Swine hepatitis E virus: cross-species infection, pork safety and chronic infection

Abstract

Swine hepatitis E virus (swine HEV) belongs to the species Orthohepevirus A within the genus Orthohepevirus in the family Hepeviridae. Four different genotypes of swine HEV within the species Orthohepevirus A have been identified so far from domesticated and wild swine population: genotypes 3 (HEV-3) and 4 (HEV-4) swine HEVs are zoonotic and infect humans, whereas HEV-5 and HEV-6 are only identified from swine. As a zoonotic agent, swine HEV is an emerging public health concern in many industrialized countries. Pigs are natural reservoir for HEV, consumption of raw or undercooked pork is an important route of foodborne HEV transmission. Occupational risks such as direct contact with infected pigs also increase the risk of HEV transmission in humans. Cross-species infection of HEV-3 and HEV-4 have been documented under experimental and natural conditions. Both swine HEV-3 and swine HEV-4 infect non-human primates, the surrogates of man. Swine HEV, predominantly HEV-3, can establish chronic infection in immunocompromised patients especially in solid organ transplant recipients. The zoonotic HEV-3, and to lesser extent HEV-4, have also been shown to cause neurological diseases and kidney injury. In this review, we focus on the epidemiology of swine HEV, host and viral determinants influencing cross-species HEV infection, zoonotic infection and its associated pork safety concern, as well as swine HEV-associated chronic infection and neurological diseases.

1. Introduction

Hepatitis E virus (HEV), the causative agent of hepatitis E, is an important pathogen in humans. According to the World Health Organization, there are an estimated 20 million HEV infections worldwide each year leading to approximately 3.3 million cases of hepatitis E and 44,000 HEV-related deaths [https://www.who.int/news-room/fact-sheets/detail/hepatitis-e]. HEV causes mostly self-limiting acute viral hepatitis but also chronic hepatitis in immunocompromised individuals (Kamar and Pischke, 2019). The first strain of swine hepatitis E virus (swine HEV) was identified from pigs in the United States (Meng et al., 1997). Since the initial discovery, several strains of swine HEV have now been reported in pigs essentially from all swine-producing countries. In addition to swine and human population, strains of HEV have also been identified from more than a dozen other animal species, such as deer, camel, mongoose, rabbit, rodent, and avian species (Meng, 2016). In this review, we focus on swine HEV and its impact on human health since the virus does not cause overt clinical disease in pigs. We discuss the epidemiology, cross-species infection and zoonosis of swine HEV. We also discuss swine HEV-3 and swine HEV-4-associated pork safety, and chronic infection in humans. In-depth reviews on the biology, structure, and replication of HEV have been published elsewhere (Cao and Meng, 2012; Kenney and Meng, 2019a).

2. Classification, and Genome Organization

HEV belongs to the family Hepeviridae, which includes two genera: Orthohepevirus and Piscihepevirus (Purdy et al., 2017). The genus Orthohepevirus comprises of four species Orthohepevirus A, B, C and D. The species Orthohepevirus A consists of at least eight distinct genotypes from various animal species including human, pig, wild boar, deer, mongoose, rabbit, moose, and camel (Smith et al., 2014). All known swine HEV strains identified thus far worldwide belong to Orthohepevirus A. Genotypes 1 and 2 HEVs (HEV-1 and HEV-2) are exclusively isolated from human causing large outbreaks of human hepatitis E. HEV-3 and HEV-4 strains have been isolated from domestic pigs and wild boars infecting pigs and humans. HEV-5 was isolated from wild boars, and can infect non-human primates (Li et al., 2019), thus possibly human. HEV-6 strains have only been isolated from wild boars thus far (Meng, 2016), and its zoonotic potential is unknown. HEV-7 infects dromedary camel and non-human primate (Li et al., 2016), as well as a human organ transplant recipient (Lee et al., 2016; Woo et al., 2014). HEV-8 infects Bactrian camel (Woo et al., 2016), and non-human primate (Wang et al., 2019a), and therefore likely human. HEV-3 is subdivided into at least 11 subgenotypes (3a-3j, and 3ra), and HEV-4 is subdivided into at least 9 subgenotypes (4a-4i) (Smith et al., 2016). The subgenotypes within HEV-3 are clustered into two different groups: group-1 including 3efg subgenotypes, and group-2 consisting of 3abchij subgenotypes.

