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The Foodborne Transmission of Hepatitis E Virus to Humans

The Foodborne Transmission of Hepatitis E Virus to Humans Globally, Hepatitis E virus (HEV) causes over 20 million cases worldwide. HEV is an emerging and endemic pathogen within economically developed countries, chiefly resulting from infections with genotype 3 (G3) HEV. G3 HEV is known to be a zoonotic pathogen, with a broad host range. The primary source of HEV within more economically developed coun- tries is considered to be pigs, and consumption of pork products is a significant risk factor and known transmission route for the virus to humans. However, other foods have also been implicated in the transmission of HEV to humans. This review consolidates the information available regarding transmission of HEV and looks to identify gaps where further research is required to better understand how HEV is transmitted to humans through food. Keywords Hepatitis E virus · Foodborne · Zoonotic · Transmission · Meat · Shellfish Introduction is probable that more infections occur worldwide than esti- mated in 2005 (Guillois et al. 2015; Yin et al. 2019), espe- Globally, hepatitis E virus (HEV), of the family Hepeviri- cially considering that this estimate was made only consider- dae, is considered the most common cause of acute viral ing G1 and G2. hepatitis. There were an estimated 20 million infections HEV was once thought only to be endemic to certain eco- worldwide annually in 2005 from genotype 1 (G1) and 2 nomically developing countries within Asia and Africa, but (G2) HEV combined (Rein et al. 2012), and 44,000 recorded research over the past decade has highlighted the emergence fatalities due to the virus in 2015 (World Health Organisa- of HEV within higher income countries. The virus spreads tion 2019). Generally, HEV causes an acute, self-limiting through a faecal-oral route, making it easily transmissible infection which resolves within a few weeks; however, in through faecally contaminated water. Indeed, this is thought some persons (such as the immunocompromised) it can to be the main transmission route within China, where G1 cause chronic infections, fulminant hepatitis (acute liver and genotype 4 (G4) HEV are dominant (Wang et al. 2001). failure) and extrahepatic manifestations (infections in other However, it is also possible for the virus to be transmit- organs), which can be fatal. Table 1 shows a summary of the ted through foodstuffs such as pork, due to the ability for pattern of infection of the different HEV genotypes, dem- some HEV genotypes to infect non-human animals. Cur- onstrating the variable clinical manifestations and factors rently, the virus is classified into eight genotypes, with G1–4 such as average age of infection and gender, where these and genotype 7 (G7) capable of infecting humans. There is, are known. There are limited data to be able to estimate however, a diverse host range for the different genotypes, the number of infections worldwide, but with a high level with G1 and G2 generally only infecting humans and non- of asymptomatic infections seen in numerous outbreaks it human primates, but genotypes 3–8 infecting many other animals, such as pigs, deer, camels, rabbits, and dolphins. A summary of the different host species of HEV was pub- * Samantha Treagus lished recently (Kenney 2019), and is summarised briefly samantha.treagus@cefas.co.uk in Table 2. Genotype 3 (G3) HEV has been found to be the Biosciences, College of Life and Environmental Sciences, most geographically diverse of the viruses thus far (Pérez- University of Exeter, Penryn Campus, Penryn, Cornwall, UK Gracia et al. 2015), and is the genotype which has emerged Seacorp Technologies Limited, Bournemouth, UK in the past 2 decades in many developed countries. The geo- graphical distribution of genotypes 1–4 can be seen in Fig. 1. Centre for Environment Fisheries and Aquaculture Science, Barrack Road, Weymouth, Dorset DT4 8UB, UK Vol.:(0123456789) 1 3 128 Food and Environmental Virology (2021) 13:127–145 1 3 Table 1 Pattern of infection for the different genotypes of HEV, adapted from Centers for Disease Control and Prevention (2020) Genotype Transmis- Transmission routes Geographical distribution Extrahepatic manifestations Age groups at higher risk Gender more commonly Lethality sion in pattern affected humans? a,b a,b c 1 Yes Faecal-oral; waterborne; Economically developed Pancreatic Differs by country Differs by country 0.5–1% ; 20% blood transfusion; organ and developing countries in pregnant d,e,f donation women 2 Yes Faecal-oral; waterborne; Economically developing Unknown Young adults Unknown 0.5–1% blood transfusion; organ countries donation 3 Yes Foodborne; blood transfu- Economically developed Chronic infections in Older adults (> 40 years) Males 0.5–1% sion; organ donation and developing countries immune-compromised patients. Neurologi- cal, haematological, immunological and renal manifestations 4 Yes Foodborne; blood transfu- Economically developed Unknown Young adults Possibly males (limited 0.5–1% sion; organ donation and developing countries data) 5 No Faecal-oral Unknown Unknown Unknown Unknown Unknown 6 No Faecal-oral Unknown Unknown Unknown Unknown Unknown 7 Yes Foodborne; Faecal-oral; Unknown Unknown Unknown Unknown Unknown blood transfusion? 8 No Faecal-oral Unknown Unknown Unknown Unknown Unknown Pathak and Barde (2017) Spina et al. (2017) Peron et al. (2007) Kumar et al. (2017) Jin et al. (2016) Kamar et al. (2014) Horvatits et al. (2019) Mizuo et al. (2005) Food and Environmental Virology (2021) 13:127–145 129 Table 2 Summary of HEV host species by genotype adapted from Kenney (2019) Genotype Hosts identified (common names) Species infected 1 Humans, Chimpanzees, Monkeys, Horses Homo sapiens, Pan troglodytes, Chlorocebus sabaeus , Chlo- a a rocebus pygerythrus , Erythrocebus patas , Macaca mulatta, Macaca radiata, Macaca fascicularis, Semnopithecus entellus, a a a Aotus trivirgatus , Saguinus mystax mystax , Saimiri sciureus , Equus caballus ferus a a 2 Humans, Monkeys Homo sapiens, Chlorocebus pygerythrus , Erythrocebus patas , Macaca mulatta, Macaca fascicularis, Aotus trivirgatus , a a Saguinus mystax mystax , Saimiri sciureus 3 Humans, Monkeys, Hares and Rabbits, Rats, Minks, Mongooses, Homo sapiens, Erythrocebus patas, Macaca mulatta, Macaca Pigs, Goats and Sheep, Deer, Dolphins, Horses, Vultures fascicularis, Macaca fuscata, Lepus europaeus, Oryctolagus cuniculus domesticus, Rattus norvegicus, Neovison vison, Herpestes javanicus, Sus scrofa, Sus scrofa domestica, Capra hircus aegagrus, Ovis aries orientalis, Cervus elaphus, Cervus nippon, Capreolus, Tursiops truncatus, Equus africanus, Equus caballus ferus, Gyps himalayensis 4 Humans, Monkeys, Gerbils, Dogs, Bears, Leopards, Pigs, Cows, Homo sapiens, Macaca fascicularis, Macaca mulatta, Meriones Goats, Deer, Cranes, Pheasants unguiculatus , Canis lupus familiaris, Ursus thibetanus, Neofe- lis nebulosa, Sus scrofa, Sus scrofa domesticus, Bos taurus primigenius, Bos grunniens, Capra hircus aegagrus, Ovis aries orientalis, Cervus nippon, Elaphodus cephalophus, Muntiacus reevesi, Balearica regulorum, Lophura nycthemera 5 Monkeys, Pigs Macaca fascicularis, Sus scrofa 6 Pigs Sus scrofa 7 Humans, Camels Homo sapiens, Camelus dromedarius, 8 Camels Camelus bactrianus Infections instigated through experimental conditions Fig. 1 The geographical distri- bution of HEV genotypes 1–4. This figure shows the genotypes of HEV which are endemic to each country, where enough data were available. For graphs which are compatible with the conditions protanopia, deutera- nopia and achromatopsia please see online resources 1 and 2. Maps created in ArcMap using the World Countries (general- ised) layer package by esri_dm and visualised in GIMP 1 3 130 Food and Environmental Virology (2021) 13:127–145 G3 HEV is thought to be spread primarily through the directly been linked to pork product consumption, including consumption of undercooked pork products from infected an outbreak in Spain linked to consumption of wild boar pigs; however, it is unknown if all transmission routes to (Rivero-Juarez et al. 2017), and another outbreak associated humans have been identified. The current theories and with consumption of a spit-roasted piglet in France (Guillois known routes of transmission can be seen in Fig. 2. This et al. 2015). review will discuss theoretical routes of HEV transmis- Consumption of pork products is now considered a sig- sion to humans through foodstuffs and identify areas which nificant risk factor for developing HEV infection, which is require further research for better understanding of the virus. concerning considering the seroprevalence levels in Euro- pean pigs (Said et al. 2014; Slot et al. 2017). Table 3 shows a non-exhaustive list of countries that have detected anti-HEV HEV in Pigs and Pork Products antibodies in pigs, and HEV RNA in pork products. Considering Table 3, it is possible that the HEV preva- Over the past 2 decades, evidence has accumulated impli- lence in pork sausages may be over- or under-estimated by cating pigs and other animals in the zoonotic transmission the fact that the sample sizes for some of these studies are of G3 HEV to humans (Tei et al. 2003; Guillois et al. 2015; relatively small. It is also important to note that different Lhomme et al. 2013; Rivero-Juarez et al. 2017). In 1998, it methods have been used between studies, some of which was shown that a HEV strain isolated from an acute HEV have been shown to be less sensitive for detecting HEV than patient in the USA was capable of infecting pigs, and that others. Interestingly, the seroprevalence of HEV in pigs is a genetically similar strain isolated from pigs was capable significantly higher than the prevalence of HEV RNA in of infecting non-human primates, suggesting a significant pork products in most cases; this discrepancy is expected as possibility that pigs could act as a zoonotic source of HEV not all pigs would be viraemic at slaughter. (Meng et al. 1998). Since this study, many countries have It has been found that HEV generally infects swine noted the emergence of HEV cases. At this stage, it was asymptomatically at an early point in their life (prior to thought that HEV was only endemic in developing countries 6 months of age (de Oya et al. 2011)), and that because (such as those in Asia, Africa and Central America), and of this, many pigs are seropositive at the time of slaughter that HEV cases in non-endemic areas were obtained through (Grierson et al. 2015; Rose et al. 2011; de Oya et al. 2011). travel. However, studies such as those by Dalton et  al. The slaughter age of pigs is normally slightly before 1 year (2007) and Fraga et al. (2017) identified that indigenous of age. The transmission between pigs is suspected to be cases of HEV were occurring in economically developed through a faecal-oral route and this is likely to be due to the countries such as the UK and Switzerland. It is now widely high shedding that is seen in pig faeces and urine (Bouw- accepted that pigs are a zoonotic source of HEV transmis- knegt et al. 2009; Halbur et al. 2001). Infection with HEV sion to humans and may be at least partially responsible early in life means that there is a lower chance of the pigs for increasing cases worldwide annually, though increased being viraemic at slaughter, and they are therefore less likely detection and awareness of HEV may also play a small role to be capable of HEV transmission to humans through the in the observed increase in cases. Outbreaks of HEV have pork food chain. However, whether HEV causes life-long Fig. 2 Theoretical and con- firmed transmission routes of HEV. The figure shows confirmed and theoretical routes of HEV transmission to humans. The theoretical routes of transmission include HEV infections contracted from the consumption of shellfish, sheep, and cows, as well as crops and drinking water, as no confirmed outbreaks from these sources have yet been identified. Illustration created using Adobe Illustrator and edited using GIMP 1 3 Food and Environmental Virology (2021) 13:127–145 131 Table 3 A list of the seroprevalence levels of anti-HEV antibodies in pigs and the percentage of pork products found to be HEV positive by RT- PCR in those locations Location Pig sample population anti-HEV antibody seroprevalence Percentage of tested foodstuffs HEV positive by RT-PCR Brazil 63.6% of 357 pigs (Vitral et al. 2005) 1.7% of 118 slaughterhouse livers (Gardinali et al. 2012) Canada 59.4% of 998 pigs (Yoo et al. 2001) 8.8% of 283 livers, 1.0% of 599 pork chops (Wilhelm et al. 2014) 47.0% of 76 pork pâtés and 10.5% of 19 retail raw pork livers (Mykytczuk et al. 2017) France 31.0% of 6565 pigs (Rose et al. 2011); 60% of 1034 pigs 4.0% of 3715 slaughterhouse livers (Rose et al. 2011) (Feurer et al. 2018) 2.8% of 1034 slaughterhouse livers (Feurer et al. 2018) 30.0% of 140 figatelli and fitone, 29.0% of 169 liver sausages, 25.0% of 55 quenelles or quenelle paste, 3.0% of 30 dried salted livers (Pavio et al. 2014) 58.3% of 12 raw liver sausage (Colson et al. 2010) Germany 49.8% of 1072 pigs (Baechlein et al. 2010) 4.0% of 200 retail livers (Wenzel et al. 2011) 20.0% of 70 raw sausages and 22.0% of 50 liver sausages (Szabo et al. 2015) Italy 45.1% of 2700 pigs (Mughini-Gras et al. 2017) 20.8% of 48 slaughterhouse livers (Di Bartolo et al. 2011) 6.0% of 33 slaughterhouse livers (Di Bartolo et al. 2012) 13.3% of 15 fresh liver sausages, 7.1% of 14 dried liver sausages (Di Bartolo et al. 2015) Japan 57.9% of 2500 pigs (Takahashi et al. 2003) 1.9% of 363 retail livers (Yazaki et al. 2003) Netherlands 89.0% of 417 organic pigs, 72% of 265 conventionally farmed 6.5% of 62 commercial pork livers (Bouwknegt et al. 2007) pigs, 76% of 164 free range pigs (Rutjes et al. 2014) 12.7% of 79 livers, 70.7% of 99 liver sausages, 68.9% of 90 liver pâté samples (Boxman et al. 2019) Spain 20.4% of 1441 pigs (de Oya et al. 2011) 6.0% of 93 sausages, 3.0% of 39 slaughterhouse livers (Di Bar- tolo et al. 2012) Switzerland 62.3% of 1001 pigs in 2006, 53.8% of 999 pigs in 2011 (Burri 1.3% of 160 slaughterhouse livers (Müller et al. 2017) et al. 2014) 11.8% of 102 raw liver sausages (Giannini et al. 2017) 11.7% of 90 pork liver and raw meat sausages (Moor et al. 2018) UK 92.8% of 629 pigs, with 20.5% viraemic at slaughter (Grierson 10.0% of 63 sausages, 3.0% of 40 slaughterhouse livers (Berto et al. 2015) et al. 2012) USA 21.9% of 182 pigs (Owolodun et al. 2013) 11.0% of 127 retail liver (Feagins et al. 2007) immunity in swine after recovery has been open to debate. strain from the same genotype could also not confer protec- In rhesus macaques and humans, the anti-HEV IgG anti- tive immunity (Huang et al. 2008). If there is genetic or bodies (characteristic of long-term immunity) wane over a environmental variation in host development of immunity to variable number of years until they are undetectable, and the HEV, and some strains of HEV may not provide protection period for which individuals may be IgG positive for varies against others, then farms with multiple circulating HEV (Arankalle et al. 1999; Lee et al. 1994). This may not be an strains could be more likely to have viraemic pigs at the time issue in pigs as they are normally slaughtered before a year of slaughter. This would therefore mean that there could be of age, so early life infections would likely allow immunity a higher likelihood of contracting HEV from undercooked against subsequent HEV challenge over their lifetime. How- pork products or food products containing raw pig compo- ever, it has been shown that animals can be re-infected by nents from these sources. different HEV strains (De Deus et al. 2008), and whether or It has been reported that 21% of pigs in the UK tested not one strain can confer protective immunity to all other positive for HEV RNA at the time of slaughter (Grierson HEV strains has also been contested. It has been shown that et al. 2015), and that in the USA 6.3% of pigs from slaugh- after infection with one strain of G3 HEV, pigs developed terhouses were HEV RNA positive (Sooryanarain et  al. some protective immunity against other strains within the 2020). As such, it is probable that the consumption of raw same genotype, and also within G4 (Sanford et al. 2011). and undercooked pork products is acting as a transmission However, in rhesus monkeys, it was shown that infection route of HEV to humans. with one strain from a genotype could not confer protective Table  4 summarises studies that have investigated the immunity to a strain in a different genotype upon subsequent thermal inactivation of HEV in non-food matrix samples. challenge, and that in some cases, infection with a different Inactivation condition combinations found sufficient to 1 3 132 Food and Environmental Virology (2021) 13:127–145 Table 4 A summary of studies investigating thermal inactivation treatments for HEV in non-food matrix samples Study Cell culture or molecular Genotype Heat treatment Growth period Inactivation/reduction detection? Tem- Time (min) perature (°C) Emerson et al. (2005) Cell culture, HepG2/C3A 1 (strain Akluj ) 56 60 5–6 days > 80% reduction cells 1 (strain SAR55 ) 56 60 5–6 days ~ 50% reduction 2 (strain Mex14 ) 60 60 5–6 days 96% reduction Huang et al. (1999) Cell culture, A549 cells 3 (strains G93-1*, 56 30 72 h < 1.0 (TCID50/0.025 ml) G93-2*, G93-3*, G93-4*) Johne et al. (2016) Cell culture, A549/D3 cells 3 (strain 47832c ) 55 1 35 days ~ 1 log reduction in focus forming units 70 2 “No infectious virus” Schielke et al. (2011) Molecular detection 3 (strain wbGER27 ) 56 15 N/A 74.07% reduction 3 (strain wbGER27 ) 56 60 N/A 99.90% reduction Tanaka et al. (2007) Cell culture, PLC/PRF/5 3 (strain JE03-1760F ) 70 10 35 days “No infectious virus” cells 3 (strain JE03-1760F ) 56 30 50 days “Still infectious” *Accession numbers unknown AF107909 M80581.1 KX578717.1 AB437319.1 FJ705359.1 KC618403.1 inactivate virus in these studies are shown in Fig. 3. Results by Huang et al. (1999), a temperature of 56 °C for 30 min vary between the different studies, and several factors make was reported to completely inactivate the virus; however, the comparison difficult. The studies use a variety of different virus was only left to grow for a relatively short period of units or expressions of reduction/inactivation. In the study time (72 h). However, it was shown that HEV was still viable Fig. 3 A summary of the reported thermal inactivation requirements for HEV from different studies. This graph summarises the observed HEV inactivation requirements for five different studies investigat- ing the effect of heat treatment over time on HEV viability, with the highest reported inacti- vation requirements being 70 °C for 10 min, and the lowest being 56 °C for 30 min. Graph created in R studio 1 3 Food and Environmental Virology (2021) 13:127–145 133 following similar heat treatments in cell culture studies with studies have all taken different approaches to heat inactiva- longer growth periods (Emerson et al. 2005; Tanaka et al. tion of the virus and the food types used, and therefore it is 2007). Schielke et al. (2011) used RNase treatment in an difficult to compare the results and form a definitive answer attempt to remove viral RNA that had broken from the cap- for the heat treatment required to inactivate the virus within sid after heat treatment, assuming this would remove RNA foodstuffs. Feagins et al. (2007) identified that boiling or from non-viable virus. However, it is unknown if any RNA stir-frying infected pig liver to an internal temperature of from viable virus could have been lost during this treatment, 71 °C for 5 min could prevent infection when the liver was and the standard deviations seen within the results were rela- then fed to pigs. However, though the pigs were not infected tively large. Without a cell culture component, it is not possi- by this oral dose, it is known that pigs commonly require a ble to say with 100% certainty that remaining detected RNA high dose of HEV to become infected through the faecal- was from viable virus. In addition to these limitations, it is oral route (Kasorndorkbua et al. 2004), of approximately known that HEV is difficult to culture effectively in vitro, 10 genome copies (Andraud et al. 2013), and therefore it is often requiring large titres of virus to begin the culture, and possible that lower doses of active virus were still present therefore it is possible that the inactivation requirements for in these food stuffs. The oral infectious dose for humans is HEV have been under-estimated as treatments sufficient to unknown. Intravenous inoculation of pigs with the cooked eliminate infectivity in vitro may not completely eliminate foodstuffs as carried out by Barnaud et al. (2012) is likely to in vivo infectivity. Some researchers have investigated dif- have provided a more accurate estimate of whether viable ferent cell lines and strains of HEV which appear to be more virus still existed, especially as intravenous inoculation of efficiently cultured in vitro due to insertions within the HEV pigs has been reported to require much lower HEV doses to genome; however, culturing these strains still requires large cause infection (Dähnert et al. 2018). Imagawa et al. (2018) titres of virus to begin the culturing process (10 copies/ml) reported similar inactivation requirements to Feagins et al. (Johne et al. 2014; Schemmerer et al. 2016). (2007). However, the limit of detection for the cell culture 4 5 It is possible that interactions of HEV with organic mol- system was 10 –10 genome copies, and therefore, as with ecules in food matrices may cause the thermal inactivation the previous study, viable virus remaining in the minced temperature to be higher. Some studies have investigated pork may not have been detected in the cell culture system. the thermal inactivation of HEV within food stuffs, such In addition, the different food preparations between the stud- as liver and pork sausage (summarised in Table 5). These ies may have influenced viral stability, as could the different Table 5 A summary of studies investigating thermal inactivation treatments for HEV in foodstuffs Study Food stuff Cooking method Temperature (°C) Time (min) Measurement of inactiva- HEV tion inacti- vated? Barnaud et al. (2012) Pâté preparation (spiked Water bath 62 5 Intravenous administration No with 10 HEV genome to pigs 20 No copies) 120 No 68 5 No 10 No 20 No 71 5 No 10 No 20 Yes Feagins et al. (2008) Pig liver (naturally infected) Incubation 56 60 Oral administration to pigs No Boiling ≥ 71 (internal) 5 Yes Stir fry ≥ 71 (internal) 5 Yes Imagawa et al. (2018) Minced meat (spiked with Boiling or roasting 63 1 Cell culture No 10 HEV genome copies) 5 No 30 Yes 65 1 No 5 Yes 70 1 No 5 Yes 1 3 134 Food and Environmental Virology (2021) 13:127–145 strains of G3 HEV used. The initial viral titres used could raw Sika deer meat contracted HEV 6–7 weeks later (typi- also have influenced the results and explain why a longer cal of the HEV incubation period), with the HEV sequence treatment time was needed in some studies. confirmed as 100% identical between the meat and infected Further research is clearly required to investigate HEV patients (Tei et al. 2003). With the presence of HEV in inactivation within foods. This may require a more effi- deer being so variable between studies (perhaps due to cient cell culturing method and an assessment of different study limitations such as sample size), further research foods, cooking methods and HEV strains. However, taking is required to identify the level of active HEV infection the results of studies conducted in non-food and food (pork within deer populations through larger prevalence studies. product) samples together, a conservative measure would be However, with the number of countries that have detected to cook pork products for longer than 20 min at temperatures HEV in deer, and the occurrence of foodborne outbreaks higher than 72 °C. from deer meat, deer could be acting as another reservoir In addition to the potential for HEV to survive some for HEV. cooking processes, raw pork products are used in some There have been reports of HEV infections in cattle (Bos consumables. Raw blood products are commonly used in taurus) from China, where both antibody seroprevalence and ready-cooked foods such as processed ham (fibrinogen) HEV RNA of G4 have been identified in multiple studies. and other blood proteins are used as food additives such as Hu and Ma (2010) showed the presence of G4 HEV RNA in emulsifiers. Spray-dried plasma powder (SDPP) is also used 8.8% of cattle from Xinjiang Autonomous Region. A subse- in domestic and farm animal foods but this has some heat quent study then identified that 37.1% of tested dairy cows in processing prior to use. SDPP is commonly fed to weaned Yunnan Province were HEV RNA positive, and that 100% of piglets; however, a previous study reported no transmission the HEV-positive cows were producing milk that contained of HEV in pigs fed spray-dried plasma products that were HEV RNA (Huang et al. 2016). A further study in Shandong positive for HEV RNA (Pujols et al. 2014), and therefore the Province also found 3% of yellow cattle to be G4 HEV RNA heating of spray-dried plasma may be sufficient to inactivate positive, with 47% seroprevalent for anti-HEV antibodies the virus present. However, as other porcine products are (Yan et al. 2016). A study in Turkey identified HEV from not subjected to heat processes before they are used, they G1, G3 and G4 in 20.3% of raw milk samples from various could constitute a transmission risk to humans through use domestic animals (cows, sheep, goats, donkeys) (Demirci in food. A study conducted in 2017 found that of 36 liquid et al. 2019). Other studies investigating HEV in cattle have porcine products derived from blood, 33 were positive for produced negative or mixed results; a study in Beijing, China HEV RNA, and seven of 24 spray-dried plasma products identified 29.4% of cattle were seroprevalent for HEV, but no were also positive for HEV RNA (Boxman et al. 2017). This HEV RNA could be detected (Chang et al. 2009). A study in is especially significant when blood products from multiple Burkina Faso also found 26.4% of 72 cattle to be seropreva- animals are commonly pooled together, meaning that prod- lent for HEV (Ouoba et al. 2019). Another study in Ger- ucts from one viraemic animal could contaminate a batch many testing 400 milk samples found no evidence of HEV and lead to widespread HEV transmission through many RNA, although G4 HEV is much less commonly reported in different food products. Europe than in Asia (Baechlein and Becher 2017). Likewise, Pork product consumption has been considered to be a a study in Belgium also found no evidence of HEV RNA major risk factor in the development of HEV due to the con- in cow milk or faeces (Vercouter et al. 2018). In the USA, nection to foodborne outbreaks and the fact that HEV in Yugo et al. (2019) identified that of 983 cows, 20.4% were pork products can reach high levels (e.g. 7 × 10 genome seroprevalent for anti-HEV antibodies; however, HEV RNA copies/g in liver pâté in the Netherlands, Boxman et  al. could not be detected in any of the cows. The authors con- 2019). However, pigs are not the only animals consumed cluded that this may have been because of an antigenically which can act as reservoirs for the virus. similar relative to HEV rather than