Orthohepevirus C: An Expanding Species of Emerging Hepatitis E Virus Variants.

Hepatitis E virus (HEV) is an emerging zoonotic pathogen that has received an increasing amount of attention from virologists, clinicians, veterinarians, and epidemiologists over the past decade. The host range and animal reservoirs of HEV are rapidly expanding and a plethora of emerging HEV variants have been recently identified, some of which have the potential for interspecies infection. In this review, the detection of genetically diverse HEV variants, classified into and presumably associated with the species Orthohepevirus C, currently comprising HEV genotypes C1 and C2, by either serological or molecular approach is summarized. The distribution, genomic variability, and evolution of Orthohepevirus C are analyzed. Moreover, the potential risk of cross-species infection and zoonotic transmission of Orthohepevirus C are discussed.


Introduction
Hepatitis E Virus (HEV) is the causative viral pathogen of hepatitis E, originally considered a self-limiting disease occurring only in developing countries with poor sanitary conditions [1]. Mortality rates in infected pregnant women can reach up to 25% during hepatitis E outbreaks in these countries [2]. In the late 1990s, hepatitis E was recognized to be a zoonotic disease and swine was found to be the natural host of HEV in the USA. Soon afterwards, a large number of autochthonous cases were reported in different Europe countries associated with the consumption of undercooked meat [3]. Hepatitis E poses a significant health risk for immunocompromised individuals, such as solid organ transplant recipients and patients with human immunodeficiency virus infection, in whom chronic infections can occur. Ribavirin is commonly administered as an off-label treatment for chronic HEV infection, but significant side-effects often limit its use [4]. Furthermore, HEV may also cause extrahepatic manifestations, including renal and neurological injuries, pancreatitis, cryoglobulinemia, and hematological disorders [5]. In light of this, hepatitis E is being increasingly recognized as a serious public health burden worldwide.
HEV was first isolated in 1983 and the viral genome was subsequently cloned and sequenced [6,7]. HEV is approximately 27-34 nm in diameter. Viral genomes are 6.4-7.2 kb long capped positive-sense single-stranded RNA containing three open readings frames (ORF1, ORF2, and ORF3), 5 -and 3untranslated regions, a 7-methylguanylate cap at the 5 end and a polyadenine tract at the 3 -end. ORF1 encodes a non-structural polyprotein containing a methyltransferase, papain-like cysteine protease, macro domain, RNA helicase, and RNA-dependent RNA polymerase. ORF2 encodes the capsid protein. ORF3 overlaps partially with ORF2 and encodes a multifunctional phosphoprotein associated with viral egress [8]. Recent studies have reported that HEV particles are non-enveloped and infectious in bile and feces, while quasi-enveloped and less infectious/non-infectious in sera [9].
HEV differs from all other human hepatitis viruses in that it is closely related to non-human viruses [10]. HEV is the prototype of the family Hepeviridae, which includes the genera Orthohepevirus and Piscihepevirus. The genus Orthohepevirus is further divided into four species Orthohepevirus A to D. Orthohepevirus A contains HEV variants isolated from human, pig, wild boar, deer, mongoose, rabbit, and camel; Orthohepevirus B from chicken; Orthohepevirus C from rat, greater bandicoot, Asian musk shrew, ferret, and mink; and Orthohepevirus D from bat. The genus Piscihepevirus occurs in cutthroat trout and possibly other fish species [11]. Based on the host range of viruses and phylogeny of viral genomic sequences, proposals are also made for the designation of HEV genotypes and subgenotypes [12]. Nevertheless, numerous novel HEV strains remain unclassified due to the large degree of divergence or the lack of complete genomic sequences.
