Hepatitis E virus (HEV) is a quasi-enveloped, single-stranded positive-sense RNA virus belongs to the family Hepeviridae (Smith et al., 2014; Nagashima et al., 2017). Hepeviridae is a highly diverse family that contains several viral species including zoonotic, anthropotropic, and animal-restricted HEV or HEV-like virus isolates (Nan and Zhang, 2016). Although the origin of Hepeviridae is a mystery, analysis of key protein domains encoded by HEV open reading frames (ORFs) and their homologs from other viruses furnish strong evidence suggesting that Hepeviridae arose as a consequence of an ancient recombination event. Specifically, recombination occurring within the junction between non-structural and structural protein encoding regions among the Alphatetraviridae and Astroviridae has been cited (Kelly et al., 2016). Of nine of 21 regions evaluated as part of a global burden of disease (GBD) study, ∼3.4 million symptomatic cases of hepatitis E have recently been observed annually, resulting in 70,000 deaths and 3,000 stillbirths (Dalton et al., 2015). Originally, HEV infection was thought to be solely restricted to humans, causing a self-limiting hepatitis with mortality ranging from 0.5 to 3% overall, but mortality in pregnant women approaching 30% (Pillot et al., 1995; Jameel, 1999). However, the discovery of HEV in swine in 1997 suggests HEV has a wider host range and is actually zoonotic (Meng, 2013).

Currently, hepatitis E cases are frequently reported in developed countries and exhibit expanded host ranges (Doceul et al., 2016; Montesano et al., 2016; Park et al., 2016; Abravanel et al., 2017; Anheyer-Behmenburg et al., 2017). Thus, it appears that HEV has become one of the most successful zoonotic viral diseases (Dalton et al., 2015) and cross-species transmission of HEV from animal reservoirs to humans is the major route for HEV transmission in those countries (Pavio et al., 2015; Salines et al., 2017). Meanwhile, serosurveillance has demonstrated a high prevalence of HEV infection in the general population, which indicates the existence of an HEV endemic which has been underestimated for a long time (Sadik et al., 2016). Moreover, chronic HEV infection, HEV-related acute hepatic failure, and extrahepatic manifestations caused by HEV have been frequently reported in recent years (Dalton et al., 2016; Feng, 2016; Geng et al., 2016; Sadik et al., 2016). These observations suggest a complicated mechanism underlying HEV-related disease, especially for zoonotic HEV. Unfortunately, our understanding of HEV are extremely limited. Moreover, HEV is still less well known publicly as compared with other hepatic viruses such as hepatitis B and C viruses. In this review, recent progress made toward understanding zoonotic HEV host range, viral pathogenesis of zoonotic HEV, cross-species transmission of zoonotic HEV, and determinants influencing HEV host tropism are reviewed in detail and new insights are discussed.

Hepatitis E virus virions contain a 7.2 kb mRNA-like genome, which is capped and poly-adenylated (Ahmad et al., 2011). Currently, three well-recognized ORFs have been identified within the HEV genome for all genotypes (Tam et al., 1991; Tsarev et al., 1992), while the presence of an additional ORF4 has only been demonstrated in genotype 1 HEV so far (Figure 1A) (Nair et al., 2016). As an mRNA-like molecule, HEV-ORF1 is translated directly from its genome and encodes all non-structural proteins (mainly a replicase), which are essential for replication. Meanwhile, ORF2 and ORF3 can be only translated from the subgenomic RNA and partially overlap with each other (or completely overlap in some species of Hepeviridae) (Figure 1B) (Graff et al., 2006). ORF2 encodes the capsid protein (the major virion component), while ORF3 encodes a multifunctional protein (probably a class I viroporin), which is essential for virion release (Mori and Matsuura, 2011). Recently, synthesis of a novel ORF4 driven by a putative internal ribosome entry site (IRES)-like sequence was identified solely in genotype 1 HEV (Nair et al., 2016). Additionally, sequence analysis of other genetic elements has suggested that the HEV genome contains two cis-reactive elements (CRE) which are required for replication (Cao et al., 2010; Parvez, 2015). The second CRE is located in the intergenic region between ORF1 and ORF2 and forms a stem-loop structure which may serve as a promoter for subgenomic RNA synthesis (Cao et al., 2010).

