Background
Hepatitis E virus (HEV) typically causes a self-limiting acute viral hepatitis with large outbreaks reported in individuals from developing countries, and sporadic and cluster cases in individuals from industrialized countries [
1,
2]. More recently, chronic hepatitis E has become a significant clinical problem in immunocompromised individuals such as organ transplant recipients [
3]. HEV is a single-stranded, positive-sense RNA virus belonging to the family
Hepeviridae [
4], which consists of two genera (
Orthohepevirus, and
Piscihepevirus) and five species. Within the species of
Orthohepevirus A, there are at least 7 genotypes: genotypes 1 and 2 are restricted to humans whereas genotypes 3 and 4 can cross species barriers infecting humans and several other animal species, and genotype 4 HEV is sometimes reportedly associated with severe acute hepatitis in humans [
5]. The genotypes 5 and 6 infect wild boar, and genotype 7 infects camels [
4]. The pig is a major animal reservoir for zoonotic transmission of HEV to humans [
6]. Indeed, sporadic and cluster cases of acute hepatitis E are caused predominantly by the zoonotic genotypes 3 and 4 HEV strains [
6,
7].
The majority of the approximately 20 million HEV infections occurred each year worldwide [
8] are asymptomatic, as only about 3 millions of these infections actually resulted in clinical cases of viral hepatitis [
9]. The mechanisms underlying the induction of liver damage by HEV remains unclear. It has been reported that the genotypes of HEV appear to be associated with disease-inducing potential [
10]. Among the 4 major genotypes of HEV that are known to infect humans, the zoonotic genotypes 3 and 4 HEV isolates are distributed worldwide and have been implicated in sporadic cases of acute hepatitis E in humans [
1,
6,
9]. Interestingly, fulminant or severe acute hepatitis was reported more frequently in humans infected with genotype 4 HEV in Japan [
5,
11] and France [
12]. Therefore, the virus genotype of the infected patient may potentially influence the severity of liver disease. However, the genetic element(s) in viral genome that are responsible for viral replication and pathogenesis remain unknown.
The genotype 3 HEV is distributed worldwide, and infects humans, pigs, deer, rabbits and other animal species. Recently, increased virulence associated with HEV genotype 3 (JIO strains) infection was reported from patients with severe hepatitis in Japan, although the course of genotype 3 HEV infection is generally asymptomatic [
13]. These JIO strains clustered together with 5 swine isolates from Japan [
14] and shared an approximately 98% to 99.8% nucleotide sequence identity with that of swine HEV, suggesting that these apparently “high virulent strains” of genotype 3 HEV may be of zoonotic origin. There is no reported recombination between the JIO strains of genotype 3 HEV and isolates of genotype 4 HEV, but 18 unique amino acid substitutions were identified. Interestingly, three of these mutations (S605P, I978V, and V1213A) located in the helicase or protease domain of HEV were found to be typical of genotype 4 viruses which are sometimes associated with more severe hepatitis. Therefore, it is logical to hypothesize that these mutations may be responsible for the increased virulence reported in these genotype 3 HEV strains in humans. Knowing the effects of these mutations on HEV replication will greatly help us understand the mechanism of HEV pathogenesis. Thus, in this study, we determined the effect of the V1213A, S605P, and I978V amino acid residue mutations on the efficiency of HEV replication using the HEV replicon systems, since currently there is a lack of an efficient cell culture system for HEV propagation.
Discussion
Genotype 3 HEV infection typically is associated with asymptomatic or mild disease, although in immunocompromised individuals genotype 3 HEV causes chronic infection [
3]. However, there was a report in Japan [
13] of severe hepatitis associated with 8 genotype 3 HEV isolates. Interestingly, three unique mutations (S605P, I978V and V1213A) identified in the ORF1 of these genotype 3 isolates were found to be characteristic of genotype 4 HEV strains, which was considered to be more likely associated with severe hepatitis [
13]. Due to the lack of a small animal model for HEV pathogenicity study and a robust cell culture system to study HEV replication, the virus replication fitness or efficiency is often used as a convenient and reliable method to estimate if the mutation(s) in viral genome has any impact on the virus virulence. Therefore, in this study, we aimed to determine if these observed mutations in these genotype 3 HEV isolates affect the replication of HEV in vitro.
