A nonhomologous recombination event involving the HEV genome and a human RNA molecule was ultimately responsible for the ability of the Kernow strain of HEV to flourish in cell culture. Such a dramatic change in virus phenotype following virus-host RNA recombination is rare, but a few cases have been reported previously. Thus, a poliovirus lethal mutation was pseudoreverted by introduction of 15 host nucleotides into a mutated cleavage site during replication in cell culture (2
). Similarly, introduction, by recombination, of 54 host nucleotides into the hemagglutinin cleavage site of an apathogenic influenza virus produced a virus variant with increased pathogenicity (14
). Even more strikingly, a rare recombination event which inserted 228 or more nucleotides of host ubiquitin gene sequence into a noncytopathogenic variant of bovine viral diarrhea virus rendered the virus cytopathogenic (19
The infectious genotype 3 cDNA clone we constructed provides an additional tool for HEV research. Since the liver is the target organ for this virus, the ability to transfect or infect human liver (HepG2/C3A) cells and to produce large quantities of viable virus may provide a more authentic model system in which to revisit numerous, well-executed studies that produced intriguing data but were limited by their reliance on overexpression of single viral proteins out of context. In addition, the ability of p6 virus to infect both human and swine cells may prove useful for identifying parameters that restrict the host range of genotype 1 and 2 strains to humans and nonhuman primates. The luciferase replicon we developed should be especially useful for some studies since it permits convenient sequential sampling and is exquisitely sensitive: since the luciferase gene is located on the subgenomic mRNA, luciferase production can act as an indirect indicator of subgenomic RNA synthesis and stability. This new model system has already provided the first evidence that the previously uncharacterized X gene region has a function in viral replication since three mutations in it contributed substantially to establishment of the infected state following transfection ( and ).
HEV is not noted for recombination and intergenotypic recombination has been reported only rarely (31
). In retrospect, this might reflect the different transmission pathways and localized geographic distribution of the four human genotypes resulting in a low number of coinfections with two or more readily distinguishable genomes; intragenotypic recombination might not be noticed unless specifically searched for. However, our discovery of three different human sequences embedded in HEV genomes from the only two patients examined suggests that HEV may undergo recombination more frequently than realized. For instance, a genotype 3 virus from France was reported to have an insert of unidentified origin of ~90 nt in the HVR (15
). Additional studies are required to determine whether insertion of ribosomal protein genes occurred by chance or reflected some unknown aspect of HEV replication.
The discovery of the human S17 gene sequence embedded in the HEV genome (28
) was especially surprising since it indicated (i) that the virus genome had recombined with host RNA and (ii) that this event had apparently imparted properties that resulted in selection of this extremely minor quasispecies virus in cell culture. This scenario was subsequently repeated with a genotype 3 strain from another chronically infected patient (21
), suggesting that illegitimate recombination by HEV is not necessarily a rare event. In the present study, we demonstrated that this recombinant virus emerged as soon as the first passage in cell culture (): its dominance in all passages thereafter strongly suggested that the insert played a critical role in cell culture adaptation. Mutagenesis studies of the infectious cDNA clone demonstrated unequivocally that the insert was a major factor in enabling efficient virus propagation in cell culture and therefore was an adaptive mutation. However, since the stepwise cloning strategy demonstrated that mutations other than the S17 insert also contributed to adaptation (), it was surprising to find an almost total elimination of enhancement of transfection by point mutations upon removal of the S17 insert from the final construct (). One possible explanation is that the inserted S17 sequence enhanced the stability/translatability of the RNA or aided the folding/processing/stability of ORF1 protein. The question of proteolytic processing has not yet been resolved for HEV: however, since introduction of synonymous mutations into 24 to 32% of the nucleotide positions in the S17 insert did not appreciably affect the level of transfection (), it seems unlikely that the viral RNA is the critical factor, but rather suggests that the effect is at the protein level. Deletion experiments have shown that decreasing the size of the standard HVR could decrease the virulence of HEV or reduce its replication in cell culture (27