After confirmation of HEV as the causative agent for hepatitis E, a prototype strain of HEV (SAR-55) originating from Pakistan was sequenced and served as the primary sequence for comparison to other isolates, such as the Burmese strain from India and several Chinese isolates (Aye et al., 1992; Tsarev et al., 1992; Bi et al., 1993). Sequences of these earlier HEV isolates shared the highest identity (greater than 90%) with each other and were classified as genotype 1 HEV (now known as HEV1 of Orthohepevirus A virus). Meanwhile, the HEV Mexican strain was originally viewed as the “New World” HEV strain due to its Central American source, while the Asian HEV isolate was viewed as “Old World” HEV (Reyes et al., 1991). Based on analysis of “Old World” and “New World” HEV sequences and excluding nucleotide sequences within the ORF1 hypervariable region, 84, 93, and 87% amino acid identity exists between the Mexican strain and the prototype Sar-55 strain for ORF1, ORF2, and ORF3, respectively (Huang et al., 1992). Subsequently, partial sequence analysis of newer HEV isolates from Africa has revealed that African HEV strains are distinct from the prototype Sar-55 strain, exhibiting greater similarity to the Mexican strain (Chatterjee et al., 1997). These novel isolates are classified as genotype 2 HEV (now HEV2 of Orthohepevirus A virus). However, only two complete sequences of genotype 2 HEV are currently available (Kaiser et al., 2017).

In 1997, a hallmark discovery affecting HEV research dramatically was the identification of swine HEV associated with human hepatitis E (Meng et al., 1997). Previously, phylogenetic analyses suggested that swine HEV is closely related to, but distinct from, previous known human HEV strains (the Asian prototype Sar-55 and the “New World” Mexican strain) (Meng et al., 1997). Subsequently, novel HEV isolates (strains US-1 and US-2) were recovered from two patients with acute hepatitis E in the United States and demonstrated close aa identity (>97%) with the 1997 US swine HEV isolate (Kwo et al., 1997; Schlauder et al., 1998). These isolates represent another HEV genotype (genotype 3 HEV, now designated HEV3 of Orthohepevirus A virus), which is zoonotic and causes cross-species infection (Kwo et al., 1997; Schlauder et al., 1998).

Next, in 1999 several new HEV isolates were obtained from Chinese HEV patients. These isolates were similar to one another but divergent from all other previously recognized HEV genotypes 1–3. Thus, they comprised a fourth HEV genotype based on phylogenetic analysis (Wang et al., 1999, 2000). Meanwhile, similar HEV isolates closely related to Chinese genotype 4 were soon reported in Japan and shown to cause indigenous acute hepatitis and consequently led to the establishment of a remarkably heterogeneous genotype 4 HEV group (Takahashi et al., 2002, 2003). Concurrently, a genotype 4 HEV isolate obtained from a sporadic acute hepatitis E patient demonstrated 99.0% sequence identity (nearly 100% identity) to the swine HEV swJ13-1 strain isolated from pigs (Nishizawa et al., 2003), supporting genotype 4 HEV as a zoonotic HEV genotype. Furthermore, although both HEV genotypes 3 and 4 are capable of infecting swine, the term “swine HEV” is confusing, since it actually neglects the existence of genotypic differences between HEV strains isolated from swine. Therefore, further clarification or genotype description is needed before using the term “swine HEV”.

In 2014, a newly proposed HEV classification system under the family Hepeviridae was published to provide a unified classification for diverse HEV isolates (Smith et al., 2014). In this proposal, two genera, Orthohepevirus (including all mammalian and avian HEV isolates) and Piscihepevirus (only trout HEV), exist under the family Hepeviridae. It is notable that all four major HEV genotypes (1–4) that infect humans are now assigned to the species Orthohepevirus A (Smith et al., 2014). Within Orthohepevirus A, two genotypes (5 and 6) represent novel HEV strains obtained from wild boar in Japan with unique viral nucleotide sequences that were previously designated as genotype 5 and genotype 6 HEVs (Takahashi et al., 2011, 2014; Smith et al., 2014). However, it should be noted that not all HEV isolates from wild boar belong to genotypes 5 and 6; indeed, most wild boar HEV isolates have been classified as genotypes 3 and 4 HEVs with the ability to infect humans (Sato et al., 2011). Moreover, other HEV strains isolated from camels in the Middle East with greater than 20% overall nucleotide difference from the other six HEV genotypes were categorized into genotype 7 within Orthohepevirus A (Smith et al., 2014; Woo et al., 2014). Notably, one patient from the Middle East who underwent liver transplantation had been infected by camel HEV, suggesting that camel HEV is zoonotic as well (Lee et al., 2016). Meanwhile, new hepatitis E virus isolates obtained from Chinese bactrian camels have recently been described and tentatively assigned to a new genotype 8, due to their genetic separation from genotype 7 camel HEV (Woo et al., 2016; Sridhar et al., 2017). However, this classification is not yet widely accepted. HEV-like isolates have constantly been discovered from additional hosts; based on the new classification system, rat and ferret HEV isolates have been categorized into Orthohepevirus C, while bat isolates have been classified into Orthohepevirus D (Lorenzo et al., 2007; Johne et al., 2010; Drexler et al., 2012; Raj et al., 2012; Krog et al., 2013; Lin et al., 2014; Smith et al., 2014; Woo et al., 2014). Notably, no zoonotic infections caused by Orthohepevirus C or D isolates have been reported to date in humans.

