PREVALENCE AND MOLECULAR CHARACTERISATION OF HEPATITIS E VIRUS IN LITHUANIAN PIGS AND WILDLIFE

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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES

Ugnė Spancernienė

PREVALENCE AND MOLECULAR

CHARACTERISATION OF HEPATITIS E

VIRUS IN LITHUANIAN

PIGS AND WILDLIFE

Doctoral Dissertation Agricultural Sciences,

Veterinary (02A)

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Dissertation has been prepared at the Laboratory of Immunology at the De-partment of Anatomy and Physiology in the Faculty of Veterinary of Veterinary Academy in Lithuanian University of Health Sciences during the period of 2014–2018.

Scientific Supervisor

Prof. Dr. Arūnas Stankevičius (Lithuanian University of Health Sciences, Agricultural Sciences, Veterinary – 02A).

Dissertation is defended at the Veterinary Research Council of Lithua-nian University of Health Sciences:

Chairperson

Prof. Dr. Albina Aniulienė (Lithuanian University of Health Sciences, Agricultural Sciences, Veterinary – 02A).

Members:

Prof. Dr. Mindaugas Malakauskas (Lithuanian University of Health Sciences, Agricultural Sciences, Veterinary – 02A);

Assoc. Prof. Dr. Juozas Kupčinskas (Lithuanian University of Health Sciences, Biomedical Sciences, Biology – 01B);

Prof. Dr. Algimantas Paulauskas (Vytautas Magnus University, Biome-dical Sciences, Biology – 01B);

Dr. Asta Tvarijonavičiūtė (University of Murcia (Spain), Agricultural Sciences, Veterinary – 02A).

Dissertation will be defended at the open session of the Veterinary Research Council of Lithuanian University of Health Sciences at 14:00 on the 18th_of December 2018, in the Dr. S. Jankauskas auditorium of the Veterinary Academy of Lithuanian University of Health Sciences.

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LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETAS

Ugnė Spancernienė

HEPATITO E VIRUSO PAPLITIMAS

IR MOLEKULINIS CHARAKTERIZAVIMAS

LIETUVOS KIAULIŲ IR LAUKINIŲ GYVŪNŲ

POPULIACIJOSE

Daktaro disertacija Žemės ūkio mokslai,

veterinarija (02A)

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Disertacija rengta 2014–2018 metais Lietuvos sveikatos mokslų universiteto Veterinarijos akademijos Veterinarijos fakulteto Anatomijos ir fiziologijos katedros Imunologijos laboratorijoje.

Mokslinis vadovas

prof. dr. Arūnas Stankevičius (Lietuvos sveikatos mokslų universitetas, žemės ūkio mokslai, veterinarija – 02A).

Disertacija ginama Lietuvos sveikatos mokslų universiteto Veterinarijos mokslo krypties taryboje:

Pirmininkė

prof. dr. Albina Aniulienė (Lietuvos sveikatos mokslų universitetas, žemės ūkio mokslai, veterinarija – 02A).

Nariai:

prof. dr. Mindaugas Malakauskas (Lietuvos sveikatos mokslų universi-tetas, žemės ūkio mokslai, veterinarija – 02A);

doc. dr. Juozas Kupčinskas (Lietuvos sveikatos mokslų universitetas, biomedicinos mokslai, biologija – 01B);

prof. dr. Algimantas Paulauskas (Vytauto Didžiojo universitetas, biome-dicinos mokslai, biologija – 01B);

dr. Asta Tvarijonavičiūtė (Mursijos universitetas, žemės ūkio mokslai, veterinarija – 02A).

Disertacija bus ginama viešame Veterinarijos mokslo krypties tarybos posė-dyje 2018 m. gruodžio 18 d. 14 val. Lietuvos sveikatos mokslų universiteto Veterinarijos akademijos Dr. S. Jankausko auditorijoje.

Disertacijos gynimo vietos adresas: Tilžės 18, LT-47181 Kaunas, Lietuva.

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LIST OF ABBREVIATIONS

ALT – alanine aminotransferase bp – a base pair

CI – confidence interval CRE – cis-reactive element

ECDC – European Centre for Disease Prevention and Control EEA – European Economic Area

EFSA – European Food Safety Authority ELISA – enzyme-linked immunosorbent assay EU – European Union

EU/EEA – European Unionand European Economic Area Grp – glucose-regulated protein

HAV – hepatitis A virus HE – hepatitis E Hel – helicase

HEV – hepatitis E virus HEV-1 – HEV genotype 1 HEV-2 – HEV genotype 2 HEV-3 – HEV genotype 3 HEV-4 – HEV genotype 4 HEV-5 – HEV genotype 5 HEV-6 – HEV genotype 6 HEV-7 – HEV genotype 7 HEV-8 – HEV genotype 8

HEV-C1 – HEV genotype detected in rats HEV-C2 – HEV genotype detected in carnivores Hsc70 – heat shock cognate protein 70

HSPG – heparan sulfate proteoglycans HSS – hepatitis-splenomegaly syndrome HVR – hypervariable region

ICTV – International Committee on Taxonomy of Viruses IgG – immunoglobulin G IgM – immunoglobulin M IV – intravenous MT – methyltransferase NCR – noncoding region nt – nucleotide

OIE – World Organisation for Animal Health OR – odds ratio

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ORF – open reading frame (s) ORF1 – open reading frame 1 ORF2 – open reading frame 2 ORF3 – open reading frame 3

Pa – a papain-like cysteine protease PCR – polymerase chain reaction PCV2 – porcine circovirus type 2

PRRSV – porcine reproductive and respiratory syndrome virus RdRp – RNA-dependent RNA polymerase

RNA ribonucleic acid

RT-nPCR – nested reverse transcription polymerase chain reaction RT-PCR – reverse transcription polymerase chain reaction

US – United States UV – ultraviolet radiation

WHO – World Health Organization

X – macro domain

Y – Y domain

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INTRODUCTION

It is clear that recognition of the emergence of zoonotic diseases (com-monly referred to as “zoonoses” – infectious diseases that are naturally trans-mitted from vertebrate animals to humans and vice versa) is on the rise. This trend is a result of increases in both the rates of emerging zoonotic infections across the globe and our enhanced ability to detect and identify these infectious agents [247]. The World Organisation for Animal Health (OIE) estimates that 60% of existing infectious diseases in humans are zoonotic (e.g., viral–rabies, avian influenza, Ebola virus, severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus;

bacte-rial–anthrax, tuberculosis, brucellosis, salmonellosis, campylobacteriosis,

Q fever; parasitic–trichinellosis, cysticercosis; protozoan–trypanosomiasis). At least 75% of emerging infectious diseases in humans have an animal origin. Moreover, five new human disease appear every year and three of them originate from animals [167]. Changes in the environment, human behaviour, and human habitat contribute to an increasing incidence of zoonoses emerging not only from domestic animals, poultry, and livestock, but also from wildlife species [247]. One cannot forget that the fight against zoonoses starts by eliminating the pathogen at its animal source [167].

The hepatitis E virus (HEV), originally named “enterically transmitted non-A and non-B hepatitis”, was discovered in humans following a hepatitis outbreak of unknown etiology in Kashmir valley (India) in 1978 [115] and was molecularly characterized in 1983 [10]. In the 40 years since its disco-very, major advances have occurred as to its causative agent, the host range in the animal kingdom, epidemiology, and modes of dispersal [114]. In 1997, Xiang-Jin Meng et al. [144] reported for the first time the detection of HEV in pigs in the United States (US). Since then, HEV has been isolated from various species such as deer, wild boar, monkey, mongoose, rabbit, ferret, trout, bat, rat, chicken, kestrel, falcon, camel, and trout [52], some of which may have zoonotic potential.