The genome of mammalian HEVs including swine HEV is a positive-sense single-stranded RNA molecule of ~7.2 kb in size, and bicistronic in nature. The HEV genome is capped at the 5’ end, and comprises of a 5’untranslated region (UTR), three partially-overlapping open reading frames (ORF) - ORF1, ORF2 and ORF3, and a 3’-UTR(Cao and Meng, 2012). The ORF1 encodes viral non-structural polyprotein responsible for virus replication. ORF2 encodes virus structural capsid protein, and a secreted form of ORF2 (ORF2^s) is translated 15 amino acids upstream of the capsid protein (Yin et al., 2018). ORF3 encodes a membrane ion channel protein (Ding et al., 2017). ORF3 overlaps with ORF2, and are expressed as a subgenomic mRNA (Graff et al., 2006). ORF1 does not overlap with ORF2 or ORF3, however the junction region between ORF1 and ORF3 contains a cis-reactive element (CRE) which is essential for subgemonic RNA expression (Cao et al., 2018; Ding et al., 2018). The HEV genome also consists of a 3’ CRE at the junction between 3’-end of ORF2 and 5’-end of the 3’-UTR, which is essential for virus replication (Cao et al., 2010).

3. Epidemiology and genotype distribution of swine HEV

Globally, prevalence of IgG anti-HEV among farmed domestic pigs is approximately 20-100%, and the prevalence of HEV RNA in domestic swine herds is approximately 0-20% (Salines et al., 2017). HEV prevalence rate varies from country-to-country, region-to-region, and farm-to-farm within a given country (Okamoto, 2007; Primadharsini et al., 2019). In the United States, approximately 41% of farmed pigs and 3% of feral swine population were seropositive for HEV antibodies (Dong et al., 2011). The HEV RNA prevalence rate amongst the U.S farmed swine herd were approximately 35% at individual animal level, and 54% at herd level (Huang et al., 2002). However, the age of the pigs is an important factor for the observed variability of HEV prevalence.

A total of 4 distinct genotypes of swine HEV have been genetically identified thus far, all belonging to Orthohepevirus A species. HEV-3 strains are endemic in Europe, Asia, and America, while HEV-4 strains have been identified mainly in Europe and Asia (Meng, 2016). HEV-5 and HEV-6 strains have only been detected thus far from wild boar population in Japan (Okamoto, 2007; Takahashi et al., 2011). The prevalent subtype strains of HEV-3 vary from country to country. In the United States, the predominant HEV-3 subtype is 3a (Meng et al., 1997), and in Japan HEV-3 subtypes 3a, 3b, and 3e (Okamoto, 2007) have been reported. In Europe, depending on geographic region and country, a diverse array of HEV-3 subtype strains is reported in the swine population, including 3a, 3b, 3c, 3e, 3f, 3h, 3i, and 3j (Oliveira-Filho et al., 2014; Rose et al., 2011; Rutjes et al., 2010). Novel HEV-3 subtypes such as subtype 3k from Japan (Miura et al., 2017), and subtype 3l from Switzerland (Wang et al., 2017) and Northern Italy (De Sabato et al., 2018) have recently been reported. It is likely that additional novel HEV-3 subtypes will continue to be discovered from pigs worldwide.

The HEV-4 is endemic mostly in Asia (Primadharsini et al., 2019; Takahashi and Okamoto, 2014), although recently it has also been reported from Europe (Tesse et al., 2012). HEV-4 subtypes 4 c and 4i strains are reportedly indigenous to Japan (Sato et al., 2011), and subtype 4b strains have been reported from Belgium (Hakze-van der Honing et al., 2011) and France (Colson et al., 2012). A recent study of global dispersal history of HEV-4 (Nakano et al., 2016) revealed that HEV-4 originated in Japan, and subsequently migrated to China, and further to other Asian countries (India, and Korea), and then to some European countries. Global swine trade was found to be coinciding with the dispersal of HEV-4 in some instances (Nakano et al., 2016). In addition to swine and humans, HEV-3 strains have also been genetically identified from other animal species such as deer, rabbit, and mongoose (Kenney and Meng, 2019b; Meng, 2016). HEV-4 strains are identified mostly from pigs, although it was reportedly identified from several other animal species as well. These epidemiological data clearly show the potential of these swine HEV-3 and HEV-4 strains to establish cross-species infection.