). Our database analysis of the 50% truncations of the S17 insert demonstrated that the size of the insert, and hence of the HVR, matters, but the experiments substituting GFP, GTPase, or S19 gene fragments (7A, B, and D) suggested that the amino acid composition of both the insert and the genomic background must also contribute to enhancement. This conclusion is in agreement with data showing decreased replication in vitro
when the HVR of a genotype 1 and a genotype 3 strain were swapped (26
Very few HEV strains have been successfully propagated in cell culture, and the selection of the S17 recombinant in cell culture raised the hope that insertion of this sequence into other genotypes and strains might increase the success rate. Certainly, the ability of Sar55-S17 to transfect human or hamster cells was promising. Unfortunately, in toto
, the data suggested that insertion of foreign sequences into the HVR of an HEV genome will not have a predictable impact and that subjection to long-term selective pressure in culture may be the only way to obtain culturable strains. On the other hand, the fact that the S17 sequence increased the ability of Sar55 genomes to replicate in cells from such an unlikely species as the hamster leads one to speculate that new syndromes, such as neurological disorders recently associated with HEV infections, may reflect the ability to infect new cell types because of changes in the HVR. Indeed, Kamar et al. have reported that genomes in the cerebrospinal fluid of a chronically infected patient had sequence differences from those that circulated in the serum (11
). Certainly, this possibility merits further exploration.
Transfection and infection experiments with human HepG2/C3A and swine LLC-PK1 cells demonstrated that the p6 virus clone retained the ability of the fecal virus quasispecies to cross species boundaries and displayed a slight preference for swine cells. In contrast, the titer of the fecal inoculum was previously reported to be up to 13-fold higher on swine cells compared to human cells (28
), which suggests that there might be other members of the fecal quasispecies that either had mutations favorable for infection of swine cells or detrimental for infection of human cells (). It is not known whether receptors or other factors determine host range. Between the p6 cloned virus and the consensus sequence of viruses in the feces, there are 4 aa differences in the capsid protein which might affect receptor interactions. Two of the four mutations were also present in the p1 virus clone which represented the first selection step for HepG2/C3A cells, so it will be interesting to determine whether reversion of any of these mutations to the consensus sequence in the feces will increase the relative titer on swine cells.
Although both p6 virus and ORF3-null virus eventually spread and infected the majority of HepG2/C3A cells in a culture, they did so relatively slowly, and the percentage of infected cells did not begin to increase until after day 7 (). In contrast, luciferase expression was detected in the culture medium as soon as day 1 posttransfection (2,163 U) and had jumped 38-fold by day 2 (A). Since the luciferase is translated from the subgenomic mRNA, viral negative-strand and subgenomic RNA synthesis must have been greatest between days 0 and 2 in this experiment, suggesting that synthesis of viral RNA and/or proteins is probably not rate limiting but rather that assembly, maturation, and/or excretion are responsible for the relatively slow production of infectious HEV virions. It is worth noting that since the luciferase construct lacks a capsid gene, it cannot spread, so the data in B suggested that translation of p6 subgenomic mRNA continued at peak rates through day 7 or 8 before declining.
Perhaps the most confounding result was the discovery that a virus unable to make ORF3 protein spread throughout the culture as efficiently as one synthesizing ORF3 protein. This result poses more questions than answers. The observed difference in specific infectivities provides an explanation of why it happened, but the question of why the specific infectivities differed remains. Okamoto has proposed that HEV egress from PLC/PRF/5 cells depends on ORF3 protein interaction with cellular protein Tsg101 (20
). So, is the null mutant exiting the HepG2/C3A cells by a different pathway than p6 uses? Is a different pathway used in different cell lines? Are any of the available culture systems truly reliable models for infection in vivo
? It now may be possible to address some of these questions.