In addition to HEV genotypes that infect mammals described above, two unique HEV-like virus isolates from chicken and cutthroat trout (Oncorhynchus clarkia) are now assigned to species Orthohepevirus B and genus Piscihepevirus (Smith et al., 2014). Avian HEV-like viruses are also known as avian HEV (Zhao et al., 2010). Avian HEVs share less than 50% nucleotide identity to known mammalian HEVs (Haqshenas et al., 2001; Zhao et al., 2010). However, their viral capsid proteins contain both unique and common antigenic epitopes when compared with mammalian HEVs (Haqshenas et al., 2001, 2002; Huang et al., 2004; Guo et al., 2006; Zhou et al., 2008; Zhao et al., 2015). Consequently, the pathogenesis of avian HEV in chicken or hens is variable and may include hepatitis-splenomegaly syndrome (Payne et al., 1999; Haqshenas et al., 2001), decreased egg production (Zhao et al., 2017), or even subclinical infection (Gerber et al., 2015).

A distant cousin of HEV, cutthroat trout virus (CTV), was originally discovered in 1988 but has not been associated with any disease in these fish (Batts et al., 2011). In a retrospective study, CTV was identified as an HEV-like virus but shares even lower sequence identity with mammalian HEV than avian HEV; therefore CTV has been classified into a separate genus, Piscihepevirus (Batts et al., 2011; Smith et al., 2014). To date, comparison of CTV with HEV isolates from other hosts has not yet been conducted. However, to date no evidence has indicated that avian HEV or CTV is capable of infecting human or mammalian hosts.

Hepatitis E was initially proposed to be a disease restricted to only developing countries (Aggarwal, 2011; Khuroo and Khuroo, 2016). However, this conclusion conflicted with the observations of sporadic hepatitis E cases in developed countries (Zaaijer et al., 1992; Coursaget et al., 1994; Tassopoulos et al., 1994; Zanetti and Dawson, 1994; Johansson et al., 1995). Although most sporadic hepatitis E cases in developed countries could be explained by transmission by travel from endemic regions or by contact with people traveling from an endemic region, HEV cases still appear and thus are not fully explained by the conventional fecal-oral transmission route. Therefore, HEV may retain a low endemicity in developed countries with unknown sources of infection (Tassopoulos et al., 1994). Meanwhile, after development of HEV diagnostic tools, serosurveillance data for anti-HEV antibodies (for detecting previous HEV infection) has revealed a drastically high proportion of the population (up to 28% in some areas) living in the United States and other developed countries where hepatitis E is not endemic (Lok et al., 1992; Quiroga et al., 1996; Mast et al., 1997; Thomas et al., 1997). Taken together, these observations imply that unknown sources of HEV infection or unrecognized non-pathogenic or lower pathogenic HEV strains are circulating in developed countries.

Although the exact causes of sporadic HEV cases in developed countries were unclear before the mid-1990s, several subsequent reports indicated that anti-HEV antibodies naturally existed in certain animal species such as primates (Arankalle et al., 1994), swine (Clayson et al., 1995), rodents (Turkestan rats, house mice, and wood mice), and sheep (Karetnyi Iu et al., 1993; Usmanov et al., 1994). Meanwhile, HEV RNA-positive samples were identified from stools of swine that were shedding virus (Clayson et al., 1995), which is consistent with an earlier report demonstrating that domestic pigs were susceptible to experimental infection by a human HEV strain of unknown genotype (Balayan et al., 1990). In other studies, because non-human primates are susceptible to human HEV infection, other animals were also tested for susceptibility to human HEV for establishment of animal models. However, only mixed data was obtained due to poor understanding of HEV genotypes and host tropism. For example, on the one hand lambs experimentally infected with a human HEV isolate (of unknown genotype, possibly genotype 1) contracted infection, with acute biochemical and histological evidence consistent with hepatitis (Usmanov et al., 1994). On the other hand, rats infected with human HEV (genotype was not mentioned) showed no clinical sign of hepatitis except evidence of HEV replication in the liver (Maneerat et al., 1996). Since viral nucleotide sequences in these animal studies were unavailable, no definitive evidence confirming the existence of a zoonotic HEV strain was reported before the mid-1990s. However, these earlier reports did imply possible HEV zoonoses had occurred.

Finally, in 1997 the hallmark of zoonotic HEV research was reported and described the identification and characterization of swine HEV isolates bearing a close relationship to human hepatitis E virus isolated from a swine farm in the United States (Meng et al., 1997). However, unlike human HEV, piglets infected with swine HEV were clinically normal except for microscopic evidence of hepatitis along with subclinical HEV viremia and seroconversion (Meng et al., 1997). Therefore, evidence for a direct link between swine HEV and human hepatitis E was emerging.