4. Cross-species infection and occupational risk of zoonosis

4.1. Experimental cross-species HEV infection

First experimental evidence of cross-species infection by swine HEV was documented in 1998, in which non-human primates, both rhesus monkeys and chimpanzee, were shown to be readily susceptible to swine HEV-3 infection (Meng et al., 1998). The infected non-human primates developed anti-HEV antibodies, viremia, and shed virus in feces from 1-5 week post-infection. Subsequent studies have also shown that human HEV-3 can readily infect conventional pigs (Cao et al., 2017; Meng et al., 1998) and gnotobiotic pigs (Yugo et al., 2018). Similarly, swine HEV-4 can readily infect non-human primates (Arankalle et al., 2006), and conversely human HEV-4 can establish infection in pigs under experimental conditions as well: the infected animals seroconverted to HEV antibodies, and had viremia, and fecal virus shedding (Cordoba et al., 2012; Feagins et al., 2008b). Infectious swine HEV-5 was successfully produced from an infectious cDNA clone, and shown experimentally to infect non-human primates as well (Li et al., 2019). These experimental transmission studies clearly demonstrate that swine HEV-3, HEV-4, and HEV-5 are capable of cross-species infection. However, attempts to experimentally infect rodent models with swine HEV-3 and HEV-4 were unsuccessful (Kenney and Meng, 2019b). However, immunocompromised mouse models, such as Balb/c nude mice (Huang et al., 2009), and mice with humanized liver (Allweiss et al., 2016) were shown to be susceptible to swine HEV-3 infection.

4.2. Viral factors potentially involved in cross-species HEV infection

The viral determinants influencing cross-species HEV infection are largely unknown. It is believed that the broad host range of HEV-3 and HEV-4 strains are probably due to its extensive genetic diversity of the viral genomes (Primadharsini et al., 2019). It has been reported that the virus-host adaptations of HEV-3 and HEV-4 strains were associated with an extensive coevolution among various amino acid residues in the genome (Lara et al., 2014). Amino acid position 1252 in ORF1 is recognized as a HEV-3 host-specific motif (Lara et al., 2014). Studies using intergenotypic chimeric viruses between genotype 1 human HEV, and genotypes 3 and 4 swine HEVs, have shown that the HEV ORF1 is likely involved in determining host-tropism, while ORF2 capsid gene does not appear to influence cross-species infection (Feagins et al., 2011; Pudupakam et al., 2009; Pudupakam et al., 2011). Insertion and/or deletion in ORF1 have been demonstrated to affect virus replication and host tropism. For example, it has been reported that insertion of host ribosomal protein sequence (RPS17) within the hypervariable region (HVR) of HEV ORF1 led to an expanded host adaptability and infection of the virus in cell cultures from different animal origins (Shukla et al., 2012). Clearly, in-depth studies are warranted in order to fine-map the region(s) within ORF1 involving in host range determination and delineate the underlying mechanism of cross-species HEV infection.

4.3. Occupational risks of swine HEV zoonotic infection

Pigs are the primary animal reservoir of zoonotic HEV-3 and HEV-4 infection. An increase in sporadic and cluster cases of human hepatitis E due to swine HEV-3 and HEV-4 infections has been steadily reported from the industrialized countries in recent years (Clemente-Casares et al., 2016). Direct contact exposure to infected animals is reported to contribute to spillover infection (Anheyer-Behmenburg et al., 2017; Schlosser et al., 2015). Contact exposure to infected animals is a major occupational risk associated with zoonotic infection of swine HEV-3 and HEV-4 in humans (De Schryver et al., 2015).