due to HEV itself, which could be possible as G4 HEV is not thought to be endemic to the USA; however, this would call into question the HEV in Other Land Animals and Animal specificity of ELISA assays and studies investigating HEV Products seroprevalence. Notwithstanding the significant number of studies with negative findings, these results are concerning In addition to pigs, deer have been reported to be infected as meat and dairy from cows are consumed worldwide by with HEV in many different countries (summarised in humans, and the possibility that cows could be a HEV reser- Table 6). It is important to identify the transmission risk voir could have a significant impact on our understanding of that deer may have to humans, as HEV outbreaks have HEV transmission to humans. More research worldwide is been directly linked to the consumption of raw deer meat. therefore needed to identify which HEV genotypes are capa- For example, in Japan, multiple people who had consumed ble of infecting cattle, and to find the prevalence of HEV in 1 3 Food and Environmental Virology (2021) 13:127–145 135 Table 6 Summary of studies investigating the prevalence of HEV in deer Study Location Deer species ELISA observed RT-PCR seroprevalence preva- lence Weger et al. (2017) Canada Odocoileus virginianus (White-tailed deer) 8.8% ND Odocoileus hemionus (Mule deer) 4.5% ND Rangifer tarandus groenlandicus (Barren- 1.7% ND ground caribou) Rangifer tarandus (Woodland caribou) 5.2% ND Zhang et al. (2015) China Cervus nippon (Sika deer) 5.4% ND Anheyer-Behmenburg et al. (2017) Germany Capreolus (Roe deer) 0.0% 6.4% Cervus elaphus (Red deer) 0.0% 3.5% Neumann et al. (2016) Cervus elaphus (Red deer) 2.5% 3.7% Capreolus (Roe deer) 6.5% 0.0% Reuter et al. (2009) Hungary Capreolus (Roe deer) ND 12.2% Di Bartolo et al. (2017) Italy Cervus elaphus (Red deer) 13.6% 11.0% Sonoda et al. (2004) Japan Cervus nippon (Sika deer) 2.0% 0.0% Matsuura et al. (2007) Cervus nippon (Sika deer) 2.6% 0.0% Tomiyama et al. (2009) Cervus nippon yesoensis (Yezo deer) 34.8% ND Spancerniene et al. (2018) Lithuania Capreolus (Roe deer) ND 22.6% Cervus elaphus (Red deer) ND 6.7% Medrano et al. (2012) Mexico Odocoileus virginianus (White-tailed deer) 62.7% ND Rutjes et al. (2010) The Netherlands Cervus elaphus (Red deer) 8.0% 15.0% Capreolus (Roe deer) 12.5% 0.0% Boadella et al. (2010) Spain Cervus elaphus (Red deer) 10.4% N/A Kukielka et al. (2016) Cervus elaphus (Red deer) 12.9% 11.1% Capreolus (Roe deer) 7.0% 0.0% Roth et al. (2016) Sweden Cervus elaphus (Red deer) 7.0% 0.0% ND not done Did not test full sample population which were tested for seropositivity cattle and dairy products. This will help to identify the risk antibodies (Ouoba et al. 2019). In the USA however, Sanford of transmission of HEV from cattle to humans. et al. (2013) suggested that a HEV-related agent was caus- Goats have also been shown to be potential reservoirs ing HEV in goats after discovering a seroprevalence of 16% for HEV infection, which is important due to goat meat, but a lack of any HEV RNA; however, this again calls into milk and cheese production. In Italy in 2016, 9.2% of goat question the accuracy of HEV ELISAs. In addition, no HEV faecal samples from six farms were found to be positive RNA was detected by conventional RT-PCR in goats that for HEV RNA, belonging to G3 strains which were highly had been experimentally infected with three different HEV related to strains found in pigs and humans (Di Martino et al. strains from G1, G3 and G4 in this study; however, the sen- 2016). Also, in Yunnan province, China, Long et al. (2017) sitivity of this PCR may be lower than previously reported found 70.3% of goat faecal samples to be positive for HEV HEV PCR assays, as assays which target a larger amplicon RNA, with milk samples from these animals also positive are generally observed to have lower sensitivity (Debode for HEV RNA. The strains obtained from these animals were et al. 2017), and qRT-PCR has generally been observed to from G4 HEV, and the Huang et al. (2016) and Long et al. be more sensitive for amplicon detection of HEV and other (2017) studies highlight that farms with mixed animals may amplicon targets (Zhao et al. 2007; Zemtsova et al. 2015). demonstrate a higher risk of HEV transmission. Another Dromedary camels have been implicated in the transmis- study in the Tai’an Region in China identified 4% of goat sion of G7 HEV to humans. In one case, a patient who regu- livers to be HEV positive, with G4 HEV that was similar to larly consumed camel meat and milk contracted chronic G7 cow HEV detected in the same region (Li et al. 2017). In HEV after a liver transplant (Lee et al. 2016). This chronicity Turkey, 18.5% of goat milk samples were reported positive is likely to have been opportunistic and influenced by immu- for HEV RNA (Demirci et al. 2019). Meanwhile in Bur- nosuppressive medication to prevent organ rejection. In a kina Faso, 28.4% of 81 goats were found to have anti-HEV separate paper, HEV was demonstrated to be seroprevalent 1 3 136 Food and Environmental Virology (2021) 13:127–145 in 23.1% of dromedary camels which originated in Sudan though rabbits are capable of carrying sub-genotype 3ra, and Saudi Arabia (El-Kafrawy et al. 2020). Due to the recent attempts to cause infections with another sub-genotype (3b) discovery of this genotype of HEV, and its implication in were unsuccessful; however, Hammerschmidt et al. (2017) human infection, further research is warranted to investi- identified a wild rabbit with HEV sub-genotype 3 g. Fur - gate how widespread camel HEV is within countries which ther research is therefore needed to identify which genotypes regularly consume camel products to determine the risk such or sub-genotypes of HEV are capable of infecting rabbits. products may have for the foodborne transmission of HEV. An interesting observation from the studies summarised in Rabbits and related species, e.g. hares, are also gaining Table 7 is that concordance in HEV prevalence between dif- increasing attention for their potential to transfer HEV to ferent samples from the same animals is often lacking. For humans through consumption of meat. In France, five cases example, Burt et al. (2016) found that 60% of liver samples of rabbit HEV (defined within Orthohepevirus A, G3ra) from 32 animals were HEV positive; however, only 16% of were identified in confirmed HEV-positive patients out of these 32 animals were faecally shedding the virus. It may 919 from 2015 to 2016 (Abravanel et  al. 2017). Several therefore be wise to identify standardised testing methods countries have identified rabbits to be seroprevalent and worldwide for identification of infected animals, with a deci- RNA positive for HEV (Table 7). The number of observa- sion made on what samples to test and which assays are best tions and the apparent ability of humans to contract rabbit to use, to avoid underestimating HEV prevalence. HEV suggest that it is a source of zoonotic HEV transmis- Despite identifying that animal product consumption is sion. However, with most human cases worldwide belonging a risk factor in the transmission of HEV, it is quite possi- to other genotypes and sub-genotypes, rabbits are likely to ble that there are other food transmission routes. One study be the cause of only a minority of cases. Studies have shown in the Netherlands showed that though seroprevalence of mixed results in terms of the ability of rabbits to carry other HEV antibodies was higher in meat-eaters (22.8%), vegetar- sub-genotypes of HEV. Zhang et al. (2017) have shown that ians still displayed a relatively high seroprevalence (13.8%) Table 7 A summary of studies identifying HEV (genotype 3ra) in rabbits and hares Country Study Seroprevalence RNA prevalence Burkina Faso Ouoba et al. (2019) 60.0% of 100 rabbits, 52.6% of 19 hares ND Canada Xie et al. (2017) ND 5.0% of 63 companion rabbit faecal samples, 0.90% of 114 commercial rabbit faecal samples China Geng et al. (2011a) 54.6% of 119 farmed rabbits 7.0% of 119 farmed rabbit serum samples Geng et al. (2011b) 15.4% of 1094 farmed rabbits 2.0% of 1094 farmed rabbit serum samples Xia et al. (2015) ND 5.0% of 492 rabbit faecal samples Li et al. (2020a) ND 15.0% of 120 rabbit faecal samples Li et al. (2020b) 7.1% of 70 farmed rabbits 11.4% of 70 farmed rabbit faecal samples France Izopet et al. (2012) ND 7.0% of 200 farmed rabbit bile samples, 23.0% of 205 wild rabbit liver samples Germany Eiden et al. (2016) 30.8% of 13 wild rabbits 30.8% of 13 wild rabbit serum samples Hammerschmidt et al. (2017) 37.3% of 164 wild rabbits, 2.2% of 669 wild 17.1% of wild rabbit serum samples, 0.0% of hares wild hare serum samples Ryll et al. (2018) 25% of 72 wild rabbits 34.7% of 72 wild rabbit liver samples Corman et al. (2019) 0.04% of 2389 wild hares 2.6% of 2389 wild hare serum samples Italy Di Bartolo et al. (2016) 3.4% of 206 farmed rabbits, 6.6% of 122 pet 0.0% of 7 IgG positive farmed rabbit serum rabbits samples, 0.0% of 122 pet rabbit serum samples The Netherlands Burt et al. (2016) ND 23.0% of 35 petting farm rabbit faecal samples, 0% of 10 farmed rabbit liver and faecal sam- ples, 60.0% of 32 wild rabbit liver samples and 16% of wild rabbit faecal samples Poland Bigoraj et al. (2020) 6.0% of 482 farmed rabbits 14.9% of 482 farmed rabbit liver samples South Korea Ahn et al. (2017) ND 6.4% of 264 rabbit faecal samples USA Cossaboom et al. (2011) 36.5% of 85 rabbits 16.5% of 85 serum samples, 15.3% of 85 faecal samples ND not done 1 3 Food and Environmental Virology (2021) 13:127–145 137 (Slot et al. 2017). This suggests that either they became could also therefore be contaminating water courses with HEV positive before becoming vegetarian through animal HEV, which is supported by studies in Italy, the Philippines meat, or they were infected through other transmission and Cambodia, showing river water contamination with HEV routes. Figure 2 shows the known and theorised routes of (Iaconelli et al. 2015; Li et al. 2014; Baez et al. 2017; Rodri- HEV transmission. One transmission route, which is much guez-Manzano et al. 2010). more tightly controlled now, was the transmission of HEV Countries within the European Union must conform to through blood transfusion (Hewitt et al. 2014), which may be EU regulations on how farm manure, animal carcasses and one way to explain the seroprevalence levels in vegetarians. digestive tract content are processed, transported, stored, Another explanation could be consumption of dairy products used as crop fertiliser, and disposed of. The sewage and such as milk. It is also possible to contract HEV through wastewater that originates on farms must either be dis- organ transplant with an infected organ (Pas et al. 2012), charged to public sewers or treated in a sewage treatment and organ transplant-associated cases commonly result in plant on the farm before the effluent can be discharged to chronic infections due to immunosuppression medication. surface waters, and a permit is required for the processing and disposal of sewage and wastewater in this way. However, it is possible for farms within EU countries to use sewage Contamination of the Aquatic Environment and slurry that has been produced on a farm to be spread on crops at the same farm, without prior processing, for the sake In addition to medical routes of transmission, it is important of fertiliser or soil quality improvement (European Com- to consider the impact that HEV within animal farm run-off mission 2001). Accordingly, the United States also allows and sewage has on the aquatic environment. It has been sug- manure originating on one farm to be spread on crops from gested that human and farm sewage may have a part to play in that farm (Environment Protection Agency 2020). However, other HEV transmission routes, potentially through farm run- manure use and farm practices are likely to be more diverse off from animal slurry stores or application of animal slurry and potentially problematic in countries within Asia, Africa to crops and through contamination of surface waters used for and South America. irrigation and shellfish farms. Raw human sewage collected at Previous studies have shown that sewage treatment pro- 2-week intervals in 2014–2015 from a sewer which serves the cesses such as long-term fermentation and composting are whole of Edinburgh was found to contain HEV in 93% of the likely to be capable of removing HEV from sewage (García samples collected (Smith et al. 2016), and many other coun- et al. 2014). A study in Switzerland also identified HEV- tries have also detected HEV in human sewage influent, such positive influent samples from wastewater treatment plants, as Spain, Switzerland, Portugal and France (Clemente-Casares but no HEV-positive effluent samples, suggesting effective et al. 2003, 2009; Rodriguez-Manzano et al. 2010; Matos et al. wastewater treatment using a cleaning and activated sludge 2018; Masclaux et al. 2013). This could therefore mean that process (Masclaux et al. 2013). However, Loisy-Hamon and when storm overflows discharge into water courses such as Leturnier (2015) detected HEV in treated pig sewage sam- rivers and seas, HEV contamination can occur. Because there ples from France that had been treated using one of saw- are many different types of wastewater treatment practices, and dust composting, slurry dehydration or anaerobic digestion. many combinations of practices between wastewater treatment Other studies have found that river water close to pig farms plants, it is difficult to know which wastewater treatment plants and pig processing plants had been contaminated with HEV, will be more effective at removing viruses from wastewater. for example, in Scotland and Italy (Crossan et al. 2012; Idolo