Hepatitis E Virus in the Food of Animal Origin: A Review

Hepatitis E virus (HEV) is a cosmopolitan foodborne pathogen. The viral agent infects humans through the consumption of contaminated food (uncooked or undercooked). Most cases of infection are asymptomatic and for this reason, this pathology is considered underdiagnosed. Domestic and wild animals are considered natural reservoirs: that is, domestic pig, wild boar, sheep, goat, deer, rabbit, and so on. Therefore, various work categories are at risk: that is, veterinarians, farmers, hunters, slaughterhouse workers, and so on. In these last decades, researchers found a high percentage of positivity to the molecular viral detection in several food matrices included: ready-to-eat products, processed meat products, milk, and shellfish. This review aims to provide an international scenario regarding HEV ribonucleic acid (RNA) detection in several foodstuffs. From this investigative perspective, the study aims to highlight various gaps of the current knowledge about technologies treatments' impact on viral loads. The purpose was also to provide an innovative point of view “One Health”-based, pointing out the strategic role of environmental safety.


Introduction
F oodborne illnesses caused by viruses are a significant global health problem. In the recent decades, knowledge regarding foodborne viral infections has increased. Furthermore, these pathogens reduce economic growth in many countries in the world (Shirazi et al., 2018).
Data regarding foodborne viral infections change every year. Foodborne viral agents are responsible for 12% of related disease outbreaks, as reported by the European Food Safety Authority (EFSA BIOHAZ Panel et al., 2017).
In the United States, norovirus (NoV) causes *58% of foodborne illnesses. However, contaminated food ingestion can also vehicle another important pathogen: hepatitis E virus (HEV).
Indeed, in the last decade there were several outbreaks in the world (Hirneisen et al., 2012).
HEV is transmitted through orofecal route. In general, contaminated water or food of animal origin ingestion (raw or undercooked products) is the main source of infection (Colson et al., 2010).
HEV belongs to the Hepeviridae family and it is classified as Orthohepevirus A (Smith et al., 2014). It is a nonenveloped single-strand ribonucleic acid (RNA) virus (Purdy et al., 2017). For this structural characteristic, Johne et al. (2016) discovered that virions maintain infectivity for up to 21 days at 37°C and 28 days at room temperature.
HEV1 and HEV2 are human specific (especially in Asiatic and African continents). These two genotypes infect humans through contaminated water ingestion. This condition is related to an inappropriate wastewaters' management (EFSA BIOHAZ Panel et al., 2017).
HEV3 is widespread in the world (including industrialized countries: European and American continents). It has been derived from both humans and other animal species (domestic swine and wild boar). In general, HEV3 causes sporadic cases in human patients (Kamar et al., 2014;Sayed et al., 2015).
However, EFSA BIOHAZ Panel et al. (2017) reported an increasing number of cases of hepatitis E infection (HEV3) among Western European countries. For this reason, many authors suggested that the epidemiological scenario is dynamic.
Consequently, a significant proportion of people in industrialized countries are seropositive (high titers of anti-HEV antibodies Abs), but without typical symptoms of acute hepatitis (Sayed et al., 2015).
HEV4 shows a zoonotic potential. It is considered an Asiatic genotype (Lu et al., 2006;Wang et al., 2021), but Colson et al. (2012) isolated this genotype in the European continent too.
In 2013, Yugo and Meng (2013) reported an expanding host range (new animal species found positive for HEV RNA) and demonstrated cross-species infections (Table 1).
There are multiple contact points between humans, wild and domestic animals, and HEV RNA . This ''viral cycle'' begins from multiple primary productions: breeding farm/mariculture (especially in extensive realities), slaughterhouses (meat products), processing meat/seafood industries, and wastewaters' management. It concludes with consumers' and environment's health.
Therefore, HEV is a global public health issue that involves humans, animals, and environment. The aim of this article was to purpose a holistic and critical point of view, focusing on the strategic role of dietary transmission that permits HEV virions diffusion. It also explains the impact of food processing technologies on the viral loads and highlights numerous gaps of knowledge concerning HEV contamination in several alimentary industries.

