In Vitro Replication of Hepatitis E Virus (HEV) Genomes and of an HEV Replicon Expressing Green Fluorescent Protein

Abstract

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 replication and interferon responses in human placental cells

“…Because infection with cell culture‐grown HEV gt1 particles is not yet possible, we next performed infection of JEG‐3 cells with a gt1 Sar55 primary isolate stool suspension not harboring the S17 insertion derived from infected rhesus macaques 31. Further controls included HEVcc gt3 particles and infection of Huh‐7 human hepatoma‐derived cell line S10‐3, which is highly permissive for HEVcc replication 31. Challenged cell lines were stained for ORF2‐encoded antigen 7 days postinfection.…”

Cross-species infections of cultured cells by hepatitis E virus and discovery of an infectious virus–host recombinant

“…Cells on eight-well chamber slides were fixed with acetone and doubly stained with chimpanzee anti-ORF2 and rabbit anti-ORF3. Stained cells or foci were visualized with a fluorescence microscope and manually counted as described previously (28) and in SI Materials and Methods.…”

Hepatitis E virus (HEV)-1 harbouring HEV-4 non-structural protein (ORF1) replicates in transfected porcine kidney cells

“…RNAdependent RNA polymerase (RdRp) mutants (GDD to GAA) were constructed for the parental full genome constructs pSK-HEV2 and HEV-4FG. Mutations in this conserved motif have been shown to abolish HEV replication (Emerson et al, 2004) and thus were used as negative controls.…”