However, other viruses such as adenovirus and norovirus are et al. 2013; Marcheggiani et al. 2015). Therefore, it is pos- commonly found in treated sewage (Bofill-Mas et al. 2006; sible that leachate (liquid leaching from solids into the envi- Campos et al. 2016). It is therefore possible that in addition to ronment) from stored manure and yard run-off from farms storm overflows, inadequate treatment of sewage could result and abattoirs may be polluting surface waters such as rivers. in HEV pollution of the aquatic environment, especially con- However, it is unknown whether all of the virus leaching into sidering that HEV is a single-stranded RNA non-enveloped the environment is viable—for example, viral RNA detected virus like norovirus. Pig farm and slaughterhouse sewage has in treated sewage may not indicate viable virus, but remain- also been found to be positive for HEV in multiple countries, ing RNA. for example, HEV RNA was detected in sewage from one of twelve slaughterhouses in Spain (Pina et al. 2000), whilst 75% of swine slurry samples collected from Italian pig farms Crop Contamination were HEV positive (La Rosa et al. 2017), and both fresh swine faecal material and pooled stored slurry from pig farms in Surface waters from sources such as rivers and groundwater the USA were shown to contain HEV (Kasorndorkbua et al. are commonly used as crop irrigation sources throughout the 2005). Human sewage, pig farm run-off and abattoir outflows world (Food and Agriculture Organisation 2011, 2016). Due 1 3 138 Food and Environmental Virology (2021) 13:127–145 to the potential contamination of such water with HEV and from commercial harvesting areas, with 73.9% of those sam- other pathogens from faecal matter (whether from human or ples giving E. coli results compliant with the end product animal sources), this could cause contamination of irrigated standard of ≤ 230 E. coli/100 g shellfish flesh. Norovirus crops. Animal waste (that can potentially be contaminated within oysters is linked to human faecal pollution that has with HEV as shown above) is also used as crop fertiliser originated from storm overflows and CSOs, or sewage that for farms. A small number of studies have found some evi- has received insufficient treatment (Campos et  al. 2013, dence of crop contamination with HEV. In France, two out 2016). CSOs release untreated sewage into surface water to of 230 herb and spice samples were positive for HEV RNA prevent overflows within mains drainage, but outfall events (Loisy-Hamon and Leturnier 2015), a study testing 125 let- can last for several hours or days and are often poorly moni- tuce samples from Greece, Serbia and Poland detected four tored (Marine Conservation Society 2011). Considering that positive samples (Kokkinos et al. 2012), and in Italy, six of farm or abattoir run-off, combined sewer overflows, and 911 “pre-washed and ready to eat” vegetable samples tested inadequately treated sewage could be polluting watercourses positive for HEV RNA (Terio et al. 2017). Another study in with HEV, it is also possible for aquatic organisms, such as four European countries (Czech Republic, Finland, Poland shellsh fi , to be affected by HEV contamination. Indeed, stud - and Serbia) also detected HEV RNA in one frozen raspberry ies around the world have found HEV to be present within sample of 38 tested (Maunula et al. 2013). However, it is bivalve shellfish, and these are summarised in Table  8. The important to note that no foodborne outbreaks of HEV from study by Rivadulla et al. (2019) also showed shellfish to have contaminated crops have been reported, and the quantities of as much as 1.1 × 10  RNA copies per gram of shellfish tis- virus found on the crops is also low enough to call into ques- sue, which is close to the pig ID50, but the human infectious tion whether they would cause illness when consumed. It is dose is still unknown. It is important to note, however, that also unknown whether the HEV RNA detected originated not all RNA found in the shellfish may have been associ- from viable virus. ated with viable virus. To date, there have been no proven foodborne outbreaks of HEV from shellfish consumption, although an outbreak of HEV on a cruise ship was theorised HEV in Bivalve Shellfish and Other Aquatic to have been caused by consumption of shellfish on the basis Animals of a retrospective risk analysis (Said et al. 2009). HEV has also been found in other aquatic organisms, Bivalve molluscs are filter feeding organisms, meaning that including dolphins, which present clinical symptoms of HEV they can accumulate and concentrate pathogens from their infection. A study of 31 dolphins at the National Aquarium, environment within their tissues. In the EU, bivalve shell- Cuba, found that 32.2% of their dolphins were seropreva- fish are tested regularly for faecal contamination, using a lent for HEV during two different studies (Villalba et al. faecal indicator,  Escherichia coli, in accordance with 2017). The cause of the infections within the dolphins was food safety regulations. However, studies have shown that unknown; however, it is possible that contamination of food though it functions well as a bacterial faecal indicator, E. items such as fish may be the cause, making an investigation coli  can be a poor indicator of the presence of  faecally of the presence of HEV in such animals important to deter- derived viruses. Lowther et al. (2012) found that norovi- mine whether there is any risk of HEV to humans from the rus RNA was present in 76.2% of total UK oyster samples consumption of fish. It may also be important to investigate Table 8 The presence of HEV in shellfish in different countries Location Study Percentage of shellfish HEV positive China Gao et al. (2015) 17.5% of 126 shellfish  samples of various species from production areas Denmark Krog et al. (2014) 0% of 29 mussel  samples from 19 production areas France Grodzki et al. (2014) 0% of 286 shellfish  samples of various species from two production areas Italy La Rosa et al. (2018) 2.6% of 384 shellfish  samples of various species from production areas Japan Li et al. (2007) 6.3% of 32 Yamato-Shijimi clam  samples Scotland Crossan et al. (2012) 85.4% of 48 individual wild mussels O’Hara et al. (2018) 2.9% of 310 retail shellfish  samples  (mussels and oysters) Spain Mesquita et al. (2016) 14.8% of 81 mussel  samples  from a production area Rivadulla et al. (2019) 24.4% of 164 mussel, clam, and cockle samples Where the study states that samples of shellfish were tested, it was either stated or assumed in each publication that each “sample” would have been formed by ten or more shellfish individuals and is therefore technically a pooled sample 1 3 Food and Environmental Virology (2021) 13:127–145 139 the presence of HEV in aquatic mammals as they are used and other seafood also become contaminated or infected. as a food source in some countries. Considering that shellfish in many countries have been found to be contaminated with HEV, this is perhaps something that warrants further investigation. Conclusion Due to mixed conclusions between and within countries about HEV presence within different hosts or matrices, it In summary, the host range of HEV appears to be diverse, appears that there needs to be not only standardised and having been found within pig, deer, rabbit, cattle, goat and improved methods for the purpose of HEV detection, but camels, amongst other animals. HEV has also been detected also that further research through larger studies around the in shellfish meat as a result of contamination of their grow - world is required to identify the full host range of HEV and ing waters. Therefore, there is a risk of contracting HEV the risk of each potential host to transmit the virus to humans from undercooked products from these animals (although (through food or other means). In particular, the sugges- it is important to note that epidemiological evidence of tion that a HEV-related virus may be causing seropreva- foodborne transmission for many of these is currently lack- lence estimates to be higher than they genuinely are requires ing), and there is also potential for other livestock species investigation. to be unidentified hosts for the virus. Generally, foodstuffs Further studies identifying both the seroprevalence and containing raw meat or shellfish products are more likely the presence of HEV through ELISA and RT-PCR tech- to cause a foodborne infection than cooked foods or crops niques, respectively (or similar techniques identifying RNA due to no thermal inactivation of the virus through cooking. presence), would be best equipped to identify both the preva- Cooking in such a way that a minimum internal temperature lence of the virus within animal populations and the amount of 72 °C is reached for at least 20 min is likely to com- of active infections within the populations at that point in pletely inactivate any HEV present; however, this is likely time. However, sequencing technologies such as nanopore to produce unwanted deterioration of organoleptic qualities RNA sequencing within human and animal populations in some risky food types, e.g. shellfish. would also be useful to identify similarities between HEV In addition to animal meat, milk from cows, sheep, goats, sequences, enabling the identification of infection sources. donkeys and camels has also been found to contain HEV Some studies investigating the evolution of the virus have in some countries, but studies investigating the presence already been performed, but are often biased by the large of HEV in milk are much more limited. Because of this, amount of HEV sequences derived from humans (Forni et al. the true risk of HEV transmission from animal milk is yet 2018). unknown and requires further research. However, if proven Investigations of HEV within food and environmental to be a prominent transmission route for the virus, a wor- matrices using whole genome sequencing approaches have rying consideration is that high-temperature short-time been limited so far due to the general observation of low (HTST) pasteurisation of milk products, which is commonly genome copy numbers and fragmented HEV RNA within used in the UK and USA, may be insuc ffi ient to reduce infec - these matrices. However, techniques utilising methods tious HEV within milk, as generally the heat treatment used such as multiplexing RNA extracted samples to obtain a for HTST pasteurisation is only 72 °C for 15 s. Other pas- full genome from multiple amplicons, followed by MinION teurisation methods, such as ultrahigh-temperature pasteuri- next-generation sequencing, which has been successfully sation, which utilise treatments of around 135 °C for 2–4 s applied to sequencing of low levels of Zika virus (Quick should be more capable of removing viable virus from milk et al. 2017), could be instrumental in future efforts to iden- products due to the higher temperature. tify low levels of HEV in a variety of matrices, including Though crops can also become contaminated with HEV, foods. it seems that the risk of contracting HEV from them is much Globally, HEV is an under-recognised viral threat, which less likely, as confirmed outbreaks from crops have not been causes an increasing case incidence annually. The best way identified, and the HEV RNA prevalence and copies of viral to tackle a virus is to understand its sources and modes of RNA present are lower for these foods. However, it may be transmission. Therefore, further research and better under- safe to conduct further research into the contamination of standing of HEV will allow a better assessment of the risk irrigation water, and the presence of HEV in crops from that animal products and other foods may have in the trans- other countries to better assess the risk of contracting HEV mission of HEV to humans. In turn, this may allow the from crop contamination. introduction of legislative controls to prevent and control It has also been shown that marine mammals can be the spread of the virus. infected with HEV, which is concerning both from an eco- Supplementary Information The online version contains supplemen- system and a seafood point of view. If marine mammals are tary material available at https:// doi.org/10.1007/s12560-02 1-09461-5 . becoming infected naturally, it could be possible that fish 1 3 140 Food and Environmental Virology (2021) 13:127–145 Acknowledgements We would like to thank the anonymous reviewers contaminations are responsible for HEV transmission in pigs. for their help with submission of this article. This study did not gener- Veterinary Research, 44(1), 102. ate any new data. Anheyer-Behmenburg, H. E., Szabo, K., Schotte, U., Binder, A., Klein, G., & Johne, R. (2017). Hepatitis E virus in wild boars and spillo- ver infection in red and roe deer, Germany, 2013–2015. Emerg- Author Contributions ST was responsible for the idea, did the literature ing Infectious Diseases, 23(1), 130. search and data analysis for the writing, as well as drafting and criti- Arankalle, V. A., Chadha, M. S., & Chobe, L. P. (1999). Long-term cally revising the work. 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The Foodborne Transmission of Hepatitis E Virus to Humans

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Copyright © The Author(s) 2021
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1867-0334
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1867-0342
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10.1007/s12560-021-09461-5
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Abstract