Categories at Risk
Veterinary food inspectors, slaughterhouse workers, farmers, and hunters Veterinary food inspectors, swine farmers, hunters, pork retailers, meat processors, and so on, are routinely exposed to the risk of HEV infection (Montagnaro et al., 2015).
Unsafe raw food, poor storage infrastructure, poor personal hygiene, improper handling methods, and crosscontamination of raw food are just some of the common causes of foodborne diseases that need to be prevented by new measures (Shirazi et al., 2018). Therefore, they are more likely to be infected with HEV than the general population by 50% .
Molecular biology (real-time PCR [RT-PCR] assay) and seroprevalence screening assays (immunoglobulin G [IgG] and/or immunoglobulin M [IgM] detection) are goldstandard methods that are used by researchers to evaluate the epidemiological scenario (De Schryver et al., 2015).
Direct contact between infected wild animals (i.e., wild boar) and humans, especially for the above-mentioned work categories, causes high prevalence of anti-HEV Abs. Carpentier et al. (2012) investigated the risk of viral seroprevalence by using enzyme-linked immunosorbent assay. They evaluated seroprevalence in 593 forestry workers (including woodcutters, game or fishing keepers, rangers, and controls) and 421 wild boars. Results evidenced anti-HEV Abs in 31% of the forestry workers and 14% of the wild boars. This study is one of the first papers in which scientists demonstrated that these worker categories are more exposed to HEV virions.
Slaughterhouses have a key role in HEV RNA diffusion. In most of cases, infected swine is asymptomatic, or it presents a self-limiting hepatitis. Therefore, veterinarians cannot identify or suspect positive animals from ante mortem evaluation. False-negative animals can enter the slaughterhouse's chain as ''healthy.'' Consequently, contaminated meat and liver handling diffuses HEV in the entire slaughter lines (Widén, 2016).
In 2019, Milojević et al. (2019) screened viral prevalence and respective genotypes in slaughtered pigs in different Serbian slaughterhouses. From the same slaughter line, they collected a total of 345 samples from liver and environmental swabs.
Environmental swabs were collected from different sites/ surfaces on the slaughter line having contact with offal and fresh meat (manipulation hooks hanging liver, inspection tables, knives, sharpener, and offal collection containers). The 53% of environmental and 34% of liver samples were positive for the presence of HEV RNA.
Therefore, cross-contaminations start from slaughterhouses and arrive as final products with repercussions on consumers' health. Krog et al. (2019) purpose a new approach of preventive medicine. It is characterized by fecal swabs collected from pigs before their arrival to the slaughterhouses. The detection of HEV RNA (through RT-PCR assay) in feces could be related to HEV positivity in organs. This approach will be able to produce satisfactory results preventing and reducing HEV diffusion in these structures.
This condition is related to the direct contact with live animals, animal blood (viremia phase), liver, and meat Mesquita et al., 2020). Therefore, more attention is required especially during evisceration phase and avoiding bile waste dispersion and cross-contamination regarding muscle masses (Milojević et al., 2019). This evidence highlights one of the crucial HEV pathogenesis principle: the entero-hepato-biliary viral cycle.
This pathogenetic aspect is strictly related to the orofecal viral transmission. It is important to remember that HEV infection is characteristically related to contaminated food ingestion.
Control groups formed by administrative staff, chemists, teachers, and so on, who have no direct contact with biohazard materials, were checked for anti-HEV Abs.
Consequently, people living in industrialized countries are generally exposed to the virus owing to the dietary factors (ingestion of contaminated foodstuffs: pork liver, and meat products) (Rivero-Juarez et al., 2017;Delage et al., 2019).
Veterinarians have an important educative role in the implementation of knowledge about zoonoses and their impact on the public health (Bahnson et al., 2001;Poizat et al., 2017). An efficient food safety service has to provide a riskbased surveillance and formative plans (improvement of slaughterhouse's personnel education), as reported by the European Law (EC No. 625/2017) .
In the near future, strict compliance between private producers and public food safety agencies will improve foodstuffs quality and safety.

Immunocompromised patients
In immunocompetent patients, HEV is usually asymptomatic (it depends on the genotype) (Weigand et al., 2018). Only a certain percentage of cases present icterus with nausea, fever, abdominal pain, vomiting, and hepatomegaly after an incubation period of 2 to 8 weeks (Park et al., 2016).