Coincidently, almost concurrent with the identification of swine HEV, two cases of human acute hepatitis E were reported in the United States. Notably, one patient had no epidemiological history of traveling from endemic regions (Kwo et al., 1997; Schlauder et al., 1998). Surprisingly, sequence of HEV (strain US-1 and US-2) recovered from these two patients demonstrated that both strains were quite distinct from previously known human HEV strains but shared highest similarity (greater than 97% aa identity of ORF1 and ORF2) to a swine HEV reference sequence (Meng et al., 1998b; Schlauder et al., 1998); this result finally confirmed an HEV zoonosis event. Moreover, both pigs and non-human primates could be experimentally infected by HEV US-2 human isolate or swine HEV isolate (Meng et al., 1998b). This result thus demonstrated that newly identified HEV isolates (swine HEV-related isolates) are zoonotic and capable of causing cross-species infection. Meanwhile, it is notable that experimental infection failed when human HEV strain Sar-55 (genotype 1) and Mexican strain (genotype 2) were used to inoculate pigs (Meng et al., 1998a). Collectively, these data confirm that at least some HEV strains are closely related to swine HEV can cross the species barrier to infect humans. On the contrary, pigs experimentally infected with either human HEV (Sar-55 and Mexican strain) or swine HEV were clinically normal. But seroconversion, fecal shedding of virus, and viremia were only observed in swine HEV-infected pigs, suggests a complete abolition of replication of human HEV (genotypes1 and 2) in pigs (Meng et al., 1998a). Taken together, these data indicate that zoonosis of HEV is genotype specific and does not apply to all HEV isolates despite the cross-reactivity of viral capsids between zoonotic HEV and human HEV.

After both genetic evidence and experimental evidence confirmed HEV zoonotic transmission, screening for HEV prevalence in various animal species, detection of new viral isolates, as well as identification of new hosts, were conducted worldwide. Consequently, the serum-positive prevalence of anti-HEV antibody was shown to be common among pig herds regardless of the HEV genotype circulating in human populations living within the same region (Chandler et al., 1999; Meng et al., 1999). Notably, HEV isolates from sporadic acute hepatitis E patients in Japan demonstrated 99.0% sequence identity (nearly 100%) to a swine HEV (swJ13-1) isolated from a pig living in the same region (Nishizawa et al., 2003). These isolates share highest homology with certain human HEV isolates from China, but were distinct from zoonotic swine HEV isolates from the United States. These results therefore suggest that zoonotic swine HEV strains are surprisingly heterogeneous and include various HEV genotypes (now known as genotypes 3 and 4 of species Orthohepevirus A) (Takahashi et al., 2002, 2003).

Meanwhile, rabbits may serve as a potential zoonotic reservoir for human hepatitis E as well (Burt et al., 2016). Based on sequence analyses of rabbit HEV strains isolated from the United States and China, rabbit HEV strains from both countries are closely related to genotype 3 HEV (Zhao et al., 2009; Cossaboom et al., 2011, 2012a). Moreover, a rabbit HEV strain identified from France matched a closely related human HEV isolate (from a French patient) and these isolates support possible zoonotic transmission of a genotype 3 rabbit HEV (Izopet et al., 2012). As further evidence, zoonosis of rabbit HEV was subsequently confirmed by experimental infection of cynomolgus macaques by rabbit HEV (Liu et al., 2013). Moreover, unlike swine HEV, experimental infection studies have suggested that rabbits are also susceptible to genotype 4 swine HEV (Ma et al., 2010; Han et al., 2014). However, no rabbit HEV isolates identified so far could be classified into genotype 4 HEV. On the contrary, although susceptible to rabbit HEV, pigs inoculated with rabbit HEV exhibited low levels of viremia and fecal virus shedding accompanied by active but weak HEV replication (Cossaboom et al., 2012b). These observations appear to be consistent with the few cases of rabbit HEV infection in humans, implying a potential species barrier separating rabbits from humans or other mammalian hosts to infection.

Deer and wild boar have been reported as potential zoonotic hosts for HEV as well (Tei et al., 2003; Takahashi et al., 2004). In one report from Japan, a full-genome genotype 3 HEV strain sequence obtained from wild boar demonstrated 99.7% sequence similarity with a previously characterized HEV strain originating from wild deer captured in the same region. The deer isolate was linked to virus from four hepatitis E patients who had eaten raw meat from a captured deer (Tei et al., 2003; Takahashi et al., 2004). Therefore, it appears that certain HEV strains of genotype 3 do not require genetic adaptation to cross the species barrier to infect new hosts. However, it is notable that no direct evidence suggesting a wild boar-to-human transmission pattern except the genetic identity of HEV recovered from patients and wild boar. Meanwhile, wild boar-specific HEV isolates (genotypes 5 and 6 of Orthohepevirus A) with unique nucleotide sequences have not yet been implicated in disease transmission to humans. Therefore, wild boar as a zoonotic host for HEV still needs further investigation.