HEV constitutes one of the five major human hepatotropic viruses along with the hepatitis A, B, C, and D viruses. HEV is unique in that among all known major hepatitis viruses, HEV is the only one with animal reservoirs [175]. Domestic pigs, wild boars, deer, rabbits, and dromedary camels com-prise the predominant reservoirs of HEV genotypes capable of infecting humans [220, 221], but other species may nevertheless also play a role. Besides well-known animal reservoirs, serological and molecular analyses have indicated infections with HEV or HEV-related viruses in a broad spect-rum of other animal species. These investigations included farmed animals,

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pets, laboratory animals, wild animals, or animals kept in captivity (e.g., zoos or animal sanctuaries). In most cases, the presence of HEV in these animals can be explained by spillover infections, but a risk of viral transmission through contact with humans cannot be excluded [219].

HEV is responsible for enterically transmitted acute hepatitis in humans and displays two distinct epidemiological patterns [64]. In endemic regions (i.e., in developing countries of Africa, East and South Asia, and Latin Ame-rica), large explosive waterborne outbreaks caused by HEV genotypes 1 (HEV-1) and 2 (HEV-2) are often linked to contamination of water supplies with human fecal material, representing a person-to-person mode of transmis-sion resulting in thousands of people affected. In contrast, in non-endemic regions (i.e., regions with high socioeconomic development), sporadic and cluster cases are caused by HEV genotypes 3 (HEV-3), 4 (HEV-4), and 7 (HEV-7) [206] that are represented in the animal kingdom [146, 175, 219, 221]. In this instance, HEV is a predominantly foodborne zoonosis, most commonly transmitted to humans consuming raw or undercooked meat and meat products or milk originating from animal reservoirs [88, 118, 125, 129, 221, 230]. Zoonotic HEV infections in humans may occur through vocational contact or via pig slurry, which leads to environmental contamination of agricultural products and seafood (e.g., shellfish) [112].

Interestingly, reservoir animals infected with HEV generally remain asymptomatic and lack visible macroscopic lesions. In experimental studies, however, piglets had microscopic evidence of hepatitis characterized by mild-to-moderate multifocal and periportal lymphoplasmacytic hepatitis with mild focal hepatocellular necrosis and, furthermore, pigs evidenced mildly-to-moderately enlarged hepatic and mesenteric lymph nodes [77, 144]. Chickens infected by avian HEV may develop a disease known as hepatitis-splenome-galy syndrome (HSS) [175]. In humans, HEV infection may be asymptomatic or cause an acute self-limiting hepatitis. However, fulminant hepatic failure can occur in pregnant women, elderly patients, or individuals suffering from underlying chronic liver disease. Immunosuppressed patients are at an increa-sed risk of developing chronic infection with severe disease progression and a fatal outcome. Hepatitis E (HE) viral infections can result in severe, fulmi-nant hepatitis as well as extrahepatic manifestations, particularly neurological and haematological disorders [52].

For the past 20 years, HEV has posed a significant health burden in many developing countries. Annually, there are an estimated 20 million infections wordwide, 3.3 million symptomatic cases, and over 56,600 HEV-related deaths [251]. Currently, there is evidence that HEV is an underrecognized pathogen in high-income countries and that the incidence of confirmed cases has been steadily increasing over the last decade [24]. According to data

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published by the European Centre for Disease Prevention and Control (ECDC), the number of confirmed HEV cases has been increasing annually, from 514 cases in 2005 to 5,617 cases in 2015, representing a ten-fold increa-se [54]. HEV is therefore a significant concern for public health worldwide.

According to Marquer et al. [137], pork is the most produced meat product in the European Union (EU); over 22 million tons of pork is produced annually, which accounts for 51% of all meat production in the EU, followed by poultry meat with 13 million tons per year (30%). Therein, pork production and manufacturing is potentially a significant source of foodborne disease transmission. The foodborne zoonotic source hypothesis is further streng-thened by the high sequence similarity of HEV gene sequences from patients with autochthonous hepatitis E and animal sources in the same region [129, 165].

To date, no significant data are available on HEV prevalence in Lithua-nian pigs and wild game species intended for human consumption. Infor-mation about the HEV prevalence in wild animals remains scarce, and the possible role of other mammals as HEV reservoirs is unknown. Likewise, few studies have been carried out to detect HEV infection in cervids (roe deer, red deer, moose) whose popularity as luxury meat products is growing globally, not only in countries around the Baltic Sea and in Scandinavia, North Ame-rica, and northern Asia where the Cervidae family is common.

Taking into consideration the aforementioned information and the increa-sing interest in the One Health Initiative (which highlights the interrelatedness of humans, animals, and the environment), we have been prompted, for the first time, to examine the findings from research undertaken between 2014−2018 in order to quantify the extent of exposure of Lithuanian pigs and wild ungulate populations to HEV infection. The main results during the work of this Ph.D. thesis are presented in the form of two published articles (see section “Copies of publications”). The results obtained fosters not only a more nuanced understanding of the modes of transmission and dynamics therein of HEV infection between wild fauna, domestic pigs, and humans within the Baltic States region, but also elucidates potential concerns for zoonosis and food safety with respect to Lithuanian pigs, wild boars, and roe deer that were actively infected with HEV or displayed evidence of previous infection.

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Scientific novelty

In Lithuania, the epidemiology of HEV infection in animals as well in humans remains largely unknown. No survey or research of HEV infection in farmed pigs and wild animals (with the exception of Lithuanian wild rats [215]) has been previously undertaken. Thus, the present work aimed to use serological, polymerase chain reaction (PCR), and sequencing assays to investigate whether HEV is present in farmed pigs and certain wild animals in Lithuania, as well as to determine the prevalent genotypes of HEV in these animals. Our research group aimed to contribute to the growing pool of knowledge about the roles of farmed pigs and various wild fauna in HEV transmission. These data are hopefully relevant both nationally and internationally as serological and molecular information remains scarce and controversial. The availability of the HEV sequences generated from this study may serve as a basis for future interdisciplinary studies comparing circulating strains in animals and humans and for tracing the source of infection in different countries. In our study, the open reading frame (ORF) 2 (ORF2) nucleotide sequences obtained from Lithuanian roe deer (Capreolus

capreolus) demonstrated for the first time that HEV subtype 3i can be found

in a novel host.

Aim and objectives

The aim of this thesis was to assess the prevalence of HEV in Lithuanian pigs and wildlife animal species using serological and molecular assays and to characterize circulating strains of HEV.

To this end, five objectives within the study were raised:

1. To identify the seroprevalence of HEV in domestic pigs (Publica-tion 1);

2. To estimate the seroprevalence of HEV in wildlife fauna species designated for human consumption (Publication 1);

3. To detect viral RNA directly and to assess the prevalence of HEV in domestic pigs samples by RT-PCR using two HEV-specific sets of primers targeting the ORF1 and ORF2 fragments of the HEV genome (Publication 2);

4. To determine the viral prevalence of HEV in wild ungulate samples by RT-PCR using two HEV-specific sets of primers targeting the ORF1 and ORF2 fragments of the HEV genome (Publication 2); and 5. To perform sequencing and phylogenetic analyses based on HEV

ORF2 for molecular characterization of circulating HEV strains in farmed Lithuanian pigs and wild ungulates (Publication 2).

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1. LITERATURE REVIEW

1.1. Classification and taxonomy

The nomenclature of HEV has been somewhat confusing [148]. HEV was first assigned to the Picornaviridae family and, later, to the Caliciviridae family based on the initial findings of its morphology and other physio-chemical properties [97, 227]. However, because the HEV genome does not share significant sequence homology with caliciviruses, the virus was subsequently declassified from the family Caliciviridae and reassigned to the newly created family Hepeviridae in 2009 by the International Committee on Taxonomy of Viruses (ICTV). Currently, HEV is placed in the sole genus

Hepevirus [221]. To date, there is a large variety of HEV-related viruses

identified in animals and humans. Identification of novel, genetically distinct strains of HEV from a number of animal species in 2015 led to the recent division of the family Hepeviridae into two genera by ICTV, namely the genus Orthohepevirus (containing all mammalian and avian HEV variants) and the genus Piscihepevirus (containing a highly divergent cutthroat trout virus placed within a single species) (Fig. 1.1.1) [52, 146, 219].