The species Orthohepevirus C is separated into two genotypes: HEV-C1 and HEV-C2. HEV-C1 contains variants derived from hosts in the orders Rodentia and Soricomorpha, and HEV-C2 from the order Carnivora [8,11]. By means of the application and development of new molecular techniques such as metagenomic sequencing, a growing number of distinct HEV variants have been discovered in a variety of animal species globally and the species Orthohepevirus C has been rapidly extended in the last years [13]. Although the zoonotic potential of Orthohepevirus C is still under debate, two recent clinical cases describing persistent hepatitis in a liver transplant patient in Hong Kong and severe acute hepatitis in an immunocompetent patient in Canada, respectively, have been attributed to infection with rat HEV [14,15]. Furthermore, seven additional rat HEV infections have been confirmed lately in Hong Kong [16]. In this review, we aim to compile and discuss the recent discoveries of HEV variants within the species Orthohepevirus C and their potential risk of cross-species infection and zoonotic transmission to humans.

Detection and Distribution of Orthohepevirus C
The first study concerning the prevalence of anti-HEV antibodies in wild rats was conducted in the USA in 1999 [17]. In 2000, another study from the USA reported on the HEV seroprevalence among 13 rodent species, providing evidence for widespread infection of Orthohepevirus C in rodents and suggesting these animals might be the natural reservoirs for this species of HEV [18]. Subsequently, Orthohepevirus C RNA was detected in feces of Norway rats in Germany in 2010 using a nested broad-spectrum reverse transcription polymerase chain reaction (RT-PCR) and the complete genomes were sequenced and found to be highly divergent from human HEV strains [19,20]. Thus far, anti-HEV or anti-rat HEV antibodies have been detected in 23 animal species, including 12 and 10 species within the families Muridae and Cricetidae of the order Rodentia, respectively, and one species within the family Soricidae of the order Soricomorpha, from eight countries (the USA, Japan, Germany, China, Vietnam, Lithuania, Indonesia, and India). Additionally, Orthohepevirus C sequences have been detected in 22 countries (the USA, Germany, France, Denmark, China, Lithuania, Hungary, Austria, Switzerland, Italy, Spain, Greece, Belgium, Czech Republic, England, Indonesia, Vietnam, Uganda, the Netherlands, Japan, Brazil, and Kenya) and 25 animal species, containing eight species within the family Muridae and 10 species within the family Cricetidae of the order Rodentia, one species within the family Ursidae and two species within the family Mustelidae of the order Carnivore, two species within the family Falconidae of the order Falconiformes, one species within the family Soricidae of the order Soricomorpha, and one species within family Hominidae of the order Primates. Worldwide detection and distribution of Orthohepevirus C is displayed in Figure 1. Although HEV serology and nucleic acid detection remains negative in several of these animal species, this may be linked to limitations of current detection assays, such as poor specificity and low sensitivity. countries with only RNA detection are in orange. World map was created by using a free and open source geographic information system Quantum Geographic Information System version 3.10 (http://qgis. osgeo.org) and free vector and raster map data from Natural Earth (http://www.naturalearthdata.com).
Antibodies against HEV were also found in multiple rodent species within the family Cricetidae. Seroprevalence rates in Clethrionomys gapperi (Southern red-backed vole), Neotoma albigula  11.0 (11/91), and 32.7 (37/113), respectively. Rodent sera were collected during 1994-1998 throughout the USA and assessed by two in-house established serological tests including antigens based on a mosaic protein composed of recombinant HEV ORF2 and ORF3 and a 55-kDa HEV ORF2 protein [18].
Additionally, seroprevalence in Suncus murinus (House shrew) within the family Soricidae of the order Soricomorpha was 10.4% (27/260) for anti-HEV IgG and 4.6% (12/260) for Immunoglobulin M (IgM), respectively, with samples collected between 2011 and 2012 in China [35]. The studies in terms of seroprevalence of anti-HEV antibodies are summarized in Table 1.
Differing seroprevalence of anti-HEV antibodies may be due to different serological tests, which have been carried out using commercial anti-HEV enzyme-linked immunosorbent assay (ELISA) kits or conducted with in-house established assays based on diverse antigens (truncated or complete human and rat HEV ORF2 protein) in numerous labs independently. Furthermore, although there is only a single serotype of HEV, the cross-reaction of other known or unknown viral pathogens cannot be excluded and may impact the results. Finally, no HEV seroprevalence studies have been performed on species of the orders Carnivora and Falconiformes.