Autochthonous HEV infection by swine HEV-3 in a slaughterhouse worker was reported from Spain (Perez-Gracia et al., 2007). Swine veterinarians in the United States were 1.51 times more likely to be seropositive for HEV antibodies as compared to age- and geography- matched control subjects (Meng et al., 2002). Similar studies have also shown a higher seroprevalence of HEV antibodies in slaughterhouse workers, pig farmers (Krumbholz et al., 2012), and hunters (Schielke et al., 2015). Recently, a meta-analysis of 32 independent studies from 16 countries comparing the prevalence of HEV infection in swine workers (including swine farmers, butchers, meat processors, pork retailers and veterinarians) and the general population (including local residents, blood donors and non-swine workers) revealed an increased prevalence of IgG anti-HEV among the swine workers, and the average prevalence ratio was estimated to be 1.52 (95% CI 1.38-1.76) with the I² being 71% (Huang et al., 2019). However, cross-sectional studies from the Netherlands (van Gageldonk-Lafeber et al., 2017) and Italy (Caruso et al., 2017) based on the general population in high-density pig farming area did not find any significant association between HEV seroprevalence and proximity to pig farm or swine workers with increased contact exposure. Therefore, in addition to occupational risks, other factors such as sanitation condition and personal hygiene practice might also influence the HEV prevalence in a given population.

The occupational risk of zoonotic HEV infection increases dependent on the exposure duration, and subject’s gender; in addition to these factors, the assay employed in a given study also determines the level of HEV prevalence (De Schryver et al., 2015). Seroprevalence was higher among men, and prevalence rate increased with the age of the individual. Due to lack of a gold-standard assay for HEV detection, it becomes problematic to determine the residual bias within each study. Sommerkorn et al compared various anti-HEV ELISA assay kits to determine prevalence of HEV antibodies in individuals with direct contact with domestic pigs (veterinarians, meat inspectors, slaughterhouse workers; N = 114) or wild boars (hunters; N = 25) (Sommerkorn et al., 2017). The results showed a kit-to-kit variability in IgG anti-HEV detection levels among the test population. Hence, development of FDA-approved standardized HEV diagnostic assays would enable the scientific community to monitor transmission of HEV especially in high risk population and more accurately compare the results from different studies.

5. Pork safety associated with swine HEV contamination: risks of foodborne zoonotic HEV infection

5.1. HEV-viremic pigs at the time of slaughter

HEV is known to be present in market-weight slaughterhouse pigs, which is the entry point to food supply chain. In addition to the age of the animal, two other main factors affecting the viremic rate in slaughterhouse pigs are: (1) occurrence of infection just before slaughter, and (2) widespread infection on the farms, i.e. higher proportion of animals shedding virus in feces thus forming a loop of continuity of new infection (Salines et al., 2017). In the United States, HEV RNA has been detected in sera of market-weight slaughterhouse pigs (Sooryanarain et al., 2020), and commercial pork products (Cossaboom et al., 2016; Feagins et al., 2007; Feagins et al., 2008a). It has been shown that approximately 40% of the pigs from U.S. slaughterhouses were seropositive for HEV antibodies, and approximately 6% (range 0%-17.4%) of them were also viremic for the zoonotic HEV-3 group-2 clade (Sooryanarain et al., 2020). Highest seropositivity and HEV viremia rate were found in sera of pigs from slaughterhouses located in high-density swine farming states such as Iowa, which was in corroboration with the finding from U.S. swine farms (Dong et al., 2011; Huang et al., 2002; Meng et al., 1997). Globally, a higher level of IgG anti-HEV seropositivity has been reported from slaughterhouse pigs from Europe [59% - 100%] (Casas et al., 2011; Di Bartolo et al., 2011; Grierson et al., 2015), and Asia [54% - 82%] (Haider et al., 2017; Li et al., 2009). A higher positivity of HEV RNA has been reported in slaughterhouse pigs from Italy [59% - 64.6%] (Di Bartolo et al., 2012; Di Bartolo et al., 2011), Scotland [44.4%] (Crossan et al., 2015), and Spain (11.5% - 39%) (Casas et al., 2011; Di Bartolo et al., 2012). The presence of HEV viremic pigs at the time of slaughter raises a food safety concern, as HEV-containing blood can contaminate pork supply chain, and thus leading to a possible foodborne HEV infection.