More recently, while HEV and HEV-like virus have been consistently isolated from various animals, HEV surveillance data has suggested a greater host range for HEV than researchers originally predicted. Additional hosts for HEV include cattle, sheep, goats, cats, dogs, chickens, mongooses, bats, ferrets, rats, camels, and even trout (Liang et al., 2014; Smith et al., 2015b; Wu et al., 2015; Park et al., 2016; Yan et al., 2016). These new HEV isolates cannot be categorized within the old classification system of HEV and appear to form new genotypes, species, and even genera within the family Hepeviridae (Smith et al., 2014), suggesting Hepeviridae is a highly diverse viral family. Meanwhile, the zoonotic potential of these new HEV isolates is still unclear. To date, only one report has demonstrated a case of chronic HEV infection caused by genotype 7 camel HEV (Woo et al., 2014; Lee et al., 2016).

To date, three zoonotic HEV genotypes have been confirmed to infect humans (Nan and Zhang, 2016). In addition to well-known HEV genotypes 3 and 4, genotype 7 of Orthohepevirus A (camel HEV) has also been recently demonstrated to infect humans (Lee et al., 2016). Moreover, analysis of the National Health and Nutrition Evaluation Survey (NHANES) 1988–1994 dataset found a relatively high seroprevalence (21%) of HEV infection in the United States general population (Ditah et al., 2014). Since most acute or chronic hepatitis E cases in developed countries have been sporadic and were caused by zoonotic HEV genotypes 3 and 4, it appears that cross-species transmission of zoonotic HEV from animal hosts to humans in developed countries depends on a variety of transmission routes other than the fecal-oral route. Nevertheless, the high stability of infectious HEV virions significantly increases the chance for cross-species transmission to humans regardless of route of transmission. Indeed, in one imported hepatitis E case reported in Japan, the HEV virus was believed to originate from an herbal product brought from China by the patient several months previously (Ishikawa et al., 1995). Furthermore, a recent study demonstrated that HEV virions obtained from cell culture maintained infectivity for up to 21 days at 37°C and for 28 days at room temperature (Johne et al., 2016). Therefore, it appears that HEV viral particles can remain highly stable in the environment (Purcell and Emerson, 2001).

After the discovery of swine HEV, the risk of zoonotic transmission has become a concern for public health. Most acute hepatitis E infections have been linked to consumption of contaminated pork or pig liver (Kamar et al., 2013; Pavio et al., 2014; Cossaboom et al., 2016). Moreover, the source of chronic hepatitis E infection in immunocompromised patients has often been unknown but is thought to be linked with pork consumption as well (Murali et al., 2015). Based on surveillance studies, approximately 2% of pig livers sold in Japan and 11% of pig livers sold in America are positive for HEV-RNA (Yazaki et al., 2003; Feagins et al., 2007). Moreover, deer have been demonstrated to be directly linked to zoonotic cases of hepatitis E in humans (Pavio et al., 2010). Meanwhile, since HAV could be taken up and concentrated by shellfish, recent studies have also supported shellfish consumption as a risk factor for HEV transmission and concentrated HEV virions have been detected in shellfish which is similar to HAV (Crossan et al., 2012; Gao et al., 2015; Huang et al., 2016). Therefore, it is important to study shellfish as a source of human HEV infection because they are filter-feeders. Such studies should be especially emphasized in more contaminated coastal areas, where consumption of raw shellfish may be a contributing factor to HEV incidence (Grodzki et al., 2014).

Contaminated milk obtained from animals might be another way for cross-species transmission of zoonotic HEV (Huang et al., 2016). Recently it has been discovered that camel HEV infection of an organ transplant recipient who regularly consumed camel meat and milk developed hepatitis, suggesting that HEV might have existed in the milk product (Lee et al., 2016). Although most milk for human consumption comes from cattle and goats, little research has been conducted to examine if HEV from cattle or goats is able to infect humans by this route. However, since both genotypes 3 and 4 HEV have been isolated from goats (Di Martino et al., 2016; Long et al., 2017), HEV transmission from goats to human is highly possible and goats should be considered as a potential animal reservoir for zoonotic HEV.

Besides foodborne transmission, direct contact with animal body fluids may be another transmission route for zoonotic HEV infection. It was reported earlier that HEV was detected in nasal and rectal swab materials in experimentally infected swine (Meng et al., 1998a). Meanwhile, during the experimental infection of rabbits with swine genotype 4 HEV, HEV-RNA was shed in the saliva of some rabbits as well (Wu et al., 2017). Moreover, a recent report documented that HEV-RNA was detected in the urine of experimentally infected pigs (Bouwknegt et al., 2009). Additionally, other data suggested that HEV could be transmitted to people by direct contact with infected animals (Andraud et al., 2013), which is consistent with observations of horizontal transmission from HEV infected wild boars that were in contact with pigs (Schlosser et al., 2014). Notably, these observations together offer a possible explanation for the rising rate of anti-HEV seroprevalence in swine workers and pork butchers (Traore et al., 2015; Lange et al., 2017; Teixeira et al., 2017).