Detection of Orthohepevirus C Genomes
Molecular biology methods, including RT-PCR amplification, Sanger sequencing and metagenomic analysis with next-generation sequencing, have facilitated the detection of Orthohepevirus C (previously designated rat HEV) RNA from liver, feces, and blood specimens of various animal species. Furthermore, comprehensive sequence and phylogenetic analyses enable genotyping of viral strains. Consequently, an increasing number of novel Orthohepevirus C variants have been discovered around the world whose assignment remain to be determined.
Additionally, Orthohepevirus C sequences have been detected in other animal species of mammals. Within the family Soricidae of the order of Soricomorpha, the species Suncus murinus (House shrew/Asian musk shrew) harbor HEV-C1 in China and Asian musk shrew have been implied as a reservoir for HEV-C1 from wild rats, while the virus in the species Crocidura olivieri (Olivier's shrew) from Kenya cannot be assigned due to increased genetic divergence [35,44,48,50].
In the order Carnivora, HEV-C1 has been identified in a Syrian brown bear (species Ursus arctos syriacus, family Ursidae) in a German zoo, most likely the result of a spillover infection from free-roaming Norway rats [42]. In 2012, HEV-C2 was firstly reported in Western polecats (species Mustela putorius) from the Netherlands [54]. Soon afterwards, HEV-C2 was detected in ferrets from the USA, Japan, and China and in minks from Denmark and China [54][55][56][57]. Both species Mustela putorius and Neovison vison belong to the carnivore family Mustelidae. Even though a short (362 nucleotide) partial ORF1 sequence was identified in a red fox (species Vulpes vulpes, family Canidae), classification into Orthohepevirus C requires comparisons based upon complete genome sequences, as recommended by the International Committee on the Taxonomy of Viruses (ICTV) Hepeviridae Study group [58].
Recently, novel HEV variants have been reported from common kestrel (species Falco tinnunculus) and red-footed falcon (species Falco vespertinus) within the family Falconidae of the order Falconiformes in Hungary, notably the viral strain from kestrel showed higher sequence similarity to members of Orthohepevirus C than to Orthohepevirus B and clustered together with aforementioned rodent HEV variants from China and Brazil; therefore, the kestrel-derived HEV strain is included in this review [59].
Finally, nine HEV-C1 strains have been reported in humans, eight being typical rat HEV sequences from Hong Kong and the other divergent rat HEV sequence derived possibly from Uganda [14][15][16]. However, the precise source and transmission pattern of these nine HEV-C1 strains remain unclear. The studies with regards to the detection of Orthohepevirus C genomes are listed in Table 2.     (Table 1). For a better comparative analysis and genomic characterization of Orthohepevirus C, the schematic description of genomic organization of representative Orthohepevirus C variants from individual animal species is depicted in Figure 2.

Genetic Variability of Orthohepevirus C
As mentioned, the majority of described Orthohepevirus C sequences are partial genomic fragments (<500 bp). In order to determine the genetic variability of HEV variants within the species Orthohepevirus C, we have included 48 currently available complete or nearly complete genomic sequences of Orthohepevirus C from GenBank. The pairwise comparisons of complete genome and deduced ORFs of Orthohepevirus C is illustrated in Table 3.
Unexpectedly, the genomic comparisons between HEV-C1 and other newly identified HEV strains derived from the rodent species Apodemus chevrieri, Apodemus agrarius, Eothenomys melanogaster, Eothenomys inez, Myodes rufocanus, Microtus gregalis, Microtus arvalis, Necromys lasiurus, Cricetulus migratorius, and Cricetulus barabensis exhibit comparable or even higher divergence than is present between HEV-C1 and HEV-C2, even though HEV-C2 strains have only been found in the carnivore species Mustela putorius. Nucleotide identities in Orthohepevirus C variants from individual rodent species vary between 48.2-67.3%. Higher identities are observed between Eothenomys melanogaster and Eothenomys Inez, ranging from 72.2 to 72.6%, and from 76.0 to 77.3% between Microtus gregalis and Microtus arvalis. Similar results have been observed for the nucleotides and Overall, the genomic length of Orthohepevirus C sequences ranges from 6.8 to 7.2 kb, excluding those without sequenced 5 or 3 ends. However, the distinct strain RtAa-HEV/JL2014 (GenBank accession no. KY432900) from Apodemus agrarius, which consists of only 6286 nucleotides and has a significant shorter ORF1 with 4334 nucleotides; moreover, RtAa-HEV/JL2014 lacks ORF3. Since it has been recently reported that the ORF3 is essential for the release of membrane-associated rat HEV particles, the absence of ORF3 in Apodemus agrarius needs further verification [60]. In addition to RtAa-HEV/JL2014, the ORF1 of the strain RtCm-HEV/XJ2016 (GenBank accession no. KY432903) from Cricetulus migratorius is only 4182 nucleotides in length. However, the short viral genome and ORF1, as well as the absence of ORF3 may be associated with errors in assembling reads to contigs and mapping assembled contigs to references in the metagenomic study [36].