By using the HEV GFP replicons system, we first demonstrated that the mutation at amino acid residue 1213 reduced the replication efficiency of both HEV genotype 1 (A1213V) and HEV genotype 3 (V1213A). Our results indicated that the amino acid residue 1213 is critical for HEV replication, which is consistent with a previous report [
13]. However, the replication level of genotype 3 HEV EGFP replicons was low and not very sensitive, as evidenced by the low number of GFP-positive cells and low density of GFP signal, which makes it difficult to more accurately evaluate the replication efficiency in GFP-positive cells. To circumvent the obstacle of the low efficiency of HEV replication, particularly with the genotype 3 HEV EGFP replicon, we subsequently constructed renilla luciferase HEV replicons, which were proved to be more sensitive and accurately reflect the HEV replication level than the HEV EGFP replicons.
The pSHEV3-Rluc genotype 3 HEV replicon system yielded similar, but more reliable, results than that of the HEV EGFP replicons system, demonstrating that the V1213A mutation significantly reduced the replication efficiency of genotype 3 HEV, whereas the A1213V mutation slightly decreased the replication efficiency of genotype 1 HEV. The amino acid residue 1213 locates in the helicase domain of the HEV ORF1, which has been considered as a critical factor for HEV replication. Since the A1213V mutation in genotype 1 HEV also reduced the viral replication efficiency, suggesting that the V1213 residue is not the optimal residue for genotype 1 HEV replication.
When we examined the effect of other mutations (S605P and I978V) that were characteristic of genotype 4 HEV on virus replication, we found that S605P and I978V mutations significantly reduced the efficiency of genotype 3 HEV replication (p < 0.001). Introduction of the V1213A mutation into the S605P-I978V double mutant further reduced the viral replication efficiency (p < 0.05), suggesting that the two sets of mutations have a cumulative, if not synergistic, effect on genotype 3 HEV replication. To further verify that the effect of mutation V1213A is caused by the amino acid, we used a different synonymous codon of the Alanine to create the V1213A mutation in the S605P + I978V mutant, and similar results in HEV replication efficiency were obtained. Taken together, the data suggested, contrary to what we initially thought, that the three mutations identified in the genotype 3 HEV isolates associated with severe hepatitis in Japan actually decreased the efficiency of replication of genotype 3 HEV. The S605P and I978V mutations are located in a relatively high variable region of the ORF1 protein, whereas the V1213A mutation is located in the helicase, an enzyme involving in viral replication. Interestingly, here we demonstrated that the Q1246H mutation identified in the high virulent genotype 3 HEV isolates, but the H residue is different from the corresponding residue typically found in the genotype 4 isolates, only decreased the replication efficiency of genotype 3 HEV at a lower level as compared to that of the V1213A mutation. Our results revealed that the V1213A mutation affects HEV replication efficiency more than that of Q1246H mutation. The V1213A mutation has more drastic effect on HEV replication efficiency, and this may likely due to the effect of the mutation on the function of viral helicase.
Interestingly, when we assessed the effect of A1213V mutation on the replication efficiency of genotype 4 HEV using the genotype 4 HEV luciferase replicon system, we found that the A1213V mutation slightly increased the replication level of genotype 4 HEV (
p < 0.05). The data suggested that the amino acid residue V1213 favors the replication of both zoonotic HEV genotypes 3 and 4, but not genotype 1 HEV, as compared to amino acid residue A1213. The V1213A mutation found in the patients infected with the genotype 3 virus isolates may reduce HEV replication level, and thus was likely not a key factor responsible for the observed severe hepatitis associated with these genotype 3 isolates containing this mutation. HEV infection causes acute viral hepatitis but can also result in chronic hepatitis E in immunocompromised patients [
20]. Genotype 4 HEV was reportedly associated with severe diseases more frequently than genotype 3 HEV [
11,
12]. An insertion of a 58 amino acid residues of human ribosomal protein S17 in the hypervariable region (HVR) of HEV ORF1 apparently enhances HEV replication efficiency in vitro [
21]. Together with our data from this study, it appears that both host-specific factors and virus genetic element(s), including virus genotype and mutations, may contribute to HEV pathogenicity.
Acknowledgements
We thank Barbara Dryman for technical assistance, Alicia Feagins for providing the infectious cDNA clone of HEV TW6196, Yao-Wei Huang for providing the pSHEV3 infectious cDNA clone, and Sue Emerson (NIAID, NIH) for providing the infectious cDNA clone of pSK-HEV2 and Huh7 S10-3 liver cells. The authors declare that there is no conflict of interest.