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 ...

Background & Aims The diagnosis of drug-induced liver injury relies upon exclusion of other causes, including viral hepatitis A, B, and C. Hepatitis E virus (HEV) infection has been proposed as another cause of suspected drug-induced liver disease. We assessed the frequency of HEV infection among patients with drug-induced liver injury in the United States. Methods The drug-induced liver injury network (DILIN) is a prospective study of patients with suspected drug-induced liver injury; clinical information and biological samples are collected to investigate pathogenesis and disease progression. We analyzed serum samples, collected from patients enrolled in DILIN, for immunoglobulin (Ig)G and IgM against HEV; selected samples were tested for HEV RNA. Results Among 318 patients with suspected drug-induced liver injury, 50 (16%) tested positive for anti-HEV IgG and 9 (3%) for anti-HEV IgM. The samples that contained anti-HEV IgM (collected 2 to 24 weeks after onset of symptoms) included 4 that tested positive for HEV RNA, genotype 3. Samples from the 6-month follow-up visit were available from 4 patients; they were negative for anti-HEV IgM, but levels of anti-HEV IgG increased with time. Patients that had anti-HEV IgM were mostly from older men (89%; mean age, 67 years) and 2 were HIV positive. Clinical reassessment of the 9 patients with anti-HEV IgM indicated that acute hepatitis E was the most likely diagnosis for 7 and might be the primary diagnosis for 2. Conclusion HEV infection contributes to a small but important proportion of cases of acute liver injury that are suspected of being drug induced. Serologic testing for HEV infection should be performed—particularly if clinical features are compatible with acute viral hepatitis.

Hepatitis E virus recombinant genomes transcribed in vitro from two cDNA clones differing by two nucleotides were infectious for chimpanzees. However, one cDNA clone encoded a virus that was attenuated for chimpanzees and unable to infect rhesus monkeys. The second cDNA clone encoded a virus that infected both chimpanzees and rhesus monkeys and caused acute hepatitis in both. One mutation differentiating the two clones identified a cisreactive element that appeared to overlap the 3 end of the capsid gene and part of the 3 noncoding region. Capping of the RNA transcripts was essential for infectivity.

Hepatitis E virus (HEV) RNA replication occurred in seven of nine primate cell cultures transfected with in vitro transcripts of an infectious cDNA clone. Cell-to-cell spread did not occur in cell cultures, but rhesus monkeys inoculated with lysates of HEV-transfected PLC/PRF/5 and Huh-7 cells became infected with HEV. A replicon with the ORF2 and ORF3 genes deleted and replaced with the green fluorescent protein gene also replicated in the same primate cells that supported the replication of the full-length genome. Fluorescenceactivated cell sorter analysis confirmed that the 7mG cap structure was critical for efficient infectivity, although replication could be initiated at a very low level in its absence. HEV virions were also able to infect a limited number of cells of certain lines.Hepatitis E virus (HEV), the prototype Hepevirus, and hepatitis A virus (HAV) together are the major etiological agents of enterically transmitted hepatitis (4,8,23). HEV has a higher mortality rate, especially in pregnant women, but the reason for this is unknown. Otherwise, the disease caused by the one virus is clinically indistinguishable from that caused by the other, and neither progresses to chronicity. Both viruses have nonenveloped capsids, and both contain a single stranded RNA genome of the positive sense which serves as an mRNA to initiate infection.In spite of the similarities, HEV and HAV have very different epidemiologies. HAV age-related seroprevalence patterns are those expected for a virus that is transmitted by the fecaloral route, whereas those of HEV are not, even though fecal contamination is the major source of transmission. In countries in which the virus is endemic, anti-HAV antibodies are generally acquired before the age of 5 years, whereas the major rise in anti-HEV seroprevalence occurs later, in young adults (3). HAV has been found only in humans and in some nonhuman primates. HEV, on the other hand, has been isolated from humans and swine (7,9,20): additionally, antibodies reactive with capsid protein from human strains of HEV have been found in many animals, including nonhuman primates (2) and multiple species of rodents including rats (6, 11). A genotype 3 strain of HEV naturally infecting swine has been passed experimentally to monkeys, and a genotype 3 strain infecting humans has been passed to swine (18). However, attempts to transmit other human strains to swine have failed (19). The question of whether HEV is a zoonosis is still open (17), but a recent cluster of hepatitis E cases in Japan was traced to ingestion of raw deer meat, suggesting that this may be the case (26).The molecular biology of HEV replication is not well un- Much of the scant knowledge concerning HEV at the molecular level has been obtained through the overexpression of recombinant proteins in vitro. In addition to identification of an active viral RNA-dependent RNA polymerase, (1), such studies have led to the demonstration of guanylyltransferase and methyltransferase activities (15), two enzymatic activities required to ...

Hepatitis E virus genotype 1 strain Sar55 replicated in subcloned Caco-2 intestinal cells and Huh7 hepatoma cells that had been transfected with in vitro transcribed viral genomes, and hepatitis E virions were released into the culture medium of both cell lines. Virus egress from cells depended on open reading frame 3 (ORF3) protein, and a proline-rich sequence in ORF3 was important for egress from cultured cells and for infection of macaques. Both intracellular ORF3 protein accumulation and virus release occurred at the apical membrane of polarized Caco-2 cells. ORF3 protein and lipids were intimately associated with virus particles produced in either cell line; ORF2 epitopes were masked in these particles and could not be immunoprecipitated with anti-ORF2.Hepatitis E virus (HEV) remains enigmatic in spite of recent advances (see references 7 and 16 for reviews). HEV is a major cause of acute hepatitis in numerous developing countries, but hepatitis E is infrequently detected in industrialized countries even though seroprevalence rates of anti-HEV as high as 20% in these countries have been reported. Although hepatitis E normally is a self-limited acute disease, recent studies have identified it as an emerging cause of chronic hepatitis in immunocompromised patients. Whereas contaminated drinking water is the source of most infections in developing countries, the sources in industrialized countries are not fully evaluated, but many, if not most, infections appear linked to eating undercooked meat, especially pork. These differences in epidemiology may reflect the fact that most infections in developing countries are caused by genotypes 1 and 2 while those in industrialized countries are mainly due to genotypes 3 and 4.

Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with epithelial-cell cancers and B cell lymphomas. An effective EBV vaccine is not available. We found that antibodies to the EBV glycoprotein gH/gL complex were the principal components in human plasma that neutralized infection of epithelial cells and that antibodies to gH/gL and gp42 contributed to B cell neutralization. Immunization of mice and nonhuman primates with nanoparticle vaccines that displayed components of the viral-fusion machinery EBV gH/gL or gH/gL/gp42 elicited antibodies that potently neutralized both epithelial-cell and B cell infection. Immune serum from nonhuman primates inhibited EBV-glycoprotein-mediated fusion of epithelial cells and B cells and targeted an epitope critical for virus-cell fusion. Therefore, unlike the leading EBV gp350 vaccine candidate, which only protects B cells from infection, these EBV nanoparticle vaccines elicit antibodies that inhibit the virus-fusion apparatus and provide cell-type-independent protection from virus infection.