A unique ORF (tentatively named ORF4), located within the 5 region of ORF1 has been proposed in certain rat and ferret HEV strains [32,55]. As shown in Figure 2, all the HEV-C1 strains from the species including Rattus norvegicus, Rattus rattus, Rattus tanezumi, and Rattus losea within the family Muridae, two HEV-C1 analogues identified from humans, HEV-C2 strains from the family Mustelidae, and the HEV variants from the species Necromys lasiurus and Microtus gregalis within the family Cricetidae harbor ORF4. In contrast, this putative ORF is not found in other species. A recent study has shown that ORF4 is not necessary for the active replication of rat HEV [60]. Therefore, the functional role of the putative ORF4 remains poorly understood. Furthermore, due to the significant genetic variability within Orthohepevirus C, the molecular evolution of ORF4 is as of yet unclear.
Typically, HEV ORF2 overlaps partially with ORF3 but not ORF1. However, three HEV variants in the species Eothenomys melanogaster, identified from two separate studies, possess ORF2 genes overlapping with ORF1. This is also the case for HEV strains in the species Eothenomys inez, Myodes rufocanus, and Microtus gregalis within the family Cricetidae [8,36,50]. Additionally, the unique motif at the 5'-untranslated regions containing the 10 nucleotides GCAACCCCG is exclusively present in HEV variants of the family Muridae [31]. Finally, Orthohepevirus C variants utilize distinct translational frames, as described in our recent study [43].
Although many novel HEV variants within the Orthohepevirus C species have been discovered in diverse wild rodents in China in a study based on metagenomic analysis, each species has only a single viral complete genomic sequence [36]. The genomic features and genetic heterogeneity of these HEV variants in individual animal species awaits the availability of more complete genome sequences.

Genetic Variability of Orthohepevirus C
As mentioned, the majority of described Orthohepevirus C sequences are partial genomic fragments (<500 bp). In order to determine the genetic variability of HEV variants within the species Orthohepevirus C, we have included 48 currently available complete or nearly complete genomic sequences of Orthohepevirus C from GenBank. The pairwise comparisons of complete genome and deduced ORFs of Orthohepevirus C is illustrated in Table 3.
Unexpectedly, the genomic comparisons between HEV-C1 and other newly identified HEV strains derived from the rodent species Apodemus chevrieri, Apodemus agrarius, Eothenomys melanogaster, Eothenomys inez, Myodes rufocanus, Microtus gregalis, Microtus arvalis, Necromys lasiurus, Cricetulus migratorius, and Cricetulus barabensis exhibit comparable or even higher divergence than is present between HEV-C1 and HEV-C2, even though HEV-C2 strains have only been found in the carnivore species Mustela putorius. Nucleotide identities in Orthohepevirus C variants from individual rodent species vary between 48.2-67.3%. Higher identities are observed between Eothenomys melanogaster and Eothenomys Inez, ranging from 72.2 to 72.6%, and from 76.0 to 77.3% between Microtus gregalis and Microtus arvalis. Similar results have been observed for the nucleotides and amino acids of the 3 coding regions. Notably, for the comparison of ORF3, most of the HEV variants in their respective group are highly divergent and exhibit low identities, especially at amino acid level (<20%). These data highlight the fact that substantial genetic variability of HEV is present within the order Rodentia, raising a need for increased efforts to characterize the variants circulating in the respective habitats. Only through this will we be able to better assess the risk potential of these viruses to animal and human health.
