 49%, respectively, but not those in feces. By sandwiching between immobilized and enzyme-labeled anti-ORF3 MAbs in ELISA, ORF3 antigen was detected in the culture media with an HEV RNA titer of >106 copies/ml and increased in parallel with the increase in HEV load. HEV progenies in the culture supernatant, with ORF3 protein on the surface, banded at a low buoyant density of 1.15 g/cm3 in sucrose. A representative anti-ORF3 MAb (TA0536) could partially neutralize the infection of cell-culture-generated HEV in a cell culture system. These results indicate that ORF3 protein, at least its C-terminal portion, is present on the surface of HEV virions released from infected cells and support a previously proposed assumption that ORF3 protein is associated with virus release from infected cells.
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References
Amon JJ, Drobeniuc J, Bower WA, Magana JC, Escobedo MA, Williams IT, Bell BP, Armstrong GL (2006) Locally acquired hepatitis E virus infection, El Paso, Texas. J Med Virol 78:741–746
Arankalle VA, Chobe LP (2000) Retrospective analysis of blood transfusion recipients: evidence for post-transfusion hepatitis E. Vox Sang 79:72–74
Balayan MS, Andjaparidze AG, Savinskaya SS, Ketiladze ES, Braginsky DM, Savinov AP, Poleschuk VF (1983) Evidence for a virus in non-A, non-B hepatitis transmitted via the fecal-oral route. Intervirology 20:23–31
Emerson SU, Anderson D, Arankalle A, Meng XJ, Purdy M, Schlauder GG, Tsarev SA (2004) Hepevirus. In: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA (eds) Virus taxonomy, the eighth report of the international committee on taxonomy of viruses. Elsevier/Academic Press, London, pp 851–855
Emerson SU, Nguyen H, Graff J, Stephany DA, Brockington A, Purcell RH (2004) In vitro replication of hepatitis E virus (HEV) genomes and of an HEV replicon expressing green fluorescent protein. J Virol 78:4838–4846
Emerson SU, Clemente-Casares P, Moiduddin N, Arankalle VA, Torian U, Purcell RH (2006) Putative neutralization epitopes and broad cross-genotype neutralization of hepatitis E virus confirmed by a quantitative cell-culture assay. J Gen Virol 87:697–704
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 80:10457–10464
Emerson SU, Purcell RH (2007) Hepatitis E virus. In: Knipe DM, Howley PM, Griffin DE, Lamb RA, Martin MA, Roizman B, Straus SE (eds) Fields virology, 5th edn. Lippincott Williams and Wilkins, Philadelphia, pp 3046–3058
Graff J, Nguyen H, Yu C, Elkins WR, St Claire M, Purcell RH, Emerson SU (2005) The open reading frame 3 gene of hepatitis E virus contains a cis-reactive element and encodes a protein required for infection of macaques. J Virol 79:6680–6689
Graff J, Torian U, Nguyen H, Emerson SU (2006) A bicistronic subgenomic mRNA encodes both the ORF2 and ORF3 proteins of hepatitis E virus. J Virol 80:5919–5926
Haagsma EB, van den Berg AP, Porte RJ, Benne CA, Vennema H, Reimerink JH, Koopmans MP (2008) Chronic hepatitis E virus infection in liver transplant recipients. Liver Transpl 14:547–553
Harrison TJ (1999) Hepatitis E virus-an update. Liver 19:171–176
Huang YW, Opriessnig T, Halbur PG, Meng XJ (2007) Initiation at the third in-frame AUG codon of open reading frame 3 of the hepatitis E virus is essential for viral infectivity in vivo. J Virol 81:3018–3026
Ijaz S, Arnold E, Banks M, Bendall RP, Cramp ME, Cunningham R, Dalton HR, Harrison TJ, Hill SF, Macfarlane L, Meigh RE, Shafi S, Sheppard MJ, Smithson J, Wilson MP, Teo CG (2005) Non-travel-associated hepatitis E in England and Wales: demographic, clinical, and molecular epidemiological characteristics. J Infect Dis 192:1166–1172
Jothikumar N, Cromeans TL, Robertson BH, Meng XJ, Hill VR (2006) A broadly reactive one-step real-time RT-PCR assay for rapid and sensitive detection of hepatitis E virus. J Virol Methods 131:65–71
