Sequence to structure analysis of the ORF4 protein from Hepatitis E virus

Hepatitis E virus (HEV) is the main cause of acute hepatitis worldwide. HEV accounts for up to 30% mortality rate in pregnant women, with highest incidences reported for genotype 1 (G1) HEV. The contributing factors in adverse cases during pregnancy in women due to HEV infection is still debated. The mechanism underlying the pathogenesis of viral infection is attributed to different genomic component of HEV, i.e., open reading frames (ORFs): ORF1, ORF2, ORF3 and ORF4. Recently, ORF4 has been discovered in enhancing the replication of GI isolates of HEV through regulation of an IRES-like RNA element. However, its characterization through computational methodologies remains unexplored. In this novel study, we provide comprehensive overview of ORF4 protein''s genetic and molecular characteristics through analyzing its sequence and different structural levels. A total of three different datasets (Human, Rat and Ferret) of ORF4 genomes were built and comparatively analyzed. Several non-synonymous mutations in conjunction with higher entropy values were observed in rat and ferret datasets, however, limited variation was observed in human ORF4 genomes. Higher transition to tranversion ratio was observed in the ORF4 genomes. Studies have reported the association of intrinsic disordered proteins (IDP) with drug discovery due to its role in several signaling and regulatory processes through protein-protein interactions (PPIs). As PPIs are potent drug target sources, thus the ORF4 protein was explored by analyzing its polypeptide structure in order to shed light on its intrinsic disorder. Pressures that lead towards preponderance of disordered-promoting amino acid residues shaped the evolution of ORF4. The intrinsic disorder propensity analysis revealed ORF4 protein (Human) as a highly disordered protein (IDP). Predominance of coils and lack of secondary structure further substantiated our findings suggesting its involvement in binding to ligand molecules. Thus, ORF4 contributes to cellular signaling processes through protein-protein interactions, as IDPs are targets for regulation to accelerate the process of drug designing strategies against HEV infections.     

Initiation at the third in-frame AUG codon of open reading frame 3 of the hepatitis E virus is essential for viral infectivity in vivo

To determine the initiation strategy of the hepatitis E virus (HEV) open reading frame 3 (ORF3), we constructed five HEV mutants with desired mutations in the ORF1 and ORF2 junction region and tested their levels of in vivo infectivity in pigs. A mutant with a C-terminally truncated ORF3 is noninfectious in pigs, indicating that an intact ORF3 is required for in vivo infectivity. Mutations with substitutions in the first in-frame AUG in the junction region or with the same T insertion at the corresponding position of HEV genotype 4 did not affect the virus infectivity or rescue, although mutations with combinations of the two affected virus recovery efficiency, and a single mutation at the third in-frame AUG completely abolished virus infectivity in vivo, indicating that the third in-frame AUG in the junction region is required for virus infection and is likely the authentic initiation site for ORF3. A conserved double stem-loop RNA structure, which may be important for HEV replication, was identified in the junction region. This represents the first report of using a unique homologous pig model system to study the molecular mechanism of HEV replication and to systematically and definitively identify the authentic ORF3 initiation site.     

Mutational analysis of the hypervariable region of hepatitis e virus reveals its involvement in the efficiency of viral RNA replication

The RNA genome of the hepatitis E virus (HEV) contains a hypervariable region (HVR) in ORF1 that tolerates small deletions with respect to infectivity. To further investigate the role of the HVR in HEV replication, we constructed a panel of mutants with overlapping deletions in the N-terminal, central, and C-terminal regions of the HVR by using a genotype 1 human HEV luciferase replicon and analyzed the effects of deletions on viral RNA replication in Huh7 cells. We found that the replication levels of the HVR deletion mutants were markedly reduced in Huh7 cells, suggesting a role of the HVR in viral replication efficiency. To further verify the results, we constructed HVR deletion mutants by using a genetically divergent, nonmammalian avian HEV, and similar effects on viral replication efficiency were observed when the avian HEV mutants were tested in LMH cells. Furthermore, the impact of complete HVR deletion on virus infectivity was tested in chickens, using an avian HEV mutant with a complete HVR deletion. Although the deletion mutant was still replication competent in LMH cells, the complete HVR deletion resulted in a loss of avian HEV infectivity in chickens. Since the HVR exhibits extensive variations in sequence and length among different HEV genotypes, we further examined the interchangeability of HVRs and demonstrated that HVR sequences are functionally exchangeable between HEV genotypes with regard to viral replication and infectivity in vitro, although genotype-specific HVR differences in replication efficiency were observed. The results showed that although the HVR tolerates small deletions with regard to infectivity, it may interact with viral and host factors to modulate the efficiency of HEV replication.     