The genus Orthohepevirus contains four species designated as

Ortho-hepevirus A to D. OrthoOrtho-hepevirus A (HEV isolates from human, domestic pig,

wild boar, rabbit, deer, mongoose, and dromedary camel) contains seven genotypes (HEV-1 to HEV-7) and a putative new genotype 8 (HEV isolates from Bactrian camel) that can infect humans and a wide variety of other mammals [125, 216, 254]. Orthohepevirus B consists of avian viruses and is divided into four proposed subtypes (I–IV) which are mainly detected in domestic chicken. Orthohepevirus C (isolates from rat, greater bandicoot, Asian musk shrew, ferret, mink, and fox) includes two genotypes primarily detected in rats (HEV-C1) and carnivores (HEV-C2). Orthohepevirus D strains have been detected in several bat species. The genus Piscihepevirus contains only one single species: Piscihepevirus A, which has been identified in cutthroat trout and related species, although its pathogenicity and full host range are unknown. Notably, additional HEV sequences identified in mam-mals (moose−Sweden) and birds (little egret–Hungary; kestrel–Hungary) are distantly related to other HEV species and remain unclassified [170].

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F ig. 1.1.1. T ax onom ic al c las si fic at ion of H EV and H EV -r el at ed v ir us es (a dapt ed f rom [216 ]) N ot e: T he gr oup ing acco rd in g to fa m ily , g en us , s peci es , g en ot yp e an d t he co m m on h os ts ar e i nd icat ed . C ol ou rs in di cat e t he zo on ot ic p ot en tial of th e ge not ype s: or ang e ( hum an -to -hu m an t ra ns m is sio n o nl y) , r ed (a ni m al -h um an t ra ns m is sio n p ro ve n) , y el lo w (a ni m al -h um an tr an sm is sio n not pr ov en , b ut m ay be pos si bl e du e t o re la tions hi p t o hum an str ai ns or s er ol og ic al e vi de nc e) a nd gr ee n ( no a ni m al -hum an t ra ns m is sion ex pect ed ). H EV -C1 – H EV g en ot yp e d et ect ed in rat s; HE V -C2 – H EV g en ot yp e d et ect ed in car ni vo res (ad ap ted fr om [2 19 ]). 13

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Four main genotypes of HEV that belong to the Orthohepevirus A species are able to infect humans (HEV-1 to HEV-4). Genotypes 1 and 2 (HEV-1 and HEV-2) infect humans exclusively and are associated with large waterborne epidemics in tropical and subtropical areas (e.g., Asia, Africa, Mexico, Cuba, and Venezuela). Genotypes 3 and 4 (HEV-3 and HEV-4) are present in hu-mans, pigs, and animals hunted for game meat such as wild boar and deer and they are responsible for the most autochthonous cases of hepatitis E in indust-rialized countries. The first HEV-related agent identified in an animal was pig HEV. HEV ribonucleic acid (RNA) and HEV-specific antibodies were first detected in domestic pig in Nepal in 1995 [32]. Two years later, a pig strain of HEV was identified in pig herds from the US and genetically characterised [144].

Pig is the major reservoir of zoonotic HEV-3 and HEV-4 worldwide and is highly prevalent in pig herds. Indeed, anti-HEV antibodies were detected in 46–100% of pig farms from many countries [174, 175, 260]. HEV-3 and HEV-4 are also able to infect wild boars, which represent, along with domestic pigs, a major reservoir of zoonotic HEV [174, 175]. HEV-3 strains have also been detected in different species of deer and in the Japanese mon-goose [131, 159, 164]. In addition, rabbit HEV-3 strains have been identified in farmed rabbits in China [263] and the US [37], in farmed and wild rabbits in France [127], and in a pet house rabbit [27]. In the Orthohepevirus A species, two other strains of HEV (HEV-5 and HEV-6) have also been iden-tified only in wild boar in Japan and are not linked to human infections [216, 225]. More recently, HEV-7 has been detected in fecal samples from camels in Middle East [255] and in an immunocompromised transplant patient who regularly consumed camel milk and meat, suggesting zoonotic potential for this genotype [125]. The new genotype (HEV-8) which has been traced to Bactrian camels, could infect humans in a similar manner as HEV-7 [24].

Additional animal species infected with HEV (HEV-related viruses) have also been described. However, the HEV strains detected in these animals are more genetically distant from human HEV strains and are classified as differ-rent Orthohepevirus species. Avian HEV (Orthohepevirus B) was first des-crybed in the US and is associated with HSS (also called big liver and spleen disease) in chickens [78, 176]. Avian HEV is enzootic in chicken flocks in the US with a seroprevalence rate of 71% [84]. A strain of HEV has been identified in rats [92] with a seroprevalence rate varying from 13−90% in many countries [174]. Other HEV variants have been identified in ferrets in the Netherlands [190] and in mink in Denmark (Orthohepevirus C) [119]. Partial sequences with the highest homology to rat HEV have also been de-tected in foxes [16]. Another species of HEV (Orthohepevirus D) was identi-fied in different bats from Central America, Africa, and Europe [53]. Finally,

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a more distant strain of HEV has been discovered in the cutthroat trout in the US and assigned to the Piscihepevirus genus [12]. Very recently, a HEV strain that might represent a novel Orthohepevirus species has been found and characterized in kestrels and falcons in Europe [195]. A HEV strain has also been characterised in Swedish moose [132] but it is still not assigned to any HEV species [52]. However, the rapid identification of novel HEV variants from various animal species and accumulating sequence information neces-sitates constant revisions of HEV taxonomy in order to keep pace with advan-ces. [221]. Despite the extensive nucleotide sequence variations between mammalian HEV and avian HEV, it appears that there exists only a single serotype [74].

1.2. Genome

The HEV virion is a small, spherical particle of approximately 27–34 nm in diameter and has a single-stranded, positive sense RNA genome surround-ded by an icosahedral capsid [101, 198]. For many years, this virus has been described as a non-enveloped virus [20, 58, 82] however, another form has recently been identified. HEV can be “masked” by the membrane of the host cells and can be resistant to antibodies when this form is in blood [30, 226]. When HEV particles were released by the cellular exosomal pathway, they appeared to be similar to a “quasi-enveloped” virus. The resistance of the “quasi-enveloped” form is caused by the absence of viral antigens on the surface [24].

The HEV genome is approximately 7.2 kb in length and contains three ORFs, named ORF1, ORF2, and ORF3, in a 5ꞌ to 3ꞌ direction (Fig. 1.2.1) [146]. Overlapping reading frames ORF2 and ORF3 encode for frameshifted products from a bicistronic subgenomic mRNA species [70]. There is inter-species variation in the location of the ORF2/ORF3 junction, with the diver-gent variant cutthroat trout virus having an ORF3 that is displaced towards the middle of ORF2 [221]. ORF1, the longest of the three ORFs and en-compassing approximately two thirds of the viral genome, encodes a set of non-structural polyproteins (with putative domains, namely methyltransfe-rase (MT), Y (Y), papain-like cysteine protease (Pa), hypervariable region (HVR), X macro (X), RNA helicase (Hel), and RNA-dependent RNA poly-merase (RdRp) domains), that are involved in viral replication [146]. ORF2 encodes the the viral capsid protein that binds to cell surface heparan sulfate proteoglycans (HSPG) in liver cells and enables virus cell entry and it also contains immunological epitopes for antibody targets (i.e., the targets for neutralizing antibodies). Moreover, immunological studies of ORF2 have

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contributed to the development of a HEV vaccine [24, 147]. ORF3 is located between ORF1 and ORF2 and partially overlaps with ORF2, but neither overlaps ORF1. It encodes phosphoproteins that interact with the cellular cytoskeleton and endosomal system and is tentatively believed to play a role in viral egress from infected cells [105, 157]. A cap structure has been identified in the 5ꞌ end of the viral genome and may play a role in the initiation of viral genome replication and protein translation [146].