Globally, Hepatitis E virus (HEV) causes over 20 million cases worldwide. HEV is an emerging and endemic pathogen within economically developed countries, chiefly resulting from infections with genotype 3 (G3) HEV. G3 HEV is known to be a zoonotic pathogen, with a broad host range. The primary source of HEV within more economically developed coun- tries is considered to be pigs, and consumption of pork products is a significant risk factor and known transmission route for the virus to humans. However, other foods have also been implicated in the transmission of HEV to humans. This review consolidates the information available regarding transmission of HEV and looks to identify gaps where further research is required to better understand how HEV is transmitted to humans through food. Keywords Hepatitis E virus · Foodborne · Zoonotic · Transmission · Meat · Shellfish Introduction is probable that more infections occur worldwide than esti- mated in 2005 (Guillois et al. 2015; Yin et al. 2019), espe- Globally, hepatitis E virus (HEV), of the family Hepeviri- cially considering that this estimate was made only consider- dae, is considered the most common cause of acute viral ing G1 and G2. hepatitis. There were an estimated 20 million infections HEV was once thought only to be endemic to certain eco- worldwide annually in 2005 from genotype 1 (G1) and 2 nomically developing countries within Asia and Africa, but (G2) HEV combined (Rein et al. 2012), and 44,000 recorded research over the past decade has highlighted the emergence fatalities due to the virus in 2015 (World Health Organisa- of HEV within higher income countries. The virus spreads tion 2019). Generally, HEV causes an acute, self-limiting through a faecal-oral route, making it easily transmissible infection which resolves within a few weeks; however, in through faecally contaminated water. Indeed, this is thought some persons (such as the immunocompromised) it can to be the main transmission route within China, where G1 cause chronic infections, fulminant hepatitis (acute liver and genotype 4 (G4) HEV are dominant (Wang et al. 2001). failure) and extrahepatic manifestations (infections in other However, it is also possible for the virus to be transmit- organs), which can be fatal. Table 1 shows a summary of the ted through foodstuffs such as pork, due to the ability for pattern of infection of the different HEV genotypes, dem- some HEV genotypes to infect non-human animals. Cur- onstrating the variable clinical manifestations and factors rently, the virus is classified into eight genotypes, with G1–4 such as average age of infection and gender, where these and genotype 7 (G7) capable of infecting humans. There is, are known. There are limited data to be able to estimate however, a diverse host range for the different genotypes, the number of infections worldwide, but with a high level with G1 and G2 generally only infecting humans and non- of asymptomatic infections seen in numerous outbreaks it human primates, but genotypes 3–8 infecting many other animals, such as pigs, deer, camels, rabbits, and dolphins. A summary of the different host species of HEV was pub- * Samantha Treagus lished recently (Kenney 2019), and is summarised briefly samantha.treagus@cefas.co.uk in Table 2. Genotype 3 (G3) HEV has been found to be the Biosciences, College of Life and Environmental Sciences, most geographically diverse of the viruses thus far (Pérez- University of Exeter, Penryn Campus, Penryn, Cornwall, UK Gracia et al. 2015), and is the genotype which has emerged Seacorp Technologies Limited, Bournemouth, UK in the past 2 decades in many developed countries. The geo- graphical distribution of genotypes 1–4 can be seen in Fig. 1. Centre for Environment Fisheries and Aquaculture Science, Barrack Road, Weymouth, Dorset DT4 8UB, UK Vol.:(0123456789) 1 3 128 Food and Environmental Virology (2021) 13:127–145 1 3 Table 1 Pattern of infection for the different genotypes of HEV, adapted from Centers for Disease Control and Prevention (2020) Genotype Transmis- Transmission routes Geographical distribution Extrahepatic manifestations Age groups at higher risk Gender more commonly Lethality sion in pattern affected humans? a,b a,b c 1 Yes Faecal-oral; waterborne; Economically developed Pancreatic Differs by country Differs by country 0.5–1% ; 20% blood transfusion; organ and developing countries in pregnant d,e,f donation women 2 Yes Faecal-oral; waterborne; Economically developing Unknown Young adults Unknown 0.5–1% blood transfusion; organ countries donation 3 Yes Foodborne; blood transfu- Economically developed Chronic infections in Older adults (> 40 years) Males 0.5–1% sion; organ donation and developing countries immune-compromised patients. Neurologi- cal, haematological, immunological and renal manifestations 4 Yes Foodborne; blood transfu- Economically developed Unknown Young adults Possibly males (limited 0.5–1% sion; organ donation and developing countries data) 5 No Faecal-oral Unknown Unknown Unknown Unknown Unknown 6 No Faecal-oral Unknown Unknown Unknown Unknown Unknown 7 Yes Foodborne; Faecal-oral; Unknown Unknown Unknown Unknown Unknown blood transfusion? 8 No Faecal-oral Unknown Unknown Unknown Unknown Unknown Pathak and Barde (2017) Spina et al. (2017) Peron et al. (2007) Kumar et al. (2017) Jin et al. (2016) Kamar et al. (2014) Horvatits et al. (2019) Mizuo et al. (2005) Food and Environmental Virology (2021) 13:127–145 129 Table 2 Summary of HEV host species by genotype adapted from Kenney (2019) Genotype Hosts identified (common names) Species infected 1 Humans, Chimpanzees, Monkeys, Horses Homo sapiens, Pan troglodytes, Chlorocebus sabaeus , Chlo- a a rocebus pygerythrus , Erythrocebus patas , Macaca mulatta, Macaca radiata, Macaca fascicularis, Semnopithecus entellus, a a a Aotus trivirgatus , Saguinus mystax mystax , Saimiri sciureus , Equus caballus ferus a a 2 Humans, Monkeys Homo sapiens, Chlorocebus pygerythrus , Erythrocebus patas , Macaca mulatta, Macaca fascicularis, Aotus trivirgatus , a a Saguinus mystax mystax , Saimiri sciureus 3 Humans, Monkeys, Hares and Rabbits, Rats, Minks, Mongooses, Homo sapiens, Erythrocebus patas, Macaca mulatta, Macaca Pigs, Goats and Sheep, Deer, Dolphins, Horses, Vultures fascicularis, Macaca fuscata, Lepus europaeus, Oryctolagus cuniculus domesticus, Rattus norvegicus, Neovison vison, Herpestes javanicus, Sus scrofa, Sus scrofa domestica, Capra hircus aegagrus, Ovis aries orientalis, Cervus elaphus, Cervus nippon, Capreolus, Tursiops truncatus, Equus africanus, Equus caballus ferus, Gyps himalayensis 4 Humans, Monkeys, Gerbils, Dogs, Bears, Leopards, Pigs, Cows, Homo sapiens, Macaca fascicularis, Macaca mulatta, Meriones Goats, Deer, Cranes, Pheasants unguiculatus , Canis lupus familiaris, Ursus thibetanus, Neofe- lis nebulosa, Sus scrofa, Sus scrofa domesticus, Bos taurus primigenius, Bos grunniens, Capra hircus aegagrus, Ovis aries orientalis, Cervus nippon, Elaphodus cephalophus, Muntiacus reevesi, Balearica regulorum, Lophura nycthemera 5 Monkeys, Pigs Macaca fascicularis, Sus scrofa 6 Pigs Sus scrofa 7 Humans, Camels Homo sapiens, Camelus dromedarius, 8 Camels Camelus bactrianus Infections instigated through experimental conditions Fig. 1 The geographical distri- bution of HEV genotypes 1–4. This figure shows the genotypes of HEV which are endemic to each country, where enough data were available. For graphs which are compatible with the conditions protanopia, deutera- nopia and achromatopsia please see online resources 1 and 2. Maps created in ArcMap using the World Countries (general- ised) layer package by esri_dm and visualised in GIMP 1 3 130 Food and Environmental Virology (2021) 13:127–145 G3 HEV is thought to be spread primarily through the directly been linked to pork product consumption, including consumption of undercooked pork products from infected an outbreak in Spain linked to consumption of wild boar pigs; however, it is unknown if all transmission routes to (Rivero-Juarez et al. 2017), and another outbreak associated humans have been identified. The current theories and with consumption of a spit-roasted piglet in France (Guillois known routes of transmission can be seen in Fig. 2. This et al. 2015). review will discuss theoretical routes of HEV transmis- Consumption of pork products is now considered a sig- sion to humans through foodstuffs and identify areas which nificant risk factor for developing HEV infection, which is require further research for better understanding of the virus. concerning considering the seroprevalence levels in Euro- pean pigs (Said et al. 2014; Slot et al. 2017). Table 3 shows a non-exhaustive list of countries that have detected anti-HEV HEV in Pigs and Pork Products antibodies in pigs, and HEV RNA in pork products. Considering Table 3, it is possible that the HEV preva- Over the past 2 decades, evidence has accumulated impli- lence in pork sausages may be over- or under-estimated by cating pigs and other animals in the zoonotic transmission the fact that the sample sizes for some of these studies are of G3 HEV to humans (Tei et al. 2003; Guillois et al. 2015; relatively small. It is also important to note that different Lhomme et al. 2013; Rivero-Juarez et al. 2017). In 1998, it methods have been used between studies, some of which was shown that a HEV strain isolated from an acute HEV have been shown to be less sensitive for detecting HEV than patient in the USA was capable of infecting pigs, and that others. Interestingly, the seroprevalence of HEV in pigs is a genetically similar strain isolated from pigs was capable significantly higher than the prevalence of HEV RNA in of infecting non-human primates, suggesting a significant pork products in most cases; this discrepancy is expected as possibility that pigs could act as a zoonotic source of HEV not all pigs would be viraemic at slaughter. (Meng et al. 1998). Since this study, many countries have It has been found that HEV generally infects swine noted the emergence of HEV cases. At this stage, it was asymptomatically at an early point in their life (prior to thought that HEV was only endemic in developing countries 6 months of age (de Oya et al. 2011)), and that because (such as those in Asia, Africa and Central America), and of this, many pigs are seropositive at the time of slaughter that HEV cases in non-endemic areas were obtained through (Grierson et al. 2015; Rose et al. 2011; de Oya et al. 2011). travel. However, studies such as those by Dalton et  al. The slaughter age of pigs is normally slightly before 1 year (2007) and Fraga et al. (2017) identified that indigenous of age. The transmission between pigs is suspected to be cases of HEV were occurring in economically developed through a faecal-oral route and this is likely to be due to the countries such as the UK and Switzerland. It is now widely high shedding that is seen in pig faeces and urine (Bouw- accepted that pigs are a zoonotic source of HEV transmis- knegt et al. 2009; Halbur et al. 2001). Infection with HEV sion to humans and may be at least partially responsible early in life means that there is a lower chance of the pigs for increasing cases worldwide annually, though increased being viraemic at slaughter, and they are therefore less likely detection and awareness of HEV may also play a small role to be capable of HEV transmission to humans through the in the observed increase in cases. Outbreaks of HEV have pork food chain. However, whether HEV causes life-long Fig. 2 Theoretical and con- firmed transmission routes of HEV. The figure shows confirmed and theoretical routes of HEV transmission to humans. The theoretical routes of transmission include HEV infections contracted from the consumption of shellfish, sheep, and cows, as well as crops and drinking water, as no confirmed outbreaks from these sources have yet been identified. Illustration created using Adobe Illustrator and edited using GIMP 1 3 Food and Environmental Virology (2021) 13:127–145 131 Table 3 A list of the seroprevalence levels of anti-HEV antibodies in pigs and the percentage of pork products found to be HEV positive by RT- PCR in those locations Location Pig sample population anti-HEV antibody seroprevalence Percentage of tested foodstuffs HEV positive by RT-PCR Brazil 63.6% of 357 pigs (Vitral et al. 2005) 1.7% of 118 slaughterhouse livers (Gardinali et al. 2012) Canada 59.4% of 998 pigs (Yoo et al. 2001) 8.8% of 283 livers, 1.0% of 599 pork chops (Wilhelm et al. 2014) 47.0% of 76 pork pâtés and 10.5% of 19 retail raw pork livers (Mykytczuk et al. 2017) France 31.0% of 6565 pigs (Rose et al. 2011); 60% of 1034 pigs 4.0% of 3715 slaughterhouse livers (Rose et al. 2011) (Feurer et al. 2018) 2.8% of 1034 slaughterhouse livers (Feurer et al. 2018) 30.0% of 140 figatelli and fitone, 29.0% of 169 liver sausages, 25.0% of 55 quenelles or quenelle paste, 3.0% of 30 dried salted livers (Pavio et al. 2014) 58.3% of 12 raw liver sausage (Colson et al. 2010) Germany 49.8% of 1072 pigs (Baechlein et al. 2010) 4.0% of 200 retail livers (Wenzel et al. 2011) 20.0% of 70 raw sausages and 22.0% of 50 liver sausages (Szabo et al. 2015) Italy 45.1% of 2700 pigs (Mughini-Gras et al. 2017) 20.8% of 48 slaughterhouse livers (Di Bartolo et al. 2011) 6.0% of 33 slaughterhouse livers (Di Bartolo et al. 2012) 13.3% of 15 fresh liver sausages, 7.1% of 14 dried liver sausages (Di Bartolo et al. 2015) Japan 57.9% of 2500 pigs (Takahashi et al. 2003) 1.9% of 363 retail livers (Yazaki et al. 2003) Netherlands 89.0% of 417 organic pigs, 72% of 265 conventionally farmed 6.5% of 62 commercial pork livers (Bouwknegt et al. 2007) pigs, 76% of 164 free range pigs (Rutjes et al. 2014) 12.7% of 79 livers, 70.7% of 99 liver sausages, 68.9% of 90 liver pâté samples (Boxman et al. 2019) Spain 20.4% of 1441 pigs (de Oya et al. 2011) 6.0% of 93 sausages, 3.0% of 39 slaughterhouse livers (Di Bar- tolo et al. 2012) Switzerland 62.3% of 1001 pigs in 2006, 53.8% of 999 pigs