戊型肝炎病毒荧光定量RT-PCR快速检测技术的建立和初步应用

利用DNAMAN软件对GenBank登录的戊型肝炎病毒四个主要基因型代表株的序列进行分析, 选择其高度保守的ORF2区域设计合成引物和探针, 并用包含有扩增区域的核苷酸片段进行体外转录制备标准品cRNA。在对荧光定量RT-PCR的反应条件优化的基础上, 建立了适用于戊型肝炎病毒主要基因型检测的荧光定量RT-PCR检测技术。该检测技术可以有效检测I型和IV型戊型肝炎阳性病料, 而对猪的其它几种疫病阳性病料则为阴性结果, 证实本技术的特异性强、可靠性好。对阳性标准品的检测结果表明, 所建立的TaqMan荧光定量RT-PCR灵敏度可达2.0×101拷贝/反应, 相比于巢式RT-PCR方法, 其灵敏度高10~100倍以上。在对54份临床样品的检测中, 进一步证实了该方法快速、灵敏且重复性好, 可满足戊型肝炎病毒早期快速诊断的需要。     

戊型肝炎病毒荧光定量RT-PCR快速检测技术的建立和初步应用

利用DNAMAN软件对GenBank登录的戊型肝炎病毒四个主要基因型代表株的序列进行分析, 选择其高度保守的ORF2区域设计合成引物和探针, 并用包含有扩增区域的核苷酸片段进行体外转录制备标准品cRNA。在对荧光定量RT-PCR的反应条件优化的基础上, 建立了适用于戊型肝炎病毒主要基因型检测的荧光定量RT-PCR检测技术。该检测技术可以有效检测I型和IV型戊型肝炎阳性病料, 而对猪的其它几种疫病阳性病料则为阴性结果, 证实本技术的特异性强、可靠性好。对阳性标准品的检测结果表明, 所建立的TaqMan荧光定量RT-PCR灵敏度可达2.0×101拷贝/反应, 相比于巢式RT-PCR方法, 其灵敏度高10~100倍以上。在对54份临床样品的检测中, 进一步证实了该方法快速、灵敏且重复性好, 可满足戊型肝炎病毒早期快速诊断的需要。     

Endoplasmic Reticulum Stress Induced Synthesis of a Novel Viral Factor Mediates Efficient Replication of Genotype-1 Hepatitis E Virus

Hepatitis E virus (HEV) causes acute hepatitis in many parts of the world including Asia, Africa and Latin America. Though self-limiting in normal individuals, it results in ~30% mortality in infected pregnant women. It has also been reported to cause acute and chronic hepatitis in organ transplant patients. Of the seven viral genotypes, genotype-1 virus infects humans and is a major public health concern in South Asian countries. Sporadic cases of genotype-3 and 4 infection in human and animals such as pigs, deer, mongeese have been reported primarily from industrialized countries. Genotype-5, 6 and 7 viruses are known to infect animals such as wild boar and camel, respectively. Genotype-3 and 4 viruses have been successfully propagated in the laboratory in mammalian cell culture. However, genotype-1 virus replicates poorly in mammalian cell culture and no other efficient model exists to study its life cycle. Here, we report that endoplasmic reticulum (ER) stress promotes genotype-1 HEV replication by inducing cap-independent, internal initiation mediated translation of a novel viral protein (named ORF4). Importantly, ORF4 expression and stimulatory effect of ER stress inducers on viral replication is specific to genotype-1. ORF4 protein sequence is mostly conserved among genotype-1 HEV isolates and ORF4 specific antibodies were detected in genotype-1 HEV patient serum. ORF4 interacted with multiple viral and host proteins and assembled a protein complex consisting of viral helicase, RNA dependent RNA polymerase (RdRp), X, host eEF1α1 (eukaryotic elongation factor 1 isoform-1) and tubulinβ. In association with eEF1α1, ORF4 stimulated viral RdRp activity. Furthermore, human hepatoma cells that stably express ORF4 or engineered proteasome resistant ORF4 mutant genome permitted enhanced viral replication. These findings reveal a positive role of ER stress in promoting genotype-1 HEV replication and pave the way towards development of an efficient model of the virus.     