Fig. 1.2.1. A schematic diagram of comparative genomic organization of mammalian, avian, and fish HEV (adapted from [146])

Note: The three ORFs are labeled and shown as boxes. ORF2 overlaps ORF3, but neither overlaps ORF1. ORF1 encodes nonstructural proteins with the putative functional domains within ORF1 indicated inside the box. ORF2 encodes the capsid protein, and ORF3 encodes a small protein that is involved in virus replication. The genome is capped (7mG-Cap) at the 5′ end and contains a poly A tail at the 3′ end. There are noncoding regions (NCR) at the 5′ and 3′ ends of the viral genome. There is a junction region between ORF1 and ORF3 for mammalian and avian HEV, which contains a stem-loop structure and a cis-reactive element (CRE). The avian HEV genome is approximately 600 bp smaller than the mammalian and fish HEV. MT – methyltransferase; Y – Y domain; Pa – a papain-like cysteine protease; HVR – hypervariable region; X – macro domain; Hel – helicase; RdRp – RNA-dependent RNA polymerase; NCR – noncoding region.

1.3. Virion properties

HEV is mainly excreted fecally in pigs, leading to an accumulation of HEV in the environment of infected livestock. It can persist in the farm environment [108] but survival parameters are largely unknown [173]. Purcell and Emerson [186] noticed that the HEV virion is sensitive to iodinated disinfectants. Cook and van der Poel [35] showed that the HEV virion could survive refrigeration (4°C) and freezing (-20°C). Experiments

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performed with HEV in cell culture supernatant indicate that infectious HEV can be detected after one month of storage at room temperature and after more than two months of storage at 4°C [93]. Recently, Emerson et al. [57] determined that the HEV virion is more heat labile than is HAV (hepatitis A virus); HAV was only 50% inactivated at 60°C for one hour but was nearly completely inactivated at 66°C. In contrast, HEV was approximately 50% inactivated at 56°C and almost completely inactivated (96%) at 60°C. It has been demonstrated that the in vivo infectivity of HEV present in commercial pig livers is completely inactivated by adequate cooking methods such as frying (five minutes at 191°C) or boiling for five minutes, however, incu-bation of the HEV-contaminated pig liver homogenates at 56°C for one hour did not abolish the virus infectivity [60].

For water samples, chlorine treatment inactivates HEV and presents an effective strategy to control the virus’ waterborne transmission. In order to achieve a 1-log HEV reduction (90% inactivation), a chlorine concentration between 0.15–0.12 mg/L × min is required [69]. It is proposed that single-stranded RNA viruses such as HEV are particularly sensitive to ultraviolet (UV) radiation, which was confirmed by a HEV inactivation of 99.99% achie-ved using low UV ranging from 195−269 J/m2_{; this means that current UV} radiation guidelines (400 J/m2_{) represent a proper disinfection treatment for} HEV [72]. Notably, it is believed that HEV is resistant to inactivation by acidic and mildly alkaline conditions in the intestinal tract, making the fecal−oral route a favourable method of transmission [186].

1.4. HEV replication

HEV lacks both a proper in vitro culture system and an animal model [197] and the mechanism of viral replication remains hypothetical (i.e., poorly studied) (Fig. 1.4.1). Systems to cultivate HEV in vitro have only recently emerged [24]. The main target organ of HEV replication in the pig is the liver. However, studies on HEV dissemination in pigs infected experimentally by intravenous (IV) inoculation have identified extrahepatic sites of HEV replication. As expected, evidence of viral replication was found in the liver but also in the colon, lymph nodes (mesenteric and hepatic), and spleen for up to 20–27 days post-inoculation [253]. The virus RNA was also detected in the stomach, small intestine, spleen, kidney, salivary glands, tonsils, or lungs (14 days post-inoculation). Viral RNA, but no viral replication, was detected in the muscles at 14 days post-infection, at which time the pigs also had viremia [253]. In a recent study, comparing the course of HEV infection in pigs via contact infection or IV inoculation, the presence

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of HEV RNA was confirmed in the longissimus, biceps, femoris, and iliopsoas muscles at 27 days post-inoculation in IV-inoculated pigs and 31 days in contact-infected pigs [19]. These HEV RNA-positive organs may result from viremia rather than from active virus replication, but they may nevertheless present a risk of HEV contamination of pork products. It has been shown that the results obtained after contact infection or IV inoculation of pigs varied, suggesting that the route of HEV infection may influence viral dissemination [175].

The cellular receptor for HEV is not yet known but the presence of HSPG appears to be necessary for the binding of the virus to target cells [99]. Additionally, experiments investigating ORF2 peptide binding suggested that the C-terminal region of ORF2 would allow the entry of the virus through binding to heat shock cognate protein 70 (Hsc70) on the cell surface [265]. Once it has bound, HEV enters the cell by endocytosis, which appears to depend on dynamin-2, clathrin, and membrane cholesterol [81, 106]. The pathway of the virus within the cell remains unknown, but heat shock protein 90 and tubulin may play a role in the intracellular transport of HEV virions [264]. Subsequently, the RNA is released in the cytoplasm of the cell but the mechanisms and precise localization of this process have not yet been delineated. Once the viral RNA is free in the cytosol, translation of the ORF1 polyprotein will occur due to its 5ꞌ cap. Following the translation of methyltransferase, protease, helicase, and RNA polymerase activities, the genomic RNA is copied into a strand of negative RNA, which then allows the synthesis of genomic and subgenomic RNAs [188].

Rehman et al. [191] have shown that the 3ꞌ end of the genome binds specifically to the viral replicase on the surface of the endoplasmic reticulum. ORF2 and ORF3 are then translated to produce the structural proteins (i.e., capsid protein and phosphoprotein) that will allow the encapsidation of the newly synthesized viral RNA strands. This makes it possible to obtain new viral particles [70, 257]. A glucose-regulated protein (Grp) belonging to the heat shock protein 70 (Hsp70) family (Grp78) may play a role in the folding and assembly of the capsid proteins [259]. The proteins encoded by ORF2 and ORF3 interact, which suggests a role for ORF3 in the assembly of viral particles [235]. HEV is not cytolytic [228]. Moreover, studies have shown the requirement of the exosomal pathway for virion release [155, 158]. Recent studies suggest that viruses secreted into the bloodstream are associated with ORF3 protein and lipids, but viruses secreted into the bile are non-enveloped [155, 156].

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Fig. 1.4.1. Putative replication cycle of HEV (adpted from [24])

Note: Step 1: HEV in the extracellular environment. Step 2: Attachment of HEV to the target cell involving heat shock protein. Step 3: Receptor binding. Step 4: Clathrin-dependent endocytosis. Step 5: Encapsidation and liberation of genomic RNA. Step 6: RNA translated into nonstructural ORF-translated protein 1 (pORF1). Step 7: Replication of RNA+ into negative-sense transcripts. Step 8a: Synthesis of subgenomic RNA. Step 8b: Synthesis of full-length positive sense transcripts. Step 9: Translation of subgenomic RNA into ORF2 and ORF3 proteins. Step 10: Capsid formation and assemblage of new virions. Step 11a and 11b: Exit of the virus via the ORF3 proteins fixed on the endoplasmic membranes or the cell wall. Step 12: Mature virions attached to ORF3 protein and lipids (quasi-enveloped form in bloodstream) or free (in bile). HSPG – heparan sulfate proteoglycans.

1.5. HEV hosts and reservoirs

Hepatitis E viruses are being identified in an increasing number of animal species. Initial detections in animals were reported in pig, chicken, and deer in 1997, 2001, and 2003, respectively [78, 144, 230]. More recently, HEV sequences have been detected in rats, wild boars, monkeys, mongooses, rabbits, ferrets, cutthroat trout, and bats [12, 53, 94, 98, 133, 164, 190, 223, 263]. All of these species are potential natural hosts of HEV but just a few of them have been identified as true reservoirs of HEVs. Defining a true reservoir as a population in which the pathogen can be permanently maintai-ned and from which it is shed to a defimaintai-ned target population [80], to date only

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domestic pig, wild boars, and deer should be regarded as true reservoirs of zoonotic HEVs [240]. The ever-expanding host range and identification of new animal reservoirs (e.g., rabbits and camels) pose a significant concern for zoonotic HEV infection but also offer new opportunities for developing useful animal models for HEV [146]. However, members of the Hepeviridae family is increasing each year with the identification of novel HEV variants from a large diversity of animal species. Therefore, it is quite probable that other animal reservoirs for HEV exist. HEV strains with extensive genetic diversity present in certain animal species may be undetectable using mole-cular techniques that are based on known HEV sequences [174].