5.2. Presence of infectious HEV in commercial pork products

Foodborne transmission of HEV is mainly attributed to consumption of contaminated raw or undercooked pork products (Meng, 2013). In the United States, HEV RNA was detected in commercial pork products sold in grocery stores such as chitterlings and raw liver (Cossaboom et al., 2016; Feagins et al., 2007; Feagins et al., 2008a). In Italy, approximately 22.2% of raw pig liver sausages, and 4.3% of the dried pig liver sausages were tested positive for HEV RNA (Di Bartolo et al., 2015). Similar results have also been reported from Germany, as 22% of liver sausages were tested positive for HEV RNA (Szabo et al., 2015). In France, about 2.8% of the pork livers were tested positive for HEV RNA (up to 1.46 10⁸ copies/g), while none of the ham muscle samples tested positive (Feurer et al., 2018). These studies clearly demonstrate that a proportion of the liver- and blood-containing pork products are contaminated by HEV. Most importantly, it has been shown that the contaminating virus from commercial pork remains infectious (Feagins et al., 2007). Taken together, these studies clearly indicate that some pork products are contaminated by HEV and thus pose a risk of foodborne HEV transmission.

5.3. Sporadic and cluster cases of foodborne HEV infection

Cluster cases of HEV infection have been definitely linked to consumption of raw figatelli, a traditional pig liver sausage in France (Colson et al., 2010). Zoonotic swine HEV-3 RNA was detected from 7 of 12 figatelli purchased in supermarkets, and the HEV-3 sequences recovered from figatelli sausages and human patients are nearly identical (Colson et al., 2010). An outbreak of HEV infection was attributed to consumption of undercooked pig liver-based stuffing in France (Guillois et al., 2016). In this outbreak, 17 cases of HEV infection were identified, of which 5 individuals developed symptomatic disease, while 12 remained asymptomatic. The HEV sequence isolated from the patients were 99% to 99.6% identical to that isolated from the local wastewater plant, and from the pig manure obtained from the pig farm (Guillois et al., 2016). Similar reports of foodborne cases of autochthonous HEV infection, due to consumption of raw or undercooked pork, have also been reported from various other countries including Australia (Yapa et al., 2016), Spain (Rivero-Juarez et al., 2017), Italy (La Rosa et al., 2011), and Germany (Faber et al., 2018; Preiss et al., 2006). A recent study from England reported an increased risk association between consumption of commercial pork products from a major supermarket and the HEV infection resulting from newly-emerging HEV-3 group-2 strains (Said et al., 2017). Direct evidence of foodborne transmission of HEV due to consumption of HEV-contaminated wild boar meat has been reported in Japan (Li et al., 2005). The HEV sequence obtained from the wild boar meat was 99.95% identical to that obtained from the human patient (Li et al., 2005). In France alone, it is estimated that there were approximately 68,000 cases of autochthonous foodborne HEV infection in humans during 2008-2013 (Van Cauteren et al., 2017). Although there is a widespread HEV prevalence amongst the U.S. swine population, only a few cases of autochthonous human hepatitis E potentially associated with swine HEV-3 have been reported from the United States (Amon et al., 2006; Curry et al., 2009; Kwo et al., 1997; Sarkar et al., 2015; Schlauder et al., 1998; Tohme et al., 2011; Tsang et al., 2000). Similarly, the risk profile of zoonotic swine HEV in Canada is found to be low as compared to Europe (Wilhelm et al., 2017). The huge difference in the incidences of foodborne HEV infection between European and North American could possibly be due to the variations in dietary habits in these countries.