戊型肝炎病毒第4基因型中国株ORF2编码蛋白的抗原表位分析

以戊型肝炎病毒(HEV)第4基因型中国株ORF2编码蛋白p166Chn制备单克隆抗体(McAbs), 同时制备20个N端或C端逐渐截短的p166Chn截短蛋白, 与7种不同基因型和亚型的p166蛋白一起, 通过ELISA、免疫印迹(Western blot)以及竞争抑制实验对主要存在于我国的HEV第4基因型毒株进行抗原表位分析。结果发现所制备的McAbs与p166Chn截短蛋白的免疫反应有两种类型, 以1G10为代表的McAbs能与N端不短于aa477、C端不短于aa613的截短蛋白反应, 其针对的抗原表位是构象依赖型表位, 依赖于aa477~aa613肽链区段; 而McAb 2F11则能与N端不短于aa474、C端不短于aa617的截短蛋白反应, 其针对的抗原表位也是构象表位, 但需依赖于较长的肽链区段(aa474~aa617)。竞争抑制实验显示两类McAbs互不抑制, 进一步证实了所发现的两个抗原表位在空间位置上的不同。更有意义的是, 两类McAbs均能与其他不同HEV基因型和亚型来源的p166重组蛋白发生阳性反应, 表明这两个抗原表位是HEV基因型共同性的, 可以在世界各国分布的不同基因型HEV毒株中诱导交叉免疫。     

The 41-amino-acid C-terminal region of the hepatitis E virus ORF3 protein interacts with bikunin, a kunitz-type serine protease inhibitor

Hepatitis E virus (HEV), a human plus-stranded RNA virus, contains three open reading frames (ORF). Of these, ORF1 encodes the viral nonstructural polyprotein, ORF2 encodes the major capsid protein, and ORF3 codes for a phosphoprotein of undefined function. Recently, using the yeast two-hybrid system to screen a human cDNA liver library, we have isolated and characterized AMBP (alpha1-microglobulin/bikunin precursor), which specifically interacts with the ORF3 protein of HEV. The ORF3 protein expedites the processing and secretion of alpha1-microglobulin. When checked individually for interaction, the second processed protein from AMBP, bikunin, strongly interacted with the full-length ORF3 protein. This protein-protein interaction has been validated by immunoprecipitation in both COS-1 and Huh7 cells and by His6 pull-down assays. In dual-labeling immunofluorescent staining, followed by fluorescence microscopy of transfected human liver cells, ORF3 colocalized with endogenously expressed bikunin. Finally, a 41-amino-acid C-terminal region of ORF3 has been found to be responsible for interacting with bikunin. The importance of this virus-host protein-protein interaction, with reference to the viral life cycle, has been discussed.     

戊型肝炎病毒基因1型和基因4型中和表位区域分子差异研究

戊型肝炎病毒(HEV)根据易感宿主的差别可以分为两大类:一类只分离自人的H(Human)类,包括HEV-1和HEV-2;一类为人畜共患的Z(Zoonosis)类,包括HEV-3和HEV-4。本研究通过比较这两类HEV的ORF2aa368~606区段,发现存在4个类保守的差异位点,均位于HEV的主要中和表位区域aa459~606,分别是aa483、aa492、aa497和aa599;对这四个位点进行定点替换突变,以一组能够捕获HEV-1和/或HEV-4的单克隆抗体比较各种突变体的免疫反应性,结果表明仅aa497的差异造成了这两类HEV中和表位构象的部分差异,提示aa497及其相关的病毒表面结构差异在H类和Z类HEV宿主选择中可能扮演重要角色。     

The PSAP motif within the ORF3 protein of an avian strain of the hepatitis E virus is not critical for viral infectivity in vivo but plays a role in virus release

The ORF3 protein of hepatitis E virus (HEV) is a multifunctional protein important for virus replication. The ORF3 proteins from human, swine, and avian strains of HEV contain a conserved PXXP amino acid motif, resembling either Src homology 3 (SH3) cell signaling interaction motifs or "late domains" involved in host cell interactions aiding in particle release. Using an avian strain of HEV, we determined the roles of the conserved prolines within the PREPSAPP motif in HEV replication and infectivity in Leghorn male hepatoma (LMH) chicken liver cells and in chickens. Each proline was changed to alanine to produce 8 avian HEV mutants containing single mutations (P64, P67, P70, and P71 to A), double mutations (P64/67A, P64/70A, and P67/70A), and triple mutations (P64/67/70A). The results showed that avian HEV mutants are replication competent in vitro, and none of the prolines in the PXXPXXPP motif are essential for infectivity in vivo; however, the second and third prolines appear to aid in fecal virus shedding, suggesting that the PSAP motif, but not the PREP motif, is involved in virus release. We also showed that the PSAP motif interacts with the host protein tumor suppressor gene 101 (TSG101) and that altering any proline within the PSAP motif disrupts this interaction. However, we showed that the ORF2 protein expressed in LMH cells is efficiently released from the cells in the absence of ORF3 and that coexpression of ORF2 and ORF3 did not act synergistically in this release, suggesting that another factor(s) such as ORF1 or viral genomic RNA may be necessary for proper particle release.     