1.6. Interspecies transmissions and zoonosis

To investigate whether the different HEV genotypes and recently identi-fied Orthohepevirus species are potentially zoonotic and transmissible to humans, animal models have been developed [174]. HEV-1 and HEV-2 within the Orthohepevirus A species have a rather limited host range and are restricted to humans because attempts to experimentally infect other species, including pigs, rats, and goats, with human HEV-1 and HEV-2 were unsuc-cessful. Lambs and Wistar rats were reportedly infected by-presumably-a genotype 1 human HEV, although others failed to infect goats or rats with HEV-1 and HEV-2 [149]. In contrast, HEV-3 and HEV-4 have a much broader host range and can infect across species barriers (Table 1.6.1).

Table 1.6.1. Host range of HEV infection and zoonotic risk [146] Natural animal

host (genus/species, Classification genotypes [gt])

Experimental hosts for

cross-species infection infection in Zoonotic humans Orthohepevirus A

Human gt 1, 2, 3, 4 Non-human primates, pigs (gt 3, 4), rabbits (gt 1, 4), lambs (gt 1), Wistar rats (gt 1) Domestic pig gt 3, 4 Non-human primates, rabbits,

Mongolian gerbils (gt 4), Balb/C mice (gt 4)

Yes

Wild boar gt 3, 4, 5, 6 Yes (gt 3, 4), likely (gt 5, 6)

Deer gt 3 Yes

Rabbit gt 3 Pigs Likely

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Table 1.6.1. Continuation Natural animal

host (genus/species, Classification genotypes [gt])

Experimental hosts for

cross-species infection infection in Zoonotic humans

Camel gt 7 Yes

Moose Unknown Not known

Orthohepevirus B

Chicken Avian HEV gt 1, 2, 3 Turkeys No

Orthohepevirus C

Rat Unlikely

Ferret Unlikely

Greater bandicoot Unlikely Asian musk shrew Unlikely

Mink Unlikely

Bat Orthohepevirus D No

Cutthroat trout Piscihepevirus No Note: gt – genotype.

Experimental infections have shown that HEV can cross the barrier from domestic pig to wild boar. Oral and IV inoculation of wild boar HEV strains to miniature or domestic pigs can lead to viremia, excretion of HEV in the feces, and seroconversion [211, 234]. In addition, wild boars naturally infected or inoculated intravenously with HEV are able to transmit the virus by contact to domestic or miniature pigs [211, 212]. These data strongly suggest that transmission via the fecal–oral route between domestic and wild pig is possible and can occur naturally when pigs and wild boars are reared in close proximity. Such interactions between domestic pigs and wild boars are common as some breeds of domestic pigs are raised outdoors in semi-open or open spaces in close contact with wildlife [173].

Under experimental conditions, pig HEV-3 and HEV-4 can readily infect non-human primates and, conversely, human HEV-3 and HEV-4 can infect pigs [9, 143]. The avian HEV from a chicken successfully infected turkeys, but failed to infect rhesus monkeys, suggesting that avian HEV is likely not zoonotic. Rabbit HEV-infected pigs, and human HEV-1 and HEV-4, also reportedly infected rabbits [148]. Genetic elements that may be important for virus cross-species contamination appear to be related to the non-structural protein (encoded by ORF1, which is likely involved in the host range). Additionally, the importance of other domestic animals such as cattle, sheep, and goats, or wild animals such as mongooses or rats for HEV transmission to humans needs to be studied in greater detail. Generally, more research on

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the HEV transmission pathways via different animal species other than domestic pigs and wild boars is required to estimate the risk of zoonotic transmission of HEV from these animals and to develop effective preventa-tive strategies for people in contact with the animals.

From a public health perspective, genotypes 3, 4, and 7 are known to cross species barriers and thus they possess the greatest zoonotic potential [219]. Furthermore, serological evidence of HEV infection has been reported in a number of other species, despite the fact that the source of seropositivity in these species has yet to be genetically identified. Anti-HEV antibodies have reportedly been detected in several ruminant species such as goats, cattle, and sheep, as well as in dogs and cats with no detection of HEV-related sequences. Definitive genetic identification of the sources for HEV seropositivity in these animal species will lead to the discovery of new animal strains of HEV and thereby further expand our understanding of the HEV host range and possi-bility of subsequent transmission to humans [146]. More data are required to clarify the reservoir status of these animal species for zoonotic strains of HEV. Various animals, such as moose, rats, ferrets, bats, and several species of birds have been reported to carry host-specific variants of HEV but there is currently no evidence for zoonotic transmission. To summarize, the disco-very of animal strains of HEV that can infect across species barriers has revolutionized the way we think about this important disease [146].

1.7. Transmission pathways to humans

The transmission pathways of HEV are complex and may involve virus transmission via fecally contaminated water, food, environment, blood pro-ducts, and direct contact with animals and other humans [111]. Some of these pathways are well established whereas others are mere speculation. Most importantly, the transmission pathways are dependent on the genotype of the virus [219]. HEV-1 and HEV-2 are mainly restricted to humans and have been responsible for large HE outbreaks in the past. According to the World Health Organization (WHO) [251], one third of the world’s population – comprising over two billion people – are living in areas highly endemic for these genotypes, including South-East Asia, the Middle East, India, Central Asia, Central America, and South America. Approximately 20 million HEV infections are reported for Africa and Eastern Asia annually [192]. Contami-nation of drinking water and food with human excretions is suspected to be the major route of transmission for HEV-1 and HEV-2. Low hygienic stan-dards and limited access to clean water therefore pose a high risk for the occurrence of HE outbreaks in developing countries. In households, sharing

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of utensils for eating and drinking during an HEV outbreak may also contri-bute to virus transmission. Direct person-to-person transmission of HEV is possible, although this has been described only rarely. Infections with HEV-1 are often associated with high mortality rates (HEV-15–25%) in pregnant women [109]. Vertical transmission from infected mothers to their babies has also been observed [110]. Since HEV exists in its non-enveloped or “quasi-enveloped” form, it is resistant in the environment and, therefore, water-borne transmission is also possible in developed countries when untreated water is ingested [24].

In keeping with the objectives established earlier in this thesis, we focu-sed our efforts more on zoonotic HEV genotypes (HEV-3 and HEV-4, for which domestic pigs, wild boar, and deer are the main reservoirs) and their possible transmission pathways to humans. Human infections occur through three methods, namely zoonotic foodborne consumption, direct contact with infected animals, and environmental contamination by animal manure run-off (Fig. 1.7.1) [260].

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F ig. 1.7.1. Zoonot ic tr an sm is si on of H E i n de ve lope d and m any de ve lopi ng c ount ri es [112 ] 24

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Wild boars, sika deer, and domestic pigs cross-transmit HEV [113] and eating their parboiled flesh or liver, considered a delicacy in many countries, may be responsible for the autochthonous cases and outbreaks of HE [152]. A more common method of the spread of HEV is through consumption of raw livers from supermarkets or eating Corsican figatelli sausage in Europe [17, 33, 67]. Approximately 2% of the commercial pig livers from local grocery stores in Japan, 4% in Germany, 6.5% in the Netherlands, and 11% in the US tested positive for the zoonotic HEV-3 RNA. Importantly, the contaminating virus in the commercial pig livers remains fully infectious and cooking the contaminated meat at a temperature similar to a medium-to-rare cooking conditions did not completely inactivate the virus. Approximately 6% of the sausages sampled at processing and at the point of sale in Spain were also positive for the zoonotic HEV-3 RNA [146].

