The RNA virus, hepatitis E virus (HEV) is the most or second-most important cause of acute clinical hepatitis in adults throughout much of Asia, the Middle East, and Africa. In these regions it is an important cause of acute liver failure, especially in pregnant women who have a mortality rate of 20–30%. Until recently, hepatitis E was rarely identified in industrialized countries, but Hepatitis E now is reported increasingly throughout Western Europe, some Eastern European countries, and Japan. Most of these cases are caused by genotype 3, which is endemic in swine, and these cases are thought to be zoonotically acquired. However, transmission routes are not well understood. HEV that infect humans are divided into nonzoonotic (types 1, 2) and zoonotic (types 3, 4) genotypes. HEV cell culture is inefficient and limited, and thus far HEV has been cultured only in human cell lines. The HEV strain Kernow-C1 (genotype 3) isolated from a chronically infected patient was used to identify human, pig, and deer cell lines permissive for infection. Cross-species infections by genotypes 1 and 3 were studied with this set of cultures. Adaptation of the Kernow-C1 strain to growth in human hepatoma cells selected for a rare virus recombinant that contained an insertion of 174 ribonucleotides (58 amino acids) of a human ribosomal protein gene.

An infectious cDNA clone of a genotype 3 strain of hepatitis E virus adapted to growth in HepG2/C3A human hepatoma cells was constructed. This virus was unusual in that the hypervariable region of the adapted virus contained a 171-nucleotide insertion that encoded 58 amino acids of human S17 ribosomal protein. Analyses of virus from six serial passages indicated that genomes with this insert, although initially rare, were selected during the first passage, suggesting it conferred a significant growth advantage. RNA transcripts from this cDNA and the viruses encoded by them were infectious for cells of both human and swine origin, the major host species for this zoonotic virus. Mutagenesis studies demonstrated that the S17 insert was a major factor in cell culture adaptation. Introduction of 54 synonymous mutations into the insert had no detectable effect, thus implicating protein, rather than RNA, as the important component. Truncation of the insert by 50% decreased the levels of successful transfection by ϳ3-fold. Substitution of the S17 sequence by a different ribosomal protein sequence or by GTPase-activating protein sequence resulted in a partial enhancement of transfection levels, whereas substitution with 58 amino acids of green fluorescent protein had no effect. Therefore, both the sequence length and the amino acid composition of the insert were important. The S17 sequence did not affect transfection of human hepatoma cells when inserted into the hypervariable region of a genotype 1 strain, but this chimeric genome acquired a dramatic ability to replicate in hamster cells.

Hepatitis E virus replicons containing the neomycin resistance gene expressed from open reading frames (ORFs) 2 and 3 were transfected into Huh-7 cells, and stable cell lines containing functional replicons were selected by constant exposure to G418 sulfate. Northern blot analyses detected full-length replicon RNA and a single subgenomic RNA. This subgenomic RNA, which was capped, initiated at nucleotide 5122 downstream of the first two methionine codons in ORF3 and was bicistronic; two closely spaced methionine codons in different reading frames were used for the initiation of ORF3 and ORF2 translation.Hepatitis E virus (HEV) was discovered in 1983 (3), but molecular characterization did not begin until the first fulllength genomic sequence was obtained by Tam et al. (19). However, lack of an efficient cell culture system for this virus has greatly hampered detailed analysis of the viral replication cycle. Therefore, many important questions about this virus remain unanswered.HEV is the sole member of the Hepeviridae family and of the genus Hepevirus (5). It is a human pathogen that causes hepatitis E, an acute self-limiting disease that does not progress to chronicity. There are four recognized genotypes that infect humans (18): genotypes 1 and 2 are thought to infect humans and nonhuman primates exclusively, whereas genotypes 3 and 4 also infect swine (2, 4). It is thought that hepatitis E may be a zoonosis, but the extent of transmission between animals and humans remains to be determined (14).The virion is 27 to 30 nm in diameter and does not possess an envelope (16). It most likely is icosahedral and is believed to be composed of a single capsid protein. The genome is a single-stranded, positive-sense RNA molecule of approximately 7.2 kb and is capped. The coding region is preceded by a short noncoding region of 25 nucleotides (nt) and is followed by a noncoding region of 65 nt and a poly(A) tract. The coding region consists of three partially overlapping open reading frames (ORFs). ORF1, consisting of approximately 5 kb, is located at the 5Ј end and encodes nonstructural proteins involved in RNA synthesis; these include guanylyl transferase, methyl transferase (13), and an RNA-dependent RNA polymerase (1, 9). ORF2, approximately 2 kb, occupies the 3Ј end of the coding region and encodes the capsid protein. ORF3 is a small reading frame of only 372 bases, with a 5Ј end that overlaps ORF1 by 4 nt and a 3Ј end that overlaps ORF2 by 331 nt; ORF3 could encode a protein with a maximum of 123 amino acids. The function(s) of ORF3 has not been fully defined, but it is postulated to interact with the ORF2 protein (22) and with cellular proteins involved in cell signaling (10, 23).Since HEV does not infect cultured cells efficiently, it has been difficult to determine how expression of the various viral proteins is regulated. Northern blot analyses of liver tissue from infected cynomolgus macaques detected genome-length RNA and two 3Ј-coterminal RNAs of 2 and 3.7 kb (19). Subsequently, two subgenomic RNAs were also ...