An ethanol extract of <Emphasis Type="Italic">Lysimachia mauritiana</Emphasis> exhibits inhibitory activity against hepatitis E virus genotype 3 replication

Hepatitis E virus (HEV) is an etiological agent of acute hepatitis E, a self-limiting disease prevalent in developing countries. HEV can cause fulminant hepatic failure with high mortality rates in pregnant women, and genotype 3 is reported to trigger chronic hepatitis in immunocompromised individuals worldwide. Screening of plant extracts for compounds with potential anti-HEV effects led to the identification of a 70% ethanol extract of Lysimachia mauritiana (LME) that interferes with replication of the swine HEV genotype 3 replicon. Furthermore, LME significantly inhibited replication of HEV genotype 3 and expression of HEV ORF2 in infected cells without exerting cytotoxic effects. Collectively, our findings demonstrate the potential utility of LME in the development of novel antiviral drugs against HEV infection.     

不同基因型戊型肝炎病毒存在多种类型抗原表位

以戊型肝炎病毒(HEV)ORF2重组蛋白p166Us为免疫原制备单克隆抗体(McAbs),采用间接ELISA和免疫印迹法,检测McAbs与不同基因型和亚型HEV重组蛋白p166Bur(Ⅰa型)、p166Pak(Ⅰb型)、p166Mor(Ⅰc型)、p166Mex(Ⅱ型)、p166Us(Ⅲ型)、p166Nz(猪HEV,Ⅲ型)和p166Chn(Ⅳ型)的反应性,采用抗原或抗体竞争ELISA分析p166蛋白与天然HEV颗粒之间抗原表位的关系。结果获得4D3、2E3、11E11、12H5、3A3和1F16株稳定分泌McAbs的杂交瘤细胞株。4D3分泌的McAb与7种p166均发生反应,其与免疫原p166Us的结合可被Ⅰ、Ⅱ、Ⅲ或Ⅳ型天然HEV颗粒或病人血清竞争抑制。2E3、11E11和12H5分泌的McAbs只与p166Us、p166Nz和p166Chn发生反应,它们与p166Us的结合仅能被Ⅲ和IV型病毒或血清所抑制。3A3分泌的McAb只与p166Us及p166Nz结合,1F1分泌的McAb只与p166Us结合,两者均能被Ⅲ型美国株竞争抑制,而Ⅰ、Ⅱ、Ⅳ型不能抑制它们与p166Us的结合。由此可见,不同基因型和亚型HEV ORF2编码蛋白p166上存在多种类型抗原表位,其中包括Ⅰ、Ⅱ、Ⅲ、Ⅳ基因型共同的,Ⅲ、Ⅳ基因型共有的和第Ⅲ基因型特异的等,这些表位与天然HEV颗粒上的抗原表位具有相同的免疫学特征。     

Genome-Wide Comparisons of Phylogenetic Similarities between Partial Genomic Regions and the Full-Length Genome in Hepatitis E Virus Genotyping

Besides the complete genome, different partial genomic sequences of Hepatitis E virus (HEV) have been used in genotyping studies, making it difficult to compare the results based on them. No commonly agreed partial region for HEV genotyping has been determined. In this study, we used a statistical method to evaluate the phylogenetic performance of each partial genomic sequence from a genome wide, by comparisons of evolutionary distances between genomic regions and the full-length genomes of 101 HEV isolates to identify short genomic regions that can reproduce HEV genotype assignments based on full-length genomes. Several genomic regions, especially one genomic region at the 3′-terminal of the papain-like cysteine protease domain, were detected to have relatively high phylogenetic correlations with the full-length genome. Phylogenetic analyses confirmed the identical performances between these regions and the full-length genome in genotyping, in which the HEV isolates involved could be divided into reasonable genotypes. This analysis may be of value in developing a partial sequence-based consensus classification of HEV species.     

