Hepatitis E

The story of the original discovery of hepatitis E virus (HEV) in Soviet Central Asia is worthy of Scheherazade. An intrepid Soviet virologist, Balayan, investigating an outbreak of hepatitis in Tashkent, volunteered himself to drink a pooled filtrate of stools from the patients; sure enough he developed hepatitis. After recovering a novel 32 nm virus from his own feces, he inoculated a filtrate of that material into monkeys, which in turn developed biochemical evidence of hepatitis and excreted in their stools a virus which he identified, by immunoelectron microscopy (IEM) using convalescent human sera, to be the same virus as was present in the original patients

Similar viruses were later recovered by others from enterically transmitted non-A, non-R hepatitis (ET-NANBH) outbreaks in India and many other countries of Asia as well as North Africa and Mexico. Bradley and colleagues then demonstrated that these several strains reacted in JEM with acute-phase sera from cases from many parts of the Third World and also with sera from monkeys in which hepatitis was induced by inoculation with geographically separated isolates.

Although the newly described agent of hepatitis E has yet to be repro-ducibly cultured, its genome has already been cloned and sequenced. Bile from the gallbladder of an experimentally infected cynomolgus macaque was assumed to be a relatively clean, high-titer source of the putative RNA virus. Nucleic acid was extracted, converted to cDNA by reverse transcriptase, amplified by PCR using random primers, cloned into the bacteriophage vector XgtlO, and expressed in bacteria. From the resulting cDNA library several cDNA fragments corresponding to genomic sequences of the putative NANBH virus were identified using labeled cDNA probes from the same original source. The nucleotide sequence of one of the clones was found to encode the RNA-dependent RNA polymerase motif characteristic of plus sense RNA viruses, and to hybridize to a 7.5 kb polyadenylated RNA from infected macaque liver and to cDNA derived by PCR from feces of human patients. By assembling a series of such overlapping cDNA fragments it finally proved possible to sequence the whole viral genome. The resulting data, together with that obtained by electron microscopic definition of the virion itself, have led to the allocation of the hepatitis E virus within the family Caliciviridae, at least for the time being.

Properties of Hepatilis E Virus

The spherical nonenveloped virion of HEV resembles that of other calici-viruses except that its icosahedral capsid with the characteristic surface depressions is perhaps more readily degraded by proteolysis. The particle is extremely labile, tending to lose its outer layer even during storage for a few days at 4°C, following freeze-thawing, during concentration by ultracentrilu-gation, or at the high salt concentration present during purification by cesium chloride equilibrium gradient centrifugation. Thus, depending on the source of the specimen and its subsequent preparation for electron microscopy, the virion can vary in diameter from 27 to 38 (mean 32) nm, and the characteristic "fuzzy loops" projecting from the surface may or may not be evident.

T he genome is a single 7.5 kb molecule of single-stranded RNA of positive sense, polyadenylated at its 3' end and having a 5' methylated cap (not a VPg protein cap). There are three separate but overlapping ORFs, the genes for the structural proteins being located in the middle of the genome. In these respects HEV is quite unlike the picomavirus causing hepatitis, hepatitis A virus Five functional domains have been identified in the nonstructural poly-protein of HEV. (1) RNA-dependent RNA polymerase, (2) RNA helicase, (3) methyltransferase, (4) "X" domain of unknown function, and (5) a papain-Iike cysteine protease. In terms of sequence homology within the five domains as well as colinearity of genome organization (except for the position of the protease domain) the HEV genome closely resembles that of rubella virus, an enveloped virus currently classified in its own genus of the family Togaviridae. It is conceivable that HEV could have evolved from rubella virus (or its progenitor), either by deletion of the envelope glycoprotein genes or by recombination with a calicivirus-like genome.

Sequencing of the genome of strains from around the world reveals that the American (Mexican) isolate is genetically distinct from the Asian isolates, suggesting evolutionary divergence in the distant past, whereas numerous more minor differences are discernible between the Asian isolates Despite this, the epitopes on the capsid protein recognized by neutralizing antibodies include one or more that are cross-reactive.

Pathogenesis and Immunity

The incubation period of hepatitis E in humans ranges from 2 to 8 weeks, with an average of 5-6 weeks, somewhat longer than for hepatitis A. Most of our limited knowledge of the pathogenesis of HEV comes from experimental studies in monkeys or chimpanzees, which are readily mfectible and in which HEV can be serially passaged. Macaques and tamarins begin to excrete virus into bile and feces about 1 month postinfection. Viral antigens can be demonstrated in the cytoplasm of hepatocytes by immunofluorescence. Serum levels of liver enzymes such as alanine aminotransferase (ALT) then begin to rise, reaching their peak at about 10 weeks. The infection then resolves; there is no evidence of chronicity. Antibodies rise slowly, do not reach high titers, and appear to fall off fairly rapidly.