In the fecal-oral transmission route, HEV are shed in the feces of infected individuals as stable, non-enveloped virions (Feng et al., 2014; Nan and Zhang, 2016). However, recent discoveries of quasi-enveloped viral particles have suggested a new challenge for HEV vaccine development. It was found that more than 90% of HEV particles from an HEV-infected individual’s serum were quasi-enveloped and unable to be neutralized by antibodies against the HEV capsid protein (the major target of current licensed HEV vaccine) (Takahashi et al., 2010). Thus, in foodborne hepatitis E cases or other transmission routes, it is possible that HEV particles causing infection are quasi-enveloped particles. Therefore, it is still uncertain that vaccine-induced antibodies are capable of neutralizing such quasi-enveloped HEV particles in vivo to subsequently confer protection in vaccine recipients under such circumstances.

Since zoonotic HEV strains have been isolated from swine, HEV and HEV-like virus have been constantly detected in a variety of mammalian hosts. Subsequently, interspecies transmission was experimentally conducted to test all HEV isolates from mammalian hosts for zoonotic characteristics or host restricted. Details regarding mammalian HEV genotypes, natural hosts, zoonotic infectivity and experimental cross-species transmission to other animals, and pathogenesis in different hosts are summarized in Table 1. Based on these data, human-restricted HEV genotypes (genotypes 1 and 2), zoonotic HEV genotypes (genotypes 3, 4, and 7), and animal-restricted HEV genotypes (Orthohepevirus C) have been observed. Therefore, it appears that both host factors and viral determinants are involved in HEV host tropism, zoonotic infection, and clinical outcomes of HEV infection.

Meanwhile, bioinformatics analysis at the complete genome level for HEV genotypes 1–4 have demonstrated that divergence of zoonotic and anthropotropic genotypes occurred approximately 536 to 1344 years ago (Purdy and Khudyakov, 2010). Genotype 1 HEV appears to be a more recent genotype than zoonotic HEV genotypes, with the estimated time to the most recent common ancestor (TMRCA) of most modern lineages of HEV-1 pinpointed to ∼87–199 years ago (Purdy and Khudyakov, 2010). Meanwhile, post-divergence evolutionary rates have appeared to differ between genotypes within Orthohepevirus A. Specifically, genotypes 3 and 4 of HEV appear to have higher aa substitution rates over time, resulting in fewer conserved aa sites than genotype 1 HEV across the entire genome (Brayne et al., 2017). These results imply that aa substitutions may be required for adaptation of viral infectivity to new non-human hosts. This is also consistent with codon usage bias of genotypes 3 and 4 HEV, whereby codon bias within their ORFs tends to be lower than observed for genotype 1 HEV (Bouquet et al., 2012). It is postulated that these findings may reflect the requirement of zoonotic HEV genotypes to adapt to multiple hosts and therefore different cell types with distinct microenvironments and codon usage preferences (Sridhar et al., 2017). Taken together, it appears that genotype 1 HEV (and possibly genotype 2) became highly adapted to humans after divergence from zoonotic HEV and thus did not undergo further diversification. Therefore, genotype 1 HEV (and possibly genotype 2) exhibits fewer aa substitutions and higher codon use bias after losing the ability to infect animal hosts other than non-human primates.

Although one report has demonstrated that rabbits infected by genotype 1 HEV exhibited no clinical signs of hepatitis other than seroconversion (Ma et al., 2010), no other similar results have been reported. In fact, available data still favors the premise that HEV genotypes 1 and 2 are human-restricted genotypes (Pavio et al., 2015). One consistent observation is that in humans infected by both genotypes 1 and 2 HEV only develop acute hepatitis, with no chronic cases reported, while acute hepatitis cases have been reported for zoonotic HEV infection (both genotypes 3 and 4). Considering that the high anti-HEV seropositive rate among the general population results from previous zoonotic HEV infection, acute hepatitis E cases caused by zoonotic HEV should consequently be very rare. This result aligns with research showing that non-human primates experimentally infected with genotype 3 swine HEV were clinically normal (Meng et al., 1998b). Moreover, a recent study based on 123 patients with clinical hepatitis E in Scotland demonstrated no evidence for an association between variants of genotype 3 HEV and disease severity. Therefore, these results suggest that host factors rather than virus variants contribute to observed clinical phenotype during genotype 3 zoonotic HEV infection (Smith et al., 2015a). Meanwhile, other studies using non-human primates to evaluate host pathogenicity during infection by four major human-HEV genotypes have demonstrated that HEV genotypes 1 and 2 are able to cause more severe disease than genotypes 3 and 4 (Krawczynski et al., 2011). Furthermore, is notable that swine infected with a genotype 3 HEV strain isolated from an acute human hepatitis E patient did not develop clinical hepatitis and were clinically normal, which is similar to observations for genotype 3 swine HEV infection (Meng et al., 1998b). However, viral determinants that underlie the above pathogenic differences between human-restricted HEV and zoonotic HEV remain unclear. Perhaps reverse genetics-based techniques will be useful for elucidating viral factors among the various HEV genotypes that trigger pathogenic outcomes.