Vocational exposure (e.g., veterinarians, pig handlers, slaughterhouse workers) to domestic pig farms, manure, or sewage is also a significant risk factor for HEV infection in many countries [18, 180, 203]. Pig veterinarians and other workers in contact with pig were 2−5 times more likely to test posi-tive for immunoglobulin G (IgG) anti-HEV than non-pig workers and the general population in many European countries [17, 66, 169, 245]. Moreover, hunters, field workers, and forestry workers in close contact with wildlife animal species displayed a significantly higher anti-HEV antibody prevalence than controls [107, 145], demonstrating that direct and indirect contact with infected animals presents a risk for HEV exposure. This raises questions about the need for screening blood products for HEV RNA prior to transfu-sion [220]. Acquisition of HEV through the use of porcine-derived medical products such as heparin, insulin, or coagulation factors has never been con-vincingly demonstrated but remains a theoretical possibility given the ubiquity of HEV in pig populations [41]. A cross-sectional survey conducted in China has also found a higher anti-HEV IgG seroprevalence in seafood processing workers who have direct contacts with raw seafood [42]. Direct contacts with contaminated food and water may then represent a risk of HEV infection.

Contact with infected pigs or their organs (e.g., pet pig, surgery training, slaughterhouse worker, butchers) was reported as a confirmed source of HEV and was recorded in three reports [34, 180, 193] but living in a pig-dense area where farm biosecurity and waste management is carefully controlled was not associated with an increased risk of contracting HEV [242]. Interestingly, traditional behaviour in some countries can potentially pose a risk for HEV exposure as novice hunters in Portugal, for instance, are called to perform the typical hunting baptism which consists of rubbing freshly collected wild boar liver with their faces [62]. Additionally, pig slurry can cause environmental

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contamination through several ways. Use of pig slurry as pasture can infect agricultural products such as raspberries, strawberries, and many vegetables [21, 248]. Run-off from outdoor pig farms causes contamination of surface water as well as produce receiving that surface water [222, 236]. Run-off from outdoor pig farms can also contaminate coastal waters and seafood such as fish and shellfish (including oysters, flat oysters, mussels, and clams), which is suspected to occur via bioaccumulation from the water and viral concent-ration in their digestive tissues [160]. In addition, HEV has been identified in commercially available mussels from several European countries [39, 51], South Korea [217], and Japan [130]. Consumption of shellfish has been iden-tified as a risk factor for HE in several case reports [23, 85, 194] and in an outbreak of HE aboard a cruise ship [205]. Thus, contaminated shellfish pose a health risk for people who eat them raw or slightly cooked. Lastly, recent data on the spread of HEV-7 through camel flesh or camel milk and detection of HEV-4 in high titers in cows’ milk in mixed animal farms from China opens up a new vista for exploring the transmission routes of HEV infection [112]. Raw or undercooked pork meat or pork liver sausages are the most frequently reported food products associated with sporadic cases or outbreaks of HEV. Food-borne transmission appears to be the major pathway for human HEV infections in Europe [56].

1.8. Geographic distribution of HEV genotypes

The global distribution of HEV has distinct epidemiological patterns based on ecology and socioeconomic factors [112]. Forni et al. [64], in order to obtain an overview of the geographic distribution of Orthohepevirus A genotypes, updated epidemiological surveys [3, 55, 68, 116, 117, 168, 172] with recent literature reports of molecularly characterised HEV infections as well as with information on HEV strains identified in animals (Fig. 1.8.1). Genotypes were assigned to countries irrespective of their prevalence. Thus, even if a single case was reported in a given country, the genotype was recorded as present. Cases that could be clearly ascribed to migration or other forms of travels were excluded [64].

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Fig. 1.8.1. Geographic distribution of the four major HEV genotypes (HEV-1 to HEV-4) (adapted from [64])

In addition, Sridhar et al. [220] created a summarized schematic map with overall geographical distributions of zoonotic HEV genotypes (i.e., HEV-3, HEV-4, HEV-7, and HEV-8) and HEV-related viruses (detected in bats, chickens, ferrets, mongooses, moose, rabbits, and rats) in terrestrial animals (Fig. 1.8.2).

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Fig. 1.8.2. Geographical distribution of zoonotic HEV (HEV-3, HEV-4, HEV-7, HEV-8) genotypes and HEV-related viruses in terrestrial animals

(adapted from [220])

Note: Pigs, wild boar, and deer have been definitively implicated in zoonotic transmission of HEV-3 and HEV-4 to humans, camels – zoonotic transmission of HEV-7 and HEV-8 (probably, more evidence is needed) to humans. The role of rats, bats and ferrets in causing human hepatitis E needs is still matter of debate and need to be studied in more detail.

1.9. Clinical outcomes in animals

The pathogenesis of HEV has thus far only been studied in pig. The majo-rity of pig become infected between the ages of 2−4 months, consistent with high seroprevalence rates in pigs older than four months [175]. Commonly, pigs are infected subclinically and do not evince macroscopic liver lesions but may display mild multifocal lymphoplasmacytic hepatitis lesions microsco-pically [170]. Pigs become infected by direct contact with feces from infected conspecifics or by exposure to HEV-contaminated drinking water or feed. Infected pigs generally have a short viremia with a duration of 1−2 weeks (Fig. 1.9.1).

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Fig 1.9.1. HEV infection response [170]

The period from time of infection to shedding of the virus via feces can range from 1−4 weeks. Anti-immunoglobulin M (IgM) antibodies can first be detected 1−3 weeks following infection and can remain for 10 weeks following the initial infection. The anti-IgG response usually becomes detectable 1−3 weeks following infection and can persist for many months or years. Most neonatal piglets have passively derived antibodies (via colostrum absorption) which persist up to 2−3 months of age and are considered protective. Once these antibodies wane, most pigs contract HEV and develop an effective immune response [170].

Hepatitis E virus infections have little impact on animal health as the animals display no evident clinical symptoms. Likewise, pigs naturally ted with pig HEV are asymptomatic. In a prospective study of naturally infec-ted piglets in a US farm, gross pathological lesions were absent in the liver and 18 other tissues and organs in four pigs necropsied during early stages of pig HEV infection [144], although all four piglets had microscopic evidence of hepatitis characterized by mild to moderate multifocal and periportal lymphoplasmacytic hepatitis with mild focal hepatocellular necrosis. Similar to natural infections, pigs experimentally infected with pig HEV and human HEV-3 exhibited no clinical signs [77], although the infected pigs did have mildly-to-moderately enlarged hepatic and mesenteric lymph nodes from 7−55 days post-inoculation. Histological lesions including mild-to-moderate multifocal lymphoplasmacytic hepatitis and focal hepatocellular necrosis were also observed [149].

Importantly, Pavio et al. [173] highlighted an additional concern of the possible effects that the presence of other pig pathogens may have on the time-course of natural HEV infections within herds. Frequent co-circulation

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of HEV with immunomodulatory viruses such as porcine reproductive and respiratory syndrome virus (PRRSV) or porcine circovirus-2 (PCV2) may influence HEV pathogenesis. A fatal disease associated with co-infection of HEV and PCV2 was recently described in piglets. General hyperthermia, hemorrhage, inflammatory cell infiltration, and necrosis were observed in the post-mortem piglet tissues [258]. In addition, experimental co-infection of pigs with HEV and PRRSV demonstrated increased and prolonged excretion of HEV in co-infected pigs. This co-infection led to an increased length of HEV excretion, estimated at 49 days relative to the 9 days’ duration typical for HEV infection alone. An exacerbated transmission, on average four times greater than in a single HEV infection, was also observed [207]. Infections of pigs by multiple concurrent pathogens must, therefore, be taken into conside-ration to develop effective preventative measures against HEV propagation and prolonged excretion that may affect the presence of HEV at the time of slaughtering [173].

The only HEV variant that causes more severe symptoms in animals is avian HEV associated with HSS and big liver and spleen disease [13]. In contrast to pig, barnyard fowl (e.g., chickens) infected with avian HEV can exhibit clinical manifestations [78, 154, 176]. Clinical signs of an acute infection with avian HEV consists of hepatomegaly, splenomegaly, kidney modifications, growth depressions, decline of laying performance, accumu-lation of abdominal blood fluid, hepatic vasculitis, amyloidosis, or increased death among poultry [4, 154].