Immunocompromised individuals such as organ transplant recipients (Kamar et al., 2008), patients with chronic liver disease (EFSA BIOHAZ Panel et al., 2011), patients affected by HIV infection (Dalton et al., 2009), and those affected by lymphoblastic leukemia (Motte et al., 2012) are some of the categories showing high risk of HEV infection.
These patients usually require the support of blood or blood product transfusions. These administrations can be responsible for interhuman HEV3 and HEV4 transmission (Matsubayashi et al., 2008), because HEV RNA resists blood cell inactivation (Hauser et al., 2014). Therefore, some industrialized countries (Ireland, United Kingdom, Japan, the Netherlands, and Germany) introduced nationwide HEV RNA screenings of blood donations (Domanović et al., 2017).
Most of screened blood samples contained low viral loads and low antibody prevalence (United States 7.7%) (Ditah et al., 2014). Data from United States and Canadian are lower than European countries, that is, IgG 8.7% and IgM 0.4%. Seroprevalence variations depend on the dietary factors (i.e., ingestion of contaminated foodstuffs) (Delage et al., 2019).
High seroprevalence values are also influenced by alimentary and geographical traditions, that is, hunting activities and game meat product consumes. It is largely performed in small communities (anti-HEV immunoglobulin over 30% in Sardinia and Abruzzo regions) (Spada et al., 2019).
Therefore, industrialized countries adopt a new and no alarmistic approach focused on ''risk-based decision making.'' It purposes molecular screenings of blood, blood products, and foodstuff destined to such vulnerable categories of patients. This system can be used if there is a condition characterized by the low seroprevalence in blood donors (Delage et al., 2019).

Meat Products
The first evidence of HEV transmission in humans was described in Japan from wild animal meat. These cases were associated with the consumption of uncooked or undercooked pork meat and venison (Matsuda et al., 2003).
In general, contamination comes from the primary production of fresh products, food handling, or water used for food production and preparation (EFSA BIOHAZ Panel et al., 2011).
In contrast to many other foodborne viruses, viral contamination of meat products is not only restricted to the food surface. HEV can also localize in the internal parts of food products (Bouwknegt et al., 2009). Viremia is responsible for HEV virion diffusion in several muscles. Diaphragm muscle may also contain small amounts of pig liver tissue owing to its contiguity. Therefore, sanitary authorities advised to stop using diaphragm muscle in unheated pork products (Bouwknegt et al., 2017).
There is little information concerning HEV survival in food matrices such as ready-to-eat and raw meat products containing swine meat or liver. Consequently, it is necessary to obtain more data about the viral infectious dose from contaminated food consumption.
In general, thermal treatments eliminate the risk of HEV infection. Conversely, there are few studies about the impact of food-processing technologies on viral loads (Sarno et al., 2017).
In North Europe, pork liver sausages are typically uncooked or cooked shortly during processing. Therefore, they still contain virions (Di Bartolo et al., 2012) and vehicle HEV to the final consumers (Kubacki et al., 2017;Giannini et al., 2018) (Table 2).
Furthermore, traditional and homemade food processing is another crucial aspect. These are typical in Mediterranean European countries (Italy, Spain, France, and Greece) (EFSA BIOHAZ Panel et al., 2011).
In Italy, Istituto Zooprofilattico Sperimentale del Mezzogiorno and Istituto Superiore di Sanità evaluated the detection of HEV RNA in typical and regional homemade meat products. Results are given in Table 2. From these data evaluation, the Italian Public Health Minister purposed new guidelines for a safe domestic production (Montone et al., 2019).
Spain is one of the principal European producers of pork from extensive farming (black Iberian pig) and bushmeat 370 FERRI AND VERGARA (deer and wild boar). Every year wild boar meat consumption level is *3.5 kg per person and it represents an important risk factor for HEV infection, especially in Southern Spain (Pineda et al., 2014). A representative case was reported by Spanish authors at the Infectious Disease Unit of the Hospital in Cordoba (Rivero-Juarez et al., 2017). A 32-year-old male patient (HIV infected) presented gastroenteric symptoms: malaise, diarrhea, jaundice, vomiting, and fever.
