Skip to main content
PMC home page
Elsevier - PMC COVID-19 Collection logoLink to Elsevier - PMC COVID-19 Collection
. 2004 Feb 23;185(1):120–131. doi: 10.1016/0042-6822(91)90760-9

Hepatitis E virus (HEV): Molecular cloning and sequencing of the full-length viral genome

Albert W Tam ∗∗, Matthew M Smith ∗∗, Martha E Guerra ∗∗, Chiao-Chain Huang ∗∗, Daniel W Bradley ∗, Kirk E Fry ∗∗, Gregory R Reyes ∗∗,1
PMCID: PMC7130833  PMID: 1926770

Abstract

We have recently described the cloning of a portion of the hepatitis E virus (HEV) and confirmed its etiologic association with enterically transmitted (waterborne, epidemic) non-A, non-B hepatitis. The virus consists of a single-stranded, positive-sense RNA genome of approximately 7.5 kb, with a polyadenylated 3' end. We now report on the cloning and nucleotide sequencing of an overlapping, contiguous set of cDNA clones representing the entire genome of the HEV Burma strain [HEV(B)]. The largest open reading frame extends approximately 5 kb from the Fend and contains the RNA-directed RNA polymerase and nucleoside triphosphate binding motifs. The second major open reading frame (ORF2) begins 37 by downstream of the first and extends approximately 2 kb to the termination codon present 65 by from the 3' terminal stretch of poly(A) residues. ORF2 contains a consensus signal peptide sequence at its amino terminus and a capsid-like region with a high content of basic amino acids similar to that seen with other virus capsid proteins. A third open reading frame partially overlaps the first and second and encompasses only 369 bp. In addition to the 7.5-kb full-length genomic transcript, two subgenomic polyadenylated messages of approximately 3.7 and 2.0 kb were detected in infected liver using a probe from the 3' third of the genome. The genomic organization of the virus is consistent with the Fend encoding nonstructural and the 3' end encoding the viral structural gene(s). The expression strategy of the virus involves the use of three different open reading frames and at least three different transcripts. HEV was previously determined to be a nonenveloped particle with a diameter of 27–34 nm. These findings on the genetic organization and expression strategy of HEV suggest that it is the prototype human pathogen for a new class of RNA virus or perhaps a separate genus within the Caliciviridae family