上海部分地区戊型肝炎病毒（HEV）基因型的分析

为了解戊型肝炎病毒 (HEV)在上海部分地区流行的基因型 ,采用RT nPCR的方法检验 35例急性散发性戊型肝炎患者中HEVRNA ,并对阳性产物进行克隆测序 ,然后对其基因型进行分析。结果显示在 35例急性散发性戊型肝炎患者中PCR阳性为 9例 ,测序证实 8例为HEV的基因序列 ;其中 1例为HEV 1型 ,7例为HEV 4型。提示在上海部分地区的急性散发性戊型肝炎中以HEV 4型感染为主     

Release of Genotype 1 Hepatitis E Virus from Cultured Hepatoma and Polarized Intestinal Cells Depends on Open Reading Frame 3 Protein and Requires an Intact PXXP Motif

Hepatitis E virus genotype 1 strain Sar55 replicated in subcloned Caco-2 intestinal cells and Huh7 hepatoma cells that had been transfected with in vitro transcribed viral genomes, and hepatitis E virions were released into the culture medium of both cell lines. Virus egress from cells depended on open reading frame 3 (ORF3) protein, and a proline-rich sequence in ORF3 was important for egress from cultured cells and for infection of macaques. Both intracellular ORF3 protein accumulation and virus release occurred at the apical membrane of polarized Caco-2 cells. ORF3 protein and lipids were intimately associated with virus particles produced in either cell line; ORF2 epitopes were masked in these particles and could not be immunoprecipitated with anti-ORF2.Hepatitis E virus (HEV) remains enigmatic in spite of recent advances (see references 7 and 16 for reviews). HEV is a major cause of acute hepatitis in numerous developing countries, but hepatitis E is infrequently detected in industrialized countries even though seroprevalence rates of anti-HEV as high as 20% in these countries have been reported. Although hepatitis E normally is a self-limited acute disease, recent studies have identified it as an emerging cause of chronic hepatitis in immunocompromised patients. Whereas contaminated drinking water is the source of most infections in developing countries, the sources in industrialized countries are not fully evaluated, but many, if not most, infections appear linked to eating undercooked meat, especially pork. These differences in epidemiology may reflect the fact that most infections in developing countries are caused by genotypes 1 and 2 while those in industrialized countries are mainly due to genotypes 3 and 4.HEV was initially classified as a calicivirus, but subsequent sequence analysis suggested that it was more closely related to the enveloped rubella virus. However, although HEV may be associated with lipids under some conditions (22), HEV virions do not possess an envelope. Four genotypes of HEV that infect humans have been identified (4). Genotypes 1 and 2 infect primates exclusively, whereas genotypes 3 and 4 are zoonotic and commonly also infect swine and rarely other nonprimates. Recent identification of a strain infecting farmed rabbits in China suggests that other reservoirs may exist (32).The capsid protein encoded by open reading frame 2 (ORF2) is able to form infectious virus particles, but these particles remain cell associated. The crystal structure of a truncated recombinant protein has been solved, but the size of the protein in mature virions is unknown (11, 15, 28, 31). The virus is not cytopathic, and it is unclear how it gets out of cells.The 7.2-kb genome of HEV is a capped mRNA that contains three ORFs that encode proteins involved in replication (ORF1), a capsid protein (ORF2), and a small protein of only 113 to 114 amino acids (ORF3). All but the 5′ terminus of ORF3 is overlapped by ORF2, and both proteins are translated from the same bicistronic subgenomic RNA (10). When overexpressed in cell culture, ORF2 is glycosylated, and ORF3 is phosphorylated (26); this phosphorylated ORF3 protein binds to nonglycosylated ORF2 protein in cell culture, but phosphorylation is not required for infection of macaques (9). The virus has been exceedingly difficult to propagate in cell culture, but recently Okamoto and colleagues reported the successful adaptation of both a genotype 3 and a genotype 4 strain to efficient growth in cultures of PLC/PRF/5 hepatoma or A549 lung cells (23, 24).The tiny ORF3 protein is particularly intriguing because it has a significant impact on virus propagation through mechanisms that have yet to be defined. Data from experiments performed with overexpressed ORF3 protein have suggested that, among other things, ORF3 may interact with cellular proteins, including signaling proteins containing Src homology 3 domains (14), bikunin (27), hemopexin (21), and microtubule proteins (13), and it may function to modulate the acute-phase disease response (3), protect cells from mitochondrial depolarization (18), and enhance expression of glycolytic pathway enzymes (17). Yet within transfected hepatoma cells in culture, virions of an ORF3 null mutant of genotype 1 were assembled in the absence of ORF3 protein and were infectious for naïve hepatoma cells (6) although this same ORF3 null mutant was unable to mount a detectable infection in rhesus monkeys (8). Also, swine transfected with genotype 3 mutant genomes encoding a truncated ORF3 protein did not get infected, indicating that an intact ORF3 protein is needed for infectivity in vivo (12). This lack of infectivity in vivo is possibly explained by the recent demonstration that the ORF3 protein of genotype 3 virus is important for export of virions out of cultured cells in vitro (30); however, this dependence on ORF3 for virion egress has not been confirmed in vivo or for strains of the other three genotypes.The four major genotypes of human HEV appear to segregate naturally into two distinct groups. One group contains genotype 1 and 2 strains that lack a zoonotic component and are spread mainly via contaminated water; in contrast, the second group contains genotype 3 and 4 strains which are able to cross species boundaries and are zoonotic since humans have been infected as a result of eating undercooked meat (16, 25). The molecular basis for the two groupings is unknown, and much more extensive comparative analyses are required to determine which variables are epidemiologically relevant. Here, for lack of an efficient cell culture system for genotype 1 or 2 strains, we have utilized an infectious cDNA clone of a genotype 1 strain in order to explore the role of the ORF3 protein in this group.     