in 2011 (Burri 1.3% of 160 slaughterhouse livers (Müller et al. 2017) et al. 2014) 11.8% of 102 raw liver sausages (Giannini et al. 2017) 11.7% of 90 pork liver and raw meat sausages (Moor et al. 2018) UK 92.8% of 629 pigs, with 20.5% viraemic at slaughter (Grierson 10.0% of 63 sausages, 3.0% of 40 slaughterhouse livers (Berto et al. 2015) et al. 2012) USA 21.9% of 182 pigs (Owolodun et al. 2013) 11.0% of 127 retail liver (Feagins et al. 2007) immunity in swine after recovery has been open to debate. strain from the same genotype could also not confer protec- In rhesus macaques and humans, the anti-HEV IgG anti- tive immunity (Huang et al. 2008). If there is genetic or bodies (characteristic of long-term immunity) wane over a environmental variation in host development of immunity to variable number of years until they are undetectable, and the HEV, and some strains of HEV may not provide protection period for which individuals may be IgG positive for varies against others, then farms with multiple circulating HEV (Arankalle et al. 1999; Lee et al. 1994). This may not be an strains could be more likely to have viraemic pigs at the time issue in pigs as they are normally slaughtered before a year of slaughter. This would therefore mean that there could be of age, so early life infections would likely allow immunity a higher likelihood of contracting HEV from undercooked against subsequent HEV challenge over their lifetime. How- pork products or food products containing raw pig compo- ever, it has been shown that animals can be re-infected by nents from these sources. different HEV strains (De Deus et al. 2008), and whether or It has been reported that 21% of pigs in the UK tested not one strain can confer protective immunity to all other positive for HEV RNA at the time of slaughter (Grierson HEV strains has also been contested. It has been shown that et al. 2015), and that in the USA 6.3% of pigs from slaugh- after infection with one strain of G3 HEV, pigs developed terhouses were HEV RNA positive (Sooryanarain et  al. some protective immunity against other strains within the 2020). As such, it is probable that the consumption of raw same genotype, and also within G4 (Sanford et al. 2011). and undercooked pork products is acting as a transmission However, in rhesus monkeys, it was shown that infection route of HEV to humans. with one strain from a genotype could not confer protective Table  4 summarises studies that have investigated the immunity to a strain in a different genotype upon subsequent thermal inactivation of HEV in non-food matrix samples. challenge, and that in some cases, infection with a different Inactivation condition combinations found sufficient to 1 3 132 Food and Environmental Virology (2021) 13:127–145 Table 4 A summary of studies investigating thermal inactivation treatments for HEV in non-food matrix samples Study Cell culture or molecular Genotype Heat treatment Growth period Inactivation/reduction detection? Tem- Time (min) perature (°C) Emerson et al. (2005) Cell culture, HepG2/C3A 1 (strain Akluj ) 56 60 5–6 days > 80% reduction cells 1 (strain SAR55 ) 56 60 5–6 days ~ 50% reduction 2 (strain Mex14 ) 60 60 5–6 days 96% reduction Huang et al. (1999) Cell culture, A549 cells 3 (strains G93-1*, 56 30 72 h < 1.0 (TCID50/0.025 ml) G93-2*, G93-3*, G93-4*) Johne et al. (2016) Cell culture, A549/D3 cells 3 (strain 47832c ) 55 1 35 days ~ 1 log reduction in focus forming units 70 2 “No infectious virus” Schielke et al. (2011) Molecular detection 3 (strain wbGER27 ) 56 15 N/A 74.07% reduction 3 (strain wbGER27 ) 56 60 N/A 99.90% reduction Tanaka et al. (2007) Cell culture, PLC/PRF/5 3 (strain JE03-1760F ) 70 10 35 days “No infectious virus” cells 3 (strain JE03-1760F ) 56 30 50 days “Still infectious” *Accession numbers unknown AF107909 M80581.1 KX578717.1 AB437319.1 FJ705359.1 KC618403.1 inactivate virus in these studies are shown in Fig. 3. Results by Huang et al. (1999), a temperature of 56 °C for 30 min vary between the different studies, and several factors make was reported to completely inactivate the virus; however, the comparison difficult. The studies use a variety of different virus was only left to grow for a relatively short period of units or expressions of reduction/inactivation. In the study time (72 h). However, it was shown that HEV was still viable Fig. 3 A summary of the reported thermal inactivation requirements for HEV from different studies. This graph summarises the observed HEV inactivation requirements for five different studies investigat- ing the effect of heat treatment over time on HEV viability, with the highest reported inacti- vation requirements being 70 °C for 10 min, and the lowest being 56 °C for 30 min. Graph created in R studio 1 3 Food and Environmental Virology (2021) 13:127–145 133 following similar heat treatments in cell culture studies with studies have all taken different approaches to heat inactiva- longer growth periods (Emerson et al. 2005; Tanaka et al. tion of the virus and the food types used, and therefore it is 2007). Schielke et al. (2011) used RNase treatment in an difficult to compare the results and form a definitive answer attempt to remove viral RNA that had broken from the cap- for the heat treatment required to inactivate the virus within sid after heat treatment, assuming this would remove RNA foodstuffs. Feagins et al. (2007) identified that boiling or from non-viable virus. However, it is unknown if any RNA stir-frying infected pig liver to an internal temperature of from viable virus could have been lost during this treatment, 71 °C for 5 min could prevent infection when the liver was and the standard deviations seen within the results were rela- then fed to pigs. However, though the pigs were not infected tively large. Without a cell culture component, it is not possi- by this oral dose, it is known that pigs commonly require a ble to say with 100% certainty that remaining detected RNA high dose of HEV to become infected through the faecal- was from viable virus. In addition to these limitations, it is oral route (Kasorndorkbua et al. 2004), of approximately known that HEV is difficult to culture effectively in vitro, 10 genome copies (Andraud et al. 2013), and therefore it is often requiring large titres of virus to begin the culture, and possible that lower doses of active virus were still present therefore it is possible that the inactivation requirements for in these food stuffs. The oral infectious dose for humans is HEV have been under-estimated as treatments sufficient to unknown. Intravenous inoculation of pigs with the cooked eliminate infectivity in vitro may not completely eliminate foodstuffs as carried out by Barnaud et al. (2012) is likely to in vivo infectivity. Some researchers have investigated dif- have provided a more accurate estimate of whether viable ferent cell lines and strains of HEV which appear to be more virus still existed, especially as intravenous inoculation of efficiently cultured in vitro due to insertions within the HEV pigs has been reported to require much lower HEV doses to genome; however, culturing these strains still requires large cause infection (Dähnert et al. 2018). Imagawa et al. (2018) titres of virus to begin the culturing process (10 copies/ml) reported similar inactivation requirements to Feagins et al. (Johne et al. 2014; Schemmerer et al. 2016). (2007). However, the limit of detection for the cell culture 4 5 It is possible that interactions of HEV with organic mol- system was 10 –10 genome copies, and therefore, as with ecules in food matrices may cause the thermal inactivation the previous study, viable virus remaining in the minced temperature to be higher. Some studies have investigated pork may not have been detected in the cell culture system. the thermal inactivation of HEV within food stuffs, such In addition, the different food preparations between the stud- as liver and pork sausage (summarised in Table 5). These ies may have influenced viral stability, as could the different Table 5 A summary of studies investigating thermal inactivation treatments for HEV in foodstuffs Study Food stuff Cooking method Temperature (°C) Time (min) Measurement of inactiva- HEV tion inacti- vated? Barnaud et al. (2012) Pâté preparation (spiked Water bath 62 5 Intravenous administration No with 10 HEV genome to pigs 20 No copies) 120 No 68 5 No 10 No 20 No 71 5 No 10 No 20 Yes Feagins et al. (2008) Pig liver (naturally infected) Incubation 56 60 Oral administration to pigs No Boiling ≥ 71 (internal) 5 Yes Stir fry ≥ 71 (internal) 5 Yes Imagawa et al. (2018) Minced meat (spiked with Boiling or roasting 63 1 Cell culture No 10 HEV genome copies) 5 No 30 Yes 65 1 No 5 Yes 70 1 No 5 Yes 1 3 134 Food and Environmental Virology (2021) 13:127–145 strains of G3 HEV used. The initial viral titres used could raw Sika deer meat contracted HEV 6–7 weeks later (typi- also have influenced the results and explain why a longer cal of the HEV incubation period), with the HEV sequence treatment time was needed in some studies. confirmed as 100% identical between the meat and infected Further research is clearly required to investigate HEV patients (Tei et al. 2003). With the presence of HEV in inactivation within foods. This may require a more effi- deer being so variable between studies (perhaps due to cient cell culturing method and an assessment of different study limitations such as sample size), further research foods, cooking methods and HEV strains. However, taking is required to identify the level of active HEV infection the results of studies conducted in non-food and food (pork within deer populations through larger prevalence studies. product) samples together, a conservative measure would be However, with the number of countries that have detected to cook pork products for longer than 20 min at temperatures HEV in deer, and the occurrence of foodborne outbreaks higher than 72 °C. from deer meat, deer could be acting as another reservoir In addition to the potential for HEV to survive some for HEV. cooking processes, raw pork products are used in some There have been reports of HEV infections in cattle (Bos consumables. Raw blood products are commonly used in taurus) from China, where both antibody seroprevalence and ready-cooked foods such as processed ham (fibrinogen) HEV RNA of G4 have been identified in multiple studies. and other blood proteins are used as food additives such as Hu and Ma (2010) showed the presence of G4 HEV RNA in emulsifiers. Spray-dried plasma powder (SDPP) is also used 8.8% of cattle from Xinjiang Autonomous Region. A subse- in domestic and farm animal foods but this has some heat quent study then identified that 37.1% of tested dairy cows in processing prior to use. SDPP is commonly fed to weaned Yunnan Province were HEV RNA positive, and that 100% of piglets; however, a previous study reported no transmission the HEV-positive cows were producing milk that contained of HEV in pigs fed spray-dried plasma products that were HEV RNA (Huang et al. 2016). A further study in Shandong positive for HEV RNA (Pujols et al. 2014), and therefore the Province also found 3% of yellow cattle to be G4 HEV RNA heating of spray-dried plasma may be sufficient to inactivate positive, with 47% seroprevalent for anti-HEV antibodies the virus present. However, as other porcine products are (Yan et al. 2016). A study in Turkey identified HEV from not subjected to heat processes before they are used, they G1, G3 and G4 in 20.3% of raw milk samples from various could constitute a transmission risk to humans through use domestic animals (cows, sheep, goats, donkeys) (Demirci in food. A study conducted in 2017 found that of 36 liquid et al. 2019). Other studies investigating HEV in cattle have porcine products derived from blood, 33 were positive for produced negative or mixed results; a study in Beijing, China HEV RNA, and seven of 24 spray-dried plasma products identified 29.4% of cattle were seroprevalent for HEV, but no were also positive for HEV RNA (Boxman et al. 2017). This HEV RNA could be detected (Chang et al. 2009). A study in is especially significant when blood products from multiple Burkina Faso also found 26.4% of 72 cattle to be seropreva- animals are commonly pooled together, meaning that prod- lent for HEV (Ouoba et al. 2019). Another study in Ger- ucts from one viraemic animal could contaminate a batch many testing 400 milk samples found no evidence of HEV and lead to widespread HEV transmission through many RNA, although G4 HEV is much less commonly reported in different food products. Europe than in Asia (Baechlein and Becher 2017). Likewise, Pork product consumption has been considered to be a a study in Belgium also found no evidence of HEV RNA major risk factor in the development of HEV due to the con- in cow milk or faeces (Vercouter et al. 2018). In the USA, nection to foodborne outbreaks and the fact that HEV in Yugo et al. (2019) identified that of 983 cows, 20.4% were pork products can reach high levels (e.g. 7 × 10 genome seroprevalent for anti-HEV antibodies; however, HEV RNA copies/g in liver pâté in the Netherlands, Boxman et  al. could not be detected in any of the cows. The authors con- 2019). However, pigs are not the only animals consumed cluded that this may have been because of an antigenically which can act as reservoirs for the virus. similar relative to HEV rather than due to HEV itself, which could be possible as G4 HEV is not thought to be endemic to the USA; however, this would call into question the HEV in Other Land Animals and Animal specificity of ELISA assays and studies investigating HEV Products seroprevalence. Notwithstanding the significant number of studies with negative findings, these results are concerning In