5.4. Food safety measures to prevent foodborne zoonotic swine HEV infection

Due to the risk of foodborne HEV infection through consumption of animal meats especially pork, it becomes pertinent to understand the thermostability of HEV virion in order to develop food processing methods to effectively inactivate the virus and prevent foodborne outbreak in susceptible populations. Studies have shown that cooking the meat at an internal temperature of 71°C for 20 min effectively inactivates HEV (Barnaud et al., 2012; Feagins et al., 2008a). Homogenates obtained from thoroughly cooked pig liver (internal temperature 71°C) were unable to establish HEV infection when inoculated into pigs, however homogenates from HEV-contaminated pig livers incubated at 56°C for 1 hr still induce active HEV infection when inoculated into pigs (Feagins et al., 2008a). Similar observations have also been reported from France: pigs inoculated with suspension of cooked pork products (71°C for 20 min) did not excrete HEV and remained uninfected (Barnaud et al., 2012). Reports using in vitro HEV cell culture model have also supported inactivation of infectious HEV at temperatures ≥ 70°C (Imagawa et al., 2018; Johne et al., 2016). Infectious HEV can remain viable for up to 56 days at 4°C, 28 days at room-temperature, or 21 days at 37°C (Johne et al., 2016). Apparently, the HEV-4 was slightly more thermostable than the HEV-3. It was reported that heating at 65°C for 3 min did not inactivate HEV-4 strain, while HEV-3 was inactivated (Imagawa et al., 2018). However, both HEV-3 and HEV-4 strains were completely inactivated at temperatures ≥70°C (Imagawa et al., 2018). Therefore, a thorough cooking of the pork products, to an internal temperature of 71°C or above, would be essential for preventing foodborne HEV infection.

6. Chronic HEV infection in humans associated with swine HEV-3 and HEV-4

Chronic hepatitis E is defined as persistent hepatitis and presence of HEV RNA in serum or stool for >3-6 months. Swine HEV-3 has been documented to establish chronic infection in immunocompromised patients including solid-organ transplant patients and HIV-infected patients (Table 1). A few cases of chronic hepatitis E caused by zoonotic HEV-4 have also been reported (Fujiwara et al., 2014; Kamar and Pischke, 2019). The major source of HEV-3-associated chronic hepatitis E in the immunocompromised patients is the consumption of raw or undercooked pork products (Meng, 2013). In addition to foodborne transmission, recent studies from Europe have shown that contaminated human blood can also increase the risk of chronic HEV-3 infection through blood transfusion amongst the patients with hematological malignancies (von Felden et al., 2019). Chronic hepatitis E cases have been documented repeatedly amongst the patients who had undergone recent heart, liver, lung or kidney transplant (Abravanel et al., 2014; Kamar et al., 2008; Pischke et al., 2014; Pischke et al., 2012). A study from France has shown that 8 out of 14 organ transplant patients with acute hepatitis E developed persistent HEV infection with a chronic elevated ALT levels, mild hepatic necrosis, and progressing to fibrosis within 12 months of infection (Kamar et al., 2008). A multicentered study from Europe showed that 66% of transplant patients with an acute HEV infection developed chronic hepatitis (Kamar et al., 2011b). In a nationwide survey from Japan, it was reported that, out of the 2625 solid-organ transplant patients (N = 99 heart transplant patients, and N = 2526 kidney transplant patients), only 12 tested positive for HEV viremia (Owada et al., 2019). Of the 12 HEV-viremic organ transplant patients, 42% of them developed chronic hepatitis (Owada et al., 2019). Sporadic cases of chronic hepatitis E in pediatric transplant patients have also been reported (Halac et al., 2012a; Halac et al., 2012b). The risk of developing a chronic hepatitis E depends on the immunosuppressive regime (Kamar and Pischke, 2019). Drugs targeting T cells, such as Tacrolimus, a calcineurin inhibitor, predisposes the patient to develop chronic hepatitis E as compared to Mycophenolic acid (Kamar et al., 2011b). Similar phenomenon was also observed in patients with hematological malignancies (a non-solid organ transplant cohort) receiving Rituximab, 45.5% of them developed chronic hepatitis (von Felden et al., 2019). Despite the fact that HEV infection cause chronic hepatitis, no fulminant liver failure has been reported amongst the HEV-infected transplant patient (Haffar et al., 2018; Kamar and Pischke, 2019). In addition to organ transplant patients, other populations with immunosuppressive conditions such as patients with pre-existing chronic liver disease (Peron et al., 2007), patients with leukemia and lymphoma (Giordani et al., 2013; Pfefferle et al., 2012), and HIV-infected patients (Dalton et al., 2011; Kuniholm et al., 2016) can also develop chronic hepatitis. Therefore, it is likely that, under immunosuppressive conditions, the host is unable to mount an adequate immune response to clear the HEV infection, and thus facilitating the establishment of chronic infection. Under experimental conditions, it has been demonstrated that pigs infected with HEV-3 and treated with an immunosuppressive regime mimicking organ transplant recipients developed chronic HEV infection, and that suppression of cell-mediated immune response is responsible for progression into chronicity (Cao et al., 2017). It has also been reported that rhesus macaques treated with an immunosuppressive drug, Tacrolimus, and infected with HEV-3 developed chronic hepatitis E (Gardinali et al., 2017).