References

  1. Andiaparidze A.G., Balayan M.S., Savinov A.P., Braginsky D.M., Poleschuk V.F., Zamyatina N.A. Fecal-oral transmitted non-A, non-B hepatitis induced in monkeys. Vopr. Virusol. 1986;1:73–80. [Google Scholar]
  2. Arankalle V.A., Ticehurst J., Sreenivasan M.A. Aetiological association of a virus-like particle with enterically transmitted non-A, non-B hepatitis. Lancet. 1988;1:550–554. doi: 10.1016/s0140-6736(88)91351-7. [DOI] [PubMed] [Google Scholar]
  3. Aviv H., Leder P. Vol. 69. 1972. Purification of biologically active globin messenger RNA by chromatography on oligo-thymidylic acid-cellulose; pp. 1408–1412. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Balayan M.S., Andiaparidze A.G., Savinskaya S.S. Evidence for a virus in non-A, non-B hepatitis transmitted via the fecal-oral route. Intervirology. 1983;20:23–31. doi: 10.1159/000149370. [DOI] [PubMed] [Google Scholar]
  5. Bradley D.W. Hepatitis non-A, non-B viruses become identified as hepatitis C and E viruses. Prog. Med. Virol. 1990;37:101–135. [PubMed] [Google Scholar]
  6. Bradley D.W. Enterically-transmitted non-A, non-B hepatitis. Br. Med. Bull. 1990;46:442–461. doi: 10.1093/oxfordjournals.bmb.a072409. [DOI] [PubMed] [Google Scholar]
  7. Bradley D.W., Balayan M.S. Virus of enterically transmitted non-A, non-B hepatitis. Lancet. 1988;1:819. doi: 10.1016/s0140-6736(88)91674-1. [DOI] [PubMed] [Google Scholar]
  8. Bradley D.W., Maynard J.E. Etiology and natural history of post-transfusion and enterically-transmitted non-A, non-B hepatitis. Sem. Liver Dis. 1986;6:56–66. doi: 10.1055/s-2008-1040794. [DOI] [PubMed] [Google Scholar]
  9. Bradley D.W., Krawczynski K., Cook E.H., Jr. Vol. 84. 1987. Enterically transmitted non-A, non-B hepatitis: serial passage of disease in cynomolgus macaques and tamarins and recovery of disease-associated 27-to 34-nm viruslike particles; pp. 6277–6281. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bradley D.W., Krawczynski K., Cook E.H., Jr. Enterically transmitted non-A, non-B hepatitis: Etiology of disease and laboratory studies in nonhuman primates. In: Zuckerman A.J., editor. Viral Hepatitis and Liver Disease. A. R. Liss; New York: 1988. pp. 138–147. [Google Scholar]
  11. Bradley D.W., Andjaparidze A., Cook E.H., Jr. Aetiological agent of enterically transmitted non-A, non-B hepatitis. J. Gen. Virol. 1988;69:731–738. doi: 10.1099/0022-1317-69-3-731. [DOI] [PubMed] [Google Scholar]
  12. Cathala G., Savonret I.-F., Mendez R. A method for isolation of intact translationally active ribonucleic acid. DNA. 1983;2:329–335. doi: 10.1089/dna.1983.2.329. [DOI] [PubMed] [Google Scholar]
  13. Choo Q.-L., Kuo G., Weiner A.J., Overby L.R., Bradley D.W., Houghton M. Isolation of cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science. 1989;244:359–362. doi: 10.1126/science.2523562. [DOI] [PubMed] [Google Scholar]
  14. Dalgarno L., Rice C.M., Strauss J.H. Ross river virus 26S RNA: Complete nucleotide sequence and deduced sequence of the encoded structural proteins. Virology. 1983;129:170–187. doi: 10.1016/0042-6822(83)90404-x. [DOI] [PubMed] [Google Scholar]
  15. Feinstone S.M., Kapikian A.Z., Purcell R.H., Alter H.J., Holland P.V. Transfusion-associated hepatitis not due to viral hepatitis type A or B. N. Engl. J. Med. 1975;292:767–770. doi: 10.1056/NEJM197504102921502. [DOI] [PubMed] [Google Scholar]
  16. Frey T.K., Mark L.D. Sequence of the region coding for virion proteins C and E2 and the carboxy terminus of the nonstructural proteins of rubella virus: Comparison with alphaviruses. Gene. 1988;62:85–99. doi: 10.1016/0378-1119(88)90582-3. [DOI] [PubMed] [Google Scholar]
  17. Fry K.E., Tam A.W., Smith M.M. Hepatitis E virus (HEV): Strain variation in the nonstructural gene region encoding consensus motifs for an RNA-dependent RNA polymerase and an ATP/GTP binding site. Virus Genes. 1991 doi: 10.1007/BF01703066. in press. [DOI] [PubMed] [Google Scholar]
