Elsevier

Virus Research

Volume 120, Issues 1–2, September 2006, Pages 57-69
Virus Research

Hepatitis E virus genotyping based on full-length genome and partial genomic regions☆

https://doi.org/10.1016/j.virusres.2006.01.013Get rights and content

Abstract

Some genomic regions for hepatitis E virus (HEV) genotyping have been reported to correlate well with the results from the phylogenetic analyses on the basis of the complete genome. However, few studies have systemically investigated the genomic regions for HEV genotyping using a combined phylogenetic and statistical approach. A consensus region for HEV genotyping has not been determined. In this study the nucleotide identities and genetic distances of 24 partial genomic regions and the complete genome sequences of 37 HEV strains were compared statistically. It was demonstrated with both one-way ANOVA and two-way ANOVA that only one genomic region in RNA-dependent RNA polymerase domain (4254–4560 nt) for which there were no significant differences when compared with the full-length genome (P > 0.05). The same four genotypes were identified by phylogenetic analysis based on this statistically predicted region identified as for the complete genome. RT-PCR amplification of HEV strains from all four genotypes confirmed conservation of the flanking primer sites of this region. Serum samples from 20 patients with a clinical diagnosis of hepatitis E were further analyzed by PCR using the same primers, 13 were positive and could be classified into genotype 4. These data strongly suggested that this newly identified region could be used for future HEV genotyping.

Introduction

Hepatitis E virus (HEV) is a major cause of enterically transmitted acute hepatitis in developing countries. Large epidemics have been reported in Asia, Africa and Latin America following fecal contamination of drinking water resources. In industrialized countries, sporadic cases of hepatitis E have been reported, either imported by travelers from endemic areas or of unspecified native origin (Emerson and Purcell, 2003).

HEV was classified recently as the sole member of the genus Hepevirus in the family Hepeviridae (Emerson et al., 2004). Its genome consists of a single-stranded, positive-sense RNA of approximately 7.2 kb, with three partially overlapping open reading frames (ORFs: ORF1, ORF2, and ORF3) (Reyes et al., 1990, Tam et al., 1991, Huang et al., 1992, Y. Wang et al., 2000). Although only one serotype has been identified to-date, HEV displays considerable genetic diversity. Based on the extensive full-length genomic variability noted among different strains, HEV has been classified into four major genotypes (genotypes 1–4). However, due to the time-consuming work of complete genome sequencing, many HEV strains were only partially sequenced. Consequently, different genotypes and sub-genotypes were proposed based on variable partial HEV genomic regions. Based on one 371-nucleotide region in ORF1 and a 147-nucleotide region in ORF2 of HEV genome, nine or more genotypes had been proposed for newly identified HEV strains (Schlauder and Mushahwar, 2001). Recently, 10 genotypes of HEV have been suggested based on a 287-nucleotide sequence analysis (Lu et al., 2004).

The use of different genomic regions for HEV genotyping by different laboratories makes it difficult to compare the results. Moreover, although some genomic regions show good correlations with the full-length genome in HEV genotyping by phylogenetic analyses, few studies have used both phylogenetic and statistical analyses to investigate the feasibility of using genomic regions to substitute the full-length genome for HEV genotyping. This lack of a consensus genomic region is an important problem for future HEV genetic classification, especially as more divergent HEV strains are included. Our recent study of the 5′ and 3′ terminal sequences of HEV showing the inconsistency in genotyping based on the 3′ UTR sequences and the complete genome sequences (Chen and Meng, 2004) strongly underlined the importance of finding a qualified consensus genomic region for HEV genotyping.

In this study, to identify a consensus genomic region that could substitute full-length genomes for HEV genotyping, 24 HEV partial genomic regions used for HEV genotyping by different laboratories worldwide were first compared statistically with complete genome sequences. Phylogenetic analyses and RT-PCR were carried out to further test the quality of the candidate genomic region. Our study reports the first statistically and phylogenetically verified genomic region for HEV genotyping.