Table 1.

Zoonotic swine HEV-3 infection in pigs and its associated disease in human in selective industrialized countries

Country	Prevalent Subgenotype	Seroprevalence in Pigs	HEV RNA prevalence in pigs	Other reservoir	Associated diseases in humans
United States	3a, and genotype-3 group-2	Farmed swine (41.2%) Slaughterhouse pigs (40%) Feral swine (2.9%)	Farmed swine (30%) Slaughterhouse pigs (6%) Pig liver from grocery stores (11%) Chitterling product from grocery store (3/12 positive)	Rabbit	Sporadic cases associated with HEV-3
Canada	3a, 3j	Farmed swine (59.4%)	Farmed swine (34%) Commercial pork-pates (47%) Commercial pig liver (10%)	Deer	Sporadic cases in transplant recipients associated with HEV-3
Europe* (France, Germany, Italy, United Kingdom, Spain)	3a, 3b, 3c, 3e, 3f, 3j, 3i and 3h	Domestic pigs (20% - 72%) Slaughterhouse pigs (59% - 100%)	Domestic pigs (0% - 73%) Slaughterhouse pigs (11.5% - 64.6%) Pig liver (2.9% - 13.5%) Commercial pork products - including pork liver sausages (0.8%- 22.2%) Commercial Figatelli (7/12 positive) Wild boars	Brown hare Deer Mussel	Autochthonous foodborne HEV infection. Chronic HEV infection in transplant patients. Neurological manifestations - Guillain-Barré syndrome. Associated renal manifestations.
Japan#	3a, 3b, and 3e	Domestic pigs (58%)	Domestic-pigs (13% - 16%) Commercial pork liver (1.9% - 5%) Wild boars	Deer	Autochthonous HEV infection. Chronic HEV infection in solid-organ transplant patients. Neurological manifestation - Guillain-Barré syndrome.

^*Nearly all chronic HEV infections in Europe are caused by HEV-3, however, fewer cases of autochthonous HEV-4 infection have been reported from some of these countries (Kamar and Pischke, 2019).

^#HEV-4 is endemic in Japan. Sporadic cases of autochthonous HEV-4 infection have been reported from Japan (Takahashi and Okamoto, 2014).

7. Neurological and kidney diseases in patients infected by swine HEV-3 and HEV-4

Extrahepatic manifestations of clinical diseases of HEV infection have been reported in patients primarily from European countries. Most of these cases are linked to the zoonotic HEV-3 and HEV-4 infections. Most notable extrahepatic manifestations during chronic HEV infection include neurological sequelae (Dalton et al., 2016), and kidney damage (Kamar and Pischke, 2019). Approximately 5.5% of the HEV-infected patients (N = 126) developed neurological complications, including inflammatory polyradiculopathy, Guillain-Barre syndrome, bilateral brachial neuritis, encephalitis, and ataxia/proximal myopathy (Kamar et al., 2011a). Independent studies have also shown an association between HEV-3 infection and neuralgic amyotrophy (Dalton et al., 2017; Fraga et al., 2018; van Eijk et al., 2014). A distinct bilateral involvement of the brachial plexus was reported in patients who had HEV-associated neuralgic amyotrophy. In one instance, bilateral facial palsy has been reported from Japan following HEV-4 infection (Yazaki et al., 2015). A prominent neurological disorder most commonly seen in HEV-infected patient is Guillain-Barré syndrome (Dalton et al., 2016). Cases of Guillain-Barré syndrome due to HEV infection have been reported from patients in many countries including Belgium (Maurissen et al., 2012), Hong Kong (Tse et al., 2012), The Netherlands (van den Berg et al., 2014), and Japan (Fukae et al., 2016). The viral or the host factors responsible for neuropathology during HEV infection remain to be defined. However, in a recent study, swine HEV-4 strain was shown to break the blood-brain barrier (BBB), and replicate in central nervous system of experimentally-infected Mongolian gerbils (Shi et al., 2016). Additionally, several human neuronal cell lines reportedly support HEV replication (Drave et al., 2016).