Among all HEV ORFs, HEV-ORF1 is the largest ORF and is considered to play an indispensable role in determining host tropism. A recent report based on gene swapping among genotypes 1 and 4 HEV infectious clones has demonstrated that HEV-ORF1 non-structural protein facilitates crossing of the species barrier (Chatterjee et al., 2016). Based on this report, chimeric virus based on a genotype 1 HEV infectious clone bearing the genotype 4 HEV-ORF1 was shown to replicate in porcine kidney cells, while the original genotype 1 HEV could not (Chatterjee et al., 2016). However, the underlying molecular mechanism that ORF1 plays to alter host cell tropism during HEV cross-species infection is not yet clear, mainly because ORF1 accounts for more than two-thirds of the entire HEV genome. Nevertheless, among all functional domains of HEV-ORF1, the hypervariable region appears to be involved in host adaptation. When the genotype 3 swine HEV and related United States human strains (US-1 and US-2) were identified and characterized, the hypervariable regions of these genotype 3 isolates were shown to be significantly larger in size than those of genotypes 1, 2, and 4 (Meng et al., 1998b). Moreover, identification of host-viral recombination events in the hypervariable region also provided further evidence for involvement of this region in cross-species infection. Notably, the novel insertion of a human ribosome protein S17 sequence into the hypervariable region of genotype 3 HEV enhanced the ability of chimeric virus to infect cells from several species (Feagins et al., 2011; Kenney and Meng, 2015b). Meanwhile, the viral RdRp-based replication complex was tested for its role in host tropism as well. In a reporter gene-based HEV replicon system comparing the replication efficiency of genotype 1 HEV in swine cells, macaque kidney and human liver cells, it appeared that translation of the ORF2 capsid gene of genotype 1 virus was severely inhibited in swine kidney cells, which led to insufficient capsid production for optimal assembly of HEV virions (Nguyen et al., 2014).

In early studies, HEV-ORF2 was initially thought unlikely to be involved in host tropism, due to its conserved status among all HEV ORFs with 85% identity within all four major HEV genotypes infect humans (Meng, 2016). Several in vivo studies conducted later suggested that genotype 3- or genotype 4-based chimeric viruses bearing ORF1 from genotype 1 failed to infect or establish a robust infection in swine (Feagins et al., 2011; Cordoba et al., 2012), demonstrating that HEV-ORF2 does not potentiate virus interspecies infectivity. However, this view was challenged by new observations based on infectious recombinant chimeras of genotypes 1 and 3 HEV isolates. Newer studies showed that transfer of genotype 1 capsid aa 456 to 605 (the putative virus receptor-binding region) to genotype 3 virus prevented the chimeric virus from infecting swine as expected. These results aligned with the fact that genotype 1 viruses are unable to enter swine cells (Nguyen et al., 2014) and implied that viral capsid proteins also determine host preference. However, since cellular receptors of HEV in humans or other animals are still unidentified, the link between capsid-dependent HEV host tropism and the cellular receptor participating in viral infection of various host species would be an attractive area of future HEV research.

Besides ORF1 and 2, other studies have indicated that HEV-ORF3 product might be involved in host tropism as well. Currently, all available data suggest that HEV-ORF3 product is a class I viroporin that functions as an ion channel essential for viral particle release during HEV infection (Ding et al., 2017). The PSAP motif located within aa 95–98 is required for HEV virion release and is highly conserved among all seven genotypes of Orthohepevirus A (Figure 2). Meanwhile, our previous research indicated the first 25 aa of genotype 1 HEV are essential for HEV-ORF3’s association with microtubule, which is also involved in virus release (Kannan et al., 2009). Alignment of the ORF3 aa sequence among all eight genotypes demonstrated the first 25 aa of ORF3 are more conserved than the remaining regions (Figure 2). Taken together, these conserved aa sequences support a conserved role of virion release promoted by HEV-ORF3 irrespective of HEV genotype. However, there is less homology elsewhere in the protein, especially in the HEV-ORF3 C-terminal half (aa 62 to aa 114) (Figure 2), which appears to be important for adaptation to various hosts. Moreover, the different genomic locations of HEV-ORF3 among the species of Orthohepevirus (either partially overlapping ORF2 or within ORF2) (Woo et al., 2014), may suggest a genotype-specific evolution pattern influencing genotype-specific function for HEV-ORF3 to shape host tropism (Figure 1B). This speculation is also consistent with our previous observation of genotype-specific enhancement of IFN induction by the HEV-ORF3 product (Nan et al., 2014). How a genotype-specific function of ORF3 product affects HEV host tropism begs further investigation. Furthermore, in addition to the three well-known ORFs, the role played by the newly identified ORF4 in host tropism has not been studied but is of interest, since this protein is only conserved in genotype 1 HEV.