Available data on clinical signs caused by HEV in wild boars remains quite sparse. In one study, no difference was found between the biometric characteristics (i.e., body length and weight) of HEV-infected and non-infected wild boars [138]. In another report, no clinical manifestations were observed in a viremic male wild boar negative for anti-HEV IgG [218]. Experimental infection of 3-month-old wild boars with a wild boar HEV-3 strain via the IV route or by contact caused subtle clinical symptoms such as reduced feed intake and mild diarrhea that were concomitant with increased bile acids, alanine aminotransferase (ALT), and gamma-glutamyl transferase (γGT) [211]. Mild lymphoplasmacytic hepatitis was also recorded. Higher viral loads of HEV were found in the feces of the infected wild boars in comparison to the feces of miniature pigs infected under the same conditions [211]. HEV RNA was also detected in the liver, gall bladder, small and large intestine, and spleen of the infected wild boars. Chronic infections in two wild boars naturally infected with HEV-3 have also been reported [212]. In these two animals, viremia or viral shedding in the feces was detected for 12–16 weeks although high titres of anti-HEV antibodies were simultaneously pre-sent in their serum. No clinical signs or histopathological lesions indicative

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of hepatitis were observed and HEV RNA was not detected in the liver or other tissues of the chronically infected wild boars [212].

Since HEV infection is asymptomatic in pig, HEV diagnosis is not routinely performed in pig farms [175]. HEV serology or detection of HEV RNA is performed only in research laboratories that have developed assays adapted for pig [14, 28, 59, 45, 91, 177]. HEV genotype 3 and 4 strains present in animals are genetically quite similar to human HEV genotypes 3 and 4 and therefore cannot be distinguished by specific RT-PCR amplification [36, 75, 96, 249]. However, increased importance should be placed on routine HEV screening in pig herds, especially in countries with large-scale industrial pig farming, to monitor the levels of contamination in this large animal reservoir [175].

1.10. Clinical outcomes in humans

HEV is one of the leading causes of acute viral hepatitis worldwide. However, the course and clinical presentation of HEV infection in humans is highly variable [24]. The incubation period ranges from 15 days to nine weeks, with an average of 40 days. [24]. The prodromal phase is quite variab-le and can manifest as asthenia, fever, and digestive disorders for several days followed by an icteric phase lasting two weeks. Accordingly, it is not surprising that most cases remain undetected at the acute stage. This can be followed by jaundice, elevated liver enzymes, abnormal liver function tests (ALT, aspartate aminotransferase, γGT, alkaline phosphatase, and bilirubin), abdominal pain, and hepatosplenomegaly [56]. Hepatitis is caused by an immune reaction directed towards the infected hepatocytes. Acute cytolytic hepatitis is the most common symptom. In most cases, the majority (> 70%) of infections are asymptomatic and people only seroconvert [73], the outcome is favourable, and biological parameters normalise within three months [163]. However, progression to severe fulminant liver failure (1–2% of cases) may occur in certain high-risk groups such as pregnant women and elderly patients with underlying liver disease [220]. The mortality rate associated with HEV infection (1–4%) is higher than that of hepatitis A (0.1–2%), and can reach up to 30% during pregnancy during outbreaks in developing countries [146, 187]. Cholestatic forms occur in 20% of cases [86]. In industrialized count-ries, symptomatic HEV infections mostly affect men over 55 years of age [136].

Hepatitis E infections affect not only the liver but also produce extra-hepatic manifestations affecting several other organ systems, causing

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logical symptoms, organ injuries, or haematological disorders [56]. Hepa-titis E infection has been associated with neurological disorders such as Guillain-Barre syndrome, Parsonage-Turner syndrome, neuralgic amyotro-phy, bilateral brachial neuritis, peripheral neuropathy, and encephalitis [43, 102, 103, 179, 181, 209, 238, 239, 231]. Other extrahepatic manifestations are renal injuries, including membranoproliferative glomerulonephritis with or without cryoglobulinaemia and membranous glomerulonephritis, acute pancreatitis, and other autoimmune manifestations such as myocarditis, arthritis, and thyroiditis [76, 100, 101]. Thrombocytopaenia and other haematological disorders have also been observed [65, 256]. Most of these presentations were described in solid-organ or bone marrow transplant recipients [1, 47, 126, 182]. Immunosuppressed patients are at risk of develo-ping chronic HEV infections with severe disease progression and fatal outcomes [56]. Hepatitis E diagnosis is confirmed by the detection of anti-HEV IgM in serum or anti-HEV RNA in serum or stool samples [56].

1.11. Food safety and prevention

Food-borne transmission of HEV occurs mainly through consumption of products originating from reservoir animals, especially consumption of raw or inadequately heat-treated meat and offal [56]. The omnipresence of HEV infection in pigs and the detection of HEV replication in the liver raised the concern for the presence of HEV in slaughtered pigs and in livers sold in grocery stores [175]. In order to study whether heating (cooking) would inactivate HEV, small pieces of HEV RNA-positive livers were cooked at different temperatures and the residual infectious virus was measured in a pig bioassay [60]. The results showed that deep frying (191°C) or boiling (five minutes) can effectively inactivate HEV, although a treatment of one hour at 56°C did not inactivate HEV [60]. These results underline that contaminated food products, if consumed raw or undercooked, could potentially transmit the virus to humans [175]. Another study revealed that efficient inactivation of HEV in food products derived from infected pork liver was only achieved after a cooking time of at least 20 minutes at an internal temperature of 71°C [11, 60]. Therefore, some food preparations, such as mid-dry liver sausage, should be consumed after cooking to minimize potential HEV infection [175]. Appropriate hygiene measures such as frequent hand and surface cleaning should also be followed when handling uncooked meat.

Many studies recommend heightened awareness of the possibility of increased risk for HEV exposure when consuming grilled wild boar meat, sashimi of sika deer, sushi (slice of raw meat), Figatelli sausage, shellfish

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(experimental bioaccumulation in oysters was successful with pig HEV-3), mussels, clam (can concentrate HEV, mostly in their digestive tissues), soft fruits (e.g., strawberries and raspberries) and vegetables irrigated with contaminated surface water, dried salted liver, quenelle paste, dried or fresh liver sausages, pork pate, blood sausage, camel-derived products (milk or flesh), and ready-to-eat processed pork pies [17, 21, 33, 51, 56, 62, 67, 112, 130, 152, 160, 194, 229, 236, 248].

Prevention of zoonotic HEV relies mainly on avoiding raw and undercooked pig, wild boar, venison meat and offal, or shellfish and cooking meat and meat products thoroughly [52]. Furthermore, risk of direct or indirect exposure to HEV can be minimised by instantiating proper workplace measures. A simple measure to prevent HEV infection for pig handlers is to wash their hands thoroughly with soap and water after handling pigs. Inte-restingly, simple preventative measures such as wearing specialized gloves and boots for pig farmers, forestry workers, or hunters are associated with reduced risk of HEV exposure [31, 210]. Moreover, hunters who used protec-tive gloves on a regular basis had a 88% lower anti-HEV prevalence as compared to hunters disembowelling wild boars in the same area but never, seldom, or sometimes wearing gloves. Therefore, wearing protective gloves during disembowelling and paying attention to abrasions on the skin when handling hunted game should be encouraged [210]. The knives for disembowelling as well as other utensils (e.g., chopping boards) for raw venison and offal should be used only for these purposes to avoid cross-contamination. Hands should be properly washed after disembowelling and handling of wild boar, venison, or their offal. In addition, pig waste should be properly eliminated and the use of pig manure as soil fertiliser should be regulated to reduce the risk of HEV contamination of crops and surface water [52]. Hunters and others handling carcasses as well as the general public should receive education about HEV transmission associated with game mammals [56]. Generally, increased public awareness and education on zoonotic HEV transmission and prevention is strongly needed.

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2. MATERIAL AND METHODS

2.1. Source and collection of samples

The study was carried out in various randomly selected geographical areas of Lithuania during 2014−2018. A subset of serum from domestic pigs was assembled by veterinarians from submissions received for official surveillance programs of infectious disease from the National Food and Veterinary Risk Assessment Institute, a subordinate body of the State Food and Veterinary Service of the Republic of Lithuania. Wildlife sampling (serum and liver) was biased towards the hunting seasons by hunters. A summarised schematic drawing of investigated samples and research workflow is depicted in Fig. 2.1.1.