addition to pigs, deer have been reported to be infected as meat and dairy from cows are consumed worldwide by with HEV in many different countries (summarised in humans, and the possibility that cows could be a HEV reser- Table 6). It is important to identify the transmission risk voir could have a significant impact on our understanding of that deer may have to humans, as HEV outbreaks have HEV transmission to humans. More research worldwide is been directly linked to the consumption of raw deer meat. therefore needed to identify which HEV genotypes are capa- For example, in Japan, multiple people who had consumed ble of infecting cattle, and to find the prevalence of HEV in 1 3 Food and Environmental Virology (2021) 13:127–145 135 Table 6 Summary of studies investigating the prevalence of HEV in deer Study Location Deer species ELISA observed RT-PCR seroprevalence preva- lence Weger et al. (2017) Canada Odocoileus virginianus (White-tailed deer) 8.8% ND Odocoileus hemionus (Mule deer) 4.5% ND Rangifer tarandus groenlandicus (Barren- 1.7% ND ground caribou) Rangifer tarandus (Woodland caribou) 5.2% ND Zhang et al. (2015) China Cervus nippon (Sika deer) 5.4% ND Anheyer-Behmenburg et al. (2017) Germany Capreolus (Roe deer) 0.0% 6.4% Cervus elaphus (Red deer) 0.0% 3.5% Neumann et al. (2016) Cervus elaphus (Red deer) 2.5% 3.7% Capreolus (Roe deer) 6.5% 0.0% Reuter et al. (2009) Hungary Capreolus (Roe deer) ND 12.2% Di Bartolo et al. (2017) Italy Cervus elaphus (Red deer) 13.6% 11.0% Sonoda et al. (2004) Japan Cervus nippon (Sika deer) 2.0% 0.0% Matsuura et al. (2007) Cervus nippon (Sika deer) 2.6% 0.0% Tomiyama et al. (2009) Cervus nippon yesoensis (Yezo deer) 34.8% ND Spancerniene et al. (2018) Lithuania Capreolus (Roe deer) ND 22.6% Cervus elaphus (Red deer) ND 6.7% Medrano et al. (2012) Mexico Odocoileus virginianus (White-tailed deer) 62.7% ND Rutjes et al. (2010) The Netherlands Cervus elaphus (Red deer) 8.0% 15.0% Capreolus (Roe deer) 12.5% 0.0% Boadella et al. (2010) Spain Cervus elaphus (Red deer) 10.4% N/A Kukielka et al. (2016) Cervus elaphus (Red deer) 12.9% 11.1% Capreolus (Roe deer) 7.0% 0.0% Roth et al. (2016) Sweden Cervus elaphus (Red deer) 7.0% 0.0% ND not done Did not test full sample population which were tested for seropositivity cattle and dairy products. This will help to identify the risk antibodies (Ouoba et al. 2019). In the USA however, Sanford of transmission of HEV from cattle to humans. et al. (2013) suggested that a HEV-related agent was caus- Goats have also been shown to be potential reservoirs ing HEV in goats after discovering a seroprevalence of 16% for HEV infection, which is important due to goat meat, but a lack of any HEV RNA; however, this again calls into milk and cheese production. In Italy in 2016, 9.2% of goat question the accuracy of HEV ELISAs. In addition, no HEV faecal samples from six farms were found to be positive RNA was detected by conventional RT-PCR in goats that for HEV RNA, belonging to G3 strains which were highly had been experimentally infected with three different HEV related to strains found in pigs and humans (Di Martino et al. strains from G1, G3 and G4 in this study; however, the sen- 2016). Also, in Yunnan province, China, Long et al. (2017) sitivity of this PCR may be lower than previously reported found 70.3% of goat faecal samples to be positive for HEV HEV PCR assays, as assays which target a larger amplicon RNA, with milk samples from these animals also positive are generally observed to have lower sensitivity (Debode for HEV RNA. The strains obtained from these animals were et al. 2017), and qRT-PCR has generally been observed to from G4 HEV, and the Huang et al. (2016) and Long et al. be more sensitive for amplicon detection of HEV and other (2017) studies highlight that farms with mixed animals may amplicon targets (Zhao et al. 2007; Zemtsova et al. 2015). demonstrate a higher risk of HEV transmission. Another Dromedary camels have been implicated in the transmis- study in the Tai’an Region in China identified 4% of goat sion of G7 HEV to humans. In one case, a patient who regu- livers to be HEV positive, with G4 HEV that was similar to larly consumed camel meat and milk contracted chronic G7 cow HEV detected in the same region (Li et al. 2017). In HEV after a liver transplant (Lee et al. 2016). This chronicity Turkey, 18.5% of goat milk samples were reported positive is likely to have been opportunistic and influenced by immu- for HEV RNA (Demirci et al. 2019). Meanwhile in Bur- nosuppressive medication to prevent organ rejection. In a kina Faso, 28.4% of 81 goats were found to have anti-HEV separate paper, HEV was demonstrated to be seroprevalent 1 3 136 Food and Environmental Virology (2021) 13:127–145 in 23.1% of dromedary camels which originated in Sudan though rabbits are capable of carrying sub-genotype 3ra, and Saudi Arabia (El-Kafrawy et al. 2020). Due to the recent attempts to cause infections with another sub-genotype (3b) discovery of this genotype of HEV, and its implication in were unsuccessful; however, Hammerschmidt et al. (2017) human infection, further research is warranted to investi- identified a wild rabbit with HEV sub-genotype 3 g. Fur - gate how widespread camel HEV is within countries which ther research is therefore needed to identify which genotypes regularly consume camel products to determine the risk such or sub-genotypes of HEV are capable of infecting rabbits. products may have for the foodborne transmission of HEV. An interesting observation from the studies summarised in Rabbits and related species, e.g. hares, are also gaining Table 7 is that concordance in HEV prevalence between dif- increasing attention for their potential to transfer HEV to ferent samples from the same animals is often lacking. For humans through consumption of meat. In France, five cases example, Burt et al. (2016) found that 60% of liver samples of rabbit HEV (defined within Orthohepevirus A, G3ra) from 32 animals were HEV positive; however, only 16% of were identified in confirmed HEV-positive patients out of these 32 animals were faecally shedding the virus. It may 919 from 2015 to 2016 (Abravanel et  al. 2017). Several therefore be wise to identify standardised testing methods countries have identified rabbits to be seroprevalent and worldwide for identification of infected animals, with a deci- RNA positive for HEV (Table 7). The number of observa- sion made on what samples to test and which assays are best tions and the apparent ability of humans to contract rabbit to use, to avoid underestimating HEV prevalence. HEV suggest that it is a source of zoonotic HEV transmis- Despite identifying that animal product consumption is sion. However, with most human cases worldwide belonging a risk factor in the transmission of HEV, it is quite possi- to other genotypes and sub-genotypes, rabbits are likely to ble that there are other food transmission routes. One study be the cause of only a minority of cases. Studies have shown in the Netherlands showed that though seroprevalence of mixed results in terms of the ability of rabbits to carry other HEV antibodies was higher in meat-eaters (22.8%), vegetar- sub-genotypes of HEV. Zhang et al. (2017) have shown that ians still displayed a relatively high seroprevalence (13.8%) Table 7 A summary of studies identifying HEV (genotype 3ra) in rabbits and hares Country Study Seroprevalence RNA prevalence Burkina Faso Ouoba et al. (2019) 60.0% of 100 rabbits, 52.6% of 19 hares ND Canada Xie et al. (2017) ND 5.0% of 63 companion rabbit faecal samples, 0.90% of 114 commercial rabbit faecal samples China Geng et al. (2011a) 54.6% of 119 farmed rabbits 7.0% of 119 farmed rabbit serum samples Geng et al. (2011b) 15.4% of 1094 farmed rabbits 2.0% of 1094 farmed rabbit serum samples Xia et al. (2015) ND 5.0% of 492 rabbit faecal samples Li et al. (2020a) ND 15.0% of 120 rabbit faecal samples Li et al. (2020b) 7.1% of 70 farmed rabbits 11.4% of 70 farmed rabbit faecal samples France Izopet et al. (2012) ND 7.0% of 200 farmed rabbit bile samples, 23.0% of 205 wild rabbit liver samples Germany Eiden et al. (2016) 30.8% of 13 wild rabbits 30.8% of 13 wild rabbit serum samples Hammerschmidt et al. (2017) 37.3% of 164 wild rabbits, 2.2% of 669 wild 17.1% of wild rabbit serum samples, 0.0% of hares wild hare serum samples Ryll et al. (2018) 25% of 72 wild rabbits 34.7% of 72 wild rabbit liver samples Corman et al. (2019) 0.04% of 2389 wild hares 2.6% of 2389 wild hare serum samples Italy Di Bartolo et al. (2016) 3.4% of 206 farmed rabbits, 6.6% of 122 pet 0.0% of 7 IgG positive farmed rabbit serum rabbits samples, 0.0% of 122 pet rabbit serum samples The Netherlands Burt et al. (2016) ND 23.0% of 35 petting farm rabbit faecal samples, 0% of 10 farmed rabbit liver and faecal sam- ples, 60.0% of 32 wild rabbit liver samples and 16% of wild rabbit faecal samples Poland Bigoraj et al. (2020) 6.0% of 482 farmed rabbits 14.9% of 482 farmed rabbit liver samples South Korea Ahn et al. (2017) ND 6.4% of 264 rabbit faecal samples USA Cossaboom et al. (2011) 36.5% of 85 rabbits 16.5% of 85 serum samples, 15.3% of 85 faecal samples ND not done 1 3 Food and Environmental Virology (2021) 13:127–145 137 (Slot et al. 2017). This suggests that either they became could also therefore be contaminating water courses with HEV positive before becoming vegetarian through animal HEV, which is supported by studies in Italy, the Philippines meat, or they were infected through other transmission and Cambodia, showing river water contamination with HEV routes. Figure 2 shows the known and theorised routes of (Iaconelli et al. 2015; Li et al. 2014; Baez et al. 2017; Rodri- HEV transmission. One transmission route, which is much guez-Manzano et al. 2010). more tightly controlled now, was the transmission of HEV Countries within the European Union must conform to through blood transfusion (Hewitt et al. 2014), which may be EU regulations on how farm manure, animal carcasses and one way to explain the seroprevalence levels in vegetarians. digestive tract content are processed, transported, stored, Another explanation could be consumption of dairy products used as crop fertiliser, and disposed of. The sewage and such as milk. It is also possible to contract HEV through wastewater that originates on farms must either be dis- organ transplant with an infected organ (Pas et al. 2012), charged to public sewers or treated in a sewage treatment and organ transplant-associated cases commonly result in plant on the farm before the effluent can be discharged to chronic infections due to immunosuppression medication. surface waters, and a permit is required for the processing and disposal of sewage and wastewater in this way. However, it is possible for farms within EU countries to use sewage Contamination of the Aquatic Environment and slurry that has been produced on a farm to be spread on crops at the same farm, without prior processing, for the sake In addition to medical routes of transmission, it is important of fertiliser or soil quality improvement (European Com- to consider the impact that HEV within animal farm run-off mission 2001). Accordingly, the United States also allows and sewage has on the aquatic environment. It has been sug- manure originating on one farm to be spread on crops from gested that human and farm sewage may have a part to play in that farm (Environment Protection Agency 2020). However, other HEV transmission routes, potentially through farm run- manure use and farm practices are likely to be more diverse off from animal slurry stores or application of animal slurry and potentially problematic in countries within Asia, Africa to crops and through contamination of surface waters used for and South America. irrigation and shellfish farms. Raw human sewage collected at Previous studies have shown that sewage treatment pro- 2-week intervals in 2014–2015 from a sewer which serves the cesses such as long-term fermentation and composting are whole of Edinburgh was found to contain HEV in 93% of the likely to be capable of removing HEV from sewage (García samples collected (Smith et al. 2016), and many other coun- et al. 2014). A study in Switzerland also identified HEV- tries have also detected HEV in human sewage influent, such positive influent samples from wastewater treatment plants, as Spain, Switzerland, Portugal and France (Clemente-Casares but no HEV-positive effluent samples, suggesting effective et al. 2003, 2009; Rodriguez-Manzano et al. 2010; Matos et al. wastewater treatment using a cleaning and activated sludge 2018; Masclaux et al. 2013). This could therefore mean that process (Masclaux et al. 2013). However, Loisy-Hamon and when storm overflows discharge into water courses such as Leturnier (2015) detected HEV in treated pig sewage sam- rivers and seas, HEV contamination can occur. Because there ples from France that had been treated using one of saw- are many different types of wastewater treatment practices, and dust composting, slurry dehydration or anaerobic digestion. many combinations of practices between wastewater treatment Other studies have found that river water close to pig farms plants, it is difficult to know which wastewater treatment plants and pig processing plants had been contaminated with HEV, will be more effective at removing viruses from wastewater. for example, in Scotland and Italy (Crossan et al. 2012; Idolo However, other viruses such as adenovirus and norovirus are et al. 2013; Marcheggiani et al. 2015). Therefore, it is pos- commonly found in treated sewage (Bofill-Mas et al. 2006; sible that leachate (liquid leaching from solids into the envi- Campos et al. 2016). It is therefore possible that in addition to ronment) from stored manure and yard run-off from