Molecular screenings confirmed HEV RNA detection. The patient's family represented an emblematic example because it comprised wild boar hunters and consumers of game meat (roasted or grilled wild boar meat).
Laboratories also screened two portions of wild boar meat (chap and loin). Both slices were positive with a viral load of 230-500 copies/mL and 155-300 copies/mL for chap and loin, respectively (Rivero-Juarez et al., 2017).
In this case, cooking processes did not completely eliminate HEV, in contrast to the previous studies (Kamar et al., 2014). However, a complete inactivation may also be dependent on the initial viral load and the exact composition of the food matrix (Cook and Van der Poel, 2015).
In the intercontinental scenario, China is the largest porkproducing and consuming country (Geng et al., 2019b).
It is common to consume hot pork (without being frozen). It means that pork products are delivered directly from the slaughterhouse to the retail markets. Pork viscera and blood (i.e., ''blood curd'' is a popular shortly cooked product) are usually consumed in Chinese popular tradition. These matrices pose at-risk consumers' health (Table 2).
There is a fragmentary knowledge about the resistance of HEV under food processing technologies, that is, fermentation, food aging, curing, drying ( salami, sausages, etc.), smoking, and cooking processes.
Therefore, food product's label should be integrated with information about the potential risk of HEV infection. In this way, the public authorities have to prevent and control possible foodborne outbreaks (Geng et al., 2019b), and protect vulnerable consumers (i.e., patients with preexisting chronic liver disease and transplant recipients) (Delage et al., 2019).
A fundamental solution comes from innovative food technologies application. Emmoth et al. (2017) provided the possibility to apply new systems to inactivate viruses (using murine NoV MNV and feline calicivirus FCV as a model for HEV) in swine food matrices.
In particular, they evaluated the effects of high-pressure processing (HPP), lactic acid (LA), and intense light pulse (ILP). Researchers applied three treatments (HPP, LA, and ILP) to fresh swine liver, dry-cured ham slices, and cold-smoked pork sausages (purchased from local grocery stores). HPP demonstrated efficacy (in the range 300-600 MPa) both on the food surfaces and on internalized viruses. LA and ILP obtained lower viral reduction. Therefore, physical parameters and properties in combination with biochemical ones will encourage new approaches to the food safety concept.

Milk
The European authorities suggest that milk is a possible source of HEV infection. Therefore, the consumption of unpasteurized and contaminated milk represents a risk for consumers (EFSA BIOHAZ Panel et al., 2017).
The pasteurization process is an important phase in dairy industries. In general, it permits to prevent and control foodborne infection (Huang et al., 2016).
However, in some cases, it is not able to inactivate all viral loads (HEV), and it is possible that consumers infect through pasteurized milk ingestion. Conversely, milk boiling process (100°C for 3 min) provides a complete sterilization (Huang et al., 2016).
Cow's milk has been largely screened in numerous research projects by scientists. However, HEV has been also detected in other dairy species: goat (Long et al., 2017), sheep, and donkey (Demirci et al., 2019) (Table 3).
There are two different epidemiological realities: on one side industrialized countries, and on the other, developing nations.
Conversely, in the African (Sayed et al., 2020) and Asiatic continents (Huang et al., 2016;Long et al., 2017;Geng et al., 2019c) it is possible to find contrasting data between rural and industrialized regions (Table 3).
In the rural regions, there are traditional small mixed farms, where different animal species (i.e., bovine, swine, ovine, caprine, and lagomorph) live in direct contact. This condition represents a particular epidemiological scenario that critically improve cross-species infections (Geng et al., 2019c). It is also strictly related to poor hygiene practices ''from the stall to the consumers.'' Therefore, this factor contributes to increase seroprevalences of anti-HEV IgG Abs.
The role of mammary glands as source of HEV RNA excretion has been investigated in humans too. High titers of anti-HEV antibodies were registered in pregnant women (5.7%, 25/439 mainly owing to the dietary factors: contaminated water and food ingestion) (Alvarado-Esquivel et al., 2014).