2.2. Serological analysis

The detection of anti-HEV bodies for both pigs (n=384) and wildlife (n=623) was conducted using the commercially available ID Screen Hepatitis E Indirect Multi-species enzyme-linked immunosorbent assay (ELISA) kit (IDvet, France) based on recombinant capsid protein of HEV-3 according to the manufacturer’s instructions. The division of domestic pigs and wild boar samples according to their age distribution is described in the “Materials and Methods” section of Publication 1.

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F ig. 2.1.1. G en er al sch em e o f r es ea rch w or kf lo w 35

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2.3. Molecular analysis

Viral RNA was isolated from pig (n=470) and wild animal (n=306) serum and wild animal liver (n=320) samples with the GeneJET RNA Purification Kit (Thermo Fisher Scientific) according to the manufacturer’s recommendations. The extracted RNA was analyzed by RT-nPCR using two HEV-specific sets of primers targeting the ORF1 and ORF2 fragments of the HEV genome. Primers used are listed in Table 1 of Publication 2. The HEV-positive ORF2 RT-nPCR products were excised from the agarose gel, purified with a GeneJET PCR Purification Kit (Thermo Fisher Scientific), and sequenced in both direc-tions using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Bio-systems) and the 3130xl Genetic Analyzer (Applied BioBio-systems) at the DNA Sequencing Centre at Vilnius University Institute of Biotechnology (Lithua-nia). All ORF2 sequences obtained in the study were deposited in GenBank with accession numbers MG739304–MG739318. Sample preparation, RNA extraction, performance of RT-nPCR, sequencing, and phylogenetic analysis are described in detail in the “Methods” section of Publication 2.

2.4. Statistical analysis

Statistical analysis was performed using Microsoft Excel 2007 and the

SPSS for Windows 15 statistics package (SPSS Inc., Chicago, IL, USA). The

results were considered significant at P < 0.05. The descriptive data are presented as percentages. Fisher’s exact test was used to test for differences in seroprevalence between different animal groups and differences in viral prevalence of HEV in the target regions. To determine the correlation between the presence of HEV in different animal species and herd size, odds ratios (OR) and their corresponding 95% confidence intervals (CI) were calculated using binary logistic regression analysis. Prevalence of HEV RNA was similarly calculated in pigs and wild animal species for the ORF1 and ORF2 sequences with 95% CIs.

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3. RESULTS AND DISSCUSION

3.1. Serological analysis

Detailed results and discussion of the prevalence of markers for past (serological assay, anti-HEV IgG antibodies) is presented in the “Results” and “Discussion” sections of Publication 1. The following is a general summary.

Domestic pigs are the main animal reservoirs of HEV worldwide. Since the discovery of pig HEV in 1997 [144], numerous publications have shown high seroprevalence rates of HEV in pig herds (up to 100%) throughout the world. Each continent is affected: Asia (China, India, Indonesia, Japan, South Korea, Mongolia, Philippines, Taiwan, Thailand, and Vietnam), North and South America (Argentina, Bolivia, Brazil, Canada, Cuba, and Mexico), Africa (Cameroon, Democratic Republic of Congo, Nigeria, and Madagas-car), Europe (Belgium, Czech Republic, Finland, France, Germany, Hungary, Italy, the Netherlands, Romania, Spain, Sweden, Switzerland, and the United Kingdom) and Oceania (Australia, New Caledonia, and New Zealand) [8, 22, 36, 46, 134, 144, 150, 171]. Seroprevalences were estimated between 5−100% [8, 22, 46, 134, 144, 150, 171]. Thus, our finding of 43.75% seroprevalance in domestic pigs (Publication 1) were not surprising given that HEV infection appears to be universal in pigs [148].

All studies converge toward the notion that infection of pigs occurs at an early age following the loss of maternal antibodies [61]. In the present study, seroprevalence was found to be age-dependent. Weaned pigs presented the highest seroprevalence (53.66%), and this value was significantly (P < 0.01) more frequent than in pigs reared for fattening (35%). In particular, the seroprevalence was significantly higher (P < 0.01) in sows (52.94%) compared to pigs reared for fattening. However, results of the age distribution of HEV infection in domestic pig populations vary by country. As described by others [44, 213], seroprevalence increases with age as a consequence of repeated contact with the virus [50]. Unexpectedly, our results indicated that seropre-valence decreased in pigs reared for fattening (35%), perhaps due to the mixing of pigs from different farms and the absence of sows in these herds or due to the absence of maternal antibodies and pigs are not yet infected with HEV.

The presence of HEV in urine, in addition to feces, may contribute to the high transmissibility of HEV between animals, which also contributes to its accumulation and persistence in the animal environment. Furthermore, in pig farms, dynamics of infection are variable as a result of differential farm mana-gement practices, hygiene (including effective disinfection of pig housing and equipment between batches), biosecurity measures, and influences of differ-rent farming systems [173]. Our results therefore support the notion put forth

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by Pavio et al. [175] that wide variations in within-herd seroprevalence rates appears to be the norm in HEV-infected herds.

Substantial evidence has been gathered in the past 20 years indicating that wild boar is also an important reservoir of HEV. It is not surprising that wild boars (Sus scrofa) are also susceptible to HEV as they are closely related to domestic pigs (Sus scrofa domesticus) [173]. The first report suggesting that HEV can also infect wild boars came from the detection of HEV anti-bodies in wild-caught pigs in Australia at the end of the 1990s [29]. Several studies have been undertaken, mainly in Japan and Europe, to determine the prevalence of HEV antibodies. Seroprevalences ranging from 1.6−41.6% and from 4.9−57.4% were found in Japan and Europe, respectively [173]. A relatively high HEV seroprevalence rate (57.05%) was detected in Lithuanian wild boar serum samples. These data clearly show that HEV infection is widely present in wild boars in Japan and in Europe [173], including Lithua-nia, and that wild boar likely represent a reservoir of HEV in these areas. Several factors such as the geographical location [2, 22, 25, 26, 79, 87, 124, 138, 139, 151, 208, 224], the year of sampling [7, 95, 151, 208, 224], the wild boar density [15, 124, 210], and the managing conditions [44, 151] can impact HEV prevalence. In our study, adult wild boar presented the highest seroprevalence (P < 0.05) rate (80.0%) compared to piglet or yearling wild boar. This finding aligns with the results reported by other groups [22, 25, 26, 95, 141, 151, 233] who usually found that higher HEV seroprevalences ten-ded to occur in adults and subadults rather than in juveniles. Interestingly, no difference in seroprevalence was observed between male and female wild boars [79, 138, 141, 151, 153, 206, 210, 214]. Comparisons of data obtained using the same serological assay have highlighted higher HEV seropreva-lences in wild boars (57.05%) than in domestic pigs (43.75%) sampled in Lithuania. These data suggest that HEV circulates at higher rates and more progressively in wild boars than in domestic pigs. The fact that the latter are reared under intensive conditions, whereas wild boars are free-ranging and reside in lower population densities, causes a unique type of aggregation not found in the wild that may account for these discrepancies. It is also possible that in domestic pigs, a weak or short-lasting protective immunity against HEV leads to chronic infections or re-infections of older animals.

HEV has also been detected in deer. Anti-HEV IgG antibodies have been found in several deer species such as red deer (Cervus elaphus), roe deer (Capreolus capreolus), sika deer (Cervus nippon), fallow deer (Dama dama), and white-tailed deer (Odocoileus virginianus) in America (Canada and Mexico), Asia (China and Japan), and Europe (Belgium, Czech Republic, France, Germany, Hungary, Italy, the Netherlands, Spain, and Sweden) [7,

PREVALENCE AND MOLECULAR CHARACTERISATION OF HEPATITIS E VIRUS IN LITHUANIAN PIGS AND WILDLIFE

Ugnė Spancernienė