In addition to viral determinants, host factors, especially host immune status, may contribute to cross-species transmission of zoonotic HEV and HEV host tropisms. This is supported by establishment of HEV chronicity is animal models by administration of immunosuppressive drugs to animals or infection of another virus impairing immune system (Salines et al., 2015; Cao et al., 2017; Gardinali et al., 2017). Conversely, infection of organ transplantation receipt by camel HEV raises an interesting question regarding whether host immune status affects cross-species transmission of HEV (Lee et al., 2016), which is also consisted with observation that genotype 3 HEV could establish chronicity in a human liver chimeric mouse model (uPA^+/+Nod-SCID-IL2Rγ^-/-)(van de Garde et al., 2016). Therefore, although a species barrier shapes the HEV host tropism, if the immune system compromised in one species (e.g., rat), reduced immunity might confer host susceptibility to another species specific-HEV, such as ferret HEV.

Finally, age might be another factor related to the severity or virulence of acute hepatitis caused by zoonotic HEV isolates both for HEV genotypes 3 and 4. In animal studies conducted to evaluate pathogenicity of swine HEV, the animals used are generally young pigs. However, in one Japanese study evaluating zoonotic HEV pathogenesis in humans, among all 37 acute hepatitis E cases caused by HEV genotypes 3 and 4, only two cases were identified in individuals younger than 30 years of age (Ohnishi et al., 2006). In another Japanese study comparing HEV sequences of sporadic acute hepatitis E patients and viruses isolated from pig livers sold in markets, none of 17 sporadic cases was younger than 40 years of age (Okano et al., 2014). Similar observations were also reported for sporadic acute hepatitis E cases caused by zoonotic HEV worldwide (Festa et al., 2014; Inagaki et al., 2015). However, while elderly people tend to develop acute hepatitis E after zoonotic HEV infection, the underlying mechanism is still unclear. Indeed, the explanation appears not to be linked with weaker immunity upon aging, since zoonotic HEV infection, especially by genotype 3 HEV, tends to become chronic in immunocompromised individuals. Therefore, the linkage of age and severity of zoonotic HEV infection in humans needs further study.

The panorama of HEV research has changed drastically with the latest findings in the field. Current research focuses on understanding virus biology, characterizing the host immune response during infection, and developing strategies for prevention of HEV as a zoonotic disease. More than two decades have passed since the report of the first complete genome sequence of the HEV prototype strain. Since then, our understanding of HEV zoonosis has remained limited. While diverse zoonotic HEV isolates exist in HEV genotypes 3 and 4, certain HEV isolates, such as genotype C1 and C2 of Orthohepevirus C, are only able to infect rats and ferrets, respectively. These results suggest an existence of host tropism and species barriers among various HEV isolates. However, except for the potential involvement of HEV ORF1 and the hypervariable region within HEV-ORF1, few studies have focused on these issues and information is still lacking to explain viral determinants and host factors involved in HEV host tropism and species barriers to interspecies HEV transmission.

Besides host tropism, HEV pathogenesis appears to involve a scenario more complex than predicted after discovery of HEV as a zoonotic pathogen. Different pathogenesis in humans or in experimental animal models induced by anthropotropic HEV (genotypes 1 and 2 which only cause acute hepatitis) or zoonotic HEV (genotypes 3 and 4, cause both acute and chronic hepatitis in humans and subclinical infections in animals) was recognized years ago. However, viral determinants influencing pathogenicity among anthropotropic and zoonotic HEV are still unidentified. Meanwhile, our understanding of chronic HEV infection in immunocompromised patients, as well as the occurrence of HEV-related ALF in both the general population and in pregnant women, requires additional research.

Although capsid proteins among all four major HEV genotypes share over 85% similarity, development of subunit vaccines based on recombinant HEV capsids was not achieved until 2012. In that year, the HEV239 vaccine was approved in China after being the only such vaccine to pass phase III trials in humans. Unfortunately, this vaccine, which is based on truncated capsid protein of genotype 1 HEV, is still unavailable to most of the world. Such vaccines are urgently needed in highly endemic HEV areas in spite of the fact that there is a high prevalence rate of HEV worldwide. Regardless, since the HEV239 vaccine is solely based on genotype 1 HEV, its efficiency for protecting against zoonotic HEV isolates is still unknown. Furthermore, recent discoveries about antigenic variation among HEV genotypes and quasi-enveloped viral particles hidden from neutralizing antibody suggest new challenges toward achieving high protective efficacy using any vaccine. Ultimately, development of a vaccine with efficacy against multiple HEV genotypes is still a challenging but a worthwhile goal.

YN prepared the main body of this manuscript. CW prepared all the figures and the table. QZ revised the manuscript. E-MZ designed this manuscript and supervised manuscript writing. All the authors contributed to the preparation of the final version of the manuscript and approved it for publication.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.