Kamar N, Selves J, Mansuy JM, Ouezzani L, Péron 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 358:811–817
Khuroo MS, Kamili S (2003) Aetiology and prognostic factors in acute liver failure in India. J Viral Hepat 10:224–231
Korkaya H, Jameel S, Gupta D, Tyagi S, Kumar R, Zafrullah M, Mazumdar M, Lal SK, Xiaofang L, Sehgal D, Das SR, Sahal D (2001) The ORF3 protein of hepatitis E virus binds to Src homology 3 domains and activates MAPK. J Biol Chem 276:42389–42400
Kwo PY, Schlauder GG, Carpenter HA, Murphy PJ, Rosenblatt JE, Dawson GJ, Mast EE, Krawczynski K, Balan V (1997) Acute hepatitis E by a new isolate acquired in the United States. Mayo Clin Proc 72:1133–1136
Lemon SM, Binn LN (1985) Incomplete neutralization of hepatitis A virus in vitro due to lipid-associated virions. J Gen Virol 66:2501–2505
Lu L, Li C, Hagedorn CH (2006) Phylogenetic analysis of global hepatitis E virus sequences: genetic diversity, subtypes and zoonosis. Rev Med Virol 16:5–36
Mansuy JM, Peron JM, Abravanel F, Poirson H, Dubois M, Miedouge M, Vischi F, Alric L, Vinel JP, Izopet J (2004) Hepatitis E in the south west of France in individuals who have never visited an endemic area. J Med Virol 74:419–424
Matsubayashi K, Nagaoka Y, Sakata H, Sato S, Fukai K, Kato T, Takahashi K, Mishiro S, Imai M, Takeda N, Ikeda H (2004) Transfusion-transmitted hepatitis E caused by apparently indigenous hepatitis E virus strain in Hokkaido, Japan. Transfusion 44:934–940
Meng XJ (2005) Hepatitis E as a zoonotic disease. In: Thomas HC, Lemon S, Zuckerman AJ (eds) Viral hepatitis, 3rd edn. Blackwell, Malden, pp 611–623
Meng XJ, Purcell RH, Halbur PG, Lehman JR, Webb DM, Tsareva TS, Haynes JS, Thacker BJ, Emerson SU (1997) A novel virus in swine is closely related to the human hepatitis E virus. Proc Natl Acad Sci USA 94:9860–9865
Meng XJ, Halbur PG, Shapiro MS, Govindarajan S, Bruna JD, Mushahwar IK, Purcell RH, Emerson SU (1998) Genetic and experimental evidence for cross-species infection by swine hepatitis E virus. J Virol 72:9714–9721
Mertens PPC, Maan S, Samuel A, Attoui H (2004) Orbivirus, reoviridae. In: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA (eds) Virus Taxonomy, the eighth report of the international committee on taxonomy of viruses. Elsevier/Academic Press, London, pp 466–483
Mitsui T, Tsukamoto Y, Yamazaki C, Masuko K, Tsuda F, Takahashi M, Nishizawa T, Okamoto H (2004) Prevalence of hepatitis E virus infection among hemodialysis patients in Japan: evidence for infection with a genotype 3 HEV by blood transfusion. J Med Virol 74:563–572
Mizuo H, Suzuki K, Takikawa Y, Sugai Y, Tokita H, Akahane Y, Itoh K, Gotanda Y, Takahashi M, Nishizawa T, Okamoto H (2002) Polyphyletic strains of hepatitis E virus are responsible for sporadic cases of acute hepatitis in Japan. J Clin Microbiol 40:3209–3218
Nakamura M, Takahashi K, Taira K, Taira M, Ohno A, Sakugawa H, Arai M, Mishiro S (2006) Hepatitis E virus infection in wild mongooses of Okinawa, Japan: demonstration of anti-HEV antibodies and a full-genome nucleotide sequence. Hepatol Res 34:137–140
Nanda SK, Ansari IH, Acharya SK, Jameel S, Panda SK (1995) Protracted viremia during acute sporadic hepatitis E virus infection. Gastroenterology 108:225–230
Nishizawa T, Takahashi M, Mizuo H, Miyajima H, Gotanda Y, Okamoto H (2003) Characterization of Japanese swine and human hepatitis E virus isolates of genotype IV with 99% identity over the entire genome. J Gen Virol 84:1245–1251
Okamoto H (2007) Genetic variability and evolution of hepatitis E virus. Virus Res 127:216–228
Okamoto H, Takahashi M, Nishizawa T, Fukai K, Muramatsu U, Yoshikawa A (2001) Analysis of the complete genome of indigenous swine hepatitis E virus isolated in Japan. Biochem Biophys Res Commun 289:929–936
Okamoto H, Takahashi M, Nishizawa T (2003) Features of hepatitis E virus infection in Japan. Intern Med 42:1065–1071