基因Ⅰ、Ⅳ型戊型肝炎病毒高灵敏度通用引物的设计和初步应用

设计针对国内流行的戊型肝炎病毒(HEV)基因Ⅰ、Ⅳ型,在这两型序列的保守区域设计了一套RT-PCR引物——E引物,并将E引物与目前较常用的3对通用引物(Meng、ConORF1和ConORF2引物)比较了检测基因Ⅰ、Ⅳ型HEV的灵敏度。对基因Ⅰ型HEV,E引物能检出的稀释度为105,参考引物能检出的稀释度为10^2～10^4;对基因Ⅳ型HEV,E引物能检出的稀释度为10^2,参考引物能检出的稀释度为10^1～10^2。在17份HEV-IgM阳性血清中,E引物检出5份,检出率为29．4％;参考引物只能检出1份或2份,检出率最高为11．8％。E引物在33份HEV-IgM阳性的隐性感染血清中检出6份,阳性率18．2％;在79份HEV-IgM阳性的临床肝炎血清中检出36份,阳性率45．6％。以上结果初步表明,对于在国内流行的基因Ⅰ、Ⅳ型HEV,E引物的检测灵敏度要高于目前常用的通用引物。     

Evolutionary history and population dynamics of hepatitis E virus

Background

Hepatitis E virus (HEV) is an enterically transmitted hepatropic virus. It segregates as four genotypes. All genotypes infect humans while only genotypes 3 and 4 also infect several animal species. It has been suggested that hepatitis E is zoonotic, but no study has analyzed the evolutionary history of HEV. We present here an analysis of the evolutionary history of HEV.

Methods and Findings

The times to the most recent common ancestors for all four genotypes of HEV were calculated using BEAST to conduct a Bayesian analysis of HEV. The population dynamics for genotypes 1, 3 and 4 were analyzed using skyline plots. Bayesian analysis showed that the most recent common ancestor for modern HEV existed between 536 and 1344 years ago. The progenitor of HEV appears to have given rise to anthropotropic and enzootic forms of HEV, which evolved into genotypes 1 and 2 and genotypes 3 and 4, respectively. Population dynamics suggest that genotypes 1, 3 and 4 experienced a population expansion during the 20^th century. Genotype 1 has increased in infected population size ∼30–35 years ago. Genotype 3 and 4 have experienced an increase in population size starting late in the 19^th century until ca.1940-45, with genotype 3 having undergone additional rapid expansion until ca.1960. The effective population size for both genotype 3 and 4 rapidly declined to pre-expansion levels starting in ca.1990. Genotype 4 was further examined as Chinese and Japanese sequences, which exhibited different population dynamics, suggesting that this genotype experienced different evolutionary history in these two countries.

Conclusions

HEV appears to have evolved through a series of steps, in which the ancestors of HEV may have adapted to a succession of animal hosts leading to humans. Analysis of the population dynamics of HEV suggests a substantial temporal variation in the rate of transmission among HEV genotypes in different geographic regions late in the 20^th Century.