Due to the high identity (> 75%) to HEV-C1 strains, two HEV variants detected in humans in Hong Kong and Uganda can be considered as HEV-C1 [14,15]. Furthermore, the kestrel HEV strain identified in Hungary shares relatively high similarity (> 83%) to the HEV strains from the species Microtus arvalis from Hungary, Germany, and the Czech Republic, which is comparable to sequence identities within common vole variants (between 81.5% and 91.9%). Therefore, it is assumed that the detection of HEV variants in birds of prey (Common kestrel and Red-footed falcon) may be linked to the consumption of wild rodents, in particular common voles [52,53,59].

Molecular Evolution of Orthohepevirus C
In order to investigate the dynamics of the molecular evolution of Orthohepevirus C, phylogenetic analysis was conducted using representative complete genomic sequences within the genus Orthohepevirus of the Hepeviridae family ( Figure 3). The discrimination of genotypes within the species Orthohepevirus A and C is in accordance with consensus proposals for the classification from ICTV Hepeviridae study group [11]. Recently, we have reported novel HEV variants in Chevrier's field mouse (species Apodemus chevrieri, family Muridae) and Père David's vole (species Eothenomys melanogaster, family Cricetidae) in China and two viral genomes of each animal species were amplified and characterized. Due to the significant divergence from HEV-C1 and HEV-C2, we have proposed these variants be classified as putative HEV-C3 and putative HEV-C4, which has been included and discussed in several recent research articles [13,50,53]. Afterwards, highly diverse HEV variants have been discovered in other wild rodent species around the world [36,51,53]. Collectively, phylogenetic tree shows that Orthohepevirus C variants form a separate branch within the genus Orthohepevirus. Predictably, HEV variants detected in the rodent genus Rattus and the carnivore species Mustela putorius cluster into two independent clades within the species Orthohepevirus C, which have already been defined as HEV-C1 and HEV-C2, respectively. Even though newly identified HEV variants are phylogenetically separate from HEV-C1 and HEV-C2, HEV variants from the rodent genus Apodemus (including species Apodemus agrarius and Apodemus chevrieri), Eothenomys (including species Eothenomys melanogaster and Eothenomys Inez), and Microtus (including the species Microtus gregalis and Microtus gregalis) clustered into three distinct clusters, which are associated with rodent taxonomy and host specificity. Taken together, it is hypothesized that co-evolutionary model, and not the host-switch pattern, might be applied regarding the species Orthohepevirus C [10]. However, the HEV variants derived from the rodent family Cricetidae, particularly RtCm-HEV/XJ2016 (GenBank accession no. KY432903) from the species Cricetulus migratorius) and RtCb-HEV/HeB2014 (GenBank accession no. KY432899) from Cricetulus barabensis, demonstrate a high degree of divergence within the Orthohepevirus C clade. With no doubt that the evolutionary history of Orthohepevirus C will be better elucidated when diversified HEV strains are discovered in insectivores and carnivores in the future.
The two HEV variants derived from a Chinese and Canadian patient, respectively, cluster within the HEV-C1 branch, providing further evidence of a potential zoonotic origin [14,15]. Concordant with pairwise comparisons of complete viral genomes and coding regions, the HEV strain (GenBank accession no. KU670940) identified in a common kestrel (Falco tinnunculus) from Hungary clusters together with the five reported HEV variants from European common voles (Microtus arvalis). Whether Orthohepevirus C variants from common voles indeed can infect common kestrel remains to be determined [52,53,59].
Thus far, all HEV variants detected in rodents have been isolated from the families Muridae and Cricetidae. Considering that the order Rodentia comprises 33 families and 2277 species (>40% of all mammal species), therefore forming the largest mammalian order, we believe that these HEV variants are only the tip of the iceberg. Future studies will undoubtably reveal further novel variants within Orthohepevirus C with new Orthohepevirus C genotypes being assigned in the coming years. Although it is evident from our analyses that HEV variants within each genotype of Orthohepevirus C (including HEV-C1, HEV-C2 and other unassigned groups) are highly divergent from one another, subtype differentiation has not yet been proposed by ICTV Hepeviridae study group [11,31,53].