High steroid hormone (estrogens) levels (in pregnant women) promote viral multiplication (Singh et al., 2019). Consequently, it is possible to isolate HEV RNA from human breast milk during acute phase of infection (Rivero-Juarez et al., (2016).
In conclusion, it is possible to consider that there is a multifocal sanitary perspective. It begins from healthy farms (through the improvement of zootechnic management during pre-and postdipping phases) and it continues in dairy industries through the introduction of new technologies (i.e., high hydrostatic pressures and ultrasounds) to provide safe food products.

FERRI AND VERGARA
HEV particles are protagonists of a particular ''water's cycle'' (Fenaux et al., 2019). It begins from virus elimination through human-and animal-infected feces that arrive to the waste treatment plants. Consequently, treated waters can be used in agriculture for fruits and crops irrigation, or they arrive to the sea in mariculture farms.
This cycle concludes with the final consumers, who are infected through contaminated waters, raw mollusks, raw or undercooked meat products, fruits and vegetables ingestion (Fenaux et al., 2019).
Aquaculture farms are frequently localized near harvested areas, that is, urban areas, slaughterhouses and meat preparation industries (O'Hara et al., 2018;Rivadulla et al., 2019), or livestock areas (Barreira et al., 2010). This localization explains the data reported in Table 4. Therefore, waste treatment plants are a critical control point. New surveillance, control measures, and structural implementations could be useful to prevent viral diffusion and infections.
A parallel development of molecular diagnostic assays will provide epidemiological and ecological information regarding HEV circulation (Di Profio et al., 2019).

Conclusions
HEV detection has been largely studied in meat production chains by numerous international research teams. Furthermore, it has been also detected in other food matrices that is, milk (cow, goat, sheep, and donkey) and mollusks.
Indeed, this review article reports the necessity of further investigations owing to the fragmentary knowledge about the efficacy of food processing technologies on viral loads.
For the future, the World Health Organization (2016) estimates and confirms that numerous foodborne cases of hepatitis E infection (caused by the following genotypes: HEV1, HEV2, and HEV4) will continue to affect endemic countries (African and Asiatic continents).
HEV3 will still be responsible for sporadic infections in Northern America and Europe. Therefore, public health authorities and private producers should enforce and improve monitoring activities in different food production chains. Regional and traditional realities will be crucial viral reservoirs.
Therefore, adequate sanitary measures are required. For symptomatic human patients, there are two possible approaches to treat and prevent HEV infections.
On the one side, there is a pharmacological approach through ribavirin administration. The British Transplantation Society supports that this molecule provide satisfactory results in transplant recipients (McPherson et al., 2018).
However, ribavirin is not recommended for pregnant women because of numerous adverse and teratogenic effects. Furthermore, emerging variants of HEV influence the efficacy and prognosis of therapies (Todt et al., 2018).
On the other side, vaccination is a valid alternative. It can be administrated in any epidemiological realities, where several outbreaks occur (endemic nations). In this way, it guarantees immunization for patients at risk.
Hecolin (Xiamen Innovax Biotech, China) is the first registered and licensed vaccine against HEV. It is a proteinbased vaccine, and Chinese scientists certified that it induces a valid immune response (Lee et al., 2015).
It provides 100% protection against HEV1 and also crossprotects against HEV4. Therefore, it is strongly recommended for fragile categories.
However, no data are available about cross-protection against HEV3 (World Health Organization, 2016).
Authors underline that is necessary to discover other viral epitopes that can produce adequate immune responses. Furthermore, it is also important that patients' immunoglobulins will provide an efficient cross-protection against different HEV genotypes. Therefore, this virus still remains an emerging and reemerging pathogen. It involves the relationships between HEV and multiple factors, that is, human and animal health, alimentary and environmental safety.
Environmental health represents a ''critical control point'' in foodborne pathogen transmission. On this concept, the European Commission built the new concept of food safety control measures (EC No. 625/2017).
Authors suggest that public authorities should incentivize new system of collaboration between private industries and public research programs. In this way, it will be possible to fill the above-mentioned gaps to obtain a complete point of view regarding HEV. This article stimulates and inspires new approaches to elaborate data in an innovative way.