Pearson WR, Wood T, Zhang Z, Miller W (1997) Comparison of DNA sequences with protein sequences. Genomics 46:24–36
Schlauder GG, Mushahwar IK (2001) Genetic heterogeneity of hepatitis E virus. J Med Virol 65:282–292
Smith JL (2001) A review of hepatitis E virus. J Food Prot 64:572–586
Sonoda H, Abe M, Sugimoto T, Sato Y, Bando M, Fukui E, Mizuo H, Takahashi M, Nishizawa T, Okamoto H (2004) Prevalence of hepatitis E virus (HEV) infection in wild boars and deer and genetic identification of a genotype 3 HEV from a boar in Japan. J Clin Microbiol 42:5371–5374
Takahashi K, Machida A, Funatsu G, Nomura M, Usuda S, Aoyagi S, Tachibana K, Miyamoto H, Imai M, Nakamura T, Miyakawa Y, Mayumi M (1983) Immunochemical structure of hepatitis B e antigen in the serum. J Immunol 130:2903–2907
Takahashi K, Kitajima N, Abe N, Mishiro S (2004) Complete or near-complete nucleotide sequences of hepatitis E virus genome recovered from a wild boar, a deer, and four patients who ate the deer. Virology 330:501–505
Takahashi M, Nishizawa T, Miyajima H, Gotanda Y, Iita T, Tsuda F, Okamoto H (2003) Swine hepatitis E virus strains in Japan form four phylogenetic clusters comparable with those of Japanese isolates of human hepatitis E virus. J Gen Virol 84:851–862
Takahashi M, Tanaka T, Azuma M, Kusano E, Aikawa T, Shibayama T, Yazaki Y, Mizuo H, Inoue J, Okamoto H (2007) Prolonged fecal shedding of hepatitis E virus (HEV) during sporadic acute hepatitis E: evaluation of infectivity of HEV in fecal specimens in a cell culture system. J Clin Microbiol 45:3671–3679
Takahashi M, Hoshino Y, Tanaka T, Takahashi H, Nishizawa T, Okamoto H (2008) Production of monoclonal antibodies against hepatitis E virus capsid protein and evaluation of their neutralizing activity in a cell culture system. Arch Virol 153:657–666
Tam AW, Smith MM, Guerra ME, Huang C, Bradley DW, Fry KE, Reyes GR (1991) Hepatitis E virus (HEV): molecular cloning and sequence of the full-length viral genome. Virology 185:120–130
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 88:903–911
Tei S, Kitajima N, Takahashi K, Mishiro S (2003) Zoonotic transmission of hepatitis E virus from deer to human beings. Lancet 362:371–373
Tyagi S, Korkaya H, Zafrullah M, Jameel S, Lal SK (2002) The phosphorylated form of the ORF3 protein of hepatitis E virus interacts with its non-glycosylated form of the major capsid protein, ORF2. J Biol Chem 277:22759–22767
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 279:29308–29319
Tyagi S, Surjit M, Lal SK (2005) The 41-amino-acid C-terminal region of the hepatitis E virus ORF3 protein interacts with bikunin, a kunitz-type serine protease inhibitor. J Virol 79:12081–12087
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 87:203–210
Yazaki Y, Mizuo H, Takahashi M, Nishizawa T, Sasaki N, Gotanda Y, Okamoto H (2003) Sporadic acute or fulminant hepatitis E in Hokkaido, Japan, may be food-borne, as suggested by the presence of hepatitis E virus in pig liver as food. J Gen Virol 84:2351–2357
Zafrullah M, Ozdener MH, Panda SK, Jameel S (1997) The ORF3 protein of hepatitis E virus is a phosphoprotein that associates with the cytoskeleton. J Virol 71:9045–9053
Zhou YH, Purcell RH, Emerson SU (2005) A truncated ORF2 protein contains the most immunogenic site on ORF2: antibody responses to non-vaccine sequences following challenge of vaccinated and non-vaccinated macaques with hepatitis E virus. Vaccine 23:3157–3165
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This study was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Ministry of Health, Labor and Welfare of Japan.
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Takahashi, M., Yamada, K., Hoshino, Y. et al. 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, 1703–1713 (2008). https://doi.org/10.1007/s00705-008-0179-6
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DOI: https://doi.org/10.1007/s00705-008-0179-6