  18. Geider K., Hoffman-Berling H. Vol. 50. 1981. Proteins controlling the helical structure of DNA; pp. 233–260. (Annu. Rev. Biochem.). [DOI] [PubMed] [Google Scholar]
  19. Gentry G.A. Vol. 82. 1985. Locating a nucleotide binding site in the thymidine kinase of vaccinia virus and of herpes simplex virus by scoring triply aligned protein sequences; pp. 6815–6819. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gorbalenya A.E., Koonin E.V., Donchenko A.P., Blinov V.M. Two related superfamilies of putative helicases involved in replication, recombination, repair and expression of DNA and RNA genomes. Nucleic Acids Res. 1989;17:4713–4730. doi: 10.1093/nar/17.12.4713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Huang C.-C., Nguyen D., Fernandez J. 1991. Hepatitis E virus: Cloning of a Mexican strain and comparison to the Burmese strain. in preparation. [Google Scholar]
  22. Kamer G., Argos P. Primary structural comparison of RNA-dependent polymerases from plant, animal, and bacterial viruses. Nucleic Acids Res. 1984;12:7269–7282. doi: 10.1093/nar/12.18.7269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Khuroo M.S. Study of an epidemic of non-A, non-B hepatitis: Possibility of another human hepatitis virus distinct from posttransfusion non-A, non-B type. Am. J. Med. 1980;68:818–824. doi: 10.1016/0002-9343(80)90200-4. [DOI] [PubMed] [Google Scholar]
  24. Klein P., Kanehisa M., Delisi C. The detection and classification of membrane-spanning proteins. Biochem. Biophys. Acta. 1985;815:468–476. doi: 10.1016/0005-2736(85)90375-x. [DOI] [PubMed] [Google Scholar]
  25. Krawczynski K., Bradley D.W. Enterically transmitted non-A, non-B hepatitis: Identification of virus-associated antigen in experimentally infected cynomolgus macaques. J. Infect. Dis. 1989;159:1042–1049. doi: 10.1093/infdis/159.6.1042. [DOI] [PubMed] [Google Scholar]
  26. Kubo Y., Takeuchi K., Boonmar S. A cDNA fragment of hepatitis C virus isolated from an implicated donor of post-transfusion non-A, non-B hepatitis in Japan. Nucleic Acids Res. 1989;17:10367–10372. doi: 10.1093/nar/17.24.10367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kuo G., Choo Q.-L., Alter H.J. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science. 1989;244:362–364. doi: 10.1126/science.2496467. [DOI] [PubMed] [Google Scholar]
  28. Maeno M., Kaminaka K., Sugimoto H. A cDNA clone closely associated with non-A, non-B hepatitis. Nucleic Acids Res. 1990;18:2685–2689. doi: 10.1093/nar/18.9.2685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. McKnight S.L., Gavis E.R., Kingsbury R. Analysis of transcriptional regulatory signals of the HSV thymidine kinase gene: Identification of an upstream control region. Cell. 1981;25:385–398. doi: 10.1016/0092-8674(81)90057-x. [DOI] [PubMed] [Google Scholar]
  30. Morch M.-D., Haenni A.L. Organization of plant virus genomes that comprise a single RNA molecule. In: Rowlands D.J., Mayo M.A., Mahy B.W.J., editors. The Molecular Biology of Positive Strand RNA Viruses. Academic Press; London: 1987. pp. 153–175. [Google Scholar]
  31. Mullis K.B., Faloona F.A. Specific synthesis of DNA in vitro via a polymerase catalysed chain reaction. In: Wu R., editor. Vol 155. Academic Press; New York: 1987. pp. 335–350. (Methods in Enzymology). [DOI] [PubMed] [Google Scholar]
  32. Neill J.D. Nucleotide sequence of a region of the feline calicivirus genome which encodes picornavirus-like RNA-dependent RNA polymerase, cysteine protease and 2C polypeptides. Virus Res. 1990;17:145–160. doi: 10.1016/0168-1702(90)90061-f. [DOI] [PubMed] [Google Scholar]
  33. Nussinov E. Eukaryotic dinucleotide preference rules and their implications for degenerate codon usage. J. Mol. Biol. 1981;149:125–131. doi: 10.1016/0022-2836(81)90264-3. [DOI] [PubMed] [Google Scholar]
  34. Ou J.-H., Rice C.M., Dalgarno L. Vol. 79. 1982. Sequence studies of several alphavlrus genomic RNAs in the region containing the start of the subgenomic RNA; pp. 5235–5239. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Prince A.M., Brotman B., Grady G.F. Long-incubation post-transfusion hepatitis without serological evidence of exposure to hepatitis B virus. Lancet. 1974;2:241–246. doi: 10.1016/s0140-6736(74)91412-3. [DOI] [PubMed] [Google Scholar]