It is a well-known fact that evolution of HEV within the species Orthohepevirus A undergoes different patterns between different genotypes (HEV-1 to HEV-8), which is associated with at least two distinct epidemiological profiles of the species [61]. Increasingly genetically diversified viruses within Orthohepevirus C have been identified; to what extent of viral evolutionary discrepancy of the species is largely unclear.

Cross-Species Transmission of Orthohepevirus C
Human hepatitis E cases in industrialized countries are mainly caused by zoonotic transmission of HEV-3 and HEV-4 within the species Orthohepevirus A; domestic swine, wild boar, and sika deer have been confirmed to be the reservoirs. Typically, HEV zoonotic transmission occurs via three routes. Firstly, population groups coming into direct contact with infected animals, such as veterinarians and workers on pig farms or slaughterhouses, who were proven to have significantly higher anti-HEV IgG seroprevalence rates than the general population and other veterinarians in some European countries; secondly, foodborne transmission via consumption of undercooked infectious meat products (mostly pork), such as sausages and liver, which is common in European countries; and thirdly, environmental contamination by animal feces, since the use of pig manure in agriculture may lead to contamination of water sources and agricultural products [3]. Furthermore, consumption of food products (milk and meat) from camels and subsequent infection with a camelid HEV-7 strain was implicated in a case of chronic hepatitis E after transplantation in a patient from the United Arab Emirates [62]. Using reverse genetic systems, the potential of zoonotic infection of HEV-7 as well as HEV-5 from wild boar have been confirmed by experimentally infecting primates in Japan [63,64]. Recently, one study has demonstrated that HEV-8 from Bactrian camels in multiple regions in China was able to cause chronic hepatitis in cynomolgus macaques (human surrogate), indicating a high risk of zoonosis [65]. Lastly, rabbit HEV-3ra might also pose a potential threat to humans, based on described cross-species infection of pigs and cynomolgus macaques [66,67].
Rodents are extraordinarily abundant and diversified in multiple continental habitats and live in close proximity to humans, domestic animals, and wild animals. This interface has resulted in the rodent origin of a plethora of zoonotic viruses, such as Lassa virus, Lymphocytic choriomeningitis virus, Sin Nombre virus, Hantaan virus, and Seoul virus, all of which have caused severe diseases in humans in the past decades [68]. Increasing amounts of evidence suggest that rodents may also play a role in the spread and epidemiology of HEV. Numerous studies from different countries have reported that rodent sera tested positive for anti-HEV antibodies were able to cross-react with human-derived HEV antigens [17,18,[21][22][23]. Initially, a 1996 study from Thailand reported experimental infection of three Wistar laboratory rats with a human pathogenic HEV strain derived from patient feces acquired during a hepatitis E outbreak in Nepal in 1992 [69]. Later, a Chinese study demonstrated the successfully infection of Balb/c nude mice with swine HEV and active infection of Sprague-Dawley after intrahepatic inoculation with infectious HEV-4 RNA [70]. A German study reported detectable HEV RNA and anti-HEV antibodies in Wistar rats inoculated with a wild boar-derived HEV-3 strain [71]. In addition, multiple rodent cell lines have been successfully infected with HEV-3 [72]. Finally, zoonotic HEV-3 was reported in wild rats in the USA, England, and Japan, and a rabbit HEV-3ra sequence was detected in a Norway rat from Belgium. However, viral RNA was mostly detected in intestines and feces and seldom in the liver in these cases [23,41,46]. In contrast, the results from a series of studies demonstrated that rats are susceptible to rat HEV but not HEV-1, HEV-2, or HEV-4 [37,73]. Therefore, the question whether rodents are truly natural reservoirs of human HEV or act as only intermediate hosts is not yet conclusively answered.