The most conspicuous pathologic feature of hepatitis E infection, in contrast to hepatitis A, is cholestasis. Histologically, the liver often shows intra-canalicular stasis of bile and rosette formation of hepatocytes and pseu-doglandular structures resembling embryonal bile ducts. It is not yet known whether HEV replicates initially in the intestine. One intriguing possibility worthy of investigation is whether the observed sensitivity of the virion to proteolysis reflects a requirement for proteolytic disruption of the capsid by trypsin or other enzymes in the intestine prior to infection of the mucosa. An important question for future research is why the disease is so severe in pregnant women (see below).

Clinical Features

Hepatitis E has so far been observed almost exclusively in the less developed parts of the world and predominantly in the 15-40 age group. Subclinical infection may be the rule in children, with icteric infection being mainly confined to young adults. Clinically, the illness closely resembles that described for hepatitis A (see Chapter 23). Bilirubin levels tend to be higher, and jaundice deeper and more prolonged. The case-fatality rate is 0.5-3%. However, the most striking feature of hepatitis E is its extraordinarily high case-fatality rate of about 10-20% in pregnant women, particularly in the final trimester. Like hepatitis A, hepatitis E does not progress to chronic hepatitis, cirrhosis, cancer, or the carrier state.

Laboratory Diagnosis

Pending the general introduction of simple diagnostic tests for hepatitis E, the first duty of the laboratory is to exclude hepatitis A (by JgM serology) and hepatitis B (HBsAg; anti-HBc IgM). Hepatitis E virus has yet to be cultured satisfactorily fir vilro. Immunoelectron microscopy, looking for aggregated calicivirus-like particles irt an acute-phase fecal specimen using polyclonal sera, was the standard approach to diagnosis and could be refined by the use of MAbs raised against clonally expressed capsid proteins or synthetic peptides, but it appears destined to be rapidly replaced by simpler and more sensitive new alternatives. Even though there seems to be relatively little antigen in feces, it maybe possible to develop an enzyme immunoassay, to be followed perhaps by a confirmatory western blot. PCR assays have been developed which amplify viral RNA that has first been adsorbed from feces or from very early acute-phase serum onto glass powder (to allow inhibitors to be washed away) before reverse transcription and amplification using primers specific for the HEV RNA polymerase gene.

Antibody was originally detected and quantified by immunofluorescence, using an assay in which the binding of fluoresceinated antibody to a liver section from an infected macaque is blocked by patient's serum, but this tedious test has now outlived its usefulness. Immunoglobulin class-specific EIAs and Western blot assays have recently been developed for detection ol antibody to a molecularly cloned fusion protein representing most of the HEV capsid antigen.


Hepatitis E virus is now recognized to be the most important cause of epidemic hepatitis in Asia. In Central Asia hepatitis E, like hepatitis A, tends to peak in autumn, while in Southeast Asia it occurs particularly during the rainy season or following extensive flooding. Transmission is fecal-oral and is mainly water-borne. The most notorious common-source epidemic resulted from fecal contamination of a drinking-water supply in New Delhi in 1955, causing 29,000 identified cases of icteric hepatitis. Ascribed to HAV at the time, the outbreak was later reinvestigated retrospectively by testing frozen stored paired sera for HAV and HBV antibodies and was demonstrated to have been caused by a non-A, non-B hepatitis virus. Many similar water-borne outbreaks have been recorded subsequently, especially on the Indian subcontinent but also in Central Asia, China, Indonesia, North Africa, and Mexico. Particularly devastating outbreaks occurred among Ethiopian refugees encamped in Somalia and Sudan during the prolonged war in the Horn of Africa. Sporadic cases have been attributed to the consumption of shellfish from sewage-polluted waters in Italy and Spain.

Clinical attack rates during epidemics range from 1 to 10%, with most of the cases occurring in young adults and most of the mortality in pregnant women. However, the secondary attack rate among household contacts of icteric patients is low (2.4% in one outbreak in Nepal, compared with 10-20% for hepatitis A in the same locality). Perhaps this is due to relatively low numbers of infectious virions shed in feces and/or their lability. The apparent absence of hepatitis E in the developed countries of the world except in returning travelers is presumably attributable to this low secondary attack rate, as well as to piped water supplies of a standard sufficient to avoid common-source water-borne outbreaks.

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