The second most common extrahepatic manifestation associated with HEV-3 and HEV-4 infection is kidney injury (Kamar and Pischke, 2019). Chronic hepatitis E has been reported from HEV-infected kidney transplant patients, and HEV RNA and antigen have been detected from urine sample obtained from a patient with chronic hepatitis E (Geng et al., 2016). A significant decrease in estimated glomerular filtration rate has been reported from HEV-infected kidney- and liver-transplant patients (Kamar et al., 2012). The common glomerular disorders observed within chronic HEV-infected organ transplant recipients include membranoproliferative glomerulonephritis and cryoglobulinemia (Guinault et al., 2016; Kamar et al., 2012; Marion et al., 2018). Deteriorated kidney function and proteinuria were significantly higher in patients with chronic HEV infection and cryoglobulinemia (Marion et al., 2018). The kidney function and associated glomerular disorder seem to improve after clearance of HEV infection (Guinault et al., 2016; Marion et al., 2018). Renal injury was one of the independent factors, along with lower triglyceride level, that was associated with acute-to-chronic liver failure in organ transplant patients with HEV-4 infection (Wang et al., 2019b).

The exact pathophysiological mechanism leading to extrahepatic manifestations of neurological and renal diseases during acute or chronic zoonotic HEV-3 and HEV-4 infections remains unknown. Therefore, more in-depth mechanistic studies would be essential to further understand the molecular pathways that influence the host response and viral replication.

8. Perspective

Of the four different genotypes of swine HEV identified from pigs worldwide thus far, the HEV-3 and HEV-4 are of greater public health importance, as they can cause cross-species HEV infection, cause neurological and renal diseases, and establish chronic hepatitis E in immunocompromised patients. Foodborne HEV transmission due to the consumption of undercooked or raw pork products remains a major route of transmission of zoonotic HEV-3 and HEV-4 in humans, and occupational risks such as direct contact with infected pigs and other animals can also increase the risk of zoonotic HEV infection in humans.

Despite the recent advances in understanding the biology of HEV, various aspects of HEV life cycle remain largely unknown. Three main areas that need further in-depth studies include (1) pathophysiology of hepatitis E, (2) factors influencing cross-species HEV infection, transmission rate, and dispersal at farm and/or herd level, and (3) development of FDA-approved gold standard HEV diagnostic assays for swine and humans. Further development of a better cell culture system for HEV (Okamoto, 2013; Todt et al., 2020) along with refinement of existing animal models would enable a better understanding of the kinetics of HEV replication and its associated pathophysiology of acute vs chronic hepatitis E. This would also enable the development of effective vaccines, and HEV-specific antivirals as per the need of the patient. Improvement of HEV diagnosis and better surveillance model would enable us more accurately to monitor the HEV circulation in swine and other animal population, and predict transmissibility rate within susceptible populations. These measures would aid in developing public health and food safety policies to prevent potential zoonotic and foodborne transmissions of HEV.

Highlights.

• Pigs are natural animal reservoir for genotypes 3, 4, 5 and 6 HEV.

• Genotypes 3 and 4 HEVs are zoonotic and infect human.

• Consumption of contaminated raw or undercooked pork leads to foodborne HEV transmission.

• Sporadic cases of chronic HEV infection occur in immunocompromised patients.