Although rodents have been confirmed to be the primary natural reservoirs of HEV variants within the species Orthohepevirus C, the potential risk of these viruses concerning cross-species infection and zoonotic transmission to humans remains a controversial topic [13]. In the past, Orthohepevirus C variants were understudied and neglected due to their genetic divergence (identities <52%) to the principle human pathogenic HEV strains (HEV-1 to HEV-4). It has been reported that pigs or non-human primates (rhesus monkeys) inoculated with rat HEV showed no evidence of infection, indicating that it is not a source of human infection [37,66]. In contrast, after screening with ELISAs based on the capsid protein of Norway rat HEV, several sera from forestry works of eastern Germany were shown to have strong reactivity [74]. In a recent study from Vietnam, IgG antibody titers against HEV-C1 antigen in three of the 99 sera of hospitalized febrile patients were more than eight-fold higher than those against HEV-1 antigen, while IgM antibodies against HEV-C1 antigen were detected in acute sera from two of the three patients using both ELISA and Western blotting and one patient developed illness with mild liver dysfunction [75]. Furthermore, in Japan, a rat HEV reverse genetics system was demonstrated to be replication competent in a human cell line, while another study reported that rat HEV from Rattus rattus could propagate efficiently in human hepatoma cell lines including PLC/PRF/5, HuH-7, and HepG2 [76]. Intriguingly, it was reported that a typical rat HEV caused persistent hepatitis in a liver transplant patient in Hong Kong and that the virus was cleared after ribavirin treatment. Additionally, a divergent rat HEV from Uganda induced severe acute hepatitis in an immunocompetent patient from Canada. These two case reports provide first evidence of a possible zoonotic potential of rat HEV [14,15]. Furthermore, based on comprehensive clinical and epidemiological analyses, seven rat HEV infections have been further identified in Hong Kong, and these strains are extremely close to an isolate from a rat captured near the residences of patients. Remarkably similar to Orthohepevirus A, rat HEV have caused chronic viral infection in immunosuppressed individuals as well as extrahepatic manifestations [16]. Jointly, it is highly presumable that rat HEV may be a cause of human hepatitis. Nevertheless, there are still many open questions regarding zoonotic transmission of Orthohepevirus C. What is the viral determinant for rat HEV cross-species infection? What is the effectivity of antiviral therapy, especially regarding ribavirin, against rat HEV infection? Will other rat HEV analogues (e.g., newly identified HEV strains from wild rodents, insectivores, and carnivores) be capable of infecting humans?
Interspecies transmission of Orthohepevirus C variants has been characterized in several studies as well. House shrews (Suncus murinus) have been reported as a reservoir of rat HEV worldwide; however, a novel divergent HEV strain has been detected in a Olivier's shrew (Crocidura olivieri) from Kenya and formed a separate monophyletic branch from HEV-C1, indicating the relationship between the viral species Orthohepevirus C and the animal order Soricomorpha should be more complex than previously considered [35,43,44,48]. Although ferret HEV (HEV-C2) can cause both acute and persistent infection in ferrets, researchers failed to infect monkeys and rats with ferret HEV [77].

Conclusions and Outlook
An increasing number of HEV variants are being detected in diverse animal species, including mammals, avian, and fish, with some of these species having been confirmed as natural reservoirs for HEV and sources of zoonotic infection. With the advancement of techniques such as high-throughput sequencing in metagenomics studies, no doubt more novel HEV variants will be discovered in the near future. The detection of numerous emerging HEV variants within the species Orthohepevirus C has expanded the known host range of HEV dramatically. This raises critical concerns about the potential risk of cross-species infection and zoonotic transmission to humans, as the biology, ecology, natural history, and pathogenesis of these viruses are largely unknown. After three recent reports identified totally nine human hepatitis E cases linked to rat HEV, the necessity to broaden our knowledge concerning the molecular epidemiology of circulating Orthohepevirus C variants is clear, and the potential risks of these viruses to public health should be reassessed. To elucidate the zoonotic potential of Orthohepevirus C variants, the patterns of transmission, mechanisms of replication, and functional roles of proteins merit further investigation.

Conflicts of Interest:
The authors declare no conflict of interest.