The objective of this study was to assess the role of the HVR in ORF1 of HEV in virus replication and/or pathogenesis. Sequence analysis of known HEV strains revealed an HVR with a high degree of variability at both amino acid and nucleotide sequence levels. This region overlaps the proline-rich hinge region of ORF1 (25
). It is known that inherent structural constraints can influence the vulnerability of genomic segments to replication errors during virus infection, resulting in the accumulation of mutations for genetic diversity (13
). The size differences in HEV genomes from different genotypes are confined mainly to the HVR of ORF1, which spanned 105 aa as originally proposed (31
). As the sequences of additional HEV isolates were published, it became clear that the first 35 aa in the originally described HVR among HEV strains (53
) is not hypervariable. Thus, the true HVR is 70 to 72 aa for genotype 1 HEV, 68 aa for genotype 2 HEV, 80 to 86 aa for genotype 3 HEV, 84 aa for all genotype 4 HEVs, and 84 aa for avian HEV (based on the corresponding region in mammalian HEVs). HEV genomes exhibited increased divergence in the HVR encompassing aa 707 to 777 for genotype 1 human HEV, aa 707 to 790 for genotype 3 swine HEV, and aa 557 to 641 for avian HEV. Extensive sequence variations observed among isolates in the four major genotypes of mammalian HEV and avian HEV, as well as within each genotype, suggested that the HVR may not be necessary for virus replication. It has been shown that a 507-nt deletion in a variable nonstructural region of rubella virus, a virus distantly related to HEV, is not required for virus replication (56
). Therefore, we hypothesize that the HVR of HEV is not required for virus infectivity.
To test our hypothesis, we first constructed two genotype 1 human HEV HVR deletion mutants using the EGFP-expressing Sar55 HEV replicon as the backbone: mutants hHVRd1 (aa 711 to 777 deleted) and hHVRd2 (aa 747 to 761 deleted). The wild-type Sar55 EGFP replicon, which was constructed in our laboratory (Huang and Meng, unpublished), was shown to be replication competent and expressed EGFP when transfected into Huh7 liver cells. The two HVR deletion mutants (hHVRd1 and hHVRd2) were tested for viability and replication competency in Huh7 cells. EGFP fluorescence signal was detected in Huh7 cells transfected with the mutant hHVRd2, as well as with the wild-type Sar55 replicon, but not in the cells transfected with the HVR deletion mutant hHVRd1. The results from this experiment showed that the mutant hHVRd2 with partial HVR deletion is viable, and thus, the HVR is dispensable for virus replication in vitro. The absence of EGFP expression for mutant hHVRd1, which contains a deletion of the nearly complete HVR, suggested that this mutant is not replication competent. Therefore, it is likely that the end sequences of the HVR for genotype 1 human HEV may be important for virus viability. Nevertheless, partial deletion of HVR sequence in the middle region, as revealed by mutant hHVRd2 (Fig. ), apparently does not affect the replication ability of the genotype 1 human HEV in vitro.
To further confirm our results from the in vitro study with genotype 1 human HEV replicon mutants, we utilized a genetically distinct chicken strain of HEV (avian HEV); we constructed three avian HEV mutants with various deletions in the HVR and tested the mutants for the ability to infect chickens. Based on the amino acid sequence alignment of avian HEV with other mammalian HEV strains, we found that the region spanning aa 557 to 641 in avian HEV is highly divergent and thus termed it the HVR for avian HEV. A total of three avian HEV HVR deletion mutants with various lengths were constructed: aHVRd1, with a partial deletion in the 5′ end of the HVR; aHVRd2, with a partial deletion in the 3′ end of the HVR; and aHVRd3, with the deletion of the nearly complete HVR (Fig. ). The infectivities of the three avian HEV mutants were tested in chickens by intrahepatically inoculating the RNA transcripts from each mutant into the livers of live chickens. The kinetics of virus replication appears to be different in chickens infected with different mutants and wild-type avian HEV (Fig. ). Although seroconversion was observed in chickens inoculated with all three mutants, only one or two out of the three inoculated chickens had seroconverted (Fig. ). Since the percutaneous intrahepatic-injection procedure used in this study to inoculate RNA transcripts into chicken livers is a blind procedure (24
), it was quite possible that the RNA transcripts were not injected into the livers of some chickens or that only a small amount was injected (24
). This may explain why not all inoculated chickens seroconverted to avian HEV antibodies. Fecal virus shedding and viremia were detected only in mutants aHVRd1 and aHVRd2. Deletions in the HVR may influence the replicative competence of the virus and thus may attenuate avian HEV. Therefore, attenuation of HVR deletion mutants to replicate at lower levels could explain why viral RNA was not detected in sera from group A chickens (aHVRd1) or in feces and sera of group C chickens (aHVRd3). Clearly, future studies are warranted to explore any potential role of the HVR in virus attenuation, which is beyond the scope of this study. The results from this avian HEV and chicken study indicated that the HVR of avian HEV is not essential for virus infectivity in vivo, although the avian HEV mutant with complete HVR deletion displayed an apparent attenuation phenotype.