farms storm overflows, inadequate treatment of sewage could result and abattoirs may be polluting surface waters such as rivers. in HEV pollution of the aquatic environment, especially con- However, it is unknown whether all of the virus leaching into sidering that HEV is a single-stranded RNA non-enveloped the environment is viable—for example, viral RNA detected virus like norovirus. Pig farm and slaughterhouse sewage has in treated sewage may not indicate viable virus, but remain- also been found to be positive for HEV in multiple countries, ing RNA. for example, HEV RNA was detected in sewage from one of twelve slaughterhouses in Spain (Pina et al. 2000), whilst 75% of swine slurry samples collected from Italian pig farms Crop Contamination were HEV positive (La Rosa et al. 2017), and both fresh swine faecal material and pooled stored slurry from pig farms in Surface waters from sources such as rivers and groundwater the USA were shown to contain HEV (Kasorndorkbua et al. are commonly used as crop irrigation sources throughout the 2005). Human sewage, pig farm run-off and abattoir outflows world (Food and Agriculture Organisation 2011, 2016). Due 1 3 138 Food and Environmental Virology (2021) 13:127–145 to the potential contamination of such water with HEV and from commercial harvesting areas, with 73.9% of those sam- other pathogens from faecal matter (whether from human or ples giving E. coli results compliant with the end product animal sources), this could cause contamination of irrigated standard of ≤ 230 E. coli/100 g shellfish flesh. Norovirus crops. Animal waste (that can potentially be contaminated within oysters is linked to human faecal pollution that has with HEV as shown above) is also used as crop fertiliser originated from storm overflows and CSOs, or sewage that for farms. A small number of studies have found some evi- has received insufficient treatment (Campos et  al. 2013, dence of crop contamination with HEV. In France, two out 2016). CSOs release untreated sewage into surface water to of 230 herb and spice samples were positive for HEV RNA prevent overflows within mains drainage, but outfall events (Loisy-Hamon and Leturnier 2015), a study testing 125 let- can last for several hours or days and are often poorly moni- tuce samples from Greece, Serbia and Poland detected four tored (Marine Conservation Society 2011). Considering that positive samples (Kokkinos et al. 2012), and in Italy, six of farm or abattoir run-off, combined sewer overflows, and 911 “pre-washed and ready to eat” vegetable samples tested inadequately treated sewage could be polluting watercourses positive for HEV RNA (Terio et al. 2017). Another study in with HEV, it is also possible for aquatic organisms, such as four European countries (Czech Republic, Finland, Poland shellsh fi , to be affected by HEV contamination. Indeed, stud - and Serbia) also detected HEV RNA in one frozen raspberry ies around the world have found HEV to be present within sample of 38 tested (Maunula et al. 2013). However, it is bivalve shellfish, and these are summarised in Table  8. The important to note that no foodborne outbreaks of HEV from study by Rivadulla et al. (2019) also showed shellfish to have contaminated crops have been reported, and the quantities of as much as 1.1 × 10  RNA copies per gram of shellfish tis- virus found on the crops is also low enough to call into ques- sue, which is close to the pig ID50, but the human infectious tion whether they would cause illness when consumed. It is dose is still unknown. It is important to note, however, that also unknown whether the HEV RNA detected originated not all RNA found in the shellfish may have been associ- from viable virus. ated with viable virus. To date, there have been no proven foodborne outbreaks of HEV from shellfish consumption, although an outbreak of HEV on a cruise ship was theorised HEV in Bivalve Shellfish and Other Aquatic to have been caused by consumption of shellfish on the basis Animals of a retrospective risk analysis (Said et al. 2009). HEV has also been found in other aquatic organisms, Bivalve molluscs are filter feeding organisms, meaning that including dolphins, which present clinical symptoms of HEV they can accumulate and concentrate pathogens from their infection. A study of 31 dolphins at the National Aquarium, environment within their tissues. In the EU, bivalve shell- Cuba, found that 32.2% of their dolphins were seropreva- fish are tested regularly for faecal contamination, using a lent for HEV during two different studies (Villalba et al. faecal indicator,  Escherichia coli, in accordance with 2017). The cause of the infections within the dolphins was food safety regulations. However, studies have shown that unknown; however, it is possible that contamination of food though it functions well as a bacterial faecal indicator, E. items such as fish may be the cause, making an investigation coli  can be a poor indicator of the presence of  faecally of the presence of HEV in such animals important to deter- derived viruses. Lowther et al. (2012) found that norovi- mine whether there is any risk of HEV to humans from the rus RNA was present in 76.2% of total UK oyster samples consumption of fish. It may also be important to investigate Table 8 The presence of HEV in shellfish in different countries Location Study Percentage of shellfish HEV positive China Gao et al. (2015) 17.5% of 126 shellfish  samples of various species from production areas Denmark Krog et al. (2014) 0% of 29 mussel  samples from 19 production areas France Grodzki et al. (2014) 0% of 286 shellfish  samples of various species from two production areas Italy La Rosa et al. (2018) 2.6% of 384 shellfish  samples of various species from production areas Japan Li et al. (2007) 6.3% of 32 Yamato-Shijimi clam  samples Scotland Crossan et al. (2012) 85.4% of 48 individual wild mussels O’Hara et al. (2018) 2.9% of 310 retail shellfish  samples  (mussels and oysters) Spain Mesquita et al. (2016) 14.8% of 81 mussel  samples  from a production area Rivadulla et al. (2019) 24.4% of 164 mussel, clam, and cockle samples Where the study states that samples of shellfish were tested, it was either stated or assumed in each publication that each “sample” would have been formed by ten or more shellfish individuals and is therefore technically a pooled sample 1 3 Food and Environmental Virology (2021) 13:127–145 139 the presence of HEV in aquatic mammals as they are used and other seafood also become contaminated or infected. as a food source in some countries. Considering that shellfish in many countries have been found to be contaminated with HEV, this is perhaps something that warrants further investigation. Conclusion Due to mixed conclusions between and within countries about HEV presence within different hosts or matrices, it In summary, the host range of HEV appears to be diverse, appears that there needs to be not only standardised and having been found within pig, deer, rabbit, cattle, goat and improved methods for the purpose of HEV detection, but camels, amongst other animals. HEV has also been detected also that further research through larger studies around the in shellfish meat as a result of contamination of their grow - world is required to identify the full host range of HEV and ing waters. Therefore, there is a risk of contracting HEV the risk of each potential host to transmit the virus to humans from undercooked products from these animals (although (through food or other means). In particular, the sugges- it is important to note that epidemiological evidence of tion that a HEV-related virus may be causing seropreva- foodborne transmission for many of these is currently lack- lence estimates to be higher than they genuinely are requires ing), and there is also potential for other livestock species investigation. to be unidentified hosts for the virus. Generally, foodstuffs Further studies identifying both the seroprevalence and containing raw meat or shellfish products are more likely the presence of HEV through ELISA and RT-PCR tech- to cause a foodborne infection than cooked foods or crops niques, respectively (or similar techniques identifying RNA due to no thermal inactivation of the virus through cooking. presence), would be best equipped to identify both the preva- Cooking in such a way that a minimum internal temperature lence of the virus within animal populations and the amount of 72 °C is reached for at least 20 min is likely to com- of active infections within the populations at that point in pletely inactivate any HEV present; however, this is likely time. However, sequencing technologies such as nanopore to produce unwanted deterioration of organoleptic qualities RNA sequencing within human and animal populations in some risky food types, e.g. shellfish. would also be useful to identify similarities between HEV In addition to animal meat, milk from cows, sheep, goats, sequences, enabling the identification of infection sources. donkeys and camels has also been found to contain HEV Some studies investigating the evolution of the virus have in some countries, but studies investigating the presence already been performed, but are often biased by the large of HEV in milk are much more limited. Because of this, amount of HEV sequences derived from humans (Forni et al. the true risk of HEV transmission from animal milk is yet 2018). unknown and requires further research. However, if proven Investigations of HEV within food and environmental to be a prominent transmission route for the virus, a wor- matrices using whole genome sequencing approaches have rying consideration is that high-temperature short-time been limited so far due to the general observation of low (HTST) pasteurisation of milk products, which is commonly genome copy numbers and fragmented HEV RNA within used in the UK and USA, may be insuc ffi ient to reduce infec - these matrices. However, techniques utilising methods tious HEV within milk, as generally the heat treatment used such as multiplexing RNA extracted samples to obtain a for HTST pasteurisation is only 72 °C for 15 s. Other pas- full genome from multiple amplicons, followed by MinION teurisation methods, such as ultrahigh-temperature pasteuri- next-generation sequencing, which has been successfully sation, which utilise treatments of around 135 °C for 2–4 s applied to sequencing of low levels of Zika virus (Quick should be more capable of removing viable virus from milk et al. 2017), could be instrumental in future efforts to iden- products due to the higher temperature. tify low levels of HEV in a variety of matrices, including Though crops can also become contaminated with HEV, foods. it seems that the risk of contracting HEV from them is much Globally, HEV is an under-recognised viral threat, which less likely, as confirmed outbreaks from crops have not been causes an increasing case incidence annually. The best way identified, and the HEV RNA prevalence and copies of viral to tackle a virus is to understand its sources and modes of RNA present are lower for these foods. However, it may be transmission. Therefore, further research and better under- safe to conduct further research into the contamination of standing of HEV will allow a better assessment of the risk irrigation water, and the presence of HEV in crops from that animal products and other foods may have in the trans- other countries to better assess the risk of contracting HEV mission of HEV to humans. In turn, this may allow the from crop contamination. introduction of legislative controls to prevent and control It has also been shown that marine mammals can be the spread of the virus. infected with HEV, which is concerning both from an eco- Supplementary Information The online version contains supplemen- system and a seafood point of view. If marine mammals are tary material available at https:// doi.org/10.1007/s12560-02 1-09461-5 . becoming infected naturally, it could be possible that fish 1 3 140 Food and Environmental Virology (2021) 13:127–145 Acknowledgements We would like to thank the anonymous reviewers contaminations are responsible for HEV transmission in pigs. for their help with submission of this article. This study did not gener- Veterinary Research, 44(1), 102. ate any new data. Anheyer-Behmenburg, H. E., Szabo, K., Schotte, U., Binder, A., Klein, G., & Johne, R. (2017). Hepatitis E virus in wild boars and spillo- ver infection in red and roe deer, Germany, 2013–2015. Emerg- Author Contributions ST was responsible for the idea, did the literature ing Infectious Diseases, 23(1), 130. search and data analysis for the writing, as well as drafting and criti- Arankalle, V. A., Chadha, M. S., & Chobe, L. P. (1999). Long-term cally revising the work. CW was involved in illustration creation and serological follow up and cross-challenge studies in rhesus mon- editing. CBA, BL and JL were responsible for editing and critically keys experimentally infected with hepatitis E virus. Journal of revising the work. Hepatology, 30(2), 199–204. Baechlein, C., & Becher, P. (2017). No evidence for zoonotic hepati- Funding Funding has been provided by Cefas, Seedcorn and the Uni- tis E virus infection through dairy milk in Germany. Hepatol- versity of Exeter for this work. Ben Longdon is supported by a Sir ogy, 65(1), 394–395. Henry Dale Fellowship jointly funded by the Wellcome Trust and the Baechlein, C., Schielke, A., Johne, R., Ulrich, R. G., Baumgaertner, Royal Society (Grant No. 109356/Z/15/Z). https ://wellc ome.ac.uk/ W., & Grummer, B. (2010). Prevalence of Hepatitis E virus- fundi ng/sir-henry -dale-fello wship s. specific antibodies in sera of German domestic pigs estimated by using different assays. 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