  36. Purcell R.H., Ticehurst J.R. Enterically transmitted non-A, non-B hepatitis: epidemiology and clinical characteristics. In: Zuckerman A.J., editor. Viral Hepatitis and Liver Disease. A. R. Liss; New York: 1988. pp. 131–137. [Google Scholar]
  37. Reyes G.R., Baroudy B.M. Molecular biology of non-A, non-B hepatitis agents: the hepatitis C and hepatitis E viruses. In: Maramorosch K., Murphy F.A., Shatkin A.J., editors. Vol. 40. Academic Press; Orlando: 1991. pp. 55–101. (Advances in Virus Research). [DOI] [PubMed] [Google Scholar]
  38. Reyes G.R. Molecular Cloning of the hepatitis E Virus. In: Hollinger F.B., Lemon S.M., Margolis H.S., editors. Viral Hepatitis and Liver Disease. Williams & Wilkens; Baltimore: 1991. pp. 514–517. [Google Scholar]
  39. Reyes G.R., Huang C.-C., Yarbough P.O. Hepatitis E virus (HEV): Epitope mapping and detection of strain variation. In: Shikata T., Purcell R.H., Uchida T., editors. Viral Hepatitis C, D and E. Elsevier; Amsterdam: 1991. pp. 237–245. [Google Scholar]
  40. Reyes G.R., Yarbough P.O., Tam A.W., Purdy M.A., Huang C.-C., Kim J.K., Bradley D.W., Fry K.E. Hepatitis E Virus (HEV): The novel agent responsible for enterically transmitted non-A, non-B hepatitis. Gastroenterol. Jpn. 1991;26(suppl 3):142–147. doi: 10.1007/BF02779285. [DOI] [PubMed] [Google Scholar]
  41. 1991.Reyes, G. R., Kim. K. P., et al. (1991d), in preparation.
  42. Reyes G.R., Purdy M.A., Kim J.P. Isolation of a cDNA from the virus responsible for enterically transmitted non-A, non-B hepatitis. Science. 1990;247:1335–1339. doi: 10.1126/science.2107574. [DOI] [PubMed] [Google Scholar]
  43. Rice C.M., Lenches E.M., Eddy S.R. Nucleotide sequence of yellow fever virus: Implications for flavivirus gene expression and evolution. Science. 1985;229:726–733. doi: 10.1126/science.4023707. [DOI] [PubMed] [Google Scholar]
  44. Saiki R.K., Scharf S., Faloona F. Enzymatic amplification of b-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985;230:1350–1354. doi: 10.1126/science.2999980. [DOI] [PubMed] [Google Scholar]
  45. Sanger F., Nicklen S., Coulson A.R. Vol. 74. 1977. DNA sequencing with chain terminating inhibitors; pp. 5463–5467. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Shepherd J.C.W. Vol. 78. 1981. Method to determine the reading frame of a protein from the purine/pyrimidine genome sequence and its possible evolutionary justification; pp. 1596–1600. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Siddell S. The organization and expression of coronavirus genomes. In: Rowlands D.J., Mayo M.A., Mahy B.W.J., editors. The Molecular Biology of the Positive Strand RNA Viruses. Academic Press; London: 1987. pp. 117–127. [Google Scholar]
  48. Strauss J.H., Strauss E.G. Evolution of RNA viruses. Annu. Rev. Microbiol. 1988;42:657–683. doi: 10.1146/annurev.mi.42.100188.003301. [DOI] [PubMed] [Google Scholar]
  49. Tabor E. The three viruses of non-A, non-B hepatitis. Lancet. 1985;1:743–745. doi: 10.1016/s0140-6736(85)91274-7. [DOI] [PubMed] [Google Scholar]
  50. Tam A.W., Smith M.M., Fry K.E., Larrick J.W. Construction of cDNA libraries from small numbers of cells using sequence independent primers. Nucleic Acids Res. 1989;17:1269. doi: 10.1093/nar/17.3.1269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wong D.C., Purcell R.H., Sreenivasan M.A., Prasad S.R., Pavri K.M. Epidemic and endemic hepatitis in India: evidence for a non-A, non-B hepatitis virus aetiology. Lancet. 1980;2:876–879. doi: 10.1016/s0140-6736(80)92045-0. [DOI] [PubMed] [Google Scholar]
  52. Yarbough P.O., Tam A.W., Fry K.E. Hepatitis E virus: Identification of type common epitopes. J. Virol. 1991 doi: 10.1128/jvi.65.11.5790-5797.1991. in press. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Virology are provided here courtesy of Elsevier

RESOURCES