In order to definitively verify our results from the avian HEV and chicken study, as well as from the in vitro genotype 1 HEV replicon mutant study, we subsequently constructed four genotype 3 swine HEV mutants with various HVR deletions: sHVRd1, with the deletion of the nearly complete HVR sequence; sHVRd2 and sHVRd3, with partial deletions of HVR sequences in the middle region; and sHVRd4, with a deletion of partial HVR sequence at the 5′ end (Fig. ). The infectivities of these four mutants were tested in pigs by intrahepatic inoculation of capped RNA transcripts from each mutant via an ultrasound-guided inoculation procedure. Similar to our observations in the chicken study, we found that mutants sHVRd2, sHVRd3, and sHVRd4, with partial deletions of the HVR sequences at the 5′ end and in the middle region, are viable and infectious in pigs. Seroconversion was observed for all HVR deletion mutants; however, there was a delayed seroconversion with no detectable viral RNA in feces or sera for pigs inoculated with mutant sHVRd1, which contains the nearly complete HVR deletion, an indication of attenuation for the sHVRd1 virus. Viral RNA was detected much later during infection, at 9 weeks p.i., in pigs (no. 291 and no. 295) infected with mutant sHVRd2, which contains a larger sequence deletion of the HVR than mutants sHVRd3 and sHVRd4. Fecal virus shedding was detected at 1 and 2 weeks p.i. in pigs inoculated with mutants sHVRd3 and sHVRd4 and lasted for 5 to 8 weeks. These results suggest that mutants sHVRd1 and sHVRd2, with larger sequence deletions of the HVR, may be attenuated to replicate at lower levels, and it appears that the lengths of HVR deletions may affect the level of virus replication and attenuation. Again, additional studies to explore the role of the HVR in HEV attenuation, which is not within the scope of this study, will provide more insights into the role of the HVR in the biology and pathogenesis of HEV.
The patterns of viremia and fecal virus shedding in experimentally infected pigs and chickens (Tables and ) are somewhat different from that observed in HEV-infected humans. In humans, viremia usually precedes fecal virus shedding, whereas in the pig and chicken studies fecal virus shedding was detected prior to viremia, which is consistent with our previous animal studies (5
). It is believed that tissues in the gastrointestinal tract are the initial sites of swine HEV and avian HEV replication, and thus, the virus is excreted to the feces before entering the bloodstream. In fact, it has recently been demonstrated that after oral injection or intravenous injection, swine HEV and avian HEV first replicate in various gastrointestinal tissues in chickens and pigs before reaching the target organ, the liver, via the bloodstream (6
), and this may explain why fecal virus shedding precedes viremia in HEV-infected pigs and chickens.
Since ORF1 contains domains essential for HEV replication, proper folding of the encoded polyprotein is essential for its role in virus replication either individually or by interacting with host alleles. The results from our in vitro, as well as in vivo, animal studies showed that the deletions in the HVR did not influence the viability of the virus, and thus, the polyprotein encoded by ORF1 appears to be properly folded in viable mutants. The HVR, aa 747 to 761 of genotype 1 human HEV, aa 557 to 585 and 612 to 641 of avian HEV, and aa 712 to 765 of genotype 3 swine HEV, apparently has no major effect on the host-mediated processing of the polyprotein, as the mutant viruses are viable and infectious in animals. Since unneeded sequences in virus genomes normally are lost rapidly during in vivo replication, it is possible that the HVR, although not essential for virus infectivity, may play a biological role in HEV pathogenesis. In fact, the results from the animal studies with limited numbers of pigs and chickens suggested that deletions of a larger or nearly complete HVR from the HEV genome apparently attenuated the virus. Therefore, additional studies with larger numbers of animals are warranted to fully evaluate the biological role of HVR in HEV replication and pathogenesis.