Section snippets

Sequence analysis

Complete or nearly complete genome sequences of 37 HEV strains were retrieved from the GenBank (Table 1). Isolates with identical sequences to the 37 isolates except for modifications at the 5′ or 3′ termini were excluded (NC-001434, M94177 are identical to L08816; L25595 is identical to L25547; AF444002 is identical to AF444003). Sequences were aligned by Clustal W (version 1.8). The alignments were analyzed using the DNADIST program (Kimura 2-parameter) of PHYLIP package (version 3.6 b) with

Phylogenetic analysis of 37 HEV strains based on full-length genome sequences

The nucleotide identities and genetic distances between the 37 HEV strains were first calculated based on both the full-length genome and the 24 partial genomic regions. Only the data based on the full-length sequence analysis was shown in Fig. 1. Examination of the nucleotide identities demonstrated there was considerable nucleotide divergence between any two strains from different major groups (the shaded boxes) that had nucleotide identities to each other of less than 76.4%. A further

Discussion

Genotyping based on an individual gene instead of on the complete genome has been successfully applied to hepatitis B virus (Bartholomeusz and Schaefer, 2004) and hepatitis C virus (Hnatyszyn, 2005). For HEV genotyping, however, studies are still in their infancy. Two prerequisites need to be established for partial genomic region genotyping. First, sufficient variability must exist between strains of the HEV in the studied genomic regions to accurately reflect the complete genome variation.

Acknowledgements

We thank Guobing Chen for his valuable input on the phylogenetic analyses and Dr. Marit Nilsen-Hamilton in Iowa State University for her critical evaluation of this manuscript. This work was supported by the national nature science foundation of China (Grant nos. 30271231, 30271212), the nature science foundation of Jiangsu Province, China (Grant no. BK2002053), the medical technology development foundation of the Department of Health, Jiangsu Province, China (Grant no. H200115), the scientific

References (56)

  • A.W. Tam et al.

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

    Virology

    (1991)
  • H. van Cuyck et al.

    Phylogenetic analysis of the first complete hepatitis E virus (HEV) genome from Africa

    FEMS Immunol. Med. Microbiol.

    (2003)
  • H.C. Worm et al.

    Hepatitis E: an overview

    Microbes Infect.

    (2002)
  • V.A. Arankalle et al.

    Phylogenetic analysis of hepatitis E virus isolates from India (1976–1993)

    J. Gen. Virol.

    (1999)
  • T.T. Aye et al.

    Complete nucleotide sequence of a hepatitis E virus isolated from the Xinjiang epidemic (1986–1988) of China

    Nucleic Acids Res.

    (1992)
  • T.T. Aye et al.

    Sequence and gene structure of the hepatitis E virus isolated from Myanmar

    Virus Genes

    (1993)
  • A. Bartholomeusz et al.

    Hepatitis B virus genotypes: comparison of genotyping methods

    Rev. Med. Virol.

    (2004)
  • J.A. Bruenn

    Relationships among the positive and double-strand RNA viruses as viewed through their RNA-dependent RNA polymerase

    Nucleic Acids Res.

    (1991)
  • R. Chatterjee et al.

    African isolates of hepatitis E virus that are distinct from Asian isolates

    J. Med. Virol.

    (1997)
  • G.B. Chen et al.

    Identification of 5′ capped structure and 3′ terminal sequence of hepatitis E virus isolated from Morocco

    World J. Gastroenterol.

    (2004)
  • Donati, M.C., Fagan, E.A., Harrison, T.J., 1997. Sequence analysis of full-length HEV clones derived directly from...
  • Emerson, S.U., Anderson, D., Arankalle, A., Meng, X.J., Purdy, M., Schlauder, G.G., Tsarev, S.A., 2004. Hepevirus, in:...
  • S.U. Emerson et al.

    Hepatitis E virus

    Rev. Med. Virol.

    (2003)
  • S.U. Emerson et al.

    Recombinant hepatitis E virus genomes infectious for primates: importance of capping and discovery of a cis-reactive element

    Proc. Natl. Acad. Sci. U.S.A.

    (2001)
  • J.C. Erker et al.

    A hepatitis E virus variant from the United States: molecular characterization and transmission in cynomolgus macaques

    J. Gen. Virol.

    (1999)
  • O. Garkavenko et al.

    Detection and characterisation of swine hepatitis E virus in New Zealand

    J. Med. Virol.

    (2001)
  • H.J. Hnatyszyn

    Chronic hepatitis C and genotyping: the clinical significance of determining HCV genotypes

    Antivir. Ther.

    (2005)
  • R. Huang et al.

    Existing variations on the gene structure of hepatitis E virus isolates from some regions of China

    J. Med. Virol.

    (1995)

    • Cited by (0)

    ☆

    The GenBank/EMBL/DDBJ accession numbers for the nucleotide sequences reported in this paper are AY789220–AY789232.

    1

    Present address: Department of Biochemistry, Biophysics and Molecular Biology, 3288 Molecular Biology Building, Iowa State University, Ames, IA 50011-3260, USA. Tel.: +1 515 294 5682.

    View full text
    Copyright © 2006 Elsevier B.V. All rights reserved.